CARP: Angiogenic Gene Therapy in Diabetic Wounds

CARP：糖尿病伤口的血管生成基因治疗

• 批准号: 7263448
• 负责人: Jeffrey M. Davidson
• 金额: $ 28.87万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7263448
• 关键词: Abbreviations; Adenovirus Protein; Affect; Affinity Chromatography; Angiogenic Proteins; Angiopoietin-1; Animal Model; Ankyrin Repeat; Apoptosis; Area; Binding; Blood Vessels; Blood capillaries; Cardiac; Cells; Defect; Deletion Mutation; Development; Diabetic mouse; Diabetic wound; Disease regression; Dose; Doxorubicin; EGF gene; EMSA; Endoribonucleases; Endothelial Cells; Epidermal Growth Factor; Epidermal Growth Factor Receptor; Exhibits; Fibroblast Growth Factor 2; Firefly Luciferases; Galactosidase; Gene Expression Profiling; Gene Targeting; Gene Transfer; Genes; Genetic Transcription; Glyceraldehyde; Green Fluorescent Proteins; Healed; Human; Hyperglycemia; Inflammatory; Internal Ribosome Entry Site; LacZ Genes; Lead; MAP Kinase Gene; MAPK14 gene; Mechanical Stress; Mediating; Mediator of activation protein; Methods; Mitogen-Activated Protein Kinases; Molecular Profiling; Morphology; Mothers; Mus; Mutation; Myocardium; Nuclear; Oxidative Stress; Oxidoreductase; Pancreatic ribonuclease; Pathway interactions; Phase; Phenotype; Phosphotransferases; Platelet-Derived Growth Factor; Polyvinyl Alcohol; Protein Activation Pathway; Protein Kinase; Protein Microchips; Protein Overexpression; Proteins; Purpose; RNA; Rattus; Reading; Research Personnel; Ribonucleases; Role; Series; Signal Pathway; Signal Transduction; Site; Small Interfering RNA; Small Nuclear Ribonucleoprotein Polypeptide F; Smooth Muscle Actin Staining Method; Smooth Muscle Myocytes; Tertiary Protein Structure; Testing; Transcription Coactivator; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Tumor Necrosis Factors; Vascular Endothelial Cell; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; Vascular blood supply; Vascular remodeling; Wound Healing; Yeasts; angiogenesis; biological adaptation to stress; capillary; cell motility; clinically relevant; connective tissue growth factor; diabetic; diabetic wound healing; extracellular; gain of function; gene delivery system; gene therapy; healing; human TNF protein; hybrid protein; improved; inorganic phosphate; neovascularization; novel; novel therapeutics; programs; protein expression; receptor; repaired; research study; transcription factor; vasculogenesis; wound; yeast two hybrid system

DESCRIPTION (provided by applicant): Gene expression profiling of murine wounds revealed marked, sustained induction of cardiac ankyrin repeat protein (CARP, ankrdl), with diminished expression in diabetic mice. Overexpression of CARP in wounds by adenoviral gene transfer produces a remarkable increase in neovascularization and thereby enhances wound healing. Under the previous aims, we established these effects in several animal models and showed that CARP induction occurs in microvascular endothelial cells (MVEC), in part, through TGF-IS mediated activation of a p38 kinase pathway. CARP also protects endothelial cells from doxorubicin-mediated apoptosis, and it enhances MVEC migration, not proliferation. To avoid overlap, the proposed studies will concentrate on gain-of-function aspects of CARP. The proposed studies will extend the original aims by delineating the CARP activation pathway as well as identifying its binding partners and direct transcriptional targets. We will test the hypothesis that hyperglycemic, oxidative stress is related to CARP induction. Experiments will determine the influence of TNF-a and possible convergent signal mechanisms. Binding partners in vascular cells will be identified by yeast two- hybrid, protein microarray, and tandem affinity purification methods. EMSA and ChIP analysis will be used to identify the transcriptional activators in MVEC and smooth muscle cells (SMC). Expression profiling will be expanded to identify key targets in both MVEC and SMC. CARP has been suggested to be a mediator of mechanical stress in cardiac muscle. Experiments will test the significance of CARP'S putative nuclear and cytoplasmic interaction sites in SMC and MVEC by deletion and mutation of discrete, functional domains of the protein, read out as migratory and stress responses of targeted cells. Partners for these sites will be identified. The clinical relevance of CARP will be investigated in a series of rat healing studies in diabetic and ischemic wounds with a novel gene delivery system. These experiments will define the optimal dose for repair, and investigate the role of CARP in vessel regression and stabilization. If the benefits and mechanisms of this form of wound therapy are validated, they could lead to the development of CARP as a novel therapeutic and to the discovery of factors that mediate CARP-enhanced neovascularization.

描述（由申请人提供）：鼠伤口的基因表达分析显示出明显的，持续诱导心动脉蛋白重复蛋白（CARP，ANKRDL），在糖尿病小鼠中表达降低。腺病毒基因转移对伤口中鲤鱼的过表达可显着增加，从而增强伤口愈合。在先前的目标下，我们在几种动物模型中确定了这些作用，并表明鲤鱼诱导发生在微血管内皮细胞（MVEC）中，部分通过TGF-IS介导的p38激酶途径的激活。鲤鱼还保护内皮细胞免受阿霉素介导的细胞凋亡的影响，并增强了MVEC迁移，而不是增殖。为了避免重叠，拟议的研究将集中于鲤鱼的功能收益方面。拟议的研究将通过描述鲤鱼激活途径以及确定其结合伙伴和直接转录目标来扩展原始目标。我们将检验以下假设：高血糖，氧化应激与鲤鱼诱导有关。实验将确定TNF-A和可能的收敛信号机制的影响。血管细胞中的结合伴侣将通过酵母两杂交，蛋白质微阵列和串联亲和纯化方法来鉴定。 EMSA和CHIP分析将用于识别MVEC和平滑肌细胞（SMC）中的转录激活剂。表达分析将扩展，以识别MVEC和SMC中的关键目标。鲤鱼被认为是心脏肌肉机械胁迫的介体。实验将通过删除和突变蛋白质的功能域的缺失和突变来测试鲤鱼推定的核和细胞质相互作用位点的重要性，并将其读取为靶向细胞的迁移和应激反应。这些网站的合作伙伴将被确定。通过新型的基因递送系统，将在一系列的糖尿病和缺血性伤口的大鼠愈合研究中研究鲤鱼的临床相关性。这些实验将定义用于修复的最佳剂量，并研究鲤鱼在血管回归和稳定中的作用。如果对这种形式的伤口疗法的好处和机制进行了验证，则可以导致鲤鱼作为一种新颖的治疗性发展，并发现介导鲤鱼增强新生血管形成的因素。