Resveratrol Reverses Endothelial Colony-Forming Cell Dysfunction in Adulthood in a Rat Model of Intrauterine Growth Restriction

Abstract

Individuals born after intrauterine growth restriction (IUGR) are at risk of developing cardiovascular diseases (CVDs). Endothelial dysfunction plays a role in the pathogenesis of CVDs; and endothelial colony-forming cells (ECFCs) have been identified as key factors in endothelial repair. In a rat model of IUGR induced by a maternal low-protein diet, we observed an altered functionality of ECFCs in 6-month-old males, which was associated with arterial hypertension related to oxidative stress and stress-induced premature senescence (SIPS). Resveratrol (R), a polyphenol compound, was found to improve cardiovascular function. In this study, we investigated whether resveratrol could reverse ECFC dysfunctions in the IUGR group. ECFCs were isolated from IUGR and control (CTRL) males and were treated with R (1 μM) or dimethylsulfoxide (DMSO) for 48 h. In the IUGR-ECFCs, R increased proliferation (5'-bromo-2'-deoxyuridine (BrdU) incorporation, p < 0.001) and improved capillary-like outgrowth sprout formation (in Matrigel), nitric oxide (NO) production (fluorescent dye, p < 0.01), and endothelial nitric oxide synthase (eNOS) expression (immunofluorescence, p < 0.001). In addition, R decreased oxidative stress with reduced superoxide anion production (fluorescent dye, p < 0.001); increased Cu/Zn superoxide dismutase expression (Western blot, p < 0.05); and reversed SIPS with decreased beta-galactosidase activity (p < 0.001), and decreased p16^ink4a (p < 0.05) and increased Sirtuin-1 (p < 0.05) expressions (Western blot). No effects of R were observed in the CTRL-ECFCs. These results suggest that R reverses long-term ECFC dysfunctions related to IUGR.

Keywords: developmental programming; endothelial colony-forming cells; intrauterine growth restriction; oxidative stress; resveratrol; stress-induced premature senescence.

Figure 1

Proliferation capacities of the CTRL ± R and IUGR ± R ECFCs isolated from six-month-old male rats using BrdU incorporation at 6 and 24 h. = 4–5 animals/group; *** p < 0.001; N.S.: not significant. R: resveratrol.

Figure 2

The capillary-like outgrowth sprouts were evaluated in 24 h Matrigel cultures of the CTRL ± R and IUGR ± R ECFCs isolated from six-month-old male rats. Magnification = 5×. These pictures are representative images from n = 4–5 animals/group. Scale bar = 25 μm. R: resveratrol.

Figure 3

Basal NO production. Measurement of NO production using DAF-2DA was performed using the CTRL ± R and IUGR ± R ECFCs isolated from six-month-old male rats under baseline conditions. Magnification = 20×. Nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI). A negative control was performed. These pictures are representative images from n = 4–5 animals/group; ** p < 0.01; N.S.: not significant. Scale bar = 100 μm. R: resveratrol.

Figure 4

Stimulated NO production using acetylcholine. Measurement of NO production using DAF-2DA was performed using the CTRL ± R and IUGR ± R ECFCs isolated from six-month-old male rats after stimulation with acetylcholine (100 mM). Magnification = 20×. Nuclei were counterstained with DAPI. These pictures are representative images from n = 4–5 animals/group; ** p < 0.01; N.S.: not significant. Scale bar = 100 μm. R: resveratrol.

Figure 5

eNOS protein expression. eNOS protein expression was measured using immunofluorescence in the CTRL ± R and IUGR ± R ECFCs isolated from six-month-old male rats. Magnification = 20×. Nuclei were counterstained with DAPI, and a negative control was performed. These pictures are representative images from n = 4–5 animals/group; *** p < 0.001; N.S.: not significant. Scale bar = 100 μm. R: resveratrol.

Figure 6

Evaluation of oxidative stress in the ECFCs. Superoxide anion production was measured using hydroethidine in the CTRL ± R and IUGR ± R ECFCs isolated from six-month-old male rats (A). Magnification = 20×. Nuclei were counterstained with DAPI and a test for autofluorescence was performed. These pictures are representative images from n = 4–5 animals/group. Scale bar = 100 μm. In addition, the antioxidant Cu/Zn SOD protein expression was evaluated using Western blotting of the CTRL ± R and IUGR ± R ECFCs isolated from six-month-old male rats (B). n = 4–5 animals/group; * p < 0.05; *** p < 0.001; N.S.: not significant. R: resveratrol.

Figure 7

SA-β-gal activity in the ECFCs. The SA-β-gal activity determined as the blue staining normalized to the total number of cells in the CTRL ± R and IUGR ± R ECFCs isolated from six-month-old male rats. Magnification = 20×. These pictures are representative images from n = 4 animals/group; *** p < 0.001. Scale bar = 100 μm. N.S.: not significant. R: resveratrol.

Figure 8

Sirtuin-1 expression. Sirtuin-1 expression was measured by immunofluorescence in the CTRL ± R and IUGR ± R ECFCs isolated from six-month-old male rats. Nuclei were counterstained with DAPI, and a negative control was performed. Magnification = 20×. These pictures are representative images from n = 4–5 animals/group; *** p < 0.001; N.S.: not significant. Scale bar = 100 μm. R: resveratrol.

Figure 9

Factors related to cellular senescence. p16^INK4a (A) and Sirtuin-1 (B) protein expressions were measured using Western blotting in the CTRL ± R and IUGR ± R ECFCs isolated from six-month-old male rats. n = 3–5 animals/group; * p < 0.05; N.S.: not significant. R: resveratrol.

Figure 10

The effects of resveratrol on the ECFC dysfunctions related to IUGR at 6 months of age.