Collaterals in Aging: Enhanced by Genetic/Cell Therapy?

衰老过程中的抵押品：基因/细胞疗法增强？

• 批准号: 7282963
• 负责人: STEPHEN E EPSTEIN
• 金额: $ 44.09万
• 依托单位: MEDSTAR HEALTH RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-26 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7282963
• 关键词: Accounting; Adenovirus Vector; Affect; Age; Aging; Angiogenic Factor; Antibodies; Area; Arterial Occlusive Diseases; Aspirate substance; Autologous; Biological; Blood flow; Bone Marrow; Bone Marrow Cells; Bromodeoxyuridine; C10; Cell Hypoxia; Cell Therapy; Cells; Clinical Trials; Complex; Condition; Conditioned Culture Media; Count; Data; Development; Endothelial Cells; Engineering; Exhibits; Family suidae; Gene Expression; Gene Targeting; Genes; Genetic; Genetically Engineered Mouse; Goals; HIF1A gene; Hindlimb; Hypoxia; Image; In Vitro; Individual; Injection of therapeutic agent; Injury; Ischemia; Label; Lasers; Ligation; Limb structure; MSC gene; Measures; Microspheres; Modeling; Molecular; Molecular Profiling; Mus; Myocardial Ischemia; Myocardium; Numbers; PECAM1 gene; Patients; Pattern; Perfusion; Phenotype; Principal Investigator; Procedures; Process; Proliferating; Protein Overexpression; Proteins; Recombinants; Recovery; Resolution; Risk Factors; Signal Transduction; Smooth Muscle Myocytes; Stromal Cells; Techniques; Testing; Time; Tissues; Transfection; Transgenes; Uncertainty; Vascular Endothelial Growth Factors; Virus; age related; aged; alpha Actin; angiogenesis; base; concept; cytokine; day; femoral artery; functional improvement; improved; indexing; novel strategies; older patient; programs; research study; response; transcription factor

DESCRIPTION (provided by applicant): Collateral development compensates for atherosclerotic obstructive arterial disease, but natural mechanisms rarely restore maximal flow capacity. Clinical trials, employing single angiogenic cytokines, have failed to improve collateral flow. However, angiogenesis is complex, requiring multiple genes to be coordinately expressed in an appropriate time-dependent manner. Bone marrow-derived cells (BMCs) express numerous angiogenesis-related cytokines. This led us to test a cell-based approach to achieve optimal angiogenesis. We found 1) freshly aspirated autologous BMCs express multiple angiogenesis factors and increase flow and function in a porcine model of myocardial ischemia, 2) cultured BM-derived stromal cells (MSCs) express multiple angiogenesis factors, and when given to an ischemic mouse hindlimb increase collateral blood flow. However, confounding factors may compromise the capacity of MSCs to enhance collaterals. BMCs from CAD pts show marked variability in secreting angiogenesis-related cytokines. Moreover, aging impairs collateral development, and MSCs derived from old mice ("old" MSCs) have reduced capacity to secrete angiogenic cytokines and to increase collateral flow. HIF-1 transactivates multiple genes involved in the cell's hypoxia responses, including angiogenesis. We show that old MSCs, when compared to "young" MSCs, exhibit reduced HIF1a levels and secrete less hypoxia-induced angiogenic cytokines. Thus, inducing MSCs to overexpress HIF-1alpha seems a particularly appropriate angiogenic strategy, given that most candidates for angiogenic Rx are older pts. Our preliminary data supplies supportive evidence: 1) in young mice collateral flow in the ischemic hindlimb is > following injection of MSCs overexpressing HIF-1a vs. non-transduced MSCs; 2) HIF-1alpha transduction of MSCs derived from CAD patients converts these low VEGF-expressing MSCs to very high VEGF-expressing cells. Our major hypothesis is: The reduced capacity of old mice to develop collaterals can be increased by injecting MSCs, but can be optimized-approaching that achieved by young mice-by genetically engineering the MSCs so they overexpress a constitutively active form of HIF-1alpha. We will determine whether: Aim 1) The gene expression profiles of old MSCs and of ischemic tissue of old mice are different from those of young mice, and whether the expression profile of each is altered by HIF1alpha-transduced MSCs toward the young expression profile; Aim 2) The recovery of blood flow and limb function of aged mice, after injection of MSCs that have been genetically altered so they overexpress a constitutively active form of HIF-1alpha, improve so they are similar to that of young mice; Aim 3) Aging markedly alters collateral phenotype and HIF1alpha transduced MSCs alter phenotype toward the young mouse phenotype (using the high resolution of micro- CT). If this novel strategy is validated, the results will be of immense value in developing individualized therapies for improving collateral flow clinically, a goal that has to date proven elusive.

描述（由申请人提供）：侧支循环的发展弥补了动脉粥样硬化性阻塞性动脉疾病，但自然机制很少恢复最大流量。采用单一血管生成细胞因子的临床试验未能改善侧支循环。然而，血管生成是复杂的，需要多个基因以适当的时间依赖性方式协调表达。 骨髓源性细胞（BMCs）表达多种血管生成相关的细胞因子。这使我们测试了一种基于细胞的方法来实现最佳的血管生成。我们发现1）新鲜抽吸的自体BMCs表达多种血管生成因子，并增加猪心肌缺血模型中的血流和功能，2）培养的BM衍生的基质细胞（MSCs）表达多种血管生成因子，并且当给予缺血小鼠后肢时增加侧支血流量。然而，混杂因素可能会损害MSC增强侧支循环的能力。来自CAD患者的BMC在分泌血管生成相关细胞因子方面显示出显著的变异性。此外，衰老损害侧支发育，并且源自老年小鼠的MSC（“老年”MSC）具有降低的分泌血管生成细胞因子和增加侧支流动的能力。 HIF-1反式激活参与细胞缺氧反应的多个基因，包括血管生成。我们发现，与“年轻”MSC相比，老年MSC表现出降低的HIF 1a水平，并分泌较少的缺氧诱导的血管生成细胞因子。因此，诱导MSC过度表达HIF-1 α似乎是一种特别合适的血管生成策略，因为大多数血管生成Rx的候选者都是老年患者。我们的初步数据提供了支持性证据：1）在年轻小鼠中，注射过表达HIF-1 α的MSC与未转导的MSC相比，缺血后肢中的侧支血流>; 2）来自CAD患者的MSC的HIF-1 α转导将这些低VEGF表达的MSC转化为非常高VEGF表达的细胞。 我们的主要假设是：通过注射间充质干细胞可以提高老年小鼠发育侧枝的能力，但是可以通过遗传工程改造间充质干细胞使其过度表达组成型活性形式的HIF-1 α来优化接近年轻小鼠的水平。我们将确定：目的1）老年小鼠缺血组织基因表达谱和老年小鼠骨髓间充质干细胞基因表达谱与青年小鼠不同，HIF 1 α转导的骨髓间充质干细胞基因表达谱是否向青年表达谱方向改变;目的2）老年小鼠血流量和肢体功能的恢复，在注射经过基因改变的MSC后，它们过度表达HIF-1 α的组成型活性形式，改善，使它们与年轻小鼠相似;目的3）衰老显著改变侧支表型，HIF 1 α转导的MSC向年轻小鼠表型改变（使用高分辨率micro-CT）。如果这种新策略得到验证，其结果将对开发临床改善侧支循环的个性化疗法具有巨大价值，而这一目标迄今为止被证明是难以捉摸的。