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To study cellular mechanisms influencing vascular reactivity, vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the rat mesenteric artery, a highly reactive, resistance-type blood vessel, and established in primary culture. Cellular binding sites for the vasoconstrictor hormone angiotensin II (All) were identified and characterized using the radioligand 125 1-angiotensin 11 . Freshly isolated VSMC, and VSMC maintained in primary culture for up to 3 wk, exhibited rapid, saturable, and specific ' 25 1-All binding similar to that seen with homogenates of the intact rat mesenteric artery . In 7-d primary cultures, Scatchard analysis indicated a single class of high-affinity binding sites with an equilibrium dissociation constant (Kd) of 2.8 ± 0.2 nM and a total binding capacity of 81 .5 ± 5 .0 fmol/mg protein (equivalent to 4.5 x 104 sites per cell) . Angiotensin analogues and antagonists inhibited 125 1-All binding to cultured VSMC in a potency series similar to that observed for the vascular All receptor in vivo . Nanomolar concentrations of native All elicited a rapid, reversible, contractile response, in a variable proportion of cells, that was inhibited by pretreatment with the competitive antagonist Sarl ,lleB-All . Transmission electron microscopy showed an apparent loss of thick (12-18 nm Diam) myofilaments and increased synthetic activity, but these manifestations of phenotypic modulation were not correlated with loss of 1251-All binding sites or hormonal responsiveness . Primary cultures of enzymatically dissociated rat mesenteric artery VSMC thus may provide a useful in vitro system to study cellular mechanisms involved in receptor activation-response coupling, receptor regulation, and the maintenance of differentiation in vascular smooth muscle .The interaction of vasoactive hormones, such as the octapeptide, angiotensin II (All), with vascular smooth muscle cells (VSMC) has important implications for normal vascular physiology (1) and the pathophysiology of hypertension (2). Although pharmacologic studies in whole animals, isolated vascular strips, and blood vessel homogenates have identified receptors for a number of vasoactive substances, the cellular localization of these receptors, biochemical correlates of their activation, and factors regulating their expression have not been well defined, in part due to the structural complexity of vascular tissues . Selective isolation and culture of the cellular components of blood vessels provides a potentially useful approach to this problem (3-12).Large fibroelastic arteries, such as the aorta, have been a preferred source of VSMC for culture (3,7,8) because the inner one-third of the medial layer of these vessels normally does not contain other cell types. However, once established in

The endothelial lining of blood vessels is constantly exposed to fluid mechanical forces generated by flowing blood. In vitro application of fluid shear stresses to cultured endothelial cells influences the expression of multiple genes, as reflected by changes in their steady-state mRNA levels. We have utilized the B chain of platelet-derived growth factor (PDGF-B) as a model to investigate the mechanisms of shear-stress-induced gene regulation in cultured bovine aortic endothelial cells (BAECs). Northern blot analysis revealed elevated endogenous PDGF-B transcript levels in BAECs, after exposure to a physiological level of laminar shear stress (10 dynes/cm2; 1 dyne = 100 mN) for 4 h. A transfected reporter gene, consisting of a 1.3-kb fragment of the human PDGF-B promoter coupled to chloramphenicol acetyltransferase (CAT), indicated a direct effect on transcriptional activity. Transfection of a series of PDGF-B-CAT deletion mutants led to the characterization of a cis-acting component within the PDGF-B promoter that was necessary for shear-stress responsiveness. In gel-shift assays, overlapping oligonucleotide probes of this region formed several protein-DNA complexes with nuclear extracts prepared from both static and shear-stressed BAECs. A 12-bp component (CTCTCAGAGACC) was identified that formed a distinct pattern of complexes with nuclear proteins extracted from shear-stressed BAECs. This shearstress-responsive element does not encode binding sites for any known transcription factor but does contain a core binding sequence (GAGACC), as defined by deletion mutation in gel-shift assays. Interestingly, this putative transcription factor binding site is also present in the promoters of certain other endothelial genes, including tissue plasminogen activator, intercellular adhesion molecule 1, and transforming growth factor p81, that also are induced by shear stress. Thus, the expression of PDGF-B and other pathophysiologically relevant genes in vascular endothelium appears to be regulated, in part, by shear-stress-induced transcription factors interacting with a common promoter element.Vascular endothelial cells form the biological interface between the blood and the various tissues and organs of the body. They are the source of multiple interacting factors that are critical to normal homeostasis and the initiation and progression of disease. These include growth stimulators and inhibitors [e.g., platelet-derived growth factor (PDGF), transforming growth factor ,31 (TGF-p1), fibroblast growth factors, and heparans], vasoconstrictors and vasodilators (e.g., endothelin and endothelial-derived relaxing factor), pro-and antithrombotic factors, fibrinolytic activators and inhibitors, adhesion molecules, and various cytokines (1).

The objective of this study was to determine whether absence of endothelial nitric oxide synthase (eNOS) affects the expression of cell surface adhesion molecules in endothelial cells. Murine lung endothelial cells (MLECs) were prepared by immunomagnetic bead selection from wild-type and eNOS knockout mice. Wild-type cells expressed eNOS, but eNOS knockout cells did not. Expression of neuronal NOS and inducible NOS was not detectable in cells of either genotype. Upon stimulation, confluent wild-type MLECs produced significant amounts of NO compared with N(omega)-monomethyl-l-arginine-treated wild-type cells. eNOS knockout and wild-type cells showed no difference in the expression of E-selectin, P-selectin, intracellular adhesion molecule-1, and vascular cell adhesion molecule-1 as measured by flow cytometry on the surface of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31)-positive cells. Both eNOS knockout and wild-type cells displayed the characteristics of resting endothelium. Adhesion studies in a parallel plate laminar flow chamber showed no difference in leukocyte-endothelial cell interactions between the two genotypes. Cytokine treatment induced endothelial cell adhesion molecule expression and increased leukocyte-endothelial cell interactions in both genotypes. We conclude that in resting murine endothelial cells, absence of endothelial production of NO by itself does not initiate endothelial cell activation or promote leukocyte-endothelial cell interactions. We propose that eNOS derived NO does not chronically suppress endothelial cell activation in an autocrine fashion but serves to counterbalance signals that mediate activation.