MECHANISM OF 12/15 -INDUCED RETINAL MICROVASCULAR DYSFUNCTION IN DIABETIC RETINOPATHY

By

Khaled El Masry

Submitted to the Faculty of the Graduate School

of Augusta University in partial fulfillment

of the Requirements of the Degree of

(Doctor of Philosophy)

May

2018

COPYRIGHT© 2018 by Khaled El Masry

ACKNOWLEDGEMENTS

I would like to thank my mentor Dr. Mohamed Al-Shabrawey for his help during my Ph.D. studies. I would like to thank all current and previous lab members, particularly

Dr. Ahmed Ibrahim and Dr. Khaled Hussein for their continuous help and support since I joined the lab. I would like to acknowledge my committee members; Dr. Ruth Caldwell,

Dr. David Fulton, Dr. Manuela Bartoli and Dr. Amany Tawfik for their great suggestions and guidance. I would like to thank my thesis reader Dr. Yutao Liu who is a great collaborator as well.

I would like to thank the Graduate School for the continuous support for the students. Special thanks to Dr. Mitchell Watsky and Dr. Patricia Cameron, who offered me a non-stop help. I would like to thank the Cell Biology and Anatomy graduate program for the extended environment of knowledge that the graduate students enjoy during their training period. I specially thank Dr. Sylvia Smith and Dr. Mark Hamrick.

I would like to thank everyone who taught me something one day since I was born. Thanks are due to all my teachers since kindergarten till my Ph.D. studies. Great appreciation is due to my family that tolerated me and supported me throughout all these years particularly my father (Dr. Mohammad Elmasry), my mother (Mrs. Hanem

Hussein) and my brothers (Dr. Moaataz Elmasry and Dr. Ahmad Elmasry). Special thanks to my dear wife (Dr. Samar Habib) and my lovely kids (Anas, Albaraa and

Aesha). I would dedicate this work and any success that I did or I will achieve to the soul of my father whom I lost one week before carrying my bags and travel to USA to start my Ph.D. My dad insisted that I have to travel to gain my Ph.D. while he was in the hospital after a major surgery and he felt that these days were his last ones. My dad was keen that I succeed and I hope I was able to make his wishes true.

ABSTRACT

KHALED EL MASRY

Mechanism of 12/15 Lipoxygenase-Induced Retinal Microvascular

Dysfunction in Diabetic Retinopathy

(Under the direction of MOHAMED Al-SHABRAWEY)

Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO- induced retinal changes in diabetic retinopathy. We used in vivo and in vitro approaches.

For in vivo studies, experimental was induced in wild-type (WT) mice and

12/15-Lo (also known as Alox15) knockout mice (12/15-Lo−/−); ER stress was then evaluated after 12-14 weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb)

(Nox2−/− mice). In vitro studies were performed using human retinal endothelial cells

(HRECs) treated with 15-HETE (0.1 µmol/l) or vehicle, with or without ER stress or

NADPH oxidase inhibitors. This was followed by evaluation of ER stress response,

NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca2+) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated

RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leukocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO-derived metabolites both in vitro and in vivo. We also found that 15-HETE increases the intracellular calcium in HRECs. ER stress contributes to 12/15-LO-induced retinal in diabetic retinopathy via activation of NADPH oxidase and VEGFR2. Perturbation of calcium homeostasis in the retina might also play a role in linking 12/15-LO to retinal ER stress and subsequent microvascular dysfunction in diabetic retinopathy.

KEY WORDS: Bioactive lipids; Calcium; Diabetic retinopathy; ; ER stress;

12-HETE; 15-HETE; 12/15-Lipoxygenase; NADPH oxidase; VEGFR2

Table of Contents

Chapter Page

1 Chapter I: INTRODUCTION ...... 2

A) Statement of the problem and specific aims of the overall project...... 2

B) Literature review ...... 7

B.1. Retina: Development, Anatomy and Physiology...... 7 B.1.1. Eye and Retina Development ...... 7 B.1.2. Macroanatomy of the Retina ...... 10 B.1.3. Microanatomy of the Retina ...... 12 B.1.4. Blood Supply of the Retina ...... 22 B.1.5. The Outer and Inner Blood Retinal Barriers ...... 24 B.1.6. Retinal Physiology ...... 25 B.2. Retinal Pathology ...... 27 B.2.1. Diabetic Retinopathy ...... 28 B.3. Bioactive Lipids ...... 40 B.3.1. Eicosanoids ...... 40 B.3.2. Bioactive Lipids and Retinopathies ...... 48 B.3.3. 12/15-LO (ALOX15) ...... 51 B.4. ER Stress ...... 59

2 Chapter II: MATERIALS AND METHODS ...... 62

3 Chapter III: RESULTS...... 70

4 Chapter IV: DISCUSSION ...... 85

5 Chapter V: SUMMARY...... 91

REFERENCES ...... 93

List of Tables

Table 1: Abbreviations list ...... 1

Table 2: Antibodies used in the experiments ...... 65

Table 3: Primers used in the experiments ...... 67

List of Figures

Figure 1: Conceptual frame of the central hypothesis of the proposed studies ...... 6

Figure 2: Eye and retinal development ...... 8

Figure 3: Eye anatomy illustration ...... 11

Figure 4: Retinal cellular organization ...... 15

Figure 5: Bioactive lipids metabolism...... 41

Figure 6: ER stress and UPR...... 61

Figure 7: Loss of 12/15-LO reduces diabetes-induced ER stress in mouse retina ...... 72

Figure 8: 15-HETE induces ER stress in HRECs ...... 74

Figure 9: ER stress induction triggers endothelial leukocyte adhesion in HRECs, while

ER stress inhibition abrogates 15-HETE-induced leukocyte adhesion ...... 76

Figure 10: Crosstalk between NADPH oxidase and ER stress

in 15-HETE-treated HRECs ...... 78

Figure 11: Nox2 deletion has no effect on 12-HETE-induced ER stress in vivo ...... 80

Figure 12: 15-HETE induces intracellular Ca2+ release in HRECs ...... 82

Figure 13: ER stress inhibition attenuates 15-HETE-induced VEGFR2

phosphorylation...... 84

Figure 14: A graphical summary ...... 92

Table 1. List of abbreviations

Abbreviation Term AA AMD Age related macular degeneration ATF Activating transcription factor BRB Blood retinal barrier CNV Choroidal neovascularization COX Cyclooxygenase DHA Decosahexaenoic acid DHE Dihydroethidium eIF2α Eukaryotic initiation factor 2α ER Endoplasmic reticulum ERAD ER-associated degradation EPA Eicosapentanoic acid HETE Hydroxyeicosatetraenoic acid HODE Hydroxyoctadecadienoic acid HREC Human retinal endothelial cell ICAM Intercellular adhesion molecule IRE1 Inositol-requiring 1 LA LT Leukotriene LO Lipoxygenase NOX NADPH oxidase PBA 4-Phenylbutyric acid PDI Protein disulfide isomerase PEDF Pigment epithelium-derived factor PERK RNA-dependent protein kinase-like ER-regulated kinase PG Prostaglandin PUFA Polyunsaturated fatty acids ROP Retinopathy of prematurity ROS RPE Retinal pigment epithelium S1P Sphingosine-1-phosphate STZ Streptozotocin TX Thromboxane UPR Unfolded protein response UPS Ubiquitin–proteasome system VEGF Vascular endothelial growth factor VEGFR VEGF receptor XBP1 X-box binding protein 1

1

I. INTRODUCTION

A: Statement of the problem and specific aims of the overall project:

Diabetic retinopathy is the leading cause of vision loss in the working-age population worldwide (Congdon et al., 2004). It is characterised by an early inflammatory response that leads to disruption of the blood retinal barrier. This ultimately leads to diabetic macular oedema and retinal neovascularisation (Leal et al., 2007). The mechanism of diabetic retinopathy has not yet been completely elucidated and the current therapies are limited by several side effects. Therefore, there is a need to investigate the underlying mechanism of diabetic retinopathy that could lead to identification of new therapeutic targets (Cheung et al., 2010).

Upregulation of adhesion molecules and subsequent leukocyte adhesion to endothelial cells (leukostasis) play a crucial role in the pathogenesis of retinal hyperpermeability and neovascularisation in diabetic retinopathy (Miyamoto et al., 1999,

Bai et al., 2003). In addition to hyperglycaemia, dyslipidaemia and increased levels of bioactive lipids were considered major contributors to microvascular dysfunction seen in diabetic retinopathy (Lupo et al., 2013). 12/15 lipoxygenase (12/15-LO) is one of the lipoxygenase superfamily that incorporates two oxygen atoms at position 12 or 15 of arachidonic acid and related substrates, generating bioactive lipid metabolites (Horn et al., 2015). Among these metabolites, 12- and 15-hydroxyeicosatetraenoic acids (HETEs) elicit pro-inflammatory and chemotactic properties in various disease models

(Li et al., 2013, Kuhn and O'Donnell, 2006) including neuronal damage (Pallast et al.,

2

2010) and diabetic retinopathy (Gubitosi-Klug et al., 2008). Our previous studies showed significant increases in 12- and 15-HETE levels in the vitreous of individuals with diabetic retinopathy and in the retinas of Akita mice, a model of type 1 diabetes (Al-

Shabrawey et al., 2011, Othman et al., 2013). In addition, we showed increased retinal permeability, inflammatory mediators such as intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1), and retinal neovascularisation in normal mice by intravitreal injection of 12-HETE (Ibrahim et al., 2017). In contrast, genetic deletion of 12/15-Lo (also known as Alox15) significantly reduced retinal permeability in diabetic mice and attenuated neovascularisation in the mouse model of oxygen-induced retinopathy (Al-Shabrawey et al., 2011, Ibrahim et al., 2015a).

Consistent with our in vivo findings, inhibition of endothelial 12/15-LO in human retinal endothelial cells (HRECs) reduced high-glucose-induced leukostasis and ICAM-1 expression (Ibrahim et al., 2017). These findings indicate that 12/15-LO plays a causal role in diabetic retinopathy; however, the underpinning mechanism is yet to be determined.

Endoplasmic reticulum (ER) is the cell organelle responsible for proper protein folding, calcium homeostasis and lipid biosynthesis. Growing evidence suggests that ER stress acts as a major sensor that regulates cell energy metabolism, inflammation, redox status and cell survival under diabetic conditions (Li et al., 2009, Zhong et al., 2012,

Wang et al., 2017, Ma et al., 2014, Zhang et al., 2015). Earlier studies have linked

12/15-LO signalling with the ER stress response (Cole et al., 2012). There are three branches of transducer proteins that recognise ER stress, triggering a cellular response known as the unfolded protein response (UPR): RNA-dependent protein kinase-like ER- 3

regulated kinase (PERK), inositol-requiring enzyme 1 (IRE1) and activating transcription factor (ATF) 6. Disturbance of any of the ER functions can trigger UPR. The first arm of

UPR is the PERK pathway, which is initiated by PERK phosphorylation. Phospho-PERK inhibits phosphorylation of eukaryotic initiation factor 2 α (eIF2-α), which decreases the protein overload in the cell followed by the selective activation of ATF4. The second pathway of UPR includes IRE1, which homodimerises and trans-auto-phosphorylates; then, by using its endonuclease activity, it splices the mRNA of X-box binding protein 1

(XBP1) to form spliced XBP1 (XBP1s). ATF6 is the third signalling pathway in UPR. It is translocated to Golgi apparatus, where it is cleaved. Activation of UPR depends to a great extent on the duration of the insult and how long the cell is exposed to the stress. These pathways cause activation of transcription factors like ATF4, XBP1s or cleaved ATF6 to translocate to the nucleus, where they stimulate the transcription of UPR genes, such as calnexin and protein disulfide isomerase (PDI) (Ma et al., 2014, Zhang et al., 2015, Cole et al., 2012).

Several reports showed that , as well as the perturbation of calcium homeostasis and elevated levels of cytosolic calcium, contribute to ER stress in different disease models (Cao and Kaufman, 2014, Fu et al., 2011). Previously, we have demonstrated NADPH oxidase (NOX) to be a major source of oxidative stress in retinas of diabetic rodents and have also demonstrated the existence of a crosstalk between

12/15-LO and the NOX system both in vitro and in vivo (Othman et al., 2013, Ibrahim et al., 2015a, Al-Shabrawey et al., 2008). However, the interplay between ER stress,

NOX and calcium homeostasis in 12/15-LO-mediated microvascular dysfunction during diabetic retinopathy remains unclear. 4

The goal of the current study was to investigate how 12/15-LO mediates microvascular dysfunction in diabetic retinopathy by portraying its causal relationship with ER stress and calcium homeostasis. Furthermore, we aimed to characterise the crosstalk between the two stress-response pathways—ER stress and NOX-mediated oxidative stress—in 12/15-LO-induced microvascular dysfunction using both genetic and pharmacological approaches.

My central hypothesis is that activation of ER stress and NADPH oxidase contributes to 12/15-LO-mediated retinal endothelial cell dysfunction in diabetic retinopathy. My hypothesis predicts a) activation of ER stress in retinal endothelial cells and mouse retina by 12/15-LO metabolites 12- or 15-HETE, b) inhibition of ER stress attenuates 12/15-LO mediated retinal endothelial cell (REC) dysfunction, c) presence of a cross talk between

12/15-LO-induced ER stress and NADPH oxidase activity in mediating REC dysfunction.

The proposed hypothesis (Fig.1) was investigated via the following specific aims:

Aim1. 12/15 LO-derived lipid metabolites activate ER stress response in retina.

Aim2. 12/15 LO-induced retinal microvascular dysfunction is mediated through ER stress/oxidative stress interaction.

5

Fig.1: Conceptual frame of the central hypothesis of the proposed studies. In the current study, we proposed to investigate if 12/15-lipoxygenase enzyme induces retinal ER stress during diabetes. We aimed to investigate if there is any interplay between 12/15-lipoxygenase-induced retinal NADPH oxidase activation during diabetes and retinal ER stress as well.

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B: Literature review:

B.1. Retina: Development, Anatomy and Physiology

 B.1.1. Eye and Retina Development:

The eye is derived from different embryonic sources. First; the retina, the optic nerve and back of the iris are derived from the neuroectoderm of the forebrain. Second; the lens and epithelial layer of the cornea are derived from the surface ectoderm. Third; the fibrous and vascular coats of the eye are derived from the mesoderm between the neuroectoderm and the surface ectoderm. Finally; the choroid, sclera and of the cornea are derived from neural crest cells (Ludwig and Czyz, 2018). Early after gastrulation (formation of the three embryonic layers; ectoderm, mesoderm and endoderm), at the center of the anterior neural plate; a single eye field is specified under the influence of eye field transcription factors such as Paired box 6 (Pax6), Retina and anterior neural fold homeobox (Rax), Sine Oculis Homeobox Homolog 3 (Six3), and

LIM Homeobox 2 (Lhx2) (Zuber et al., 2003). This single eye field will be divided later into two eye fields under the influence of sonic hedgehog (SHH) and Six3 transcription factors (Geng et al., 2008, Jeong et al., 2008).

The human eye development starts at day 22 of the embryonic development by the appearance of the optic groove on the sides of the forebrain which is derived from the cranial end of the neural tube of the developing embryo (Fig.2A). Then with closure of the cranial end of the neural tube, these grooves change into optic vesicles (Fig.2B) which are continuous with the forebrain cavity (Heavner and Pevny, 2012). The connection of the optic vesicles and the forebrain will form optic stalks which will give rise to optic nerves later. The optic vesicles come in contact with the surface ectoderm 7

with subsequent induction of its thickening to form the lens placode (Fig.2B). The lens placode invaginates and forms lens pits. Lens pits fuse to form lens vesicles (Fig.2C).

The lens vesicles then lose their connection with the surface ectoderm. Simultaneously the optic vesicles invaginate to form double layered optic cups connected to the forebrain via the optic stalks. The outer layer of the optic cup will form the retinal pigmented epithelium while the inner layer will form the neural retina (Eiraku et al., 2011)

Fig.2: Eye and retinal development. The eye develops from the forebrain (A) which is derived from the cranial end of the developing embryo. (B) Formation of the optic vesicles on the sides of the forebrain which remain connected with the cavity of the forebrain. Contact of the optic vesicles to the surface ectoderm induces its thickening to form the lens placode. (C) invagination of the surface ectoderm into the optic vesicle forms the lens vesicle and the double layered optic cup. (D) separation of the lens vesicle from the surface ectoderm to form the primordium lens. The double layered optic cup will form the retina and RPE. 8

(Fig.2D).

The adult retina has six different types of neuronal cells (retinal ganglion cells, cone and rod photoreceptors, amacrine cells, bipolar cells, horizontal cells) derived from multipotent retinal progenitor cells. Moreover, retina has glial cells which are believed to arise from retinal progenitor cells as well. In mouse retina; retinal ganglion cells, horizontal cells, cone photoreceptors, and some of amacrine cells are developed during the first wave of retinogenesis which occurs prenatally from embryonic (E) day 11 to day

18 (E11–E18). While, during the second wave of retinogenesis that occurs postnatally

(P0–P7), rod photoreceptors, bipolar interneurons, and Müller glial cells are developed

(Rancic et al., 2017).

Angioblasts develop from a group of precursors that exist in the developing human retina and then migrate to the nerve fiber layer. It was reported that stem cell factor (SCF) and stromal derived factor-1 (SDF-1) are associated with the retinal precursors’ differentiation into angioblasts and their migration later to the sites where the vessels will develop (Hasegawa et al., 2008). During development, the retinal vascularisation starts from the central region of the retina and then extends centrifugally towards the periphery of the retina. It is initiated via the process of vasculogenesis where new endothelial cells are derived from vascular precursor cells. This is followed by the formation of new blood vessels from already existing endothelial cells by the process of angiogenesis. The retinal vascularization is mediated mainly via vascular endothelial growth factor (VEGF) signalling which is essential for the development, remodelling, and preservation of the retinal circulation (Lutty and McLeod, 2018, Gariano, 2003).

9

 B.1.2. Macroanatomy of the Retina:

The retina is the light-sensitive part at the back of the eye which converts the light signals into neuronal signals (Fig.3). The retina can be divided anatomically into two parts; central retina and peripheral retina. The central retina (also known as the macula) is located in adult human retina nearly 3 mm temporal to the optic disc and is about 1.5 mm in diameter. At the central part of the macula there is a depression called the fovea which represents the area of maximum visual acuity in the retina, because it has the highest density of cone photoreceptors. The fovea contains only cone photoreceptors and some

Müller cells. The fovea has a Rod-free zone (where there are no Rod photoreceptors) and a foveal avascular zone (where there are no retinal capillaries). So, the fovea is dependent on choriocapillaris for its blood supply. The remaining part of the retina is called the peripheral retina. The ora serrata is the part which demarcates the end of the sensory retina and the commencement of the ciliary body. At the ora serrata the sensory retina is reduced to a single layer of cells which becomes anteriorly the non-pigmented ciliary epithelium while the retinal pigment epithelium is switched to pigmented ciliary epithelium (Kolb, 1995b).

10

Fig.3: Eye anatomy illustration. The eye is divided into an anterior part and a posterior part. The vitreous and the retina occupy the posterior portion of the eye. Retinal branches of the central retinal artery that enters the eye within the substance of the optic nerve ramify on the vitreal side of the retina to form the retinal circulation. The magnified part is to show that the retina is formed of more than one layer.

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 B.1.3. Microanatomy of the Retina:

Retinal cells and organization of retinal layers:

The main cellular components of the retina are: the retinal pigment epithelium, the photoreceptors, the interneurons, the ganglion cells, the glial cells and the vascular cells.

The retina is arranged into 10 layers (Fig.4) which are -from outside inward-

1. Retinal Pigment Epithelium (RPE)

RPE is derived from the neuro-ectoderm. RPE cells are hexagonal in shape and form a single layer of cuboidal epithelium. It is separated from the chorio-capillaries by

Bruch’s membrane. The tight junctions between this epithelial monolayer form the outer blood retinal barrier (BRB) which selectively separates the retinal photoreceptors from blood in the choroid (Bonilha, 2014, Rizzolo, 2014).

2. Photoreceptor Layer

The light-sensitive part of the retina in which rods and cones form a single layer of photoreceptors (Anderson and Fisher, 1976). The photoreceptors are the sensory retinal neurones responsible for the photo-transduction process in which light photons converted into neuronal signals. Two types of photoreceptors are present in the human retina, rods and cones. Rods are responsible for dark vision (also known as scotopic vision) (Sharpe et al., 1989). Cones are responsible for colour vision (also known as photopic vision). A single photoreceptor is made up of an outer segment, an inner segment, a nucleus, an inner fibre and a synaptic terminal (Swaroop et al., 2010). The outer segment has the photo-pigment that captures the photons while the inner segment contains the endoplasmic reticulum, the mitochondria and the microtubules which are necessary for the high metabolic activity of the photoreceptors (Molday and Moritz, 2015). Both 12

segments are connected via a non-motile cilium. The plasma membrane of the outer segments is arranged into hundreds of horizontal discs. Glutamate was described as an important neurotransmitter at the synaptic terminals of photoreceptors during the photo- transduction process (Yau, 1994).

3. Outer Limiting Membrane

It is formed by junctional complexes of neighbouring Müller cells in addition to the junctional complexes between Müller and photoreceptor cells (Omri et al., 2010).

4. Outer Nuclear Layer

It is formed of the nuclei of the photoreceptor cells (Kolb, 1995b).

5. Outer Plexiform Layer

It is the site of the synaptic connections between photoreceptor cells and the connecting neurones including the bipolar and horizontal cells (Brzezinski and Reh,

2015).

6. Inner Nuclear Layer

It contains the nuclei of the horizontal, the bipolar, the amacrine, the inter-plexiform, and the Müller cells. They have specific organization where the horizontal cells are closer to the outer plexiform layer, amacrine cells closer to the inner plexiform layer, while the bipolar, inter-plexiform, and Müller cells are in between (Kolb, 1995b). The connecting neurons (bipolar, horizontal, amacrine, and interplexiform cells) form the inner nuclear layer of the retina. They transmit the visual signals via connecting the photoreceptor layer with the ganglion cell layer. Their synapses form the outer plexiform layer with the synaptic terminals of photoreceptors while they form the inner plexiform layer with the dendrites of the ganglion cells. Bipolar cells receive signals from both rods and cones. 13

Rod bipolar cells may be connected up to 70 rods, but cone bipolar cells may be connected to only one cone (Vecino et al., 2016).

7. Inner Plexiform Layer

It is the site of the synaptic connections between bipolar and amacrine cells in the inner nuclear layer and ganglion cells in the ganglion cell layer (Kolb, 1995b, Kolb,

1995a).

8. Ganglion Cell Layer

It contains ganglion cells along with other cells, including some amacrine cells, pericytes, endothelial cells, and astrocytes. The ganglion cells are collecting the visual information from the retinal photoreceptors and project these data to the parvocellular and magnocellular layers of the lateral geniculate nucleus of the brain. Their cell bodies form the ganglion cell layer while their dendrites participate in the inner plexiform layer where they connect with bipolar and amacrine cells. Their axons collect in the nerve fibre layer and form the optic nerve. Due to the long distance between the retina and the brain, the axonal transport of the ganglion cells is highly active and requires ATP which is supplied by the active axonal mitochondria (Yokota et al., 2015, Yu et al., 2013,

Nuschke et al., 2015).

9. Nerve Fibre Layer

Axons of the ganglion cells collect in this layer travelling to the optic nerve head.

10. Inner Limiting Membrane

The inner processes of Müller cells expand on the vitreal surface of the retina to form this membrane in which the collagen fibres of the vitreous are inserted (Kolb, 1995b,

Kolb, 1995a). 14

10.

9. Nerve fiber layer 8.

7. Inner plexiform layer

6. Inner nuclear layer

5. Outer plexiform layer

4. Outer nuclear layer 3. Outer limiting 2. membrane

1.

Fig.4: Retinal cellular organization.

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Retinal Glial cells:

There are different types of glial cells in the retina including Müller cells, astrocytes and micoglia. Müller cells are the most numerous type of glial cells that exist in the retina (Reichenbach and Bringmann, 2013). Their cell bodies are located in the inner nuclear layer while their cell processes extend across the whole retina. These processes extend proximally to form the inner limiting membrane which separates the retina from the vitreous (Vecino et al., 2016). Distally, they form the outer limiting membrane. The distal processes of Müller cells are in contact with each other. They are attached to the inner segments of the photoreceptors as well. This connection is formed by continuous adherence junctions that together form the outer limiting membrane.

Studies suggested the outer limiting membrane as a barrier that can be interrupted during diseases as diabetic retinopathy (Omri et al., 2010). Laterally, they surround the retinal neurons and retinal blood vessels. Müller cells are important for supporting retinal neurons and maintaining the integrity of blood retinal barrier. Astrocytes are believed to be derived from optic nerve stem cells. They surround retinal blood vessels, ganglion cells, and nerve fibres in the superficial layers of the retina (Jiang et al., 1994, Escartin et al., 2007). Microglial cells are the retinal resident . They are derived from progenitor cells found in yolk sac during early embryonic development (Elmore et al.,

2014, Kierdorf et al., 2013). An interesting recent study suggested the presence of additional two extra retinal origins for the retinal microglia that include residual microglia at the optic nerve and macrophages of the ciliary body or the iris (Huang et al.,

2018)

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Glia cells are highly important for the proper function of the retina. They display several important functions. For instance, processes of glia cells form boundaries separating blood vessels from neural retina. They actively participate in the retinal blood barrier as their processes surround the blood vessels controlling the movement of different materials across the BRB. Moreover, they form sheaths around neuronal cells of the retina ensuring their interaction and support of the neural retina (Vecino et al., 2016).

Their structural support of the neural retina is suggested during the developmental stage because of their elastic properties. The retina stretches during development with an increase in the intraocular pressure. The retina continues to grow after birth and be stretched more. The components of the retina should be elastic during that time. The retinal neurons are considerably firmer than the glial cells. The elastic properties of retinal glial cells help to support the retinal neurons during this developmental period (Lu et al., 2013, Lu et al., 2006).

The ability of Müller cells to regulate the homeostasis of extracellular pH and

K+ ions enable them to play a critical role in maintaining the integrity of the BRB. Müller cells get rid of the increased extracellular K+ particularly in the inner and outer plexiform layers, by releasing K+ into either the blood or the vitreous. Thus, Müller cells prevent possible K+ - induced depolarization of retinal neurons (Reichenbach et al., 2007,

Reichenbach and Bringmann, 2013). Müller cells and astrocytes regulate the extracellular space volume, metabolites clearance, K+ buffering, and calcium-related signalling via aquaporin molecules particularly aquaporin 4 (AQP4) which are located in their cell membranes (Lan et al., 2017).

17

Glial cells are metabolically active. Müller cells can tolerate hypoxia and hypoglycemic circumstances via activating anaerobic glycolysis and by oxidizing other substrates, for instance, lactate, glutamate or glutamine for gaining energy through ATP

(Kitano et al., 1996, Reichenbach and Bringmann, 2013). The retina utilizes more

ATP in comparison with other tissues via Warburg effect or aerobic glycolysis in which lactate is produced from glucose via oxidative phosphorylation in spite of the presence of oxygen (Casson et al., 2013, Ng et al., 2015).

Glia cells are also implicated in lipid metabolism. It was suggested that Müller glia cells have a role in the intra-retinal lipids transport (Tserentsoodol et al., 2006). For example, Müller cells are capable of decosahexaenoic acid (DHA) uptake (Gordon and

Bazan, 1990), that incorporate into glial phospholipids and finally transport to the photoreceptors (Politi et al., 2001). Previous research demonstrated that DHA is essential for photoreceptors discs regeneration. Studies reported that DHA has both anti-apoptotic and anti-inflammatory properties (Bazan, 2007, Bazan et al., 2011) particularly in retinal diseases such as diabetic retinopathy via inhibiting peroxisome proliferator-activated receptor γ (PPARγ)/NFκB-induced microglial activation (Wang et al., 2015).

Moreover, Müller cells are also activated by lipid metabolites that lead to retinal vascular dysfunction. For example, Al-Shabrawey et al., demonstrated that bioactive lipids products of 12/15-lipoxygenase (12/15-LO) could regulate retinal neovascularization via disrupting the delicate balance between VEGF and pigment epithelium-derived factor (PEDF) produced by retinal Müller cells (Al-Shabrawey et al.,

2011). 12-hydroxyeicosatetraenoic acid (12-HETE), a lipid product of 12/15-LO upregulated VEGF and downregulated PEDF expression in cultured rat Müller cells. 18

Furthermore, our recent studies showed that restoration of retinal PEDF levels counteracts the retinal angiogenic and inflammatory effects of 12-HETE and hence underscoring its beneficial effect in the treatment of diabetic retinopathy (Ibrahim et al.,

2015b).

In addition, glia cells produce extracellular matrix (ECM) which contributes to the inner limiting membrane, providing a significant support for the retina. Glia cells produce for instance, laminin a major constituent of the inner limiting membrane (Kohno et al.,

1987), collagen (Ponsioen et al., 2008, Keenan et al., 2012), Fibronectin (Jiang et al.,

1994), heparan sulphate and proteoglycans (Clark et al., 2011, Keenan et al., 2012).

These ECM components have a role in retinal angiogenesis as well (Jiang et al., 1994,

Keenan et al., 2012).

Müller cells are located between blood vessels and retinal ganglion cells, forming a barrier through which they provide nutrients, remove excess glutamate and secrete critical elements to the retinal ganglion cells (Bringmann et al., 2009). Interestingly,

Müller cells secrete variable cytokines, under different stress conditions. For instance, interleukin-8 (Liu et al., 2014) and interleukin-6 (Yoshida et al., 2001) are released from

Müller cells in response to infection, inflammation or injury (Mesquida et al., 2014).

These cytokines can induce stimulation of , endothelial cells or macrophages leading to chemokines production and recruitment of immune cells to the retina with subsequent inflammatory reaction (Liu et al., 2015b).

One of the most important factors released by Müller cells is VEGF. Basal levels of VEGF are neuroprotective; however, higher levels of VEGF are associated with vascular disorders (Zheng et al., 2012). VEGF can induce neuronal regeneration and 19

angiogenesis after ischemia/reperfusion injury (Ma et al., 2011) via direct activation of both neurons and endothelial cells (Oosthuyse et al., 2001). Nevertheless, prolonged and excessive exposure of the retinal cells including the retinal vasculature to VEGF is injurious that may even causes eventually retinal neurodegeneration (Tolentino et al.,

2002). Furthermore, high glucose induces upregulation of HIF-1α and VEGF in Müller cells in a calcium-dependent manner, which was suggested as a possible mechanism in developing diabetic retinopathy(Li et al., 2012).

Moreover, beside VEGF, glial cells synthesize other neurotrophic factors including PEDF (Unterlauft et al., 2012, Zhou et al., 2009) , brain-derived neurotrophic factor (BDNF) (Dai et al., 2012), ciliary neurotrophic factor (CNTF) (Escartin et al.,

2007) and glial cell-derived neurotrophic factor (GDNF) (Harada et al., 2003, Hauck et al., 2006).

The therapeutic potential of Müller cells has attracted several vision scientists. For example, Müller cells derived from human retina when transplanted into the sub-retinal space of rodents could migrate to the retina and expressed neural stem cell markers.

These cells were able to de-differentiate and re-enter cell cycle (Lawrence et al., 2007).

Moreover, Müller cells have the capability to express genes resembling retinal stem cells

(Roesch et al., 2008, Blackshaw et al., 2004). Recently, Müller cells treated with embryonic stem cells-derived extracellular vesicles including stem cells-derived exosomes have been de-differentiated and showed a pluripotent potential. (Katsman et al., 2012).

Retinal microglia were suggested to contribute to retinal inflammation, cell death and cell survival via their released cytokines (Schafer et al., 2013). It was reported that 20

major histocompatibility complex (MHC class 1), glutamate receptors and ATP signaling are involved in the microglial phagocytic function in the neural retina (Miyamoto et al.,

2013). Microglial cells are the major cellular component of the innate immunity in the retina and their activation is strictly regulated by different inhibitory pathways (Copland et al., 2010, Chen et al., 2012a). Additionally, although complement proteins are expressed in the retina under normal physiological circumstances (Luo et al., 2011), complement activation was observed under different pathological conditions in the retina such as inflammation (Chen et al., 2010) or ageing (Chen et al., 2007). Studies suggested that targeting the complement system and microglia could be beneficial in reducing retinal inflammation (Song et al., 2017, Xu and Chen, 2016).

Activated retinal microglia secrete mediators that affect the neural retina leading to increased neuronal apoptosis (Altmann and Schmidt, 2018). Hyperglycemia and glycated albumin activated retinal microglia during diabetes (Ibrahim et al., 2011).

Prolonged activation of microglial cells in diabetic retina was suggested to be more towards the pro-inflammatory action rather than the anti-inflammatory action. This generates a status of chronic inflammation that may be related to retinal affection during diabetic retinopathy (Arroba and Valverde, 2017). Research demonstrated that prolonged stimulation of the diabetic retina by damage-associated molecular patterns

(DAMPs) leads to dysregulation of the retinal innate immune system which may contribute to development of DR. In the progressive stages of diabetic retinopathy, recruited immune cells infiltrating the retina were suggested to participate in retinal chronic inflammation with subsequent retinal vascular and neuronal damage (Xu and

Chen, 2017). 21

 B.1.4. Blood Supply of the Retina:

The retina has a double blood supply, the outer retina (RPE and the photoreceptors) is supplied by the choroidal circulation while the inner retina is supplied by the retinal circulation. However, the fovea and the extreme peripheral retina are avascular and receive their nutrition via diffusion from the choroidal circulation (Delaey and Van De

Voorde, 2000).

 Choroidal Circulation

The choroidal circulation is derived from short and long posterior ciliary arteries of the ophthalmic branch of the internal carotid artery. The choroid gets 80% of the blood supply of the eye (Spector et al., 2015). The chorio-capillaries represent the innermost layer of the choroid. It is formed of a single layer of tightly settled capillaries resting on

Bruch’s membrane that separate the chorio-capillaries from the RPE. They are fenestrated capillaries to facilitate the diffusion process. Healthy chorio-capillaries as well as healthy RPE are required for optimum function (Hirata and Nishiwaki, 2006).

 Retinal Circulation

The retinal circulation is derived from the central retinal artery of the ophthalmic branch of the internal carotid artery. Central retinal artery arises directly after the ophthalmic artery enters the orbit, it pierces the dura and the arachnoid matters, then enters the optic nerve substance (Moss, 2015). In the humans, the central retinal artery appears from within the optic cup where it is divided into four main branches; the superior temporal, inferior temporal, superior nasal and inferior nasal retinal arteries.

These branches will form the retinal circulation (Kornfield and Newman, 2014). The arteries become arterioles due to the loss of the elastic fibres and the internal elastic 22

membrane. The arterioles give rise to retinal capillaries. The retinal capillaries supply the whole retina directly except the photoreceptors and RPE which are supplied by the choroid via diffusion. The retinal capillaries are arranged in a laminar meshwork to guarantee satisfactory perfusion to all retinal cells. Retinal capillaries are formed of endothelial cells surrounded by pericytes at a ratio of 1:1 (Shin et al., 2014).

The venous drainage follows a similar path starting from the retinal capillaries to the retinal venules which drain into the central retinal vein. Central retinal vein drains into the ophthalmic vein or the cavernous sinus. The disruption of any artery of the retinal circulation may lead to the loss of arterial supply to that part supplied by it due to the fact that retinal arteries are end arteries. The crossing points of retinal arteries and veins are important clinically because an artery may compress a vein or a vein may occlude an artery due to their physical proximity (Thapa et al., 2017, Cehofski et al., 2017).

23

 B.1.5. The Outer and Inner Blood Retinal Barriers (BRB):

The BRB is divided into an inner and an outer BRB. The barrier is formed by tight junctions of endothelial cells, basal lamina and end feet of astrocytes (Yao et al., 2014).

The inner BRB is formed by the tight junctions between the non-fenestrated retinal endothelial cells, which are surrounded by the processes of pericytes and Müller cells.

Retinal endothelium, pericytes and Müller cells together form the inner BRB which is critical for the nutrition and protection of the inner two-thirds of the retina (Hosoya and

Tachikawa, 2012). On the other hand, the outer BRB, which is important for homeostatic maintenance of outer third of the retina comprises of the tight junctions of retinal pigment epithelial cells (Cunha-Vaz et al., 2011, Campbell and Humphries,

2012). Tight junction proteins of both barriers include occludins, claudins, zonula occludin (ZO) proteins, and junctional adhesion molecules (JAMs) (Zhang et al., 2014).

The barrier prevents water-soluble molecules from entering the retina. The fenestrated choriocapillaris is permeable to macromolecules and does not appear to have a considerable importance in the blood-retinal barrier. Bruch’s membrane, situated between the choriocapillaris and RPE, represents a diffusion barrier only to macromolecules. A greater oncotic pressure in the choroid than in the retina is generated due to the high protein permeability of the choriocapillaris which results in fluid absorption from the retinal extracellular spaces into the choroid. The blood retinal barrier is affected in retinal pathologies (Cunha-Vaz et al., 2011, Campbell and Humphries, 2012).

24

 B.1.6. Retinal Physiology

1. Phototransduction process:

This the process by which the light received by the photoreceptors of the eye is transformed into a nerve impulse transmitted to the brain for processing (Stryer, 1991).

The process starts when the light hits the photopigment (retinal) in the photoreceptors.

This leads to conformational changes in the retinal molecule which dissociates from its to the opsin protein. This will cause further conformational changes in the opsin (called rhodopsin in rods and photopsin in cones) protein. The new confirmation of opsin causes activation of an opsin-associated G-protein called transducin. Transducin is formed of three subunits (α, β and γ). The α subunit of transducin subsequently dissociates and activates c-GMP-phosphodiestrase enzyme which will break down c-

GMP into 5` GMP. As a result of the decrease in the cytoplasmic availability of c-GMP, c-GMP-gated sodium channels will be shut down and hence hyperpolarization of the photoreceptor membrane occurs. The voltage gated calcium channels in the hyperpolarized membrane will be affected decreasing the calcium influx and affecting the release of the neurotransmitters at the synaptic terminal of the photoreceptors. This alteration of the neurotransmitter release will affect the postsynaptic neurones and the nerve impulse will start its propagation from the photoreceptors to the subsequent neurons in the visual pathway (Yau, 1994).

Method of Assessment of Retinal Neuronal Function:

The electroretinogram (ERG) is used to give an idea about the function of the retinal cells. It is composed of an a-wave representing the photoreceptor hyperpolarization, b- wave representing Müller cells depolarization, c-wave representing RPE apical 25

polarization, the oscillatory potentials representing the activity of amacrine cells and scotopic threshold response (STR) components as an indicator of retinal ganglion cells function (Smith et al., 2014).

2. Regulation of retinal blood flow

The choroidal circulation is controlled via the autonomic nervous system via the sympathetic innervation, with little or no autoregulation. In contrary, the retinal circulation is controlled via autoregulation as it is not supplied by the sympathetic nervous system. Central mechanisms regulating the retinal blood flow include direct autonomic innervation in addition to the indirect effects of hormonal factors such as norepinephrine, angiotensin or vasopressin. Local autoregulation of the retinal blood flow occurs in response to changes in hydrostatic pressure, O2 levels, CO2 levels, temperature, pH, and ATP levels (Pournaras and Riva, 2013, Yu et al., 2014, Moss, 2015).

26

 B.2. Retinal Pathology:

The causes of retinal pathology could include congenital, metabolic, traumatic, inflammatory and neoplastic retinal disorders. However, most of the retinal pathologies are multifactorial.

1. Congenital retinal disorders: these disorders mostly present since birth. for

example; congenital hypertrophy of the RPE (Youhnovska et al., 2013),

coloboma (Lingam, 2013) and Persistent Hyperplastic Primary Vitreous (PHPV)

(Jain, 2009).

2. Metabolic Retinal Diseases: for instance, diabetic retinopathy (Al-Shabrawey et

al., 2015), hyperhomocysteinemia (Ibrahim et al., 2016, Elmasry et al., 2018b).

3. Traumatic retinal disorders: ocular trauma can cause retinal tears or retinal

detachment (Stryjewski et al., 2014, Orban et al., 2016). Retinal detachment can

be caused by other pathologies as well such as diabetic retinopathy (Chang et al.,

2017, McElnea et al., 2018, Kapoor et al., 2014).

4. Inflammatory retinal disorders: They may be caused by infections or non-

infectious retinal inflammation. Emerging role of inflammation in retinal diseases

suggested the anti-inflammatory drugs as a possible therapy. Diabetic retinopathy

(Tang and Kern, 2011, Semeraro et al., 2015, Zhang et al., 2011, Rangasamy

et al., 2012, Al-Shabrawey et al., 2015, Elmasry et al., 2018a), age related

macular degeneration (Parmeggiani et al., 2012, Kauppinen et al., 2016),

retinitis pigmentosa (Nash et al., 2015) and retinal vein occlusion were classified

as inflammatory retinal diseases as well. These diseases have multiple aetiologies

other than the inflammation. 27

5. Neoplastic retinal disorders: for instance, retinoblastoma (Dimaras et al., 2015)

and vascular tumours of the retina (Shanmugam and Ramanjulu, 2015).

Here, we will discuss in more details, diabetic retinopathy as it is the disease of our research interest.

B.2.1. Diabetic Retinopathy:

 Definition:

Diabetic retinopathy is a neurovascular complication affecting the retina of diabetic patients which remains one of the most common causes of blindness all over the world. It affects 100% of patients with type 1 diabetes and approximately 60% of patients with type 2 diabetes (Al-Shabrawey et al., 2015, Cheung et al., 2010, Duh et al., 2017,

Joussen et al., 2004, Rangasamy et al., 2012).

 Epidemiology:

World Health Organization has added diabetic retinopathy on the priority list of eye conditions that can be prevented or treated. It was reported that there are approximately

285 million diabetes patients in 2010 worldwide. Nearly 100 million diabetic patients have signs of diabetic retinopathy (Lee et al., 2015). In USA, diabetic retinopathy cases represented 30% among individuals with diabetes aged 40 years and older (Zhang et al.,

2010). According to the NEI (National Eye Institute), by 2050, the number of Americans with diabetic retinopathy is expected to nearly double from 7.7 million to 14.6 million.

28

 Aetiology and pathophysiology:

Hyperglycaemia remains the driving force for the development of neurovascular dysfunction in diabetic retinopathy. The longer the duration of diabetes, the higher the risk for diabetic retinopathy development. However, other risk factors such as dyslipidaemia and hypertension were reported as well (Lupo et al., 2013, Duh et al.,

2017).

a) Retinal microvascular dysfunction:

Retinal micro vessels are affected either by occlusion or leakage. Retinal blood barrier is interrupted during diabetes (Ibrahim et al., 2015a, Ibrahim et al., 2017).

These leaky blood vessels cause macular edema. Retinal capillaries are degenerated in diabetes causing hypoxic retina which induces retinal neovascularization or angiogenesis, the newly formed blood vessels are weak and leaky as well which can cause retinal haemorrhage (Al-Shabrawey et al., 2013, Al-Shabrawey et al., 2011, Al-Shabrawey et al., 2015, Cheung et al., 2010, Duh et al., 2017, Evans et al., 2014, Ishida et al., 2003,

Joussen et al., 2002).

b) Retinal neuronal dysfunction:

Diabetes induces apoptosis of retinal neurones including retinal ganglion cells and inner nuclear layer neurones. Glutamate toxicity affects retinal neurones as diabetes impairs glutamate metabolism in retinal neurones (Ishikawa, 2013, Lau et al., 2013,

Santiago et al., 2009).

c) Role of inflammation:

It was reported that during diabetes retinal microglia are activated. This is associated with an increased expression of ICAM 1 and leukocyte adhesion in retinal endothelial 29

cells. Impaired blood retinal barrier in diabetic retinopathy contributes to the migration of blood into the retina to become macrophages. Migrated immune cells in the retina contribute to the retinal inflammatory response during diabetes and increased vascular permeability (Ibrahim et al., 2017, Ibrahim et al., 2011, Elmasry et al.,

2018a).

 Molecular mechanisms of diabetic retinopathy:

There is a strong association between chronic hyperglycemia during diabetes and the development of diabetic retinopathy, however the underlying mechanisms of this association are still ambiguous (Cheung et al., 2010). Some biochemical pathways have been suggested as possible links between hyperglycemia and diabetic retinopathy which include; the polyol pathway dysregulation, accumulation of advanced glycation end products (AGEs), protein kinase C (PKC) activation, increased expression of growth factors as VEGF and -like growth factor-1 (IGF-1), hemodynamic alterations, , oxidative stress, ER stress, renin-angiotensin-aldosterone system (RAAS) activation, chronic inflammation and endothelial-leukocytes adhesion.

The polyol pathway involves reduction of glucose molecules by aldose reductase

(AR) enzyme using NADPH as a into sorbitol. Then sorbitol dehydrogenase

(SDH) enzyme using NAD+ as a cofactor converts sorbitol to fructose. Sorbitol cannot diffuse via the plasma membrane so, during hyperglycemia, sorbitol level increases and sorbitol accumulates in the tissues including the retina which can cause osmotic and oxidative damage of these tissues (Oates, 2002, Gabbay, 1973). Studies suggested targeting AR as possible targets for therapy to prevent diabetic retinopathy progression

(Drel et al., 2008, Obrosova et al., 2010). Furthermore, other studies proposed targeting 30

SDH will be more efficient in diabetic retinopathy prevention than AR. Research showed that SDH upregulation is toxic to the pericytes in the retina via induction of oxidative stress (Amano et al., 2002). Genetic studies identified SDH as possible player in diabetic retinopathy development as well (Amano et al., 2003).

Moreover, accumulated AGEs and their interaction with specific receptors have been linked to the development of diabetic retinopathy (Chen et al., 2013, Zong et al.,

2011). Studies showed that prolonged hyperglycemia in experimental murine models of diabetes showed excessive accumulation of AGEs in the retina which was associated with retinal capillaries and pericytes loss as well. Pharmacological inhibition of AGEs formation significantly decreased retinal AGEs accumulation and protected the retina against capillary and pericytes loss (Hammes et al., 1991, Stitt et al., 2002).

Interestingly, another way was proposed to decrease AGEs-induced retinal complications via drugs with the ability to break the AGE crosslinks such as a drug known as

Alagebrium (Thallas-Bonke et al., 2004).

Protein kinase C (PKC) is a serine/threonine kinase involved in signal transduction pathways responding to different stimuli. The key activator of PKC is diacylglycerol (DAG) which is upregulated during diabetes under hypoglycemia via activated glycolytic pathway. Additionally, β1/2 isoform of PKC were reported to be closely related with the development of diabetic retinopathy and have been shown to be upregulated in diabetic patients (Koya and King, 1998, Wang, 2006). PKC activation has been associated with increased retinal endothelial permeability, retinal expression of

VEGF and retinal endothelial-leukocyte adhesion (leukostasis) (Koya and King, 1998,

Aiello et al., 1997, Aiello et al., 2006). PKC upregulation contributes to the pathogenesis 31

of diabetic retinopathy via directly affecting other pathways such as inflammation, angiogenesis, and retinal haemodynamic alterations, which consequently contribute to the pathogenesis of diabetic retinopathy. Interestingly, inhibition of PKC reduced leukostasis in diabetic patients, significantly reduced the progression of diabetic retinopathy and significantly reduced vision loss (Clarke and Dodson, 2007, Nonaka et al., 2000, Joy et al., 2005).

Furthermore, hypertension contributes to the progression of diabetic retinopathy via the hypertension-induced sheer stress on retinal endothelial cells and higher blood viscosity with subsequent endothelial dysfunction (Simonson, 1988, Patel et al., 1992).

In addition, the renin-angiotensin-aldosterone system (RAAS) which is involved in regulation of the blood pressure is involved in the pathogenesis of diabetic retinopathy as well. Intriguingly, independent of blood pressure, retinal levels of RAAS components including; renin, angiotensin converting I and II (ACE I and ACE II) and angiotensin receptors were reported to be upregulated in Proliferative diabetic retinopathy

(Wilkinson-Berka, 2006, Funatsu et al., 2002). Moreover, ACE inhibitors being approved as anti-hypertensive drugs, reduced angiogenesis in type 1 diabetic mouse model (Ebrahimian et al., 2005). It was suggested that RAAS-induced retinal affection in diabetic retinopathy may be via PKC activation or VEGF signaling pathway (Otani et al., 1998).

VEGF is one of the most studied factors involved in the pathogenesis of diabetic retinopathy. VEGF; causes retinal endothelial activation, disruption of the blood-retinal barrier, an increase of the retinal vascular permeability and promoting retinal angiogenesis (Zhang et al., 2009, Ishida et al., 2003). Several studies have established a 32

role for VEGF in the development and progression of diabetic retinopathy (Wirostko et al., 2008). VEGF-induced retinal vascular dysfunction is mediated through the activation of two membrane bound tyrosine kinase VEGF receptors (VEGFR1/2) (Takahashi and

Shibuya, 2005). The binding of VEGF to its receptors activates two signalling pathways including either opening of calcium channels or a mitogen activating protein kinase

(MAPK) signalling pathway with subsequent affection of blood retinal barrier and increased retinal vascular permeability (Bates and Curry, 1997). VEGF was reported to upregulate ICAM-1 and nitric oxide (NO) synthase resulting in increased leukocyte adhesion to retinal endothelial cells and promoting inflammation during diabetic retinopathy. Increased retinal levels of VEGF were associated with increased vascular permeability which may lead to haemorrhage, exudates, and vascular leakage noticed in non-proliferative diabetic retinopathy or may lead to angiogenesis noticed in proliferative diabetic retinopathy (Joussen et al., 2002, Nguyen et al., 2006).

Moreover, Carbonic anhydrases (CAs), a group of enzymes rapidly convert carbon dioxide to bicarbonate and protons, have been reported to be upregulated during diabetes and inhibiting CAs was associated with a reduction of the progression of diabetic retinopathy (Weiwei and Hu, 2009). CA inhibitors suggested to be beneficial in diabetic retinopathy via improving retinal blood flow, reducing platelet aggregation and decreasing retinal vascular permeability (Gao et al., 2007).

Several studies established the role of inflammation in diabetic retinopathy.

Retinal inflammation enhances the impact of hyperglycaemia, oxidative stress, ER stress,

AGEs, and hypertension via activation of downstream signalling pathways such as VEGF signalling, NF-κB signalling or NO signalling and via cytokines or adhesion molecules as 33

well. Consequently, retinal subclinical inflammation upregulates endothelial nitric oxide synthase (eNOS), causes retinal neovascularization and induces leukostasis (van Hecke et al., 2005, Spijkerman et al., 2007, Klein et al., 2009). Leukostasis is an important event in pathogenesis of diabetic retinopathy, causing retinal capillaries occlusion with subsequent retinal hypoxia and ischemia. Leukostasis facilitates ROS-mediated cell death and amplify the local retinal inflammatory response (Schroder et al., 1991, Lutty et al.,

1997, Chibber et al., 2000, Chibber et al., 2003). Accumulated body of evidence showed that the increase in serum pro-inflammatory cytokines, adhesion molecules such as ICAM-1, and immune cells activation during diabetes are associated with the progression of diabetic retinopathy. Interestingly, increased levels of interleukin-8, chemoattractant protein-1 and interleukin-10 have been reported in the vitreous of patients with proliferative diabetic retinopathy (Kaul et al., 2010, Hernandez et al.,

2005). Increased levels of pro-inflammatory cytokines and adhesion molecules as well as endothelial dysfunction contribute to leukostasis by enhancing leukocyte-endothelial cell interaction (Takami et al., 1998, Larson and Springer, 1990). This was suggested by studies where knocking out of adhesion molecules significantly decreased leukocyte adhesion in retinal endothelial cells (Joussen et al., 2004). Studies showed that increased

O-glycosylation of carbohydrate chains on the surface of leukocytes causes leukocyte activation and significantly increase leukocyte rolling and adhesion to retinal endothelial cells. This subsequently causes leukocyte dysfunction and increased leukostasis

(Chibber et al., 2000, Kaul et al., 2010). Local retinal inflammation implicating leukostasis and activation of retinal immune cells including retinal microglia and macrophages with subsequent increase in cytokines production or ROS generation was 34

suggested to play an important role in the pathogenesis of diabetic retinopathy

(Langmann, 2007). These studies suggested the introduction of anti-inflammatory drugs that have been reported to decrease VEGF expression, improve vascular permeability and decrease leukostasis as possible therapies for prevention of diabetic retinopathy (Gillies et al., 2006, Kuppermann et al., 2007, Kern et al., 2007).

Oxidative stress is a term describing the imbalance between levels of reactive oxygen species (ROS) and the cellular antioxidant factors including thioredoxin, glutathione (GSH), and vitamin E, superoxide (SOD), catalase, , and thioredoxin reductase. This may happen by either increased production of ROS or reduced activity of the antioxidants (Baynes, 1991). Oxidative stress-induced tissue damage was reported to be implicated in the pathogenesis of diabetic retinopathy with a strong correlation between hyperglycaemia, dysregulated redox homeostasis, and oxidative stress (Cui et al., 2006, van Anken and Braakman, 2005). ROS generation was reported to be increased in vivo in diabetic retinas of animal models and in vitro in high glucose treated retinal cells (Kowluru, 2001, Kowluru and Abbas, 2003, Othman et al., 2013). Moreover, endogenous antioxidants activity was reported to be downregulated in diabetic retinas as well (Stadler, 2012, Delmastro and Piganelli,

2011). Different sources have been suggested for diabetes-induced ROS. Mitochondrial- derived ROS have been reported to induce DNA strand breaks that activate poly-(ADP- ribose)-polymerase (PARP) which inhibits glyceraldehyde phosphate dehydrogenase

(GAPDH) activity with subsequent accumulation of glycolytic metabolites. These glycolytic metabolites have the ability to activate polyol, AGE OR PKC related signalling pathways (Brownlee, 2005). Additional suggested source for retinal ROS 35

generation was NADPH oxidase-derived ROS, which was suggested to work through induced phosphorylation of VEGFR2 (Al-Shabrawey et al., 2008, Elmasry et al.,

2018a, Ibrahim et al., 2015a, Othman et al., 2013).

 Clinically, Diabetic retinopathy is classified into 2 types:

1. Non-proliferative diabetic retinopathy with the following signs:

Micro aneurysms (the earliest sign which occur because of the pericyte loss and consequent weakness of the micro-vessels walls), micro haemorrhage, macular edema

(the main cause of visual impairment), vascular tortuosity and beading. Cotton-wool spots occur when precapillary arterioles are occluded leading to infarctions of the nerve fiber layer (Duh et al., 2017, Iwase et al., 2017).

2. Proliferative diabetic retinopathy with the following features:

Retinal angiogenesis, vitreous neovascularization, haemorrhage or retinal detachment

(Al-Shabrawey et al., 2011, Al-Shabrawey et al., 2015).

 Current Treatment Modalities of Diabetic Retinopathy

1. Tight glycaemic control:

Hyperglycemia is an important determinant of diabetic microvascular complications such as diabetic retinopathy. Thus, tight glycaemic control is effective in reducing diabetic retinopathy incidence and progression (Diabetes et al., 2015). The long-term beneficial effect of glycemic control has been examined by two large studies: The Diabetes Control and Complications Trial (DCCT) in Type 1 diabetes (Diabetes et al., 2015, Diabetes et al., 1993) and the United Kingdom Prospective Diabetes Study (UKPDS) in Type 2 diabetes (1998). Both DCCT and the UKPDS have demonstrated that tight glycemic control that maintains the levels of HbA1c ≤7% reduced development and progression of 36

diabetic retinopathy, with the beneficial effects of tight glycemic control persisting up to

10–20 years.

2. Intravitreal injection of anti-VEGF therapies:

The introduction of intravitreal injection of anti-VEGF therapies have changed the goal of diabetic retinopathy treatment from stabilization to improvement of vision. Anti-

VEGF therapies significantly improve the visual acuity of patients with diabetic macular edema. There are Four FDA approved anti-VEGF drugs that have been used in the treatment of diabetic retinopathy. The first one is bevacizumab (with the trade name

Avastin, Genentech, South San Francisco, CA). It is a recombinant humanized anti-

VEGF monoclonal antibody. The second drug is ranibizumab (trade name is Lucentis,

Genentech, South San Francisco, CA) which is a recombinant antibody fragment (Fab) against VEGF-A. The third anti-VEGF drug is called aflibercept (trade name is Eylea or known also as VEGF Trap-Eye, Regeneron, Tarrytown, NY) which is acting as a VEGF- receptor capable of binding to both circulating VEGF-A and VEGF-B. It has VEGF- binding domains of VEGF receptors 1 and 2. The fourth drug is pegaptanib (Macugen,

Valeant, Madison, NJ) which binds specifically to the main pathological VEGF isomer in the eye which is the VEGF-A165 (Osaadon et al., 2014, Vaziri et al., 2015).

3. Laser photocoagulation:

This intervention is widely used for treatment of diabetic retinopathy, in which a laser light is applied to the retina to stop new blood vessels formation and growth. This procedure is used to treat macular edema in non-proliferative diabetic retinopathy. In proliferative diabetic retinopathy, pan-retinal photocoagulation is used where laser is applied to the whole retina except the macula (Evans et al., 2014). 37

4. Intravitreal injection of corticosteroids:

Intravitreal injection of synthetic corticosteroids including triamcinolone acetonide

(Bressler et al., 2013), dexamethasone (Callanan et al., 2013) and fluocinolone

(Pearson et al., 2011) have been associated with reduced progression of diabetic retinopathy particularly when combined with other lines of treatment (photocoagulation or anti-VEGF) . However, this procedure was associated with adverse effects such as cataract formation or glaucoma progression. But, they were less associated with vitreous hemorrhage, retinal detachment or possibility of endophthalmitis (Sampat and Garg,

2010).

5. Surgical removal of the vitreous (vitrectomy):

This procedure is used in proliferative diabetic retinopathy when vitreous haemorrhage or retinal detachment occur. Vitrectomy improved visual acuity in patients suffering from diabetic macular edema, with a low rate of postoperative complications (Diabetic

Retinopathy Clinical Research Network Writing et al., 2010).

6. Combined Therapy:

Due to the fact that the pathogenesis of diabetic retinopathy is complex and multifactorial, there was a rationale to use more than one line of treatment in the same patient hoping to achieve a better response. Studies showed that when intravitreal corticosteroids (Kang et al., 2006, Avitabile et al., 2005, Lam et al., 2007, Gillies et al.,

2011) or anti-VEGF (Mitchell et al., 2011, Elman et al., 2011, Nguyen et al., 2009, Lee et al., 2011) were used as a combined therapy along with retinal laser photocoagulation, the outcome was better with a more improved visual acuity in those patients who received the combined therapy. 38

Although there are significant improvements in the therapeutic modalities for diabetic retinopathy, additional therapeutic strategies are urgently needed. Current therapies are invasive, and directed mainly toward late stages of diabetic retinopathy, after significant and irreversible damage has ensued; hence, preventative strategies that address early pathological events and neuronal dysfunction are highly desirable. Studies suggested new early players in the pathogenesis of diabetic retinopathy such as bioactive lipids and ER stress which will be discussed in more details.

39

B.3. Bioactive Lipids:

Lipids are the main elements of cell membranes of all organisms. Bioactive lipids are lipid molecules synthesized from cell membrane lipids with a relatively short half- life. However, they are engaged in lipid-protein interactions and function as intercellular and intracellular signalling mediators. They regulate multiple cellular functions. Those lipids are dependent on diet and can be modified by dietary supplementation.

Bioactive lipids include mainly; classical eicosanoids, phospholipids, sphingolipids, endocannabinoids and specialized pro-resolving lipid mediators (SPMs).

B.3.1. Eicosanoids:

Eicosanoids are locally acting bioactive lipids derived from polyunsaturated fatty acids (PUFAs). They are related to various diseases and regulate variable cellular processes. Phospholipase A2 enzyme releases PUFAs such as linoleic acid (LA), arachidonic acid (AA), DHA and eicosapentanoic acid (EPA) from the cell membrane lipid layers. Those released lipids undergo either a cyclic metabolism via

Cyclooxygenases (COX) enzymes or a linear metabolism via Cytochrome P450 (CYP) enzyme or (LO) enzymes (Fig.5).

40

Fig.5: Bioactive lipids metabolism. Polyunsaturated fatty acids liberated from the membrane lipid bilayer by the action of phospholipase A2. Different enzymes (COX, LO & CYP) act on different substrates liberating different types of bioactive lipids. The diagram is modified from (Klawitter et al., 2013).

There are two main COX isoforms which are cyclooxygenase-1 (COX-1) and

cyclooxygenase-2 (COX-2). Both convert AA into (PGH2), which then

converted by appropriate synthases, into prostaglandins (PGs) and thromboxanes (TXs)

which are collectively known as prostanoids. Prostanoids are considered as tissue

hormones synthesized from long chain polyunsaturated fatty acids, mainly AA (Sales et

al., 2008). The most important prostanoids include: prostaglandin E2 (PGE2),

prostaglandin F2α (PGF2α), prostaglandin D2 (PGD2), PGI2, and

(TxA2). PGE2 is a key pro-inflammatory prostaglandin which has anti-apoptotic

properties with important functions in the immunity and cancer development. PGF2α is

identified as an autocrine growth factor related to endometrial carcinoma (Sales et al.,

41

2008, Woodward et al., 2011). TxA2 (synthesized by COX-1) and PGI2 (synthesized by

COX-2) (Catella-Lawson et al., 1999) have important roles in both the physiology and pathology of blood vessels. TxA2 causes platelets aggregation and vasoconstriction, while PGI2 has opposite effects (Meyer-Kirchrath et al., 2004, Caughey et al., 2001a,

Caughey et al., 2001b).

Interestingly, PG signalling was reported to be involved in chronic inflammatory diseases for instance or Crohn’s disease. This may happen via their ability to enhance the release of pro-inflammatory cytokines, their ability to activate pro- inflammatory T cells, or macrophages activation (Claar et al., 2015, Wallace, 2018).

Cytochrome P450s metabolize AA to epoxyeicosatrienoic acids (EETs), dihydroxyeicosatrienoic acids (DHETs), and 20- hydroxyeicosatetraenoic acid (20

HETE). Cytochrome P450-derived eicosanoids are formed in a tissue and cell-specific way with abundant biological functions. Interestingly, EETs and 20-HETE have opposite actions within the vasculature. EETs have powerful vasodilatory effect while 20-HETE is a strong vasoconstrictor. In addition, cytochrome P450 eicosanoids have been suggested to have a role in blood pressure regulation denoted by their expression’s deregulation in hypertensive rats. They have been reported to have a role in cell proliferation and inflammation as well (Cook et al., 2016, Christmas, 2015, Capdevila, 2007).

Lipoxygenases (LOs) are non-heme iron-containing lipid peroxidizing enzymes which catalyze dioxygenation of PUFAs such as LA and AA, liberating a wide variety of bioactive lipids (Kuhn et al., 2015). The phylogenetic distribution of LOs demonstrates their wide distribution across both the plant and animal kingdoms. The contains one functional LO located on 10 (ALOX5) and five genes 42

located on (ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3), which encode for six different LO isoforms (Funk et al., 2002). The ALOX15 gene encodes for 12/15-LO, the ALOX15B gene encodes for 15-LO2, the ALOX12 gene encodes for the platelet-type 12-LO, the ALOX12B gene, encodes for a 12R-LO enzyme, the ALOXE3 gene encodes for two epidermal LO isoforms, and finally the ALOX5 gene encodes for 5-LO enzyme. The corresponding mouse genes were located on 6 (Alox5) and 11 (other Lo isoforms). These genes encode for slightly different LO enzymes in mice (Meruvu et al., 2005, Kinzig et al., 1999, Yu et al.,

2006).

Indeed, mouse Alox12, Alox12b, Aloxe3 and Alox5 are highly conserved with similar enzymatic characteristics with the human LOs. However, mouse Alox15-encoded enzyme has a 12-LO enzyme activity converting AA to 12S-HpETE. Moreover, mouse

Alox15b-encoded LO converts AA into 8S-HpETE and not 15S-HpETE like what the human ortholog does. Mammalian LO is a protein formed of a single polypeptide chain.

It has an N-terminal domain formed of several β-sheets and C-terminal domain which is the catalytic domain containing non-heme iron in the substrate-binding site (Chen et al.,

1994).

Mammalian LO isoforms have diverse biological functions. Different LO isoforms are involved in RBCs synthesis, differentiation and via their ability to oxidize esterified membrane lipids modifying their structural and functional proprieties (Sun and Funk, 1996, Capra et al., 2013, Chen et al., 1994,

Kinzig et al., 1999). LOs are implicated in affecting different cellular functions via their

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ability to interrupt the cellular redox status. LOs form lipid peroxides which affects the intracellular redox balance and implicated in oxidative stress induction (Elmasry et al.,

2018a, Othman et al., 2013). LOs are involved in cell proliferation and tumorigenesis, however their role is still controversial. For instance, 5-HETE and 12-HETE, the bioactive lipid products of 5-LO and 12-LO respectively were reported to be tumorigenic

(Nie et al., 1998). However, 13-hydroxyoctadecadienoic acid (HODE) which is a bioactive lipid product of 15-LO from linoleic acid substrate was reported to have anti- tumorigenic properties in colorectal carcinoma (Shureiqi et al., 2003). Interestingly, overexpression of ALOX15 in human prostate cancer cells increased cellular proliferation and was considered a pro-cancerous factor (Kelavkar et al., 2001).

LOs were described to be involved in inflammation. Some of the bioactive lipid products of LOs have pro-inflammatory properties while others have anti-inflammatory properties. Leukotrienes (LTs) derived from the ALOX5 pathway are known pro- inflammatory factors. ALOX5, catalyzes dioxygenation of arachidonic acid to 5S-HpETE and then formation of LTA4. LTs are synthesized in different types of leukocytes and other immune cells (Gubitosi-Klug et al., 2008, Haeggstrom and Funk, 2011). Other bioactive lipid products of LOs have anti-inflammatory properties. For instance,

(Ryan and Godson, 2010), (Spite et al., 2014), protectins and

(Serhan et al., 2015b) are pro-resolving lipid mediators. Those are mostly products of

LOs using EPA or DHA as substrates.

Moreover, LOs have been linked to cardiovascular system pathologies including hypertension and atherosclerosis. ALOX5 and ALOX15B are present extensively in atherosclerotic plaques (Wuest et al., 2014, Gertow et al., 2011). ALOX5 and 44

leukotriene signaling were associated with pathogenesis of vascular inflammation. Alox5 knockout mice under high fat diet was not protected from lipid accumulation in the vascular wall but however had a higher tendency for aortic aneurysms (Zhao et al.,

2004). The function of ALOX15 in atherosclerosis is still debated (Kuhn et al., 2007,

Wittwer and Hersberger, 2007). 13S-HODE which is a 15-LO derivative from linoleic acid was detected in atherosclerotic lesions of rabbits (Kuhn et al., 1994) and humans

(Kuhn et al., 1997), but its exact source is still unknown. While, several studies utilizing

Alox15 knockout mice supported a pro-atherogenic impact of ALOX15 (Zhao et al.,

2005), other studies utilized ALOX15 overexpression proposed anti-atherogenic functions via synthesis of pro-resolving lipid mediators (Shen et al., 1996, Merched et al., 2008). Interestingly, human platelets are a main source of ALOX12 and its lipid metabolite 12-HETE which were reported to have both pro- and antithrombotic actions.

Studies using Alox12 knockout mice showed attenuated platelet integrin activity.

Moreover, although human platelets don’t express ALOX15B, it has recently been connected to platelet function regulation and thrombus formation (Vijil et al., 2014).

In addition, as the retina is considered a neurovascular organ and diabetic retinopathy is considered a neurovascular disease, so it was interesting to find out that

LOs have both physiological and patho-physiological roles in the CNS. ALOX15 but not

ALOX12 is expressed at low but significant levels under normal circumstances in canine and rodents’ brain (Nishiyama et al., 1993, Hada et al., 1994). However, 12-HETE has been implicated as a signaling mediator in axonal growth (Ross et al., 2000, Mikule et al., 2002). ALOX15 impacts synaptic signaling via 12-HpETE that acts on L-type

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calcium channels (DeCostanzo et al., 2010). However, Alox15 knockout mice in spite of generating low levels of 12-HETE, they did not present any obvious behavioral problems, probably due to compensatory mechanisms that develops in the knockout mice

(Feinmark et al., 2003).

LOs are main players in ischemia, and neurodegenerative disorders via their interplay with oxidative stress, mitochondrial dysfunction or apoptosis (Pallast et al.,

2010, Pallast et al., 2009). A bulky body of evidence has accumulated to reveal the contribution of ALOX15 in stroke induced-brain insult (van Leyen, 2013, Schewe et al.,

1986, Seiler et al., 2008, Brinckmann et al., 1998, van Leyen et al., 2006, Jin et al.,

2008, Xu et al., 2010, Cui et al., 2010). Alox15 gene knockout but not Alox5

(Kitagawa et al., 2004) had a protective effect against stroke, and blood–brain barrier dysfunction (Jin et al., 2008, Xu et al., 2010, Cui et al., 2010). On the other hand, lipoxins and protectins derived from LOs were reported to have a neuroprotective effect in stroke. LO-facilitated conversion of DHA to neuroprotectin D1 and LO-dependent formation of A4 have been reported to have neuroprotective effects in experimental stroke (Sobrado et al., 2009, Eady et al., 2014). Previous studies showed upregulation of ALOX15 and increased levels of 12- and 15-HETE in the brains or CSF of Alzheimer's patients (Pratico et al., 2004). In contrary, others showed downregulation of ALOX15 (Lukiw et al., 2005) and upregulation of ALOX5 (Firuzi et al., 2008) in the hippocampus of Alzheimer's patients. This discrepancy in the data among different groups may be related to the complexity of the disease or presence of a differential expression of those enzymes in different brain regions.

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A number of LO derived-bioactive lipids and some LO isoforms have been associated with the pathogenesis of multiple metabolic disorders including diabetes both type 1 and type 2. Interestingly, 12-HETE derived from either ALOX15 or ALOX12 and

ALOX5 derived- were elevated in diabetic patients (Issan et al., 2013).

Particularly for ALOX15, a significant body of evidence has gathered to connect the enzyme to the pathogenesis of diabetes (Laybutt et al., 2002, Sears et al., 2009, Bleich et al., 1998, Bleich et al., 1999, Chen et al., 2005a, Nunemaker et al., 2008).

Expression of ALOX15 was significantly increased in both cell culture and animal models of diabetes. This may be related in part to LO-induced oxidative stress (Dobrian et al., 2011, Zhang et al., 2013).

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B.3.2. Bioactive Lipids and Retinopathies:

Bioactive lipids such as eicosanoids, pro-resolving mediators, sphingolipids were reported to have a role in the pathogenesis of retinopathies including retinopathy of prematurity (ROP), age related macular degeneration (AMD) or diabetic retinopathy as we will discuss to some extent below.

ROP is a disease affecting the retinas of premature babies. An interesting study reported a positive correlation between low serum levels of adiponectin (APN) and development of ROP. The researchers identified a significant reduction of retinal angiogenesis in ROP by an increase of dietary uptake of ω-3 PUFAs which was associated with an increase of serum APN. They suggested that this effect may be via reduced retinal-ER stress (Fu et al., 2015). Additionally, sphingolipids (Skoura et al.,

2007) and ALOX15 (Al-Shabrawey et al., 2011) were involved in retinal neovascularization in the oxygen-induced retinopathy which is a mouse model for ROP.

In early stages of AMD, lipid-containing deposits called drusen appear in Bruch's membrane underlying RPE. This is suggesting a major role of lipids in the pathogenesis of AMD (Shen et al., 2016). Earlier studies demonstrated that ω-3 PUFAs as EPA are able to significantly reduce choroidal neovascularization (CNV) associated with AMD and thus a diet rich in ω-3 PUFAs may be protective against AMD development and other pathological retinal angiogenesis as well (Koto et al., 2007, Connor et al., 2007).

The protective effect was suggested to be because of the 5-LO-dependent oxidation of

DHA to form 4-hydroxy-decosahexaenoic acid (4-HDHA) which inhibits endothelial proliferation and angiogenesis in a PPARγ-dependent mechanism (Sapieha et al., 2011).

Interestingly, in patients with wet-AMD; a combined therapy of anti-VEGF and ω-3 48

PUFAs supplementation was associated with a more profound reduction of VEGF levels in the vitreous of those patients (Rezende et al., 2014). Additionally, it was shown that photoreceptors were protected from oxidative stress via ω-3 PUFAs such as EPA and

DHA (Sala et al., 2010). Recently, it was reported that LA induces CNV which was inhibited via DHA supplementation. Moreover, eyes derived from AMD donors had a significantly lower level of very long chain PUFAs which is supporting for the notion of the protective effect of these lipids in AMD (Gorusupudi et al., 2016).

Furthermore, Oxidized phospholipids were reported to be increased in the eyes of

AMD patients, induce CNV in mice (Suzuki et al., 2007) and upregulate VEGF via

ATF4-dependent mechanism in RPE cells (Pollreisz et al., 2013). The sphingolipids including (Cer), sphingosine (Sph) or sphingosine-1-phosphate (S1P) have been shown to be implicated in AMD as well. Although it was suggested that S1P and Cer-1- phosphate have a protective effect on photoreceptors and involved in their differentiation and development (Miranda et al., 2009, Miranda et al., 2011), others showed that S1P can induce angiogenesis in either the choroid or the retina (Caballero et al., 2009, Xie et al., 2009). Moreover, accumulated cholesterol in retinal endothelial cells, macrophages and RPE cells has been shown to be involved in the pathogenesis of AMD (Saadane et al., 2014, Omarova et al., 2012).

Diabetic retinopathy is a neurovascular multifactorial retinal disease that represents a socioeconomic burden and hence, necessitates finding out new therapeutic targets to optimize the outcome of current therapies. Not only that the retina has a unique lipids profile but also, diabetes induces specific changes in retinal lipid metabolism. Diet rich in beneficial lipids such as DHA or EPA has been suggested as 49

an alternative therapeutic strategy to prevent diabetic retinopathy (Tikhonenko et al.,

2010, Connor et al., 2007). The retina is rich in DHA, however there is a significant reduction of the amount of DHA in diabetic retina. DHA express anti-inflammatory effect on retinal endothelial cells (Chen et al., 2005b) and it has been shown to have a protective effect against pathological angiogenesis of the retina (Connor et al., 2007).

Studies highlighted that the lipidomic profile of the diabetic retina is different from the circulation indicating specific lipid content of the retina. In these studies, the bioactive lipid products of 12/15-LO synthetic pathway were dominant in the retina while the products of cytochrome P-450 pathway were dominant in diabetic circulation

(Ibrahim et al., 2015a, Ibrahim et al., 2017). 5-LO (ALOX5) and 12/15-LO

(ALOX15) were both reported to have a role in the pathogenesis of DR. However, the findings suggested a differential role of the two enzymes. Retinas of diabetic 5-Lo-/- mice and not 12/15-Lo -/- showed less retinal capillaries degeneration after 9 months of diabetes while retinas of both diabetic 5-Lo -/- and 12/15-Lo -/- showed less leukocyte adhesion at three months’ time point (Gubitosi-Klug et al., 2008). Moreover, 12/15-

LO- derived bioactive lipids were significantly increased in the vitreous of patients with proliferative diabetic retinopathy (Al-Shabrawey et al., 2011) and in epiretinal membranes as well (Augustin et al., 1997).

Furthermore, several studies suggested a role of sphingolipids in the development of diabetic retinopathy (Fox et al., 2006, Masson et al., 2005).

Sphingomyelinase has been reported to be activated in diabetic retina. DHA was capable of inhibiting sphingomyelinases activity in both in vitro or in vivo models of diabetic retinopathy, which resulted in reducing both diabetes-induced inflammation 50

and neovascularization (Opreanu et al., 2010, Opreanu et al., 2011). In addition, pharmacological inhibitors of sphingosine kinase attenuated VEGF-induced activation of retinal endothelial cells and reduced retinal vascular leakage in a rat model of diabetic retinopathy (Maines et al., 2006).

B.3.3. 12/15-LO (ALOX15):

LO genes are functionally conserved in human and mouse genomes except

Aloxe12 gene which is dysfunctional in human genome. Mouse Alox15 converts arachidonic acid to 12-H(p)ETE while human ALOX15 converts AA to 15-H(p)ETE.

However, mouse Alox15 represents the corresponding functional gene of human

ALOX15 (Funk et al., 2002). The same observation was made in rats as well

(Watanabe and Haeggstrom, 1993, Pekarova et al., 2015). The old traditional LOs classification was dependent on which carbon atom in the fatty acid backbone will be oxygenated by the corresponding enzyme. For example, human ALOX15 oxygenates arachidonic acid at carbon 15 (Sloane et al., 1991). However, mouse Alox15 oxygenates arachidonic acid at carbon 12 (Sun and Funk, 1996). That is why the new classification of LOs depends on their similarity to the human isoforms rather than the fatty acid’s carbon dioxygenated.

ALOX15 has three catalytic activities:

1. Lipoxygenase activity: which leads to the formation of hydroperoxy lipids.

2. Lipohydroperoxidase activity: which converts lipid hydroperoxides to secondary

lipid peroxidation products.

3. Leukotriene synthase activity: which is considered a combination of its

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and its lipohydroperoxidase activities (Brash et al., 1989, Schewe, 2002).

Several studies reported ALOX15 activation in a calcium dependent manner

(Brinckmann et al., 1998, Walther et al., 2004) by membrane binding to either cell membrane or even mitochondrial, endoplasmic reticulum (Kuhn et al., 1990) or nuclear membranes as well (Radmark et al., 2015).

LA, AA, EPA and DHA are possible substrates for ALOX15. The enzyme binding site for the fatty acid substrates is a hydrophobic pocket close to the non-heme iron into which the substrate slides with its methyl end ahead (Kuhn et al., 1986, Rickert and

Klinman, 1999). Phospholipids (Schewe et al., 1975) and cholesterol (Belkner et al., 1991) containing polyunsaturated fatty acids have been shown to be possible substrates to ALOX15 as well. The ability of ALOX15 to oxygenate phospholipids was unexpected because the phospholipid molecule is too big to fit in the binding site of

ALOX15. This can only be explained if the enzyme shows a high degree of motional flexibility permitting rearrangement to facilitate phospholipid binding.

Moreover, membrane lipids, lipids of low-density lipoproteins and lipoproteins were reported to be additional substrates for ALOX15 (Belkner et al., 1993, Kuhn et al.,

1990). This activity of ALOX15 suggested a mechanism for explaining how the bio- membranes are reformed during the process of maturational degradation of cellular organelles (van Leyen et al., 1998).

When LA is the ALOX15 substrate, the main products of the enzymatic reaction are

13-HODE or 9-HODE. While if arachidonic acid is the substrate, ALOX15 produces 15-

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HETE or 12-HETE. These different products are due to the dual positional specificity of

ALOX15.

The human ALOX15 gene is located on the short arm of chromosome 17 in a gene cluster along with the genes encoding for other LOs except for ALOX5 which is localized on the long arm of chromosome 10. The corresponding mouse gene () is localized on chromosome 11. ALOX15 gene spans more than 11 kbp and contains 14 and 13 . The promoter region of the human ALOX15 gene and potential transcription factors’ binding sites were recognized. Human ALOX15 gene showed some genetic variability with 78 single nucleotide polymorphisms and 8 nonsense have been identified (Kelavkar et al., 1998).

Human ALOX15 is constitutively expressed at high levels in immature red blood cells, and airway epithelial cells (Nadel et al., 1991). Lower expression levels have been reported for polymorphonuclear leukocytes of different species

(Narumiya et al., 1982, Vanderhoek and Bailey, 1984), alveolar macrophages (Levy et al., 1993), vascular cells (Takayama et al., 1987), uterus (Lei and Rao, 1992), the male reproductive system (Fischer et al., 2005), various parts of the brain (van Leyen et al.,

2006, Han et al., 2015) and for atherosclerotic lesions (Yla-Herttuala et al., 1990).

Human peripheral blood monocytes do not express ALOX15. However, interleukin-4 and interleukin-13 (IL4, IL13) upregulate ALOX15 in those cells (Conrad et al., 1992,

Nassar et al., 1994).

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3D Crystal structure of ALOX15:

15-LO protein consists of N-terminal domain, C-terminal catalytic domain. The

N-terminal domain has 8 β-sheets which are involved in the membrane binding ability of the enzyme and regulate the catalytic activity of the enzyme as well (Walther et al.,

2004). The C-terminal catalytic domain of ALOX15 comprises 21 helices, with a small

β-sheet sub- domain. The center of the C-terminal domain has two long helices, carrying four of the five protein iron ligands. The substrate-binding pocket is a boot-shaped cavity.

Arachidonic acid substrate slides into the ALOX15 substrate-binding pocket with its methyl end ahead and binds at the active site by hydrophobic interactions. Substrate binding at the active site induces major enzyme structural changes (Gillmor et al., 1997).

Functions of ALOX15:

ALOX15 is able to display its physiological bioactivity via its bioactive lipid products. It oxidizes PUFA containing ester lipids even if they are incorporated in bio- membranes or lipoproteins and regulates the cellular redox equilibrium. ALOX15 has physiological functions in RBCs maturation, brain development /synaptic signaling and adipocyte differentiation. Moreover, ALOX15 has been implicated in tissue growth via

15-HETE -induced angiogenesis (Soumya et al., 2013, Ibrahim et al., 2015a).

ALOX15 has been associated with the regulation of the endothelial permeability which is reported to be involved in the pathogenesis of vascular diseases. 15S-HETE, the major ALOX15 metabolite of arachidonic acid in human, induced endothelial barrier permeability via phosphorylation of zonula occludens-1 and -2, which leads to disruption

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of the tight junctions causing a decrease in the integrity of the endothelial barrier and increasing the permeability (Kundumani-Sridharan et al., 2013, Ibrahim et al., 2015a,

Chattopadhyay et al., 2014). Moreover, alox15 was reported to be involved in high-fat diet-induced endothelial tight junction disruption (Chattopadhyay et al., 2015,

Kundumani-Sridharan et al., 2013). Othman et al reported that ALOX15 bioactive lipids induce retinal endothelial cell barrier dysfunction in retinal endothelial cells via

NADPH-oxidase dependent mechanisms, which include inhibition of protein tyrosine phosphatase and activation of the VEGF-receptor 2 signaling pathway (Othman et al.,

2013). The involvement of NADPH oxidase in ALOX15-induced retinal endothelial barrier dysfunction highlighted the importance of the cellular redox state for the endothelial barrier integrity. Other reports suggested for the first time a role of ALOX15 in the process of autophagy where cells lacking ALOX15 showed dysfunctional autophagy (Morgan et al., 2015).

Several studies elaborated a possible role of ALOX15 in the pathogenesis of inflammation (Radmark et al., 2015, Elmasry et al., 2018a, Ibrahim et al., 2015a,

Othman et al., 2013). The effect of ALOX15 during inflammation depends to a high extent on the substrate as it can exhibit both pro- or anti-inflammatory properties. For instance, the main arachidonic acid/linoleic acid oxygenation products of ALOX15 (15-

HETE, 12-HETE, 13-HODE) exhibit pro-inflammatory activities in various inflammation models (Kuhn, 1996). Reports suggested that the pro-inflammatory properties of

ALOX15 may be in part via affecting the cellular redox state and induction of oxidative stress (Elmasry et al., 2018a, Ibrahim et al., 2015a, Othman et al., 2013,

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Chattopadhyay et al., 2015, Kundumani-Sridharan et al., 2013).

On the other hand, ALOX15 showed anti-inflammatory properties when the substrates are either DHA or EPA via synthesis of pro-resolving bioactive lipids that have the ability of inflammatory resolution (Sala et al., 2010, Serhan et al., 2015a). During the active process of resolution of inflammation, the pro-inflammatory mediators are downregulated whereas the anti-inflammatory mediators are upregulated. These

ALOX15 anti-inflammatory products include lipoxins (Ryan and Godson, 2010), resolvins (Spite et al., 2014), protectins and maresins (Serhan et al., 2015b). These mediators provoke some anti-inflammatory procedures including leukocyte migration inhibition (Freire and Van Dyke, 2013), vascular permeability stabilization (Ereso et al., 2009), apoptosis of pro-inflammatory (El Kebir and Filep, 2013) and macrophages polarization (Ohira et al., 2010). However, it is important to note that

(Kuhn, 1996) suggested that even products of linoleic and arachidonic acid oxygenation

[13S- H(p)ODE, 15S-H(p)ETE] may show anti-inflammatory activities during inflammation as well.

Interestingly, ALOX15 was reported to be implicated in the pathogenesis of different metabolic disorders including obesity and diabetes. Studies showed upregulation of ALOX15 expression is both, in vitro and in vivo animal models of diabetes (Dobrian et al., 2011). In addition, others showed that 12/15-Lo-/- mice have been more resistant to

STZ-induced type 1 diabetes (Bleich et al., 1999). Although, 12/15 LO have been implicated in the pathogenesis of diabetes (Laybutt et al., 2002, Sears et al., 2009,

Bleich et al., 1998, Bleich et al., 1999, Chen et al., 2005a, Nunemaker et al., 2008) but,

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the molecular mechanisms for its contribution are not fully understood. 12/15 LO bioactive products might act as signaling molecules but other mechanisms have also been suggested. For instance, 12/15 LO-induced oxidative stress and subsequent mitochondrial dysfunction might be a suggested mechanism in the pathogenesis of diabetic vascular disorders because of the lipid peroxidation products of 12/15 LO (Boudina and Abel,

2007).

To study biological effects of 12/15-LO metabolites, pharmacological inhibitors have been developed. However, many 12/15-LO inhibitors used (NDGA, CDC, AA861, baicalein, PD146176) did not show a definite isoform-specificity (Rai et al., 2010,

Kenyon et al., 2011). Moreover, these inhibitors display a species-specificity which means some inhibitors that effectively inhibit human ALOX15 may not inhibit orthologous enzymes of other species. In addition, those inhibitors had off-target effects.

12/15-LO inhibitors have anti-oxidative properties and they affect the cellular redox homeostasis. It is difficult to distinguish which of the two functions (LO inhibition vs. redox homeostasis) is the main cause for the detected biological result (Goswami, 2013,

Kim et al., 2013). Therefore, results acquired with these inhibitors must be taken with care to prevent misinterpretation. To avoid these problems, the inhibitor studies should continuously be confirmed by another method as for example the use of the 12/15-Lo siRNA or 12/15-Lo -/- mice.

Finally, we want to highlight that the biological significance of variable positional specificity of ALOX15 orthologs (major 15-lipoxygenating vs. major 12- lipoxygenating

ALOX15 orthologs) still a matter of discussion.

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Regulation of ALOX15:

The promoter of ALOX15 gene includes putative binding sites for STAT6 (Liu et al., 2012). Studies showed that phosphorylation and acetylation of STAT6 have been implicated in ALOX15 regulation (Shankaranarayanan et al., 2001). Moreover, phosphorylation of Jak2 / Tyk2, p38 MAPK induced phosphorylation of STAT1 and

STAT3 and activation of PKCd were reported to be involved in ALOX15 regulation as well (Roy and Cathcart, 1998, Xu et al., 2004). In addition, ERK1/2 protein, the

Elk1/Egr-1/CREB transcription factors and histone deacetylase-induced epigenetic modifications have been involved in the IL4- and IL13-induced ALOX15 upregulation

(Zuo et al., 2009, Bhattacharjee et al., 2013).

The human ALOX15 mRNA contains around 2700 nucleotide bases with an open reading frame encoding for about 662 amino acids. The degree of amino acid product identity of the murine alox15 mRNA to the human ALOX15 is 75%. ALOX15 mRNA has a sequence motif called differentiation control element (DICE) (Reimann et al.,

2002, Messias et al., 2006). Regulatory proteins as hnRNP K and hnRNP E1 have been suggested to bind to the DICE sequence in the 3′-UTR of the ALOX15 mRNA (Ostareck et al., 1997). Recent studies suggested the interaction of DEAD-box RNA helicase 6

(DDX6) with hnRNP K/E1 in a DICE-dependent manner. However, Alox15 mRNA in mice and rats doesn’t have the DICE element. Alternatively, they have 3–6 repetitive

CCCC or UCCC elements (Naarmann et al., 2010).

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B.4. ER Stress:

ER is a very important cell organelle which is formed of a network of branching tubules interconnected via the ER lumen. It is separated from cytoplasm by the ER membrane which is formed of a single lipid bilayer that controls the passage of molecules to and from the ER lumen (Fagone and Jackowski, 2009). ER has critical functions such as proper protein folding, lipid synthesis and calcium homeostasis. Proteins are synthesized in ER-membrane bound ribosomes, then the synthesized proteins are targeted to the ER lumen for being folded into their 3D conformation by molecular chaperones that are abundant in the ER lumen (Anelli and Sitia, 2008). Proteins in ER lumen undergo posttranslational modifications as well. These posttranslational modifications include glycosylation and disulfide bond formation occurs in the ER lumen via ER- resident enzymes such as glycosylating enzymes or as PDI (Tu and

Weissman, 2004). Interestingly, ER has higher calcium concentration and a higher oxidizing redox potential (van Anken and Braakman, 2005).

The protein folding capacity of the ER is surprisingly low as less than 20% of the proteins targeted to the ER lumen are properly folded and ready for their final destination in the secretory pathway. The cell has zero tolerance for the improperly folded proteins and they must be degraded as they may be mediating critical cellular function. This happens via the ER-associated degradation (ERAD) process as a tight protein quality control function (McCracken and Brodsky, 2003). The famous example of a misfolded protein which is malfunctioning causing a serious disease is the one happening in disease. In cystic fibrosis there is a genetic causing generation of a

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misfolded protein lacking an essential chloride ion channel in the epithelium (Rowe et al., 2005).

ER protein-folding ability may be defective with exceeding amount of accumulated misfolded proteins, and thus cells will be undergoing ER stress (Tabas and

Ron, 2011). The protein folding capacity of the ER can be affected by a wide variety of cellular disturbances such as starvation, hypoxia, protein aggregation or disturbed calcium homeostasis (Ma and Hendershot, 2004a). As we mentioned earlier, accumulation of misfolded proteins will cause translocation of membrane bound BIP protein, liberating three transmembrane located proteins (ATF6, PERK, IRE1) from BIP- inhibitory effect and initiating signaling pathways known as UPR. The UPR function initially to restore ER homeostasis via ERAD (Elmasry et al., 2018a, Ma et al., 2014).

However, if ERAD was unable to restore the homeostasis inside the ER lumen, apoptosis will be induced which mainly occurs via PERK pathway activation (Harding et al.,

2000) (Fig.6).

Several studies linked ER stress with different diseases including type 1 diabetes

(Tersey et al., 2012), type 2 diabetes (Cole et al., 2012), diabetic retinopathy (Elmasry et al., 2018a, Li et al., 2009), neurodegenerative diseases (Wang et al., 2011, Vidal et al., 2011), cardiovascular diseases (Minamino et al., 2010, Myoishi et al., 2007) and cancer (Ma and Hendershot, 2004b, Moenner et al., 2007). ER stress represents a new suggested target for therapy of these diseases and studies involving the role of ER stress may pave the way for upcoming clinical trials for drugs targeting ER stress to be used in treatment of different diseases.

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Fig.6: ER stress and UPR. Accumulation of unfolded proteins induces ER stress response with activation of three signaling pathways known as three arms of UPR. The signaling pathways activation results in nuclear translocation of transcriptional factors which are able to upregulate transcription of UPR genes which include molecular chaperones responsible for proper protein folding, ERAD genes or apoptosis pathway. The cell fate decision depends to a large extent on the duration and degree of the ER stress.

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II. MATERIALS AND METHODS

Experimental animals

Animals were used in accordance with the Association for Research in Vision and

Opthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision

Research and all procedures were approved by the Institutional Animal Care and Use

Committee (IACUC) of Augusta University. We used 8-week-old male wild-type (WT),

12/15-Lo knockout mice (12/15-Lo−/−; B6.129S2-Alox15tm1Fun/J; stock no: 002778,

Jackson Laboratories, Bar Harbor, ME, USA), or Nox2-deficient mice (Nox2−/−; B6.129S-

Cybbtm1Din/J; stock No: 002365, Jackson Laboratories) on C57BL/6J background.

Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) (Sigma, St

Louis, MO, USA) at 50 mg/kg for 3-5 days until the mice developed diabetes (random blood glucose ≥ 13.9 mmol/l). Intravitreal injections were performed in non-diabetic mice as previously described using 12-HETE (Cayman Chemical, Ann Arbor, MI, USA)

(Ibrahim et al., 2011, Ibrahim et al., 2015b). To avoid uncontrolled intraocular pressure increase, the volume of intravitreal injections was limited to 1 μl of 12-HETE that was dissolved in ethanol and a working solution of 10× was prepared by diluting 0.32 μl of stock solution (312 μmol/l) to 100 μl with PBS, supposing the vitreous volume of mouse eye is ∼10 μl (Remtulla and Hallett, 1985). Then by injecting 1 μl of this working solution, a 0.1 μmol/l vitreal concentration of 12-HETE was achieved. The dose of 12-

HETE was selected based on what was identified earlier in the vitreous of patients with

DR, 50 ng/ml (∼0.1 μmol/l) (Al-Shabrawey et al., 2011).

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Culture of HRECs

HRECs were purchased from Cell Systems Corporation (Kirkland, WA, USA) and were grown at 37°C under 5% CO2 in Endothelial Basal Medium-2 (EBM-2) (Lonza,

Walkersville, MD, USA) supplemented with 10% FBS (vol./vol.) (Atlantic Biological,

Norcross, GA, USA) and 1% penicillin/streptomycin (vol./vol.) (Corning, Tewksbury,

MA, USA). These cells were negative for mycoplasma and the majority were >95% positive for von Willebrand factor/factor VIII and CD31 (endothelial markers), whereas the contaminating astrocytes (stained with glial fibrillary acidic protein [GFAP]) and pericytes (stained with neural/glial antigen 2 [NG2]) were insignificant <1%. After

HRECs reached 90% confluence, the medium was changed to be serum free, followed by different treatment with 15-HETE (0.1 μmol/l, Cayman Chemical) or vehicle (ethanol) in the presence or absence of 4-phenylbutyric acid (PBA, 30 μmol/l, Santa Cruz, Dallas,

TX, USA), VAS2870 (10 μmol/l, Sigma), or apocynin (30 μmol/l, Calbiochem, La Jolla,

CA, USA). Of note, species-specific divergence between orthologous LO isoforms has been reported for mouse 12-LO, which is arachidonate 15-LO in humans. This difference implies that caution should be taken if experimental data on LO activity are being translated from one species to others (Funk et al., 2002, Kuhn et al., 2015). Therefore,

12-HETE has been used in all mouse experiments while 15-HETE has been used in all human retinal endothelial cell culture experiments. The doses of 12- and 15-HETE were selected according to what was previously detected in the vitreous of individuals with diabetic retinopathy, 50 ng/ml (approximately 0.1–0.2 μmol/l) (Al-Shabrawey et al.,

2011).

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Western blot analysis and immunoprecipitation

Western blot analysis was conducted on retinal or cell lysates using antibodies for

XBP1s, XBP1, p-PERK, PERK, p-eIF2α, eIF2α, ATF4, ATF6, PDI, gp91phox, P47phox, vascular endothelial growth factor receptor-2 (VEGFR2), β-actin and glyceraldehyde-3- phosphate dehydrogenase (GAPDH) (Table 2) according to the previously described method (Ibrahim et al., 2015b). To assess the phosphorylation of VEGFR2 in HRECs, immunoprecipitation was performed by the addition of an anti-VEGFR2 antibody and protein A agarose beads (Cell Signaling Technology, Beverly, MA, USA) per manufacturer’s instructions. Briefly, HRECs were homogenized in 25 mM Tris-HCl (pH

7.4) containing 150 mmol/l NaCl, 1% NP-40, 1 mmol/l EDTA, 5% glycerol, and

1 mmol/l Na3VO4 and protease inhibitor. An equal amount of protein from the vehicle- treated and each treatment group was gently rocked overnight at 4 °C with anti-VEGFR2 antibody (Cell Signaling Technology, Beverly, MA). Prewashed protein A agarose beads

(20 μl of 50% bead slurry) were then added to the mixture and gently rocked overnight at

4 °C. The mixtures were centrifuged; then the immunoprecipitates were washed five times with cell lysis buffer. After washing, the resultant pellets were re-suspended in

20 μl of 3X SDS sample buffer and then denatured for 5 min at 95 °C, loaded on SDS-

PAGE gel, and transferred to polyvinyl difluoride membranes. This was followed by immunoblotting using anti-phosphotyrosine-PY20 antibody (BD Transduction

Laboratories, San Diego, CA, USA). To evaluate loading of an equal amount of protein, the blots were stripped and incubated with the anti-VEGFR2 antibody.

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Table 2. Antibodies Used in the Experiments

Antigen Source Molecular Dilution Company Catalog weight No. (kDa) XBP1s rabbit 60 1:1000 Cell Signaling 12782 Technology, Beverly, MA

XBP1 rabbit ~ 40 1:1000 Invitrogen, Eugene, OR PA5- 25010 phospho- rabbit 170 1:1000 Cell Signaling 3179 PERK Technology

PERK rabbit 140 1:1000 Cell Signaling 5683 Technology phospho- rabbit 37 1:1000 Cell Signaling 9721 EIF2α Technology

EIF2α rabbit 37 1:1000 Cell Signaling 5324 Technology

ATF4 rabbit 49 1:1000 Abcam, Cambridge, MA ab184909

ATF6 rabbit 66-90 1:1000 Invitrogen, Eugene, OR PA5- 20216

PDI mouse 57 1:1000 Abcam ab2792

NOX2 or mouse 91 1:1000 BD transduction 611415 gp91phox laboratories, San Diego, CA

P47phox mouse 47 1:1000 BD transduction 610355 laboratories

VEGFR2 rabbit 210,230 1:1000 Cell Signaling 2479 Technology

β-actin mouse 42 1:2000 Abcam ab8226

GAPDH rabbit ~ 40 1:1000 Abcam ab9485

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Quantitative real-time RNA PCR

The mRNA levels of various ER stress markers were measured using TaqMan primers for Eif2a, Atf6, Chop (also known as Ddit3), Xbp1, Pdi (also known as Pdia3), calnexin and 18s for mouse samples, or IRE1α, XBP1, PERK (also known as EIF2AK3),

ATF4, ATF6, CHOP (also known as DDIT3), PDI (also known as PDIA2), BIP (also known as HSPA5) and calnexin for HREC samples (Table 3) according to the previously described method (Ibrahim et al., 2017). PCR amplification was performed using

StepOnePlus Real-time PCR System (Applied Biosystem, Foster City, CA, USA). The thermocycling program consisted of 50°C for 2 min and 95°C for 2 min, then 40 cycles at

95°C for 3 seconds, and 60°C for 3 minutes.

Enzyme-linked immunosorbent assay (ELISA)

Vascular endothelial growth factor (VEGF) level in HREC conditioned media was estimated using VEGF ELISA kits (R&D Systems, Minneapolis, MN, USA) per the manufacturer’s instructions. First, Assay Diluent was added to each well (50 μl of Assay

Diluent RD1W). Then, the standards and samples were added in each well (200 μl).

Plates were incubated for 2 hours at room temperature. Wells were aspirated and washed with Wash Buffer (400 μl) 3 times. Conjugates (Monoclonal antibody specific for VEGF and conjugated to horseradish peroxidase) were then added (200 μl) to each well. The plate was incubated for additional 2 hours at room temperature. Then washing was done again. Substrate Solution was added (200 μl) to each well and protected from light.

Incubation with substrate was done at room temperature (20 minutes). Lastly, 50 μl of

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Stop Solution was added to each well. The optical density was detected in each well within 30 minutes, using a microplate reader set to 450 nm and corrected at 540 nm.

Table 3. Primers Used in the Experiments

Primers for Species ID Company

Eif2α Mouse Mm01289723

Atf6 Mouse Mm01295319_m1

Ddit3 or CHOP Mouse Mm01135937_g1

XBP1 Mouse Mm00457357_m1

Pdia3 Mouse Mm00433130_m1

Calnexin Mouse Mm00500330_m1

18s Mouse Mm03928990_g1

IRE1 α Human Hs00176385_m1 Applied Biosystem, XBP1 Human Hs00231936_m1 Foster City, CA,

PERK Human Hs00984006_m

ATF4 Human Hs00909569_g1

ATF6 Human Hs00232586_m1

CHOP Human Hs01090850_m1

PDI Human Hs00607126_m1

BIP Human Hs00607129_gH

Calnexin Human Hs01558409_m1

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In vitro leukocyte adhesion assay

This assay was done according to our previous procedure (Ibrahim et al., 2015a) where adherent leukocytes were counted under an inverted fluorescence microscope

(excitation/emission = 480/520 nm). HRECs were grown to confluence in 12 well plates then shifted to serum free media overnight. Treatment was added to HRECs for 24 h.

Following the treatment, leukocytes (300,000–400,000 cells/well) (Sanguine Bioscience,

Valencia, CA, USA) labelled with diluted LeukoTracker solution (Cell Biolabs, Inc, San

Diego, CA, USA) for 60 minutes at 370C. the labelled leukocytes were added to the confluent monolayer of HRECs and incubated for 90 min at 37 0C. Immediately before the assay, non-adherent cells were removed by washing three times with Endothelial

Basal Medium-2 (EBM-2). The fluorescent attached leukocytes were counted under the inverted fluorescence microscope from three separate fields per well.

Superoxide measurement using dihydroethidium

HRECs were incubated with dihydroethidium (DHE) (20 µmol/l, Invitrogen, Eugene,

OR, USA; catalogue no. D11347) in Earle’s balanced salt solution (EBSS) for 30 min.

The medium was then replaced by EBSS containing NADPH (100 µmol/l) as an enzyme substrate for NOX followed by 15-HETE treatment in the presence or absence of

VAS2870 (NADPH oxidase inhibitor, 10 µmol/l, Sigma) or PBA (30 µmol/l, Santa Cruz).

A microplate reader (BioTek Instruments, Winooski, VT, USA) was used to detect the fluorescence intensity at different time intervals.

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Live imaging of intracellular calcium

Intracellular calcium (Ca2+) mobilisations were detected by the use of Cal-520, AM

(AAT Bioquest, Sunnyvale, CA, USA; catalogue no. 21130) according to manufacturer’s instructions. Measurements were performed using a Zeiss 780 Inverted Confocal microscope (Zeiss 780 Inverted Confocal, Imaging Core Facility, Augusta University) at

(excitation/emission = 490/525 nm)

Statistical data analysis

The results are expressed as means ± SEM. Differences among different experimental groups were assessed by one-way ANOVA or two-tailed t test. Results were considered significant for p <0.05.

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III. RESULTS

12/15-LO plays a causal role in diabetes-induced retinal endoplasmic reticulum stress

To explore the role of the 12/15-LO pathway in diabetes-induced retinal ER stress, we used two complementary approaches. The first approach was to test whether deletion of 12/15-Lo reduces retinal ER stress induced by diabetes. To achieve this, we induced diabetes in 12/15-Lo−/− mice and their matched congenic controls by STZ injection. After 12-14 weeks in diabetes, the animals were killed and retinas were collected to screen for ER stress markers by quantitative real-time RNA PCR (q-RT-

PCR). Absence of the 12/15-Lo gene in the retina of 12/15-Lo−/− mice was confirmed by genotyping according to the protocol of the Jackson Laboratory (data not shown).

Diabetes caused upregulation of the mRNA of many ER stress marker genes, including

Xbp1 (p< 0.01), Eif2α (p= 0.06), Atf6, Pdi and Chop (p< 0.05) in retinas of diabetic WT mice compared with the non-diabetic littermates. These markers were significantly lower in retinas of diabetic 12/15-Lo−/− compared with diabetic WT mice, in particular, Xbp1

(p< 0.001), Atf6 (p< 0.05), Pdi (p< 0.01) and Chop (p< 0.01) (Fig. 7a).

Further evidence for the involvement of 12/15-LO in the induction of retinal ER stress was acquired from the second approach in which normal mice were injected

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intravitreally with the 12/15-LO-derived predominant murine metabolite 12-HETE. The effects of 12-HETE on retinal ER stress marker mRNA and proteins were examined by qRT-PCR and western blot, respectively. Retinal levels of ER stress markers mRNA were upregulated one week after intravitreal injection of 12-HETE (0.1 µmol/l) including

Eif2α (p< 0.01), calnexin (p< 0.05), Chop (p<0.001) and Pdi (p= 0.06), compared with retinas of vehicle-injected animals (Fig. 7b). Moreover, western blot analysis (Fig.7 c) revealed marked increases in p-PERK/PERK, PDI and ATF4 levels in retinas of 12-

HETE-injected mice compared with the vehicle-injected controls, suggesting a causal relationship between 12/15-LO and ER stress induction in diabetic retinopathy.

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Fig.7: Loss of 12/15-LO reduces diabetes-induced ER stress in mouse retina. (a) q-RT-PCR analysis of relative mRNA expression of ER stress markers in retinas of WT non-diabetic mice (white circles), WT diabetic mice (black squares), 12/15-Lo−/− non-diabetic mice (white triangles) and 12/15-Lo−/− diabetic mice (black triangles). Data represent individual data points with means ± SEM. (b) q-RT-PCR analysis of relative mRNA expression of ER stress markers in retinas of WT mice that received intravitreal injection of vehicle (white circles) or 12-HETE (0.1 µmol/l) (black squares). Data represent individual data points with means ± SEM. (c) Representative western blots and densitometry of ER stress protein levels in retinas isolated from WT mice that received intravitreal injection of vehicle or 12-HETE (0.1 µmol/l). Data represent means ± SEM; *p<0.05, **p<0.01, ***p<0.001

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12/15-LO-induced endothelial dysfunction is ER-stress-dependent

The aforementioned data provided a rationale to study in depth the direct relationship between 12/15-LO and ER stress in mediating endothelial dysfunction using

HRECs. Initially, we sought to confirm whether 12/15-LO-derived bioactive lipids could induce ER stress in vitro in HRECs. For this purpose, HRECs were treated with 15-

HETE (0.1 µmol/l) or vehicle and cell lysates were collected at different time points (1, 4 and 24 h). Western blot analysis showed an increase in the levels of PDI by 15-HETE starting after 1 h of treatment and reached a significant difference after 4 h (Fig. 8a).

Concordantly, WB analyses for other ER stress markers (Fig. 8b) revealed that p-

PERK/PERK and ATF6 showed significant increases in their levels compared with vehicle-treated cells. In (Fig. 8c), RT-PCR array of ER stress genes was utilised to assess

UPR activation in HRECs treated with 15-HETE for 3 h. There was a significant upregulation of CHOP by 15-HETE compared with the vehicle-treated group (p< 0.05).

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Fig.8: 15-HETE induces ER stress in HRECs. (a) Representative western blots and densitometry for PDI protein level in HRECs treated with vehicle or 15-HETE (0.1 µmol/l) at different time points (0-24 h). Data represent means ± SEM. (b) Representative western blots and densitometry for ER stress proteins in HRECs treated with vehicle or 15-HETE (0.1 µmol/l) for 4 h. Data represent means ± SEM. (c) q-RT-PCR of relative mRNA expression of ER stress markers in HRECs treated with vehicle or 15-HETE (0.1 µmol/l) for 3 h. Data represent means ± SEM; *p<0.05, **p<0.01

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To functionally evaluate the impact of ER stress on 15-HETE-mediated endothelial dysfunction, we tested the effect of ER stress inhibition on 15-HETE-induced leukostasis in HRECs. Initial studies were conducted to confirm the contribution of ER stress in endothelial-leukocyte interaction using tunicamycin (Sigma), a known ER stress inducer.

HRECs were treated with tunicamycin (5 µg/ml) for 24 h before adding fluorescently labelled human leukocytes for 90 min, followed by washing to remove the non-adherent leukocytes. As shown in Fig. 9, ER stress induction by tunicamycin led to a significant increase in the number of adherent leukocytes to the HRECs in comparison with the vehicle-treated cells (p< 0.001). To investigate the involvement of ER stress in 15-HETE- induced leukostasis, ER stress inhibitor (PBA, 30 µmol/l) was added to HRECs before

15-HETE treatment. PBA significantly reduced (p< 0.01) the number of adherent leukocytes induced by 15-HETE. At the concentration of PBA used, HREC viability was not affected (90 ± 6%) as assessed by (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) (MTT) assay, indicating that the decrease in the number of adherent leukocytes to 15-HETE-activated HRECs was due to the inhibition of ER stress, but not to cell death.

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Fig.9: ER stress induction triggers endothelial leukocyte adhesion in HRECs, while ER stress inhibition abrogates 15-HETE-induced leukocyte adhesion. (a) In vitro leukocyte adhesion assay showing fluorescently labelled leukocytes adherent to HRECs under the influence of different treatments: control (vehicle), 15-HETE in the presence or absence of lipopolysaccharide (LPS), tunicamycin (TM), VEGF and ER stress inhibitor (PBA). (b) Quantification of the fold change of the mean number of the adherent leukocytes in each group showing a significant increase by the ER stress inducer tunicamycin, LPS, VEGF and 15-HETE compared with the vehicle-treated group. ER stress inhibition using PBA significantly reduced 15-HETE-induced endothelial leukocyte adhesion. Data represent means ± SEM; *p<0.05, **p<0.01; ***p<0.001; scale bar, 50 µm

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Crosstalk between NADPH oxidase and ER stress

We previously reported that NADPH oxidase is implicated in 12/15-LO-induced retinal endothelial cell dysfunction (Ibrahim et al., 2015a, Othman et al., 2013). Here, the crosstalk between NADPH oxidase and ER stress in 12/15-LO-induced retinal microvascular dysfunction was also examined. HRECs were treated with 15-HETE with or without the NADPH oxidase inhibitor apocynin (30 µmol/l). The ER stress marker

PDI was upregulated by 15-HETE in the presence or absence of apocynin (p= 0.008 vs control) (Fig. 10a). On the contrary, ER stress inhibition with PBA significantly reduced

15-HETE-induced expression of NOX2 and P47phox (Fig.10b), which are the catalytic and regulatory subunits of NADPH oxidase, respectively. Furthermore, 15-HETE significantly increased superoxide production after 15 min (p< 0.001) and 30 min (p<

0.01) vs control that declined at 45 min. ER stress inhibition by PBA significantly reduced 15-HETE-induced superoxide production (p< 0.05 vs 15-HETE treated HRECs) and this effect was comparable to the effect of NADPH oxidase inhibition (Fig. 10c).

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Fig.10: Crosstalk between NADPH oxidase and ER stress in 15-HETE-treated HRECs. (a) Representative western blots and densitometry for PDI protein level in HRECs treated for 24 h with vehicle (control), 15-HETE (0.1 µmol/l) or 15-HETE + NADPH oxidase inhibitor (apocynin, 30 µmol/l). Data represent means ± SEM. (b) Representative western blots and densitometry for NOX2 protein or P47phox protein in HRECs treated for 24 h with vehicle, 15-HETE (0.1 µmol/l) or 15-HETE + ER stress inhibitor (PBA, 30 µmol/l). Data represent means ± SEM. (c) Superoxide measurement using DHE staining in HRECs treated with vehicle (white circles), 15- HETE (0.1 µmol/l), 15-HETE (0.1 µmol/l) + VAS2870 (NADPH oxidase inhibitor, 10 µmol/l) or 15-HETE (0.1 µmol/l) + PBA (30 µmol/l). Data represent means ± SEM

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This proof of concept was then extended to be tested in vivo using Nox2−/− mice.

Non-diabetic Nox2−/− mice were injected intravitreally with 12-HETE (0.1 µmol/l). One week after the injection, retinas were collected and both q-RT-PCR and western blotting were performed to screen the changes in retinal levels of ER stress genes and proteins, respectively. Intravitreal injection of 12-HETE in Nox2−/− mice induced increases in RNA levels of Xbp1 (p< 0.001), Eif2α (p< 0.05), Atf6 (p< 0.01), Pdi (p<001), Chop (p< 0.05) and calnexin (p= 0.09) (Fig. 11a). Furthermore, 12-HETE significantly upregulated protein levels of retinal PDI (p= 0.03 vs vehicle-injected) and ATF4 (p= 0.02 vs vehicle- injected) in Nox2−/− mice and WT mice compared with the vehicle-injected group (Fig.

10b). Taken together, our in vivo and in vitro data suggest that NADPH oxidase does not have a regulatory role in 12/15-LO-mediated ER stress in the retina or retinal endothelial cells.

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Fig.11: Nox2 deletion has no effect on 12-HETE-induced ER stress in vivo. (a) q-RT-PCR analysis of relative mRNA expression of ER stress markers in retinas isolated from Nox2−/− mice that had received intravitreal injection of vehicle or 12-HETE (0.1 µmol/l). Data represent means ± SEM. (b) Representative western blots and densitometry of ER stress proteins PDI and ATF4 in retinas isolated from WT or Nox2−/− mice after intravitreal injection of 12-HETE (0.1 µmol/l). Data represent means ± SEM. *p<0.05, **p<0.01; ***p<0.001

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15-HETE induces intracellular calcium release

To find out a possible mechanism by which 12/15-LO induces ER stress, we were interested in testing whether 12/15-LO-derived lipid products dysregulate calcium homeostasis, a known regulator of ER stress in HRECs (Fu et al., 2011). To this end,

HRECs were loaded with a Ca2+-detecting dye followed by adding vehicle or 15-HETE

(0.1 µmol/l). Real-time imaging showed a marked increase in the level of cytosolic calcium after adding 15-HETE compared with the control group. The ability of 15-HETE to induce intracellular calcium release may underscore a possible mechanism for 12/15-

LO-induced ER stress (Fig. 12).

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Fig.12: 15-HETE induces intracellular Ca2+ release in HRECs. (a) Real-time imaging of intracellular calcium in HRECs treated with vehicle or 15- HETE (0.1 µmol/l). HRECs were loaded with a calcium-detecting dye, and then treated with vehicle or 15-HETE under the inverted confocal microscope. Treatment of HRECs with 15-HETE caused a significant increase in the fluorescence intensity, suggesting increased intracellular cytosolic Ca2+ levels. (b) Quantification of normalised calcium fluorescence in HRECs treated with vehicle (blue line) or 15-HETE (0.1 µmol/l) (red line) over 30 s from the beginning of the treatment. Data represent means ± SEM. Statistical analysis of the averaged normalised fluorescence intensity of at least three independent experiments shows that 15-HETE treatment significantly increases HRECs intracellular cytosolic calcium levels compared with the vehicle-treated HRECs (*p<0.05). Data represent means ± SEM; scale bars, 200 µm

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Inhibition of ER stress attenuates 15-HETE-induced VEGFR2 phosphorylation

We measured VEGF levels in the supernatant of HRECs treated with 15-HETE or vehicle. 15-HETE had no effect on VEGF release from HRECs when compared with that of the vehicle-treated group (n=4, p> 0.05) (Fig. 13a). We previously showed that inhibition of NADPH oxidase reduced VEGFR2 phosphorylation in retinal endothelial cells treated with 12/15-LO-derived metabolites (Othman et al., 2013). To investigate whether ER stress inhibition elicits a similar effect, HRECs were treated with 15-HETE for 5 min in the presence or absence of PBA (30 µmol/l), then immunoprecipitation was used to detect VEGFR2 phosphorylation (Fig. 13b). 15-HETE induced VEGFR2- phosphorylation (p< 0.05), which was significantly reduced by the ER stress inhibitor

(PBA) and the NADPH oxidase inhibitor (apocynin) (n=4, p< 0.05) (Fig. 7c). These data suggest that ER stress could play a role in 12/15-LO-induced retinal microvascular complications via activation of VEGFR2 signalling.

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Fig.13: ER stress inhibition attenuates 15-HETE-induced VEGFR2 phosphorylation. (a) ELISA of VEGF levels in supernatant of HRECs treated with vehicle or 15- HETE for 24 h showing no significant difference in the VEGF levels. Data represent means ± SEM. (b) Immunoprecipitation (IP) of VEGFR2 in HRECs treated with 15- HETE in the presence or absence of PBA or apocynin for 5 min, followed by immunoblotting (IB) of phosphotyrosine (pTyr). Normalisation was done by re- blotting the membrane for VEGFR2. Densitometry analysis shows a significant increase in VEGFR2 phosphorylation by 15-HETE compared with the control. ER stress inhibitor (PBA) and NADPH oxidase inhibitor (apocynin) reduced the effect of 15-HETE on VEGFR2 phosphorylation. Data represent means ± SEM; *p< 0.05

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IV. DISCUSSION

Our previous studies established 12/15-LO as a potential player in the development of microvascular dysfunction in diabetic retinopathy via activating NADPH oxidase and VEGFR2 signalling (Ibrahim et al., 2015a, Ibrahim et al., 2017, Othman et al., 2013). The current study explored the role of ER stress in mediating the effect of

12/15-LO lipid metabolites on retinal endothelial dysfunction during diabetic retinopathy.

Our major findings include: (1) deletion of 12/15-Lo significantly attenuated the diabetes- induced ER stress in mouse retina; (2) 15-HETE, the major product of 12/15-LO in humans, induced ER stress in HRECs; (3) inhibition of ER stress reduced 15-HETE- induced leukocyte adhesion; (4) inhibition of NADPH oxidase or deletion of its catalytic subunit NOX2 did not reduce 12/15-LO-induced ER stress; (5) inhibition of ER stress reduced NADPH oxidase activity and expression of its catalytic and regulatory subunits

NOX2 and P47phox, respectively; (6) 15-HETE induced an increase in intracellular calcium levels in HRECs; (7) inhibition of ER stress reduced 15-HETE-induced

VEGFR2 phosphorylation.

Accumulated evidence demonstrated that ER stress is a crucial player in the development of retinal microvascular dysfunction in diabetic retinopathy (Ma et al.,

2014, Zhang et al., 2015). However, the implication of ER stress in 12/15-LO-mediated retinal endothelial cell activation has not been investigated yet. The current study portrays a mechanistic axis that links hyperglycaemia to retinal endothelial cell

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dysfunction. This axis starts from hyperglycaemia-induced activation of 12/15-LO, followed by ER stress and NADPH oxidase activation, and ends with increased VEGFR2 phosphorylation. Consistent with previous reports, our in vivo experiments showed that diabetes induces ER stress in mouse retina (Li et al., 2009). Our RNA data from the

12/15-Lo−/− mice showed a significant reduction in ER stress markers in diabetic 12/15-

Lo−/− retina vs diabetic WT mouse retina. Moreover, intravitreal injection of 12-HETE, the main bioactive lipid product of 12/15-LO in rodents, into the eyes of non-diabetic WT mice activated retinal ER stress, suggesting ER stress as an important player in mediating the pro-inflammatory and permeability effects of 12/15-LO in the retina in diabetes.

Although our diabetes model was type 1, another study utilised 12/15-Lo−/− mice placed on a high-fat diet (as a model for pre-type-2 diabetes) (Cole et al., 2012) showed a significant reduction in ER stress markers as well, compared with WT mice fed a normal diet. Interestingly, ER stress markers that changed with a high-fat diet were different among different tissues, indicating the complexity and variability of the ER stress response across different tissues.

We also used an in vitro model utilising HRECs treated with 15-HETE and we detected activation of the ER stress response by 15-HETE. Lipoxygenases catalyse oxygen insertion into polyunsaturated fatty acids. There are different LO analogues such as 5-LO, 12-LO, and 15-LO. The different numbers indicate the carbon at which the oxygen is added. Interestingly, a previous study reported that deletion of 5- or 12/15-Lo dampens diabetes-induced leukostasis after 3 months of diabetes. However, 5-Lo deletion only reduced capillary degeneration after 9 months of diabetes, suggesting a differential

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role for each enzyme at different stages of diabetic retinopathy (Gubitosi-Klug et al.,

2008). Our data showed that ER stress inhibition significantly reduced 15-HETE-induced leukostasis in HRECs, suggesting the early involvement of ER stress in the 12/15-LO- induced inflammatory reaction.

Others have reported the presence of interaction between ER and oxidative stresses in endothelial cell dysfunction. Recently, mRNA-sequencing data showed ER stress-induced downregulation of endogenous antioxidant genes (Han et al., 2013). Also,

ER stress induces endothelial dysfunction via NADPH oxidase. This notion has been supported by studies done both in vitro utilising primary coronary endothelial cells and in vivo using p47phox−/− mice injected with tunicamycin (Galan et al., 2014). We previously reported that 12/15-LO induced retinal microvascular complications in an NADPH- oxidase-dependent mechanism (Ibrahim et al., 2015a, Ibrahim et al., 2017, Othman et al., 2013). In the current study, we looked at the relationship between ER stress and

NADPH oxidase in retinal endothelial cell dysfunction induced by 12/15-LO-derived metabolites. 12/15-LO-derived bioactive lipids induced ER stress despite the deletion or inhibition of NADPH oxidase, suggesting that either ER stress and NADPH oxidase are working independently, or ER stress is an upstream effector of NADPH oxidase. To uncover which of these two possibilities is involved, we measured superoxide production and the expression of NADPH oxidase subunits, NOX2 and P47phox in HRECs after 15-

HETE treatment in the presence or absence of a ER stress inhibitor. ER stress inhibition significantly reduced 15-HETE-induced superoxide production and expression of NOX2, and P47phox; in HRECs subjected to 15-HETE treatment. These findings suggest that ER

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stress is an upstream effector of NADPH oxidase in the signalling pathway induced by activated retinal 12/15-LO in diabetes.

PDI has been suggested as an organiser of NADPH oxidase activation in leukocytes (de et al., 2011), vascular smooth muscles and endothelial cells (Laurindo et al., 2008, Janiszewski et al., 2005). 12/15-LO-derived lipid metabolites increased levels of PDI in HRECs and mouse retina, which was not abrogated by either inhibition or deletion of NOX2. Moreover, diabetes-induced PDI upregulation was reduced in diabetic

12/15-LO−/− mouse retina. Interestingly, it was shown that PDI is associated with

NADPH oxidase and acts as a redox-dependent enzyme complex regulator with a supportive effect on the activity of NADPH oxidase (de et al., 2011). Thus, the ability of

12/15-LO-derived bioactive lipids to upregulate PDI both in vitro and in vivo suggests a mechanistic link between 12/15-LO-induced ER stress and NADPH oxidase activation during diabetic retinopathy.

It was reported that disruption of both lipid metabolism and calcium homeostasis causes activation of ER stress in the liver (Fu et al., 2011). Moreover, thapsigargin, a widely used drug for ER stress induction, is an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), leading to elevated intracellular cytosolic Ca2+ levels and induction of ER stress (Rogers et al., 1995). In obesity, it was reported that lipids inhibit SERCA and cause disturbance of Ca2+ homeostasis with subsequent activation of

ER stress (Fu et al., 2011), which might be a possible mechanism of 12/15-LO-induced

ER stress. In addition, the role of NOX2 in ER stress-oxidative stress interplay is Ca2+ dependent (Li et al., 2010, Santos et al., 2009).

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We reported in this study the 12/15-LO-induced upregulation of ATF4.

Interestingly, ATF4 is not only involved in ER stress-induced oxidative stress (Han et al.,

2013), but in addition, ATF4 binds to the promoter of human VEGF and upregulates both

VEGF and cytokine production in retinas of diabetic rodents. Recently, ATF4 was reported to have a critical role in the regulation of the inflammatory response in brain and retinal endothelial cells (Huang et al., 2015, Chen et al., 2012b, Wang et al., 2017). The role of ER stress in diabetic retinopathy has been also linked to activation of VEGF signalling (Zhong et al., 2012, Chen et al., 2012b). The link between ER stress and

VEGF is supported by other reports that ER stress inhibition reduces VEGF expression in retinas of diabetic mice (Li et al., 2009). On the other hand, VEGF activates ER stress signalling in endothelial cells independent of the accumulation of misfolded proteins within the ER lumen (Karali et al., 2014), suggesting that both ER stress and VEGF have positive feedback. Taking together the accumulated evidence of ER stress–VEGF crosstalk, and our previous study (Othman et al., 2013), which demonstrated activation of VEGFR2 in retinal endothelial cells by 12/15-LO-derived metabolites, we examined whether ER stress is involved in activating VEGF signalling by 12/15-LO metabolites.

Consistent with our previous report (Othman et al., 2013), our data showed that, while

15-HETE has no effect on VEGF expression in HRECs, it significantly activated

VEGFR2. This effect was significantly attenuated by PBA and apocynin, the ER stress and NADPH oxidase inhibitors, respectively. These data support the potential role of both

ER stress and NADPH oxidase in mediating the effect of 15-HETE on retinal endothelial function via activating VEGF signalling.

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The ubiquitin–proteasome system (UPS) is an important member of the protein quality control machinery (Shang and Taylor, 2004, Ye, 2005). UPS dysfunction is implicated in ER stress-induced apoptosis (Casas et al., 2007), oxidative stress induction

(Ishii et al., 2005, Shang and Taylor, 2012), disturbed calcium homeostasis (Liu et al.,

2015a) and retinal diseases including diabetic retinopathy (Campello et al., 2013).

Recently, defective UPS was reported to cause ER stress in diabetic retina (Shruthi et al., 2017). However, other studies showed that ER stress itself can cause defective UPS

(Menendez-Benito et al., 2005). The discrepancy noticed in RNA and protein levels of

ER stress markers can be explained in part through disturbed UPS. Studying the impact of 12/15-LO on UPS may provide new insights into the mechanism of 12/15-LO-induced

ER stress.

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V. SUMMARY

In summary, our previous and current data suggest that 12/15-LO is implicated in retinal microvascular dysfunction in diabetic retinopathy via activation of the ER stress/NADPH oxidase/VEGFR2 signalling pathway, and that disruption of Ca2+ homeostasis could be an essential step in initiating this signalling pathway (Fig. 13). Thus, the 12/15-LO/ER stress signalling loop may represent an early player that could be targeted to prevent progression of microvascular dysfunction in diabetic retinopathy.

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Fig.14: A graphical summary. Hyperglycaemia increases the activity of 12/15-LO enzyme with a subsequent increase of its bioactive lipid products, 12- and 15-HETE, in retinal endothelial cells. 12/15-LO lipid metabolites increase endothelial intracellular calcium levels that may activate ER stress and in turn NADPH oxidase. These intracellular activated pathways lead to increased phosphorylation of VEGFR2 and subsequent retinal endothelial dysfunction.

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VI. REFERENCES

1998. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). UK Prospective Diabetes Study (UKPDS) Group. Lancet, 352, 837- 53. AIELLO, L. P., BURSELL, S. E., CLERMONT, A., DUH, E., ISHII, H., TAKAGI, C., MORI, F., CIULLA, T. A., WAYS, K., JIROUSEK, M., SMITH, L. E. & KING, G. L. 1997. Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta- isoform-selective inhibitor. Diabetes, 46, 1473-80. AIELLO, L. P., CLERMONT, A., ARORA, V., DAVIS, M. D., SHEETZ, M. J. & BURSELL, S. E. 2006. Inhibition of PKC beta by oral administration of ruboxistaurin is well tolerated and ameliorates diabetes-induced retinal hemodynamic abnormalities in patients. Invest Ophthalmol Vis Sci, 47, 86-92. AL-SHABRAWEY, M., BARTOLI, M., EL-REMESSY, A. B., MA, G., MATRAGOON, S., LEMTALSI, T., CALDWELL, R. W. & CALDWELL, R. B. 2008. Role of NADPH oxidase and Stat3 in statin-mediated protection against diabetic retinopathy. Invest Ophthalmol Vis Sci, 49, 3231-8. AL-SHABRAWEY, M., ELSHERBINY, M., NUSSBAUM, J., OTHMAN, A., MEGYERDI, S. & TAWFIK, A. 2013. Targeting Neovascularization in Ischemic Retinopathy: Recent Advances. Expert Rev Ophthalmol, 8, 267-286. AL-SHABRAWEY, M., MUSSELL, R., KAHOOK, K., TAWFIK, A., ELADL, M., SARTHY, V., NUSSBAUM, J., EL-MARAKBY, A., PARK, S. Y., GUREL, Z., SHEIBANI, N. & MADDIPATI, K. R. 2011. Increased expression and activity of 12-lipoxygenase in oxygen-induced ischemic retinopathy and proliferative diabetic retinopathy: implications in retinal neovascularization. Diabetes, 60, 614- 24. AL-SHABRAWEY, M., ZHANG, W. & MCDONALD, D. 2015. Diabetic retinopathy: mechanism, diagnosis, prevention, and treatment. Biomed Res Int, 2015, 854593. ALTMANN, C. & SCHMIDT, M. H. H. 2018. The Role of Microglia in Diabetic Retinopathy: Inflammation, Microvasculature Defects and Neurodegeneration. Int J Mol Sci, 19. AMANO, S., YAMAGISHI, S., KATO, N., INAGAKI, Y., OKAMOTO, T., MAKINO, M., TANIKO, K., HIROOKA, H., JOMORI, T. & TAKEUCHI, M. 2002. Sorbitol dehydrogenase overexpression potentiates glucose toxicity to cultured retinal pericytes. Biochem Biophys Res Commun, 299, 183-8.

93

AMANO, S., YAMAGISHI, S., KODA, Y., TSUNEOKA, M., SOEJIMA, M., OKAMOTO, T., INAGAKI, Y., YAMADA, K. & KIMURA, H. 2003. Polymorphisms of sorbitol dehydrogenase (SDH) gene and susceptibility to diabetic retinopathy. Med Hypotheses, 60, 550-1. ANDERSON, D. H. & FISHER, S. K. 1976. The photoreceptors of diurnal squirrels: outer segment structure, disc shedding, and protein renewal. J Ultrastruct Res, 55, 119-41. ANELLI, T. & SITIA, R. 2008. Protein quality control in the early secretory pathway. EMBO J, 27, 315-27. ARROBA, A. I. & VALVERDE, A. M. 2017. Modulation of microglia in the retina: new insights into diabetic retinopathy. Acta Diabetol, 54, 527-533. AUGUSTIN, A. J., GRUS, F. H., KOCH, F. & SPITZNAS, M. 1997. Detection of eicosanoids in epiretinal membranes of patients suffering from proliferative vitreoretinal diseases. Br J Ophthalmol, 81, 58-60. AVITABILE, T., LONGO, A. & REIBALDI, A. 2005. Intravitreal triamcinolone compared with macular laser grid photocoagulation for the treatment of cystoid macular edema. Am J Ophthalmol, 140, 695-702. BAI, N., TANG, S., MA, J., LUO, Y. & LIN, S. 2003. Increased expression of intercellular adhesion molecule-1, vascular cellular adhesion molecule-1 and leukocyte common antigen in diabetic rat retina. Yan Ke Xue Bao, 19, 176-83. BATES, D. O. & CURRY, F. E. 1997. Vascular endothelial growth factor increases microvascular permeability via a Ca(2+)-dependent pathway. Am J Physiol, 273, H687-94. BAYNES, J. W. 1991. Role of oxidative stress in development of complications in diabetes. Diabetes, 40, 405-12. BAZAN, N. G. 2007. Omega-3 fatty acids, pro-inflammatory signaling and neuroprotection. Curr Opin Clin Nutr Metab Care, 10, 136-41. BAZAN, N. G., MUSTO, A. E. & KNOTT, E. J. 2011. Endogenous signaling by omega- 3 -derived mediators sustains homeostatic synaptic and circuitry integrity. Mol Neurobiol, 44, 216-22. BELKNER, J., WIESNER, R., KUHN, H. & LANKIN, V. Z. 1991. The oxygenation of cholesterol esters by the lipoxygenase. FEBS Lett, 279, 110-4. BELKNER, J., WIESNER, R., RATHMAN, J., BARNETT, J., SIGAL, E. & KUHN, H. 1993. Oxygenation of lipoproteins by mammalian lipoxygenases. Eur J Biochem, 213, 251-61. BHATTACHARJEE, A., SHUKLA, M., YAKUBENKO, V. P., MULYA, A., KUNDU, S. & CATHCART, M. K. 2013. IL-4 and IL-13 employ discrete signaling pathways for target in alternatively activated monocytes/macrophages. Free Radic Biol Med, 54, 1-16.

94

BLACKSHAW, S., HARPAVAT, S., TRIMARCHI, J., CAI, L., HUANG, H., KUO, W. P., WEBER, G., LEE, K., FRAIOLI, R. E., CHO, S. H., YUNG, R., ASCH, E., OHNO-MACHADO, L., WONG, W. H. & CEPKO, C. L. 2004. Genomic analysis of mouse retinal development. PLoS Biol, 2, E247. BLEICH, D., CHEN, S., GU, J. L. & NADLER, J. L. 1998. The role of 12-lipoxygenase in pancreatic -cells (Review). Int J Mol Med, 1, 265-72. BLEICH, D., CHEN, S., ZIPSER, B., SUN, D., FUNK, C. D. & NADLER, J. L. 1999. Resistance to type 1 diabetes induction in 12-lipoxygenase knockout mice. J Clin Invest, 103, 1431-6. BONILHA, V. L. 2014. Retinal pigment epithelium (RPE) cytoskeleton in vivo and in vitro. Exp Eye Res, 126, 38-45. BOUDINA, S. & ABEL, E. D. 2007. Diabetic cardiomyopathy revisited. Circulation, 115, 3213-23. BRASH, A. R., YOKOYAMA, C., OATES, J. A. & YAMAMOTO, S. 1989. Mechanistic studies of the and leukotriene synthase activities of the porcine leukocyte 12S-lipoxygenase. Arch Biochem Biophys, 273, 414-22. BRESSLER, S. B., QIN, H., MELIA, M., BRESSLER, N. M., BECK, R. W., CHAN, C. K., GROVER, S., MILLER, D. G. & DIABETIC RETINOPATHY CLINICAL RESEARCH, N. 2013. Exploratory analysis of the effect of intravitreal ranibizumab or triamcinolone on worsening of diabetic retinopathy in a randomized clinical trial. JAMA Ophthalmol, 131, 1033-40. BRINCKMANN, R., SCHNURR, K., HEYDECK, D., ROSENBACH, T., KOLDE, G. & KUHN, H. 1998. Membrane translocation of 15-lipoxygenase in hematopoietic cells is calcium-dependent and activates the oxygenase activity of the enzyme. Blood, 91, 64-74. BRINGMANN, A., PANNICKE, T., BIEDERMANN, B., FRANCKE, M., IANDIEV, I., GROSCHE, J., WIEDEMANN, P., ALBRECHT, J. & REICHENBACH, A. 2009. Role of retinal glial cells in neurotransmitter uptake and metabolism. Neurochem Int, 54, 143-60. BROWNLEE, M. 2005. The pathobiology of diabetic complications: a unifying mechanism. Diabetes, 54, 1615-25. BRZEZINSKI, J. A. & REH, T. A. 2015. Photoreceptor cell fate specification in vertebrates. Development, 142, 3263-73. CABALLERO, S., SWANEY, J., MORENO, K., AFZAL, A., KIELCZEWSKI, J., STOLLER, G., CAVALLI, A., GARLAND, W., HANSEN, G., SABBADINI, R. & GRANT, M. B. 2009. Anti-sphingosine-1-phosphate monoclonal antibodies inhibit angiogenesis and sub-retinal fibrosis in a murine model of laser-induced choroidal neovascularization. Exp Eye Res, 88, 367-77. CALLANAN, D. G., GUPTA, S., BOYER, D. S., CIULLA, T. A., SINGER, M. A., KUPPERMANN, B. D., LIU, C. C., LI, X. Y., HOLLANDER, D. A., 95

SCHIFFMAN, R. M., WHITCUP, S. M. & OZURDEX, P. S. G. 2013. Dexamethasone intravitreal implant in combination with laser photocoagulation for the treatment of diffuse diabetic macular edema. Ophthalmology, 120, 1843- 51. CAMPBELL, M. & HUMPHRIES, P. 2012. The blood-retina barrier: tight junctions and barrier modulation. Adv Exp Med Biol, 763, 70-84. CAMPELLO, L., ESTEVE-RUDD, J., CUENCA, N. & MARTIN-NIETO, J. 2013. The ubiquitin-proteasome system in retinal health and disease. Mol Neurobiol, 47, 790-810. CAO, S. S. & KAUFMAN, R. J. 2014. Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease. Antioxid Redox Signal, 21, 396-413. CAPDEVILA, J. H. 2007. Regulation of ion transport and blood pressure by cytochrome p450 . Curr Opin Nephrol Hypertens, 16, 465-70. CAPRA, V., BACK, M., BARBIERI, S. S., CAMERA, M., TREMOLI, E. & ROVATI, G. E. 2013. Eicosanoids and their drugs in cardiovascular diseases: focus on atherosclerosis and stroke. Med Res Rev, 33, 364-438. CASAS, S., GOMIS, R., GRIBBLE, F. M., ALTIRRIBA, J., KNUUTILA, S. & NOVIALS, A. 2007. Impairment of the ubiquitin-proteasome pathway is a downstream endoplasmic reticulum stress response induced by extracellular human islet amyloid polypeptide and contributes to pancreatic beta-cell apoptosis. Diabetes, 56, 2284-94. CASSON, R. J., CHIDLOW, G., HAN, G. & WOOD, J. P. 2013. An explanation for the Warburg effect in the adult mammalian retina. Clin Exp Ophthalmol, 41, 517. CATELLA-LAWSON, F., MCADAM, B., MORRISON, B. W., KAPOOR, S., KUJUBU, D., ANTES, L., LASSETER, K. C., QUAN, H., GERTZ, B. J. & FITZGERALD, G. A. 1999. Effects of specific inhibition of cyclooxygenase-2 on sodium balance, hemodynamics, and vasoactive eicosanoids. J Pharmacol Exp Ther, 289, 735-41. CAUGHEY, G. E., CLELAND, L. G., GAMBLE, J. R. & JAMES, M. J. 2001a. Up- regulation of endothelial cyclooxygenase-2 and prostanoid synthesis by platelets. Role of thromboxane A2. J Biol Chem, 276, 37839-45. CAUGHEY, G. E., CLELAND, L. G., PENGLIS, P. S., GAMBLE, J. R. & JAMES, M. J. 2001b. Roles of cyclooxygenase (COX)-1 and COX-2 in prostanoid production by human endothelial cells: selective up-regulation of prostacyclin synthesis by COX-2. J Immunol, 167, 2831-8. CEHOFSKI, L. J., HONORE, B. & VORUM, H. 2017. A Review: Proteomics in Retinal Artery Occlusion, Retinal Vein Occlusion, Diabetic Retinopathy and Acquired Macular Disorders. Int J Mol Sci, 18. CHANG, Y. S., WENG, S. F., CHANG, C., WANG, J. J., CHEN, H. I., KO, S. Y., TU, I. T., CHIEN, C. C., WANG, J. J., WANG, C. M. & JAN, R. L. 2017. Risk of 96

serous retinal detachment in patients with end-stage renal disease on dialysis. PLoS One, 12, e0180133. CHATTOPADHYAY, R., DYUKOVA, E., SINGH, N. K., OHBA, M., MOBLEY, J. A. & RAO, G. N. 2014. Vascular endothelial tight junctions and barrier function are disrupted by 15(S)-hydroxyeicosatetraenoic acid partly via protein kinase C epsilon-mediated zona occludens-1 phosphorylation at threonine 770/772. J Biol Chem, 289, 3148-63. CHATTOPADHYAY, R., TINNIKOV, A., DYUKOVA, E., SINGH, N. K., KOTLA, S., MOBLEY, J. A. & RAO, G. N. 2015. 12/15-Lipoxygenase-dependent ROS production is required for diet-induced endothelial barrier dysfunction. J Lipid Res, 56, 562-77. CHEN, M., CURTIS, T. M. & STITT, A. W. 2013. Advanced glycation end products and diabetic retinopathy. Curr Med Chem, 20, 3234-40. CHEN, M., FORRESTER, J. V. & XU, H. 2007. Synthesis of complement factor H by retinal pigment epithelial cells is down-regulated by oxidized photoreceptor outer segments. Exp Eye Res, 84, 635-45. CHEN, M., MUCKERSIE, E., LUO, C., FORRESTER, J. V. & XU, H. 2010. Inhibition of the alternative pathway of complement activation reduces inflammation in experimental autoimmune uveoretinitis. Eur J Immunol, 40, 2870-81. CHEN, M., YANG, Z. D., SMITH, K. M., CARTER, J. D. & NADLER, J. L. 2005a. Activation of 12-lipoxygenase in proinflammatory cytokine-mediated beta cell toxicity. Diabetologia, 48, 486-95. CHEN, M., ZHAO, J., LUO, C., PANDI, S. P., PENALVA, R. G., FITZGERALD, D. C. & XU, H. 2012a. Para-inflammation-mediated retinal recruitment of bone marrow-derived myeloid cells following whole-body irradiation is CCL2 dependent. Glia, 60, 833-42. CHEN, W., ESSELMAN, W. J., JUMP, D. B. & BUSIK, J. V. 2005b. Anti-inflammatory effect of docosahexaenoic acid on cytokine-induced adhesion molecule expression in human retinal vascular endothelial cells. Invest Ophthalmol Vis Sci, 46, 4342-7. CHEN, X. S., KURRE, U., JENKINS, N. A., COPELAND, N. G. & FUNK, C. D. 1994. cDNA cloning, expression, mutagenesis of C-terminal isoleucine, genomic structure, and chromosomal localizations of murine 12-lipoxygenases. J Biol Chem, 269, 13979-87. CHEN, Y., WANG, J. J., LI, J., HOSOYA, K. I., RATAN, R., TOWNES, T. & ZHANG, S. X. 2012b. Activating transcription factor 4 mediates hyperglycaemia-induced endothelial inflammation and retinal vascular leakage through activation of STAT3 in a mouse model of type 1 diabetes. Diabetologia, 55, 2533-45. CHEUNG, N., MITCHELL, P. & WONG, T. Y. 2010. Diabetic retinopathy. Lancet, 376, 124-36. 97

CHIBBER, R., BEN-MAHMUD, B. M., COPPINI, D., CHRIST, E. & KOHNER, E. M. 2000. Activity of the glycosylating enzyme, core 2 GlcNAc (beta1,6) , is higher in polymorphonuclear leukocytes from diabetic patients compared with age-matched control subjects: relevance to capillary occlusion in diabetic retinopathy. Diabetes, 49, 1724-30. CHIBBER, R., BEN-MAHMUD, B. M., MANN, G. E., ZHANG, J. J. & KOHNER, E. M. 2003. Protein kinase C beta2-dependent phosphorylation of core 2 GlcNAc-T promotes leukocyte-endothelial cell adhesion: a mechanism underlying capillary occlusion in diabetic retinopathy. Diabetes, 52, 1519-27. CHRISTMAS, P. 2015. Role of Cytochrome P450s in Inflammation. Adv Pharmacol, 74, 163-92. CLAAR, D., HARTERT, T. V. & PEEBLES, R. S., JR. 2015. The role of prostaglandins in allergic lung inflammation and asthma. Expert Rev Respir Med, 9, 55-72. CLARK, S. J., KEENAN, T. D., FIELDER, H. L., COLLINSON, L. J., HOLLEY, R. J., MERRY, C. L., VAN KUPPEVELT, T. H., DAY, A. J. & BISHOP, P. N. 2011. Mapping the differential distribution of glycosaminoglycans in the adult human retina, choroid, and sclera. Invest Ophthalmol Vis Sci, 52, 6511-21. CLARKE, M. & DODSON, P. M. 2007. PKC inhibition and diabetic microvascular complications. Best Pract Res Clin Endocrinol Metab, 21, 573-86. COLE, B. K., KUHN, N. S., GREEN-MITCHELL, S. M., LEONE, K. A., RAAB, R. M., NADLER, J. L. & CHAKRABARTI, S. K. 2012. 12/15-Lipoxygenase signaling in the endoplasmic reticulum stress response. Am J Physiol Endocrinol Metab, 302, E654-65. CONGDON, N., O'COLMAIN, B., KLAVER, C. C., KLEIN, R., MUNOZ, B., FRIEDMAN, D. S., KEMPEN, J., TAYLOR, H. R., MITCHELL, P. & EYE DISEASES PREVALENCE RESEARCH, G. 2004. Causes and prevalence of visual impairment among adults in the United States. Arch Ophthalmol, 122, 477- 85. CONNOR, K. M., SANGIOVANNI, J. P., LOFQVIST, C., ADERMAN, C. M., CHEN, J., HIGUCHI, A., HONG, S., PRAVDA, E. A., MAJCHRZAK, S., CARPER, D., HELLSTROM, A., KANG, J. X., CHEW, E. Y., SALEM, N., JR., SERHAN, C. N. & SMITH, L. E. H. 2007. Increased dietary intake of omega-3-polyunsaturated fatty acids reduces pathological retinal angiogenesis. Nat Med, 13, 868-873. CONRAD, D. J., KUHN, H., MULKINS, M., HIGHLAND, E. & SIGAL, E. 1992. Specific inflammatory cytokines regulate the expression of human monocyte 15- lipoxygenase. Proc Natl Acad Sci U S A, 89, 217-21. COOK, D. J., FINNIGAN, J. D., COOK, K., BLACK, G. W. & CHARNOCK, S. J. 2016. Cytochromes P450: History, Classes, Catalytic Mechanism, and Industrial Application. Adv Protein Chem Struct Biol, 105, 105-26.

98

COPLAND, D. A., HUSSAIN, K., BAALASUBRAMANIAN, S., HUGHES, T. R., MORGAN, B. P., XU, H., DICK, A. D. & NICHOLSON, L. B. 2010. Systemic and local anti-C5 therapy reduces the disease severity in experimental autoimmune uveoretinitis. Clin Exp Immunol, 159, 303-14. CUI, L., ZHANG, X., YANG, R., LIU, L., WANG, L., LI, M. & DU, W. 2010. Baicalein is neuroprotective in rat MCAO model: role of 12/15-lipoxygenase, mitogen- activated protein kinase and cytosolic phospholipase A2. Pharmacol Biochem Behav, 96, 469-75. CUI, Y., XU, X., BI, H., ZHU, Q., WU, J., XIA, X., QIUSHI, R. & HO, P. C. 2006. Expression modification of uncoupling proteins and MnSOD in retinal endothelial cells and pericytes induced by high glucose: the role of reactive oxygen species in diabetic retinopathy. Exp Eye Res, 83, 807-16. CUNHA-VAZ, J., BERNARDES, R. & LOBO, C. 2011. Blood-retinal barrier. Eur J Ophthalmol, 21 Suppl 6, S3-9. DAI, M., XIA, X. B. & XIONG, S. Q. 2012. BDNF regulates GLAST and glutamine synthetase in mouse retinal Muller cells. J Cell Physiol, 227, 596-603. DE, A. P. A. M., VERISSIMO-FILHO, S., GUIMARAES, L. L., SILVA, A. C., TAKIUTI, J. T., SANTOS, C. X., JANISZEWSKI, M., LAURINDO, F. R. & LOPES, L. R. 2011. Protein disulfide isomerase redox-dependent association with p47(phox): evidence for an organizer role in leukocyte NADPH oxidase activation. J Leukoc Biol, 90, 799-810. DECOSTANZO, A. J., VOLOSHYNA, I., ROSEN, Z. B., FEINMARK, S. J. & SIEGELBAUM, S. A. 2010. 12-Lipoxygenase regulates hippocampal long-term potentiation by modulating L-type Ca2+ channels. J Neurosci, 30, 1822-31. DELAEY, C. & VAN DE VOORDE, J. 2000. Regulatory mechanisms in the retinal and choroidal circulation. Ophthalmic Res, 32, 249-56. DELMASTRO, M. M. & PIGANELLI, J. D. 2011. Oxidative stress and redox modulation potential in type 1 diabetes. Clin Dev Immunol, 2011, 593863. DIABETES, C., COMPLICATIONS TRIAL /EPIDEMIOLOGY OF DIABETES, I., COMPLICATIONS RESEARCH, G., LACHIN, J. M., WHITE, N. H., HAINSWORTH, D. P., SUN, W., CLEARY, P. A. & NATHAN, D. M. 2015. Effect of intensive diabetes therapy on the progression of diabetic retinopathy in patients with type 1 diabetes: 18 years of follow-up in the DCCT/EDIC. Diabetes, 64, 631-42. DIABETES, C., COMPLICATIONS TRIAL RESEARCH, G., NATHAN, D. M., GENUTH, S., LACHIN, J., CLEARY, P., CROFFORD, O., DAVIS, M., RAND, L. & SIEBERT, C. 1993. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. N Engl J Med, 329, 977-86.

99

DIABETIC RETINOPATHY CLINICAL RESEARCH NETWORK WRITING, C., HALLER, J. A., QIN, H., APTE, R. S., BECK, R. R., BRESSLER, N. M., BROWNING, D. J., DANIS, R. P., GLASSMAN, A. R., GOOGE, J. M., KOLLMAN, C., LAUER, A. K., PETERS, M. A. & STOCKMAN, M. E. 2010. Vitrectomy outcomes in eyes with diabetic macular edema and vitreomacular traction. Ophthalmology, 117, 1087-1093 e3. DIMARAS, H., CORSON, T. W., COBRINIK, D., WHITE, A., ZHAO, J., MUNIER, F. L., ABRAMSON, D. H., SHIELDS, C. L., CHANTADA, G. L., NJUGUNA, F. & GALLIE, B. L. 2015. Retinoblastoma. Nat Rev Dis Primers, 1, 15021. DOBRIAN, A. D., LIEB, D. C., COLE, B. K., TAYLOR-FISHWICK, D. A., CHAKRABARTI, S. K. & NADLER, J. L. 2011. Functional and pathological roles of the 12- and 15-lipoxygenases. Prog Lipid Res, 50, 115-31. DREL, V. R., PACHER, P., ALI, T. K., SHIN, J., JULIUS, U., EL-REMESSY, A. B. & OBROSOVA, I. G. 2008. Aldose reductase inhibitor fidarestat counteracts diabetes-associated cataract formation, retinal oxidative-nitrosative stress, glial activation, and apoptosis. Int J Mol Med, 21, 667-76. DUH, E. J., SUN, J. K. & STITT, A. W. 2017. Diabetic retinopathy: current understanding, mechanisms, and treatment strategies. JCI Insight, 2. EADY, T. N., KHOUTOROVA, L., OBENAUS, A., MOHD-YUSOF, A., BAZAN, N. G. & BELAYEV, L. 2014. Docosahexaenoic acid complexed to albumin provides neuroprotection after experimental stroke in aged rats. Neurobiol Dis, 62, 1-7. EBRAHIMIAN, T. G., TAMARAT, R., CLERGUE, M., DURIEZ, M., LEVY, B. I. & SILVESTRE, J. S. 2005. Dual effect of angiotensin-converting enzyme inhibition on angiogenesis in type 1 diabetic mice. Arterioscler Thromb Vasc Biol, 25, 65- 70. EIRAKU, M., TAKATA, N., ISHIBASHI, H., KAWADA, M., SAKAKURA, E., OKUDA, S., SEKIGUCHI, K., ADACHI, T. & SASAI, Y. 2011. Self-organizing optic-cup morphogenesis in three-dimensional culture. Nature, 472, 51-6. EL KEBIR, D. & FILEP, J. G. 2013. Modulation of Neutrophil Apoptosis and the Resolution of Inflammation through beta2 Integrins. Front Immunol, 4, 60. ELMAN, M. J., BRESSLER, N. M., QIN, H., BECK, R. W., FERRIS, F. L., 3RD, FRIEDMAN, S. M., GLASSMAN, A. R., SCOTT, I. U., STOCKDALE, C. R., SUN, J. K. & DIABETIC RETINOPATHY CLINICAL RESEARCH, N. 2011. Expanded 2-year follow-up of ranibizumab plus prompt or deferred laser or triamcinolone plus prompt laser for diabetic macular edema. Ophthalmology, 118, 609-14. ELMASRY, K., IBRAHIM, A. S., SALEH, H., ELSHERBINY, N., ELSHAFEY, S., HUSSEIN, K. A. & AL-SHABRAWEY, M. 2018a. Role of endoplasmic reticulum stress in 12/15-lipoxygenase-induced retinal microvascular dysfunction in a mouse model of diabetic retinopathy. Diabetologia. 100

ELMASRY, K., MOHAMED, R., SHARMA, I., ELSHERBINY, N. M., LIU, Y., AL- SHABRAWEY, M. & TAWFIK, A. 2018b. Epigenetic modifications in hyperhomocysteinemia: potential role in diabetic retinopathy and age-related macular degeneration. Oncotarget, 9, 12562-12590. ELMORE, M. R., NAJAFI, A. R., KOIKE, M. A., DAGHER, N. N., SPANGENBERG, E. E., RICE, R. A., KITAZAWA, M., MATUSOW, B., NGUYEN, H., WEST, B. L. & GREEN, K. N. 2014. Colony-stimulating factor 1 receptor signaling is necessary for microglia viability, unmasking a microglia progenitor cell in the adult brain. Neuron, 82, 380-97. ERESO, A. Q., CURETON, E. L., CRIPPS, M. W., SADJADI, J., DUA, M. M., CURRAN, B. & VICTORINO, G. P. 2009. Lipoxin a(4) attenuates microvascular fluid leak during inflammation. J Surg Res, 156, 183-8. ESCARTIN, C., PIERRE, K., COLIN, A., BROUILLET, E., DELZESCAUX, T., GUILLERMIER, M., DHENAIN, M., DEGLON, N., HANTRAYE, P., PELLERIN, L. & BONVENTO, G. 2007. Activation of astrocytes by CNTF induces metabolic plasticity and increases resistance to metabolic insults. J Neurosci, 27, 7094-104. EVANS, J. R., MICHELESSI, M. & VIRGILI, G. 2014. Laser photocoagulation for proliferative diabetic retinopathy. Cochrane Database Syst Rev, CD011234. FAGONE, P. & JACKOWSKI, S. 2009. Membrane phospholipid synthesis and endoplasmic reticulum function. J Lipid Res, 50 Suppl, S311-6. FEINMARK, S. J., BEGUM, R., TSVETKOV, E., GOUSSAKOV, I., FUNK, C. D., SIEGELBAUM, S. A. & BOLSHAKOV, V. Y. 2003. 12-lipoxygenase metabolites of arachidonic acid mediate metabotropic glutamate receptor- dependent long-term depression at hippocampal CA3-CA1 synapses. J Neurosci, 23, 11427-35. FIRUZI, O., ZHUO, J., CHINNICI, C. M., WISNIEWSKI, T. & PRATICO, D. 2008. 5- Lipoxygenase gene disruption reduces amyloid-beta pathology in a mouse model of Alzheimer's disease. FASEB J, 22, 1169-78. FISCHER, K. A., VAN LEYEN, K., LOVERCAMP, K. W., MANANDHAR, G., SUTOVSKY, M., FENG, D., SAFRANSKI, T. & SUTOVSKY, P. 2005. 15- Lipoxygenase is a component of the mammalian sperm cytoplasmic droplet. Reproduction, 130, 213-22. FOX, T. E., HAN, X., KELLY, S., MERRILL, A. H., 2ND, MARTIN, R. E., ANDERSON, R. E., GARDNER, T. W. & KESTER, M. 2006. Diabetes alters sphingolipid metabolism in the retina: a potential mechanism of cell death in diabetic retinopathy. Diabetes, 55, 3573-80. FREIRE, M. O. & VAN DYKE, T. E. 2013. Natural resolution of inflammation. Periodontol 2000, 63, 149-64.

101

FU, S., YANG, L., LI, P., HOFMANN, O., DICKER, L., HIDE, W., LIN, X., WATKINS, S. M., IVANOV, A. R. & HOTAMISLIGIL, G. S. 2011. Aberrant lipid metabolism disrupts calcium homeostasis causing liver endoplasmic reticulum stress in obesity. Nature, 473, 528-31. FU, Z., LOFQVIST, C. A., SHAO, Z., SUN, Y., JOYAL, J. S., HURST, C. G., CUI, R. Z., EVANS, L. P., TIAN, K., SANGIOVANNI, J. P., CHEN, J., LEY, D., HANSEN PUPP, I., HELLSTROM, A. & SMITH, L. E. 2015. Dietary omega-3 polyunsaturated fatty acids decrease retinal neovascularization by adipose- endoplasmic reticulum stress reduction to increase adiponectin. Am J Clin Nutr, 101, 879-88. FUNATSU, H., YAMASHITA, H., NAKANISHI, Y. & HORI, S. 2002. Angiotensin II and vascular endothelial growth factor in the vitreous fluid of patients with proliferative diabetic retinopathy. Br J Ophthalmol, 86, 311-5. FUNK, C. D., CHEN, X. S., JOHNSON, E. N. & ZHAO, L. 2002. Lipoxygenase genes and their targeted disruption. Prostaglandins Other Lipid Mediat, 68-69, 303-12. GABBAY, K. H. 1973. The sorbitol pathway and the complications of diabetes. N Engl J Med, 288, 831-6. GALAN, M., KASSAN, M., KADOWITZ, P. J., TREBAK, M., BELMADANI, S. & MATROUGUI, K. 2014. Mechanism of endoplasmic reticulum stress-induced vascular endothelial dysfunction. Biochim Biophys Acta, 1843, 1063-75. GAO, B. B., CLERMONT, A., ROOK, S., FONDA, S. J., SRINIVASAN, V. J., WOJTKOWSKI, M., FUJIMOTO, J. G., AVERY, R. L., ARRIGG, P. G., BURSELL, S. E., AIELLO, L. P. & FEENER, E. P. 2007. Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation. Nat Med, 13, 181-8. GARIANO, R. F. 2003. Cellular mechanisms in retinal vascular development. Prog Retin Eye Res, 22, 295-306. GENG, X., SPEIRS, C., LAGUTIN, O., INBAL, A., LIU, W., SOLNICA-KREZEL, L., JEONG, Y., EPSTEIN, D. J. & OLIVER, G. 2008. Haploinsufficiency of Six3 fails to activate Sonic hedgehog expression in the ventral forebrain and causes holoprosencephaly. Dev Cell, 15, 236-47. GERTOW, K., NOBILI, E., FOLKERSEN, L., NEWMAN, J. W., PEDERSEN, T. L., EKSTRAND, J., SWEDENBORG, J., KUHN, H., WHEELOCK, C. E., HANSSON, G. K., HEDIN, U., HAEGGSTROM, J. Z. & GABRIELSEN, A. 2011. 12- and 15-lipoxygenases in human carotid atherosclerotic lesions: associations with cerebrovascular symptoms. Atherosclerosis, 215, 411-6. GILLIES, M. C., MCALLISTER, I. L., ZHU, M., WONG, W., LOUIS, D., ARNOLD, J. J. & WONG, T. Y. 2011. Intravitreal triamcinolone prior to laser treatment of diabetic macular edema: 24-month results of a randomized controlled trial. Ophthalmology, 118, 866-72. 102

GILLIES, M. C., SUTTER, F. K., SIMPSON, J. M., LARSSON, J., ALI, H. & ZHU, M. 2006. Intravitreal triamcinolone for refractory diabetic macular edema: two-year results of a double-masked, placebo-controlled, randomized clinical trial. Ophthalmology, 113, 1533-8. GILLMOR, S. A., VILLASENOR, A., FLETTERICK, R., SIGAL, E. & BROWNER, M. F. 1997. The structure of mammalian 15-lipoxygenase reveals similarity to the lipases and the determinants of substrate specificity. Nat Struct Biol, 4, 1003-9. GORDON, W. C. & BAZAN, N. G. 1990. Docosahexaenoic acid utilization during rod photoreceptor cell renewal. J Neurosci, 10, 2190-202. GORUSUPUDI, A., LIU, A., HAGEMAN, G. S. & BERNSTEIN, P. S. 2016. Associations of human retinal very long-chain polyunsaturated fatty acids with dietary lipid biomarkers. J Lipid Res, 57, 499-508. GOSWAMI, S. K. 2013. Cellular redox, epigenetics and diseases. Subcell Biochem, 61, 527-42. GUBITOSI-KLUG, R. A., TALAHALLI, R., DU, Y., NADLER, J. L. & KERN, T. S. 2008. 5-Lipoxygenase, but not 12/15-lipoxygenase, contributes to degeneration of retinal capillaries in a mouse model of diabetic retinopathy. Diabetes, 57, 1387- 93. HADA, T., HAGIYA, H., SUZUKI, H., ARAKAWA, T., NAKAMURA, M., MATSUDA, S., YOSHIMOTO, T., YAMAMOTO, S., AZEKAWA, T., MORITA, Y. & ET AL. 1994. Arachidonate 12-lipoxygenase of rat pineal glands: catalytic properties and primary structure deduced from its cDNA. Biochim Biophys Acta, 1211, 221-8. HAEGGSTROM, J. Z. & FUNK, C. D. 2011. Lipoxygenase and leukotriene pathways: biochemistry, biology, and roles in disease. Chem Rev, 111, 5866-98. HAMMES, H. P., MARTIN, S., FEDERLIN, K., GEISEN, K. & BROWNLEE, M. 1991. Aminoguanidine treatment inhibits the development of experimental diabetic retinopathy. Proc Natl Acad Sci U S A, 88, 11555-8. HAN, J., BACK, S. H., HUR, J., LIN, Y. H., GILDERSLEEVE, R., SHAN, J., YUAN, C. L., KROKOWSKI, D., WANG, S., HATZOGLOU, M., KILBERG, M. S., SARTOR, M. A. & KAUFMAN, R. J. 2013. ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death. Nat Cell Biol, 15, 481- 90. HAN, J., SUN, L., XU, Y., LIANG, H. & CHENG, Y. 2015. Activation of PPARgamma by 12/15-lipoxygenase during cerebral ischemia-reperfusion injury. Int J Mol Med, 35, 195-201. HARADA, C., HARADA, T., QUAH, H. M., MAEKAWA, F., YOSHIDA, K., OHNO, S., WADA, K., PARADA, L. F. & TANAKA, K. 2003. Potential role of glial cell line-derived neurotrophic factor receptors in Muller glial cells during light- induced retinal degeneration. Neuroscience, 122, 229-35. 103

HARDING, H. P., ZHANG, Y., BERTOLOTTI, A., ZENG, H. & RON, D. 2000. Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol Cell, 5, 897-904. HASEGAWA, T., MCLEOD, D. S., PROW, T., MERGES, C., GREBE, R. & LUTTY, G. A. 2008. Vascular precursors in developing human retina. Invest Ophthalmol Vis Sci, 49, 2178-92. HAUCK, S. M., KINKL, N., DEEG, C. A., SWIATEK-DE LANGE, M., SCHOFFMANN, S. & UEFFING, M. 2006. GDNF family ligands trigger indirect neuroprotective signaling in retinal glial cells. Mol Cell Biol, 26, 2746-57. HEAVNER, W. & PEVNY, L. 2012. Eye development and retinogenesis. Cold Spring Harb Perspect Biol, 4. HERNANDEZ, C., SEGURA, R. M., FONOLLOSA, A., CARRASCO, E., FRANCISCO, G. & SIMO, R. 2005. Interleukin-8, monocyte chemoattractant protein-1 and IL-10 in the vitreous fluid of patients with proliferative diabetic retinopathy. Diabet Med, 22, 719-22. HIRATA, Y. & NISHIWAKI, H. 2006. The choroidal circulation assessed by laser- targeted angiography. Prog Retin Eye Res, 25, 129-47. HORN, T., ADEL, S., SCHUMANN, R., SUR, S., KAKULARAM, K. R., POLAMARASETTY, A., REDANNA, P., KUHN, H. & HEYDECK, D. 2015. Evolutionary aspects of lipoxygenases and genetic diversity of human leukotriene signaling. Prog Lipid Res, 57, 13-39. HOSOYA, K. & TACHIKAWA, M. 2012. The inner blood-retinal barrier: molecular structure and transport biology. Adv Exp Med Biol, 763, 85-104. HUANG, H., JING, G., WANG, J. J., SHEIBANI, N. & ZHANG, S. X. 2015. ATF4 is a novel regulator of MCP-1 in microvascular endothelial cells. J Inflamm (Lond), 12, 31. HUANG, Y., XU, Z., XIONG, S., QIN, G., SUN, F., YANG, J., YUAN, T. F., ZHAO, L., WANG, K., LIANG, Y. X., FU, L., WU, T., SO, K. F., RAO, Y. & PENG, B. 2018. Dual extra-retinal origins of microglia in the model of retinal microglia repopulation. Cell Discov, 4, 9. IBRAHIM, A. S., EL-REMESSY, A. B., MATRAGOON, S., ZHANG, W., PATEL, Y., KHAN, S., AL-GAYYAR, M. M., EL-SHISHTAWY, M. M. & LIOU, G. I. 2011. Retinal microglial activation and inflammation induced by amadori- glycated albumin in a rat model of diabetes. Diabetes, 60, 1122-33. IBRAHIM, A. S., ELSHAFEY, S., SELLAK, H., HUSSEIN, K. A., EL-SHERBINY, M., ABDELSAID, M., RIZK, N., BEASLEY, S., TAWFIK, A. M., SMITH, S. B. & AL-SHABRAWEY, M. 2015a. A lipidomic screen of hyperglycemia-treated HRECs links 12/15-Lipoxygenase to microvascular dysfunction during diabetic retinopathy via NADPH oxidase. J Lipid Res, 56, 599-611.

104

IBRAHIM, A. S., MANDER, S., HUSSEIN, K. A., ELSHERBINY, N. M., SMITH, S. B., AL-SHABRAWEY, M. & TAWFIK, A. 2016. Hyperhomocysteinemia disrupts retinal pigment epithelial structure and function with features of age- related macular degeneration. Oncotarget, 7, 8532-45. IBRAHIM, A. S., SALEH, H., EL-SHAFEY, M., HUSSEIN, K. A., EL-MASRY, K., BABAN, B., SHEIBANI, N., WANG, M. H., TAWFIK, A. & AL- SHABRAWEY, M. 2017. Targeting of 12/15-Lipoxygenase in retinal endothelial cells, but not in monocytes/macrophages, attenuates high glucose-induced retinal leukostasis. Biochim Biophys Acta, 1862, 636-645. IBRAHIM, A. S., TAWFIK, A. M., HUSSEIN, K. A., ELSHAFEY, S., MARKAND, S., RIZK, N., DUH, E. J., SMITH, S. B. & AL-SHABRAWEY, M. 2015b. Pigment epithelium-derived factor inhibits retinal microvascular dysfunction induced by 12/15-lipoxygenase-derived eicosanoids. Biochim Biophys Acta, 1851, 290-8. ISHIDA, S., USUI, T., YAMASHIRO, K., KAJI, Y., AHMED, E., CARRASQUILLO, K. G., AMANO, S., HIDA, T., OGUCHI, Y. & ADAMIS, A. P. 2003. VEGF164 is proinflammatory in the diabetic retina. Invest Ophthalmol Vis Sci, 44, 2155-62. ISHII, T., SAKURAI, T., USAMI, H. & UCHIDA, K. 2005. Oxidative modification of proteasome: identification of an oxidation-sensitive subunit in 26 S proteasome. Biochemistry, 44, 13893-901. ISHIKAWA, M. 2013. Abnormalities in glutamate metabolism and excitotoxicity in the retinal diseases. Scientifica (Cairo), 2013, 528940. ISSAN, Y., HOCHHAUSER, E., GUO, A., GOTLINGER, K. H., KORNOWSKI, R., LESHEM-LEV, D., LEV, E., PORAT, E., SNIR, E., THOMPSON, C. I., ABRAHAM, N. G. & LANIADO-SCHWARTZMAN, M. 2013. Elevated level of pro-inflammatory eicosanoids and EPC dysfunction in diabetic patients with cardiac ischemia. Prostaglandins Other Lipid Mediat, 100-101, 15-21. IWASE, T., KOBAYASHI, M., YAMAMOTO, K., RA, E. & TERASAKI, H. 2017. Effects of photocoagulation on ocular blood flow in patients with severe non- proliferative diabetic retinopathy. PLoS One, 12, e0174427. JAIN, T. P. 2009. Bilateral persistent hyperplastic primary vitreous. Indian J Ophthalmol, 57, 53-4. JANISZEWSKI, M., LOPES, L. R., CARMO, A. O., PEDRO, M. A., BRANDES, R. P., SANTOS, C. X. & LAURINDO, F. R. 2005. Regulation of NAD(P)H oxidase by associated protein disulfide isomerase in vascular smooth muscle cells. J Biol Chem, 280, 40813-9. JEONG, Y., LESKOW, F. C., EL-JAICK, K., ROESSLER, E., MUENKE, M., YOCUM, A., DUBOURG, C., LI, X., GENG, X., OLIVER, G. & EPSTEIN, D. J. 2008. Regulation of a remote Shh forebrain enhancer by the Six3 homeoprotein. Nat Genet, 40, 1348-53.

105

JIANG, B., LIOU, G. I., BEHZADIAN, M. A. & CALDWELL, R. B. 1994. Astrocytes modulate retinal vasculogenesis: effects on fibronectin expression. J Cell Sci, 107 ( Pt 9), 2499-508. JIN, G., ARAI, K., MURATA, Y., WANG, S., STINS, M. F., LO, E. H. & VAN LEYEN, K. 2008. Protecting against cerebrovascular injury: contributions of 12/15-lipoxygenase to edema formation after transient focal ischemia. Stroke, 39, 2538-43. JOUSSEN, A. M., POULAKI, V., LE, M. L., KOIZUMI, K., ESSER, C., JANICKI, H., SCHRAERMEYER, U., KOCIOK, N., FAUSER, S., KIRCHHOF, B., KERN, T. S. & ADAMIS, A. P. 2004. A central role for inflammation in the pathogenesis of diabetic retinopathy. FASEB J, 18, 1450-2. JOUSSEN, A. M., POULAKI, V., QIN, W., KIRCHHOF, B., MITSIADES, N., WIEGAND, S. J., RUDGE, J., YANCOPOULOS, G. D. & ADAMIS, A. P. 2002. Retinal vascular endothelial growth factor induces intercellular adhesion molecule-1 and endothelial nitric oxide synthase expression and initiates early diabetic retinal leukocyte adhesion in vivo. Am J Pathol, 160, 501-9. JOY, S. V., SCATES, A. C., BEARELLY, S., DAR, M., TAULIEN, C. A., GOEBEL, J. A. & COONEY, M. J. 2005. Ruboxistaurin, a protein kinase C beta inhibitor, as an emerging treatment for diabetes microvascular complications. Ann Pharmacother, 39, 1693-9. KANG, S. W., SA, H. S., CHO, H. Y. & KIM, J. I. 2006. Macular grid photocoagulation after intravitreal triamcinolone acetonide for diffuse diabetic macular edema. Arch Ophthalmol, 124, 653-8. KAPOOR, K. G., HODGE, D. O., ST SAUVER, J. L. & BARKMEIER, A. J. 2014. Oral fluoroquinolones and the incidence of rhegmatogenous retinal detachment and symptomatic retinal breaks: a population-based study. Ophthalmology, 121, 1269- 73. KARALI, E., BELLOU, S., STELLAS, D., KLINAKIS, A., MURPHY, C. & FOTSIS, T. 2014. VEGF Signals through ATF6 and PERK to promote endothelial cell survival and angiogenesis in the absence of ER stress. Mol Cell, 54, 559-72. KATSMAN, D., STACKPOLE, E. J., DOMIN, D. R. & FARBER, D. B. 2012. Embryonic stem cell-derived microvesicles induce gene expression changes in Muller cells of the retina. PLoS One, 7, e50417. KAUL, K., HODGKINSON, A., TARR, J. M., KOHNER, E. M. & CHIBBER, R. 2010. Is inflammation a common retinal-renal-nerve pathogenic link in diabetes? Curr Diabetes Rev, 6, 294-303. KAUPPINEN, A., PATERNO, J. J., BLASIAK, J., SALMINEN, A. & KAARNIRANTA, K. 2016. Inflammation and its role in age-related macular degeneration. Cell Mol Life Sci, 73, 1765-86.

106

KEENAN, T. D., CLARK, S. J., UNWIN, R. D., RIDGE, L. A., DAY, A. J. & BISHOP, P. N. 2012. Mapping the differential distribution of proteoglycan core proteins in the adult human retina, choroid, and sclera. Invest Ophthalmol Vis Sci, 53, 7528- 38. KELAVKAR, U., WANG, S., MONTERO, A., MURTAGH, J., SHAH, K. & BADR, K. 1998. Human 15-lipoxygenase gene promoter: analysis and identification of DNA binding sites for IL-13-induced regulatory factors in monocytes. Mol Biol Rep, 25, 173-82. KELAVKAR, U. P., NIXON, J. B., COHEN, C., DILLEHAY, D., ELING, T. E. & BADR, K. F. 2001. Overexpression of 15-lipoxygenase-1 in PC-3 human prostate cancer cells increases tumorigenesis. Carcinogenesis, 22, 1765-73. KENYON, V., RAI, G., JADHAV, A., SCHULTZ, L., ARMSTRONG, M., JAMESON, J. B., 2ND, PERRY, S., JOSHI, N., BOUGIE, J. M., LEISTER, W., TAYLOR- FISHWICK, D. A., NADLER, J. L., HOLINSTAT, M., SIMEONOV, A., MALONEY, D. J. & HOLMAN, T. R. 2011. Discovery of potent and selective inhibitors of human platelet-type 12- lipoxygenase. J Med Chem, 54, 5485-97. KERN, T. S., MILLER, C. M., DU, Y., ZHENG, L., MOHR, S., BALL, S. L., KIM, M., JAMISON, J. A. & BINGAMAN, D. P. 2007. Topical administration of nepafenac inhibits diabetes-induced retinal microvascular disease and underlying abnormalities of retinal metabolism and physiology. Diabetes, 56, 373-9. KIERDORF, K., ERNY, D., GOLDMANN, T., SANDER, V., SCHULZ, C., PERDIGUERO, E. G., WIEGHOFER, P., HEINRICH, A., RIEMKE, P., HOLSCHER, C., MULLER, D. N., LUCKOW, B., BROCKER, T., DEBOWSKI, K., FRITZ, G., OPDENAKKER, G., DIEFENBACH, A., BIBER, K., HEIKENWALDER, M., GEISSMANN, F., ROSENBAUER, F. & PRINZ, M. 2013. Microglia emerge from erythromyeloid precursors via Pu.1- and Irf8- dependent pathways. Nat Neurosci, 16, 273-80. KIM, G. H., RYAN, J. J. & ARCHER, S. L. 2013. The role of redox signaling in epigenetics and cardiovascular disease. Antioxid Redox Signal, 18, 1920-36. KINZIG, A., HEIDT, M., FURSTENBERGER, G., MARKS, F. & KRIEG, P. 1999. cDNA cloning, genomic structure, and chromosomal localization of a novel murine epidermis-type lipoxygenase. Genomics, 58, 158-64. KITAGAWA, K., MATSUMOTO, M. & HORI, M. 2004. Cerebral ischemia in 5- lipoxygenase knockout mice. Brain Res, 1004, 198-202. KITANO, S., MORGAN, J. & CAPRIOLI, J. 1996. Hypoxic and excitotoxic damage to cultured rat retinal ganglion cells. Exp Eye Res, 63, 105-12. KLAWITTER, J., ZAFAR, I., KLAWITTER, J., PENNINGTON, A. T., KLEPACKI, J., GITOMER, B. Y., SCHRIER, R. W., CHRISTIANS, U. & EDELSTEIN, C. L. 2013. Effects of lovastatin treatment on the metabolic distributions in the Han:SPRD rat model of polycystic kidney disease. BMC Nephrol, 14, 165. 107

KLEIN, B. E., KNUDTSON, M. D., TSAI, M. Y. & KLEIN, R. 2009. The relation of markers of inflammation and endothelial dysfunction to the prevalence and progression of diabetic retinopathy: Wisconsin epidemiologic study of diabetic retinopathy. Arch Ophthalmol, 127, 1175-82. KOHNO, T., SORGENTE, N., ISHIBASHI, T., GOODNIGHT, R. & RYAN, S. J. 1987. Immunofluorescent studies of fibronectin and laminin in the human eye. Invest Ophthalmol Vis Sci, 28, 506-14. KOLB, H. 1995a. Inner Plexiform Layer. In: KOLB, H., FERNANDEZ, E. & NELSON, R. (eds.) Webvision: The Organization of the Retina and Visual System. Salt Lake City (UT). KOLB, H. 1995b. Simple Anatomy of the Retina. In: KOLB, H., FERNANDEZ, E. & NELSON, R. (eds.) Webvision: The Organization of the Retina and Visual System. Salt Lake City (UT). KORNFIELD, T. E. & NEWMAN, E. A. 2014. Regulation of blood flow in the retinal trilaminar vascular network. J Neurosci, 34, 11504-13. KOTO, T., NAGAI, N., MOCHIMARU, H., KURIHARA, T., IZUMI-NAGAI, K., SATOFUKA, S., SHINODA, H., NODA, K., OZAWA, Y., INOUE, M., TSUBOTA, K., OIKE, Y. & ISHIDA, S. 2007. is anti- inflammatory in preventing choroidal neovascularization in mice. Invest Ophthalmol Vis Sci, 48, 4328-34. KOWLURU, R. A. 2001. Diabetes-induced elevations in retinal oxidative stress, protein kinase C and nitric oxide are interrelated. Acta Diabetol, 38, 179-85. KOWLURU, R. A. & ABBAS, S. N. 2003. Diabetes-induced mitochondrial dysfunction in the retina. Invest Ophthalmol Vis Sci, 44, 5327-34. KOYA, D. & KING, G. L. 1998. Protein kinase C activation and the development of diabetic complications. Diabetes, 47, 859-66. KUHN, H. 1996. Biosynthesis, metabolization and biological importance of the primary 15-lipoxygenase metabolites 15-hydro(pero)XY-5Z,8Z,11Z,13E-eicosatetraenoic acid and 13-hydro(pero)XY-9Z,11E-octadecadienoic acid. Prog Lipid Res, 35, 203-26. KUHN, H., BANTHIYA, S. & VAN LEYEN, K. 2015. Mammalian lipoxygenases and their biological relevance. Biochim Biophys Acta, 1851, 308-30. KUHN, H., BELKNER, J. & WIESNER, R. 1990. Subcellular distribution of lipoxygenase products in rabbit reticulocyte membranes. Eur J Biochem, 191, 221-7. KUHN, H., BELKNER, J., ZAISS, S., FAHRENKLEMPER, T. & WOHLFEIL, S. 1994. Involvement of 15-lipoxygenase in early stages of atherogenesis. J Exp Med, 179, 1903-11. KUHN, H., CHAITIDIS, P., ROFFEIS, J. & WALTHER, M. 2007. Arachidonic Acid metabolites in the cardiovascular system: the role of lipoxygenase isoforms in 108

atherogenesis with particular emphasis on vascular remodeling. J Cardiovasc Pharmacol, 50, 609-20. KUHN, H., HEYDECK, D., HUGOU, I. & GNIWOTTA, C. 1997. In vivo action of 15- lipoxygenase in early stages of human atherogenesis. J Clin Invest, 99, 888-93. KUHN, H. & O'DONNELL, V. B. 2006. Inflammation and immune regulation by 12/15- lipoxygenases. Prog Lipid Res, 45, 334-56. KUHN, H., SCHEWE, T. & RAPOPORT, S. M. 1986. The stereochemistry of the reactions of lipoxygenases and their metabolites. Proposed nomenclature of lipoxygenases and related enzymes. Adv Enzymol Relat Areas Mol Biol, 58, 273- 311. KUNDUMANI-SRIDHARAN, V., DYUKOVA, E., HANSEN, D. E., 3RD & RAO, G. N. 2013. 12/15-Lipoxygenase mediates high-fat diet-induced endothelial tight junction disruption and monocyte transmigration: a new role for 15(S)- hydroxyeicosatetraenoic acid in endothelial cell dysfunction. J Biol Chem, 288, 15830-42. KUPPERMANN, B. D., BLUMENKRANZ, M. S., HALLER, J. A., WILLIAMS, G. A., WEINBERG, D. V., CHOU, C., WHITCUP, S. M. & DEXAMETHASONE, D. D. S. P. I. I. S. G. 2007. Randomized controlled study of an intravitreous dexamethasone drug delivery system in patients with persistent macular edema. Arch Ophthalmol, 125, 309-17. LAM, D. S., CHAN, C. K., MOHAMED, S., LAI, T. Y., LEE, V. Y., LIU, D. T., LI, K. K., LI, P. S. & SHANMUGAM, M. P. 2007. Intravitreal triamcinolone plus sequential grid laser versus triamcinolone or laser alone for treating diabetic macular edema: six-month outcomes. Ophthalmology, 114, 2162-7. LAN, Y. L., CHEN, J. J., HU, G., XU, J., XIAO, M. & LI, S. 2017. Aquaporin 4 in Astrocytes is a Target for Therapy in Alzheimer's Disease. Curr Pharm Des, 23, 4948-4957. LANGMANN, T. 2007. Microglia activation in retinal degeneration. J Leukoc Biol, 81, 1345-51. LARSON, R. S. & SPRINGER, T. A. 1990. Structure and function of leukocyte integrins. Immunol Rev, 114, 181-217. LAU, J. C., KROES, R. A., MOSKAL, J. R. & LINSENMEIER, R. A. 2013. Diabetes changes expression of genes related to glutamate neurotransmission and transport in the Long-Evans rat retina. Mol Vis, 19, 1538-53. LAURINDO, F. R., FERNANDES, D. C., AMANSO, A. M., LOPES, L. R. & SANTOS, C. X. 2008. Novel role of protein disulfide isomerase in the regulation of NADPH oxidase activity: pathophysiological implications in vascular diseases. Antioxid Redox Signal, 10, 1101-13. LAWRENCE, J. M., SINGHAL, S., BHATIA, B., KEEGAN, D. J., REH, T. A., LUTHERT, P. J., KHAW, P. T. & LIMB, G. A. 2007. MIO-M1 cells and similar 109

muller glial cell lines derived from adult human retina exhibit neural stem cell characteristics. Stem Cells, 25, 2033-43. LAYBUTT, D. R., SHARMA, A., SGROI, D. C., GAUDET, J., BONNER-WEIR, S. & WEIR, G. C. 2002. Genetic regulation of metabolic pathways in beta-cells disrupted by hyperglycemia. J Biol Chem, 277, 10912-21. LEAL, E. C., MANIVANNAN, A., HOSOYA, K., TERASAKI, T., CUNHA-VAZ, J., AMBROSIO, A. F. & FORRESTER, J. V. 2007. Inducible nitric oxide synthase isoform is a key mediator of leukostasis and blood-retinal barrier breakdown in diabetic retinopathy. Invest Ophthalmol Vis Sci, 48, 5257-65. LEE, R., WONG, T. Y. & SABANAYAGAM, C. 2015. Epidemiology of diabetic retinopathy, diabetic macular edema and related vision loss. Eye Vis (Lond), 2, 17. LEE, S. J., KIM, E. T. & MOON, Y. S. 2011. Intravitreal bevacizumab alone versus combined with macular photocoagulation in diabetic macular edema. Korean J Ophthalmol, 25, 299-304. LEI, Z. M. & RAO, C. V. 1992. The expression of 15-lipoxygenase gene and the presence of functional enzyme in cytoplasm and nuclei of pregnancy human myometria. Endocrinology, 130, 861-70. LEVY, B. D., ROMANO, M., CHAPMAN, H. A., REILLY, J. J., DRAZEN, J. & SERHAN, C. N. 1993. Human alveolar macrophages have 15-lipoxygenase and generate 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid and lipoxins. J Clin Invest, 92, 1572-9. LI, G., SCULL, C., OZCAN, L. & TABAS, I. 2010. NADPH oxidase links endoplasmic reticulum stress, oxidative stress, and PKR activation to induce apoptosis. J Cell Biol, 191, 1113-25. LI, J., RAO, J., LIU, Y., CAO, Y., ZHANG, Y., ZHANG, Q. & ZHU, D. 2013. 15- Lipoxygenase promotes chronic hypoxia-induced pulmonary artery inflammation via positive interaction with nuclear factor-kappaB. Arterioscler Thromb Vasc Biol, 33, 971-9. LI, J., WANG, J. J., YU, Q., WANG, M. & ZHANG, S. X. 2009. Endoplasmic reticulum stress is implicated in retinal inflammation and diabetic retinopathy. FEBS Lett, 583, 1521-7. LI, J., ZHAO, S. Z., WANG, P. P., YU, S. P., ZHENG, Z. & XU, X. 2012. Calcium mediates high glucose-induced HIF-1alpha and VEGF expression in cultured rat retinal Muller cells through CaMKII-CREB pathway. Acta Pharmacol Sin, 33, 1030-6. LINGAM, G. 2013. Pattern of blood vessels in eyes with coloboma. Indian J Ophthalmol, 61, 743-8. LIU, C., SCHAIN, F., HAN, H., XU, D., ANDERSSON-SAND, H., FORSELL, P., CLAESSON, H. E., BJORKHOLM, M. & SJOBERG, J. 2012. Epigenetic and

110

transcriptional control of the 15-lipoxygenase-1 gene in a Hodgkin lymphoma cell line. Exp Cell Res, 318, 169-76. LIU, K., LYU, L., CHIN, D., GAO, J., SUN, X., SHANG, F., CACERES, A., CHANG, M. L., ROWAN, S., PENG, J., MATHIAS, R., KASAHARA, H., JIANG, S. & TAYLOR, A. 2015a. Altered ubiquitin causes perturbed calcium homeostasis, hyperactivation of calpain, dysregulated differentiation, and cataract. Proc Natl Acad Sci U S A, 112, 1071-6. LIU, X., YE, F., XIONG, H., HU, D., LIMB, G. A., XIE, T., PENG, L., YANG, W., SUN, Y., ZHOU, M., SONG, E. & ZHANG, D. Y. 2014. IL-1beta Upregulates IL-8 Production in Human Muller Cells Through Activation of the p38 MAPK and ERK1/2 Signaling Pathways. Inflammation, 37, 1486-95. LIU, X., YE, F., XIONG, H., HU, D. N., LIMB, G. A., XIE, T., PENG, L., ZHANG, P., WEI, Y., ZHANG, W., WANG, J., WU, H., LEE, P., SONG, E. & ZHANG, D. Y. 2015b. IL-1beta induces IL-6 production in retinal Muller cells predominantly through the activation of p38 MAPK/NF-kappaB signaling pathway. Exp Cell Res, 331, 223-31. LU, Y. B., FRANZE, K., SEIFERT, G., STEINHAUSER, C., KIRCHHOFF, F., WOLBURG, H., GUCK, J., JANMEY, P., WEI, E. Q., KAS, J. & REICHENBACH, A. 2006. Viscoelastic properties of individual glial cells and neurons in the CNS. Proc Natl Acad Sci U S A, 103, 17759-64. LU, Y. B., PANNICKE, T., WEI, E. Q., BRINGMANN, A., WIEDEMANN, P., HABERMANN, G., BUSE, E., KAS, J. A. & REICHENBACH, A. 2013. Biomechanical properties of retinal glial cells: comparative and developmental data. Exp Eye Res, 113, 60-5. LUDWIG, P. E. & CZYZ, C. N. 2018. Embryology, Eye, Malformations. StatPearls. Treasure Island (FL). LUKIW, W. J., CUI, J. G., MARCHESELLI, V. L., BODKER, M., BOTKJAER, A., GOTLINGER, K., SERHAN, C. N. & BAZAN, N. G. 2005. A role for docosahexaenoic acid-derived neuroprotectin D1 in neural cell survival and Alzheimer disease. J Clin Invest, 115, 2774-83. LUO, C., CHEN, M. & XU, H. 2011. Complement gene expression and regulation in mouse retina and retinal pigment epithelium/choroid. Mol Vis, 17, 1588-97. LUPO, G., MOTTA, C., GIURDANELLA, G., ANFUSO, C. D., ALBERGHINA, M., DRAGO, F., SALOMONE, S. & BUCOLO, C. 2013. Role of phospholipases A2 in diabetic retinopathy: in vitro and in vivo studies. Biochem Pharmacol, 86, 1603-13. LUTTY, G. A., CAO, J. & MCLEOD, D. S. 1997. Relationship of polymorphonuclear leukocytes to capillary dropout in the human diabetic choroid. Am J Pathol, 151, 707-14.

111

LUTTY, G. A. & MCLEOD, D. S. 2018. Development of the hyaloid, choroidal and retinal vasculatures in the fetal human eye. Prog Retin Eye Res, 62, 58-76. MA, J. H., WANG, J. J. & ZHANG, S. X. 2014. The unfolded protein response and diabetic retinopathy. J Diabetes Res, 2014, 160140. MA, Y. & HENDERSHOT, L. M. 2004a. ER chaperone functions during normal and stress conditions. J Chem Neuroanat, 28, 51-65. MA, Y. & HENDERSHOT, L. M. 2004b. The role of the unfolded protein response in tumour development: friend or foe? Nat Rev Cancer, 4, 966-77. MA, Y., QU, Y. & FEI, Z. 2011. Vascular endothelial growth factor in cerebral ischemia. J Neurosci Res, 89, 969-78. MAINES, L. W., FRENCH, K. J., WOLPERT, E. B., ANTONETTI, D. A. & SMITH, C. D. 2006. Pharmacologic manipulation of sphingosine kinase in retinal endothelial cells: implications for angiogenic ocular diseases. Invest Ophthalmol Vis Sci, 47, 5022-31. MASSON, E., TRONCY, L., RUGGIERO, D., WIERNSPERGER, N., LAGARDE, M. & EL BAWAB, S. 2005. a-Series gangliosides mediate the effects of advanced glycation end products on pericyte and mesangial cell proliferation: a common mediator for retinal and renal microangiopathy? Diabetes, 54, 220-7. MCCRACKEN, A. A. & BRODSKY, J. L. 2003. Evolving questions and paradigm shifts in endoplasmic-reticulum-associated degradation (ERAD). Bioessays, 25, 868-77. MCELNEA, E., STEPHENSON, K., GILMORE, S., O'KEEFE, M. & KEEGAN, D. 2018. Paediatric retinal detachment: aetiology, characteristics and outcomes. Int J Ophthalmol, 11, 262-266. MENENDEZ-BENITO, V., VERHOEF, L. G., MASUCCI, M. G. & DANTUMA, N. P. 2005. Endoplasmic reticulum stress compromises the ubiquitin-proteasome system. Hum Mol Genet, 14, 2787-99. MERCHED, A. J., KO, K., GOTLINGER, K. H., SERHAN, C. N. & CHAN, L. 2008. Atherosclerosis: evidence for impairment of resolution of vascular inflammation governed by specific lipid mediators. FASEB J, 22, 3595-606. MERUVU, S., WALTHER, M., IVANOV, I., HAMMARSTROM, S., FURSTENBERGER, G., KRIEG, P., REDDANNA, P. & KUHN, H. 2005. Sequence determinants for the reaction specificity of murine (12R)-lipoxygenase: targeted substrate modification and site-directed mutagenesis. J Biol Chem, 280, 36633-41. MESQUIDA, M., LESZCZYNSKA, A., LLORENC, V. & ADAN, A. 2014. Interleukin- 6 blockade in ocular inflammatory diseases. Clin Exp Immunol, 176, 301-9. MESSIAS, A. C., HARNISCH, C., OSTARECK-LEDERER, A., SATTLER, M. & OSTARECK, D. H. 2006. The DICE-binding activity of KH domain 3 of hnRNP K is affected by c-Src-mediated tyrosine phosphorylation. J Mol Biol, 361, 470- 81. 112

MEYER-KIRCHRATH, J., DEBEY, S., GLANDORFF, C., KIRCHRATH, L. & SCHROR, K. 2004. Gene expression profile of the Gs-coupled prostacyclin receptor in human vascular smooth muscle cells. Biochem Pharmacol, 67, 757-65. MIKULE, K., GATLIN, J. C., DE LA HOUSSAYE, B. A. & PFENNINGER, K. H. 2002. Growth cone collapse induced by semaphorin 3A requires 12/15- lipoxygenase. J Neurosci, 22, 4932-41. MINAMINO, T., KOMURO, I. & KITAKAZE, M. 2010. Endoplasmic reticulum stress as a therapeutic target in cardiovascular disease. Circ Res, 107, 1071-82. MIRANDA, G. E., ABRAHAN, C. E., AGNOLAZZA, D. L., POLITI, L. E. & ROTSTEIN, N. P. 2011. Ceramide-1-phosphate, a new mediator of development and survival in retina photoreceptors. Invest Ophthalmol Vis Sci, 52, 6580-8. MIRANDA, G. E., ABRAHAN, C. E., POLITI, L. E. & ROTSTEIN, N. P. 2009. Sphingosine-1-phosphate is a key regulator of proliferation and differentiation in retina photoreceptors. Invest Ophthalmol Vis Sci, 50, 4416-28. MITCHELL, P., BANDELLO, F., SCHMIDT-ERFURTH, U., LANG, G. E., MASSIN, P., SCHLINGEMANN, R. O., SUTTER, F., SIMADER, C., BURIAN, G., GERSTNER, O., WEICHSELBERGER, A. & GROUP, R. S. 2011. The RESTORE study: ranibizumab monotherapy or combined with laser versus laser monotherapy for diabetic macular edema. Ophthalmology, 118, 615-25. MIYAMOTO, A., WAKE, H., MOORHOUSE, A. J. & NABEKURA, J. 2013. Microglia and synapse interactions: fine tuning neural circuits and candidate molecules. Front Cell Neurosci, 7, 70. MIYAMOTO, K., KHOSROF, S., BURSELL, S. E., ROHAN, R., MURATA, T., CLERMONT, A. C., AIELLO, L. P., OGURA, Y. & ADAMIS, A. P. 1999. Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition. Proc Natl Acad Sci U S A, 96, 10836-41. MOENNER, M., PLUQUET, O., BOUCHECAREILH, M. & CHEVET, E. 2007. Integrated endoplasmic reticulum stress responses in cancer. Cancer Res, 67, 10631-4. MOLDAY, R. S. & MORITZ, O. L. 2015. Photoreceptors at a glance. J Cell Sci, 128, 4039-45. MORGAN, A. H., HAMMOND, V. J., SAKOH-NAKATOGAWA, M., OHSUMI, Y., THOMAS, C. P., BLANCHET, F., PIGUET, V., KISELYOV, K. & O'DONNELL, V. B. 2015. A novel role for 12/15-lipoxygenase in regulating autophagy. Redox Biol, 4, 40-7. MOSS, H. E. 2015. Retinal Vascular Changes are a Marker for Cerebral Vascular Diseases. Curr Neurol Neurosci Rep, 15, 40. MYOISHI, M., HAO, H., MINAMINO, T., WATANABE, K., NISHIHIRA, K., HATAKEYAMA, K., ASADA, Y., OKADA, K., ISHIBASHI-UEDA, H., 113

GABBIANI, G., BOCHATON-PIALLAT, M. L., MOCHIZUKI, N. & KITAKAZE, M. 2007. Increased endoplasmic reticulum stress in atherosclerotic plaques associated with acute coronary syndrome. Circulation, 116, 1226-33. NAARMANN, I. S., HARNISCH, C., MULLER-NEWEN, G., URLAUB, H., OSTARECK-LEDERER, A. & OSTARECK, D. H. 2010. DDX6 recruits translational silenced human reticulocyte 15-lipoxygenase mRNA to RNP granules. RNA, 16, 2189-204. NADEL, J. A., CONRAD, D. J., UEKI, I. F., SCHUSTER, A. & SIGAL, E. 1991. Immunocytochemical localization of arachidonate 15-lipoxygenase in erythrocytes, leukocytes, and airway cells. J Clin Invest, 87, 1139-45. NARUMIYA, S., SALMON, J. A., FLOWER, R. J. & VANE, J. R. 1982. Purification and properties of arachidonate-15-lipoxygenase from rabbit peritoneal polymorphonuclear leukocytes. Adv Prostaglandin Thromboxane Leukot Res, 9, 77-82. NASH, B. M., WRIGHT, D. C., GRIGG, J. R., BENNETTS, B. & JAMIESON, R. V. 2015. Retinal dystrophies, genomic applications in diagnosis and prospects for therapy. Transl Pediatr, 4, 139-63. NASSAR, G. M., MORROW, J. D., ROBERTS, L. J., 2ND, LAKKIS, F. G. & BADR, K. F. 1994. Induction of 15-lipoxygenase by interleukin-13 in human blood monocytes. J Biol Chem, 269, 27631-4. NG, S. K., WOOD, J. P., CHIDLOW, G., HAN, G., KITTIPASSORN, T., PEET, D. J. & CASSON, R. J. 2015. Cancer-like metabolism of the mammalian retina. Clin Exp Ophthalmol, 43, 367-76. NGUYEN, Q. D., SHAH, S. M., HEIER, J. S., DO, D. V., LIM, J., BOYER, D., ABRAHAM, P., CAMPOCHIARO, P. A. & GROUP, R.-S. 2009. Primary End Point (Six Months) Results of the Ranibizumab for Edema of the mAcula in diabetes (READ-2) study. Ophthalmology, 116, 2175-81 e1. NGUYEN, Q. D., TATLIPINAR, S., SHAH, S. M., HALLER, J. A., QUINLAN, E., SUNG, J., ZIMMER-GALLER, I., DO, D. V. & CAMPOCHIARO, P. A. 2006. Vascular endothelial growth factor is a critical stimulus for diabetic macular edema. Am J Ophthalmol, 142, 961-9. NIE, D., HILLMAN, G. G., GEDDES, T., TANG, K., PIERSON, C., GRIGNON, D. J. & HONN, K. V. 1998. Platelet-type 12-lipoxygenase in a human prostate carcinoma stimulates angiogenesis and tumor growth. Cancer Res, 58, 4047-51. NISHIYAMA, M., WATANABE, T., UEDA, N., TSUKAMOTO, H. & WATANABE, K. 1993. Arachidonate 12-lipoxygenase is localized in neurons, glial cells, and endothelial cells of the canine brain. J Histochem Cytochem, 41, 111-7. NONAKA, A., KIRYU, J., TSUJIKAWA, A., YAMASHIRO, K., MIYAMOTO, K., NISHIWAKI, H., HONDA, Y. & OGURA, Y. 2000. PKC-beta inhibitor

114

(LY333531) attenuates leukocyte entrapment in retinal microcirculation of diabetic rats. Invest Ophthalmol Vis Sci, 41, 2702-6. NUNEMAKER, C. S., CHEN, M., PEI, H., KIMBLE, S. D., KELLER, S. R., CARTER, J. D., YANG, Z., SMITH, K. M., WU, R., BEVARD, M. H., GARMEY, J. C. & NADLER, J. L. 2008. 12-Lipoxygenase-knockout mice are resistant to inflammatory effects of obesity induced by Western diet. Am J Physiol Endocrinol Metab, 295, E1065-75. NUSCHKE, A. C., FARRELL, S. R., LEVESQUE, J. M. & CHAUHAN, B. C. 2015. Assessment of retinal ganglion cell damage in glaucomatous optic neuropathy: Axon transport, injury and soma loss. Exp Eye Res, 141, 111-24. OATES, P. J. 2002. Polyol pathway and diabetic peripheral neuropathy. Int Rev Neurobiol, 50, 325-92. OBROSOVA, I. G., MAKSIMCHYK, Y., PACHER, P., AGARDH, E., SMITH, M. L., EL-REMESSY, A. B. & AGARDH, C. D. 2010. Evaluation of the aldose reductase inhibitor fidarestat on ischemia-reperfusion injury in rat retina. Int J Mol Med, 26, 135-42. OHIRA, T., ARITA, M., OMORI, K., RECCHIUTI, A., VAN DYKE, T. E. & SERHAN, C. N. 2010. E1 receptor activation signals phosphorylation and phagocytosis. J Biol Chem, 285, 3451-61. OMAROVA, S., CHARVET, C. D., REEM, R. E., MAST, N., ZHENG, W., HUANG, S., PEACHEY, N. S. & PIKULEVA, I. A. 2012. Abnormal vascularization in mouse retina with dysregulated retinal cholesterol homeostasis. J Clin Invest, 122, 3012-23. OMRI, S., OMRI, B., SAVOLDELLI, M., JONET, L., THILLAYE-GOLDENBERG, B., THURET, G., GAIN, P., JEANNY, J. C., CRISANTI, P. & BEHAR-COHEN, F. 2010. The outer limiting membrane (OLM) revisited: clinical implications. Clin Ophthalmol, 4, 183-95. OOSTHUYSE, B., MOONS, L., STORKEBAUM, E., BECK, H., NUYENS, D., BRUSSELMANS, K., VAN DORPE, J., HELLINGS, P., GORSELINK, M., HEYMANS, S., THEILMEIER, G., DEWERCHIN, M., LAUDENBACH, V., VERMYLEN, P., RAAT, H., ACKER, T., VLEMINCKX, V., VAN DEN BOSCH, L., CASHMAN, N., FUJISAWA, H., DROST, M. R., SCIOT, R., BRUYNINCKX, F., HICKLIN, D. J., INCE, C., GRESSENS, P., LUPU, F., PLATE, K. H., ROBBERECHT, W., HERBERT, J. M., COLLEN, D. & CARMELIET, P. 2001. Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat Genet, 28, 131-8. OPREANU, M., LYDIC, T. A., REID, G. E., MCSORLEY, K. M., ESSELMAN, W. J. & BUSIK, J. V. 2010. Inhibition of cytokine signaling in human retinal

115

endothelial cells through downregulation of sphingomyelinases by docosahexaenoic acid. Invest Ophthalmol Vis Sci, 51, 3253-63. OPREANU, M., TIKHONENKO, M., BOZACK, S., LYDIC, T. A., REID, G. E., MCSORLEY, K. M., SOCHACKI, A., PEREZ, G. I., ESSELMAN, W. J., KERN, T., KOLESNICK, R., GRANT, M. B. & BUSIK, J. V. 2011. The unconventional role of acid sphingomyelinase in regulation of retinal microangiopathy in diabetic human and animal models. Diabetes, 60, 2370-8. ORBAN, M., ISLAM, Y. F. & HADDOCK, L. J. 2016. Timing and Outcomes of Vitreoretinal Surgery after Traumatic Retinal Detachment. J Ophthalmol, 2016, 4978973. OSAADON, P., FAGAN, X. J., LIFSHITZ, T. & LEVY, J. 2014. A review of anti-VEGF agents for proliferative diabetic retinopathy. Eye (Lond), 28, 510-20. OSTARECK, D. H., OSTARECK-LEDERER, A., WILM, M., THIELE, B. J., MANN, M. & HENTZE, M. W. 1997. mRNA silencing in erythroid differentiation: hnRNP K and hnRNP E1 regulate 15-lipoxygenase translation from the 3' end. Cell, 89, 597-606. OTANI, A., TAKAGI, H., SUZUMA, K. & HONDA, Y. 1998. Angiotensin II potentiates vascular endothelial growth factor-induced angiogenic activity in retinal microcapillary endothelial cells. Circ Res, 82, 619-28. OTHMAN, A., AHMAD, S., MEGYERDI, S., MUSSELL, R., CHOKSI, K., MADDIPATI, K. R., ELMARAKBY, A., RIZK, N. & AL-SHABRAWEY, M. 2013. 12/15-Lipoxygenase-derived lipid metabolites induce retinal endothelial cell barrier dysfunction: contribution of NADPH oxidase. PLoS One, 8, e57254. PALLAST, S., ARAI, K., PEKCEC, A., YIGITKANLI, K., YU, Z., WANG, X., LO, E. H. & VAN LEYEN, K. 2010. Increased nuclear apoptosis-inducing factor after transient focal ischemia: a 12/15-lipoxygenase-dependent organelle damage pathway. J Cereb Blood Flow Metab, 30, 1157-67. PALLAST, S., ARAI, K., WANG, X., LO, E. H. & VAN LEYEN, K. 2009. 12/15- Lipoxygenase targets neuronal mitochondria under oxidative stress. J Neurochem, 111, 882-9. PARMEGGIANI, F., ROMANO, M. R., COSTAGLIOLA, C., SEMERARO, F., INCORVAIA, C., D'ANGELO, S., PERRI, P., DE PALMA, P., DE NADAI, K. & SEBASTIANI, A. 2012. Mechanism of inflammation in age-related macular degeneration. Mediators Inflamm, 2012, 546786. PATEL, V., RASSAM, S., NEWSOM, R., WIEK, J. & KOHNER, E. 1992. Retinal blood flow in diabetic retinopathy. BMJ, 305, 678-83. PEARSON, P. A., COMSTOCK, T. L., IP, M., CALLANAN, D., MORSE, L. S., ASHTON, P., LEVY, B., MANN, E. S. & ELIOTT, D. 2011. Fluocinolone acetonide intravitreal implant for diabetic macular edema: a 3-year multicenter, randomized, controlled clinical trial. Ophthalmology, 118, 1580-7. 116

PEKAROVA, M., KUHN, H., BEZAKOVA, L., UFER, C. & HEYDECK, D. 2015. Mutagenesis of triad determinants of rat Alox15 alters the specificity of fatty acid and phospholipid oxygenation. Arch Biochem Biophys, 571, 50-7. POLITI, L. E., ROTSTEIN, N. P. & CARRI, N. G. 2001. Effect of GDNF on neuroblast proliferation and photoreceptor survival: additive protection with docosahexaenoic acid. Invest Ophthalmol Vis Sci, 42, 3008-15. POLLREISZ, A., AFONYUSHKIN, T., OSKOLKOVA, O. V., GRUBER, F., BOCHKOV, V. N. & SCHMIDT-ERFURTH, U. 2013. Retinal pigment epithelium cells produce VEGF in response to oxidized phospholipids through mechanisms involving ATF4 and protein kinase CK2. Exp Eye Res, 116, 177-84. PONSIOEN, T. L., VAN LUYN, M. J., VAN DER WORP, R. J., PAS, H. H., HOOYMANS, J. M. & LOS, L. I. 2008. Human retinal Muller cells synthesize collagens of the vitreous and vitreoretinal interface in vitro. Mol Vis, 14, 652-60. POURNARAS, C. J. & RIVA, C. E. 2013. Retinal blood flow evaluation. Ophthalmologica, 229, 61-74. PRATICO, D., ZHUKAREVA, V., YAO, Y., URYU, K., FUNK, C. D., LAWSON, J. A., TROJANOWSKI, J. Q. & LEE, V. M. 2004. 12/15-lipoxygenase is increased in Alzheimer's disease: possible involvement in brain oxidative stress. Am J Pathol, 164, 1655-62. RADMARK, O., WERZ, O., STEINHILBER, D. & SAMUELSSON, B. 2015. 5- Lipoxygenase, a key enzyme for leukotriene biosynthesis in health and disease. Biochim Biophys Acta, 1851, 331-9. RAI, G., KENYON, V., JADHAV, A., SCHULTZ, L., ARMSTRONG, M., JAMESON, J. B., HOOBLER, E., LEISTER, W., SIMEONOV, A., HOLMAN, T. R. & MALONEY, D. J. 2010. Discovery of potent and selective inhibitors of human reticulocyte 15-lipoxygenase-1. J Med Chem, 53, 7392-404. RANCIC, A., FILIPOVIC, N., MARIN LOVRIC, J., MARDESIC, S., SARAGA- BABIC, M. & VUKOJEVIC, K. 2017. Neuronal differentiation in the early human retinogenesis. Acta Histochem, 119, 264-272. RANGASAMY, S., MCGUIRE, P. G. & DAS, A. 2012. Diabetic retinopathy and inflammation: novel therapeutic targets. Middle East Afr J Ophthalmol, 19, 52-9. REICHENBACH, A. & BRINGMANN, A. 2013. New functions of Muller cells. Glia, 61, 651-78. REICHENBACH, A., WURM, A., PANNICKE, T., IANDIEV, I., WIEDEMANN, P. & BRINGMANN, A. 2007. Muller cells as players in retinal degeneration and edema. Graefes Arch Clin Exp Ophthalmol, 245, 627-36. REIMANN, I., HUTH, A., THIELE, H. & THIELE, B. J. 2002. Suppression of 15- lipoxygenase synthesis by hnRNP E1 is dependent on repetitive nature of LOX mRNA 3'-UTR control element DICE. J Mol Biol, 315, 965-74.

117

REMTULLA, S. & HALLETT, P. E. 1985. A schematic eye for the mouse, and comparisons with the rat. Vision Res, 25, 21-31. REZENDE, F. A., LAPALME, E., QIAN, C. X., SMITH, L. E., SANGIOVANNI, J. P. & SAPIEHA, P. 2014. Omega-3 supplementation combined with anti-vascular endothelial growth factor lowers vitreal levels of vascular endothelial growth factor in wet age-related macular degeneration. Am J Ophthalmol, 158, 1071-78. RICKERT, K. W. & KLINMAN, J. P. 1999. Nature of hydrogen transfer in soybean lipoxygenase 1: separation of primary and secondary isotope effects. Biochemistry, 38, 12218-28. RIZZOLO, L. J. 2014. Barrier properties of cultured retinal pigment epithelium. Exp Eye Res, 126, 16-26. ROESCH, K., JADHAV, A. P., TRIMARCHI, J. M., STADLER, M. B., ROSKA, B., SUN, B. B. & CEPKO, C. L. 2008. The transcriptome of retinal Muller glial cells. J Comp Neurol, 509, 225-38. ROGERS, T. B., INESI, G., WADE, R. & LEDERER, W. J. 1995. Use of thapsigargin to study Ca2+ homeostasis in cardiac cells. Biosci Rep, 15, 341-9. ROSS, S., ESSARY, B., DE LA HOUSSAYE, B. A., PAN, Z., MIKULE, K., MUBARAK, O. & PFENNINGER, K. H. 2000. Thrombin causes pseudopod detachment via a pathway involving cytosolic phospholipase A2 and 12/15- lipoxygenase products. Cell Growth Differ, 11, 19-30. ROWE, S. M., MILLER, S. & SORSCHER, E. J. 2005. Cystic fibrosis. N Engl J Med, 352, 1992-2001. ROY, B. & CATHCART, M. K. 1998. Induction of 15-lipoxygenase expression by IL-13 requires tyrosine phosphorylation of Jak2 and Tyk2 in human monocytes. J Biol Chem, 273, 32023-9. RYAN, A. & GODSON, C. 2010. Lipoxins: regulators of resolution. Curr Opin Pharmacol, 10, 166-72. SAADANE, A., MAST, N., CHARVET, C. D., OMAROVA, S., ZHENG, W., HUANG, S. S., KERN, T. S., PEACHEY, N. S. & PIKULEVA, I. A. 2014. Retinal and nonocular abnormalities in Cyp27a1(-/-)Cyp46a1(-/-) mice with dysfunctional metabolism of cholesterol. Am J Pathol, 184, 2403-19. SALA, A., FOLCO, G. & MURPHY, R. C. 2010. Transcellular biosynthesis of eicosanoids. Pharmacol Rep, 62, 503-10. SALES, K. J., GRANT, V. & JABBOUR, H. N. 2008. Prostaglandin E2 and F2alpha activate the FP receptor and up-regulate cyclooxygenase-2 expression via the cyclic AMP response element. Mol Cell Endocrinol, 285, 51-61. SAMPAT, K. M. & GARG, S. J. 2010. Complications of intravitreal injections. Curr Opin Ophthalmol, 21, 178-83.

118

SANTIAGO, A. R., GASPAR, J. M., BAPTISTA, F. I., CRISTOVAO, A. J., SANTOS, P. F., KAMPHUIS, W. & AMBROSIO, A. F. 2009. Diabetes changes the levels of ionotropic glutamate receptors in the rat retina. Mol Vis, 15, 1620-30. SANTOS, C. X., TANAKA, L. Y., WOSNIAK, J. & LAURINDO, F. R. 2009. Mechanisms and implications of reactive oxygen species generation during the unfolded protein response: roles of endoplasmic reticulum oxidoreductases, mitochondrial electron transport, and NADPH oxidase. Antioxid Redox Signal, 11, 2409-27. SAPIEHA, P., STAHL, A., CHEN, J., SEAWARD, M. R., WILLETT, K. L., KRAH, N. M., DENNISON, R. J., CONNOR, K. M., ADERMAN, C. M., LICLICAN, E., CARUGHI, A., PERELMAN, D., KANAOKA, Y., SANGIOVANNI, J. P., GRONERT, K. & SMITH, L. E. 2011. 5-Lipoxygenase metabolite 4-HDHA is a mediator of the antiangiogenic effect of omega-3 polyunsaturated fatty acids. Sci Transl Med, 3, 69ra12. SCHAFER, D. P., LEHRMAN, E. K. & STEVENS, B. 2013. The "quad-partite" synapse: microglia-synapse interactions in the developing and mature CNS. Glia, 61, 24-36. SCHEWE, T. 2002. 15-lipoxygenase-1: a prooxidant enzyme. Biol Chem, 383, 365-74. SCHEWE, T., HALANGK, W., HIEBSCH, C. & RAPOPORT, S. M. 1975. A lipoxygenase in rabbit which attacks phospholipids and intact mitochondria. FEBS Lett, 60, 149-52. SCHEWE, T., RAPOPORT, S. M. & KUHN, H. 1986. Enzymology and physiology of reticulocyte lipoxygenase: comparison with other lipoxygenases. Adv Enzymol Relat Areas Mol Biol, 58, 191-272. SCHRODER, S., PALINSKI, W. & SCHMID-SCHONBEIN, G. W. 1991. Activated monocytes and granulocytes, capillary nonperfusion, and neovascularization in diabetic retinopathy. Am J Pathol, 139, 81-100. SEARS, D. D., MILES, P. D., CHAPMAN, J., OFRECIO, J. M., ALMAZAN, F., THAPAR, D. & MILLER, Y. I. 2009. 12/15-lipoxygenase is required for the early onset of high fat diet-induced inflammation and in mice. PLoS One, 4, e7250. SEILER, A., SCHNEIDER, M., FORSTER, H., ROTH, S., WIRTH, E. K., CULMSEE, C., PLESNILA, N., KREMMER, E., RADMARK, O., WURST, W., BORNKAMM, G. W., SCHWEIZER, U. & CONRAD, M. 2008. Glutathione peroxidase 4 senses and translates oxidative stress into 12/15-lipoxygenase dependent- and AIF-mediated cell death. Cell Metab, 8, 237-48. SEMERARO, F., CANCARINI, A., DELL'OMO, R., REZZOLA, S., ROMANO, M. R. & COSTAGLIOLA, C. 2015. Diabetic Retinopathy: Vascular and Inflammatory Disease. J Diabetes Res, 2015, 582060.

119

SERHAN, C. N., CHIANG, N. & DALLI, J. 2015a. The resolution code of acute inflammation: Novel pro-resolving lipid mediators in resolution. Semin Immunol, 27, 200-15. SERHAN, C. N., DALLI, J., COLAS, R. A., WINKLER, J. W. & CHIANG, N. 2015b. Protectins and maresins: New pro-resolving families of mediators in acute inflammation and resolution bioactive metabolome. Biochim Biophys Acta, 1851, 397-413. SHANG, F. & TAYLOR, A. 2004. Function of the ubiquitin proteolytic pathway in the eye. Exp Eye Res, 78, 1-14. SHANG, F. & TAYLOR, A. 2012. Roles for the ubiquitin-proteasome pathway in protein quality control and signaling in the retina: implications in the pathogenesis of age-related macular degeneration. Mol Aspects Med, 33, 446-66. SHANKARANARAYANAN, P., CHAITIDIS, P., KUHN, H. & NIGAM, S. 2001. Acetylation by histone acetyltransferase CREB-binding protein/p300 of STAT6 is required for transcriptional activation of the 15-lipoxygenase-1 gene. J Biol Chem, 276, 42753-60. SHANMUGAM, P. M. & RAMANJULU, R. 2015. Vascular tumors of the choroid and retina. Indian J Ophthalmol, 63, 133-40. SHARPE, L. T., STOCKMAN, A. & MACLEOD, D. I. 1989. Rod flicker perception: scotopic duality, phase lags and destructive interference. Vision Res, 29, 1539-59. SHEN, J., HE, J. & WANG, F. 2016. Association of lipids with age-related macular degeneration. Discov Med, 22, 129-145. SHEN, J., HERDERICK, E., CORNHILL, J. F., ZSIGMOND, E., KIM, H. S., KUHN, H., GUEVARA, N. V. & CHAN, L. 1996. -mediated 15- lipoxygenase expression protects against atherosclerosis development. J Clin Invest, 98, 2201-8. SHIN, E. S., SORENSON, C. M. & SHEIBANI, N. 2014. Diabetes and retinal vascular dysfunction. J Ophthalmic Vis Res, 9, 362-73. SHRUTHI, K., REDDY, S. S. & REDDY, G. B. 2017. Ubiquitin-proteasome system and ER stress in the retina of diabetic rats. Arch Biochem Biophys, 627, 10-20. SHUREIQI, I., JIANG, W., ZUO, X., WU, Y., STIMMEL, J. B., LEESNITZER, L. M., MORRIS, J. S., FAN, H. Z., FISCHER, S. M. & LIPPMAN, S. M. 2003. The 15- lipoxygenase-1 product 13-S-hydroxyoctadecadienoic acid down-regulates PPAR-delta to induce apoptosis in colorectal cancer cells. Proc Natl Acad Sci U S A, 100, 9968-73. SIMONSON, D. C. 1988. Etiology and prevalence of hypertension in diabetic patients. Diabetes Care, 11, 821-7. SKOURA, A., SANCHEZ, T., CLAFFEY, K., MANDALA, S. M., PROIA, R. L. & HLA, T. 2007. Essential role of sphingosine 1-phosphate receptor 2 in pathological angiogenesis of the mouse retina. J Clin Invest, 117, 2506-16. 120

SLOANE, D. L., DIXON, R. A., CRAIK, C. S. & SIGAL, E. 1991. Expression of cloned human 15-lipoxygenase in eukaryotic and prokaryotic systems. Adv Prostaglandin Thromboxane Leukot Res, 21A, 25-8. SMITH, B. J., WANG, X., CHAUHAN, B. C., COTE, P. D. & TREMBLAY, F. 2014. Contribution of retinal ganglion cells to the mouse electroretinogram. Doc Ophthalmol, 128, 155-68. SOBRADO, M., PEREIRA, M. P., BALLESTEROS, I., HURTADO, O., FERNANDEZ- LOPEZ, D., PRADILLO, J. M., CASO, J. R., VIVANCOS, J., NOMBELA, F., SERENA, J., LIZASOAIN, I. & MORO, M. A. 2009. Synthesis of lipoxin A4 by 5-lipoxygenase mediates PPARgamma-dependent, neuroprotective effects of rosiglitazone in experimental stroke. J Neurosci, 29, 3875-84. SONG, D., SULEWSKI, M. E., JR., WANG, C., SONG, J., BHUYAN, R., STERLING, J., CLARK, E., SONG, W. C. & DUNAIEF, J. L. 2017. Complement C5a receptor knockout has diminished light-induced microglia/macrophage retinal migration. Mol Vis, 23, 210-218. SOUMYA, S. J., BINU, S., HELEN, A., REDDANNA, P. & SUDHAKARAN, P. R. 2013. 15(S)-HETE-induced angiogenesis in adipose tissue is mediated through activation of PI3K/Akt/mTOR signaling pathway. Biochem Cell Biol, 91, 498- 505. SPECTOR, R., KEEP, R. F., ROBERT SNODGRASS, S., SMITH, Q. R. & JOHANSON, C. E. 2015. A balanced view of choroid plexus structure and function: Focus on adult humans. Exp Neurol, 267, 78-86. SPIJKERMAN, A. M., GALL, M. A., TARNOW, L., TWISK, J. W., LAURITZEN, E., LUND-ANDERSEN, H., EMEIS, J., PARVING, H. H. & STEHOUWER, C. D. 2007. Endothelial dysfunction and low-grade inflammation and the progression of retinopathy in Type 2 diabetes. Diabet Med, 24, 969-76. SPITE, M., CLARIA, J. & SERHAN, C. N. 2014. Resolvins, specialized proresolving lipid mediators, and their potential roles in metabolic diseases. Cell Metab, 19, 21-36. STADLER, K. 2012. Oxidative stress in diabetes. Adv Exp Med Biol, 771, 272-87. STITT, A., GARDINER, T. A., ALDERSON, N. L., CANNING, P., FRIZZELL, N., DUFFY, N., BOYLE, C., JANUSZEWSKI, A. S., CHACHICH, M., BAYNES, J. W. & THORPE, S. R. 2002. The AGE inhibitor pyridoxamine inhibits development of retinopathy in experimental diabetes. Diabetes, 51, 2826-32. STRYER, L. 1991. Visual excitation and recovery. J Biol Chem, 266, 10711-4. STRYJEWSKI, T. P., ANDREOLI, C. M. & ELIOTT, D. 2014. Retinal detachment after open globe injury. Ophthalmology, 121, 327-33. SUN, D. & FUNK, C. D. 1996. Disruption of 12/15-lipoxygenase expression in peritoneal macrophages. Enhanced utilization of the 5-lipoxygenase pathway and diminished oxidation of low density lipoprotein. J Biol Chem, 271, 24055-62. 121

SUZUKI, M., KAMEI, M., ITABE, H., YONEDA, K., BANDO, H., KUME, N. & TANO, Y. 2007. Oxidized phospholipids in the macula increase with age and in eyes with age-related macular degeneration. Mol Vis, 13, 772-8. SWAROOP, A., KIM, D. & FORREST, D. 2010. Transcriptional regulation of photoreceptor development and homeostasis in the mammalian retina. Nat Rev Neurosci, 11, 563-76. TABAS, I. & RON, D. 2011. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat Cell Biol, 13, 184-90. TAKAHASHI, H. & SHIBUYA, M. 2005. The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions. Clin Sci (Lond), 109, 227-41. TAKAMI, S., YAMASHITA, S., KIHARA, S., KAMEDA-TAKEMURA, K. & MATSUZAWA, Y. 1998. High concentration of glucose induces the expression of intercellular adhesion molecule-1 in human umbilical vein endothelial cells. Atherosclerosis, 138, 35-41. TAKAYAMA, H., GIMBRONE, M. A., JR. & SCHAFER, A. I. 1987. Vascular lipoxygenase activity: synthesis of 15-hydroxyeicosatetraenoic acid from arachidonic acid by blood vessels and cultured vascular endothelial cells. Thromb Res, 45, 803-16. TANG, J. & KERN, T. S. 2011. Inflammation in diabetic retinopathy. Prog Retin Eye Res, 30, 343-58. TERSEY, S. A., NISHIKI, Y., TEMPLIN, A. T., CABRERA, S. M., STULL, N. D., COLVIN, S. C., EVANS-MOLINA, C., RICKUS, J. L., MAIER, B. & MIRMIRA, R. G. 2012. Islet beta-cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model. Diabetes, 61, 818- 27. THALLAS-BONKE, V., LINDSCHAU, C., RIZKALLA, B., BACH, L. A., BONER, G., MEIER, M., HALLER, H., COOPER, M. E. & FORBES, J. M. 2004. Attenuation of extracellular matrix accumulation in diabetic nephropathy by the advanced glycation end product cross-link breaker ALT-711 via a protein kinase C-alpha- dependent pathway. Diabetes, 53, 2921-30. THAPA, R., BAJIMAYA, S., PAUDYAL, G., KHANAL, S., TAN, S., THAPA, S. S. & VAN RENS, G. 2017. Prevalence, pattern and risk factors of retinal vein occlusion in an elderly population in Nepal: the Bhaktapur retina study. BMC Ophthalmol, 17, 162. TIKHONENKO, M., LYDIC, T. A., WANG, Y., CHEN, W., OPREANU, M., SOCHACKI, A., MCSORLEY, K. M., RENIS, R. L., KERN, T., JUMP, D. B., REID, G. E. & BUSIK, J. V. 2010. Remodeling of retinal Fatty acids in an animal model of diabetes: a decrease in long-chain polyunsaturated fatty acids is

122

associated with a decrease in fatty acid elongases Elovl2 and Elovl4. Diabetes, 59, 219-27. TOLENTINO, M. J., MCLEOD, D. S., TAOMOTO, M., OTSUJI, T., ADAMIS, A. P. & LUTTY, G. A. 2002. Pathologic features of vascular endothelial growth factor- induced retinopathy in the nonhuman primate. Am J Ophthalmol, 133, 373-85. TSERENTSOODOL, N., SZTEIN, J., CAMPOS, M., GORDIYENKO, N. V., FARISS, R. N., LEE, J. W., FLIESLER, S. J. & RODRIGUEZ, I. R. 2006. Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor- mediated process. Mol Vis, 12, 1306-18. TU, B. P. & WEISSMAN, J. S. 2004. Oxidative protein folding in eukaryotes: mechanisms and consequences. J Cell Biol, 164, 341-6. UNTERLAUFT, J. D., EICHLER, W., KUHNE, K., YANG, X. M., YAFAI, Y., WIEDEMANN, P., REICHENBACH, A. & CLAUDEPIERRE, T. 2012. Pigment epithelium-derived factor released by Muller glial cells exerts neuroprotective effects on retinal ganglion cells. Neurochem Res, 37, 1524-33. VAN ANKEN, E. & BRAAKMAN, I. 2005. Versatility of the endoplasmic reticulum protein folding factory. Crit Rev Biochem Mol Biol, 40, 191-228. VAN HECKE, M. V., DEKKER, J. M., NIJPELS, G., MOLL, A. C., HEINE, R. J., BOUTER, L. M., POLAK, B. C. & STEHOUWER, C. D. 2005. Inflammation and endothelial dysfunction are associated with retinopathy: the Hoorn Study. Diabetologia, 48, 1300-6. VAN LEYEN, K. 2013. Lipoxygenase: an emerging target for stroke therapy. CNS Neurol Disord Drug Targets, 12, 191-9. VAN LEYEN, K., DUVOISIN, R. M., ENGELHARDT, H. & WIEDMANN, M. 1998. A function for lipoxygenase in programmed organelle degradation. Nature, 395, 392-5. VAN LEYEN, K., KIM, H. Y., LEE, S. R., JIN, G., ARAI, K. & LO, E. H. 2006. Baicalein and 12/15-lipoxygenase in the ischemic brain. Stroke, 37, 3014-8. VANDERHOEK, J. Y. & BAILEY, J. M. 1984. Activation of a 15- lipoxygenase/leukotriene pathway in human polymorphonuclear leukocytes by the anti-inflammatory agent ibuprofen. J Biol Chem, 259, 6752-6. VAZIRI, K., SCHWARTZ, S. G., RELHAN, N., KISHOR, K. S. & FLYNN, H. W., JR. 2015. New Therapeutic Approaches in Diabetic Retinopathy. Rev Diabet Stud, 12, 196-210. VECINO, E., RODRIGUEZ, F. D., RUZAFA, N., PEREIRO, X. & SHARMA, S. C. 2016. Glia-neuron interactions in the mammalian retina. Prog Retin Eye Res, 51, 1-40. VIDAL, R., CABALLERO, B., COUVE, A. & HETZ, C. 2011. Converging pathways in the occurrence of endoplasmic reticulum (ER) stress in Huntington's disease. Curr Mol Med, 11, 1-12. 123

VIJIL, C., HERMANSSON, C., JEPPSSON, A., BERGSTROM, G. & HULTEN, L. M. 2014. Arachidonate 15-lipoxygenase enzyme products increase platelet aggregation and thrombin generation. PLoS One, 9, e88546. WALLACE, J. L. 2018. Eicosanoids in the GI Tract. Br J Pharmacol. WALTHER, M., WIESNER, R. & KUHN, H. 2004. Investigations into calcium- dependent membrane association of 15-lipoxygenase-1. Mechanistic roles of surface-exposed hydrophobic amino acids and calcium. J Biol Chem, 279, 3717- 25. WANG, L., CHEN, K., LIU, K., ZHOU, Y., ZHANG, T., WANG, B. & MI, M. 2015. DHA inhibited AGEs-induced retinal microglia activation via suppression of the PPARgamma/NFkappaB pathway and reduction of signal transducers in the AGEs/RAGE axis recruitment into lipid rafts. Neurochem Res, 40, 713-22. WANG, L., POPKO, B. & ROOS, R. P. 2011. The unfolded protein response in familial amyotrophic lateral sclerosis. Hum Mol Genet, 20, 1008-15. WANG, Q. J. 2006. PKD at the crossroads of DAG and PKC signaling. Trends Pharmacol Sci, 27, 317-23. WANG, Y., GAO, S., ZHU, Y. & SHEN, X. 2017. Elevated Activating Transcription Factor 4 and Glucose-Regulated 78 Kda Protein Levels Correlate with Inflammatory Cytokines in the Aqueous Humor and Vitreous of Proliferative Diabetic Retinopathy. Curr Eye Res, 42, 1202-1208. WATANABE, T. & HAEGGSTROM, J. Z. 1993. Rat 12-lipoxygenase: mutations of amino acids implicated in the positional specificity of 15- and 12-lipoxygenases. Biochem Biophys Res Commun, 192, 1023-9. WEIWEI, Z. & HU, R. 2009. Targeting carbonic anhydrase to treat diabetic retinopathy: emerging evidences and encouraging results. Biochem Biophys Res Commun, 390, 368-71. WILKINSON-BERKA, J. L. 2006. Angiotensin and diabetic retinopathy. Int J Biochem Cell Biol, 38, 752-65. WIROSTKO, B., WONG, T. Y. & SIMO, R. 2008. Vascular endothelial growth factor and diabetic complications. Prog Retin Eye Res, 27, 608-21. WITTWER, J. & HERSBERGER, M. 2007. The two faces of the 15-lipoxygenase in atherosclerosis. Prostaglandins Leukot Essent Fatty Acids, 77, 67-77. WOODWARD, D. F., JONES, R. L. & NARUMIYA, S. 2011. International Union of Basic and Clinical Pharmacology. LXXXIII: classification of prostanoid receptors, updating 15 years of progress. Pharmacol Rev, 63, 471-538. WUEST, S. J., HORN, T., MARTI-JAUN, J., KUHN, H. & HERSBERGER, M. 2014. Association of polymorphisms in the ALOX15B gene with coronary artery disease. Clin Biochem, 47, 349-55.

124

XIE, B., SHEN, J., DONG, A., RASHID, A., STOLLER, G. & CAMPOCHIARO, P. A. 2009. Blockade of sphingosine-1-phosphate reduces macrophage influx and retinal and choroidal neovascularization. J Cell Physiol, 218, 192-8. XU, B., BHATTACHARJEE, A., ROY, B., FELDMAN, G. M. & CATHCART, M. K. 2004. Role of protein kinase C isoforms in the regulation of interleukin-13- induced 15-lipoxygenase gene expression in human monocytes. J Biol Chem, 279, 15954-60. XU, H. & CHEN, M. 2016. Targeting the complement system for the management of retinal inflammatory and degenerative diseases. Eur J Pharmacol, 787, 94-104. XU, H. & CHEN, M. 2017. Diabetic retinopathy and dysregulated innate immunity. Vision Res, 139, 39-46. XU, Y. W., SUN, L., LIANG, H., SUN, G. M. & CHENG, Y. 2010. 12/15-Lipoxygenase inhibitor baicalein suppresses PPAR gamma expression and nuclear translocation induced by cerebral ischemia/reperfusion. Brain Res, 1307, 149-57. YAO, H., WANG, T., DENG, J., LIU, D., LI, X. & DENG, J. 2014. The development of blood-retinal barrier during the interaction of astrocytes with vascular wall cells. Neural Regen Res, 9, 1047-54. YAU, K. W. 1994. Phototransduction mechanism in retinal rods and cones. The Friedenwald Lecture. Invest Ophthalmol Vis Sci, 35, 9-32. YE, Y. 2005. The role of the ubiquitin-proteasome system in ER quality control. Essays Biochem, 41, 99-112. YLA-HERTTUALA, S., ROSENFELD, M. E., PARTHASARATHY, S., GLASS, C. K., SIGAL, E., WITZTUM, J. L. & STEINBERG, D. 1990. Colocalization of 15- lipoxygenase mRNA and protein with epitopes of oxidized low density lipoprotein in macrophage-rich areas of atherosclerotic lesions. Proc Natl Acad Sci U S A, 87, 6959-63. YOKOTA, S., TAKIHARA, Y., ARIMURA, S., MIYAKE, S., TAKAMURA, Y., YOSHIMURA, N. & INATANI, M. 2015. Altered Transport Velocity of Axonal Mitochondria in Retinal Ganglion Cells After Laser-Induced Axonal Injury In Vitro. Invest Ophthalmol Vis Sci, 56, 8019-25. YOSHIDA, S., SOTOZONO, C., IKEDA, T. & KINOSHITA, S. 2001. Interleukin-6 (IL- 6) production by cytokine-stimulated human Muller cells. Curr Eye Res, 22, 341- 7. YOUHNOVSKA, P., TOFFOLI, D. & GAUTHIER, D. 2013. Congenital hypertrophy of the retinal pigment epithelium complicated by a choroidal neovascular membrane. Digit J Ophthalmol, 19, 24-7. YU, D. Y., CRINGLE, S. J., BALARATNASINGAM, C., MORGAN, W. H., YU, P. K. & SU, E. N. 2013. Retinal ganglion cells: Energetics, compartmentation, axonal transport, cytoskeletons and vulnerability. Prog Retin Eye Res, 36, 217-46.

125

YU, D. Y., YU, P. K., CRINGLE, S. J., KANG, M. H. & SU, E. N. 2014. Functional and morphological characteristics of the retinal and choroidal vasculature. Prog Retin Eye Res, 40, 53-93. YU, Z., SCHNEIDER, C., BOEGLIN, W. E. & BRASH, A. R. 2006. Human and mouse eLOX3 have distinct substrate specificities: implications for their linkage with lipoxygenases in skin. Arch Biochem Biophys, 455, 188-96. ZHANG, C., WANG, H., NIE, J. & WANG, F. 2014. Protective factors in diabetic retinopathy: focus on blood-retinal barrier. Discov Med, 18, 105-12. ZHANG, H. J., SUN, C. H., KUANG, H. Y., JIANG, X. Y., LIU, H. L., HUA, W. F., LIU, Z. J., ZHOU, H., SUI, H. & QI, R. 2013. 12S-hydroxyeicosatetraenoic acid levels link to coronary artery disease in Type 2 diabetic patients. J Endocrinol Invest, 36, 385-9. ZHANG, S. X., MA, J. H., BHATTA, M., FLIESLER, S. J. & WANG, J. J. 2015. The unfolded protein response in retinal vascular diseases: implications and therapeutic potential beyond protein folding. Prog Retin Eye Res, 45, 111-31. ZHANG, W., LIU, H., ROJAS, M., CALDWELL, R. W. & CALDWELL, R. B. 2011. Anti-inflammatory therapy for diabetic retinopathy. Immunotherapy, 3, 609-28. ZHANG, X., SAADDINE, J. B., CHOU, C. F., COTCH, M. F., CHENG, Y. J., GEISS, L. S., GREGG, E. W., ALBRIGHT, A. L., KLEIN, B. E. & KLEIN, R. 2010. Prevalence of diabetic retinopathy in the United States, 2005-2008. JAMA, 304, 649-56. ZHANG, X. L., WEN, L., CHEN, Y. J. & ZHU, Y. 2009. Vascular endothelial growth factor up-regulates the expression of intracellular adhesion molecule-1 in retinal endothelial cells via reactive oxygen species, but not nitric oxide. Chin Med J (Engl), 122, 338-43. ZHAO, L., MOOS, M. P., GRABNER, R., PEDRONO, F., FAN, J., KAISER, B., JOHN, N., SCHMIDT, S., SPANBROEK, R., LOTZER, K., HUANG, L., CUI, J., RADER, D. J., EVANS, J. F., HABENICHT, A. J. & FUNK, C. D. 2004. The 5- lipoxygenase pathway promotes pathogenesis of hyperlipidemia-dependent aortic aneurysm. Nat Med, 10, 966-73. ZHAO, L., PRATICO, D., RADER, D. J. & FUNK, C. D. 2005. 12/15-Lipoxygenase gene disruption and vitamin E administration diminish atherosclerosis and oxidative stress in apolipoprotein E deficient mice through a final common pathway. Prostaglandins Other Lipid Mediat, 78, 185-93. ZHENG, X. R., ZHANG, S. S., YIN, F., TANG, J. L., YANG, Y. J., WANG, X. & ZHONG, L. 2012. Neuroprotection of VEGF-expression neural stem cells in neonatal cerebral palsy rats. Behav Brain Res, 230, 108-15. ZHONG, Y., LI, J., CHEN, Y., WANG, J. J., RATAN, R. & ZHANG, S. X. 2012. Activation of endoplasmic reticulum stress by hyperglycemia is essential for

126

Muller cell-derived inflammatory cytokine production in diabetes. Diabetes, 61, 492-504. ZHOU, X., LI, F., KONG, L., CHODOSH, J. & CAO, W. 2009. Anti-inflammatory effect of pigment epithelium-derived factor in DBA/2J mice. Mol Vis, 15, 438-50. ZONG, H., WARD, M. & STITT, A. W. 2011. AGEs, RAGE, and diabetic retinopathy. Curr Diab Rep, 11, 244-52. ZUBER, M. E., GESTRI, G., VICZIAN, A. S., BARSACCHI, G. & HARRIS, W. A. 2003. Specification of the vertebrate eye by a network of eye field transcription factors. Development, 130, 5155-67. ZUO, X., MORRIS, J. S., BROADDUS, R. & SHUREIQI, I. 2009. 15-LOX-1 transcription suppression through the NuRD complex in colon cancer cells. Oncogene, 28, 1496-505. VII.

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