Biomedical subjects

I Vlodavsky

Publications and source records attributed to I Vlodavsky.

At least 163 records · Page 9Linked to original sources

PubMed1985

Production and characterization of interferon from endothelial cells.

The capacity of cultured bovine aortic, capillary, and corneal endothelial cells as well as of human umbilical cord endothelial cells to produce interferon (IFN) was investigated. The endothelial cells of the two species produced significant amounts of IFN in response to various viruses and poly (I) poly (C). The IFN produced by human umbilical cord endothelial cells was a mixture of alpha- and beta-IFN, as determined by neutralization with antibodies directed against these two types of IFNs as well as by measuring the antiviral activity on heterologous cells. Bovine endothelial cells also produced a mixture of at least two IFN subspecies, one of them labile at pH 2 and active on human cells and the other stable at pH 2 and inactive on human cells.

Animals↗

PubMed1985

The antiproliferative effect of interferon and the mitogenic activity of growth factors are independent cell cycle events. Studies with vascular smooth muscle cells and endothelial cells.

We studied the antagonistic effects of interferon (IFN) and growth factors in G0/G1-arrested normal bovine aortic smooth muscle cells (SMC) which were stimulated by serum, or purified platelet derived growth factor (PDGF), supplemented with plasma-derived serum (PDS). The growth response, measured as [3H]thymidine incorporation into DNA, was dependent on the concentration of the mitogen. Human IFN alpha, recombinant human IFN alpha 2, or a crude bovine-IFN preparation prepared from virus-infected bovine aortic endothelial cells, inhibited SMC growth induced by either serum or PDGF with PDS. The extent of IFN inhibition was inversely related to the concentration of the mitogenic stimulus. We also investigated whether IFN inhibited the early events in G1 phase, stimulated by the competence factor PDGF, or the progression of the cell into the S phase induced by PDS. The results indicated that IFN inhibited these two stages of the G1 phase independently. In addition, we investigated the antiproliferative effect of IFN on bovine aortic endothelial cells (BAEC), which do not respond to PDGF but to the mitogenic activity of fibroblast growth factor (FGF). IFN inhibited the mitogenic activity of FGF in a dose-dependent manner. The results indicate that the anti-proliferative activity of IFN and the mitogenic effects of different growth factors are independent.

Animals↗

PubMed1985

Differential structural requirements for the induction of cell attachment, proliferation and differentiation by the extracellular matrix.

The subendothelial extracellular matrix (ECM) mediates the attachment of both human Ewing's sarcoma and colon carcinoma cells. Attachment and flattening of the sarcoma cells was sensitive to heat treatment but not to periodate oxidation of the ECM, whereas the colon carcinoma cells attached and flattened over heated but not periodate-treated ECM. Such differential sensitivity to heat treatment and periodate oxidation was also observed using purified fibronectin and laminin, respectively, but the inhibition of cell attachment was greater than with a similarly treated ECM. It is therefore conceivable that fibronectin and laminin specifically mediate the attachment and flattening of Ewing's sarcoma and colon carcinoma cells to the ECM, but that other constituents may support this attachment either directly or via interaction and stabilization of adhesive glycoproteins in the ECM. The ECM-mediated morphological differentiation of adult rat oligodendrocytes was sensitive to periodate oxidation to a much higher extent than to heat treatment of the ECM. In contrast, both treatments had only a small effect on the ECM-induced proliferation of vascular endothelial cells. These results indicate that different constituents of the ECM may be held responsible for its effects on different parameters of cell behavior, and that various cell types respond differently to a given modification of the ECM.

Animals↗

PubMed1985

Cell interaction with the extracellular matrices produced by endothelial cells and fibroblasts.

The extracellular matrices (ECM) produced by cultured bovine corneal endothelial cells and chick embryo fibroblasts were compared for their induction of cell attachment, proliferation and differentiation. The corneal endothelial ECM (cECM) induced a comparable and rapid attachment and flattening of both human Ewing's sarcoma and colon carcinoma cells which utilize fibronectin and laminin as adhesive glycoproteins, respectively. In contrast, the ECM produced by fibroblasts (fECM) readily supported the attachment and flattening of Ewing's sarcoma cells but had only a small effect on the carcinoma cells. Vascular endothelial cells were stimulated to proliferate by both types of matrices, but to a lesser extent by the fECM. In contrast, the formation of a closely apposed, non-overlapping and contact-inhibited endothelial cell monolayer was only dictated by the cECM. Vascular endothelial cells cultured on fECM grew on top of each other and incorporated [3H]thymidine even late at confluency. Neurite outgrowth (ciliary ganglion cells) and network formation (adult rat oligodendrocytes) were promoted by both types of matrices but in a more consistent manner with the cECM. It is likely that the small amounts of laminin deposited by chick embryo fibroblasts into their ECM are responsible for its efficient induction of neurite outgrowth and for the limited degree of carcinoma cell attachment and flattening. It is thus demonstrated that differences in chemical composition and supramolecular arrangement between cECM and fECM result not only in differences in the attachment, spreading and proliferative responses of cells but also in the expression of their characteristic morphological appearance and differentiated functions.

Animals↗

PubMed1985

Characterization of an extracellular matrix-degrading protease derived from a highly metastatic tumor cell line.

A proteolytic activity associated with the microsomal fraction of L-5178Y/Esb tumor cells has been characterized. The enzyme has a molecular weight of 80-90 kD as determined by affinity-labelling with [3H]DFP and SDS-gel electrophoresis. It cleaves ester substrates at the carboxyl position of lysine and arginine and can activate the proenzyme plasminogen. The enzyme is found to be associated with the plasma membranes of high and low metastatic tumor cell lines and is shed in high-molecular-weight form mainly by the high metastatic variant. The pH optimum for esterase and protease activities was 7.5-8.5. Although similar to trypsin in substrate specificity, the enzyme was not inhibited by lima-bean trypsin inhibitor but was inhibited by DFP, PMSF, aprotinin and leupeptin. Partially purified preparations of the protease can alone degrade 125I-labelled endothelial cell extracellular matrix, pointing at the putative role of this enzyme in tumor invasion.

Animals↗

PubMed1985

Platelet-tumor cell interaction with the subendothelial extracellular matrix: relationship to cancer metastasis.

Dissemination of neoplastic cells within the body involves invasion of blood vessels by tumor cells. This requires adhesion of blood-borne cells to the luminal surface of the vascular endothelium, invasion through the endothelial cell layer and local dissolution of the subendothelial basement membrane. Platelets may participate in each of these steps and thus play a role in the pathogenesis of tumor cell metastasis. To learn more about the possible involvement of platelets we studied the interaction of platelets and tumor cells with cultured vascular endothelial cells and their secreted basement membrane-like extracellular matrix (ECM). Whereas the apical surface of the vascular endothelium lacks adhesive glycoproteins and hence protect the vessel wall against platelet and tumor cell adhesion, the underlying ECM constitute a highly adhesive and thrombogenic surface. Interaction of platelets with this ECM was associated with platelet activation, aggregation and degradation of heparan sulfate in the ECM by means of the platelet heparitinase. The activity of a similar enzyme has been previously correlated with the metastatic potential of various tumor sublines. Biochemical and scanning electron microscopy (SEM) studies have demonstrated that platelets may detect even minor gaps between adjacent endothelial cells and degrade the ECM heparan sulfate. This may expose a larger area of the subendothelium and facilitate subsequent adhesion of blood borne tumor cells. Platelets were also shown to recruit lymphoma cells into minor gaps in the vascular endothelium, that otherwise do not constitute a preferential site of invasion. It is suggested that the platelet heparitinase is involved in the impairment of the integrity of the vessel wall and thus play a role in tumor cell metastasis.

Animals↗

PubMed1985

Degradation of heparan sulfate in the subendothelial extracellular matrix by a readily released heparanase from human neutrophils. Possible role in invasion through basement membranes.

Freshly isolated human neutrophils were investigated for their ability to degrade heparan sulfate proteoglycans in the subendothelial extracellular matrix (ECM) produced by cultured corneal and vascular endothelial cells. The ECM was metabolically labeled with Na2(35S)O4 and labeled degradation products were analyzed by gel filtration over Sepharose 6B. More than 90% of the released radioactivity consisted of heparan sulfate fragments 5-6 times smaller than intact heparan sulfate side chains released from the ECM by either papain or alkaline borohydride. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of either heparin or serine protease inhibitors. In contrast, degradation of soluble high molecular weight heparan sulfate proteoglycan, which was first released from the ECM, was inhibited by heparin but there was no effect of protease inhibitors. These results indicate that interaction of human neutrophils with the subendothelial ECM is associated with degradation of its heparan sulfate by means of a specific, newly identified, heparanase activity and that this degradation is facilitated to a large extent by serine proteases. The neutrophil heparanase was readily and preferentially released (15-25% of the cellular content in 60 min) by simply incubating the cells at 4 degrees C in the absence of added stimuli. Under these conditions, less than 5% of the cellular content of lactate dehydrogenase, lysozyme, and globin degrading proteases was released. Further purification of the neutrophil heparanase was achieved by its binding to heparin-Sepharose and elution at 1 M NaCl. It is suggested that heparanase activity is involved in the early events of extravasation and diapedesis of neutrophils in response to a threshold signal from an extravascular inflamed organ.

Basement Membrane↗

PubMed1985

Serum reduces the TSH content in rat pituitary cells in culture.

Euthyroid rat pituitary cells were plated on a natural extracellular matrix (ECM) and cultured in either a medium supplemented with hormones and growth factors (SM) or in an unsupplemented medium (USM). Hormone supplementation resulted in a marked increase in the number of cells and in a less pronounced increase in the TSH content. Addition of 10% dehormonized serum (DHS) to the medium caused a further increase in the cell number as well as a significant elevation in the GH content but, surprisingly, it caused also a dramatic decrease in the TSH content. Time course experiments revealed that the serum doubled the rate of suppression of the TSH. DHS prepared from rat serum was as effective as that prepared from foetal calf serum and heat-inactivation of the serum did not change its suppressive effect on the production of TSH. This effect was confined to the serum fraction which was precipitable with 45% ammonium sulphate but did not bind to Con-A. It is suggested that a high molecular weight protein fraction of serum reduces the number of thyrotrophs in the culture and/or decreases the rate of TSH production by these cells.

Animals↗

PubMed1985

Platelet interaction with subendothelial extracellular matrix: platelet-fibrinogen interactions are essential for platelet aggregation but not for the matrix-induced release reaction.

Cultured endothelial cells produce an extracellular matrix (ECM) to which platelets adhere and spread, ultimately resulting in platelet aggregation, thromboxane B2 production, and serotonin release. We have investigated the role of fibrinogen binding to the platelet GPIIb/IIIa complex in these reactions by comparing normal platelet-rich plasma (PRP), PRP from patients with Glanzman's thrombasthenia (whose platelets lack the GPIIb/IIIa complex), PRP in the presence of a monoclonal antibody that blocks the binding of fibrinogen to the GPIIb/IIIa complex, platelets washed free of fibrinogen, and washed platelets to which fibrinogen was added. Although platelet aggregation was virtually completely inhibited in the samples in which the normal interaction between fibrinogen and GPIIb/IIIa was impaired, adhesion of platelets to the matrix, spreading, and release of [14C]-serotonin were not affected. All of the platelet preparations released significant amounts of T X B2 with time, but there was a decrease in the amount produced by both the thrombasthenic and antibody-treated platelets. We conclude that the interaction of fibrinogen with platelet GPIIb/IIIa is not required for platelet adhesion to ECM or for adhesion-induced shape change or serotonin release. On the other hand, the platelet-fibrinogen interaction may play some role in augmenting adhesion-induced T X B2 production, and it is absolutely required for adhesion-induced platelet aggregation.

Animals↗

PubMed1985

Megakaryocyte interaction with subendothelial extracellular matrix is associated with adhesion, platelet-like shape change, and thromboxane A2 production.

We have examined the morphological and secretory behavior of rat and guinea pig megakaryocytes exposed for up to 24 hours to extracellular matrix produced by cultured bovine endothelial cells. By phase-contrast microscopy of living cells and in more detail by scanning electron microscopy, the megakaryocytes showed a nonreversible adherence, an extensive formation of filopodia around the periphery like the rays of the sun, and a tendency toward flattening. These filopodia were generally linear with attenuated tips and were larger than, but resembled the filopodia of, rat or guinea pig platelets exposed to this extracellular matrix. In contrast, isolated megakaryocytes on glass or on uncoated plastic surfaces did not show these responses; adherence, in the face of gentle agitation before fixation, was minimal, with rare filopodia and no flattening. Megakaryocytes that interacted with the extracellular matrix produced significant amounts of thromboxane A2, but this did not occur on uncoated surfaces and could not be attributed to other contaminating cells in the megakaryocyte suspensions. The appearance in megakaryocytes of these typical platelet responses indicates that megakaryocytes acquire the functional capabilities of platelets by the synthesis and assembly of platelet substances and organelles. Thromboxane production by megakaryocytes stimulated by the extracellular matrix is a readily quantifiable measure of this capacity.

Animals↗

PubMed1984

Adult rat oligodendrocytes grown in vitro upon an extracellular matrix have the ability to proliferate.

The use of extracellular matrix (ECM) as a natural substrate for cell culture has markedly improved the growth and morphological differentiation of isolated adult rat oligodendrocytes. ECM-grown oligodendrocytes exhibited cyclic nucleotide phosphodiesterase (CNP)-activity which increased with time in culture and network formation. As much as 50-70% of the cells incorporated [3H]thymidine as visualized by the high labeling index of galactocerebroside (GalC)-positive cells. Chemical and enzymatic modifications of the ECM suggested that laminin in conjunction with other ECM constituents, plays a role in the induction of proliferation and/or differentiation responses in mature oligodendrocytes.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1984

Interaction of T lymphocytes and macrophages with cultured vascular endothelial cells: attachment, invasion, and subsequent degradation of the subendothelial extracellular matrix.

Circulating macrophages and T lymphocytes can invade the vascular endothelium and migrate from the circulatory system to an extravascular compartment such as inflammatory organs. In an in vitro model system we have examined the capacity of murine T lymphocytes and peritoneal macrophages to attach and invade a confluent vascular endothelial cell monolayer and to degrade sulfated proteoglycans in the subendothelial extracellular matrix. Concanavalin A and antigen-specific (egg albumin) activated T lymphocytes labeled with [3H]thymidine attached to the apical surface of the vascular endothelium in a time-dependent manner. A subsequent invasion of the endothelial cell monolayer was observed by scanning electron microscopy. Both activated T lymphocytes and murine macrophages degraded the [35S]O4 = -containing fragments in a process which required cell-matrix contact but was not dependent on serum proteases. Sulfated glycosaminoglycan chains produced from matrix proteoglycans by treatment with papain or alkaline borohydride were 3-4 times larger than the cell-mediated degradation fragments. This suggests that both macrophages and T lymphocytes elaborate upon stimulation an endoglicosidase capable of cleaving glycosaminoglycans specifically and releasing heparan sulfate-rich fragments. The ability of activated cells of the immune system to attach and invade the vascular endothelium and to degrade sulfated proteoglycans is very similar to that reported for highly metastatic tumor cells.

Animals↗

PubMed1984

Growth characteristics of human first trimester decidual cells cultured in serum-free medium: production of prolactin, prostaglandins and fibronectin.

A procedure for the establishment of pure human first trimester decidual cells in primary cultures has been developed. The high rate of success in obtaining such cultures resulted from the elimination of fibroblasts by appropriate enzyme dissociation and filtration of the initial tissue sample, and subsequent maintenance of the cells in a serum-free medium supplemented with insulin, fibroblast growth factor (FGF), estradiol, progesterone, hydrocortisone, transferrin and sodium selenite. Under these culture conditions, we obtained pure and actively dividing decidual cells forming tightly packed and nonoverlapping epithelioid cell monolayers covering more than 75% of the culture area. The cultured decidual cells retained their in vivo capacity to produce prolactin and various prostaglandins (PGs), primarily PGE2. There was a marked reduction in hormone production after 20 days in culture. A massive network of fibrillar surface fibronectin was detected by indirect immunofluorescence staining of the cultured cells. The production of prolactin and PGs together with the secretion of fibronectin may play a role in the implantation and subsequent growth of the embryo. The described procedure of obtaining fibroblast-free decidual cell monolayers will promote studies on the hormonal regulation of this tissue at the time of early intrauterine life.

Cells, Cultured↗

PubMed1984

Binding of hormonal steroids to isolated oligodendroglia and astroglia grown in vitro on a naturally produced extracellular matrix.

The ability of glial cells, grown on extracellular matrix (ECM), to bind corticosterone (CS), dexamethasone (Dex), and testosterone (T) was investigated. Isolated oligodendrocytes were able to specifically bind CS as well as T. Bound [3H]CS was partially displaced by excess T, whereas no displacement of [3H]T was observed with excess CS. Oligodendrocytes were not able to bind [3H]Dex. Astrocytes derived from hippocampal or hypothalamic slices showed no binding of either [3H]CS or [3H]Dex. The results demonstrate that ECM-grown oligodendrocytes possess receptors for CS and T but not for Dex. Furthermore, the binding sites for CS were less specific than the comparable sites for T. These results substantiate the notion that oligodendrocytes are involved in the action of steroid hormones within the brain.

Animals↗

PubMed1984

Interferon enhances prostacyclin production by cultured vascular endothelial cells.

The effects of interferon (IFN) on the arachidonate metabolism and physiological functions of cultured endothelial cells and blood platelets have been examined. Cultured bovine aortic endothelial cells were found to be sensitive to the antiviral and antiproliferative activities of human leukocyte (alpha) IFN and to increase their capacity to synthesize prostacyclin (PGI2) upon exposure to IFN. Several observations indicate that IFN stimulates PGI2 synthesis at the level of the enzymes phospholipase A2 and cyclooxygenase: (a) PGI2 production was dependent upon the supply of exogenous arachidonic acid or the liberation of endogenous cellular arachidonate by ionophore A23187, but was not observed when IFN-treated cells were exposed to the endoperoxide prostaglandin H2. (b) IFN had no effect on the spontaneous release of PGI2 into the culture medium during the incubation period (24-72 h). (c) The stimulatory effect of IFN on PGI2 production was inhibited by both glucocorticoids and indomethacin. The effect of IFN on platelet prostaglandin metabolism was also investigated. Incubation of platelet-rich plasma with IFN had no effect on platelet aggregation and thromboxane A2 production. The biological significance of the findings presented in this paper may be considered in view of the protective role of PGI2 in the vessel wall and the fact that infection with certain viruses induces endothelial damage both in man and experimental animal models.

Animals↗

PubMed1984

Degradation of sulfated proteoglycans in the subendothelial extracellular matrix by human platelet heparitinase.

Cultured vascular and corneal endothelial cells produce an underlying extracellular matrix (ECM) which induces platelet adherence, aggregation, and release reaction. Incubation of a metabolically (35S)O = 4-labeled ECM with platelet-rich plasma or washed platelets, but not with platelet-poor plasma, resulted in degradation of its heparan sulfate-containing proteoglycans into labeled fragments four to five times smaller than intact glycosaminoglycan side chains. These fragments were sensitive to deamination with nitrous acid and were not produced in the presence of heparin, indicating that heparan sulfate in the ECM is susceptible to cleavage by the platelet heparitinase. This degradation required adhesion of platelets to the ECM rather than aggregation since it was not inhibited by aspirin, which prevented platelet aggregation but not adherence. The enzyme was not released during aggregation of platelets on the ECM but was readily liberated upon their exposure to thrombin. This liberation was inhibited in the presence of prostacyclin (PGI2). Isolated high molecular weight proteoglycans first released from the ECM by incubation with platelet poor plasma served as a substrate for further degradation by the platelet heparitinase, suggesting a cascade mechanism for degradation of heparan sulfate in the ECM. Heparitinase, although to a lower level, was also active when washed platelets were added on top of a confluent endothelial cell monolayer covering the (35S)O = 4-labeled ECM. It is suggested that the platelet heparitinase may be involved in the impairment of the integrity of the vessel wall and thus facilitate the extravasation of blood-borne cells.

Blood Platelets↗

PubMed1983

High-density lipoprotein and extracellular matrix promotes growth and plating efficiency of normal human mammary epithelial cells in serum-free medium.

A routine procedure has been developed for the establishment in culture of normal primary and secondary human mammary epithelial cells. The high (80-100%) rate of success resulted from the combined use of a serum-free medium supplemented with high-density lipoprotein (HDL) and of cell plating on a naturally produced extracellular matrix (ECM). Plating on ECM greatly improved cell attachment, plating efficiency and initial outgrowth. HDL supported epithelial cell proliferation and prevented their detachment and degeneration while the omission of serum prevented the growth of stromal fibroblasts. Under these conditions we obtained from each specimen, and regardless of the patient's age, pure and actively dividing epithelial cell cultures forming a tightly packed and non-overlapping cell monolayer covering the entire area of the culture dish. These epithelial cultures could be easily dissociated and subcultured at a split ratio of 1:10. The described procedure will promote studies on the role of hormones and growth factors in the proliferation and differentiation of human mammary epithelial cells and on the susceptibility of human breast epithelial cells to various transforming agents and anti-cancer treatments.

Blood↗

PubMed1983

Enhanced growth and morphological differentiation of isolated adult rat oligodendrocytes in vitro: use of a naturally produced extracellular matrix.

Attachment, growth and morphological differentiation of isolated adult rat oligodendrocytes cultured on a naturally produced basement membrane-like extracellular matrix (ECM) occurred much faster than on poly-L-lysine (PLL) coated tissue culture dishes. In each individual trial the cells cultured on ECM exhibited, within 3-5 days in culture, a massive outgrowth of long and branched cytoplasmic processes. Outgrowth to such an extent, using PLL or plastic tissue culture dishes, was not observed even after 2 weeks in culture. The successful high plating efficiency, rapid growth and network formation as well as its resemblance to the in vivo environment of cells make this naturally produced substrate a superior substitute for PLL and therefore more attractive for studying the behavior and function of oligodendrocytes in vitro.

Animals↗