NF- kappa B independent suppression of endothelial vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 gene expression by inhibition of flavin binding proteins and superoxide production.

Oxidation-reduction (redox) coupled mechanisms play an important role in the regulation of cell surface adhesion molecule expression. In endothelial cells membrane-bound NADH/NADPH oxidase is a significant source of intracellular superoxide (O(2)(-)) production. We explored the role of flavin containing proteins such as NADH/NADPH oxidase in the induction of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) gene expression in human aortic endothelial cells (HAECs) and human dermal microvascular endothelial cells (HMECs). Treatment of HAECs by tumor necrosis factor- alpha (TNF- alpha, 100 U/ml) for 1 h induced a 31% increase in O(2)(-)production within 5 min as determined by lucigenin chemiluminescence analysis of whole cells (n=4, P<0.05). Pretreatment with the NADH/NADPH oxidase inhibitor diphenylene iodonium (DPI, 40 microm) for 1 h inhibited O(2)(-)production. DPI also inhibited TNF and LPS-induced VCAM-1 and ICAM-1 cell surface expression and TNF- alpha, LPS, or IL-1 beta induced VCAM-1 and ICAM-1 mRNA accumulation. However, DPI did not inhibit TNF- alpha -induced activation of nuclear NF- kappa B-like binding activity in HAECs and HMECs. Furthermore, DPI did not inhibit TNF- alpha induced transactivation of NF- kappa B-driven VCAM-1 and HIV-LTR promoter gene constructs in transiently transfected HMECs. These data suggest that flavin binding proteins such as NADH/NADPH oxidase can regulate VCAM-1 gene expression independent of NF- kappa B. Furthermore, intracellular O(2)(-)generation is not necessary for NF- kappa B activation or for transactivation of NF- kappa B driven promoters.

Angiotensin II induces vascular cell adhesion molecule-1 expression in rat vasculature: A potential link between the renin-angiotensin system and atherosclerosis.

BACKGROUND: Cardiovascular ischemic events may occur more frequently in hypertensive patients with activated renin-angiotensin systems. We tested the hypothesis that angiotensin II (Ang II) may contribute to atherosclerosis by increasing expression of vascular inflammatory genes such as vascular cell adhesion molecule-1 (VCAM-1). METHODS AND RESULTS: Rats infused with norepinephrine or Ang II for 6 days developed similar hypertensive responses, but only Ang II-treated rats exhibited significant increases in aortic VCAM-1 protein and mRNA expression. Oral losartan treatment (50 mg. kg(-1). d(-1)) inhibited Ang II-induced hypertension and aortic VCAM-1 mRNA expression. Ang II treatment significantly increased VCAM-1 mRNA expression in cultured rat aortic smooth muscle cells (RASMCs). Ang II also induced nuclear NF-kappaB-like binding activity and transactivated an NF-kappaB-driven VCAM-1 promoter. Losartan and proteasome inhibitors blocked Ang II-induced NF-kappaB activation and VCAM-1 mRNA accumulation. IkappaB-alpha overexpression in RASMCs inhibited Ang II-induced VCAM-1 promoter transactivation. CONCLUSIONS: Ang II may contribute to atherogenesis by activation of VCAM-1 through proteasome dependent, NF-kappaB-like transcriptional mechanisms.

Angiotensin II induces monocyte chemoattractant protein-1 gene expression in rat vascular smooth muscle cells.

Monocyte infiltration into the vessel wall, a key initial step in the process of atherosclerosis, is mediated in part by monocyte chemoattractant protein-1 (MCP-1). Hypertension, particularly in the presence of an activated renin-angiotensin system, is a major risk factor for the development of atherosclerosis. To investigate a potential molecular basis for a link between hypertension and atherosclerosis, we studied the effects of angiotensin II (Ang II) on MCP-1 gene expression in rat aortic smooth muscle cells. Rat smooth muscle cells treated with Ang II exhibited a dose-dependent increase in MCP-1 mRNA accumulation that was prevented by the AT1 receptor antagonist losartan. Ang II also activated MCP-1 gene transcription. Inhibition of NADH/NADPH oxidase, which generates superoxide and H2O2, with diphenylene iodonium or apocynin decreased Ang II-induced MCP-1 mRNA accumulation. Induction of MCP-1 gene expression by Ang II was inhibited by catalase, suggesting a second messenger role for H2O2. The tyrosine kinase inhibitor genistein and the mitogen-activated protein kinase kinase inhibitor PD098059 inhibited Ang II-induced MCP-1 gene expression, consistent with a mitogen-activated protein kinase-dependent signaling mechanism. Ang II may thus promote atherogenesis by direct activation of MCP-1 gene expression in vascular smooth muscle cells.

E-selectin gene expression in vascular smooth muscle cells. Evidence for a tissue-specific repressor protein.

E-Selectin is an inducible, endothelium-specific, cell surface adhesion molecule that mediates inflammatory responses in the vasculature. Nonendothelial cell types such as cultured human aortic smooth muscle cells (HASMCs) lack the ability to express E-selectin. We tested the hypothesis that HASMCs express a negative regulatory factor that inhibits E-selectin gene expression. E-Selectin mRNA and gene transcription were not detected in HASMCs after treatment with tumor necrosis factor-alpha (TNF-alpha) by Northern and nuclear runoff analyses, respectively. However, both E-selectin mRNA and gene transcription were dramatically induced by TNF-alpha in the same cells pretreated with the protein synthesis inhibitor cycloheximide. Lipopolysaccharide demonstrated similar effects. Furthermore, E-selectin was detected on the cell surface of HASMCs after washing out of cycloheximide. Cycloheximide pretreatment enabled immortalized human dermal microvascular endothelial cells that have lost the ability to express E-selectin to induce both E-selectin mRNA and gene transcription in response to TNF-alpha. Induction of E-selectin mRNA by lipopolysaccharide or TNF-alpha in cycloheximide-treated HASMCs was inhibited by the antioxidant pyrrolidinedithiocarbamate and the serine protease inhibitor N alpha-L-tosyl-L-phenylalanine chloromethyl ketone, suggesting that a nuclear factor-kappa B-like mechanism may play an important role in E-selectin gene expression in HASMCs. These data strongly suggest that a labile repressor protein(s) plays an important role in inhibiting E-selectin gene expression in HASMCs likely at the level of gene transcription. Except for this repressor, HASMCs and endothelial cells may share similar regulatory mechanisms for controlling E-selectin expression.

Alterations in the secretion of atrial natriuretic factor in atria from aged rats.

We measured plasma atrial natriuretic factor levels and atrial natriuretic factor secretion by isolated left atria from aging rats to determine the secretory response to stretch and adrenergic stimulation. Systolic arterial pressure and right atrial pressure were measured in vivo. Twenty-four hours later, atria were removed and studied in vitro in a perifusion system. After removal, stabilization at 0.7 g tension, and equilibration for 65 minutes, atria were stretched by increasing external tension for 20 minutes. After reequilibration atria were perifused with phenylephrine, 10(-5) M, for an additional 30 minutes. Right atrial pressure was not different between young (3 months) and aged (16-24 months) rats. Aged rats had higher plasma atrial natriuretic factor levels (52 +/- 8 versus 21 +/- 6 pmol/l; p less than 0.05) than young rats. Basal atrial natriuretic factor secretory rate in vitro was greater in atria from aged rats than young rats (875 +/- 35 versus 402 +/- 22 pg/min; p less than 0.05). Atria from aged rats had an increased response to phenylephrine compared with young rats (1,687 +/- 143 versus 788 +/- 113 pg/min; p less than 0.05) when means were adjusted for basal secretory rate. The secretory response to stretch was less than that of young rats (673 +/- 37 versus 773 +/- 27 pg/min), although this difference was not significant (p = 0.07). Atrial natriuretic factor secretion in response to adrenergic stimulation is increased with aging, and these secretory responses may contribute to increased plasma levels that occur during aging. In contrast to increased adrenergic responses, atrial natriuretic factor secretion after external stretch is not increased in aging rats.