Contraction of collagen lattices by skin fibroblasts: drug-induced changes

Summary A range of dermatologically useful drugs were added to human skin fibroblasts cultured in collagen lattices to assess possible effects on the rate of lattice contraction. Vitamin C, Vitamin E, phenytoin, sodium salicylate, d-penicillamine and dibutyryl c-AMP had no significant effect. Chlorhexidine acetate at 10 g/ml arrested contraction after 24 h but this was related to its cytotoxicity. The antibiotics griseofulvin (2–16 g/ml) and cycloheximide (5–30 g/ml) caused dose-related inhibitions of contraction without affecting fibroblast viability. Four corticosteroids at 10 g/ml inhibited contraction, clobetasol propionate having the greatest effect. On the other hand 4 retinoids at 10^-5 M enhanced contraction by up to 20%. As lattice contraction appears to model the contraction of skin wounds and there are broad parallels between the effects shown here of antiseptics, corticosteroids and the retinoids, and their reported influence on healing wounds, the lattice system may be a useful pharmacological screen for new compounds.     

Contraction of collagen lattices by skin fibroblasts from dystrophic recessive epidermolysis bullosa and other dermatoses

Skin fibroblasts incorporated into a lattice of hydrated collagen contract it to form a tissue-like structure. Fibroblasts from patients with skin diseases, including psoriasis, epidermolysis bullosa, and scleroderma, and from control subjects, all showed a similar ability to contract the lattice. The established skin epithelial cell line NCTC 2544, melanoma cells, and 3T6 mouse fibroblasts produced little contraction. Contraction required the presence of serum and was unaffected by the addition of fibronectin (10-20 micrograms/ml). Hyaluronic acid at 50-500 micrograms/ml had no effect, but contraction was inhibited at 1 mg/ml.     

Changes in collagen connective tissue and fibroblasts in aging

Some of the characteristic features of aging skin, such as wrinkling, loss of elasticity, and atrophy, can largely be attributed to dermal changes. The amount of collagen in the skin decreases, while the cross linking increases, and the solubility of collagen is reduced. The total number of fibroblasts decreases, and their metabolism shows characteristic alterations. Some of the functions of fibroblasts in aging skin, such as the synthesis of protein and collagen, but also proliferation and chemotaxis, can be investigated by means of in vitro models. In addition, various syndromes of premature aging (e.g. progeria, Werner's syndrome) have proved useful models and have contributed considerably to the understanding of aging processes.     

Ocular and cutaneous manifestations of heritable disorders of collagen and elastic tissue.

Ocular and cutaneous stigmata are hallmarks of the heritable disorders of collagen and elastic tissue. Awareness of the oculocutaneous features is essential both to aid diagnosis and to direct management of affected patients. Although the angioid streaks of PXE, blue sclerae of OI, ocular fragility of EDS and ectopia lentis of Marfan syndrome are the best-known ocular signs of these disorders, other relevant ophthalmologic symptoms often accompany such classic findings.     

Decreased collagenase production by regional fibroblasts cultured from skin of a patient with connective tissue nevi of the collagen type

A 10-yr-old female presented with cerebriform tumors covering the plantar surfaces of both feet. Histologically, the lesions consisted of thick collagen fibers and the content of collagen per surface area of skin was increased about 8-fold. Examination of the collagen by SDS-polyacrylamide gel electrophoresis, after limited pepsin proteolysis, showed that the lesions consisted almost exclusively of type I collagen, the predominant collagen type in human skin. Thus, a diagnosis of connective tissue nevi of the collagen type was made. Fibroblast cultures were established from the affected and normal-appearing areas of the skin, and examined for the rate of collagen synthesis, production of collagenase and growth kinetics of the cells. Cell cultures derived from the lesion and from control skin synthesized procollagen at the same rate and in a normal type I/type III procollagen ratio. However, the production of enzymatically active and immunologically detectable collagenase was reduced by 70-82% in the cultures derived from the lesion as compared to controls (p less than 0.005). Fibroblasts derived from the lesions also displayed a mean population doubling time of 1.17 +/- 0.08 days compared to 1.83 +/- 0.24 and 1.92 +/- 0.09 days for control cell strains and cells derived from normal skin of the patient, respectively (p less than 0.025). These results suggest that the excessive deposition of collagen in this case may have resulted from decreased local degradation of collagen. Enhanced proliferative capacity of the regional fibroblasts may have contributed to the accumulation of collagen in these lesions.     

Contractile forces generated by striae distensae fibroblasts embedded in collagen lattices

Striae distensae are characterized by linear, smooth bands of atrophic-appearing skin that are reddish at first and finally white. They are due to stretching of the skin, as in rapid weight gain, or mechanical stress, as in weight lifting. The pathogenesis of striae distensae is unknown but probably relates to changes in the fibroblast phenotype. In order to characterize striae distensae fibroblasts, alpha-smooth muscle actin expression and contractile forces were studied. Five healthy women with early erythematous striae and five healthy women with older striae were selected. Paired biopsies were taken from the center of lesional striae and adjacent normal skin. Fibroblasts were obtained by an explant technique and expanded in vitro in Dulbecco’s modified Eagle‘s medium. Contractile forces generated by fibroblasts in collagen lattices were measured with the Glasbox device developed in our laboratory. Alpha-smooth muscle actin expression was studied by immunofluorescence labeling of cells and by flow cytometry. Fibroblasts from early striae distensae were the richest cells in alpha-smooth muscle actin filaments and generated the highest contractile forces. Their peak contractile force was 26% greater than normal fibroblasts. There was a 150% higher level of alpha-smooth muscle actin content in fibroblasts from early striae distensae compared with fibroblasts from normal skin. In contrast, there was no significant difference in force generation between old striae fibroblasts and normal fibroblasts with cells expressing no alpha-smooth muscle actin. The contractile properties of fibroblasts from striae distensae varies depending on the stage of the disease. In early striae distensae, fibroblasts acquire a more contractile phenotype, corresponding to that of myofibroblasts.     

Cosmetical treatments of connective tissue disorders

Connective tissue disorders (CTDs) are chronic inflammatory conditions that can lead to scarring and disfiguration. Although conventional methods are often of little benefit in cutaneous manifestations, the use of cosmetic procedures is still controversial. Concerns have also been raised concerning cosmetic treatments in CTDs, and particularly regarding lasers and fillers, due to photosensitivity and potential reactivation. This article reviews the cosmetic treatment of various CTDs under three headings ‐ lasers, fillers, and botulinum toxin.     

Induction of matrix metalloproteinase‐1 by small interfering RNA targeting connective tissue growth factor in dermal fibroblasts from patients with systemic sclerosis

Please cite this paper as: Induction of matrix metalloproteinase‐1 by small interfering RNA targeting connective tissue growth factor in dermal fibroblasts from patients with systemic sclerosis. Experimental Dermatology 2010; 19 : e111–e116. Abstract: We aimed to evaluate the effect of small interfering RNA (siRNA) targeting CTGF on extracellular matrices (ECMs) metabolism in normal and SSc fibroblasts. Normal and SSc fibroblasts were transfected with CTGF‐specific siRNAs to silence CTGF synthesis. After silencing CTGF, production of type I collagen and matrix metalloproteinase (MMP)‐1 by fibroblasts stimulated with TGF‐β was examined. Then quantitative analyses of protein production or mRNA expression of type I collagen, MMP‐1,‐2,‐9 and tissue inhibitor of metalloproteinase (TIMP)‐1 with TGF‐β stimulation were carried out. Furthermore, after silencing CTGF, proliferations of normal and SSc fibroblasts were investigated. CTGF‐specific siRNA significantly reduced CTGF production. The production of type I collagen was significantly reduced by CTGF silencing in normal fibroblasts. The CTGF silencing significantly increased the production of MMP‐1 and decreased the production of TIMP‐1 in SSc fibroblasts. The mRNA expression of MMP‐1 was increased in CTGF‐silenced SSc fibroblasts, but not in normal fibroblasts. There were no significant changes in the production or mRNA expression of other ECM‐related genes in normal and SSc fibroblasts. Fibroblast proliferations were suppressed by CTGF silencing in normal and SSc fibroblasts. Our data showed that MMP‐1 was increased by CTGF‐specific siRNA transfection only in SSc fibroblasts. RNAi targeting CTGF could be a novel therapeutic strategy for SSc.     

Melanoma cell-derived factors stimulate glycosaminoglycan synthesis by fibroblasts cultured as monolayers and within contracted collagen lattices

BACKGROUND: Various tumours exhibit glycosaminoglycan rich, and in particular hyaluronan rich matrices surrounding them that facilitate tumour growth and invasion. In many tumours, this matrix is predominantly synthesized by fibroblasts following stimulation by tumour cell-derived factors. OBJECTIVES: To determine what effect tumour cell-conditioned medium has upon fibroblast glycosaminoglycan synthesis when cells were cultured as monolayers and within contracted collagen lattices. METHODS: Serum-free conditioned medium from melanoma cell lines (C8161, MV3, A375 and Hs294T) was examined for its ability to stimulate the incorporation of 3H-glucosamine and 35SO4 into glycosaminoglycans synthesized by fibroblasts. RESULTS: Conditioned medium from all four melanoma cell lines exhibited potent glycosaminoglycan-stimulating activity. In monolayer culture, C8161-conditioned medium stimulated a 4.2-fold increase in fibroblast hyaluronan, and a 9.9-fold increase in sulphated glycosaminoglycan synthesis, while 35SO4 incorporation was increased only 2.1-fold. In collagen lattice cultures, C8161-conditioned medium stimulated a 4.9-fold increase in hyaluronan synthesis, a 5.4-fold increase in sulphated glycosaminoglycans, and a 1.3-fold increase in 35SO4 incorporation. CONCLUSIONS: Melanoma cells produce factors that are potent stimulators of fibroblast glycosaminoglycan synthesis, in both monolayer culture and within contracted collagen lattices. Synthesis of both hyaluronan and sulphated glycosaminoglycans with a reduced degree of polymer sulphation is stimulated. Such changes are likely to promote tumour cell proliferation and migration.     

The metabolism of the main substance of connective tissue and of collagen proteins in psoriasis patients

Blood and serum glucosaminoglycanes, serum glycosaminoglycane hydrolases, hydroxyprolinuria were measured in 140 patients, 60 of these suffering from psoriasis and 80 from psoriatic arthropathy. A relationship between the severity of impairments of connective tissue metabolism and dissemination of the skin process, as well as activity of articular and visceral inflammations, was examined. The detected regularities are discussed in the light of psoriasis pathogenesis; differential diagnostic potentialities of these regularities are analyzed.     

Inhibition of collagen production in scleroderma fibroblast cultures by a connective tissue glycoprotein extracted from normal dermis

It was shown in a previous paper that a connective tissue glycoprotein (CTGP) extracted from normal rabbit dermis was able to inhibit total protein and collagen syntheses by normal dermis fibroblast cultures. In the present study, the effects of CTGP on scleroderma fibroblasts were investigated. [14C]Proline incorporation into total proteins of the supernatant was not significantly different from that found in controls. By contrast, the amount of collagen, expressed as percentage of total secreted protein, was far higher in scleroderma cultures than in normal ones (14.4% +/- 6.0% vs 4.6% +/- 0.9%). Addition of CTGP to the medium induced a concentration-dependent inhibition of [14C]proline incorporation into proteins from both control and scleroderma cells. In control cultures, no significant decrease of the percentage of collagen was observed, but over 60 micrograms/ml, both cytotoxic effects and inhibition of protein synthesis occurred. In scleroderma cultures, the inhibition was twice as effective on collagen as on noncollagen protein synthesis. The inhibition of collagen secretion was not related either to changes in collagen hydroxylation or to the intracellular catabolism of newly synthesized procollagen.     

Chronological ageing and photoageing of the fibroblasts and the dermal connective tissue

In recent years, the exposure of human skin to environmental and artificial UV irradiation has increased dramatically. This is due not only to increased solar UV irradiation as a consequence of stratospheric ozone depletion, but also to inappropriate social behaviour with the use of tanning salons still being very popular in the public view. Besides this, leisure activities and a lifestyle that often includes travel to equatorial regions add to the individual annual UV load. In addition to the common long-term detrimental effects such as immunosuppression and skin cancer, the photo-oxidative damage due to energy absorption of UV photons in an oxygenized environment leads to quantitative and qualitative alterations of cells and structural macromolecules of the dermal connective tissue responsible for tensile strength, resilience and stability of the skin. The clinical manifestations of UV/reactive oxygen species (ROS)-induced disturbances result in photoaged skin with wrinkle formation, laxity, leathery appearance as well as fragility, impaired wound healing capacities and higher vulnerability. Strategies to prevent or at least minimize ROS-induced photo-ageing and intrinsic ageing of the skin necessarily include protection against UV irradiation and antioxidant homeostasis.     

Effects of retinoids on glycosaminoglycan synthesis by human skin fibroblasts grown as monolayers and within contracted collagen lattices

Fibroblasts grown within contracted collagen lattices synthesize substantially less glycosaminoglycans than fibroblasts grown as monolayers on a plastic substrate. [3H]glucosamine incorporation into hyaluronate was reduced by 70%, and incorporation into sulphated glycosaminoglycans was reduced by 40%. However, incorporation into heparan sulphate and chondroitin sulphates was reduced by 14 and 49%, respectively, resulting in a substantial change in the proportions of the individual glycosaminoglycans. On the basis of [³H]glucosamine incorporation, hyaluronate constituted 80% of the total glycosaminoglycans synthesized in monolayer cultures, but only 67% in collagen lattice cultures. Incorporation of ³⁵SO₄ into chondroitin sulphates was reduced by 22%, whereas no change was observed in heparan sulphates following culture within collagen lattices. Exposure of the fibroblast cultures to retinotc acid (10^?6mol/l) and retinyl propionate (2 × 10^?6mol/l) resulted in a decrease in the incorporation of [³H]glucosamine into hyaluronate by up to 41 % in monolayer cultures, and 25% in collagen lattice cultures. The retinoids stimulated the incorporation of [3H]glucosamine into heparan sulphate by up to 72%, and chondroitin sulphates by up to 30%, whereas ³⁵SO₄ incorporation remained essentially unaltered. Only modest changes in the incorporation of both isotopes into tibroblasl sulphated glycosaminoglycans were observed following exposure to the retinoids in lattice cultures. Q-Sepharose ion-exchange chromatography at pH 2–0 revealed that there was no change in the degree of polymer sulphation of either chondroitin sulphate or heparan sulphate isolated from collagen lattice cultures compared with monolayer cultures. Retinoic acid (10^?6mol/l) treatment did, however, reduce the degree of polymer sulphation of heparan sulphates and chondroitin sulphates in both monolayer and lattice cultures. The molecular mass of the sulphated glycosaminoglycans was unaffected by culture conditions, but hyaluronate isolated from collagen lattice cultures had a higher molecular mass than that from monolayer cultures. Retinoic acid treatment had no effect on the molecular mass of the glycosaminoglycans isolated from either culture system.