Redox Control of Wound Healing

描述(由申请人提供)： 长期以来，伤口组织氧合受损一直被认为是愈合的关键限制因素。因此，氧稳态一直是创伤愈合研究的主要关注焦点。目前的研究结果表明，伤口处的很大一部分氧气被用来产生活性氧物种(ROS)。ROS被经典地描述为意外的代谢副产品，通常被认为是有害的。吞噬细胞NADPH氧化酶故意在瞬间爆发时产生ROS来杀死病原体。临床上用于伤口消毒的是高浓度的过氧化氢(等于或超过1%)。然而，人们普遍认为，在这些剂量下，过氧化氢对再生细胞有刺激性，可能不利于整体愈合。最近发现的另一个NADPH氧化酶家族，即Nox/DUOX家族，提供了伤口处非吞噬细胞故意产生ROS的更多例子。在诱导后，这些细胞持续产生低水平的ROS。最近的数据支持，如此低浓度的ROS可以调节特定的关键氧化还原敏感信号过程，其中大多数可以直接与伤口愈合有关。这一建议基于我们惊人的观察结果，即遗传和药物方法传递低浓度的ROS促进真皮伤口血管生成、收缩和闭合。如此低浓度的ROS并不影响伤口感染状态。在伤口处分解ROS的策略损害了愈合。综上所述，这些观察结果导致了一种假设，即抵抗感染并不是伤口部位ROS的唯一作用，ROS驱动氧化还原信号以支持愈合。事实上，人类NADPH氧化酶的先天缺陷会导致伤口愈合受损，而仅靠抗生素无法纠正这种缺陷。我们观察到，NADPH氧化酶缺陷转基因小鼠即使在无感染的条件下也会受到皮肤愈合的损害；这种损害可以通过低剂量的ROS传递来纠正。我们的工作假设是创伤相关细胞产生的ROS(由NOx产生的低ROS；以及吞噬细胞呼吸爆发后残留的ROS)通过诱导氧化还原敏感的信号转导通路支持早期急性伤口愈合。我们的长期目标是了解伤口组织氧稳态的这一新方面。我们试图阐明氧化还原敏感过程在伤口愈合中的重要性，并设计基于氧化还原的策略来促进愈合。为了验证上述假设，我们利用小鼠急性皮肤创伤的标准模型和真皮微血管内皮细胞的体外培养，提出了以下三个特定的目标：目的1.研究ROS在真皮创面血管生成、收缩和修复中的作用；目的2.确定创伤部位NADPH氧化酶的意义；以及目的3.表征氧化还原敏感的调控伤口血管生成的机制。

DESCRIPTION (provided by applicant): Compromised wound tissue oxygenation has been long recognized as a key limiting factor in healing. Oxygen homeostasis has therefore been the center of major attention in wound healing research. Current findings indicate that a significant fraction of oxygen at the wound site is utilized to generate reactive oxygen species (ROS). ROS are classically described as accidental metabolic by-products, and are generally thought to be deleterious. The phagocyte NADPH oxidase deliberately generates ROS in transient bursts to kill pathogens. High concentrations of H2O2 (equal to or more than 1%) are used clinically for wound disinfection. However, there is a general agreement that at these doses H2O2 is harsh to regenerating cells and may not benefit overall healing. The recent discovery of another family of NADPH oxidases, the Nox/Duox family, provides additional examples of deliberate generation of ROS by non-phagocytic cells at the wound site. Upon induction, these cells generate low-levels of ROS on a sustained basis. Recent data support that such low concentration of ROS can regulate specific key redox-sensitive signaling processes most of which can be directly linked to wound healing. This proposal rests on our striking observation that genetic as well as pharmacological approaches to deliver low concentrations of ROS promote dermal wound angiogenesis, contraction and closure. Such low concentrations of ROS did not influence wound infection status. Strategies to decompose ROS at the wound site impaired healing. Taken together, these observations led to the hypothesis that resisting infection is not the sole role of ROS at the wound-site, and that ROS drives redox-signaling to support healing. Indeed, congenital defect in human NADPH oxidase results in impaired wound healing and antibiotics alone cannot correct such defect. We have observed that NADPH oxidase deficient transgenic mice suffer from impaired dermal healing even under infection-free conditions; the impairment is corrected by low-dose ROS delivery. Our working hypothesis is that ROS generated by wound-related cells (low-ROS by Nox; and residual ROS in the aftermath of phagocyte respiratory burst) support earlyphase acute wound healing by inducing redox-sensitive signal transduction pathways. Our long-term objective is to understand this new aspect of wound tissue oxygen homeostasis. We seek to illuminate the significance of redoxsensitive processes in wound healing, and to design redox-based strategies to promote healing. To test the stated hypothesis we propose the following three specific aims using standard models of murine acute dermal wound and in vitro culture of dermal microvascular endothelial cells: Aim 1. Investigate the role of ROS in excisional dermal wound vascularization, contraction and repair; Aim 2. Determine the significance of NADPH oxidases at the wound site; and Aim 3. Characterize the redox-sensitive mechanisms that regulate wound angiogenesis.