Kontrollmechanismen der endothelialen NO-Synthase-Expression

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An insufficient production or excessive degradation of nitric oxide (NO) in endothelial cells has been linked to the pathogenesis of atherosclerosis. Consequently, a single nucleotide polymorphism (SNP) in the promoter of the gene encoding endothelial cell NO synthase (NOS-3) that apparently causes a decline in enzyme expression has been associated with cardiovascular disease. According to our preparatory work, homozygous replacement of the thymidine at position -786 by a cytosine results in the tight binding of an as yet unidentified protein, termed factor X, to the promoter of the nos-3 gene which prevents not only the nearby binding of STAT-3 but also that of a putative shear stress-sensitive transcription factor. While STAT-3 mediates the stimulatory effect of the anti-inflammatory cytokine interleukin-10 on NOS-3 expression, laminar shear stress is the physiologically most important stimulus for maintaining expression of the enzyme in vivo. Consistent with the decreased NO-synthesizing capacity of endothelial cells in individuals homozygous for the -786C/T-SNP (up to 15% of the Caucasian population), we could further demonstrate that this SNP is associated with a significantly increased risk to develop coronary artery disease as well as rheumatoid arthritis. Based on these findings, the proposed project has three objectives: (i) To identify factor X by using a combination of affinity chromatography and mass spectrometry analysis; (ii) to characterize the hitherto elusive shear stress-sensitive transcription factor and its binding motif by exploiting the fact that shear stress sensitivity of the nos-3 gene is mediated through the promoter sequence around the -786C/T SNP; and (iii) to set-up an animal experimental proof of concept model (cardiac transplant vasculopathy) to demonstrate efficacy of nucleic acid-based drugs, developed to block factor X or boost NOS-3 expression, for functional correction of the -786C/T SNP. Since this SNP does not seem to occur in other mammals, two knock in mouse strains harboring the human C-type and, for comparison, T-type promoter will be generated for this purpose.