Collagen transport and related pathways in Osteogenesis Imperfecta.

Osteogenesis Imperfecta (OI) comprises a heterogeneous group of patients who share bone fragility and deformities as the main characteristics, albeit with different degrees of severity. Phenotypic variation also exists in other connective tissue aspects of the disease, complicating disease classification and disease course prediction. Although collagen type I defects are long established as the primary cause of the bone pathology, we are still far from comprehending the complete mechanism. In the last years, the advent of next generation sequencing has triggered the discovery of many new genetic causes for OI, helping to draw its molecular landscape. It has become clear that, in addition to collagen type I genes, OI can be caused by multiple proteins connected to different parts of collagen biosynthesis. The production of collagen entails a complex process, starting from the production of the collagen Iα1 and collagen Iα2 chains in the endoplasmic reticulum, during and after which procollagen is subjected to a plethora of posttranslational modifications by chaperones. After reaching the Golgi organelle, procollagen is destined to the extracellular matrix where it forms collagen fibrils. Recently discovered mutations in components of the retrograde transport of chaperones highlight its emerging role as critical contributor of OI development. This review offers an overview of collagen regulation in the context of recent gene discoveries, emphasizing the significance of transport disruptions in the OI mechanism. We aim to motivate exploration of skeletal fragility in OI from the perspective of these pathways to identify regulatory points which can hint to therapeutic targets.

Severe disruption and disorganization of dermal collagen fibrils in early striae gravidarum.

BACKGROUND: Striae gravidarum (SG), or stretch marks of pregnancy, begin as erythematous streaks and mature into hypopigmented atrophic bands. OBJECTIVES: In order to investigate molecular alterations that may promote atrophy of SG, we investigated dermal type I collagen fibrils, which provide human skin with support. METHODS: We obtained skin samples of recently developed, erythematous abdominal SG from pregnant women. To examine the organization of collagen fibrils, second-harmonic generation imaging was performed using multiphoton microscopy. Immunostaining was used to determine protein expression and localization of type I procollagen, the precursor of type I collagen fibrils. Real-time polymerase chain reaction was used to determine gene expression levels. RESULTS: In control (hip) and stretched normal-appearing perilesional abdominal skin, dermal collagen fibrils were organized as tightly packed, interwoven bundles. In SG, collagen bundles appeared markedly separated, especially in the mid-to-deep dermis. In the spaces separating these bundles, loosely packed wavy collagen fibrils lacking organization as bundles were present. These disorganized fibrils persisted into the postpartum period and failed to form densely packed bundles. Numerous large fibroblasts displaying type I procollagen expression were in close proximity to the disorganized fibrils, suggesting that the fibrils are newly synthesized. Supporting this possibility, immunostaining and gene expression of type I procollagen were increased throughout the dermis of SG. CONCLUSIONS: Early SG display marked separation of collagen bundles and emergence of disorganized collagen fibrils that fail to form bundles. These alterations may reflect ineffective repair of collagen bundles disrupted by intense skin stretching. Persistent disruption of the collagenous extracellular matrix likely promotes formation and atrophy of SG.

Beneficial impact of zinc supplementation on the collagen in the bone tissue of cadmium-exposed rats.

Cadmium (Cd) is a toxic metal that damages bone tissue by affecting its mineral and organic components. The organic matrix is mainly (90%) composed of collagen, which determines the biomechanical strength of bone. The aim of this study was to evaluate the effect of zinc (Zn) supplementation (30 or 60 mg l-1 ) under moderate and relatively high exposure to Cd (5 and 50 mg l-1 ) on collagen in the rat tibia proximal epiphysis and diaphysis (regions abundant in trabecular and cortical bone, respectively). Significant decrease in collagen type I biosynthesis was found in both regions of the tibia in Cd-treated rats, whereas the supplementation with Zn provided significant protection against this effect. Western blot confirmed the presence of the major type I collagen in the tibia epiphysis and diaphysis, but collagen type II was revealed only in the epiphysis. Acetic acid- and pepsin-soluble collagen concentration in the tibia epiphysis and diaphysis was significantly increased due to the exposure to Cd, whereas the supplementation with Zn protected, partially or totally, from these effects, depending on the used concentration. The supplementation with Zn also provided protection from unfavorable Cd impact on the maturation of the bone collagen, as the ratio of cross-links to monomers was higher compared to the Cd-treated group. This report confirms our previous findings on the preventive action of Zn against harmful effects of Cd on bone, but additionally, and to the best of our knowledge for the first time, explains the possible mechanism of the beneficial influence of this bioelement.

HSP47 and FKBP65 cooperate in the synthesis of type I procollagen.

Osteogenesis imperfecta (OI) is a genetic disorder that results in low bone mineral density and brittle bones. Most cases result from dominant mutations in the type I procollagen genes, but mutations in a growing number of genes have been identified that produce autosomal recessive forms of the disease. Among these include mutations in the genes SERPINH1 and FKBP10, which encode the type I procollagen chaperones HSP47 and FKBP65, respectively, and predominantly produce a moderately severe form of OI. Little is known about the biochemical consequences of the mutations and how they produce OI. We have identified a new OI mutation in SERPINH1 that results in destabilization and mislocalization of HSP47 and secondarily has similar effects on FKBP65. We found evidence that HSP47 and FKBP65 act cooperatively during posttranslational maturation of type I procollagen and that FKBP65 and HSP47 but fail to properly interact in mutant HSP47 cells. These results thus reveal a common cellular pathway in cases of OI caused by HSP47 and FKBP65 deficiency.

Preparation, Characterization, and Biological Activities of Topical Anti-Aging Ingredients in a Citrus junos Callus Extract.

In this study, we prepared and characterized a callus extract from Citrus junos and assessed its utility as a source of topical anti-aging ingredients. Callus extract was produced by aqueous extraction from Citrus junos grown on Murashige and Skoog medium with picloram as a growth regulator. After measuring the total phenolic and flavonoid contents, the major phenolic compound in calli was identified as p-hydroxycinnamoylmalic acid (1) by spectroscopic analysis. The total phenol content in the extract was determined to be 24.50 ± 0.43 mg/g of gallic acid equivalents; however, the total flavonoid content of the extract was not determined. The biological activities of the callus extract, in terms of skin anti-aging, were assessed by measuring the anti-tyrosinase activity in, and melanogenesis by, melanoma cells; and proliferation of, and procollagen synthesis by, human fibroblasts. The callus extract was incorporated into nanoliposomes (NLs) to improve its percutaneous absorption. Addition of the callus extract resulted in a 1.85-fold decrease in the melanin content of melanocytes compared with that with arbutin. The extract (500 µg/mL) significantly promoted the proliferation of, and procollagen synthesis by, fibroblasts (by 154% and 176%, respectively). In addition, the flux through the human epidermis of Citrus junos callus extract incorporated into NLs was 17.67-fold higher than that of the callus extract alone. These findings suggest that Citrus junos callus extract-loaded NLs have promise as an anti-aging cosmetic, as well as having a skin-lightening effect.

The interferon type I signature is present in systemic sclerosis before overt fibrosis and might contribute to its pathogenesis through high BAFF gene expression and high collagen synthesis.

BACKGROUND: Interferon (IFN) signature has been reported in definite systemic sclerosis (SSc) but it has not been characterised in early SSc (EaSSc). We aim at characterising IFN type I signature in SSc before overt skin fibrosis develops. METHODS: The expression of 11 IFN type I inducible genes was tested in whole-blood samples from 30 healthy controls (HCs), 12 subjects with primary Raynaud's phenomenon (RP), 19 patients with EaSSc, 7 patients with definite SSc without cutaneous fibrosis, 21 limited cutaneous SSc and 10 diffuse cutaneous SSc subjects. The correlation between IFN activity in monocytes, B cell activating factor (BAFF) mRNA expression and type III procollagen N-terminal propeptide (PIIINP) serum levels was tested. RESULTS: In all the SSc groups, higher IFN scores were observed compared with HC. An IFN score ≥7.09 discriminated HCs from patients with SSc (sensitivity=0.7, specificity=0.88, area under receiving operating characteristic (AUROC)=0.82); the prevalence of an elevated IFN score was: HC=3.3%; RP=33.3%, EaSSc=78.9%, definite SSc=100%, limited cutaneous SSc=42.9%, diffuse cutaneous SSc=70.0%. In monocytes an IFN score ≥4.12 distinguished HCs from patients with fibrotic SSc (sensitivity=0.62, specificity=0.85, AUROC=0.76). Compared with IFN-negative subjects, IFN-positive subjects had higher monocyte BAFF mRNA levels (19.7±5.2 vs 15.20±4.0, p=2.1×10(-5)) and serum PIIINP levels (median=6.0 (IQR 5.4-8.9) vs median=3.9 (IQR 3.3-4.7), p=0.0004). CONCLUSIONS: An IFN type I signature is observed in patients with SSc from the earliest phases of the disease, even before overt skin fibrosis. The presence of IFN type I signature in monocytes is correlated with BAFF mRNA expression and serum PIIINP levels, supporting a contribution in the pathogenesis and progression of SSc.

Conditioned medium from human bone marrow-derived mesenchymal stem cells promotes skin moisturization and effacement of wrinkles in UVB-irradiated SKH-1 hairless mice.

BACKGROUND: Mesenchymal stem cells (MSCs) are promising therapeutic agents for various diseases. AIMS: To investigate the effects of conditioned medium from human bone marrow-derived mesenchymal stem cells (MSC-CdM) on pro-collagen production and wrinkle formation, we performed in vitro and in vivo experiments. METHODS: We assessed the effects of MSC-CdM on proliferation and photo-aging in human dermal fibroblasts after UVB exposure using enzyme activity assays for collagen type I secretion and MMP-1. To determine the effect of topically applied MSC-CdM on wrinkle formation, MSC-CdM (1% and 10%) and vehicle (propylene glycol: ethanol, 7 : 3) were applied to the dorsal skin of UVB-irradiated hairless mice for 8 weeks. We examined the effects on wrinkle formation by assessing visual skin grading, replica, tape stripping, transepidermal water loss (TEWL), and skin hydration measurement. We also examined histology of the lesions using hematoxylin-eosin, Masson's trichrome, and immunohistochemical staining. RESULTS: MSC-CdM markedly reduced UV-induced matrix metalloproteinase-1 expression and increased pro-collagen synthesis in a dose-dependent manner. Our findings suggest that MSC-CdM induces repair of dermal damage and effacement of wrinkles on UVB-irradiated hairless mice through protective effect of hydration. CONCLUSION: These results support an anti-wrinkle effect of MSC-CdM that involves increased collagen synthesis and suggest that MSC-CdM might be a potential candidate for preventing UV-induced skin damage.

Ageing does not result in a decline in cell synthetic activity in an injury prone tendon.

Advancing age is a well-known risk factor for tendon disease. Energy-storing tendons [e.g., human Achilles, equine superficial digital flexor tendon (SDFT)] are particularly vulnerable and it is thought that injury occurs following an accumulation of micro-damage in the extracellular matrix (ECM). Several authors suggest that age-related micro-damage accumulates due to a failure of the aging cell population to maintain the ECM or an imbalance between anabolic and catabolic pathways. We hypothesized that ageing results in a decreased ability of tendon cells to synthesize matrix components and matrix-degrading enzymes, resulting in a reduced turnover of the ECM and a decreased ability to repair micro-damage. The SDFT was collected from horses aged 3-30 years with no signs of tendon injury. Cell synthetic and degradative ability was assessed at the mRNA and protein levels. Telomere length was measured as an additional marker of cell ageing. There was no decrease in cellularity or relative telomere length with increasing age, and no decline in mRNA or protein levels for matrix proteins or degradative enzymes. The results suggest that the mechanism for age-related tendon deterioration is not due to reduced cellularity or a loss of synthetic functionality and that alternative mechanisms should be considered.

Anti-wrinkle effects of Seungma-Galgeun-Tang as evidenced by the inhibition of matrix metalloproteinase-I production and the promotion of type-1 procollagen synthesis.

BACKGROUND: Seungma-Galgeun-Tang (SMGGT), a traditional herbal medicinal formula, has been used to treat various skin problems such as inflammation and rashes in Korean traditional medicine. In order to clarify the scientific evidence for the biological efficacy of SMGGT on the prevention of skin aging and in particular wrinkle formation, molecular anti-wrinkle parameters were evaluated in cultured human dermal fibroblasts. METHODS: Standard SMGGT was prepared from KFDA-certified herbal medicines and the chemical fingerprint of SMGGT was verified by HPLC-ESI-MS to insure the quality of SMGGT. To evaluate the inhibitory effects of SMGGT on the synthesis of matrix metalloproteinase-1 (MMP-1) and type-1 procollagen, the content of MMP-1 and type-1 procollagen synthesizing enzymes in cultured human dermal fibroblasts were measured using an ELISA kit and Western Blot, respectively. RESULTS: The treatment of SMGGT water extract significantly inhibited the production of MMP-1 and promoted type-1 procollagen synthesis concentration dependently. CONCLUSIONS: These results suggest that SMGGT has the potential to prevent wrinkle formation by down-regulating MMP-1 and up-regulating type-1 procollagen in human dermal fibroblasts.

Development and characterization of a eukaryotic expression system for human type II procollagen.

BACKGROUND: Triple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required. RESULTS: Here, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils. CONCLUSIONS: Using a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation.

Acute bone response to whole body vibration in healthy pre-pubertal boys.

The skeleton responds to mechanical stimulation. We wished to ascertain the magnitude and speed of the growing skeleton's response to a standardised form of mechanical stimulation, vibration. 36 prepubertal boys stood for 10 minutes in total on one of two vibrating platforms (high (>2 g) or low (<1 g) magnitude vibration) on either 1, 3 or 5 successive days (n=12 for each duration); 15 control subjects stood on an inactive platform. Blood samples were taken at intervals before and after vibration to measure bone formation (P1NP, osteocalcin) and resorption (CTx) markers as well as osteoprotegerin and sclerostin. There were no significant differences between platform and control groups in bone turnover markers immediately after vibration on days 1, 3 and 5. Combining platform groups, at day 8 P1NP increased by 25.1% (CI 12.3 to 38.0; paired t-test p=0.005) and bone resorption increased by 10.9% (CI 3.6 to 18.2; paired t-test p=0.009) compared to baseline. Osteocalcin, osteoprotogerin and sclerostin did not change significantly. The growing skeleton can respond quickly to vibration of either high or low magnitude. Further work is needed to determine the utility of such "stimulation-testing" in clinical practice.

Differential effects of fluticasone on extracellular matrix production by airway and parenchymal fibroblasts in severe COPD.

Chronic obstructive pulmonary disease (COPD) is characterized by abnormal repair in the lung resulting in airway obstruction associated with emphysema and peripheral airway fibrosis. Because the presence and degree of airways disease and emphysema varies between COPD patients, this may explain the heterogeneity in the response to treatment. It is currently unknown whether and to what extent inhaled steroids can affect the abnormal repair process in the airways and lung parenchyma in COPD. We investigated the effects of fluticasone on transforming growth factor (TGF)-ß- and cigarette smoke-induced changes in mothers against decapentaplegic homolog (Smad) signaling and extracellular matrix (ECM) production in airway and parenchymal lung fibroblasts from patients with severe COPD. We showed that TGF-ß-induced ECM production by pulmonary fibroblasts, but not activation of the Smad pathway, was sensitive to the effects of fluticasone. Fluticasone induced decorin production by airway fibroblasts and partly reversed the negative effects of TGF-ß treatment. Fluticasone inhibited biglycan production in both airway and parenchymal fibroblasts and procollagen 1 production only in parenchymal fibroblasts, thereby restoring the basal difference in procollagen 1 production between airway and parenchymal fibroblasts. Our findings suggest that the effects of steroids on the airway compartment may be beneficial for patients with severe COPD, i.e., restoration of decorin loss around the airways, whereas the effects of steroids on the parenchyma may be detrimental, since the tissue repair response, i.e., biglycan and procollagen production, is inhibited. More research is needed to further disentangle these differential effects of steroid treatment on the different lung compartments and its impact on tissue repair and remodeling in COPD.

The ginsenoside Rg1 prevents transverse aortic constriction-induced left ventricular hypertrophy and cardiac dysfunction by inhibiting fibrosis and enhancing angiogenesis.

BACKGROUND: Ginsenoside Rg1, an important and active ingredient of Panax ginseng, has been shown to exert cardioprotective effects in vivo. The present study aimed to test the hypothesis that ginsenoside Rg1 attenuates cardiac dysfunction in a transverse aortic constriction (TAC)-induced left ventricular hypertrophy in vivo via proangiogenic and antifibrotic effects. METHODS: This study investigated the effects of ginsenoside Rg1 in a rat model of TAC-induced left ventricular hypertrophy. Cardiac function was assessed by echocardiography. The antifibrotic and proangiogenic effects were assessed by histopathology and mRNA expression of procollagen I, III, and vascular endothelial growth factor (VEGF) through quantitative real-time PCR. The expression of phosphorylation of Akt, p38 mitogen-activated protein kinase (MAPK), hypoxia inducible factor-1 (HIF-1), and VEGF proteins were examined by Western blotting. RESULTS: Ginsenoside Rg1 treatment significantly decreased TAC-induced myocardial fibrosis and left ventricular hypertrophy, and preserved cardiac function. Ginsenoside Rg1 administration enhanced angiogenesis by increasing the expression of HIF-1 and VEGF. These cardioprotective effects of ginsenoside Rg1 are partially related to the activation of phospho-Akt and inhibition of p38 MAPK. CONCLUSIONS: Ginsenoside Rg1 exhibited protective effect against TAC-induced left ventricular hypertrophy and cardiac dysfunction, which is potentially associated with phospho-Akt activation and p38 MAPK inhibition.

The potential effect of botulinum toxin type A on human dermal fibroblasts: an in vitro study.

BACKGROUND: The use of botulinum toxin type A (BoNT) continues to expand. Some physicians have noted a face-lifting effect after intradermal injection of BoNT, although the effects are controversial. OBJECTIVE: To investigate the in vitro effects of BoNT on human dermal fibroblasts. METHODS: The proliferation and toxic effects of BoNT on human dermal fibroblasts were measured. To understand the mechanism of BoNT on collagen production of fibroblasts, procollagen type I carboxy-terminal peptide (PIP) was measured using enzyme-linked immunosorbent assay, and collagen production was monitored using Western blotting. To examine the effect of BoNT on collagen degradation, we evaluated matrix metalloproteinase (MMP) production using gelatin zymography. RESULTS: BoNT did not stimulate the proliferation of or show toxic effects on human dermal fibroblasts. Levels of PIP increased significantly in fibroblasts grown in the presence of BoNT, and BoNT upregulated the expression of type I collagen and decreased the production of some MMPs in fibroblasts that prevent collagen degradation. CONCLUSIONS: This study shows interesting effects of BoNT on collagen production and degradation of human dermal fibroblasts in vitro. This research provides the experimental background for using intradermal BoNT injection for remodeling of dermal tissues in aged skin.

[Effects of matrix metallopeptidase 9 on the proliferation, apoptosis, type I and III procollagen synthesis of rat dermal fibroblasts].

OBJECTIVE: To explore the effects of matrix metallopeptidase 9 (MMP-9) on the proliferation, apoptosis, type I and III procollagen synthesis of rat dermal fibroblasts. METHODS: Lipopolysaccharide (LPS) at 0.1 and 1.0 µg/ml was used to stimulate fibroblasts to up-regulate the expression of MMP-9 for 18 and 48 h. And then RNA interference was employed to inhibit the high expression of MMP-9. Cell proliferation was tested by CCK-8, cell apoptosis by flow cytometry and type I and III procollagen expressions by quantitative reverse transcriptase PCR (qRT-PCR). RESULTS: The MMP-9 expression of fibroblasts increased after the stimulation of LPS. And the 1.0 µg/ml LPS stimulation of 48 hours was 14.25 times of mRNA expression and 2.31 times of protein expression versus that of the normal group (both P < 0.05). The RNA interference obviously inhibited the high expression of MMP-9. The mRNA expression was 1/8 and protein expression 1/3 (both P < 0.05) as compared with the control group. Cell proliferation decreased with the rising expression of MMP-9 to some extent [(1.08 ± 0.08) vs (1.18 ± 0.09), P < 0.05] and improved after the inhibition of high expression of MMP-9 [(1.78 ± 0.17) vs (1.53 ± 0.15), P < 0.01]. There was no change of apoptosis accompanied with high expression of MMP-9 (P > 0.05). Apoptosis decreased after the inhibition of high expression of MMP-9 for 48 hours (3.53% ± 0.22% vs 4.47% ± 0.46%, P < 0.05). The synthesis of type I procollagen was the same no matter up-regulation or down-regulation of MMP-9 expression (P > 0.05). As the expression of MMP-9 increased, the synthesis of type III procollagen decreased (P < 0.01), but not increased by 1.02 folds after the inhibited expression of MMP-9 (P > 0.05). CONCLUSION: MMP-9 can affect the biological behaviors of rat dermal fibroblasts.

Peritoneal repairing cells: a type of bone marrow derived progenitor cells involved in mesothelial regeneration.

The peritoneal mesothelium exhibits a high regenerative ability. Peritoneal regeneration is concomitant with the appearance, in the coelomic cavity, of a free-floating population of cells whose origin and functions are still under discussion. We have isolated and characterized this cell population and we have studied the process of mesothelial regeneration through flow cytometry and confocal microscopy in a murine model lethally irradiated and reconstituted with GFP-expressing bone marrow cells. In unoperated control mice, most free cells positive for mesothelin, a mesothelial marker, are green fluorescent protein (GFP). However, 24 hrs after peritoneal damage, free mesothelin(+)/GFP(+) cells appear in peritoneal lavages. Cultured lavage peritoneal cells show colocalization of GFP with mesothelial (mesothelin, cytokeratin) and fibroblastic markers. Immunohistochemical staining of the peritoneal wall also revealed colocalization of GFP with mesothelial markers and with procollagen-1 and smooth muscle α-actin. This was observed in the injured area as well as in the surrounding not-injured peritoneal surfaces. These cells, which we herein call peritoneal repairing cells (PRC), are very abundant 1 week after surgery covering both the damaged peritoneal wall and the surrounding uninjured area. However, they become very scarce 1 month later, when the mesothelium has completely healed. We suggest that PRC constitute a type of monocyte-derived cells, closely related with the tissue-repairing cells known as 'fibrocytes' and specifically involved in peritoneal reparation. Thus, our results constitute a synthesis of the different scenarios hitherto proposed about peritoneal regeneration, particularly recruitment of circulating progenitor cells and adhesion of free-floating coelomic cells.

Selenium supplementation attenuates procollagen-1 and interleukin-8 production in fat-loaded human C3A hepatoblastoma cells treated with TGFbeta1.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and hepatic steatosis. Non-alcoholic steatohepatitis (NASH) is a serious consequence of NAFLD where chronic tissue damage and inflammation result in fibrosis which may progress to cirrhosis. Transforming growth factor beta1 (TGFbeta1), proinflammatory cytokines and oxidative stress are thought to play crucial roles in the pathogenesis of these conditions. The contributions of individual liver cell types to fibrogenesis remain controversial and the influence of selenium status has not been investigated. METHODS: In this study we have used a cell culture model of fat-loading using oleate-treated human hepatoblastoma (C3A) cells to investigate how fat-loading and selenium status might influence the production of collagen in response to TGFbeta1. The secretion of inflammatory cytokines was also investigated, together with the epithelial character of the treated cells. RESULTS: We found that in response to treatment with TGFbeta1, C3A cells produced mRNA encoding the pro-alphaI chain of procollagen I, secreted procollagen I peptide, up-regulated production of the proinflammatory cytokine interleukin-8 (IL-8) and the mesenchymal marker vimentin, and down-regulated albumin production. Most of these responses were considerably enhanced when cells were fat-loaded with oleate and were attenuated by selenium addition at a dose that optimised the expression of thioredoxin reductase and glutathione peroxidase. CONCLUSIONS: Our data establish that both fat-loading and suboptimal selenium status enhance collagen and IL-8 production by C3A hepatocytes in response to TGFbeta1, possibly as part of an epithelial to mesenchymal transition. GENERAL SIGNIFICANCE: These findings suggest that the hepatocyte may be an important contributor to the pathogenesis of fibrosis associated with NAFLD.

CC chemokine receptor 5 deletion impairs macrophage activation and induces adverse remodeling following myocardial infarction.

Post-myocardial infarction (MI), chemokine homing of inflammatory cells into the injured left ventricle (LV) regulates ventricular remodeling, in part by stimulating the extracellular matrix response. The CC chemokine receptor 5 (CCR5) is a key chemokine receptor expressed on macrophages, and CCR5 ligands are highly upregulated post-MI. We hypothesized that deletion of CCR5 would attenuate adverse remodeling by decreasing inflammatory cell recruitment. Accordingly, we examined LV function, macrophage recruitment and activation, and collagen content in wild-type (WT, n = 25) and CCR5 null (n = 33) mice at 7 days post-MI. Both groups had similar infarct sizes (44 ± 2% in WT and 42 ± 2% in CCR5 null; P = 0.37). However, the LV remodeling index (end diastolic volume/LV mass) increased to a larger extent in CCR5 null (1.28 ± 0.08 µl/mg for CCR5 null and 1.02 ± 0.06 µl/mg for WT; P < 0.05). Although numbers of infiltrated macrophages were similar in WT and CCR5 null mice, CCR5-deficient macrophages isolated from the infarct zone displayed >50% decrease in gene expression levels of proinflammatory activation markers (interleukin-1ß, interleukin-6, and tumor necrosis factor-α), as well as anti-inflammatory activation markers (arginase 1, CD163, mannose receptor, and transforming growth factor-ß1) compared with WT (all P < 0.05). Concomitant with the reduced macrophage activation, heat shock protein-47 and collagen type I precursor levels in the infarct region decreased in the CCR5 null (1.2 ± 0.3 units in the CCR5 null and 2.3 ± 0.4 units in the WT; P < 0.05), while collagen fragments increased (88.3 ± 5.9 units in the CCR5 null and 32.7 ± 8.5 units in the WT; P < 0.05). We conclude that CCR5 deletion impairs LV remodeling by hindering macrophage activation, which stimulates an imbalance in collagen metabolism and increases the remodeling index.

Receptors and effects of gut hormones in three osteoblastic cell lines.

BACKGROUND: In recent years the interest on the relationship of gut hormones to bone processes has increased and represents one of the most interesting aspects in skeletal research. The proportion of bone mass to soft tissue is a relationship that seems to be controlled by delicate and subtle regulations that imply "cross-talks" between the nutrient intake and tissues like fat. Thus, recognition of the mechanisms that integrate a gastrointestinal-fat-bone axis and its application to several aspects of human health is vital for improving treatments related to bone diseases. This work analysed the effects of gut hormones in cell cultures of three osteoblastic cell lines which represent different stages in osteoblastic development. Also, this is the first time that there is a report on the direct effects of glucagon-like peptide 2, and obestatin on osteoblast-like cells. METHODS: mRNA expression levels of five gut hormone receptors (glucose-dependent insulinotropic peptide [GIP], glucagon-like peptide 1 [GLP-1], glucagon-like peptide 2 [GLP-2], ghrelin [GHR] and obestatin [OB]) were analysed in three osteoblastic cell lines (Saos-2, TE-85 and MG-63) showing different stages of osteoblast development using reverse transcription and real time polymerase chain reaction. The responses to the gut peptides were studied using assays for cell viability, and biochemical bone markers: alkaline phosphatase (ALP), procollagen type 1 amino-terminal propeptides (P1NP), and osteocalcin production. RESULTS: The gut hormone receptor mRNA displayed the highest levels for GIP in Saos-2 and the lowest levels in MG-63, whereas GHR and GPR39 (the putative obestatin receptor) expression was higher in TE-85 and MG-63 and lower in Saos-2. GLP-1 and GLP-2 were expressed only in MG-63 and TE-85. Treatment of gut hormones to cell lines showed differential responses: higher levels in cell viability in Saos-2 after GIP, in TE-85 and MG-63 after GLP-1, GLP-2, ghrelin and obestatin. ALP showed higher levels in Saos-2 after GIP, GHR and OB and in TE-85 after GHR. P1NP showed higher levels after GIP and OB in Saos-2. Decreased levels of P1NP were observed in TE-85 and MG-63 after GLP-1, GLP-2 and OB. MG-63 showed opposite responses in osteocalcin levels after GLP-2. CONCLUSIONS: These results suggest that osteoblast activity modulation varies according to different development stage under different nutrition related-peptides.

A glycosidic spinasterol from Koreana stewartia promotes procollagen production and inhibits matrix metalloproteinase-1 expression in UVB-irradiated human dermal fibroblasts.

Methanol extract of Koreana stewartia leaves (SKE) stimulated collagen production in ultraviolet-B (UVB)-irradiated human fibroblast cells. An active compound was isolated from SKE by successive partitioning and chromatography, and the chemical structure was determined to be 3-O-ß-D-glucopyranosylspinasterol (spinasterol-Glc) by spectroscopic characterization. Spinasterol-Glc increased collagen production in the supernatant of UVB-irradiated dermal fibroblast cell cultures in a dose-dependent manner. The effects of spinasteol-Glc on expression of procollagen and matrix metalloproteinase-1 (MMP-1) were further evaluated. We found that the compound stimulated collagen production in UVB-treated fibroblasts than in vehicle-treated control cells by about 3-fold. In addition, we also demonstrate that the compound increased the mRNA and protein levels of procollagen in UVB-treated fibroblast cells, while it inhibited expression of MMP-1. These results indicate that spinasterol-Glc protects fibroblast cells from the adverse effects of UV radiation via stimulation of procollagen synthesis as well as inhibition of MMP-1 expression. Spinasterol-Glc may be useful in the future development of therapeutic and cosmetic applications.