Lamellar Mitogen-Activated Protein Kinase and Hypoxia Signaling in a Sepsis-Related Laminitis Model and a Novel Supporting Limb Laminitis Model

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University

Alison K Gardner DVM, BS, MS

Graduate Program in Comparative and Veterinary Medicine The Ohio State University 2015

Thesis Committee Members James Belknap DVM PhD, Advisor Prosper Boyaka PhD Teresa Burns DVM PhD Ramiro Toribio DVM PhD

Abstract

The term laminitis refers to dysadhesion of the lamellar basilar epithelial

cell (LBEC) from the basement membrane, resulting in distal displacement of the

third phalanx. Multiple etiologies of laminitis exist, which can be grouped into

three broad categories: equine metabolic syndrome-associated laminitis, sepsis- related laminitis (SRL) and supporting limb laminitis (SLL). This thesis centers on two different laminitis models, the first being based on sepsis-related laminitis and the second being a novel model for supporting limb laminitis.

Sepsis-related laminitis, an often fatal sequela in critical equine patients

secondary to endotoxemia and sepsis-potentiating diseases, appears to be due

to aberrant laminar cell signaling reportedly involving inflammatory and possibly

other signaling pathways. The only documented effective treatment for sepsis-

related laminitis is regional deep hypothermia (RDH, foot submerged in ice

water). Mitogen-activated protein kinases (MAPKs), activated in inflammation or

downstream of growth factor signaling, are potential therapeutic targets for many

disease processes. Our objectives were to assess MAPK signaling in laminar

tissue in a model of sepsis-related laminitis and to determine the effect of RDH

on MAPK signaling. Lamellar concentrations of MAPKs were assessed from two

groups of horses receiving a carbohydrate-overload with samples collected at different time points versus a control. Another set was taken from a carbohydrate-overload model where one front limb was treated with RDH while the other remained at ambient temperature. Lamellar concentrations and cellular localization of the MAPKs and the signaling proteins of the interconnected protein kinase B (Akt)/Phosphoinositide 3 kinase (PI3k)/mammalian target of rapamycin

(mTOR) pathway were assessed. Whereas no change in lamellar p38 MAPK was found in the CHO models, lamellar concentrations of growth factor-related signaling molecules including the phosphorylated/activated MAPK, extracellular signal-regulated kinase (ERK) 1/2 and its downstream effector ribosomal protein

S6 (RPS6) were increased (p<0.05) at the onset of laminitis, as was the MAPK stress-activated protein kinase/c-jun N terminal (SAPK/JNK) 1/2, the cellular negative-energy balance signaling protein AMP-activated protein kinase α

(AMPKα), and Akt. Hypothermia did not inhibit ERK 1/2, but did cause a decrease in RPS6 phosphorylation/activation but an increase in SAPK/JNK 1/2 phosphorylation/activation. Signaling related to growth factor-related pathways should be further investigated in SRL, especially since it has been implicated in cell-cell dysadhesion and upregulation of the ERK 1/2 pathway.

The pathophysiology of equine supporting limb laminitis (SLL), a common and often fatal complication of equine orthopedic disease, is poorly understood.

Often horses survive the initial catastrophic trauma, e.g. fracture, only to succumb to fulminant failure of the contralateral limb. Suggested causes of

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lamellar failure include inflammatory injury, hypoxia and mechanical strain. We

hypothesized that lamellar hypoxia occurs in the supporting limb (SL) resulting in

increase in lamellar hypoxia-inducible factor-1α (HIF-1α). A novel model of SLL was used in this study in which a custom shoe insert causing instability to the sole surface upon weight bearing was placed on one forelimb of horses resulting in excessive weighting of the contralateral forelimb (SL). Lamellae were harvested and immediately snap-frozen from all four limbs 48 h post-application of the shoe. Western immunoblotting and real time-quantitative PCR (qPCR) were used to assess markers of hypoxia (HIF-1α) inflammation and stretch. The only change noted was an increase (P<0.05) in lamellar HIF-1α protein concentrations in the SL compared to one hindlimb and a trending increase in the other. Genes including those indicated in stretch, metabolism and inflammation were not upregulated, nor was HIF-1α mRNA. These results indicate that lamellar hypoxia and HIF-1α may play a central role in SLL and future research should focus on therapeutic options, including novel shoeing options on the SL which may allow increased vascular oxygen delivery to the distal limb. HIF-1α may have value as a biomarker of lamellar hypoxia and be used to assess the efficacy of SLL treatments.

The two models (CHO and SLL) exhibit differences between the arms of sepsis-related and supporting limb laminitis. However, further work must be done on cellular growth factor signaling and metabolic pathways, as recent evidence shows that while SRL and SLL have differing properties in MAPK,

Akt/PI3k/mTOR, and HIF-1α activation as well as pro-inflammatory cyotokine gene expression, RPS6 may play a role in dysadhesion of the epidermal lamellae.

Dedication

To my family, who have sacrificed so much.

To my mother, who reminds me daily what strength and grace can overcome.

Acknowledgements

First and foremost, I have to thank Dr. Jim Belknap, my advisor, for his guidance both through the research process and through my residency. I have always felt I have had his support, even on the thirtieth time he had to explain calculating fold change. It is difficult training a nascent pony doctor to be well- versed in cellular-signaling, but he certainly is willing to try, always with a sense of humor. Thank you to my committee members, Drs. Prosper Boyaka, Teresa

Burns and Ramiro Toribio for being on my committee.

I am equally indebted to Mauria Watts. She has been my tutor in bench top protocols, my editor and a guide both in the lab and out. Mauria is a good friend and amazing asset, and any student who works under her is lucky to do so.

I would also like to thank Dr. Teresa Burns for her guidance and help with nearly every step in the process. Dr. Burns is always available and so very kind, whether I am asking a stupid internal medicine question or equally ignorant statistical evaluation one. Thanks also to Heather Lane and Dr. Cassie Quinlan for sample collection, and to Dr. Carlin Kelly for the research she had started on p38 MAPK. Thanks also to Dr. Andrew Van Eps and Dr. Britta Leise for

vii lamellar tissue used in the CHO and RDH models. Thank you to Tim Vojt for his work on the HIF-1α figures.

Thank you also to my family and my partner, Mark, for all their patience and fortitude. Thank you to my other clinicians, my resident mates and the wonderful technicians for their patience with me during this residency.

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Vita

Ohio State University, graduate research associate…June 2012-present

Colorado State University, DVM…………...... ….………...May 2011

Colorado State University, MS……………………..………….August 2007

Colorado State University, BS…………………………………….May 2006

Publications

A.K. Gardner, M.R. Watts, T. A. Burns, J. K. Belknap. Abstract: Examining

laminar signaling in a model of supporting limb laminitis. Journal of Equine

Veterinary Science, 2013 v33 n10: 862-863 presented at the 2013 ACVS

Symposium

B.S. Leise, R.R. Faleiros, T.A. Burns, A.K. Gardner, M.A. Watts, S.J. Black, R.

Geor, L.J. McCutcheon, A. van Eps, C.C. Pollitt, S. Eades, P.J. Johnson, and

J.K. Belknap. Inflammation in laminitis: the “itis” in laminitis may not pertain to

all.Journal of Equine Veterinary Science, 2013 v33 n10: 860

Fields of Study

Major Field: Comparative and Veterinary Medicine

Table of Contents

Abstract…………………………………………………………………………………...ii Dedication……………………………………………………………………………….vi Acknowledgements…………………………………………………………………….vii Vita……………………………………………………………………………………….ix List of Figures…………………………………………………………………………..xii

CHAPTER 1. Introduction………………………………………………………………1 1.1 Structure and Function of the Equine Lamellae………………...………1 1.2 Structural Failure in Laminitis……………………………………………..9 1.3 Equine Metabolic-Syndrome-Associated Laminitis…………………...11 1.4 Sepsis-Related Laminitis………………………………………………...12 1.5 Mitogen-Activated Protein Kinase Signaling (MAPK) and the Akt/PI3/mTOR Signaling Pathways…………………………………….16 1.6 Supporting Limb Laminitis……………………………………………….25

CHAPTER 2. MAPK and the Akt/PI3/mTOR Signaling Pathways in a Carbohydrate-Overload (CHO) Model and in Response to Regional Deep Hypothermia (RDH)……………………………………………………………………30 2.1 Materials and Methods: CHO and RDH Models……………………….30 2.2 Results: CHO and RDH Models…………………………………………34 2.3 Discussion: CHO and RDH Models……………………………………..53

CHAPTER 3.Hypoxia Signaling in a Novel Supporting Limb Laminitis (SLL) Model……………………………………………………………………………………59 3.1 Materials and Methods: SLL Model……………………………………..59 3.2 Results: SLL Model……………………………………………………….66 3.3 Discussion: SLL Model…………………………………………………...71

CHAPTER 4: Discussion and Conclusions…………………………………………75 4.1 Overview of Results from Two Laminitis Models……………………...75 4.2 MAPK Data………………………………………………………………..77 4.3 Moving Away from Pro-Inflammatory Gene Expression as Sole Causation in Sepsis-Related Laminitis……………………………………………..80

4.4 A Potential Role of the Ras/ERK 1/2 PI3/Akt/mTOR Pathway and RPS6……………………………………………………………………………………81 4.5 Hypoxia in the SLL Model………………………………………………..83 4.6 Further Directions in Hemidesmosome and Cytoskeletal Regulation……………………………………………………………….85

REFERENCES…………………………………………………………………………86

Appendix A: Western Immunoblotting and Immunofluorescence/Immunohisotchemistry Primary Antibodies………………..99

Appendix B: PCR Primers……………….………………………………………….100

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List of Figures

Figure 1: Lamellar Organization Within the Equine Digit……………………………2

Figure 2: Forces Upon Lamellae………………………………………………………4

Figure 3: Primary and Secondary Epidermal Lamellae……………………………..5

Figure 4: Hemidesmosome…………………………………………………………….7

Figure 5: MAPK Signaling Pathway………………………………………………….18

Figure 6: HIF-1α Under Normoxic Conditions………………………………………27

Figure 7: HIF-1α Under Hypoxic Conditions………………………………………..28

Figure 8: p38 MAPK Western Immunoblotting……………………………………..36

Figure 9: p38 MAPK Immunohistochemistry……………………………………….37

Figure 10: ERK 1/2 Western Immunoblotting………………………………………40

Figure 11: ERK 1/2 Immunofluorescence…………………………………………..40

Figure 12: RPS6 (235/236) Western Immunoblotting……………………………..42

Figure 13: RPS6 (240/244) Western Immunoblotting……………………………..44

Figure 14: Akt Western Immunoblotting…………………………………………….46

Figure 15: AMPKα Western Immunoblotting……………………………………….48

Figure 16: mTOR Western Immunoblotting………………………………………...50

Figure 17: SAPK/JNK 1/2 Western Immunoblotting……………………………….52

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Figure 18: Supporting Limb Laminitis Model Construct…………………………..60

Figure 19: Supporting Limb Laminitis Model Limb Designation Schematic…….61

Figure 20: Gene Expression in the SLL Model…………………………………….67

Figure 21: HIF-1α Western Immunoblotting………………………………………..69

Figure 22: HIF-1α Immunofluorescence…………………………………………….70

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Chapter 1. Introduction

1.1 Structure and Function of the Equine Lamellae

The entirety of the equid’s weight is borne upon the single digit of each limb, each of which is expected to carry even a much greater physiologic burden when the horse is moving at speed. The distal phalanx, upon which rests the whole of the appendicular force of its limb, is suspended within the hoof capsule by a series of convolutions or infoldings of the deepest layer of this modified epidermis, called lamellae. Each epidermal infolding is adhered to a partner dermal out-folding [1] (Figure 1).

There are approximately 600 larger, keratinized tubular-like infoldings descending down from coronary band to ground called primary epithelial lamellae

(PELs) [2]. The cells of the PEL are attached to each other by desmosomes, whose mediated dissolution and reformation are purported to allow for normal hoof growth in a ratchet-like motion [3]. Equine neonates have circumferential uniformity of PEL density, but division and reorientation of each lamellae occurs so that density is highest at the toe and least at the quarters and heels by adulthood [1, 4] , which may be due to the need to combat the distracting forces on the dorsal lamellae due to both the toe acting as a lever arm from the center of rotation in the coffin joint and the deep digital flexor tendon at its attachment to the palmar/plantar aspect of the distal phalanx (Figure 2).

Furthermore, PEL density may be altered in pathology, such as in a low-

heel conformation [4]. Therefore, we must consider the equine lamellae to be

metabolically active and consistently evolving, albeit at a very slow pace.

The most important aspect of combatting the weight of the animal and

ground-reaction forces in the equine dermoepidermal junction is the non-

keratinized secondary epithelial lamellae (SELs), of which there are about 100-

200 per PEL [1, 2]. These increase the surface area of epidermal and dermal contact to about two meters2 per hoof, and allows for appropriation of strain and 4 stress of the equine body weight during movement [1, 2]. These SELs, like other epidermal cell layers, are anchored to the underlying dermis via their basal cell layer to a basement membrane (Figure 3).

Hemidesmosomes span the basement membrane to firmly anchor the laminar basal epidermal cells (LBECs) to underlying corium [5, 6]. A secondary dermal lamella corresponds to each SEL, and arteriovenous anastamoses and microcapillaries supply oxygen and nutrients through diffusion across the basement membrane to each PEL via its corresponding primary dermal lamella

[3, 7].

The literal lynchpin in the construct is therefore the hemidesmosome

(Figure 4). Two types exist; type I is found in epidermis and is more rigid, while type II are found in viscera, such as in gastrointestinal epithelium. The type II hemidesmosome is a multiprotein complex composed of a core of the heterodimer integrin α6β4, which binds to laminin-332, and P1a plectin, which forms a bridge to the keratin intermediate filament network [8]. This filament network distributes forces to protect the cell from mechanical stresses [9, 10].

Type I hemidesmosomes have the above protein components and also have bullous pemphigoid antigen (BPAG or BP230) 1 isoform e and BPAG2, aka

BP180 or type XVII collagen as well as tetraspanin CD 151. BPAG1 isoform e also binds the keratin intermediate filament network for more stability [8]. Normal hemidesmosome dissolution occurs in wound healing as is necessary for cell proliferation and migration, but several carcinomas, including pancreatic, take advantage of this mechanism for tumor growth and metastasis [8-10].

An absence of integrin α6β4 is lethal in knockout mice, but blistering is milder and

manifests similarly to epidermolysis bullosa diseases in humans in BP180 or

BP230 knockout mice [8].

Figure 4: Hemidesmosome

Hemidesmosome dissolution occurs when the β4 subunit of integrin α6β4 is

phosphorylated on serine and threonine residues, which causes disruption

between β4 and plectin 1a [9, 10]. β4 protein was found in a lower quantity of

7 lamellar samples from horses subjected to carbohydrate-overload in one study compared with controls [11]. Growth factors are most notoriously implicated in this phosphorylation, including epidermal growth factor, hepatocyte growth factor and macrophage stimulating protein [12]. Phosphorylation sites causing hemidesomosome dissolution are well preserved across species, including humans, mice and horses [8]. Upon dissolution, hemidesmosomes translocate to the lamellopodia of the migrating cell or the cytoplasm rather than being concentrated at the plasma membrane at the site of cell-cell adhesion [8].

1.2 Structural Failure in Laminitis

The term laminitis corresponds to almost any disease process where there is failure of the lamellar dermoepidermal interdigitations from coronary band to toe, resulting in loss of the suspension of the horse’s bony column. This can manifest as rotation of the distal phalanx when lamellar failure is at the dorsal lamellae, causing forces of the moment arm created by the toe and bodyweight to outweigh those of the ground-force reactions, as sinking, when lamellar failure is concentric, or as a combination of the two [13, 14]. At a more cellular level, disintegration of the basement membrane and loss of attachment of LBECs to the underlying basement membrane are the two events most associated with laminitis histopathology, with some signaling arising directly from the LBEC [2,

15, 16]. This may result in loss of hemidesmosomes. A decrease in hemidesmosomes with an increased distance between the lamina densa and plasmolemma of the underlying cell was visualized in a laminitis model [5, 6].

This separation purportedly leads to loss of dermoepidermal adhesion first at the

SELs and the PEL tip, resulting in lengthening of the epidermal lamellae without obvious necrosis, seen both in histological preparations [2] and on magnetic resonance imaging [17]. In a hyperinsulinemic-euglycemic model mimicking equine metabolic syndrome-associated laminitis, elongation of SEL and PEL axis occurred as soon as 6 hours after induction of laminitis [18]. Severity of these histopathologic signs is closely correlated to clinical progression of disease [15].

Clinical laminitis has been categorized into grades first described by Obel in

1948. Grade 1 is a horse that “shifts” his feet to distribute weight fairly constantly 9 and has an appreciably shortened bilateral forelimb cranial phase of stride at the trot but not the walk. Grade 2 is a horse with appreciable lameness at a walk but will still permit a forelimb being lifted. Grade 3 is a horse that reluctantly bears weight on one forelimb when the contralateral forelimb is lifted. Finally, grade 4 is a horse that refuses to move and adopts a “sawhorse” stance, often rocking back to redistribute as much weight as possible to the hind limbs [19, 20].

Local pro-inflammatory cytokines as well as increased systemic activation

and immigration of leukocytes, particularly neutrophils into lamellar

microvasculature and dermis prior to basement membrane separation [21-24]

has been shown in certain laminitis models, but inflammation is not seen in the

acute stages of all types of laminitis and therefore the “itis” may be a misnomer

[25, 26]. Indeed, it is important to touch on the term “laminitis” as being an umbrella to three histologically similar but etiologically wholly different disease processes: equine metabolic syndrome-associated laminitis and the focus of this thesis; sepsis-related laminitis and supporting limb laminitis.

1.3 Equine Metabolic Syndrome-Associated Laminitis

Endocrinopathies are implicated in laminitis, and are mentioned here for sake of completeness, as they represent the most frequent etiology of laminitis

[27]. Laminitis caused by equine metabolic syndrome affects all four hooves, such as in sepsis-related laminitis but not supporting limb laminitis. It most often has a more chronic progression than supporting limb laminitis, and management involves frequent but often long-term care by the owner and clinician. Obesity and insulin-resistance have been implicated in endocrinopathic-associated laminitis, but pro-inflammatory cytokines are not upregulated in the lamellar tissue of obese ponies fed a high non-structural carbohydrate diet [26].

Hyperinsulenemic models of equine metabolic syndrome-associated laminitis have had conflicting results as to whether pro-inflammatory mediators have increased protein concentrations in affected horses [28]. A hyperinsulinemic- euglycemic clamp model was noted to serendipitously incite laminitis within 72 hours in ponies and 48 hours in horses as part of an ancillary study, therefore paving the way for more elucidation of this poorly understood but often researched and encountered disease [27, 29, 30]. Interestingly, in these samples, leukocyte emigration from vasculature into dermis preceded basement membrane dissolution, but fewer than in carbohydrate-overload models [18, 25].

1.4 Sepsis-Related Laminitis

Sepsis-related laminitis can affect all four feet, like in equine metabolic

syndrome-associated laminitis, but is more fulminant and quickly manifests, often while the patient is still in the hospital [31]. Signs usually manifest in the forefeet

more rapidly and severely, but this is thought to be a function of the horse’s

increased weight bearing on the front rather than any difference in systemic

response [32]. Comorbidities for acute laminitis in a hospital population include

septic precursors such as pleuropneumonia, acute diarrhea, surgical colic,

duodenitis and proximal jejunitis, as well as other gastrointestinal disease [31,

33]. Signs of endotoxemia and manifestation of the systemic inflammatory

response syndrome (SIRS) (fever, neutropenia or neutrophilia, tachycardia and

congested mucous membranes) precede laminitis events in both clinically septic

patients and in sepsis-related laminitis models.

Several equine models have been developed to address sepsis-related laminitis, including the cornstarch and wood flour carbohydrate-overload (CHO) and the oligofructose-overload (OF) model. Horses in these two models develop fever, diarrhea and tachypnea prior to lameness [34, 35]. An overgrowth of both gram-positive and gram-negative bacteria in the cecum is seen. Endotoxemia, as well as other signs of sepsis, is preceded by a drop in cecal pH [36, 37]. The cecal acidosis is thought to cause both die-off of acid-labile bacteria, causing lipopolysaccharide (LPS) and other pathogen-associated molecular pattern

(PAMP) release, as well as a decrease in the transepithelial resistance of the viscera, allowing for bacterial translocation [37, 38]. The CHO model follows 12 clinical cases in that treated horses gradually progress to Obel grade 1-2 lameness over 24-36 hours, but between 20-30% of horse do not respond to

CHO administration [32]. The OF model is the most reliable in that 90-100% of subjects respond in a predictable and clinically analogous timeline [35].

Sepsis/endotoxemia in the human and veterinary literature has been proposed to be a product of an uncontrollable SIRS resulting in multiple organ dysfunction syndrome (MODS) and eventual death [39-42]. The definition of

SIRS has been modified in horses to include at least two of the following: fever

(temperature >38.5°C), tachycardia (>50 beats per minute), tachypnea

(respiratory rate> 25 breaths per minute), leukocytosis (>14.5 x 109 cells/L) or

leukopenia (<5 x 109 cells/L) [33]. It has been shown that pro-inflammatory

cytokine gene upregulation such as interleukin (IL) -1β, IL-6, and cyclooxygenase

(COX)-2 precedes laminitis in sepsis-related models both systemically and

localized to lamellar tissue [16, 32, 38, 43, 44]. However, a causative effect has

not been proven [43, 45-48]. Indeed, intravenous lipopolysaccharide

administration has not been shown to cause laminitis even with other systemic

signs of transient sepsis such as elevated heart rate and temperature [45],

suggesting that some other etiologic factor is necessary for development. This

mirrors current concerns regarding inflammation in SIRS as the sole causative

event of disease, as anti-inflammatory therapies have proven to be ineffective in

mitigating sepsis in intensive care units, and no reliable link has been proposed

between SIRS and eventual MODS [49, 50].

Therapeutic hypothermia has been used systemically in human intensive care 13 units, usually in treatment of an injury to reduce post-resuscitation disease, reperfusion injury and secondary brain injury. Hypothermia has recently regained popularity after more than 30 years of quiescence after it was found that mild to moderate hypothermia (31-35°C) still produced beneficial effects without severe side effects associated with deep hypothermia (<30°C) [51, 52]. The mechanism of hypothermia as a proposed inflammatory moderator through a decrease in expression of tumor necrosis factor (TNF)-α, IL-6, and concentration of upstream effectors such as the mitogen-activated protein kinases (p38 MAPK, extracellular-regulated kinase (ERK) 1/2, and stress-activated protein/c-jun-N terminal kinase (SAPK/JNK)) [53] in cell culture as well as in vivo systemically- treated mouse models [54, 55] has been postulated. Hypothermia also reduces size of ischemic lesion due to decrease in apoptosis as well as providing a decrease in metabolic activity and therefore oxygen demand on affected tissues in models of hippocampal ischemia [51, 52].

Regional deep hypothermia (RDH) of 5-10°C has been used as both a prophylactic therapy and treatment for clinical cases of laminitis, cited as anecdotally therapeutic in 1963 [56], but has only recently been proven to have a positive effect. Regional deep hypothermia has been proposed to work in three interconnected spheres: as an analgesic by increasing nociceptive threshold and decreasing neuronal transduction speed, by effectively squashing inflammatory mediator release, and finally by slowing metabolism, therefore pushing cells into a protective senescent state [51, 57, 58]. In a multi-center retrospective study by

Kullman et.al. in 2014, at-risk horses (colitis, Potomac horse fever, 14 pleuropneumonia etc.) were ten times less likely to develop laminitis when they were immersed in ice-water up over the fetlock than those untreated [33]. Ice-

water submersion showed a decrease in pro-inflammatory cytokines such as

COX-2 and IL-6 in horses subjected to OF overload [57, 58].

Ice-water submersion is performed with the foot constantly submerged in a

bag or specialized boot with the level of the ice-water above the fetlock [33, 57].

In the clinical study by Kullman et.al., feet were submerged for at least 48 hours

[33]. Horses in this study were ten times less likely to develop laminitis if regional

limb hypothermia was applied, and no detrimental effects were reported.

Subsequent carbohydrate-overload studies showed that regional deep

hypothermia was extremely effective at maintaining the dermoepidermal lamellar

junction and decreased the expression of matrix metalloproteinases and pro-

inflammatory cytokines previously identified in lamellar tissue from carbohydrate-

overload models [57-59]. Therefore, it was our goal to assess upstream effectors

of the pro-inflammatory cytokines mitigated by regional deep hypothermia, that

is, the cytokines IL-6, IL-1β, IL-8; the chemokines CXCL1 and 6, and endothelian

cell adhesion molecules ICAM-1 and E-selectin.

1.5 Mitogen-Activated Protein Kinase Signaling (MAPK) and the

Akt/PI3K/mTOR Signaling Pathways

Our goal in the mitogen-activated protein kinase study was to assess these

upstream effectors of pro-inflammatory cytokines and regulators of growth,

mitosis, cell survival, apoptosis and proliferation [60-62]. MAPKs are

serine/threonine kinases well preserved across eukaryotic species [61]. They

are varied in function, but each responds to extracellular signal to cause

intracellular signal transduction. While there are 14 different MAPKs, the family

has three main and extensively studied members, p38 MAPK, ERK 1/2 and

SAPK/JNK 1/2/3. These MAPKs are activated by a myriad of factors, including

growth factors, heat shock-proteins, pro-inflammatory cytokines, PAMPs,

damage-associated molecular proteins such as DNA (DAMPs) and

environmental stressors [61, 63].

Each classical MAPK signaling pathway follows the same formula, where

extracellular signaling through different receptors result in phosphorylation and

activation of a member usually of the Ras/Rho family, which in turn

phosphorylates a MAPK kinase kinases (MAPKKK) to act upon MAPK kinases

(MAPKK) prior to the specific MAPK in question [60]. Some MAPKKKs are

shared by all three pathways, such as tumor progression locus (Tpl) 2, making

upstream effector evaluation difficult [62]. For example, Tpl2 is necessary for

ERK 1/2 activation following LPS-stimulated macrophages but not for JNK activation [64]. The MAPKs then mostly translocate to the nucleus while also exerting control on cytosolic cell processes to act on transcription factors for 16 further gene expression to activate cell motility, cell differentiation, apoptosis, metabolism or inflammation depending on the signaling pathway activated [61,

65]. Ancillary scaffolding proteins, such as docking sites on MAPK effector proteins, provide organization and specificity to the signaling cascades [61, 62].

These signaling pathways confer specificity to which MAPK is activated by which commonly shared activator, but their behavior is poorly understood at this time

[62]. However, therapies aimed directly at the MAPK docking site rather than

MAPK total inhibition show promise. For example, mice with inflammation- associated hyperalgesia in a high-dose carageenan study were much more comfortable after the p38 MAPK docking groove inhibitor FGA-19 was injected intrathecally [66].

p38 MAPK

p38 MAPK was the first and is still the most routinely implicated MAPK as a

cause of cellular inflammation and fibrosis [66, 67], and is activated mostly by

stressors including ultraviolet radiation, inflammatory mediators, especially TNF-

α, and ischemia [61, 65]. p38 MAPK is divided into four separate isomers,

α,β,γ,and δ but most research has been performed in the ubiquitously expressed

α variant [65]. IL-1β and TNF- α , specifically, recruit TNF receptor associated factor (TRAF) to intracellularly activate p38 MAPKKKs, but the p38 pathway may

18 also be activated by Rho family guanosine triphosphatases (GTPases) and G- protein coupled receptors [61]. p38 MAPKKKs (e.g. mitogen-activated ERK

kinase-kinases (MEKK1-3), mixed-lineage kinase (MLK 2/3), apoptosis-

regulating signal kinase (ASK1), Tpl2, transforming growth factor-β activated

kinase (TAK1), and thousand and one (TAO) 1/2) have some overlap with the

other MAPK pathways in that the upstream effectors may activate ERK 1/2 or

SAPK/JNK as well as p38 MAPK [61]. These then act upon the p38 MAPKKs

MKK 3/4, which, in turn, activate p38 MAPK. Inactivated p38 is present in both

cytoplasm and nucleus, but nuclear translocation occurs upon activation and

enhanced transcriptional factor activity of p53, ETS domain containing protein

(Elk-1) and others is subsequent to p38 activation. Cytosolic activation of

calcium dependent phospholipase (cPLA2), MAPK-interacting kinases (MNKs)

and Bax also occurs secondarily to p38 MAPK activation [61]. The MNKs are

implicated in the translation of pro-inflammatory cytokines secondary to LPS-

mediated p38 MAPK activation [61].

In mouse models of sepsis, p38 MAPK activation has been shown to be

necessary for the pro-inflammatory cytokine transcription factor nuclear factor

kappa light chain enhancer of activated B cells (NF-κB) activation and contributes

to lethality [68]. It specifically causes increased gene expression of TNF-α, IL-1,

IL-6 and IL-8 pro-inflammatory cytokines [66]. p38 MAPK has been reported to be necessary for activation of circulating neutrophils and pro-inflammatory cytokines (particularly TNF-α, IL-1 and IL-6) in in vitro equine cultured cells [69] but has not been investigated in lamellae. Therefore, our first goal was to 19 evaluate p38 MAPK signaling in a carbohydrate-overload model of laminitis, and secondly, if p38 MAPK expression was changed in a regional deep hypothermia model.

ERK 1/2

ERK 1 was the first identified MAPK and shares an 83% homology with

ERK2, and both are coupled together here due to their similar molecular weight and activation of effector pathways [61, 62]. In addition to being activated by inflammatory and oxidative stress in the same manner as p38 activation through receptor tyrosine kinases, ERK 1/2 is also activated by growth factors platelet- derived growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), insulin and potentially insulin-like growth factor (IGF) [61, 62, 70].

Tyrosine kinase cell membrane receptor activation by extracellular growth factor signaling resulting in son-of-sevenless (SOS) guanine nucleotide exchange mediated Ras activation, is the most classic activator of the ERK 1/2 pathway [62]. Ras then goes on to act upon many proteins, including Raf, for cell motility and proliferation. The Raf pathway, A-Raf, B-Raf and Raf-1, comprises the most ERK 1/2-specific MAPKKK pathway described [61, 62]. MEKK1, Mos, and Tpl2/Cot may also act as ERK 1/2 MAPKKK in specific circumstances [61].

The Raf pathway, once activated, phosphorylates MEK1 and MEK2, which go on to phosphorylate ERK 1 and ERK 2. Once activated, ERK 1/2 then localizes to the nucleus to cause transcription of factors necessary for cell dysadhesion, growth and proliferation, or remains in the cytoskeleton to activate cytosolic 20 proteins, including membrane proteins CD120a, Syk and calnexin, cytoskeletal neurofillaments and paxillin, and tuberous sclerosis complex 2 (TSC2), ribosomal

S6 kinase (RSK), and MNK [61, 62, 71]. ERK 1/2 activation is implicated in psoriasis, a keratinocyte disease in humans and animals not without parallels to laminitis in that keratinocyte dysadhesion at the basement membrane results in pathology [71]. ERK 1/2 activates and phosphorylates p90 ribosomal-S6 kinase

(P90RSK) which then activates the (ribosomal protein S6) RPS6 pathway at the

phosphorylation site serine 235/236 only [72, 73]. RPS6 is also activated by the

mammalian target of rapamycin (mTOR)/phosphatidylinositol 3-kinase

(PI3K)/protein kinase B (PKB or Akt) pathway, both at the serine 240/244 site through p70 RSK activation [60, 71, 73, 74] and at the serine 235/236 site, but to a lesser degree than p90RSK [73]. Therefore, in addition to exploring ERK 1/2 expression in the same fashion as p38 MAPK, a tertiary experiment was performed to evaluate whether ERK 1/2 also activated RPS6 for downstream transcription.

RPS6

RPS6 was first identified as a 40S ribosomal subunit protein correlated with epithelial neoplasms [74]. It is activated/phosphorylated by several ribosomal S6 kinases (RSKs) downstream of the P90RSK/ERK 1/2 pathway (a member of the MAPK-activated protein kinase family [73]) or the P70RSK mTOR/PI3/Akt pathway [74]. RPS6 influences the majority of the ribosomal biosynthesis including transcription factor ternary complex factor (TCF) for 21 immediate early gene (IEG) transcriptions as well as late-response genes to promote cell division, survival and motility [74]. Specifically to our research, along with phosphorylating and activating RPS6, p90RSK 1/2 causes phosphorylation of the integrin at β4 to disrupt interaction of the anchoring filaments to plectin 1a [9, 10]. This has been suggested to also be phosphoinositide dependent kinase 1 (PDK1) mediated, another downstream effector of ERK 1/2 [10]. There has been suggestion that phosphorylation is ERK

1/2 dependent, as phosphorylation of the β4 complex was inhibited when the

ERK 1/2 inhibitor P2YR G-coupled protein receptor was used on cultured cells

[75]. β4 protein was found in a lower quantity of lamellar samples from horses subjected to carbohydrate-overload in one study compared with controls [11].

ERK 1/2 was found to drive lamellipodia protrusion to commence cell migration

through RPS6 by activating the WAVE2 regulatory complex [60]. In an

endocrinopathic laminitis model, phospho-RPS6 concentrations were increased

in lamellar samples in obese ponies given a high non-structural carbohydrate diet

(Belknap laboratory, unpublished research). Due to this previous research and

previous research performed on the relationship between ERK 1/2 and

hemidesmosomes, our main hypothesis was that phospho-RPS6 concentrations

would be increased in horses subjected to a carbohydrate overload and

exhibiting laminitis.

mTOR

While the activation of RPS6 through p90RSK and ERK 1/2 has been a fairly 22 recent development [72, 73], the largest amount of research has been in regards to its activation through the mammalian target of rapamycin (mTOR) pathway

[71, 74] through phosphorylation of RPS6 by the RSK P70. mTOR is a master- cell regulator, activating many anabolic pathways while decreasing catabolic ones in an extracellular energy positive state [74]. It is activated by growth factors, energy status and cellular stress [74]. Protein kinase B/phosphoinositide

3-kinase (Akt/PI3K) activates mTOR through growth factor signaling such as epidermal growth factor (EGF), insulin and IGF signaling. [74, 76, 77]. Akt/PI3 itself can be activated by mTOR, integrin-linked kinase 1, DNA-dependent protein kinase and PDK1 [77]. However, multiple levels of cross-talk exist between Akt/PI3K and mTOR, where one can either activate or inhibit the other depending on the context [77]. Even more confoundingly, mTOR can also be activated through the downstream actions of ERK 1/2 [60]. Activated P70RSK and mTOR concentrations were found to be positively correlated to insulin concentrations in ponies subjected to a high non-structural carbohydrate diet.

Therefore, we also evaluated concentrations of these phospho-proteins in our study.

SAPK/JNK 1/2

Stress signaling similar to ERK 1/2 and most p38 MAPKKKs stimulate the stress-activated protein kinase/c-jun n-terminal kinase (SAPK/JNK), which has three members [63]. It is thought that 1 and 2 share most cell tropisms and perform similar functions, but JNK 3 has so far been localized to only neural and 23 testicular tissue and cardiac myocytes [61, 78]. MKK4/SEK1 and MKK7 cooperate to activate SAPK/JNK, which, like the ERK 1/2 and p38 MAPK, translocate to the nucleus for upregulation of transcription factors, such as phosphorylation of c-Jun at ser63/73, while also activating cytosolic signaling pathways for cell proliferation [61, 62].

In regards to current laminitis literature, JNK activates the transcription factor signal transducer and activator of transcription 3 (STAT3) [61], which has been shown to be up regulated in laminar tissue from a carbohydrate overload laminitis model [79]. Also interesting is that an IL-1β-dependent increase in SAPK/JNK concentration has been linked to pancreatic carcinoma migration [80]. Several studies on pro-inflammatory cytokine expression have reported increases in ERK

1/2 and JNK 1/2 without a commensurate increase in p38 MAPK [61, 63, 81]. In a recent study on the pro-inflammatory cytokine TNF-α upregulation, Fas (CD95),

Fas initiated signaling through ERK 1/2 and JNK to activate NFκB for increased in vitro IL-8 gene expression [82]. Therefore, we assessed lamellar proteins

related to this MAPK as well.

1.6 Supporting Limb Laminitis

Supporting limb laminitis (SLL) is a very different process than laminitis caused by sepsis or endocrinopathies, both due to the fact that only one limb is affected and the apparent lack of need for a systemic disease process to initiate the local lamellar disease process. In SLL, one limb undergoes prolonged excessive weight bearing due to, for example, focal sepsis or trauma in the contralateral limb. A challenge both to the clinician and researcher is the incredibly variable length of time between the onset of severe lameness in one limb and the development of SLL in the opposite limb. Support limb laminitis had a mean developmental time of ~37 days in one study [83], but a range of 17 to

134 days was found in that study. This prolonged developmental time may be artificially increased by the fact that lameness of the laminitic supporting limb must supersede lameness of the unloaded limb to be clinically apparent. In my clinical experience, I have had patients completely lose dermoepidermal adhesion within 72 hours of non-weight bearing due to injury, meaning cellular

signaling events leading to dysadhesion must have commenced nearly

immediately. Rapid catastrophic lamellar failure is a common entity once clinical

signs are present in spite of a prolonged developmental period. A recent study

stated that euthanasia was necessary for humane reasons in 50% of cases that

developed SLL when the primary injury required casting [84] but other studies

have higher rates of mortality. In a study of SLL secondary to contralateral limb

cellulitis, 75% of horses necessitated euthanasia after development [85].

The etiology of supporting limb laminitis has been proposed to be either 25 vascular disturbances or tissue ischemia arising from supra-physiologic and sustained weight bearing on the affected limb causing occlusion of arterioles and arteriovenous anastamoses [86, 87], mechanical stretch signaling leading to dysadhesion of cell-to-cell junctions as reported in other dermoepidermal dysadhesion pathologies [88, 89] or inflammatory signaling due to ischemia/reperfusion injury [87, 89]. Causation of lamellar injury by circulating pro-inflammatory cytokines is less likely due to the fact only one limb is affected,

but concurrent systemic inflammation either due to a sterile SIRS or

endocrinopathy may contribute to lamellar failure in a dermoepidermal support

system already weakened by overweighting and increased shear forces [87, 90].

A secondary goal in this thesis was to develop a SLL model and evaluate hypoxia through hypoxia-inducible factor 1α (HIF-1α), stretch-signaling through mitogen-activated protein kinases unregulated in response to mechanical stretch as well as inflammation and other factors and pro-inflammatory cytokines previously evaluated in a sepsis-related laminitis model.

In normoxia, the hypoxic mediator hypoxia inducible factor (HIF-1α) is hydroxylated and marked for ubiquitination and lysosomal proteosomal degradation by prolylyl hydroxylases, which are activated by oxygen (PHDs) and von Hippau-Landeau factor (vHL), respectively (Figure 6) [91].

However, upon a hypoxic environment of less than 5% oxygen, down- regulation of PHDs occurs with subsequent concentration-dependent nuclear translocation of HIF-1α. This causes activation of transcriptional factors, most notably the hypoxic response element (HRE) and transcription and translation of angiogenic and metabolic regulatory proteins such as vascular-endothelian

27 growth factor (VEGF), glucose-transporter (Glut)1 and PGK1 [92-94] (Figure 7).

A previous study of HIF-1α in horses subjected to carbohydrate overload exhibited hypoxia in lamellar tissue relative to other organs (liver, small intestines and skin) [95],which means lamellae over any other organ may be most susceptible to changes in extracellular oxygen concentration.

We hypothesized SLL may be manifested by a primarily hypoxic event, and

28 therefore a more increased HIF-1α concentration would be found in lamellae found in feet subjected to increased weight-bearing over control feet.

Chapter 2. MAPK and Growth Factor Signaling in a Carbohydrate-Overload

Model and in Response to Regional Deep Hypothermia

2.1 Materials and Methods: CHO and RDH Models

Carbohydrate Overload Sample Collection

Archived lamellar samples were taken from two previous studies, one in which horses underwent the carbohydrate overload (CHO) laminitis model using nasogastric administion of a bolus of a corn starch/wood flour gruel [32, 34] and one using nasogastric administration of a bolus of oligofructose (OF) overload with one hoof subjected to regional deep hypothermia and the other maintained at ambient temperature [58, 59].

All animal protocols were approved by the University of Queensland Animal

Ethics Committee, the Institutional Animal Care and Use Committees of the

University of Missouri, and the Ohio State University. Briefly, as previously

described [32], lamellae harvested from the CHO model from 24 clinically normal horses, where laminitis was induced in 16 animals by cornstarch and wood flour

overload (85% starch, 15% wood flour, 17.6g/kg bwt.). Six horses that received

the CHO bolus were euthanized after development of a fever (>38.9oC) between

12-22 hours post CHO (DEV group) while another six that received the CHO

bolus were euthanized at the onset of OG1 lameness (occurring between 20 and 30

48 hours) (OG1 group). Two horses were removed from each group after they did not respond to CHO-overload with a fever or lameness, and therefore were labeled non-responders. Six control (CON) animals received a sham treatment of 6L water via nasogastric tube, and were euthanized at 24 hours post water administration. Limbs were removed by disarticulation of the metocarpophalangeal joint and 1.5 cm thick sagittal sections were cut and lamina rapidly dissected then immediately snap frozen in liquid nitrogen or preserved and frozen in OCT media. Samples were stored at -80oC until use or fixed in

10% phosphate buffered formalin and paraffin embedded for immunohistochemistry. In the regional deep hypothermia experiment, the horses received an overdose of OF (10 g/kg bwt). Immediately after the OF administration, one randomly assigned limb (ICE) was immersed in 50% ice and

50% water and continuously cooled to 5°C up to the carpus for the duration of the

experiment. The opposite limb (AMB) was maintained at ambient temperature.

As such, each horse acted as its own control. Tissue from the hypothermia

experiment was harvested at the onset of OG1 lameness in the same fashion as

the CHO study.

Protein Extraction and Western Immunoblotting

Protein was extracted from snap frozen lamellae that were first pulverized

on dry ice, then homogenized in M-Per lysis buffer, with the addition of 4M NaCl,

protease and phosphatase cocktail inhibitors (Halt), and PMSF. After 30 minutes of incubation on ice, the supernatant was centrifuged and collected. Protein

31 concentration was determined using Bradford reagent on a spectrophotometer.

Samples were aliquotted and stored at −80oC until Western Blot hybridization.

Lamellar protein from all three laminitis models was used to assess proteins at both the phosphorylated (indicating activation) and total protein (phosphorylated and un-phosphorylated protein) vs. β-Actin. Proteins assessed were the mitogen- activated protein kinases p38 MAPK, ERK 1/2 and SAPK/JNK; and RPS6, mTOR, Akt and AMPK. As previously described [95], 30 μg protein from individual animals was loaded on a SDS-PAGE gel, separated by electrophoresis the transferred onto a polyvinyldifluoridine (PVDF) membrane, blocked in 5% milk in Tris-buffered saline and Tween 20 (TBST) 1 hour at room temperature, then hybridized at 4oC overnight first in the phosphorylated antibody, washed in

TBST, incubated with the appropriate horseradish peroxidase (HRP) linked

secondary antibody and the chemilumenescent signal captured on Kodak

Biomax light film. After phospho detection, the membranes were stripped for 15

minutes in Restore, a commercially available buffer and subsequently probed in

the same manner for the total protein and finally to β-Actin for protein

normalization. Band intensities were calculated with Image-J

(http://rsb.info.nih.gov/ij) and relative intensity was determined against β-Actin.

Immunofluorescence

To visualize cellular localization of phosphorylated and total Erk 1/2 and

RPS6 in lamellar tissues, 10 μm frozen sections were cut from archived lamellar

tissue preserved in OCT and affixed to slides, the sections were fixed for 15 32 minutes in 4% formaldehyde, washed in phosphate buffered saline (PBS) then blocked for 1 hour at room temperature in PBS containing 5% normal goat serum and 0.3% Triton X-100. Sections were hybridized at 4oC overnight in a 1:100 dilution of primary antibody in PBS containing 1% bovine serum albumin and

0.3% Triton X (separate slides for phosphorylated and total). After three washes, slides were incubated with 1:200 dilution of an Alexafluor fluorochrome conjugated secondary antibody at room temperature for 1 hour 30 minutes. The slides were then washed, air dried and cover slipped with a mounting media containing DAPI, a nuclear counterstain, cured overnight then imaged on a Leica

DM IRE laser assisted confocal microscope equipped with digital imaging software.

Immunohistochemistry

Slides were prepared as previously described [32] from paraffin embedded lamellae from both experiments, the sections were deparaffinized, rehydrated, simmered for 15 minutes in EDTA for antigen retrieval, blocked 1 hour at room temperature in a PBS solution containing 2.5% BSA, 2% normal goat serum, 0.1% triton X-100, 0.05% tween-20. The samples were hybridized overnight at 4oC to phospho-p38MAPK, then incubated with a biotinylated

secondary antibody and a peroxidase substrate to visualize immunolocalization.

The slides were counterstained with Harris hematoxylin and coverslipped. Digital

images were captured at 40 x with the Aperio digital scanning system.

Data Analysis

Protein sample concentration was assessed using Image J software.

Relative density of each band was calculated using the area under the curve method. Phosphorylated/activated and total protein concentrations were compared against β-Actin housekeeping protein prior to statistical analysis.

Normality was assessed using the Kolmogorov-Smirnov test. Non-normal data populations were transformed in multiple ways, including squaring the data and performing a log function. If samples were normally distributed or could be transformed, a one-way ANOVA was performed with a Tukey’s post hoc test. If samples could not be normalized, a Kruskal-Wallis test was used to compare each group (CON, DEV and OG1) with a Dunn’s multiple comparison test applied post hoc. A paired t-test was used to compare ICE versus AMB in the OF regional-deep hypothermia group after assuring normality using a Kolmogorov-

Smirnov test. An α-error of 5% (p<0.05) was designated as being statistically

significant.

2.2 Results p38 MAPK

No difference was present in lamellar protein concentrations of either phospho-p38 MAPK nor total p38 MAPK between control (CON), developmental

(DEV) or animals having developed Obel grade 1 laminitis (OG1) in the traditional starch gruel CHO model (Figure 8a). Likewise, there was no difference in lamellar protein concentrations between limbs subjected to regional deep hypothermia (ICE) or those kept at ambient temperature (AMB) in the OF

CHO model (Figure 8b).

Immunohistochemistry of lamellar samples in both the CHO and OF

models for phospho-p38 MAPK was relegated mostly to the epidermal lamellae

(brown stain), with some stain uptake in lamellar vasculature in the lamellar dermis (Figure 9).

ERK 1/2

A log function was performed of the ERK CHO data for normalization, followed by an ANOVA and Tukey’s post hoc for statistical analysis. Lamellar phospho-ERK 1/2 concentrations were increased (p=0.0005) in OG1 samples

compared to CON samples and in DEV samples compared to OG1 samples (Fig.

10a).

No difference in lamellar concentration of phospho-ERK 1/2 (red stain) was present between the cooled or ambient limb samples in the regional deep hypothermia OF model (Fig. 10b).

Upon immunofluorescence of lamellar samples in both the CHO and OF models for phospho-ERK 1/2, signal was localized primarily to the epidermal lamellae in all samples (Fig. 11). Qualitative analysis of the immunofluorescence images indicates a greater cytoplasmic concentration in the lamellar basal epithelium in OG1 compared to CON samples (Fig. 11).

As images were taken with confocal microscopy and therefore point illumination allows for more optimal depth resolution of 0.5 μm slices (in contrast to standard microscopy where all signal within the section is summated in the image), nuclear signal was also qualitatively assessed as all signal present within the nuclear membrane is likely to truly be nuclear (e.g. within that “slice” of the nucleus) and not from any signal in the cytoplasm above or below the nucleus as can occur with standard microscopy. Nuclear signal for phospho-ERK 1/2 appeared consistently increased from the CON samples to the OG1 samples in the lamellar samples from the starch gruel model (Fig. 4aii & bii, insets, CHO

Model); quantitative assessment of nuclear vs. cytoplasmic ERK 1/2 concentration was not performed. Although nuclear signal for phospho-ERK 1/2 appeared qualitatively less in iced limbs (vs. ambient, Fig. 11cii & dii, insets,

RDH) in some horses in the RDH protocol, this was not consistent.

RPS6 serine 235/236 phosphorylation

A log function of the RPS6 serine 235/236 moeity was performed to ensure normality. Lamellar phospho-RPS6 (235/236) concentrations were not only increased in OG1 samples compared to control samples (p=0.0006) but also increased significantly in OG1 samples compared to the developmental time point in the carbohydrate-overload experiment (Figure 12a).

While lamellar samples kept at ambient temperatures in the regional digital hypothermia study were increased over those iced, this was not significant

(p=0.06) (Figure 12b).

RPS6 serine 240/244 phosphorylation

Normality existed in the lamellar phospho-RPS6 (240/244) concentrations,

so no transformation was needed. These samples were also increased in OG1

samples compared to control samples (p=0.05) in the carbohydrate-overload

experiment (Figure 13a). A similar trend was seen to RPS6 235/236 in that

RPS6 240/244 was also increased in limbs kept at ambient temperatures compared to those iced in the RDH study, but this was significant (p=0.05).

Akt

A measurable increase of phospho-Akt was seen in the OG1 samples compared to the control samples in the first study (p=0.05) after data was transformed by squaring activated Akt/β-Actin ratios (Figure 14a). There was no change in activated Akt between ambient and iced limbs in the RDH study

(Figure 14b).

AMPKα

Similar to Akt, an increase was seen in the OG1 samples compared to the

control samples in the carbohydrate overload study (p=0.002) (Figure 15a). The

data could not be normalized even after several transformations, so a Kruskal-

Wallis test was performed. There was no difference between phospho-AMPKα

or total AMPKα in ambient limbs versus iced limbs in the RDH study (Figure

15b).

48 mTOR

There was no change exhibited between phospho-activated mTOR nor total mTOR in either the first carbohydrate overload study between the control and different time point samples, nor the ambient and iced limbs in the regional deep hypothermia study (Figure 16).

SAPK/JNK 1/2

Lamellar phospho-SAPK/JNK 1/2 concentrations were increased (p=0.005) in OG1 samples compared to CON samples (Figure 17a). Interestingly, RDH also induced increased lamellar SAPK/JNK 1/2 concentrations, with lamellar phospho-SAPK/JNK 1/2 higher in cooled (ICE) limbs than in ambient (AMB) limbs (p=0.03). Immunofluorescence for SAPK/JNK was uninterpretable due to poor fidelity of antibodies on our equine lamellar samples (Figure 17b).

2.3 Discussion

The induction of a broad array of pro-inflammatory signaling in the lamellae

[16, 32, 38, 43, 44] and a subsequent dysadhesion of the lamellar basal epithelial cell layer from the underlying lamellar dermis and basement membrane [5, 25] are two events well-documented to occur in sepsis-related laminitis. Thus, in order to study this disease, investigators must take into account the scientific literature concerning both the cellular inflammatory signaling documented to occur in target (visceral) organs in models of human sepsis, and epithelial diseases which result in dysregulation of adhesion properties to the underlying matrix. MAPKs are integral in signaling pathways downstream of multiple types of cell-surface receptors responsive to a broad array of stimuli ranging from growth factors to inflammatory molecules [61, 63, 81, 96]. The MAPKs are therefore likely candidates to play roles in the pathophysiology of laminitis both due to their demonstrated roles in sepsis and in epithelial diseases with cellular signaling related to that occurring in laminitis including dysregulation of adhesion and inflammatory signaling (including epithelial cancers with dysregulation of growth factor signaling or psoriasis) [8, 76, 97]. In the current study, we not only quantified lamellar concentrations of the active (phosphorylated) forms of the

MAPKs with immunoblotting, but also attempted immunolocalization techniques on lamellar sections both to establish the cell types positive for the activated forms of the MAPK proteins of interest and to assess nuclear versus cytoplasmic localization of the phospho-proteins.

Phospho-p38 MAPK was determined to be present in multiple lamellar cell types 53 including the lamellar epithelium. However, the lack of a difference in lamellar concentrations of phospho-p38 MAPK between groups in the CHO model of SRL and no difference in lamellar concentrations between the iced limb versus the limb at ambient temperature in the RDH study indicate that p38 MAPK activation may not play a central role in SRL. Additionally, there was no histologic evidence of differences in nuclear versus cytoplasmic signal in either the CHO

SRL model or the RDH study, again indicating a lack of increased p38 MAPK activation, as p38 MAPK must translocate to the nucleus to exert most of its inflammatory effects [63]. Thus, whereas p38 MAPK has been a signaling molecule of interest in human and equine sepsis due to its reported activation in models of sepsis and other pro-inflammatory diseases, we did not find evidence of p38 MAPK activation in affected lamellae in the CHO model of sepsis-related laminitis or that its regulation plays a role in the protective effect of RDH.

In contrast to p38 MAPK, we found a significant increase in lamellar concentrations of phospho-ERK 1/2 by the onset of OG1 laminitis in the CHO model. These data, combined with immunofluorescence results primarily localizing ERK 1/2 to the LBEC and epidermal lamellae, indicate a possible role of ERK 1/2 signaling in sepsis-related laminitis. Although the ERK pathway is mainly associated with growth factor signaling involved in cellular growth and proliferation, ERK 1/2 activation has been reported in several models of endotoxemia and sepsis [96, 98, 99]. Of interest to the events leading to dysadhesion of the LBEC layer from the underlying matrix in laminitis, ERK 1/2 plays an active role in many epithelial cancers in which dysregulation of cellular 54 adhesion dynamics occurs [61]. In addition to its nuclear role, ERK 1/2, is the only MAPK reported to effect hemidesmosome dissolution through phosphorylation of the cell-adhesion molecule integrin β4 [9, 10]. As the hemidesmosome is the primary adhesion complex responsible for firm adhesion of keratinocytes in all animals including horses, and there is evidence of their dissolution in a model of sepsis-related laminitis [5, 6], signaling related to ERK

1/2 needs to be further investigated in SRL. In the face of this compelling data indicating a role of ERK 1/2 signaling in SRL, lamellar concentrations of phospho-ERK 1/2 did not did not differ between the cooled limb and limb at

ambient temperature in samples in the RDH study. Although these data indicate

that the therapeutic efficacy of RDH in sepsis-related laminitis is not through an effect on ERK signaling, targeting of ERK signaling may provide an ancillary therapy to RDH if proven to play a pathologic role in sepsis-related laminitis.

Furthermore, a downstream effector of ERK 1/2, RPS6 activated and phosphorylated at the serine 235/236 site as well as the 240/244 site, was also significantly increased in the CHO model. The 240/244 and 234/236 sites may be phosphorylated through the Akt/mTOR pathway, but recent literature shows that ERK 1/2 is also a possible phosphorylator of the serine 235/236 site. RPS6 also trended in an increased activated concentration in ambient-limbs versus those iced in the RDH model. We believe limitations of sample size to be the reason this increase was not significant in the 235/236 moeity, as dramatic differences between treated limbs is apparent graphically (see Fig. 12b). RPS6 is the phospho-protein directly implicated in cell to cell dysadhesion in the blood- 55 testis barrier, and may occur in other tissues [100], but may also be a harbinger of upstream ERK 1/2 activation or mTORC1 activation.

Surprisingly, phospho-Akt was also significantly increased in the CHO model, which could account for the increase in RPS6 240/244. It is reported that in a positive energy balance Akt activates mTOR, partially through negatively regulating phospho-AMPK activation, which is a positive regulator of the mTOR inhibitor, tuberous sclerosis complex (TSC) 2 [101] and activated mTOR

However, in this study, we found the opposite. Similarly to other studies

performed on lamellae from obese ponies subjected to a high non-structural

carbohydrate (NSC) diet [102], phospho-AMPK levels actually decreased. This is quite the opposite of what is a normal tissue response to a positive energy state, such as that which is induced with a NSC diet. Indeed, in this study, lean ponies exhibited a normal decrease of activated AMPK when placed on a high

NSC diet [102]. More research must be performed, such as evaluating insulin

levels in the CHO samples, to explain the contradicting increased p-AMPK and p-

Akt and unchanged mTOR from OG1 samples in comparison to controls.

The results of lamellar SAPK/JNK 1/2 signaling are less compelling, primarily due to the poor fidelity of antibodies resulting in poor but acceptable immunoblots but unacceptable results for immunolocalization. Overall, phospho-JNK concentrations, similar to ERK1/2, were also increased in OG1 lamellar samples.

However, we could not determine the lamellar cell types undergoing the increase in JNK activation. Interestingly, lamellar JNK concentrations were also increased in the cooled limb (compared to ambient) in the RDH samples, possibly indicating 56 that JNK may have a protective role. Supporting this possibility, JNK activation has been reported with the use of a milder hypothermia in an experimental model of brain trauma; the investigators reported that JNK plays a protective role in part by decreasing inflammatory signaling [78]. Further studies with optimized reagents are needed to confirm our findings with JNK.

While p38 MAPK has historically been the mitogen-activated protein kinase

associated with sepsis [68, 98, 103], several sepsis studies have found similar

results to ours, in that ERK 1/2 and SAPK/JNK 1/2 are activated while no change

is found in p38 MAPK [61, 63, 81]. One reason for the difference in signaling

compared to many reports may be that lamellar tissue is epidermal in contrast to

the tissues and cell types (commonly leukocytes) used in many sepsis studies

[69, 104]. In a recent report on exposure of another epithelial cell type (bronchial

epithelium) to LPS, the same pattern of MAPK activation-activated ERK 1/2 and

JNK but not p38 MAPK was reported as found in the laminae in our study [99].

In that study, inhibition of these MAPKs (ERK 1/2 and JNK) also led to decreased

expression of pro-inflammatory cytokines [99]. The results of the current study

not only warrant further investigation into the ERK and JNK pathway signaling as

potential therapeutic targets in models of SRL, but also investigation of other

pathways induced by the same types of cell surface receptors that lead to

activation of these MAPK pathways. For example, the PI3K/Akt pathway,

commonly induced by the same signaling that leads to ERK 1/2 signaling [30],

has not been reported to be activated in sepsis [88, 105], but has also been

implicated in hemidesmosome dysregulation [88]. Therefore, not only does this 57 study suggest a potential treatment for laminitis through control of the ERK 1/2 and SAPK/JNK pathways, but will hopefully lead to further exploration of cellular metabolic dysregulation in laminitis and other sepsis-related diseases.

CHAPTER 3. Hypoxia Signaling in a Novel Supporting Limb Laminitis (SLL)

Model

3.1 Materials and Methods: SLL Study

Supporting Limb Laminitis Sample Collection

The experimental method was approved by the Ohio State University

Institutional Animal Care and Use Committee. Eight Standardbred horses destined for abattoir were used. They were aged 3-15 years old, three being geldings and five mares. Each horse was acclimated to the hospital environment for one week before commencement of experiment (T0 hr) to obviate effects of shipping after 3 weeks of quarantine in a separate facility. Temperature, heart rate, GI sounds, digital pulses and respiration (TPR+) were monitored daily.

Lateromedial radiographs of both front feet were performed to determine if there was any indication of chronic laminitis. Gait examination at the trot and walk as well as hoof tester application was performed to rule out lameness or foot sensitivity. Each horse was fed grass hay and given water ad libitum. Horses were stalled in a 12’ x 12’ stall with fresh shavings rising no higher than one inch above the rubber flooring. A custom aluminum shoe with two bolt-holes at the 3

o’clock and 9 o’clock position was applied one week before time 0.

Horses were monitored for 48 hours prior to device placement (T0-48hrs). 59

Horses were monitored every hour and TPR+ was recorded every 6 hours.

This schedule continued until cessation of the experiment (T96 hr). The device was applied at T48 hr. It consisted of a v- shaped metal bar with the apex pointing towards the floor and each arm of the v- secured to the aluminum shoe

with bolts (Figure 18a). The construct necessitated overweighting of the

contralateral forelimb due to the animal only being able to place the toe (Figure

18b) or the heel of the shod foot on the ground (Figure 18c), but never the entire

sole. The device did not contact the subject’s hoof.

This limb was designated “unloaded limb (UL).” Its contralateral limb was labeled “supporting limb (SL)”. The hind limbs were named in accordance with their position against the SL: “contralateral limb (CL)” and “ipsilateral limb (IL)”

(Figure 19).

FORE

HIND

Horses were tied to prohibit lying down and to mitigate movement. A full hay

61 net and water bucket was kept in front of horses at all times. Horses were monitored hourly with a physical exam performed every 6 hours to ensure comfort, device position and that animals were not in distress. At T72 hrs, the device was removed for not more than 2 minutes and hoof testers were applied rapidly to the SL to rule out discomfort in this hoof. At T96 hrs, horses were given detomidine (0.01mg/kg) and an overdose of barbituates for humane euthanasia in the stall. Each of the SL, CL and IL limbs were disarticulated at the metacarpophalangeal joint in random order. The UL limb was taken from 3 subjects for comparison, but time limitations (sampling within 20 minutes of euthanasia) prevented collection from all horses. The device was removed from

these prior to sectioning. Each foot was sagittally sectioned using a band saw

lamellar samples were snap-frozen, ensuring both dermal and epidermal lamellae were included in the sections. Time from euthanasia to snap-freezing of

samples was less than 20 minutes in all eight horses save for the UL, which took

no longer than an extra 10 minutes. Lamellar samples were stored at −80°C.

Gene Expression Quantification

Real-time quantitative polymerase chain reaction (PCR) procedure

The lamellar sample previously stored at -80°C was pulverized over dry ice

prior to total RNA extraction (Agilent Absolutely MiniPrep) with a DNAse step to

degrade any genomic DNA contamination. Poly (A) RNA (mRNA) was isolated

from total mRNA using Streptavidin magnetic beads (Roche mRNA isolation kit).

Integrity of the mRNA sample was assured using a Nanodrop 62 spectrophotometer. 400 ng of mRNA was used in complementary (c)DNA synthesis via reverse-transcriptase (Ambion Retroscript) PCR on a standard thermocycler. cDNA synthesis was performed with reverse transcriptase buffer

(4μg), nucleotides (8μg), RNAse inhibitor (2μL) and reverse transcriptase (2μL).

The cDNA was stored at -20°C until ready for real-time quantitative PCR.

Prior to real-time quantitative PCR, external standards were created from primers (see Appendix). A known PCR product was transformed into E. coli

(TOPO TA cloning kit). After growth overnight, an individual colony was isolated and the plasmid product with PCR product was amplified through Miniprep or

Maxiprep. The resulting plasmids were were sequenced to confirm the correct sequence and gel electrophoresis was performed for size verification. cDNA was linearized enzymatically (HINDIII) and serial dilutions of a known copy number were then made from 101 to 106.

Real-time quantitative PCR (RT-qPCR) was performed using a Lightcycler

2.0 thermocycler and quantified with external standards with the SYBR Green I

dye fluorescent format as previously described [32, 44]. Briefly, primers for

ADAMTS-4, IL-6, MMP-2, MMP-9, COX-2, MMP13, IL-1 β were used due to their

previous up-regulation in a sepsis-related laminitis model [32]. In regards to hypoxia signaling by HIF-1α and its downstream effectors, PGK-1, VEGF, NOS-

2, GLUT-1, and HIF-1α were designed from equine-specific sequences

(Appendix B). Each lamellar cDNA sample was diluted 1:5 for ADAMTS-4,

MMP9, MMP2, IL-1 β, IL-6, VEGF, iNOS, COX-2; 1:50 for GLUT1; and 1:500 for

HIF-1α, PGK1, and for housekeeping genes β-Actin, GAPDH and B2M according 63 to the dilution that would best fit in the standard curve.

RT-qPCR reactions were performed at 5 μL cDNA dilution to 15 μL of PCR

Master Mixture, composed of forward and reverse DNA primers, PCR nucleotide plus, 1:10000 dilution SYBR Green, Taq polymerase (1 unit), 0.2 units uracil-N-

glycosylase to prevent PCR product carryover (0.2 units) and PCR buffer

(20mmol/L Tris-HCl). All reactions were performed in duplicate and included a

standard curve and water as a negative control.

RT-qPCR was carried out as previously described [32] with uracil-N-

glycosylase for 2 minutes at 50°C to prevent carryover contamination followed by

a uracil-N-glycosylase inactivation step at 95°C for 2 minutes, amplification for

40-45 cycles at 1-5°C below the melting temperature of the specific primer

optimized per PCR primer and finally a melting temperature stage performed

step-wise up to 95°C for 15 minutes for fluorescence acquisition. Variances in

step length and temperature were adjusted for optimal gene expression product

quantification.

Data Analysis: Gene Expression

The average copy number was analyzed against the housekeeping genes

using geNorm (Ghent University, Belgium). β-Actin was chosen as the

housekeeping gene, as it had the least amount of variance among samples.

geNorm corrected copy samples were divided by the normalization factor

supplied by the geNorm adjusted β-Actin normalization factor for the same

sample, which indicated fold change. Normality was assessed via the 64

Kolmogorov-Smirnov test for normality. Normally distributed samples were assessed using a one-way repeated measures ANOVA with Dunnett’s multiple comparisons post-hoc and the non-normal distributed data were assessed using a Kruskal-Wallis test with Dunn’s multiple comparisons post-hoc. An α-error of

5% (p<0.05) was designated as being statistically significant.

Protein Extraction and Western Immunoblotting

Protein concentrations of phosphorylated and total forms of RPS6 (240-

44), p38 MAPK, ERK 1/2, SAPK/JNK 1/2, AMPK were normalized against β-

Actin under the same protocol as the MAPK and growth-factor signaling carbohydrate overload study (Chapter 2.1) in the SL and hind limbs (CL and IL).

Total HIF-PHD2 and HIF-1α concentrations were assessed only due to the fact that these proteins are not primarily regulated by phosphorylation events; they are constitutively active and mainly regulated by degradation. Data were assessed in the same manner as the MAPK and growth factor signaling data.

Hindlimb and forelimb lamellar samples from the previously described carbohydrate-overload model were used to perform an ancillary HIF-1α western immunoblot to ensure changes in protein concentration were not merely a normal physiologic difference between hind and forelimb weight bearing.

Immunofluorescence

HIF-1α concentrations were assessed in the same manner under confocal

microscopy as the MAPK and growth factor-signaling carbohydrate overload

data.

3.2 Results

Gene Expression Quantification

There were no changes in expression of ADAMTS-4, IL-6, MMP-2, MMP-

9, COX-2, MMP13, IL-1 β, ADAMTS-4, MMP9, MMP2, IL-1 β, IL-6, VEGF, iNOS,

COX-2 or GLUT1 between SL, CL and IL samples (Figure 20).

Figure 20: Gene Expression in the SLL Model 68

Protein Extraction and Western Immunoblotting

There were no significant changes in activated/phosphorylated and total p38 MAPK, ERK 1/2, SAPK/JNK 1/2 and AMPK protein concentrations when assessed against β-Actin from the supporting limb samples to the hindlimb (CL and IL samples).

There was an increase in HIF-1α protein concentrations in the supporting limb (SL) individual lamellar samples compared to the contralateral hindlimb samples (CL) with a repeated measures ANOVA p value of 0.013 after normality was assured. This was not significant in the SL versus the IL limbs (Figure 21).

There was no difference in HIF-1α protein concentrations between fore and hind limbs in the carbohydrate-overload model.

Immunofluorescence

Upon immunofluorescence of lamellar samples in both the supporting limb and hind feet for HIF-1α, signal was localized primarily to the epidermal lamellae in all samples (Figure 22).

3.3 Discussion

Our results show that, in direct contrast to a carbohydrate-overload model of sepsis-related laminitis, there is not an increase in pro-inflammatory cytokine

gene expression nor is there a change in phosphorylated/activated protein

concentrations of any MAPKs in affected feet compared to controls in a

supporting limb laminitis model [32, 38, 44, 45]. This model was created to

mimic the acute, non-painful over-weighting of a front limb in response to a

pathological inability to bear weight on the contralateral forelimb. These results

somewhat parallel those in a model of equine metabolic syndrome-associated

laminitis, where pro-inflammatory cytokines were not found in an increased

concentration in obese ponies subjected to a high NSC diet [26]. However, in

contrast to both sepsis-related laminitis and equine metabolic syndrome-

associated laminitis, supporting-limb laminitis usually manifests in only one limb.

Also contrary to cell signaling in sepsis-related laminitis, a change between

HIF-1α was noted between the supporting limb and a control hindlimb. HIF-1 α

can be induced and/or positively regulated at the post-transcriptional level by hypoxia, stretch signaling or inflammation. However, previous data show that while the equine lamellae is at a constant state of hypoxia with a larger concentration of HIF-1 α than other equine tissues, cultured equine keratinocytes had an increased concentration of HIF-1 α only after being subjected to hypoxia,

but showed no change attributable to translation when exposed to LPS [106] This

is consistent with the findings in our study, as pro-inflammatory cytokines did not

have an increased expression in the supporting limb. 72

In this study an increase in HIF-1α from supporting limbs to contralateral hind limbs was significant, while an increase from supporting limbs to ipsilateral hind limbs appeared present on immunoblotting but was not significant. This suggests a preponderance of hypoxia in the supporting limb compared to at least one hind limb. While the fact that only one hind limb was significantly different from the supporting limb may be a problem with sample size, a lack of concurrent decrease in the HIF-1α-inhibitor PHD2 or an increase in hypoxia-effector gene

expression beg the need for further research, such as nanostring gene assay for

an increased sensitivity and spectrum of gene probing and a kinome array to

establish other kinase effectors of HIF-1α.

In the end, this study was a proof-of-concept model as a non-painful supporting limb laminitis model. Limitations included an outbred population of horses, a very small sample size and an inability to measure differential weight bearing between limbs. Furthermore, the time period was short to ensure that the acute cell-signaling pathways were elucidated and that the model was non-

painful.

Further research after completion of this study utilizing a similar supporting limb laminitis model suggests that limb cycling is critical to perfusion and acceptable glucose to lactate ratios [107]. These horses had one forelimb suspended, and so were totally weight bearing on the supporting limb, but interestingly enough this study was done with the horse over-weighting the supporting limb for only one hour. The dialysate indicating perfusion and metabolism was collected via microdialysate catheter threaded between 73 lamellae, and further research could be done on our model to assess hypoxia- parameters over our longer time period in this model.

Chapter 4. Discussion and Conclusions

4.1 Overview of Results from Two Laminitis Models

These two models of laminitis highlight differences in cell signaling that lead

to similar histopathologic changes in equine lamellae. In the carbohydrate

overload model, previous work has shown an increase in lamellar pro-

inflammatory molecule cytokine gene expression, specifically IL-1 , IL-6, IL-8,

COX-2, STAT3, CXCL1 and 6, and endothelin adhesion moleculesβ at the time of

onset of lameness [38, 44, 45, 48, 79] and at least some of this expression is

localized to the epidermal lamellae [16]. The non-painful model of supporting

limb laminitis showed no increase in pro-inflammatory cytokines in the acute

periods, but did suggest hypoxia was associated with the supporting limb. The

third arm of the triad of laminitic diseases, equine metabolic-associated lamintitis,

shares some similarities and differences with the other two arms. While equine

metabolic associated laminitis often affects all four feet, similar to sepsis-related

laminitis, a recent study showed no upregulation in pro-inflammatory cytokines in

obese ponies subjected to a high non-structural carbohydrate diet [26]. A study

of hyperinsulinemia found an increase in TNF-α protein concentrations, whose

gene expression was not increased in carbohydrate-overload sepsis-related

models of laminitis, but IL-6 was unchanged [28], causing the authors to call

75 results conflicting and a role for lamellar inflammation in equine metabolic syndrome-associated laminitis unknown. While a correlation between the acute periods of equine metabolic syndrome-associated laminitis and lamellar pro- inflammatory cytokine up regulation has not been definitely found, systemic inflammation may play a role, as obesity has been correlated with increased circulating inflammatory cytokines and chemokines in humans and other animals

[28]. We cannot rule out circulating inflammation in episodes of supporting limb laminitis, as what may be found in injuries causing sterile SIRS or focal sites of infection causing non-weight bearing of one limb, but we did not find any lamellar pro-inflammatory gene expression in our model in the face of evidence of hypoxia.

4.2 MAPK Data

Therefore, implicating inflammation as causation in sepsis-related laminitis is appealing especially after inflammatory cytokines were directly localized to the lamellar basilar epithelial cell in a recent laser capture study [16]. By assessing

MAPKs, we attempted to investigate central signaling mechanisms upstream of

the inflammatory molecules as potential therapeutic targets. This led us to

elucidate the role of upstream effectors of pro-inflammatory cytokine gene

expression, as well as other pathways that may be implicated in

hemidesmosome and growth factor signaling regulation.

p38 MAPK is the most historically correlated the mitogen-activated protein

kinase most commonly associated with inflammation, probably due to its close

association with the transcription factor NF B, often implicated in transcription of

pro-inflammatory molecular factors [61, 65,휅 68]. p38 MAPK has been shown to

be necessary for IL-6 and IL-1 activation in circulating equine leukocytes [69], but

our results did not find an increase in phosphorylated lamellar activated p38

MAPK protein. More recently, the other two central MAPKs, ERK 1/2 and

SAPK/JNK, have also been implicated in inflammatory diseases, even without

concurrent activation of p38 MAPK [77, 99]. In cultured bronchoepithelial cells,

SAPK/JNK and ERK 1/2 were necessary for NF B transcription factor activation,

causing increases in IL-8 [99], a cytokine/chemokine휅 also increased in lamellar

samples from horses subjected to carbohydrate overload. This parallels our

results, where lamellar concentrations of activated p38 MAPK were not increased

but SAPK/JNK and ERK 1/2 exhibited increases in lamellar tissue from horses 77 subjected to carbohydrate overload at the advent of lameness.

Interestingly, regional deep hypothermia reduces inflammation in the acute period of the carbohydrate overload model [58]. In a carbohydrate overload

study, distal limbs submerged in ice water to 5-10°C for 72 hours after

carbohydrate overload then removed ameliorated histopathologic signs of

laminitis when the horses were euthanized 7 days after CHO induction [108].

Regional deep hypothermia interestingly has no effect on phospho-ERK 1/2 concentration in lamellar tissue, but an increase in nuclear localization could be visualized in some ambient samples relative to the limbs receiving ice treatment.

The fact that regional deep hypothermia is very effective in reducing pro- inflammatory cytokine gene expression in lamellar cells but not in regulating phospho-ERK 1/2 led us to investigate cofactors associated with the Ras/ERK pathway and its downstream targets as fellow contributors to basilar lamellar epithelial cell dysadhesion from the basement membrane.

SAPK/JNK concentrations were higher in iced limbs relative to ambient limbs. SAPK/JNK may therefore be protective, if its activation is part of the reason regional deep hypothermia is effective at mitigating sepsis-related laminitis, neutral with no effect on dermoepidermal viability, or possibly detrimental. If it is the latter, targeting SAPK/JNK or a SAPK/JNK docking site may potentiate regional deep hypothermia efficacy in treatment. However, given recent research on regional deep hypothermia in in vitro traumatic brain injury mouse models, it is important to discern if SAPK/JNK is indeed protective [78].

In these models, traumatic brain injury to neuronal cells was mitigated by mild 78

(33°C) hypothermia. Phospho-JNK concentrations were increased in these tissues, leading the authors to believe that SAPK/JNK may have an acute neuroprotective effect [78]. SAPK/JNK is the most sensitive MAPK to environmental stressors [63], but may also regulate cell survival [78].

4.3 Moving Away from Pro-Inflammatory Gene Expression as Sole

Causation in Sepsis-Related Laminitis

Historically, sepsis in human medicine had been thought of as an imbalanced systemic inflammatory response followed by a compensatory anti- inflammatory response [49]. However, spectacular failure of treating sepsis with inflammatory mediator drugs such as the anti-TNFα afelimomab, the recombinant activated protein c (Drotrecogin alfa) and anti -p38 MAPK drugs has caused

hesitation in human medicine for treating solely anti-inflammatory pathways [66,

109]. Some argue that inflammation in sepsis is a positive response, and anti-

inflammatory mediators in critical disease may contribute to morbidity rather than

treatment [109]. Further evidence for aberrancy in metabolism is the fact that

many humans become insulin-resistant in sepsis [110], and that management of

hyperglycemia significantly increases survival. This theory is similar to pathology

in equine metabolic syndrome-associated laminitis. While equine lamellae do not

have insulin receptors, circulating insulin may act through insulin-like growth

factor receptors [111]. Insulin is itself anti-inflammatory [110], but horses

subjected to a hyperinsulinemic euglycemic clamp develop laminitis [18, 25, 27,

28, 30]. Therefore, insulin may itself potentiate signaling of dermoepidermal

dysadhesion through signaling unrelated to inflammation.

4.4 A Potential Role of the Ras/ERK 1/2, PI3/Akt/mTOR Pathway and RPS6

Recent research has suggested ERK and its downstream effectors P90RSK and PDK1 may potentiate dysadhesion of the epidermal cell from the basement membrane by phosphorylation of the β4 integrin subunit on of the hemidesmosome [8-10]. In our research, we found an increased concentration of activated ERK 1/2, Akt and AMPKα in horses subjected to carbohydrate overload. We did not find an increase in mTOR concentrations, which is interesting as ERK 1/2 can activate mTOR in certain circumstances [60].

However, there is immense crosstalk between the pathways; Akt and mTOR can

activate or deactivate each other depending on the upstream signaling events

[77]. Akt can also prolong phosphorylation of ERK by inhibiting p38 MAPK-

dependent dephosphorylation of ERK [77], thereby maximizing ERK activity.

RPS6 is a protein regulated downstream of mTORC1 which was first

studied primarily as part of a ribosomal subunit responsible for transcription of

genes necessary for cell mobility, proliferation and survival [99], being

responsible for 70% of ribosomal biogenesis products in cell growth [112]. RPS6

has also recently been implicated in tight junction break down in the blood testis

barrier by Ras/ERK and Akt-mediated production of matrix metalloproteinase 9

[100]. RPS6 has also been implicated in localized transcritiption promoting cell

migration of cancer cells when co-localized to paxillin at the borders of cellular

lamellopodia [113]. Its increase in lamellar samples from horses subjected to

carbohydrate overload may be phospho-ERK 1/2 mediated, as P90RSK is

phosphorylated by ERK 1/2 and then goes on to phosphorylate and activate 81

RPS6. Interestingly, RPS6 had a trend towards a lower concentration in limbs treated with regional deep hypothermia in the second carbohydrate overload study, while as ERK 1/2 did not. Further work would have to be done to evaluate if this finding is real but statistical significance was not achieved due to small sample size. Also, further evaluation of other proteins activated by P90RSK, such as PDK1, would be necessary to further the hypothesis that ERK 1/2 activation may be involved in hemidesmosome dysadhesion. Finally, it would be prudent to evaluate the concentrations of the activated forms of RSKs P70 and

P90 as a middle step in RPS6 activation. Nuclear localization of ERK 1/2 and further kinome data to assess a possible activator of RPS6 other than mTOR and

ERK 1/2 would also further this preliminary research.

Also similar to an equine metabolic syndrome-associated laminitis model,

AMPKα concentrations were increased in lamellar tissue subjected to a carbohydrate overload. In ponies, an increase in NSC diet leads to a decrease in activated AMPKα concentrations as expected, as it is the protein responsible for cell survival in starvation [102]. Many of these ponies developed laminitis in this model. These former results in the NSC study may indicated activated AMPK to

be neutral in the development of laminitis in our study, but our results in this

sepsis-related model suggest that there is metabolic aberrancy in that growth

factor-dependent kinases ERK 1/2 and Akt are increased in active concentrations

along with the starvation protein AMPKα.

4.5 Hypoxia in the SLL Model

In our supporting limb laminitis model, we based our hypothesis that hypoxia may play a role in supporting limb laminitis on a study first performed by van Kraayenberg and then repeated by van Eps in a computed tomography model. In these experiments, blood flow to the lamella was interrupted in cadaver limbs subjected to supra-physiologic loads [86, 87]. Hypoxia triggers a

decrease in eventual degradation of the protein HIF-1α, where, when in sufficient

quantities, it dimerizes to HIF-1β and acts on the hypoxic response element

(HRE) in the nucleus for activation of transcription factors related to glucose

harvesting regulation and angiogenesis [114]. HIF-1α expression can be

increased by pro-inflammatory cytokines [91], so we performed quantitative real-

time PCR on inflammatory genes found to be upregulated in carbohydrate-

overload studies. These were not increased in the supporting limb relative to

hind limbs, nor was HIF-1α increased in lamellar samples from a carbohydrate

overload model in another study [95].

HIF-1α concentrations can be increased with growth factor and mTOR

activation in certain cancers or normoxia under certain conditions [91], but there

was no increase in concentrations of activated ERK 1/2, JNK, p38 MAPK or

mTOR in this study. HIF-1 α was increased in concentration in the supporting

limb lamellae relative to the contralateral hind foot, and trended towards an

increase in the ipsilateral hind foot. This study has many limitations, one of

which is sample size. There are no data regarding how much weight a horse

bore on each leg, which may explain the difference between hind limb 83 concentrations.

Preliminary data indicate that phospho-RPS6 240/244 may be increased in

SLL feet. RPS6 is phosphorylated at the serine sites 240/244 by P70RSK, which is activated by mTOR. Not only does this suggest a role in mTOR in SLL, it provides a link in cellular signaling to the other types of laminitis.

4.6 Further Directions in Hemidesmosome and Cytoskeletal Regulation

The data in this project propose a relationship between cellular signaling separate to pro-inflammatory cytokine upregulation in the dysregulation of dermoepidermal basement membrane stability While ERK 1/2 and SAPK/JNK have been implicated in pro-inflammatory cytokine gene expression [61, 80, 99], activated ERK 1/2 was unchanged in RDH and activated SAPK/JNK actually increased, suggesting a role separate from inflammation. Pro-inflammatory cytokines were not found to have increased gene expression in our supporting limb laminitis model. Recent research suggests that the hemidesmosome dysadheres in normal situations of cell mobility, and that this is ERK 1/2 mediated [8-10]. A recent experiment in carbohydrate-overload lamellar samples shows a lower concentration of β4 integrin in laminitis [11], which suggests some loss of hemidesmosome stability. Furthermore, signaling pathways related to activated Akt and RPS6 have been implicated in loss of cell to cell adhesion with concurrent Ras/ERK pathway activation [100], whose activation in the sepsis-related laminitis model may also be important for LBEC dysadhesion from the basement membrane. While many of these proteins were not increased in our supporting limb laminitis model, HIF-1α was increased in supporting limbs relative to a control hind limb. HIF-1α is partially regulated by mTOR [114], and prelimary data in the supporting limb laminitis model exhibits an increase in activated P70RSK protein, suggesting a role of the mTOR pathway.

A kinome array is in progress for the carbohydrate-overload and supporting 85 limb laminitis samples to further elucidate cell signaling pathways involved in

growth factor signaling and cell mobility that may signal dissolution of the

hemidesmosome or loss of the LBEC to the underlying basement membrane.

Furthermore, an RNA-Seq was performed in the SLL lamellar samples. This

returned several RNAs and microRNAs that were significantly over or under-

expressed in the supporting limb relative to the hind limbs, which may indicate a

role of mTOR, P70RSK or RPS6 and which genes are manipulated by the

increase in HIF-1α.

Our eventual goal is to discover the cellular signaling pathways of the markedly different arms of laminitis, each of which converges on the event of laminar basilar epithelial cell dysadhesion from the basement membrane. This will allow for further treatment invention, as well as explaining the cellular basis for regional deep hypothermia as treatment in sepsis-related laminitis.

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Appendix A: Western Immunoblotting and Immunofluorescence/Immunohisotchemistry Primary Antibodies

Protein Phospho- 2ndary Total 2ndary Β-Actin B-Actin Antibody phospho- protein phospho- antibody 2ndary antibody antibody antibody antibody p38 MAPK CS9211 CS7074 CS9212 CS7074 SC1616 SC2961

ERK 1/2 CS4376 CS7074 CS4695 CS7074 SC1616 SC2961

RPS6 CS2211 CS7074 CS2217 CS7074 SC1616 SC2961 235/236

RPS6 CS2217 CS7074: CS2217 CS7074 SC1616 SC2961 240/244

Akt CS9271 CS7074 CS4691 CS7074 SC1616 SC2961 mTOR CS2971 CS7064 CS2972 CS7064 SC1616 SC2961

AMPK CS 2535 CS7074 CS5831 CS7074 SC1616 SC2961

SAPK/JNK LT700031 CS7074 CS9252 CS7074 SC1616 SC2961 1/2 HIF-2α N/A N/A NB100-122 CS7074 SC1616 SC2961

PHD2 N/A N/A SC34920 SC2961 SC1616 SC2961

HIF-1α N/A N/A NB100-449 CS7074 SC1616 SC2961

CS: Cell Signaling; Beverly, Massachusetts LT: Invitrogen; Camarillo, CA NB: Novus Biologicals; Oakville, ON

Appendix B: PCR Primers

Gene Sequence (5’-3’)

ADAMTS4 GAA TCC CAG TCC CAG GCC CCG AAG AG (A disintegrin and metalloproteinase with thromobspondin motifs 4) PGK1 CTG GGC CGG CCT GAT GGT GTC C (Phosphoglycerate kinase 1) HIF-1α AAG CTT TGG ATG GTT TTG TTA T (Hypoxia-inducible factor-1 α) GLUT1 CTC GCC CTG CTG CCA TTG CTG TCT CT (Glucose transporter 1) IL-1β CCA GAC GCG GCC GGG ACA TAA C (Interleukin-1β) IL-6 CAC CGT CAC TCC AGT TGC CTT (Interleukin-6) IL-8 TGG GCC ACA CTG CGA AAA CTC (Interleukin-8) MMP-2 ATC GCT GCG GCC TGT GTC TGT G (Matrix metalloproteinase-2) MMP-9 CCT GTA CCG CTA TGG CTA CAC (Matrix metalloproteinase 9) COX-2 GTA TCC GCC CAC AGT CAA AGA (Cyclooxygenase 2) VEGF AGT ACG TTC GTT TAA CTC AAG CTG (Vascular endothelin growth factor) β-Actin GGG AAA TCG TGC GTG ACA T (β-Actin housekeeping gene) MMP-13 GTC CCT GAT GTG GGT GAA TAC (Matrix metalloproteinase 13) iNOS CAG GGG AGC ATC TTG GGG TTT TC (inducible isoform nitric oxide synthase)

100