Shh通过Rho激酶依赖的非经典信号通路诱导发育肺高氧暴露后的血管生成

Disruption of angiogenesis in pulmonary vasculature plays a crucial role in the mechanism of bronchopulmonary dysplasia of premature infants. Sonic hedgehog (Shh) and the ligand Ptch1 act as morphogens in multiple tissues during embryonic development. The role of Shh in vasculogenesis has been documented in a variety of contexts. Recent studies show that Sonic hedgehog induces angiogenesis by a non-canonical pathway via Rho kinase (ROCK)-dependent signaling in endothelial cells. Our previous studies demonstrate prolonged exposure of high concentration of oxygen may cause impairment of lung vascular development in neonatal rats and the expression of Shh, Ptch1, SMO and Ang-1 in the lung are inhibited. In this study, we will further investigate: (1) the effect of exogenous Shh protein on Shh/Ptch1/Gli pathway and RhoA/ROCK in the neonatal rat lungs after exposed to hyperoxia. (2) in human umbilical endothelial cell (HUVEC), changs of cell apoptosis, migration and formation of capillary-like structures, active RhoA content, and expression of matrix metallopeptidase 9 (MMP-9) and osteopontin (OPN) after Shh signaling or ROCK is activated or inhibited, respectively.(3) After silencing of the Shh receptor, Patched-1 mRNA, using small interfering RNA, the effect of Shh activation on cell apoptosis, migration and formation of capillary-like structures in HUVEC. In summary, the objective of this study is to investigate the effect of Shh and ROCK signaling pathway in the pathogenesis of BPD and to provide approaches to the novel therapeutic tool of BPD.

血管发育受阻在早产儿支气管肺发育不良（BPD）的发病机制中占有重要地位。Sonic hedgehog（Shh）通路是血管生成中重要信号通路之一。近年有研究表明，Rho激酶（ROCK）介导Shh非经典信号通路而诱导血管生成。本项目前期研究发现，新生大鼠高氧暴露14天，肺血管发育受阻，肺组织Shh、Ptch1、SMO和血管生成素-1的表达下降。本项目拟进一步观察高氧暴露后外源性补充Shh蛋白对新生大鼠肺组织Shh/Ptch1/Gli和RhoA/ROCK信号通路的影响；以人脐静脉内皮细胞（HUVEC）为研究对象，分别观察活化和抑制Shh通路、活化和抑制ROCK活性及沉默HUVEC的Ptch1基因表达后，HUVEC的凋亡、迁移、小管形成能力和RhoA、MMP-9、OPN表达的变化。通过本课题的研究，旨在探讨Shh和Rho激酶信号通路在BPD血管发育受阻中的作用机制，探索防治早产儿BPD的新途径。

支气管肺发育不良的肺血管发育受阻的机制尚不清楚。本课题组前期研究发现，高氧能抑制新生鼠肺血管Ang-1的表达。本课题探讨Shh信号通路在高氧导致肺血管发育受阻中的作用。高氧可能通过抑制肺组织血管内皮转录因子Sox17的表达而影响肺血管的发育。高氧导致新生小鼠肺组织Shh和Gli1的表达代偿性升高，同时增加了肺间质胶原蛋白的表达。在人脐静脉内皮细胞（HUVECs），高氧对HUVECs迁移能力有明显的抑制作用，但不影响小管形成能力；不管空气或高氧组，Shh可以显著的增加HUVEC的小管形成能力及细胞迁移能力，同时高氧组Y-27632特异性阻断RhoA可明显减低HUVEC的细胞迁移能力，继续加用Shh可改善Y-27632对细胞迁移的抑制作用，但对小管形成的抑制作用无明显改善。结论：高氧可能诱导Shh通过RhoA途径调节HUVEC的迁移，从而参与肺血管发育。