Experimental Study Of The Effects Of Raloxifene On Cardiovascular System And The Molecular Mechanisms Involved

Many of the cardiovascular effects of estrogen would be expected to inhibit the development and progression of CAD. It was reported that long-term exposure to estrogen inhibits vascular smooth cell proliferation, enhance endothelial cell proliferation and reduces activation of the inflammatory system, and short-term exposure to estrogen enhances endothelium-dependent production of nitric oxide, resulting in enhanced vasodilation. In vitro, estrogen inhibits calcium channels in smooth muscle cells. In vivo, estrogen prevents neointimal thickening after balloon injury and also ameliorates the lesions occurring in atherosclerotic conditions.However, estrogen may increase the risks of breast cancer and uterus cancer. Raloxifene belongs to a class of drugs recently described as selective estrogen receptor modulators (SERMs) . Tissue selectivity of raloxifene (which is ER ligand) may be achieved through several mechanisms: the ligand structure, interaction of the ligand with different receptor subtypes in various tissues, and intracellular events after ligand binding. Raloxifene has estrogen-agonist effects on bone and lipids and estrogen antagonist effects on the breast and uterus. In addition to its well established effects on osteoporosis, recent preclinical and clinical findings suggest that raloxifene may also possess beneficial effects on cardiovascular system without an increased risk of cancer or other side effects. Raloxifene has been shown to reduce total and low-density lipoprotein cholesterol concentrations in plasma, an effect similar to that produced by estrogens. Unlike estrogens, however, raloxifene does not increase high-density lipoprotein cholesterol level in plasma. Although estrogens remain the drugs of choice in the hormonal therapy of most postmenopausal women, raloxifene may represent estrogens and become an alternative in postmenopausal women who are at risk of coronary artery disease. However, the cardiovascular properties of raloxifene and the molecular mechanisms involved remain not fully understood until now. Thus, we performed this experimental study to clarify the direct effects of raloxifene on the vascular cells including vascular smooth muscle cells (VSMCs) and endothelial cells and the mechanisms involved.Part I Effects of raloxifene on the proliferation and apoptosis and cyclinD1mRNA and protein expressions in vascular smooth muscle cellsObjectiveTo assess the effect of raloxifene on VSMCs growth and the role of cyclinD1 in cell cycle distribution.MethodsRat aortic smooth muscle cells (RASMCs) were isolated from the aorta of 8-week-old female Wistar rats by enzymatic digestion. The cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/ml) and streptomycin (100g/ml) at 37℃in a humidified atmosphere of 95% air and 5% CO₂. Dextran-coated charcoal-stripped FCS (DCC-FCS) and phenol red-free DMEM were used to avoid contaminations of steroids and estrogen receptor agonist. Subcultured RASMCs (4-10 passages) were used in the following experiments. RASMCs were randomly assigned to 1 of 4 treatment groups: raloxifene alone (10^-8,10^-7 and 10^-6 mol/L) ,ICI 182,780 alone (10^-7,10^-6 and 10^-5 mol/L), raloxifene (10^-8mol/L)plus ICI 182,780(10^-7mol/L),or vehicle; treatment continued for 24 hours. The effect of raloxifene on VSMC growth was determined by MTT assay. The effect of raloxifene on the expression of proliferating cell nuclear antigen (PCNA) was determined by immunocytochemistry assay. Flow cytometry was also used to examine the profile of cell cycle distribution and apoptosis. RT-PCR was performed for cyclinD1 mRNA in RASMC, and Western blot analysis was also performed for cyclinD1 protein, and the cyclinD1 promo tor activity in RASMCs was detected by a cyclinD1 promotor-luciferase gene reporter plasmidsystem.Results(1)Cell viability was measured by MTT and cell count method. Raloxifeneinhibited the growth of RASMCs. Nonselective ER antagonist (10^-7mol/L ICI 182,780) demolished the effects of raloxifene , and promoted the proliferation of RASMCs at a high concentration 10^-5mol/L).(2)Raloxifene decreased the expression of PCNA in RASMCs.(3)Flow cytometry demonstrated that raloxifene introduced the apoptosis of RASMCs, and blocked the translation of G1 to S phase.(4)Raloxifene downregulated the mRNA and protein expressions of cyclinD1,and significantly inhibited the cyclinD1 promotor activity in RASMCs .ConclusionsRaloxifene significantly inhibits vascular smooth muscle cell growth and introduces the apoptosis of VSMCs, blocks the translation of G1 to S phase. These effects of raloxifene on VSMCs are ER mediated, and may be partly due to the downregulation of cyclinD1 mRNA and protein expressions in VSMCs by raloxifene .Part II Effects of raloxifene on caveolin-1 mRNA and protein expression in vascular smooth muscle cellsObjectiveCaveolin-1 is regulated by estrogen in vascular smooth muscle cells. However, the relationship between raloxifene and caveolin-1 is still unknown. Therefore, this study was designed to determine if raloxifene regulates caveolin-1 expression and if so, whether such regulation is mediated by an estrogen receptor.MethodsSubcultured RASMCs (4-10 passages) were used in the following experiments.Rat aortic smooth muscle cells were cultured in the absence and presence of raloxifene (10^-8 to 10^-6 mol/L) for 12 or 24 h. RT-PCR was performed for caveolin-1 mRNA in RASMC, and Western blot analysis was also performed for caveolin-1 protein.ResultsCompared with controls, the mRNA and protein expressions of caveolin-1 were upregulated significantly by raloxifene (10^-8 to 10^-6 mol/L) after 24 h but not after 12 h of treatment, as well as 17p-estrodial (10^-7 mol/L). The pure estrogen receptor antagonist ICI 182780 (10^-5 mol/L) inhibited the increase of caveolin-1 mRNA and protein expressions in response to raloxifene treatment for 24 h.ConclussionsResults of this study suggest that raloxifene stimulates caveolin-1 transcription and translation through estrogen receptor mediated mechanisms.Part III Effects of raloxifene on the gene expression of endothelin-1 in vascular endothelial cells and the relationship with estrogen receptorObjectiveTo investigate the effect of raloxifene on the mRNA expression of endothelin-1 in vascular endothelial cells and assess the role of estrogen receptor.MethodsBovine carotid endothelial cells were pretreated with 10^-8mol/L raloxifene for 24 hours, then total RNA was harvested and Northern blotting was performed to investigate the effect of raloxifene on the mRNA expression of endothelin-1. Furthermore, estrogen receptor inhibitor, 10^-7 mol/L ICI 182,780 was used to pretreat the cells together with raloxifene and the expression of endothelin-1 was observed too.ResultsThe mRNA expression of endothelin-1 in bovine carotid endothelial cells was inhibited significantly by pretreatment with raloxifene and this effect could be blocked by ICI 182,780.Conclusions The selective estrogen receptor modulator, raloxifene may inhibit the gene expression of endothelin-1 in vascular endothelial cells via estrogen receptor.Part IV Effects of raloxifene on the ERE transcription activity and its relationship with estrogen receptor subtypes in vascular endothelial cellsObjectiveTo investigate the effect of raloxifene on the estrogen-response element (ERE) transcription activity and its relationship with estrogen receptor subtypes in vascular endothelial cells.MethodsERE-TK-Luc, which contains a firefly luciferase reporter vector, was used as a reporter plasmid. Vascular endothelial cells were transfected with ERE-TK-Luc and PRL-SV40 control plasmid via FuGENE 6. Two kinds of luciferase activity were measured by dual-luciferase reporter assay system. The luciferase activity in transfected cells treated with raloxifene was compared with that in estrogen-treated cells and control. Replication-deficient adenovirus vectors carrying the coding region of human ERαor ERβwere constructed and transferred into vascular endothelial cells, and the luciferase activities of raloxifene-treated and -untreated cells were measured and compared.ResultsIn cells transferred with ERα, the luciferase activity decreased significantly after treatment with raloxifene (P<0.001). But in cells transferred with ERβ, no significant change was found in the luciferase activity after treatment with raloxifene (P>0.05).ConclusionsRaloxifene inhibits the ERE transcription activity induced by ERαin vascular endothelial cells, but not affect the ERE transcription activity induced by ERβ.