Stem cell biology in cardiovascular regeneration

血管创伤修复受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。最近，对损伤的局部反应占主导地位的经典教条受到了循环血管祖细胞鉴定的挑战。这些血管祖细胞在血管再生中的作用及其与局部血管细胞和浸润免疫细胞的相互作用知之甚少。我的实验室已经采取了许多方法来了解这些细胞如何促进整体血管损伤反应，以及它们最终如何被治疗操纵。 我们已经使用了各种小鼠模型，以更好地了解是什么调节了损伤后局部和循环细胞的相互作用。我们最初的方法之一是研究p21 Cip 1表达是否调节局部血管细胞和循环免疫细胞之间的相互作用。我们发现，与p21+/+小鼠相比，p21-/-小鼠股动脉的机械损伤加速了血管平滑肌细胞增殖，并增加了循环免疫细胞的浸润。p21-/-和p21+/+小鼠之间的骨髓移植表明p21 Cip 1表达在常驻血管细胞中的重要作用。在血管创伤修复过程中，p21 Cip 1的缺失增加了血管中的细胞凋亡和SDF-1水平。类似地，SDF-1受体CXCR 4的拮抗作用在体内减少了p21-/-小鼠中单核细胞的浸润和新生内膜的形成。 我们还开展了一些研究，以确定参与血管重塑的祖细胞的起源和命运。我们正在使用基于Cre-Lox的谱系跟踪系统来遗传跟踪体内血管平滑肌和内皮细胞的命运。我们还利用Tie 2Cre/R26 RLacZ细胞命运定位系统在小鼠动脉-静脉移植模型中研究内皮源性细胞在新生内膜形成中的作用。此外，由于长期标记保留细胞（LRC）的方法已经成功地识别干细胞群体内的小生境，我们已经采用这种方法的血管壁。初步结果表明，一小群LRC存在于血管附近。我们相信这些细胞可能代表常驻体干/祖细胞，因此，我们目前正在确定这些新发现的和潜在的独特细胞的表面标记表型。 我们还探索了胚胎干细胞（ESCs）作为研究血管细胞和心肌细胞分化的体外模型的实用性。根据内皮细胞分化的发展计划，我们已经成功地将小鼠胚胎干细胞分化为血管和心脏中胚层前体细胞及其后代。我们已经确定了独特的表面标志物，以进一步区分心脏和血管祖细胞，目前正在测试这些纯化的细胞类型在心血管疾病的小鼠模型。 局部和循环血管祖细胞的鉴定也为干/祖细胞用于心血管疾病的治疗应用提供了理论基础。多能成体祖细胞（MAPCs）是最早描述的从成年人、小鼠和大鼠的骨髓中纯化的成体多能祖细胞之一。我们从大鼠骨髓中分离MAPC衍生的祖细胞（MDPCs），并将其移植到大鼠后肢模型的缺血肌肉中。四周后，MDPC保留在缺血腿的血管周围区域。虽然MDPC没有分化成血管细胞，灌注增加通过旁分泌机制和局部调节炎症反应。 总之，我们正在使用广泛的方法，包括研究靶向小鼠遗传模型，开发细胞命运图谱和细胞谱系耗竭策略，以及分析小鼠胚胎干细胞（mESC），所有这些都是为了探索血管再生和重塑过程中发生的复杂细胞相互作用和调控途径。我们的主要目标是增加我们在血管生物学这一领域的知识，并最终将这种理解转化为临床环境。

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 have used a variety of mouse models to better understand what regulates the interplay of local and circulating cells following injury. One of our initial approaches was to investigate whether p21Cip1 expression modulates the interactions between local vascular cells and circulating immune cells. We found that mechanical injury of the femoral artery in p21-/- mice accelerated vascular smooth muscle cell proliferation and increased infiltration of circulating immune cells compared to p21+/+ mice. Bone marrow transplantations between p21-/- and p21+/+ mice indicated an important role of p21Cip1 expression in resident vascular cells. Deletion of p21Cip1 increased apoptosis and SDF-1 level in the vasculature during vascular wound repair. Similarly, antagonism of the SDF-1 receptor, CXCR4, in vivo decreased infiltration of monocytes and neointima formation in p21-/- mice. We have also embarked on a number of studies to determine the origin and fate of progenitor cells involved in vascular remodeling. We are using a Cre-Lox-based lineage tracking system to genetically track the fate of vascular smooth muscle and endothelial cells in vivo. We are also utilizing a Tie2Cre/R26RLacZ cell fate mapping system in a murine arterial-vein graft model to investigate the role of endothelial-derived cells in neointima formation. In addition, because long-term label-retaining cell (LRC) approaches have been previously successful in identifying stem cell populations within their niche, we have employed this approach to the vessel wall. Preliminary results suggest that a small population of LRCs exist in close proximity to blood vessels. We believe these cells may represent resident somatic stem/progenitor cells and, as such, we are currently determining the surface marker phenotypes of these newly discovered and potentially unique cells. We have also explored the utility of embryonic stem cells (ESCs) as an in vitro model to study the differentiation of vascular cells and cardiomyocytes. Following developmental programs for endothelial cell differentiation, we have successfully differentiated murine ESCs into hematovascular and cardio-mesodermal precursor cells and their progenies. We have identified unique surface markers to further distinguish the cardiac and vascular progenitor cells, and are currently testing these purified cell types in murine models of cardiovascular disease. The identification of local and circulating vascular progenitor cells also provides the rationale for therapeutic application of stem/progenitor cells for cardiovascular disease. Multipotent adult progenitor cells (MAPCs) were one of the first described adult pluripotent progenitor cells purified from the bone marrow of adult humans, mice and rats. We isolated MAPC-derived progenitor cells (MDPCs) from rat bone marrow and transplanted them into the ischemic muscle of a rat hind limb model. After four weeks, MDPCs were retained in the perivascular area of the ischemic leg. Although MDPCs did not differentiate into vascular cells, perfusion was increased through the paracrine mechanism and by local modulation of the inflammatory response. In summary, we are using a wide range of approaches including studying targeted mouse genetic models, developing cell fate mapping and cell lineage depletion strategies, and analyzing murine embryonic stem cells (mESCs), all in an effort to explore the complex cellular interactions and regulatory pathways that occur during vascular regeneration and remodeling. Our main goal is to increase our knowledge in this area of vascular biology and eventually translate this understanding to the clinical setting.