Human Retinal Capillary Endothelial Cells Research Articles

The role of endoplasmic reticulum stress in the early stage of diabetic retinopathy

The aim of this study was to evaluate the role of endoplasmic reticulum (ER) stress in diabetic retinopathy (DR) using in vitro and in vivo models. For the in vivo studies, diabetes was induced in rats, and retinal expression of glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), and vascular endothelial growth factor (VEGF) in groups of rats at 1, 3, and 6 months was assessed. For the in vitro studies, human retinal capillary endothelial cells (HRCECs) were cultured in the presence of varying glucose concentrations, and the expression of mRNA and protein for GRP78, ATF4, CHOP, and VEGF was assessed. The chosen glucose concentrations were reflective of those apparent in diabetic patients. Expression of VEGF and CHOP mRNA levels were significantly increased in diabetic rats compared to control rats at 1, 3, and 6 months (P < 0.05). In HRCECs cultured in the presence of high as well as variable glucose concentrations, CHOP expression and apoptosis were significantly increased (P < 0.05). However, GRP78 and ATF4 expression levels were unchanged. Our findings suggest that early progression of DR may be mediated by ER stress, but probably does not involve changes in ATF4 or GRP78.

Primary Culture of Human Blood–Retinal Barrier Cells and Preliminary Study of APOBEC3 Expression: An In Vitro Study

To develop methods for primary culture of human blood-retinal barrier (BRB) cells and to explore the expression of APOBEC3 (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3) family gene, novel host-defense factors to HIV-1. Cellular components of human BRB (human retinal capillary endothelial cells [HRCECs], human retinal capillary pericytes, and human retinal pigment epithelial cells) were isolated separately and subjected to primary culture according to procedures modified in our laboratory. Immunocytochemistry and immunofluorescence were used to identify specific markers of the primary cells and to analyze their purity by flow cytometry. RNA of the three different cells was isolated, and primers were designed to probe expression of the APOBEC3 gene by reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. For further confirmation, APOBEC3F and APOBEC3G proteins were detected in the cultured cells and fresh retina tissue through Western blot analysis. In the end, HRCECs were treated with IFN-gamma, and change of APOBEC3G expression was displayed. Pure BRB cells (>95% purity) were primary cultured according to procedures modified in our laboratory. Qualitative test of RT-PCR and semiquantitative examination of real-time PCR demonstrated the presence of APOBEC3B, -3C, -3F, and -3G genes and the absence of APOBEC3A and -3D genes in all cellular components of the BRB. Finding of the APOBEC3G and APOBEC3F proteins expressed in the three primary cultured cells and different layers of retinal tissue by Western blot analysis further confirmed the PCR results. Moreover, IFN-gamma could upregulate the expression of APOBEC3G in HRCECs. Major cellular components of human BRB could be primary cultured in vitro according to procedures optimized in our laboratory. Different expression of APOBEC3 in human blood-retinal barrier gives a clue to further research in intrinsic antiviral immunity in HIV-1-related retinopathy.

Remodeling Retinal Neovascularization by ALK1 Gene Transfection In Vitro

To explore a novel strategy for balancing retinal neovascularization by assessing the role activin-like kinase receptor 1 (ALK1) plays in neovascularization in vascular endothelial growth factor (VEGF)-stimulated human retinal capillary endothelial cells (HRCECs). HRCECs were transfected with an ALK1 gene-encoding plasmid or a pSIREN-ALK1 RNAi vector and stimulated with VEGF. The mRNA and protein expression levels of ALK1, occludin, ANG2, and ALK5 were evaluated by real-time PCR and/or Western blot analysis. Microscopy techniques and flow cytometry were used to assess the effects of enhanced levels of ALK1 on migration and proliferation and the formation of tubelike structures of HRCECs. The level of ALK1 in ALK1-transfected cells was significantly increased compared with that in control cells. ALK1-transfected cells exhibited increased expression of occludin and decreased expression of ANG2 and ALK5, compared with expression in the control cells. HRCECs transfected with pSIREN-ALK1 RNAi exhibited decreased expression of ALK1 and occludin and increased expression of ANG2 and ALK5 compared with the control cells. Transfection with ALK1 affected the migration and proliferation of VEGF-stimulated HRCECs. ALK1 also inhibited the formation of endothelial tubelike structures, but did allow the formation of entire vessel structures. Overexpression of ALK1 promoted remodeling of newly formed blood vessels and prevented further angiogenesis. These findings provide insight into the control of retinal neovascularization and demonstrate a novel strategy for maintaining a stable phase of vessel formation, allowing for effective retinal neovascularization without the common adverse effects seen in patients with diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion.

Enhanced anti‐angiogenic effect of a deletion mutant of plasminogen kringle 5 on neovascularization

Kringle 5 (K5), a proteolytic fragment of plasminogen, has been proved to be an angiogenic inhibitor. Previously, we have evaluated the effect of K5 on the vascular leakage and neovascularization in a rat model of oxygen-induced retinopathy. In this study, we expressed K5 and a deletion mutant of K5 (K5 mutant) in a prokaryocyte expression system and purified them by affinity chromatography. K5 mutant was generated by deleting 11 amino acids from K5 while retaining the three disulfide bonds. The anti-angiogenic activity of intact K5 and K5 mutant were compared in endothelial cells and retinal neovascularization rat model. K5 mutant inhibited the proliferation of primary human retinal capillary endothelial cells (HRCEC) in a concentration-dependent manner, with an apparent EC50 of approximate 35 nmol/L, which is twofold more potent than intact K5. In the even higher concentration range, K5 mutant did not inhibit pericytes from the same origin of HRCEC, which suggested an endothelial cell-specific inhibition. K5 mutant had no effect on normal liver cells and Bel7402 hepatoma cells even at high concentration range either. Intravitreal injection of the K5 and mutant in the oxygen-induced retinopathy rat model both resulted in significantly fewer neovascular tufts and nonperfusion area than controls with PBS injection, as shown by fluorescein angiography. Furthermore, K5 mutant exhibited more strong inhibition effect on neovascularization than intact K5 by quantification of vascular cells. These results suggest that this K5 deletion mutant is a more potent angiogenic inhibitor than intact K5 and may have therapeutic potential in the treatment of those disorders with neovascularization, such as solid tumor, diabetic retinopathy, age-related macular degeneration, rheumatoid arthritis, and hyperplasia of prostate.

Culture and characterization of human retinal capillary endothelial cell

目的 建立人视网膜血管内皮细胞培养的简便方法.方法胰蛋白酶消化供角膜移植术后留下的眼球,获得新鲜视网膜血管,用5%胎牛血清的人内皮细胞培养基培养(HE-SFM BGM),添加生长因子和胰岛素-转铁蛋白-硒添加物,并用纤维连接蛋白促进内皮细胞贴壁.第Ⅷ因子相关抗原鉴定内皮细胞.结果内皮细胞第1 d贴壁,第6 d形成克隆,第15 d细胞融合.传至7～8代转化为成纤维细胞.第Ⅷ因子相关抗原鉴定纯净度达95%.结论利用5%胎牛血清的人内皮细胞培养基,添加生长因子和胰岛素-转铁蛋白-硒添加物,并用纤维连接蛋白包被培养瓶,能简单有效地培养人视网膜血管内皮细胞。

The expression of plasminogen activators and their inhibitors in cultured human retinal capillary endothelial cells.

The expression of plasminogen activators and their inhibitors in cultured human retinal capillary endothelial cells.