BackgroundPhagocytosis of apoptotic cells by macrophages induces a suppressor phenotype. Previous data from our group suggested that this occurs via Platelet-activating factor receptor (PAF-R)-mediated pathways. In the present study, we investigated the impact of apoptotic cell inoculation or induction by a chemotherapeutic agent (dacarbazine, DTIC) on tumour growth, microenvironmental parameters and survival, and the effect of treatment with a PAF-R antagonist (WEB2170). These studies were performed in murine tumours: Ehrlich Ascitis Tumour (EAT) and B16F10 melanoma.MethodsTumour growth was assessed by direct counting of EAT cells in the ascitis or by measuring the volume of the solid tumour. Parameters of the tumour microenvironment, such as the frequency of cells expressing cyclo-oxygenase-2 (COX-2), caspase-3 and galectin-3, and microvascular density, were determined by immunohistochemistry. Levels of vascular endothelium growth factor (VEGF) and prostaglandin E2 (PGE2) were determined by ELISA, and levels of nitric oxide (NO) by Griess reaction. PAF-R expression was analysed by immunohistochemistry and flow cytometry.ResultsInoculation of apoptotic cells before EAT implantation stimulated tumour growth. This effect was reversed by in vivo pre-treatment with WEB2170. This treatment also reduced tumour growth and modified the microenvironment by reducing PGE2, VEGF and NO production. In B16F10 melanoma, WEB2170 alone or in association with DTIC significantly reduced tumour volume. Survival of the tumour-bearing mice was not affected by WEB2170 treatment but was significantly improved by the combination of DTIC with WEB2170. Tumour microenvironment elements were among the targets of the combination therapy since the relative frequency of COX-2 and galectin-3 positive cells and the microvascular density within the tumour mass were significantly reduced by treatment with WEB2170 or DTIC alone or in combination. Antibodies to PAF-R stained the cells from inside the tumour, but not the tumour cells grown in vitro. At the tissue level, a few cells (probably macrophages) stained positively with antibodies to PAF-R.ConclusionsWe suggest that PAF-R-dependent pathways are activated during experimental tumour growth, modifying the microenvironment and the phenotype of the tumour macrophages in such a way as to favour tumour growth. Combination therapy with a PAF-R antagonist and a chemotherapeutic drug may represent a new and promising strategy for the treatment of some tumours.

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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.

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 alpha-smooth muscle actin (alpha-SMA). During pregnancy, the pubic symphysis cells always expressed alpha-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.

Proteoglycans were accurately localized in mouse pubic symphyseal tissues using the cuprolinic blue method. Specific glycosaminoglycans degradative enzymes, together with chondroitin sulfate and decorin antibodies, allowed the identification of glycosaminoglycans. Chondroitin sulfate proteoglycans were the main proteoglycans observed in hyaline cartilage, fibrocartilage, and dense connective tissue. Ultrastructurally, they were seen as electron-dense granules and filaments. The granules, rich in chondroitin sulfate chains, were exclusively found in hyaline cartilage, whereas filaments were present in cartilage, fibrocartilage, and dense connective tissue. The latter were classified by size and susceptibility to enzyme digestion into F1, F2 and F3 filaments: F1 filaments were small, thin, and collagen fibril-associated; F2 filaments were thick, heavily stained, and localized around individual collagen fibrils and between bundles of collagen fibrils; and F3 filaments were scattered throughout elastic fiber surfaces. Considering their localization, susceptibility to chondroitinase AC and immunohistochemical detection, the symphysial F1 filaments were found to be preferentially decorin substituted with chondroitin sulfate side chains. The F2 filaments were also susceptible to chondroitinase AC treatment, whereas F3 filaments could be digested by heparitinase. The data thus obtained on the localization and identification of pubic symphyseal proteoglycans in virgin mice may be useful in the study of structural modifications that occur throughout pregnancy.

Marked changes in mice pubic symphysis occur by the end of pregnancy. Tissue remodeling involves a dynamic balance between cell proliferation and programmed cell death as well as changes in the extracellular matrix components. Therefore, it is important to consider both of these cellular behaviors when investigating the mechanism that regulates interpubic tissue remodeling, growth during late pregnancy and partus ensuring involution during the postpartum period. Proliferating and programmed death cells were identified by immunohistochemistry (proliferating cell nuclear antigen and TUNEL detection, respectively) and the rates at which these processes occurred were determined by morphometric analysis. The results demonstrated that cellular proliferation was intense during the period of ligament formation, from D15 to D18, thereafter abruptly declining on D19. From parturition (D19) onwards, an ever-increasing decline in the cellular proliferation levels could be observed. The quantitative analyses of cellular death showed opposite results when compared to cellular proliferation. During early pregnancy the cycle of cellular renovation was clearly proliferative and during late mouse pregnancy the cycle was directed by programmed cellular death. Although the high levels of cellular death during postpartum involution could be shown by the TUNEL-positive cells, we were unable to observed picnotic nucleus at the light microscopy.

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.

SummaryCurrent literature concerning smooth muscle blood vessels has shown versican as the main proteoglycan (PG) component of the matrix. To show whether smooth muscle matrix has the same PG distribution when present in organs, other than the blood vessels, the inner circular smooth muscle layer of the small intestine was obtained by dissection as a highly purified tissue and analyzed by biochemical and cytochemical methods. The smooth muscle layer PGs were extracted from dog small intestine with 4 M guanidineHCl in the presence of proteinase inhibitors, purified by charge equilibrium, isolated by equilibrium CsCl density gradients, and analyzed in terms of anion exchange, size, and glycosaminoglycan (GAG) distribution. Proteoheparan sulfate itself represented 91.5% of the PGs present in this tissue. The remainder was proteodermatan sulfate. Cytochemical analyses using the cationic dye cuprolinic blue associated with enzymatic treatments with chondroitinases ABC and heparitinase III showed the arrangement and identification of PGs in basal lamina and intramuscular connective tissues. The PGs in the basal lamina were proteoheparan sulfate, and those associated with collagen fibrils in the endomysium and perimysium were rich in dermatan sulfate. In contrast to the blood vessels, inner circular muscle smooth tissue in intestine has, as the main PG, perlecan.