Characterization of dermatan sulfate proteoglycan 3 (DSPG3) and cartilage oligomeric matrix protein (COMP)

This dissertation describes the identification and characterization of human dermatan sulfate proteoglycan 3 (DSPG3) and the characterization of the transcriptional regulation of human cartilage oligomeric matrix protein (COMP) in cartilage, ligament, and tendon cells. DSPG3 and COMP are two extracellular matrix proteins. The function of these ECM proteins is unknown.^ DSPG3 was cloned, sequenced, and shown to be expressed in cartilage, ligament, and placenta. DSPG3 was mapped to human chromosome 12q21, and the genomic structure was identified. 1.6 kb of the promoter region has been sequenced, and several putative SOX9 sites were identified as well as 3 TATA sites. Furthermore, an evolutionary tree of the SLRP gene family, which includes DSPG3, is presented.^ The promoter region of COMP was cloned and sequenced. Several putative transcription factor binding sites were identified including multiple AP2 and SP1 sites. Three transcription start sites were found to be located directly downstream of one of the SP1 sites. In addition, the expression of COMP was demonstrated to be higher in tendon than in cartilage and ligament by both Northern and Western blot analysis, and several regions of the COMP promoter were shown to contain cell-specific regulatory elements. Analysis of the proximal 370bp region of the COMP promoter has also identified distinct patterns of nuclear protein binding for the three tissues, and two SP1 sites may play a role in the tissue-specific expression of COMP. ^

The heparan sulfate proteoglycan (HSPG) glypican-3 mediates commitment of MC3T3-E1 cells toward osteogenesis

Heparan sulfate (HS) sugar chains attached to core proteoglycans (PGs) termed HSPGs mediate an extensive range of cell-extracellular matrix (ECM) and growth factor interactions based upon their sulfation patterns. When compared with non-osteogenic (maintenance media) culture conditions, under established osteogenic culture conditions, MC3T3-E1 cells characteristically increase their osteogenic gene expression profile and switch their dominant fibroblast growth factor receptor (FGFR) from FGFR1 (0.5-fold decrease) to FGFR3 (1.5-fold increase). The change in FGFR expression profile of the osteogenic-committed cultures was reflected by their inability to sustain an FGF-2 stimulus, but respond to BMP-2 at day 14 of culture. The osteogenic cultures decreased their chondroitin and dermatan sulfate PGs (biglycan, decorin, and versican), but increased levels of the HS core protein gene expression, in particular glypican-3. Commitment and progress through osteogenesis is accompanied by changes in FGFR expression, decreased GAG initiation but increased N- and O-sulfation and reduced remodeling of the ECM (decreased heparanase expression) resulting in the production of homogenous (21 kDa) HS chain. With the HSPG glypican-3 expression strongly upregulated in these processes, siRNA was used to knockdown this gene to examine the effect on osteogenic commitment. Reduced glypican-3 abrogated the expression of Runx2, and thus differentiation. The reintroduction of this HSPG into Runx2-null cells allowed osteogenesis to proceed. These results demonstrate the dependence of osteogenesis on specific HS chains, in particular those associated with glypican-3.

Involvement of heparin-like glycosaminoglycans and expression of a novel heparan sulfate binding protein (p24) during human placentation and in a model for human implantation

Previous studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. To investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were selected to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell-cell adhesion assay showed that initial binding between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAG) with heparin-like properties, i.e., heparan sulfate and dermatan sulfate. Furthermore, a single class of highly specific, protease-sensitive heparin/heparan sulfate binding sites exist on the surface of RL95 cells. Three heparin binding, tryptic peptide fragments were isolated from RL95 cell surfaces and their amino termini partially sequenced. Reverse transcription-polymerase chain reaction (RT-PCR) generated 1 to 4 PCR products per tryptic peptide. Northern blot analysis of RNA from RL95 cells using one of these RT-PCR products identified a 1.2 Kb mRNA species (p24). The amino acid sequence predicted from the cDNA sequence contains a putative heparin-binding domain. A synthetic peptide representing this putative heparin binding domain was used to generate a rabbit polyclonal antibody (anti-p24). Indirect immunofluorescence studies on RL95 and JAR cells as well as binding studies of anti-p24 to intact RL95 cells demonstrate that p24 is distributed on the cell surface. Western blots of RL95 membrane preparations identify a 24 kDa protein (p24) highly enriched in the 100,000 g pellet plasma membrane-enriched fraction. p24 eluted from membranes with 0.8 M NaCl, but not 0.6 M NaCl, suggesting that it is a peripheral membrane component. Solubilized p24 binds heparin by heparin affinity chromatography and $\sp{125}$I-heparin binding assays. Furthermore, indirect immunofluorescence studies indicate that cytotrophoblast of floating and attached villi of the human fetal-maternal interface are recognized by anti-p24. The study also indicates that the HSPG, perlecan, accumulates where chorionic villi are attached to uterine stroma and where p24-expressing cytotrophoblast penetrate the stroma. Collectively, these data indicate that p24 is a cell surface membrane-associated heparin/heparan sulfate binding protein found in cytotrophoblast, but not many other cell types of the fetal-maternal interface. Furthermore, p24 colocalizes with HSPGs in regions of cytotrophoblast invasion. These observations are consistent with a role for HSPGs and HSPG binding proteins in human trophoblast-uterine cell interactions. ^

IgG4 Autoantibodies to DNA in Systemic Lupus erythematosus Patients

The presence of DNA-specific IgG4 antibodies was demonstrated in the sera of patients with systemic lupus erythematosus (SLE) by a microtiter solid-phase radioimmunoassay. A patient with distal inter-phalangeal swelling and extensive ulcers in the oral cavity, seronegative for anti-DNA antibodies of the IgG isotype, was found to have anti-DNA autoantibodies exclusively of the IgG4 subclass. These autoantibodies directed against the dsDNA conformation cross-reacted with chondroitin sulfate, dermatan sulfate and heparin.

Composição da matriz extracelular do ligamento cardinal de mulheres na pós-menopausa com e sem prolapso uterino

O prolapso uterino tem sua incidência aumentada na pós-menopausa. O objetivo deste estudo é identificar as alterações na matriz extracelular do ligamento cardinal associadas à menopausa e ao prolapso uterino. Ligamento cardinal de três diferentes grupos de mulheres, pré-menopausa, prolapso uterino e pós-menopausa, foram identificados e biopsiados durante 57 histerectomias abdominais ou vaginais. As amostras foram processadas por métodos bioquímicos para caracterização e quantificação de glicosaminoglicanos sulfatados e colágeno. As concentrações relativas de glicosaminoglicanos foram obtidas por eletroforese. Procedimentos histológicos foram feitos para identificar fibras elásticas (Weigert), distribuição de colágeno (Picro Sirius) e decorin (imunohistoquímica). Nossos resultados mostraram aumento na concentração de GAG de 72,2%, redução na concentração de colágeno de 37% e diminuição de 22% de fibras elásticas no grupo de prolapso uterino quando comparado ao grupo da pós-menopausa (p<0,05, p<0,04 e p<0,05 respectivamente). As concentrações relativas de glicosaminoglicanos sulfatados para condroitin sulfato, heparan sulfato e dermatan sulfato não mostraram diferenças entre os três grupos. A organização do colágeno foi similar entre os três grupos e a marcação do decorin pareceu estar diminuída no grupo de prolapso uterino. Nossos resultados indicam alterações no metabolismo do tecido conjuntivo. O ligamento cardinal da mulher na pós-menopausa possui uma matriz extracelular mais densa. Esta alteração não ocorre na mulher com prolapso uterino.

Polissacarídeos sulfatados de interesse farmacológico no camarão litopenaeus schimitti

Sulfated Polysaccharides with unique chemical structures and important biological activities has been found in a diversity of sea invertebrates. For that, to exist a huger interest on the biotechnology field in the research theses sulfated compounds isolated from sea organisms. Despite the privileged brazilian position for these compounds attainment, there are still a few scientific informations about the isolated substances and their biological activities. A head the displayed, the present work has for objectives, to evaluate the pharmacological properties of the glycosaminoglycans isolated from the sea shrimp Litopenaeus schimitti on homeostasis, blood coagulation, leukocytes migration and platelet/leukocyte adhesion. For this, yhe glycosaminoglycans were extracted from crustacean tissues by proteolysis, fractionation with acetone and later submitted to pharmacological assays. The crustacean tissues showed compounds heparin-like, with anticoagulant activity of 45 IU/mg and 90 IU/mg, respectively. These molecules showed low residual hemorrhagic effects in the tested concentration (100 µg/mL), when compared to unfractionated commercial heparin (UFH). Another dermatan sulfate-like compound, predominately constituted for disulfated disaccharides, was isolated from crustacean abdomen. This compound showed an efficient effect on leukocytes migration inhibition, in the concentration of 15 µg/mL, reducing the cellular infiltration in 65% when compared to the controlled animals. In this same concentration, the DS reduced in 60% the protein concentration of the peritoneal exudates. In the concentration, this compound of 0.5 mg/mL, it was capable to reduce in 40% platelet/leukocytes adhesion. Our data demonstrate that these sulfated polysaccharides isolated from the shrimp L. schimitti will can be used as bioactive compounds, appearing as active principles for pharmacological development, anticoagulants and inflammatory response regulators

Effect of amniotic membrane transplantation on corneal healing and proteoglycan expression in an experimental model of limbal deficiency in rabbits

PURPOSE. Amniotic membrane transplantation (AMT) has been used as a graft or as a dressing in ocular surface reconstruction, facilitating epithelization, maintaining normal epithelial phenotype, and reducing inflammation, vascularization, and scarring. The corneal transparency is due, at least in part, to the arrangement in orthogonal lamellae of collagen fibrils, surrounded by proteoglycans (PGs). These PGs regulate fibrilogenesis, the matrix assembly, and ultimately the corneal transparency. The purpose of the present study was to investigate the effects of AMT upon the corneal PGs after severe limbal injury.METHODS. Experiments were performed on the right corneas of 22 New Zealand female albino rabbits, and their left corneas were used as matched controls. These animals were divided into 3 groups: G1 (n = 10): total peritomy and keratolimbectomy, followed by application of 0.5 M NaOH; G2 (n = 10): submitted to the same trauma as G1, and treated by AMT; G3: no trauma, only AMT (n = 2). The right corneas of G2 and G3 were covered by DMSO 4 cryopreserved human amniotic membrane, fixed by interrupted 9-0 mononylon sutures, with its stromal face toward the ocular surface. After 7 or 30 days, the corneas were removed and PGs were extracted.RESULTS. Normal corneas contained approximately 9 mg of PGs per gram of dry tissue. AMT on intact cornea (G3) did not cause any changes in the concentration of PGs. In contrast, injured corneas contained much less PGs, both on the seventh and on the 30th day posttrauma. The PG concentration was even lower in injured corneas treated by AMT. This decrease was due almost exclusively to dermatan sulfate PGs, and the structure of dermatan sulfate was also modified, indicating changes in the biosynthesis patterns.CONCLUSIONS. Although beneficial effects have been observed on clinical observation and concentration of soluble proteins after AMT, the normal PG composition of cornea was not attained, even 30 days postinjury, indicating that the normal ocular surface reconstruction, if possible, is a long-term process. (Eur J Ophthalmol 2010; 20: 290-9)

Glycosaminoglycans in components of the rabbit eye: synthesis and characterization

Purpose. To trace the eye components involved in proteoglycan synthesis and to characterize the sulfated glycosaminoglycans which are associated to these macromolecules.Methods. Sodium [S-35]-sulfate was injected intravitreally and the rabbits were killed at different time intervals after the injection. The glycosaminoglycans of choroid, ciliary body, cornea, iris, lens capsule, retina and sclera were extracted and processed for estimations of their specific activities, and for electrophoresis plus autoradiography with or without previous treatment with specific enzymes. In addition, methacrylate sections of the eyes were analysed by autoradiography.Results. The peak of specific activities of the glycosaminoglycans of all eye components occurred at 2 days after the intravitreal injection of [S-35]-sulfate. The autoradiography of the agarose gels revealed three types of glycosaminoglycans, namely, heparan-, chondroitin- and dermatan sulfate, only in the retina. The other eye components contained heparan sulfate and either chondroitin or dermatan sulfate. Tissue autoradiography together with the biochemical techniques contributed to unravel the origin of the glycosaminoglycans in the eye components.Conclusions. The results of the present investigation have shown that heparan sulfate, contrasting to chondroitin sulfate and dermatan sulfate, is synthesized in all eye components studied and that the glycosaminoglycan composition differs according to the tissue of origin.

Effect of different glycosaminoglycans in a guinea-pig carotid-artery thrombosis model

Heparin is the most frequently used drug for the prevention and treatment of thrombosis. Its use, however, is restricted by its side-effects. To study the efficacy of other glycosaminoglycans that could substitute heparin in the management of arterial thrombosis, 60 guinea-pigs were randomly allocated into 6 groups: G1= control, G2= heparin (150 IU/kg), G3= heparan sulfate from beef pancreas (2.5 mg/kg), G4= heparan sulfate from beef lung (2.5 mg/kg), G5= N-acetylated heparan from beef pancreas, G6= dermatan sulfate from beef intestine (2.5 mg/kg). Ten minutes after intravenous injection of the drugs, thrombosis was induced by the injection of a 50% glucose solution into a segment of the right carotid artery isolated between 2 thread loops during 10 minutes. Three hours later the artery was re-exposed and if a thrombus was present it was measured, withdrawn and weighed. Thrombin time and activated partial thromboplastin time were measured in all animals. Thrombus developed in 90% of the animals in the control group, 0% in G2 and G3, 62.5% in G4, 87.5% in G5 and G6. Only in the animals treated with heparin the coagulation tests were prolonged. In conclusion, in the used dose only the heparan sulfate from beef pancreas presented an antithrombotic effect similar to heparin in this experimental model.

Extracellular matrix of porcine pericardium: Biochemistry and collagen architecture

Pericardial tissue has been used to construct bioprostheses employed in the repair of different kinds of injuries, mostly cardiac. However, calcification and mechanical failure have been the main causes of the limited durability of cardiac bioprostheses constructed with bovine pericardium. In the course of this work, a study was conducted on porcine fibrous pericardium, its microscopic structure and biochemical nature. The general morphology and architecture of collagen were studied under conventional light and polarized light microscopy. The biochemical study of the pericardial matrix was conducted according to the following procedures: swelling test, hydroxyproline and collagen dosage, quantification of amino acids in soluble collagen, component extraction of the extracellular matrix of the right and left ventral regions of pericardium with different molarities of guanidine chloride, protein and glycosaminoglycan (GAG) dosage, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and total GAG analysis. Microscopic analysis showed collagen fibers arranged in multidirectionally oriented layers forming a closely knit web, with a larger number of fibers obliquely oriented, initiating at the lower central region toward the upper left lateral relative to the heart. No qualitative differences were found between proteins extracted from the right and left regions. Likewise, no differences were found between fresh and frozen material. Protein dosages from left frontal and right frontal pericardium regions showed no significant differences. The quantities of extracted GAGs were too small for detection by the method used. Enzymatic digestion and electrophoretic analysis showed that the GAG found is possibly dermatan sulfate. The proteoglycan showed a running standard very similar to the small proteoglycan decorin.

Glycosaminoglycan profiles in the uterus of adult ovariectomized rats treated with estrogen and progestagen

Objectives: To evaluate the effects of conjugated equine estrogens (CE) alone or in combination with medroxyprogesterone acetate (MPA) on glycosaminoglycans (GAGs) in the cervix and horns of the rat uterus. Study design: Thirty days after ovariectomy, adult rats were randomly divided into four groups: Cl, control (treated with drug vehicle); GII CE (50 mu g/kg per day); GIII, MPA (0.2 mg/kg per day), and GIV, CE + MPA (doses as in GII and Gill). Drugs and vehicle were given by gavage during 28 days. Afterwards the animals were anesthetized, the cervix and uterine horns were dissected out and the middle portion fixed in 10% formaldehyde solution; other portions were fixed in acetone for histological examination and glycosaminoglycan quantification, respectively. Agarose gel electrophoresis was used for sulfated GAG analyses, and hyaluronic acid was assayed with an ELISA-like method. Statistical analysis was done by the Student's t test and the Tukey-Kramer test (P < 0.05). Results: The cervix and uterine horn structures presented signs of atrophy in the control group (GI). The other groups, mainly groups III and IV, had histological aspects of proliferation. In all groups the concentration of sulfated GAGs (especially dermatan sulfate) was higher than that of non-sulfated GAGs, both in cervix and in uterine horns. Estrogens increased sulfated GAG concentration at the cervix and the horn, whereas in uterine horns the amounts of sulfated GAGs were decreased after estrogens plus MPA treatment. The concentration of hyaluronic acid in uterine horns was higher than in cervices. Conclusions: The profiling and amounts of glycosaminoglycans in the two portions of the rat uterus are uneven. Dermatan sulfate occurs in higher concentrations in both cervix and uterine horns. Sulfated GAGs in rat cervix were increased by estrogens plus MPA, but were decreased by MPA alone in uterine horns. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Charakterisierung des Rezeptors, des Aufnahmeweges und der Neutralisation humanpathogener Papillomviren

Im Rahmen dieser Arbeit wurden Pseudovirionen mit gfp als Reportergen generiert und zur Charakterisierung verschiedener Aspekte der HPV-Infektion verwendet. Es konnte gezeigt werden, daß Heparansulfatproteoglykane für die Infektion mit HPV16- und HPV33-Pseudovirionen essentiell sind, da: (i) Heparin, nicht aber Chondroitin- oder Dermatansulfat die Infektion inhibiert, (ii) Heparinase I oder chloratbehandelte Zellen vollständig bzw. teilweise resistent gegen eine Infektion sind, (iii) monoklonale Antikörper Pseudovirionen neutralisieren, indem sie die Bindung an Heparin verhindern. Ein intakter C-Terminus des L1-Proteins ist für die Heparinbindung nicht notwendig. Alpha-6 Integrin ist kein obligater HPV-Rezeptor. Die Neutralisation gebundener Pseudovirionen durch ein neutralisierendes Antiserum einerseits und durch Heparin andererseits demonstriert, daß die Aufnahme von Pseudovirionen sehr langsam verläuft und daß die Bindung von einem heparinsensitiven zu einem heparinresistenten Zustand übergeht, möglicherweise unter Beteiligung weiterer Rezeptoren. Die Wirkung von verschiedenen Inhibitoren auf die Pseudoinfektion lassen schließen, daß Pseudovirionen über eine mikrofilamentabhängige und energieabhängige Endozytose mit anschließender Penetration durch saure Vesikel aufgenommen werden. Caveolen sind an der Aufnahme nicht beteiligt. Untersuchungen zur Neutralisation von HPV16-, HPV18- und HPV33-Pseudovirionen durch Antiseren gegen HPV16, 18, 31, 33, 35, 39 und 45 zeigen eine Kreuzneutralisation zwischen HPV31 und HPV33 einerseits und zwischen HPV18 und HPV45 andererseits.

Selective matrix remodeling to increase interstitial flow in lymphedema

Lymphedema is a disease characterized by swelling resulting from the accumulation of fluid in the extracellular matrix (ECM) of the skin. In order to alleviate this swelling, the authors sought to selectively degrade certain hydrophilic molecules in the ECM called glycosaminoglycans (GAGs). GAGs are long unbranched sugar molecules present in the ECM that attract water to their numerous negative charges. The authors hypothesized that the density of GAGs would increase in lymphedema and inhibit fluid from leaving the tissue. An existing mouse tail model of experimental lymphedema that reproduced important features of the human condition was used to evaluate GAG content in swollen tissue. In this model, a surgical excision of tissue was made circumferentially around the tail that caused swelling distal to the wound site. Tissue distal to the wound site was analyzed via two assays; one that measured hyaluronan (an unsulfated GAG) and another that measured sulfated GAGs (including Dermatan Sulfate and Chondroitin Sulfate), at various timepoints post surgical intervention. Hyaluronan (HA) levels were significantly higher than control (tissues with no surgical intervention) by day 5 (p

Colocalization of a large heterodimeric proteoglycan with basement membrane proteins in cultured cells.

A novel large heterodimeric dermatan sulfate proteoglycan with core proteins of 460 and 300 kDa, respectively, had been described as a secretory product of human fetal skin fibroblasts (Breuer et al., J. Biol. Chem. 266, 13224-13232 (1991)). Pulse-chase experiments showed a preferential association of the proteoglycan with the cell membrane. Immunogold labeling indicated its localization in fibrils on the cell surface as well as in fibrillar extensions from the cell body. Immunofluorescence studies yielded a fibrillar and punctate staining pattern which was also seen in cultured human and porcine endothelial cells. Dot-like structures were observed in transformed human keratinocytes. Various immunocytochemical double-labeling experiments indicated a remarkable colocalization of the proteoglycan with fibronectin, laminin, perlecan, and type IV collagen whereas only occasionally a colocalization with chondroitin-6-sulfate was found. No evidence for an enrichment of the proteoglycan in vinculin-containing structures was obtained. These results suggest that the proteoglycan is a widely distributed macromolecule which can associate with basement membrane components. Preliminary findings in rat cornea supported this conclusion.