Angiotensin II mediates the high-glucose-induced endothelial-to-mesenchymal transition in human aortic endothelial cells

Abstract

Background: Substantial evidence suggests that high glucose (HG) causes endothelial cell damage; however, the potential mechanism therein has yet to be clarified. The aim of this study was to investigate the influence of HG on the endothelial-to-mesenchymal transition (EndMT) and its relevance to the activation of the renin-angiotensin system.

Methods: Primary human aortic endothelial cells (HAECs) were divided into three groups: a normal glucose (NG) group, HG group, and irbesartan (1 microM)-treated (HG+irbesartan) group. The concentration of angiotensin II in the supernatant was detected by radioimmunoassay. Pathological changes were investigated using fluorescence microscopy and electron microscopy. Immunofluorescence staining was performed to detect the co-expression of CD31 and fibroblast markers, such as fibroblast-specific protein 1 (FSP1). The expressions of FSP1 and alpha-SMA were detected by RT-PCR and Western blot.

Results: The treatment of HAECs in the HG group resulted in significant increases in the expressions of FSP1 and angiotensin II in dose-and time-dependent manners. The incubation of HAECs exposure to HG resulted in a fibroblast-like phenotype, wherein increased microfilamentation and a roughened endoplasmic reticulum structure were observed in the cytoplasm. The expressions of FSP1 and alpha-SMA were significantly increased in the HG group, and these changes were inhibited by irbesartan treatment (P < 0.05). Double staining of the HAECs indicated a co-localization of CD31 and FSP1 and that some cells acquired spindle-shaped morphologies and a loss of CD31 staining; however, treatment with irbesartan attenuated the expression of EndMT (P < 0.05).

Conclusions: These findings suggest a novel mechanism in HG-induced endothelial damage via the mediation of the EndMT by angiotensin II, which was inhibited by Irbesartan.

Figure 1

The dose and time dependency of HG-stimulated Ang II synthesis in HAECs. A: HAECs grown in a culture medium consisting of 5.5 mM, 15 mM, or 30 mM glucose for 48 h. Mannitol was used as a control for hyperosmolarity. B: HAECs grown in a 30 mM glucose medium for 6-72 h. Ang II was measured in the supernatant using radioimmunoassay. C: High glucose stimulated Ang II synthesis in the supernatant in the absence or presence of irbesartan in HAECs. HAECs that were grown in a serum-free normal glucose medium (NG, 5.5 mM) for 24 h and exposed to high glucose (HG, 30 mM) or in 5.5 mM glucose + 24.5 mM mannitol (NG+M) in the absence or presence of 1 μM irbesartan for 48 h. Irbesartan partially inhibited Ang II production in the culture medium. The depicted values are the means mean ± SD from three independent experiments that were performed in duplicate. # P < 0.05 vs. mannitol (A) or NG (B). *P < 0.05 vs. HG without inhibitor.

Figure 2

The effect of irbesartan on the mRNA expression of FSP1 and α-SMA. ^#P < 0.05 vs. NG. and *P < 0.05 vs. HG. NG: normal glucose. HG: high glucose. HI+Irb: high glucose + Irbesartan.

Figure 3

Western blot analysis of the effect of HG on the synthesis of FSP1 and α-SMA protein in HAECs. A: HAECs were grown in a culture medium that contained 5.5-30 mM glucose for 48 h. Mannitol was used as a control for hyperosmolarity. B: HAECs were incubated with HG (30 mM) for 0, 12, 24, and 48 h. (0 h: 0.04 ± 0.001, 12 h: 0.65 ± 0.04, 24 h: 0.98 ± 0.04, and 48 h: 1.22 ± 0.02; ^#P < 0.05 vs. the control). C: Western blot analysis of the effect of HG on the synthesis of α-SMA and FSP1 protein in HAECs. Values are means ± SD; ^#P < 0.01 vs. NG and * P < 0.05 vs. HG. NG: normal glucose. HG: high glucose. HI+Irb: high glucose + Irbesartan. Experiments were repeated three times. Irb: Irbesartan

Figure 4

Irbesartan inhibited the high glucose-induced EndMT in HAECs according to laser-scanning confocal microscopy. Representative immunofluorescence staining of CD31 (green) and FSP1 (red) were observed. A merging of both images reveals populations of cells acquired FSP1 expression and lost CD31 expression (arrows, B). The administration of irbesartan reduced the number of co-localization of CD31 and FSP1 (C, P < 0.05). A: normal glucose as controls; B: Treated with HG (30 mM) for 48 h. C: Treated with HG (30 mM) + irbesartan (1 μM) for 48 h. Experiments were repeated three times. NG: normal glucose. HG: high glucose. HI+Irb: high glucose + Irbesartan.

Figure 5

Immunofluorescence staining of HAECs with CD31 in various groups. The Incubation of HAECs with high glucose (30 mM) for 48 h resulted in a fibroblast-like phenotype (B). Treatment with irbesartan could significantly prevent the morphological changes (C). NG: normal glucose. HG: high glucose. HI+Irb: high glucose + Irbesartan.

Figure 6

Cellular ultrastructure following HG treatment. Transmission electron microscopy depicts the change in cellular ultrastructure following HG (30 mM) exposure (magnification × 6,000). It can be seen that normal HAECs present with few microfilaments and a rough endoplasmic reticulum (A). After exposure to HG, microfilamentation and a swollen rough endoplasmic reticulum appeared in the cytoplasm (B). These changes were attenuated by treatment with irbesartan (C). 1 bar = 4 μm.