Diabetes Volume 63, September 2014 3057

Sung Wook Park,1,2 Jang-Hyuk Yun,3,4 Jin Hyoung Kim,1 Kyu-Won Kim,5 Chung-Hyun Cho,3,4 and Jeong Hun Kim1,2,6

Angiopoietin 2 Induces Pericyte Apoptosis via a3b1 Signaling in Diabetic Retinopathy

Diabetes 2014;63:3057–3068 | DOI: 10.2337/db13-1942

Pericyte loss is an early characteristic change in diabetic microvascular complication in diabetic patients despite retinopathy (DR). Despite accumulating evidence that the recent improvement in the management of DR via hyperglycemia-induced angiopoietin 2 (Ang2) has a cen- glycemic control and photocoagulation (1). Macular edema tral role in pericyte loss, the precise molecular mecha- (leakage) and neovascularization () both cause nism has not been elucidated. This study investigated severe vision loss in DR (2), and pericyte loss is one of the the role of Ang2 in pericyte loss in DR. We demonstrated earliest and most characteristic changes of DR (3). that pericyte loss occurred with Ang2 increase in the The pericyte plays two major important clinical roles in COMPLICATIONS diabetic mouse retina and that the source of Ang2 could DR. First, the pericyte enwraps endothelial cells to keep be the endothelial cell. Ang2 induced pericyte apoptosis the integrity of inner blood–retinal barrier (BRB) with the via the p53 pathway under high glucose, whereas Ang2 role of microvascular autoregulation (4). Thus, pericyte alone did not induce apoptosis. Integrin, not Tie-2 re- loss could weaken the inner BRB, even when endothelial ceptor, was involved for Ang2-induced pericyte apopto- cells are intact, and lead to capillary instability and vas- sis under high glucose as an Ang2 receptor. High glucose changed the integrin expression pattern, which cular leakage in macular edema. Second, microaneurysm increased integrin a3 and b1 in the pericyte. Further- and neovascularization occur in proliferating endothelial more, Ang2-induced pericyte apoptosis in vitro was ef- cells at the site of pericyte loss (5). Because pericyte loss is fectively attenuated via p53 suppression by blocking an early diabetic change, preventing pericyte loss for the fi integrin a3 and b1. Although intravitreal injection of primary prevention of DR would be bene cial. Ang2 induced pericyte loss in C57BL/6J mice retina in Although pericyte loss is important in early DR, the vivo, intravitreal injection of anti-integrin a3 and b1 anti- mechanism by which hyperglycemia leads to pericyte loss bodies attenuated Ang2-induced pericyte loss. Taken to- remains largely unknown. However, Ang2 plays a critical role gether, Ang2 induced pericyte apoptosis under high in pericyte loss in DR. Hyperglycemia causes pericyte glucose via a3b1 integrin. Glycemic control or blocking apoptosis and, ultimately, pericyte loss (6–8). Ang2 increases Ang2/integrin signaling could be a potential therapeutic in the vitreous of patients with proliferative DR (9). In ad- target to prevent pericyte loss in early DR. dition, Ang2 is upregulated by hyperglycemia in the diabetic retina and in endothelial cells (10–12). Ang2 induces pericyte loss in normal mice retina and in mice overexpressing Ang2 Diabetic retinopathy (DR) is the leading cause of visual (10,13). Thus, we postulated that hyperglycemia increases loss in working-aged people and the most common Ang2, which in turn induces pericyte apoptosis in DR.

1Fight against Angiogenesis-Related Blindness Laboratory, Biomedical Research Corresponding authors: Jeong Hun Kim, [email protected]; and Chung-Hyun Institute, Seoul National University Hospital, Seoul, Korea Cho, [email protected]. 2 Department of Biomedical Sciences, College of Medicine, Seoul National Uni- Received 24 December 2013 and accepted 31 March 2014. versity, Seoul, Korea This article contains Supplementary Data online at http://diabetes 3Department of Pharmacology and Ischemic/Hypoxic Disease Institute, College of .diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1942/-/DC1. Medicine, Seoul National University, Seoul, Korea 4Cancer Research Institute, College of Medicine, Seoul National University, Seoul, S.W.P. and J.-H.Y. contributed equally to this work. Korea © 2014 by the American Diabetes Association. Readers may use this article as 5Department of Pharmacy, Seoul National University, Seoul, Korea long as the work is properly cited, the use is educational and not for profit, and 6Department of Ophthalmology, Seoul National University Hospital, Seoul, Korea the work is not altered. 3058 Ang2 Induces Pericyte Loss via a3b1 Integrin Diabetes Volume 63, September 2014

The role of Ang2 in pericyte loss has been studied MAB1973), anti-integrin a3 (clone P1B5, MAB1952), (10,11,13–15). Apoptosis and migration are both sug- anti-integrin b1 (clone 6S6, MAB2253), and a-integrin gested mechanisms of pericyte loss by Ang2; however, blocking and immunohistochemistry (a1-6, v) were pur- the precise mechanism by which Ang2 induces pericyte chased from Millipore and used for functional blocking. loss has not yet been fully elucidated. Ang2 has been Small interfering (si)RNAs for p53 were purchased from known to bind to the endothelial-specific Tie-2 tyrosine Bioneer (Daejeon, Korea). fi receptor with similar af nity to Ang1 (16). Ang2 acts in Animals an autocrine manner in angiogenesis. This endothelial cell– All animal experiments in this study were in strict derived antagonistic ligand of vessel maturation and agreement with the Association for Research in Vision remodeling controls the Ang1–Tie-2 signaling axis (17). Al- and Ophthalmology Statement for the Use of Animals though Ang2 has been postulated to naturally bind to the in Ophthalmic and Vision Research and the guidelines Tie-2 receptor in the pericyte as the Ang-Tie system (11,13), of the Seoul National University Animal Care and Use whether Tie-2 indeed serves as a receptor for Ang2-induced Committee. Eight-week-old, pathogen-free male C57BL/6J pericyte loss is not clear. Integrin was recently found to mice were purchased from Central Laboratory Animal mediate platelet-derived -BB–induced pericyte Inc. After an 8-h fast, diabetes was induced by one loss in tumor vessels (18). Also, Ang2 binds to integrin intraperitoneal injection of freshly prepared streptozotocin and regulates angiogenesis through integrin signaling (STZ; Sigma-Aldrich) at a concentration of 180 mg/kg (19). Thus, we hypothesized that Ang2 induces pericyte body weight in 10 mmol/L citrate buffer (pH 4.5). Age- apoptosis via integrin signaling. matched controls received citrate buffer only. Mice with In this study, we demonstrated that Ang2 induced blood glucose levels .300 mg/dL 4 days after STZ in- pericyte apoptosis via the p53 pathway under high glucose. jection were deemed diabetic. Interestingly, integrin, not Tie-2 receptor, was important Diabetic and nondiabetic mice were killed 6 months after for Ang2-induced pericyte apoptosis under high glucose. diabetes induction. Eyes were collected under deep anesthe- High glucose increased integrin a3b1 in pericytes. Further- sia and immediately frozen at 280°C for ELISA or were fixed more, our results showed Ang2-induced pericyte apoptosis in 4% paraformaldehyde for retinal digestion. Glucose levels was effectively attenuated by blocking integrin a3b1in and body weight were monitored consecutively, and glycated vitro and in vivo. Taken together, Ang2 induced pericyte hemoglobin was determined before mice were killed. apoptosis via a3b1 integrin signaling in DR. Retinal Digest Preparations RESEARCH DESIGN AND METHODS Vascular preparations of whole-mount retinas were per- fl Cell Cultures formed using a trypsin digestion technique. Brie y, retinas fi Human umbilical vein endothelial cells (HUVECs; Lonza), were xed for at least 24 h in 4% paraformaldehyde, human retina microvascular endothelial cells (HRMECs; incubated in water for 1 h, and then digested in 2.5% ACBRI), human brain astrocytes (ACBRI), and human trypsin (Gibco) at 37°C for 1 h. After careful removal of the pericytes (PromoCell) were maintained in EBM-2, M199 inner limiting membrane, the retinal vessels were isolated fi medium, DMEM with 20% FBS, and pericyte media by careful irrigation with ltered water. The retinal digest containing growth factors (PromoCell), respectively. All samples were dried and stained with periodic acid Schiff cells were cultured at 37°C in an incubator with a humid- base for 15 min and hematoxylin. fi i ed atmosphere of 95% O2 and 5% CO2. Morphological Quantification of Pericytes and Acellular Capillaries Reagents and Antibodies To determine numbers of retinal pericytes and acellular Recombinant mouse and human Ang2, human integrin capillary, retinal digest preparations (n =7–8) were ana- a b – 3 1, and phycoerythrin (PE)-conjugated anti Tie-2 and lyzed. Pericytes were identified according to the morphol- mouse IgG antibodies were purchased from R&D Systems. ogy and relative location to capillaries. Total pericytes – Other reagents and antibodies were anti Bcl-2 family were counted in 10 randomly selected areas (original mag- – – antibodies (Epitomics); anti phospho-p53, anti poly nification 3400) in the middle one-third of the retinal ADP ribose polymerase (PARP), anti-cleaved caspase-3, capillary area. The number of pericytes and acellular capil- b anti-integrin 1 antibodies (Cell Signaling Technology); laries were standardized to the capillary area (numbers of anti-p53, anti-Tie1, peroxidase-conjugated secondary anti- cells or acellular capillaries/mm2 capillary area). The cap- bodies (Santa Cruz Biotechnology); anti-Tie2 antibody and illary area was calculated using the NIS-Elements AR 3.2 H-Gly-Arg-Gly-Asp-Ser-OH (GRGDS) (Millipore); program (Nikon, Tokyo, Japan). Samples were evaluated MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in a masked fashion. bromide; Sigma-Aldrich); fluorescein isothiocyanate– conjugated annexin V/propidium iodide assay kit (BD Intravitreal Injection of Ang2 and Anti-Integrin Biosciences); anti–neuron-glial antigen 2 (NG2), anti- Antibodies integrin a3b1, anti-Ang2 antibodies (Abcam); and TUNEL Normal 6-week-old male mice were used. An intravitreal fluorescein kit (Roche). Anti-integrin a1(cloneFB12, injection was performed under deep anesthesia with 1 mL diabetes.diabetesjournals.org Park and Associates 3059 containing 100 ng Ang2 and 500 ng anti-integrin a3 TTGAGAGCA-39); ITGA3 (forward: 59-AAGGGACCTTCAG (clone P1B5) or anti-integrin b1 (clone 6S6). Sterile PBS GTGCA-39 and reverse: 59-TGTAGCCGGTGATTTACCAT-39); was injected for control. After 10 days, retinas underwent ITGB1 (forward: 59-GAAGGGTTGCCCTCCAGA-39 and digestion preparation. reverse: 59-GCTTGAGCTTCTCTGCTGTT-39); and b-actin (forward: 59-GCCGCCAGCTCACCAT-39 and reverse: 59- Transferase-Mediated TUNEL Assay and 9 Immunofluorescence TCGATGGGGTACTTCAGGGT-3 ). A mean quantity was Retinal digestion preparations were incubated with anti- calculated from triplicate qPCR for each sample and nor- rabbit NG2 antibody (1:100) and a TUNEL fluorescein kit. malized to the control gene. Nuclei were counterstained with DAPI. TUNEL and NG2 Different primers were used for Tie-2 RT-PCR (forward: 9 9 9 double-positive cells were evaluated with a fluorescence 5 -TGTTCCTGTGCCACAGGCTG-3 and reverse: 5 -CACT 9 microscope (Nikon). GTCCCATCCGGCTTCA-3 ). PCR products were separated on 1% agarose gels and visualized using SYBR Safe DNA Cell Viability Assay Gel Stain (Invitrogen) under ultraviolet transillumination. In all experiments, 2.5 3 104 cells were seeded into 96-well plates. After 24 h, cells were treated with Ang1 Immunoprecipitation and Immunoblotting a b (300 ng/mL), Ang2 (300 ng/mL) under normal glucose For immunoprecipitation, Ang2 (500 ng), integrin 3 1 a b m (5 mmol/L glucose), high glucose (25 mmol/L glucose), (500 ng), and anti-integrin 3 1 antibodies (2 g) were and high mannitol (5 mmol/L glucose and 20 mmol/L incubated with G-Sepharose beads at 4°C overnight (19). mannitol) as an osmotic control for 48 h. Cell viability Immune complexes were collected by centrifugation and was determined by MTT assay according to the manufac- washed with buffer three times. For immunoblotting, cells turer’s instructions. Three independent experiments were were harvested and lysed in radioimmunoprecipitation as- performed for each experimental condition. say buffer with a protease inhibitor cocktail. lysates were resolved by SDS-PAGE and transferred onto nitrocel- FACS Analysis lulose membrane. The membranes were incubated with To evaluate apoptosis, 5 3 105 cells were treated with primary antibodies (1:1,000) at 4°C overnight and second- Ang2 (300 ng/mL) under normal glucose, high mannitol, ary antibodies (1:5,000) at room temperature for 1 h. The and high glucose at 37°C for 48 h. To determine the effect membranes were incubated with enhanced chemilumines- of integrin blocking, cells were treated with anti–integrin cent substrate (Pierce) and exposed to film. blocking antibodies (5 mg/mL) 1 h before the addition of Ang2. The cells were harvested and washed twice in PBS. Statistical Analysis Cells were stained with fluorescein isothiocyanate annexin-V Statistical analyses were performed using the standard and propidium iodide (PI) for 15 min and analyzed by flow two-tailed Student t test assuming unequal variances, and , fi cytometry. Annexin V–positive/PI-negative cells were de- P 0.01 was considered statistically signi cant. Quanti- 6 fi termined to be apoptotic. tative data are given as mean SD. Data in gures are 6 To evaluate Tie-2 expression, pericytes and HUVECs depicted as mean SE. (1 3 106) were suspended in the complete media (25 mL) for each experiment. PE-conjugated anti-Tie2 antibody was RESULTS added to the each sample at 4°C for 1 h. PE-conjugated Retinal Capillary Pericyte Numbers Are Decreased in mouse IgG antibody was used as a control. Diabetic Mice Retinas The numbers of pericytes and acellular capillaries in retinal Quantitative RT-PCR digestion were compared between mice with 6-month STZ- All RNA was collected and isolated from cells using the induced diabetes and age-matched nondiabetic control mice RNeasy Plus Mini kit (Qiagen). cDNAs were prepared (Fig. 1A). On one hand, the number of retinal capillary m m from RNAs (1 g) using 2.5 mol/L oligo-dT primers, pericytes was significantly decreased in the STZ-induced 1 mmol/L deoxyribonucleotide triphosphates, and murine diabetic mice retinas (932.6 6 70.3) compared with that leukemia virus RT. Quantitative (q)PCR assays were per- of the control group (1,341.8 6 55.9, P , 0.001; Fig. 1B). formed in qPCR Master Mix for SYBR Green PCR Master On the other hand, the number of retinal acellular capil- Mix (Applied Biosystems) using 7900HT real-time PCR laries was significantly increased in the STZ-induced dia- (Applied Biosystems). Reaction conditions were 50 cycles betic mice retinas (93.0 6 22.5) compared with that of the of 95°C for 5 s and 60°C for 20 s for qPCR. Quantitative control group (28.1 6 11.5, P , 0.001; Fig. 1C). Table 1 real-time PCR was performed using the following primers: reports the metabolic and physical parameters of the ex- 9 9 Ang2 (forward: 5 -ACTGTGTCCTCTTCCACCAC-3 and perimental groups. reverse: 59-GGATGTTTAGGGTCTTGCTTT-39); Tie-2 (for- ward: 59-GCTTGCTCCTTTCTGGAACTGT-39 and reverse: Ang2 Is Increased in Diabetic Retinas, and the Source 59-CGCCACCCAGAGGCAAT-39) (20); Tie-1 (forward: 59- of Ang2 Could Be Endothelial Cells AGAACCTAGCCTCCAAGATT-39 and reverse: 59-ACTGTA The effect of hyperglycemia on Ang2 expression in STZ- GTTCAGGGACTCAA-39); ITGA1 (forward: 59-GGTTCCT induced diabetic retinas was evaluated by qRT-PCR. In ACTTTGGCAGTATT-39 and reverse: 59- AACCTTGTCTGA diabetic retinas at 6 months, Angpt2 mRNA increased 3060 Ang2 Induces Pericyte Loss via a3b1 Integrin Diabetes Volume 63, September 2014

Figure 1—The number of retinal capillary pericytes is decreased in the diabetic mouse retina. Pericytes were identified in retinal digest preparations by morphologic criteria (shape, staining intensity, and relative position in the capillary) and quantitated in 6-month STZ- induced diabetic mice (DM) and age-matched controls (Con). A: Representative examples of periodic acid Schiff– and hematoxylin-stained retinal digest preparations of nondiabetic and diabetic mice after 6 months of diabetes are shown. The arrows indicate pericytes, and the arrowheads indicate acellular capillaries (original magnification 3400; scale bar = 20 mm). The number of retinal pericytes (B) and acellular capillaries (C) are shown normalized to the area of capillaries (mm2) in which they were counted (n = 8 mice in each group). The bar graph represents mean 6 SE. *P < 0.01 by Student t test.

1.87-fold compared with nondiabetic normal retinas (P = Ang2 Plays Synergistic Role in Pericyte Apoptosis 0.004; Fig. 2A). Also, Angpt1 mRNA increased 2.54-fold Under High-Glucose Conditions (P = 0.004) and Vegfa mRNA increased 1.6-fold (P = We determined the effect of Ang2 on the cell viability and 0.027; Supplementary Fig. 1A and B). apoptosis of pericytes under high glucose. Ang2 alone did Next, to determine the source of Ang2 in the diabetic not affect cell viability in pericytes (97.6 6 3.6%, P = retinas, we examined the effects of high glucose on in 0.221). High glucose reduced cell viability in pericytes vitro ANGPT2 mRNA transcription by qRT-PCR in three (89.4 6 7.9%, P = 0.020; Fig. 3A). Interestingly, Ang2 major components of the BRB: HRMECs, pericytes, and astrocytes. On one hand, high glucose increased ANGPT2 Table 1—Metabolic and physical parameters of the mRNA level in HRMECs more than 1.5-fold (1.52 6 0.09, STZ-induced diabetic and age-matched nondiabetic P = 0.003) compared with normal glucose (Fig. 2B). On control mice at 6 months the other hand, high mannitol, an osmotic control, did Nondiabetic Diabetic P value not increase ANGPT2 mRNA (1.21 6 0.13, P = 0.094) in Body weight (g) 32.46 6 3.22 18.97 6 0.69 ,0.001 HRMECs (Fig. 2B). Also, high glucose did not increase Blood glucose ANGPT2 mRNA in pericytes (1.13 6 0.22, P = 0.418) (mmol/L) 10.11 6 1.48 32.83 6 1.34 ,0.001 6 and astrocytes (1.13 0.22, P = 0.418; Fig. 2B and C). HbA1c (%) 5.33 6 0.88 8.90 6 1.10 ,0.001 These data demonstrate that Ang2 increases in the dia- HbA1c (mmol/mol) 34.75 6 9.74 73.75 6 11.95 betic retina and that the source of Ang2 increase is retinal Data are presented as mean 6 SD. microvascular endothelial cells. diabetes.diabetesjournals.org Park and Associates 3061

Figure 2—Ang2 is increased in diabetic retinas, and the source of Ang2 could be endothelial cells. A: Angpt2 mRNA levels were determined in 6-month STZ-induced diabetic mice (DM) retinas compared with age-matched control (Con) mice by qRT-PCR and normalized to Rn18s mRNA. Angpt2 expression increased in 6-month STZ-induced DM retinas (n = 6 mice in each group). ANGPT2 mRNA transcription is induced by high glucose (HG; 25 mmol/L) in HRMECs (B) but not in pericytes (C) or astrocytes (D). HRMEC, pericytes, and astrocytes were incubated for 48 h under 25 mmol/L HG, 20 mmol/L mannitol plus 5 mmol/L glucose (HM), or 5 mmol/L glucose (NG) as an osmotic control. ANGPT2 mRNA transcription was assessed by qRT-PCR, with actin as an internal control. ANGPT2 mRNA levels were normalized to ACTIN mRNA and reported as fold-induction compared with cells exposed to 5 mmol/L glucose. Bar graph represents mean 6 SE. *P < 0.01, #P > 0.05 by Student t test.

aggravated cell death under high glucose in pericytes normal glucose (Fig. 4A). Next, we aimed to identify the (72.4 6 2.9%, P = 0.002; Fig. 3A). Next, pericyte apoptosis mechanism that mediates Ang2-induced pericyte apopto- was assessed by annexin-V/PI flow cytometric analysis. sis under high glucose. We found that Ang2 induced p53 Of importance, the number of apoptotic pericytes was in- phosphorylation (Fig. 4B) and, subsequently, p53 accumu- creased under high glucose (7.7 6 0.3%, P , 0.001) com- lation (Fig. 4C) under high glucose. Then, to determine pared that of the control group (2.4 6 0.4%). Furthermore, the role of the p53 pathway for the observed Ang2- Ang2 significantly aggravated high glucose–induced pericyte mediated pericyte apoptosis, we treated pericytes with apoptosis (25.9 6 0.4%, P , 0.001), whereas Ang2 alone control siRNA or two different p53 siRNAs. The p53 did not induce apoptosis (2.7 6 0.1%, P = 0.298) under siRNAs effectively downregulated p53 expression (Fig. 4D) normal glucose (Fig. 3B and Supplementary Fig. 2A). and also attenuated Ang2-induced pericyte apoptosis under Next, we determined the effect of Ang2 on the cell high glucose (Fig. 4E). Interestingly, Ang2 phosphorylated viability and apoptosis of HRMECs under high-glucose extracellular signal–related kinase (ERK), but not Akt conditions. Ang1 and Ang2 increased cell viability in (Fig. 4F). This ERK phosphorylation by Ang2 was HRMECs under high glucose (114.4 6 8.8% [P = 0.014] inhibited by PD98059 (ERK inhibitor), and the ERK and 109.5 6 2.3% [P = 0.040], respectively; Fig. 3C). As inhibitor attenuated Ang2-induced p53 phosphoryla- expected, Ang2 significantly decreased the apoptotic cell tion (Fig. 4G). These data suggest that Ang2 induces population in HRMECs (Fig. 3D, and Supplementary Fig. pericyte apoptosis via the p53 pathway under high- 2B). These data suggested that Ang2 plays a synergistic glucose conditions. role in pericyte apoptosis under a high-glucose condition Integrin, Not Tie-2, Is Important for Ang2-Induced and has a protective effect on the endothelial cell. Pericyte Apoptosis Under High Glucose As an Ang2 Ang2 Induces Pericyte Apoptosis via the p53 Pathway Receptor Under High-Glucose Conditions To determine whether the Tie-2 receptor is related with Western blot studies confirmed that Ang2 induced the Ang2-induced pericyte apoptosis, Western blot analysis apoptosis pathway with an increase of Bax, cleaved PARP, (Fig. 5A)andRT-PCR(Fig.5B) for Tie-2 and Tie-1 were and cleaved caspase-3 under high glucose but not under performed on lysates obtained from HUVECs and pericytes. 3062 Ang2 Induces Pericyte Loss via a3b1 Integrin Diabetes Volume 63, September 2014

Figure 3—Ang2 plays synergistic role in pericyte apoptosis under high glucose (HG; 25 mmol/L glucose). The effects of Ang2 on the cell viability and apoptosis of pericytes and HRMECs under HG conditions were determined. Pericytes and HRMECs were incubated for 48 h with and without Ang1 (300 ng/mL) or Ang2 (300 ng/mL) under HG and compared with control (Con). A and C: Cell viability was assessed by MTT assay. Ang2 induced cell death under HG in pericytes (A) but not in HRMECs (C). Pericytes (B) and HRMECs (D) were stained with annexin-V fluorescein isothiocyanate and PI and analyzed by flow cytometry. Cell apoptosis was expressed as the percentage of apoptotic cells in total cell populations. B: HG induced pericyte apoptosis, and Ang2 aggravated that apoptosis. D: Ang2 showed a protective effect on HRMEC apoptosis. The bar graph represents the mean 6 SE of three independent experiments. HM, high mannitol (5 mmol/L glucose and 20 mmol/L mannitol); NG, normal glucose (5 mmol/L glucose). *P < 0.01 by Student t test.

Interestingly, pericytes did not express Tie-2 or Tie-1, changing the integrin pattern. We screened the integrin a whereas HUVECs expressed Tie-2 and Tie-1. These data subunits (a1–6 and av) to determine which integrin sub- were confirmed by qRT-PCR. TIE2 mRNA and TIE1 mRNA unit is responsible for Ang2-induced pericyte apoptosis. levels were significantly lower in pericytes than in HUVECs Integrin a1 and a3–blocking antibodies attenuated Ang2- (Fig. 5C). Compared with HUVECs with Tie-2 expression, induced p53 phosphorylation (Fig. 6A). pericytes did not express Tie-2 in FACS analysis (Fig. 5D). Next, to determine whether high glucose changes To test whether may serve as receptors for the integrin expression pattern, we performed qRT-PCR Ang2 in pericyte apoptosis under high glucose, we and Western blot studies for a1, a3, and b1. The choice incubated pericytes under high glucose for 15 min with was made because integrin a1anda3 can form the het- Ang2 and Gly-Arg-Gly-Asp-Ser (GRGDS) , which erodimer with only integrin b1 (22). High glucose in- can inhibit integrins that bind the Arg-Gly-Asp (RGD) creased mRNA levels at 24 and 48 h for ITGa1 (1.86- sequence (21). GRGDS (0.5 mg/mL) attenuated Ang2- fold and 2.20-fold, P , 0.01; Fig. 6B), ITGa3 (1.50-fold induced p53 phosphorylation (Fig. 5E). This result showed and 1.83-fold, P , 0.01; Fig. 6C), and ITGb1 (1.50-fold that integrin signaling is involved in Ang2-induced and 1.43-fold, P , 0.01; Fig. 6D), respectively. In addi- p53 phosphorylation. Of importance, these data sug- tion, high glucose increased integrin a1, a3, and b1 ex- gested that integrin, not Tie-2, is important for Ang2- pression (Fig. 6E). However, the integrin a1 was rarely induced pericyte apoptosis under high glucose as an Ang2 expressed compared with integrin a3. In this regard, we receptor. performed a coimmunoprecipitation assay to show direct a b a a b binding of Ang2 to integrin 3 1. Indeed, Ang2 directly High Glucose Increases Integrin 1, 3, and 1in a b Pericytes bound to integrin 3 1 (Fig. 6F). As shown in Fig. 3C and Fig. 5E, Ang2 induced pericyte Integrin a3b1 Suppression Inhibits Ang2-Induced apoptosis via the integrin receptor under high glucose but Pericyte Apoptosis not under normal glucose. We hypothesized that high From the result of integrin expression in pericytes under glucose preconditioned pericytes susceptible to Ang2 by high glucose, integrin a1b1ora3b1 were supposed to be diabetes.diabetesjournals.org Park and Associates 3063

Figure 4—Ang2 induces pericyte apoptosis via the p53 pathway under high glucose (HG; 25 mmol/L glucose). A: Western blot analysis for Bax, Bcl-2, Bcl-xL, cleaved caspase-3, and cleaved PARP were performed on lysates obtained from pericytes treated with Ang2 (300 ng/mL) under normal glucose (NG; 5 mmol/L glucose), high mannitol (HM; 5 mmol/L glucose and 20 mmol/L mannitol), and HG for 48 h compared with control (Con). B: Western blot analysis for phospho (p)-p53 (Ser15) was performed on lysates obtained from pericytes treated with Ang2 for 15, 30, and 60 min under HG (25 mmol/L). C: Western blot analysis for p53 was performed on lysates obtained from pericytes treated with Ang2 for 24 and 48 h under HG (25 mmol/L). D: After the pericyte transfection with control siRNA or p53 siRNA, Western blot analysis for p53 was performed on cell lysates with Ang2 for 48 h under HG (25 mmol/L), with b-tubulin used as a loading control. Data represent three independent experiments. E: Apoptotic cell counts were assessed by FACS analysis 48 h after Ang2 treatment in siRNA-transfected pericytes. The bar graph represents the mean 6 SE of three independent experiments. *P < 0.01 by Student t test. F: Ang2 was treated for 5, 15, 30, and 60 min under HG. Determination of p-ERK, ERK, p-Akt, and Akt was by Western blot. G: Pericytes were preincubated with Wortmannin (1 mmol/L) or PD98059 (20 mmol/L) for 1 h and treated with Ang2 for 15 min under HG. Determination of p-p53, p53, p-ERK, ERK, p-Akt, and Akt was by Western blot, with b-tubulin used as a loading control.

the possible receptor of Ang2. To determine whether integrin a1 antibody (Fig. 7A, and Supplementary Fig. Ang2 induced pericyte apoptosis through integrin a1b1 3A). These results were confirmed for p53 expression on or a3b1, pericytes were incubated under 25 mmol/L high Western blot analysis (Fig. 7B). Ang2 significantly in- glucose with Ang2 and anti-integrin a1, a3, and b1 anti- creased p53 expression under high glucose (2.7 6 0.2, bodies for 48 h. Interestingly, Ang2-induced pericyte P , 0.01) but was significantly attenuated by anti– apoptosis under high glucose was attenuated by anti– integrin a1, a3, and b1 antibodies (1.5 6 0.2, 1.0 6 integrin a3 and b1 antibodies but not by the anti– 0.1, and 0.7 6 0.1, respectively; P , 0.01). 3064 Ang2 Induces Pericyte Loss via a3b1 Integrin Diabetes Volume 63, September 2014

Figure 5—Integrin, not Tie-2, is important for Ang2-induced pericyte apoptosis under high glucose as an Ang2 receptor. Western blot (A) and RT-PCR (B) for Tie-2 and Tie-1 expression were performed on lysates obtained from HUVECs and pericytes. b-Tubulin was used as an internal control. C: TIE2 and TIE1 mRNA transcriptions were assessed by qRT-PCR. Actin was used as an internal control. TIE2 and TIE1 mRNA levels were normalized to ACTIN mRNA and reported as fold induction compared with HUVECs. *P < 0.01 by Student t test. D: HUVECs and pericytes were analyzed by flow cytometry for Tie-2 expression. E: Western blot analysis for phospho (p)-p53 (ser15) and p53 were performed on lysates obtained from pericytes treated with Ang2 (300 ng/mL) or GRGDS (0.5 mg/mL) under 25 mmol/L high glucose (HG) for 15 min. b-Tubulin was used as a loading control. Data represent three independent experiments. Con, control.

On the basis of in vitro experiments, we next intra- induced pericyte survival and recruitment (11); however, vitreously injected normal mice with 100 ng Ang2 with there was no direct evidence that Ang2 induced pericyte and without 500 ng anti-a3 or 500 ng anti-b1 antibodies. apoptosis via Tie-2. Unlike the previous study (11), peri- Ten days after the intravitreal injection, the isolated ret- cytes did not express Tie-2 mRNA or protein in our study. inas were digested with trypsin for pericyte evaluation Although Ang2 alone did not induce apoptosis in vitro (Fig. 7C). The in vivo intravitreal injection of Ang2 in- (11), Ang2 induced pericyte apoptosis under high glucose duced pericyte loss in the retina compared with that in via the p53 pathway (Fig. 4). Thus, we postulated that PBS injected control mice (1,047 6 52 and 1,401 6 109 Ang2 would induce Tie-2–independent apoptosis in peri- cells/mm2, P , 0.001). Ang2-induced pericyte loss was cytes. Accumulating evidence supports the role of integrin significantly attenuated by anti-a3 or anti-b1 antibodies in Ang2 activity in endothelial cells (19); however, little is (1,354 6 148 and 1,380 6 66 cells/mm2, P , 0.001; known about the Ang2/integrin system in the pericyte. Fig. 7D). In addition, Ang2-induced TUNEL and NG2 We thus focused on the integrin receptors on pericytes. double-positive pericytes were decreased in mice injected Integrins are cell adhesion molecules that are expressed with anti-a3 or anti-b1 antibodies (Fig. 7E). These in vivo on the surface of endothelial cells and pericytes. The data suggest that Ang2 induced pericyte loss via an contribution of endothelial and mural cell integrins to apoptotic mechanism in the retina. Overall, these data angiogenesis has been studied. Pericytes express integ- demonstrate that integrin a3 and b1 are important for rins, including the collagen receptors a1b1anda2b1; Ang2-induced pericyte apoptosis via the p53 pathway. the laminin receptors a3b1, a6b1, a6b4, and a7b1; the fibronectin receptors a4b1anda5b1; and the osteoponin DISCUSSION receptors a8b1anda9b1 (22). and extracellular In this study, we demonstrated that pericyte loss occurred matrix change integrin subtypes in pericytes (23). On the with Ang2 increase in diabetic mice retinas (10,11,13). basis of the differential response to Ang2 in the pericytes Previously, Tie-2 was known to be related with Ang- depending on the glucose concentration (Fig. 3B and 4A), diabetes.diabetesjournals.org Park and Associates 3065

Figure 6—High glucose (HG; 25 mmol/L) increases integrin a1, a3, and b1 in pericytes. A: Western blot analysis for phospho (p)-p53 (Ser15) and p53 were performed on lysates obtained from pericytes treated with Ang2 (300 ng/mL) or various integrin-blocking antibodies (5 mg/mL, a1, a2, a3, a4, a5, a6, and av) under HG for 15 min. b-Tubulin was used as a loading control. Data represent three independent experiments. B–D: Pericyte were incubated under HG for 24 and 48 h. ITGa1 (B), ITGa3 (C), and ITGb1 (D) mRNA transcriptions were assessed by qRT-PCR. Actin was used as an internal control. ITGa1, ITGa3, and ITGb1 mRNA levels were normalized to actin mRNA and reported as fold induction compared with control. *P < 0.01 by Student t test. E: Western blot analyses for integrin a1, a3, and b1 were performed on lysates obtained from pericytes incubated under HG and normal glucose (NG; 5 mmol/L) for 48 h. b-Tubulin was used as a loading control. Data represent three independent experiments. F: After incubation of Ang2 (500 ng), integrin a3b1 (500 ng), and integrin a3b1 antibody, immune complexes were coimmunoprecipitated to show direct binding of Ang2 to integrin a3b1 and then were analyzed for Ang2 and integrin b1. The same amounts of recombinant Ang2 and integrin a3b1 were used as an input.

we hypothesized that high glucose could change the integ- mRNA was more than six times higher than ITGa1or rin subtype more susceptible to Ang2. During qRT-PCR ITGa2 mRNA in pericytes even under normal glucose con- array for screening the integrin subtype change response ditions. Thus, we concluded that integrin a3andb1are to high glucose, only ITGa1, ITGa2, ITGa3,andITGb1 both predominant and high-glucose inducible integrins in mRNA increased more than 1.5-fold among ITGa1, pericytes. The proportion of integrin a1 in pericytes is too ITGa2, ITGa3, ITGa4, ITGa5, ITGa6, ITGa7, ITGav, and minor to be responsible for Ang2-induced pericyte apopto- ITGb1 mRNA (data not shown). In addition, basal ITGa3 sis (Fig. 6E and 7B). In addition, many integrins, including 3066 Ang2 Induces Pericyte Loss via a3b1 Integrin Diabetes Volume 63, September 2014

Figure 7—Ang2-induced pericyte apoptosis is inhibited by suppression of integrin a3b1. A and B: Pericytes were incubated under 25 mmol/L high glucose (HG) with Ang2 (300 ng/mL) and anti-integrin a1, a3, and b1 antibodies (Ab; 5 mg/mL) for 48 h. A: Pericyte apoptosis was analyzed by FACS and expressed as the percentage of apoptotic cells in total cell populations. B: Western blot analysis for p53 was performed on lysates obtained from pericyte. b-Tubulin was used as a loading control. Data represent three independent experiments. (ImageJ quantitation; n = 3, mean 6 SD). *P < 0.01 compared with Ang2 treatment by two-tailed Student t test. C: Ang2 (100 ng), with and without anti-a3 or anti-b1 antibodies (500 ng), was intravitreously injected to normal mice (n = 8 mice in each group). Eyes were enucleated 10 days after the intravitreal injection, and the isolated retinas were digested with trypsin for pericyte evaluation. Pericytes were identified in periodic acid Schiff– and hematoxylin-stained retinal digest preparations by morphologic criteria. Representative examples of retinal digest preparations are shown. Arrows indicate representative pericytes. D: The number of pericytes was normalized to the area of capillaries (mm2) in which they were counted. *P < 0.01 by Student t test. E: Retinal digest preparations were immunostained with NG2 (red), TUNEL (green), and DAPI (blue). White arrows indicate TUNEL-positive pericytes. C and E: Original magnification 3400. Scale bar = 20 mm. Bar graph represents mean 6 SE. diabetes.diabetesjournals.org Park and Associates 3067 avb3, a5b1, aIIbb3, avb6, and a3b1, recognize the tri- induced by STZ injection as early as 3 days. Then, Ang2 peptide RGD in their ligands (24). However, integrin a1b1 had increased at 3 months with pericyte loss, and finally, recognizes a configuration of residues formed by arginine acellular capillary at 6 months (10,44). The mechanism by and aspartic acid residues, not RGD. Attenuation of p53 by which capillaries become acellular is largely unknown. GRGDS supported that Ang2-induced pericyte apoptosis Apoptosis preceding the formation of acellular capillar- was effectively attenuated by blocking integrin a3b1(Fig. ies in retinas from diabetic rats and humans is one 5E and 7B). Integrin receptors induced by hyperglycemia in mechanism by which endothelial cells could be elimi- DR could make the pericyte more susceptible to the Ang2, nated from the diabetic capillary (8). Retinal capillary and in turn, lead to the pericyte apoptosis by the Ang2/ coverage with pericytes is crucial for the survival of en- integrin signaling pathway. dothelial cells. Loss of pericytes is related to the increase The laminin receptor a3b1 integrin is expressed in of acellular capillaries from hyperglycemic injury (5). Our vascular endothelial cells and acts to suppress pathological data indicate Ang2 did not directly induce endothelial angiogenesis in the retina and other organs (25,26). How- cell death (Fig. 3C and D); thus, we postulated that an ever, a role for integrin a3b1 in the pericyte is yet to be acellular capillary could cause endothelial cell death sec- determined (22). Inhibition of b1 integrin induced ondary to pericyte loss by Ang2. a rounded morphology of the pericytes, suggesting peri- In conclusion, we demonstrated that pericyte loss cyte adhesive properties were affected or that these cells occurred with Ang2 increase in the diabetic mice retinas. were undergoing apoptosis (27). In this study, we sug- Ang2 induced pericyte apoptosis via the p53 pathway gested that integrin a3b1 was important for Ang2- under high glucose. High glucose increased integrin a3b1 induced apoptosis. This is notably different from the in pericytes. Interestingly, integrin was involved in Ang2- classical concept that Tie-2 mediates the effect of Ang2 induced pericyte apoptosis. Furthermore, Ang2-induced as an Ang2 receptor. Absence of Tie-2 in the human peri- pericyte apoptosis was effectively attenuated by blocking cytes used in this study was confirmed by various experi- integrin a3b1 both in vitro and in vivo. Taken together, ments, including RT-PCR with two different primers, Ang2 induced p53-dependent pericyte apoptosis via a3b1 Western blot, and FACS analysis (Fig. 5A–D). In addition, integrin signaling in DR. We suggest that glycemic control Ang1 and Ang2 can both directly bind to some integrins or blocking Ang2/integrin signaling could be a potential and thereby signal in the absence of Tie-2 (28–31). Thus, therapeutic target to prevent pericyte loss in early DR. we suggested that integrin mediated Ang2-induced apo- ptosis at least in Tie-2–negative pericytes. Previous studies have shown that vascular endothelial Funding. This study was supported by the Seoul National University Research cells are the primary source of Ang2 (32–35). High glucose Grant (800-20130338 to Je.H.K.), the Seoul National University Hospital Research Fund (03-2013-0070 to Je.H.K.), the Pioneer Research Center Program of the increases Ang2 mRNA in HRMECs (36). The endothelial National Research Foundation for the Ministry of Education, Science and Tech- cell is regarded as potential source of Ang2 in the retina nology (NRF/MEST; 2012-0009544 to Je.H.K.), the Bio-Signal Analysis Technol- by transient rapid release from the Weibel-Palade body ogy Innovation Program of NRF/MEST (2009-0090895 to Je.H.K.), the Global and chronic upregulation of Ang2 (32,37,38). Chronic hy- Research Laboratory Program of NRF/MEST (2011-0021874 to K.-W.K.) and the perglycemia upregulates Ang2 in the diabetic retina NRF of Korea grant funded by the Korea government (2012R1A2A2A01012400, (36,39), and Ang2 upregulation is causally involved in 2011-0030739 to C.-H.C. and 2012-0006019 to Je.H.K.). the pathogenesis of pericyte loss in DR (10). Hyperglyce- Duality of Interest. No potential conflicts of interest relevant to this article mia induces pericyte loss not only by increasing Ang2 in were reported. endothelial cells but also by changing the integrin subtype Author Contributions. S.W.P. and J.-H.Y. performed the experiments prone to Ang2 in pericytes. Our data support that glyce- and wrote the manuscript. Ji.H.K. and K.-W.K. analyzed the data and reviewed mic control is important to prevent Ang2-induced peri- the manuscript. C.-H.C. and Je.H.K. designed the study and critically revised the manuscript. Je.H.K. is the guarantor of this work, and, as such, had full access to cyte loss in early DR. Glycemic control is the effective – all the data in the study and takes responsibility for the integrity of the data and treatment to reduce the progression of DR (40 42). the accuracy of the data analysis. We showed that Ang2 induced pericyte apoptosis under high glucose via the p53 pathway. Furthermore, References Ang2-induced pericyte apoptosis was effectively attenu- 1. Antonetti DA, Klein R, Gardner TW. Diabetic retinopathy. N Engl J Med 2012; ated in vitro and in vivo by blocking integrin a3b1. Integ- 366:1227–1239 rin a3 and b1 blockers reduced p53 signaling by Ang2. 2. Moss SE, Klein R, Klein BE. The incidence of vision loss in a diabetic – Ser-15 of p53 is phosphorylated by a mitogen-activated population. Ophthalmology 1988;95:1340 1348 3. Cogan DG, Toussaint D, Kuwabara T. Retinal vascular patterns. IV. Diabetic protein kinase (ERK1/2)-dependent pathway, and this retinopathy. 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