Regulation of Extracellular Matrix Homeostatsis in Skin Aging

描述（由申请人提供）：拟议研究的长期目标是了解皮肤结缔组织衰老的分子基础和功能影响。皮肤，像所有的人体器官一样，会随着时间的流逝而发生有害的变化。皮肤的自然老化主要表现为变薄，主要是由于真皮层中I型胶原蛋白的损失。I型胶原蛋白是皮肤中最丰富的蛋白质，赋予皮肤结构、强度和弹性。随着年龄的增长，胶原蛋白的流失会导致皮肤的脆弱性增加，从而使皮肤更容易受到瘀伤，并阻碍伤口愈合。美国人口的老龄化使得对脆弱皮肤的医疗护理日益成为公共卫生问题。皮肤除了是人体最大的器官外，还很容易用于研究。皮肤的这些独特特性为研究人类衰老的分子机制提供了机会。衰老的自由基理论认为，自然衰老是由有氧代谢产生的活性氧（ROS）氧化引起的细胞损伤所驱动的。我们发现体内衰老的人皮肤成纤维细胞中ROS水平升高。成纤维细胞是产生I型胶原蛋白的主要细胞类型。此外，我们发现TGF-2/SMAD/CTGF轴是驱动皮肤中I型胶原生成的主要调控网络，在衰老的人类皮肤中受损。这种损伤是由于SMAD3的表达减少，SMAD3是TGF-2作用的下游效应，结缔组织生长因子（CTGF）的表达减少，结缔组织生长因子是一种与TGF-2协同作用调节I型胶原表达的多功能蛋白。此外，我们发现原代培养的人皮肤成纤维细胞轻度、短期的氧化暴露会导致永久性的细胞改变，这种改变与体内衰老皮肤成纤维细胞中观察到的变化非常相似；即ROS增加，SMAD3减少，CTGF减少，I型胶原表达减少。基于这些观察结果，我们假设ROS的增加会降低SMAD3和CTGF的表达，从而导致衰老人类皮肤成纤维细胞中I型胶原蛋白的产生减少。我们提出了四个具体目标来检验这一假设：1)确定体内人皮肤成纤维细胞中ROS、SMAD3、CTGF和I型胶原生成的年龄相关变化；2)确定局部抗氧化剂降低体内衰老人皮肤中ROS水平、减轻TGF-2/SMAD/CTGF轴损伤和诱导I型胶原生成的能力；3)确定氧化暴露降低人皮肤成纤维细胞中SMAD3、CTGF和I型胶原表达的分子机制。4)确定CTGF调控I型胶原表达的分子机制。这些研究的结果将在以下方面提供重要的见解：1)人类皮肤衰老的开始年龄，2)局部抗氧化剂的分子作用，3)氧化暴露调节TGF-2/SMAD/CTGF轴的机制，以及4)CTGF与TGF-2协同调节I型胶原表达的分子基础。公共卫生相关性：拟议研究的长期、广泛目标是了解皮肤结缔组织衰老的分子基础。随着年龄的增长，皮肤胶原蛋白的流失会导致皮肤更加脆弱，从而使皮肤更容易受到瘀伤，并阻碍伤口愈合。美国人口的老龄化使得脆弱皮肤的医疗保健日益成为公共卫生问题。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand molecular basis and functional impact of skin connective tissue aging. Skin, like all human organs, undergoes deleterious alterations as a consequence of the passage of time. Natural aging of skin is manifested primarily by thinning, largely due to loss of type I collagen in the dermis. Type I collagen is the most abundant protein in skin and confers structure, strength and resiliency. Age-dependent loss of collagen causes increased fragility and thereby makes skin more susceptible to bruising and impedes wound healing. Aging of the US population makes medical care of fragile skin a growing public health concern. In addition to being the largest human organ, skin is readily accessible for study. These unique properties of skin provide the opportunity study molecular mechanisms of aging in humans. The free radical theory of aging posits that natural aging is driven by cellular damage that results from oxidation by reactive oxygen species (ROS) that are generated as a consequence of aerobic metabolism. We find that ROS levels are elevated in aged human skin fibroblasts in vivo. Fibroblasts are the major cell type that produces type I collagen. In addition, we find that the TGF-2/SMAD/CTGF axis, which is the major regulatory network that drives type I collagen production in skin, is impaired in aged human skin. This impairment results from decreased expression of SMAD3, which is a downstream effector of TGF-2 actions, and reduced expression of connective tissue growth factor (CTGF), which is a multi-functional protein that acts in concert with TGF-2 to regulate type I collagen expression. Furthermore, we find that mild, short-term oxidative exposure of primary cultured human dermal fibroblasts causes permanent cellular alterations that closely mimic those observed in fibroblasts in aged skin in vivo; namely, increased ROS, reduced SMAD3, reduced CTGF, and reduced type I collagen expression. Based on these observations, we hypothesize that increased ROS, reduces expression of SMAD3 and CTGF, which results in reduction of type I collagen production, in fibroblasts in aged human skin. We propose four Specific Aims to test this hypothesis: 1) determine age-related alterations of ROS, SMAD3, CTGF, and type I collagen production, in human skin fibroblasts in vivo, 2) determine the ability of topical anti-oxidant to reduce ROS levels, mitigate impairment of the TGF-2/SMAD/CTGF axis, and induce type I collagen production, in aged human skin in vivo, 3) determine molecular mechanisms by which oxidative exposure reduces SMAD3, CTGF and type I collagen expression in human ski fibroblasts, and 4) determine molecular mechanisms by which CTGF regulates type I collagen expression. The results from the proposed studies will provide important insights regarding 1) the age of onset of human skin aging, 2) molecular actions of topical antioxidant, 3) mechanisms by which oxidative exposure regulates the TGF-2/SMAD/CTGF axis, and 4) molecular basis by which CTGF cooperates with TGF-2 in the regulation of type I collagen expression. PUBLIC HEALTH RELEVANCE: The long-term, broad goal of the proposed research is to understand the molecular basis of skin connective tissue aging. Age-dependent loss of skin collagen causes increased skin fragility and thereby makes skin more susceptible to bruising and impedes wound healing. The aging of the US population makes medical care of fragile skin a growing public health concern.