Molecular Dynamics-Based Comparative Analysis of Chondroitin and Dermatan Sulfates.

Glycosaminoglycans (GAGs) are a class of linear anionic periodic polysaccharides containing disaccharide repetitive units. These molecules interact with a variety of proteins in the extracellular matrix and so participate in biochemically crucial processes such as cell signalling affecting tissue regeneration as well as the onset of cancer, Alzheimer's or Parkinson's diseases. Due to their flexibility, periodicity and chemical heterogeneity, often termed "sulfation code", GAGs are challenging molecules both for experiments and computation. One of the key questions in the GAG research is the specificity of their intermolecular interactions. In this study, we make a step forward to deciphering the "sulfation code" of chondroitin sulfates-4,6 (CS4, CS6, where the numbers correspond to the position of sulfation in NAcGal residue) and dermatan sulfate (DS), which is different from CSs by the presence of IdoA acid instead of GlcA. We rigorously investigate two sets of these GAGs in dimeric, tetrameric and hexameric forms with molecular dynamics-based descriptors. Our data clearly suggest that CS4, CS6 and DS are substantially different in terms of their structural, conformational and dynamic properties, which contributes to the understanding of how these molecules can be different when they bind proteins, which could have practical implications for the GAG-based drug design strategies in the regenerative medicine.

Glycomics by ion mobility tandem mass spectrometry of chondroitin sulfate disaccharide domain in biglycan.

Biglycan (BGN), a small leucine-rich repeat proteoglycan, is involved in a variety of pathological processes including malignant transformation, for which the upregulation of BGN was found related to cancer cell invasiveness. Because the functions of BGN are mediated by its chondroitin/dermatan sulfate (CS/DS) chains through the sulfates, the determination of CS/DS structure and sulfation pattern is of major importance. In this study, we have implemented an advanced glycomics method based on ion mobility separation (IMS) mass spectrometry (MS) and tandem MS (MS/MS) to characterize the CS disaccharide domains in BGN. The high separation efficiency and sensitivity of this technique allowed the discrimination of five distinct CS disaccharide motifs, of which four irregulated in their sulfation pattern. For the first time, trisulfated unsaturated and bisulfated saturated disaccharides were found in BGN, the latter species documenting the non-reducing end of the chains. The structural investigation by IMS MS/MS disclosed that in one or both of the CS/DS chains, the non-reducing end is 3-O-sulfated GlcA in a rather rare bisulfated motif having the structure 3-O-sulfated GlcA-4-O-sulfated GalNAc. Considering the role played by BGN in cancer cell spreading, the influence on this process of the newly identified sequences will be investigated in the future.

Concentration of sulfated glycosaminoglycans in the mammary tissue of female rats with the aging and about hormonal influence.

It was to evaluate the concentration of sulfate glycosaminoglycans (GAG) in mammary tissue of the young and adult female rats and ovariectomized females rats after hormonal stimulation. For this purpose, 60 female rats were divided into six groups with 10 animals/each: nonovariectomized groups: G1 (5 months), and G2 (15 months) and ovariectomized groups: OG (vehicle); EG: (estradiol, 7 days of treatment), PG (progesterone acetate, 23 days of treatment) and EPG: (estradiol (7 days of treatment) and next progesterone acetate (23 days of treatment). Twenty-four hours after the last treatment, all animals were euthanized, the mammary tissue removed, processed for biochemical evaluation and quantification of the GAG. The comparison between groups showed that the concentration dermatan sulfate (DS) G1 was lower compared to G2, OG, EG (p < .05) and G2 was lower compared to OG (p < .05), and OG was higher compared to EG, GP, EPG (p < .05); and heparan sulfate (HS) G1 was higher compared to G2 (p < .05), and G2 was higher compared to OG, EP, PG and EPG (p < .05). These changes in the extracellular matrix might explain, at least in part, hormonal influence about sulfated glycosaminoglycans in response to physiological state/age, and in response to hormonal treatment in the mammary tissues.

Incomplete biomarker response in mucopolysaccharidosis type I after successful hematopoietic cell transplantation.

BACKGROUND: Residual disease, primarily involving musculoskeletal tissue, is a common problem in patients with neuronopathic mucopolysaccharidosis type I (MPS I, Hurler or severe Hurler-Scheie phenotype) after a successful hematopoietic cell transplantation (HCT). The concentration of the GAG derived biomarkers heparan sulfate (HS) and dermatan sulfate (DS), may reflect residual disease and is used for monitoring biochemical response to therapies. This study investigates the response of HS and DS in blood and urine to HCT in MPS I patients. METHODS: In 143 blood- and urine samples of 17 neuronophatic MPS I patients, collected prior and post successful HCT, the concentration of the disaccharides derived after full enzymatic digestion of HS and DS were analyzed by multiplex liquid chromatography tandem-mass spectrometry (LC-MS/MS). RESULTS: Median follow up after HCT was 2.4years (range 0-11years). HCT led to a rapid decrease of both HS and DS. However, only 38% of the patients reached normal HS levels in blood and even less patients (6%) reached normal DS levels. In none of the patients normalization of HS or DS was observed in urine. CONCLUSIONS: Biomarker response after HCT is incomplete, which may reflect residual disease activity. Novel therapeutic strategies should aim for full metabolic correction to minimize clinical manifestations.

Glycomics expression analysis of sulfated glycosaminoglycans of human colorectal cancer tissues and non-neoplastic mucosa by electrospray ionization mass spectrometry / Análise da expressão de glicosaminoglicanos sulfatados no tecido humano neoplásico colorretal e na mucosa não neoplásica por espectrometria de massa por ionização por electrospray

ABSTRACT Objective To determine the presence of glycosaminoglycans in the extracellular matrix of connective tissue from neoplastic and non-neoplastic colorectal tissues, since it has a central role in tumor development and progression. Methods Tissue samples from neoplastic and non-neoplastic colorectal tissues were obtained from 64 operated patients who had colorectal carcinoma with no distant metastases. Expressions of heparan sulphate, chondroitin sulphate, dermatan sulphate and their fragments were analyzed by electrospray ionization mass spectrometry, with the technique for extraction and quantification of glycosaminoglycans after proteolysis and electrophoresis. The statistical analysis included mean, standard deviation, and Student’st test. Results The glycosaminoglycans extracted from colorectal tissue showed three electrophoretic bands in agarose gel. Electrospray ionization mass spectrometry showed characteristic disaccharide fragments from glycosaminoglycans, indicating their structural characterization in the tissues analyzed. Some peaks in the electrospray ionization mass spectrometry were not characterized as fragments of sugars, indicating the presence of fragments of the protein structure of proteoglycans generated during the glycosaminoglycan purification. The average amount of chondroitin and dermatan increased in the neoplastic tissue compared to normal tissue (p=0.01). On the other hand, the average amount of heparan decreased in the neoplastic tissue compared to normal tissue (p= 0.03). Conclusion The method allowed the determination of the glycosaminoglycans structural profile in colorectal tissue from neoplastic and non-neoplastic colorectal tissue. Neoplastic tissues showed greater amounts of chondroitin sulphate and dermatan sulphate compared to non-neoplastic tissues, while heparan sulphate was decreased in neoplastic tissues.

RESUMO Objetivo Determinar a presença de glicosaminoglicanos na matriz extracelular do tecido conjuntivo colorretal neoplásico e não neoplásico, tendo em vista seu papel central no desenvolvimento e na progressão dos tumores. Métodos Amostras de tecidos colorretais neoplásicos e não neoplásicos foram obtidas de 64 pacientes operados com carcinoma colorretal sem metástases a distância. As expressões de heparan sulfato, sulfato de condroitina e sulfato de dermatan e seus fragmentos foram analisadas por espectrometria de massa por ionização por electrospray, com técnica de extração e quantificação de glicosaminoglicanos após proteólise e eletroforese. Para análise estatística, utilizaram-se média, desvio padrão e teste t de Student. Resultados Em gel de agarose, os glicosaminoglicanos extraídos de tecido colorretal mostraram três bandas eletroforéticas. A espectrometria de massa por ionização por electrospray mostrou fragmentos de dissacarídeos característicos de glicosaminoglicanos e indicou sua característica estrutural. Alguns picos na espectrometria de massa por ionização por electrospray não foram caracterizados como fragmentos de açúcares, sugerindo a presença de fragmentos de proteínas estruturais dos proteoglicanos, formadas durante a purificação dos glicosaminoglicanos. A quantidade média de condroitina e dermatan aumentou no tecido neoplástico em relação ao tecido normal (p=0,01). Por outro lado, a quantidade média de heparan foi menor no tecido neoplásico em relação ao tecido normal (p=0,03). Conclusão O método empregado permitiu determinar o perfil estrutural dos glicosaminoglicanos nas amostras. Tecidos neoplásicos apresentaram maiores quantidades de sulfato de condroitina e sulfato de dermatan em comparação com os não neoplásicos, enquanto o sulfato de heparan foi encontrado em menores quantidades nos tecidos neoplásicos.

Glycomics expression analysis of sulfated glycosaminoglycans of human colorectal cancer tissues and non-neoplastic mucosa by electrospray ionization mass spectrometry.

OBJECTIVE: To determine the presence of glycosaminoglycans in the extracellular matrix of connective tissue from neoplastic and non-neoplastic colorectal tissues, since it has a central role in tumor development and progression. METHODS: Tissue samples from neoplastic and non-neoplastic colorectal tissues were obtained from 64 operated patients who had colorectal carcinoma with no distant metastases. Expressions of heparan sulphate, chondroitin sulphate, dermatan sulphate and their fragments were analyzed by electrospray ionization mass spectrometry, with the technique for extraction and quantification of glycosaminoglycans after proteolysis and electrophoresis. The statistical analysis included mean, standard deviation, and Student'st test. RESULTS: The glycosaminoglycans extracted from colorectal tissue showed three electrophoretic bands in agarose gel. Electrospray ionization mass spectrometry showed characteristic disaccharide fragments from glycosaminoglycans, indicating their structural characterization in the tissues analyzed. Some peaks in the electrospray ionization mass spectrometry were not characterized as fragments of sugars, indicating the presence of fragments of the protein structure of proteoglycans generated during the glycosaminoglycan purification. The average amount of chondroitin and dermatan increased in the neoplastic tissue compared to normal tissue (p=0.01). On the other hand, the average amount of heparan decreased in the neoplastic tissue compared to normal tissue (p= 0.03). CONCLUSION: The method allowed the determination of the glycosaminoglycans structural profile in colorectal tissue from neoplastic and non-neoplastic colorectal tissue. Neoplastic tissues showed greater amounts of chondroitin sulphate and dermatan sulphate compared to non-neoplastic tissues, while heparan sulphate was decreased in neoplastic tissues.

Carbohydrate-containing molecules as potential biomarkers in colon cancer.

Glycans play a critical role in physiological and pathological processes through interaction with a variety of ligands. Altered expression and dysregulation of these molecules can cause aberrant cellular function such as malignancy. Glycomics provide information of the structure and function of glycans, glycolipids, and glycoproteins such as proteoglycans, and may help to predict cancer development and progression as biomarkers. In this report, we compared the expression of proteoglycans, the content and structure of glycosaminoglycans and glycolipids between patient-matched normal and cancer tissues obtained from colon cancer patients. Tumor-related proteoglycans, glypican-3, and syndecan-1 showed downregulation in cancer tissues compared to normal tissues. In cancer tissue, the total amount of chondroitin sulfate (CS)/dermatan sulfate and heparan sulfate were lower and, interestingly, the level of disaccharide units of both 4S6S (CS-E) and 6S (CS-C) were higher compared to normal tissue. Also, overall lipids including glycolipids, a major glycomics target, were analyzed by hydrophilic interaction liquid chromatography mass spectrometry. Increase of lyso-phosphatidylcholine (phospholipid), sphingomyelin (sphigolipid), and four types of glycolipids (glucosylceramide, lactosylceramide, monosialic acid ganglioside, and globoside 4) in cancer tissue showed the possibility as potential biomarkers in colon cancer. While requiring the need for careful interpretation, this type of broad investigation gives us a better understanding of pathophysiological roles on glycosaminoglycans and glycolipids and might be a powerful tool for colon cancer diagnosis.

Dermatan sulphate in methoxy polyethylene glycol-polylactide-co-glycolic acid scaffolds upregulates fibronectin gene expression but has no effect on in vivo osteochondral repair.

PURPOSE: The purpose of the study was to investigate the effect of dermatan sulphate (DS) addition to biodegradable methoxy polyethylene glycol (MPEG) substituted polylactide-co-glycolic acid (PLGA) scaffolds for cartilage repair in vitro and in vivo. METHODS: Human chondrocytes from eight patients undergoing anterior cruciate ligament reconstruction were isolated and cultured in 5% oxygen on MPEG-PLGA scaffolds±DS for one, three, seven and 14 days. Analyses were performed using quantitative gene expression analysis for chondrogenic and cell attachment markers. An osteochondral drill hole defect was created in the intertrochlear groove of the distal femur in 20 New Zealand white rabbits (defects n=20). When bleeding was observed, the defects were treated with MPEG-PLGA scaffolds±DS. Twelve weeks after surgery the rabbits were sacrificed and the defects were analysed using histological grading with O'Driscoll scoring. RESULTS: DS addition to MPEG-PLGA scaffolds resulted in a significant upregulation of fibronectin gene expression on day 1. No differences were observed in chondrogenic gene expression. There were no differences between the two groups in histological grading (+DS 10.3 and -DS 9.6). CONCLUSIONS: Upregulation of fibronectin in vitro indicating early cell-scaffold interaction and attachment did not result in improved cartilage repair in an osteochondral defect model in rabbits.

Simultaneous analysis of heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan disaccharides by glycoblotting-assisted sample preparation followed by single-step zwitter-ionic-hydrophilic interaction chromatography.

Glycosaminoglycans (GAGs) play important roles in cell adhesion and growth, maintenance of extracellular matrix (ECM) integrity, and signal transduction. To fully understand the biological functions of GAGs, there is a growing need for sensitive, rapid, and quantitative analysis of GAGs. The present work describes a novel analytical technique that enables high throughput cellular/tissue glycosaminoglycomics for all three families of uronic acid-containing GAGs, hyaluronan (HA), chondroitin sulfate (CS)/dermatan sulfate (DS), and heparan sulfate (HS). A one-pot purification and labeling procedure for GAG Δ-disaccharides was established by chemo-selective ligation of disaccharides onto high density hydrazide beads (glycoblotting) and subsequent labeling by fluorescence. The 17 most common disaccharides (eight comprising HS, eight CS/DS, and one comprising HA) could be separated with a single chromatography for the first time by employing a zwitter-ionic type of hydrophilic-interaction chromatography column. These novel analytical techniques were able to precisely characterize the glycosaminoglycome in various cell types including embryonal carcinoma cells and ocular epithelial tissues (cornea, conjunctiva, and limbus).

Estudo bioquímico do glicosaminoglicano dermatam sulfato em homens adultos portadores de hérnia inguinal tipo II de Nyhus / Biochemical study of dermatan sulfate glycosaminoglycan in adult male patients with Nyhus type II inguinal hernia

OBJETIVO: Comparar a quantidade do glicosaminoglicano dermatam sulfato entre pacientes homens, portadores de hérnia inguinal tipo II de Nyhus e, indivíduos sem hérnia inguinal, com idade entre 20 e 40 anos. MÉTODOS: Foram constituídos dois grupos. Um de 15 pacientes do sexo masculino com hérnia inguinal tipo II de Nyhus e idade entre 20 e 40 anos, com risco ASA I e II, e um grupo controle com dez indivíduos, também do sexo masculino entre 20 e 40 anos, que morreram em período de até 24 h. Foram excluídos os pacientes do sexo feminino, diabéticos, portadores de doença do tecido conjuntivo, tabagistas e com risco cirúrgico ASA III e IV. Foi retirada uma amostra de 1cm² da fáscia transversal na parte intermediária do trígono inguinal, e 1cm² na bainha anterior do músculo reto abdominal na região inguinal correspondente e quantificados os glicosaminoglicanos dermatam sulfato por densitometria, após eletroforese em gel de agarose. RESULTADOS: A quantidade de dermatam sulfato não apresentou diferença estatisticamente significante entre os pacientes com hérnia inguinal e os indivíduos sem hérnia inguinal, tanto na fáscia transversal (p=0,108) quanto na bainha anterior do músculo reto abdominal (p=0,292). CONCLUSÃO: Não se encontrou diferença na quantidade do glicosaminoglicano dermatam sulfato entre os pacientes portadores de hérnia inguinal tipo II de Nyhus e indivíduos sem hérnia inguinal em homens adultos.

OBJECTIVE: To compare the amount of the dermatan sulfate glycosaminoglycan between male patients with Nyhus type II inguinal hernias and subjects without inguinal hernia, aged between 20 and 40 years. METHODS: Two groups were formed: One with 15 male patients with Nyhus type II inguinal hernia and aged between 20 and 40 years with ASA risk I and II, and a control group of ten individuals, also males between 20 and 40, who had died up to 24 h before. We excluded female patients, diabetic patients with connective tissue disease, smokers and surgical risk ASA III and IV. We resected a sample of 1 cm² of the transversalis fascia in the middle of the inguinal trigone, and 1 cm² of the anterior sheath of the rectus abdominis muscle in the groin for the quantification of dermatan sulfate glycosaminoglycans by densitometry after agarose gel electrophoresis. RESULTS: The amount of dermatan sulfate showed no statistically significant difference between patients with inguinal hernia and individuals without inguinal hernia in both the transverse fascia (p = 0.108) and anterior sheath of the rectus abdominis muscle (p = 0.292). CONCLUSION: There was no difference in the amount of the dermatan sulfate glycosaminoglycan among patients with Nyhus type II inguinal hernias and subjects without inguinal hernia in adult males.

Quantification of chondroitin sulfate and dermatan sulfate in danaparoid sodium by (1)H NMR spectroscopy and PLS regression.

Danaparoid sodium (the active pharmaceutical ingredient in Orgaran; Merck Sharp and Dohme) is a biopolymeric non-heparin drug used as anticoagulant and antithrombotic agent approved for the prophylaxis of post-operative deep-vein thrombosis, which may lead to pulmonary embolism in patients undergoing, e.g., elective hip replacement surgery. It consists of a mixture of three glycosaminoglycans (GAGs): heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate (CS). Currently, the CS and DS content are quantified by means of a time-consuming enzymatic method. In this paper the use of (1)H NMR in combination with multivariate regression (partial least-squares, PLS) is proposed as a new method. In order to evaluate the proposed method, a series of danaparoid sodium samples were analyzed and the results were compared with those obtained by the enzymatic method (reference method). The results showed that the proposed (1)H NMR method is a good alternative for analysis of CS and DS in danaparoid sodium. Accuracy of ±0.7% (w/w) and ±1.1% (w/w) for CS and DS was obtained by the (1)H NMR method and accuracy of ±1.0% (w/w) and ±1.3% (w/w) by the enzymatic method. Furthermore, the use of (1)H NMR in combination with PLS results in a fast quantification. The analysis time is reduced to 35 min per sample instead of 60 h for a maximum of 16 samples.

Analysis of glycosaminoglycans in stem cell glycomics.

Glycosaminoglycans (GAGs) play a critical role in the binding and activation of growth factors in cell signal transduction required for biological development. A glycomics approach can be used to examine GAG content, composition, and structure in stem cells in order to characterize their general differentiation. Specifically, this method may be used to evaluate chondrogenic differentiations by profiling for the GAG content of the differentiated cells. Here, embryonic-like teratocarcinoma cells, NCCIT, a developmentally pluripotent cell line, were used as a model for establishing GAG glycomic methods, but will be easily transferrable to embryonic stem cell cultures.

Glycosaminoglycan distribution in the rat uterine cervix during the estrous cycle.

OBJECTIVE: To analyze the amount of glycosaminoglycans in the uterine cervix during each phase of the rat estrous cycle. DESIGN: Based on vaginal smears, forty female, regularly cycling rats were divided into four groups (n = 10 for each group): GI - proestrous, GII - estrous, GIII - metaestrous and GIV - diestrous. Animals were sacrificed at each phase of the cycle, and the cervix was immediately removed and submitted to biochemical extraction and determination of sulfated glycosaminoglycans and hyaluronic acid. The results were analyzed by ANOVA followed by the Bonferroni post-hoc test. RESULTS: The uterine cervix had the highest amount of total sulfated glycosaminoglycans and dermatan sulfate during the estrous phase (8.90 +/- 0.55 mg/g of cetonic extract, p<0.001; and 8.86 +/- 0.57 mg/g of cetonic extract, p<0.001). In addition, there was more heparan sulfate at the cervix during the proestrous phase (0.185 +/- 0.03 mg/g of cetonic extract) than during any other phase (p<0.001). There were no significant changes in the concentration of hyaluronic acid in the uterine cervix during the estrous cycle. CONCLUSION: Our data suggest that the amount of total sulfated glycosaminoglycans may be influenced by hormonal fluctuations related to the estrous cycle, with dermatan sulfate and heparan sulfate being the glycosaminoglycans most sensitive to hormonal change.

Glycosaminoglycan distribution in the rat uterine cervix during the estrous cycle

OBJECTIVE: To analyze the amount of glycosaminoglycans in the uterine cervix during each phase of the rat estrous cycle. DESIGN: Based on vaginal smears, forty female, regularly cycling rats were divided into four groups (n = 10 for each group): GI - proestrous, GII - estrous, GIII - metaestrous and GIV - diestrous. Animals were sacrificed at each phase of the cycle, and the cervix was immediately removed and submitted to biochemical extraction and determination of sulfated glycosaminoglycans and hyaluronic acid. The results were analyzed by ANOVA followed by the Bonferroni post-hoc test. RESULTS: The uterine cervix had the highest amount of total sulfated glycosaminoglycans and dermatan sulfate during the estrous phase (8.90 ± 0.55 mg/g of cetonic extract, p<0.001; and 8.86 ± 0.57 mg/g of cetonic extract, p<0.001). In addition, there was more heparan sulfate at the cervix during the proestrous phase (0.185 ± 0.03 mg/g of cetonic extract) than during any other phase (p<0.001). There were no significant changes in the concentration of hyaluronic acid in the uterine cervix during the estrous cycle. CONCLUSION: Our data suggest that the amount of total sulfated glycosaminoglycans may be influenced by hormonal fluctuations related to the estrous cycle, with dermatan sulfate and heparan sulfate being the glycosaminoglycans most sensitive to hormonal change.

Alterations of glycosaminoglycan disaccharide content and composition in colorectal cancer: structural and expressional studies.

The glycosaminoglycans are implicated in many processes important in the growth and progression of malignant tumors. In the present study glycosaminoglycans were purified from healthy, macroscopically normal and cancerous specimens of different anatomic sites and different stages of cancer and analyzed by FACE after chondroitinases and sulfatases digestion. The cancerous samples contained increased levels of 6-sulfated unsaturated disaccharides compared to macroscopically normal and healthy samples, the increase being stage-related. The differences in sulfation were found to be related to the anatomic site and the stage of cancer. RT-PCR analysis of 4-sulfotransferase mRNA revealed its presence in decreasing amounts as the stage of the cancer increased. Furthermore, the percent content of hyaluronan disaccharides was elevated in macroscopically normal samples compared to the cancerous, and in addition, it was much more elevated than that of healthy samples. Haluronan levels increase with stage in cancerous tissues. Therefore, it could be concluded that the glycosaminoglycans in colorectal cancer are biosynthetically directed to contribute in different ways depending on the cancer stage and anatomical site.

[Evaluation of glycosaminoglycans of periurethral tissue in patients with and without pelvic organ prolapse]. / Avaliação dos glicosaminoglicanos do tecido periuretral de pacientes com e sem prolapso genital.

OBJECTIVE: To characterize and quantify periurethral tissue sulphated glycosaminoglycans (GAGs) in women with and without pelvic organ prolapse. STUDY DESIGN: Periurethral tissue was obtained from 35 women who underwent surgery for pelvic organ prolapse, for stress urinary incontinence, or for other gynecological benign conditions. Patients were submitted to a clinical history, physical and urodynamic examination and were divided in two groups according to genital prolapse. The standard biopsy with 1.0 x 1.0 cm was taken from periurethral tissue during surgery and assessed by biochemical methods. The GAGs were obtained by proteolysis and precipitated by trichloroacetic acid. The relative concentration of sulfated GAGs was determined by densitometry of toluidine blue stained gel using a spectrophotometer with a 525 nm wavelength. Data were compared using analysis of variance (ANOVA). RESULTS: In the two groups dermatan sulphate (DS) was the predominant glycosaminoglycan (85%), followed by chondroitin sulphate (CS) and heparan sulphate (HS). Women with pelvic organ prolapse had significantly more total GAGs, DS and HS. Differences in CS were not observed. CONCLUSIONS: This study showed altered biochemical characteristics in the extracellular matrix of periurethral tissue and also accumulation of GAGs, DS and CS, in women with pelvic organ prolapse.

Avaliação dos glicosaminoglicanos do tecido periuretral de pacientes com e sem prolapso genital / Evaluation of glycosaminoglycans of periurethral tissue in patients with and without pelvic organ prolapse

OBJETIVOS: Caracterizar e quantificar os subtipos de glicosaminoglicanos sulfatados (GAGs) existentes no tecido peri-uretral de pacientes com e sem prolapso genital. METODOS: Foram incluídas 35 pacientes que se submeteram a cirurgia vaginal para correção de distopias genitais e/ou incontinência urinária de esforço ou por outra condição benigna. As pacientes foram avaliadas por anamnese padronizada, exame físico e urodinâmico e agrupadas segundo a existência do prolapso genital. Durante o procedimento cirúrgico, amostras de aproximadamente 1,0 x 1,0 cm do tecido periuretral foram retiradas para avaliação. Os GAGs foram extraídos do tecido por proteólise e precipitação por ácido tricloroacético e caracterizados por eletroforese em gel de agarose. A quantificação foi feita por meio de densitometria a 525 nm do gel corado com azul de toluidina. Compararam-se os dados pela análise de variância (ANOVA). RESULTADOS: Nos grupos estudados, houve maior predomínio de dermatam sulfato (DS), em torno de 85 por cento do total de GAGs, seguido do condroitim sulfato (CS) e do heparam sulfato (HS). Observou-se aumento significativo dos GAGs totais, do DS e do HS em mulheres com prolapso genital. Não se observou diferença significante com relação ao CS. CONCLUSÃO: Este estudo demonstrou diferenças na matriz extracelular do tecido periuretral com aumento de GAGs totais, DS e HS nas mulheres com prolapso genital.

OBJECTIVE: To characterize and quantify periurethral tissue sulphated glycosaminoglycans (GAGs) in women with and without pelvic organ prolapse. STUDY DESIGN: Periurethral tissue was obtained from 35 women who underwent surgery for pelvic organ prolapse, for stress urinary incontinence, or for other gynecological benign conditions. Patients were submitted to a clinical history, physical and urodynamic examination and were divided in two groups according to genital prolapse. The standard biopsy with 1.0 x 1.0 cm was taken from periurethral tissue during surgery and assessed by biochemical methods. The GAGs were obtained by proteolysis and precipitated by trichloroacetic acid. The relative concentration of sulfated GAGs was determined by densitometry of toluidine blue stained gel using a spectrophotometer with a 525 nm wavelength. Data were compared using analysis of variance (ANOVA). RESULTS: In the two groups dermatan sulphate (DS) was the predominant glycosaminoglycan (85 percent), followed by chondroitin sulphate (CS) and heparan sulphate (HS). Women with pelvic organ prolapse had significantly more total GAGs, DS and HS. Differences in CS were not observed. CONCLUSIONS: This study showed altered biochemical characteristics in the extracellular matrix of periurethral tissue and also accumulation of GAGs, DS and CS, in women with pelvic organ prolapse.

Commercial extracellular matrix scaffolds for rotator cuff tendon repair. Biomechanical, biochemical, and cellular properties.

BACKGROUND: We are not aware of any in vitro study comparing the biomechanical, biochemical, and cellular properties of commercial extracellular matrix materials marketed for rotator cuff tendon repair. In this study, the properties of GraftJacket, TissueMend, Restore, and CuffPatch were quantified and compared with each other. The elastic moduli were also compared with that of normal canine infraspinatus tendon. METHODS: Samples were tested from different manufacturing lots of four materials: GraftJacket (ten lots), TissueMend (six), Restore (ten), and CuffPatch (six). The Kruskal-Wallis test was used to compare thickness, stiffness, and modulus as well as hydroxyproline, chondroitin/dermatan sulfate glycosaminoglycan, hyaluronan, and DNA contents among these matrices. The moduli of the extracellular matrices were also compared with those of normal canine infraspinatus tendon. RESULTS: All four extracellular matrices required 10% to 30% stretch before they began to carry substantial load. Their maximum moduli were realized in their linear region at 30% to 80% strain. The elastic moduli of all four commercial matrices were an order of magnitude lower than that of canine infraspinatus tendon. TissueMend had significantly higher DNA content than the other three matrices (p<0.0001), although both Restore and GraftJacket also had measurable amounts of DNA. CONCLUSIONS: Our data demonstrate chemical and mechanical differences among the four commercial extracellular matrices that we evaluated. Probably, the source (dermis or small intestine submucosa), species (human, porcine, or bovine), age of the donor (fetal or adult), and processing of these matrices all contribute to the unique biophysical properties of the delivered product. The biochemical composition of commercial extracellular matrices is similar to that of tendon. However, the elastic moduli of these materials are an order of magnitude lower than that of tendon, suggesting a limited mechanical role in augmentation of tendon repair.

A tandem mass spectrometric approach to determination of chondroitin/dermatan sulfate oligosaccharide glycoforms.

Dermatan sulfate (DS) chains are variants of chondroitin sulfate (CS) that are expressed in mammalian extracellular matrices and are particularly prevalent in skin. DS has been implicated in varied biological processes including wound repair, infection, cardiovascular disease, tumorigenesis, and fibrosis. The biological activities of DS have been attributed to its high content of IdoA(alpha1-3)GalNAc4S(beta1-4) disaccharide units. Mature CS/DS chains consist of blocks with high and low GlcA/IdoA ratios, and sulfation may occur at the 4- and/or 6-position of GalNAc and 2-position of IdoA. Traditional methods for the analysis of CS/DS chains involve differential digestion with specific chondroitinases followed by steps of chromatographic isolation of the products and di-saccharide analysis on the individual fraction. This work reports the use of tandem mass spectrometry to determine the patterns of sulfation and epimerization of CS/DS oligosaccharides in a single step. The approach is first validated and then applied to a series of skin DS samples and to decorins from three different tissues. DS samples ranged from 74 to 99% of CSB-like repeats, using this approach. Decorin samples ranged from 30% CSB-like repeats for those samples from articular cartilage to 75% for those from sclera. These values agree with known levels of glucuronyl C5-epimerase in these tissues.

Glycosoaminoglycans in nasal polyps.

CONCLUSION: The qualitative and quantitative compositions of GAGs were comparable in all the polyps examined. OBJECTIVE: Glycosoaminoglycans (GAGs) are an integral component of proteoglycans, which are constituents of connective tissue. The qualitative and quantitative compositions of GAGs occurring in proteoglycans determine their biological role. In this work, individual fractions of GAGs occurring in nasal polyps were isolated and estimated. MATERIAL AND METHODS: Polyps were obtained over a 2-year period from 31 patients (18 males, 13 females; age range 28-70 years) who underwent polypectomy and evaluated using routine histopathology. RESULTS: The amount of hyaluronic acid in nasal polyps was high, the amounts of dermatane sulphate and chondroitine-6-sulphate were slightly lower and the amounts of chondroitine-4-sulphate, heparin, heparan sulphate and keratan sulphate were the lowest.