Response Of Human Skin Fibroblasts Research Articles

Ketotifen directly modifies the fibrotic response of human skin fibroblasts

Fibrosis is a destructive, end-stage disease process. In the skin, it is associated with systemic sclerosis and scarring with considerable health burden. Ketotifen is a clinical antihistamine and mast cell stabilizer. Studies have demonstrated mast cell-dependent anti-fibrotic effects of ketotifen but direct effects on fibroblasts have not been determined. Human dermal fibroblasts were treated with pro-fibrotic transforming growth factor-β1 (TGFβ) followed by ketotifen or control treatments to determine direct effects on fibrotic fibroblasts. Ketotifen impaired TGFβ-induced α-smooth muscle actin gene and protein responses and decreased cytoskeletal- and contractility-associated gene responses associated with fibrosis. Ketotifen reduced Yes-associated protein phosphorylation, transcriptional coactivator with PDZ binding motif transcript and protein levels, and phosphorylation of protein kinase B. In a fibroblast-populated collagen gel contraction assay, ketotifen reduced the contractile activity of TGFβ-activated fibroblasts. In a murine model of bleomycin-induced skin fibrosis, collagen density and dermal thickness were significantly decreased in ketotifen-treated mice supporting in vitro findings. These results support a novel, direct anti-fibrotic activity of ketotifen, reducing pro-fibrotic phenotypic changes in fibroblasts and reducing collagen fibres in fibrotic mouse skin. Together, these findings suggest novel therapeutic potential and a novel mechanism of action for ketotifen in the context of fibrosis.

Synergistic amalgamation of shellac with self-antibacterial hydroxyapatite and carboxymethyl cellulose: An interactive wound dressing for ensuring safety and efficacy in preliminary in vivo studies

This study focuses on the synergistic formulation of environmentally friendly blended materials based on carboxymethyl cellulose (CMC) for advanced interactive wound dressing. New CMC hydrogels were prepared with two degrees of functionalization and chemically crosslinked with citric acid (CA) to fine-tune their properties. Additionally, CMC-based hybrids were created by blending with shellac (SHL) and incorporating self-antibacterial hydroxyapatite (HA) to inhibit bacterial growth and promote wound healing. The results demonstrate the successful production of superabsorbent hydrogels with typical swelling degrees ranging from 81% in water to 82% in phosphate-buffered saline (PBS). These hydrogels exhibit distinct morphological features and remarkable improvements in surface mechanical properties, specifically in their tensile properties, which show a significant increase from approximately 0.03 to 2.2 N/mm2 due to the formation of CMC-SHL-HA hybrid nanostructures. Furthermore, the cytocompatibility of these CMC-based hydrogels was investigated by assessing the in vitro cell viability responses of human skin fibroblasts. The results reveal the cell viability responses over 91%, indicating their biocompatibility with human cells. Moreover, the characteristics of surgical wounds were assessed before and after the application of the hydrogel on dogs, and no signs of infection were observed at any of the surgical sites post-surgery.

Response of Human Skin Fibroblasts to Mechanical Stretch in Wound Healing Process Analyzed Using a Three-Dimensional Culture Model

Response of Human Skin Fibroblasts to Mechanical Stretch in Wound Healing Process Analyzed Using a Three-Dimensional Culture Model

Stress‐induced responses of human skin fibroblasts in vitro reflect human longevity

Unlike various model organisms, cellular responses to stress have not been related to human longevity. We investigated cellular responses to stress in skin fibroblasts that were isolated from young and very old subjects, and from offspring of nonagenarian siblings and their partners, representatives of the general population. Fibroblasts were exposed to rotenone and hyperglycemia and assessed for senescence-associated beta-galactosidase (SA-beta-gal) activity by flow cytometry. Apoptosis/cell death was measured with the Annexin-V/PI assay and cell-cycle analysis (Sub-G1 content) and growth potential was determined by the colony formation assay. Compared with fibroblasts from young subjects, baseline SA-beta-gal activity was higher in fibroblasts from old subjects (P = 0.004) as were stress-induced increases (rotenone: P < 0.001, hyperglycemia: P = 0.027). For measures of apoptosis/cell death, fibroblasts from old subjects showed higher baseline levels (Annexin V+/PI+ cells: P = 0.040, Sub-G1: P = 0.014) and lower stress-induced increases (Sub-G1: P = 0.018) than fibroblasts from young subjects. Numbers and total size of colonies under nonstressed conditions were higher for fibroblasts from young subjects (P = 0.017 and 0.006, respectively). Baseline levels of SA-beta-gal activity and apoptosis/cell death were not different between fibroblasts from offspring and partner. Stress-induced increases were lower for SA-beta-gal activity (rotenone: P = 0.064, hyperglycemia: P < 0.001) and higher for apoptosis/cell death (Annexin V+/PI- cells: P = 0.041, Annexin V+/PI+ cells: P = 0.008). Numbers and total size of colonies under nonstressed conditions were higher for fibroblasts from offspring (P = 0.001 and 0.024, respectively) whereas rotenone-induced decreases were lower (P = 0.008 and 0.004, respectively). These data provide strong support for the hypothesis that in vitro cellular responses to stress reflect the propensity for human longevity.

High glucose-induced alterations of extracellular matrix of human skin fibroblasts are not dependent on TSP-1–TGFβ1 pathway

Elevated glucose level is the main cause of extracellular matrix (ECM) derangement in various tissues in diabetes mellitus. The development of diabetic nephropathy is considered to be dependent on profibrotic cytokine, transforming growth factor-β1 (TGFβ1). Its excessive activation due to the up-regulation of thrombospondin-1 (TSP-1) in mesangial cells exposed to high glucose contributes to ECM accumulation. However, the role of TSP-1–TGFβ1 pathway in the development of glucose-induced imbalance of ECM homeostasis in skin connective tissue is not studied. We investigated the response of human skin fibroblasts to elevated glucose level (11.0 and 30.0 mM) in terms of: (1) the expression and secretion of fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1); (2) the accumulation of hyaluronic acid (HA) in pericellular matrix and in the conditioned medium; (3) TGFβ1 expression, secretion and activation; (4) TSP-1 expression and secretion. We demonstrated the up-regulation of FN and PAI-1 by elevated glucose and the stimulation of HA accumulation in both cellular compartments. However, we failed to demonstrate the increase of expression, secretion and activation of TGFβ1, and the increase of TSP-1 expression and secretion in fibroblasts exposed to high glucose. These results show that ECM derangement in skin fibroblasts due to high glucose is not determined by TGFβ1 and its activation by TSP-1.

Cell cycle arrest and apoptosis provoked by UV radiation-induced DNA damage are transcriptionally highly divergent responses.

DNA damage caused by UV radiation initiates cellular recovery mechanisms, which involve activation of DNA damage response pathways, cell cycle arrest and apoptosis. To assess cellular transcriptional responses to UVC-induced DNA damage we compared time course responses of human skin fibroblasts to low and high doses of UVC radiation known to induce a transient cellular replicative arrest or apoptosis, respectively. UVC radiation elicited >3-fold changes in 460 out of 12,000 transcripts and 89% of these represented downregulated transcripts. Only 5% of the regulated genes were common to both low and high doses of radiation. Cells inflicted with a low dose of UVC exhibited transcription profiles demonstrating transient regulation followed by recovery, whereas the responses were persistent after the high dose. A detailed clustering analysis and functional classification of the targets implied regulation of biologically divergent responses and suggested involvement of transcriptional and translational machinery, inflammatory, anti-proliferative and anti-angiogenic responses. The data support the notion that UVC radiation induces prominent, dose-dependent downregulation of transcription. However, the data strongly suggest that transcriptional repression is also target gene selective. Furthermore, the results demonstrate that dose-dependent induction of cell cycle arrest and apoptosis by UVC radiation are transcriptionally highly distinct responses.

Effect of antioxidant supplementation on the adaptive response of human skin fibroblasts to UV-induced oxidative stress

The effect of supplementation with substances having antioxidant properties on the adaptive responses of human skin fibroblasts to UV-induced oxidative stress was studied in vitro. UVR was found to induce a substantial oxidative stress in fibroblasts, resulting in an increased release of superoxide anions and an increase in lipid peroxidation (shown by an elevated malonaldehyde content). Sub-lethal doses of UVR were also found to induce adaptive responses in the fibroblast antioxidant defences, with a transient rise in catalase and superoxide dismutase activities followed by a slower, large increase in cellular glutathione content. Supplementation of the fibroblasts with the antioxidants, Trolox (a water soluble analogue of α-tocopherol), ascorbic acid or β-carotene, had differential effects on these responses. Trolox supplementation reduced the UVR-induced cellular oxidative stress and adaptive response in a predictable concentration-dependant manner. This was in contrast to ascorbic acid which increased superoxide release from fibroblasts. At low doses, ascorbate supplements also reduced the magnitude of the adaptive increases in catalase and superoxide dismutase activities and increase in glutathione content. β-Carotene had a similar effect to ascorbic acid, reducing the extent of the adaptations to UVR at lower doses while simultaneously increasing superoxide release and malonaldehyde content. These in vitro data indicate that only the vitamin E analogue suppressed UVR-induced oxidative stress in a predictable manner and suggest that common dietary antioxidants may not be equally effective in reducing the potential deleterious effects of UVR-induced oxidative stress in skin.

Alteration of the Chemotactic Response of Human Skin Fibroblasts to PDGF by Growth Factors

Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for fibroblastic cells. In the early stage of wound healing, PDGF is released from aggregated platelets and it is believed that its chemotactic activity may play a key role in the influx of connective tissue cells into wound sites. Using the Boyden chamber assay, we investigated factors that alter the migratory response of human skin fibroblasts to PDGF. The response was related to the growth state of cells; that is, growing cells at low cell density exhibited a greater migratory response to PDGF than density-arrested quiescent cells. The level of random migration was also elevated in growing cells, compared to quiescent cells. The chemotactic response after 3-h exposure to serum was markedly decreased (>50%). Three-hour preincubation of the cells with transforming growth factor-β (TGF-β) or with epidermal growth factor reduced the migratory response to approximately half that in the nonstimulated control. In contrast, cells treated with TGF-β or basic fibroblast growth factor for 24 h exhibited a two- to threefold greater chemotactic response to PDGF than control cells. This stimulatory effect of TGF-β on the fibroblast migration induced by PDGF suggests that TGF-β acts synergistically with PDGF on the influx of connective tissue cells into human wound sites and may explain the potent wound healing activity of TGF-β in vivo.

Alteration of the chemotactic response of human skin fibroblasts to PDGF by growth factors.

Abstract Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant for fibroblastic cells. In the early stage of wound healing, PDGF is released from aggregated platelets and it is believed that its chemotactic activity may play a key role in the influx of connective tissue cells into wound sites. Using the Boyden chamber assay, we investigated factors that alter the migratory response of human skin fibroblasts to PDGF. The response was related to the growth state of cells; that is, growing cells at low cell density exhibited a greater migratory response to PDGF than density-arrested quiescent cells. The level of random migration was also elevated in growing cells, compared to quiescent cells. The chemotactic response after 3-h exposure to serum was markedly decreased (>50%). Three-hour preincubation of the cells with transforming growth factor-β (TGF-β) or with epidermal growth factor reduced the migratory response to approximately half that in the nonstimulated control. In contrast, cells treated with TGF-β or basic fibroblast growth factor for 24 h exhibited a two- to threefold greater chemotactic response to PDGF than control cells. This stimulatory effect of TGF-β on the fibroblast migration induced by PDGF suggests that TGF-β acts synergistically with PDGF on the influx of connective tissue cells into human wound sites and may explain the potent wound healing activity of TGF-β in vivo.

Influence of the extracellular matrix on the proliferative response of human skin fibroblasts to serum and purified platelet-derived growth factor.

The culture of adult human skin fibroblasts on reconstituted bovine type 1 fibrillar collagen gels, ranging in concentration from 2.5-35.0 mg/ml, results in a reduction in proliferation rate by 40%-60% as measured by (3H) thymidine incorporation. The suppressive effect was noted when cells were cultured in both human and bovine serum. Drying the gels into thin films abolishes the suppressive effect of the fibrillar collagen on cell proliferation. Cell attachment studies showed that differences in the proliferation rate of cells on the various substrata were not simply due to differences in initial attachment. Studies with purified platelet-derived growth factor (PDGF) demonstrated that the reduced responsiveness of cells to this factor, when cultured on collagen gels as compared to plastic, was largely responsible for the reduced proliferative activity of the cells when cultured in the presence of serum. The reduced proliferative activity of fibroblasts in response to PDGF, when cultured on collagen gels, was confirmed by total DNA determination. It was shown that the reduced responsiveness of cells to PDGF was not simply because the factor bound to the fibrillar collagen gel or was inaccessible to the cells. The data indicate that the reduced proliferation rate of fibroblasts cultured on collagen gels is a direct result of the influence of the extracellular matrix on the cells' ability to respond to a soluble mitogenic mediator.

Complementary adhesive responses of human skin fibroblasts to the cell-binding domain of fibronectin and the heparan sulfate-binding protein, platelet factor-4

Plasma fibronectin (pFN) contains binding domains for an unidentified receptor on the surface of fibroblasts and for heparan sulfate chains of proteoglycans on these same cells. A series of experiments were designed to assess the relative importance of these activities in mediating substratum adhesion of human skin fibroblasts (strain 4449) grown in the absence of ascorbate (asc −) or in its presence (asc +) to minimize or maximize collagen production-maturation, respectively. The cell-binding fragment (CBF) of pFN was purified from chymotryptic digests free of any heparan sulfate-binding activity. The responses of cells to CBF were then compared with those mediated by the heparan sulfate-binding protein, platelet factor-4 (PF4). At early time points when cells had spread effectively on pFN, both asc − or asc + cells extended spiky projections on PF4 and long projections on CBF with actively ruffling membranes at their tips. By 4 h, asc + cells had spread much more effectively on CBF than asc + cells on PF4 or asc − cells on either binding activity. Mixtures ( w w ) of CBF:PF4 between 1:1 and 9:1 generated a more physiologically normal response than to either of the binding proteins alone, particularly for asc + cells. Examination of cytoskeletal reorganization by fluorescence analysis with an antibody to 7S tubulin (for microtubules) and NBD-phallacidin (for F-actin) revealed condensations of microfilaments at the ruffling edges of asc − cells on CBF or on PF4 and for asc − cells on PF4; in contrast, asc + cells on CBF generated long bundles of microfilaments in their spreading lamellae within 4 h. Microtubule networks reorganized very well on CBF but only partially on PF4 with either cell type. Microfilament reorganization was comparable to that on intact pFN with CBF:PF4 mixtures of 1:1 and 9:1 for asc + cells, whereas asc − cells generated condensations of microfilaments but little bundling. These studies reveal that the adhesive responses to mixtures of these two binding activities are significantly greater than to the individual activities and that the responses of asc + cells approach the properties of cells on intact pFN, whereas asc − cells remain incapable of forming stress fiber-like bundles of microfilaments under all conditions.