Numerous immunotoxins and many vascular pathologies are associated with the release of inflammatory mediators such as bacterial lipopolysaccharide (LPS), interleukin-1 (IL-1) and tumor necrosis factor (TNF). Activation of inflammatory cytokines has opposite effects on the two types of vascular nitric oxide synthase (NOS): downregulation of the consititutive form (cNOS) and upregulation of the inducible form (iNOS). In the proposed studies we will identify major cellular and molecular mechanisms involved in the upregulation of iNOS and downregulation of cNOS in vascular cells during immunological challenges. On the basis of the recently discovered NF-kappaB consensus sequence in the cloned promoter region of macrophage iNOS and our preliminary data regarding the effects of the antioxidant and NF-kappaB inhibitor N-acetyl-L-cysteine and various inhibitors of protein tyrosine kinase (PTK) and microtubule assembly on iNOS expression in vascular smooth muscle cells (VSMC), we hypothesize that NF-kappaB is present in VSMC and its activation by inflammatory mediators (via oxidant generation which triggers a pathway involving a tyrosine kinase) leads to cytoskeleton-dependent iNOS upregulation. On the basis of data that immunomodulators attenuate endothelial (EC) cNOS, that NF-kappaB is present in endothelial cells and that downregulation of cNOS mRNA by TNF involves the induction of an anti-cNOS protein, we further hypothesize that a similar NF-kappaB dependent signal transduction path way is responsible for immunomodulator-induced endothelial cNOS downregulation. To test these hypotheses, we will l) quantify LPS, IL-1- and TNF-induced oxidant stress and thiol depletion and determine the contribution of oxidants to iNOS expression in VSMC, 2) investigate the kinetics and mechanisms of tyrosine kinase activation and contribution of protein tyrosine phosphorylation to iNOS expression in VSMC, 3) investigate the kinetics and mechanisms of NF-kappaB activation in VSMC 4) investigate whether superinduction of iNOS by protein synthesis inhibitors is mediated through a labile repressor of NF-kappaB. 5) investigate the role of the cytoskeleton in iNOS expression, 6) quantify LPS-, IL-1- and TNF-induced oxidant stress and thiol depletion in EC, 7) investigate the kinetics and mechanism of activation of protein kinases and NF-kappaB, cytoskeletal reorganization and determine the contribution of these mechanisms to cNOS downregulation in EC, 8) investigate the contribution of oxidants, cellular thiols, protein kinases and the cytoskeleton to altered NO regulation in an animal model of endotoxin shock, in vivo and ex-vivo. Results should help elucidate mechanisms underlying an important pathological vascular response.
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