Calcium dobesilate may alleviate diabetes‑induced endothelial dysfunction and inflammation

Abstract

Diabetic kidney disease (DKD) is a leading cause of end‑stage renal disease. However, the pathogenesis of DKD remains unclear, and no effective treatments for the disease are available. Thus, there is an urgent need to elucidate the pathogenic mechanisms of DKD and to develop more effective therapies for this disease. Human umbilical vein endothelial cells (HUVECs) were cultured using different D‑glucose concentrations to determine the effect of high glucose (HG) on the cells. Alternatively, HUVECs were incubated with 100 µmol/l calcium dobesilate (CaD) to detect its effects. The authors subsequently measured HUVEC proliferation via cell counting kit‑8 assays. In addition, HUVEC angiogenesis was investigated via migration assays and fluorescein isothiocyanate (FITC)‑labelled bovine serum albumin (BSA) permeability assays. The content or distribution of markers of endothelial dysfunction [vascular endothelial growth factor (VEGF), VEGF receptor (R) and endocan) or inflammation [intercellular adhesion molecule (ICAM)‑1, monocyte chemotactic protein (MCP)‑1 and pentraxin‑related protein (PTX3)] was evaluated via reverse transcription‑quantitative polymerase chain reaction and western blotting. HG treatment induced increased in VEGF, VEGFR, endocan, ICAM‑1, MCP‑1 and PTX3 mRNA and protein expression in HUVECs. HG treatment for 24 to 48 h increased cell proliferation in a time‑dependent manner, but the cell proliferation rate was decreased at 72 h of HG treatment. Conversely, CaD inhibited abnormal cell proliferation. HG treatment also significantly enhanced HVUEC migration compared to the control treatment. In contrast, CaD treatment partially inhibited HUVEC migration compared to HG exposure. HG‑treated HUVECs exhibited increased FITC‑BSA permeability compared to control cells cultured in medium alone; however, CaD application prevented the HG‑induced increase in FITC‑BSA permeability and suppressed HG‑induced overexpression of endothelial markers (VEGF, VEGFR‑2, endocan) and inflammation markers (ICAM‑1, MCP‑1, PTX3) in HUVECs. CaD has angioprotective properties and protects endothelial cells partly by ameliorating HG‑induced inflammation. The current results demonstrated the potential applicability of CaD to the treatment of diabetic nephropathy, particularly during the early stages of this disease.

Figures

Figure 1.

mRNA expression of VEGF, VEGFR, endocan, ICAM, MCP-1 and PTX3 mRNA in high glucose conditions in human umbilical vein endothelial cells. mRNA expression gradually increased. *P

Figure 2.

Western blot analysis of VEGF, VEGFR, endocan, ICAM, MCP-1 and PTX3 protein expression. Expression gradually increased under high glucose conditions in human umbilical vein endothelial cells. *P

Figure 3.

Effects of different cell treatments on HUVEC proliferation. Cell Counting kit-8 assay was performed to analysed effect on cell proliferation. HG (35 mmol/l) treatment induced increased cell proliferation at 24 and 48 h. CaD (100 µmol/l) application significantly attenuated abnormal HUVEC proliferation. *P

Figure 4.

CaD prevents migration of HUVECs, which partially induced by diabetes or HG. Transwell assay analysing migration of HUVECs following cell treatment. The stained cells were counted in 10 randomly chosen fields at ×200 magnification. *P&P<0.05 vs. mannitol group. CaD, calcium dobesilate; HG, high glucose; HUVECs, human umbilical vein endothelial cells.

Figure 5.

HG increased the permeability of HUVECs, which inhibited by CaD. Permeability assay analysing permeability of HUVECs following cell treatment. *P&P<0.05 vs. mannitol group. HG, high glucose; HUVECs, human umbilical vein endothelial cells; CaD, calcium dobesilate.

Figure 6.

HG (35 mmol/l) increased the mRNA levels of VEGF, VEGFR-2, endocan in a time-dependent manner from 12 to 48 h. However, the growth trend began to slow down in 72 h. 100 µmol/l CaD prevented the increase of VEGF, VEGFR-2 and endocan levels induced by HG. HG also increased the mRNA levels of ICAM, MCP-1 and PTX3 in a time-dependent manner from 12 to 72 h. 100 µmol/l CaD prevented the increase the expression of these inflammatory factors induced by HG. (A) HUVECs were incubated with control or experimental medium for 12 h. (B) HUVECs were incubated with control or experimental medium for 24 h. (C) HUVECs were incubated with control or experimental medium for 48 h. (D) HUVECs were incubated with control or experimental medium for 72 h. *P

Figure 7.

HG (35 mmol/l) increased the protein levels of VEGF, VEGFR-2, endocan in a time-dependent manner from 12 to 48 h. However, the growth trend began to slow down in 72 h. 100 µmol/l CaD prevented the increase of VEGF, VEGFR-2 and endocan levels induced by HG. HG also increased the protein levels of ICAM, MCP-1 and PTX3 in a time-dependent manner from 12 to 72 h. 100 µmol/l CaD prevented the increase the expression of these inflammatory factors induced by HG. (A) HUVECs were incubated with control or experimental medium for 12 h. (B) HUVECs were incubated with control or experimental medium for 24 h. (C) HUVECs were incubated with control or experimental medium for 48 h. (D) HUVECs were incubated with control or experimental medium for 72 h. *P&P<0.05 vs. control group. HG, high glucose; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; ICAM, intercellular adhesion molecule; MCP-1, monocyte chemotactic protein 1; PTX3, pentraxin 3; CaD, calcium dobesilate; HUVECs, human umbilical vein endothelial cells.