Page 1 of 52 Diabetes
Plasma Kallikrein-Kinin System as a VEGF-Independent Mediator of Diabetic
Macular Edema
Takeshi Kita1, Allen C. Clermont1, Nivetha Murugesan1 Qunfang Zhou1, Kimihiko
2 2 1,3 1,4 Fujisawa , Tatsuro Ishibashi , Lloyd Paul Aiello , Edward P. Feener
1 Joslin Diabetes Center, Harvard Medical School, One Joslin Place, Boston, MA 02215,
USA
2 Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University,
3 1 1 Maidashi, Higashi Ku, Fukuoka 812 8582, Japan.
3 Beetham Eye Institute, Department of Ophthalmology, Harvard Medical School, One Joslin
Place, Boston, MA 02215, USA
4 Department of Medicine, Harvard Medical School, One Joslin Place, Boston, MA 02215,
USA
Corresponding Author: Edward P. Feener, Ph.D., Joslin Diabetes Center, Harvard Medical
School, One Joslin Place, Boston, MA 02215, USA.
Phone: 617 307 2599, Fax:617 307 2637.
e mail: [email protected]
1
Diabetes Publish Ahead of Print, published online May 15, 2015 Diabetes Page 2 of 52
Abstract:
This study characterizes the kallikrein kinin system in vitreous from individuals with diabetic macular edema (DME) and examines mechanisms contributing to retinal thickening and retinal vascular permeability (RVP). Plasma prekallikrein (PPK) and plasma kallikrein (PKal) were increased 2 and 11.0 fold (both p<0.0001), respectively, in vitreous from subjects with DME compared to those with macular hole (MH). While vascular endothelial growth factor (VEGF) was also increased in DME vitreous, PKal and VEGF concentrations do not correlate (r=0.266, p=0.112). Using mass spectrometry based proteomics we identified 167 vitreous proteins, including 30 that were increased in DME (> 4 fold, p< 0.001 vs. MH). The majority of proteins associated with DME displayed a higher correlation with PPK than with VEGF concentrations. DME vitreous containing relatively high levels of PKal and low VEGF induced RVP when injected into the vitreous of diabetic rats, a response blocked by bradykinin receptor antagonism but not by bevacizumab. Bradykinin induced retinal thickening in mice was not affected by blockade of VEGF R2. Diabetes induced RVP was decreased by up to 78% (p<0.001) in Klkb1 (PPK) deficient mice compared with wild type controls. B2 and B1 receptor induced RVP in diabetic mice was blocked by endothelial nitric oxide synthase (eNOS) and inducible NOS deficiency, respectively. These findings implicate the PKal pathway as a VEGF independent mediator of DME.
2 Page 3 of 52 Diabetes
Introduction
Diabetic macular edema (DME) is a leading cause of vision loss among
working aged adults with a global prevalence of approximately 16% for people with
20 years or more of diabetes (1). This sight threatening disease is characterized by
thickening of the macula due to accumulation of intra retinal and/or subretinal fluid and
exudates, with associated impairment of central visual acuity. The pathogenesis of DME has
been primarily attributed to retinal vascular hyperpermeability, which results in extravasation
of plasma proteins and lipids into the macula, and increased hydrostatic pressure across the
blood retinal barrier (2). Although DME can occur at any stage of diabetic retinopathy (DR),
its incidence is higher in patients with more advanced stages of the disease, including severe
nonproliferative diabetic retinopathy (NDPR) and proliferative diabetic retinopathy (PDR)
(3). These observations suggest that underlying retinal pathologies associated with DR, in
combination with systemic factors including hyperglycemia, hypertension and dyslipidemia,
contribute to DME (4).
Vascular endothelial growth factor (VEGF) contributes to macular thickening and
visual impairment associated with DME (5). VEGF concentrations in vitreous and aqueous
humor are increased in PDR and DME compared with controls without advanced DR (6 8).
Intravitreal injection of therapies that block VEGF actions, including ranibizumab,
bevacizumab, and aflibercept have emerged as effective primary treatments for DME (9).
However, most studies to date have reported that a significant proportion of DME patients,
up to approximately 50%, do not fully respond to anti VEGF therapy (10;11).
Diabetic retinopathy exhibits aspects of inflammation, including vascular
hyperpermeability, immune cell recruitment, and elevated expression of proinflammatory
3 Diabetes Page 4 of 52
cytokines, which have been implicated in the development and progression retinal pathologies (4). The kallikrein kinin system (KKS) contributes to the inflammatory response to vascular injury and is a clinically significant mediator of vasogenic edema associated with hereditary angioedema (12;13). Reports from our group and others have demonstrated that the KKS mediates vascular hyperpermeability, leukostasis, cytokine production, and retinal thickening in rodent models of DR (14 18). The pro inflammatory effects of the KKS are mediated by plasma kallikrein (PKal); a serine protease derived from the abundant circulating zymogen plasma prekallikrein (PPK). Zymogen activation by FXIIa cleaves PPK into disulfide linked heavy and light chains of catalytically active PKal, which in turn cleaves FXII to FXIIa to provide positive feedback and amplification of the KKS. The physiological mechanisms that mediate FXII and PPK activation are not fully understood, however hemorrhage, activated platelets, and unfolded proteins (19 21) have been shown to activate the KKS. PKal cleaves its substrate high molecule weight kininogen (HK) to release the nonapeptide hormone bradykinin (BK). BK activates the bradykinin B2 receptor (B2R) and is also proteolytically processed by carboxypeptidase N (CPN) into des Arg9 Bradykinin
(DABK), which is a bradykinin B1 receptor (B1R) agonist (22). Both B1R and B2R are G protein coupled receptors that are highly expressed in the retina (23), and retinal expression of B1R is increased in diabetes (15;16). While the plasma KKS has been implicated in DR
(24;25), the levels of KKS components in DME vitreous and mechanisms that mediate the effects of the KKS on retinal vascular permeability (RVP) and retinal thickening are not yet available.
In this report, we quantified KKS components and VEGF, and utilized mass
4 Page 5 of 52 Diabetes
spectrometry based proteomics to characterize vitreous samples from patients with DME.
The effects of the KKS on retinal vascular function and thickness in diabetic rodents were
examined using Klkb1, iNOS, and eNOS knockout mice, and measurements of retinal B1R
and B2R responses. Our results suggest that up regulation of the intraocular KKS may
contribute to a VEGF independent mechanism of DME.
Methods
Analysis of KKS components, VEGF, and proteome in human vitreous. Human vitreous
samples were obtained from patients undergoing pars plana vitrectomy during surgery for
macular hole (MH) and various stages of diabetic retinopathy, including DME without any
PDR findings such as preretinal proliferative membrane or neovascularization; and PDR
without vitreous hemorrhage. VEGF levels were measured by enzyme linked
immunosorbent assay (R&D systems, Minneapolis, MN). Undiluted vitreous samples were
separated by SDS PAGE (vitreous volumes used: 5µl, 1µl and 1µl for PPK/PK, FXII/FXIIa
and Kininogen heavy chain (HC), respectively) and immunoblotted using primary antibodies
against Kallikrein 1B (Novus Biologicals, #NBP1 87711), FXII (anti FXII heavy chain,
#ab1007, AbCam), HK (anti Kininogen HC (2B5), #sc 23914, Santa Cruz). Results were
visualized by enhanced chemiluminescence (20X LumiGLO, Cell Signaling) and quantified
using Image J, 1.48v (National Institutes of Health) image analysis software. Western blot
analysis of purified standards PPK, FXII, and HK (Enzyme Research Laboratories) were also
performed along with vitreous samples for the quantification. Vitreous proteomics was
performed by tandem mass spectrometry as described previously (26;27). Protein abundance
was calculated using total spectral counting for each protein from the X!Tandem search
5 Diabetes Page 6 of 52
results. For statistical comparisons between groups, samples that did not have 2 unique peptides for an identified protein were assigned values of 1, corresponding to half of the minimum detection threshold of 2 unique peptides. Protein IDs were converted to gene symbols using DAVID (28). This study was carried out with approval from the Institutional
Review Board and performed in accordance with the ethical standards of the 1989
Declaration of Helsinki.
Animal models and diabetes induction. Diabetes (DM) was induced by intraperitoneal injection of streptozotocin (STZ; Sigma Aldrich, Milwaukee, WI) in male Sprague Dawley rats (8 weeks old, Taconic Farms, Hudson, NY). Blood glucose values >250 mg/dL were considered diabetic. Experiments were conducted after 4 weeks diabetes and compared with age matched nondiabetic (NDM) controls.
Klkb1 knockout mice were described previously (29) and backcrossed against
C59Bl/6 for 9 generations. Mice deficient in eNOS (B6.129P2 Nos3tm1Unc/J) and iNOS
(B6;129P2 Nos2tm1Lau/J) and age matched wild type (WT) mice were purchased from
Jackson Laboratories (Bar Harbor, Maine). Diabetes was induced by intraperitoneal injection of STZ (45 mg/kg) every 24hrs for 5 days. Mice with blood glucose levels >250 mg/dl were considered diabetic and retinal measurements were performed on eNOS and iNOS at 8 weeks and Klkb1 / at 12 weeks of diabetes duration. Approval for the animal protocols was obtained from the Joslin Diabetes Center Institutional Animal Care and Use Committee. The procedures adhered to the guidelines from the Association for Research in Vision and
Ophthalmology (ARVO) for animal use in research.
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Measurements of retinal vascular permeability. Intravitreal injections of BK, DABK and
VEGF in rats were performed and RVP was measured by vitreous fluorophotometry as
described previously (14). Briefly, the eyes were dilated and received intravitreal injection of
BK (2 µM, Sigma Aldrich), DABK (2 µM, Sigma Aldrich), VEGF (0.02 ng/ml in vitreous)
(rhVEGF 165, R&D Systems), or phosphate buffered saline (PBS). Ten minutes after the
injection the rats were infused with 10% sodium fluorescein (300 µl/kg) through a jugular
vein catheter. After an additional 30 minutes, vitreous fluorescein levels were measured by
vitreous fluorophotometry as previously described (19). L NAME (100 mg/l), a nitric oxide
synthesis inhibitor, was administered for 3 days prior to intravitreal injection via the drinking
water. B1R antagonist des Arg10 Hoe140 and B2R antagonist Hoe140 (AnaSpec, Fremont,
CA) were systemically administrated via micro osmotic pump (Alzet model 1003D,
subcutaneously implanted in the dorsal flank) at 1 µg/µl/hr, initiated 24 hrs prior to
intravitreal injection. Bevacizumab (12.5 µg/eye) was administered to rats by intravitreal
injection in the absence or presence of VEGF or human vitreous. RVP in mice was measured
using Evans blue permeation (30). Mice were anesthetized, received intravitreal injections
with 1 µl of 20 µM BK or DABK or PBS, and infused with Evans blue dye (45 mg/kg)
through a catheter in the jugular vein. The dye was allowed to circulate for 1 hr prior to
sacrifice. After tissue fixation by 10% formalin, the eyes were enucleated and retinas were
extracted. Retinas were incubated with dimethyl formamide over night at 72oC after drying,
and the resultant supernatant was used to determine Evans blue dye content (14). Results
were normalized by retinal tissue weight and levels of the dye from plasma.
7 Diabetes Page 8 of 52
Measurement of retinal thickness by spectral domain-optical coherence tomography.
Retinal thickness was analyzed using a commercial spectral domain optical coherence tomography (SD OCT) system (840SDOCT System; Bioptigen, Durham, NC) as previously described (14). Animals received intravitreal injections as described above. In addition, a subset of mice received intravitreal injections with VEGF and DC101 (Genetex). In brief, rectangular volumes of each retina were obtained consisting 1000 A scans by 100 B scans over a 1.5x1.5 mm area centered upon optic nerve head. Thickness was measured at 500 µm relative to the optic nerve head. A total of 4 caliper measurements were taken from each site corresponding to the temporal, nasal, superior, and inferior quadrants of the retina. The measurements from the retinal pigmented epithelium to the nerve fiber layer were averaged to produce a single thickness value for each retina.
Cell culture and western blot analysis
Bovine eyes were obtained from a local abattoir and neonate rats were purchased from
Taconic Farms (NY). BRECs and ASCs were isolated as previously reported (31;32). ASCs were confirmed by immunocytochemical staining for glial fibrillary acidic protein (GFAP).
Cultured BRECs and ASCs were used between passages 4 to 7 and 3 to 5, respectively.
Sub confluent cells were starved with endothelial basal medium (EBM, LONZA
Walkersville) for BRECs or DMEM/F 12 for ASCs, containing 1% calf serum and subsequently treated with DMEM containing 25 mM glucose, which were re fed with fresh media every 24 hours up to 5 days. Total cell lysates were extracted and western blot analysis
8 Page 9 of 52 Diabetes
was performed to detect B1R Enzo Biosciences (Farmingdale, NY.), B2R (BD biosciences
(San Jose, CA), iNOS (Ab3523, AbCam), eNOS (anti phospho eNOS (Ser1177), #9571,
anti eNOS, #9572, Cell Signaling), and GAPDH (Santa Cruz Biotechnology). Band
intensities were quantified and expressed as the percentage ratio to GAPDH.
Statistics. All results were expressed as mean ± SEM. The statistical significance of
differences between groups was analyzed by 2 tailed Student’s t test or Mann Whitney
U test. For in vivo experiments, group conditions were compared by One Way ANOVA
using Fisher LSD method for pairwise multiple comparisons. Differences were considered
significant at p<0.05.
Results
Intravitreal KKS and VEGF in DME
Concentrations of KKS components and VEGF were measured in vitreous samples obtained
by pars plana vitrectomy from patients with NPDR and DME (NPDR/DME), PDR, and
macular hole (MH) (Supplemental Table 1). Within the PDR group, patients with combined
PDR and DME vs. PDR alone were not examined separately. Levels of PPK, FXII, and HK
in undiluted vitreous samples were quantified by western blotting and comparison of band
intensities for corresponding purified protein standards (Figures 1A-E, Supplemental
Figures 1A,B). Western blots of PPK and FXII revealed bands at approximately 75 kDa and
50 kDa, which are consistent with the zymogens and heavy chains of the activated enzymes,
respectively (Supplemental Figures 1A,B). PPK levels were significantly elevated in the
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vitreous from eyes with DME and PDR by approximately 2 fold compared with MH
(p<0.001) (Figure 1B). Activated PKal, measured by heavy chains levels, were increased by
11.0 and 10.4 fold in the DME and PDR samples respectively, relative to the average MH level. (Figure 1C). Comparable increases in FXII and HK heavy chain were observed in
NPDR/DME and PDR compared with MH vitreous (Figures 1D,E, Supplemental Figure
1B). In addition, increased levels of CPN were detected in vitreous from patients with
NPDR/DME and PDR compared with MH, and hemoglobin was increased in PDR vitreous
(Supplemental Figure 1C). Concentrations of VEGF in vitreous with NPDR/DME
(1063.1±285.3pg/ml, p<0.0001, n=20) and PDR (1107.8±260.7pg/ml, p<0.0001, n=24) were elevated compared with VEGF in vitreous from MH, which was below the detection limit for the assay (Figure 1F).
The correlations among levels of KKS components and VEGF in the vitreous from
NPDR/DME and MH subjects were examined. PPK levels correlated with its substrate HK
(PPK vs. HK: r=0.656, p<0.001, Supplemental Figure 2A) but not with VEGF (VEGF vs.
PPK: r=0.168, p=0.499 Figure 1G). DME vitreous samples were ranked in order of their
VEGF concentrations and the levels of PKal in these samples were shown in Figure 1H.
PKal and VEGF levels in DME vitreous do not correlate (r=0.266, p=0.112). Moreover, these results revealed 3 groups of samples, including samples (ID#s 6 to 20) with high PKal and low VEGF, samples (ID# 9 to 10 and 14 to 3) with elevated levels of both VEGF and
PKal, and sample ID#13 with high VEGF and low PKal (Figure 1H). Similarly, the correlations among the other KKS were higher than their correlations with VEGF (PPK vs.
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FXII: r=0.329, p=0.157, FXII vs. HK: r=0.592, p=0.006 VEGF vs. FXII: r=0.130, p=0.585,
and VEGF vs. HK: r=0.020, p=0.934) (Supplemental Figure 2B-E).
Vitreous proteome in DME
Vitreous proteomic abnormalities have been implicated in DME (19;26). We
utilized proteomics to further characterize the DME vitreous proteome and correlations with
PPK and VEGF concentrations. Mass spectrometry based proteomics was performed on 10
vitreous samples from DME patients and 5 vitreous samples from MH patients. DME
samples used for proteomics are indicated in Figure 1H. The list of proteins and
corresponding numbers of spectral/peptide counts for each sample are shown in
Supplemental Table 2. We identified a total of 167 proteins, including 39 proteins only
detected in DME vitreous and 10 proteins only in MH vitreous (Figure 2A). This proteome
contained 138 proteins that were identified in 3 or more independent vitreous samples. The
numbers of total spectral peptide matches were used to compare the abundance of these
proteins between DME and MH samples. Thirty proteins were increased in DME (> 4 fold,
p< 0.001 vs. MH) in vitreous from DME compared with MH samples (Table 1). The
concentrations of PKal and VEGF in the 10 DME vitreous samples used for proteomics are
indicated in Figure 1H. Correlation analyses for spectral peptide counts for these proteins
and PPK and VEGF concentrations were performed. This analysis shows that most of the
proteins in Table 1 displayed higher correlative R values for comparison with PPK than with
VEGF concentrations (Figure 2B). In addition, we show that the concentrations of PPK and
total protein correlate in DME vitreous (r=0.618, p=0.001), whereas VEGF concentration do
11 Diabetes Page 12 of 52
not correlate with total vitreous protein concentration (r=0.115, p=0.499) (Figures 2C,D).
These data reveal marked heterogeneity in the concentrations of KKS components, VEGF, and total protein in vitreous samples from patients with NPDR and DME.
Retinal B1R and B2R expression in diabetes.
To further characterize the effects of diabetes on the intraocular KKS, we examined retinal B1R and B2R expression in rats with 4 weeks of STZ induced diabetes (DM) and age matched nondiabetic controls (NDM). B1R levels were increased by 2.2 fold in DM rats compared with NDM controls (p=0.001), whereas B2R was similarly expressed in DM and
NDM rat retina (Supplemental Figure 3A). Next we examined the effects of extracellular glucose concentration on B1R and B2R levels in cultured bovine retinal capillary endothelial cells (BRECs) and rat brain astrocytes. B1R expression was increased by 2 to 3 fold in
BRECs and astrocytes cultured in media containing 25 mM glucose compared with cells maintained in media containing 5mM glucose (p<0.01, n=3) (Supplemental Figures 3B,C).
In contrast, incubation of BRECs and astrocytes in the presence of 25 mM glucose did not alter B2R levels.
Effects of BK and DABK on retinal vascular permeability and thickness
Since effects of the KKS on vascular permeability are primarily mediated by the bradykinin system (22), we examined the effects of BK and DABK on RVP and retinal thickness in rats with 4 weeks of DM along with age matched NDM controls. The effects of intravitreal injection of BK and DABK in DM rats were examined over a time course of 40
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min to 48 hrs (data not shown). BK rapidly increased RVP (at 40 min), and this response
gradually decreased at later time points. In contrast, the effect of DABK on RVP was delayed
and highest at 48 hours post intravitreal injection. The effects of BK and DABK on RVP were
further characterized at 40 min and 48 hrs, respectively. We observed a small increase in
RVP in DM rats compared with NDM rats at 40 minutes (p<0.0001) and 48 hours (p<0.0001)
after intravitreal injections with PBS vehicle. Intravitreal injection of BK increased RVP after
40 minutes in both NDM (3.6 fold, p=0.001) and DM (2.1 fold, p<0.0001) rats compared
with PBS injected controls and returned to baseline levels at 48 hours post injection (Figure
3A). In contrast, intravitreal injection of DABK did not acutely affect RVP at 40 minutes but
increased RVP at 48 hrs post injection in DM rats (2 fold, p<0.0001), an effect not observed
at 48 hrs in NDM controls. The effects of BK and DABK on RVP in DM rats were blocked
by Hoe140 (B2R antagonist) and des Arg10 Hoe140 (B1R antagonist) administered by
subcutaneously implanted mini osmotic pumps (Figure 3B). The effects of intravitreal
injection of VEGF on RVP were also investigated. VEGF increased RVP by 2.6 fold
compared with vehicle injected eyes at 40 min (p=0.004) and this response returned to
baseline at 48 hrs post injection (Figure 3C). The effect of VEGF on RVP measured at 40
min post injection was blocked by co administration with bevacizumab but not by combined
B1R/B2R antagonism.
The effects of intravitreal injection of BK and DABK on retinal thickness and retinal
vessels in DM rats were examined by SD OCT imaging at 6, 24, and 48hrs post injection
compared with baseline. Representative B scans show retinal structure at 24 and 48hrs after
BK and DABK injections respectively, compared with vehicle injected eyes (Figure 3D).
13 Diabetes Page 14 of 52
Intravitreal injection of saline vehicle produced a small increase in retinal thickness at 24 and
48hrs post injection (5.46% and 5.81% increase, respectively, p<0.05, n=5). Intravitreal injection of BK rapidly increased the retinal thickness at 6 and 24 hrs after injection (20.1% increase, p<0.01) and thickness trended to decrease at 48hrs but was still thicker than baseline (11.9 % increase, p<0.01). DABK increased retinal thickness at 24 hrs (13.7%) and
48hrs (15.8%) after injection (Figure 3D).
Effects of human vitreous on retinal vascular permeability in rats.
The potential effects of VEGF and bradykinin peptides in human vitreous on the
RVP were examined in the rat model of RVP described above. We selected two human vitreous DME samples, as indicated in Figure 1H; patient ID#12 with relatively high PKal and low VEGF (PK↑ vit: PPK 91.6 nM [18.1 fold increase in PKal], VEGF: 114 pg/ml) and patient ID#13 with low PKal and high VEGF (VEGF↑ vit: PPK 25.2 nM [2.3 fold increase in
PKal], VEGF: 2864 pg/ml), and a control MH vitreous with low levels of both PKal and
VEGF. Ten µl of human vitreous fluid was injected into the vitreous of DM rats in the presence or absence of co injections with bevacizumab or the combination of Hoe140 and des Arg10Hoe140. At 40 min after injection, PK↑ vit did not increase RVP, whereas VEGF↑ vit significantly enhanced RVP (1.41 fold, p<0.05) compared with vitreous from MH patient.
The VEGF↑ vit induced response was blocked by bevacizumab (p<0.01) but not affected by
Hoe140/des Arg10Hoe140 (Figure 4A). In contrast, at 48 hrs, RVP was increased by intravitreal injection of PKal↑ vit (1.6 fold, p<0.01), which was suppressed by
Hoe140/des Arg10Hoe140 (p<0.01), but not by bevacizumab (N.S.). RVP that was increased
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at 40 min by intravitreal injection of VEGF↑ vit returned to baseline at 48 hrs post injection
(N.S. vs. MH) (Figure 4B).
Effects of VEGFR2 blockade on VEGF- and BK-induced retinal thickening
Next we tested the effects of VEGFR2 blockade using DC101, an anti VEGFR2 neutralizing
antibody, on both VEGF and BK induced retinal thickening in wild type C57Bl/6 mice.
Retinal thickness was measured by SD OCT at baseline and then mice received 1 µl
intravitreal injections of VEGF (10 ng) and BK (20 µM), in the absence or presence of
DC101. At 24 hours post injection, retinal OCT measurements were repeated and the
changes in thickness were determined. VEGF and BK similarly increased retinal thickness at
24 hours by 15.2% and 13.6%, respectively (p<0.001) as compared to baseline (Figure 5).
Co injection of DC101 at doses of 0.36 and 0.73 µg reduced VEGF induced retinal thickness
in a dose responsive manner to 12.3% (p<0.001) and 2.3% (NS) compared to baseline,
respectively. Co injection of 0.73 µg DC101 did not alter BK’s effects on retinal thickness
(12.3%, p<0.001) as compared to baseline. Co injection of DC101 did not alter retinal
thickness in control eyes receiving PBS vehicle.
Effects of Klkb1 deficiency on RVP and retinal thickness in diabetic mice
PPK, encoded by the Klkb1 gene, is the major source of proinflammatory levels of BK (22).
Previous studies have shown that PKal inhibition reduces RVP in diabetic rats (14). To
further evaluate the role of PPK in diabetic retinopathy, we examined the effects of Klkb1
deficiency on RVP and retinal thickness in WT and Klkb1 / mice with 3 months diabetes
15 Diabetes Page 16 of 52
(DM) compared with nondiabetic (NDM) controls. Klkb1 deficiency did not alter body weight or blood glucose levels for DM or NDM groups, compared with age matched WT littermate controls (Supplemental Figures 4A,B). RVP, quantified using Evan’s blue dye permeation, was increased in DM female (400±63%, p<0.001) and male (138±31%, p<0.001) WT mice compared to NDM WT controls (Figure 6A,B). NDM WT and NDM
Klkb1 / mice of the same gender displayed similar RVP however we observed increased basal RVP in NDM male mice compared with NDM female mice. RVP in female DM
Klkb1 / mice was 78±6% (p<0.001) less than female DM WT mice (Figure 6A). Similarly, the diabetes induced increase in RVP in male Klkb1 mice was 58±11% (p=0.033) less than male DM WT mice (Figure 6B).
Previous studies have reported that diabetes causes retinal neurodegeneration in mice (33).
Using SD OCT, we observed 3.4±0.6% and 5.8±0.8% decreases (p<0.01) in retinal thickness in DM WT mice compared with NDM WT for female and male mice, respectively (Figure
6C,D). The diabetes associated decrease in retinal thickness in female Klkb1 / mice was
1.6±0.6%, which was thicker than that observed in female WT mice (204.9±1.3 vs.
198.9±1.2 µm, p=0.002). In contrast, retinal thickness in male DM Klkb1 / and DM WT mice were similar.
Involvement of eNOS and iNOS in BK- and DABK-induced vascular permeability
Increased nitric oxide contributes to bradykinin’s action and vascular permeability in diabetic retinopathy (17;34;35), however the role of eNOS and iNOS in the effects of the
16 Page 17 of 52 Diabetes
KKS on the retina are not yet available. We examined the role of nitric oxide in BK and
DABK induced RVP using L NG Nitroarginine Methyl Ester (L NAME), which is
hydrolyzed to L NNA; a competitive inhibitor of eNOS, iNOS and nNOS. Systemic
administration of L NAME in DM rats blocked both the acute effect of BK and the delayed
effect of DABK on RVP (Figure 7A). The effects of BK and DABK on eNOS
phosphorylation were examined using bovine retinal endothelial cells (BREC). Addition of
BK to the culture media increased eNOS phosphorylation at Ser 1177 within 5 min and
maximum phosphorylation was observed after 30 min. In contrast, incubation of BREC with
DABK did not alter eNOS phosphorylation at this site (Figure 7B). The potential role of
eNOS on BK induced RVP was examined in WT and eNOS knockout mice in the absence
and presence of 8 weeks of diabetes. In WT mice, BK increased RVP by 2.1 fold and 1.7 fold
at 40 min after intravitreal injection compared with vehicle injected eyes (p<0.01) in NDM
and DM mice, respectively, whereas intravitreal injection of BK did not alter RVP in eNOS
deficient mice (with/without DM)(Figure 7C).
Since DABK effects on RVP were blocked by L NAME but did not alter eNOS
phosphorylation (Figures 7A,B), we examined the effects of DABK on iNOS. Intravitreal
injection of DABK increased iNOS expression in DM rat retina compared with vehicle
injected eye. This effect of DABK on iNOS was not observed in NDM rats (Figure 7D).
Furthermore, we examined the effect of DABK on iNOS protein levels in astrocytes in vitro.
DABK induced increased expression of iNOS in astrocytes by 36 hrs and 48hrs stimulation
(p<0.01 compared with untreated control) (Figure 7E). To examine the potential role of
iNOS on DABK induced RVP, we examined the effects of intravitreal injection of DABK in
17 Diabetes Page 18 of 52
DM iNOS knockout mice. Intravitreal injection of DABK increased RVP at 48 hrs post injection in WT mice (2.0 fold vs. vehicle treated WT mice, p<0.01), whereas DABK did not alter RVP in iNOS deficient mice (Figure 7F).
Discussion
In this report, we show that the KKS is activated in vitreous from DME patients.
Vitreous from subjects with relatively high concentrations of PKal and low VEGF induce
RVP via the bradykinin pathway whereas VEGF mediates this response for vitreous with high VEGF concentrations and low PKal. These data demonstrate heterogeneity among a cohort of DME subjects in regards to vitreous protein composition and activity of vitreous fluid. Since KKS components are abundant plasma proteins, and PPK levels correlate with other abundant plasma proteins in the vitreous, it is likely that the increases in DME vitreous are due to plasma protein extravasation as a result of blood retinal barrier dysfunction. Indeed the proteins that we identified to be increased in DME vitreous (Table 1) have been previously identified in human plasma (http://www.plasmaproteomedatabase.org) (36). The concentrations of these proteins in normal plasma have been estimated by Farrah et al (37) using spectral counts and are listed in Table 1. We show that PPK, but not VEGF, concentrations correlate with most vitreous protein increases identified by proteomics and total vitreous protein concentration. Moreover, PKal is elevated in most DME vitreous samples whereas VEGF displays a greater range of concentrations among these vitreous samples. Increased intraocular concentrations of VEGF have been largely attributed to retinal ischemia/hypoxia, and there is a known wide range of VEGF concentrations among DME
18 Page 19 of 52 Diabetes
vitreous samples (7;38). The wide variability among vitreous VEGF concentrations and
extent of retinal edema implies that additional factors may play important roles in mediating
some cases of DME (9;19). Increased RVP and plasma protein extravasation in diabetic
retinopathy has been attributed to a variety of mechanisms, which are not limited to VEGF
(2). Intravitreal injection of KKS components increases RVP and retinal thickening in
diabetic animals (14;16;19). While bradykinin generated from HK is a primary effector
pathway of the KKS, the DME vitreous also contains components of the complement and
coagulations systems, such as complement C3, thrombin, and plasminogen, which may
contribute to proinflammatory effects downstream of PKal and FXIIa (39). The temporal
events and relative contributions of the VEGF and PKal pathways in DME may depend on
the underlying retinal pathologies, such as levels of retinal ischemic and the extent of
vascular injury and inflammation. We propose that KKS extravasation in diabetic retinopathy
is mediated by multiple factors and high levels of intraocular KKS activity exacerbates RVP,
which overwhelms fluid resorptive mechanisms resulting in retinal thickening. Moreover,
our findings suggest that increased intraocular PKal may contribute to incomplete or delayed
response to anti VEGF therapies for patients where intravitreal KKS activity is high.
Additional VEGF independent pathways and retinal structural changes such as
disorganization of the retinal inner layers are also likely to contribute to the range of clinical
responses observed anti VEGF treatment of DME (40 42).
Elevated nitric oxide production been implicated in the pathogenesis of diabetic
retinopathy via multiple mechanisms (43). Mice deficient in eNOS display increased iNOS
expression, nitric oxide levels, and diabetic retinopathy compared with diabetic wild type
19 Diabetes Page 20 of 52
controls (35). Mice with iNOS deficiency are protected from diabetes induced RVP and retinal capillary degeneration (44;45). B1R and B2R are expressed in vascular, glial, and neuronal cells and the mechanisms that couple the KKS to nitric oxide production differ at the cellular level. BK stimulates eNOS phosphorylation in BREC whereas the B1R agonist,
DABK, induces iNOS expression in astrocytes. Astrocytes are distributed on the surface of the inner retina, and retinal capillaries are surrounded by astrocytes’ foot process, which can swell and release inflammatory cytokines that increase blood retinal barrier permeability
(46). These findings suggest that the KKS may exert its effects on RVP by inducing nitric oxide production in both glial and vascular cells. We show that BK similarly induces RVP in
DM and NDM rat, suggesting that the B2R may contribute to RVP in the presence of KKS activation in the vitreous. In addition, we show that B1R levels and DABK responses in the retina are increased in diabetic rats compared with NDM controls. These findings are consistent with previous reports showing that B1R mRNA expression and responses are increased in diabetes and inflammation (15;16;47). In diabetes, proinflammatory cytokines may contribute to increased DABK action via up regulation of B1R and iNOS expression, which could provide additive effects to the B2R/eNOS pathway on RVP (34). Although selective inhibition of B1R could potentially provide some protective effect for retinal dysfunction in DR, this approach would not address the potential contributions of B2R or effects mediated by PKal substrates other than HK (18;29). However, blockade of PKal could reduce the proinflammatory effects of the intravitreal KKS while preserving the potential beneficial ocular effects of BK generated by tissue kallikrein (Klk1) (48).
Taken together, our results suggest that the PKal mediated KKS exerts effects on
20 Page 21 of 52 Diabetes
DR at two levels. In early stages of DR, a low level of KKS activation increases RVP via both
retinal B1R and B2R. This diabetes induced vascular hyperpermeability is blocked by Klkb1
deficiency and administration of either a PKal inhibitor (14) or B1R antagonists (15;17).
Although this early increase in RVP may contribute to the pathogenesis of DR, it is not
sufficient to cause retinal thickening. The transition to retinal edema, including DME,
appears to require further up regulation of vascular permeability factors, such VEGF and
PKal, which are markedly increased in the vitreous during advanced stages of DR compared
to diabetes alone (6). Since many individuals are not fully responsive to anti VEGF DME
therapy (10), PKal inhibition in these patients may provide an opportunity to further reduce
macular thickness and improve vision.
In summary, we show that the KKS is increased in human DME vitreous, Klkb1
deficiency in mice decreases diabetes induced RVP, and the effects of the BK on retinal
thickening do not require VEGF R2. Both B1R and B2R contribute to effects of the KKS on
retinal thickening and RVP, and these responses are mediated by eNOS and iNOS,
respectively. These findings suggest that the intraocular KKS may contribute to DME via a
VEGF independent mechanism.
Acknowledgements
The project described was supported in part by JDRF grant 17 2011 251, Massachusetts
Lions Eye Research Fund, and NIH Award Numbers EY019029, DK36836, and
T32EY007145, and Japan Society for the Promotion of Science (JSPS) Postdoctoral
21 Diabetes Page 22 of 52
Fellowships for Research Abroad. We thank Leilei Sun and Xiaohong Chen for their assistance with the proteomic analyses.
Edward P. Feener is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
E.P.F and L.P.A are cofounders of KalVista Pharmaceuticals Ltd. The Joslin Diabetes Center has a US patent 8,658,685 on methods for treatment of kallikrein related disorders. No potential conflicts of interest relevant to this article for T.K., A.C.C, N.M., Q.Z. K.F., and T.I. were reported.
Author Contributions
T.K. designed and performed experiments and assisted in writing the manuscript. ACC, NM, and QZ performed experiments and edited the manuscript. KF contributed to collection vitreous samples. TI supervised clinical work. LPA contributed to study design, review the data, and edited the manuscript. EPF conceived and designed the overall study, reviewed data, and wrote the manuscript.
Abbreviations: Bovine retinal endothelial cells, BREC; bradykinin, BK;
22 Page 23 of 52 Diabetes
des Arg9 Bradykinin, DABK; diabetes mellitus, DM; diabetic macular edema, DME;
diabetic retinopathy, DR; kallikrein kinin system, KKS; no diabetes mellitus, macular hole,
MH; NDM; plasma kallikrein, PKal; plasma prekallikrein, PPK; proliferative diabetic
retinopathy, PDR; retinal vascular permeability, RVP; vascular endothelial growth factor,
VEGF.
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Figure Legends:
Figure 1. Concentrations of KKS components and VEGF in human vitreous. Western blot analyses of plasma prekallikrein (PPK), plasma kallikrein (PKal), and high molecular weight kininogen heavy chain (HK) in vitreous samples from subjects with macular hole
(MH), diabetic macular edema (DME), and proliferative diabetic retinopathy (PDR) (A,D).
Concentrations of PPK (B), PKal (C), HK (E), and VEGF (F) in undiluted vitreous samples.
Correlations of PPK and VEGF is r=0.168, p=0.499 in DME vitreous samples (G). Levels of
PKal in DME vitreous samples ranked in ascending order of VEGF concentration. Samples used for proteomics are circled (H). **p<0.0001 vs. MH (Mann Whitney U test).
Figure 2. Proteomics of DME vitreous and correlations with PKal and VEGF. Venn diagram summary of total proteins identified in DME and MH vitreous by mass spectroscopy based proteomics (A). Correlations of proteins that are increased (> 3 fold, p<0.001) in DME vitreous with respective intravitreal PPK and VEGF concentrations. The
10 proteins that displayed the largest fold increase in DME compared with MH vitreous are
34 Page 35 of 52 Diabetes
labeled with gene symbols (B). Correlations of total vitreous protein concentration with PPK
(C) and VEGF (D) concentrations.
Figure 3. Effects of intravitreal injection of bradykinin (BK) and des-Arg9 bradykinin
(DABK) on retinal vascular permeability and retinal thickness in diabetic rats. Rat with
4 weeks of STZ induced diabetes and age matched nondiabetic controls received intravitreal
injections with BK (final concentration: 2µM), DABK (final concentration: 2 µM), and
saline vehicle (PBS), as indicated. Retinal vascular permeability was measured at 40 minutes
and 48 hours post intravitreal injection by vitreous fluorophotometry (VFP) (A C). Rats were
pretreated with Hoe140 or des Arg10Hoe140 (1 µg/kg/day) prior to IVT injections as shown
in panels B&C. Co intravitreal injections with bevacizumab were performed in panel C.
Representative SD OCT B scans images of rat retina following intravitreal injections with
PBS, BK, and DABK. Retinal thickness was quantified by SD OCT (D). *p<0.05, **p<0.01
vs. baseline.
Figure 4. Effects of intravitreal injection of human DME vitreous on retinal vascular
permeability in diabetic rats. Human vitreous samples, including DME patient ID#12 with
relatively high PKal and low VEGF (PK↑ vit: PPK 91.6 nM (18.1 fold increase in PKal),
VEGF: 114pg/ml), DME patient ID#13 with low PKal and high VEGF (VEGF↑ vit: PPK
25.2 nM (2.3 fold increase in PKal), VEGF: 2864 pg/ml), and control MH vitreous with low
levels of both PKal and VEGF, were injected into rat vitreous. Co injections were performed
with Hoe140/desArg10Hoe140 and bevacizumab (Bev) as indicated and RVP was measured
35 Diabetes Page 36 of 52
by VFP at 40 min (A) and 48 hrs (B) post injection.
Figure 5. Effect of VEGFR2 blockade by DC101 on VEGF- and BK- induced retinal thickening. Retinal thickness was quantified by SD OCT in WT mice at baseline and at 24 hours post injection of PBS,VEGF, and BK with or without DC101 co injections. VEGF and
BK increased retinal thickness by 15.2 and 13.6% compared to baseline, respectively (* p<0.001). Co injection of VEGF with DC101 at 0.36 and 0.73 µg reduced VEGF induced thickness by 14.7 (NS) and 90% (** p<0.001) compared to VEGF alone, respectively.
Co injection of DC101 with BK had no effect upon BK induced thickness.
Figure 6. Effects of PPK deficiency (Klkb1-/-) on RVP and retinal thickness in diabetic mice. Diabetes increased RVP in female (A) and male (B) wild type (WT) mice by 138±31% and 400±63%, respectively, compared to NDM control (** p<0.001). Diabetes induced RVP was decreased in Klkb1 / mice by 58±11% (* p=0.033) in male mice and 78±6% (* p<0.001) in female mice. Diabetes decreased retinal thickness in female WT mice by
3.4±0.6% (* p=0.001) and female Klkb1 / mice by 1.6±0.6% (NS). The diabetes associated decrease in retinal thickness in female Klkb1 / mice was less than that observed in female
WT mice (** p=0.002). (C). Diabetes decreased total retinal thickness in male WT mice by
5.8±0.8% (* p<0.001) and male Klkb1 / mice by 3.7±0.8% (** p=0.004) (D).
Figure 7. Role of nitric oxide synthase (NOS) on retinal vascular permeability (RVP)
36 Page 37 of 52 Diabetes
induced by bradykinin peptides. Effect of L NAME on DABK and BK induced RVP
measured using vitreous fluorophotometry (A). Phosphorylation state of eNOS by bovine
retinal endothelial cells (BRECs) in the presence of BK (100 nM) or DABK (100 nM) (B).
Effects of BK on RVP in wild type (WT) and eNOS deficient mice (C). Western blot analysis
of iNOS normalized to GAPDH in diabetic and nondiabetic mice injected with DABK (D).
Effect DABK on iNOS levels in astrocytes (E). Effects of DABK on RVP in WT and iNOS
deficient mice (F). ** p<0.01.
Supplemental Figure 1. Quantification of plasma KKS components in vitreous.
Concentrations of plasma prekallikrein (PPK) (A) and FXII (B) in vitreous were quantified
using a standard curve of band intensities of purified proteins visualized by western blotting.
Controls showing PKal from normal human plasma and FXIIa from a mixture of PPK, FXII
and HK were generated by incubation for 1 hr with kaolin (K) at room temperature (24oC)
prior to gel loading. Western blot analyses of carboxypeptidase N (CPN) and hemoglobin in
vitreous (C). Representative vitreous samples for patients with macular hole (MH), diabetic
macular edema (DME), and proliferative diabetic retinopathy (PDR) are shown.
Supplemental Figure 2. Comparisons among KKS components and VEGF in DME
vitreous. Concentration correlations for PPK and HK (A), PPK and FXII (B), FXII and HK
(C), FXII and VEGF (D), and HK and VEGF (E).
37 Diabetes Page 38 of 52
Supplemental Figure 3. B1R and B2R levels in retina from diabetic (DM) and nondiabetic (NDM) rats, and bovine retinal endothelial cells (BREC) and rat astrocytes exposed to high glucose. (A) Total retinal protein was extracted from 4 weeks STZ induced diabetic rats (DM, n=4) and age matched non diabetic controls (NDM, n=3), and analyzed by Western blotting. Total cell lysate was extracted from BRECs (n=3) (B) and astrocytes
(n=3) (C), which were untreated or treated with media containing high glucose (25mM) for the indicated period. B1R and B2R levels were normalized to GAPDH levels. Signal intensities were quantified as percentages of B1R/GAPDH or B2R/GAPDH ratio compared with NDM or day 0 (untreated). **p<0.01, compared with NDM or day 0.
Supplemental Figure 4. Blood glucose and body weight in diabetic (DM) and nondiabetic (NDM) wild type (WT) and Klkb1-/- mice. Blood glucose of female (A) and male (B) WT and Klkb1 / mice at DM onset and 3 months post induction of diabetes and age matched NDM controls. Body weight of female (C) and male (D) NDM and DM WT and Klkb1 / mice at baseline and after 3 months of diabetes. For body weight * p<0.001 compared to baseline and ** p=0.001 NDM 3 month body weight compared to DM mice. For blood glucose * P<0.01 DM onset blood glucose compared to NDM mice and ** p<0.001
DM 3 month blood glucose compared to NDM mice.
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Molecular Weight DME10 DME11 DME13 DME14 DME15 DME1 DME2 DME4 DME6 DME8 MH9 MH3 MH4 MH5 MH7 Average in DME Average in MH Average DME/Average MH Gene Symbol Description A1BG alpha‐1‐B glycoprotein 54261 72 51 53 52 68 66 70 52 72 70 11 11 17 30 9 62.6 15.6 4.0 A2M alpha‐2‐macroglobulin 163287 258 554 151 181 174 476 95 460 269 252 137 282 208 350 100 287 215.4 1.3 ABI3BP ABI family, member 3 (NESH) binding protein 118713 3 3 6 12 8 14 14 1.4 10.8 0.1 ACTB2 actin, beta 41710 4 7 8 10 4 7 7 3.8 3.4 1.1 ACTBL2 actin, beta‐like 2 41990 4 3 3 1.5 1.4 1.1 AFM afamin 69061 28 36 20 24 32 24 12 20 17 29 3 4 8 24.2 3.4 7.1 AGA aspartylglucosaminidase 37181 6 7 2.1 1 2.1 AGT angiotensinogen (serpin peptidase inhibitor, clade A, member 8) 53147 13 9 15 14 8 13 8 15 2 13 5 10 18 17 4 11 10.8 1.0 ALB albumin 69357 3938 4523 3277 3445 6246 4565 6180 5039 5132 8058 2082 1712 1973 2611 1443 5040.3 1964.2 2.6 AHSG alpha‐2‐HS‐glycoprotein 39313 18 11 8 12 33 22 15 14 14 28 5546 17.5 4.2 4.2 AMBP alpha‐1‐microglobulin/bikunin precursor 38986 58 90 52 86 65 55 247 61 33 72 4 2 7 17 81.9 6.2 13.2 APCS amyloid P component, serum 25372 8 10 11 7 4 4 5 5.2 1 5.2 APLP1 amyloid beta (A4) precursor‐like protein 1 72245 4 5 5 4 3 7 7749 3.25.60.6 APLP2 amyloid beta (A4) precursor‐like protein 2 80716 4 7 15 12 24751299537277.7 19.4 0.4 APOA1 apolipoprotein A‐I 30766 577 708 417 466 774 601 749 783 642 926 139 142 87 176 34 664.3 115.6 5.7 APOA2 apolipoprotein A‐II 11159 50 69 17 43 101 105 79 46 64 69 6 14 10 4 64.3 7 9.2 APOA4 apolipoprotein A‐IV 45368 94 202 105 144 90 102 423 97 82 191 25 41 36 49 7 153 31.6 4.8 APOB apolipoprotein B 515632 36 92 15 16 10 164 53 2 5 39.4 1 39.4 APOC1 apolipoprotein C‐I 9315 3 3 1.4 1 1.4 APOC2 apolipoprotein C‐II 11268 11 16 6 6 12 11 6 8 3 8 1 8.0 APOC3 apolipoprotein C‐III 12799 23 19 10 6 13 9 12 4 4 3 10.3 1 10.3 APOD apolipoprotein D 21260 13 21 14 21 22 13 24 8 14 12 4 16.2 1.6 10.1 APOE apolipoprotein E 36146 226 200 137 224 188 192 137 189 161 148 138 188 35 107 55 180.2 104.6 1.7 APOH apolipoprotein H (beta‐2‐glycoprotein I) 30323 6 14 4 5 14 10 13 10 5 21 5 4 10.2 2.4 4.3 APOL1 apolipoprotein L, 1 43962 5 9 6 4 3 1 3.0 APP amyloid beta (A4) precursor protein 84814 5 2 8 1.5 2.4 0.6 ASAH1 N‐acylsphingosine amidohydrolase (acid ceramidase) 1 46490 367345 415 3.24.40.7 ATP6AP1 ATPase, H+ transporting, lysosomal accessory protein 1 52016 2 9 9 3 1.1 4.6 0.2 ATRN attractin 150812 4 7 2334 2.7 1 2.7 AZGP1 alpha‐2‐glycoprotein 1, zinc‐binding 34247 103 110 111 128 80 108 207 103 93 103 46 62 41 53 10 114.6 42.4 2.7 B2M beta‐2‐microglobulin 13697 18 17 22 23 22 15 33 6 15 14 14 21 10 9 18.5 11 1.7 B3GNT1 UDP‐GlcNAc:betaGal beta‐1,3‐N‐acetylglucosaminyltransferase 1 47109 7 10 8 8 5346 823 2 5.37 0.8 CALM1 calmodulin 1 (phosphorylase kinase, delta) 17147 2 3 5 2 1.8 1 1.8 C1QA complement component 1, q subcomponent, A chain 26004 4 7 11 4 7 6 4.3 1 4.3 C1QB complement component 1, q subcomponent, B chain 24365 5 10 7 7 5 19 2 13 4 10 7 8.2 2.2 3.7 C1QC complement component 1, q subcomponent, C chain 25759 10 12 10 10 12 14 10 10 10 16 6 13 4 11.4 5 2.3 C1R complement component 1, r subcomponent 81886 3 3 2 3 1.7 1 1.7 C1S complement component 1, s subcomponent 75896 2 4 1 1.8 0.6 C3 complement component 3 187163 581 471 279 371 494 582 260 562 368 441 73 87 67 215 28 440.9 94 4.7 C4B complement component 4B (Chido blood group) 192892 213 280 222 295 225 265 209 253 154 207 19 250 55 109 22 232.3 91 2.6 C5 complement component 5 188339 30 23 16 36 6 30 30 11 7 5 19 1.8 10.6 C6 complement component 6 105751 23 17 14 27 30 31 26 11 12 9 19.2 2.6 7.4 C7 complement component 7 93514 13 11 4 5 18 20 8 13 6 8 2 3 10.6 1.6 6.6 C8G complement component 8, gamma polypeptide 22265 31 31 8 8 32 15 33 15 19 27 7 6 21.9 3.2 6.8 C9 complement component 9 63162 5 7 22 18 6 7 13 2 4 4 2 8.8 1.2 7.3 CA1 carbonic anhydrase I 28855 2 16 3 18 17 20 2.8 11.4 0.2 CD14 CD14 molecule 40064 2 2 4 1.2 1.6 0.8 CDH2 cadherin 2, type 1, N‐cadherin (neuronal) 99807 4 7 13 4 10 8 14 6 12 4 15 11 3 7 8.2 7.4 1.1 CFB complement factor B 140947 58 7 50 53 65 48 77 50 49 61 9 20 12 26 4 51.8 14.2 3.6 CFD complement factor D (adipsin) 27765 6355243 5 1 5.0 CFH complement factor H 139095 43 63 24 34 51 43 26 81 36 32 8 3 17 3 43.3 6.4 6.8 CFHR2 complement factor H‐related 2 27883 4 5 4 2 1 2.0 CFI complement factor I 66687 42 57 34 36 30 34 25 36 29 33 16 40 2 22 35.6 16.2 2.2 CHI3L1 chitinase 3‐like 1 (cartilage glycoprotein‐39) 42614 29 22 6 30 6 24 10 34 30 19 12 32 9 25 2 21 16 1.3 CHL1 cell adhesion molecule L1‐like 135028 2 7 1.1 2.2 0.5 CLEC3B C‐type lectin domain family 3, member B 22553 28 24 18 23 49 28 31 20 20 36 6 6 27.7 3 9.2 CLSTN1 calsyntenin 1 107812 11 13 28 19 7 13 10 12 18 7 34 42 15 49 9 13.8 29.8 0.5 CLU clusterin 52357 326 394 224 402 274 417 459 342 447 574 248 597 143 286 93 385.9 273.4 1.4 COL18A1 collagen, type XVIII, alpha 1 178169 3 8 1.2 2.4 0.5 COL2A1 collagen, type II, alpha 1 40939 2 2 4 2 4 5 1.6 2.4 0.7 CP ceruloplasmin (ferroxidase) 122201 434 431 294 360 425 436 332 375 329 399 123 228 111 236 44 381.5 148.4 2.6 CPE carboxypeptidase E 63664 5 3 16 23 8 10 6 3 13 2 14 20 5 10 8.9 10 0.9 CRYAA crystallin, alpha A 19894 13 13 8 15 19 4.1 7.4 0.6 CRYAB crystallin, alpha B 17909 4 10 24 2.2 5.6 0.4 CRYBA1 crystallin, beta A1 23177 4 3 1.3 1.4 0.9 CRYBB1 crystallin, beta B1 28009 7 8 7 1.6 3.6 0.4 CRYBB2 Beta‐crystallin B2 23367 5 26 23 17 15 7 39 7.7 12.6 0.6 CRYGS crystallin, gamma S 14249 11 31 23 5 23 4 5 6 2 23 10.5 6.6 1.6 CST3 cystatin C 15783 40 49 21 32 11 37 48 32 56 46 38 61 29 39 15 37.2 36.4 1.0 CTSD cathepsin D 44540 19 26 12 23 11 13 9 18 24 17 24 96 29 30 9 17.2 37.6 0.5 CTSL 0 gene was found in Genecards 25487 3 7 1.2 2.2 0.5 CUTA cutA divalent cation tolerance homolog (E. coli) 19100 4 4 1.6 1 1.6 DAG1 dystroglycan 1 (dystrophin‐associated glycoprotein 1) 97575 3 2 3 5 6 4 1.9 2.6 0.7 DKK3 dickkopf WNT signaling pathway inhibitor 3 35203 6 9 6 25 8 6 2 5 13 30 108 18 20 6 8.1 36.4 0.2 EFEMP1 EGF containing fibulin‐like extracellular matrix protein 1 39185 2 2 13 4 2 2 1.2 4.4 0.3 ENPP2 ectonucleotide pyrophosphatase/phosphodiesterase 2 101959 9 9 16 13 3586318810137.3100.7 F12 coagulation factor XII (Hageman factor) 67813 4 2 2 1.5 1 1.5 F2 coagulation factor II (thrombin) 70029 18 21 17 18 19 13 20 19 21 12 4 4 3 17.8 2.6 6.8 FAM3C family with sequence similarity 3, member C 24665 2 3 14 7 15 6 4 14 5 5 9 11 7.1 5.4 1.3 FBLN1 fibulin 1 77255 2 6 5 2 3 3 6 2 4 3 1.8 1.7 FCGBP Fc fragment of IgG binding protein 572150 19 18 6 34 20 27 9 11 10 17 14.6 6 2.4 FCN3 ficolin (collagen/fibrinogen domain containing) 3 31663 5 2 3 3 1.9 1 1.9 FGA fibrinogen alpha chain 69747 224 391 74 147 216 422 117 262 94 291 25 223.8 5.8 38.6 FGB fibrinogen beta chain 55919 72 129 21 56 60 183 4 143 47 200 5 5 26 68 4 91.5 21.6 4.2 FGG fibrinogen gamma chain 50313 120 158 26 76 79 280 23 164 103 256 4 46 111 2 128.5 32.8 3.9 FN1 fibronectin 1 249329 18 51 10 16 41 84 22 70 10 20 9 26 7 23 34.2 13.2 2.6 FRZB frizzled‐related protein 36239 7 7662 66 12 4.33.21.3 FSTL5 follistatin‐like 5 94595 5 3 1 2.2 0.5 GAPDH glyceraldehyde-3-phosphate dehydrogenase 31533 8 19 8 11 9 11 5 4 7 2 8.3 1.2 6.9 GC group‐specific component (vitamin D binding protein) 52905 101 79 93 109 112 95 136 82 81 122 36 35 37 51 7 101 33.2 3.0 GPX3 glutathione peroxidase 3 (plasma) 25491 65 92 73 97 93 85 66 77 68 102 57 83 50 51 22 81.8 52.6 1.6 GSN gelsolin 85694 71 74 87 102 101 87 106 79 56 75 2 68 30 63 9 83.8 34.4 2.4 HBA1 hemoglobin, alpha 1 15109 13 3 10 37 2 7 5 30 4 9 137 63 78 11.2 57.6 0.2 HBB hemoglobin, beta 15850 50 20 17 100 6 19 23 22 84 18 17 297 159 171 35.9 129 0.3 HBD hemoglobin, delta 9789 3 2 1 1.6 0.6 HP haptoglobin 45193 230 317 198 242 454 288 230 235 143 273 49 23 43 82 20 261 43.4 6.0 HPX hemopexin 51664 188 203 174 206 212 180 222 209 154 255 88 79 56 97 49 200.3 73.8 2.7 HRG histidine‐rich glycoprotein 59568 38 67 30 25 46 35 30 44 41 52 37 25 11 5 40.8 15.8 2.6 HRNR hornerin 282458 14 7 2.9 1 2.9 HS3ST6 heparan sulfate (glucosamine) 3‐O‐sulfotransferase 6 37173 4 2 1.4 1 1.4 HSPG2 heparan sulfate proteoglycan 2 468899 2 23 3 2 4 3.6 1.6 2.3 IGFALS insulin‐like growth factor binding protein, acid labile subunit 70212 2 2 3 4 1.7 1 1.7 IGFBP6 insulin‐like growth factor binding protein 6 25310 3 10 2.1 1 2.1 IGFBP7 insulin‐like growth factor binding protein 7 29116 3 7 1.2 2.2 0.5 ITIH1 inter‐alpha‐trypsin inhibitor heavy chain 1 101391 31 40 35 23 37 35 16 60 5 26 6 2 3 19 4 30.8 6.8 4.5 ITIH2 inter‐alpha‐trypsin inhibitor heavy chain 2 105216 65 88 36 70 70 86 37 77 24 43 4 7 3 24 59.6 7.8 7.6 ITIH3 inter‐alpha‐trypsin inhibitor heavy chain 3 99323 4 10 6 7362 4.1 1 4.1 ITIH4 inter‐alpha‐trypsin inhibitor heavy chain family, member 4 103360 72 94 69 118 92 79 54 120 70 46 8 9 13 32 81.4 12.6 6.5 KNG1 kininogen 1 47891 18 25 7 16 14 20 15 5 7 12 6 13.9 2 7.0 LCN1 lipocalin 1 19234 3 2 1 1.6 0.6 LMAN2 lectin, mannose‐binding 2 40214 2 4 4 2 1.8 1 1.8 LGALS3BP Lectin, Galactoside‐Binding, Soluble, 3 Binding Protein1 46409 4 3 1 2 0.5 LRG1 leucine‐rich alpha‐2‐glycoprotein 1 38168 13 17 28 23 27 21 18 12 11 11 3 3 9 18.1 3.4 5.3 LRP2 low density lipoprotein receptor‐related protein 2 522006 2 5 3833 1.53.60.4 LUM lumican 38415 5 8 5 8 18 6 10 4 7 6.3 2.8 2.3 LYZ lysozyme 16521 58444 16 4 4.81 4.8 MFAP4 microfibrillar‐associated protein 4 33100 15 8 10 7 5 7 5.6 1 5.6 NPC2 Niemann‐Pick disease, type C2 22395 6 3 1.5 1.4 1.1 NRCAM neuronal cell adhesion molecule 131718 3 4 2 9 1.5 2.8 0.5 OAF OAF homolog (Drosophila) 30676 11 14 17 27 17 18 16 10 10 10 5 12 2 3 15 4.6 3.3 OMG oligodendrocyte myelin glycoprotein 49597 6 4 1.8 1 1.8 OPTC opticin 37247 7 5 16 11 7 8 10 10 23 3 35 30 21 30 10 10 25.2 0.4 ORM1 orosomucoid 1 23525 152 82 112 142 88 114 236 65 56 131 53 26 47 41 11 117.8 35.6 3.3 PAPPA2 pappalysin 2 198550 2 4 1 1.8 0.6 PCSK1N proprotein convertase subtilisin/kexin type 1 inhibitor 27358 6669263445 6 5.12 2.6 PEBP1 phosphatidylethanolamine binding protein 1 17310 2 4 1.4 1 1.4 PGLYRP2 peptidoglycan recognition protein 2 67992 4 10 49434 8 4.81 4.8 PLG plasminogen 90563 36 33 34 46 43 29 31 30 15 44 3 9 10 14 34.1 7.4 4.6 PON1 paraoxonase 1 39735 13 11 4 12 10 15 5 13 5 13 10.1 1 10.1 PROS1 protein S (alpha) 60346 3 2 1.3 1 1.3 PRSS3 protease, serine, 3 26682 4 7 1.9 1 1.9 PRDX2 peroxiredoxin 2 20090 3 4 5 13 5 5 1.9 5 0.4 PTGDS prostaglandin D2 synthase 21kDa (brain) 22821 206 243 241 202 182 282 173 160 255 216 118 374 168 188 36 216 176.8 1.2 RARRES2 retinoic acid receptor responder (tazarotene induced) 2 18603 673858775 2 5.71.24.8 RBP3 retinol binding protein 3, interstitial 135366 241 203 305 271 231 239 140 273 352 235 298 684 215 319 129 249 329 0.8 RBP4 retinol binding protein 4, plasma 22995 133 175 203 241 237 195 408 127 107 85 13 18 11 23 191.1 13.2 14.5 RS1 retinoschisin 1 25578 4 6 6 5 2 15 24 5 5 2.8 10 0.3 RTBDN retbindin 24600 7 19 17 17 14 16 11 10 18 8 13 6 13 5.8 2.2 SAA4 serum amyloid A4, constitutive 14790 19 27 22 25 27 14 10 23 11 9 18.7 1 18.7 SEMA7A semaphorin 7A, GPI membrane anchor (John Milton Hagen bloo 74818 4 4 1 2.2 0.5 SERPINA1 serpin peptidase inhibitor, clade A (alpha‐1 antiproteinase, antit 46725 283 409 383 453 429 424 448 309 298 333 117 145 175 312 58 376.9 161.4 2.3 SERPINA3 serpin peptidase inhibitor, clade A (alpha‐1 antiproteinase, antit 47639 58 90 81 91 90 105 38 50 26 46 27 71 42 54 12 67.5 41.2 1.6 SERPINA4 serpin peptidase inhibitor, clade A (alpha‐1 antiproteinase, antit 48530 2 3 1.3 1 1.3 SERPINA5 serpin peptidase inhibitor, clade A (alpha‐1 antiproteinase, antit 45692 3 3 6 3 2937 3.81 3.8 SERPINA6 serpin peptidase inhibitor, clade A (alpha‐1 antiproteinase, antit 45130 3 2 1.2 1.2 1.0 SERPINA7 serpin peptidase inhibitor, clade A (alpha‐1 antiproteinase, antit 46313 3 5 3 1 2.6 0.4 SERPINC1 serpin peptidase inhibitor, clade C (antithrombin), member 1 52590 28 35 28 43 39 29 23 22 15 23 15 12 23 23 9 28.5 16.4 1.7 SERPIND1 serpin peptidase inhibitor, clade D (heparin cofactor), member 1 57061 3 10 53335 4 3.81 3.8 SERPINF1 serpin peptidase inhibitor, clade F (alpha‐2 antiplasmin, pigment 46330 84 103 101 113 78 77 97 83 160 95 124 401 212 123 49 99.1 181.8 0.5 SERPINF2 serpin peptidase inhibitor, clade F (alpha‐2 antiplasmin, pigment 47901 255652 737 4.31 4.3 SERPING1 serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 59484 74 80 63 82 48 93 57 65 70 57 37 63 41 57 11 68.9 41.8 1.6 SERPINI1 serpin peptidase inhibitor, clade I (neuroserpin), member 1 46415 4 7 16 7 4 1.3 7 0.2 SEZ6 seizure related 6 homolog (mouse) 94068 8 3 1 2.8 0.4 SHBG sex hormone‐binding globulin 43769 3 10 2.1 1 2.1 SOD1 superoxide dismutase 1, soluble 15788 4 6 6 3 2.5 1 2.5 SOD3 superoxide dismutase 3, extracellular 25837 8 2 3 4 3 2 2 1.0 SPARCL1 SPARC‐like 1 (hevin) 64134 4 4485 521011 7 3.56 0.6 SPON1 spondin 1, extracellular matrix protein 90969 4 9 9 10 862959173126.38.40.8 SPP1 secreted phosphoprotein 1 33003 7 2 7 3 11 10 15 37 7 6 4.4 13.2 0.3 SRP68 signal recognition particle 68kDa 70725 2 2 1.2 1 1.2 TF transferrin 77039 1030 965 982 965 1368 1285 1488 1368 1182 1542 697 1166 856 759 553 1217.5 806.2 1.5 TTR transthyretin 20181 599 738 316 293 465 531 739 440 580 786 290 669 298 217 102 548.7 315.2 1.7 UBC ubiquitin C 77029 18 54 1 15 0.1 VASN vasorin 71706 2 2 1.2 1 1.2 VCAN versican 369719 13 18 14 2 3.9 3.8 1.0 VTN vitronectin 54296 34 16 23 28 51 22 18 29 23 37 6 3 12 28.1 4.6 6.1 WIF1 WNT inhibitory factor 1 42466 11 14 30 24 14 30 17 6 18 12 6 25 7 9 17.6 9.6 1.8