Molecular Mechanisms of Impaired Angiogenesis in Aging Gastric Mucosa

老化胃粘膜血管生成受损的分子机制

• 批准号: 8259072
• 负责人: ANDRZEJ S TARNAWSKI
• 金额: --
• 依托单位: VETERANS HEALTH ADMINISTRATION
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8259072
• 关键词: 5' -AMP-activated protein kinase; Acetylation; Acute; Age Reporting; Age-Years; Aging; Albumins; American; Angiogenesis Inhibition; Angiogenesis Inhibitors; Angiogenic Factor; Angiostatins; Antibodies; Apoptosis; Area; Arthritis; Asses; Binding; Biochemical; Blood Vessels; Blood capillaries; CREB1 gene; Cell Nucleus; Cell physiology; Cells; Characteristics; Chronic; Cicatrix; Clinical; Complementary DNA; Connective Tissue; DNA Binding; Dermal; Down-Regulation; Duodenal Ulcer; Economics; Endostatins; Endothelial Cells; Enzymes; Epithelial; Equilibrium; Ethanol; Exhibits; Extravasation; Fluorescein; Funding; Gastric mucosa; Gastric ulcer; Gastritis; Gelatinase B; Gene Activation; Gene Expression; Genes; Genetic Transcription; Goals; Granulation Tissue; HIF1A gene; Healed; Health; Heat shock proteins; Heat-Shock Proteins 90; Human; Hypoxia; Hypoxia Inducible Factor; Image; Imagery; Impaired wound healing; Impairment; Importins; In Vitro; Incidence; Indomethacin; Infarction; Injury; Investigation; Literature; Mediating; Molecular; Morbidity - disease rate; Myocardium; Natural regeneration; Non-Steroidal Anti-Inflammatory Agents; Nuclear; Nutrient; Organ; Oxygen; Pain; Patients; Peptic Ulcer; Pericytes; Permeability; Phosphorylation; Physiological; Plasmids; Population; Predisposition; Prevalence; Properdin; Proteins; Publications; Rattus; Relative (related person); Research; Research Design; Resolution; Role; STAT3 gene; Serum; Site; Small Interfering RNA; Stimulus; Stomach; Structure; Techniques; Technology; Testing; Therapeutic Intervention; Time; Tissues; Transcriptional Activation; Ulcer; Up-Regulation; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; Vascular Permeabilities; Veterans; Work; Wound Healing; age related; alcohol response; alpha Karyopherins; angiogenesis; annexin A5; autocrine; base; capillary; density; gene therapy; healing; high risk; hypoxia inducible factor 1; improved; in vivo; indexing; injured; instrument; mortality; mouse model; mutant; neutralizing antibody; novel; novel therapeutic intervention; novel therapeutics; overexpression; patient population; promoter; protein expression; protein kinase P; receptor; response; response to injury; stem; transcription factor; treatment strategy

DESCRIPTION (provided by applicant): ABSTRACT Background/Rationale: Gastric mucosa of aging humans and rats (aging gastric mucosa) exhibits increased susceptibility to injury and delayed healing. Healing of gastric injury requires re-establishment of mucosal blood vessels through the formation of new vessels (angiogenesis). Previous studies showed that healing of gastric injury in aging rats is delayed and impaired vs. young rats; but the underlying mechanisms are not explained, and angiogenesis in aging gastric mucosa has not been examined before. Our long-term objectives are: 1) to identify the molecular mechanisms of impaired angiogenesis in aging gastric mucosa and based on these findings 2) to develop new therapeutic strategies for treatment and reversing aging- related impaired angiogenesis and delayed healing. Our overall hypothesis is that an imbalance between angiogenic and anti-angiogenic factors suppresses angiogenesis and inhibits healing of injury in aging gastric mucosa. In this project, we will examine the mechanisms responsible for this imbalance and its consequences, ultimately demonstrating that manipulating this imbalance can improve angiogenesis and healing. We hypothesize that the mechanisms underlying impaired angiogenesis in aging gastric mucosa are: 1) Aging-related alterations in gastric microvascular endothelial cell (EC) function - reduced transcriptional activation of the VEGF gene due to: A) downregulation of importin and B) downregulation of the transcriptional factors P-CREB and P-STAT3, 2) Angiogenic imbalance due to reduced pro-angiogenic VEGF and concomitant increased anti-angiogenic endostatin in aging gastric mucosa leads to inhibition of angiogenesis, and 3) Therapeutic interventions aimed at the mechanisms: A) local VEGF gene therapy, B) upregulation and activation of importin with AICAR treatment, or C) specific neutralizing antibody against endostatin will significantly reverse impaired angiogenesis, and will improve microvascular regeneration and healing of chronic gastric ulcers in aging rats. The rationale for these hypotheses stems from our preliminary work that demonstrated in gastric mucosa of aging (vs. young) rats significantly decreased VEGF, increased endostatin, reduced angiogenesis in response to injury, and distorted and impaired mucosal healing. Furthermore, we showed that EC isolated from the gastric mucosa of aging rats exhibit reduced VEGF gene activation, impaired in vitro angiogenesis and reduced angiogenic response to hypoxia. We also demonstrated that importin is an essential requirement for VEGF gene activation and for angiogenesis since its silencing with specific siRNA significantly reduced angiogenesis in young EC. Conversely, activation of importin by treatment with AICAR significantly reversed impaired angiogenesis in aging EC. Research Design: We will examine the above hypotheses in EC isolated from gastric mucosa of young and aging rats and in vivo in gastric mucosa of aging vs. young rats following injury and ulceration. In vitro studies will include analysis of: expression of proteins involved in angiogenesis - VEGF, VEGFR2, P-VEGFR2 as well as HSP90, HIF1, HIF1, importin , AMPK, P-CREB and P-STAT3; their interactions and binding of HIF1, P-CREB and P- STAT3 to the VEGF gene. For in vivo studies, we will examine in gastric mucosa of rats, injury (erosion and ulcer) and will quantitatively asses angiogenesis during gastric ulcer healing, and determine expression of endostatin, MMP9 & others listed proteins. Significance: In 2006, ~39 million Americans (12% of population) were 65 years of age or older, and this number is projected reach ~70 million by 2030. This population is at high risk of tissue and organ injuries. Impaired angiogenesis is a key factor in delayed healing, morbidity and mortality. The central topic of our project is angiogenesis, a critical requirement for "wound healing", which is one of the VA priority areas. PUBLIC HEALTH RELEVANCE: Relevance to Veterans Health. In 2006 nearly 39 million Americans (12% of population) were 65 years of age or older, and this number is projected to grow to about 70 million by 2030. The veteran patient population is aging and patients 70 years or older constitute a significant proportion. This population is at high risk of tissue and organ injuries. Impaired angiogenesis is a key factor in delayed tissue injury healing, morbidity, and mortality. Impaired healing of wounds, both internal, such as gastric and duodenal ulcers or external, such as non-healing dermal ulcers constitute important clinical and economic problems. Gastric injury - gastritis and ulcers are frequently encountered in VA patients often as a result of chronic treatment NSAIDs for arthritis or pain. Lifetime prevalence of gastroduodenal ulcers in the US population is 3-6% but their incidence among the VA population is 2.5-fold higher. The central topic of our proposed investigations is directly related to angiogenesis that is a critical requirement for "wound healing", which is one of the VA priority areas.

描述(由申请人提供)： 摘要背景/原理：人和大鼠衰老的胃粘膜(衰老的胃粘膜)表现出对损伤的敏感性增加和愈合延迟。胃损伤的修复需要通过形成新的血管来重建粘膜血管(血管生成)。先前的研究表明，与青年大鼠相比，老龄大鼠胃损伤的愈合延迟和受损；但其潜在的机制尚不清楚，而且衰老胃粘膜中的血管生成也没有被研究过。我们的长期目标是：1)确定衰老胃粘膜血管生成受损的分子机制；2)开发新的治疗策略，以治疗和逆转衰老相关的血管生成受损和愈合延迟。我们的总体假设是，血管生成因子和抗血管生成因子之间的失衡抑制了血管生成，抑制了老化胃粘膜损伤的愈合。在这个项目中，我们将研究导致这种失衡的机制及其后果，最终证明操纵这种失衡可以促进血管生成和愈合。我们推测，衰老胃粘膜血管生成障碍的机制是：1)与衰老相关的胃微血管内皮细胞功能改变--血管内皮生长因子基因转录活性降低，其原因是：A)Importin下调，B)转录因子P-CREB和P-STAT3下调，2)促血管生成血管生成因子减少和老年胃粘膜中抗血管生成内皮抑素的增加导致血管生成失衡，3)针对以下机制的治疗干预：A)局部血管生成基因治疗，B)Importin的上调和激活与AICAR治疗，或C)针对内皮抑素的特异性中和抗体将显著逆转血管生成和血管生成。并能促进衰老大鼠慢性胃溃疡的微血管再生和愈合。这些假说的基本原理源于我们的初步工作，即在老年(与年轻相比)大鼠的胃粘膜中，血管内皮生长因子显著降低，内皮抑素增加，损伤后血管生成减少，并扭曲和损害粘膜愈合。此外，我们还发现，从衰老大鼠胃粘膜分离的EC表现出血管内皮生长因子基因活性降低，体外血管生成受损，对低氧的血管生成反应降低。我们还证明了Importin是血管生成所必需的，因为它与特定的siRNA一起沉默，显著减少了年轻EC的血管生成。相反，用AICAR处理importin的激活可显著逆转衰老EC中受损的血管生成。研究设计：我们将从幼年和老年大鼠的胃粘膜中分离出EC，并在损伤和溃疡后的老龄大鼠和幼年大鼠的胃粘膜中检验上述假说。体外研究将包括：与血管生成有关的蛋白质--血管内皮生长因子、血管内皮生长因子2、P-血管内皮生长因子R2以及HSP90、HIF1、HIF1、Importin、AMPK、P-CREB和P-STAT3的表达；它们之间的相互作用以及HIF1、P-CREB和P-STAT3与血管生长因子基因的结合。对于活体研究，我们将检测大鼠胃粘膜损伤(糜烂和溃疡)，并将定量评估胃溃疡愈合过程中的血管生成，并测定内皮抑素、MMP9等列出的蛋白的表达。意义：2006年，约有3900万美国人(占总人口的12%)年龄在65岁或以上，预计到2030年，这一数字将达到约7000万。这一人群组织和器官损伤的风险很高。血管生成障碍是延迟愈合、发病率和死亡率的关键因素。我们项目的中心主题是血管生成，这是“伤口愈合”的关键要求，也是退伍军人管理局的优先领域之一。 公共卫生相关性： 与退伍军人健康的相关性。2006年，近3900万美国人(占总人口的12%)年龄在65岁或以上，预计到2030年，这一数字将增长到约7000万。退伍军人患者群体正在老龄化，70岁及以上的患者占相当大的比例。这一人群组织和器官损伤的风险很高。血管生成障碍是导致组织损伤延迟愈合、发病率和死亡率的关键因素。创面愈合受损，无论是内部创面，如胃和十二指肠溃疡，还是外部创面，如无法愈合的真皮溃疡，都构成了重要的临床和经济问题。胃损伤--VA患者经常会出现胃炎和溃疡，通常是由于慢性治疗关节炎或疼痛的非类固醇抗炎药造成的。胃十二指肠溃疡在美国人口中的终生患病率为3-6%，但在退伍军人中的发病率要高出2.5倍。我们提出的研究的中心话题是与血管生成直接相关的，血管生成是创伤愈合的关键要求，而创伤愈合是退伍军人管理局优先考虑的领域之一。