The goal of the project is to understand the role of NOTCH3 signaling in the genesis of pulmonary hypertension. The long-term objectives are to apply these discoveries and enable the translation of these findings into clinical practice. Recently, we have shown that human pulmonary arterial hypertension is characterized by overexpression of NOTCH3, a receptor of the NOTCH gene family, and HES-5, a downstream effector in the NOTCH pathway, in small pulmonary artery smooth muscle cells. We have found that the severity of pulmonary hypertension in humans and rodents correlates with the amount of NOTCH3 or HES-5 in the lung. We have demonstrated that mice with homozygous deletion of Notch3 do not develop pulmonary hypertension in response to hypoxic stimulation and that pulmonary hypertension can be successfully treated in mice and rats by administration of a gamma-secretase inhibitor drug that blocks the cleavage and activation of Notch3 in pulmonary vascular smooth muscle cells. Building on these results, we have demonstrated a mechanistic link from NOTCH3 receptor signaling through HES-5, to small pulmonary artery smooth muscle cell proliferation and a shift to an undifferentiated smooth muscle cell phenotype. Recently, we have found evidence of signal integration and regulation between NOTCH3 and BMPR pathways in vascular smooth muscle cells from individuals with pulmonary arterial hypertension, with the discovery that HES-5 binds to specific ID proteins, which function as downstream effectors of BMPR-2 signaling. Thus, we hypothesize that the NOTCH3-HES-5 signaling pathway is crucial for the pathologic remodeling of small vessels in the lung in pulmonary hypertension and that targeting this pathway may be a useful strategy to treat this disease. To test this hypothesis, we propose the following specific aims: 1) determine whether different forms of pulmonary hypertension are linked by the same pattern of overexpression of NOTCH3 and HES-5 in pulmonary vascular smooth muscle cells and examine whether the proliferative/anti-apoptotic capacities of these cells in patients with different World Health Organization Classifications of pulmonary hypertension is dependent on constitutive expression of NOTCH3 and HES-5. We will also examine NOTCH ligand expression on pulmonary vascular endothelium from patients with different World Health Organization forms of disease, 2) investigate NOTCH downstream signaling as a key regulator of downstream BMPR signaling in pulmonary hypertension, by determining if modulation of levels or function of ID proteins by HES-5 in vascular smooth muscle cells is obligate for this disease, and 3) develop a therapeutic approach targeting the Notch signaling pathway to treat pulmonary hypertension, using two independent methods: gamma-secretase inhibitor treatment and Notch3-specific antibody treatment in rodent and pig models of disease. Information gained from these proposed experiments should elucidate a molecular mechanism for pulmonary hypertension and suggest possible treatment strategies for this disease.