Regulation of endothelial cell adhesion by profilin.

BACKGROUND: Although profilin is believed to be an essential regulator of the actin cytoskeleton in most cells, its precise role in mammalian cells remains unknown. We have used replication-incompetent adenovirus carrying the human profilin I cDNA as a means rapidly to increase the concentration of profilin in human aortic endothelial cells 12-31-fold above baseline--levels never before achieved in mammalian cells. RESULTS: The concentration of filamentous actin was not detectably affected by profilin overexpression. Actin stress fibers were, however, absent from areas of high profilin content in overexpressing cells, and the bulk of filaments was located at the periphery of the cells. We observed a gradient in the distribution of overexpressed profilin in migrating endothelial cells, with most profilin molecules concentrated near the advancing edge where focal contacts are being formed and focal adhesion proteins are located. Profilin overexpression resulted in increased recruitment of fibronectin receptors to the plasma membrane. Adhesion of endothelial cells to fibronectin was markedly and selectively increased by profilin overexpression. CONCLUSIONS: We conclude that an important role for profilin in mammalian cells may be its contribution to the formation of focal contacts, particularly those involving the fibronectin receptor.

Methylation of the estrogen receptor gene is associated with aging and atherosclerosis in the cardiovascular system.

OBJECTIVE: Methylation of the promoter region of the estrogen receptor gene alpha (ER alpha) occurs as a function of age in human colon, and results in inactivation of gene transcription. In this study, we sought to determine whether such age-related methylation occurs in the cardiovascular system, and whether it is associated with atherosclerotic disease. METHODS: We used Southern blot analysis to determine the methylation state of the ER alpha gene in human right atrium, aorta, internal mammary artery, saphenous vein, coronary atherectomy samples, as well as cultured aortic endothelial cells and smooth muscle cells. RESULTS: An age related increase in ER alpha gene methylation occurs in the right atrium (range 6 to 19%, R = 0.36, P < 0.05). Significant levels of ER alpha methylation were detected in both veins and arteries. In addition, ER alpha gene methylation appears to be increased in coronary atherosclerotic plaques when compared to normal proximal aorta (10 +/- 2% versus 4 +/- 1%, P < 0.01). In endothelial cells explanted from human aorta and grown in vitro, ER alpha gene methylation remains low. In contrast, cultured aortic smooth muscle cells contain a high level of ER alpha gene methylation (19-99%). CONCLUSIONS: Methylation associated inactivation of the ER alpha gene in vascular tissue may play a role in atherogenesis and aging of the vascular system. This potentially reversible defect may provide a new target for intervention in heart disease.

Protein tyrosine phosphorylation mediates TNF-induced endothelial-neutrophil adhesion in vitro.

Proinflammatory cytokines initiate the vascular inflammatory response via the upregulation of adhesion molecules on the luminal endothelial surface. We investigated directly the role of protein tyrosine phosphorylation in the upregulation of the endothelial adhesion molecules, intercellular adhesion molecule 1 (ICAM-1) and E-selectin, and the consequent adhesion of neutrophils, after tumor necrosis factor (TNF)-alpha-stimulation of human aortic endothelial cells in vitro. Time- and dose-dependent TNF-alpha-stimulated ICAM-1 and E-selectin upregulation and neutrophil adhesion each were suppressed by tyrosine kinase inhibitors, including genistein (200 microM), but not genistein, its isoflavone analog without tyrosine kinase inhibitory activity. Tyrphostin AG 126, a synthetic selective tyrosine kinase inhibitor, also suppressed ICAM-1 and E-selectin upregulation and neutrophil adhesion, each in a dose-dependent manner, whereas tyrphostin AG 1288 had no effect. Tyrosine phosphorylation of two proteins (85 and 145 kDa in the cytoskeleton fraction) found minutes after TNF-alpha-stimulation was also inhibited by genistein. These findings suggest that, in endothelial cells, TNF-alpha upregulates ICAM-1 and E-selectin expression and consequent neutrophil adhesion via protein tyrosine phosphorylation.

17beta-estradiol inhibits apoptosis of endothelial cells.

Endothelial cells provide an antithrombotic and anti-inflammatory barrier for the normal vessel wall. Dysfunction of endothelial cells has been shown to promote atherosclerosis, and normalization of previously dysfunctional endothelial cells can inhibit the genesis of atheroma. In normal arteries, endothelial cells are remarkably quiescent. Acceleration of the turnover rate of endothelial cells can lead to their dysfunction. Apoptosis is a physiological process that contributes to vessel homeostasis, by eliminating damaged cells from the vessel wall. However, increased endothelial cell turnover mediated through accelerated apoptosis may alter the function of the endothelium and therefore, promote atherosclerosis. Apoptotic endothelial cells can be detected on the luminal surface of atherosclerotic coronary vessels, but not in normal vessels. This finding links endothelial cell apoptosis and the process of atherosclerosis, although a causative role for apoptosis in this process remains hypothetical. Estrogen metabolites have been shown to be among the most potent anti-atherogenic agents available to date for post-menopausal women. The mechanism of estrogen's protective effect is currently incompletely characterized. Here we show that 17beta-estradiol, a key estrogen metabolite, inhibits apoptosis in cultured endothelial cells. Our data support the hypothesis that 17beta-estradiol's anti-apoptotic effect may be mediated via improved endothelial cell interaction with the substratum, increased tyrosine phosphorylation of pp125 focal adhesion kinase, and a subsequent reduction in programmed cell death of endothelial cells. Inhibition of apoptosis by estrogens may account for some of the anti-atherogenic properties of these compounds.

Superoxide-mediated actin response in post-hypoxic endothelial cells.

The mechanism leading to changes in the superstructure of endothelial cells exposed to ischemia and reperfusion remains uncharacterized. We show that in post-hypoxic endothelial cells, the simple re-addition of oxygen induces a profound reorganization of the actin cytoskeleton. The total filamentous actin pool increases by 41% and translocation of actin filaments to the submembranous network is observed. Concurrent with the actin polymerization, increased tyrosine phosphorylation of endothelial cell substrates is detected on Western blots. Overexpression of superoxide dismutase using replication incompetent adenovirus inhibits the actin and tyrosine phosphorylation responses to reoxygenation. Inhibition of tyrosine kinases with the isoflavone genistein also suppressed the actin polymerization response to reoxygenation, but unlike superoxide dismutase, genistein also induced the collapse of the superstructure of endothelial cells upon reoxygenation. These experiments support the concept that reoxygenation following a period of hypoxia can induce the remodeling of the actin cytoskeleton in endothelial cells. Such a response requires the intact coupling of superoxide producing pathway(s) with tyrosine kinase pathway(s).