Targeting peptide Lv and its downstream signaling against ocular neovascularization

项目摘要 抗血管内皮生长因子（VEGF）疗法广泛用于治疗具有以下特征的眼部疾病： 血管新生，但近30%的受折磨的患者对抗VEGF剂无应答。此外，本发明还提供了一种方法， 阻断复发性新血管形成所需的重复抗VEGF治疗通常导致非预期的血管生成。 副作用包括视网膜脱落甚至失明。因此，寻找VEGF非依赖性血管生成因子， 这些因素并揭示其潜在机制成为帮助对抗复发的迫切临床需求 这些疾病中的新血管形成和对抗VEGF的抗性。Lv肽是一种新发现的 在早期增殖性糖尿病视网膜病变（PDR）患者视网膜中上调的血管生成肽 以及患有氧诱导的视网膜病变（OIR）的小鼠，其特征在于眼部病理性新血管形成。 虽然它与VEGF家族成员和VEGF受体（VEGFRs）没有序列同源性，但肽Lv 在体外和体内促进血管生成。抗Lv肽的抗体，anti-Lv，不仅抑制VEGF， 引起内皮细胞增殖，还有效阻断OIR小鼠的病理性新生血管形成， 以及激光诱导的脉络膜新生血管形成。此外，肽Lv不仅协同促进 VEGF诱导的血管生成，但也增强VEGF/VEGFR 2/一氧化氮非依赖性血管舒张。慢性 现有血管的血管舒张导致血管通透性增加，刺激血管生成，并促进血管生成。 新生血管形成血管扩张剂与约70%的早期年龄相关性黄斑病变发病率增加有关 退行性变（AMD）。因此，Lv肽可能通过促进PDR和湿性AMD的发病机制， VEGF/VEGFR 2依赖性和非依赖性血管生成和血管舒张。然而， 肽Lv介导VEGF非依赖性血管生成和血管舒张是完全未知的。肽Lv是 能够增加中电导钙依赖性钾通道（IKCa）的蛋白质表达 在培养的内皮细胞中。内皮细胞K+通道的激活，尤其是IKCa的激活，在血管生成中是重要的 其可以是VEGF/VEGFR 2非依赖性的。开放这些K+通道也可以导致VEGF非依赖性 血管舒张在此，目的是揭示肽Lv在VEGF/VEGFR 2 - 1中的新的细胞机制。 独立的血管生成和血管舒张。中心假设是Lv肽激活并增强IKCa 在视网膜内皮细胞中导致VEGF/VEGFR 2非依赖性血管生成和血管舒张， 抗VEGF抗体耐药的潜在机制。结合体外、离体和体内方法的两个目的 为了解读VEGF/VEGFR 2非依赖性血管生成和血管舒张中的肽Lv-IKCa轴，将通过以下方式进行： 使用肽Lv null（肽Lv-/-）、IKCa null（Kcnn 4-/-）和血管内皮细胞特异性敲除VEGFR 2 （VEGFR 2 i ΔEC）小鼠。完成拟议的研究将揭示新的机制，肽Lv在VEGF/ VEGFR 2非依赖性血管生成和血管舒张，这将对未来的发展产生直接影响。 阻断肽LV-下游靶点作为针对眼部病理性新血管形成的治疗工具。

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 VEGFR2 (VEGFR2iΔEC) mice. Completion of the proposed research will unveil new mechanisms of peptide Lv in VEGF/ VEGFR2-independent angiogenesis and vasodilation, which will have a direct impact on the future development of blocking peptide Lv-downstream targets as a therapeutic tool against ocular pathological neovascularization.