A computational model of ureteral peristalsis and an investigation into ureteral reflux.

The aim of this study is to create a computational model of the human ureteral system that accurately replicates the peristaltic movement of the ureter for a variety of physiological and pathological functions. The objectives of this research are met using our in-house fluid-structural dynamics code (CgLes-Y code). A realistic peristaltic motion of the ureter is modelled using a novel piecewise linear force model. The urodynamic responses are investigated under two conditions of a healthy and a depressed contraction force. A ureteral pressure during the contraction shows a very good agreement with corresponding clinical data. The results also show a dependency of the wall shear stresses on the contraction velocity and it confirms the presence of a high shear stress at the proximal part of the ureter. Additionally, it is shown that an inefficient lumen contraction can increase the possibility of a continuous reflux during the propagation of peristalsis.

Anticoagulant heparan sulfate proteoglycans expression in the rat ovary peaks in preovulatory granulosa cells.

Ovarian granulosa cells synthesize anticoagulant heparan sulfate proteoglycans (aHSPGs), which bind and activate antithrombin III. To determine if aHSPGs could contribute to the control of proteolytic activities involved in follicular development and ovulation, we studied the pattern of expression of these proteoglycans during the ovarian cycle. aHSPGs were localized on cells and tissues by (125)I-labeled antithrombin III binding followed by microscopic autoradiography. Localization of aHSPGs has shown that cultured granulosa cells, hormonally stimulated by gonadotropins to differentiate in vitro, up-regulate their synthesis and release of aHSPGS: In vivo, during gonadotropin-stimulated cycle, aHSPGs are present on granulosa cells of antral follicles and are strongly labeled in preovulatory follicles. These data demonstrate that aHSPG expression in the ovarian follicle is hormonally induced to culminate in preovulatory follicles. Moreover, we have shown that five heparan sulfate core proteins mRNA (perlecan; syndecan-1, -2, and -4; and glypican-1) are synthesized by granulosa cells, providing attachment for anticoagulant heparan sulfate chains on the cell surface and in the extracellular matrix. These core proteins are constantly expressed during the cycle, indicating that modulations of aHSPG levels observed in the ovary are likely controlled at the level of the biosynthesis of anticoagulant heparan sulfate glycosaminoglycan chains. This expression pattern enables aHSPGs to focus serine protease inhibitors in the developing follicle to control proteolysis and fibrin formation at ovulation.

Posttranscriptional stimulation of endothelial cell matrix metalloproteinases 2 and 1 by endothelioma cells.

Matrix metalloproteinases (MMPs) play a critical role in the development of hemangioma-like vascular tumors in mice injected with murine eEnd.1 endothelioma cells. The current study was designed to (a) characterize the presence of MMPs in the vascular tumor, (b) define whether these MMPs originate from the transformed cells or from the recruited stromal cells and (c) study the stimulatory effect of eEnd.1 cells on the production of MMPs by endothelial cells. Several gelatinases were present in the eEnd.1 tumor extract, including latent and activated MMP-2 (72-kDa gelatinase A, EC 3.4.24. 24) and MMP-9 (92-kDa gelatinase B, EC 3.4.24.35). Immunohistochemical analysis of the tumor revealed focal reactivity for MMP-2. No gelatinase was produced by cultured eEnd.1 cells, or by six of nine related endothelioma cell lines, suggesting that stroma cells, particularly endothelial cells recruited by the tumor cells, rather than eEnd.1 cells themselves, are the source of the gelatinases observed in the tumors in vivo. The conditioned medium of eEnd.1 cells stimulated the release of MMP-2 and MMP-1 (interstitial collagenase, EC 3.4.24.7) by endothelial cells, but not of the inhibitor TIMP-2. The increased production of MMP-2 and MMP-1, observed at the protein level (zymogram and Western blot analysis), occurred through a posttranscriptional mechanism, since no increase in mRNA was observed and the stimulation was not prevented by inhibitors of protein synthesis. The inhibitory effects of monensin and brefeldin A, inhibitors of protein secretion, and the decrease in cell-associated MMP-2 in stimulated endothelial cells indicated that regulation occurred mostly at the level of protease secretion. MMPs are known to be regulated at different levels; this study indicates that, in endothelial cells, the stimulation of MMPs can also occur at the level of secretion, a mechanism that provides a rapid mobilization of these crucial enzymes in the early phases of angiogenesis.

Constitutively active mitogen-activated protein kinase kinase MEK1 disrupts morphogenesis and induces an invasive phenotype in Madin-Darby canine kidney epithelial cells.

During certain developmental processes, as well as during tumor progression, polarized epithelial cells integrated within multicellular structures convert into scattered, freely migrating fibroblast-like cells. Despite the biological and clinical importance of this phenomenon, the intracellular biochemical cascades that control the switch between the epithelial and mesenchymal phenotypes have not been elucidated. Using Madin-Darby canine kidney (MDCK) cells (clone C7) as a model system, we have assessed the potential role of the mitogen-activated protein kinase (MAPK)/ extracellular signal-regulated kinase (ERK) cascade in the modulation of epithelial plasticity. When grown in three-dimensional collagen gels, MDCK-C7 cells form spherical cysts composed of polarized epithelial cells circumscribing a central lumen. This morphogenetic behavior is profoundly subverted in MDCK-C7 cells expressing a constitutively active MAPK/ERK kinase 1 (caMEK1) mutant (C7-caMEK1 cells). When suspended in collagen gels, C7-caMEK1 cells assume an elongated fibroblastoid shape and are unable to generate multicellular cysts. In addition, when seeded onto the surface of a collagen gel, C7-caMEK1 cells penetrate extensively into the underlying matrix, unlike wild-type and mock-transfected MDCK-C7 cells, which remain confined to the surface of the gel. Similar changes in morphogenetic and invasive properties are observed in MDCK-C7F cells, a nontransfected, stably dedifferentiated derivative of MDCK-C7 cells that expresses substantially increased ERK2 activity. Both C7-caMEK1 and MDCK-C7F cells but not wild-type or mock-transfected MDCK-C7 cells express activated M(r) 72,000 gelatinase A [matrix metalloproteinase (MMP)-2] as well as elevated levels of membrane type-1 MMP. Synthetic MMP inhibitors as well as recombinant tissue inhibitor of metalloproteinases 2 and 3 suppress the invasion of collagen gels and restore the capacity of C7-caMEK1 cells to form cysts, thereby implicating the membrane type-1 MMP/MMP-2 proteolytic system in epithelial cell invasiveness and loss of multicellular organization. Taken together, our data demonstrate that increased activity of the MEK1-ERK2 signaling module in MDCK-C7 cells is associated with failure of morphogenesis and expression of a highly invasive phenotype. Sustained activation of the MAPK cascade therefore results in the destabilization of the three-dimensional architecture and the conversion of polarized epithelial cells into migrating mesenchymal-like cells.

Effect of plasma from patients with idiopathic nephrotic syndrome on proteoglycan synthesis by human and rat glomerular cells.

In vivo and in vitro findings have shown that plasma of patients with idiopathic nephrotic syndrome (INS) contain factors that increase glomerular permeability to proteins. The effects of these factors on proteoglycan synthesis by glomerular cells are unknown. To investigate the effect of plasma from patients with INS (n = 23) and other glomerulopathies (n = 12) on the amount of proteoglycans synthesized by cultured rat mesangial cells and human glomerular epithelial cells, glomerular cells were cultured for 24 h with plasma from patients or control subjects, and incorporation of Na2(35)SO4 in chondroitin dermatan sulfate and heparan sulfate was assessed using a cationic nylon membrane. The mean ratio of glycosaminoglycan produced by rat mesangial cells when in contact with plasma (5%) from INS patients to the amount produced when in contact with control plasma was 0.70+/-0.06. The mean ratio of heparan sulfate was 0.58+/-0.08. The decrease of heparan sulfate production was present in the cellular and in the extracellular fraction. It was observed when the cells were in contact with plasma from patients in relapse but not when in remission. No decrease of heparan sulfate production was observed with four of the five patients with membranous glomerulonephritis (ratio of 1.27+/-0.03), IgA nephropathy (n = 5, ratio of 1.27+/-0.03), and membranoproliferative glomerulonephritis (n = 2, ratio of 1.39+/-0.34). When human glomerular epithelial cells were exposed to 5% plasma from INS patients in relapse (n = 9), the mean ratio of heparan sulfate was 0.62+/-0.06 in the cellular fraction and 0.72+/-0.08 in the medium. When in contact with plasma from patients in remission, no difference of glycosaminoglycan production was observed. A factor present in plasma from patients with INS during initial episodes or relapses is able to decrease the proteoglycan production of glomerular cells.

Characterization and hormonal modulation of anticoagulant heparan sulfate proteoglycans synthesized by rat ovarian granulosa cells.

Anticoagulant heparan sulfate proteoglycans endow the vascular endothelium with antithrombotic properties, but their role outside the vascular bed is unknown. Granulosa cells form an avascular compartment in the ovarian follicle, in which a heparin-like activity has been described. At ovulation extravascular coagulation occurs around ovulatory follicles, and after expulsion of the oocyte, a fibrin clot forms in the antral cavity. Granulosa cells synthesize two major heparan sulfate proteoglycans, whose anticoagulant nature has not been investigated. The purpose of this study was to characterize anticoagulant heparan sulfate proteoglycans synthesized by rat ovarian granulosa cells. Affinity purified 35S-labeled anticoagulant heparan sulfate glycosaminoglycans represent 6.5% of the total heparan sulfate synthesized, and they contain 13% 3-O-sulfated disaccharides that are markers of the antithrombin-binding site of heparin. The biological activity of granulosa cell heparan sulfate proteoglycans was demonstrated by their ability to bind antithrombin and to accelerate the formation of thrombin-antithrombin complexes. The impact of hormonal stimulation on granulosa cell anticoagulant heparan sulfate proteoglycans was studied using 125I-antithrombin binding assays. Follicle-stimulating hormone induced a redistribution of anticoagulant heparan sulfate proteoglycans from the granulosa cell layer to the culture medium, indicating that their distribution could be modulated according to the stage of follicular development. These results suggest that anticoagulant heparan sulfate might be critically located in the follicle to maintain fluidity around the oocyte until its expulsion at ovulation.