Molecular Mechanisms of Impaired Angiogenesis in Aging Gastric Mucosa

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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.