Netrin-1 and Netrin-1 Preconditioned EPCs in Vascular Protection

摘要 本申请旨在确定netrin-1和netrin-1预处理的潜在作用和分子机制。 内皮祖细胞（EPCs）在血管保护中的作用，特别是与其抗再狭窄和抗 动脉粥样硬化效应。四个全新的假设将被解决：1）Netrin-1预处理保护 氧化应激诱导的EPCs通过PI 3 K依赖的p70 s6激酶激活和Bim抑制诱导凋亡; 此外，通过调节新的调节miRNA依赖性增强EPC存活的机制途径， netrin-1将被完全描绘。通过靶向这些新机制，可以增强EPC功能， 减少再狭窄和动脉粥样硬化，导致新疗法的开发。2)Netrin-1抑制 以UNC 5 B（netrin-1受体的排斥类）依赖性方式激活单核细胞; UNC 5 B是 由p47 phox依赖性机制创新地调节。因此，抑制p47 phox可能会增强 netrin-1通过减弱UBC 5 B表达限制再狭窄和动脉粥样硬化的有益作用， UNC 5 B依赖性单核细胞活化。Netrin-1还通过抑制VSMC的迁移和增殖， NO/cGMP/PKG/p38 MAPK依赖机制。3)用netrin-1或netrin-1预处理的EPC施用 在高脂喂养的apoE缺失和LDLR缺陷小鼠中减弱动脉粥样硬化。这些保护作用至少在 部分归因于增强的EPC功能和废除的单核细胞活化，以及减弱的VSMC 扩散和迁移。4)内源性netrin-1信号传导对再狭窄具有生理保护作用， 动脉粥样硬化，动脉粥样硬化的丧失会加重血管病变。我们假设股动脉损伤/高脂血症 喂食netrin-1或netrin-1/apoE双敲除小鼠将导致过度的再狭窄/动脉粥样硬化。 总的假设是，给予netrin-1和netrin-1预处理的EPCs显著减弱了 再狭窄和动脉粥样硬化通过增强EPC功能的机制（增加存活，增殖和 归巢），导致快速再内皮化，减弱单核细胞和VSMC活化，以及减少 血脂异常内源性netrin-1信号传导是生理性血管保护性的，其放大与 netrin-1的外源性给药对于产生足够的保护作用是必要的，而netrin-1的缺乏 netrin-1的内源性信号传导（即由于遗传缺陷）易于恶化血管病变。四 具体目标的设计是为了充分解决这些假设，目标的实现可以建立 netrin-1、netrin-1预处理的EPCs和相关通路的调节剂作为新的治疗选择， 再狭窄和动脉粥样硬化的血管病理学。

ABSTRACT The application aims to identify potential roles and molecular mechanisms of netrin-1 and netrin-1 pre-conditioned endothelial progenitor cells (EPCs) in vascular protection, specifically related to their anti-restenosis and anti- atherosclerosis effects. Four entirely novel hypotheses will be addressed: 1) Netrin-1 pre-conditioning protects EPCs from oxidative stress induced apoptosis through PI3K-dependent p70s6 kinase activation and Bim inhibition; In addition, mechanistic pathways mediating novel regulatory miRNAs-dependent enhancement of EPC survival by netrin-1 will be fully delineated. By targeting these novel mechanisms, EPC functions can be augmented to attenuate restenosis and atherosclerosis, leading to development of novel therapeutics. 2) Netrin-1 inhibits monocyte activation in a UNC5B (repellant class of netrin-1 receptor)-dependent fashion; and that UNC5B is innovatively regulated by a p47phox-dependent mechanism. Therefore, inhibition of p47phox may potentiate the beneficial effects of netrin-1 in limiting restenosis and atherosclerosis via attenuation of UBC5B expression and UNC5B-dependent monocyte activation. Netrin-1 also inhibits VSMC migration and proliferation via a NO/cGMP/PKG/p38MAPK-dependent mechanism. 3) Administration with netrin-1 or netrin-1 preconditioned EPCs attenuates atherosclerosis in high-fat fed apoE null and LDLR deficient mice. These protective effects are at least in part attributed to augmented EPC function and abrogated monocyte activation, as well as attenuated VSMC proliferation and migration. 4) Endogenous netrin-1 signaling is physiologically protective against restenosis and atherosclerosis, loss of which exaggerates vascular pathologies. We hypothesize that femoral artery injury/high-fat feeding into the netrin-1 or netrin-1/apoE double knockout mice will result in exaggerated restenosis/atherosclerosis. The overall hypothesis is that administration of netrin-1 and netrin-1 preconditioned EPCs remarkably attenuate restenosis and atherosclerosis via mechanisms of augmented EPC function (increased survival, proliferation and homing) to lead to rapid re-endothelialization, attenuated monocyte and VSMC activation, as well as diminished dyslipidemia. Endogenous netrin-1 signaling is physiologically vascular protective, amplification of which with exogenous administration of netrin-1 is necessary to result in sufficient protection, whereas deficiency in the endogenous signaling of netrin-1 (i.e. due to genetic defects) is prone to deteriorated vascular pathologies. Four specific aims are designed to fully address these hypotheses, and accomplishments of the aims may establish netrin-1, netrin-1 pre-conditioned EPCs, and modulators of related pathways as novel therapeutic options for vascular pathologies of restenosis and atherosclerosis.