Effect of α-Klotho on macrophage-vascular endothelial cell crosstalk in diabetic oxidative stress environment

Abstract:

AIM:To investigate the effects of overexpressing α-Klotho(KL)in RAW264.7 cells stimulated by oxidative stress on the proliferation, migration, tube-formation and tight junction of human umbilical vein endothelial cells(HUVECs).

METHODS:RAW264.7 cells were categorized into control, 4-hydroxynonenal(4HNE), and 4HNE+KL groups, with F4/80 expression assessed via immunofluorescence staining. Three groups of conditional media were prepared for HUVECs and culture divided into Mø-NC, Mø-4HNE, and Mø-4HNE+KL groups. Cell proliferation was evaluated using CCK8 assay, while scratch test and Transwell assays were employed to measure cell migration. Additionally, tube-formation assay was conducted to assess cell tubule formation, and Western blot assay was utilized to detect the protein expression levels of Claudin 5, Occludin and ZO 1.

RESULTS:The results of immunofluorescence staining showed that the fluorescence intensity of F4/80 of RAW264.7 cells in the 4HNE group was significantly enhanced compared with the control group, while that of F4/80 in the 4HNE+KL group was significantly decreased compared with the 4HNE group(all P<0.05). The CCK8 assay results revealed a significant increase in the proliferation of HUVECs in the Mø-4HNE group compared with the Mø-NC group. Conversely, the proliferation of the Mø-4HNE+KL group exhibited a significant decrease compared with that in the Mø-4HNE group(all P<0.01). The results of scratch test and Transwell assays demonstrated a significant increase in the migration of HUVECs in the Mø-4HNE group compared with the Mø-NC group, while the migration of the Mø-4HNE+KL group exhibited a significant decrease compared with the Mø-4HNE group(all P<0.01). In the tube-formation assay, it was observed that the number of tubes formed by HUVECs in the Mø-4HNE group was significantly increased compared with the Mø-NC group, while that of tubes formed in the Mø-4HNE+KL group was significantly decreased compared with the Mø-4HNE group(all P<0.01). Additionally, the Western blot results revealed a significant decrease in the relative expression levels of Claudin 5, Occludin, and ZO 1 in the Mø-4HNE group compared with the Mø-NC group. Conversely, in the Mø-4HNE+KL group, there was a significant increase in the relative expression levels of Claudin 5, Occludin, and ZO 1 compared to the Mø-4HNE group(all P<0.01).

CONCLUSIONS: KL inhibits the proliferation, migration, and tube-formation of HUVECs while enhancing the tight junction by changing the activation state of macrophages in the diabetic oxidative stress environment.

Key words:oxidative stress; macrophage; vascular endothelial cells; proliferation; migration; tube-formation; tight junction; retinal neovascularization