Figure 2

Protein kinase C (PKC) plays a role in the high-concentration glucose-induced disruption of endothelial adherens junctions. A, Treatment of human umbilical vein endothelial cells (HUVECs) with glucose for 24 h led to an increase in total PKC activity. Total PKC activity was measured spectrophotometrically at 492 nm by using a colorimetric enzyme-linked immunosorbent assay technique. For each sample shown, three independent experiments were performed. B, Treatment of HUVECs with glucose for 24 h increased ERK1/2 phosphorylation. C, Treatment of HUVECs with 10 μM PKC inhibitor, bisindolylmaleimide I (Bis), for 2 h attenuated high-concentration glucose-induced tyrosine phosphorylation of VE-cad. HUVECs were first treated with 20 mM of glucose for 24 h. D, Treatment of HUVECs for 1 and 48 h increased and inhibited PKC activity, respectively. Total PKC activity was measured spectrophotometrically at 492 nm by using a colorimetric enzyme-linked immunosorbent assay technique. E, Treatment of HUVECs with 50 nM of phorbol myristate acetate (PMA) for 48 h attenuated high-concentration glucose-induced transendothelial migration (TEM) of THP-1 cells. HUVECs were washed with the cell culture medium before addition of THP cells. The TEM of THP-1 cells were evaluated by using a transwell assay. Calcein-labeled THP-1 cells were quantified by using a fluorescent plate reader. Data are expressed as the mean ± SD from triplicate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control. Each experiment was independently performed 3 to 4 times.