Increased Procollagen Synthesis Research Articles

Antioxidant and Pro-collagen Synthesis Activities of the Eriobotrya japonica Leaf Extract Using Enzyme

Purpose: We conducted a study that examined the physicochemical properties of Eriobotrya japonica leaf enzyme-assisted extract (EJLE) from Jeju Island.Methods: EJLE was prepared using the pepsin enzyme. The antioxidant and antiaging effects of EJLE were investigated to determine its utility as a functional material.Results: In the analysis of total amino acids, 16 kinds of components were isolated from EJLE. The DPPH and ABTS radical scavenging activities of EJLE were found to be 97.19±0.24% and 96.91±0.02% respectively in 500 μg/mL. Inhibition activities of elastase by EJLE were confirmed. In fibroblasts, EJLE at 500 μg/mL demonstrated a procollagen synthesis activity of approximately 52%.Conclusion: Our results confirmed that EJLE induced antioxidants in cosmetic materials and increased procollagen synthesis on the skin.

The protective effect of alfalfa (Medicago sativa L.) seed extract containing polysaccharides on human keratinocytes and fibroblasts

To protect the body against environmental threats, cosmetics can play important antiinflammatory and anti-aging roles. Many researchers have developed cosmetic ingredients using natural plant extracts or the active compounds of such extracts. In this study, we assessed the ability of extracts from the seeds of alfalfa (Medicago sativa L.) to affect skin cells. In a keratinocyte-derived cell line (HaCaT cells), alfalfa polysaccharide extract (APE) treatment triggered cornified envelope formation and increased the gene expression levels of filaggrin and involucrin, suggesting that APE increased epidermal cell differentiation. In addition, APE dramatically increased the mRNA and protein levels of claudin1, suggesting that it enhances tight junction formation. Finally, APE exerted antiinflammatory effects in keratinocytes by decreasing the LPSinduced production of proinflammatory cytokines. In primary normal human dermal fibroblasts (NHDF), APE increased procollagen synthesis and decreased elastase activity and metalloproteinase-1 (MMP) secretion. APE also showed superoxide dismutase (SOD)-like activity in a cell-free system, resulting in antioxidant effects. Finally, we found that the polysaccharides from alfalfa seeds used in our experiments consist mainly of galactose and mannose. Based on our findings, we conclude that APE may have potent skin-protective and skin-improving effects and could be developed as an important ingredient in cosmetics and medicines.

Pheophorbide a Derivatives Exert Antiwrinkle Effects on UVB-Induced Skin Aging in Human Fibroblasts.

Pheophorbide a is a chlorophyll metabolic breakdown product. This study investigated the antiwrinkle effect of pheophorbide a (PA) and its derivatives, including pyropheophorbide a (PyroPA) and pyropheophorbide a methyl ester (PyroPA-ME), on ultraviolet (UV) B-stimulated CCD-986sk fibroblasts. PA, PyroPA, and PyroPA-ME effectively suppressed reactive oxygen species accumulation in UVB-exposed CCD-986sk fibroblasts. All three pheophorbides also reduced UVB-induced matrix metalloproteinase (MMP)-1 secretion and mRNA expression of MMP-1, MMP-2, and MMP-9. Treatment with pheophorbides resulted in increased procollagen synthesis, and this required enhancement of procollagen type I C-peptide content and mRNA expression of collagen type I alpha 1 (COL1A1) and COL1A2 in CCD-986sk cells. These antiwrinkle effects were more potent with PA and PyroPA than with PyroPA-ME. Furthermore, PA and PyroPA suppressed UVB-induced phosphorylation of extracellular signal-regulated protein kinase and c-Jun N-terminal kinase but not p38. Moreover, all three pheophorbides inhibited NF-κB p65 phosphorylation. Therefore, these pheophorbides, especially PA and PyroPA, can be used as antiwrinkle agents, and PA- or PyroPA-rich natural resources can be used in functional cosmetics.

ROS Scavenging and Anti-Wrinkle Effects of Clitocybin A Isolated from the Mycelium of the Mushroom Clitocybe aurantiaca.

Clitocybin A, an isoindolinone from Clitocybe aurantiaca, was investigated to assess its anti-wrinkle properties, through reactive oxygen species (ROS)-scavenging and elastase inhibitory activities, procollagen synthesis, and matrix metalloproteinase-1 (MMP-1) expression, in human primary dermal fibroblast-neonatal (HDF-N) cells. Clitocybin A exhibited no significant cytotoxicity up to 10 ppm in HDF-N cells, with cell viability and cell proliferation activity greater than 94.6% and 91.9%, respectively. Strong and concentration-dependent ROS radical scavenging activities of clitocybin A were observed following irradiation with UVB at 30 mJ/cm2. Furthermore, clitocybin A treatment of cells at 0.1, 1, and 10 ppm exhibited decreased elastase activity, in a concentration-dependent manner, by 1.97%, 6.6%, and 8.31%, respectively, versus the control group. The effects of clitocybin A on procollagen synthesis and MMP-1 expression were investigated. Clitocybin A treatment of cells at 1, 5, and 10 ppm increased procollagen synthesis, by 67.9%, 74.4%, and 112.9%, respectively, versus the control group. At these concentrations, MMP-1 expression decreased significantly following UV irradiation. Together, these findings suggest that clitocybin A may be an effective ingredient for use in anti-wrinkle cosmetic products.

Development of an in vitro model of menopause using primary human dermal fibroblasts

To overcome the current lack of in vitro models to specifically reproduce hormonal skin ageing in women, and in search of active ingredients with innovative efficacy claim for cosmetic skin care, we developed a cell culture-based model by simulating menopause's hormonal decline and assessed several parameters of collagen metabolism. Human dermal fibroblasts were incubated with media containing 17β-oestradiol, progesterone, dehydroepiandrosterone, growth hormone and insulin-like growth factor-1 at concentrations corresponding to those of non-menopausal women's sera and then of menopausal women's sera. We measured cell proliferation [by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)], matrix metalloproteinase-1 and metalloproteinase-3 (MMPs) release (by enzyme-linked immunosorbent assay - ELISA), total collagen deposition (by Sirius red staining), types I and III collagen deposition (by ELISA), and types I and III procollagen gene expression (by real-time q-RT-PCR). Our results showed a significant decrease over time in cell proliferation, collagen deposition and type III/type I collagen ratio, together with an increase in MMP release, when cells were incubated in media containing sex hormones at menopausal levels. This is consistent with in vivo data from menopausal women available in the literature. Surprisingly, procollagen gene expression was only reduced within the first hours and increased afterwards when compared with non-menopausal culture conditions. Our results demonstrated that the increased procollagen synthesis with menopausal conditions was not sufficient to compensate for the MMPs' catabolic effects and/or the impaired procollagen protein maturation, resulting in a decrease in extracellular collagen content. These findings add to the overall understanding of hormone-dependent skin behaviour and highlight the suitability of this in vitro model for cosmetic actives testing aiming to underpin claims of anti-ageing efficacy, specifically for menopausal women, regarding collagen metabolism and balance of types, for maintenance of dermal mechanical properties.

Inhibitory effects of sea buckthorn (Hippophae rhamnoides L.) seed on UVB-induced Photoaging in human dermal fibroblasts

Sea buckthorn (Hippophae rhamnoides L.) seed, which has very high nutraceutical, cosmetoceutical and therapeutic properties, has been widely used to treat diseases in Tibetan and Mongolian traditional medicines. In this study, we measured the antioxidant activities of the solvent-dependent SBS (Sea buckthorn seed) extracts using the DPPH and Folin-Ciocalteu assays and we investigated the efficacy of SBS in protecting skin against UVB-induced damage using cultured human dermal fibroblasts. In addition, the inhibitory effects of SBS on UVB-induced skin photoaging were examined by determining the level of Metalloproteinase (MMP)-1 and procollagen after UVB-irradiation. The antioxidant capacity of the SBSE (alcoholic) extract was significantly higher than the SBSH (hexane) and SBSW (water) extracts, as measured based on the free-radical scavenging activity and total phenolic content. SBSE was chosen as the most suitable candidate antioxidant. In human dermal fibroblasts, the cell viability of SBSE extract at doses of 2.5, 5, and 10 μg/mL was higher than the UVB-treated control. By RT-PCR and Western blot, SBSE treatment inhibited UVB-induced IL-1β expression in cultured cells. In addition, SBSE restrained UVB-induced IL-6 and COX-2 gene expression in cultured fibroblasts in a dose-dependent manner. Treatment with the SBSE extract after UVB irradiation in human dermal fibroblasts significantly reduced MMP-1 expression and increased procollagen synthesis when compared with UVB-irradiation only. In this study, SBSE was shown to increase the synthesis of procollagen, decrease the expression of MMP-1, and inhibit the production of IL-1β, IL-6, and COX-2 in UVB-irradiated human fibroblasts. These findings suggest that the SBSE extract may be a potential therapeutic agent for preventing and treating skin photoaging.

A Translational Approach to Hypertensive Heart Disease

The spectrum of the cardiac complications of arterial hypertension includes heart failure (HF), sudden death, and cardiac dysrhythmias, as well as the exacerbation of coincidental diseases (ie, atherosclerosis and chronic renal disease).1 However, knowledge of the cardiac impact of chronic elevation of arterial pressure has so evolved that hypertensive heart disease (HHD) may be thought as those consequences derived from left ventricular (LV) responses to fundamental disease mechanisms triggered by mechanical overload and neurohumoral stimuli.2 For instance, the long-held views are that LV hypertrophy (LVH), as the result of cardiomyocyte growth in response to pressure overload, serves to restore heart muscle economy back to normal and to preserve LV function.3 However, a number of alterations of the cardiomyocyte and the noncardiomyocyte components of the myocardium (including apoptosis, fibrosis, and changes in the microcirculation) also develop in HHD that lead to pathological myocardial remodeling not only in the left ventricle but also the left atrium and right ventricle.2 These alterations may explain the overall risk of LVH and its associated cardiac and noncardiac complications in hypertensive patients.4 Although LVH may be detected early and accurately in hypertensive patients by electrocardiography and echocardiography, newer cardiac imaging methods and the monitoring of several circulating biomarkers hold promise as noninvasive tools for the diagnosis of myocardial remodeling. Numerous clinical studies have shown that effective long-term antihypertensive treatment may be associated with a decreased LV mass (LVM), which has been attributed to diminished risk. However, no large study (or meta-analysis) has demonstrated that diminished risk from the contemporary reduction of arterial pressure by virtue of its a priori design. Therefore, because overall risk remains unacceptably high, especially from HF, new therapeutic strategies aimed not only to decrease arterial pressure and LVM but also to repair and even prevent myocardial remodeling …

GATA4, a new regulator of cardiac fibroblasts, is sensitive to natriuretic peptides

Cardiac fibroblasts are the most prevalent cell type in the heart. These cells exert a critical role in regulating normal myocardial function and in adverse myocardial remodelling that occur with hypertension, myocardial infarction, and heart failure.1 In normal cardiac tissue, fibroblasts are found throughout the cardiac tissue in parallel to the prevailing direction of muscle fibres. In essence, every cardiac myocyte is therefore in direct contact with fibroblasts. Adverse myocardial remodelling includes enlargement of myocytes, proliferation of fibroblasts, and increased procollagen synthesis. Natriuretic peptides, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), released from myocytes have been shown to act as autocine/paracrine factors that reduce myocyte hypertrophy and exert antifibrotic and antiproliferative effects on cardiac fibroblasts.2,3 Antifibrotic effects are attributed to C-type natriuretic peptide (CNP) released from endothelial cells and cardiac fibroblasts.4 Natriuretic peptides initiate their signalling via guanylyl cyclase receptors (NPR-A, NPR-B) and C-type receptor (NPR-C) all expressed in cardiac fibroblasts.3,4 These effects may be of particular importance during pathological conditions, when the fibrosis and increased deposition of collagen results in increased myocardial stiffness and diastolic dysfunction. Myocardial hypertrophy is also characterized by activation of transcription factors. A number of stimuli that induce myocyte hypertrophy are known to enhance GATA4 transcriptional activity through … *Corresponding author. Tel: +1 514 890 8000; fax: +1 514 412 7204; E-mail address : marek.jankowski{at}umontreal.ca

Molecular Markers in Patients with Chronic Wounds to Guide Surgical Debridement

Chronic wounds, such as venous ulcers, are characterized by physiological impairments manifested by delays in healing, resulting in severe morbidity. Surgical debridement is routinely performed on chronic wounds because it stimulates healing. However, procedures are repeated many times on the same patient because, in contrast to tumor excision, there are no objective biological/molecular markers to guide the extent of debridement. To develop bioassays that can potentially guide surgical debridement, we assessed the pathogenesis of the patients' wound tissue before and after wound debridement. We obtained biopsies from three patients at two locations, the nonhealing edge (prior to debridement) and the adjacent, nonulcerated skin of the venous ulcers (post debridement), and evaluated their histology, biological response to wounding (migration) and gene expression profile. We found that biopsies from the nonhealing edges exhibit distinct pathogenic morphology (hyperproliferative/hyperkeratotic epidermis; dermal fibrosis; increased procollagen synthesis). Fibroblasts deriving from this location exhibit impaired migration in comparison to the cells from adjacent nonulcerated biopsies, which exhibit normalization of morphology and normal migration capacity. The nonhealing edges have a specific, identifiable, and reproducible gene expression profile. The adjacent nonulcerated biopsies have their own distinctive reproducible gene expression profile, signifying that particular wound areas can be identified by gene expression profiling. We conclude that chronic ulcers contain distinct subpopulations of cells with different capacity to heal and that gene expression profiling can be utilized to identify them. In the future, molecular markers will be developed to identify the nonimpaired tissue, thereby making surgical debridement more accurate and more efficacious.

Increase in Collagen Turnover But Not in Collagen Fiber Content Is Associated with Flow-Induced Arterial Remodeling

Background: Degradation and synthesis of collagen are common features in arterial geometrical remodeling. Previous studies described an association between arterial remodeling and an increase in collagen fiber content after balloon injury. However, this does not exclude that the association between collagen content and remodeling depends on arterial injury since the association of collagen fiber content and arterial remodeling, without arterial injury, has not been investigated. The aim of the present study was to study the relation between flow-induced arterial geometrical remodeling, without arterial injury, and collagen synthesis and degradation, collagen fiber content and cell-migration-associated moesin levels. Methods and Results: In 23 New Zealand White rabbits an arteriovenous shunt (AV shunt) was created in the carotid and femoral artery to induce a structural diameter increase or a partial ligation (n = 27 rabbits) to induce a diameter decrease. In both models, arterial remodeling was accompanied by increased procollagen synthesis, reflected by increased procollagen mRNA or Hsp47 protein levels. In both models, however, no changes were detected in collagen fiber content. Active MMP-2 and moesin levels were increased after AV shunting. Conclusions: Collagen synthesis and MMP-2 activation were associated with arterial remodeling. However, a change in collagen fiber content was not observed. These results suggest that, during flow-induced geometrical arterial remodeling, increases in collagen synthesis are used for matrix collagen turnover and cell migration but not to augment collagen fiber content.

Optimum force and duration of toothbrushing to enhance gingival fibroblast proliferation and procollagen type I synthesis in dogs.

Toothbrushing enhances gingival fibroblast proliferation, which promotes wound healing. Optimum force and duration of toothbrushing for stimulation of fibroblast proliferation are key factors in maximizing effects of toothbrushing on periodontal wound healing. We therefore evaluated the effects of different durations and forces of toothbrushing on proliferative activity and procollagen synthesis of gingival fibroblasts. Twelve dogs were used. In each dog, buccal gingivae of 12 teeth were examined for 3 weeks. Nine of these 12 teeth were each assigned to 1 of 9 different combinations of brushing force (0.98, 1.96, or 2.45 N) and duration (10, 20, or 40 seconds). The remaining 3 teeth received plaque removal without brushing, via a scaler. Force and duration of toothbrushing affected both proliferating cell nuclear antigen (PCNA)-positive and procollagen Type I C-peptide (PIP)-positive fibroblast ratios (P < 0.05). The highest ratio of PCNA-positive fibroblasts was produced by brushing at 1.96 N for 20 seconds. The highest ratio of PIP-positive fibroblasts was produced by brushing at 1.96 N for 10 seconds. Toothbrushing at certain forces and durations enhanced the proliferative activity and procollagen synthesis of gingival fibroblasts. The toothbrushing duration that increased procollagen synthesis (10 seconds) was shorter than that which increased fibroblast proliferative activity (20 seconds).

Myocardial fibrosis in DOCA-salt hypertensive rats: effect of endothelin ET(A) receptor antagonism.

To test the hypothesis that endothelin-1 contributes to cardiac fibrosis, cardiac collagen deposition was studied in deoxycorticosterone acetate-salt (DOCA-salt) hypertensive rats, in which the endothelin system is activated. The effects of the ET(A)-selective endothelin receptor antagonist A-127722 were evaluated. A-127722 (30 mg/kg per day) was administered for 4 weeks. Myocardial fibrosis was evaluated after Sirius red F3BA staining. Systolic blood pressure was 103+/-1.6 mm Hg in unilaterally nephrectomized rats (Uni-Nx), 202+/-3.2 mm Hg in DOCA-salt rats (P:<0.01 versus Uni-Nx), and 182+/-3.1 mm Hg in ET(A) antagonist-treated DOCA-salt rats (P:<0.01 versus DOCA-salt or Uni-Nx). In DOCA-salt rats, interstitial and perivascular collagen density was increased in the subendocardial and midmyocardial regions of the left ventricle (3- to 4-fold, P:<0.05), whereas in subepicardial myocardium, the increase was predominantly perivascular. The ET(A) antagonist prevented cardiac fibrosis in DOCA-salt rats. Procollagen I and III mRNA, which were increased in hearts of DOCA-salt rats, were normalized by ET(A) antagonist treatment. TGF-beta(1) mRNA and TGF-beta(1) protein increased at 1 week in DOCA-salt rats and were lowered in ET(A) antagonist-treated rats. ET(A) receptor-mediated collagen deposition in hearts of DOCA-salt rats results from increased procollagen synthesis associated with an initial increment in expression of TGF-beta(1). These results support the hypothesis of a role for endothelin-1 in cardiac collagen deposition in mineralocorticoid hypertension, which may have pathophysiological and pharmacological implications in hypertensive heart disease.

In vivo collagen turnover during development of thyroxine-induced left ventricular hypertrophy

Cardiac fibroblasts synthesize large amounts of procollagens, yet only a small fraction of mature collagens accumulate in the extracellular matrix. To determine the roles of intracellular degradation of newly synthesized procollagens and extracellular degradation of mature collagens during normal growth and during thyroxine-induced left ventricular hypertrophy, in vivo left ventricular procollagen synthetic rates were assessed in control rats and rats treated with L-thyroxine for 1, 2, 4, and 8 wk (1 mg.kg-1.day-1). A modification of the flooding infusion method was developed using measurements of cardiac prolyl-tRNA, and tissue-free and protein-bound hydroxyproline specific radioactivities 60 min after intravenous administration of a massive dose of [3H]proline. Degradative rates of newly synthesized procollagens and mature collagens were then derived as the difference between rates of procollagen synthesis and collagen accumulation. Left ventricular procollagen synthetic rates were markedly increased after 1 wk of hormone administration (256 +/- 16 and 166 +/- 13 micrograms/day per left ventricle for thyroxine-treated and control animals, respectively; P less than 0.01). An even greater increase in procollagen synthetic rates was observed after 8 wk (438 +/- 46 and 202 +/- 18 micrograms/day for thyroxine-treated and control animals, respectively; P less than 0.01). Despite increased procollagen synthesis, disproportionate accumulation of fibrillar collagens (assessed as the relative concentration of protein-bound hydroxyproline in left ventricular tissue) did not occur. Derived left ventricular degradative rates for newly synthesized procollagens as well as for mature collagens were increased in thyroxine-treated animals. Increased procollagen synthesis, enhanced flux of newly synthesized procollagens through intracellular degradative pathways, and extensive extracellular matrix remodeling without disproportionate collagen accumulation are characteristics of this form of "physiological" left ventricular hypertrophy.

Interferon-α and interferon-γ reduce excessive collagen synthesis and procollagen mRNA levels of scleroderma fibroblasts in culture

The effects of interferon-α and interferon-γ on collagen synthesis and mRNA levels of type I and type III procollagens were studied in skin fibroblasts cultured from affected and unaffected skin sites of two patients with localized scleroderma (morphea). Both scleroderma cell lines exhibited elevated type I and type III procollagen mRNA levels to account for the increased procollagen synthesis, when compared to the unaffected controls. Interferon-γ treatment resulted in a dose-dependent reduction in collagen synthesis and procollagen mRNA levels in scleroderma fibroblasts. A 72-h exposure to interferon-γ reduced procollagen mRNA levels in the scleroderma fibroblast lines to the levels exhibited by the unaffected control fibroblasts. The suppressive effect of interferon-α on procollagen mRNA levels was somewhat weaker than that of interferon-γ. The results suggest potential use of interferon-γ in treatment and prevention of human fibrotic conditions.

Bleomycin-induced synthesis of type I procollagen by human lung and skin fibroblasts in culture

Bleomycin is a chemotherapeutic agent sometimes associated with pulmonary fibrosis and skin lesions in patients undergoing treatment. We examined the mechanisms of increased collagen deposition in bleomycin-induced fibrosis by incubating human lung and skin fibroblast cultures with [ 14C]proline; the synthesis of [ 14C]hydroxyproline relative to DNA or cell protein was taken as an index of procollagen formation. Procollagen synthesis by lung cells in the presence of 0.1 and 1.0 μg/ml bleomycin was significantly increased and similar results were obtained with skin fibroblasts. The relative synthesis of genetically distinct types of collagen was measured by isolating the newly synthesized type I and type III procollagens by DEAE-cellulose chromatography. The proportion of type III procollagen of total newly synthesized procollagen in control lung fibroblasts cultures was 17.4 ± 0.6% (mean ± S.E.) while the corresponding value in cells incubated in 1 μg/ml bleomycin wa 12.5 ± 0.6% ( n = 6, P < 0.01). Similar results were obtained when the ratios of newly synthesized type I and type III collagens were estimated by interrupted polyacrylamide disc gel electrophoresis in sodium dodecyl sulfate after a limited proteolytic digestion with pepsin. The results indicate that the increased procollagen synthesis induced by bleomycin in fibroblast cultures is predominantly directed towards the synthesis of type I procollagen.