Optimizing Therapeutic Revascularization by Endothelial Cell Transplantation

 描述(由申请人提供)：器官移植是治疗器官衰竭的最有效的方法，目前严重缺乏可用的供体器官。组织工程学是一种很有前途的解决器官短缺的方法。构建复杂替代器官的障碍是在组织工程移植物中创建一个功能良好的微血管系统，以提供足够的血流。人内皮细胞(ECs)植入免疫缺陷小鼠体内可自组装成微血管管道，但要进化成一个功能齐全的微血管系统，需要血管重塑和壁细胞，特别是周细胞(PC)的募集，通过形成内皮细胞间紧密连接(TJ)来控制细胞旁的泄漏，以及器官不同部分之间的适当吻合。为了使器官衰竭患者受益，移植很可能必须提前从同种异体细胞构建。但人体内皮细胞是移植物灌流所必需的，会引发同种异体移植物排斥反应。我们将测试几种解决这些悬而未决问题的策略，使用改变移植微环境或通过基因工程改变内皮细胞的创新方法，利用我们开发的用于修饰来自脐血祖细胞(HCBECs)的内皮细胞的CRISPR/Cas9方法。我们将使用体外和体内微血管形成模型来测试我们的方法，微血管形成的模型可以是自组装的单个EC悬浮液，也可以是EC球体的萌发。在目标1a中，我们将首先使用Bcl2转导来优化EC管的血管复杂性和PC投资。我们还将通过改变RAS信号来增加EC管的形成，或通过增加EC产生PDGF-BB来增加PC投资，来研究从藻酸盐微粒中传递VEGF-A或通过改变RAS信号来增敏HCBECs对VEGF-A的影响。在特定的目标1b中，我们将确定是否可以通过持续释放Tie2激活剂或通过cAMP诱导剂靶向cAMP/Epac1/Rap1/Rac-1屏障增强途径来增加HCBECs的TJ形成。或者，我们也可以通过修饰HCBEC来增强其对Tie-2激动剂的敏感性或模拟对cAMP的反应。在目标1c中，我们将通过控制VEGF-A、信号素3a和信号素3c信号的平衡，或通过改变神经毛细管内皮细胞对这些药物的反应来优化人脐静脉内皮细胞来源的微血管对联合移植的大鼠肾小球的灌流。在目标2a中，我们将改变凝胶微环境，通过持续释放免疫抑制药物雷帕霉素或免疫抑制细胞因子IL-10，或通过共植入生理上在血管壁上创建免疫豁免部位的被包裹的平滑肌细胞来创建免疫豁免区域。在目标2b中，我们将通过基因改变HCBEC来移除T细胞激活所必需的信号，即MHC分子和CD58，或过度表达抑制信号，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.