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PubMed1993

Phenotypic and functional modulation of interleukin-2-activated peripheral blood mononuclear cells by anti-CD3 and anti-CD28 antibody.

For effective adoptive cancer immunotherapy, not only killer lymphocytes (cytotoxic T cells or NK cells) but also helper T cells are considered to be beneficial. The conventional culture of human lymphocytes with high-dose of recombinant interleukin -2 (rIL-2) yields a high proportion of killer cells (CD8+ or CD56+ cells) but is usually accompanied by a decrease in the proportion of CD4+ cells. Peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 monoclonal antibody (10 ng/ml) in the absence of rIL-2 during the initial 48-hr period of cultivation followed by the addition of rIL-2 (200 Japan reference units/ml) (referred to as anti-CD3-rIL-2) resulted in CD4+ cells expanding 8.0- to 63.3-fold at day 12-14. When PBMC were cultivated with rIL-2 alone, or with rIL-2 together with anti-CD3 initially, CD4+ cells expanded 1.7-6.2- or 5.7-36.9-fold, respectively. The expansion of CD4+ cells from PBMC in the anti-CD3-rIL-2 cultures was enhanced by the addition of anti-CD28 monoclonal antibody at the initiation of cultures (500 ng/ml) (referred to as anti-CD3/anti-CD28-rIL-2) (12.1- to 127.8-fold expansion). The anti-CD3-rIL-2 and anti-CD3/anti-CD28-rIL-2 cultures also demonstrated a lesser expansion of CD3-CD56+ cells (NK cells). The PBMC in the anti-CD3/anti-CD28-rIL-2 cultures showed low cytotoxic activity per cell; however, the cytotoxic activity per total culture was comparable to that of PBMC expanded with rIL-2 alone because of the larger total expansion of the former culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1999

In vitro modulation of macrophage phenotype and inhibition of polymer degradation by dexamethasone in a human macrophage/Fe/stress system.

A new in vitro accelerated biological model, the macrophage-FeCl2-stress system was used for the evaluation of dexamethasone (DEX)-polymer formulations. This model combines the effects of cells (macrophages), transition metal ions (Fe2+), and polymer stress to promote material biodegradation. The cell and material effects of DEX, either in solution or incorporated into a polyetherurethane matrix (DEX/PEU), were monitored. Cell morphology and hydroperoxide formation in the polymer during cell culturing were characterized. After a subsequent treatment with FeCl2 the development of environmental stress cracking in the polymer was evaluated. We attempted to duplicate the biodegradation of PEU in terms of environmental stress cracking (ESC). Our results support the direct involvement of macrophages in polyetherurethane oxidation, probably by inducing hydroperoxide formation in the polymer structure. Under the influence of stress or strain, polymers with sufficient hydroperoxides degrade in the presence of Fe2+ metal ions in a manner that closely resembles the stress cracking that is observed in vivo. By contrast, polymers treated with either agents that inhibit cell activation and/or the oxidative burst, or with cells with no oxidative burst did not show signs of the biodegradative process. We demonstrated a reduction in hydroperoxide formation and no later ESC development in macrophage-cultured PEU in the presence of DEX in solution or in DEX-loaded PEU. We believe the prevention of initial polymer oxidation by reducing the cell's potential to produce oxidative stress at the tissue-biomaterial interface can directly inhibit the ESC degradation of chronically implanted polymers. The in vitro macrophage-Fe-stress system is a valuable tool for reliable assessment and cost-effective evaluation of biomaterials.

Adult↗
PubMed1998

The gp200-MR6 molecule which is functionally associated with the IL-4 receptor modulates B cell phenotype and is a novel member of the human macrophage mannose receptor family.

The human gp200-MR6 molecule has previously been shown to have either an antagonistic or agonistic effect on IL-4 function, demonstrated by inhibition of IL-4-induced proliferation of T cells or mimicking of IL-4-induced maturation of epithelium, respectively. We now show that gp200-MR6 ligation can also mimic IL-4 and have an anti-proliferative pro-maturational influence within the immune system, causing up-regulation of co-stimulatory molecules on B lymphocytes. Biochemical analysis and cDNA cloning reveal that gp200-MR6 belongs to the human macrophage mannose receptor family of multidomain molecules. It comprises 1722 amino acids in toto (mature protein, 1695 amino acids; signal sequence, 27 amino acids) organized into 12 external domains (an N-terminal cysteine-rich domain, a fibronectin type II domain and 10 C-type carbohydrate recognition domains), a transmembrane region and a small cytoplasmic C terminus (31 amino acids) containing a single tyrosine residue (Y1679), but no obvious kinase domain. Strong amino acid sequence identity (77%) suggests that gp200-MR6 is the human homologue of the murine DEC-205, indicating that this molecule has much wider functional activity than its classical endocytic role. We also show that the gp200-MR6 molecule is closely associated with tyrosine kinase activity; the link between gp200-MR6 and the IL-4 receptor may therefore be via intracellular signaling pathways, with multifunctionality residing in its extracellular multidomain structure.

Amino Acid Sequence↗
PubMed1990

Modulation of membrane phenotype, matrix adhesion and microinvasiveness of metastatic tumour cells by HUdR.

The effect of HUdR, proved to be anti-metastatic in vivo, was studied in vitro on cell proliferation, nucleoside uptake, membrane fluidity, expression of galactosylated glycans and proteoglycans in metastatic HM tumour cells. The observed increase in membrane fluidity and the suppression of nucleoside transport were early events of the HUdR action followed by decrease of galactosylated glycan and HSPG expression. However, these changes did not influence the proliferation capacity of the cells at the concentrations studied. As a consequence of the membrane alterations a reduced adhesiveness and spreading on extracellular matrix components was detected. In addition, the HUdR treated HM cells showed reduced capacity to invade fibroblast monolayers in vitro. Based on these observations, HUdR could be the prototype of new anti-metastatic agents acting at the level of tumour-host interaction.

Animals↗
PubMed1994

Scatter factor modulates the metastatic phenotype of the EMT6 mouse mammary tumor.

EMT6 is a transplantable mouse mammary tumor cell line that has been utilized widely as a model system to study the effects of various treatments on local tumor growth and pulmonary metastasis. In this study, we examined the cellular mechanisms by which scatter factor (SF), a fibroblast-derived cytokine that stimulates epithelial cell motility, may contribute to tumor-cell dissemination, using the EMT6 model system. In vitro, SF stimulated EMT6 cell motility, invasiveness and cell-surface expression of urokinase (an enzyme required for cell migration through tissue). SF differentially stimulated EMT6 cell adhesion to and migration onto surfaces coated with collagen I and laminin. EMT6 cells treated in vitro with SF and injected i.v. into isogeneic BALB/c-Rw mice showed a small but significant increase (1.7-fold) in lung colony formation as compared with control cells. For EMT6 cells in vitro, SF had no effect on DNA synthesis, cell proliferation, cell size distribution, or in vitro colony-forming ability. Thus, the increase in lung colonization may be due to enhanced ability of SF-treated cells to adhere to subendothelial basement membrane or to invade through tissue. Studies of the tissue distribution of SF in BALB/c-Rw mice demonstrated high levels of active factor in the lung. Thus, the presence of endogenous pulmonary SF may have reduced the degree to which SF treatment stimulated EMT6 lung colonization. Significant SF activity was also found in extracts of EMT6 tumors. Cultured EMT6 cells did not produce SF, but did produce high titers of a soluble low-molecular-weight protein activity that is capable of stimulating SF production in human fibroblasts 3- to 5-fold. EMT6 tumor extracts contained high titers of a similar SF-inducing activity. These observations suggest that SF may contribute to the invasive and metastatic phenotype of EMT6 cells via a paracrine mechanism in which tumor cells induce the production of SF in stromal fibroblasts.

Animals↗
PubMed2002

Modulation of podocyte phenotype in collapsing glomerulopathies.

Podocytes are well-differentiated postmitotic cells whose function is largely based on their complex cytoskeletal architecture. In diseases with proteinuria, podocytes undergo morphologic changes. Podocytes react to an injurious stimulus by a reorganization of their foot process architecture that is independent of the primary injury and the cause of the proteinuria. Collapsing glomerulopathies, including the idiopathic and secondary forms due to HIV infection, have been previously considered a part of the focal sclerosing glomerulosclerosis (FSGS) spectrum. However, in contrast to FSGS, both forms of collapsing glomerulopathy are characterized by segmental and global collapse of the glomerular basement membrane (GBM) and by characteristic ultrastructural alterations in podocytes. These alterations include loss of the actin-based cytoskeleton, a dysregulated/dedifferentiated phenotype, cellular hypertrophy, and cell proliferation. These observations raise the following questions: 1) What mechanism causes glomerular collapse and do podocytes have a role? We recently proposed that in collapsing glomerulopathies the composition of the GBM is altered and contains more immature forms of collagen IV. These observations suggest that dedifferentiated/dysregulated podocytes may participate in remodeling the GBM composition, producing fetal collagen isoforms. 2) What is the pathomechanism underlying podocyte dysregulation? Although it is still unclear which etiologic factors are responsible for the idiopathic forms of collapsing glomerulopathy, in situ hybridization studies in a transgenic mouse model of HIV-associated collapsing glomerulopathy and on renal biopsies of patients with HIV-associated collapsing glomerulopathy demonstrated the presence of the HIV-1 RNA in podocytes and tubular epithelial cells. These findings suggest a direct link between viral gene expression and the dysregulation of the podocyte phenotype. 3) Another open question is how podocytes become infected in HIV-associated collapsing glomerulopathy. HIV-1 typically uses CD4 and a co-receptor such as CCR5 or CXCR4 to enter cells. So far, there is no demonstration of the expression of these receptors in podocytes. These negative findings, however, do not exclude the possibility that in the kidney another, CD4 independent, co-receptor may be used for viral cell entry. Finally, is it important to mention that collapsing glomerulopathies have a high prevalence in black patients, suggesting a link between racial background and the virus-related podocyte injury.

Cell Size↗
PubMed1995

Modulation of cell phenotype in human osteoblast-like cells by the simian virus 40.

At present, the majority of in vitro research into bone metabolism is performed on either primary cultures of bone or osteosarcoma lines. A better model of the behaviour of normal bone cells would be a cell line derived from normal, adult bone that retained osteoblast-like characteristics. We infected a culture of bone cells from adult humans with simian virus 1613, a variant of the simian virus 40, and obtained 12 clones of variable morphology. The clones were maintained in culture for as long as 6 months. Population doubling times, synthesis of alkaline phosphatase and osteocalcin, secretion of mineral, morphology, and ability to withstand freezing were examined. SV/EC cell morphology varied from the polygonal, osteoblast-like to the bipolar, fibroblast-like. Population doubling times ranged from 0.55 to 2.8 days (compared with 3.9 days for the nontransformed human osteoblast-like cells). Synthesis of alkaline phosphatase varied but was less than that by the human osteoblast-like cells. With the exception of clone 11, all of the transformed clones synthesised mineral in vitro under mineralising conditions. Four clones showed increased synthesis of alkaline phosphatase and increased population doubling times after passaging. All of the clones were successfully frozen and thawed, but, unlike normal human osteoblast-like cells, none responded to stimulation with parathyroid hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗
PubMed2005

Mast cell tryptase may modulate endothelial cell phenotype in healing myocardial infarcts.

Mast cells and macrophages infiltrate healing myocardial infarcts and may play an important role in regulating fibrous tissue deposition and extracellular matrix remodelling. This study examined the time-course of macrophage and mast cell accumulation in healing infarcts and studied the histological characteristics and protease expression profile of mast cells in a canine model of experimental infarction. Although macrophages were more numerous than mast cells in infarct granulation tissue, macrophage density decreased during maturation of the scar, whereas mast cell numbers remained persistently elevated. During the inflammatory phase of infarction, newly recruited leucocytes infiltrated the injured myocardium and appeared to be clustered in close proximity to degranulating cardiac mast cells. During the proliferative phase of healing, mast cells had decreased granular content and were localized close to infarct neovessels. In contrast, macrophages showed no selective localization. Mast cells in healing canine infarcts were alcian blue/safranin-positive cells that expressed both tryptase and chymase. In order to explain the pro-inflammatory and angiogenic actions of tryptase--the major secretory protein of mast cells--its effects on endothelial chemokine expression were examined. Chemokines are chemotactic cytokines that play an important role in leucocyte trafficking and angiogenesis and are highly induced in infarcts. Tryptase, a proteinase-activated receptor (PAR)-2 agonist, induced endothelial expression of the angiogenic chemokines CCL2/MCP-1 and CXCL8/IL-8, but not the angiostatic chemokine CXCL10/IP-10. Endothelial PAR-2 stimulation with the agonist peptide SLIGKV induced a similar chemokine expression profile. Mast cell tryptase may exert its angiogenic effects in part through selective stimulation of angiogenic chemokines.

Animals↗
PubMed2006

Biomechanical modulation of endothelial phenotype: implications for health and disease.

The functional phenotypic plasticity of the vascular endothelium relies on the ability of individual endothelial cells to integrate and transduce both humoral and biomechanical stimuli from their surrounding environments. Increasing evidence strongly suggests that biomechanical stimulation is a critical determinant of endothelial gene expression and the functional phenotypes displayed by these cells in several pathophysiological conditions. Herein we discuss the types of biomechanical forces that endothelial cells are constantly exposed to within the vasculature, explain how these biomechanical stimuli serve as regulators of endothelial function and discuss the increasing evidence that "atherosclerosis-protective" or "atherosclerosis-prone" haemodynamic environments can be important causative factors for atherogenesis via the differential regulation of endothelial transcriptional programmes.

Animals↗
PubMed2002

Familial factors and hearing impairment modulate the neuromotor phenotype in Turner syndrome.

In Turner syndrome (TS), an X-chromosomal anomaly characterised by total or partial loss of the second X-chromosome, muscle hypotonia, and lower proficiency in fine and gross motor skills have been described. It is assumed that the neuromotor phenotype in TS is linked with X-chromosomal factors (individual mosaicism) and with the oestrogen deficiency due to streak gonads. From studies in normal populations, a further risk factor may be recurrent otitis media (OM), which occurs frequently in TS, often in combination with hearing impairment (HI). It is also likely that familial factors are involved. The aim of our study was to specify the respective impact of the interindividual varying status of mosaicism and of hypergonadotropic hypogonadism as well as of the risk factors recurrent OM and HI in comparison to familial coinfluences on the neuromotor proficiency (MOP) in TS. We used the Bruininks-Oseretsky Test of Motor Proficiency to examine 105 subjects with TS (mean age 9.4 years; SD 3.3 years) and 52 sisters (mean age 9.8 years; SD 3.7 years). Significant correlations were found for familial covariance regarding the relation between TS subjects and their sisters ( r=0.42, P<0.01) and HI and MOP ( r=-0.39, P<0.01) CONCLUSION: we conclude that the familial influences and risk factors such as recurrent otitis media in combination with hearing impairment serve primarily as important predictors of the individual neuromotor phenotype in Turner syndrome.

Adolescent↗
PubMed1987

Cell lineages in peripheral nervous system ontogeny: medium-induced modulation of neuronal phenotypic expression in neural crest cell cultures.

Neural crest, taken from cephalic and trunk levels of quail embryos, was grown in vitro in conventional tissue culture medium (Dulbecco's modified Eagle's medium containing 15% fetal calf serum and either 2 or 15% chick embryo extract (CEE] or in a chemically defined serum- and CEE-free medium. Depending on the conditions employed, different types of neuronal or neuronlike cells developed in the cultures. Thus, in medium containing 15% CEE, adrenergic cells (identified by tyrosine hydroxylase immunoreactivity and catecholamine histofluorescence) emerged after 5-6 days. These cells lacked tetanus toxin binding sites and did not react with an antibody directed against 70-kDa neurofilament protein. In the fully defined medium, a neuronal cell type exhibiting neurofilament and substance P (SP) immunoreactivity differentiated from noncycling precursors within 1 or 2 days of culture. If serum was added to the medium, the neurites disintegrated and the neuronal cells ultimately died. By sequentially culturing neural crest, first in the wholly synthetic medium for 1-3 days and then in the conventional medium supplemented with serum and 15% CEE, the disappearance of the SP-positive neurons was followed, several days later, by the emergence of adrenergic cells. The majority of these cells and/or their precursors were found to undergo cell division in culture. We conclude that the cells expressing the adrenergic phenotype (characteristic of the sympathetic nervous system) and those displaying SP immunoreactivity, comparable to a category of neurons in dorsal root and cranial sensory ganglia, derive from distinct sets of precursors. Our results reinforce the contention, deduced from in ovo transplantation experiments (see N. M. Le Douarin, (1984) In Cellular and Molecular Biology of Neuronal Development (I. Black, Ed.), pp. 3-28. Plenum, New York), that at least two lineages, from which sensory and autonomic cell types are derived respectively, are segregated early during neural crest ontogeny and have extremely different survival and trophic requirements.

Acetylcholine↗
PubMed1991

The neuropeptide VIP modulates the neurotransmitter phenotype of cultured chick sympathetic neurons.

The present report provides evidence for a novel function for the neuropeptide vasoactive intestinal peptide (VIP). We demonstrate that VIP increases the cholinergic and the noradrenergic properties of cultured chick sympathetic neurons without changing neuronal survival and metabolism. VIP induces a 10- to 15-fold increase in the activity of choline acetyltransferase and an approximately twofold increase in the activity of tyrosine hydroxylase. Forskolin, an activator of adenylate cyclase, mimics all the effects of VIP on these cells. In addition, the effects of forskolin and VIP at optimal concentrations are not additive. Furthermore, VIP induces a rapid increase in the intracellular cAMP levels. Thus VIP acts via a cAMP-dependent pathway to enhance the cholinergic and noradrenergic properties of cultured chick sympathetic neurons.

Animals↗
PubMed1994

Modulation of the phenotype and function of bovine afferent lymph cells during infection with Trypanosoma congolense.

Alterations in the phenotype and function of cells isolated from bovine afferent lymph were studied following tsetse-transmitted Trypanosoma congolense infection. Little alteration was observed in the output of the CD2+ T cells in the lymph, and within this population the CD4:CD8 ratio remained relatively constant. By contrast, a marked decrease was observed in the output of gamma delta T cells over the first 7 days following infection. The number of B cells increased between 2 and 6 days post-infection, and thereafter returned to pre-infection values. Little change was observed within the afferent lymph veiled cell population. Examination of activation markers on the lymphocyte fraction of afferent lymph revealed a decrease in the number of cells expressing the Interleukin-2 receptor alpha-chain from Day 5 post-infection. At this time the expression of ACT 1, another early activation marker, was seen to increase. Afferent lymph cells collected pre-infection and on the first 4 days post-infection proliferated in response to stimulation with Concanavalin A in vitro. This response to mitogenic stimulation was completely abrogated from day five post-infection. However, these cells were not capable of suppressing the capacity of normal peripheral blood mononuclear cells to respond to mitogenic stimulus in co-culture assays. These studies suggest that although a degree of lymphocyte activation occurs in the afferent lymph following tsetse-transmitted infection with T. congolense, this may be sub-optimal owing to the immunosuppression which appears to operate at the level of the skin and the lymph nodes.

Animals↗
PubMed1995

Calorie restriction modulates lymphocyte subset phenotype and increases apoptosis in MRL/lpr mice.

Defective expression of the Fas apoptotic gene may account for overproduction of CD4- CD8- B220+ cells (double-negative) in MRL/MpJ-lpr/lpr (lpr) mice. Previous studies have shown that calorie restriction (CR) inhibits the development of autoimmune disease and extends life span in these animals. The present studies describe the effects of CR on the distribution of lymphocyte phenotypes, lymphocyte proliferative response, and cytokine release. The effects of CR on dexamethasone (DEX)-induced apoptosis were also studied using propidium iodide (PI) uptake and DNA fragmentation in splenocytes and lymph node (LN) cells. Weanling female mice were fed a nutritionally adequate semipurified diet either ad libitum (AL) or with 40% fewer calories than AL (CR), and killed at 5 months of age. CR mice had fewer palpable lymph nodes, and decreased serum anti-dsDNA antibodies. Mitogen (ConA, anti-CD3, and LPS) and superantigen (SEB)-induced proliferative response was significantly lower in lymphoid cells from AL fed animals. FACS analysis of cells from CR animals showed decreased CD4- CD8- cells in spleen (1.7-fold, P < 0.025) and LN (1.6-fold P < 0.01) and significantly higher CD4+ (spleen, 1.7-fold, P < 0.0001; LN, 2.6-fold, P < 0.025) and CD8+ (spleen, 1.6-fold, P < 0.001; LN, 5.2-fold, P < 0.005) cells. ConA-stimulated IL-2 release was increased in CR animals (splenocytes, 7.5-fold, P < 0.001; LN cells, 6.1-fold, P < 0.01). Finally, apoptosis in response to Dex was increased in CR animals as indicated by the presence of more PI-positive cells (spleen, 15.8%; LN, 10.7%; P < 0.01) and increased DNA fragmentation. In summary, the amelioration of autoimmune disease in MRL/lpr mice by CR is accompanied by prevention of the rise in 'double-negative' T cells and by maintenance of lymphocyte responsiveness to mitogens and DEX-induced apoptosis at higher levels.

Animals↗
PubMed1996

Pharmacological modulations of adrenergic phenotype in medullary C2 and C3 cell groups of adult rat.

The adrenergic phenotype was analysed in the rat's rostral dorsomedial medulla under normal conditions and 3 days after a single intraperitoneal injection of an eburnamine derivative, RU 24722, which increases tyrosine hydroxylase protein expression in the rostral portion of the nucleus tractus solitarius. This approach was investigated by a double immunofluorescence labelling of tyrosine hydroxylase and phenylethanolamine N-methyltransferase proteins. Under normal conditions, most adrenergic cell bodies are anatomically distributed in the dorsal and rostral medulla oblongata between the rostral part of the dorsal motor nucleus of the vagus nerve and the medial longitudinal fasciculus. Adrenergic neurons detected in this medullar region were distributed between both cell groups. Three days after the pharmacological RU 24722 treatment, an upregulation in tyrosine hydroxylase and phenylethanolamine N-methyltransferase protein expression was detected in both cell groups characterized by a highly increased number of tyrosine hydroxylase- and phenylethanolamine N-methyltransferase-containing cell bodies. The number of TH-mRNA containing neurons was also increased, indicating the transcriptional level of this regulation. These results demonstrated a particular neuronal plasticity of adrenergic phenotype in the medullary cell groups of adult rat.

Analysis of Variance↗
PubMed2000

Modulation of metastasis phenotypes of non-small cell lung cancer cells by 17-allylamino 17-demethoxy geldanamycin.

BACKGROUND: Cancer cells that overexpress c-erbB oncogenes exhibit resistance to chemotherapy, enhanced tumorigenicity, as well as increased propensity for metastasis. The aim of this study was to investigate if depletion of erbB-1/EGFR and erbB-2/HER2neu oncogene products by 17-allylamino 17-demethoxy Geldanamycin (17AAGA) could diminish the metastatic potential of non-small cell lung cancer (NSCLC) cells that express varying levels of the erbB1/erbB2 oncogenes. METHODS: NSCLC cell lines (H460, H358, H322, or H661) were assayed for expression of erbB1 and erbB2, the cell adhesion molecule E-cadherin, secretion of the matrix metalloproteinase 9 (MMP-9), and vascular endothelial cell growth factor (VEGF), as well as their ability to invade Matrigel after 48-hour exposure to 17AAGA. RESULTS: 17AAGA significantly depleted erbB1 or erbB2 levels in NSCLC cells expressing high levels of these proteins, and effectively inhibited their growth with IC50 values ranging from 50 to 90 nmol/L. Moreover, drug treatment enhanced E-cadherin expression in H322 and H358 cells, and inhibited secretion of MMP-9 and VEGF secretion by tumor cells. 17AAGA diminished hypoxia-induced upregulation of VEGF expression as well as growth factor-mediated augmentation of MMP-9 secretion, and profoundly inhibited the ability of H322 and H358 cells to migrate through Matrigel in response to chemoattractants. CONCLUSIONS: In addition to its known antiproliferative and chemosensitization effects, 17AAGA inhibits the metastatic phenotype of lung cancer cells. 17AAGA may be a novel pharmacologic agent for specific molecular intervention in lung cancer patients.

Benzoquinones↗
PubMed2001

Nitric oxide modulates the angiogenic phenotype of middle-T transformed endothelial cells.

The role of nitric oxide (NO) in the induction of angiogenesis was evaluated in a murine heart endothelioma cell line (H.end.FB) carrying the mT oncogene. Two clonal derivatives of H.end.FB, H80 and H73, exhibiting different NO synthase (NOS) activities were selected and used in the study. The relationship among NOS activity and tumor cell behaviour (growth, and angiogenic capacity) and the molecular control of gene expression were investigated. H.end.FB and H80 on one side and H73 on the other side exhibited the highest and lowest NOS activity, respectively. Cell growth was inversely correlated to the amount of NO produced by the cell lines. Conversely, in the avascular rabbit cornea assay, H.end.FB and H80 cells were strongly angiogenic, while H73 were poorly angiogenic, indicating that the ability of the cells to induce neovascularization was associated with the extent of NO produced. Consistently, systemic administration to rabbits of the NOS inhibitor N(w)-nitro-L-arginine methyl ester (L-NAME) significantly reduced the angiogenicity of H.end.FB cells. RT-PCR evidenced that H.end.FB expressed mRNA for TGF-beta1 and all VEGF isoforms, VEGF165 being predominantly expressed. NOS inhibition reduced the basal expression of VEGF isoforms, while it markedly potentiated TGF-beta1 expression. These results indicate that the endogenous production of NO in tumor cells can serve as an autocrine/paracrine signalling mechanism of progression, by controlling angiogenic factor/modulator expression.

Animals↗