Mechanisms of Vascular Neotissue Formation In Tissue Engineered Vascular Grafts

描述(申请人提供)：这项建议的首要目标是研究组织工程血管移植物(TEVG)中血管新生组织形成的细胞和分子机制，特别强调导致植入后TEVG狭窄的机制。我们设计并开发了一种专门用于先天性心脏病手术的TEVG。我们的TEVG是通过将自体骨髓来源的单个核细胞(BM-MNC)种植到可生物降解的管状支架上，并在植入作为血管管道之前在自体血清中短暂孵育来创建的。我们已经进行了第一次临床试验，评估了我们的TEVG作为连接下腔静脉和肺动脉的管道在需要改良Fontan手术的儿童中的使用。这项初步研究表明，我们的方法既安全又有效。本研究还表明，狭窄是移植物失败的主要模式。改良的“第二代”血管移植物的合理设计将基于我们对TEVG狭窄机制的理解。这项研究是基于我们使用小鼠模型获得的初步数据，该模型忠实地概括了人类血管新生组织的形成和TEVG狭窄的发展，如我们的人类临床试验所展示的那样。我们假设BM-MNC种植在可生物降解的管状支架上，作为血管植入移植物产生单核细胞趋化蛋白-1(MCP-1)，它将循环中的单核细胞招募到植入的TEVG中。单核细胞分化为巨噬细胞，并渗透到移植物中。随后，这些巨噬细胞通过血小板衍生生长因子(PDGF)依赖机制从邻近的血管壁招募平滑肌细胞，并通过血管内皮生长因子(VEGF)依赖机制招募内皮细胞。这些过程中的改变可用于调节新生组织的形成，并促进或抑制形成狭窄。我们将使用转基因小鼠模型来验证这些假设，以研究以下特定目的：目的1：确定循环中的单核细胞是否是MCP-1/CCR2信号的细胞靶点，以及由此产生的巨噬细胞在TEVG中的巨噬细胞浸润是否对血管新组织的形成和TEVG狭窄的发展至关重要。目的2：确定形成TEVG中层的平滑肌细胞的来源；确定巨噬细胞产生PDGF-B在这一过程中的作用；以及巨噬细胞产生PDGF的调节是否会影响TEVG狭窄的形成。目的3：确定形成新生血管内膜的细胞的来源和特性；确定巨噬细胞产生的血管内皮生长因子-A在这一过程中的作用；以及确定巨噬细胞产生的血管内皮生长因子-A的调节是否会影响TEVG狭窄。 公共卫生相关性：先天性心脏畸形是最常见的出生缺陷，也是新生儿期死亡的主要原因。先天性心脏畸形最有效的治疗方法是重建手术。不幸的是，使用目前可用的血管导管引起的并发症是术后发病率和死亡率的重要原因。组织工程化血管移植物的开发，由个人自身的细胞创造，具有生长、修复和重塑的能力，对于推动先天性心脏手术领域的发展和改善需要手术干预的婴儿的预后具有巨大的前景。

DESCRIPTION (provided by applicant): The overriding aim of this proposal is to investigate the cellular and molecular mechanisms of vascular neotissue formation in tissue engineered vascular grafts (TEVG), with special emphasis placed on the mechanisms contributing to post-implantation TEVG stenosis. We have designed and developed a TEVG specifically for use in congenital heart surgery. Our TEVG is created by seeding autologous bone marrow derived-mononuclear cells (BM-MNC) onto a biodegradable tubular scaffold and briefly incubating the seeded scaffolds in autologous serum prior to implantation as a vascular conduit. We have performed the first clinical trial evaluating the use of our TEVG as a conduit connecting the inferior vena cava to the pulmonary artery in children requiring modified Fontan surgery. This pilot study demonstrated that our method is both safe and effective. This study also demonstrated that stenosis is the primary mode of graft failure. The rational design of improved "second-generation" vascular grafts will be predicated on our understanding of the mechanisms underlying TEVG stenosis. This research is based upon our preliminary data obtained using a murine model that faithfully recapitulates human vascular neotissue formation and the development of TEVG stenosis as exhibited in our human clinical trial. We hypothesize that BM-MNC seeded onto a tubular biodegradable scaffold and implanted as a vascular interposition graft produce monocyte chemoattractant protein-1 (MCP-1), which recruits circulating monocytes to the implanted TEVG. The monocytes differentiate into macrophages and infiltrate the graft. Subsequently, these macrophages recruit smooth muscle cells from the adjacent vessel wall through a platelet derived growth factor (PDGF)-dependent mechanism and endothelial cells via a vascular endothelial growth factor (VEGF)-dependent mechanism. Alterations in these processes can be used to modulate neotissue formation and to either promote or inhibit the formation stenosis. We will use transgenic mouse models to test these hypotheses in order to investigate the following specific aims: Aim 1: Determine if circulating monocytes are the cellular targets of MCP-1/CCR2 signaling and if the resulting macrophage infiltrate in TEVG is critical to the formation of vascular neotissue and development of TEVG stenosis. Aim 2: Determine the source of the smooth muscle cells that form the medial layer of the TEVG; determine the role of macrophage production of PDGF-B in mediating this process; and determine if modulation of macrophage production of PDGF will affect the formation of TEVG stenosis. Aim 3: Determine the source and identity of the cells that form the intimal layer of the neovessel; determine the role of macrophage production of VEGF-A in mediating this process; and determine if modulation of macrophage production of VEGF-A will affect TEVG stenosis. PUBLIC HEALTH RELEVANCE: Congenital cardiac anomalies are the most common birth defect and a leading cause of death in the newborn period. The most effective treatment for congenital cardiac anomalies is reconstructive surgery. Unfortunately, complications arising from the use of currently available vascular conduits are a significant cause of postoperative morbidity and mortality. The development of a tissue engineered vascular graft, created from an individual's own cells, with the ability to grow, repair, and remodel, holds great promise for advancing the field of congenital heart surgery and improving the outcomes of infants requiring surgical intervention.