Molecular Mechanisms of Impaired Angiogenesis in Aging Gastric Mucosa

描述（由申请人提供）： 摘要背景/依据：老年人和大鼠的胃粘膜（老年胃粘膜）表现出对损伤和延迟愈合的敏感性增加。胃损伤的愈合需要通过形成新血管（血管生成）重建粘膜血管。先前的研究表明，与年轻大鼠相比，老年大鼠胃损伤的愈合延迟和受损;但其潜在机制尚未得到解释，并且以前未研究过老年胃粘膜中的血管生成。我们的长期目标是：1）确定衰老胃粘膜中血管生成受损的分子机制，并基于这些发现2）开发新的治疗策略用于治疗和逆转衰老相关的血管生成受损和延迟愈合。我们的总体假设是，血管生成和抗血管生成因子之间的不平衡抑制血管生成，抑制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不平衡及其后果的机制，最终证明操纵这种不平衡可以改善血管生成和愈合。我们假设衰老胃粘膜中血管生成受损的潜在机制是：1）胃微血管内皮细胞（EC）功能的衰老相关改变-VEGF基因的转录激活减少，原因是：A）输入蛋白的下调和B）转录因子P-CREB和P-STAT 3的下调，2）在老化的胃粘膜中由于减少的促血管生成VEGF和伴随的增加的抗血管生成内皮抑制素导致的血管生成不平衡导致血管生成的抑制，和3）针对以下机制的治疗性干预：A）局部VEGF基因治疗，B）AICAR治疗上调和激活importin，或C）针对内皮抑制素的特异性中和抗体将显着逆转受损的血管生成，并改善微血管再生和衰老大鼠慢性胃溃疡的愈合。这些假设的基本原理源于我们的初步工作，即在老年（与年轻）大鼠胃粘膜中，VEGF显著降低，内皮抑制素增加，对损伤的血管生成减少，粘膜愈合扭曲和受损。此外，我们发现，EC从胃粘膜中分离的老龄大鼠表现出减少VEGF基因的激活，受损的体外血管生成和减少缺氧的血管生成反应。我们还证明了importin是VEGF基因激活和血管生成的必要条件，因为用特异性siRNA沉默importin显著降低了年轻EC中的血管生成。相反，通过AICAR治疗激活importin显著逆转了衰老EC中受损的血管生成。研究设计：我们将在从年轻和老年大鼠胃粘膜分离的EC中以及在损伤和溃疡后的老年大鼠与年轻大鼠的胃粘膜中检查上述假设。体外研究将包括分析：参与血管生成的蛋白质- VEGF、VEGFR 2、P-VEGFR 2以及HSP 90、HIF 1、HIF 1、输入蛋白、AMPK、P-CREB和P-STAT 3的表达;它们的相互作用以及HIF 1、P-CREB和P-STAT 3与VEGF基因的结合。对于体内研究，我们将检查大鼠胃粘膜损伤（糜烂和溃疡），并定量评估胃溃疡愈合过程中的血管生成，并确定内皮抑素、MMP 9和其他列出的蛋白质的表达。重要性：2006年，约3900万美国人（占人口的12%）年龄在65岁或以上，预计到2030年这一数字将达到约7000万。这一人群的组织和器官损伤风险很高。血管生成受损是延迟愈合、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VA的优先领域之一。 公共卫生相关性： 与退伍军人健康相关。2006年，近3900万美国人（占人口的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。老年患者人群正在老龄化，70岁或以上的患者占很大比例。这一人群的组织和器官损伤风险很高。受损的血管生成是延迟组织损伤愈合、发病率和死亡率的关键因素。伤口的愈合受损，无论是内部的，如胃和十二指肠溃疡，还是外部的，如不愈合的皮肤溃疡，都构成了重要的临床和经济问题。胃损伤-胃炎和溃疡在VA患者中经常发生，通常是由于长期治疗关节炎或疼痛的NSAID。美国人群中胃十二指肠溃疡的终生患病率为3-6%，但VA人群中的发病率高出2.5倍。我们提出的研究的中心主题与血管生成直接相关，血管生成是“伤口愈合”的关键要求，这是VA的优先领域之一。

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.