RESUMO
Diabetic retinopathy (DR) is a microvascular complication of diabetes mellitus, and its main clinical manifestation is retinal vascular dysfunction. DR causes blindness and is a problem with significant global health implications. However, treating DR is still challenging. In this study, we aimed to explore the role of polo-like kinase-3 (PLK-3) and the potential regulatory mechanism in DR. Sprague-Dawley rats were injected intraperitoneally with streptozotocin (STZ, 60 mg/kg) to induce a rat model of DR, and rat retinal microvascular endothelial cells (RRMECs) were treated with high glucose (HG, 25 mmol/L glucose) to develop a cell model of DR. We found that PLK-3 was significantly downregulated in the retinal tissues of STZ-induced diabetic rats and HG-induced RRMECs. Lentivirus-mediated PLK-3 overexpression alleviated the histological damages in DR rats. After HG stimulation, cell proliferation, migration, and angiogenesis in RRMECs were inhibited after PLK-3 upregulation. By using label-free proteomics, we identified 82 differentially expressed proteins downstream of PLK-3, including BRCA1-associated protein 1 (BAP1), which was significantly upregulated in PLK-3-overexpressed RRMECs compared to control cells under the HG condition. In vivo and in vitro assays indicated that the forced expression of PLK-3 increased the phosphorylation of BAP1 at serine 592 and caspase-8 expression. Detailed evidence showed that BAP1-shRNA-mediated knockdown restored the cell function in HG-treated RRMECs when PLK-3 was overexpressed. Collectively, this study shows that PLK-3 alleviates retinal vascular dysfunction in DR by inhibiting the phosphorylation of BAP1. Thus, PLK-3 may develop as a promising target for the therapy of DR.
RESUMO
Diabetic retinopathy (DR), a microvascular complication characterized by abnormal angiogenesis, is the most common reason for irreversible blindness. Glycoprotein non-metastatic melanoma protein B (GPNMB), as a transmembrane protein, was found to be associated with angiogenesis. This study aims to investigate the role of GPNMB in DR. The levels of GPNMB and Integrin ß1 were detected by real-time PCR and western blot and were found to be increased in human retinal microvascular endothelial cells (HRMECs) with high glucose (HG, 25 mmol/L) treatment. Knockdown of GPNMB was mediated by lentivirus carrying shRNA targeting GPNMB in vivo and in vitro. Functional experiments, including cell counting kit-8 (CCK-8), scratch, and tube formation assays, showed the anti-proliferative, anti-migrative, and anti-angiogenic roles of GPNMB knockdown in HRMECs using the lentivirus system following HG challenge. Additionally, increased GPNMB levels were detected in the retina of DR rats induced by a single intraperitoneal injection of streptozotocin (60 mg/kg) using real-time PCR, western blot, and immunofluorescence assays. Downregulation of GPNMB inhibited the angiogenesis and vascular endothelial growth factor production in the retina of rats with DR. Furthermore, overexpression of Integrin ß1 led to increased angiogenesis in DR. Integrin ß1 was indicated as a target protein of GPNMB. Upregulated-Integrin ß1 restored GPNMB knockdown-induced inhibition of cell viability, migration, and tube formation in HRMECs. In conclusion, we provide evidence for the angiogenic role of GPNMB and demonstrate that silencing GPNMB may represent a therapeutic potential in the treatment of DR.
