SB-431542 inhibits TGF-beta-induced contraction of collagen gel by normal and keloid fibroblasts

BACKGROUND: Transforming growth factor (TGF)-beta induces fibroblast contraction, which is implicated in wound healing and keloid formation. SB-431542 is a novel specific inhibitor of TGF-beta type I receptor kinase activity. OBJECTIVE: We sought to determine whether SB-431542 inhibited TGF-beta-induced fibroblast contraction. METHODS: We used an in vitro type I collagen gel contraction assay with normal or keloid dermal fibroblasts incorporated. RESULTS: TGF-beta induced contraction of collagen gels with normal dermal fibroblasts incorporated, which was efficiently suppressed by SB-431542. Keloid fibroblasts showed higher basal contraction of collagen gels in the absence of TGF-beta than normal fibroblasts, which was enhanced by addition of TGF-beta. SB-431542 suppressed both the basal and TGF-beta-enhanced contraction of collagen gels by keloid fibroblasts. These inhibitory effects of SB-431542 were associated with suppression of TGF-beta-induced alpha-smooth muscle actin (alpha-SMA) expression and phosphorylation of Smad2 in normal and keloid fibroblasts. CONCLUSION: SB-431542 can suppress TGF-beta-induced contraction of collagen gel by normal and keloid dermal fibroblasts. Importantly, SB-431542 can inhibit basal contraction of collagen gel by keloid fibroblasts. These results suggest that an inhibitor of TGF-beta type I receptor kinase activity may have therapeutic potential for excessive skin contraction as observed in keloid.     

The suitability of cells from different tissues for use in tissue-engineered skin substitutes

Tissue-engineered skin substitutes may be a future remedy for burn wounds and chronic wounds, as wound contraction and scar formation cannot be prevented with the current standard treatment. The aim of this study therefore was to identify readily available sources of fibroblasts suitable for dermal substitution. Three different tissues were studied: dermal tissue from split-skin graft, subcutaneous fat tissue and eschar tissue obtained through debridement of burn wounds. We determined the cellular profile and the cell numbers immediately after isolation and after 2 and 14 days of fibroblast culture using flow cytometry and cell counting with a cytometer. In addition, parts of the isolated cell suspensions were seeded directly into a porous collagen dermal substitute to investigate contraction over time. Various cell types were isolated from the three different tissues, but after 14 days of culturing predominantly fibroblasts (>90%) were detected. Keratinocytes, granulocytes and macrophages, if present, disappeared within 14 days. In the cell populations derived from dermal tissue, the percentage of myofibroblasts had decreased significantly by day 14 (from 8% to 3%, P=0.028). In contrast, this percentage had increased in the cell populations derived from fat and eschar (from 23% to 40% and from 20% to 38%, respectively). The fibroblast yield from dermal tissue after 2 weeks of culturing (50 x 10(6) cells/g of tissue) was significantly higher than the yield from fat and eschar tissue (2 x 10(6) cells/g of each tissue, P=0.029). Immunohistochemistry of collagen matrices seeded and cultured with fat- and eschar-derived cells revealed a high prevalence of myofibroblasts, whereas hardly any myofibroblasts were detected in the matrices seeded with dermal cells. The contraction of the eschar matrices was highest (74+/-6% remaining area), whereas dermal matrices contracted significantly less (92+/-7% remaining area, P=0.029) with intermediate contraction for fat matrices. We conclude that fibroblast cultures can be established from dermal tissue, fat tissue and eschar tissue. Dermis is the best fibroblast source for use in skin substitutes as it yields the highest numbers of fibroblasts with minimal numbers of myofibroblasts.     

Cryotherapy modifies synthetic activity and differentiation of keloidal fibroblasts in vitro

In order to obtain a persuasive explanation for the beneficial clinical effect of cryotherapy on keloids, we developed a reproducible model to apply freezing temperatures on cell cultures, and investigated their influence on proliferation, viability, synthetic activity and differentiation of dermal fibroblasts in vitro. Cell cultures were established from 13 untreated keloids and 10 healthy skin specimens matched for age and skin localization to the donors. No significant influence of cell freezing on the proliferation rates of both keloidal and normal fibroblasts was documented, but mechanical cell destruction with a wide variation in lethality rates (29% average lethal effect on keloidal fibroblasts and 41% on normal ones) was observed. When comparing specimens of keloidal and normal tissue derived from the same four donors, the keloidal fibroblasts were similar regarding their synthetic activity but presented enhanced tenascin-C expression compared with the normal fibroblasts. After cryotherapy, delayed collagen III increase was detected in both cell types (P = 0.03). The collagen II/collagen I ratio increased from 1.6 to 2.8 in the keloidal and only from 1.9 to 2.2 in the normal fibroblasts after subcultivation. Normal fibroblasts exhibited a significantly lasting increase in fibronectin synthesis after freezing (P = 0.03). The intensity of staining against tenascin-C was decreased in five of nine keloidal fibroblast cultures after cryotherapy (P < 0.05) but increased in four of five normal fibroblast cultures (P = 0.016), so that the intensity of tenascin-C staining after freezing became identical in both cell types. Immunoblot studies in four patients and two controls confirmed a temporary decrease of tenascin-C in keloidal but not in normal fibroblasts immediately after freezing. Significantly decreased staining with two markers of myogenic differentiation, myosin in keloidal fibroblasts (P = 0.002) and desmin (P = 0.007) in normal fibroblasts, could also be detected after treatment. In summary, with the help of a model for controlled cell freezing in vitro, cryotherapy was found to modify collagen synthesis and differentiation of keloidal fibroblasts.     

Effect of vitamin A and its derivatives on collagen production and chemotactic response of fibroblasts

Vitamin A and several other retinoids were added to fibroblast cultures in order to study possible alterations in biochemical properties and cellular responsiveness. The proliferation of cells was inhibited as the concentration of retinoids increased from 10(-9) to 10(-5) mol/l. Synthesis of non-collagenous proteins and production of both type I and type III collagen were decreased. The onset of type III collagen synthesis by tendon fibroblasts in culture was delayed. Furthermore, the chemotactic response of fibroblasts to fibroblast-conditioned medium was markedly reduced in the presence of retinoids (10(-6) to 10(-12) mol/l).     

Liposome-encapsulated ursolic acid increases ceramides and collagen in human skin cells

Skin wrinkling and xerosis associated with aging result from decreases in dermal collagen and stratum corneum ceramide content. This study demonstrated that ursolic acid incorporated into liposomes (URA liposomes) increases both the ceramide content of cultured normal human epidermal keratinocytes (NHEK), and the collagen content of cultured normal human dermal fibroblasts. In addition, URA liposomes increased the ceramide content of the skin of human subjects, with increases in hydroxy ceramides occurring after only 3 days of treatment. Both URA liposomes and retinoic acid decreased markers of keratinocyte differentiation (keratin 1, keratin 10 and involucrin) in cultured NHEK. Thus, URA liposomes have effects on keratinocyte differentiation and dermal fibroblast collagen synthesis similar to those of retinoids. However, this study showed that URA liposomes increase ceramides in NHEK, in contrast to the decreases previously shown to be caused by retinoids. URA liposomes have the potential to be used alone or in combination with other agents to restore or maintain skin ceramide and collagen content.     

Effect of tretinoin on collagen gel contraction induced by mouse 3T3 fibroblasts

Balb/3T3 fibroblasts were cultured in type I collagen gel and the effects of tretinoin (all-trans-retinoic acid) were examined on cell growth and the gel contraction produced by cells. Cell proliferation was suppressed and the degree of gel contraction was enhanced by the addition of 10(-7) and 10(-6) M tretinoin. Growth and gel contractility of transformed cells derived from the Balb/3T3 cells were not influenced by this agent. Addition of 12-O-tetradecanoylphorbol ester, which is known to antagonize tretinoin in several biological processes, enhanced gel contraction synergistically with tretinoin. These results suggest that tretinoin influences cell-to-collagen interactions.     

Smads regulate collagen gel contraction by human dermal fibroblasts

BACKGROUND: Transforming growth factor (TGF)-beta induces fibroblast contraction that is implicated in efficient wound healing. The Smad family of proteins mediates signal transduction of the TGF-beta superfamily. However, its role in fibroblast contraction remains unclear. OBJECTIVES: To determine whether Smad proteins regulate fibroblast contraction. METHODS: We used an in vitro type I collagen gel contraction assay with human dermal fibroblasts infected with adenoviruses carrying Smads. RESULTS: Overexpression of Smad3, a major signal transducer in the Smad family, enhanced collagen gel contraction by fibroblasts when compared with fibroblasts overexpressing a control lacZ. Addition of a very low concentration of TGF-beta1 that did not affect the collagen gel contraction by itself enhanced the contraction by fibroblasts overexpressing Smad3. In contrast, TGF-beta1-mediated collagen gel contraction was suppressed by overexpression of Smad7, a major inhibitory regulator in the Smad family, in fibroblasts. In addition, inhibitors of the Erk and p38 pathways, PD98059 and SB203580, did not affect TGF-beta1-mediated collagen gel contraction by dermal fibroblasts. CONCLUSIONS: Modulation of Smad3 or Smad7 expression in dermal fibroblasts affected their contraction of collagen gels possibly by regulating TGF-beta signalling in fibroblasts.     

Collagen telopeptides (cross-linking sites) play a role in collagen gel lattice contraction

Solubilized interstitial collagens will form a fibrillar, gel-like lattice when brought to physiologic conditions. In the presence of human dermal fibroblasts the collagen lattice will contract. The rate of contraction can be determined by computer-assisted planemetry. The mechanisms involved in contraction are as yet unknown. Using this system it was found that the rate of contraction was markedly decreased when collagen lacking telopeptides was substituted for native collagen. Histidinohydroxylysinonorleucine (HHL) is a major stable trifunctional collagen cross-link in mature skin that involves a carboxyl terminal, telopeptide site 16c, the sixteenth amino acid residue from the carboxy terminal of the telopeptide region of alpha 1 (I) in type I collagen. Little, if any, HHL was present in native, purified, reconstituted, soluble collagen fibrils from 1% acetic acid-extracted 2-year-old bovine skin. In contrast, HHL cross-links were present (0.22 moles of cross-link per mole of collagen) in lattices of the same collagen contracted by fibroblasts. However, rat tail tendon does not contain HHL cross-links, and collagen lattices made of rat tail tendon collagen are capable of contraction. This suggests that telopeptide sites, and not mature HHL cross-links per se, are essential for fibroblasts to contract collagen lattices. Beta-aminopropionitrile fumarate (BAPN), a potent lathyrogen that perturbs collagen cross-linking by inhibition of lysyl oxidase, also inhibited the rate of lattice cell contraction in lattices composed of native collagen. However, the concentrations of BAPN that were necessary to inhibit the contraction of collagen lattices also inhibited fibroblast growth suggestive of cellular toxicity. In accordance with other studies, we found no inhibition of the rate of lattice contraction when fibronectin-depleted serum was used. Electron microscopy of contracted gels revealed typical collagen fibers with a characteristic axial periodicity. The data provide evidence that collagen telopeptide sites play a role in collagen gel lattice contraction.     

Peripheral anchorage of dermal equivalents

Human fibroblasts can induce collagen gel contraction with different kinetics depending on the number of cells and on the collagen concentration within this lattice, which has been considered as a dermal equivalent. Skin equivalent is a combined culture of dermo-epidermal layers which may be of therapeutic value in the treatment of burn patients. However, the current production of the dermal equivalent component gives results that present many drawbacks for their eventual clinical use as a first step in obtaining a skin equivalent. These include: (i) final surfaces which are very small; less than 20% of the initial size (ii) excessive thickness which may hamper successful graft take (iii) fibroblasts that do not have an arrangement comparable with normal dermal tissue. We propose, as a solution to these problems, the utilization of a 5-mm-wide fibre-glass filter ring peripherally attached to the surface of the Petri dishes to prevent inordinate contraction while the fibroblasts reorganize the collagen gel. Using this technique the initial surface was preserved and the dermal equivalent contracted only in thickness. Histological analysis of these anchored equivalents confirmed an alignment of fibroblasts and collagen fibres resembling normal dermal tissue. We consider this method useful in the development of dermo-epidermal sheets for clinical purposes.     

Differential expression of collagen integrin receptor on fetal vs. adult skin fibroblasts: implication in wound contraction during healing

BACKGROUND: Fetal skin wound healing is characterized by an absence of contraction and scar formation, two important observations associated with adult healing often leading to pathological problems. OBJECTIVES: We have studied the capacity of adult and fetal human skin fibroblasts to contract collagen gels, collagen being the major structural component of dermal matrix. METHODS: In parallel with collagen gel contraction studies, we have used fluorescence-activated cell sorter analysis to study the levels of collagen receptors expressed at the surface of fibroblasts derived from fetal or adult skin samples. RESULTS: Strong differences were detected between freshly isolated fetal and adult fibroblasts. Fetal fibroblasts had a very low capacity to contract collagen gel, whereas adult cells significantly contracted gels in the same conditions. The expression of alpha1, alpha2 and alpha3 integrin subunits was also significantly different depending of the donor age: alpha1 and alpha3 integrin subunit expression was lower in fetal cells compared with adult cells, whereas alpha2 integrin subunit expression was higher. When grown in monolayers, adult cells showed rapid changes in their contractile capacity and integrin expression while fetal cells were only affected after several passages. CONCLUSIONS: These observations indicate that intrinsic differences between fetal and adult fibroblasts can strongly influence the quality of wound repair.     

Gap-junctional protein connexin 43 is expressed in dermis and epidermis of human skin: differential modulation by retinoids.

Retinoids are effective modulators of proliferation and differentiation of keratinocytes in vivo and in vitro. In mouse 10T1/2 cells, retinoid action on proliferation and neoplastic transformation is correlated with the upregulation of gap-junctional communication and expression of connexin 43 (Cx43). In the present study we have determined if retinoids induce similar effects on gene expression in human skin. Studies were conducted in intact skin and on cultured keratinocytes and dermal fibroblasts. In a clinical study, 2 weeks of treatment with 0.05% all-trans retinoic acid resulted in increased expression of Cx43 mRNA and protein in epidermis. Expression occurred predominantly in the suprabasal layer. Cultured cells exhibited a differential response to retinoic acid. In keratinocytes, increased expression of Cx43 occurred at low (10(-11) M) concentrations, whereas inhibition occurred at high (10(-7) M) concentrations; however, junctional communication, measured by dye transfer, was not altered over this concentration range. Dermal fibroblasts, in contrast, exhibited a dose-dependent increased expression of Cx43 at concentrations up to 10(-7) M retinoic acid and proportionately increased their junctional communication over this dose range. These data indicate that control of Cx43 gene expression by retinoids in human skin cells is complex. The production of gradients of junctional channels could play a role in the control of growth and differentiation in epidermis.     

Tumor necrosis factor alpha induces invasiveness of human skin fibroblasts in vitro

The chemotactic response of fibroblasts plays an important role during wound healing and fibrosis. Several substances have been found to mediate fibroblast migration in vitro. In the tissue, however, fibroblasts have also the potential to pass through connective tissue barriers following a chemotactic stimulus. Since tumor necrosis factor alpha (TNF alpha) is a cytokine released by mononuclear cells during wound healing, we have been interested in studying its effect on the regulation of fibroblast chemotaxis and invasive migration. TNF alpha did not attract skin fibroblasts nor did it alter their chemotactic response towards other chemoattractants like fibroblast conditioned medium or fibronectin. However, whereas normal skin fibroblasts did not invade a collagen I gel, preincubation of the cells with TNF alpha markedly induced their invasive migration into the gel. This seems to be associated with a specific degradation of type I collagen, because TNF alpha did not promote the invasion of skin fibroblasts through a reconstituted basement membrane (Matrigel).     

Hypertrophic scar cells fail to undergo a form of apoptosis specific to contractile collagen-the role of tissue transglutaminase

Failure of apoptosis has been postulated to cause the hypercellularity and thus excess scar-tissue formation of hypertrophic scars (HTS). Here, we have examined the susceptibility of fibroblasts derived from normal or HTS to apoptosis induced during collagen-gel contraction, a wound-healing model. Normal scar (NS) fibroblasts underwent significant apoptosis (>40% total) in contractile collagen, whereas apoptosis was not detected in HTS cells. This inability was specific to apoptosis induced by contractile collagen because apoptosis could be induced using diverse modalities. Since chronic fibrotic tissue is known to be excessively cross-linked, we next examined whether collagen matrix that had been conditioned by HTS fibroblasts became refractory to enzymatic breakdown and indeed, found that it is resistant to breakdown by both collagenase D and matrix metalloproteinase-2. Newly formed extracellular matrix is stabilized by the enzyme, tissue transglutaminase, which we demonstrated to be overexpressed by HTS fibroblasts in vivo and in vitro. Reducing tissue transglutaminase activity in collagen gels containing HTS fibroblasts permitted induction of apoptosis on gel contraction, whereas increasing enzymic activity in NS cell-containing gels completely abrogated collagen-contraction-induced-apoptosis. Together, these observations show that HTS fibroblasts exhibit resistance to a specific form of apoptosis elicited by contraction of collagen gels, and that this phenomenon is dependent on excess activity of cell surface tissue transglutaminase.     

Retinoids suppress cysteine-rich protein 61 (CCN1), a negative regulator of collagen homeostasis, in skin equivalent cultures and aged human skin in vivo

Alterations in connective tissue collagen are prominent features of both chronologically aged and photoaged (ageing because of sun exposure) human skin. These age-related abnormalities are mediated in part by cysteine-rich protein 61 (CCN1). CCN1 is elevated in the dermis of both chronologically aged and photoaged human skin in vivo and promotes aberrant collagen homeostasis by down-regulating type I collagen, the major structural protein in skin, and promoting collagen degradation. Vitamin A and its metabolites have been shown to improve chronologically aged and photoaged skin by promoting deposition of new collagen and preventing its degradation. Here, we investigated regulation of CCN1 expression by retinoids in skin equivalent cultures and chronologically aged and photoaged human skin in vivo. In skin equivalent cultures, all-trans retinoic acid (RA), the major bioactive form of vitamin A in skin, significantly increased type I procollagen and reduced collagenase (matrix metalloproteinases-1, MMP-1). Addition of recombinant human CCN1 to skin equivalent cultures significantly reduced type I procollagen and increased MMP-1. Importantly, RA significantly reduced CCN1 expression in skin equivalent cultures. Topical treatment with retinol (vitamin A, 0.4%) for 7days significantly reduced CCN1 mRNA and protein expression in both chronologically aged (80+years) and photoaged human skin in vivo, compared to vehicle-treated skin. These data indicate that the mechanism by which retinoids improve aged skin, through increased collagen production, involves down-regulation of CCN1.     

Collagenase-3 (MMP-13) enhances remodeling of three-dimensional collagen and promotes survival of human skin fibroblasts

Collagenase-3 (MMP-13) is a matrix metalloproteinase capable of cleaving a multitude of extracellular matrix proteins in addition to fibrillar collagens. Human MMP-13 is expressed by fibroblasts in chronic cutaneous ulcers, but not in normally healing adult skin wounds. However, MMP-13 is produced by fibroblasts in adult gingival and in fetal skin wounds characterized by rapid collagen remodeling and scarless healing. Here, we have examined the role of human MMP-13 in remodeling of three-dimensional (3D) collagenous matrix by primary adult human skin fibroblasts. The high level of human MMP-13 expression by fibroblasts achieved by adenoviral gene delivery resulted in potent enhancement of remodeling and contraction of 3D collagen. Fibroblasts expressing MMP-13 in 3D collagen possessed altered filamentous actin morphology with patch-like actin distribution in cell extensions. The expression of MMP-13 promotes survival and proliferation of fibroblasts in floating collagen gel, and results in activation of Akt and extracellular signal-regulated kinase-1/2 by these cells. The results provide evidence for a novel role for human MMP-13 in regulating dermal fibroblast survival, proliferation, and interaction in 3D collagen, which may be an important survival mechanism for fibroblasts in chronic skin ulcers and contribute to scarless healing of adult gingival and fetal skin wounds.     

Construction of low contracted 3D skin equivalents by genipin cross‐linking

Continuous contraction of 3D skin equivalents in construction and use restricts their applications in clinical and pharmaceutical practices. So far, no effective method has been developed to inhibit such contraction. Hence, low cytotoxic cross‐linkers, 1‐ethyl‐3‐3‐dimethylaminopropylcarbodiimide hydrochloride (EDC) and genipin, are investigated to reduce the contraction in this study. As found, both genipin and EDC at 0.2 and 0.4 mmol/L are nontoxic to collagen‐entrapped fibroblasts and upregulate the extracellular matrix expression of fibroblasts in cross‐linked collagen. Particularly, collagen cross‐linking by intermediate concentrations of genipin, specifically 0.4 mmol/L, greatly reduces the contraction of 3D skin equivalents from 87% to 28% (n = 9, P < 0.05), while the collagen after EDC cross‐linking at 0.4 mmol/L still presented severe contraction of 64% over a 21‐day follow‐up period. The inhibited contraction might relate to the increased gel stiffness and slowed collagen degradation. Moreover, the genipin cross‐linking does not impair the formation of epidermal layers and improves the epidermal‐dermal junction of skin equivalents as well. In this regard, genipin cross‐linking might facilitate the applications of 3D skin equivalents in clinical practices and pharmacology testing.