Skin Dermal Fibroblasts Research Articles

Effects of Intense Pulsed Light on the Biological Properties and Ultrastructure of Skin Dermal Fibroblasts: Potential Roles in Photoaging

The aim of this study was to evaluate the effects of intense pulsed light (IPL) irradiation on skin fibroblasts in vitro, as well as to explore the biomolecular mechanisms and ultrastructure changes of the IPL effect. IPL is frequently used to improve telangiectasias, lentigos, and skin texture. Clinical studies have demonstrated that IPL has significant photorejuvenation effects on photoaged skin. However, the biomolecular mechanisms underlying the photorejuvenation of IPL treatment remain largely unknown. Human skin fibroblasts cultured in vitro were irradiated with IPL via double pulses of 4 ms and 6 ms with a pulse interval of 20 ms and fluences of 18, 23, 28, and 33 J/cm(2). Twenty-four hours after irradiation, cell viability, cell cycle distribution, mRNA and protein levels of procollagen (types I and III), and changes of ultrastructure were evaluated using methyl thiazole tetrazolium (MTT) assay, flow cytometry, real-time reverse transcriptase polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assays (ELISA), and transmission electron microscopy, respectively. IPL irradiation resulted in the improvement of cell viability of skin fibroblasts in a dose-dependent manner. There were obvious changes in ultrastructure of fibroblasts, compared with the control group. The percentage of fibroblasts at the S and G(2)/M stage of the cell cycle was significantly higher, whereas the percentage at the G(0)/G(1) stage was lower. IPL irradiation increased the mRNA level of collagen I considerably, to 123%, 154%, 172%, and 141% of the control, and that of collagen III to 120%, 141%, 164%, and 132%. IPL irradiation was able to upregulate expression of collagen (types I and III) at the mRNA and protein level. The present study demonstrates that IPL irradiation imparts stimulatory effects on skin fibroblasts in vitro. This provides valuable evidence of the photorejuvenation effect of IPL in vivo.

Localization, Age- and Site-Dependent Expression, and Regulation of 11β-Hydroxysteroid Dehydrogenase Type 1 in Skin

Glucocorticoids (GCs) are highly detrimental to skin integrity and function both when applied topically for anti-inflammatory treatments and during conditions of circulating excess, e.g., Cushing's syndrome. Within target tissues, GC availability is regulated at a prereceptor level, independently of systemic levels, by isozymes of 11β-hydroxysteroid dehydrogenase (11β-HSD) that interconvert active cortisol and inactive cortisone. Many of the adverse effects of GCs on skin are also reminiscent of the natural aging process. 11β-HSD1 (which activates cortisol), but not 11β-HSD2 (which inactivates cortisol), was expressed in epidermal keratinocytes and dermal fibroblasts in human skin and also in outer hair follicle root sheath cells in murine skin. 11β-HSD1 activity was present ex vivo in both species and increased with age in human skin tissue explants. In primary human dermal fibroblasts (HDF) from both photoprotected and photoexposed sites, 11β-HSD1 also increased with donor age. Additionally, photoexposed HDF displayed higher 11β-HSD1 mRNA expression than donor-matched photoprotected HDF. GC treatment of HDF caused upregulation of 11β-HSD1 mRNA levels independent of donor age or site. The age- and site-associated increase in dermal 11β-HSD1, and the ensuing increased local GC activation, may contribute to the adverse changes in skin morphology and function associated with chronological aging and photoaging.

Interpenetrated Chitosan-Poly(Acrylic Acid-Co-Acrylamide) Hydrogels. Synthesis, Characterization and Sustained Protein Release Studies

Interpenetrated polymer networks of chitosan (CHI), polyacrylic acid (PAA) and polyacrylamide (PAM) were prepared by free radical polymerization. These hydrogels were either washed with double distilled water (CHI/PAA/PAM) A or hydrolyzed with 1M sodium hydroxide (NaOH), (CHI/PAA/PAM) S. Both types of hydrogels were characterized by infrared spectroscopy, microstructural techniques and compressive mechanical testing. Finally, hydrogels were loaded with bovine serum albumin (BSA) and release followed at different pHs. Infrared spectra analysis showed correspondence between hydrogels and monomer feed compositions. Hydrolyzed hydrogels, had increased water content and pH swelling dependence. Compression modulus of swelled hydrolyzed hydrogels decreased with increasing equilibrium water content. Higher BSA loadings were achieved on hydrolyzed hydrogels due to their high water content and porosity. Protein release from hydrogels was low (≤ 20% after 10 hours) at pH 1.2, but sustained release was observed at pH 6.8 and 7.4. The integrity of the protein released at 6.8 and 7.4 by hydrolyzed hydrogels was unaffected. The hydrogles showed no cytotoxic effects on human skin dermal fibroblasts as determined by MTT assay except for two compositions of (CHI/PAA/PAM) A samples, which after seven days presented a viability lower than 80% respect to the control.

Akt‐mediated mechanotransduction in murine fibroblasts during hypertrophic scar formation

Although numerous factors are implicated in skin fibrosis, the exact pathophysiology of hypertrophic scarring remains unknown. We recently demonstrated that mechanical force initiates hypertrophic scar formation in a murine model, potentially enhancing cellular survival through Akt. Here, we specifically examined Akt-mediated mechanotransduction in fibroblasts using both strain culture systems and our murine scar model. In vitro, static strain increased fibroblast motility, an effect blocked by wortmannin (a phosphoinositide-3-kinase/Akt inhibitor). We also demonstrated that high-frequency cyclic strain was more effective at inducing Akt phosphorylation than low frequency or static strain. In vivo, Akt phosphorylation was induced by mechanical loading of dermal fibroblasts in both unwounded and wounded murine skin. Mechanically loaded scars also exhibited strong expression of α-smooth muscle actin, a putative marker of pathologic scar formation. In vivo inhibition of Akt increased apoptosis but did not significantly abrogate hypertrophic scar development. These data suggest that although Akt signaling is activated in fibroblasts during mechanical loading of skin, this is not the critical pathway in hypertrophic scar formation. Future studies are needed to fully elucidate the critical mechanotransduction components and pathways which activate skin fibrosis.

Fibroblast Response to Gadolinium

The purpose of this study was to assess the effects of gadolinium (Gd3+), provided as gadolinium chloride, on fibroblast function. Human dermal fibroblasts in monolayer culture and intact skin in organ culture were exposed to the lanthanide metal (1-20 μM). Increased proliferation was observed, in association with upregulation of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1, without an apparent increase in production of type I procollagen. A platelet-derived growth factor (PDGF) receptor-blocking antibody inhibited fibroblast proliferation in response to Gd3+ as did inhibitors of signaling pathways--that is, mitogen-activated protein kinase and phosphatidylinositol-3 kinase pathways--that are activated by PDGF. The responses to gadolinium chloride are similar to responses previously seen with chelated Gd3+ in clinically used magnetic resonance imaging contrast agents. Fibroblast responses appear to reflect Gd3+-induced PDGF receptor activation and downstream signaling. Increased dermal fibroblast proliferation in conjunction with effects on matrix metalloproteinase-1 and tissue inhibitor of metalloproteinases-1 could contribute to the fibroplastic/fibrotic changes seen in the lesional skin of individuals with nephrogenic systemic fibrosis.

Hyaluronan Used for Intradermal Injections Is Incorporated into the Pericellular Matrix and Promotes Proliferation in Human Skin Fibroblasts in vitro

Background: Over the past decade injectable hyaluronan (HA) formulations have been widely used to decrease the visibility of skin aging. However, little basic research has been performed to address their effect on dermal skin fibroblasts. Objective: The aim of the present study was to investigate whether human skin fibroblasts are affected by exogenous non-cross-linked HA with respect to proliferation, migration and extracellular matrix composition. Methods: The effect of a non-cross-linked HA on proliferation, migration and gene expression of human dermal fibroblasts was determined. Furthermore, affinity histochemistry of pericellular HA was performed. Results: Proliferation was significantly stimulated by HA whereas migration was not affected. Importantly, exogenous HA was incorporated into fine HA filaments of the pericellular fibroblast matrix. Conclusion: This is the first evidence that a HA formulation used in humans for cosmetic reasons stimulates fibroblast proliferation and is utilized to assemble a supramolecular pericellular structure.

Eotaxins and CCR3 Interaction Regulates the Th2 Environment of Cutaneous T-Cell Lymphoma

CC chemokine receptor 3 (CCR3), the sole receptor for eotaxins, is expressed on eosinophils and T helper type 2 (Th2) cells. In Hodgkin's disease, eotaxin-1 secreted by fibroblasts collects Th2 cells and eosinophils within the tissue. Similarly, many Th2 cells infiltrate the lesional skin of cutaneous T-cell lymphoma (CTCL). In this study, we investigated the role of eotaxins in the development of the Th2 environment of CTCL. We revealed that fibroblasts from lesional skin of CTCL expressed higher amounts of eotaxin-3 messenger RNA (mRNA) compared with those from normal skin. Lesional skin of CTCL at advanced stages contained significantly higher levels of eotaxin-3 and CCR3 mRNA, compared with early stages of CTCL. IL-4 mRNA was expressed in some cases at advanced stages. Immunohistochemistry revealed that keratinocytes, endothelial cells, and dermal fibroblasts in lesional skin of CTCL showed a stronger expression of eotaxin-3 than did normal skin. CCR3(+) lymphocytes and IL-4 expression were observed in some cases of advanced CTCL. Furthermore, both serum eotaxin-3 and eotaxin-1 levels of CTCL patients at advanced stages were significantly higher than those of healthy individuals. The concentrations of these chemokines correlated with serum soluble IL-2 receptor levels. These results suggest that interaction of eotaxins and CCR3 regulates the Th2-dominant tumor environment, which is closely related to the development of CTCL.

Ellagitannins from Phyllanthus muellerianus: geraniin stimulates keratinocytes differentiation and collagen synthesis of skin dermal fibroblasts – new concept for improved wound-healing

Ellagitannins from Phyllanthus muellerianus: geraniin stimulates keratinocytes differentiation and collagen synthesis of skin dermal fibroblasts – new concept for improved wound-healing

Sesamol inhibits UVB-induced ROS generation and subsequent oxidative damage in cultured human skin dermal fibroblasts

The exposure of cells to ultraviolet B radiation (UVB) can induce the production of reactive oxygen species (ROS) which damage cellular components. Free radical scavengers and antioxidants can interfere with the production of ROS. We studied cytotoxicity, intracellular ROS levels, lipid peroxidation, antioxidant status and oxidative DNA damage in cultured human skin dermal fibroblast adult cells (HDFa) exposed to UVB in the presence of sesamol, a natural phenolic compound. The levels of cytotoxicity, intracellular ROS, lipid peroxidation, oxidative DNA damage and apoptotic morphological changes were significantly increased in UVB irradiated HDFa cells. We also observed that the activities of enzymatic antioxidants (superoxide dismutase, catalase and glutathione peroxidase) and the levels of non-enzymatic antioxidant status (GSH) were significantly decreased in UVB irradiated cells. On the other hand, sesamol pretreatment significantly decreased cytotoxicity, intracellular ROS, lipid peroxidation, oxidative DNA damage and apoptotic morphological changes in sesamol-pretreated and UVB-irradiated HDFa cells. We have also observed increased enzymatic and non-enzymatic antioxidants status in sesamol plus UVB-irradiated cells. Among the different doses tested, 80 μM of sesamol shows maximum protection for UVB-induced oxidative damage. In conclusion, UVB-induced ROS formation, cell fatality, lipid peroxidation, antioxidant depletion and oxidative DNA damage in HDFa cells is inhibited by sesamol, which, probably through its ROS scavenging activity.

Cigarette smoke-induced Egr-1 represses TβR-II expression in human skin dermal fibroblasts

Tobacco smoking is one of the many factors that contribute to premature skin aging, but the exact mechanism by which smoking induces facial wrinkling is still poorly understood. To investigate the regulatory potential of early growth response-1 (Egr-1) on the premature skin aging by smoking, this study examined the hypothesis that cigarette smoke-induced Egr-1 represses TβR-II expression in human skin dermal fibroblasts (HSDFs). The protein and mRNA expressions of Egr-1 and TβR-II were detected using Western blot and real-time RTPCR in HSDFs after exposure to cigarette smoke extract (CSE). Egr-1 and TβR-II promoter activities were analyzed in CSE-exposed fibroblasts using luciferase assay. TβR-II promoter activity was also evaluated in HSDFs to be transfected with Egr-1 overexpression vector. To investigative Egr-1-specific effects, we utilized Egr-1 small interfering RNA (siRNA) to inhibit Egr-1 expression. The expressions of Egr-1 protein and mRNA were increased in a time and dose-dependent manner. CSE also induced Egr-1 at the transcription level. Egr-1 was induced though phosphorylation of Erk1/2 after CSE exposure in HSDFs. We also observed the immunostained Egr-1 proteins were mainly localized from the cytoplasm to the nucleus after CSE treatment by immunocytochemical analyzes. Furthermore, TβR-II protein and mRNA levels were decreased in a dose-dependent manner by CSE and TβR-II promoter activity was significantly repressed by CSE. HSDFs transfected with Egr-1 overexpression vector showed significantly reduced TβR-II promoter activity. In addition, TβR-II mRNA levels were upregulated in HSDFs transfected with Egr-1 siRNA, suggesting that TβR-II expressional downregulation by CSE is induced via an Egr-1-dependent mechanism. This study suggests that the downregulation of TβR-II expression by cigarette smoke-induced Egr-1 may contribute to smoking-induced premature skin aging.

The Skin Cancer Chemotherapeutic Agent Ingenol-3-Angelate (PEP005) Is a Substrate for the Epidermal Multidrug Transporter (ABCB1) and Targets Tumor Vasculature

Ingenol-3-angelate (Ing3A), extracted from Euphorbia peplus, is currently in clinical trials for eradicating basal cell carcinoma, actinic keratosis, and squamous cell carcinoma (SCC) in situ by topical application. Although structurally related to phorbol esters and a protein kinase C activator, topical Ing3A, but not phorbol 12-myristate 13-acetate (PMA), inhibited the growth of subcutaneous tumors derived from PAM212 (mouse SCC) and B16 (mouse melanoma). Ing3A and PMA both induced acute neutrophilic inflammation on mouse skin, but only Ing3A caused subcutaneous hemorrhage and vascular damage. Both Ing3A and PMA activated extracellular signal-regulated kinase 1/2 (ERK1/2) in epidermis, but Ing3A also activated ERK1/2 in skin dermal fibroblasts and endothelial cells. Pretreatment with topical cyclosporin A (CsA), verapamil, or XR9576, modulators of P-glycoprotein (P-gp), prevented Ing3A-induced hemorrhage but not neutrophil infiltration. CsA also impaired the anticancer activity of Ing3A, whereas the anti-inflammatory dexamethasone did not. Ing3A, but not PMA, blocked photoaffinity labeling of human P-gp with [(125)I]iodoaryazidoprazosin and inhibited P-gp-mediated drug resistance to HCT-15 cells. The intracellular levels of Ing3A were significantly lower in P-gp-expressing cells, and treatment with XR9576 increased the levels to those of cells that do not express P-gp, showing that Ing3A binds to and is transported by P-gp. Taken together, our results suggest that P-gp-mediated absorptive transport, dermal penetration, and vascular damage contribute to the anticancer activity of Ing3A in vivo.

Abstract 1777: The skin cancer chemotherapeutic agent PEP005 is a substrate for the epidermal multidrug transporter (ABCB1) and targets tumor vasculature

Abstract Ingenol-3-angelate (PEP005), extracted from Euphorbia peplus, is currently in clinical trials for eradicating basal cell carcinoma (BCC), actinic keratosis and squamous cell carcinoma (SCC) in situ by topical application. Although structurally related to phorbol esters and a PKC activator, topical PEP005, but not phorbol 12-myristate 13-acetate (PMA), inhibited the growth of subcutaneous tumors derived from PAM212 (mouse SCC) and B16 (mouse melanoma). PEP005 and PMA both induced acute neutrophilic inflammation on mouse skin, but only PEP005 caused subcutaneous hemorrhage and vascular damage. Both PEP005 and PMA activated Erk1/2 in epidermis, but PEP005 also activated Erk1/2 in skin dermal fibroblasts and endothelial cells. Pretreatment with topical cyclosporin A (CsA), verapamil or XR9576, modulators of P-glycoprotein (P-gp), prevented PEP005-induced hemorrhage but not neutrophil infiltration. CsA also impaired Ing3A's anti-cancer activity while the anti-inflammatory dexamethasone did not. PEP005, but not PMA, blocked photoaffinity labeling of human P-gp with [125I]-Iodoaryazidoprazosin and inhibited P-gp mediated drug resistance to HCT-15 cells. The intracellular levels of PEP005 were significantly lower in P-gp expressing cells and treatment with XR9576 increased the levels to those of cells that do not express P-gp, demonstrating that PEP005 binds to and is transported by P-gp. Taken together, our results suggest that P-gp mediated absorptive transport, dermal penetration and vascular damage contribute to the anti-cancer activity of PEP005 in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1777.

IL-18 Downregulates Collagen Production in Human Dermal Fibroblasts via the ERK Pathway

Excessive accumulation of collagen contributes to the fibrotic process. Several experimental studies have shown that IFN-gamma is effective in preventing fibrogenesis. IL-18, originally identified as an IFN-gamma-inducing factor, is a key mediator of inflammation and host defense responses. In this study, we investigated the regulatory effect of IL-18 on the expression of type I and III collagen genes in dermal fibroblasts. The exposure of human dermal fibroblasts (HDFs) to IL-18 resulted in a reduction of collagen gene expression and production. Also, IL-18 inhibited the fibrogenic cytokine transforming growth factor (TGF)-beta-induced collagen gene expression. Next, to determine the molecular mechanism involved in this regulation, we showed that IL-18-regulated collagen expression was blocked by small interfering RNA (siRNA)-mediated Ets-1 knockdown. Furthermore, we showed that IL-18 induced phosphorylation of extracellular signal-regulated kinase (ERK) within 10 minutes and that the ERK inhibitor PD98059 blocked the inhibitory effect of IL-18. IL-18 also inhibited the production of collagen in systemic sclerosis (SSc) dermal fibroblasts. Our data indicate that IL-18 downregulates collagen production in HDF directly via Ets-1 and the ERK pathway, suggesting that IL-18 may exert antifibrotic activities in dermal fibroblasts.

Nicotine induces the expression of early growth response‐1 in human skin dermal fibroblasts

Egr-1 is known to play an important role in cell growth, differentiation, and cell survival. Human skin dermal fibroblasts (HSDF) may be an important target for adverse effects of tobacco components. However, nicotinic effect for early growth response-1 (Egr-1) expression is unknown in HSDF. Cytotoxicity of nicotine was assessed by cell viability test in HSDF. The expression of Egr-1 protein and mRNA after nicotine treatment was evaluated by Western blotting and real-time reverse transcription-polymerase chain reaction. We also measured the promoter activity of Egr-1 in HSDF after nicotine exposure. Early growth response-1 protein and mRNA levels were increased in dermal fibroblasts exposed to nicotine. Nicotine treatment stimulated the promoter activity of Egr-1 in cultured human fibroblasts. In this study, we demonstrate that Egr-1 expression is markedly induced in HSDF after exposure to nicotine.

Intense Pulsed Light Effects on the Expression of Extracellular Matrix Proteins and Transforming Growth Factor Beta-1 in Skin Dermal Fibroblasts Cultured within Contracted Collagen Lattices

Intense Pulsed Light Effects on the Expression of Extracellular Matrix Proteins and Transforming Growth Factor Beta-1 in Skin Dermal Fibroblasts Cultured within Contracted Collagen Lattices

Fucoidan-Vitamin C complex suppresses tumor invasion through the basement membrane, with scarce injuries to normal or tumor cells, via decreases in oxidative stress and matrix metalloproteinases

Fucoidan (Fucdn) and vitamin C (VC) were saturatedly dissolved in water and lyophilized and thoroughly ethanol-rinsed until no detection for supernatant vitamin C to form the Fucdn-VC (1:0.23 wt/wt) inclusion body (Fucdn-VC-IB). Fucdn-VC-IB increased not only VC-stabilizing at 37 degrees C, but also hydroxyl-radical scavenging as shown by ESR/spin-trap method, more markedly than a mere mixture of Fucdn:VC (1:0.23 wt/wt). Invasion of human fibrosarcoma cells HT-1080 through the basement membrane was repressed by Fucdn-VC-IB of non-cytotoxic concentrations without significant inhibition to human skin dermal fibroblasts DUMS-16 cells. Fucdn-VC-IB suppressed the invasiveness-related gelatinases MMP-2/9, and diminished reactive oxygen species inside the cytoplasm around the nucleus, in HT-1080 cells as shown by electrophoretic zymography and the redox indicator NBT assay, respectively. Thus Fucdn-VC-IB markedly exhibits antioxidant and MMP-suppressing activities and preferentially inhibited tumor invasion without cytotoxicity to normal cells, and is suggested as a potent tumor-invasion suppressor.

The Relationship between the Aging- and Photo-Dependent T414G Mitochondrial DNA Mutation with Cellular Senescence and Reactive Oxygen Species Production in Cultured Skin Fibroblasts

Mutations in the mitochondrial genome (mtDNA) are thought to be one of the causes of age-dependent cellular decline through their detrimental effects on respiration or reactive oxygen species (ROS) production. However, for many mutations, this link has not been clearly established. This study aimed to further investigate the phenotypic importance of a T414G mutation within the control region of mtDNA, previously shown to accumulate in both chronologically and photoaged human skin. We demonstrate that during dermal skin fibroblast replication in vitro in five separate cultures obtained from elderly individuals, the T414G mutant load can either increase or decrease during progressive cell division, implying the absence of consistent selection against the mutation in this context. In support of this, by utilizing a cell-sorting approach, we demonstrate that the level of the T414G mutation does not directly correlate with increased or decreased mtDNA copy number, or markers of cellular ageing including lipofuscin accumulation or ROS production. By consequence, the mutation can be distributed with a bias towards either the proliferating or senescent cell populations depending on the cell line. In conclusion, we propose that this particular mutation may have little effect on ROS production and the onset of cellular senescence in cultured fibroblasts.

Periostin localizes to cells in normal skin, but is associated with the extracellular matrix during wound repair

Epidermal tissue repair represents a complex series of temporal and dynamic events resulting in wound closure. Matricellular proteins, not normally expressed in quiescent adult tissues, play a pivotal role in wound repair and associated extracellular matrix remodeling by modulating the adhesion, migration, intracellular signaling, and gene expression of inflammatory cells, pericytes, fibroblasts and keratinocytes. Several matricellular proteins show temporal expression during dermal wound repair, but the expression pattern of the recently identified matricellular protein, periostin, has not yet been characterized. The primary aim of this study was to assess whether periostin protein is present in healthy human skin or in pathological remodeling (Nevus). The second aim was to determine if periostin is expressed during dermal wound repair. Using immunohistochemistry, periostin reactivity was detected in the keratinocytes, basal lamina, and dermal fibroblasts in healthy human skin. In pathological nevus samples, periostin was present in the extracellular matrix. In excisional wounds in mice, periostin protein was first detected in the granulation tissue at day 3, with levels peaking at day 7. Periostin protein co-localized with α-smooth muscle actin-positive cells and keratinocytes, but not CD68 positive inflammatory cells. We conclude that periostin is normally expressed at the cellular level in human and murine skin, but additionally becomes extracellular during tissue remodeling. Periostin may represent a new therapeutic target for modulating the wound repair process.

Intense Pulsed Light Effects on the Expression of Extracellular Matrix Proteins and Transforming Growth Factor Beta-1 in Skin Dermal Fibroblasts Cultured within Contracted Collagen Lattices

Emerging clinical evidence suggests that intense pulsed light (IPL) treatment may exert some beneficial effects on photoaged skin. The molecular mechanisms underlying this IPL effect have not been fully elucidated. To examine the effects of IPL irradiation on normal human dermal fibroblasts grown in contracted collagen lattices. Human skin fibroblasts cultured in contracted collagen lattices were irradiated with IPL with triple pulses of 7 ms with a pulse interval of 70 ms and fluences of 20, 50, and 75 J/cm(2). Twenty-four hours after the irradiation, cell viability, messenger RNA (mRNA), and protein levels of extracellular matrix proteins (e.g., collagen I, collagen III, and fibronectin) and transforming growth factor beta-1 (TGF-beta1) were evaluated using dye exclusion, real-time reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay, respectively. A dose-dependent increase in viable cells was demonstrated after the IPL irradiation. There was no significant change in mRNA levels of collagen I and fibronectin. Upregulated expression of collagen III and TGF-beta1 in dermal fibroblasts was verified. The analytical results presented here provide a potential mechanistic explanation for the mechanism of clinical photorejuvenation effects of IPL that involves the increase of extracellular matrix construction by upregulating the gene expressions of collagen III and TGF-beta1.

Wound‐healing factors secreted by epidermal keratinocytes and dermal fibroblasts in skin substitutes

Full-skin substitutes, epidermal substitutes, and dermal substitutes are currently being used to heal deep burns and chronic ulcers. In this study, we investigated which wound-healing mediators are released from these constructs and whether keratinocyte-fibroblast interactions are involved. Autologous skin substitutes were constructed from human keratinocytes, fibroblasts, and acellular donor dermis. Full-thickness skin was used to represent an autograft. Secretion of wound-healing mediators was investigated by means of protein array, enzyme-linked immunosorbent assay, neutralizing antibodies, and conditioned culture supernatants. Full-skin substitutes and autografts produce high amounts of inflammatory/angiogenic mediators (IL-6, CCL2, CXCL1, CXCL8, and sST2). Epidermal and dermal substitutes produced less of these proteins. Epidermal-derived proinflammatory cytokines interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) were found to mediate synergistically the secretion of these wound-healing mediators (with the exception of sST2) from fibroblasts in dermal substitutes. The secretion of proinflammatory cytokines (IL-1alpha, TNF-alpha), chemokine/mitogen (CCL5) and angiogenic factor (vascular endothelial growth factor) by epidermal substitutes and tissue remodeling factors (tissue inhibitor of metalloproteinase-2, hepatocyte growth factor) by dermal substitutes was not influenced by keratinocyte-fibroblast interactions. The full-skin substitute has a greater potential to stimulate wound healing than epidermal or dermal substitutes. Both epidermal-derived IL-1alpha and TNF-alpha are required to trigger the release of dermal-derived inflammatory/angiogenic mediators from skin substitutes.