Ultrastructural, immunohistochemical and biochemical analysis of glycosaminoglycans and proteoglycans in the mouse pubic symphysis during pregnancy

During pregnancy, an interpubic ligament is formed in the mouse pubic symphysis. In late stages, this ligament undergoes "relaxation" to allow proper delivery, which is expected on the 19th day. Proteoglycans and hyaluronic acid play an important role in the remodeling of the extracellular matrix in these tissues. Glycosaminoglycans and proteoglycans were studied by electron microscopic, immunohistochemical and biochemical methods in samples of mouse pubic symphysis from the 12th to 18th day of pregnancy. At the ultrastructural level, using cuprolinic blue and enzymatic digestion by chondroitin lyases, two types of proteoglycan filaments were observed in the fibrocartilage on the 12th day, as well as in D 15, D 17 and D 18 pubic ligaments. The only sulfated glycosaminoglycan in these filaments was chondroitin sulfate, as shown by chondroitin lyase treatment. Their electrophoretic mobility, before and after enzymatic degradation, corroborated this inference. The ratio of chondroitin sulfate/dry weight of symphysis showed two phases of increase: between D12 and D 15, and between D 17 and D 18. We suggest that the first corresponds mainly to an increase in decorin when the ligament is formed, and the second to versican, during "relaxation". Versican and hyaluronic acid, working as water holding molecules would be responsible for the hydration of the ligament at the end of pregnancy, allowing an increase in resiliency. The presence of hyaluronic acid was confirmed by labeling with HA-probe in the perichondrium, fibrocartilage and ligament. The role of collagen fibers as physical restrictors of the complete expansion of glycosaminoglycans and hyaluronic acid in tissue is discussed.     

High iNOS mRNA and protein localization during late pregnancy suggest a role for nitric oxide in mouse pubic symphysis relaxation

Remodeling and relaxation of the mouse pubic symphysis (PS) are central events in parturition. The mouse PS remodels in a hormone-controlled process that involves the modification of the fibrocartilage into an interpubic ligament (IpL), followed by its relaxation prior to parturition. It is recognized that nitric oxide synthase (NOS) and consequently nitric oxide (NO) generation play important roles in extracellular matrix modification, and may promote cytoskeleton changes that contribute to the remodeling of connective tissue, which precedes the onset of labor. To our knowledge, no studies thus far have investigated inducible nitric oxide synthase (iNOS) expression, protein localization, and NO generation in the mouse PS during pregnancy. In this work, we used a combination of the immunolocalization of iNOS, its relative mRNA expression, and NO production to examine the possible involvement of iNOS in remodeling and relaxation of the mouse IpL during late pregnancy. The presence of iNOS was observed in chondrocytes and fibroblast-like cells in the interpubic tissues. In addition, iNOS mRNA and NO production were higher during preterm labor on Day 19 of pregnancy (D19) than NO production on D18 or in virgin groups. The significant increase in iNOS mRNA expression and NO generation from the partially relaxed IpL at D18 to the completely relaxed IpL at D19 may indicate that NO plays an important role in late pregnancy during relaxation of the mouse IpL.     

Recovery of the pubic symphysis on primiparous young and multiparous senescent mice at postpartum

It has been observed that parturition has a significant effect on female skeletal architecture and that age alters musculoskeletal tissues and their functions. We therefore hypothesized that multiparity affects the recovery of the pubic symphysis in senescent mice at postpartum and the morphology of the interpubic tissues. The pubic symphysis of primiparous young, virgin senescent (VS) and multiparous senescent (MS) Swiss mice was examined by light microscopy, transmission electron microscopy, morphometric analysis and immunohistochemistry. The mouse pubic symphysis was remodeled during the first pregnancy: the cellular phenotype and morphology changed to ensure a structurally safe birth canal, followed by recovery of the interpubic articulation after birth. The morphology of the pubic symphysis in the VS group was maintained in a state similar to that observed in virgin young mice. In contrast, MS mice exhibited an interpubic ligament characterized by extended fibrocyte-like cells, an opened interpubic articulation gap, compacted and thin collagen fibrils and scarce galectin-3-positive cells. Thus, we found that the cellular and extracellular characteristics of the pubic symphysis were altered by multiparity in senescent mice. These particular tissue characteristics of the MS group might be associated with an impaired recovery process at postpartum. Thus, a better understanding of the alterations that occur in the birth canal, including the pubic symphysis, due to multiparity in reproductively aged mice may contribute to our comprehension of the biological mechanisms that modify the skeleton and pelvic ligaments and even play a role in the murine model of pelvic organ prolapse.     

A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease

The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4^−/− mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection.     

Reprint of: A rapid increase in macrophage-derived versican and hyaluronan in infectious lung disease

The goals of this study were to characterize the changes in chondroitin sulfate proteoglycans and hyaluronan in lungs in acute response to gram-negative bacterial infection and to identify cellular components responsible for these changes. Mice were treated with intratracheal (IT) live Escherichia coli, E. coli lipopolysaccharide (LPS), or PBS. Both E. coli and LPS caused rapid selective increases in mRNA expression of versican and hyaluronan synthase (Has) isoforms 1 and 2 associated with increased immunohistochemical and histochemical staining for versican and hyaluronan in the lungs. Versican was associated with a subset of alveolar macrophages. To examine whether macrophages contribute to versican and hyaluronan accumulation, in vitro studies with primary cultures of bone marrow-derived and alveolar macrophages were performed. Unstimulated macrophages expressed very low levels of versican and hyaluronan synthase mRNA, with no detectible versican protein or hyaluronan product. Stimulation with LPS caused rapid increases in versican mRNA and protein, a rapid increase in Has1 mRNA, and concomitant inhibition of hyaluronidases 1 and 2, the major hyaluronan degrading enzymes. Hyaluronan could be detected following chloroquine pre-treatment, indicating rapid turnover and degradation of hyaluronan by macrophages. In addition, the effects of LPS, the M1 macrophage classical activation agonist, were compared to those of IL-4/IL-13 or IL-10, the M2a and M2c alternative activation agonists, respectively. Versican and Has1 increased only in response to M1 activation. Finally, the up-regulation of versican and Has1 in the whole lungs of wild-type mice following IT LPS was completely abrogated in TLR-4^−/− mice. These findings suggest that versican and hyaluronan synthesis may play an important role in the innate immune response to gram-negative lung infection.     

Hyaluronan involvement in the changes of mouse interpubic tissue during late pregnancy and post-partum

The present work quantifies hyaluronan (HA) during the late pregnancy and post-partum in order to provide a better understanding of the role of HA in the adaptations that occur in the pubic symphysis during this period. HA was quantified in situ (histochemically) and in interpubic tissue extracts by fluorimetric assay. Samples were taken from virgin mice and from pregnant animals at various stages of pregnancy: 12th-18th days into pregnancy, the day of delivery (D19) and the 3rd and 5th day post-partum. The quantitative fluorimetric analysis indicated a gradual increase of HA in the interpubic tissue throughout late pregnancy (2.4-14.6 microg/mg dry weight). This was followed by a decrease beginning on D19 (12.4 microg/mg), reaching close to virgin levels (2.2 microg/mg) on the 5th day post-partum. The same optical density changes could be seen in the HA staining. Furthermore, the histochemical analysis demonstrated the presence of HA both in the extracellular matrix of the tissue and within its cells. Such results indicate that the extracellular presence of HA may contribute to the transformation of the symphysis into a flexible structure. In addition, HA's intracellular presence (until the 18th day of pregnancy) may contribute to cellular proliferation. Finally, during parturition and on the 5th day post-partum, HA may contribute to the maintenance of the myofibroblastic phenotype of ligament cells, aiding the ligament involution after parturition.     

Hyaluronan and its binding proteins during cervical ripening and parturition: Dynamic changes in size, distribution and temporal sequence

The uterine cervix undergoes changes during pregnancy and labor that transform it from a closed, rigid, collagen dense structure to one that is distensible, has a disorganized collagen matrix, and dilates sufficiently to allow birth. To protect the reproductive tract from exposure to the external environment, the cervix must be rapidly altered to a closed, undistensible structure after birth. Preparturition remodeling is characterized by increased synthesis of hyaluronan, decreased expression of collagen assembly genes and increased distribution of inflammatory cells into the cervical matrix. Postpartum remodeling is characterized by decreased hyaluronan (HA) content, increased expression of genes involved in assembly of mature collagen and inflammation. The focus of this study is to advance our understanding of functions HA plays in this dynamic process through characterization of HA size, structure and binding proteins in the mouse cervix. Changes in size and structure of HA before and after birth were observed as well as cell specific expression of HA binding proteins. CD44 expression is localized to the pericellular matrix surrounding the basal epithelia and on immune cells while inter alpha trypsin inhibitor (IalphaI) and versican are localized to the stromal matrix. Colocalization of HA and IalphaI is most pronounced after birth. Upregulation of the versican degrading protease, ADAMTS1 occurs in the cervix prior to birth. These studies suggest that HA has multiple, cell specific functions in the cervix that may include modulation of tissue structure and integrity, epithelial cell migration and differentiation, and inflammatory responses.     

A Role for Versican in the Development of Leiomyosarcoma

Leiomyosarcoma (LMS) is a mesenchymal cancer that occurs throughout the body. Although LMS is easily recognized histopathologically, the cause of the disease remains unknown. Versican, an extracellular matrix proteoglycan, increases in LMS. Microarray analyses of 80 LMSs and 24 leiomyomas showed a significant elevated expression of versican in human LMS versus benign leiomyomas. To explore the importance of versican in this smooth muscle cell tumor, we used versican-directed siRNA to knock down versican expression in a LMS human cell line, SK-LMS-1. Decreased versican expression was accompanied by slower rates of LMS cell proliferation and migration, increased adhesion, and decreased accumulation of the extracellular matrix macromolecule hyaluronan. Addition of purified versican to cells expressing versican siRNA restored cell proliferation to the level of LMS controls, increased the pericellular coat and the retention of hyaluronan, and decreased cell adhesion in a dose-dependent manner. The presence of versican was not only synergistic with hyaluronan in increasing cell proliferation, but the depletion of versican decreased hyaluronan synthase expression and decreased the retention of hyaluronan. When LMS cells stably expressing versican siRNA were injected into nude mice, the resulting tumors displayed significantly less versican and hyaluronan staining, had lower volumes, and had reduced levels of mitosis as compared with controls. Collectively, these results suggest a role for using versican as a point of control in the management and treatment of LMS.     

Platelet-derived growth factor stimulates the formation of versican-hyaluronan aggregates and pericellular matrix expansion in arterial smooth muscle cells

Hyaluronan and versican-rich pericellular matrices form around arterial smooth muscle cells (ASMC) preferentially during the detachment phase of proliferation and migration. PDGF is a potent mitogen and chemotactic agent for ASMC and also stimulates the production of extracellular matrix molecules which may regulate the proliferative and migratory capacity of the cells. We have examined the effect of PDGF on the formation of hyaluronan-dependent pericellular matrices, and on the synthesis and interaction of several major pericellular coat constituents. As demonstrated using a particle exclusion assay, PDGF stimulated the formation of pericellular matrices and was seen both in an increased proportion of cells with a coat and a greater coat size. This increase was accompanied by a transient increase in hyaluronan synthase 2 (HAS2) expression and an increase in hyaluronan synthesis and polymer length. PDGF also increased the synthesis of versican and link protein as measured at the mRNA and protein levels. The amount of native versican-hyaluronan aggregates and link-stabilized aggregate was also increased following PDGF treatment. Time lapse imaging showed that pericellular matrix formation occurred around trailing cell processes prior to their detachment. These data suggest that PDGF modulates the synthesis and organization of ASMC pericellular coat-forming molecules such as versican, hyaluronan, and link protein, which leads to extracellular matrix expansion and alterations in ASMC phenotype.     

Phenotypic modulation of fibroblastic cells in mice pubic symphysis during pregnancy,partum and postpartum

In many species, the cartilaginous pubic symphysis of the pregnant female is gradually replaced by a fibrous connective tissue, forming a flexible and elastic interpubic ligament. This newly formed ligament is responsible for the separation of the pubic bones, enabling safe delivery of the young. Following labor, the ligament undergoes rapid involution. To our knowledge, no previous work has focused on the phenotypic modulation that is responsible for the changes present at the interpubic ligament throughout the relaxation and closing of the symphysis. The purpose of this study was to investigate the ultrastructural features and immunophenotype of the peculiar cell type found in the pubic symphysis of cycling, pregnant and postpartum mice. In particular, immunohistochemistry studies were conducted on the expressions of the cytoskeletal proteins desmin, vimentin and -smooth muscle actin (-SMA). During pregnancy, the pubic symphysis cells always expressed -SMA, whereas the expression of vimentin and desmin was transient from early pregnancy to postpartum. Furthermore, the expression patterns of these three cytoskeletal proteins were distinct. Cells present in the medial region of the mouse symphysis in cycling and at D12 displayed ultrastructural features characteristic of a typical fibroblast. In contrast, during the last week of pregnancy and in postpartum these cells acquired ultrastructural features representative of a myofibroblast; for example, a fibronexus and a contractile apparatus were found to be present lying in close contact with the extracellular collagenous and elastic system fibrils. Taken together, these results strongly suggest a contractile function for these cells which might contribute to support of the varying mechanical stresses present during pubic bone movement.This work was supported by grants from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). This paper is part of a thesis submitted to the Department of Histology and Embryology of the Biology Institute State University of Campinas, in partial fulfillment of the requirements for the Masters degree.     

Expression of versican in relation to chondrogenesis-related extracellular matrix components in canine mammary tumors

Versican plays a role in tumor cell proliferation and adhesion and may also regulate cell phenotype. Furthermore, it is one of the pivotal proteoglycans in mesenchymal condensation during prechondrogenesis. We have previously demonstrated accumulation of versican protein in myoepithelial-like spindle cell proliferations and myxoid tissues of complex and mixed mammary tumors of dogs. The objective of this study was to investigate whether the high expression of versican relates to prechondrogenesis in these tissues. Therefore, we aimed to identify cartilage markers, such as collagen type II and aggrecan both at mRNA and protein level in relation to versican. The neopitope of chondoitin-6-sulphate (3B3) known to be generated in developing cartilage has been investigated by immunohistochemisty and a panel of antibodies were used to characterize the phenotype of cells that are involved in cartilage formation. In addition, co-localization of versican with hyaluronan and link protein was studied. RT-PCR revealed upregulation of genes of versican, collagen type II and aggrecan in neoplastic tissues, especially in complex and mixed tumors. Immunohistochemistry showed the expression of cartilage biomarkers not only in the cartilagenous tissues of mixed tumors, but also in myoepitheliomas and in the myoepithelial-like cell proliferations and myxoid areas of complex and mixed tumors. The results show the cartilagenous differentiation of complex tumors and myoepitheliomas and indicate that the myxoid tissues and myoepithelial-like cell proliferations are the precursor tissues of the ectopic cartilage in mixed tumors. Furthermore, we suggest that cartilage formation in canine mammary tumors is a result of (myo)epithelial to mesenchymal transition.     

Effects of the hormone relaxin on the metabolism of the glycosaminoglycans in the mouse symphysis pubis

The influence of the peptide hormone relaxin on the glycosaminoglycan (GAG) metabolism was investigated in the pubic ligament of the symphysis pubis and in serum of the virgin mouse. Fresh weight DNA and GAG content per 1 ligament is significantly increased, the level of water soluble protein is not affected. A shift in the electrophoretic GAG pattern by an increasing amount of hyaluronic acid and a decreasing amount of chondroitin sulfate and dermatan sulfate can be observed. Concerning GAG-splitting enzymes (N-acetylglucosaminidase, arylsulfatase, beta-glucuronidase) the N-acetylglucosaminidase reveals a significant increase of its activity in the interpubic ligament and in the serum. The data demonstrate that relaxin treatment induces some changes in the GAG metabolism.     

Elastic fiber assembly in the adult mouse pubic symphysis during pregnancy and postpartum

Impairment of pelvic organ support has been described in mice with genetic modifications of the proteins involved in elastogenesis, such as lysyl oxidase-like 1 (LOXL1) and fibulin 5. During pregnancy, elastic fiber-enriched pelvic tissues are modified to allow safe delivery. In addition, the mouse pubic symphysis is remodeled in a hormone-controlled process that entails the modification of the fibrocartilage into an interpubic ligament (IpL) and the relaxation of this ligament. After first parturition, recovery occurs to ensure pelvic tissue homeostasis. Because ligaments are the main supports of the pelvic organs, this study aimed to evaluate elastogenesis in the IpL during mouse pregnancy and postpartum. Accordingly, virgin, pregnant, and postpartum C57BL/6 mice were studied using light, confocal, and transmission electron microscopy as well as Western blots and real-time PCR. Female mice exhibited the separation of the pubic bones and the formation, relaxation, and postpartum recovery of the IpL. By the time the IpL was formed, the elastic fibers had increased in profile length and diameter, and they consisted of small conglomerates of amorphous material distributed among the bundles of microfibrils. Our analyses also indicated that elastin/tropoelastin, fibrillin 1, LOXL1/Loxl1, and fibulin 5 were spatially and temporally regulated, suggesting that these molecules may contribute to the synthesis of new elastic fibers during IpL development. Overall, this work revealed that adult elastogenesis may be important to assure the elasticity of the pelvic girdle during preparation for parturition and postpartum recovery. This finding may contribute to our understanding of pathological processes involving elastogenesis in the reproductive tract.     

Accumulation and loss of extracellular matrix during shear stress-mediated intimal growth and regression in baboon vascular grafts.

The composition of extracellular matrix during growth and regression of the neointima was analyzed during healing in a baboon aorto-iliac polytetrafluoroethylene graft. Graft neointimal thickening can be modulated by altering blood flow by construction of downstream arteriovenous fistulas. Normal flow with normal shear stress induces neointimal thickening, whereas high flow with high shear stress upstream of a fistula induces regression of established neointima. The neointima formed under normal shear stress is enriched in hyaluronan and proteoglycans, particularly versican. On the other hand, the neointima near the graft material is enriched in collagen and biglycan. Neointimal regression in response to high shear stress is associated with a loss of proteoglycans as detected by histochemical staining. Immunostaining with an antibody against an ADAMTS cleavage neoepitope of versican increases after switching to high flow, although immunostaining for versican core protein is not appreciably changed by high flow. The present data demonstrate that the graft neointima is enriched with proteoglycans, particularly versican and hyaluronan, as well as collagen, and there is a differential distribution of each. Neointimal atrophy occurs with an apparent loss of proteoglycans and evidence of versican degradation.     

Link protein has greater affinity for versican than aggrecan

The function of link protein in stabilizing the interaction between aggrecan and hyaluronan to form aggrecan aggregates, via the binding of link protein to the aggrecan G1 domain and hyaluronan, is well established. However, it is not known whether link protein can function with similar avidity with versican, another member of the large hyaluronan-binding proteoglycan family that also binds to hyaluronan via its G1 domain. To address this issue, we have compared the interaction of the versican and aggrecan G1 domains with link protein and hyaluronan using recombinant proteins expressed in insect cells and BIAcore analysis. The results showed that link protein could significantly improve the binding of both G1 domains to hyaluronan and that its interaction with VG1 is of a higher affinity than that with AG1. These observations suggest that link protein may function as a stabilizer of the interaction, not only between aggrecan and hyaluronan in cartilage, but also between versican and hyaluronan in many tissues.     

Hyaluronan and versican in the control of human T-lymphocyte adhesion and migration

The ability of lymphocytes to migrate freely through connective tissues is vital to efficient immune function. How the extracellular matrix (ECM) may affect T-cell adhesion and migration is not well understood. We have examined the adhesion and migration of activated human T-lymphocytes on ECM made by fibroblast-like synoviocytes and lung fibroblasts. These cells were minimally interactive until treated with a viral mimetic, Poly I:C. This treatment promoted myofibroblast formation and engendered a higher-order structured ECM, rich in versican and hyaluronan, to which T-cells avidly adhered in a hyaluronidase-sensitive manner. This Poly I:C-induced matrix impeded T-cell spreading and migration on and through synoviocyte monolayers, while hyaluronidase treatment or adding versican antibody during matrix formation reversed the effect on T-cell migration. Hyaluronidase also reversed the spread myofibroblast morphology. These data suggest that the viscous hyaluronan- and versican-rich matrix binds and constrains T-lymphocytes. Using purified matrix components and solid state matrices of defined composition, we uncovered a role for versican in modulating hyaluronan-T-cell interactions. Versican prevented T-cell binding to soluble hyaluronan, as well as the amoeboid shape change on hyaluronan-coated dishes and T-cell penetration of collagen gels. Together, these data suggest that hyaluronan and versican play a role in T-cell trafficking and function in inflamed tissues.