CCN Family: Matricellular Proteins in Cartilage and Bone Development

The extracellular matrix is the intricate scaffolding which surrounds and supports cells and helps to organize them into tissues and organs. The CCN family of matricellular proteins helps to regulate and modulate production, degradation, and remodeling of the extracellular matrix. In this chapter, we review the extracellular matrix of cartilage and bone, including an overview of chondrogenesis and skeletogenesis, and summarize the importance of the CCN proteins in establishment of the skeletal system. CCN proteins have both positive and negative regulatory roles in skeletal development, and their abnormal expression is related to the pathogenesis of several diseases observed in cartilage and bone that arise when inflammation or tissue injury becomes chronic, including fibrosis, arthritis, and cancer. Understanding the biological functions of the CCN proteins within this context offers opportunities for developing therapeutics by targeting CCN functions

A cell culture model using rat coronary artery adventitial fibroblasts to measure collagen production

<p>Abstract</p> <p>Background</p> <p>We have developed a rat cell model for studying collagen type I production in coronary artery adventitial fibroblasts. Increased deposition of adventitial collagen type I leads to stiffening of the blood vessel, increased blood pressure, arteriosclerosis and coronary heart disease. Although the source and mechanism of collagen deposition is yet unknown, the adventitia appears to play a significant role. To demonstrate the application of our cell model, cultured adventitial fibroblasts were treated with sex hormones and the effect on collagen production measured.</p> <p>Methods</p> <p>Hearts (10–12 weeks) were harvested and the left anterior descending coronary artery (LAD) was isolated and removed. Tissue explants were cultured and cells (passages 2–4) were confirmed as fibroblasts using immunohistochemistry. Optimal conditions were determined for cell tissue harvest, timing, proliferation and culture conditions. Fibroblasts were exposed to 10^-7M testosterone or 10^-7M estrogen for 24 hours and either immunostained for collagen type I or subjected to ELISA.</p> <p>Results</p> <p>Results showed increased collagen staining in fibroblasts treated with testosterone compared to control and decreased staining with estrogen. ELISA results showed that testosterone increased collagen I by 20% whereas estrogen decreased collagen I by 15%.</p> <p>Conclusion</p> <p>Data demonstrates the usefulness of our cell model in studying the specific role of the adventitia apart from other blood vessel tissue in rat coronary arteries. Results suggest opposite effects of testosterone and estrogen on collagen synthesis in the rat coronary artery adventitial fibroblasts.</p

Ultrastructural and cytochemical evaluation of sepsis-induced changes in the rat pulmonary intravascular mononuclear phagocytes

Sepsis stimulates an increase in the number and activity of mononuclear phagocytes in systemic host-defence organs. The present study was conducted to define the ultrastructural and cytochemical characteristics of the mononuclear phagocytes that sequester in the lung microvasculature of septic rats. Fourteen rats were challenged with a single intraperitoneal injection of saline (0.5 ml/100 g), E. coli (2×10(7)/100 g) or glucan (4 mg/100 g), and euthanased 2, 4, or 7 d later. The lungs were inflation fixed and processed for transmission electron microscopy. Cellular morphology was used to identify the intravascular mononuclear phagocytes and acid phosphatase (AcPase) expression was monitored as an index of cellular differentiation and activation. Control rats contained a limited number of monocytes in the pulmonary vasculature. In contrast, large numbers of activated mononuclear phagocytes were seen in the microvasculature within 48 h of treatment with either microbial product. The recruited pulmonary intravascular mononuclear phagocytes (PIMP) exhibited AcPase-reactive Golgi complexes, accumulation of secretory vesicles and other features of cell activation consistent with enhanced biosynthetic activity. Subsequent electron microscopy, conducted 4 and 7 d posttreatment, suggested that a progressive decline in the number and activity of PIMPs then occurred. In order to quantify the sepsis-induced accumulation of AcPase-positive PIMP, the experimental challenges were repeated in 11 rats and, 48 h later, tissue samples were evaluated by light microscopy for tartrate-insensitive acid phosphatase. Control rats exhibited 0.148±0.107 AcPase-positive PIMP/alveoli. E. coli and glucan challenged animals exhibited significant (P<0.01) increases in AcPase-positive mononuclear phagocytes, with 0.782±0.073 and 0.636±0.170 PIMP/alveoli respectively. The results demonstrate that focal sepsis stimulates a significant, but transient, recruitment of activated mononuclear phagocytes into the rat pulmonary microvasculature

Removal of left anterior descending coronary artery (LAD) from SHR male rat

<p><b>Copyright information:</b></p><p>Taken from "A cell culture model using rat coronary artery adventitial fibroblasts to measure collagen production"</p><p>http://www.biomedcentral.com/1471-2261/7/13</p><p>BMC Cardiovascular Disorders 2007;7():13-13.</p><p>Published online 8 May 2007</p><p>PMCID:PMC1885448.</p><p></p> A) intact LAD, B) heart with LAD removed, C) isolated LAD, D) LAD length, E) LAD diameter, and F) fibroblasts migrating from LAD explant

Collagen Type I (μg) measured by ELISA in LAD fibroblasts treated with 10M testosterone or 10M estrogen

<p><b>Copyright information:</b></p><p>Taken from "A cell culture model using rat coronary artery adventitial fibroblasts to measure collagen production"</p><p>http://www.biomedcentral.com/1471-2261/7/13</p><p>BMC Cardiovascular Disorders 2007;7():13-13.</p><p>Published online 8 May 2007</p><p>PMCID:PMC1885448.</p><p></p> Testosterone significantly increased the amount of collagen deposited (2.912 ± 0.247 μg) compared to control values (2.475 ± 0.211 μg) (* p < 0.05, 1 tailed t-test). Estrogen decreased the amount of collagen (2.103 ± 0.262 μg) compared to control (p = 0.058, 1 tailed t-test). Collagen was measured in wells with a 0.32 cmgrowth area from a 96 well culture plate

Advanced Osteoarthritis in Humans Is Associated With Altered Collagen VI Expression and Upregulation of ER-stress Markers Grp78 and Bag-1

To test the hypothesis that a perturbation of endoplasmic reticulum (ER) function is involved in the pathogenesis of osteoarthritis (OA), articular cartilage was isolated from non-OA patients secondary to resection of osteo- or chondrosarcomas. Intra-joint samples of minimal and advanced osteoarthritic cartilage were isolated from patients undergoing total knee arthroplasty and scored for disease severity. Glucose-regulated protein-78 (grp78) and bcl-2–associated athanogene-1 (bag-1) were detected via immunofluorescence as markers of non-homeostatic ER function. Additionally, the expression of type VI collagen and its integrin receptor, NG2, was determined to examine cartilage matrix health and turnover. There was an upregulation of grp78 in advanced OA, and variable expression in minimal OA. Non-OA cartilage was consistently grp78 negative. The downstream regulator bag-1 was also upregulated in OA compared with normal cartilage. Collagen VI was mainly cell-associated in non-OA cartilage, with a more widespread distribution observed in OA cartilage along with increased intracellular staining intensity. The collagen VI integral membrane proteoglycan receptor NG2 was downregulated in advanced OA compared with its patient-matched minimally involved cartilage sample. These results suggest that chondrocytes exhibit ER stress during OA, in association with upregulation of a large secreted molecule, type VI collagen. (J Histochem Cytochem 57:923–931, 2009