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: 7472456
• 负责人: STEPHEN E EPSTEIN
• 金额: $ 32.37万
• 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 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7472456
• 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(申请人提供)：侧支发育可以补偿动脉粥样硬化性阻塞性动脉疾病，但自然机制很少能恢复最大血流能力。使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方式协调表达。 骨髓来源的细胞(BMC)表达大量与血管生成相关的细胞因子。这导致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)新鲜吸入的自体BMCs在猪心肌缺血模型中表达多种血管生成因子，增加血流量和功能；2)培养的BM来源的基质细胞表达多种血管生成因子，当移植到缺血的小鼠后肢时，侧支血流量增加。然而，混杂因素可能会损害MSCs增强抵押品的能力。冠心病患者的骨髓细胞在分泌与血管生成相关的细胞因子方面表现出明显的变异性。此外，衰老会损害侧支循环的发育，来自老年小鼠的MSCs(“老年”MSCs)分泌血管生成细胞因子的能力降低，侧支循环增加。 HIF-1反式激活多个参与细胞缺氧反应的基因，包括血管生成。我们发现，与年轻的MSCs相比，年长的MSCs表现出HIF1a水平降低，并分泌更少的缺氧诱导的血管生成细胞因子。因此，诱导MSCs过度表达HIF-1α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的候选对象都是年龄较大的患者。我们的初步数据提供了支持证据：1)在幼鼠缺血后肢的侧支循环是&GT；注射高表达HIF-1a的MSCs与未转导的MSCs；2)转导来自CAD患者的MSCs可将这些低表达VEGF的MSCs转化为高表达VEGF的细胞。 我们的主要假设是：注射MSCs可以增加老年小鼠发育侧支的能力，但可以通过对MSCs进行基因工程，使其过度表达一种构成上具有活性的HIF-1α形式，来优化-接近年轻小鼠实现的水平。我们将确定：目的1)老年小鼠和缺血组织的基因表达谱是否与年轻小鼠不同，以及HIF1α转导的MSCs是否使各自的表达谱向年轻小鼠改变；目的2)注射经过基因改变的MSCs后，老年小鼠血流和肢体功能的恢复，使其过度表达HIF-1α，使其与年轻小鼠相似；目的3)衰老显著改变侧支表型，HIF1α转导的MSCs向年轻小鼠表型转变(利用Micro-CT的高分辨率)。如果这一新策略得到验证，结果将在临床上开发改善侧支循环的个体化治疗方法方面具有巨大的价值，这一目标迄今已被证明是难以实现的。