| Background and Objection:Aging is a biological process that changes structural integrity and physiological function. The resulting skin changes include dyschromia, roughness, and fine rhytids followed by persistent deeper folds. Structurally this is explained by dermal atrophy, decreased collagen, loss of subcutaneous fat, loss of inherent elasticity, and increased melanogenesis.Several theories have been developed to comprehend this progressive process, which include accumulation of mutations in the genome, accumulation of toxic metabolites, hormonal deprivation, increased formation of free radicals (oxidative damage), and cross-linking of macromolecules by glycation.Glycation is a non-enzymatically driven reaction between free amine groups like those of amino acids in proteins and reducing sugars like glucose. This reaction, also called the Maillard reaction, eventually leads to the formation Advanced Glycation End products (AGEs) such as carboxymethyl-L-Lysine, pentosidine and others, which can be responsible for the formation of cross-links between macromolecules by covalent bonding. This reaction therefore preferentially affects tissues in which macromolecular structures have a slow turnover rate, and is therefore thought to play an important role in aging. Accumulating evidence indicates that AGEs exacerbate and accelerate the aging process and contribute to the early phases of age-related diseases, including neurodegenerative disease, cataracts, renal failure, arthritis, and age-related macular degeneration. Moreover, AGEs and their precursors usually contain reactive carbonyl groups, which can be generated by the actions of reactive oxygen species (ROS), and lipid peroxidation is an important biological consequence of oxidative cellular damage. Therefore, the level of SOD and malondialdehyde (MDA) could be used to examine the relationship between the ageing process and defence antioxidantand lipid peroxidation.Besides the glycation, the alterations of skin collagen content and dermal vascularization also play key roles in aging process. As the process of ageing advances, collagen fibers become thinner and change their aspect, at advanced age, the lysis of collagen fibers and their thickening in the deep dermis is present. Moreover, a progressive reduction of dermis vasculature is present, due to a reduction in the number and size of vascular vessels, which is in its turn associated with the progressive alterations of vascular walls components, changes that advance until the function of the vessel ceases.Previous studies indicated that adipose tissue transplantation could improve the skin quality of recipient site, in addition to volume improvement. This unexpected result of adipose tissue transplantation may probably due to the effect of Mesenchymal stem cells (MSCs) within the stromal-vascular fraction of subcutaneous adipose tissue, or adipose-derived stem cells (ASCs). ASCs exhibit multi-lineage developmental plasticity and are similar to bone-marrow-derived MSCs in terms of surface markers and gene profiling. In addition,many clinical studies and animal experiments have confirmed that injection of these cells has favorable effects on wound repair, immunomodulation and anti-apoptosis via a paracrine effect or differentiation. The production and secretion of cytokines are an essential function of ASCs, and the cytokines have diverse pharmacological effects. Moreover, recent studies also have reported that ASCs improve wrinkles resulting from photo-aging and promote collagen synthesis and epidermal thickening of photo-aged fibroblasts in vitro.However, the underlying mechanisms of the anti-aging effects of ASCs have not been well studied. Therefore, in an attempt to further understand the mechanisms, we designed an experimental animal study on theskin aging mouse model induced by D-galactose (D-gal). The goal of the study was to use histologic and immunohistologic analyses to discover the suppression of glycation and potential to restore the functional capacity of the skin by both direct and indirect ways of ASCs.Methods and materialsIsolation and culture of ASCsMouse inguinal fat pad adipose tissue samples were acquired from Six-week-old green fluorescent protein (GFP)expressing mice, the mice were provided by Model Animal Research Center of Nanjing University(Nanjing, China).The obtained samples were cut into piecesand then digested with0.075%type I collagenase (Sigma-Aldrich, St. Louis, MO) under gentle agitation for45min at37℃. Mature adipocytes and connective tissue were separated from pellets by centrifugation (800g for10minutes) and then discarded. The pellets were re-suspended in phosphate-buffered saline (PBS) and filtered through a200μm mesh followed by centrifugation (800g for10minutes) to spin down stromal vascular fraction cell pellets.The retrieved cell fraction was cultured overnight at37℃/5%CO2in control medium (Dulbecco’s modified Eagle media,10%fetal bovine serum,100units/ml penicillin,100mg/ml streptomycin). The resulting cell population was maintained over3to5days until confluence. ASCs were cultured and expanded in control medium. The cells from P3to P5were used in the following experiment.D-galactose (D-gal) induced aging model and Animal experimentChronic administration of a low dose of D-gal induces have been widely used as the animals aging models for investigating skin aging or anti-aging pharmacology. AGEs is considered to account for the underlying mechanism of aging because the AGE inhibitor aminoguanidine (AG) could block most of the aging phenotypes in this mouse model.80Six-week-old nude mice (gender unregarded) were provided by the Southern Medical University Experimental Animal Center (Guangzhou, China). Mice were randomly divided into4groups (n=20each). Three groups of animals received daily subcutaneous injections of D-gal (1000mg/kg, subcutaneously) for8weeks.2weeks later, these three groups received a subcutaneous injection of106GFP-expressing ASCs, AG (100mg/kg, intragastrically) or PBS at the midline of the dorsum. After the injection,all4groups of mice were housed for another4weeks. All the animals wereallowed free access to water and a chow diet and were observed daily. Mice were killed at the end of treatment, and the skin tissue was immediately collected or stored at-70℃.Survival of ASCsAfter the injectingof GFP-expressing ASCs, mice were anesthetized with isoflurane and underwent fluorescence live imaging by use of the Kodak In-Vivo Imaging System F (Carestream Health, Inc. Rochester, NY, US) at days1,3,7,14and28after injection.Histological examinationSkin tissue from all the4groups were fixed in4%paraformaldehyde, dehydrated and paraffin-embedded for haematoxylin and eosin (H&E).Tissue blocks were serially sectioned(6-μm sections), mounted onto an APES-treated glass slide, then assessed under an Olympus BX51microscope and photographed by use of an Olympus DP71digital camera. The dermal thickness of the skin samples was measured.Collagen quantificationFor determination of total collagen, samples obtained at all four groups were stained with Masson’strichrome. Briefly, sections were deparaffinized in xylene, rehydrated in graded ethanol and post-fixed in Bouin’s fixative for one hour at55℃. The nuclei and collagen were sequentially stained with equal volumes of ferric chloride solution and alcoholic hematoxylin and trichrome solution, respectively. Total collagen content was reported as a percentage of the aniline blue staining divided by the total tissue area of the section using Image J software.Immunohistochemistry for CD31and vascular endothelial growth factor (VEGF)Immunohistochemistry was used for confirming the angiogenesis of the samples. The sections obtained from each groupwere examined using CD31antibody(Abcam, Cambridge,UK) and VEGF antibody(Abcam, Cambridge, UK). Paraffin sections were dewaxed and hydrated before immunohistochemical staining. Slides were washed with PBS and incubated in3%H2O2for10mins, after another wash in PBS then in protein block for30mins. Then thesections were incubated with primary antibody at4°Covernight.After three wash steps, tissues were incubated with the biotinylated secondary antibody. Following30min incubation with the complex of avidin and biotinylated horseradish peroxidase, enzyme activity was visualized using3,3’-diaminobenzidine. Slides were scored by two independent observers using anOlympus BX51microscope and photographed by use of an Olympus DP71digital camera. Then the number of CD31-positive vessels was counted and positive area of VEGF was quantified using Sigma Scan software across five nonconsecutive tissue sections for each image.Measurement of superoxide dismutase (SOD) activity and lipid peroxidationTissue homogenization:Part of theskin tissue samples were weighed and homogenized in normal saline, and homogenates of5%were obtained. Homogenates were sonicated twice at30-sec intervals. Homogenization and sonication were performed at4℃. After sonication, homogenates were centrifuged at3000rpm for10min and12000rpm for15min. Aliquots of supernatants were used for studies. Protein content of the aliquots was determined by use of a BCA protein assay kit (Pierce Chemical Co.).SOD activity in skin was examined by the xanthine oxidase method with use of a kit (Nanjing Jiancheng Bioengineering Institute, China) as described. The assay involves the xanthine-xanthine oxidase system to produce superoxide ions, which react with2-(4-iodophenyl)-3-(4-nitrophenol-5-phenlyltet-razolium chloride) to form a red formazan dye, with absorbance at550nm. Protein concentration was determined by a BCA protein assay kit (Pierce Chemical Co.), with one unit of SOD defined as the amount of SOD inhibiting the rate of reaction by50%at25℃. Lipid peroxidation was evaluated by MDA content according to the thiobarbituric acid (TBA) method as recommended (Nanjing Jiancheng Bioengineering Institute, China). The method is based on spectrophotometric measurement of the color produced during the MDA reaction with TBA. MDA concentrations were calculated by the absorbance of TBA reactive substances (TBARS) at532nm.Inhibition of formation of AGEs in vitroAGE-modified bovine serum albumin (BSA) was prepared as described. Briefly, BSA (100mg/mL) was incubated under sterile conditions with0.5M D-gal in0.2M PBS (pH7.4) at37℃for8weeks. For ASCs treatment or AG inhibition, AGEs-modified BSA samples were incubated with ASCs (1×106) or AG (100mM) under identical conditions. The control BSA sample was incubated under identical conditions but without D-gal. Samples were dialyzed (10-kDa cut-off) against PBS and then BSA-AGEs content was determined by use of a commercial ELISA kit as described previously.Statistical analysisIn vitro data are representative of3or more independent experiments. One-way ANOVA was used for statistical analysis of in vivo data. P<0.05was considered statistically significant.Results:Characterization of ASCsASCs expanded easily in vitro and showed fibroblast-like morphologic features.The retention rate of transplanted ASCs in mouse aging modelGFP signals were detected by fluorescence live imaging in mice throughout the experiment. Signals were limited to the dorsum at day1, and the injection site also showed the strongest signal. At day3, the signal of the injection area decreased but remained strong. From days7to14, the signal in the dorsum area began to gradually decrease and was weak at day28.Effect of ASCs on formation of AGEs in miceWith ASCs treatment, visual inspection revealed no major abnormalities in mice.All groups gained weight normally throughout the study. As expected, mice treated with D-gal showed a remarkably increased level of skin AGEs as compared with control mice (P<0.05), and AGEs formation inhibition with AG significantly reversed the increased level of AGEs in D-gal-treated mice (P<0.05) ASCs treatment was efficacious, significantly blocking the increase in AGEs level (P<0.05), which suggests that ASCs had an inhibitory effect on BSA-AGE formation.Effect of ASCs on antioxidant enzyme activity and lipid peroxidation in miceTo further confirm that ASCs could protect skin by antioxidant action, we measured SOD activity and MDA level in mouse skin tissue. As expected, SOD activity decreased, while MDA level, an indicator of lipid peroxidation, increased significantly; however, treatment with ASCs increased SOD activity and decreased that of MDA in D-gal-treated mouse skin.Histological observationH&E staining showed great changes in skin appendages in the sample of D-gal-treated mice.Moreover,the dermal thicknesswas significantly decreased in D-gal-treated mice compared with control group, and that of ASCs treated group was significantly increased. Quantification showed that ASCs treated group also had the higher collagen content compared with D-gal-treated group.ASCs increase VEGF levels and enhance skin tissue angiogenesisTo further confirm that ASCs could increaseangiogenesis of the skin, we measured CD31-positivemicrovessels and VEGF expression in skin tissue.As we expected, the ASCs treated group had a higher microvessel density and VEGF expression than those of D-galactose-treated group.DiscussionsStem cells have various potential uses achieved through their differentiation and paracrine effects in most medical areas. In particular, ASCs have an advantage in clinical application because they are easy to harvest and abundant in the human body, and thus have no implied ethical problems. In this study, we examined the effect of ASCson anti-aging, particularly suppressing the glycation reaction and restoring the functional capacity of the skin in an accelerated aging mouse model induced by D-gal. Our findings can be summarized as follows:(1) ASCs could survive till28days after injecting into dermal.(2) ASCs could decrease the AGEs levels, thus reversing the aging phenotype, similar to the effect of AG, the inhibitor of AGEs and ASCs also reversed the expression of senescence-associated markers such as SOD and MDA.(3) ASCs could significantly increase the dermal thickness and collagen content of skin.(4) ASCs could enhance the expression of VEGF and increase the vessel density of the skin, which may have a skin trophic effect.Skin aging occurs through intrinsic and extrinsic pathways. Intrinsic aging, so-called normal aging, was confirmed by changes in levels of senescence-associated molecular markers. Previous study demonstrated that D-gal injection led to an accelerated aging phenotype, with changes in AGE level and senescence markers such as SOD and MDA activity. In our study, nude mice treated with D-gal showed significant changes resembling normal aging.And the AGEs inhibitor, AG, could prevent the accelerated aging process.These results strongly suggest that AGEs are a crucial mediator in our D-gal-induced aging model.An overwhelming issue in cell therapy remains the low engraftment rate of transplanted cells, which diminishes the efficiency of cell therapy. Some previous studies suggested that transplanted ASCs have a very low retention in the later stages of transplan, though combination of pro-survival factors favors their survival. Remarkably, in our study, the GFP signal of injected ASCs could not be detected till28days. The low survival rate of transplanted ASCs may due to the phagocytosis of local immune cells.However, it also indicates that despite the direct contribution of the transplanted cells, they cannot be entirely responsible for the beneficial effect, so the paracrine effect is more likely the mechanism explaining the functional results.Excess AGEs intake and chronic accumulation of AGE-related glycated proteins in tissues are suggested to further potentiate the aging process resulting in impaired mitochondrial function and decreased life span in Caenorhabditiselegans and mice. Our results showed that ASCs treatment could inhibit the AGEs formation, though ASCs had less inhibitory effect on the formation of AGEs, in contrast to AG,suggesting ASCs treatment had an inhibitory effect on BSA-AGE formation. Cells in the body possess a wide range of inter-linked antioxidant defense mechanisms to protect themselves against damage by ROS. Among these mechanisms, antioxidant enzymes, including SODs, are important in scavenging ROS remaining in the cells. SODs are metalloenzymes that catalyze the dismutation of superoxide anion to molecular O2and H2O2and thus form a crucial part of the cellular antioxidant defense mechanism. Three forms of SOD exist in cells and tissues:cytosolic Cu/Zn-SOD (SOD1), mitochondrial Mn-SOD (SOD2) and extracellular SOD (SOD3). Cu/Zn-SOD and Mn-SOD may be important in defense against oxygen toxicity. Proteomic analysis revealed112proteins, including SOD, upregulated with ASCs treatment. Representative proteins showing antioxidant effects on epithelial cells from previous study. As an important finding of this study, we have successfully detected ASCs could reverse the effects of D-gal-induced oxidative stress in mouse skin, as seen by activity of senescence-associated molecular markers such as SOD and MDA. Secretary proteins from ASCs such as SOD and some cytokines may mediate the protective effects and play key roles in vivo. Stem cells may have potent antioxidant action as suggested by ASCs reversing the expression of senescence-associated molecular markers. This finding is similar to the role of AG, the inhibitor of AGEs, for a role of ASCs and their secretory factors in wound healing and skin anti-aging.Kim et al. suggested that ASCs conditioned medium enhanced typel collagen secretion from human dermal fibroblasts andfibroblast migration in an in vitro wound-healing model. In another study, wrinkles induced by UVBirradiation, were significantly improved by subcutaneous injection of ASCs into hairless mice. In addition, dermalthickness and collagen contents were increased in the ASCs injection groups.Our histological observations showed decreased expression of collagen and dermal thickness in our D-gal-induced mice, which was reversed with ASCs injection.Though some studies suggested that ASCs could secret collagen, the low retention rate of ASCs indicated the increasing of collagen expression was more likely due to the up-regulation of collagen expression from local fibroblast caused by ASCs paracrine.Another potential role of ASCs treatment in skin anti-aging is angiogenesis. Substantial evidence indicates that ASCs could increase the angiogenesis of tissue through secreting angiogenic factors such as VEGF, hepatocyte growth factor. In this study,our results support thatASCs transplantation strongly induced revascularization of the skin tissue along with secretion of the VEGF, which was clearly detected in the transplanted cells and adds support to the trophic hypothesis. CD31staining studies further confirm this mechanism. Although previous studies indicated that ASCs could differentiate into vascular endothelial cells, considering the GFP positive ASCs could be hardly detected after28day, the angiogenic effect was mainly due to the paracrine of ASCs. ConclusionIn summary, we examined the suppression glycation by ASCs in a mouse aging skin model induced by D-gal. ASCs may have potential to contribute to the regeneration of skin with aging and provide a functional benefit. ASCs injection reversed the expression of senescence-associated molecular markers. Similar to the role of AG, the inhibitor of AGEs formation, ASCs inhibited D-gal-increased AGEs levels, thus reversing the aging phenotype in our mouse model. Activities of SOD and MDA in skin were enhanced with ASCs injection, so ASCs may suppress glycation activity in skin. ASCs may be good candidates for control and prevention of skin damage from glycation in various skin conditions, including wounding and aging. |
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