RESUMEN
PURPOSE: To characterize the vitreous intrinsic proteoglycans, investigate their dynamics, and examine their role in the supramolecular organization of the vitreous. METHODS: Vitreous from normal rabbits was collected and processed for observation with the transmission electron microscope after treatment with glycosidases. Also, rabbits were injected intravitreally with [35S]-sodium sulfate and sacrificed at several time intervals after the injection. Proteoglycans (PGs) were assayed in the vitreous supernatant or in whole samples extracted with guanidine hydrochloride by polyacrylamide or agarose gel electrophoresis, followed respectively by fluorography or autoradiography, and ion-exchange chromatography and gel-filtration chromatography, combined with glycolytic treatment of the samples. The sulfated glycosaminoglycans (GAGs) were characterized by agarose gel electrophoresis after treating vitreous samples with protease and specific glycosidases. RESULTS: The electron microscopic study revealed a network with hyaluronic acid (HA) as thin threads coating and connecting collagen fibrils. The elimination of the HA coat showed chondroitin sulfate granules (8-25 nm) arranged at regular intervals on the fibril surface. The chondroitinase ABC digestion, besides removing the granules, also caused the formation of thicker bundles of the collagen fibrils. The PG and GAG analysis indicated that there are three renewable PGs in the vitreous (e.g., one heparan- and two chondroitin-sulfate ones). CONCLUSIONS: At least one of the chondroitin sulfate PGs is involved in the interactions that occur in the vitreous structure, mainly by providing adequate spacing between the collagen fibrils, a condition that is probably required for the transparency of the vitreous.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/análisis , Cuerpo Vítreo/química , Animales , Proteoglicanos Tipo Condroitín Sulfato/aislamiento & purificación , Proteoglicanos Tipo Condroitín Sulfato/ultraestructura , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Colágeno/análisis , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Glicósido Hidrolasas/farmacología , Proteoglicanos de Heparán Sulfato/análisis , Proteoglicanos de Heparán Sulfato/aislamiento & purificación , Ácido Hialurónico/análisis , Microscopía Electrónica , Conejos , Cuerpo Vítreo/efectos de los fármacos , Cuerpo Vítreo/ultraestructuraRESUMEN
It has been suggested that airway remodelling is responsible for the persistent airway obstruction and decline in lung function observed in some asthmatic patients. The small airways are thought to contribute significantly to this functional impairment. Proteoglycans (PGs) are important components of the extracellular matrix (ECM) in the lungs. Besides controlling biophysical properties of the ECM, they play important roles in the regulation of some cytokines. Increased subepithelial PG deposition in the airways of mild asthmatics has been reported. However, there are no data on the PG content in small airways in asthma. This study has compared the content and distribution of PGs in large and small airways of patients who died of asthma with those in control lungs. Immunohistochemistry and image analysis were used to determine the content of lumican, decorin, biglycan, and versican in large (internal perimeter >6 mm) and small (internal perimeter < or =6 mm) airways of 18 patients who had died of asthma (A) and ten controls (C). The results were expressed as PG area (microm2)/epithelial basement membrane length (microm). The main differences between asthmatics and controls were observed in the small airways. There was a significant decrease in decorin and lumican contents in the external area of small airways in asthmatics (decorin: A = 1.05 +/- 0.27 microm, C = 3.97 +/- 1.17 microm, p = 0.042; lumican: A = 1.97 +/- 0.37 microm, C = 5.66 +/- 0.99 microm, p = 0.002). A significant increase in versican content in the internal area of small and large airways in asthmatics was also observed (small: A = 7.48 +/- 0.84 microm, C = 5.16 +/- 0.61 microm, p = 0.045; large: A = 18.38 +/- 1.94 microm, C = 11.90 +/- 2.86 microm, p = 0.028). The results show that PGs are differentially expressed in the airways of fatal asthma and may contribute to airway remodelling. These data reinforce the importance of the small airways in airway remodelling in asthma.
Asunto(s)
Pulmón/química , Proteoglicanos/análisis , Estado Asmático/metabolismo , Adolescente , Adulto , Anciano , Autopsia , Proteoglicanos Tipo Condroitín Sulfato/análisis , Decorina , Proteínas de la Matriz Extracelular , Femenino , Humanos , Técnicas para Inmunoenzimas , Sulfato de Queratano/análisis , Lectinas Tipo C , Lumican , Pulmón/patología , Masculino , Persona de Mediana Edad , Estado Asmático/patología , VersicanosRESUMEN
Preparation for embryo implantation requires extensive adaptation of the uterine microenvironment. This process consists of cell proliferation and cell differentiation resulting in the transformation of endometrial fibroblasts into a new type of cell called decidual cell. In the present study, we followed the space-time distribution of versican and hyaluronan (HA) in different tissues of the uterus before and after embryo implantation. Fragments of mouse uteri obtained on the fourth, fifth, sixth and seventh days of pregnancy were fixed in Methacarn, embedded in Paraplast and cut into 5-microm thick sections. HA was detected using a biotinylated fragment of the proteoglycan aggrecan, which binds to this glycosaminoglycan with high affinity and specificity. Versican was detected by a polyclonal antibody. Both reactions were developed by peroxidase methods. Before embryo implantation, both HA and versican were present in the endometrial stroma. However, after embryo implantation, HA disappeared from the decidual region immediately surrounding the implantation chamber, whereas versican accumulated in the same region. The differences observed in the expression of HA and versican suggest that both molecules may participate in the process of endometrial decidualization and/or embryo implantation.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/análisis , Implantación del Embrión/fisiología , Ácido Hialurónico/análisis , Útero/química , Animales , División Celular , Decidua/citología , Decidua/metabolismo , Femenino , Lectinas Tipo C , Ratones , Embarazo , Preñez , Células del Estroma , VersicanosRESUMEN
Remodelling of the extracellular matrix (ECM) occurs during decidualization of the endometrium in mice. Previously we have documented the appearance of large-diameter collagen fibrils around mature decidual cells between day 5 and day 7 of pregnancy. Proteoglycans are important in the regulation of collagen fibrillogenesis, and the present study analysed four members (decorin, biglycan, lumican and fibromodulin) of the family of small leucine-rich proteoglycans (SLRPs) in the uterus from day 1 to day 7 of pregnancy. Decorin was present together with lesser amounts of lumican in the stroma before the onset of decidualization, whereas biglycan and fibromodulin were almost absent. Biglycan and, less significantly, lumican were expressed in decidualized regions of the endometrium, but decorin was absent. Fibromodulin was weakly expressed in the non-decidualized stroma, but only after implantation. Decorin and lumican were strongly expressed in the undifferentiated interimplantation site stroma, whereas biglycan and fibromodulin were expressed only weakly. These results indicate that the SLRP profile of the uterine ECM alters with differentiation of endometrial stromal cells. The large decidual collagen fibrils are thought to arise by lateral association of smaller diameter fibrils. As decorin has been shown to inhibit lateral association of collagen fibrils, its disappearance between day 2 and day 5 of pregnancy may be a prerequisite for the formation of large fibrils in decidua in mice.
Asunto(s)
Proteínas de la Matriz Extracelular , Miometrio/química , Preñez/metabolismo , Proteoglicanos/análisis , Animales , Biglicano , Proteínas Portadoras/análisis , Proteoglicanos Tipo Condroitín Sulfato/análisis , Decorina , Implantación del Embrión , Desarrollo Embrionario , Femenino , Fibromodulina , Edad Gestacional , Técnicas para Inmunoenzimas , Sulfato de Queratano/análisis , Lumican , Ratones , Ratones Endogámicos , EmbarazoRESUMEN
After arriving at the limb bud, migrating myogenic precursor cells express transcription factors responsible for the induction of terminal skeletal muscle differentiation. One such factor is myogenin, a member of the basic helix-loop-helix family, known to activate the expression of muscle-specific genes. The extracellular signals involved in activating the myogenic program in the muscle precursor cells that reach the limb in vivo are not known. However, in vitro, it has been shown that proteoglycans, macromolecules composed of a core protein and glycosaminoglycan chains, modulate the triggering of myogenin activity. To understand the role of proteoglycans during limb muscle development, we assessed the synthesis of proteoglycans in limb bud explants at 10.5 days post coitum, when migrating cells arrive, evaluated the expression and nature of these macromolecules during in vivo early limb bud formation, and determined the colocalization of myoblasts expressing myogenin with specific proteoglycans. We found that the expression of myogenin was temporally and spatially coincident with the expression of syndecan-3 and decorin, two essential proteoglycans in the modulation of skeletal muscle differentiation. This article is the first report of myogenic activation and proteoglycan expression during limb muscle formation.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/genética , Regulación del Desarrollo de la Expresión Génica , Proteoglicanos de Heparán Sulfato/genética , Músculo Esquelético/embriología , Animales , Linaje de la Célula/fisiología , Proteoglicanos Tipo Condroitín Sulfato/análisis , Decorina , Proteínas de la Matriz Extracelular , Femenino , Proteoglicanos de Heparán Sulfato/análisis , Esbozos de los Miembros/citología , Esbozos de los Miembros/embriología , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Ratones , Músculo Esquelético/química , Músculo Esquelético/citología , Embarazo , Proteoglicanos/análisis , Proteoglicanos/genética , ARN Mensajero/análisis , Células Madre/química , Células Madre/fisiología , Sindecano-3RESUMEN
Aortic dissections (AD) are characterized by the separation of the artery into two sheets, possibly due to fragility of the vessel wall. A mucoid histological pattern, imparted to the tissues mainly by hyaluronan and proteoglycans, can be seen in "cysts" and, in chronic cases, in a band of repair tissue. We studied the localization of hyaluronan, versican, decorin and biglycan in situ in aortas of 21 patients with recent AD, 8 with chronic AD and in 15 control cases. None of these substances was increased in the areas of mucoid "cysts" that possibly contain anomalous material. Similar distributions were seen in normal and dissected aortas: versican and hyaluronan were more prominent in the external half of the medial layer where the dissection usually occurs. Since these molecules play a role in resistance to compression, disorders not detected by our method may be involved in aortic dissection. Hyaluronan was seen adjacent to fibrin at the dissection tear, probably as an early wound repair phenomenon. Biglycan, hyaluronan and mostly versican are seen during advanced repairing. The mucoid deposits may represent various compounds which reflect different disorders in vascular biology.
Asunto(s)
Aorta/química , Aneurisma de la Aorta/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/análisis , Dermatán Sulfato/análisis , Ácido Hialurónico/análisis , Enfermedad Aguda , Adulto , Anciano , Aorta/patología , Aneurisma de la Aorta/patología , Biglicano , Enfermedad Crónica , Proteínas de la Matriz Extracelular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteoglicanos/análisisRESUMEN
We have analyzed the immunohistochemical expression of chondroitin sulfate proteoglycan (CSPG), fibronectin (FN), laminin (LN), tenascin (TN), and glial fibrillary acidic protein (GFAP) along the anterior commissure (AC) of hamster embryos (n=175; from embryonic day (E)12 to E16). Frozen sections were cut at different planes from embryonic brains between E12 and E16, treated for immunohistochemistry, and observed under epifluorescence microscopy. During the pre-crossing stage (E12-E13), CSPG was expressed as a sagittal stratum between the interhemispheric fissure and the prospective AC region. TN appeared rostral to the third ventricle and along the medial subventricular zone of the lateral ventricles. LN and FN both presented a faint expression, and GFAP was not detected. Although AC axons started crossing the midline region (E13.5-E14), CSPG, FN, LN, and, much less intensely, GFAP circumscribed the AC bundle, forming a tunnel through which AC fibers elongate. TN was no longer seen at the midplane but remained visible laterally. During the post-crossing stage (E14.5-E16), CSPG and TN were no longer seen at the midline, although both could be observed between the AC limbs, seeming to form boundaries for AC lateral growth. LN and FN were then absent near the AC bundle. During this late stage, GFAP expression became most intense, forming a distinct tunnel around the AC. We have shown that the expression of extracellular matrix molecules and GFAP follow a time- and space-regulated course related to AC development, plausibly representing influential factors for growth and guidance of commissural fibers.
Asunto(s)
Axones/fisiología , Corteza Cerebral/embriología , Cuerpo Calloso/embriología , Cricetinae/embriología , Animales , Axones/química , Corteza Cerebral/química , Corteza Cerebral/citología , Proteoglicanos Tipo Condroitín Sulfato/análisis , Cuerpo Calloso/química , Cuerpo Calloso/citología , Femenino , Técnica del Anticuerpo Fluorescente , Proteína Ácida Fibrilar de la Glía/análisis , Embarazo , Tenascina/análisisRESUMEN
Heparan sulphate proteoglycan is the predominant proteoglycan synthesized by the parenchymal cells of the rat submandibular gland. A polyclonal antibody was used to localize this proteoglycan in the adult rat submandibular gland. Localization was accomplished by indirect immunoperoxidase cytochemistry at the light and electron microscopic levels. Heparan sulphate proteoglycan was localized in a continuous, linear pattern in the lamina densa of the basement membrane surrounding all of the epithelial components of the gland as well as the basement membrane of the capillaries and small arterioles in the glandular stroma. In addition, heparan sulphate proteoglycan was seen in vesicles and pits along the acinar cell basal plasmalemma adjacent to the basement membrane and in the endoplasmic reticulum and Golgi apparatus of the acinar cells.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/análisis , Heparitina Sulfato/análisis , Glándula Submandibular/química , Animales , Arteriolas/química , Membrana Basal/química , Capilares/química , Membrana Celular/química , Proteoglicanos Tipo Condroitín Sulfato/inmunología , Retículo Endoplásmico/química , Epitelio/química , Aparato de Golgi/química , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/inmunología , Técnicas para Inmunoenzimas , Masculino , Microscopía Inmunoelectrónica , Ratas , Ratas Endogámicas , Glándula Submandibular/ultraestructuraRESUMEN
Rats infected with the helminth Nippostrongylus brasiliensis were injected i.p. with 2 mCi of [35S] sulfate on days 13, 15, 17, and 19 after infection. The intestines were removed from animals on day 20 or 21 after infection, the intestinal cells were obtained by collagenase treatment and mechanical dispersion of the tissue, and the 35S-labeled mucosal mast cells (MMC) were enriched to 60 to 65% purity by Percoll centrifugation. The cell-associated 35S-labeled proteoglycans were extracted from the MMC-enriched cell preparation by the addition of detergent and 4 M guanidine HCl and were partially purified by density gradient centrifugation. The isolated proteoglycans were of approximately 150,000 m.w., were resistant to pronase degradation, and contained highly sulfated chondroitin sulfate side chains. Analysis by high-performance liquid chromatography of chondroitinase ABC-treated 35S-labeled proteoglycans from these rat MMC revealed that the chondroitin sulfate chains consisted predominantly of disaccharides with the disulfated di-B structure (IdUA-2SO4----GalNAc-4SO4) and disaccharides with the monosulfated A structure (G1cUA----GalNAc-4SO4). The ratio of disaccharides of the di-B to A structure ranged from 0.4 to 1.6 in three experiments. Small amounts of chondroitin sulfate E disaccharides (GlcUA----GalNAc-4,6-diSO4) were also detected in the chondroitinase ABC digests of the purified rat MMC proteoglycans, but no nitrous acid-susceptible heparin/heparan sulfate glycosaminoglycans were detected. The presence in normal mammalian cells of chondroitin sulfate proteoglycans that contain such a high percentage of the unusual disulfated di-B disaccharide has not been previously reported. The rat intestinal MMC proteoglycans are the first chondroitin sulfate proteoglycans that have been isolated from an enriched population of normal mast cells. They are homologous to the chondroitin sulfate-rich proteoglycans of the transformed rat basophilic leukemia-1 cell and the cultured interleukin 3-dependent mouse bone marrow-derived mast cell, in that these chondroitin sulfate proteoglycans as well as rat serosal mast cell heparin proteoglycans are all highly sulfated, protease-resistant proteoglycans.