Regulation of Extracellular Matrix Homeostatsis in Skin Aging

描述(申请人提供)：拟议研究的长期目标是了解皮肤结缔组织老化的分子基础和功能影响。皮肤，就像所有的人体器官一样，会随着时间的推移而发生有害的变化。皮肤的自然老化主要表现为皮肤变薄，主要是由于真皮中I型胶原的丧失。I型胶原是皮肤中含量最丰富的蛋白质，赋予皮肤结构、强度和弹性。与年龄相关的胶原蛋白丢失会增加皮肤的脆弱性，从而使皮肤更容易擦伤，阻碍伤口愈合。美国人口老龄化使得脆弱皮肤的医疗护理日益成为公共卫生问题。皮肤除了是人类最大的器官外，也很容易被研究。皮肤的这些独特性质为研究人类衰老的分子机制提供了机会。衰老的自由基理论假设，自然衰老是由有氧代谢产生的活性氧物种(ROS)氧化造成的细胞损伤所驱动的。我们发现在活体内老化的人皮肤成纤维细胞中ROS水平升高。成纤维细胞是产生I型胶原的主要细胞类型。此外，我们还发现，在老年人皮肤中，驱动I型胶原产生的主要调控网络--转化生长因子-2/SMAD/CTGF轴受损。这种损伤是由于Smad3的表达减少和结缔组织生长因子(CTGF)的表达减少所致，Smad3是转化生长因子-2作用的下游效应因子，结缔组织生长因子是一种多功能蛋白，与转化生长因子-2协同作用调节I型胶原的表达。此外，我们发现，原代培养的人真皮成纤维细胞在温和、短期的氧化暴露下，会引起永久性的细胞变化，这与体内老年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，我们推测，在老年人皮肤成纤维细胞中，ROS增加，Smad3和CTGF的表达减少，从而导致I型胶原产生减少。我们提出了四个特定的目标来验证这一假说：1)确定在活体人皮肤成纤维细胞中与年龄相关的ROS、SMAD3、CTGF和I型胶原产生的变化，2)确定局部抗氧化剂降低ROS水平的能力，减轻体内老年人皮肤中转化生长因子-2/SMAD/CTGF轴的损伤和诱导I型胶原的产生，3)确定氧化暴露降低人SKI成纤维细胞中SMAD3、CTGF和I型胶原表达的分子机制，以及4)确定CTGF调节I型胶原表达的分子机制。这项研究的结果将为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)氧化暴露调节转化生长因子-2/SMAD/CTGF轴的机制；以及4)转化生长因子与转化生长因子-2合作调节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，皮肤胶原蛋白的丧失会增加皮肤的脆弱性，从而使皮肤更容易受到瘀伤的影响，阻碍伤口愈合。美国人口老龄化使得脆弱皮肤的医疗护理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.