Project Summary Anti-vascular endothelial growth factor (VEGF) therapies are widely used for treating ocular diseases with neovascularization, but nearly 30% of afflicted patients are non-responders to anti-VEGF agents. In addition, repetitive anti-VEGF treatments that are needed to block the recurring neovascularization often lead to unintended side-effects including retinal detachment and even blindness. Thus, searching for VEGF-independent angiogenic factors and unveiling their underlying mechanisms become an imperative clinical need to help combat the recurrence of neovascularization and the resistance to anti-VEGFs in these diseases. Peptide Lv is a newly discovered angiogenic peptide that is upregulated in the retinas of patients with early proliferative diabetic retinopathy (PDR) and of mice subjected to oxygen-induced retinopathy (OIR), featuring ocular pathological neovascularization. Although it has no sequence homology to members of the VEGF family and VEGF receptors (VEGFRs), peptide Lv promotes angiogenesis in vitro and in vivo. An antibody against peptide Lv, anti-Lv, not only dampens VEGF- elicited endothelial cell proliferation, it also effectively blocks pathological neovascularization in mice with OIR, as well as laser-induced choroidal neovascularization. Furthermore, peptide Lv not only cooperatively promotes VEGF-induced angiogenesis, but also elicits VEGF/VEGFR2/nitric oxide-independent vasodilation. Chronic vasodilation of existing vessels causes increased vascular permeability, stimulates angiogenesis, and promotes neovascularization. Vasodilators are associated with a ~70% increased incidence of early age-related macular degeneration (AMD). Thus, peptide Lv might be involved in the pathogenesis of PDR and wet AMD via promoting both VEGF/VEGFR2-dependent and -independent angiogenesis and vasodilation. However, the mechanistic role of peptide Lv in mediating VEGF-independent angiogenesis and vasodilation is completely unknown. Peptide Lv is able to augment the protein expression of intermediate conductance calcium-dependent potassium channels (IKCa) in cultured endothelial cells. Activation of K+ channels in the endothelium, especially IKCa, is important in angiogenesis that can be VEGF/VEGFR2-independent. Opening these K+ channels can also lead to VEGF-independent vasodilation. Herein, the objective is to unveil novel cellular mechanisms of peptide Lv in VEGF/VEGFR2- independent angiogenesis and vasodilation. The central hypothesis is that peptide Lv activates and augments IKCa in retinal endothelial cells that consequently leads to VEGF/VEGFR2-independent angiogenesis and vasodilation, the underlying mechanism of resistance to anti-VEGFs. Two Aims combining in vitro, ex vivo, and in vivo approaches to decipher peptide Lv-IKCa axis in VEGF/VEGFR2-independent angiogenesis and vasodilation will be carried out by using peptide Lv null (peptide Lv-/-), IKCa null (Kcnn4-/-), and vascular endothelial cell-specific knockout of V...