Apoptosis Of Retinal Pericytes Research Articles

Upregulation of Epac1 Promotes Pericyte Loss by Inducing Mitochondrial Fission, Reactive Oxygen Species Production, and Apoptosis.

The pathogenic mechanisms behind the development of ischemic retinopathy are complex and poorly understood. This study investigates the involvement of exchange protein directly activated by cAMP (Epac)1 signaling in pericyte injury during ischemic retinopathy, including diabetic retinopathy, a disease that threatens vision. Mouse models of retinal ischemia-reperfusion injury and type 1 diabetes induced by streptozotocin were used to investigate the pathogenesis of these diseases. The roles of Epac1 signaling in the pathogenesis of ischemic retinopathy were determined by an Epac1 knockout mouse model. The cellular and molecular mechanisms of Epac1-mediated pericyte dysfunction in response to high glucose were investigated by specific modulation of Epac1 activity in primary human retinal pericytes using Epac1-specific RNA interference and a pharmacological inhibitor. Ischemic injury or diabetes-induced retinal capillary degeneration were associated with an increased expression of Epac1 in the mouse retinal vasculature, including both endothelial cells and pericytes. Genetic deletion of Epac1 protected ischemic injury-induced pericyte loss and capillary degeneration in the mouse retina. Furthermore, high glucose-induced Epac1 expression in retinal pericytes was accompanied by increased Drp1 phosphorylation, mitochondrial fission, reactive oxygen species production, and caspase 3 activation. Inhibition of Epac1 via RNA interference or pharmacological approaches blocked high glucose-mediated mitochondrial dysfunction and caspase 3 activation. Our study reveals an important role of Epac1 signaling in mitochondrial dynamics, reactive oxygen species production, and apoptosis in retinal pericytes and identifies Epac1 as a therapeutic target for treating ischemic retinopathy.

R-(+)-WIN55212-2 protects pericytes from ischemic damage and restores retinal microcirculatory patency after ischemia/reperfusion injury

Background and purposeCannabinoids are vasoactive substances that act as key regulators of arterial tone in the blood vessels supplying peripheral tissues and the central nervous system. This study aimed to investigate the potential of R-(+)-WIN55212-2 (WIN), a cannabinoid receptor 1 agonist (CB1), as a treatment for retinal ischemia/reperfusion (I/R) injury. Experimental approachMale Wistar rats were subjected to retinal I/R injury by increasing intraocular pressure in the anterior chamber. The rats were randomly divided into four groups: normal control, I/R, vehicle (pre-treated with dimethyl sulfoxide [DMSO] via intraperitoneal injection), and experimental (pre-treated with WIN at a dose of 1 ml/kg via intraperitoneal injection). The rats were sacrificed at different time points of reperfusion (1 hour, 3 hours, 6 hours, and 1 day) after inducing retinal I/R injury, and their retinas were collected for analysis. Oxygen-glucose deprived/reperfusion (OGD/R) was performed by initially perfusing the retinas with oxygenated artificial cerebrospinal fluid (ACSF), then switching to an OGD solution to simulate ischemia, followed by another perfusion with ACSF. Pericyte contraction and the "no-reflow" phenomenon were observed using infrared differential interference contrast (IR-DIC) microscopy and immunohistochemistry. Western blot, enzyme-linked immunosorbent assay (ELISA), and nitric oxide (NO) detection were used to explore the potential mechanism. Key resultsIn both the OGD/R and I/R models, retinal pericytes exhibited persistent contraction even after reperfusion. The ability of WIN to regulate the tone of retinal pericytes and capillaries was specifically blocked by the BKCa inhibitor iberiotoxin (100 nM). WIN demonstrated a protective effect against retinal I/R injury by preserving blood flow in vessels containing pericytes. Pretreatment with WIN alleviated the persistent contraction and apoptosis of retinal pericytes in I/R-induced rats, accompanied by a reduction in intracellular calcium ion (Ca2+) concentration. The expression of CB1 decreased in a time-dependent manner in the I/R group. After I/R injury, endothelium-derived nitric oxide (eNOS) levels were reduced at all time points, which was successfully reversed by WIN therapy except for the 1 day group. Additionally, the downregulation of cyclic guanosine monophosphate (cGMP) and BKCa expression at 3 hours, 6 hours, and 1 day after I/R injury was restored by pretreatment of WIN. Conclusions & implicationsWIN exerted its protective effects on retinal I/R injury by inhibiting the contraction and apoptosis of pericytes through the CB1-eNOS-cGMP-BKCa signaling pathway, thus ameliorated the occlusion of retinal capillaries.

A TGFβ1‐BIGH3‐Apoptosis Axis Comprising Peptidase and Integrin α3β1 Promotes Renal Cell Death

Transforming growth factor‐β1 (TGFβ1) helps contribute to diabetes complications in various organs including the renal system. TGFβ1 signaling activates genes encoding various extracellular matrix (ECM) molecules that accumulate and promote renal cell injury, increasing the probability of end stage renal disease (ESRD). The mechanisms underlying development of ESRD are complex and not known completely. We have shown previously that TGFβ1 strongly stimulates renal proximal tubule epithelial (PTE) cells to express the gene that encodes an ECM protein called BIGH3. This newly‐synthesized endogenous BIGH3 protein increased PTE cell apoptosis. Treating PTE cells with exogenous recombinant BIGH3 protein increased apoptosis. Anti‐BIGH3 antibody blocked endogenous and exogenous BIGH3‐mediated apoptosis. These findings signify a TGFβ1‐BIGH3‐Apoptosis axis that augments kidney disease progression. BIGH3’s unstructured C‐terminus encodes integrin‐binding sequences EPDIM and RGD. In the kidney, BIGH3 C‐terminal proteolysis (CTP) provides bioactive C‐terminal fragments (BCTF) that comprise EPDIM and RGD and account for PTE cell apoptosis. The EPDIM motif in BIGH3 induced two‐fold greater apoptosis of PTE cells when compared to apoptosis induced by the RGD motif. In order to provide evidence that BIGH3 CTP and integrin interaction with EPDIM is required for PTE cell apoptosis, we used recombinant BIGH3, PTE cells, BIGH3 mutagenesis, protease inhibitors, TUNEL, immunoblots, integrin α3 antibody and siRNA. Homology modeling and molecular docking software Raptor X, ZDOCK and AutoDock Vina were used to generate monomeric and heterodimer models of α3β1 integrin; followed by in silico analysis of EPDIM interaction with α‐integrin subunit. Results show that a serine peptidase accomplishes BIGH3 CTP, which we regard as mechanistic in the proposed axis. A 21% chemical inhibition of BIGH3 CTP resulted in a 43% decrease in PTE cell apoptosis. In silico analysis indicates that the BCTF encoding EPDIM, bound to α3 subunit, prevents full activation and disrupts integrin‐mediated pro‐survival signals. We conclude that BIGH3 mediates at least some of the destructive arm of TGFβ1 signaling in diabetic nephropathy. BIGH3 CTP provides local mediator BCTFs that act through paracrine or autocrine pathways to block α3β1 pro‐survival signaling leading to PTE cell apoptosis. The data indicate that molecules of the axis pathway, including BIGH3, BIGH3 peptidase, and BIGH3 BCTFs are potential therapeutic targets to treat diabetic nephropathy. In a broader perspective, we have evidence that a similar mechanism is active in retinal endothelial and retinal pericyte apoptosis in diabetic complications of the ocular system.

DJ-1/PARK7 inhibits high glucose-induced oxidative stress to prevent retinal pericyte apoptosis via the PI3K/AKT/mTOR signaling pathway

Reactive oxygen species (ROS) act through multiple pathways to induce apoptosis of retinal capillary pericytes, which is an early marker and the primary cause of the progression of diabetic retinopathy. However, the specific molecular mechanisms behind ROS-induced retinal capillary pericyte loss in diabetic retinopathy remains elusive. In this study, we investigated the molecular regulation and effects of DJ-1/PARK7 on oxidative stress and injury of rat retinal pericytes (RRPs). To perform the research, RRPs were isolated from rat retina and cultured in medium with for 2 days: control group (5.6 mM glucose), high glucose group (30 mM glucose), hypertonic group (5.6 mM glucose + 24.4 mM mannitol). We found decreased expression of DJ-1 and increased apoptosis of RRPs in high glucose group. To further study the role of DJ-1, four groups were divided as follows: normal control group (5.6 mM glucose), high glucose (30 mM glucose), empty vector control group (pcDNA3.1,30 mM glucose), DJ-1 overexpression group (pcDNA3.1-myc-DJ-1,30 mM glucose). DJ-1, P53, p-P53, cleaved caspase-3, manganese superoxide dismutase (MnSOD), catalase (CAT) and PI3K/Akt/mTOR signaling pathway in each group was detected by Western Blot. RRPs apoptosis was detected by Terminal-deoxynucleoitidyl Transferase mediated Nick End Labeling (TUNEL) and 4′6- diamidino-2-phenylindole (DAPI). Mitochondrial function was detected by jc-1 and fluorescent probes DCFH-DA was used to determine reactive oxygen species (ROS). We found that high glucose (30 mM) lasting two days can induce significant apoptosis of RRPs, increase ROS production and expressions of p-p53 and active caspase-3, impair mitochondrial function, decrease the activities of MnSOD and CAT, and decrease expression of DJ-1, p-AKT and p-mTOR. In contrast, DJ-1/PARK7 overexpression significantly increases expression of DJ-1, p-AKT and p-mTOR, increases expression and activities of MnSOD and CAT, improves mitochondrial function, decreases expression of apoptotic gene protein p-p53 and active caspase-3, reduces ROS production and reduces the apoptotic rate of RRPs induced by high glucose. These results suggest that DJ-1 may play a role in protecting RRPs from high glucose induced-oxidative injury. DJ-1 might improve mitochondrial function, inhibit ROS production and enhance antioxidant capacity to reduce apoptosis of retinal pericytes through the PI3K/AKT/mTOR signaling pathway which may be related to early pathogenesis of diabetic retinopathy.

Asymmetric dimethylarginine aggravates blood-retinal barrier breakdown of diabetic retinopathy via inhibition of intercellular communication in retinal pericytes.

Blood-retinal barrier breakdown is the main pathological characteristics of diabetic retinopathy (DR). Asymmetric dimethylarginine (ADMA) was reported to be elevated in DR patients. In this study, we observed the dynamic profile of ADMA, retinal morphology and permeability of BRB at 2, 4 or 8week of diabetic rats induced by a single intraperitoneal injection of streptozocin (60mg/kg) and in cultured rat retinal pericytes pretreated with D-glucose (30mM) for 1, 3, 5 and 7days or ADMA (3, 10, 30 μM) for 24, 48 and 72h, trying to explore the effects of ADMA on blood-retinal barrier in DR. Gap junction intercellular communication (GJIC) and the expression of blood-retinal barrier-specific component connexin 43 (Cx43) were examined in diabetic rats or cultured retinal pericytes to elucidate whether ADMA impacted blood-retinal barrier function via damaging Cx43-GJIC. The results showed that with increasing duration of diabetes, the ultrastructure of blood-retinal barrier of diabetic rats appeared cell junction damage, apoptosis of retinal pericytes and breakdown of barrier successively. The increases in retinal permeability, ADMA levels and Cx43 expression, and abnormal GJIC were observed in diabetic rats and retinal pericytes exposed to D-glucose (30mM). A glucose-like effect was seen using ADMA or another L-arginine analogue NG-monomethyl-L-arginine or dimethylarginine dimethylaminohydrolases (DDAHs) siRNA, implicating that ADMA aggravated the breakdown of blood-retinal barrier via damaging Cx43-GJIC.

The Extract of Aster Koraiensis Prevents Retinal Pericyte Apoptosis in Diabetic Rats and Its Active Compound, Chlorogenic Acid Inhibits AGE Formation and AGE/RAGE Interaction.

Retinal capillary cell loss is a hallmark of early diabetic retinal changes. Advanced glycation end products (AGEs) are believed to contribute to retinal microvascular cell loss in diabetic retinopathy. In this study, the protective effects of Aster koraiensis extract (AKE) against damage to retinal vascular cells were investigated in streptozotocin (STZ)-induced diabetic rats. To examine this issue further, AGE accumulation, nuclear factor-kappaB (NF-κB) and inducible nitric oxide synthase (iNOS) were investigated using retinal trypsin digests from streptozotocin-induced diabetic rats. In the diabetic rats, TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling)-positive retinal microvascular cells were markedly increased. Immunohistochemical studies revealed that AGEs were accumulated within the retinal microvascular cells, and this accumulation paralleled the activation of NF-κB and the expression of iNOS in the diabetic rats. However, AKE prevented retinal microvascular cell apoptosis through the inhibition of AGE accumulation and NF-κB activation. Moreover, to determine the active compounds of AKE, two major compounds, chlorogenic acid and 3,5-di-O-caffeoylquinic acid, were tested in an in vitro assay. Among these compounds, chlorogenic acid significantly reduced AGE formation as well as AGE/RAGE (receptor for AGEs) binding activity. These results suggest that AKE, particularly chlorogenic acid, is useful in inhibiting AGE accumulation in retinal vessels and exerts a preventive effect against the injuries of diabetic retinal vascular cells.

TGFβ induces BIGH3 expression and human retinal pericyte apoptosis: a novel pathway of diabetic retinopathy.

PurposeOne of the earliest hallmarks of diabetic retinopathy is the loss of retinal pericytes. However, the mechanisms that promote pericyte dropout are unknown. In the present study, we propose a novel pathway in which pericyte apoptosis is mediated by macrophages, TGFβ and pro-apoptotic BIGH3 (TGFβ-induced Gene Human Clone 3) protein.Patients and methodsTo elucidate this pathway, we assayed human retinal pericyte (HRP) apoptosis by TUNEL assay, BIGH3 mRNA expression by qPCR, and BIGH3 protein expression by western blot analysis. HRP were treated with BIGH3 protein, TGFβ1 and TGFβ2 and inhibition assays were carried out by blocking with antibodies against BIGH3. The distribution of BIGH3 and CD68+ macrophages were compared in a post-mortem donor eye with 7-year history of Type II diabetes and histopathogically confirmed non-proliferative diabetic retinopathy (NPDR).ResultsTGFβ induced a significant increase in BIGH3 mRNA and protein expression, and HRP apoptosis. BIGH3 treatment showed HRP undergo apoptosis in a dose-dependent manner. At 5 μg/ml, BIGH3 induced 3.5-times more apoptosis in HRP than in retinal endothelial cells. TGFβ induced apoptosis was inhibited by blocking with antibodies against BIGH3. In an example of NPDR, BIGH3 accumulated within the walls of the inner retina arterioles. Macrophage infiltrates were frequently associated with these vessels and the inner nuclear layer.ConclusionTogether with our previously published results on macrophage-induced retinal endothelial cell apoptosis, the present study supports a novel inflammatory pathway mediated by macrophages and the BIGH3 protein leading to HRP apoptosis. As shown in human post-mortem globes, these observations are clinically relevant, suggesting a new mechanism underlying pericyte dropout during NPDR.

High Glucose-induced Retinal Pericyte Apoptosis Depends on Association of GAPDH and Siah1

Diabetic retinopathy (DR) is a leading cause of blindness worldwide, and its prevalence is growing. Current therapies for DR address only the later stages of the disease, are invasive, and have limited effectiveness. Retinal pericyte death is an early pathologic feature of DR. Although it has been observed in diabetic patients and in animal models of DR, the cause of pericyte death remains unknown. A novel pro-apoptotic pathway initiated by the interaction between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the E3 ubiquitin ligase, seven in absentia homolog 1 (Siah1), was recently identified in ocular tissues. In this article we examined the involvement of the GAPDH/Siah1 interaction in human retinal pericyte (hRP) apoptosis. HRP were cultured in 5 mm normal glucose, 25 mm l- or d-glucose for 48 h (osmotic control and high glucose treatments, respectively). Siah1 siRNA was used to down-regulate Siah1 expression. TAT-FLAG GAPDH and/or Siah1-directed peptides were used to block GAPDH and Siah1 interaction. Co-immunoprecipitation assays were conducted to analyze the effect of high glucose on the association of GAPDH and Siah1. Apoptosis was measured by Annexin V staining and caspase-3 enzymatic activity assay. High glucose increased Siah1 total protein levels, induced the association between GAPDH and Siah1, and led to GAPDH nuclear translocation. Our findings demonstrate that dissociation of the GAPDH/Siah1 pro-apoptotic complex can block high glucose-induced pericyte apoptosis, widely considered a hallmark feature of DR. Thus, the work presented in this article can provide a foundation to identify novel targets for early treatment of DR.

Gemigliptin, a dipeptidyl peptidase-4 inhibitor, inhibits retinal pericyte injury in db/db mice and retinal neovascularization in mice with ischemic retinopathy

Retinal pericyte loss and neovascularization are characteristic features of diabetic retinopathy. Gemigliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, has shown robust blood-glucose lowering effects in type 2 diabetic patients, but its effects on diabetic retinopathy have not yet been reported. We evaluated the efficacy of gemigliptin on retinal vascular leakage in db/db mice, which is an animal model for type 2 diabetes, and neovascularization in oxygen-induced retinopathy (OIR) mice, which is an animal model for ischemic proliferative retinopathy. Gemigliptin (100mg/kg/day) was orally administered to the db/db mice for 12weeks. C57BL/6 mice on postnatal day 7 (P7) were exposed to 75% hyperoxia for 5days, followed by exposure to room air from P12 to P17 to induce OIR. Gemigliptin (50mg/kg/day) was intraperitoneally injected daily from P12 to P17. Retinal neovascularization was analyzed in flat-mounted retinas on P17. We determined the efficacy and possible mechanism of gemigliptin on high glucose-induced apoptosis of primary human retinal pericytes. The oral administration of gemigliptin for 4months significantly ameliorated retinal pericyte apoptosis and vascular leakage in the db/db mice. Gemigliptin also ameliorated retinal neovascularization in the OIR mice. Gemigliptin attenuated the overexpression of plasminogen activator inhibitor-1 (PAI-1) in the retinas of diabetic and OIR mice. Gemigliptin and PAI-1 siRNA significantly inhibited pericyte apoptosis by inhibiting the overexpression of PAI-1, which is induced by high glucose. Our results suggest that gemigliptin has potent anti-angiogenic and anti-apoptotic activities via suppressing DPP-4 and PAI-1, and the results support the direct retinoprotective action of gemigliptin.

Identification of O-GlcNAc modification targets in mouse retinal pericytes: implication of p53 in pathogenesis of diabetic retinopathy.

Hyperglycemia is the primary cause of the majority of diabetes complications, including diabetic retinopathy (DR). Hyperglycemic conditions have a detrimental effect on many tissues and cell types, especially the retinal vascular cells including early loss of pericytes (PC). However, the mechanisms behind this selective sensitivity of retinal PC to hyperglycemia are undefined. The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is elevated under hyperglycemic condition, and thus, may present an important molecular modification impacting the hyperglycemia-driven complications of diabetes. We have recently demonstrated that the level of O-GlcNAc modification in response to high glucose is variable in various retinal vascular cells. Retinal PC responded with the highest increase in O-GlcNAc modification compared to retinal endothelial cells and astrocytes. Here we show that these differences translated into functional changes, with an increase in apoptosis of retinal PC, not just under high glucose but also under treatment with O-GlcNAc modification inducers, PUGNAc and Thiamet-G. To gain insight into the molecular mechanisms involved, we have used click-It chemistry and LC-MS analysis and identified 431 target proteins of O-GlcNAc modification in retinal PC using an alkynyl-modified GlcNAc analog (GlcNAlk). Among the O-GlcNAc target proteins identified here 115 of them were not previously reported to be target of O-GlcNAc modification. We have identified at least 34 of these proteins with important roles in various aspects of cell death processes. Our results indicated that increased O-GlcNAc modification of p53 was associated with an increase in its protein levels in retinal PC. Together our results suggest that post-translational O-GlcNAc modification of p53 and its increased levels may contribute to selective early loss of PC during diabetes. Thus, modulation of O-GlcNAc modification may provide a novel treatment strategy to prevent the initiation and progression of DR.

Stroke Literature Synopses: Basic Science

<i>Stroke</i> Literature Synopses: Basic Science

STAT1-mediated Bim expression promotes the apoptosis of retinal pericytes under high glucose conditions

Hyperglycemia impacts different vascular cell functions and promotes the development and progression of various vasculopathies including diabetic retinopathy. Although the increased rate of apoptosis in pericytes (PCs) has been linked to increased oxidative stress and activation of protein kinase C-δ (PKC-δ) and SHP-1 (Src homology region 2 domain-containing phosphatase-1) tyrosine phosphatase during diabetes, the detailed mechanisms require further elucidation. Here we show that the rate of apoptosis and expression of proapoptotic protein Bim were increased in the retinal PCs of diabetic Akita/+ mice and mouse retinal PCs cultured under high glucose conditions. Increased Bim expression in retinal PCs under high glucose conditions required the sustained activation of signal transducer and activator of transcription 1 (STAT1) through production of inflammatory cytokines. PCs cultured under high glucose conditions also exhibited increased oxidative stress and diminished migration. Inhibition of oxidative stress, PKC-δ or Rho-associated protein kinase I/II was sufficient to protect PCs against apoptosis under high glucose conditions. Furthermore, PCs deficient in Bim expression were protected from high glucose-mediated increased oxidative stress and apoptosis. However, only inhibition of PKC-δ lowered Bim levels. N-acetylcysteine did not affect STAT1 levels, suggesting that oxidative stress is downstream of Bim. PCs cultured under high glucose conditions disrupted capillary morphogenesis of retinal endothelial cells (ECs) in coculture experiments. In addition, conditioned medium prepared from PCs under high glucose conditions attenuated EC migration. Taken together, our results indicate that Bim has a pivotal role in the dysfunction of retinal PCs under high glucose conditions by increasing oxidative stress and death of PCs.

Critical role of TXNIP in oxidative stress, DNA damage and retinal pericyte apoptosis under high glucose: Implications for diabetic retinopathy

Diabetic retinopathy (DR) is characterized by early loss of retinal capillary pericytes and microvascular dysfunction. We recently showed that pro-oxidative stress and pro-apoptotic thioredoxin interacting protein (TXNIP) is significantly up-regulated in rat retinas in experimental diabetes and mediates inflammation and apoptosis. Therefore, we hypothesize here that TXNIP up-regulation in pericyte plays a causative role in oxidative stress and apoptosis under sustained high glucose exposure in culture. We maintained a rat retinal capillary pericyte cell line (TR-rPCT1) for 5 days under low glucose (LG, 5.5mM) or high glucose (HG, 25mM) with or without anti-oxidant N-acetylcysteine (5mM, NAC), Azaseine (2μM, AzaS), an inhibitor of TXNIP, and TXNIP siRNA (siTXNIP3, 20nM). The results show that HG increases TXNIP expression in TR-rPCT1, which correlates positively with ROS generation, protein S-nitrosylation, and pro-apoptotic caspase-3 activation. Furthermore, pericyte apoptosis is demonstrated by DNA fragmentation (alkaline comet assay) and a reduction in MTT survival assay. Treatment of TR-rPCT1 with NAC or an inhibition of TXNIP by AzaS or siTXNIP3 each reduces HG-induced ROS, caspase-3 activation and DNA damage demonstrating that TXNIP up-regulation under chronic hyperglycemia is critically involved in cellular oxidative stress, DNA damage and retinal pericyte apoptosis. Thus, TXNIP represents a novel gene and drug target to prevent pericyte loss and progression of DR.

KIOM-79 Protects AGE-Induced Retinal Pericyte Apoptosis via Inhibition of NF-kappaB Activation In Vitro and In Vivo

KIOM-79 is an herbal mixture of parched Puerariae radix, gingered Magnoliae cortex, Glycyrrhizae radix and Euphorbiae radix. In the present study, we determined the efficacy and possible mechanism of KIOM-79 on the advanced glycation end product (AGE)-modified bovine serum albumin (BSA)-induced apoptosis of cultured bovine retinal pericytes and rat retinal pericytes in Zucker diabetic fatty (ZDF) rats. Seven-week-old male ZDF rats were treated with KIOM-79 (50 mg/kg body weight) once a day orally for 13 weeks. KIOM-79 significantly inhibited pericyte apoptosis which were induced by the AGE-BSA treatment. The KIOM-79 treatment markedly suppressed the activation of nuclear factor-kappaB (NF-κB) through the inhibition of inhibitory κB kinase complex. In addition, the oral administration of KIOM-79 inhibited the changes in retinal vasculature (vascular hyperpermeability, acellular capillary). KIOM-79 strongly inhibited pericyte apoptosis, NF-κB activation and the expression of pro-apoptotic Bax and tumor necrosis factor-α. Our results suggest that KIOM-79 may exert inhibitory effects on AGE-induced pericyte apoptosis by blocking NF-κB activation, thereby ameliorating retinal microvascular dysfunction.

Puerarin inhibits the retinal pericyte apoptosis induced by advanced glycation end products in vitro and in vivo by inhibiting NADPH oxidase-related oxidative stress

Retinal pericyte loss is one of the histopathological hallmarks of early diabetic retinopathy. Puerarin (4′-7-dihydroxy-8-beta-d-glucosylisoflavone), which is an isoflavone-C-glucoside, causes various pharmacological effects that include antihyperglycemic and anti-inflammatory activities. In the present study, we determined the efficacy and possible mechanism of puerarin on the advanced glycation end product (AGE)-modified bovine serum albumin (BSA)-induced apoptosis of cultured bovine retinal pericytes and rat retinal pericytes in intravitreally AGE-modified rat serum albumin (RSA)-injected eyes. Puerarin significantly inhibited pericyte apoptosis, the generation of reactive oxygen species (ROS), and NADPH oxidase activity by inhibiting the phosphorylation of p47phox and Rac1 which were induced by the AGE-BSA treatment. The puerarin treatment markedly suppressed the activation of nuclear factor-kappaB (NF-κB). In addition, the in vivo apoptosis of the retinal pericyte of rats that was stimulated by the intravitreal injection of AGE-RSA was evidently attenuated by the puerarin treatment. These results demonstrate that puerarin may exert inhibitory effects on AGE-induced pericyte apoptosis by interfering with the NADPH oxidase-related ROS pathways and blocking NF-κB activation, thereby ameliorating retinal microvascular dysfunction.

High Glucose Induces Mitochondrial Morphology and Metabolic Changes in Retinal Pericytes

Mitochondrial dysfunction is known to play a role in retinal vascular cell loss, a prominent lesion of diabetic retinopathy. High glucose (HG) has been reported to induce mitochondrial fragmentation and dysfunction in retinal endothelial cells, contributing to apoptosis. In this study, the effects of HG on mitochondrial morphology, membrane potential, and metabolic changes and whether they could contribute to HG-induced apoptosis in retinal pericytes were investigated. Bovine retinal pericytes (BRPs) were grown in normal or HG medium for 7 days. Both sets of cells were double stained with mitochondrial membrane potential-independent dye and tetramethylrhodamine-ethyl-ester-perchlorate (TMRE) and imaged by confocal microscopy. The images were analyzed for average mitochondria shape, by using form factor and aspect ratio values, and membrane potential changes, by using the ratio between the red and green dye. BRPs grown in normal or HG medium were analyzed for transient changes in oxygen consumption and extracellular acidification with a flux analyzer and apoptosis by TUNEL assay. BRPs grown in HG media exhibited significant fragmentation of mitochondria and increased membrane potential heterogeneity compared with the BRPs grown in normal medium. Concomitantly, BRPs grown in HG showed reduced steady state and maximum oxygen consumption and reduced extracellular acidification. Number of TUNEL-positive pericytes was increased in HG condition as well. In HG condition, mitochondria of retinal pericytes display significant fragmentation, metabolic dysfunction, and reduced extracellular acidification. The detrimental effects of HG on mitochondrial function and cellular metabolism could play a role in the accelerated apoptosis associated with the retinal pericytes in diabetic retinopathy.

Pigment epithelium-derived factor inhibits advanced glycation end-products-induced cytotoxicity in retinal pericytes

Aim This study investigated the effects of pigment epithelium-derived factor (PEDF) on advanced glycation end-product (AGE)-induced cytotoxicity in porcine retinal pericytes and the signalling mechanism involved. Methods Retinal pericytes were isolated from porcine eyes and characterized by immunocytochemistry. The effect of AGEs and PEDF on cell proliferation was determined by bromodeoxyuridine (BrdU) assay. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was analyzed by luminescence assay. Reactive oxygen species (ROS), nitric oxide (NO), superoxide dismutase (SOD) and glutathione peroxidase (GSH) were determined by biochemical assays. Induction of apoptosis was determined by caspase-3 colorimetric assay and DNA fragmentation analysis. Src activity was assessed by transient transfection analysis, and the status of Src phosphorylation at Y419 was analyzed by a competitive ELISA method. Results AGEs significantly increased intracellular ROS generation in pericytes via NADPH oxidase and induced cell death via caspase-3 enzyme activation, whereas PEDF increased cell proliferation in a dose-dependent manner. In addition, PEDF inhibited AGE-induced ROS generation by increasing levels of SOD and GSH, and also blocked the activation of caspase-3. Furthermore, PEDF induced cell survival via the Src pathway by Src phosphorylation at Y419, as evidenced by a pharmacological inhibitor and Src mutants. Conclusion These results suggest that PEDF abrogates AGE-induced oxidative stress and apoptosis in retinal pericytes via the Src pathway, thereby suggesting that PEDF is an effective therapeutic agent for the treatment of loss of pericytes in early diabetic retinopathy.

Activation of AMP-Activated Protein Kinase Prevents Lipotoxicity in Retinal Pericytes

The recent FIELD study demonstrated that the lipid-lowering agent fenofibrate significantly reduces the development and progression of diabetic retinopathy (DR). These results suggest that lipids may play a causal role in DR. They also suggest that AMP-activated protein kinase (AMPK) activation could account for these findings given that fenofibrate is an AMPK activator. The authors previously demonstrated that free fatty acids, in addition to hyperglycemia, can induce apoptosis in retinal pericytes (PCs), the first cells lost in the diabetic retina. Incubation with the saturated fatty acid palmitate, but not the monounsaturated fatty acid oleate, elicited cytotoxicity in a manner dependent on oxidative stress, NF-κB activation, and ceramide accumulation. In this study, the authors explored whether AMPK can downregulate these pathways and, in doing so, protect PCs from apoptosis. PCs were incubated with palmitate or oleate to determine whether the factors previously linked to lipotoxicity were uniquely increased by palmitate. The effects of AMPK activation on these parameters and on apoptosis were concurrently examined. Only palmitate increased NF-κB activation, ceramide and diacylglycerol mass, and apoptosis. Activation of AMPK with AICAR or, where used, expression of a constitutively active AMPK prevented all these effects. In contrast, both palmitate and oleate markedly increased oxidative stress, and the activation of AMPK did not prevent this. AMPK activation prevents the metabolic abnormalities and apoptosis specifically caused by palmitate in cultured PCs. Pharmacologic agents that activate AMPK in the diabetic retina may warrant consideration as a therapeutic option to avert PC apoptosis and to maintain microvascular homeostasis.

Elevated Nε-(Carboxymethyl)lysine Is Associated with Apoptosis of Retinal Pericytes in Streptozotocin-Induced Diabetic Rats

Advanced glycation end products including NΕ-(carboxymethyl)lysine (CML) are believed to contribute to retinal pericyte loss in diabetic retinopathy. Nuclear factor-ĸB (NF-ĸB) activation has been considered as a potential cytotoxic modulator of retinal pericytes. Herein, we investigated whether CML accumulation can trigger NF-ĸB activation and apoptosis of retinal pericytes in streptozotocin (STZ)-induced diabetic rats. Seven-week-old Sprague-Dawley rats were made diabetic (STZ, 60 mg/kg). After 5 months, CML level and NF-ĸB activation were measured in trypsin-digested retinal vessels. In diabetic rats, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling)-positive and caspase 3-positive retinal pericytes were significantly increased. CML and NF-ĸB activation was also markedly increased in diabetic retinal vessels. Moreover, the immunoreactivity of NF-ĸB was localized within the region where CML were accumulated. Apoptosis occurred in CML-accumulating retinal pericytes. These results suggest that NF-ĸB could be activated in CML-accumulating pericytes from diabetic retina. CML accumulation is responsible, at least in part, for the apoptosis of retinal pericytes.

High glucose‐induced apoptosis in bovine retinal pericytes is associated with transforming growth factor β and βIG‐H3: βIG‐H3 induces apoptosis in retinal pericytes by releasing Arg‐Gly‐Asp peptides

Transforming growth factor beta (TGF-beta) plays an important role in diabetic retinopathy. betaIG-H3 is a downstream target molecule of TGF-beta that may participate in the pathogenesis of diabetic retinopathy and in particular in the loss of pericytes during early pathological changes. We observed bovine retinal pericytes apoptosis and the increased expression of TGF-beta and betaIG-H3 induced by high concentrations of glucose in the cell culture media. An anti-TGF-beta antibody was used to block glucose-induced retinal pericytes apoptosis. Retinal pericytes were also transfected with cDNA encodings either wild-type or mutant betaIG-H3 lacking Arg-Gly-Asp (RGD) sequences in order to validate the effects of betaIG-H3 and RGD signalling on retinal pericytes apoptosis. A cell death-detecting enzyme-linked immunosorbent assay revealed that 25 mM glucose significantly increased cell death compared with 5.5 mM glucose after 5 or 7 days of exposure (P < 0.01). High glucose significantly increased the TGF-beta levels as compared with 5.5 mM glucose after 5 days, and betaIG-H3 levels after 3, 5 and 7 days of exposure (P < 0.01). TGF-beta increased cell death and betaIG-H3 levels in a dose-dependent manner, with a maximal effect observed at 1 ng/mL. An anti-TGF-beta antibody nearly completely blocked high glucose-induced cell death. Wild-type betaIG-H3-transfected cells showed a significant increase in cell death as compared with mutant betaIG-H3-transfected (Mycb-c) cells, untransfected or mock-transfected cells. These results suggest that hyperglycaemia-induced expression of TGF-beta and betaIG-H3 contributes to accelerated retinal pericytes apoptosis. betaIG-H3 induces pericytes apoptosis through its RGD motif, which may constitute an important pathogenic mechanism leading to pericytes loss in diabetic retinopathy.