Fi02 and Blood Vessel Formation in Adult Lung

描述(由申请人提供)： 在成人肺中引发小血管和毛细血管丧失的损伤，以及存活的肺壁过度生长，最终会限制血流，增加血管阻力和压力，从而导致肺动脉高压(PH)。本研究的长期目标是获得有可能在成人肺中形成新的血管单位的循环血源性细胞的数据，以及调节血管形成的RTK-配体信号系统，作为促进血管节段(重新)生长和阻止壁过度生长的策略的合理基础。我们最近在PH血管重塑的活体模型中的数据表明，FiO2触发血管和毛细血管单位的退化和生长，使血管密度与组织的功能需求相匹配。由于正常肺泡-毛细血管膜的血管形成了毛细血管网络的延续，许多是简单的内皮衬里通道，几乎没有壁细胞。当高氧(FiO2 0.87)修剪这些节段时，壁细胞在存活的节段周围发展，首先导致壁上支撑，然后导致壁上过度生长。返回空气的早期阶段(代表对高氧适应肺的相对低氧)触发了壁细胞的进一步发展，并在整个肺泡毛细血管膜上形成新的血管和毛细血管。根据初步数据，假设是循环内皮祖细胞(CEP)形成这些新的血管网络，FiO2刺激间质成纤维细胞表达PDGF的RTKs(PDGFR-α和R-β)和局部内皮细胞表达Ang-1(Tie-2)，诱导壁细胞发育，CEPs和内皮细胞表达的RTKs(VEGFR-1，VEGFR-2和Tie-2)增加，形成血管单位。血液来源的细胞将通过细胞谱系分析来表征。PDGFR-β对壁细胞发育的需求将通过功能抑制抗体研究来证明。将通过细胞死亡分析来评估FiO2诱导的血管细胞退化、单位修剪和细胞凋亡，并调查血管内皮生长因子提供的保护作用。通过最先进的高分辨率免疫金标记法和数字成像技术，将获得以下定量数据：(I)荧光素标记/荧光素酶转基因的血液来源细胞和(Ii)RTK及其配体(PDGF-AA、PDGF-BB、VEGF、Ang-land Ang-2)在由FiO2引发的生长反应中形成血管单位的内皮细胞和壁细胞中的表达水平。这些方法将展示血源性细胞和RTK信号在“异常”FiO2触发的作用，导致新的血管单位形成和管壁过度生长，并为预防或纠正导致人类患者严重疾病的血管重塑的治疗方法提供数据基础。

DESCRIPTION (provided by applicant): Injuries that trigger loss of small vessels and capillaries in the adult lung, and wall overgrowth in ones that survive ultimately restrict blood flow and increase vascular resistance and pressure to cause pulmonary hypertension (PH). The long-term goals of the present studies are to obtain data for circulating blood-derived cells that have the potential to form new vascular units in the adult lung, and the RTK-ligand signaling systems that regulate blood vessel formation, as a rational basis for strategies to promote (re)growth of vascular segments and block wall overgrowth. Our recent data in an in vivo model of vascular remodeling in PH indicate that the FiO2 triggers regression and growth of vessel and capillary units to match vascular density to the functional demands of the tissue. Because vessels of the normal alveolar-capillary membrane form the continuation of capillary networks many are simple endothelial-lined channels with few, if any, mural cells. As hyperoxia (FiO2 0.87) prunes these segments, mural cells develop around ones that survive, resulting first in wall support and then in wall overgrowth. The early stage of return to air (representing relative hypoxia to the hyperoxia-adapted lung) triggers further mural cell development and the formation of new vessels and capillaries throughout the alveolar-capillary membrane. Based on preliminary data, the hypotheses tested are that circulating endothelial progenitors (CEPs) form these new vascular networks, and that FiO2 -triggered increase in expression of RTKs for PDGF (PDGFR-alpha and R-beta) by interstitial fibroblasts and ANG- 1 (TIE-2) by local endothelial cells induces mural cell development, and increase in expression of RTKs for VEGF and ANG-2 (VEGFR-1, VEGFR-2 and TIE-2) by CEPs and endothelial cells the formation of vascular units. Blood-derived cells will be characterized by cell lineage analysis. The requirement of PDGFR-beta for mural cell development will be demonstrated by function-inhibition antibody studies. FiO2--induced vascular cell regression, unit pruning, by apoptosis, will be assessed by cell death assays, and the protection afforded by VEGF investigated. Quantitative data will be obtained by state-of-the-art high resolution immunogold labeling and digital imaging techniques for (i) fluorescein labeled/luciferase transfected blood-derived cells and (ii) RTK and ligand (PDGF-AA, PDGF-BB, VEGF, ANG-land ANG-2) expression levels in endothelial and mural cells of forming vascular units in growth responses triggered by the FiO2. These approaches will demonstrate a role for blood derived cells and RTK signaling triggered by an 'abnormal' FiO2 that lead to the formation of new vascular units and wall overgrowth, and provide data on which to base therapeutic approaches to prevent or correct the vascular remodeling that results in severe illness in human patients.