PROJECT SUMMARY/ABSTRACT Hypertension constitutes the most preventable risk factor for cardiovascular disease, affecting nearly 1 out of every 3 Americans. Cardiovascular diseases are the leading causes of death each year across the globe, creating an average economic burden of nearly $1 billion dollars/year in direct healthcare costs and loss of economic productivity. Healthy vascular function is maintained by the nitric oxide (NO) signaling pathway, which plays a necessary role in allowing for proper dilation of blood vessels in response to higher blood pressure. Within vascular smooth muscle cells (SMC), the NO receptor, soluble guanylyl cyclase (sGC) plays a pivotal role in producing the second messenger molecule, cyclic guanosine-3’,5’-monophosphate (cGMP), to induce downstream relaxation of SMC. Despite the integral role that sGC plays in the regulation of vascular tone, the mechanisms that regulate sGC gene expression in SMC remain unknown. We recently found evidence that the Forkhead box class O (FoxO) transcription factors are capable of binding on both the sGCa and b promoters. Our preliminary data indicate that pharmacological inhibition of FoxO transcription factors in rat aortic SMC with AS1842856 results in a 90-95% loss of sGCα and sGCβ mRNA expression, a 70-80% loss of sGCα and sGCβ protein expression, and a 90% loss of cGMP production after stimulation by the NO-donor, DEA-NONOate. Likewise, treatment of isolated murine aortic rings with the FoxO inhibitor resulted in a 48% loss of sGCβ protein expression as assessed by immunofluorescence and significantly blunted NO-dependent vasorelaxation. Therefore, we sought to identify which of the three FoxO transcription factors expressed in SMC (FoxO1, FoxO3a, and FoxO4) is responsible for the regulation of sGC expression in SMC by developing adenoviral FoxO shRNA constructs to transiently knock down expression of each transcription factor. Our preliminary data show that sGC expression increases following treatment by either FoxO1 or FoxO3a shRNA. Conversely, treatment of rat aortic SMC with FoxO4 shRNA resulted in a 50% loss of sGCα and sGCβ mRNA and sGCβ protein expression. These preliminary data have led to the formation of the following aims: 1) Elucidate whether FoxO4 is responsible for sGC transcriptional regulation in SMC and how this pathway is modulated, and 2) Determine the consequences of manipulating FoxO4 expression on NO-mediated vasoreactivity ex vivo vasorelaxation. In Aim 1, we will attempt to 1.1) determine the downstream sGC signaling effects following FoxO4 shRNA knockdown, 1.2) generate sGC promoter-luciferase constructs and utilize chromatin immunoprecipitation (ChIP) experiments to determine whether FoxO4 is capable of binding and initiating transcription of sGC within SMC, and 1.3) determine if transient knockdown/rescue of FoxO proteins is sufficient to modulate sGC mRNA and protein expression. To assess Aim 2, we will 2.1) treat isolated mouse aortic rings with ...