Syndecan-2 is a novel target of insulin-like growth factor binding protein-3 and is over-expressed in fibrosis

Extracellular matrix deposition and tissue scarring characterize the process of fibrosis. Transforming growth factor beta (TGFβ) and Insulin-like growth factor binding protein-3 (IGFBP-3) have been implicated in the pathogenesis of fibrosis in various tissues by inducing mesenchymal cell proliferation and extracellular matrix deposition. We identified Syndecan-2 (SDC2) as a gene induced by TGFβ in an IGFBP-3-dependent manner. TGFβ induction of SDC2 mRNA and protein required IGFBP-3. IGFBP-3 independently induced production of SDC2 in primary fibroblasts. Using an ex-vivo model of human skin in organ culture expressing IGFBP-3, we demonstrate that IGFBP-3 induces SDC2 ex vivo in human tissue. We also identified Mitogen-activated protein kinase-interacting kinase (Mknk2) as a gene induced by IGFBP-3. IGFBP-3 triggered Mknk2 phosphorylation resulting in its activation. Mknk2 independently induced SDC2 in human skin. Since IGFBP-3 is over-expressed in fibrotic tissues, we examined SDC2 levels in skin and lung tissues of patients with systemic sclerosis (SSc) and lung tissues of patients with idiopathic pulmonary fibrosis (IPF). SDC2 levels were increased in fibrotic dermal and lung tissues of patients with SSc and in lung tissues of patients with IPF. This is the first report describing elevated levels of SDC2 in fibrosis. Increased SDC2 expression is due, at least in part, to the activity of two pro-fibrotic factors, TGFβ and IGFBP-3. © 2012 Ruiz et al

A Human Skin Model Recapitulates Systemic Sclerosis Dermal Fibrosis and Identifies <em>COL22A1</em> as a TGFβ Early Response Gene that Mediates Fibroblast to Myofibroblast Transition

: Systemic sclerosis (SSc) is a complex multi-system autoimmune disease characterized by immune dysregulation, vasculopathy, and organ fibrosis. Skin fibrosis causes high morbidity and impaired quality of life in affected individuals. Animal models do not fully recapitulate the human disease. Thus, there is a critical need to identify ex vivo models for the dermal fibrosis characteristic of SSc. We identified genes regulated by the pro-fibrotic factor TGFβ in human skin maintained in organ culture. The molecular signature of human skin overlapped with that which was identified in SSc patient biopsies, suggesting that this model recapitulates the dermal fibrosis characteristic of the human disease. We further characterized the regulation and functional impact of a previously unreported gene in the setting of dermal fibrosis, COL22A1, and show that silencing COL22A1 significantly reduced TGFβ-induced ACTA2 expression. COL22A1 expression was significantly increased in dermal fibroblasts from patients with SSc. In summary, we identified the molecular fingerprint of TGFβ in human skin and demonstrated that COL22A1 is associated with the pathogenesis of fibrosis in SSc as an early response gene that may have important implications for fibroblast activation. Further, this model will provide a critical tool with direct relevance to human disease to facilitate the assessment of potential therapies for fibrosis

Beneficial effect of mechanical stimulation on the regenerative potential of muscle-derived stem cells is lost by inhibiting vascular endothelial growth factor

OBJECTIVE - : We previously reported that mechanical stimulation increased the effectiveness of muscle-derived stem cells (MDSCs) for tissue repair. The objective of this study was to determine the importance of vascular endothelial growth factor (VEGF) on mechanically stimulated MDSCs in a murine model of muscle regeneration. APPROACH AND RESULTS - : MDSCs were transduced with retroviral vectors encoding the LacZ reporter gene (lacZ-MDSCs), the soluble VEGF receptor Flt1 (sFlt1-MDSCs), or a short hairpin RNA (shRNA) targeting messenger RNA of VEGF (shRNA-VEGF MDSCs). Cells were subjected to 24 hours of mechanical cyclic strain and immediately transplanted into the gastrocnemius muscles of mdx/scid mice. Two weeks after transplantation, angiogenesis, fibrosis, and regeneration were analyzed. There was an increase in angiogenesis in the muscles transplanted with mechanically stimulated lacZ-MDSCs compared with nonstimulated lacZ-MDSCs, sFlt1-MDSCs, and shRNA -VEGF MDSCs. Dystrophin-positive myofiber regeneration was significantly lower in the shRNA-VEGF-MDSC group compared with the lacZ-MDSC and sFlt1-MDSC groups. In vitro proliferation of MDSCs was not decreased by inhibition of VEGF; however, differentiation into myotubes and adhesion to collagen were significantly lower in the shRNA-VEGF-MDSC group compared with the lacZ-MDSC and sFlt1-MDSC groups. CONCLUSIONS - : The beneficial effects of mechanical stimulation on MDSC-mediated muscle repair are lost by inhibiting VEGF. © 2013 American Heart Association, Inc

Optimization of a murine and human tissue model to recapitulate dermal and pulmonary features of systemic sclerosis

<div><p>The murine bleomycin (BLM)-induced fibrosis model is the most widely used in systemic sclerosis (SSc) studies. It has been reported that systemic delivery of BLM via continuous diffusion from subcutaneously implanted osmotic minipumps can cause fibrosis of the skin, lungs, and other internal organs. However, the mouse strain, dosage of BLM, administration period, and additional important features differ from one report to the next. In this study, by employing the pump model in C57BL/6J mice, we show a dose-dependent increase in lung fibrosis by day 28 and a transient increase in dermal thickness. Dermal thickness and the level of collagen in skin treated with high-dose BLM was significantly higher than in skin treated with low dose BLM or vehicle. A reduction in the thickness of the adipose layer was noted in both high and low dose groups at earlier time points suggesting that the loss of the fat layer precedes the onset of fibrosis. High-dose BLM also induced dermal fibrosis and increased expression of fibrosis-associated genes <i>ex vivo</i> in human skin, thus confirming and extending the <i>in vivo</i> findings, and demonstrating that a human organ culture model can be used to assess the effect of BLM on skin. In summary, our findings suggest that the BLM pump model is an attractive model to analyze the underlying mechanisms of fibrosis and test the efficacy of potential therapies. However, the choice of mouse strain, duration of BLM administration and dose must be carefully considered when using this model.</p></div

Interferon-gamma inducible protein-10 as a potential biomarker in localized scleroderma

Introduction\ud The purpose of this study was to evaluate the presence and levels of interferon-gamma inducible protein-10 (IP-10) in the plasma and skin of pediatric localized scleroderma (LS) patients compared to those of healthy pediatric controls and to determine if IP-10 levels correlate to clinical disease activity measures.\ud \ud Methods\ud The presence of IP-10 in the plasma was analyzed using a Luminex panel in 69 pediatric patients with LS and compared to 71 healthy pediatric controls. Of these patients, five had available skin biopsy specimens with concurrent clinical and serological data during the active disease phase, which were used to analyze the presence and location of IP-10 in the skin by immunohistochemistry (IHC).\ud \ud Results\ud IP-10 levels were significantly elevated in the plasma of LS patients compared to that of healthy controls and correlated to clinical disease activity measures in LS. Immunohistochemistry staining of IP-10 was present in the dermal infiltrate of LS patients and was similar to that found in psoriasis skin specimens, the positive disease control.\ud \ud Conclusions\ud Elevation of IP-10 levels in the plasma compared to those of healthy controls and the presence of IP-10 staining in the affected skin of LS patients indicates that IP-10 is a potential biomarker in LS. Furthermore, significant elevation of IP-10 in LS patients with active versus inactive disease and correlations between IP-10 levels and standardized disease outcome measures of activity in LS strongly suggest that IP-10 may be a biomarker for disease activity in LS

Expression levels of fibrosis-related genes.

<p>Mice treated with BLM (60 U/kg) were sacrificed on days 7, 10, 14, 21, and 28. Expression levels of fibrosis-related genes were measured in skin tissues on the indicated days. (A) Coll1α1mRNA levels. (B) Fibronectin mRNA levels. (C) Ctgf mRNA levels. (D) Tgfb1 mRNA levels.</p

Summary of previous reports describing dermal and pulmonary fibrosis using the pump model.

<p>Summary of previous reports describing dermal and pulmonary fibrosis using the pump model.</p

Ameliorating Fibrosis in Murine and Human Tissues with END55, an Endostatin-Derived Fusion Protein Made in Plants

Organ fibrosis, particularly of the lungs, causes significant morbidity and mortality. Effective treatments are needed to reduce the health burden. A fragment of the carboxyl-terminal end of collagen XVIII/endostatin reduces skin and lung fibrosis. This fragment was modified to facilitate its production in plants, which resulted in the recombinant fusion protein, END55. We found that expression of END55 had significant anti-fibrotic effects on the treatment and prevention of skin and lung fibrosis in a bleomycin mouse model. We validated these effects in a second mouse model of pulmonary fibrosis involving inducible, lung-targeted expression of transforming growth factor β1. END55 also exerted anti-fibrotic effects in human lung and skin tissues maintained in organ culture in which fibrosis was experimentally induced. The anti-fibrotic effect of END55 was mediated by a decrease in the expression of extracellular matrix genes and an increase in the levels of matrix-degrading enzymes. Finally, END55 reduced fibrosis in the lungs of patients with systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF) who underwent lung transplantation due to the severity of their lung disease, displaying efficacy in human tissues directly relevant to human disease. These findings demonstrate that END55 is an effective anti-fibrotic therapy in different organs

BLM-induced fibrosis in human skin <i>ex vivo</i>.

<p>Human skin from three different donors was injected with, or immersed in, media containing BLM (1 or 10 mU/ml) and maintained in culture for one week. (A) The amount of collagen in skin injected with BLM was quantified using hydroxyproline assay. (B) Representative images of H&E (upper)–and MT (lower)–stained sections of BLM–injected human skin (original magnification ×25). (C) The amount of collagen in skin immersed in BLM was quantified using hydroxyproline assay. (D) Representative images of H&E (upper)–and MT (lower)–stained sections of BLM–immersed human skin (original magnification ×25). (E) Expression levels of fibrosis-related genes were measured in human skin treated with 10 mU/ml BLM for 48 hours.</p

The effect of low dose BLM on dermal and pulmonary fibrosis.

<p>Mice treated with BLM (1 U/kg) were sacrificed on days 7 (N = 7), 10 (N = 4), 14 (N = 4), 21 (N = 4), and 28 (N = 4). (A) Hydroxyproline levels were measured in lungs using hydroxyproline assay. (B) Dermal thickness was measured in dorsal skin. (C) Subcutaneous adipose layer thickness was measured in dorsal skin.</p