Optimizing Therapeutic Revascularization by Endothelial Cell Transplantation

通过内皮细胞移植优化治疗性血运重建

基本信息

批准号：
9759975
负责人：
JORDAN S POBER
金额：
$ 40.18万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-08-15 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9759975
关键词：
ANGPT1 gene Address Agonist Alginates Allogenic Anastomosis - action Astrocytes BCL2 gene Blood Vessels Blood capillaries CD58 gene CRISPR/Cas technology Cell Line Cell Transplantation Cell Transplants Cells Clone Cells Complex Cyclic AMP Development Encapsulated Endothelial Cells Endothelium Engineering Engraftment Environment Equilibrium Evolution Funding Gel Generations Genetic Engineering Goals Graft Rejection Homologous Transplantation Human Human Engineering Immune Immunodeficient Mouse Immunologics Immunosuppressive Agents Implant In Vitro Interleukin-10 Investigation Investments Kidney Liquid substance Longevity Mediating Methods Modeling Modification Neuropilins Organ Organ Donor Organ Transplantation Organ failure Outcome PDCD1LG1 gene Paracrine Communication Pathway interactions Patients Performance Perfusion Pericytes Pharmaceutical Preparations Physiological Polymers Process Production Proteins Rattus Reproducibility Secretory Cell Semaphorin-3 Semaphorin-3A Semaphorins Signal Transduction Sirolimus Site Smooth Muscle Myocytes Stem cells Structure Suspensions System T cell response T-Cell Activation T-Lymphocyte Technology Testing Therapeutic Tight Junctions Time Tissue Engineering Transplantation Tube Umbilical Cord Blood Umbilical vein Vascular Endothelial Growth Factors Vascular System Work allograft rejection base cytokine effective therapy end-stage organ failure extracellular genetic manipulation human tissue implantation improved in vivo in vivo Model innovation macromolecule nanoparticle overexpression platelet-derived growth factor BB public health relevance recruit response self assembly solute vascular bed

项目摘要

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.

描述（由申请人提供）：器官移植是治疗器官衰竭的最有效方法，目前由于严重缺乏可用的供体器官而受到限制。组织工程是解决器官短缺的一种有希望的解决方案，但正在为构建复杂的替代器官带来障碍。组织工程移植物内的功能性微血管系统可提供足够的灌注，当植入免疫缺陷小鼠体内时，人内皮细胞（EC）可以自组装成微血管导管，但进化为功能齐全的微血管系统涉及血管重塑。壁细胞，特别是周细胞（PC）的募集，通过内皮间紧密连接（TJ）的形成控制细胞旁渗漏，以及器官不同部分之间的适当吻合为了使器官衰竭的患者受益，移植物可能必须这样做。但移植物灌注所需的人类 ECs 会引发同种异体移植排斥反应，我们将使用改变移植物的创新方法来测试几种解决这些未解决问题的策略。微环境或通过基因工程改变 EC，利用我们开发的对脐带血祖细胞 (HCBEC) 衍生的 EC 进行 CRISPR/Cas9 修饰的方法，我们将使用微血管形成的体外和体内模型来测试我们的方法。自组装单个 EC 悬浮液或通过 EC 球体发芽在目标 1a 中，我们将优化 EC 管的容器复杂性和 PC 投资，最初使用 Bcl-2。我们还将检查藻酸盐微粒递送 VEGF-A 的效果，或通过改变 Ras 信号传导以增加 EC 管形成，或通过增强 EC 产生 PDGF-BB 来增加 PC 投资，从而使 HCBEC 对 VEGF-A 敏感。，我们将确定是否可以通过持续释放 Tie2 激活药物或通过 cAMP 诱导剂来增加 HCBEC 的 TJ 形成，以靶向cAMP/Epac1/Rap1/Rac-1 屏障强化途径或者，我们将修改 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，我们将优化共移植大鼠肾小球的灌注。通过人 HCBEC 衍生的微血管通过操纵 VEGF-A、信号蛋白 3a 和信号蛋白 3c 信号的平衡，或通过通过改变神经毡蛋白表达来改变 HCBEC 对这些药物的反应在目标 2a 中，我们将通过持续释放雷帕霉素（一种免疫抑制药物）或 IL-10（一种免疫抑制药物）来改变凝胶微环境，以创建免疫豁免区。在目标 2b 中，我们将通过细胞因子或共同植入封装的平滑肌细胞，在生理学上在血管壁中创建免疫特权位点。基因改变 HCBEC 以消除 T 细胞激活所需的信号，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广泛适用于组织工程的方法。

项目成果

期刊论文数量（9）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Pericytes modulate endothelial sprouting.

周细胞调节内皮出芽。

DOI：
10.1093/cvr/cvt215
发表时间：
2013-12-01
期刊：
Cardiovascular research
影响因子：
10.8
作者：
William G. Chang;J. W. Andrejecsk;Martin S Kluger;W. Saltzman;J. Pober
通讯作者：
J. Pober

In vitro self-assembly of human pericyte-supported endothelial microvessels in three-dimensional coculture: a simple model for interrogating endothelial-pericyte interactions.

三维共培养中人周细胞支持的内皮微血管的体外自组装：用于询问内皮-周细胞相互作用的简单模型。

DOI：
发表时间：
2013
期刊：
影响因子：
1.7
作者：
Waters, J P;Kluger, M S;Graham, M;Chang, W G;Bradley, J R;Pober, J S
通讯作者：
Pober, J S

Engineering of multifunctional gels integrating highly efficient growth factor delivery with endothelial cell transplantation.

将高效生长因子递送与内皮细胞移植相结合的多功能凝胶工程。

DOI：
发表时间：
2008-08
期刊：
影响因子：
0
作者：
Jay, Steven M;Shepherd, Benjamin R;Bertram, James P;Pober, Jordan S;Saltzman, W Mark
通讯作者：
Saltzman, W Mark

Differential functional roles of fibroblasts and pericytes in the formation of tissue-engineered microvascular networks in vitro.

成纤维细胞和周细胞在体外组织工程微血管网络形成中的不同功能作用。

DOI：
发表时间：
2020-01-06
期刊：
影响因子：
0
作者：
Kosyakova, Natalia;Kao, Derek D;Figetakis, Maria;López;Spindler, Susann;Graham, Morven;James, Kevin J;Won Shin, Jee;Liu, Xinran;Tietjen, Gregory T;Pober, Jordan S;Chang, William G
通讯作者：
Chang, William G

Controlled protein delivery in the generation of microvascular networks.

微血管网络生成中的受控蛋白质递送。