RESUMO
OBJECTIVES: It has been demonstrated that 17beta-estradiol (E2) has an inhibitory effect on the proliferation of vascular smooth muscle cells (VSMCs) through an estrogen receptor (ER)-dependent pathway. Both ER subtypes, classical ER (ERalpha) and the newly identified ER subtype (ERbeta), are expressed in VSMCs. However, it remains unknown which receptor plays the critical role in the inhibitory effect on VSMC proliferation. METHODS AND RESULTS: We constructed replication-deficient adenoviruses bearing the coding region of human ERalpha, ERbeta, and the dominant-negative form of ERbeta (designated AxCAERalpha, AxCAERbeta, and AxCADNERbeta, respectively). Prior to infection with the adenoviruses, 100 nmol/l E2 attenuated DNA synthesis by up to 14% and transactivated the estrogen-induced expression of the desired mRNA in rat VSMCs. This was accompanied by increased transcriptional activity of estrogen responsive element in response to E2, and the increase was comparable between AxCAERalpha and AxCAERbeta. When VSMCs were infected with AxCAERbeta at a multiplicity of infection of 5 or higher, DNA synthesis as well as cell number decreased by 50% in response to E2, and the effect was abolished by co-infection with AxCADNERbeta. In contrast, when VSMCs were infected with AxCAERalpha, the reduction in DNA synthesis was minimal. CONCLUSIONS: Our results indicate that ERbeta is more potent than ERalpha in the inhibitory effect on VSMC proliferation.
Assuntos
Estradiol/farmacologia , Músculo Liso Vascular/metabolismo , Receptores de Estrogênio/metabolismo , Adenoviridae/genética , Animais , Northern Blotting/métodos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Ciclina A/metabolismo , Depressão Química , Receptor alfa de Estrogênio , Receptor beta de Estrogênio , Vetores Genéticos/administração & dosagem , Luciferases/análise , Luciferases/genética , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de Estrogênio/análise , Receptores de Estrogênio/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução Genética/métodosRESUMO
The current regimens of hormone replacement therapy for postmenopausal women, estrogen combined with progestogen, have failed to show beneficial effects for the prevention of atherosclerotic disease. Although the relatively higher dose of estrogen contained in those regimens exerted adverse effects, there are few data examining a lower dose of estrogen in an atherosclerosis model. Therefore, we investigated experimentally whether lower doses of estrogen could inhibit neointimal formation after balloon injury of the rat carotid artery. Ten-week-old Wistar rats were subjected to ovariectomy or sham-operation (n=7). Four days after ovariectomy, rats were implanted with an osmotic mini-pump containing 17-beta estradiol (0.2, 1, 2, 10 and 20 microg/kg/day; n=6, 4, 8, 6 and 5, respectively) or placebo (n=10). After 3 days of hormone therapy, balloon injury was performed in the left common carotid artery. Neointimal formation was histologically evaluated 2 weeks after injury. Cross-sectional intimal area and the ratio of intimal area to medial area were dose-dependently reduced by estrogen replacement compared with those in ovariectomized rats without estrogen replacement. The effects of estrogen replacement were identical to those of an angiotensin II type 1 receptor blocker, candesartan. Interestingly, the effect was significant even in rats receiving lower doses of estrogen, in which plasma estradiol concentrations were not increased and the hyperplastic response of the uterus was minimal. These results suggest the efficacy of low-dose estrogen therapy for the protection of atherosclerosis.
Assuntos
Artérias Carótidas/efeitos dos fármacos , Estenose das Carótidas/prevenção & controle , Cateterismo/efeitos adversos , Estrogênios/administração & dosagem , Túnica Íntima/efeitos dos fármacos , Animais , Artérias Carótidas/patologia , Estenose das Carótidas/patologia , Esquema de Medicação , Feminino , Ovariectomia , Ratos , Ratos Wistar , Túnica Íntima/patologiaRESUMO
Estrogen has diverse effects on the vasculature, such as vasodilation, endothelial growth and inhibition of vascular smooth muscle cell (VSMC) proliferation and migration. However, little is known about the genes that are regulated by estrogen in the vascular wall. Wistar rats were ovariectomized or sham-operated (Sham group), and 2 weeks after the operation, were subjected to subcutaneous implantation of placebo pellets (OVX + V group) or estradiol pellets (OVX + E group). Endothelium-denuded aortic tissue was examined 2 weeks after implantation. By applying high-density oligonucleotide microarray analysis, the expression of approximately 7000 genes was analyzed. Among the genes with different expression levels between the OVX + E group and the OVX + V group, those that have been reported to be expressed in the vasculature or muscle tissue, were chosen. Finally, four genes, caveolin-1, two LIM proteins (enigma and SmLIM) and Id3a, were identified. Microarray as well as real-time polymerase chain reaction showed that the expression levels of these genes were significantly higher in the OVX + E group than in the OVX + V group. To clarify whether estrogen directly upregulates these genes in the vascular wall, Northern blot analysis was performed using cultured rat VSMC. Addition of 100 nmol/L estradiol for 24 hours increased the mRNA levels of all four genes. Although the precise mechanism remains unclear, regulation of these genes by estrogen might contribute to its effect on VSMC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Transporte/genética , Caveolinas/genética , Estradiol/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Musculares/genética , Músculo Liso Vascular/efeitos dos fármacos , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Animais , Aorta Torácica , Northern Blotting , Proteínas de Transporte/metabolismo , Caveolina 1 , Caveolinas/metabolismo , Proteínas do Citoesqueleto , Feminino , Proteínas Inibidoras de Diferenciação , Proteínas com Domínio LIM , Proteínas Musculares/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Ovariectomia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
The effect of 17 beta-estradiol (E2) on the proliferation of cardiac fibroblasts (CFs) remains controversial. This study investigated which subtype of estrogen receptor (ER), ER alpha or ER beta, mediated the effect of E2 on CF growth by the gain of function analysis using an adenovirus vector. One hundred nanomoles per liter of E2 attenuated DNA synthesis by up to 10%, and transactivated the estrogen-responsive element determined by luciferase assay in rat neonatal CFs. We constructed replication-deficient adenoviruses bearing the coding region of human ER alpha, ER beta, or the dominant-negative form of ER beta (designated AxCAER alpha, AxCAER beta, and AxCADNER beta, respectively). When CFs were infected with AxCAER alpha or AxCAER beta at multiplicity of infection of 20 or higher, DNA synthesis was decreased by 50% in response to E2 and the effect was abolished by co-infection with AxCADNER beta. Similarly, transcriptional activity of ER in CFs infected with AxCAER alpha or AxCAER beta was markedly enhanced and co-infection with AxCADNER beta abolished the effects. These results suggest that E2 inhibits CF growth and that both ER subtypes mediate the effect comparably and redundantly.