RESUMO
Dupuytren's disease (DD) is a prevalent fibroproliferative disorder of the hand, shaped by genetic, epigenetic, and environmental influences. The extracellular matrix (ECM) is a complex assembly of diverse macromolecules. Alterations in the ECM's content, structure and organization can impact both normal physiological functions and pathological conditions. This study explored the content and organization of glycosaminoglycans, proteoglycans, and collagen in the ECM of patients at various stages of DD, assessing their potential as prognostic indicators. This research reveals, for the first time, relevant changes in the complexity of chondroitin/dermatan sulfate structures, specifically an increase of disaccharides containing iduronic acid residues covalently linked to either N-acetylgalactosamine 6-O-sulfated or N-acetylgalactosamine 4-O-sulfated, correlating with the disease's severity. Additionally, we noted an increase in versican expression, a high molecular weight proteoglycan, across stages I to IV, while decorin, a small leucine-rich proteoglycan, significantly diminishes as DD progresses, both confirmed by mRNA analysis and protein detection via confocal microscopy. Coherent anti-Stokes Raman scattering (CARS) microscopy further demonstrated that collagen fibril architecture in DD varies importantly with disease stages. Moreover, the urinary excretion of both hyaluronic and sulfated glycosaminoglycans markedly decreased among DD patients.Our findings indicate that specific proteoglycans with galactosaminoglycan chains and collagen arrangements could serve as biomarkers for DD progression. The reduction in glycosaminoglycan excretion suggests a systemic manifestation of the disease.
Assuntos
Colágeno , Decorina , Contratura de Dupuytren , Proteoglicanas , Humanos , Contratura de Dupuytren/metabolismo , Contratura de Dupuytren/patologia , Colágeno/metabolismo , Proteoglicanas/metabolismo , Decorina/metabolismo , Matriz Extracelular/metabolismo , Masculino , Progressão da Doença , Feminino , Dermatan Sulfato/metabolismo , Pessoa de Meia-Idade , Idoso , Versicanas/metabolismo , Versicanas/genética , Glicosaminoglicanos/metabolismo , Sulfatos de Condroitina/metabolismo , PolissacarídeosRESUMO
Acute and chronic dermatological injuries need rapid tissue repair due to the susceptibility to infections. To effectively promote cutaneous wound recovery, it is essential to develop safe, low-cost, and affordable regenerative tools. Therefore, we aimed to identify the biological mechanisms involved in the wound healing properties of the glycosaminoglycan dermatan sulfate (DS), obtained from ascidian Styela plicata, a marine invertebrate, which in preliminary work from our group showed no toxicity and promoted a remarkable fibroblast proliferation and migration. In this study, 2,4-DS (50 µg/mL)-treated and control groups had the relative gene expression of 84 genes participating in the healing pathway evaluated. The results showed that 57% of the genes were overexpressed during treatment, 16% were underexpressed, and 9.52% were not detected. In silico analysis of metabolic interactions exhibited overexpression of genes related to: extracellular matrix organization, hemostasis, secretion of inflammatory mediators, and regulation of insulin-like growth factor transport and uptake. Furthermore, in C57BL/6 mice subjected to experimental wounds treated with 0.25% 2,4-DS, the histological parameters demonstrated a great capacity for vascular recovery. Additionally, this study confirmed that DS is a potent inducer of wound-healing cellular pathways and a promoter of neovascularization, being a natural ally in the tissue regeneration strategy.
Assuntos
Dermatan Sulfato , Urocordados , Animais , Camundongos , Dermatan Sulfato/metabolismo , Dermatan Sulfato/farmacologia , Camundongos Endogâmicos C57BL , Urocordados/metabolismo , Cicatrização , Recursos NaturaisRESUMO
Mucopolysaccharidosis type I is a rare autosomal recessive genetic disease caused by deficient activity of α-L-iduronidase. As a consequence of low or absent activity of this enzyme, glycosaminoglycans accumulate in the lysosomal compartments of multiple cell types throughout the body. Mucopolysaccharidosis type I has been classified into 3 clinical subtypes, ranging from a severe Hurler form to the more attenuated Hurler-Scheie and Scheie phenotypes. Over 200 gene variants causing the various forms of mucopolysaccharidosis type I have been reported. DNA isolated from dried blood spot was used to sequencing of all exons of the IDUA gene from a patient with a clinical phenotype of severe mucopolysaccharidosis type I syndrome. Enzyme activity of α-L-iduronidase was quantified by fluorimetric assay. Additionally, a molecular dynamics simulation approach was used to determine the effect of the Ser633Trp mutation on the structure and dynamics of the α-L-iduronidase. The DNA sequencing analysis and enzymatic activity shows a c.1898C>G mutation associated a patient with a homozygous state and α-L-iduronidase activity of 0.24 µmol/L/h, respectively. The molecular dynamics simulation analysis shows that the p.Ser633Trp mutation on the α-L-iduronidase affect significant the temporal and spatial properties of the different structural loops, the N-glycan attached to Asn372 and amino acid residues around the catalytic site of this enzyme. Low enzymatic activity observed for p.Ser633Trp variant of the α-L-iduronidase seems to lead to severe mucopolysaccharidosis type I phenotype, possibly associated with a perturbation of the structural dynamics in regions of the enzyme close to the active site.
Assuntos
Anormalidades Múltiplas/genética , Dermatan Sulfato/química , Heparitina Sulfato/química , Iduronidase/química , Mucopolissacaridose I/genética , Mutação Puntual , Anormalidades Múltiplas/enzimologia , Anormalidades Múltiplas/patologia , Anormalidades Múltiplas/terapia , Domínio Catalítico , Cristalografia por Raios X , Dermatan Sulfato/metabolismo , Terapia de Reposição de Enzimas/métodos , Expressão Gênica , Heparitina Sulfato/metabolismo , Humanos , Iduronidase/genética , Iduronidase/metabolismo , Lactente , Masculino , Simulação de Dinâmica Molecular , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/patologia , Mucopolissacaridose I/terapia , Análise de Componente Principal , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Especificidade por SubstratoRESUMO
Proteoglycan accumulation within the arterial intima has been implicated in atherosclerosis progression in humans. Nevertheless, hypercholesterolaemia is unable to induce intimal thickening and atheroma plaque development in rats. The study was performed to analyse proteoglycans modifications in rats fed with a high-cholesterol diet to understand whether vascular wall remodelling protects against lesions. Sections obtained from rat aortas showed normal features, in intimal-to-media ratio and lipid accumulation. However, focal endothelial hyperplasia and neo-intima rearrangement were observed in high-cholesterol animals. Besides, hypercholesterolaemia induced an inflammatory microenviroment. We determined the expression of different proteoglycans from aortic cells by Western blot and observed a diminished production of decorin and biglycan in high-cholesterol animals compared with control (P < 0.01 and P < 0.05, respectively). Versican was increased in high-cholesterol animals (P < 0.05), whereas perlecan production showed no differences. No modification of the total content of glycosaminoglycans (GAGs) was found between the two experimental groups. In contrast, the chondroitin sulphate/dermatan sulphate ratio was increased in the high-cholesterol group as compared to the control (0.56 and 0.34, respectively). Structural alterations in the disaccharide composition of galactosaminoglycans were also detected by HPLC, as the ratio of 6-sulphate to 4-sulphate disaccharides was increased in high-cholesterol animals (P < 0.05). Our results suggest that attenuation of decorin and biglycan expression might be an effective strategy to inhibit the first step in atherogenesis, although specific GAG structural modification associated with the development of vascular disease took place. Results emphasize the potential application of therapies based on vascular matrix remodelling to treat atherosclerosis.
Assuntos
Aterosclerose/prevenção & controle , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Dermatan Sulfato/metabolismo , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Hipercolesterolemia/fisiopatologia , Placa Aterosclerótica/prevenção & controle , Animais , Aorta/citologia , Aorta/metabolismo , Aterosclerose/fisiopatologia , Colesterol/sangue , Proteoglicanas de Sulfatos de Condroitina/química , Dermatan Sulfato/química , Dieta Aterogênica/efeitos adversos , Modelos Animais de Doenças , Glicosaminoglicanos/química , Cabras , Humanos , Hipercolesterolemia/metabolismo , Lipídeos/sangue , Masculino , Coelhos , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
INTRODUCTION AND HYPOTHESIS: This study aims to evaluate the effects of simulated birth trauma and vaginal and Cesarean delivery on glycosaminoglycans (GAGs) in the vagina of female rats. METHODS: One hundred ten rats were divided into six groups: A (control), B (vaginal trauma), C (Cesarean delivery), D (Cesarean delivery followed by vaginal trauma), E (vaginal delivery), and F (20th day of gestation). In each group, half of the animals were killed 4 days after the procedure (time 1) and 12 weeks later (time 2). GAGs were extracted, isolated, and identified by using agarose gel electrophoresis and quantified by densitometry. Statistical analysis was performed using Kruskal-Wallis and Mann-Whitney tests. RESULTS: We observed a significant decrease in total GAGs and dermatan sulfate (DS) at time 1. Evaluation at time 2 showed a significant increase in total GAGs, DS, and heparan sulfate. CONCLUSIONS: Levels of sulfated GAGs in the rat vagina are affected by delivery and simulated birth trauma.
Assuntos
Glicosaminoglicanos/metabolismo , Parto/metabolismo , Distúrbios do Assoalho Pélvico/metabolismo , Período Pós-Parto/metabolismo , Prenhez/metabolismo , Vagina/metabolismo , Animais , Cesárea , Dermatan Sulfato/metabolismo , Feminino , Heparitina Sulfato/metabolismo , Modelos Animais , Gravidez , Ratos , Ratos WistarRESUMO
PURPOSE: We evaluated extracellular matrix remodeling in human fetal and cryptorchidic gubernacula. METHODS: Gubernacula were obtained from 40 normal human fetuses aged 15-29 weeks postconception (WPC) and from 39 children aged 1.3-10 years who had been submitted to orchiopexy. Total collagen and glycosaminoglycan (GAG) concentrations were assessed as µg hydroxyproline and µg hexuronic acid per mg dry gubernacular tissue, respectively, and proportions of sulfated GAG were determined by agarose gel electrophoresis. RESULTS: Fetal age correlated with collagen (r = 0.77, P < 0.001) and GAG (r = -0.83, P < 0.01) concentrations, which varied from 10 to 50 µg/mg and 7.1-2.5 µg/mg, respectively. Collagen and GAG concentrations in cryptorchidic gubernacula varied from 80.0 to 120.0 µg/mg and from 0.8 to 1.0 µg/mg, respectively. These values did not correlate with patient's age, but when the testis was located more proximally, collagen content was lower. At 15-18 WPC, GAG consisted of 57.3% ± 13.5% chondroitin sulfate and 28.2% ± 10.1% dermatan sulfate, and at 25-28 WPC, 42.7% ± 8.7% and 71.8% ± 11.3%, respectively. GAG in postnatal gubernacula consisted mostly of dermatan sulfate. CONCLUSIONS: From the 15th to the 29th WPC, the extracellular matrix of the gubernaculum undergoes extensive remodeling and this may contribute to testicular descent. Cryptorchidic gubernacula are markedly more fibrous than the corresponding fetal tissue, change little after birth, and have a lower collagen concentration when the undescended testis is abdominal in position.
Assuntos
Criptorquidismo/metabolismo , Matriz Extracelular/metabolismo , Mesoderma/embriologia , Mesoderma/metabolismo , Testículo/embriologia , Testículo/metabolismo , Criança , Pré-Escolar , Colágeno/metabolismo , Criptorquidismo/cirurgia , Dermatan Sulfato/metabolismo , Desenvolvimento Fetal/fisiologia , Feto/metabolismo , Glicosaminoglicanos/metabolismo , Humanos , Lactente , Masculino , OrquidopexiaRESUMO
BACKGROUND: Previously, we have reported the presence of highly sulfated dermatans in solitary ascidians from the orders Phlebobranchia (Phallusia nigra) and Stolidobranchia (Halocynthia pyriformis and Styela plicata). Despite the identical disaccharide backbone, consisting of [â4IdoA(2S)ß-1â3GalNAcß-1â], those polymers differ in the position of sulfation on the N-Acetyl galactosamine, which can occur at carbon 4 or 6. We have shown that position rather than degree of sulfation is important for heparin cofactor II activity. As a consequence, 2,4- and 2,6-sulfated dermatans have high and low heparin cofactor II activities, respectively. In the present study we extended the disaccharide analysis of ascidian dermatan sulfates to additional species of the orders Stolidobranchia (Herdmania pallida, Halocynthia roretzi) and Phlebobranchia (Ciona intestinalis), aiming to investigate how sulfation evolved within Tunicata. In addition, we analysed how heparin cofactor II activity responds to dermatan sulfates containing different proportions of 2,6- or 2,4-disulfated units. RESULTS: Disaccharide analyses indicated a high content of disulfated disaccharide units in the dermatan sulfates from both orders. However, the degree of sulfation decreased from Stolidobranchia to Phlebobranchia. While 76% of the disaccharide units in dermatan sulfates from stolidobranch ascidians are disulfated, 53% of disulfated disaccharides are found in dermatan sulfates from phlebobranch ascidians. Besides this notable difference in the sulfation degree, dermatan sulfates from phlebobranch ascidians contain mainly 2,6-sulfated disaccharides whereas dermatan sulfate from the stolidobranch ascidians contain mostly 2,4-sulfated disaccharides, suggesting that the biosynthesis of dermatan sulfates might be differently regulated during tunicates evolution. Changes in the position of sulfation on N-acetylgalactosamine in the disaccharide [â4IdoA(2-Sulfate)ß-1â3GalNAcß-1â] modulate heparin cofactor II activity of dermatan sulfate polymers. Thus, high and low heparin cofactor II stimulating activity is observed in 2,4-sulfated dermatan sulfates and 2,6-sulfated dermatan sulfates, respectively, confirming the clear correlation between the anticoagulant activities of dermatan sulfates and the presence of 2,4-sulfated units. CONCLUSIONS: Our results indicate that in ascidian dermatan sulfates the position of sulfation on the GalNAc in the disaccharide [â4IdoA(2S)ß-1â3GalNAcß-1â] is directly related to the taxon and that the 6-O sulfation is a novelty apparently restricted to the Phlebobranchia. We also show that the increased content of [â4IdoA(2S)ß-1â3GalNAc(4S)ß-1â] disaccharide units in dermatan sulfates from Stolidobranchia accounts for the increased heparin cofactor II stimulating activity.
Assuntos
Dermatan Sulfato/química , Dermatan Sulfato/metabolismo , Cofator II da Heparina/metabolismo , Filogenia , Urocordados/metabolismo , Animais , Antitrombinas/química , Antitrombinas/metabolismo , Sequência de Carboidratos , Condroitina ABC Liase/metabolismo , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Evolução Molecular , Ácidos Hexurônicos/metabolismo , Humanos , Dados de Sequência Molecular , Tempo de Tromboplastina Parcial , Especificidade da Espécie , Urocordados/genéticaRESUMO
Decorin and biglycan proteoglycans play important roles in the organization of the extracellular matrix, and in the regulation of cell adhesion and migration. Given morphological and functional endothelial heterogeneity, information is needed regarding whether endothelial cells (ECs) from different vascular beds possess different profiles of proteoglycan constituents of the basement membranes. Here, we report that endothelia from different murine organs and EC lines derived thereof produce and secrete different patterns of proteoglycans. A faint colocalization between decorin and PECAM/CD31 was found on tissue sections from mouse heart, lung and kidney by immunofluorescence. Three EC lines derived from these organs produced decorin (100-kDa) and its core protein (45-kDa). Extracellular decorin recognition in culture supernatant was only possible after chondroitin lyase digestion suggesting that the core protein of secreted proteoglycan is more encrypted by glycosaminoglycans than the intracellular one. Heart and lung ECs were able to produce and release decorin. Kidney ECs synthesized the proteoglycan and its core protein but no secretion was detected in culture supernatants. Although biglycan production was recorded in all EC lines, secretion was almost undetectable, consistent with immunofluorescence results. In addition, no biglycan secretion was detected after EC growth supplement treatment, indicating that biglycan is synthesized, secreted and quickly degraded extracellularly by metalloproteinase-2. Low molecular-mass dermatan sulfate was the predominant glycosaminoglycan identified bound to the core protein. ECs from different vascular beds, with differences in morphology, physiology and cell biology show differences in the proteoglycan profile, extending their heterogeneity to potential differences in cell migration capacities.
Assuntos
Biglicano/metabolismo , Decorina/metabolismo , Células Endoteliais/metabolismo , Animais , Biomarcadores/metabolismo , Western Blotting , Linhagem Celular , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/metabolismo , Dermatan Sulfato/metabolismo , Células Endoteliais/citologia , Matriz Extracelular/metabolismo , Rim/citologia , Rim/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/citologia , Miocárdio/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismoRESUMO
BACKGROUND: The pathogenesis of abdominal aortic aneurysm is associated with changes of several components of arterial wall. Vascular glycosaminoglycans contribute to the non-thrombogenic activity of blood vessels. We investigated whether modifications of glycosaminoglycans in human abdominal aortic aneurysm affect their anticoagulant properties. METHODS: Glycosaminoglycans were extracted from abdominal aortic aneurysms (n=11) derived from reconstitution surgeries, human abdominal aortas (n=9) from normal organ transplant donors and from preserved (n=10) and atherosclerotic (n=17) segments obtained from autopsy of an old patient. Glycosaminoglycan composition, concentration and anticoagulant activity were determined. RESULTS: Glycosaminoglycans extracted from aneurysms have a more potent anticoagulant activity than those from normal arteries of young adults, mostly due to a relative enrichment of dermatan sulfate, which potentiates heparin cofactor II inhibition of thrombin. Arterial segments of aged patient with severe atherosclerosis showed a glycosaminoglycan composition similar to aneurysms samples. Glycosaminoglycans extracted from these regions showed also a more potent heparin cofactor II-dependent anticoagulant activity than lesion-free areas due to the relative enrichment of dermatan sulfate. CONCLUSION: The anticoagulant activity from abdominal aortic aneurysms is preserved. No modifications particular to the aneurysms were dissociated from those observed in atherosclerosis.
Assuntos
Aneurisma da Aorta Abdominal/metabolismo , Aterosclerose/metabolismo , Coagulação Sanguínea , Dermatan Sulfato/metabolismo , Heparitina Sulfato/metabolismo , Adolescente , Adulto , Idoso , Envelhecimento/metabolismo , Aorta Abdominal/metabolismo , Aorta Abdominal/fisiologia , Aorta Abdominal/fisiopatologia , Aneurisma da Aorta Abdominal/fisiopatologia , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Humanos , Pessoa de Meia-Idade , Adulto JovemRESUMO
OBJECTIVE: The objective was to determine the sulfated glycosaminoglycans (GAGs) of the extracellular matrix (ECM) in pre- and postmenopausal women. STUDY DESIGN: Periurethral tissue was obtained from 44 consecutive women who underwent surgery for urinary incontinence, for pelvic organ prolapse, or for other gynecologic benign conditions. Biopsy specimens were assessed by biochemical methods to characterize and quantify sulfated GAG. Measurements were made of total GAG, chondroitin sulfate, dermatan sulfate and of heparan sulfate. Data were compared using the t-test. RESULTS: Patients were divided into two groups (pre- and postmenopausal groups) and dermatan sulfate was the most predominant glycosaminoglycan. Postmenopausal women had significantly less total sulfated glycosaminoglycans (p<0.01), dermatan sulfate (p<0.01) and chondroitin sulfate (p<0.05) than premenopausal women. We did not observe any differences in heparan sulfate. CONCLUSIONS: Postmenopausal women showed quantitative differences in the biochemical characteristics of the ECM in periurethral tissue by analysis of sulfated GAG.
Assuntos
Tecido Conjuntivo/metabolismo , Glicosaminoglicanos/metabolismo , Pós-Menopausa/metabolismo , Pré-Menopausa/metabolismo , Uretra/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Sulfatos de Condroitina/metabolismo , Tecido Conjuntivo/patologia , Dermatan Sulfato/metabolismo , Matriz Extracelular/metabolismo , Feminino , Heparitina Sulfato/metabolismo , Humanos , Pessoa de Meia-Idade , Uretra/patologiaRESUMO
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.
Assuntos
Glicosaminoglicanos/análise , Uretra/química , Incontinência Urinária por Estresse/metabolismo , Prolapso Uterino/metabolismo , Adulto , Idoso , Análise de Variância , Sulfatos de Condroitina/análise , Sulfatos de Condroitina/metabolismo , Dermatan Sulfato/análise , Dermatan Sulfato/metabolismo , Feminino , Glicosaminoglicanos/metabolismo , Humanos , Pessoa de Meia-Idade , Uretra/metabolismo , Incontinência Urinária por Estresse/patologia , Incontinência Urinária por Estresse/cirurgia , Prolapso Uterino/patologia , Prolapso Uterino/cirurgia , Adulto JovemRESUMO
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.
Assuntos
Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Adulto Jovem , Glicosaminoglicanos/análise , Uretra/química , Incontinência Urinária por Estresse/metabolismo , Prolapso Uterino/metabolismo , Análise de Variância , Sulfatos de Condroitina/análise , Sulfatos de Condroitina/metabolismo , Dermatan Sulfato/análise , Dermatan Sulfato/metabolismo , Glicosaminoglicanos/metabolismo , Uretra/metabolismo , Incontinência Urinária por Estresse/patologia , Incontinência Urinária por Estresse/cirurgia , Prolapso Uterino/patologia , Prolapso Uterino/cirurgia , Adulto JovemRESUMO
The electrogenic tissue of the electric eel Electrophorus electricus (L.) is distributed in three well-defined electric organs, the Main electric organ, Sach's organ and Hunter's organ. Sulfated glycosaminoglycan (GAG) composition was characterized in the three electric organs of the electric eel. Sulfated GAGs were analyzed in the electric organs using metachromatic staining, biochemical analysis including electrophoresis before and after specific enzymatic or chemical degradations, and immunostaining with an antibody against chondroitin sulfate (CS). Our results showed in the three electric organs that CS was the main sulfated GAG species detected, accompanied by small and diminutive amounts of CS/dermatan sulfate hybrid chains and heparan sulfate (HS), respectively. However, HS was not detected in the Sach's organ. CS was predominantly detected in the innervated membrane face of the electroplaques in the three electric organs. Our findings extend previous observations on the GAG composition in the electric organs of E. electricus and provide new information regarding the tissue distribution and location of CS.
Assuntos
Sulfatos de Condroitina/metabolismo , Órgão Elétrico/metabolismo , Electrophorus/metabolismo , Animais , Dermatan Sulfato/metabolismo , Glicosaminoglicanos/metabolismo , Heparitina Sulfato/metabolismo , Imuno-HistoquímicaRESUMO
Decorin is an extracellular matrix dermatan sulfate/chondroitin sulfate proteoglycan found in a variety of vertebrate species. In the extracellular matrix of mammals, decorin interacts with fibrillar collagen and regulates its morphology. We report here the occurrence and distribution of collagen type I and the peptide, CEASGIGPEVPDDRD, which is present in the human decorin proteoglycan, in the extracellular matrix of different tissues of the primitive invertebrate chordate Styela plicata. The content of collagen was estimated by hydroxyproline, and its distribution in the tissues by histochemistry. Collagen was detected biochemically in intestine, heart, pharynx and mantle, occurring in higher amounts in the heart, followed by pharynx, mantle and intestine. Histochemical analysis with Sirius red indicates that collagen is present in the extracellular matrix of intestine and pharynx. Further ultrastructural immuno-gold assays using polyclonal antibodies raised against the decorin-specific peptide CEASGIGPEVPDDRD and collagen type I showed a co-localization of these molecules. These data suggest the occurrence of a protein containing a decorin-like peptide sequence, which may be interacting with fibrillar collagen in this primitive chordate.
Assuntos
Colágeno Tipo I/metabolismo , Proteínas da Matriz Extracelular/química , Proteoglicanas/química , Urocordados/metabolismo , Animais , Decorina , Dermatan Sulfato/metabolismo , Guanidina/farmacologia , Imuno-Histoquímica , Microscopia Eletrônica , Ligação Proteica , Estrutura Terciária de Proteína , Distribuição Tecidual , Extratos de TecidosRESUMO
BACKGROUND: Heparin exerts beneficial effects in different experimental models of nephropathy, as observed by the preservation of the structural morphology of the kidney after heparin therapy. Here we investigate molecular and cellular events involved in the protective effects of heparin in the progression of renal disease after unilateral ureteral obstruction. METHODS: Thirty-six rats were divided into six groups: group C (control) was not subjected to any surgical manipulation; group S (sham) was subjected to surgical manipulation but without ureteral ligation; group UUO was subjected to ureteral obstruction and received no treatment; group UUO + S was subjected to ureteral obstruction and received saline subcutaneously (s.c.) once daily; group UUO + H was subjected to ureteral obstruction and received low molecular weight heparin (LMW-Hep; 4 mg/kg) s.c. once daily; and group C + H was not subjected to any surgical manipulation and received LMW-Hep (4 mg/kg) s.c. once daily. After 14 days, the content of collagen, fibronectin, total glycosaminoglycans (GAGS), chondroitin sulfate/dermatan sulfate proteoglycans (CS/DSPGs), transforming growth factor-beta (TGF-beta) and cellular infiltration were determined in the kidneys by immunohistochemical and biochemical techniques. RESULTS: Collagen, fibronectin, total GAGS, CS/DSPGs, TGF-beta and cellular infiltration increased significantly in group UUO. LMW-Hep treatment reduced collagen, fibronectin and TGF-beta, but induced an increase in the content of total GAGS, CS/DSPGs and macrophage infiltration in group UUO + H when compared with group UUO. CONCLUSIONS: LMW-Hep diminishes fibrosis in obstructed kidneys by downregulating the synthesis of collagen, fibronectin and TGF-beta. The mechanisms underlying the overproduction of CS/DSPGs and the increase in cellular infiltration upon LMW-Hep administration remain to be elucidated.
Assuntos
Biomarcadores/análise , Heparina de Baixo Peso Molecular/farmacologia , Obstrução Ureteral/tratamento farmacológico , Animais , Biópsia por Agulha , Movimento Celular/efeitos dos fármacos , Condroitina/metabolismo , Proteoglicanas de Sulfatos de Condroitina/efeitos dos fármacos , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Colágeno/efeitos dos fármacos , Colágeno/metabolismo , Dermatan Sulfato/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Fibronectinas/efeitos dos fármacos , Fibronectinas/metabolismo , Imuno-Histoquímica , Rim/efeitos dos fármacos , Rim/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Masculino , Probabilidade , Distribuição Aleatória , Ratos , Ratos Wistar , Valores de Referência , Sensibilidade e Especificidade , Fator de Crescimento Transformador beta/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima , Obstrução Ureteral/patologiaRESUMO
OBJECTIVE: The objective was to determine sulfated glycosaminoglycans (GAG) of the extracellular matrix (ECM) in women with and without stress urinary incontinence according to genital prolapse stage. STUDY DESIGN: Periurethral tissue was obtained from 30 women who underwent surgery for urinary incontinence, for pelvic organ prolapse, or for other benign gynecologic conditions. Biopsy specimens were assessed by biochemical methods to characterize and quantify sulfated glycosaminoglycans. Measurements were made of total glycosaminoglycans, chondroitin sulfate, dermatan sulfate, and of heparan sulfate. Data were compared using the t-test. RESULTS: In two groups, dermatan sulfate was the most predominant glycosaminoglycan. Women with stress urinary incontinence had significantly more total sulfated glycosaminoglycans (p<0.05) and dermatan sulfate (p<0.05) than women without stress urinary incontinence. We did not observe any differences in chondroitin sulfate and heparan sulfate. CONCLUSIONS: Women with stress urinary incontinence showed quantitative and qualitative differences in the biochemical characteristics of the extracellular matrix in periurethral tissue by analysis of sulfated glycosaminoglycans, according to genital prolapse stage.
Assuntos
Dermatan Sulfato/metabolismo , Glicosaminoglicanos/metabolismo , Uretra/metabolismo , Incontinência Urinária por Estresse/metabolismo , Prolapso Uterino/metabolismo , Adulto , Idoso , Biomarcadores/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Índice de Gravidade de Doença , Incontinência Urinária por Estresse/patologia , Prolapso Uterino/patologiaRESUMO
Sulfated polysaccharides are capable of binding with proteins at several levels of specificity. As highly acidic macromolecules, they can bind non-specifically to any basic patch on a protein surface at low ionic strength, and such interactions are not likely to be physiologically significant. On the other hand, several systems have been identified in which very specific substructures of sulfated polysaccharides confer high affinity for particular proteins; the best-known example of this is the pentasaccharide in heparin with high affinity for antithrombin, but other examples may be taken from the study of marine invertebrates: the importance of the fine structure of dermatan sulfate (DS) to its interaction with heparin cofactor II (HCII), and the involvement of sea urchin egg-jelly fucans in species specific fertilization. A third, intermediate, kind of specific interaction is described for the cell-surface glycosaminoglycan heparan sulfate (HS), in which patterns of sulfate substitution can show differential affinities for cytokines, growth factors, and morphogens at cell surfaces and in the intracellular matrix. This complex interplay of proteins and glycans is capable of influencing the diffusion of such proteins through tissue, as well as modulating cellular responses to them.
Assuntos
Polissacarídeos/metabolismo , Proteínas/metabolismo , Sulfatos/metabolismo , Animais , Antitrombinas/metabolismo , Dermatan Sulfato/química , Dermatan Sulfato/metabolismo , Interações Medicamentosas , Substâncias de Crescimento/metabolismo , Heparina/química , Heparina/metabolismo , Polissacarídeos/química , Proteínas/química , Ouriços-do-Mar , Sulfatos/químicaRESUMO
Os polissacarídeos sulfatados são capazes de se ligar às proteínas com diferentes níveis de especificidade. São macromoléculas altamente ácidas que podem se ligar de forma inespecífica a qualquer domínio básico da superfície de uma proteína em soluções com baixa força iônica, contudo tais interações não parecem ser fisiologicamente significativas. Por outro lado, foram identificados vários sistemas nos quais componentes estruturais muito específicos dos polissacarídeos sulfatados conferem alta afinidade para algumas proteínas. O exemplo mais conhecido é o pentassacarídeo da heparina com alta afinidade pela antitrombina. Outros exemplos podem ser observados no estudo de invertebrados marinhos, tais como a importância da estrutura fina do dermatam sulfato para sua interação com o cofator II da heparina e o envolvimento defucanas sulfatadas encontradas no gel que envolve osóvulos dos ouriços-do-mar na espécie especificidade da fertilização. Um terceiro exemplo de interação específica é aquele descrito para o glicosaminoglicano heparam sulfato encontrado na superfície celular. Neste caso, o padrão de sulfatação pode determinar diferentes afinidades do carboidrato por citoquinas, fatores de crescimento e outras proteínas encontradas na superfície celular e na matriz extracelular. Estas interações complexas entre proteínas e carboidratos são capazes de influenciar a difusão das proteínas através dos tecidos, assim como modelar a resposta celular a estas moléculas.
Assuntos
Animais , Polissacarídeos/metabolismo , Proteínas/metabolismo , Sulfatos/metabolismo , Antitrombinas/metabolismo , Interações Medicamentosas , Dermatan Sulfato/química , Dermatan Sulfato/metabolismo , Substâncias de Crescimento/metabolismo , Heparina/química , Heparina/metabolismo , Polissacarídeos/química , Proteínas/química , Ouriços-do-Mar , Sulfatos/químicaRESUMO
The role of different glycosaminoglycan species from the vessel walls as physiological antithrombotic agents remains controversial. To further investigate this aspect we extracted glycosaminoglycans from human thoracic aorta and saphenous vein. The different species were highly purified and their anticoagulant and antithrombotic activities tested by in vitro and in vivo assays. We observed that dermatan sulfate is the major anticoagulant and antithrombotic among the vessel wall glycosaminoglycans while the bulk of heparan sulfate is a poorly sulfated glycosaminoglycan, devoid of anticoagulant and antithrombotic activities. Minor amounts of particular a heparan sulfate (< 5% of the total arterial glycosaminoglycans) with high anticoagulant activity were also observed, as assessed by its retention on an antithrombin-affinity column. Possibly, this anticoagulant heparan sulfate originates from the endothelial cells and may exert a significant physiological role due to its location in the interface between the vessel wall and the blood. In view of these results we discuss a possible balance between the two glycosaminoglycan-dependent anticoagulant pathways present in the vascular wall. One is based on antithrombin activation by the heparan sulfate expressed by the endothelial cells. The other, which may assume special relevance after vascular endothelial injury, is based on heparin cofactor II activation by the dermatan sulfate proteoglycans synthesized by cells from the subendothelial layer.
Assuntos
Anticoagulantes/metabolismo , Dermatan Sulfato/metabolismo , Endotélio Vascular/metabolismo , Fibrinolíticos/metabolismo , Cofator II da Heparina/fisiologia , Anticoagulantes/isolamento & purificação , Aorta Torácica/citologia , Aorta Torácica/metabolismo , Dermatan Sulfato/isolamento & purificação , Fibrinolíticos/isolamento & purificação , Heparitina Sulfato/química , Heparitina Sulfato/isolamento & purificação , Heparitina Sulfato/metabolismo , Humanos , Veia Safena/citologia , Veia Safena/metabolismo , Trombose/metabolismoRESUMO
Mucopolysaccharidosis I (MPS I) is a lysosomal disorder characterized by a deficiency of the enzyme alpha-L: -iduronidase (IDUA), which is responsible for the degradation of glycosaminoglycans (GAGs). This deficiency leads to the accumulation of dermatan and heparan sulphate in lysosomes. Presently available treatments include bone marrow transplantation and enzyme replacement therapies, both of which are limited in their effects. In this work, knockout (KO) MPS I mice were treated with a nonviral vector containing the human IDUA cDNA. KO mice were transfected by hydrodynamic injection of pRIDUA in the caudal vein (i.v., n = 3) or by intraperitoneal injection of pRIDUA/Superfect complexes (i.p., n = 3). GAG concentration and IDUA activity were analysed in the kidneys, spleen, lungs, brain and liver. The expression of IDUA in the organs of i.v.- and i.p.-treated mice was also analysed by real-time reverse-transcription (RT) PCR and compared by relative quantification. The concentration of GAGs in the organs differed between KO and wild-type mice. In the spleen and liver, GAG levels were lower in i.v.- and i.p.-treated KO mice than in control nontreated animals. Real-time RT-PCR showed that the transgene is expressed in all the analysed organs of i.p.- and i.v.-treated KO mice. Enzyme activity was similarly observed in all the organs analysed. Our data suggest that this kind of transfection may be a useful tool for studies of nonviral protocols for gene therapy of MPS.