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Biomedical subjects

J Varani

Publications and source records attributed to J Varani.

At least 91 records · Page 5Linked to original sources
PubMed1992

Monocyte diapedesis through an in vitro vessel wall construct: inhibition with monoclonal antibodies to thrombospondin.

Human peripheral blood monocytes were examined for migration across an endothelial cell monolayer in an in vitro vessel wall construct. Few monocytes invaded in the absence of a chemotactic gradient, despite significant adhesion to the endothelial monolayer. However, the addition of zymosan-activated human plasma to the lower compartment, to create a chemotactic gradient across the vessel wall, resulted in significantly enhanced monocyte migration. Pretreatment of the monocytes with monoclonal antibodies to thrombospondin (TSP) dramatically inhibited monocyte diapedesis into the vessel wall. The same treatment inhibited monocyte adhesion to endothelial cells in two-dimensional monolayer cultures as well as in vessel wall constructs (no chemotactic gradient). Of interest, however, the monoclonal antibodies had no inhibitory effect on monocyte migration into collagen gels devoid of endothelial cells in response to the same chemotactic gradient, suggesting the importance of TSP in monocyte-endothelial cell interactions. Monoclonal antibodies to fibronectin and normal mouse immunoglobulin G did not inhibit migration in this model of a vessel wall. Furthermore, monoclonal antibodies to TSP showed no inhibition of human peripheral blood neutrophil migration. Previous studies have shown that monocytes synthesize TSP and express this moiety on their surface. The present data suggest that monocytes may utilize TSP to interact with endothelial cells lining the vessel wall during diapedesis.

Antibodies, Monoclonal↗
PubMed1992

Synergism among oxidants, proteinases, phospholipases, microbial hemolysins, cationic proteins, and cytokines.

A striking similarity exists between the pathogenetic properties of group A streptococci and those of activated mammalian professional phagocytes (neutrophils, macrophages). Both types of cells are endowed by the ability to adhere to target cells; to elaborate oxidants, hydrolases, and membrane-active agents (hemolysins, phospholipases); and to freely invade tissues and destroy cells. From the evolutionary point of view, streptococci might justifiably be considered the forefathers of "modern" leukocytes. Our earlier findings that synergy between a streptococcal hemolysin (streptolysin S, SLS) and a streptococcal thiol-dependent proteinase and between cytotoxic antibodies+complement and streptokinase-activated plasmin readily killed tumor cells, led us to hypothesize that by analogy to the pathogenetic mechanisms of streptococci, the mechanisms of tissue destruction initiated by activated leukocytes in inflammatory sites, as well as in tissues undergoing episodes of ischemia and reperfusion, might also be the result of the synergistic effects among leukocyte-derived oxidants, phospholipases, proteinases, cytokines, and cationic proteins. The current report extends our previous synergy studies with endothelial cells to two additional cell types--monkey kidney epithelial cells and rat beating heart cells. Monolayers of 51Cr-labeled cells that had been treated by combinations of sublytic amounts of hydrogen peroxide (generated either by glucose oxidase, xanthine-xanthine oxidase, or by paraquat) and with sublytic amounts of a variety of membrane-active agents (streptolysin S, phospholipases A2 and C, lysophosphatides, histone, chlorhexidine) were killed in a synergistic manner (double synergy). Crystalline trypsin markedly enhanced cell killing by combinations of oxidant and the membrane-active agents (triple synergy). Injury to the cells was characterized by the appearance of large membrane blebs that detached from the cells and floated freely in the media, looking like lipid droplets. Cytotoxicity induced by the various combinations of agonists was depressed, to a large extent, by scavengers of hydrogen peroxide (catalase, dimethyl thiourea, and by Mn2+) but not by SOD or by deferoxamine. When cationic agents were employed together with hydrogen peroxide, polyanions (heparin, polyanethole sulfonate) were also found to inhibit cell killing. It is proposed that in order to effectively combat the deleterious toxic effects of leukocyte-derived agonists on cells and tissues, antagonistic "cocktails" comprised of cationized catalase, cationized SOD, dimethylthiourea, Mn(2+)+glycine, proteinase inhibitors, putative inhibitors of phospholipases, and polyanions might be concocted. The current literature on synergistic phenomena pertaining to mechanisms of cell and tissue injury in inflammation is selectively reviewed.

Animals↗
PubMed1992

Expression of plasminogen activator and plasminogen activator inhibitor mRNA in human fibroblasts grown on different substrates.

mRNA levels for urokinase type plasminogen activator (uPA), tissue type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator inhibitor-2 (PAI-2) were examined in human diploid (neonatal foreskin) fibroblasts grown in 200-ml microcarrier suspension culture. Four different substrates were used. These included gelatin-coated polystyrene plastic, DEAE-dextran, glass-coated polystyrene plastic and uncoated polystyrene plastic. Our previous studies have shown that culture fluids from diploid fibroblasts grown on DEAE-dextran contained higher levels of plasminogen-dependent fibrinolytic activity than culture fluids from the same cells grown on other substrates. The increased plasminogen activator activity was due largely to elevated amounts of tPA (In Vitro Cell. Develop. Biol. 22: 575-582, 1986). The present study shows that there is a corresponding elevation of tPA mRNA in diploid fibroblasts cultured on DEAE-dextran relative to the other substrates. There does not appear to be any difference in uPA mRNA or in mRNA for PAI-1 or PAI-2 produced by the same cells on the four substrates. These data suggest that the influence of the substrate on plasminogen activator production is mediated at the genetic level.

Cell Division↗
PubMed1992

Mesangial cell killing by leukocytes: role of leukocyte oxidants and proteolytic enzymes.

Mesangial cells from human and rat kidney were examined for sensitivity to killing by neutrophils. Cells from both species were sensitive to killing by phorbol myristate acetate-stimulated neutrophils. Catalase was highly protective while superoxide dismutase was less protective and a number of protease inhibitors were not protective. Strong protection was also observed with the iron chelators, deferoxamine and phenanthroline, and with the hydroxyl radical scavengers, dimethylthiourea and 5,5-dimethyl-1-pyrroline N-oxide. Pretreatment of the mesangial cells with deferoxamine followed by washing also provided protection. Mesangial cells were also killed by reagent hydrogen peroxide (H2O2) but were much less sensitive to injury by direct application of proteolytic enzymes. The ability of H2O2 to injure mesangial cells was prevented by pre-incubation of the H2O2 with human leukocyte myeloperoxidase. These data suggest that killing is due primarily to the generation of H2O2 by the stimulated neutrophils and its further reduction in an iron-catalyzed reaction. The hydroxyl radical may be the reduction product that actually mediates lethal injury but lack of scavenger specificity prevents definitively concluding this. Mesangial cell killing by activated neutrophils could be significantly inhibited by monoclonal antibodies to CD11/CD18 molecules, suggesting that close contact between the target and effector cells is required for cytotoxicity. Although qualitatively similar to endothelial cells, the mesangial cells appeared to be quantitatively more oxidant sensitive than previously examined human and rat endothelial cells. Taken together, these data show that mesangial cells from rat and human are sensitive to leukocyte-induced injury and that injury results via an oxidant pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

FGF promotes corneal stromal fibroblast motility.

It is well-known that growth factors accelerate wound healing by stimulating mitosis. Growth factors may also directly stimulate the motility of individual cells. We employed two different experimental methods to determine if fibroblast growth factor (FGF) enhances the individual motility of corneal stromal fibroblasts (independent of mitogenic effects). The effects of FGF on the motility of tissue-cultured rabbit corneal stromal fibroblasts were investigated in a modified Boyden chamber and by the agarose drop motility assay. Both assays showed a significant enhancement of stromal fibroblast motility by FGF at 100 ng/ml. It appears, therefore, that FGF may promote corneal stromal wound healing not only by increased cellular proliferation, but also by increased cellular motility.

Animals↗
PubMed1992

Modulation of growth in normal and malignant melanocytic cells by all-trans retinoic acid.

Human epidermal melanocytes were examined for proliferation under various conditions in the presence or absence of all-trans retinoic acid (RA). Under conditions which supported proliferation, RA at concentrations of 0.25-1.0 microgram/ml inhibited cell growth but was not cytotoxic. When melanocytes were cultured under conditions which by themselves did not support growth, RA did not overcome the growth limitation. Treatment of melanocytes with RA altered their morphological appearance. Alterations included retraction of dendritic processes, increased flattening, and a slight darkening of the cytoplasm in some of the cells. However, when examined biochemically, there was no significant change in the amount of malanin per cell or in tyrosinase activity. RA also inhibited proliferation of six different malignant melanoma lines. Inhibition was observed over the same RA concentrations and over the same time course in the melanoma cells as was seen in melanocytes. Inhibition of melanocyte and melanoma cell proliferation was slowly reversed following removal of RA from the culture medium. These results indicate that RA can inhibit proliferation of melanocytic cells.

Animals↗
PubMed1992

Modulation of Ca2+ levels in keratinocytes by all-trans retinoic acid.

Human epidermal keratinocytes, that have been growth-arrested by removal of epidermal growth factor from the culture medium, are stimulated to proliferate by all-trans retinoic acid (RA). The same treatment inhibits the onset of differentiated features and reduces cell-substrate adhesion. In the present study we show that the same treatment results in a decrease in total cell-associated Ca2+ as measured by changes in the amount of 45Ca2+ bound to cells at equilibrium following RA treatment and by a decrease in intracellular free Ca2+ levels as measured with the Ca(2+)-sensitive dye, Indo-1. The alterations in Ca2+ levels were evident within an hour after RA treatment, were in the range of 30-35% and occurred over the same RA concentration range that stimulated proliferation (i.e., 0.25-1.0 micrograms/ml). When the extracellular Ca2+ concentration was elevated from the normal level of 0.15-1.4 mM, intracellular free Ca2+ increased by a factor of 2 while total cell-associated Ca2+ increased approximately 6-fold. Even under conditions of high extracellular Ca2+, RA was able to reduce cell-associated and intracellular free Ca2+. These data indicate that RA has the capacity to lower Ca2+ levels in keratinocytes concomitantly with its effects on biological behavior.

Calcium↗
PubMed1992

Human umbilical vein endothelial cell killing by activated neutrophils. Loss of sensitivity to injury is accompanied by decreased iron content during in vitro culture and is restored with exogenous iron.

First passage human umbilical vein endothelial cells (HUVECs) were sensitive to killing by activated neutrophils and reagent hydrogen peroxide (H2O2). Catalase and deferoxamine prevented killing whereas soybean trypsin inhibitor and superoxide dismutase did not. In these regards, HUVECs are similar to previously characterized endothelial cells from bovine and rat. Although first passage HUVECs were killed by activated neutrophils, sensitivity fell off rapidly as the cells were maintained in culture. At passage 2 (four population doublings), and beyond, HUVECs were highly resistant. The cells also became resistant to killing by reagent H2O2. The acquisition of resistance to killing was not accompanied by a failure to up-regulate neutrophil adhesion molecules or to support neutrophil adhesion. Levels of intracellular anti-oxidants (total thiols, though not glutathione, glutathione peroxidase or catalase activity) increased as a function of passage in culture. However, levels of glutathione and total thiols in late passage (resistant) HUVECs were similar to levels in late passage rat pulmonary artery endothelial cells, that were sensitive to killing by activated neutrophils. Cell-associated iron in HUVECs fell as a function of time in culture. By passage 2, the amount of total iron measurable with the Ferrozine reagent was only about 30% of the amount recovered from first passage HUVECs. The loss of iron from the cells may underlie much of the concomitant resistance to killing because when the cells were pretreated with iron under conditions in which it could be taken up, sensitivity to killing by activated neutrophils and by H2O2 was restored.

Cells, Cultured↗
PubMed1992

Protection of human umbilical vein endothelial cells by glycine and structurally similar amino acids against calcium and hydrogen peroxide-induced lethal cell injury.

Cultured human umbilical vein endothelial cells treated with either the calcium ionophore, ionomycin, or ionomycin plus cyanide-m-chlorophenylhydrazone had immediate severe depletion of adenosine triphosphate, (ATP) and increases of cytosolic free calcium (Caf) and then sustained lethal cell injury as manifested by release of lactate dehydrogenase and failure to exclude vital dyes within 15 minutes. Inclusion of glycine in the experimental medium prevented the enzyme leakage for at least 60 minutes without altering the ATP depletion or increases of Caf. The physiologic glycine concentration of 0.25 mmol/l gave 50% protection, and protection was complete at 1 mmol/l. Several other small neutral amino acids, L- and D-alanine, beta-alanine, 1-aminocyclopropane-1-carboxylate, alpha-aminoisobutyrate, and L-serine, had effects similar to glycine, but other amino acids and metabolic substrates did not. The endothelial cells were relatively resistant to damage from hydrogen peroxide, but sensitivity could be increased by preloading with Fe2+. In both non-loaded and Fe(2+)-loaded cells, hydrogen-peroxide-induced lactate dehydrogenase (LDH) release developing over 180 minutes was prevented by glycine in a fashion analogous to that seen with ionomycin damage. Mn2+ also partially protected against hydrogen peroxide injury but was not required for glycine's effects. These data demonstrate that striking modulatory effects of glycine and structurally similar amino acids that have previously been characterized in most detail using kidney tubule cells are strongly expressed in human umbilical vein endothelial cells and are involved in their response to Ca2+ and oxidant-mediated damage. These amino acid effects must be considered in the design of in vitro studies of endothelial cell injury and may contribute to endothelial cell pathophysiology in vivo.

Amino Acids↗
PubMed1991

Production of fibronectin by human tumor cells and interaction with exogenous fibronectin: comparison of cell lines obtained from colon adenocarcinomas and squamous carcinomas of the upper aerodigestive tract.

Cell lines derived from 13 different human colon adenocarcinomas were examined for production of fibronectin by ELISA and for cell-surface expression of fibronectin by indirect immunofluorescence. Two squamous epithelial cell lines obtained from tumors of the upper aerodigestive tract were used as controls. None of the 13 colon carcinoma lines produced detectable amounts of fibronectin or showed detectable cell-surface staining with anti-fibronectin. The 2 squamous epithelial cell lines, in contrast, produced large amounts of fibronectin which could be detected in the culture medium and bound to the substratum. The squamous carcinoma cells also stained brightly when examined in the viable state by immunofluorescence with anti-fibronectin. In addition to being studied for fibronectin production, each cell line was also examined for the ability to interact with exogenous fibronectin in an adhesion assay. None of the colon carcinoma cells were adherent to fibronectin-coated culture dishes while the 2 squamous carcinoma cells rapidly attached and spread on this substratum. These data suggest that cell lines derived from adenocarcinomas of the colon are deficient in production of fibronectin and in their ability to interact with exogenous fibronectin. In their degree of deficiency, the colon carcinoma cells are significantly different from several different types of human tumor cell. The failure of the colon carcinoma cells to synthesize detectable amounts of fibronectin endogenously or to interact with exogenous fibronectin may explain, in part, the low degree of adhesive interaction which these cells have for their substratum. This, in turn, may influence the in vitro and in vivo properties of colon carcinoma cells.

Adenocarcinoma↗
PubMed1991

Hydrogen peroxide-induced cell and tissue injury: protective effects of Mn2+.

Recent evidence indicates that under in vitro conditions, superoxide anion and hydrogen peroxide (H2O2) are unstable in the presence of manganese ion (Mn2+). The current studies show that in the presence of Mn2+, H2O2-mediated injury of endothelial cells is greatly attenuated. A source of bicarbonate ion and amino acid is required for Mn2+ to exert its protective effects. Injury by phorbol ester-activated neutrophils is also attenuated under the same conditions. EDTA reverses the protective effects. Acute lung injury produced in vivo in rats by intratracheal instillation of glucose-glucose oxidase is almost completely blocked in rats treated with Mn2+ and glycine. Conversely, treatment of rats with EDTA, a chelator of Mn2+, markedly accentuates lung injury caused by glucose-glucose oxidase. These data are consistent with the findings of others that Mn2+ can facilitate direct oxidation of amino acids with concomitant H2O2 disproportionation. This could form the basis of a new therapeutic approach against oxygen radical-mediated tissue injury.

Animals↗
PubMed1991

Identification of laminin domains involved in branching morphogenesis: effects of anti-laminin monoclonal antibodies on mouse embryonic lung development.

We recently found that polyclonal antibodies to laminin, a basement membrane-related glycoprotein, inhibited murine lung morphogenesis when added to organ cultures of mouse embryonic lung. Using a series of monoclonal anti-laminin antibodies with previously characterized subunit specificity (termed AL-1, AL-2, AL-3, AL-4, and AL-5), the deposition and functional involvement of different laminin domains in the developing lung were investigated. By immunohistochemistry the antibodies' reactivity was largely localized to the basement membrane, but was also present diffusely in the extracellular matrix throughout the mesenchyme. Organ cultures of lung explants from Day 12 embryos were cultured for 3 days in the presence of 50-100 micrograms/ml of each antibody or in the presence of the same concentration of immunoglobulins G and M, laminin-neutralized antibody, or medium alone. Cultures were monitored by phase-contrast microscopy, light microscopy, and immunofluorescence. Although all antibodies penetrated the tissues in culture, only two of them inhibited branching activity. These two antibodies were AL-1, which binds on or near the cross region of laminin, and AL-5, which binds to the lateral short arms at the globular end regions of the B chain of laminin. Inhibition of branching with these two antibodies was dose-dependent and statistically significant for the two concentrations used. AL-2, AL-3, AL-4, laminin-neutralized antibodies and control immunoglobulins did not alter lung morphogenesis. The two domains of laminin that promote lung branching morphogenesis have been reported by others to promote the attachment of a variety of cells and/or bind heparin. These domains of laminin may promote branching morphogenesis by facilitating cell attachment and, consequently, cell proliferation.

Animals↗
PubMed1991

Thrombospondin production and thrombospondin-mediated adhesion in U937 cells.

U937 cells have low levels of surface thrombospondin (TSP) under control conditions but express higher levels after treatment for 1 day with 100 nM phorbol myristate acetate (PMA). Increased surface expression is due, in part, to increased biosynthesis. Untreated U937 cells do not adhere to TSP-coated plastic culture dishes but adhere strongly to TSP after stimulation with PMA. Untreated U937 cells also adhere weakly to endothelial cell monolayers while PMA-treated U937 cells attach strongly to monolayers of rat pulmonary artery endothelial cells. Endothelial cell adhesion appears to be mediated, in part, by TSP since antibodies to TSP partially inhibit.

Animals↗
PubMed1991

Differential expression of glycoproteins containing alpha-D-galactosyl groups on normal human breast epithelial cells and MCF-7 human breast carcinoma cells.

Cell surface glycoproteins were isolated from the lysates of 125I-labeled normal human mammary epithelial cells (NHMEC) and from the human breast carcinoma cell line MCF-7, of blood-group O phenotype, by affinity chromatography on Griffonia simplicifolia I lectin-Sepharose. Specific elution of glycoproteins from the column with methyl alpha-D-galactoside suggests the presence of alpha-D-galactosyl groups on these moieties. SDS-PAGE analysis of isolated glycoproteins revealed both quantitative and qualitative differences between glycoproteins from normal and malignant cells. Three major glycoproteins of Mr 180 kDa, 85 kDa and the 44 kDa were obtained from MCF-7 cells. The 180-kDa glycoprotein was absent in NHMEC and the 44-kDa glycoprotein was very weakly expressed in these cells. The only glycoprotein which was found in almost equal amount in the lysate from both normal and malignant cells was the 85-kDa glycoprotein. These results indicate differences between normal human mammary epithelial cells and one kind of malignant human mammary epithelial cells, in the expression of glycoproteins containing alpha-D-galactosyl groups, irrespective of blood-group phenotype; they also demonstrate that alpha-D-galactosyl group are expressed in a very restrictive manner on the surface of this tumor cell line.

Breast↗
PubMed1991

Halothane and isoflurane increase pulmonary artery endothelial cell sensitivity to oxidant-mediated injury.

Volatile anesthetics inhibit phagocytic cell function, yet little is known about their effects on target tissues or on the target tissue response to stimulated phagocytes. Experiments were performed to determine how exposure to halothane and isoflurane changes rat pulmonary artery endothelial cell (RPAEC) viability in response to the toxic oxygen metabolites produced by stimulated phagocytic cells. RPAECs were grown in monolayer culture. The monolayers were treated with phorbol myristate acetate (PMA) -stimulated human neutrophils at an effector-to-target ratio of 20:1 after equilibration with 0.4% or 1.7% halothane or 0.7% or 2.8% isoflurane. As measured by percent-specific release of incorporated 51Cr label (mean +/- SE), cytotoxicity in the presence of 1.7% halothane (75.3 +/- 3.4%) was significantly greater (P less than 0.02) than cytotoxicity in 5% CO2 in air (44.7 +/- 3.3%) and in 0.4% halothane (57.3 +/- 4.7%). Also, cytotoxicity in 1.7% halothane was significantly greater than in 0.4% halothane (P less than 0.02). The authors found that RPAECs incubated in isoflurane exhibited significantly greater release of 51Cr than cells incubated in the MAC equivalent concentrations of halothane: 78.2 +/- 2.6% in 0.7% isoflurane (P = 0.0004) and 83.8 +/- 1% in 2.8% isoflurane (P = 0.005). Because early neutrophil cytotoxicity has been found to be mediated primarily by hydroxyl radical (HO.) and hydrogen peroxide (H2O2), the authors measured H2O2 production by similar numbers of PMA-stimulated neutrophils under similar exposure conditions. In carrier gas, PMA-stimulated neutrophils produced 20.5 +/- 1.3 nmol H2O2.10(6) cells-1.h-1. At the higher concentrations of halothane, H2O2 production actually was inhibited in comparison with carrier gas (15.4 +/- 1.4 nmol H2O2.10(6) cells-1.h-1 in 1.7% halothane and 16.8 +/- 0.8 in 2.8% halothane), but the degree of inhibition did not reach statistical significance. In isoflurane, however, H2O2 production was not different from that seen in carrier gas. In other experiments, the monolayers were treated with 0, 200, 500, and 1,000 microM H2O2 after equilibration with 0.4%, 1.7%, and 2.8% halothane or 0.7%, 2.8%, and 5% isoflurane in 5% CO2 in air. Efficiency of replating was used to measure degree of injury. Both halothane and isoflurane enhance the sensitivity of the RPAEC monolayers to injury by H2O2. The sensitizing effect of halothane was reversed by removing the anesthetic. Halothane and isoflurane thus enhance RPAEC sensitivity to injury by both H2O2 and PMA-stimulated neutrophils. In increasing RPAEC sensitivity to injury by oxygen metabolites, halothane and isoflurane may be inhibiting processes involved in intracellular antioxidant defenses.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Induction of proliferation of growth-inhibited keratinocytes and fibroblasts in monolayer culture by sodium lauryl sulfate: comparison with all-trans retinoic acid.

We have previously shown that all-trans retinoic acid (RA) has the capacity to stimulate proliferation of growth-inhibited human epidermal keratinocytes and growth-inhibited human dermal fibroblasts. The same treatment also stimulates extracellular matrix synthesis by fibroblasts (J Invest Dermatol 93:449; 94:717). In the present study we have examined the capacity of sodium lauryl sulfate to stimulate keratinocyte and fibroblast proliferation under the same conditions. Our data show that both cell types are stimulated to proliferate. Sodium lauryl sulfate is less potent than RA; it requires a higher molar concentration to achieve optimal stimulation and the number of responding cells at optimal concentrations is less with sodium lauryl sulfate than with RA. Further, there is a rapid onset of toxicity at concentrations of sodium lauryl sulfate that are only slightly higher than the optimal stimulatory concentration. Finally, sodium lauryl sulfate is less effective than RA in stimulating production of extracellular matrix (fibronectin, thrombospondin, and laminin) by dermal fibroblasts. Despite its ability to partially mimic RA as a stimulant of keratinocyte and fibroblast growth, sodium lauryl sulfate does not activate chloramphenicol acetyl transferase in cells co-transfected with retinoic acid receptors and a retinoic acid responsive element linked to the chloramphenicol acetyl transferase gene.

Cell Division↗