Collagen Synthesis In Skin Fibroblasts Research Articles

N-phenethyl caffeamide and photodamage: Protecting skin by inhibiting type I procollagen degradation and stimulating collagen synthesis

Skin is mainly damaged by genetic and environmental factors such as ultraviolet (UV) light and pollutants. UV light is a well-known factor that causes various types of skin damage and premature aging. Reactive oxygen species (ROS) are commonly involved in the pathogenesis of skin damage by activating the metalloproteinases that break down type I collagen. This study investigated the antioxidant and antiphotodamage activity and mechanisms of N-phenethyl caffeamide (K36) in human skin fibroblasts. The results indicated that K36 demonstrated strong 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) scavenging activity, which dose-dependently reduced the production of UVB-induced intracellular ROS in human dermal fibroblasts. K36 prevented UVB-irradiation-induced type I collagen degradation by inhibiting the expression of matrix metalloproteins-1, -3, and -9 and the phosphorylation of mitogen-activated protein (MAP) kinases. Furthermore, K36 elevated collagen synthesis in skin fibroblasts by inhibiting UVB-induced Smad7 overexpression. K36 downregulated the expression of the transcription factor, activator protein-1 (AP-1). Our results indicated that K36 exhibited antioxidant properties and prevented skin collagen degradation caused by UV exposure and the stimulation of collagen synthesis, which suggests the potential use of K36 in preventing photodamage.

Tropisetron suppresses collagen synthesis in skin fibroblasts via α7 nicotinic acetylcholine receptor and attenuates fibrosis in a scleroderma mouse model

There is increasing evidence that serotonin (5-hydroxytryptamine [5-HT]) and distinct 5-HT receptors are involved in the pathogenesis of systemic sclerosis. The aim of this study was to test the hypothesis that tropisetron, a routinely used antiemetic agent previously characterized as a 5-HT(3/4) receptor-modulating agent, can directly affect collagen synthesis in vitro and attenuate experimentally induced fibrosis in vivo. Functional in vitro studies were performed using human dermal fibroblasts (HDFs). Signal transduction studies included immunofluorescence analysis, Western immunoblotting, promoter reporter assays, cAMP/Ca(2+) measurements, and use of pharmacologic activators and inhibitors. Gene silencing was performed using small interfering RNA. Putative receptors of tropisetron were detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. The murine model of bleomycin-induced scleroderma was used to assess the antifibrogenic and antifibrotic effects of tropisetron in vivo. Collagen expression in vitro, ex vivo, and in situ was determined by real-time RT-PCR analysis, Western immunoblotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunohistochemical analysis. Tropisetron suppressed collagen synthesis induced by transforming growth factor β1 (TGFβ1). This effect was independent of 5-HT(3/4) receptor but was mediated via α7 nicotinic acetylcholine receptor (α7nAChR). Suppression of TGFβ1-induced collagen synthesis occurred via an unknown molecular mechanism not involving modulation of the Smad, cAMP, Akt, c-Jun, or MAPK pathway. In vivo, tropisetron not only prevented skin fibrosis but also reduced the collagen content in established dermal fibrosis induced by bleomycin. Tropisetron directly reduces collagen synthesis in HDFs via an α7nAChR-dependent mechanism. The antifibrogenic and antifibrotic effects of this agent observed in a mouse model of bleomycin- induced scleroderma indicate the future potential of tropisetron in the treatment of fibrotic diseases such as scleroderma.

Screening for phytoestrogens and their anti-skin aging effect in vitro

Objective To screen phytoestrogens and investigate their effect on collagen synthesis in skin fibroblasts. MethodsSixty-four kinds of plant extracts were screened for phytoestrogens by determining their effect on estrogen receptor(ER)-positive MCR-7 cell proliferation with MTT assay.ER-negative MDA-MB-231 cells and ER antagonist ICI 182780 were used to validate if the stimulatory effect on MCF-7 cell proliferation was mediated by ER.By using dot blot method,the effect on procollagen-1(PC-1) synthesis in neonatal foreskin fibroblasts was also assessed. ResultsAmong the selected 64 plant extracts and active fractions,soybean isoflavonoids and the ethanol extracts of Radix Puerariae,Spica Humuli Lupuli,Semen Cassiae,Folium Mori and Radix Glycyrrhizae effectively induced the proliferation of MCF-7 cells,but showed no effect on MDA-MB-231 cells.Moreover,their effect on MCF-7 cell proliferation was inhibited by ICI 182780.Genistein and puerarin significantly increased PC-1 synthesis in skin fibroblasts(P0.01),and puerarin showed more potent effect than genistein.In contrast,daidzein significantly reduced PC-1 synthesis(P0.01). ConclusionThe extracts of Radix Puerariae and soybean isoflavnoids have relatively potent estrogenic activity.Puerarin and genistein,their main active ingredients,stimulate collagen synthesis of human skin fibroblasts,suggesting their potential anti-wrinkle and anti-skin aging effect.

Trichostatin A inhibits collagen synthesis and induces apoptosis in keloid fibroblasts

Keloid, a fibro-proliferative benign tumor of skin, is characterized by an enriched milieu of growth factors and an abundant accumulation of extracellular matrix (ECM). Transforming growth factor (TGF)-β1 is well known as the crucial fibrogenic cytokine promoting ECM production and tissue fibrosis in keloid forming. Epigenetic modifications have been shown to play a role in the pathogenesis of cancer as well as autoimmune and inflammatory disorders. Recent publication reports epigenetic modifications in keloid fibroblasts that include an altered pattern of DNA methylation and histone acetylation. Therefore, the field of epigenetics may provide a new therapeutic idea for keloid treatment strategies. Currently, there is some evidence from experimental studies that histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) causes abrogation of TGF-β1 induced collagen synthesis in skin fibroblasts. Furthermore, TSA could suppress proliferation and induce apoptosis in a broad spectrum of tumor cells both in vitro and in vivo. These findings suggest that TSA could also cause abrogation of TGF-β1 induced collagen synthesis and induce apoptosis of proliferating keloid fibroblasts.

Proof of new collagen synthesis in lid skin after Er:YAG laser skin resurfacing with in‐situ hybridization

Abstract Purpose: The aim of the study is to prove new collagen synthesis in lid skin fibroblasts and correlate it to laser energy fluence after Er:YAG laser skin resurfacing, using the method of in situ hybridization. Methods: Lid skin of 9 patients with blepharochalasis was treated with laser at day 7 and 21 before elective blepharoplasty, when it was excised for further investigation. We used a 2940 nm Er:YAG laser (Fidelis by Fotona) with smooth mode parameters: laser fluence 0.75 – 2.00 J/cm2, spot size 5 mm, repetition rate 20 Hz, pulse length 550 μs, 6 pulses per packet, packet length 250 ms, no overlapping. We designed 2 oligonucleotide RNA probes for pro‐alpha 1 and pro‐alpha 2 collagen and labeled them with radioactive Sulphur 35. At time of surgery the excised tissue was frozen in dry ice. Tissue sections were incubated with the probes and marked with film emulsion for 10 days. We counted silver grains over specific cells, which were identified with imunohistochemistry. Results: We noticed significant elevation of pro‐alpha‐collagen mRNA expression in the laser treated areas of lid skin compared to untreated areas. Pro‐alpha‐collagen mRNA expression was particularly dense in cells, identified as fibroblasts by imunohistochemistry. Pro‐alpha‐collagen mRNA expression in these cells correlated with laser fluence used in treatment of lid skin. Conclusions: The method of in situ hybridization brings a definitive proof of new collagen synthesis in skin fibroblasts after Er:YAG laser skin resurfacing; and it is possible to measure the rate of new collagen synthesis as a function of the applied laser fluence.

Collagen synthesis in rat skin and ileum fibroblasts is affected differently by diabetes-related factors.

Untreated diabetes reduces wound strength: a concomitant reduction in collagen deposition has been found in cutaneous wounds but not in intestinal anastomoses. This raises the question if collagen synthesis in fibroblasts from skin and intestine reacts differently to the diabetic state. Fibroblast lines were established from healthy rat skin and ileum. Diabetic rat serum was collected from hyperglycaemic rats 3 days after intravenous injection of streptozotocin (200 mg/kg). Fibroblast cultures were grown to confluency in foetal calf serum and maintained in various concentrations of glucose, insulin, normal or diabetic rat serum. Collagen synthesis was measured by incorporation of [3H]-proline into Collagenase-Digestible-Protein. Collagen synthesis in fibroblasts from both skin and ileum was not affected by increasing glucose concentrations. Insulin strongly and specifically stimulated collagen synthesis in skin fibroblasts whereas in ileum fibroblasts only a nonspecific increase of total protein synthesis was observed. In skin fibroblasts, diabetic rat serum stimulated collagen synthesis to a significantly lesser extent than normal rat serum, whereas in ileum fibroblasts stimulation by serum was far less explicit and no difference was observed between normal and diabetic serum. The fact that ileum fibroblasts respond less strongly to culture in diabetic serum than skin fibroblasts may explain our prior finding that would collagen accumulation in intestinal anastomoses is virtually unaffected during diabetes and supports the existence of tissue-specific healing responses.

Incorporation of triterpenic derivatives within an o/w/o multiple emulsion: structure and release studies

Triterpenic derivatives stimulate the total collagen synthesis of skin fibroblasts. Their incorporation within an o/ w/o multiple emulsion should protect them against oxidation, particularly for unsaturated derivatives and should lead to a modulated release after topical application. The influence of the incorporation of madecassic acid, asiaticoside and asiatic acid on the viscosity and the stability of a multiple o/w/o emulsion was studied. Rheological analyses were carried out by flow experiments in order to obtain the apparent viscosity and by the oscillatory technique to calculate the basic visceolastic parameters. The comparative stability study was assessed by the tracer release method with phthalic acid diethyl ester previously incorporated in the internal phase of the emulsions. Six months storage at room temperature and at 40 degrees C did not affect the stability of emulsions with triterpenic substances. The in vitro percutaneous absorption of triterpenic derivatives was investigated by Franz diffusion cells in hairless rats. Triterpenic derivatives were analysed in stratum corneum, epidermis and dermis, by liquid chromatography. Correlations were established between the triterpenic derivatives structure (glycosylated or not) and their percutaneous absorption. O/w/o multiple emulsion seems to be an efficient vehicle to preserve these active substances and to control their distribution in the skin.

Effect of (+)-catechin on purified prolyl hydroxylase and on collagen synthesis in skin fibroblasts in culture

The flavonoid, (+)-catechin. inhibited the activity of purified rabbit prolyl hydroxylase, or the crude enzyme present in liver homogenate. (+)-Catechin at a concentration of 10 −4 M inhibited both forms of enzyme 50 percent. The inhibition was partially reversed when excessive amounts of Fe 2+ (10 −3 M) or ascorbate (10 −2 M) were added to the reaction mixture. A higher recovery of the original activity was obtained when Fe 2+ and ascorbate were added together to the same inhibited system. In addition, (+)-catechin revealed a competitive type of inhibition with respect to varying concentrations of enzyme. These results suggest that the drug and the enzyme compete for the free-radicals produced by the interaction of Fe 2+, ascorbate and molecular oxygen. The effects of (+)-catechin on collagen synthesis and on proline hydroxylation were examined in confluent cultures of human skin fibroblasts. The activity of prolyl hydroxylase was significantly (P < 0.01) reduced (50 percent of the control) when the cells were incubated for 7 hrs in the presence of 2 × 10 −4 M (+)-catechin. When exposed to the same inhibitor concentration for 24 hr, collagen, and non-collagen, protein synthesis, as measured by the collagenase digestion method, were also significantly reduced (P < 0.01). However, the significant (P < 0.01) decrease in the proportion of collagen synthesized to total protein showed that the drug is more effective on the pathway of collagen production. The ratio of hydroxyproline/proline in total protein was also significantly decreased (P < 0.01) in the cultures exposed to (+)-catechin (2 × 10 −4 M) for 24 hr.