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R Montesano

Publications and source records attributed to R Montesano.

At least 19 recordsLinked to original sources
PubMed1992

Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor or vasculotropin, is a recently characterized endothelial-specific mitogen which is angiogenic in vivo. Here we demonstrate that VEGF is angiogenic in vitro: when added to microvascular endothelial cells grown on the surface of three-dimensional collagen gels, VEGF induces the cells to invade the underlying matrix and to form capillary-like tubules, with an optimal effect at approximately 2.2nM (100ng/ml). When compared to basic fibroblast growth factor (bFGF) at equimolar (0.5nM) concentrations, VEGF was about half as potent. The most striking effect was seen in combination with bFGF: when added simultaneously, VEGF and bFGF induced an in vitro angiogenic response which was far greater than additive, and which occurred with greater rapidity than the response to either cytokine alone. These results demonstrate that like bFGF, VEGF induces an angiogenic response via a direct effect on endothelial cells, and that by acting in concert, these two cytokines have a potent synergistic effect on the induction of angiogenesis in vitro. We suggest that the synergism between VEGF and bFGF plays an important role in the control of angiogenesis in vivo.

Adrenal Cortex↗
PubMed1992

Hepatocyte growth factor increases urokinase-type plasminogen activator (u-PA) and u-PA receptor expression in Madin-Darby canine kidney epithelial cells.

We have recently demonstrated that fibroblast-conditioned medium induces Madin-Darby canine kidney (MDCK) epithelial cells to form branching tubules when grown in three-dimensional collagen or fibrin gels (Montesano, R., Schaller, G., and Orci, L. (1991) Cell 66, 697-711), and that this morphogenetic effect is mediated by hepatocyte growth factor (HGF), also known as scatter factor (Montesano, R., Matsumoto, K., Nakamura, T., and Orci, L. (1991) Cell 67, 901-908). In fibrin gels, this effect is inhibited by addition of exogenous serine protease inhibitors, which suggests a role for plasminogen activators (PAs) in the matrix remodeling required for tubulogenesis. In the studies reported in this paper, we have investigated the effect of fibroblast-conditioned medium (CM) and HGF on the production of PAs by MDCK cells. We have found that urokinase-type PA (u-PA) activity and mRNA are increased 4.9-fold by CM from human MRC-5 fibroblasts, which has tubulogenic activity, but not by CM from human Detroit-550 fibroblasts, which lacks tubulogenic activity. The u-PA inductive property of MRC-5 CM was completely inhibited by preincubation with antibodies to recombinant human HGF (rhHGF). Exogenously added rhHGF also increased u-PA activity and mRNA 5.9-fold in MDCK cells, with an optimal effect at approximately 10 ng/ml. MRC-5 CM also increased u-PA receptor mRNA 34.9-fold in MDCK cells, an effect which was inhibited by 71% by preincubating the CM with antibodies to rhHGF, and which was mimicked by exogenously added rhHGF (31.3-fold increase). These results demonstrate that HGF, which induces tubulogenesis by MDCK cells in vitro, also increases u-PA and u-PA receptor expression in these cells. Taken together with our previous observations, this suggests that the resulting increase in extracellular proteolysis, appropriately localized to the cell surface, is required for epithelial morphogenesis.

Animals↗
PubMed1992

Plasminogen activator inhibitor-1 is induced in migrating endothelial cells.

It has been proposed that a finely tuned protease-anti-protease equilibrium must be maintained during processes of cell migration in order to limit extracellular proteolysis to the close proximity of the cell surface, and thereby to prevent excessive extracellular matrix degradation. We have previously shown that urokinase-type plasminogen activator (u-PA) activity is induced in microvascular endothelial cells migrating from the edges of a wounded monolayer in vitro (Pepper et al., J. Cell Biol., 105:2535-2541, 1987). By Northern analysis, we now demonstrate that plasminogen activator inhibitor 1 (PAI-1) mRNA is increased in multiple-wounded monolayers of bovine microvascular (BME) or aortic (BAE) endothelial cells, with a maximal 7- and 9-fold increase 4 h after wounding, respectively. By in situ hybridization, we demonstrate that the increase in PAI-1 mRNA is localized to cells at the edge of a wounded BME or BAE cell monolayer. The increase in PAI-1 mRNA observed in BME cells is independent of cell division and is inhibited by antibodies to basic fibroblast growth factor (bFGF), suggesting that PAI-1 induction in migrating cells is mediated by the autocrine activity of bFGF. Taken together with our previous observations on the induction of u-PA, these results support the hypothesis that the proteolytic balance in the pericellular environment of migrating cells is regulated through the concomitant production of proteases and protease inhibitors.

Animals↗
PubMed1992

Field studies of aflatoxin exposure, metabolism and induction of genetic alterations in relation to HBV infection and hepatocellular carcinoma in The Gambia and Thailand.

The relative contribution of aflatoxins (AF) and hepatitis B virus (HBV) to the aetiology of liver cancer remains to be determined, as does the mechanism of any interaction between these two factors. Methods to measure individual exposure to AF permit the assessment of this possible interaction in field studies. The measurement of AF covalently bound to albumin in peripheral blood has been particularly useful in this respect. In east and west African countries the majority (75-100%) of individuals has been found positive (> 5 pg AFB1-lysine eq./mg albumin) for the AF-albumin adduct with levels ranging up to 720 pg/mg. Levels of adduct to date have been age- and sex-independent, although marked seasonal variations were seen in The Gambia. Exposure also occurs in utero, with the AF-adduct being found in umbilical cord blood. In a study in The Gambia involving 323 children (age 3-8 years) the AF-albumin adduct levels were examined with respect to HBV infection and ethnic group. Over 95% of all sera contained detectable adduct but children positive for HBV surface antigen (HBsAG) had significantly higher adduct levels than children with markers of past infection or who had never been infected (mean (log) AF-albumin adduct levels 4.41 +/- 0.95, 4.04 +/- 0.99, and 4.05 +/- 1.03 respectively, p = 0.04). In addition, there were highly significant differences in adduct levels between the three major ethnic groups (Wollof 4.41 +/- 0.69: Fula 4.05 +/- 1.1; Mandinka 3.7 +/- 1.14). Wollof children were also more likely to be HBsAg positive than the other two groups. These data suggest that ethnic group and HBV infection can influence AF metabolism and this is being examined in this population with respect to genetic polymorphisms in cytochrome P450 and glutathione-S-transferase enzymes. In addition, these biomarkers are being compared to the nature and frequency of mutations in somatic and tumour cells.

Aflatoxins↗
PubMed1992

Workshop on DNA repair.

A workshop on DNA repair with emphasis on eukaryotic systems was held, under the auspices of the EC Concerted Action on DNA Repair and Cancer, at Noordwijkerhout (The Netherlands) 14-19 April 1991. The local organization of the meeting was done under the auspices of the Medical Genetic Centre South-West, The Netherlands (MGC), c/o Department of Radiation Genetics and Chemical Mutagenesis, University of Leiden (The Netherlands). Local organizers were: D. Bootsma (chairman), W. Ferro, J.H.J. Hoeijmakers, A.R. Lehmann, P.H.M. Lohman, L. Mullenders, and A.A. van Zeeland (secretarial assistance: Mrs. C. Escher-van Heerden and Mrs. R. Bontre). Over 190 scientists participated, and the format of the meeting followed that of the 1987 workshop on the 'Molecular Aspects of DNA Repair' (Friedberg et al., 1987). Plenary review talks in the mornings were followed, in the afternoon, by poster viewing in three or four parallel sessions. Groups of 15-20 posters were discussed in detail, and later on, in plenary sessions, chairpersons of the poster discussions reviewed the afternoons' posters. The principal themes of the meeting were the isolation and characterisation of repair genes and proteins, repair in specific sequences, consequences of defective DNA repair, and new methods for detecting DNA damage and repair. Remarkable progress has been made recently in all of these areas, and many exciting new results were presented. It is impossible to summarize all contributions to this (intensive) one-week meeting. Therefore, and for the sake of coherence, presentations that did not fit easily into any of the general themes of the meetings have not been included.

Animals↗
PubMed1992

Aflatoxin M1 in human breast milk from The Gambia, west Africa, quantified by combined monoclonal antibody immunoaffinity chromatography and HPLC.

Maternal to child exposure of aflatoxin M1 in breast milk is an underevaluated risk factor from dietary exposure to aflatoxin B1. A molecular dosimetry study in The Gambia, West Africa, was initiated to explore the relationships between dietary intake of aflatoxins during a 1 week period and a number of aflatoxin biomarkers including aflatoxin metabolite excretion into breast milk. For the breast milk study, five lactating women were identified and milk samples were collected by hand expression once a day during days 3-7 for three women and during days 3-6 for the two other women. Aflatoxin M1 (AFM1) in human milk was measured in all five subjects by a preparative monoclonal antibody immunoaffinity column/HPLC method. In three of the five women, aflatoxin G1 was found. Estimates of the percentage of aflatoxin in the diet excreted as AFM1 in milk ranged from 0.09 to 0.43%. Thus, these data indicate that a rapid methodology exists to assess the levels of AFM1 excretion in human milk and to use this approach as a biomarker for exposure of children to this carcinogen.

Aflatoxin M1↗
PubMed1992

Predictors of poliomyelitis case confirmation at initial clinical evaluation: implications for poliomyelitis eradication in the Americas.

In 1985, the Pan American Health Organization adopted the goal of eradication of poliomyelitis from the Americas by 1990. Strategies to accomplish this included high vaccination coverage, aggressive outbreak control, and active surveillance for acute flaccid paralysis (AFP). Although the sensitivity of AFP surveillance for detecting paralytic poliomyelitis cases is high, studies have shown the specificity to be low. In 1990, 2497 notified cases of AFP were investigated in the Americas of which 2146 had stool specimens collected. However, only 18 were confirmed as poliomyelitis by isolation of wild poliovirus from stool specimens, 71 were classified as being compatible with poliomyelitis. Cases of AFP due to causes other than poliomyelitis result in extensive but unnecessary outbreak control measures. To predict, at initial clinical evaluation, the likelihood of future confirmation as a case of poliomyelitis, likelihood ratios (LR) were calculated for different combinations of clinical characteristics of AFP cases (249) from Mexico in 1989 and 1990. The best predictors in a child with AFP were proximal muscle involvement which progressed '4 days together with fever at onset of paralysis, and proximal and unilateral involvement with either fever at onset or paralysis which progressed '4 days. The odds would increase by 12 that the child would eventually be confirmed as poliomyelitis (19), based on a stool culture positive for wild poliovirus (95% confidence interval (CI) 2.6-55.9). A guide for use in the field is proposed whereby local health officials, often with little training in neurological evaluation, can predict at initial clinical examination the likelihood that an AFP case will subsequently be confirmed as poliomyelitis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1992

Coupling and connexin 43 expression in microvascular and large vessel endothelial cells.

Endothelial cells of the microvasculature differ both structurally and functionally from endothelial cells of larger vessels. To assess whether these cells also differ in terms of direct cell-to-cell communication, we compared gap junction-mediated intercellular coupling and connexin (Cx) expression in monolayer cultures of bovine microvascular and large vessel (aortic and pulmonary artery) endothelial cells. In confluent monolayers, junctional communication (as assessed by transfer of Lucifer Yellow) was greater between large vessel than between microvascular endothelial cells. Basal levels of connexin 43 (Cx43) and Cx43 mRNA were also greater in large vessel than in microvascular endothelial cells. When monolayers of microvascular endothelial cells were mechanically wounded, junctional communication was increased between migrating cells at the wound edge. In contrast, coupling between large vessel endothelial cells was not increased after wounding. The wound-induced increase in coupling between microvascular endothelial cells was accompanied by an increase in Cx43 and Cx43 mRNA. In contrast, Cx43 expression was unaltered after wounding monolayers of large vessel endothelial cells. These studies revealed differences in basal and wound-induced levels of coupling and Cx43 expression in microvascular and large vessel endothelial cells in vitro, raising the possibility that the role of coupling in endothelial cell function may be different in these different cell types.

Animals↗
PubMed1991

Vascular endothelial growth factor (VEGF) induces plasminogen activators and plasminogen activator inhibitor-1 in microvascular endothelial cells.

Extracellular proteolysis is believed to be an essential component of the angiogenic process. The effects of VEGF, a recently described angiogenic factor, were assessed on PA activity and PA and PAI-1 mRNA levels in microvascular endothelial cells. u-PA and t-PA activity were increased by VEGF in a dose-dependent manner, with maximal induction at 30 ng/ml. u-PA and t-PA mRNAs were increased 7.5- and 8-fold respectively after 15 hours, and PAI-1 mRNA 4.5-fold after 4 hours exposure to VEGF. At equimolar concentrations (0.5 nM), VEGF was a more potent inducer of t-PA mRNA than bFGF, while bFGF was a more potent inducer of u-PA and PAI-1 mRNAs. In addition, VEGF induced u-PA and PAI-1 mRNAs with kinetics similar to those previously demonstrated for bFGF. These results demonstrate the regulation of PA and PAI-1 production by VEGF in microvascular endothelial cells and are in accord with the hypothesis that extracellular proteolysis, appropriately balanced by protease inhibitors, is required for normal capillary morphogenesis.

Animals↗
PubMed1991

Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor.

We have previously shown that Madin-Darby canine kidney (MDCK) epithelial cells grown in collagen gels in the presence of fibroblasts or fibroblast-conditioned medium (CM) form branching tubules, instead of the spherical cysts that develop under control conditions. We now report that the fibroblast-derived molecule responsible for epithelial tubulogenesis is hepatocyte growth factor (HGF). First, addition of exogenous HGF to cultures of MDCK cells induces formation of epithelial tubules. Second, the tubulogenic activity of fibroblast CM is completely abrogated by antibodies to HGF. These results demonstrate that HGF, a polypeptide that was identified as a mitogen for cultured hepatocytes, has the properties of a paracrine mediator of epithelial morphogenesis, and suggest that it may play important roles in the formation of parenchymal organs during embryonic development.

Animals↗
PubMed1991

Induction of epithelial tubular morphogenesis in vitro by fibroblast-derived soluble factors.

We have designed an in vitro system in which Madin-Darby canine kidney (MDCK) epithelial cells are cocultured in collagen gels with fibroblasts under conditions precluding heterocellular contact. Using this experimental approach, we have obtained evidence that fibroblast-derived soluble factors play a crucial role in the control of epithelial morphogenesis. First, MDCK cells suspended alone in collagen gels form spherical cysts, whereas in the presence of fibroblasts they form branching tubules. Second, MDCK cells grown as a monolayer on fibroblast-containing collagen gels invade the underlying matrix, within which they form a network of tubules. Third, fibroblast-conditioned medium mimics the effects of coculture by eliciting tubulogenesis by MDCK cells. These results demonstrate the involvement of diffusible paracrine factors in morphogenetic epithelial-mesenchymal interactions and provide a strategy for their molecular characterization.

Animals↗
PubMed1991

Genetic analysis of human esophageal tumors from two high incidence geographic areas: frequent p53 base substitutions and absence of ras mutations.

Esophageal squamous cell carcinoma (ESC) samples from patients residing in Uruguay and in Normandy, France, where alcoholic beverages and tobacco smoke are major risk factors, were analyzed for point mutations in the p53 tumor suppressor gene. Among 34 tumors (15 from Normandy and 19 from Uruguay) 15 point mutations in the p53 gene that result in amino acid substitutions or chain termination were identified by polymerase chain reaction amplification of exons 5-8 and direct DNA sequencing. Base substitutions in ESC from these high-incidence areas are dispersed over the midregion of the p53 gene. There are differences between ESC and other types of gastrointestinal cancer in the nature of frequent base substitutions. CpG to TpG transitions were far less prevalent in these ESC than in colorectal tumors, whereas G to T transversions, rarely found in colon cancers, were found in one-fourth of the ESC samples. Base substitutions at A:T pairs constitute an important fraction of ESC p53 mutations, in contrast to mutation patterns in most other types of solid tumors. In contrast to the frequent mutation of the p53 gene in these samples, no mutations in the H-, K-, or N-ras genes were found in 16 tumors from Uruguay by direct sequencing of exons in which transforming mutations are known to occur. A previous study on ras mutations in ESC from France was also negative (M. C. Hollstein et al., Cancer Res., 48: 5119-5123, 1988). The role of distinct etiological factors in generating these differences and the potential for linking patient exposure histories with patterns of p53 mutations in high risk populations are considered.

Adult↗