This is a proposal to investigate the cellular/molecular mechanisms underlying the heme oxygenase (HO )-mediated regulation of hemoglobin- stimulated vascular endothelial cell (EC) activation and toxicity. EC activation by an injurious stimulus is manifested by the release of many mediators such as cytokines expression of adhesion molecules and alteration in cell permeability. This response, if it persists, leads to changes in EC function (vasoreactivity, proliferation) and ultimately, cell death. Hemoglobin (perhaps via its degradation to heme):"""""""" may operate in a similar manner. HO, as the rate-limiting enzyme in heme degradation, may be a key factor in regulating hemoglobin-induced EC activation and toxicity. Our hypothesis implies that induction of HO-I, following hemoglobin injury provides a protective mechanism to the cell. Thus an increase in endothelial HO-I, activity promotes ferritin synthesis to sequester free iron; production of antioxidant metabolite, bilirubin; and production of vasodilator metabolites. Conversely, hemoglobin itself may be instrumental in activating (via specific transcriptional factors) activating protooncogene c-fos expression and subsequently cellular mediator production such as IL-6. Elimination of heme by HO-I may attenuate this response. Thus manipulation of HO-I on EC activation (proliferation, oncogene expression and cytokine release) and viability in the presence and absence of hemoglobin toxicity (viability) and EC activation, 2) To study transcriptional regulation of HO-I and c-fos genes in response to hemoglobin by a) identifying nuclear factors involved in the transcriptional regulation of HO-I and nuclear oncogens by hemoglobin, and b) investigating the involvement of hemoglobin in the physical interactions of transcriptional factors regulating HO-I and c-fos; 3) To evaluate whether suppression or stimulation of HO-I gene expression using triplex-forming oligonucleotides affects hemoglobin-induced Ec activation and toxicity. The proposed experiments are expected to define the role and significance of HO-I in hemoglobin-induced EC activation and the release of various cytokines and whether trhey have relevance to the overall function of normal and activated EC during incur and the inflammatory response.
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