School of Pharmacy & Life Sciences, Institute for Health & Welfare Research, Robert Gordon University, Schoolhill, Aberdeen AB10 1FR, UK.
Microvasc Res. 2013 Jul;88:61-9. doi: 10.1016/j.mvr.2013.03.008. Epub 2013 Apr 3.
Delayed wound healing is characteristic of those affected by both Type 1 and Type 2 diabetes. We have developed a novel assay to investigate endothelial cell migration using primary microvascular endothelial cells of dermal origin. Endothelial cell migration was determined using defined monolayers of cells. Net migration or migration at a wounded edge was recorded after 24 or 48 h following incubation in either 20% or 5% oxygen in combination with either 5 mmol/l or 20 mmol/l glucose. Specific intracellular inhibitors of p42/44 MAPK, Pi3 kinase and protein kinase CβII were used. Hypoxia inducible factor type 1 alpha protein was detected using immunocytochemical staining. Cell migration was increased in the presence of hypoxia and decreased with high glucose concentration (p<0.001). The newly developed wound healing assay revealed that re-endothelialisation occurred at a greater rate (p<0.001) than endothelialisation. Inhibition of p42/44MAPK significantly reduced endothelial cell migration at both the intact and the wounded edge in 20 mmol/l glucose but not 5 mmol/l glucose. Inhibition of Pi3 kinase significantly (p<0.001) reduced migration in all test conditions, while inhibition of PKCβ restored glucose mediated impaired migration (p>0.05). HIF-1α protein levels did not significantly reduce in the presence of a PKCβ inhibitor at the wounded edge of cells in 20 mmol/l glucose. In conclusion, we have established a novel assay to determine endothelial cell migration that is robust and reproducible. Impaired cell migration mediated by high glucose concentration was restored using an inhibitor of the PKCβII pathway which correlated with an increase in the level of HIF1α protein.
1 型和 2 型糖尿病患者均存在伤口愈合延迟的特征。我们开发了一种新的分析方法，使用源自皮肤的原代微血管内皮细胞来研究内皮细胞迁移。使用定义明确的单层细胞确定内皮细胞迁移。在 20%或 5%氧气中孵育 24 或 48 小时后，在存在 5 mmol/l 或 20 mmol/l 葡萄糖的情况下，记录到净迁移或在伤口边缘的迁移。使用特定的细胞内 p42/44 MAPK、Pi3 激酶和蛋白激酶 CβII 抑制剂。使用免疫细胞化学染色检测缺氧诱导因子 1α 蛋白。缺氧时细胞迁移增加，高葡萄糖浓度时细胞迁移减少（p<0.001）。新开发的伤口愈合分析显示，再内皮化的速度比内皮化更快（p<0.001）。在 20 mmol/l 葡萄糖中，但在 5 mmol/l 葡萄糖中，p42/44MAPK 的抑制显著降低了完整和伤口边缘的内皮细胞迁移。在所有测试条件下，Pi3 激酶的抑制显著降低了迁移（p<0.001），而 PKCβ 的抑制恢复了葡萄糖介导的迁移受损（p>0.05）。在存在 20 mmol/l 葡萄糖中细胞伤口边缘的 PKCβ 抑制剂时，HIF-1α 蛋白水平没有显著降低。总之，我们建立了一种新的分析方法来确定内皮细胞迁移，该方法具有稳健性和可重复性。使用 PKCβII 途径抑制剂恢复了由高葡萄糖浓度介导的受损细胞迁移，这与 HIF1α 蛋白水平的增加相关。
