Sex steroids and the endothelium.

Gonadal steroids clearly influence the course of atherosclerotic cardiovascular disease in women. This observation has suggested that these hormones have beneficial effects on the physiology of the vascular wall. Increased arterial vascular caliber after estrogen treatment, decreased lipid levels in subjects receiving hormone replacement therapy, and the markedly decreased extent of atherosclerotic plaque formation in young women as compared with young men support a cardioprotective effect of ovarian steroids. Generally, it appears that the effects of 17beta-estradiol are particularly beneficial, and the mechanism of action is targeted largely to the endothelial cell. This review describes the evidence for positive effects of estrogens on endothelial cell biology and considers potential mechanisms for estrogen actions on endothelial cell signal transduction.

TGF-beta1 activates MAP kinase in human mesangial cells: a possible role in collagen expression.

BACKGROUND: Although the pathogenic relevance of transforming growth factor-beta (TGF-beta) to glomerular sclerosis has been established, the intracellular mechanisms by which TGF-beta induces extracellular matrix accumulation are not fully understood. We examined whether the mitogen-activated protein (MAP) kinase pathway is involved in TGF-beta1-induced collagen expression by cultured human mesangial cells. METHODS: The activation of MAP kinase pathways by TGF-beta1 was assessed by immunoblot with anti-phospho-ERK or -JNK antibodies and by transfection of plasmids expressing pathway-specific transcription activators fused to the DNA-binding domain of GAL4, as well as a GAL4 response element-luciferase reporter gene. The role of MAP kinase was assessed using biochemical inhibitors and transiently expressed dominant negative mutant constructs. The effects on TGF-beta1-induced alpha1(I) collagen expression were evaluated by Northern blot and by activation of a transiently transfected alpha1(I) promoter-luciferase reporter construct. RESULTS: ERK and JNK phosphorylation occurred 30 minutes and one hour, respectively, after TGF-beta1 treatment. A biochemical blockade of the ERK pathway inhibited TGF-beta1-induced alpha1(I) collagen expression. A dominant negative mutant of ERK1 but not of JNK decreased alpha1(I) gene promoter activation. Activation of the TGF-beta-responsive p3TP-Lux construct was partially inhibited by cotransfection of an ERK1 dominant negative mutant. CONCLUSION: These data indicate that MAP kinase pathways can be activated by TGF-beta1 in mesangial cells and that the ERK MAP kinase plays a role in TGF-beta-stimulated collagen I expression. Because we have shown previously that SMADs mediate TGF-beta1-stimulated collagen I expression, our findings raise the possibility of interactions between the MAP kinase and the SMAD pathways.

Increased expression of extracellular matrix proteins and decreased expression of matrix proteases after serial passage of glomerular mesangial cells.

The cellular events causing pathological extracellular matrix (ECM) accumulation in vivo are not well understood. Prolonged serial passage of several cell types in culture leads to increased production of extracellular matrix (ECM) proteins, but the mechanism for these putative fibrotic changes is not known. Here, human fetal glomerular mesangial cells were subjected to serial passage (P) in culture and the expression of ECM proteins, proteases and protease inhibitors was comprehensively evaluated. From P11 through P14, a series of phenotypic changes occurred. Steady-state expression of mRNA for alpha 1 chains of type III and type IV (but not type I) collagen, and for laminin beta 1 and gamma 1, increased 2- to 8-fold, while expression of mRNA for interstitial collagenase (MMP-1) and gelatinase A (MMP-2) virtually ceased. Expression of tissue-type plasminogen activator (tPA) mRNA also decreased markedly. Expression of mRNA for the tissue inhibitor of metalloproteinases (TIMP)-1, and of the smaller of two mRNA species for the PA inhibitor PAI-1, ceased by P14. There was a switch in expression of the two species of TIMP-2 mRNA: whereas the ratio of signal intensity comparing the 3.5 kb mRNA species to the 1.0 kb species was 5:1 up to P11, it was reversed (1:5) at P14 and later. Serial passage also led to changes in protein expression, with increased type IV collagen and laminin, but decreased interstitial collagenase and gelatinase A. The cells showed a progressive increase in staining for type IV collagen. These findings define the appearance of a matrix-accumulating phenotype in later-passage mesangial cells. Matrix expansion in vivo has been associated with increased transforming growth factor (TGF)-beta synthesis; the cells were found to show at least 5-fold increased expression of TGF-beta 1 mRNA from P8 to P16. However, treatment of P9 or P10 cells with graded doses of TGF-beta 1 increased expression of both collagen IV and gelatinase A mRNA and did not alter the ratio of signal intensity for TIMP-2 mRNA species. Thus, assumption of a matrix-accumulating phenotype by these cultured fetal glomerular mesangial cells is not accelerated by exogenous TGF-beta. These data describe an in vitro model of mesangial cell matrix turnover in which matrix accumulation could result from a concerted increase in ECM synthesis and decrease in ECM degradation.

Expression of an estrogen receptor by human coronary artery and umbilical vein endothelial cells.

BACKGROUND: Premenopausal women have much lower susceptibility to coronary artery disease than do men or postmenopausal women. It has been proposed that estrogen plays a role in cardioprotection, but little information is available regarding the mechanism by which estrogen may help to protect the vasculature. Here, we describe an estrogen receptor (ER) in human coronary artery and umbilical vein endothelial cells. METHODS AND RESULTS: Human umbilical vein endothelial cells and human coronary artery endothelial cells were cultured in hormone-free medium for 48 hours before experiments. Estradiol (3.7 nmol/L) added to cultures promoted proliferation by a mechanism that is inhibited by the specific ER antagonist ICI182,780. Estradiol-treated cells incorporated twice the [3H]thymidine of hormone-free cells; this increase was prevented by ICI182,780. Endothelial cells from both sources stained in a nuclear pattern with an ER-specific antibody. Ribonuclease protection assay detected mRNA for the ER. Ligand-binding studies estimated 2 x 10(4) to 8 x 10(4) receptors per cell and a Kd of approximately 5 nmol/L. Interaction of ERs with a consensus estrogen response element was shown by an electrophoretic mobility shift assay. In addition, an antibody against the ER supershifted the protein-DNA complex. CONCLUSIONS: These studies define the presence of an ER in human coronary artery and umbilical vein endothelial cells. They support the hypothesis that cardioprotective effects of estrogen are mediated, at least in part, through a classic steroid hormone receptor mechanism.