Maltodextrin/ascorbic acid stimulates wound closure by increasing collagen turnover and TGF-β1 expression in vitro and changing the stage of inflammation from chronic to acute in vivo

It has been reported that carbohydrates confer physicochemical properties to the wound environment that improves tissue repair. We evaluated in vitro and in vivo wound healing during maltodextrin/ascorbic acid treatment. In a fibroblast monolayer scratch assay, we demonstrated that maltodextrin/ascorbic acid stimulated monolayer repair by increasing collagen turnover coordinately with TGF-β1 expression (rising TGF-β1 and MMP-1 expression, as well as gelatinase activity, while TIMP-1 was diminished), similar to in vivo trends. On the other hand, we observed that venous leg ulcers treated with maltodextrin/ascorbic acid diminished microorganism population and improved wound repair during a 12 week period. When maltodextrin/ascorbic acid treatment was compared with zinc oxide, almost four fold wound closure was evidenced. Tissue architecture and granulation were improved after the carbohydrate treatment also, since patients that received maltodextrin/ascorbic acid showed lower type I collagen fiber levels and increased extracellular alkaline phosphatase activity and blood vessels than those treated with zinc oxide. We hypothesize that maltodextrin/ascorbic acid treatment stimulated tissue repair of chronic wounds by changing the stage of inflammation and modifying collagen turnover directly through fibroblast response.     

A fibroblast cell line cultured from a hypertrophic scar displays selective downregulation of collagen gene expression: barely detectable messenger RNA levels of the proα1(III) chain of type III collagen

The present study was designed to investigate the expression of type I, III and VI collagens by a fibroblast cell line initiated from a hypertrophic scar. The same tissue has previously been demonstrated to display markedly elevated expression of type I and III collagen mRNAs in vivo. Unexpectedly, slot-blot and Northern hybridizations revealed a barely detectable steady-state level of pro1(III) collagen chain mRNA in cultured hypertrophic scar fibroblasts. The levels of pro1(I) and 2(VI) collagen chain mRNAs were essentially the same in fibroblasts cultured from hypertrophic scar and in fibroblasts cultured from normal skin. However, Northern blot analyses indicated that the ratio of 5.8 kb to 4.8 kb species of pro1(I) collagen mRNA was slightly reduced in fibroblasts originating from the hypertrophic scar compared to that in normal fibroblasts. When normal fibroblasts were incubated in conditioned medium from hypertrophic scar cultures, the expression of pro1(III) collagen chain mRNA decreased to a markedly lower level. Our studies suggest that collagen synthesis by fibroblasts in hypertrophic scars is stimulated by humoral factors which are active only in vivo. Furthermore, the results suggest that fibroblasts cultured from hypertrophic scar display a selective downregulation of different collagen genes and that this downregulation is exerted through an autocrine mechanism.     

Oleanolic acid regulates the proliferation and extracellular matrix of keloid fibroblasts by mediating the TGF-β1/SMAD signaling pathway

Background

Keloid (KD) is a unique pathological fibroproliferative disease that seriously affects the appearance of patients. This study investigated the effect of oleanolic acid (OA) on the proliferation of keloid fibroblasts (KFs) and the expression of extracellular matrix (ECM)-related proteins.

Methods

The proliferation of KFs was evaluated using an MTT assay. The effects of OA on intra- and extracellular levels of fibronectin (FN), procollagen I, matrix metalloproteinase-1 (MMP-1), and α-smooth muscle actin (α-SMA) were evaluated using Western blotting. To simulate the KD microenvironment, TGF-β1 was added to the serum-free culture medium, and KFs were incubated with TGF-β1 and OA for 24 h. The intra- and extracellular levels of the ECM-related proteins and the effect of OA on TGF-β1-induced phosphorylation of the SMAD2 and SMAD3 proteins were evaluated using Western blotting.

Results

OA inhibited the proliferation of KFs in a concentration- and time-dependent manner. Furthermore, OA treatment of KFs reduced the intra- and extracellular levels of FN, procollagen I, and α-SMA and increased those of MMP-1. OA also reduced TGF-β1-induced increases in the intra- and extracellular levels of FN, procollagen I, and α-SMA and increased the levels of the MMP-1 protein. Additionally, OA significantly reduced TGF-β1-induced phosphorylation of SMAD2 and SMAD3 in KFs.

Conclusions

OA inhibited KF proliferation and reduced ECM deposition through the TGF-β1/SMAD pathway, which suggests that OA may be an effective drug for the prevention and treatment of KD.     

Circulating γ/δ T cells in systemic sclerosis exhibit activated phenotype and enhance gene expression of proalpha2(I) collagen of fibroblasts

BackgroundSystemic sclerosis (SSc) is a systemic inflammatory and fibrotic disease characterized by activation of circulating T lymphocytes.ObjectiveTo determine phenotypic abnormalities of γ/δ T cells and whether γ/δ T cells influence fibroblasts activation in SSc patients.MethodsNumber and proportion of peripheral γ/δ T lymphocytes, and their expressions of cell surface molecules were evaluated by flow cytometry. Isolated γ/δ T cells were cocultured with fibroblast, and mRNA expressions of proalpha1(I) collagen and proalpha2(I) collagen (COL1A2) of fibroblasts were analyzed by real time RT-PCR. γ/δ T cell infiltrations in the skin were examined histopathologically.ResultsNo significant difference in number and proportion of γ/δ T cells was observed in SSc patients compared to healthy controls (HCs). Geometric mean fluorescence intensity (GMFI) of CD16 and CD69 on γ/δ T cells was significantly increased in patients with diffuse cutaneous SSc (dcSSc) compared to HCs. CD62L expression was significantly decreased in patients with dcSSc compared to HCs. The percentage of CD69 positive γ/δ T cells was significantly higher in patients with SSc than HCs. COL1A2 mRNA expression was significantly higher in fibroblasts cocultured with γ/δ T cells from SSc than that from HCs in cell contact independent manner. Additionally, γ/δ T cell infiltrations were observed in SSc patients’ skin.ConclusionOur results suggest that γ/δ T cells showed activated phenotype in SSc and suggest that SSc γ/δ T cells may play an important role on fibrotic process by upregulation of COL1A2 mRNA expression in fibroblasts.     

TNF-α and serum induce SKALP/elafin gene expression in human keratinocytes by a p38 MAP kinase-dependent pathway

Keratinocytes of inflamed epidermis (psoriasis, wound healing) are hyperproliferative and display an abnormal differentiation programme. This regenerative differentiation pathway is characterized by the induction of genes that are not expressed by keratinocytes in normal skin, such as the cytokeratins CK6, CK16, CK17, and the proteinase inhibitor SKALP/elafin. In the study reported here we investigated the induction and regulation of SKALP expression as a marker for regenerative differentiation in epidermal keratinocytes. Various cytokines and growth factors known to be present in psoriatic epidermis were examined for their ability to induce SKALP gene expression in cultured human keratinocytes. Tumour necrosis factor-alpha (TNF-alpha) and serum were found to be potent inducers of SKALP expression at both the mRNA and the protein levels. SB202190 or SB203580, two specific p38 MAP kinase inhibitors almost completely blocked the induction of SKALP expression by TNF-alpha and serum. These results suggest that in keratinocytes, p38 activity is crucial for the induction of SKALP gene expression. These findings could be relevant for the elucidation of the mechanisms involved in normal and disturbed epidermal differentiation.     

TGF-β1 up-regulates transglutaminase two and fibronectin in dermal fibroblasts: a possible mechanism for the stabilization of tissue inflammation

Transglutaminase (TGase) has been reported to stabilize tissue inflammation via the mediation of the polymerization of extracellular matrix proteins. A set of cytokines has been implicated in wound healing processes in the dermis. This study was undertaken in order to evaluate the effects of these cytokines on the expression of TGase 2 in human dermal fibroblasts (hDFs), in that TGase 2 is known to be the principal TGase in the dermis. In Western blot analysis, TGF-β1 (1 ng/ml) treatment was found to steadily up-regulate TGase 2 expression for up to 7 days. However, such increases were not observed when the cells were treated with IL-1β, IL-2, and TNF-α. In the enzyme assay, total TGase activities were closely related to the levels of TGase 2 expression. TGase 2 mRNA expression was up-regulated as the result of TGF-β treatment in competitive RT-PCR. In the denatured SDS-PAGE, TGF-β1 treatment resulted in marked induction of an approximately 220 kDa protein, which was revealed to be a fibronectin (FN) via western immunoblotting with an anti-FN antibody. Next, when the hDFs were treated with TGF-β1 (1 ng/ml), FN expression was induced beginning at the third day after treatment. The immunoprecipitants generated by anti-FN antibody were positive for the anti-TGase 2 antibody, and the immune complexes were identified at molecular weights of 92 kDa. Collectively, TGF-β1 stimulates the polymerization of FN via the action of TGase 2, which is supposed to to be an important mechanism in the stabilization of the inflammatory dermis.     

Subcutaneous administration of polymerized type I collagen downregulates interleukin (IL)-17A, IL-22 and transforming growth factor-β1 expression, and increases Foxp3-expressing cells in localized scleroderma

Background. Localized scleroderma (LS) is a disfiguring inflammatory autoimmune disease of the skin and underlying tissue. As in systemic sclerosis, a key feature is the presence of T cells in inflammatory lesions. Aim. To evaluate the effect of polymerized type I collagen vs. methylprednisolone (MP) in LS, and to determine the influence of this polymerized collagen (PC) on CD4+ peripheral T cells expressing interleukin (IL)‐4, IL‐17A, interferon‐γ and Forkhead box protein (Foxp)3, and on cells expressing transforming growth factor (TGF)‐β1, IL‐17A, IL‐22 and Foxp3 in the skin. Methods. In total, 16 patients with LS were treated for 3 months with monthly subcutaneous intralesional injections of 0.1 mL MP (giving a total dose of 20 mg/mL each month) and 15 patients were treated, with weekly subcutaneous intralesional injections of PC, ranging from 0.2 mL (equivalent to 1.66 mg collagen) for a lesion of 50 mm in size, up to a maximum of 1.0 mL (8.3 mg collagen) for a lesion > 100 mm in size, and followed up for a further 6 months. Skin biopsies were obtained from lesions at baseline (before treatment) and 9 months later (6 months after treatment end). Tissue sections were evaluated by histology and immunohistochemistry (IL‐17A, IL‐22, TGF‐β1 and Foxp3). CD4+ T‐cell subsets were determined in peripheral blood by flow cytometry. Results. Abnormal tissue architecture was seen in the biopsies taken from patients treated with MP, whereas the PC treatment restored normal skin architecture. PC downregulated pro‐inflammatory/profibrotic cytokine expression in peripheral cells, and upregulated the number of regulatory T cells (Tregs) in skin. PC was safe and well tolerated. Conclusions. PC is not only an antifibrotic/fibrolytic agent but also an immunomodulator biodrug that restores the balance between T helper (Th)1, Th2, Th17 and Tregs, downregulates production of pro‐inflammatory or profibrogenic cytokines (IL‐17A, IL‐22 and TGF‐β1), and renews skin architecture, without adverse effects.     

Aging decreases collagen IV expression <Emphasis Type="Italic">in vivo</Emphasis> in the dermo-epidermal junction and <Emphasis Type="Italic">in vitro</Emphasis> in dermal fibroblasts: possible involvement of TGF-β1

Background

Collagen IV is a major component of the dermo-epidermal junction (DEJ).

Objectives

To study expression of collagen IV upon aging in the DEJ and dermal fibroblasts isolated from the same patients. A model of senescent fibroblasts was developed in order to identify biological compounds that might restore the level of collagen IV.

Materials & methods

Skin fragments of women (30 to 70 years old) were collected. Localisation of collagen IV expression in the DEJ was studied by immunofluorescence. Fibroblast collagen IV expression was studied by real-time PCR, ELISA, and western blotting. Premature senescence was simulated by exposing fibroblasts to subcytotoxic H₂O₂ concentrations.

Results

Collagen IV decreased in the DEJ and fibroblasts relative to age. TGF-β1 treatment significantly increased collagen IV gene and protein expression in fibroblasts and restored expression in the model of senescence. Addition of TGF-β1-neutralizing antibody to fibroblast cultures decreased collagen IV expression.

Conclusion

Taken together, the results suggest that the decrease in collagen IV in the DEJ, relative to age, could be due to a decrease in collagen IV expression by senescent dermal fibroblasts and may involve TGF-β1 signalling.

     

Suppression of TGF-β1/SMAD pathway and extracellular matrix production in primary keloid fibroblasts by curcuminoids: its potential therapeutic use in the chemoprevention of keloid

Keloid is a fibrotic disease characterized by abnormal accumulation of extracellular matrix (ECM) in the dermis. It is a late spreading skin overgrowth and may be considered a plastic surgeon’s nightmare. In nature, curcuminoid is composed of curcumin, demethoxycurcumin (DMC) and bisdemethoxycurcumin (bDMC). Curcuminoids have been found to inhibit fibrosis. However, their role in the synthesis of ECM in the keloid fibroblasts (KFs) has remained unclear. In this series of studies, a total of seven primary KFs cultures were used as the KFs model for investigating the inhibitory effect of curcuminoids on the expression of ECM and TGF-β1. A sensitive and reproducible HPLC method was developed to provide a quantitative analysis on the cellular uptake of curcuminoids onto the KF cells. The level of ECM in the primary KFs was elevated. The elevation of ECM and TGF-β1/p-SMAD-2 level was substantially blocked by the cellular uptake of curcumin in a dose-dependent manner in all the seven primary KFs. The results have led to the conclusion that the excessive production of ECM in the KF cells could be blocked and/or rapidly decreased by curcumin.     

Hair follicle stem cell differentiation is inhibited through cross-talk between Wnt/β-catenin and androgen signalling in dermal papilla cells from patients with androgenetic alopecia

Summary Background  Hair follicle (HF) regeneration begins when signals from the mesenchyme‐derived dermal papilla cells (DPC) reach multipotent epidermal stem cells in the bulge region. Wnt/β‐catenin signalling is known to affect mammalian hair growth positively. In androgenetic alopecia (AGA), androgens cause HF miniaturization through a mechanism that remains unclear. Circulating androgens act on DPC and alter paracrine factors that influence hair epithelial cells. Objectives  To elucidate the role of androgens in dermal papilla‐induced differentiation of HF stem cells. Methods  HF stem cell differentiation was evaluated in a coculture model with DPC or culturing with media conditioned by DPC after activation of androgen and Wnt/β‐catenin signalling pathways. To study the molecular cross‐talk between the androgen and Wnt signalling pathway in DPC, we analysed the expression and activation of downstream Wnt signalling molecules in the presence of androgens. Results  In a coculture model with human DPC from patients with AGA and HF stem cells, we observed that androgens abrogate hair differentiation evaluated by hair‐specific keratin 6 expression. Wnt signalling activation restored the ability of androgen‐treated DPC to induce differentiation. Androgen treatment revealed a significant decrease in the cytoplasmic/total β‐catenin protein ratio and upregulation of the activity of glycogen synthase kinase‐3β in DPC, indicative of canonical Wnt pathway inhibition. Conclusions  These results suggest that androgens deregulate DPC‐secreted factors involved in normal HF stem cell differentiation via the inhibition of the canonical Wnt signalling pathway.     

Fermentable metabolite of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> controls collagen reduction in photoaging skin by improving TGF-β/Smad signaling suppression

Solar ultraviolet (UV) irradiation causes damages on human skin and premature skin aging (photoaging). UV-induced reduction of type I collagen in dermis is widely considered primarily induction of wrinkled appearance of photoaging skin. Type I procollagen synthesis is reduced under UV irradiation by blocking transforming growth factor-β (TGF-β)/Smad signaling; more specifically, it is down-regulation of TGF-β type II receptor (TβRII). Therefore, preventing UV-induced loss of TβRII results decreased type I collagen reduction in photoaging skin. Zymomonas mobilis is an alcohol fermentable, gram-negative facultative anaerobic bacterium whose effect on skin tissue is scarcely studied. We investigated the protective effects of fermentable metabolite of Z. mobilis (FM of Z. mobilis) against reduction of type I procollagen synthesis of UV-induced down-regulation of TβRII in human dermal fibroblasts FM of Z. mobilis was obtained from lyophilization of bacterium culture supernatant. The levels of TβRII and type I procollagen mRNA in human dermal fibroblasts were measured by quantitative real-time RT-PCR, and TβRII protein levels were assayed by western blotting. TβRII, type I procollagen, and type I collagen proteins in human dermal fibroblasts or hairless mouse skin were detected by immunostaining. FM of Z. mobilis inhibited down regulation of TβRII mRNA, and protein levels in UVB irradiated human dermal fibroblasts consequently recover reduced type I procollagen synthesis. These results indicate UVB irradiation inhibits type I procollagen synthesis by suppression of TGF-β/Smad signaling pathway, and FM of Z. mobilis has inhibitory effect on UVB-induced reduction of type I procollagen synthesis. While short period UVB irradiation decreased both TβRII and type I procollagen protein levels in hairless mouse skin, topical application of FM of Z. mobilis prevented this decrease. Wrinkle formation in hairless mouse skin surface was accelerated by continuous 5 month UVB irradiation along with a reduction of type I collagen in the dermis, but this change was prevented by topical application of FM of Z. mobilis. From this experimental data, it is suggested that FM of Z. mobilis is effective for suppression of wrinkle formation in photoaging skin by inhibition of type I procollagen synthesis reduction.     

Non-M protein(s) on the cell wall and membrane of group A streptococci induce(s) IFN-γ production by dermal CD4+ T cells in psoriasis

Abstract Recently we have demonstrated that a disease-specific subpopulation of CD4⁺ T cells isolated from skin lesions of chronic plaque psoriasis produces interferon-γ in response to group A streptococcal (GAS) antigens. To determine if these T cells recognize M or non-M protein, extracts from cell wall of type M6 GAS (M6W) and its isogenic M gene deletion mutant (M-W), M6 membrane extract (M6M) and recombinant M6 protein (rM6) were used to stimulate GAS-reactive T-cell lines from nine patients with chronic plaque psoriasis. T-cell lines were incubated with or without streptococcal extracts for 18 h in the presence of a transport inhibitor, stained for surface CD4 and intracellular cytokine expression, and analysed by flow cytometry. Variable numbers (0.2–34%) of CD4⁺ T cells produced interferon-γ, in all but one of the T-cell lines tested, in response to M6W, M-W and M6M extracts. No significant difference between the response to M6W and M-W extracts was detected. In addition, rM6 protein failed to increase CD4⁺/interferon-γ⁺ T-cell numbers in seven of nine T-cell lines compared to medium alone. For the group, there was a highly significant correlation between the responses to the three extracts (M6W vs M-W, P = 0.0005; M6W vs M6M, P = 0.0003; M-W vs M6M, P = 0.0001). Low or minimal numbers of interleukin-4- and interleukin-10-producing CD4⁺ T cells were occasionally induced. These findings suggest that a subpopulation of CD4⁺ T cells isolated from skin lesions of chronic plaque psoriasis patients produces interferon-γ in response to non-M protein(s) present on the cell wall and membrane of GAS. Received: 11 September 2000 / Revised: 20 December 2000 / Accepted: 3 February 2001