Estrogen inhibits cuff-induced intimal thickening of rat femoral artery: effects on migration and proliferation of vascular smooth muscle cells

doi:10.1016/S0021-9150(96)06023-6

Atherosclerosis

Volume 130, Issues 1–2, April 1997, Pages 1-10

https://doi.org/10.1016/S0021-9150(96)06023-6 Get rights and content

Abstract

The present study was performed to elucidate the mechanism underlying the anti-atherogenic action of estrogen. We investigated the effect of estrogen on intimal thickening of the rat femoral artery induced by cuff placement and further examined the effect of estrogen on migration and proliferation of vascular smooth muscle cells (VSMCs) in culture. Intimal thickening was significantly greater in males than in control females. Intimal thickening in females was increased to the level in males by ovariectomy. Estrogen replacement to ovariectomized rats reversed this effect. Proliferating cell nuclear antigen immunohistochemistry showed that in vivo proliferation of VSMCs contributed to the difference in intimal thickening. There was no difference in blood pressure and serum lipids, suggesting that estrogen directly acted on artery and inhibited intimal thickening. 17β-Estradiol (E2, 1–100 nmol/l) inhibited migration of cultured rat VSMCs, assayed using a microchemotaxis chamber, in a concentration-dependent manner. E2 (0.01–100 nmol/l), but not progesterone or testosterone, also inhibited [³H]thymidine incorporation in rat VSMCs in a concentration-dependent manner. Indomethacin, N^G-monomethyl-l-arginine and methylene blue did not influence the inhibitory action of E2 on [³H]thymidine incorporation, suggesting that prostanoids and nitric oxide are not involved in the action of E2. E2 did not provoke VSMC injury, as measured by the release of incorporated [³H]2-deoxy-d-glucose. These results suggest that the inhibition of migration and proliferation of VSMCs contributes to the inhibitory effect of estrogen on intimal thickening.

Introduction

It is an established epidemiological observation that the incidence of atherosclerosis is lower in premenopausal women than in men and increases after menopause 1, 2. Estrogen replacement therapy is effective to protect women from coronary heart disease 2, 3, 4. Estrogen has also been reported to inhibit the development of atherosclerosis in animal experiments 5, 6, 7. These epidemiological and experimental findings suggest that estrogen has an anti-atherogenic action. The mechanism, however, is not fully understood.

A number of investigators have reported that lipid metabolism is improved by estrogen administration 5, 6, 8, 9, 10. Estrogen increases serum high-density lipoprotein cholesterol level and decreases serum low-density lipoprotein cholesterol level. On the other hand, the existence of functional estrogen receptors in vascular endothelial cells [11]and vascular smooth muscle cells (VSMCs) 12, 13, 14suggests that estrogen acts directly on the arterial wall. Estrogen appears to have both endothelium-independent 15, 16and endothelium-dependent 17, 18, 19vasodilator actions. A calcium-antagonistic effect 15, 16, regulation of prostacyclin 20, 21and nitric oxide production [22]may be involved in these effects.

Migration and proliferation of VSMCs are fundamental steps in the development of atherosclerosis [23]and neointimal formation after endothelial injury [24]. However, the effect of estrogen on migration of cultured VSMCs is not known. Moreover, the effect of estrogen on proliferation of VSMCs is controversial. Fischer-Dzoga et al. [25]and Vargas et al. [26]reported an inhibitory effect of estrogen on VSMC proliferation, while Farhat et al. [27]demonstrated a stimulatory effect of estrogen.

We first aimed at investigating the effect of estrogen on cuff-induced intimal thickening of the rat femoral artery. The reason we used this model was that lipid metabolism was not involved in the development of intimal thickening by cuff placement. We further examined the effect of estrogen on migration and proliferation of cultured VSMCs to investigate the mechanism underlying the effect on intimal thickening.

Section snippets

Chemicals

Estradiol dipropionate was obtained from Teikoku Hormone (Tokyo, Japan). Dulbecco's modified Eagle's medium (DMEM) was purchased from GIBCO BRL (Gaithersburg, MD) and fetal bovine serum (FBS) from ICN Biomedicals (Osaka, Japan). Bovine serum albumin (BSA), trichloroacetic acid (TCA) and Triton X-100 were purchased from Wako (Osaka, Japan). Phenol red-free DMEM, 17β-estradiol (E2), 17α-estradiol, progesterone, testosterone, endothelin-1, human basic fibroblast growth factor (bFGF), indomethacin,

Sex-related difference in intimal thickening

Cuff placement induced diffuse intimal thickening of the rat femoral artery, while no intimal thickening was observed in the control rats without cuff placement. We checked the time-related change in the development of intimal thickening. The I:M ratio was significantly higher 2 weeks after than 1 week after cuff placement (P<0.05) and then tended to decrease until 4 weeks after cuff placement: 1 week, 0.11±0.01 (n=8); 2 weeks, 0.33±0.07 (n=9); 3 weeks, 0.25±0.05 (n=8); 4 weeks, 0.21±0.02 (n

Discussion

In the present study, we showed that intimal thickening of the rat femoral artery induced by cuff placement was greater in males than that in control females. Intimal thickening of females was increased by ovariectomy and this increase was reversed by estrogen replacement.

Arterial intimal thickening induced by a polyethylene cuff has been reported in the rabbit femoral artery [38]and in the rabbit carotid artery [28]. Histological observations in these studies elucidate that cuff placement

Acknowledgements

We would like to thank Masae Watanabe and Hitomi Yamaguchi for their excellent technical assistance. This work was supported by grants from the Uehara Memorial Foundation and the Sankyo Life Science Research Foundation. This work was presented in part at the 10th International Symposium on Atherosclerosis in Montréal, October 9–14, 1994.

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