XXIX Italian Society for the Study of Connective Tissues (SISC) Meeting,October 29–30, 2009, Alghero,Italy

Soluble substances in the bovine nuchal ligament were removed by preliminary extractions with a buffer and dilute alkali solution. The insoluble residue was then extracted with 6 M guanidine HCl and with 6 M guanidine HCl containing a reducing agent (20 mM dithiothreitol) successively. Each of the two extracts contained a glycoprotein; that in the first extract was designated Glycoprotein A and that in the second Glycoprotein B. They were purified by ion-exchange chromatography and gel filtration till essentially homogeneous. Both proteins had similar molecular weights of 35,000 in SDS-polyacrylamide gel electrophoresis and by gel filtration, and their chemical compositions resembled each other closely. It is suggested that Glycoprotein B was present in the native state as a disulfide-bonded, aggregated form of Glycoprotein A. The compounds also showed similarity with the microfibrillar glycoprotein(s) previously reported in bovine nuchal ligament extracts.     

Degradation of cartilage by macrophages in culture: Evidence for the involvement of an enzyme which is associated with the cell surface

A cell culture system is described in which purified mononuclear phagocytes may be cultured with a cartilage substrate which is radiolabelled in its proteoglycan. Resident mouse peritoneal macrophages degraded this substrate, and did so more avidly if cultured in direct contact with it. There was no evidence for complete intralysosomal degradation of the proteoglycan of the cartilage. Lysates were found to contain considerable activity at pH 7, which was inhibited by the presence of 10% serum, or by boiling the lysate.

Proximity of macrophages to the substrate did not induce selective release of the lysosomal marker enzyme hexosaminidase, and concentrated enzymes secreted from the macrophages after treatment with the lysosomotropic agent ammonium chloride were ineffective in degrading cartilage at neutral pH.

The active enzyme in macrophage lysates at neutral pH was found to be sedimentable by 100,000 × g centrifugation for 1 hour, in absence of lysosomal protective agents. There is evidence for a cell membrane-associated process in the degradation of cartilage by these cells, which may be a proteolytic, endoglycosidic or free radical-mediated event.     

Differences in mRNA and protein expression of small proteoglycans in vaginal wall tissue from women with and without stress urinary incontinence

BACKGROUND: To investigate changes in mRNA and protein levels of biglycan (BGN), decorin (DCN) and fibromodulin (FMOD) in vaginal wall tissue from women with stress urinary incontinence (SUI) compared to menstrual-cycle matched continent women. METHODS: We determined mRNA expressions of BGN, DCN and FMOD by quantitative real-time PCR. They were localized in vaginal wall tissue by immunohistochemistry. We performed western blot analysis to examine protein expression. RESULTS: BGN, DCN and FMOD co-localized with collagen and elastin in the extracellular matrix (ECM) of vaginal wall tissue from both groups. The mRNA expression of FMOD was significantly lower in cases versus controls in the proliferative phase (P = 0.03). DCN mRNA expression in cases was higher in the proliferative (P = 0.05) and secretory phases (P = 0.02) versus controls. BGN mRNA expression showed no significant differences in either phase. Protein expression of FMOD in cases was lower in the proliferative phase versus controls (six out of nine pairs), whereas DCN and BGN protein expression in the secretory phase in cases was higher (seven out of nine pairs). CONCLUSION: BGN, DCN and FMOD expressions in vaginal wall tissue differ in women with SUI and are hormonally modulated. Differences in small proteoglycans may contribute to the altered pelvic floor connective tissues found in these women.     

核心蛋白聚糖在Ishikawa细胞中的表达及意义

目的观察表皮生长因子受体（EGFR）信号通路抑制剂RG-14260（RG）单用及RG联合mTOR信号通路抑制剂亚罗奠司（RA）体外对子宫内膜癌Ishikawa细胞增殖及核心蛋白聚糖（DCN）表达的影响。方法子宫内膜癌Ishikawa细胞经RG及RG联合RA作用后,采用免疫组织化学法检测细胞增殖细胞核抗原（PCNA）的表达,反转录酶-聚合酶链式反应（RT-PCR）及免疫印迹法分别检测细胞内DCN基因和蛋白表达的变化。结果RG单用及联合RA均可降低Ishikawa细胞PCNA阳性细胞表达率,促进细胞DCN基因和蛋白的表达,但二者合用效果更为显著。结论单独和联合EGFR及mTOR信号通路抑制剂均可在基因和蛋白水平上上调Ishikawa细胞内DCN的表达,而升高的DCN反过来又可增强信号通路抑制剂对细胞增殖的抑制作用。     

Improvement of muscle healing through enhancement of muscle regeneration and prevention of fibrosis

Skeletal muscle is able to repair itself through regeneration. However, an injured muscle often does not fully recover its strength because complete muscle regeneration is hindered by the development of fibrosis. Biological approaches to improve muscle healing by enhancing muscle regeneration and reducing the formation of fibrosis are being investigated. Previously, we have determined that insulin-like growth factor-1 (IGF-1) can improve muscle regeneration in injured muscle. We also have investigated the use of an antifibrotic agent, decorin, to reduce muscle fibrosis following injury. The aim of this study was to combine these two therapeutic methods in an attempt to develop a new biological approach to promote efficient healing and recovery of strength after muscle injuries. Our findings indicate that further improvement in the healing of muscle lacerations is attained histologically by the combined administration of IGF-1 to enhance muscle regeneration and decorin to reduce the formation of fibrosis. This improvement was not associated with improved responses to physiological testing, at least at the time-points tested in this study.     

Decorin mimic promotes endothelial cell health in endothelial monolayers and endothelial–smooth muscle co‐cultures

Non‐specific cytotoxins, including paclitaxel and sirolimus analogues, currently utilized as anti‐restenotic therapeutics, affect not only smooth muscle cells (SMCs) but also neighbouring vascular endothelial cells (ECs). These drugs inhibit the formation of an intact endothelium following vessel injury, thus emphasizing the critical need for new candidate therapeutics. Utilizing our in vitro models, including EC monolayers and both hyperplastic and quiescent EC–SMC co‐cultures, we investigated the ability of DS–SILY₂₀, a decorin mimic, to promote EC health. DS–SILY₂₀ increased EC proliferation and migration by 1.5‐ and 2‐fold, respectively, which corresponded to increased phosphorylation of ERK‐1/2. Interestingly, IL‐6 secretion and the production of both E‐selectin and P‐selectin were reduced in the presence of 10 μm DS–SILY₂₀, even in the presence of the potent pro‐inflammatory cytokine platelet‐derived growth factor (PDGF). In hyperplastic and quiescent EC–SMC co‐cultures, DS–SILY₂₀ treatment reduced the secretion of IFNγ, IL‐1β, IL‐6 and TNFα, corresponding to a 23% decrease in p38 phosphorylation. E‐selectin and P‐selectin expression was further reduced following DS–SILY₂₀ treatment in both co‐culture models. These results indicate that DS–SILY₂₀ promotes EC health and that this decorin mimic could serve as a potential therapeutic to promote vessel healing following percutaneous coronary intervention (PCI). Copyright © 2015 John Wiley & Sons, Ltd.     

蛋白聚糖与糖尿病微血管病变

蛋白聚糖在糖尿病微血管病变的发生发展过程中起重要的作用 ,大量研究表明其在糖尿病微血管病变时出现质和量的变化。糖尿病肾病和糖尿病视网膜病变时硫酸软骨素、透明质酸、硫酸皮肤素等多种蛋白聚糖在纤维化细胞外基质组织局部表达明显升高 ,而硫酸类肝素蛋白聚糖无明显变化或表达下降 ,在糖尿病视网膜病变时表达明显下降。此外 ,由于硫酸软骨素蛋白聚糖中小分子核心蛋白聚糖decorin能结合并中和转化生长因子 β1的活性 ,为糖尿病肾病的治疗提供一种可能的方法