Aged human skin is fragile because of fragmentation and loss of type I collagen fibrils, which confer strength and resiliency. We report here that dermal fibroblasts express increased levels of collagen-degrading matrix metalloproteinases-1 (MMP-1) in aged (>80 years old) compared with young (21 to 30 years old) human skin in vivo. Transcription factor AP-1 and ␣2␤1 integrin, which are key regulators of MMP-1 expression, are also elevated in fibroblasts in aged human skin in vivo. MMP-1 treatment of young skin in organ culture causes fragmentation of collagen fibrils and reduces fibroblast stretch, consistent with reduced mechanical tension , as observed in aged human skin. Limited fragmentation of three-dimensional collagen lattices with exogenous MMP-1 also reduces fibroblast stretch and mechanical tension. Furthermore, fibroblasts cultured in fragmented collagen lattices express elevated levels of MMP-1, AP-1, and ␣2␤1 integrin. Importantly, culture in fragmented collagen raises intracellular oxidant levels and treatment with antioxidant MitoQ 10 significantly reduces MMP-1 expression. These data identify positive feedback regulation that couples age-dependent MMP-1-catalyzed collagen fragmentation and oxidative stress. We propose that this self perpetuating cycle promotes human skin aging. These data extend the current understanding of the oxidative theory of aging beyond a cellular-centric view to include extracellular matrix and the critical role that connective tissue microenvironment plays in the biology of aging. Skin connective tissue (dermis) provides structural support for the skin's vasculature, appendages, and epidermis, which are vital to the function of skin. Structural integrity and function of the dermis are primarily dependent on its extracellular matrix, which is primarily composed of type I collagen fibrils. Type I collagen is the most abundant structural protein in skin, 1 and fragmented collagen fibrils are prominent, characteristic features of aged human skin in vivo.2-4 This fragmentation seriously impairs both the mechanical properties of skin, and the functions of cells that reside within the dermis. Clinically, this impairment manifests as delayed wound healing, reduced vascularization, propensity to bruise, and thin skin. Failure of normal functional interactions among dermal cells and their extracellular matrix microenvironment underlie these age-dependent phenotypic alterations. 6Damage to the collagenous extracellular matrix of the dermis can be observed at both the histological and ultrastructural level. 5,[7][8][9] In young dermis, intact, tightly packed, well-organized, long collagen fibrils are abundant. In contrast, in aged dermis, collagen fibrils are fragmented, disorganized, and sparse, resulting in the appearance of amorphous open space. Quantitative biochemical analysis reveals that the amount of fragmented collagen is 4.3-fold greater in aged (Ͼ80 years old) compared with young (21 to 30 years old) human dermis in vivo.

Stromal cells provide a crucial microenvironment for overlying epithelium. Here we investigated the expression and function of a stromal cell-specific protein, stromal cell-derived factor-1 (SDF-1), in normal human skin and in the tissues of diseased skin. Immunohistology and laser capture microdissection (LCM)-coupled quantitative real-time RT-PCR revealed that SDF-1 is constitutively and predominantly expressed in dermal stromal cells in normal human skin in vivo. To our surprise, an extremely high level of SDF-1 transcription was observed in the dermis of normal human skin in vivo, evidenced by much higher mRNA expression level than type I collagen, the most abundant and highly expressed protein in human skin. SDF-1 was also upregulated in the tissues of many human skin disorders including psoriasis, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). Double immunostaining for SDF-1 and HSP47 (heat shock protein 47), a marker of fibroblasts, revealed that fibroblasts were the major source of stroma-cell-derived SDF-1 in both normal and diseased skin. Functionally, SDF-1 activates the ERK (extracellular-signal-regulated kinases) pathway and functions as a mitogen to stimulate epidermal keratinocyte proliferation. Both overexpression of SDF-1 in dermal fibroblasts and treatment with rhSDF-1 to the skin equivalent cultures significantly increased the number of keratinocyte layers and epidermal thickness. Conversely, the stimulative function of SDF-1 on keratinocyte proliferation was nearly completely eliminated by interfering with CXCR4, a specific receptor of SDF-1, or by knock-down of SDF-1 in fibroblasts. Our data reveal that extremely high levels of SDF-1 provide a crucial microenvironment for epidermal keratinocyte proliferation in both physiologic and pathologic skin conditions.Electronic supplementary materialThe online version of this article (doi:10.1007/s13238-015-0198-5) contains supplementary material, which is available to authorized users.

BackgroundReduced cell spreading is a prominent feature of aged dermal fibroblasts in human skin in vivo. Mitochondrial DNA (mtDNA) common deletion has been reported to play a role in the human aging process, however the relationship between age-related reduced cell spreading and mtDNA common deletion has not yet been reported.ResultsTo examine mtDNA common deletion in the dermis of aged human skin, the epidermis was removed from full-thickness human skin samples using cryostat. mtDNA common deletion was significantly elevated in the dermis of both naturally aged and photoaged human skin in vivo. To examine the relationship between age-related reduced cell spreading and mtDNA common deletion, we modulated the shape of dermal fibroblasts by disrupting the actin cytoskeleton. Reduced cell spreading was associated with a higher level of mtDNA common deletion and was also accompanied by elevated levels of endogenous reactive oxygen species (ROS). Boosting cellular antioxidant capacity by using antioxidants was found to be protective against mtDNA common deletion associated with reduced cell spreading.ConclusionmtDNA common deletion is highly prevalent in the dermis of both naturally aged and photoaged human skin in vivo. mtDNA common deletion in response to reduced cell spreading is mediated, at least in part, by elevated oxidative stress in human dermal fibroblasts. These data extend current understanding of the mitochondrial theory of aging by identifying the connection between mtDNA common deletion and age-related reduction of cell spreading.

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We investigated the effects of mesenchymal stem cells (MSCs) on wound healing using a three-dimensional (3D) collagen gel scaffold. Three circular full-thickness skin defects were created on the back of Sprague-Dawley rats. One site was covered with a 3D collagen gel containing 2 × 106 MSCs (MSCs+/3D collagen+). Another site was replaced with a 3D collagen gel without MSCs and the third site was left empty. The wound size was significantly reduced in the MSCs+/3D collagen+ sites. MSCs+/3D collagen+ sites exhibited the most neovascularization. FISH showed that Y-chromosome possessing cells were found within the dermis of MSCs+/3D collagen+ sites. Gelatin zymography revealed that the most intense expression of MMP-9 was detected early in the MSCs+/3D collagen+ sites. Our results indicate that MSCs upregulate the early expression of MMP-9 which induces the early mobilization of VEGF. Thus, MSCs appear to accelerate significantly wound healing via early activation of MMP-9 and VEGF.