Murine models of vascular remodeling

血管重塑的小鼠模型

• 批准号: 8158040
• 负责人: Manfred Boehm
• 金额: $ 99.09万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8158040
• 关键词: Acute; Binding; Cellularity; Clinical; Data; Development; Disease; Endothelial Cells; Environment; Inflammation; Inflammatory Response; Injury; Knock-out; Knockout Mice; Laboratories; Lesion; Lung; Maps; Medial; Mesenchymal; Modeling; Mus; Phase; Physiologic intraventricular pressure; Process; Pulmonary Hypertension; Rag1 Mouse; Right Ventricular Hypertrophy; STAT3 gene; Small Interfering RNA; Smooth Muscle Myocytes; Stenosis; Structure of jugular vein; Vascular Endothelial Cell; Vascular remodeling; femoral artery; in vivo; macrophage; prevent; pulmonary arterial hypertension

Vascular wound repair is controlled by a complex interaction between local vascular cells and circulating immune and non-immune cells. Recently, the classic dogma of a predominantly local response to injury has been challenged by the identification of circulating vascular progenitor cells. The role of these vascular progenitor cells in vascular regeneration and their interaction with local vascular cells and infiltrating immune cells is poorly understood. My laboratory has undertaken a number of approaches to understand how these cells contribute to the overall vascular injury response and how they might ultimately be therapeutically manipulated. We studied events immediately following arterial injury and identified an acute STAT3- and NF-B (p65 subunit)-dependent upregulation of RANTES production by medial VSMCs, leading to early T cell and macrophage recruitment - processes also under the higher-order regulation of p21Cip1. Unique to VSMCs, RANTES production was initiated by TNF but not IL-6/gp130, and was dependent on binding of a p65-STAT3 complex to NF-B binding sites within the RANTES promoter, with shRNA knockdown of either STAT3 or p65 markedly attenuating RANTES production. In vivo, acute NF-B and STAT3 activation in medial VSMCs was identified, with acute RANTES production after injury significantly reduced in TNF-/- mice compared to controls. Finally, we generated mice with smooth muscle cell-specific conditional STAT3 knockout (STAT3fl/fl;SM22-Cre) and confirmed the STAT3-dependence of acute RANTES production by VSMCs. Together, these observations unify inflammatory events after vascular injury, demonstrating that VSMCs orchestrate the arterial inflammatory response program via acute RANTES production and subsequent inflammatory cell recruitment. Veins grafted into an arterial environment undergo vascular remodeling, a complex process of major clinical importance. Using lineage tracing experiments, we provide unprecedented evidence that cells of endothelial origin from the vein graft contribute to mesenchymal cellularity; during neointimal formation. In murine jugular veins grafted to femoral arteries, we found that endothelial cells lose their nascent markers and over time gain smooth muscle cell markers, indicative of endothelial to mesenchymal transition (EndoMT). This process is dependent on TGF- signaling, with early Smad activation. Antagonism of TGF- signaling by TGF- neutralizing antibody, siRNA-mediated Smad3 knockdown, or Smad3 haploinsufficiency resulted in decreased EndoMT and favorable vascular remodeling. Hence, we have identified EndoMT as a novel and pivotal mechanism underlying the stenosis-producing neointimal overgrowth of vein grafts, implying a potential a new therapeutic target to prevent vein graft pathology. Pulmonary hypertension is a vascular proliferative disease characterized by pulmonary artery remodeling due to dysregulated endothelial and smooth muscle cell proliferation. While the role inflammation plays in the development of the disease is not well defined, plexogenic lesions in human disease have been characterized by perivascular inflammation composed in part by T cells. We sought to explore the role of T cell infiltration on pulmonary vascular remodeling following endothelial cell damage. We induced endothelial cell damage using monocrotaline and isolated the role of T cells by utilizing Rag1tm1Mom mice and performing adoptive T cell transfer. We found that monocrotaline causes a pulmonary vascular endothelial cell injury which is followed by a perivascular inflammatory response. The infiltration of inflammatory cells is made up primarily of CD4+ T cells and leads to a progressive muscularization of small (<30m) arterioles. Pulmonary vascular proliferative changes were accompanied by progressive and persistent elevations in right ventricular pressure and right ventricular hypertrophy. Supporting the central role of CD4+ T cells in the inflammatory response, Rag1tm1Mom (Rag1-/-) mice which are devoid of T and B cells were protected from the development ofvascular injury when exposed to monocrotaline. Introduction of T cells from control mice into Rag1-/- mice reproduced the vascular injury phenotype. These data indicate that following endothelial cell damage CD4+ T cell infiltration participates in pulmonary vascular remodeling. This suggests that a CD4+ T cell immune response may contribute to the pathogenesis of inflammatory vascular lesions seen in some forms of pulmonary hypertension.

血管损伤修复是由局部血管细胞与循环免疫和非免疫细胞之间的复杂相互作用控制的。最近，对损伤主要是局部反应的经典教条受到了循环血管前体细胞鉴定的挑战。这些血管前体细胞在血管再生中的作用以及它们与局部血管细胞和浸润性免疫细胞的相互作用还知之甚少。我的实验室采用了许多方法来了解这些细胞如何对整体血管损伤反应做出贡献，以及它们最终可能如何被治疗操纵。 我们研究了动脉损伤后立即发生的事件，发现内侧VSMCs的RANTES产生依赖于STAT3和NF-B(p65亚基)的急性上调，导致早期T细胞和巨噬细胞募集过程也受到p21Cip1的高阶调控。与VSMCs不同，RANTES的产生是由肿瘤坏死因子而不是IL-6/gp130启动的，并且依赖于p65-STAT3复合体与RANTES启动子内的NF-B结合位点的结合，而STAT3或p65的shRNA敲除显著抑制RANTES的产生。在体内，内侧VSMCs中发现了急性的NF-B和STAT3的激活，与对照组相比，损伤后的TNF-/-小鼠的急性RANTES的产生显著减少。最后，我们建立了平滑肌细胞特异性条件性STAT3基因敲除的小鼠(STAT3fl/fl；SM22-Cre)，并证实了VSMCs产生的急性RANTES依赖于STAT3。总之，这些观察统一了血管损伤后的炎症事件，表明VSMCs通过急性RANTES的产生和随后的炎症细胞募集来协调动脉炎症反应程序。 移植到动脉环境中的静脉经历血管重塑，这是一个复杂的过程，具有重要的临床意义。利用谱系追踪实验，我们提供了前所未有的证据，表明来自静脉移植物的内皮细胞在新生内膜形成过程中有助于间充质细胞的形成。在移植到股动脉上的小鼠颈静脉中，我们发现内皮细胞失去了它们的新生标记，随着时间的推移，获得了指示内皮细胞向间充质转化(EndoMT)的平滑肌细胞标记。这一过程依赖于转化生长因子信号，早期激活Smad。转化生长因子中和抗体拮抗转化生长因子信号、siRNA介导的Smad3基因敲除或Smad3单倍体缺失可导致内源性MT降低，有利于血管重塑。因此，我们已确定EndoMT是导致移植静脉狭窄的新生内膜过度生长的一个新的和关键的机制，这意味着一个潜在的新的治疗靶点来预防静脉移植的病理。 肺动脉高压是一种血管增生性疾病，其特征是由于内皮细胞和平滑肌细胞增殖失调而导致的肺动脉重塑。虽然炎症在疾病发展中的作用尚不清楚，但人类疾病的丛源性病变的特点是部分由T细胞组成的血管周围炎症。我们试图探讨T细胞浸润在内皮细胞损伤后肺血管重塑中的作用。我们使用野百合碱诱导内皮细胞损伤，并通过利用Rag1tm1Ma小鼠和进行过继T细胞转移来分离T细胞的作用。我们发现野百合碱引起肺血管内皮细胞损伤，继而引起血管周围炎症反应。炎症细胞的渗透主要由CD4+T细胞组成，并导致小动脉(30m)的进行性肌化。肺血管增生性改变伴有进行性和持续性的右室压力升高和右室肥厚。支持CD4+T细胞在炎症反应中的中心作用，缺乏T和B细胞的Rag1tm1Ma(Rag1-/-)小鼠在暴露于野百合碱时可以保护血管免受损伤。将对照小鼠的T细胞导入Rag1-/-小鼠，重现了血管损伤表型。提示内皮细胞损伤后，CD4+T细胞的浸润参与了肺血管重塑。这表明，CD4+T细胞免疫反应可能参与了某些形式的肺动脉高压中炎性血管病变的发病机制。