page 1 of 52 diabetesand klkb1-/- at 12 weeks of diabetes duration. approval for the animal...

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Plasma Kallikrein-Kinin System as a VEGF-Independent Mediator of Diabetic

Macular Edema

Takeshi Kita1, Allen C. Clermont

1, Nivetha Murugesan

1 Qunfang Zhou

1, Kimihiko

Fujisawa2, Tatsuro Ishibashi

2, Lloyd Paul Aiello

1,3, Edward P. Feener

1,4

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]

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Diabetes Publish Ahead of Print, published online May 15, 2015

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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.

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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

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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

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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

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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.

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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

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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

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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).

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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-Arg10

Hoe140. 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-Arg10

Hoe140 (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-Arg10

Hoe140 (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

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(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

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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

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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

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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

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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

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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

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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;

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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

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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-Arg10

Hoe140 (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/desArg10

Hoe140 and bevacizumab (Bev) as indicated and RVP was measured

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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)

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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).

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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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Gene Symbol Description 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

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 5 5 4 6 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 7 7 4 9 3.2 5.6 0.6

APLP2 amyloid beta (A4) precursor‐like protein 2 80716 4 7 15 12 2 4 7 5 12 9 9 53 7 27 7.7 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 3 6 7 3 4 5 4 15 3.2 4.4 0.7

ATP6AP1 ATPase, H+ transporting, lysosomal accessory protein 1 52016 2 9 9 3 1.1 4.6 0.2

ATRN attractin 150812 4 7 2 3 3 4 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 5 3 4 6 8 23 2 5.3 7 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 6 3 5 5 24 3 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 3 5 8 6 3 18 8 10 13 7.3 10 0.7

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 7 6 6 2 6 6 12 4.3 3.2 1.3

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 7 3 6 2 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 3 8 3 3 1.5 3.6 0.4

LUM lumican 38415 5 8 5 8 18 6 10 4 7 6.3 2.8 2.3

LYZ lysozyme 16521 5 8 4 4 4 16 4 4.8 1 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 6 6 6 9 2 6 3 4 4 5 6 5.1 2 2.6

PEBP1 phosphatidylethanolamine binding protein 1 17310 2 4 1.4 1 1.4

PGLYRP2 peptidoglycan recognition protein 2 67992 4 10 4 9 4 3 4 8 4.8 1 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 6 7 3 8 5 8 7 7 5 2 5.7 1.2 4.8

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 2 9 3 7 3.8 1 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 5 3 3 3 5 4 3.8 1 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 2 5 5 6 5 2 7 3 7 4.3 1 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 4 4 8 5 5 2 10 11 7 3.5 6 0.6

SPON1 spondin 1, extracellular matrix protein 90969 4 9 9 10 8 6 2 9 5 9 17 3 12 6.3 8.4 0.8

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

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