Platelet-derived growth factor inhibits basic fibroblast growth factor angiogenic properties in vitro and in vivo through its alpha receptor

Basic fibroblast growth factor (bFGF) and platelet-derived growth factor-BB (PDGF-BB) modulate vascular wall cell function in vitro and angiogenesis in vivo. The aim of the current study was to determine how bovine aorta endothelial cells (BAECs) respond to the simultaneous exposure to PDGF-BB and bFGF. It was found that bFGF-dependent BAEC migration, proliferation, and differentiation into tubelike structures on reconstituted extracellular matrix (Matrigel) were inhibited by PDGF-BB. The role played by PDGF receptor alpha (PDGF-Ralpha) was investigated by selective stimulation with PDGF-AA, by blocking PDGF-BB-binding to PDGF-Ralpha with neomycin, or by transfecting cells with dominant-negative forms of the receptors to selectively impair either PDGF-Ralpha or PDGF-Rbeta function. In all cases, PDGF-Ralpha impairment abolished the inhibitory effect of PDGF-BB on bFGF-directed BAEC migration. In addition, PDGF-Ralpha phosphorylation was increased in the presence of bFGF and PDGF, as compared to PDGF alone, whereas mitogen-activated protein kinase phosphorylation was decreased in the presence of PDGF-BB and bFGF compared with bFGF alone. In vivo experiments showed that PDGF-BB and PDGF-AA inhibited bFGF-induced angiogenesis in vivo in the chick embryo chorioallantoic membrane assay and that PDGF-BB inhibited bFGF-induced angiogenesis in Matrigel plugs injected subcutaneously in CD1 mice. Taken together these results show that PDGF inhibits the angiogenic properties of bFGF in vitro and in vivo, likely through PDGF-Ralpha stimulation.     

Acidosis inhibits endothelial cell apoptosis and function and induces basic fibroblast growth factor and vascular endothelial growth factor expression

Endothelial cells are exposed to an acidotic environment in a variety of pathological and physiological conditions. However, the effect of acidosis on endothelial cell function is still largely unknown, and it was evaluated in the present study. Bovine aortic endothelial cells (BAECs) were grown in bicarbonate buffer equilibrated either with 20% CO(2) (pH 7.0, acidosis) or 5% CO(2) (pH 7.4, control). Acidosis inhibited BAEC proliferation in 10% FCS, whereas by day 7 in serum-free medium, cell number was 3-fold higher in acidotic cells than in control cells. Serum deprivation enhanced BAEC apoptosis, and apoptotic cell death was markedly inhibited by acidosis. Additionally, acidosis inhibited FCS-stimulated migration in a modified Boyden chamber assay and FCS-stimulated differentiation into capillary-like structures on reconstituted basement membrane proteins. Conditioned media from BAECs cultured for 48 hours either at pH 7.0 or pH 7.4 enhanced BAEC proliferation and migration at pH 7.4, and both effects were more marked with conditioned medium from BAECs grown in acidotic than in control conditions. Acidosis enhanced vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) mRNA expression as well as bFGF secretion, and a blocking bFGF antibody inhibited enhanced BAEC migration in response to conditioned medium from acidotic cells. These results show that acidosis protects endothelial cells from apoptosis and inhibits their proangiogenic behavior despite enhanced VEGF and bFGF mRNA expression and bFGF secretion.     

Lysophosphatidylcholine increases the secretion of matrix metalloproteinase 2 through the activation of NADH/NADPH oxidase in cultured aortic endothelial cells

Matrix metalloproteinases (MMPs) play a pivotal role in angiogenesis, atherogenesis, vascular remodeling after vascular injury, and instability of atherosclerotic plaque. The present study was undertaken to investigate the effect of lysophosphatidylcholine, a major component of oxidized low density lipoprotein (LDL), on the regulation of MMPs in cultured bovine aortic endothelial cells (BAECs). Furthermore, we explored the potential role of oxidative stress in the regulation of MMP. LPC increased the secretion of gelatinolytic activity, as well as, protein of MMP-2 from BAECs. The stimulation of BAEC with superoxide increased the production of MMP-2 and it also induced its activation. Electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin trap agent demonstrated that lysophosphatidycholine (LPC) induced generation of reactive oxygen (ROS) species from BAECs. The inhibition of NADH/NADPH oxidase, one of the potential sources of superoxide in endothelial cells, attenuated the effect of LPC. Our findings suggest that LPC might activate the endothelial NADH/NADPH oxidase to enhance superoxide production, and it might, in turn, enhance MMP-2 induction.     

Retinoic acid is a negative regulator for the differentiation of cord blood-derived human mast cell progenitors

We examined the effects of retinoids on the human mast cell development using a serum-deprived culture system. When 10-week cultured mast cells derived from CD34(+) cord blood cells were used as target cells, both all-trans retinoic acid (ATRA) and 9-cis RA inhibited the progeny generation under stimulation with stem cell factor (SCF) in a dose-dependent manner (the number of progeny grown by SCF plus RA at 10(-7) mol/L was one tenth of the value obtained by SCF alone). The early steps in mast cell development appear to be less sensitive to RA according to the single CD34(+)c-kit(+) cord blood cell culture study. The optimal concentration of RAs also reduced the histamine concentration in the cultured mast cells (3.00 +/- 0.47 pg per cell in SCF alone, 1.44 +/- 0.18 pg per cell in SCF+ATRA, and 1.41 +/- 0.10 pg per cell in SCF+9-cis RA). RT-PCR analyses showed the expression of RARalpha, RARbeta, RXRalpha, and RXRbeta messenger ribonucleic acid (mRNA) in 10-week cultured mast cells. The addition of an RAR-selective agonist at 10(-10) mol/L to 10(-7) mol/L decreased the number of mast cells grown in SCF, whereas an RXR-selective agonist at up to 10(-8) mol/L was inactive. Among RAR subtype selective retinoids used at 10(-9) mol/L to 10(-7) mol/L, only the RARalpha agonist was equivalent to ATRA at 10(-7) mol/L in its ability to inhibit mast cell growth. Conversely, the addition of excess concentrations of a RARalpha antagonist profoundly counteracted the retinoid-mediated suppressive effects. These results suggest that RA inhibits SCF-dependent differentiation of human mast cell progenitors through a specific receptor. (Blood. 2000;95:2821-2828)     

In vitro treatment with retinoids decreases bcl-2 protein expression and enhances dexamethasone-induced cytotoxicity and apoptosis in multiple myeloma cells

All-trans retinoic acid (ATRA) has been shown to inhibit in vitro growth of multiple myeloma (MM) cells, and this effect can be further potentiated by the addition of Dexamethasone (DEX). We here extended this study by testing the activity of 9-cis retinoic acid (9-cis RA) and 13-cis retinoic acid (13-cis RA), both alone and in combination with DEX, in two MM cell lines, U266 and RPMI 8226. Furthermore, we aimed at investigating the mechanisms involved in the interactions of retinoids and DEX in this setting. 9-cis RA appeared to be the most active agent in U266 cell line (IC50 = 1.2 mumol/l vs 10.5 and 9.8 mumol/l obtained with ATRA and 13-cis RA, respectively) while, in RPMI 8226 cell line, 9-cis RA and 13-cis RA were almost equally cytotoxic (IC50 = 1 and 0.8 mumol/l) and ATRA was less effective. Co-incubation with DEX resulted in a synergistic cytotoxic activity in both the cell lines except for the combinations DEX + 9-cis RA in U266 cell line and DEX + 13-cis RA in RPMI 8226 cell line, where the effect was merely additive. A synergistic cytotoxic effect of retinoids and DEX was also observed on fresh MM cells obtained from 7 patients. Both retinoids and DEX are known to be inducers of apoptosis; we verified that the combined inhibitory activity of retinoids and DEX could be attributed to an increased induction of apoptosis. This effect may be mediated by a reduced intracellular expression of BCL-2 protein, which indeed observed after prolonged in vitro treatment with retinoids. It has been described recently that an enhanced expression of BCL-2 protein can contribute to the occurrence of early chemoresistance; the downregulation of BCL-2 protein induced by retinoids could thus be exploited, by means of novel chemotherapy plus retinoids combinations, in order to improve the efficacy of conventional chemotherapy in MM.     

Neridronate inhibits angiogenesis in vitro and in vivo

The effects of the amino-bisphosphonate neridronate on endothelial cell functions involved in angiogenesis, namely, proliferation and morphogenesis on Matrigel were tested in vitro, whereas its effects on angiogenesis were studied in vivo, by using the chick embryo chorioallantoic membrane (CAM) assay. In vitro, neridronate inhibited endothelial cell proliferation in a dose-dependent fashion, peaking at 30 μM. At the same concentration, neridronate inhibited fibroblast growth factor-2 (FGF-2)-induced capillary-like tube formation in the morphogenesis assay on Matrigel. In vivo, when tested in the CAM assay, neridronate again displayed the capability to inhibit FGF-2-induced angiogenesis. Overall, these results suggest that anti-angiogenesis by neridronate could be used to treat a wide spectrum of angiogenesis-dependent diseases, including certain chronic inflammatory diseases and cancer.     

Retinoids (all-trans and 9-cis retinoic acid) stimulate production of macrophage colony-stimulating factor and granulocyte-macrophage colony- stimulating factor by human bone marrow stromal cells

Retinoic acids (RAs) exert pleiotropic effects on cellular growth and differentiation. All-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), a stereoisomer of ATRA, induce differentiation of leukemic cell lines and cells from patients with acute myelogenous leukemia (AML) in vitro. Despite information on the effects of RAs on hematopoietic cells, little is known about how RAs act on the hematopoietic microenvironment, especially on bone marrow stromal cells. Based on recent observations that various cytokines produced mainly by bone marrow stromal cells regulate hematopoiesis, we analyzed the effects of RAs on cytokine production by these cells. ATRA or 9-cis RA treatment of human bone marrow stromal cell line KM101, which produces macrophage colony-stimulating factor (M-CSF) and granulocyte- macrophage colony-stimulating factor (GM-CSF) constitutively, enhanced mRNA levels of both cytokines in a dose-dependent manner. Both RAs also stimulated M-CSF production from primary cultures of human bone marrow stromal cells. Both retinoic acid receptor (RAR)-alpha and retinoid X receptor (RXR)-alpha were expressed constitutively in KM101 cells. ATRA did not affect the expression of either receptor, whereas 9-cis RA increased RXR-alpha mRNA expression in a dose-dependent manner, but did not affect levels of RAR-alpha mRNA. These findings may have important biologic implications for both the role of RAs in hematopoiesis and the therapeutic effects of ATRA on the hematopoietic microenvironment in patients with acute promyelocytic leukemia (APL).     

Extracellular angio-associated migratory cell protein plays a positive role in angiogenesis and is regulated by astrocytes in coculture

The extracellular form of angio-associated migratory cell protein (AAMP), a recently discovered protein, plays a positive role in angiogenesis and can be regulated by astrocytes. Angiogenic activities are inhibited by an affinity-purified, polyclonal antibody generated to recombinant AAMP. Inhibition of endothelial cell tube formation was previously shown and now endothelial cell migration assays using this antibody show dose-dependent inhibition (75%) of endothelial cell migration. Also, antisense inhibition has been used to determine the effects of reducing total AAMP (extracellular and intracellular forms). An AAMP-specific antisense oligonucleotide that targets a region near its amino terminus, anti-MES, inhibits (45%) total AAMP production by bovine aortic endothelial cells (BAECs), compared to a negative control oligonucleotide. Paradoxically, comparable use of antisense-MES results in a 27% increase in BAEC motility. Decreased cellular production of total AAMP (via antisense) that results in an increase of endothelial migration contrasts with antibody inhibition of extracellular AAMP that decreases migration. This indicates compartment-specific roles for AAMP in angiogenesis. Transwell cocultures of human astrocytes and BAECs increase (53%) the amount of extracellular AAMP found associated with endothelial cells. Therefore, regulation of extracellular AAMP by astrocytes is hypothesized to aid in angiogenesis of the nervous system. Extracellular AAMP's positive role may be either as a promoter or as a permissive protein in this process.     

Superior effect of 9‐cis retinoic acid (RA) compared with all‐trans RA and 13‐cis RA on the inhibition of clonogenic cellgrowth and the induction of apoptosis in OCI/AML‐2 subclones: is the p53 pathway involved?

In the present study, the effects of 9-cis retinoic acid (RA) and 13-cis RA on acute myeloblastic leukaemia (AML) cell growth and the induction of apoptosis as well as its relationship with bcl-2 and p53 were compared with those of all-trans RA (ATRA). The study was performed with the subclones of the retinoid-sensitive OCI/AML-2 cell line. The most prominent inhibitory effect on clonogenic cell growth and morphological apoptosis was shown by 9-cis RA. In addition, Western blotting revealed the most obvious translocation of p53 from cytosol to nucleus in the case of 9-cis RA, which was the only retinoid able to change the conformation of p53 from mutational to wild type, as demonstrated by flow cytometry. There was no difference between the retinoids in the downregulation of bcl-2 as analysed by Western blotting and flow cytometry. The RA receptor (RAR)-alpha antagonist had no effect on apoptosis in any of the three retinoids studied using the annexin V method. In conclusion, this study shows that 9-cis RA was a more potent agent than ATRA or 13-cis RA in inducing growth arrest and apoptosis in the OCI/AML-2 subclones. The effect was associated with the downregulation of bcl-2 and was hardly mediated through the RAR-alpha receptor, but might be related to the activation of p53.     

All-trans retinoic acid at low concentration directly stimulates normal adult megakaryocytopoiesis in the presence of thrombopoietin or combined cytokines.

In order to investigate the direct effects of retinoids on normal adult hematopoietic progenitors, purified CD34+ cells were seeded in serum-free cultures in the presence of pharmacological (10(-6)) M or physiological (10(-12)) M concentrations of all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA) plus combinations of specific cytokines. 10(-6) M ATRA and 9-cis RA significantly decreased the number of granulomacrophagic, erythroid and megakaryocytic (CFU-meg) progenitors. On the other hand, 10(-12) M ATRA significantly promoted the growth of CFU-meg, in the presence either of thrombopoietin or of IL-3+ GM-CSF, and induced a reproducible stimulation of the immature CD34+DR- subset. In conclusion, our findings suggest that retinoic acids probably play a direct role in normal adult hematopoietic development at both physiological and pharmacological concentrations. The stimulatory effect on megakaryocytopoiesis should be considered in the perspective of a potential use of low-dose ATRA, combined with thrombopoietin or other cytokines, in pathological conditions where the megakaryocytic compartment is impaired and the stimulation of megakaryocytopoiesis is requested.     

Kinetics of placenta growth factor/vascular endothelial growth factor synergy in endothelial hydraulic conductivity and proliferation

Vascular endothelial growth factor (VEGF) was originally discovered as vascular permeability factor because of its ability to increase microvascular permeability to plasma proteins. Since then, it has been shown to induce proliferation and migration in endothelial cells. Placenta growth factor (PlGF) is a member of the VEGF family of growth factors, but has little or undetectable mitogenic activity on endothelial cells. Intriguingly, however, PlGF was able to potentiate the action of low concentrations of VEGF on endothelial cell growth and macromolecule permeability in vitro. Here we show that PlGF can potentiate the effects of VEGF on the hydraulic conductivity of certain endothelial cells and that the duration of pretreatment with PlGF determines the resulting response. Hydraulic conductivity (Lp) was calculated from the water flux across the monolayer of human umbilical vein endothelial cells (HUVECs) or bovine aortic endothelial cells (BAECs). After 2 h of exposure to VEGF(165), the Lp of BAEC monolayers increased threefold, but the Lp of HUVEC monolayers did not increase. PlGF alone induced a small (63%) increase in Lp in BAECs, but not in HUVECs. BAEC, but not HUVEC, monolayers exposed first to PlGF and then to VEGF exhibited a seven- to eightfold increase in Lp. This enhancement in BAEC Lp could be observed for 4 h after the administration of PlGF. PlGF also potentiated the effect of VEGF on BAEC proliferation. Thus, augmentation of VEGF action by PlGF depends on the duration of PlGF exposure and on the origin of endothelial cells.     

Dehydroepiandrosterone stimulates endothelial proliferation and angiogenesis through extracellular signal-regulated kinase 1/2-mediated mechanisms

Dehydroepiandrosterone (DHEA) activates a plasma membrane receptor on vascular endothelial cells and phosphorylates ERK 1/2. We hypothesize that ERK1/2-dependent vascular endothelial proliferation underlies part of the beneficial vascular effect of DHEA. DHEA (0.1-10 nm) activated ERK1/2 in bovine aortic endothelial cells (BAECs) by 15 min, causing nuclear translocation of phosphorylated ERK1/2 and phosphorylation of nuclear p90 ribosomal S6 kinase. ERK1/2 phosphorylation was dependent on plasma membrane-initiated activation of Gi/o proteins and the upstream MAPK kinase because the effect was seen with albumin-conjugated DHEA and was blocked by pertussis toxin or PD098059. A 15-min incubation of BAECs with 1 nm DHEA (or albumin-conjugated DHEA) increased endothelial proliferation by 30% at 24 h. This effect was not altered by inhibition of estrogen or androgen receptors or nitric oxide production. There was a similar effect of DHEA to increase endothelial migration. DHEA also increased the formation of primitive capillary tubes of BAECs in vitro in solubilized basement membrane. These rapid DHEA-induced effects were reversed by the inhibition of either Gi/o-proteins or ERK1/2. Additionally, DHEA enhanced angiogenesis in vivo in a chick embryo chorioallantoic membrane assay. These findings indicate that exposure to DHEA, at concentrations found in human blood, causes vascular endothelial proliferation by a plasma membrane-initiated activity that is Gi/o and ERK1/2 dependent. These data, along with previous findings, define an important vascular endothelial cell signaling pathway that is activated by DHEA and suggest that this steroid may play a role in vascular function.     

Differential regulation of the fibroblast growth factor (FGF) family by alpha(2)-macroglobulin: evidence for selective modulation of FGF-2-induced angiogenesis

The fibroblast growth factor (FGF) family has an important role in processes such as angiogenesis, wound healing, and development in which precise control of proteinase activity is important. The human plasma proteinase inhibitor alpha(2)-macroglobulin (alpha(2)M) regulates cellular growth by binding and modulating the activity of many cytokines and growth factors. These studies investigate the ability of native and activated alpha(2)M (alpha(2)M*) to bind to members of the FGF family. Both alpha(2)M and alpha(2)M* bind specifically and saturably to FGF-1, -2, -4, and -6, although the binding to alpha(2)M* is of significantly higher affinity. Neither alpha(2)M nor alpha(2)M* bind to FGF-5, -7, -9, or -10. FGF-2 was chosen for more extensive study in view of its important role in angiogenesis. It was demonstrated that FGF-2 binds to the previously identified TGF-beta binding site. The alpha(2)M* inhibits FGF-2-dependent fetal bovine heart endothelial cell proliferation in a dose-dependent manner. Unexpectedly, alpha(2)M* does not affect FGF-2-induced vascular tubule formation on Matrigel basement membrane matrix or collagen gels. Further studies demonstrate that FGF-2 partitions between fluid-phase alpha(2)M* and solid-phase Matrigel or collagen. These studies suggest that the ability of alpha(2)M* to modulate the activity of FGF-2 is dependent on an interplay with extracellular matrix components. (Blood. 2001;97:3450-3457)     

Metformin decreases intracellular production of reactive oxygen species in aortic endothelial cells

Beyond its antidiabetic activity justifying its use in the treatment of the type 2 diabetes, metformin (MET [dimethylguanidine, Glucophage]) has been shown to exhibit antioxidant properties in vitro, which could contribute to limit the deleterious vascular complications of diabetes. We investigated whether MET, at the pharmacological level of 10 -5 mol/L, was able to modulate intracellular production of reactive oxygen species (ROS) both in quiescent bovine aortic endothelial cells (BAECs) and in BAECs stimulated by a short incubation with high levels of glucose (30 mmol/L, 2 hours) or angiotensin II (10 -7 mol/L, 1 hour). Intracellular ROS production was measured by fluorescence of the DCF (2,7-dichlorodihydrofluorescein) probe. Our results showed that MET was able to reduce the intracellular production of ROS in both nonstimulated BAECs (-20%, P < .05) and BAEC stimulated by high levels of glucose or angiotensin II (-28% and -72%, respectively, P < .01). Experiments performed in the presence of the nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase inhibitor apocynin or the respiratory mitochondrial chain inhibitor rotenone indicated that MET exerted its effect partly through an inhibition of the formation of ROS produced mainly by NAD(P)H oxidase and also, to a lesser extent, by the respiratory mitochondrial chain.     

Additive Inhibitory Effect of Experimentally Induced Hepatic Cirrhosis by Agonists of Peroxisome Proliferator Activator Receptor γ and Retinoic Acid Receptor

Peroxisome proliferator activator receptor (PPAR) ligands prevent liver fibrosis, while the role of all-trans retinoic acid (ATRA) and its metabolite 9-cis retinoic acid (9-cis RA) is less clear. We have investigated the ability of the combination of PPARγ ligand rosiglitazone (RSG) and of ATRA to prevent liver fibrosis. In vivo treatment with RSG or ATRA reduced fibrotic nodules, spleen weight, and hydroxyproline levels in rat model of thioacetamide-induced liver fibrosis. The combination of ATRA + RSG caused the strongest inhibition, accompanied by decreased expression of collagen I, α-smooth muscle actin, TGFβ1, and TNFα. In vitro studies showed that PPARγ ligand 15-deoxy-Δ12,14-prostaglandinJ(2)[PJ(2)] and RXR ligand 9-cis RA or PJ(2) and ATRA inhibited proliferation of hepatic stellate cells HSC-T6. 9-cis RA inhibited c-jun levels and also inhibited expression of its receptor RXRα in HSC-T6 cells. The combination of PPAR-γ and RAR agonists demonstrated an additive effect in the inhibition of TAA-induced hepatic fibrosis, due to inhibition of HSC proliferation and reduction of profibrotic TGFβ1 and proinflammatory TNFα.     

Activin A as a critical mediator of capillary formation: interaction with the fibroblast growth factor action

The present study was conducted to elucidate the role of activin A in capillary formation. When bovine aortic endothelial cells (BAEC) were cultured in a collagen gel, basic fibroblast growth factor (FGF-2) induced tube formation. Activin A also induced tube formation and the addition of two factors together was more effective. BAEC produced both FGF-2 and activin A as autocrine factors. Exogenous FGF-2 did not affect the production of activin A but instead upregulated the type II activin receptor. On the other hand, activin A increased the expression of FGF-2 as well as the FGF receptor. Most importantly, when the action of endogenous activin A was blocked by adding follistatin, the tubulogenic action of FGF-2 was nearly completely inhibited. Activin-induced tubulogenesis was markedly inhibited by overexpression of Smad7, an inhibitory Smad. Similarly, an inhibitor of p44/42 mitogen-activated protein (MAP) kinase attenuated the activin-mediated tubulogenesis, whereas an inhibitor of p38 MAP kinase had no effect. These results indicate that FGF-2 and activin A enhance their signals each other in BAEC, and endogenous activin A is critical for FGF-2-induced capillary formation.     

Ubiquitinated or sumoylated retinoic acid receptor alpha determines its characteristic and interacting model with retinoid X receptor alpha in gastric and breast cancer cells

Retinoic acid receptor alpha (RARalpha) plays an important role in mediating all-trans retinoic acid (ATRA) signals. In this study, we found that ATRA up-regulated RARalpha mRNA and protein expression in gastric cancer BGC-823 cells. However, in breast cancer MCF-7 cells it down-regulated RARalpha protein expression with no effect on its RARalpha mRNA. Immunoprecipitation/Western blot analysis showed that, although sumoylated and ubiquitinated RARalpha existed simultaneously in both cancer cell lines, ATRA exerted different regulatory effects on sumoylation and ubiquitination of RARalpha. In MCF-7 cells, ATRA treatment enhanced the ubiquitination of RARalpha and the subsequent degradation of RARalpha through the ubiquitin/proteasome pathway. This resulted in a reduction in the DNA binding activity of RARalpha/retinoid X receptor alpha (RXRalpha) heterodimer, the separation of RXRalpha from RARalpha and the translocation of RXRalpha from the nucleus to the cytoplasm. By contrast, in BGC-823 cells, ATRA augmented sumoylation, not ubiquitination, of RARalpha. The stability of sumoylated RARalpha was significantly stronger than in non-sumoylated RARalpha. These results also showed an increase in the DNA binding activity of the RARalpha/RXRalpha heterodimer and the stability of nuclear localization of this heterodimer, which normally facilitates the ATRA signal transduction. In conclusion, our results reveal a novel mechanism for the regulation of RARalpha-dependent signal transduction through the ubiquitin/proteasome pathway in breast cancer cells and the sumoylation pathway in gastric cancer cells.     

Oncostatin M induces angiogenesis and cartilage degradation in rheumatoid arthritis synovial tissue and human cartilage cocultures

OBJECTIVE: To investigate the role of oncostatin M (OSM) in cell adhesion, angiogenesis, and matrix degradation in rheumatoid arthritis (RA) synovial tissue and normal human cartilage. METHODS: Human dermal microvascular endothelial cell (HDMEC) and RA synovial fibroblast (RASF) proliferation and intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression were assessed by a bromodeoxyuridine proliferation assay and flow cytometry. HDMEC tubule formation and migration were assessed by Matrigel culture and migration assay. Production of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases 1 (TIMP-1) in RA synovial explants, and proteoglycan/glycosaminoglycan (GAG) release, vascular endothelial growth factor (VEGF), and angiopoietin 2 production from RASF/normal cartilage cocultures were assessed by enzyme-linked immunosorbent assay and immunohistology. RESULTS: HDMEC/RASF proliferation was induced by OSM and interleukin-1beta (IL-1beta), alone and in combination. OSM enhanced cell surface expression of ICAM-1, but not VCAM-1, on endothelial cells and RASFs. OSM increased endothelial cell tubule formation and migration. In RA synovial explants, OSM induced production of MMP-1 and TIMP-1. When OSM was combined with IL-1beta, however, the MMP-1:TIMP-1 ratio was significantly increased. OSM potentiated IL-1beta-induced MMP-1 and MMP-13 expression in normal human cartilage/RASF cocultures, resulting in a significant increase in the MMP:TIMP ratio. In OSM/IL-1beta- stimulated cocultures, cartilage sections demonstrated significant proteoglycan depletion that was paralleled by a significant increase in GAG release in supernatants. Finally, compared with either cytokine alone, the combination of OSM and IL-1beta significantly induced VEGF production in RASF/cartilage cocultures. CONCLUSION: These data suggest that OSM promotes angiogenesis and endothelial cell migration and potentiates the effects of IL-1beta in promoting extracellular matrix turnover and human cartilage degradation. Furthermore, the induction of VEGF in cocultures supports the hypothesis of a link between angiogenesis and cartilage degradation.