Endotoxic shock is a life-threatening consequence of severe Gram-negative infection characterized by vascular smooth muscle cell relaxation and severe hypotension. The production of nitric oxide (NO), through the inducible NO synthase pathway, has been implicated as a major contributor in this process. We now demonstrate that heme oxygenase (HO), an enzyme that generates carbon monoxide (CO) in the course of heme metabolism, may also be involved in the hemodynamic compromise of endotoxic shock. Inducible HO (HO-1) mRNA levels are dramatically increased in aortic tissue from rats receiving endotoxin, and this increase in vascular HO-1 message is associated with an 8.9-fold increase in HO enzyme activity in vivo. Immunocytochemical staining localizes an increase in HO-1 protein within smooth muscle cells of both large (aorta) and small (arterioles) blood vessels. Furthermore, zinc protoporphyrin IX, an inhibitor of HO activity, abrogates endotoxin-induced hypotension in rats. Studies performed in rat vascular smooth muscle cells in vitro show that the induction of HO-1 mRNA is regulated at the level of gene transcription, and this induction is independent of NO production. Taken together, these studies suggest that the up-regulation of HO-1, and the subsequent production of CO, contributes to the reduction in vascular tone during endotoxic shock.Endotoxemia leading to shock is a detrimental consequence of severe Gram-negative bacterial infection. Endotoxic shock is initiated by the release of bacterial cell wall-derived lipopolysaccharide (LPS) 1 and the subsequent production of cytokines and vasoactive mediators that result in vascular smooth muscle cell relaxation and hypotension (1, 2). One of the most important cytokines in the cascade of events leading to LPSinduced hypotension is interleukin (IL)-1␤ (1, 3). We have demonstrated previously that IL-1␤ stimulates the inducible isoform of nitric oxide synthase (NOS) and increases the production of NO in vascular smooth muscle cells (4). NO is a labile, free radical gas that acts as a potent vasodilator (5, 6). The importance of NO in the pathogenesis of endotoxic shock has been emphasized by recent studies demonstrating that mice carrying a disrupted inducible NOS gene have an attenuated hypotensive-response to LPS (7) and are resistant to LPS-induced death (7,8). However, the study by MacMicking and colleagues (7) also suggested that an inducible NOS-independent pathway contributes to LPS-induced hypotension and death, and we hypothesize that one potential pathway involves heme oxygenase (HO).HO is the enzyme that generates carbon monoxide (CO) and biliverdin (subsequently reduced to bilirubin) in the course of heme metabolism (9). CO is a gas molecule that shares some of the properties of NO, inasmuch as CO binds to the heme moiety of cytosolic guanylyl cyclase to produce cGMP (10). Two distinct forms of heme oxygenase have been identified (9): HO-1 (an inducible isozyme) and HO-2 (a non-inducible isozyme). Morita and colleagues (11) have demonstrated that HO-...

Homocysteine is an important and independent risk factor for arteriosclerosis. We showed previously that homocysteine stimulates vascular smooth muscle cell proliferation, a hallmark of arteriosclerosis. We show here that homocysteine and serum increased DNA synthesis synergistically in both human and rat aortic smooth muscle cells (RASMCs). Treatment of quiescent RASMCs with 1 mM homocysteine or 2% calf serum for 36 h increased cyclin A mRNA levels by 8-and 14-fold, respectively, whereas homocysteine plus serum increased cyclin A mRNA levels by 40-fold, indicating a synergistic induction of cyclin A mRNA. Homocysteine did not increase the half-life of cyclin A mRNA (2.9 h), but it did increase the transcriptional rate of the cyclin A gene in nuclear run-on experiments. The positive effect of homocysteine on cyclin A gene transcription was confirmed by our finding that homocysteine increased cyclin A promoter activity and ATF-binding protein levels in RASMCs.

Nitric-oxide synthases (NOS) utilize L-arginine to produce NO, a potent vasodilator that contributes to the regulation of vascular tone. We demonstrated previously that transforming growth factor (TGF)-␤1 downregulates inducible NOS after its induction by interleukin (IL)-1␤ by decreasing the rate of inducible NOS gene transcription. In the present study we transfected reporter plasmids containing various lengths of the inducible NOS 5 -flanking region into primary cultured rat aortic smooth muscle cells and stimulated the cells with IL-1␤ or vehicle. IL-1␤ increased the activity of the plasmid containing ؊1485 to ؉31 of the inducible NOS gene by more than 10-fold, indicating the presence of IL-1␤-responsive elements. Further deletion analysis revealed that a construct containing ؊234 to ؉31 of the inducible NOS gene contained the majority of promoter/enhancer activity after IL-1␤ stimulation. Mutation of the NF-B site within this region partially reduced IL-1␤-inducible activity; however, a large portion of activity remained independent of the NF-B site. TGF-␤1 suppressed promoter/enhancer activity after IL-1␤ stimulation, and this suppression was complete in the construct with a mutated NF-B site. In addition, TGF-␤1 did not decrease the binding of nuclear proteins to the NF-B site. These data suggest that the ability of TGF-␤1 to suppress inducible NOS promoter/enhancer activity occurs through a site(s) other than the NF-B motif in vascular smooth muscle cells.