Optimizing Therapeutic Revascularization by Endothelial Cell Transplantation

 描述（由申请人提供）：器官移植是器官衰竭的最有效疗法，目前受到严重缺乏可用供体器官的限制。组织工程是解决器官短缺的一个有前途的办法。构建复杂替代器官的障碍是在组织工程移植物内创建功能性微血管系统以提供足够的灌注。当植入免疫缺陷小鼠时，人内皮细胞（EC）可以自组装成微血管导管，但进化成全功能的微血管系统涉及血管重塑和壁细胞（特别是周细胞（PC））的募集，通过形成内皮间紧密连接（TJ）控制细胞旁渗漏，以及器官不同节段之间的适当微血管化。为了使器官衰竭的患者受益，移植物可能必须提前从同种异体细胞中构建。但是，移植物灌注所需的人类EC会引发同种异体移植物排斥反应。我们将测试几种策略来解决这些未解决的问题，使用改变移植物微环境或通过基因工程改变EC的创新方法，利用我们开发的一种方法对来自脐带血祖细胞（HCBEC）的EC进行CRISPR/Cas9修饰。我们将测试我们的方法，无论是通过自组装单EC悬浮液或从EC球体发芽微血管形成的体外和体内模型。在目标1a中，我们将优化EC管的血管复杂性和PC投资，最初使用Bcl-2转导。我们还将研究VEGF-A从藻酸盐微粒递送或通过改变Ras信号以增加EC管形成来使HCBECs对VEGF-A敏感的影响，或通过增强PDGF-BB的EC产生来增加PC投资。在具体目标1b中，我们将确定HCBEC的TJ形成是否可以通过持续释放Tie 2激活药物或通过cAMP诱导剂靶向屏障强化的cAMP/Epac 1/Rap 1/Rac-1途径来增加。或者，我们将修饰HCBEC以增强其对Tie 2激动剂的敏感性或模拟对cAMP的反应。在目标1c中，我们将通过操纵VEGF-A，semaphorin 3a和semaphorin 3c信号的平衡，或通过改变神经纤毛蛋白表达来改变HCBEC对这些药物的反应，来优化人HCBEC衍生微血管对共移植大鼠肾小球的灌注。在目标2a中，我们将改变凝胶微环境，以通过持续释放雷帕霉素（一种免疫抑制药物）或IL-10（一种免疫抑制细胞因子），或通过在血管壁中生理上产生免疫豁免位点的包囊平滑肌细胞的共植入来产生免疫豁免区。在目标2b中，我们将通过基因改变HCBEC来去除T细胞活化所需的信号，即MHC分子和CD 58，或过表达抑制信号，即PD-L1和PD-L2。这些研究的成功结果将确定可能广泛适用于组织工程的方法。

 DESCRIPTION (provided by applicant): Organ transplantation, the most effective therapy for organ failure, is currently limited by a severe lack of available donor organs. Tissue engineering is a promising solution to organ shortage. A barrier to construction of complex replacement organs is creating a functional microvascular system within the tissue engineered graft to provide adequate perfusion. Human endothelial cells (ECs) can self-assemble into microvascular conduits when implanted into immunodeficient mice, but evolution into a fully functional microvascular system involves vessel remodeling and recruitment of mural cells, particularly pericytes (PCs), control of paracellular leak through formation of inter-endothelial tight junctions (TJs), and appropriate anastomoses between different segments of the organ. To benefit patients with organ failure, grafts will likely have to be constructed in advance from allogeneic cells. But human ECs, which are required for graft perfusion, initiate allograft rejection. We will test several strategies to address these unsolved issues, using innovative approaches that alter the graft microenvironment or that alter the ECs through genetic engineering, exploiting a method we developed for CRISPR/Cas9 modification of ECs derived from umbilical cord blood progenitor cells (HCBECs). We will test our approaches using in vitro and in vivo models of microvessel formation either by self-assembly single EC suspensions or by sprouting from EC spheroids. In aim 1a, we will optimize vessel complexity and PC investment of EC tubes, initially using Bcl-2 transduction. We will also examine the effects of VEGF-A delivery from alginate microparticles or sensitizing HCBECs to VEGF-A by altering Ras signaling to increase EC tube formation, or increasing PC investment by enhancing EC production of PDGF-BB. In specific aim 1b, we will determine if TJ formation by HCBECs can be increased by sustained release of a Tie2 activating drug or by a cAMP-inducing agent to target the cAMP/Epac1/Rap1/Rac-1 pathways of barrier strengthening. Alternatively, we will modify HCBECs to enhance their sensitivity to Tie 2 agonists or to mimic the response to cAMP. In aim 1c, we will optimize perfusion of co-engrafted rat glomeruli by human HCBEC-derived microvessels by manipulating the balance of VEGF-A, semaphorin 3a and semaphorin 3c signals, or by altering the responses of HCBECs to these agents through changes in neuropilin expression. In aim 2a, we will alter the gel microenvironment to create a zone of immune-privilege through sustained release of rapamycin, an immunosuppressive drug, or of IL-10, an immunosuppressive cytokine, or by co-engraftment of encapsulated smooth muscle cells that physiologically create a site of immune-privilege in the vessel wall. In Aim 2b, we will geneticall alter HCBECs to remove signals necessary for T cell activation, namely MHC molecules and CD58, or overexpress inhibitory signals, namely PD-L1 and PD-L2. Successful outcomes of these investigations will identify approaches that may be broadly applicable to tissue engineering.