Introduction and Objective: Septic shock marks the point in a severe infection when cascading responses overwhelm compensatory mechanisms resulting in overt cardiovascular failure. The appearance of vasopressor requiring hypotension substantially increases the risk of death from infection. Up to 60% of septic shock non-survivors die in refractory shock during the first 7-10 days of illness. In refractory septic shock both vascular relaxation and constriction ultimately become impaired, acute abnormalities analogous to endothelial dysfunction and injury in chronic atherosclerosis.? ? This investigation is exploring the mediators, signal transduction pathways, and underlying mechanisms of endothelial dysfunction and vascular inflammation.? ? Proposed Course of Work: ? Explore nitric oxide-triggered signal transduction pathways in a human hybrid endothelial cell line and in primary human endothelial cells. Initial studies demonstrated that exogenous nitric oxide inhibits proteosome function and activates p38 MAPK. Other work has shown potentially important interactions between nitric oxide signaling and peroxisome proliferator-activated receptors (PPARs). Like nitric oxide, (PPARs) have been associated with endothelial protection and other aspects of vascular health. Nitric oxide was found to activate PPARgamma and thereby regulate downstream target genes that contain PPAR-response elements. ? ? Investigate interactions between Nitric oxide signaling and inflammatory responses in human primary pulmonary microvascular endothelial cells. ? ? Develop an in vitro model of endothelial cell dysfunction using the RNA interference pathway in primary cells. Endothelial dysfunction has been associated with reduced expression or function of a number of genes including eNOS, PPARs, and BMPR2 in a wide variety of models and clinical settings including sepsis and atherosclerosis. Gene knockdown using small interfering RNA (siRNA) will be followed by phenotypic characterization using Western blots, immunofluorescent staining, and oligonucleotide microarrays in the presence and absence of inflammatory-mediator activation (TNFalpha). Data from this in vitro work will be examined and analyzed in the context of clinical samples from a protocol (with Michael Solomon, M.D.) enrolling patients with primary pulmonary hypertension.? ? Examine the ability of anthrax toxins (lethal toxin and edema toxin) to cause dysfunction and injury in endothelial cells and moncytes. Explore molecular mechanisms such as nitric oxide bioavailability that might protect cells from the harmful effects of anthrax toxins. Test whether interventions based on these mechanisms might have theraeutic value in anthrax infections.? ? Progress:? Transfection of monoblastoid U937 cells with human eNOS resulted in a cell line that produced nitric oxide in response to calcium ionophore, but not in the resting state (Blood, 1997). However, after differentiation with phorbol-12-acetate-13-myristate, eNOS expressing cells produced increased amounts of both TNFalpha and reactive oxygen species by mechanisms that were independent of nitric oxide.? ? Neither Nw-methyl-L-arginine, a NOS inhibitor, nor mutation of the L-arginine binding site of eNOS, rendering it incapable of producing nitric oxide, blocked the ability of eNOS to upregulate TNFalpha. Conversely, co-transfection with superoxide dismutase or deletion of the NADPH binding site of eNOS completely prevented eNOS from upregulating TNFalpha production. These results suggested that eNOS can regulate inflammatory responses through both nitric oxide (J Immunol, 1994; J Biol Chem, 1997) and reactive oxygen species-based signal transduction pathways (J Biol Chem, 2000). Superoxide produced by eNOS was shown to upregulate TNFalpha via p42/44 MAPK activation (J Biol Chem, 2001).? ? More recent work has shown that nitric oxide can activate PPAgamma and thereby regulate genes through a P38 MAPK- dependent pathway. This work suggests that some endothelial protective effects of nitric oxide may be mediated by PPAgamma activation. Current experiments are focused on identifying PPAgamma co-factors that are regulated by nitric oxide. ? ? In a related study, the comparative effects of nitric oxide and carbon monoxide on kinase activation is being studied in endothelial cells and monocytes. These low molecular weight gases share signaling transduction pathways, but are otherwise chemically dissimilar.
