Retinal Vascular Cell Apoptosis Research Articles

Downregulation of Drp1 and Fis1 Inhibits Mitochondrial Fission and Prevents High Glucose-Induced Apoptosis in Retinal Endothelial Cells.

Diabetic retinopathy is a prevalent microvascular complication characterized by apoptotic vascular cell loss in the retina. Previous studies have shown that high glucose (HG)-induced mitochondrial fragmentation plays a critical role in promoting retinal vascular cell apoptosis. Here, we investigated whether downregulation of mitochondrial fission genes, Fis1 and Drp1, which are overexpressed in HG condition, prevents mitochondrial fragmentation, preserves mitochondrial function, and protects retinal endothelial cells from apoptosis. Rat retinal endothelial cells (RRECs) were grown in normal (5 mM glucose) or HG (30 mM glucose) medium; in parallel, cells grown in HG medium were transfected with either Fis1 siRNA or Drp1 siRNA, or both siRNAs in combination, or scrambled siRNA as control. Live-cell confocal imaging showed decreased mitochondrial fission in cells transfected with Fis1 siRNA or Drp1 siRNA concomitant with reduced TUNEL-positive cells and a decrease in the expression of pro-apoptotic proteins, Bax and cleaved caspase 3, under HG condition. Importantly, the combined siRNA approach against Fis1 and Drp1 prevented HG-induced changes in the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). The findings from this study indicate that reducing HG-induced overexpression of mitochondrial fission genes preserves mitochondrial morphology and prevents retinal vascular cell apoptosis associated with diabetic retinopathy.

Bcl-2 Expression in Pericytes and Astrocytes Impacts Vascular Development and Homeostasis

B-cell lymphoma 2 (Bcl-2) protein is the founding member of a group of proteins known to modulate apoptosis. Its discovery set the stage for identification of family members with either pro- or anti-apoptotic properties. Expression of Bcl-2 plays an important role during angiogenesis by influencing not only vascular cell survival, but also migration and adhesion. Although apoptosis and migration are postulated to have roles during vascular remodeling and regression, the contribution of Bcl-2 continues to emerge. We previously noted that the impaired retinal vascularization and an inability to undergo pathologic neovascularization observed in mice globally lacking Bcl-2 did not occur when mice lacked the expression of Bcl-2 only in endothelial cells. To further examine the effect of Bcl-2 expression during vascularization of the retina, we assessed its contribution in pericytes or astrocytes by generating mice with a conditional Bcl-2 allele (Bcl-2Flox/Flox) and Pdgfrb-cre (Bcl-2PC mice) or Gfap-cre (Bcl-2AC mice). Bcl-2PC and Bcl-2AC mice demonstrated increased retinal vascular cell apoptosis, reduced numbers of pericytes and endothelial cells and fewer arteries and veins in the retina. Bcl-2PC mice also demonstrated delayed advancement of the superficial retinal vascular layer and aberrant vascularization of the deep vascular plexus and central retina. Although pathologic neovascularization in oxygen-induced ischemic retinopathy (OIR) was not affected by lack of expression of Bcl-2 in either pericytes or astrocytes, laser-induced choroidal neovascularization (CNV) was significantly reduced in Bcl-2PC mice compared to littermate controls. Together these studies begin to reveal how cell autonomous modulation of apoptosis in vascular cells impacts development and homeostasis.

Aster koraiensis extract prevents diabetes-induced retinal vascular dysfunction in spontaneously diabetic Torii rats

BackgroundAster koraiensis extract (AKE) is a standard dietary herbal supplement. The aim of this study is to investigate the inhibitory effects of AKE on diabetes-induced retinal vascular dysfunction in Spontaneously Diabetic Torii (SDT) rats.MethodsAKE (50 and 100 mg/kg body weight/day) was administered for 16 weeks. The effects of orally administered AKE on blood glucose levels, retinal vascular leakage, apoptosis, and accumulation of advanced glycation end products (AGEs) in the retina were evaluated.ResultsSDT rats exhibited hyperglycemia and retinal vascular leakage, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was clearly detected apoptosis in the retinal microvasculature. Immunofluorescence staining revealed the accumulation of AGEs in the retinal vasculature of the SDT rats. However, oral administration of AKE for 16 weeks blocked diabetes-induced blood–retinal barrier (BRB) breakdown and the loss of occludin, which is an important tight junction protein. Apoptosis of retinal vascular cells and AGE accumulation were significantly inhibited after AKE treatment.ConclusionThese results indicate that, as a dietary herbal supplement, AKE may have beneficial effects on patients with diabetic retinopathy.

The effects of Curcumin on the cellular apoptosis induced by high glucose in rat retinal vascular endothelial cells

Objective To observe the effects of Curcumin on the cellular apoptosis of rat retinal vascular endothelial cells (RRVEC) induced by high glucose. Methods Generation 4 cultured RRVEC were used in this experiment, and identified with anti-vWF factor antibody by immunochemistry and immunofluorescence. The RRVEC were divided into control group (5.5 mmol/L glucose), high glucose group (30 mmol/L glucose), and treatment group (30 mmol/L glucose+30 μmol/L Curcumin), respectively. Flow cytometry was used to measure the cellular reactive oxygen species (ROS) level and apoptosis. The expression intensity and location of nuclear factor (NF)-κB p65 in the cells of the three groups were detected by immunochemistory. The expression of Bcl-2 and Bax protein was detected by Western blot test. Results Immunostaining showed that RRVEC were positive for vWF factor. The flow cytometry showed that the cellular ROS level in treatment group was higher than that in the control group (t=8.677, P=0.000), but less than that in the high glucose group (t=40.957, P=0.000). Compared with the high glucose group, the cellular ROS level in the treatment group was decreased significantly (t=6.568, P=0.000). The cellular apoptosis were significantly different among the three groups (F=325.137, P=0.000). Compared with the high glucose group, the cellular apoptosis in the treatment group was decreased significantly (t=12.818, P=0.000). Immunochemistry showed that NF-κB p65 was expressed strongly in the cellular nuclei and cytoplasm in the high glucose group than that in the control group and the treatment group with the significant differences (t=8.322, P=0.000). Western blot results demonstrated that compared with the control group, the expression of Bcl-2 of RRVEC and Bcl-2/Bax ratio decreased (t=4.362, 6.449; P=0.005, 0.001) and Bax increased (t=3.813, P=0.009) in the high glucose group, with statistically significant differences. Compared with the high glucose group, the expression of NF-κB and Bax decreased (t=2.577, 3.059; P=0.042, 0.022) and Bcl-2/Bax ratio increased significantly (t=3.831, P=0.009) in the treatment group. Conclusion Curcumin could suppress the cellular apoptosis of RRVEC induced by high glucose. The mechanism of Curcumin protecting RRVEC may be via regulating NF-κB signal pathway. Key words: Curcumin; Retinal Vessels/pathology; Endothelial cells; Apoptosis; NF-κB

The effects of interleukin 17 on the proliferation, migration and apoptosis of human retinal vascular endothelial cells

Objective To address the effect and mechanism of interleukin 17 (IL-17) on the proliferation, migration and apoptosis of human retinal vascular endothelial cells (HREC). Methods IL-17 receptor (IL-17R) mRNA and protein expression in human retinal vascular endothelial cells (HREC) were quantified by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation of HREC was examined using CCK-8 assay in the presence of different concentrations of IL-17. Cell migration of HREC was detected using wound scratch assay. Flow cytometry was used to test the effect of IL-17 on the apoptosis of HREC. The effects of IL-17 on HREC expression of basic fibroblast growth factor (bFGF), Caspase-3 and thrombospondin-1 (TSP-1) were quantified by reverse transcriptase polymerase chain reaction (RT-PCR). The effect of IL-17 on HREC expression of Caspase-3 was examined using Western blot. Results IL-17 receptor (IL-17R) expressed in HREC as quantified by RT-PCR and Western blot. The proliferation of HREC in the presence of IL-17 was promoted in a dosage-dependent manner (t=-3.235, -6.276; P=0.032, 0.000). Wound scratch assay showed a significant increase in the migrated distance of HREC with IL-17 stimulation under the concentration of 100 μg/L(t=-3.551, -2.849; P=0.006, 0.019), 200 μg/L(t=-10.347, -4.519; P=0.000, 0.001) and 500 μg/L (t=-3.541, -2.607; P=0.008, 0.036). The intervention of 200 μg/L IL-17 can effectively inhibit the apoptosis of HREC, compared with the control group using flow cytometry (t=5.682, P=0.047). RT-PCR results showed that IL-17 can promote the expression of bFGF and inhibit the expression of Caspase-3 and TSP-1. Western blot result also showed that IL-17 can suppress the protein expression of Caspase-3. Conclusion The mechanism of IL-17 promoting proliferation, migration but suppress apoptosis of HREC may via regulating the expression of bFGF and Caspase-3. Key words: Interleukin-17; Endothelial cells/pathophysiology; Cell proliferation; Cell movement; Apoptosis

Epicatechin breaks preformed glycated serum albumin and reverses the retinal accumulation of advanced glycation end products

The accumulation of advanced glycation end products (AGEs) is associated with many of the complications of diabetes mellitus, including diabetic retinopathy. AGE-breakers, such as N-phenacylthiazolium and alagebrium, have been proposed as therapeutic agents for reversing the increase in protein crosslinking in diabetes. (−)-Epicatechin is a major dietary flavonoid with a wide range of health-promoting biological activities. The aim of this study was to determine the potential effect of (−)-epicatechin in reducing the burden of AGEs in vitro and in vivo and to evaluate whether the reduced AGE burden could translate into improvement in retinal vascular function in exogenously AGE-injected rats. Glycated human serum albumin was purified from patients with diabetes. The breakdown of the already formed AGEs was studied by treating glycated human serum albumin with (−)-epicatechin. To study the effect of (−)-epicatechin on retinal vascular function, exogenously AGE-injected rats were treated with (−)-epicatechin (50 and 100mg/kg i.p.) for two weeks. Apoptosis of retinal vascular cells was quantified using TUNEL staining. The AGE load in the retinas was determined via immunohistochemical staining and western blot analysis. (−)-Epicatechin was able to break preformed glycated human serum albumin in vitro as well as reduce AGE accumulation in retinas in vivo in a dose dependent manner. In exogenously AGE-injected rats, treatment with (−)-epicatechin was evidenced by an improved retinal vascular apoptosis. AGE burden in retinas was also reduced upon treatment. This study suggests that (−)-epicatechin could represent a valuable drug for the treatment of diabetic retinopathy by reducing the AGE burden.

Reduced Connexin 43 Expression and Its Effect on the Development of Vascular Lesions in Retinas of Diabetic Mice

PURPOSE. To examine whether diabetes-induced connexin 43 downregulation promotes retinal vascular lesions characteristic of diabetic retinopathy (DR). METHODS. Two animal models, streptozotocin-induced diabetic mice and Cx43 heterozygous knockout (Cx43(+/-)) mice, were studied to directly assess whether diabetes reduces the expression of retinal Cx43, which, in turn, contributes to retinal vascular cell loss by apoptosis. Retinal Cx43 protein levels were assessed in nondiabetic control mice, diabetic mice, and Cx43(+/-) mice by Western blot analysis, and Cx43 localization and distribution in the retinal vascular cells were studied by immunostaining of retinal trypsin digests (RTDs). In parallel, RTDs were stained with hematoxylin and periodic acid Schiff to determine pericyte loss (PL) and acellular capillaries (AC), and TUNEL assays were performed to determine retinal vascular cell apoptosis. RESULTS. Western blot analysis indicated significant reductions in retinal Cx43 protein levels in diabetic mice and Cx43(+/-) mice compared with those of nondiabetic mice. Similarly, a significant reduction in Cx43 immunostaining was observed in the retinal capillaries of diabetic mice and Cx43(+/-) mice compared with those of control mice. Both diabetic and age-matched Cx43(+/-) mice exhibited increased amount of PL, AC, and TUNEL-positive cells compared with control mice. CONCLUSIONS. Diabetes-induced inhibition of Cx43 expression contributes to vascular cell apoptosis in retinas of diabetic mice. This suggests that reduced Cx43 expression plays a critical role in the development of AC and PL associated with DR.

Delivery of Antioxidant Enzyme Genes to Protect against Ischemia/Reperfusion-Induced Injury to Retinal Microvasculature

Retinal ischemia/reperfusion (I/R) injury results in the generation of reactive oxygen species (ROS). The aim of this study was to investigate whether delivery of the manganese superoxide dismutase gene (SOD2) or the catalase gene (CAT) could rescue the retinal vascular damage induced by I/R in mice. I/R injury to the retina was induced in mice by elevating intraocular pressure for 2 hours, and reperfusion was established immediately afterward. One eye of each mouse was pretreated with plasmids encoding manganese superoxide dismutase or catalase complexed with cationic liposomes and delivered by intravitreous injection 48 hours before initiation of the procedure. Superoxide ion, hydrogen peroxide, and 4-hydroxynonenal (4-HNE) protein modifications were measured by fluorescence staining, immunohistochemistry, and Western blot analysis 1 day after the I/R injury. At 7 days after injury, retinal vascular cell apoptosis and acellular capillaries were quantitated. Superoxide ion, hydrogen peroxide, and 4-HNE protein modifications increased at 24 hours after I/R injury. Administration of plasmids encoding SOD2 or CAT significantly reduced levels of superoxide ion, hydrogen peroxide, and 4-HNE. Retinal vascular cell apoptosis and acellular capillary numbers increased greatly by 7 days after the injury. Delivery of SOD2 or CAT inhibited the I/R-induced apoptosis of retinal vascular cell and retinal capillary degeneration. Delivery of antioxidant genes inhibited I/R-induced retinal capillary degeneration, apoptosis of vascular cells, and ROS production, suggesting that antioxidant gene therapy might be a treatment for I/R-related disease.

Antioxidants and an inhibitor of advanced glycation ameliorate death of retinal microvascular cells in diabetic retinopathy

Pericyte ghosts and acellular capillaries are well known as early histological changes resulting from diabetic retinopathy. These histological changes mean that the cell death of retinal microvessels has accelerated. It was reported that apoptosis of retinal microvascular cells (RMCs) was increased in diabetic patients. Therefore, we investigated apoptosis of RMCs in Goto-Kakizaki (GK) rats, a type 2 diabetic model, and involvement with antioxidants (a combination of vitamins C and E) or a novel inhibitor of advanced glycation, OPB-9195. GK rats were treated with the antioxidants combination or OPB-9195 for 36 weeks. We obtained isolated preparations of the vascular network from their retinas by trypsin digestion. Apoptosis of retinal vascular cells was detected with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. We found that apoptosis of RMCs was increased in the diabetic GK rats. Furthermore, a combination of vitamins C and E and an advanced glycation end-products inhibitor mostly inhibited this increased apoptosis. We concluded that apoptosis of RMCs was a good marker that indicates the progression of diabetic retinopathy in GK rats. Both oxidative stress and the accumulation of advanced glycation end-products appears to promote the apoptosis of retinal microvascular cells, and antioxidants or advanced glycation end-products inhibitors might ameliorate diabetic retinopathy.