DIABETIC NEUROPATHY: IMPLICATIONS FOR WOUND REPAIR

糖尿病患者的周围神经病变与皮肤不愈合溃疡的发展密切相关。在美国，每年有2万到3万例患有慢性无法愈合溃疡的糖尿病患者进行截肢手术，这对医疗费用、损伤和生活质量产生了重大影响。确定导致糖尿病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。我们推测，在糖尿病患者中，微血管内皮细胞和角化细胞1)不能产生感觉神经纤维生长所需的神经营养因子，2)不能对神经源性炎症介质做出正常反应，这些异常导致伤口愈合受损。我们预计高血糖会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。由于长期高血糖，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。另外，通过增加酶中性内多肽酶水平对P物质进行蛋白水解降解可能会减少神经炎症。我们将通过解决以下问题来验证我们的假设：具体目标1：确定高血糖是否会减弱皮肤细胞对P物质的反应。我们将比较正常和高血糖条件下微血管内皮细胞和角质形成细胞在P物质诱导下产生的NGF。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。特异性目的2：确定基质分子糖基化是否会干扰皮肤细胞对p物质的反应。我们将确定基质分子糖基化是否会减少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。特异性目的3：探讨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。我们将确定高血糖或基质分子糖基化是否会增加皮肤细胞的中性内肽酶活性。我们将确定高血糖或基质分子糖基化是否会增加微血管内皮细胞或角化细胞中性内肽酶的表达和活性。特异性目的4：确定神经肽或神经营养素的恢复是否能改善糖尿病小鼠的伤口修复（db/db）。采用高血糖小鼠切除模型，我们将替代P物质，替代NGF或抑制中性内肽酶活性，以评估神经肽和NGF在伤口修复中的作用。

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.