Apoptosis Of Retinal Pericytes Research Articles

Cytotoxic role of methylglyoxal in rat retinal pericytes: Involvement of a nuclear factor-kappaB and inducible nitric oxide synthase pathway

Methylglyoxal (MGO), a cytotoxic metabolite, is produced from glycolysis. Elevated levels of MGO are observed in a number of diabetic complications, including retinopathy, nephropathy and cardiomyopathy. Loss of retinal pericyte, a hallmark of early diabetic retinal changes, leads to the development of formation of microaneurysms, retinal hemorrhages and neovasculization. Herein, we evaluated the cytotoxic role of MGO in retinal pericytes and further investigated the signaling pathway leading to cell death. Rat primary retinal pericytes were exposed to 400 μM MGO for 6 h. Retinal vessels were prepared from intravitreally MGO-injected rat eyes. We demonstrated apoptosis, nuclear factor-kappaB (NF-κB) activation and inducible nitric oxide synthase (iNOS) induction in cultured pericytes treated with MGO and MGO-injected retinal vessels. In MGO-treated pericytes, TUNEL-positive nuclei were markedly increased, and NF-κB was translocalized into the nuclei of pericytes, which paralleled the expression of iNOS. The treatment of pyrrolidine dithiocarbamate (an NF-κB inhibitor) or l-N6-(1-iminoethyl)-lysine (an iNOS inhibitor) prevented apoptosis of MGO-treated pericytes. In addition, in intravitreally MGO-injected rat eyes, TUNEL and caspase-3-positive pericytes were significantly increased, and activated NF-κB and iNOS were highly expressed. These results suggest that the increased expression of NF-κB and iNOS caused by MGO is involved in rat retinal pericyte apoptosis.

KIOM-79 prevents methyglyoxal-induced retinal pericyte apoptosis in vitro and in vivo

Aims of study The purpose of this study was to investigate the protective effects of KIOM-79, a combination of four plant extracts, on retinal pericytes loss, which is one of the histopathological hallmarks of early diabetic retinopathy. Materials and methods To investigate the protective effect of KIOM-79 on pericyte apoptosis, we have used methylglyoxal (MGO)-treated primary rat retinal pericytes and retinal vessels from intravitreally MGO-injected eyes. Primary rat retinal pericytes were exposed to 400 μM MGO for 6 h with or without KIOM-79. 400 μM final intravitreal concentration of MGO with or without KIOM-79 (10 μg/ml final intravitreal concentration) were intravitreally injected into the right eyes of rats for 2 days. The left eyes were received 3 μl of saline only. Retinal vessels were then isolated. We examined apoptosis, ROS productions, 8-OHdG in cultured rat retinal pericytes and retinal vessels. Results In CCK-8 assay and TUNEL staining, MGO-induced apoptosis of rat retinal pericytes was markedly inhibited by KIOM-79. KIOM-79 significantly reduced intracellular ROS productions and oxidative damage induced by MGO. In addition, the intravitreal injection of KIOM-79 prevented apoptosis of retinal microvascular cells, MGO accumulation and oxidative DNA damage in intravitreally MGO-injected eye. Conclusion These observations suggest that KIOM-79 acts through an antioxidant mechanism to protect against oxidative stress-induced apoptosis in retinal pericytes.

Role of the Polyol Pathway in High Glucose–Induced Apoptosis of Retinal Pericytes and Proliferation of Endothelial Cells

The selective degeneration of pericytes and the proliferation of endothelial cells (ECs) appear to be associated with microaneurysm formation, an initial deficit in the early stage of diabetic retinopathy. The preventive effect of aldose reductase (AR) inhibitor (ARI) for high glucose-induced pericyte loss and EC growth was investigated. The effect of high glucose (30 mM) exposure in the presence or absence of ARI for the cell growth of porcine pericytes and ECs was examined with the use of an in vitro coculture system to mimic the interaction between pericytes and ECs. To determine the role of transforming growth factor (TGF)-beta, its amount in culture media was measured, and the effects of the treatment of TGF-beta or neutralizing antibody on EC growth were examined. Abundant expression of AR and increased levels of polyol and apoptosis induced by high glucose were observed in pericytes, but not in ECs. ECs overexpressing AR cultured in high-glucose medium showed decreased cell viability. The growth-inhibitory effect of ECs on coculture with pericytes was attenuated by exposure to a high glucose concentration. Biochemical assays disclosed that the levels of active TGF-beta in media were linked to EC growth. Supply of active TGF-beta to coculture medium containing 30 mM D-glucose restored the inhibitory activity on EC growth. ARI rescued pericytes from high glucose-induced apoptosis and maintained the levels of TGF-beta, resulting in the prevention of cocultured EC growth.

Effects of Generated trans-Arachidonic Acids on Retinal Capillary during Nitrative Stress in Diabetic Rats

Purpose: To investigate whether the trans-arachidonic acids (TAAs) change in diabetic conditions and the effect on capillary cells and its possible mechanism. Methods: Diabetes was induced by intraperitoneal injection of streptozotocin at 60 mg kg^–1 body weight. The amount of TAAs and arachidonic acid (AA) in serum of diabetic rats was measured by the gas chromatography and mass spectrometry method and the ratio of the peak area of TAAs to AA with a selected ion of 79 was estimated by group t test. The expression level of thrombospondin-1 (TSP-1) in the retina was examined by Western blot. The pericyte apoptotic index was investigated by flow cytometry. Result: The ratio of TAAs to AA significantly increased in serum at the time points of 8, 12 and 16 weeks after streptozotocin induction (p < 0.05). The expression of TSP-1 in the retina of diabetic rats elevated with longer duration of diabetes (checking the time points of 2-, 4-, 8-, 12- and 16-week diabetic rats). The expression of TSP-1 tended to be physiologically elevated in relation to age but less significantly than in the diabetic group. TAAs could not induce the apoptosis of retinal pericytes (p > 0.05). Conclusion: TAAs were generated associated with nitrative stress in diabetic rats and a possible mechanism of microvascular injury exits in diabetic conditions.

Matrix Metalloproteinase‐2 Expression and Apoptogenic Activity in Retinal Pericytes

Diabetic retinopathy (DR) commences as a basement membrane disorder with a dramatic loss of the innate retinal vascular autoregulation. In this process, retinal pericytes, which regulate endothelial cell proliferation and survival, undergo morphometric changes consistent with apoptosis. The ability of retinal pericytes to survive is dependent on their interaction with extracellular matrix (ECM) proteins, which are susceptible to rapid degradation by matrix metalloproteinases (MMPs). Here, we examined the potential involvement of MMPs and a membrane-type MMP in retinal pericyte death in experimental diabetes and in cultured retinal pericytes. Our data showed that chemically induced diabetes of 6 months' duration significantly increased the expression and activity of both MMP-2 and its physiological activator MT1-MMP. TdT-mediated dUTP nick end labeling (TUNEL)-positive pericytes and endothelial cells were concomitantly detected within the retinal capillaries of diabetic animals. In situ zymography showed a weak MMP activity in control retinas but an intense perivascular MMP activity in retinas from diabetic animals. In vitro studies showed that hyperglycemia-induced retinal pericyte apoptosis in vitro was attenuated by a specific MMP inhibitor. Incubation of pericytes with purified MMP-2 significantly increased the number of apoptotic cells. Our data suggest that increased MMP-2 activity compromises retinal pericyte survival possibly through MMP-2 action on ECM proteins and/or direct association of MMP-2 with integrins, which promotes apoptosis/anoikis by loss of cell contact with an appropriate ECM.

Cyclic Stretch–Induced Reactive Oxygen Species Generation Enhances Apoptosis in Retinal Pericytes Through c-Jun NH 2 -Terminal Kinase Activation

Hypertension is known to exacerbate diabetic complications, such as retinopathy and nephropathy. Apoptosis of retinal vascular pericytes has been well established as the earliest conceivable change in diabetic retinopathy. In this study, we investigated the contribution of cyclic stretch, which mimics a hypertensive state to pericyte apoptosis. A 48-hour cyclic stretch induced DNA fragmentation in porcine retinal pericytes and increased the number of TUNEL+ cells at a pathophysiologically relevant extension level (10%/60 cycles per minute). Stretch also increased intracellular reactive oxygen species generation and increased c-Jun NH(2)-terminal kinase phosphorylation in a time- and magnitude-dependent manner, which were reduced by the nicotinamide-adenine dinucleotide phosphate oxidase inhibitor diphenylene iodonium or dominant-negative protein kinase C-delta. Stretch activated protein kinase C-delta and increased its association with p47phox. Stretch induced cleavage of caspase-9 and -3 and increased caspase-3 activity. Protein kinase C-delta or c-Jun NH(2)-terminal kinase inhibition normalized stretch-induced caspase-3 activity and prevented stretch-induced apoptosis. These data indicate that cyclic stretch induces apoptosis in porcine retinal pericytes by activation of the reactive oxygen species-c-Jun NH(2)-terminal kinase-caspase cascades, suggesting a novel molecular mechanism to explain the exacerbation of early diabetic retinopathy by concomitant hypertension.

Effects of mechanical stress and high glucose on pericyte proliferation, apoptosis and contractile phenotype

Pericyte loss is an early step of diabetic retinopathy. High glucose induces apoptosis in retinal pericytes, but systemic and capillary hypertension are also believed to be important in the onset and progression of diabetic retinopathy. The haemodynamic insult of retinal capillary hypertension can be mimicked by exposing pericytes to mechanical stretch. We investigated the effect of stretch combined with high glucose on pericyte proliferation/apoptosis and morphology. Bovine retinal pericytes, cultured in either normal or high glucose concentrations in flexible-base plates, were exposed to mechanical stretch for 48/72 h. Cell replication was determined by both cell counting and DNA synthesis, apoptosis by ELISA, cell morphology and actin cytoskeleton distribution by immunofluorescence. Both reduction in cell proliferation and increase in apoptosis were confirmed in high glucose alone. When cells were subjected to stretch, proliferation was reduced and apoptosis increased in both normal and high glucose in comparison with unstretched controls. In both cases, a synergistic effect of hyperglycaemia combined with stretch was shown. Cell morphology showed modifications of cytoskeleton in all experimental conditions; in particular, cells subjected to stretch showed a clear elongation and translocation of actin fibres. In conclusion, our results show that stretch, alone or combined with high glucose, reduces cell proliferation, increases apoptosis and induces morphological changes in pericyte cytoskeleton. Further elucidations of the mechanisms on the basis of reduced proliferation of pericytes subjected to high glucose and stretch could help to clarify the effects of combined hyperglycaemia and hypertension in the pathogenesis of diabetic retinopathy.

Glyoxalase I Is Critical for Human Retinal Capillary Pericyte Survival under Hyperglycemic Conditions

Retinal capillary pericytes undergo premature death, possibly by apoptosis, during the early stages of diabetic retinopathy. The alpha-oxoaldehyde, methylglyoxal (MGO), has been implicated as a cause of cell damage in diabetes. We have investigated the role of MGO and its metabolizing enzyme, glyoxalase I, in high glucose-induced apoptosis (annexin V binding) of human retinal pericyte (HRP). HRP incubated with high glucose (30 mm d-glucose) for 7 days did not undergo apoptosis despite accumulation of MGO. However, treatment with a combination of high glucose and S-p-bromobenzylglutathione cyclopentyl diester, a competitive inhibitor of glyoxalase I, resulted in apoptosis along with a dramatic increase in MGO. Overexpression of glyoxalase I in HRP protected against S-p-bromobenzylglutathione cyclopentyl diester-induced apoptosis under high glucose conditions. Incubation of HRP with high concentrations of MGO resulted in an increase of apoptosis relative to untreated controls. We found an elevation of nitric oxide (NO.) in HRP that was incubated with high glucose when compared with those incubated with either the l-glucose or untreated controls. When HRP were incubated with an NO. donor, DETANONOATE ((Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), we observed both decreased glyoxalase I expression and activity relative to untreated control cells. Further studies showed that HRP underwent apoptosis when incubated with DETANONOATE and that apoptosis increased further on co-incubation with high glucose. Our findings indicate that glyoxalase I is critical for pericyte survival under hyperglycemic conditions, and its inactivation and/or down-regulation by NO. may contribute to pericyte death by apoptosis during the early stages of diabetic retinopathy.

Effects of Modified Low‐Density Lipoproteins on Human Retinal Pericyte Survival

According to a current paradigm cardiovascular diseases can be initiated by exposure of vascular cells to qualitatively modified low-density lipoproteins (LDL). Capillary leakage, an early feature of diabetic retinopathy, results in the exposure of retinal pericytes to modified LDL, including glycated (G-LDL) and heavily oxidized glycated LDL (HOG-LDL). We demonstrate here that modified LDL inhibits the proliferation and survival of cultured human retinal pericytes. Modified LDL also induced DNA fragmentation in bovine retinal pericytes. Overall, HOG-LDL produced a significantly higher extent of cytotoxicity and apoptosis in retinal pericytes. These results indicate that exposure of pericytes to HOG-LDL could be implicated in the development of diabetic retinopathy.

Substrates modified by advanced glycation end-products cause dysfunction and death in retinal pericytes by reducing survival signals mediated by platelet-derived growth factor

Premature death of retinal pericytes is a pathophysiological hallmark of diabetic retinopathy. Among the mechanisms proposed for pericyte death is exposure to AGE, which accumulate during diabetes. The current study used an in vitro model, whereby retinal pericytes were exposed to AGE-modified substrate and the mechanisms underlying pericyte death explored. Pericytes were isolated from bovine retinal capillaries and propagated on AGE-modified basement membrane (BM) extract or non-modified native BM. The extent of AGE modification was analysed. Proliferative responses of retinal pericytes propagated on AGE-modified BM were investigated using a 5-bromo-2-deoxy-uridine-based assay. The effect of extrinsically added platelet-derived growth factor (PDGF) isoforms on these proliferative responses was also analysed alongside mRNA expression of the PDGF receptors. Apoptotic death of retinal pericytes grown on AGE-modified BM was investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling labelling, mitochondrial membrane depolarisation and by morphological assessment. We also measured both the ability of PDGF to reverse Akt dephosphorylation that was mediated by AGE-modified BM, and increased pericyte apoptosis. Retinal pericytes exposed to AGE-modified BM showed reduced proliferative responses in comparison to controls (p<0.05-0.01), although this effect was reversed at low-AGE modifications. PDGF mRNA levels were differentially altered by exposure to low and high AGE levels, and AGE-modified BM caused significantly increased apoptosis in retinal pericytes. Pre-treatment of AGE-modified BM with PDGF-AA and -BB reversed the apoptosis (p<0.05-0.001) and restored Akt phosphorylation in retinal pericytes. Evidence suggests that substrate-derived AGE such as those that occur during diabetes could have a major influence on retinal pericyte survival. During diabetic retinopathy, AGE modification of vascular BM may reduce bioavailability of pro-survival factors for retinal pericytes.

Involvement of caspase-10 in advanced glycation end-product-induced apoptosis of bovine retinal pericytes in culture

Apoptosis appears to be the death mechanism of pericyte loss observed in diabetic retinopathy. We have previously shown that advanced glycation end-products (AGE-MGX) induce apoptosis of retinal pericytes in culture associated with diacylglycerol (DAG)/ceramide production. In the present study, we investigated possible caspase involvement in this process. Bovine retinal pericytes (BRP) were cultured with AGE-MGX and apoptosis examined after annexin V staining. Effects of peptidic inhibitors of caspases were determined on DAG/ceramide production and apoptosis. Pan-caspase inhibitor z-VAD-fmk (50 μM) was able to inhibit both DAG/ceramide production and apoptosis, whereas caspase-3-like inhibitor z-DEVD-fmk (50 μM) or caspase-9 inhibitor z-LEHD-fmk (50 μM) was only active on apoptosis. This differential effect strongly suggests involvement of initiator caspase(s) upstream and effector caspase(s) downstream DAG/ceramide production in AGE-mediated apoptosis. Pericyte treatment with caspase-8 inhibitor z-IETD-fmk (50 μM) did not protect cells against AGE-induced apoptosis and we failed to detect caspase-8 in pericytes by immunoblotting assay. Interestingly, one inhibitor of caspase-10 and related caspases z-AEVD-fmk (50 μM) inhibited both AGE-MGX-induced apoptosis and DAG/ceramide formation in pericytes. Cleavage of caspase-10 precursor into its active subunits was demonstrated by immunoblotting assay in pericytes incubated with AGE-MGX. These results strongly suggest that caspase-10, but not caspase-8, might be involved in the early phase of AGE-induced pericyte apoptosis, in contrast to caspase-9 and -3-like enzymes involved after DAG/ceramide production. This finding may provide new therapeutic perspectives for early treatment in diabetic retinopathy.

Involvement of caspase-10 in advanced glycation end-product-induced apoptosis of bovine retinal pericytes in culture.

Apoptosis appears to be the death mechanism of pericyte loss observed in diabetic retinopathy. We have previously shown that advanced glycation end-products (AGE-MGX) induce apoptosis of retinal pericytes in culture associated with diacylglycerol (DAG)/ceramide production. In the present study, we investigated possible caspase involvement in this process. Bovine retinal pericytes (BRP) were cultured with AGE-MGX and apoptosis examined after annexin V staining. Effects of peptidic inhibitors of caspases were determined on DAG/ceramide production and apoptosis. Pan-caspase inhibitor z-VAD-fmk (50 microM) was able to inhibit both DAG/ceramide production and apoptosis, whereas caspase-3-like inhibitor z-DEVD-fmk (50 microM) or caspase-9 inhibitor z-LEHD-fmk (50 microM) was only active on apoptosis. This differential effect strongly suggests involvement of initiator caspase(s) upstream and effector caspase(s) downstream DAG/ceramide production in AGE-mediated apoptosis. Pericyte treatment with caspase-8 inhibitor z-IETD-fmk (50 microM) did not protect cells against AGE-induced apoptosis and we failed to detect caspase-8 in pericytes by immunoblotting assay. Interestingly, one inhibitor of caspase-10 and related caspases z-AEVD-fmk (50 microM) inhibited both AGE-MGX-induced apoptosis and DAG/ceramide formation in pericytes. Cleavage of caspase-10 precursor into its active subunits was demonstrated by immunoblotting assay in pericytes incubated with AGE-MGX. These results strongly suggest that caspase-10, but not caspase-8, might be involved in the early phase of AGE-induced pericyte apoptosis, in contrast to caspase-9 and -3-like enzymes involved after DAG/ceramide production. This finding may provide new therapeutic perspectives for early treatment in diabetic retinopathy.

The role of polyol pathway in glucose-induced apoptosis of cultured retinal pericytes

The pathogenesis of pericyte loss, an initial deficit in the early stage of diabetic retinopathy, remains unclear. Recent studies have suggested that polyol pathway hyperactivity and apoptosis may be involved in pericyte loss. The mechanisms of the glucose-induced apoptosis in retinal pericytes were investigated to evaluate the pathogenesis of diabetic retinopathy. Under the 20 mM glucose condition, intracellular calcium concentrations and caspase-3 activities were significantly increased, and reduced glutathione (GSH) contents were significantly decreased compared with those under the 5.5 mM glucose condition. These abnormalities were all significantly prevented by an aldose reductase inhibitor, SNK-860. Glucose-induced apoptosis was partially but significantly prevented by SNK-860, an inhibitor of calcium-dependent cysteine protease, calpain, or GSH supplementation, and completely normalized by a caspase-3 inhibitor. These observations suggest that glucose-induced apoptosis in retinal pericytes, as one of the pathogenic factors of diabetic retinopathy, would be mediated through an aldose reductase-sensitive pathway including calcium–calpain cascade and increased oxidative stress, and that caspase-3 would be located furthest downstream of these apoptotic signals.