Enhanced angiogenesis characteristic of SPARC-null mice disappears with age

The impairment of angiogenesis in aging has been attributed, in part, to alterations in proteins associated with the extracellular matrix (ECM). SPARC (secreted protein acidic and rich in cysteine/osteonectin/BM-40) is a matricellular protein that regulates endothelial cell function as well as cell-ECM interactions. We have previously shown that angiogenesis, as reflected by fibrovascular invasion into subcutaneously implanted polyvinyl alcohol (PVA) sponges, is increased in SPARC-null mice (6-9 months of age) relative to their wild-type (WT) counterparts. In this study, we define the influence of aging on (a) the expression of SPARC and (b) fibrovascular invasion into sponge implants in SPARC-null and WT mice. The expression of SPARC in fibroblasts and endothelial cells derived from young donors (humans mean age less than 30 years and mice 4-6 months of age) and old donors (humans mean age over 65 years and mice 22-27 months of age) decreased 1.6 to 2.3-fold with age. Analysis of fibrovascular invasion into sponges implanted into old (22-27 months) SPARC-null and WT mice showed no differences in percent area of invasion or collagenous ECM. Moreover, sponges from old SPARC-null and WT mice contained similar levels of VEGF that were significantly lower than those from young (4-6 months) mice. In contrast to fibroblasts from young SPARC-null mice, dermal fibroblasts from old SPARC-null mice did not migrate farther, proliferate faster, or produce greater amounts of VEGF relative to their old WT counterparts. However, when stimulated with TGF-beta1, primary cells isolated from the sponge implants, and dermal fibroblasts from both old SPARC-null and WT mice, showed marked increases in VEGF secretion. These data indicate that aging results in a loss of enhanced angiogenesis in SPARC-null mice, as a result of the detrimental impact of age on cellular functions, collagen deposition, and VEGF synthesis. However, the influence of aging on these processes may be reversed, in part, by growth factor stimulation.     

Impaired wound healing in mice deficient in a matricellular protein SPARC (osteonectin, BM-40)

Background  

SPARC is a matricellular protein involved in cell-matrix interactions. From expression patterns at the wound site and in vitro studies, SPARC has been implicated in the control of wound healing. Here we examined the function of SPARC in cutaneous wound healing using SPARC-null mice and dermal fibroblasts derived from them.     

Exogenous Tryptophan Promotes Cutaneous Wound Healing of Chronically Stressed Mice through Inhibition of TNF-α and IDO Activation

Stress prolongs the inflammatory response compromising the dermal reconstruction and wound closure. Acute stress-induced inflammation increases indoleamine 2, 3-dioxygenase-stimulated tryptophan catabolism. To investigate the role of indoleamine 2, 3-dioxygenase expression and tryptophan administration in adverse effects of stress on cutaneous wound healing, mice were submitted to chronic restraint stress and treated with tryptophan daily until euthanasia. Excisional lesions were created on each mouse and 5 or 7 days later, the lesions were analyzed. In addition, murine skin fibroblasts were exposed to elevated epinephrine levels plus tryptophan, and fibroblast activity was evaluated. Tryptophan administration reversed the reduction of the plasma tryptophan levels and the increase in the plasma normetanephrine levels induced by stress 5 and 7 days after wounding. Five days after wounding, stress-induced increase in the protein levels of tumor necrosis factor-α and indoleamine 2, 3-dioxygenase, and this was inhibited by tryptophan. Stress-induced increase in the lipid peroxidation and the amount of the neutrophils, macrophages and T cells number was reversed by tryptophan 5 days after wounding. Tryptophan administration inhibited the reduction of myofibroblast density, collagen deposition, re-epithelialization and wound contraction induced by stress 5 days after wounding. In dermal fibroblast culture, the tryptophan administration increased the cell migration and AKT phosphorylation in cells treated with high epinephrine levels. In conclusion, tryptophan-induced reduction of inflammatory response and indoleamine 2, 3-dioxygenase expression may have accelerated cutaneous wound healing of chronically stressed mice.     

SPARC-null mice exhibit accelerated cutaneous wound closure.

Expression of SPARC (secreted protein acidic and rich in cysteine; osteonectin, BM-40), an extracellular matrix (ECM) associated protein, is coincident with matrix remodeling. To further identify the functions of SPARC in vivo, we have made excisional wounds on the dorsa of SPARC-null and wild-type mice and monitored closure over time. A significant decrease in the size of the SPARC-null wounds, in comparison to that of wild-type, was observed at Day 4 and was maximal at Day 7. Although substantial differences in the percentage of proliferating cells were not apparent in SPARC-null relative to wild-type wounds, primary cultures of SPARC-null dermal fibroblasts displayed accelerated migration, relative to wild-type fibroblasts, in wound assays in vitro. Although the expression of collagen I mRNA in wounds, as measured by in situ hybridization (ISH), was not significantly different in SPARC-null vs wild-type mice, the collagen content of unwounded skin appeared to be substantially lower in the SPARC-null animals. By hydroxyproline analysis, the concentration of collagen in SPARC-null skin was found to be half that of wild-type skin. Moreover, we found an inverse correlation between the efficiency of collagen gel contraction by dermal fibroblasts and the concentration of collagen within the gel itself. We propose that the accelerated wound closure seen in SPARC-null dermis results from its decreased collagen content, a condition contributing to enhanced contractibility.     

The essential involvement of cross-talk between IFN-gamma and TGF-beta in the skin wound-healing process

Several lines of in vitro evidence suggest the potential role of IFN-gamma in angiogenesis and collagen deposition, two crucial steps in the wound healing process. In this report, we examined the role of IFN-gamma in the skin wound healing process utilizing WT and IFN-gamma KO mice. In WT mice, excisional wounding induced IFN-gamma mRNA and protein expression by infiltrating macrophages and T cells, with a concomitant enhancement of IL-12 and IL-18 gene expression. Compared with WT mice, IFN-gamma KO mice exhibited an accelerated wound healing as evidenced by rapid wound closure and granulation tissue formation. Moreover, IFN-gamma KO mice exhibited enhanced angiogenesis with augmented vascular endothelial growth factor mRNA expression in wound sites, compared with WT mice, despite a reduction in the infiltrating neutrophils, macrophages, and T cells. IFN-gamma KO mice also exhibited accelerated collagen deposition with enhanced production of TGF-beta1 protein in wound sites, compared with WT mice. Furthermore, the absence of IFN-gamma augmented the TGF-beta1-mediated signaling pathway, as evidenced by increases in the levels of total and phosphorylated Smad2 and a reciprocal decrease in the levels of Smad7. These results demonstrate that there is crosstalk between the IFN-gamma/Stat1 and TGF-beta1/Smad signaling pathways in the wound healing process.     

Delayed cutaneous wound closure in HO‐2 deficient mice despite normal HO‐1 expression

Impaired wound healing can lead to scarring, and aesthetical and functional problems. The cytoprotective haem oxygenase (HO) enzymes degrade haem into iron, biliverdin and carbon monoxide. HO‐1 deficient mice suffer from chronic inflammatory stress and delayed cutaneous wound healing, while corneal wound healing in HO‐2 deficient mice is impaired with exorbitant inflammation and absence of HO‐1 expression. This study addresses the role of HO‐2 in cutaneous excisional wound healing using HO‐2 knockout (KO) mice. Here, we show that HO‐2 deficiency also delays cutaneous wound closure compared to WT controls. In addition, we detected reduced collagen deposition and vessel density in the wounds of HO‐2 KO mice compared to WT controls. Surprisingly, wound closure in HO‐2 KO mice was accompanied by an inflammatory response comparable to WT mice. HO‐1 induction in HO‐2 deficient skin was also similar to WT controls and may explain this protection against exaggerated cutaneous inflammation but not the delayed wound closure. Proliferation and myofibroblast differentiation were similar in both two genotypes. Next, we screened for candidate genes to explain the observed delayed wound closure, and detected delayed gene and protein expression profiles of the chemokine (C‐X‐C) ligand‐11 (CXCL‐11) in wounds of HO‐2 KO mice. Abnormal regulation of CXCL‐11 has been linked to delayed wound healing and disturbed angiogenesis. However, whether aberrant CXCL‐11 expression in HO‐2 KO mice is caused by or is causing delayed wound healing needs to be further investigated.     

GADD34 suppresses wound healing by upregulating expression of myosin IIA

Wound healing consists of sequential steps of tissue repair, and cell migration is particularly important. In order to analyze the potential function of growth arrest and DNA damage inducible protein 34 (GADD34) in tissue repair, we performed in vitro and in vivo wound healing experiments. In an in vitro scratch assay, GADD34 knockout (KO) mouse embryonic fibroblasts (MEFs) had higher migration rates than did wild type (WT) MEFs. Furthermore, the rate of wound closure was faster in GADD34 KO MEFs than in WT MEFs. Using in vivo punch biopsy assays, GADD34 KO mice had accelerated wound healing compared to WT mice. WT mice expressed higher amounts of myosin IIA in migrating macrophages and myofibroblasts than did GADD34 KO mice. These results indicate that GADD34 negatively regulates cell migration in wound healing via expression of myosin IIA.     

Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-β1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.     

Effects of cardiotonic steroids on dermal collagen synthesis and wound healing.

We previously reported that cardiotonic steroids stimulate collagen synthesis by cardiac fibroblasts in a process that involves signaling through the Na-K-ATPase pathway (Elkareh et al. Hypertension 49: 215-224, 2007). In this study, we examined the effect of cardiotonic steroids on dermal fibroblasts collagen synthesis and on wound healing. Increased collagen expression by human dermal fibroblasts was noted in response to the cardiotonic steroid marinobufagenin in a dose- and time-dependent fashion. An eightfold increase in collagen synthesis was noted when cells were exposed to 10 nM marinobufagenin for 24 h (P < 0.01). Similar increases in proline incorporation were seen following treatment with digoxin, ouabain, and marinobufagenin (10 nM x 24 h, all results P < 0.01 vs. control). The coadministration of the Src inhibitor PP2 or N-acetylcysteine completely prevented collagen stimulation by marinobufagenin. Next, we examined the effect of digoxin, ouabain, and marinobufagenin on the rate of wound closure in an in vitro model where human dermal fibroblasts cultures were wounded with a pipette tip and monitored by digital microscopy. Finally, we administered digoxin in an in vivo wound healing model. Olive oil was chosen as the digoxin carrier because of a favorable partition coefficient observed for labeled digoxin with saline. This application significantly accelerated in vivo wound healing in rats wounded with an 8-mm biopsy cut. Increased collagen accumulation was noted 9 days after wounding (both P < 0.01). The data suggest that cardiotonic steroids induce increases in collagen synthesis by dermal fibroblasts, as could potentially be exploited to accelerate wound healing.     

Amniotic mesenchymal stem cells enhance wound healing in diabetic NOD/SCID mice through high angiogenic and engraftment capabilities

Although human amniotic mesenchymal stem cells (AMMs) have been recognised as a promising stem cell resource, their therapeutic potential for wound healing has not been widely investigated. In this study, we evaluated the therapeutic potential of AMMs using a diabetic mouse wound model. Quantitative real-time PCR and ELISA results revealed that the angiogenic factors, IGF-1, EGF and IL-8 were markedly upregulated in AMMs when compared with adipose-derived mesenchymal stem cells (ADMs) and dermal fibroblasts. In vitro scratch wound assays also showed that AMM-derived conditioned media (CM) significantly accelerated wound closure. Diabetic mice were generated using streptozotocin and wounds were created by skin excision, followed by AMM transplantation. AMM transplantation significantly promoted wound healing and increased re-epithelialization and cellularity. Notably, transplanted AMMs exhibited high engraftment rates and expressed keratinocyte-specific proteins and cytokeratin in the wound area, indicating a direct contribution to cutaneous closure. Taken together, these data suggest that AMMs possess considerable therapeutic potential for chronic wounds through the secretion of angiogenic factors and enhanced engraftment/differentiation capabilities.     

IDH2 deficiency impairs cutaneous wound healing via ROS-dependent apoptosis

Dermal fibroblasts are mesenchymal cells found between the skin epidermis and subcutaneous tissue that play a pivotal role in cutaneous wound healing by synthesizing fibronectin (a component of the extracellular matrix), secreting angiogenesis factors, and generating strong contractile forces. In wound healing, low concentrations of reactive oxygen species (ROS) are essential in combating invading microorganisms and in cell-survival signaling. However, excessive ROS production impairs fibroblasts. Mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) is a key enzyme that regulates the mitochondrial redox balance and reduces oxidative stress-induced cell injury through the generation of NADPH. In the present study, the downregulation of IDH2 expression resulted in an increase in cell apoptosis in mouse skin through ROS-dependent ATM-mediated p53 signaling. IDH2 deficiency also delayed cutaneous wound healing in mice and impaired dermal fibroblast function. Furthermore, pretreatment with the mitochondria-targeted antioxidant mito-TEMPO alleviated the apoptosis induced by IDH2 deficiency both in vitro and in vivo. Together, our findings highlight the role of IDH2 in cutaneous wound healing in association with mitochondrial ROS.     

Alcohol extract of Echinacea pallida reverses stress-delayed wound healing in mice

Healing of open skin wounds begins with an inflammatory response. Restraint stress has been well documented to delay wound closure, partially via glucocorticoid (GC)-mediated immunosuppression of inflammation. Echinacea, a popular herbal immunomodulator, is purported to be beneficial for wound healing. To test the hypothesis, an alcohol extract of E. pallida was administrated orally to mice for 3 days prior to, and 4 days post wounding with a dermal biopsy on the dorsum. Concominantly, mice were exposed to 3 cycles of daily restraint stress prior to, and 4 cycles post wounding. Echinacea accelerated wound closure in the stressed mice, but had no apparent wound healing effect for the non-stressed mice when compared to their respective controls. To test if the positive healing effect is through modulation of GC release, plasma corticosterone concentrations were measured in unwounded mice treated with restraint stress and the herbal extract for 4 days. Plasma GC in restraint stressed mice gavaged with Echinacea was not different from mice treated with restraint only, but was increased compared to the vehicle control. This data suggests that the improved wound healing effect of Echinacea in stressed mice is not mediated through modulation of GC signaling.     

Heme Oxygenase-1 Accelerates Cutaneous Wound Healing in Mice

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild type mice, maximal expression of HO-1 in the skin was observed on the 2^nd and 3^rd days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer.     

Nod-Like Receptor Protein-3 Inflammasome Plays an Important Role during Early Stages of Wound Healing

The Nod-like receptor protein (NLRP)-3 inflammasome/IL-1β pathway is involved in the pathogenesis of various inflammatory skin diseases, but its biological role in wound healing remains to be elucidated. Since inflammation is typically thought to impede healing, we hypothesized that loss of NLRP-3 activity would result in a downregulated inflammatory response and accelerated wound healing. NLRP-3 null mice, caspase-1 null mice and C57Bl/6 wild type control mice (WT) received four 8 mm excisional cutaneous wounds; inflammation and healing were assessed during the early stage of wound healing. Consistent with our hypothesis, wounds from NLRP-3 null and caspase-1 null mice contained lower levels of the pro-inflammatory cytokines IL-1β and TNF-α compared to WT mice and had reduced neutrophil and macrophage accumulation. Contrary to our hypothesis, re-epithelialization, granulation tissue formation, and angiogenesis were delayed in NLRP-3 null mice and caspase-1 null mice compared to WT mice, indicating that NLRP-3 signaling is important for early events in wound healing. Topical treatment of excisional wounds with recombinant IL-1β partially restored granulation tissue formation in wounds of NLRP-3 null mice, confirming the importance of NLRP-3-dependent IL-1β production during early wound healing. Despite the improvement in healing, angiogenesis and levels of the pro-angiogenic growth factor VEGF were further reduced in IL-1β treated wounds, suggesting that IL-1β has a negative effect on angiogenesis and that NLRP-3 promotes angiogenesis in an IL-1β-independent manner. These findings indicate that the NLRP-3 inflammasome contributes to the early inflammatory phase following skin wounding and is important for efficient healing.     

CD44-dependent inflammation,fibrogenesis, and collagenolysis regulates extracellular matrix remodeling and tensile strength during cutaneous wound healing

Cutaneous wound healing consists of three main phases: inflammation, re-epithelialization, and tissue remodeling. During normal wound healing, these processes are tightly regulated to allow restoration of skin function and biomechanics. In many instances, healing leads to an excess accumulation of fibrillar collagen (the principal protein found in the extracellular matrix - ECM), and the formation of scar tissue, which has compromised biomechanics, tested using ramp to failure tests, compared to normal skin (Corr and Hart, 2013 [1]). Alterations in collagen accumulation and architecture have been attributed to the reduced tensile strength found in scar tissue (Brenda et al., 1999; Eleswarapu et al., 2011). Defining mechanisms that govern cellular functionality and ECM remodeling are vital to understanding normal versus pathological healing and developing approaches to prevent scarring. CD44 is a cell surface adhesion receptor expressed on nearly all cell types present in dermis. Although CD44 has been implicated in an array of inflammatory and fibrotic processes such as leukocyte recruitment, T-cell extravasation, and hyaluronic acid (the principal glycosaminoglycan found in the ECM) metabolism, the role of CD44 in cutaneous wound healing and scarring remains unknown. We demonstrate that in an excisional biopsy punch wound healing model, CD44-null mice have increased inflammatory and reduced fibrogenic responses during early phases of wound healing. At wound closure, CD44-null mice exhibit reduced collagen degradation leading to increased accumulation of fibrillar collagen, which persists after wound closure leading to reduced tensile strength resulting in a more severe scarring phenotype compared to WT mice. These data indicate that CD44 plays a previously unknown role in fibrillar collagen accumulation and wound healing during the injury response.     

SPARC Deficiency Results in Improved Surgical Survival in a Novel Mouse Model of Glaucoma Filtration Surgery

Glaucoma is a disease frequently associated with elevated intraocular pressure that can be alleviated by filtration surgery. However, the post-operative subconjunctival scarring response which blocks filtration efficiency is a major hurdle to the achievement of long-term surgical success. Current application of anti-proliferatives to modulate the scarring response is not ideal as these often give rise to sight-threatening complications. SPARC (secreted protein, acidic and rich in cysteine) is a matricellular protein involved in extracellular matrix (ECM) production and organization. In this study, we investigated post-operative surgical wound survival in an experimental glaucoma filtration model in SPARC-null mice. Loss of SPARC resulted in a marked (87.5%) surgical wound survival rate compared to 0% in wild-type (WT) counterparts. The larger SPARC-null wounds implied that aqueous filtration through the subconjunctival space was more efficient in comparison to WT wounds. The pronounced increase in both surgical survival and filtration efficiency was associated with a less collagenous ECM, smaller collagen fibril diameter, and a loosely-organized subconjunctival matrix in the SPARC-null wounds. In contrast, WT wounds exhibited a densely packed collagenous ECM with no evidence of filtration capacity. Immunolocalization assays confirmed the accumulation of ECM proteins in the WT but not in the SPARC-null wounds. The observations in vivo were corroborated by complementary data performed on WT and SPARC-null conjunctival fibroblasts in vitro. These findings indicate that depletion of SPARC bestows an inherent change in post-operative ECM remodeling to favor wound maintenance. The evidence presented in this report is strongly supportive for the targeting of SPARC to increase the success of glaucoma filtration surgery.     

Spatiotemporal expression of periostin during skin development and incisional wound healing: lessons for human fibrotic scar formation

Differentiation of fibroblasts to myofibroblasts and collagen fibrillogenesis are two processes essential for normal cutaneous development and repair, but their misregulation also underlies skin-associated fibrosis. Periostin is a matricellular protein normally expressed in adult skin, but its role in skin organogenesis, incisional wound healing and skin pathology has yet to be investigated in any depth. Using C57/BL6 mouse skin as model, we first investigated periostin protein and mRNA spatiotemporal expression and distribution during development and after incisional wounding. Secondarily we assessed whether periostin is expressed in human skin pathologies, including keloid and hypertrophic scars, psoriasis and atopic dermatitis. During development, periostin is expressed in the dermis, basement membrane and hair follicles from embryonic through neonatal stages and in the dermis and hair follicle only in adult. In situ hybridization demonstrated that dermal fibroblasts and basal keratinocytes express periostin mRNA. After incisional wounding, periostin becomes re-expressed in the basement membrane within the dermal-epidermal junction at the wound edge re-establishing the embryonic deposition pattern present in the adult. Analysis of periostin expression in human pathologies demonstrated that it is over-expressed in keloid and hypertrophic scars, atopic dermatitis, but is largely absent from sites of inflammation and inflammatory conditions such as psoriasis. Furthermore, in vitro we demonstrated that periostin is a transforming growth factor beta 1 inducible gene in human dermal fibroblasts. We conclude that periostin is an important ECM component during development, in wound healing and is strongly associated with pathological skin remodeling.     

The role of nuclear hormone receptors in cutaneous wound repair

The cutaneous wound repair process involves balancing a dynamic series of events ranging from inflammation, oxidative stress, cell migration, proliferation, survival and differentiation. A complex series of secreted trophic factors, cytokines, surface and intracellular proteins are expressed in a temporospatial manner to restore skin integrity after wounding. Impaired initiation, maintenance or termination of the tissue repair processes can lead to perturbed healing, necrosis, fibrosis or even cancer. Nuclear hormone receptors (NHRs) in the cutaneous environment regulate tissue repair processes such as fibroplasia and angiogenesis. Defects in functional NHRs and their ligands are associated with the clinical phenotypes of chronic non‐healing wounds and skin endocrine disorders. The functional relationship between NHRs and skin niche cells such as epidermal keratinocytes and dermal fibroblasts is pivotal for successful wound closure and permanent repair. The aim of this review is to delineate the cutaneous effects and cross‐talk of various nuclear receptors upon injury towards functional tissue restoration. Copyright © 2014 John Wiley & Sons, Ltd.     

Characterization of Smad3 knockout mouse derived skin cells

TGF-β plays an important role in skin wound healing process, in which Smad3 acts as a signaling molecule. Smad3 knockout mice exhibit enhanced wound healing and less inflammatory process, but the intrinsic properties of the mouse derived skin cells are generally unexplored. The purpose of this study is to characterize the biological behavior of skin cells derived from Smad3 knockout mice and thus to define the mechanism of this particular wound healing process. Keratinocytes and dermal fibroblasts were harvested from the skin of Smad3 knockout (Smad3 KO) and wild-type (WT) mice and in vitro cultured for one and two passages for various experiments. The results showed that KO mouse serum contained significantly higher levels of TGF-β1 and lower level of IL-6 and IL-10 than WT mouse serum (p < 0.05), which were also supported by the same findings of more TGF-β1 and less IL-6 and IL-10 in the supernatant of cultured KO dermal fibroblasts than those of WT cells (p < 0.05). At gene levels, IL-6, IL-10, and TGF-β1 were significantly less expressed in KO fibroblasts than in WT fibroblasts (p < 0.05). In addition, KO dermal fibroblasts also exhibited stronger migration and proliferation potentials than WT fibroblasts (p < 0.05). Moreover, both KO fibroblasts and keratinocytes showed higher colony-forming efficiency than WT counterparts with significant difference (p < 0.05). These findings indicate that both systemic factors and intrinsic properties of skin cells contribute to enhanced wound healing and less inflammatory reaction observed in Smad3 knock-out mice.