DIABETIC NEUROPATHY--IMPLICATIONS FOR WOUND REPAIR

糖尿病患者的周围神经病变与皮肤不愈合性溃疡的发生密切相关。在美国，每年对患有慢性非愈合性溃疡的糖尿病患者进行两万至三万次截肢手术，显著影响医疗成本、损伤和生活质量。确定导致糖尿病性神经病变和伤口愈合受损的细胞事件为治疗干预提供了机会。我们假设在糖尿病患者中，微血管内皮细胞和角质形成细胞1）不产生感觉神经纤维生长所必需的神经营养因子，2）对神经源性炎症介质没有正常反应，这些异常导致伤口愈合受损。我们预期，高血糖症阻止皮肤细胞和感觉神经纤维之间的正常信号传导。这可能是由于神经支配减少导致P物质减少所致。长期高血糖导致的P物质、细胞表面受体或基质分子的糖基化可能抑制正常的神经炎症。或者，通过增加中性内肽酶水平的P物质的蛋白水解降解可以减少神经炎症。我们将测试我们的假设，解决以下问题：具体目标1：以确定是否高血糖钝化皮肤细胞的反应，P物质。我们将比较P物质诱导的神经生长因子生产的微血管内皮细胞和角质形成细胞在正常和高血糖条件下。我们将评估高血糖对P物质诱导的内皮细胞整合素表达和细胞骨架组织变化的影响。具体目标二：为了确定是否基质分子糖基化干扰皮肤细胞对P物质的反应，我们将确定是否基质分子糖基化降低P物质诱导的NGF合成或内皮细胞细胞骨架组织和/或整合素表达的变化。具体目标3：确定高血糖和基质分子糖基化对皮肤细胞中性内肽酶表达和活性的影响。我们将确定是否高血糖或基质分子糖化增加中性内肽酶活性的皮肤细胞。我们将确定是否高血糖或基质分子糖化增加微血管内皮细胞或角质形成细胞的中性内肽酶表达和活性。特异性im 4：确定神经肽或神经营养因子的恢复是否改善糖尿病（db/db）小鼠的伤口修复。采用高血糖db/db小鼠的切创模型，我们将替代P物质、替代NGF或抑制中性内肽酶活性，以评价神经肽和NGF在创伤修复中的作用。

Peripheral neuropathy in patients with diabetes mellitus is closely associated with development of cutaneous non-healing ulcers. Twenty to thirty thousand amputations performed annually in the United States on diabetic patients with chronic non-healing ulcers significantly impact medical costs, impairment and quality of life. Determining cellular events leading to diabetic neuropathy and impaired wound healing provides an opportunity for therapeutic intervention. We hypothesize that in patients with diabetes mellitus, microvascular endothelial cells and keratinocytes 1) do not produce necessary neurotrophic factors for sensory nerve fiber growth and 2) do not respond normally to nerve derived inflammatory mediators and these abnormalities contribute to impaired wound healing. We anticipate that hyperglycemia prevents normal signaling between cutaneous cells and sensory nerve fibers. This may result from decreased substance P due to the reduced innervation. Glycosylation of substance P, cell surface receptors or matrix molecules due to prolonged hyperglycemia may inhibit normal neuroinflammation. Alternatively, proteolytic degradation of substance P by increased levels of the enzyme neutral endopeptidase may reduce neuroinflammation. We will test our hypothesis by addressing the following: Specific Aim 1: To determine whether hyperglycemia blunts the response of cutaneous cells to substance P. We will compare substance P- induced NGF production by microvascular endothelial cells and keratinocytes under normal and hyperglycemic conditions. We will evaluate the effect of hyperglycemia on substance P-induced changes in endothelial cell integrin expression and cytoskeleton organization. Specific Aim 2: To determine whether matrix molecule glycosylation interferes with response of cutaneous cells to substance P. We will determine whether matrix molecule glycation decrease substance P-induced NGF synthesis or changes in endothelial cell cytoskeletal organization and/or integrin expression. Specific Aim 3: To determine the effect of hyperglycemia and matrix molecule glycosylation on neutral endopeptidase expression and activity by cutaneous cells. We will determine whether hyperglycemia or matrix molecule glycation increases neutral endopeptidase activity by cutaneous cells. We will determine whether hyperglycemia or matrix molecule glycation increases neutral endopeptidase expression and activity by microvascular endothelial cells or keratinocytes. Specific im 4: To determine whether restoration of neuropeptides or neurotrophins improves wound repair in diabetic (db/db) mice. Using an excisional would model in hyperglycemia db/db mice, we will replace substance P, replace NGF or inhibit neutral endopeptidase activity to evaluate the roles of neuropeptides and NGF in wound repair.