The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression.

Inverted CCAAT box-binding protein of 90 kDa (ICBP90) is over-expressed in several types of cancer, including breast, prostate and lung cancers. In search for proteins that interact with the set and ring-associated (SRA) domain of ICBP90, we used the two-hybrid system and screened a placental cDNA library. Several clones coding for a new domain of DNMT1 were found. The interaction, between the ICBP90 SRA domain and the DNMT1 domain, has been confirmed with purified proteins by glutathione-S-transferase pull-down experiments. We checked whether ICBP90 and DNMT1 are present in the same macro-molecular complexes in Jurkat cells and immortalized human vascular smooth muscle cells (HVTs-SM1). Co-immunoprecipitation experiments showed that ICBP90 and DNMT1 are present in the same molecular complex, which was further confirmed by co-localization experiments as assessed by immunocytochemistry. Downregulation of ICBP90 and DNMT1 decreased VEGF gene expression, a major pro-angiogenic factor, whereas those of p16(INK4A) gene and RB1 gene were significantly enhanced. Together, these results indicate that DNMT1 and ICBP90 are involved in VEGF gene expression, possibly via an interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 and an upregulation of p16(INK4A). They further suggest a new role of ICBP90 in the relationship between histone ubiquitination and DNA methylation in the context of tumoral angiogenesis and tumour suppressor genes silencing.

[Culture and characterization of porcine coronary endothelial cells]. / Culture et caractérisation des cellules endothéliales d'artère coronaire de porc.

INTRODUCTION: Vascular endothelium possesses biological properties that are involved in important physiological functions such vascular permeability, vascular tone regulation and angiogenesis. The difficulty of culture and long-term maintenance of sufficient amount of normal endothelial cells has proven to be the limitation for the understanding of the biological function of the endothelium. Therefore, the aim of this study was to culture and characterize the porcine coronary endothelial cells. MATERIAL AND METHODS: The endothelial cells were isolated by collagenase treatment and cultured in culture dishes coated with collagen, prepared from rat tail, containing medium RPMI1640/M199 and 15% fetal calf serum supplemented with antibiotics and fungizon. The cells were maintained to grown at 37 degrees C. The medium was changed one day after and then every two day. The cells were incubated with Dil-labeled-acetylated-LDL for determination of acetylated-LDL uptake. Confluence cultures of cells were examined by phase-contrast and confocal microscopy. RESULTS: After a day of culture, the endothelial cells adhere to the collagen and began to grow. While multiplying themselves, they colonize little by little the body of the surface of culture to form to confluence a monolayer of flat cells relatively homogenous. To confluence, the proliferation of the endothelial cells is inhibited by the contact and the cells present a polygonal aspect. Our results show that all the cultivated cells were strongly positive for acetylated-LDL markers. The endothelial cells, cultivated until the second passage corresponding to the second culture of the primary cultures, continued to present a good fluorescence. CONCLUSION: Porcine coronary endothelial cells represent a useful in vitro model to study biological and physiopathological properties of vascular endothelium.

Culture et caracterisation des cellules endotheliales d'artere coronaire de porc

Introduction : Les proprietes biologiques de l'endothelium vasculaire lui conferent un role majeur dans le controle de la permeabilite; du tonus vasculaire et l'angiogenese; des fonctions physiologiques cruciales a l'homeostasie vasculaire. La comprehension du ou des mecanisme(s) d'action sous-jacent(s) a ces multiples proprietes a longtemps ete retardee par la difficulte technique d'obtenir des quantites suffisantes de ce tissu permettant de realiser les etudes in vitro. Ce travail a pour objectif de proceder a la mise en culture et a la caracterisation des cellules endotheliales d'artere coronaire de porc. Materiels et Methodes : La couche de cellules endotheliales a ete prelevee par passage de la solution de collagenase a travers la lumiere du vaisseau. Les cellules ont ete ensuite ensemencees dans des flacons de culture; prealablement recouverts de collagene de queue de rat dans un milieu de culture RPMI1640/M199 auquel a ete rajoute 15de serum de veau fotal; des antibiotiques et de la fungizone. Les cultures ont ete maintenues a 37oC dans un incubateur. La sonde fluorescente constituee de lipoproteines acetylees de faible densite (Dil-Ac-LDL) a ete utilisee comme marqueur biologique specifique par recapture par les cellules endotheliales. Les cellules confluentes ont ete repiquees dans des chambres de culture LAB-TEK puis examinees au microscope photonique et confocal. Resultats : Apres 24 heures de mise en culture; les cellules endotheliales adherent au collagene et forment des rosettes. En se multipliant; elles colonisent peu a peu l'ensemble de la surface de la boite de culture pour former a confluence un tapis pavimenteux relativement homogene. A confluence; la proliferation des cellules endotheliales est inhibee par le contact et elle presente un aspect polygonal. Nos resultats montrent que les cellules cultivees presentent un intense marquage au Dil-Ac-LDL. Conclusion : Les cellules endotheliales de l'artere coronaire de porc en culture constituent un bon outil pour l'etude des fonctions endotheliales dans des conditions physiologiques et pathologiques.Introduction : Les proprietes biologiques de l'endothelium vasculaire lui conferent un role majeur dans le controle de la permeabilite; du tonus vasculaire et l'angiogenese; des fonctions physiologiques cruciales a l'homeostasie vasculaire. La comprehension du ou des mecanisme(s) d'action sous-jacent(s) a ces multiples proprietes a longtemps ete retardee par la difficulte technique d'obtenir des quantites suffisantes de ce tissu permettant de realiser les etudes in vitro. Ce travail a pour objectif de proceder a la mise en culture et a la caracterisation des cellules endotheliales d'artere coronaire de porc. Materiels et Methodes : La couche de cellules endotheliales a ete prelevee par passage de la solution de collagenase a travers la lumiere du vaisseau. Les cellules ont ete ensuite ensemencees dans des flacons de culture; prealablement recouverts de collagene de queue de rat dans un milieu de culture RPMI1640/M199 auquel a ete rajoute 15de serum de veau fotal; des antibiotiques et de la fungizone. Les cultures ont ete maintenues a 37oC dans un incubateur. La sonde fluorescente constituee de lipoproteines acetylees de faible densite (Dil-Ac-LDL) a ete utilisee comme marqueur biologique specifique par recapture par les cellules endotheliales. Les cellules confluentes ont ete repiquees dans des chambres de culture LAB-TEK puis examinees au microscope photonique et confocal. Resultats : Apres 24 heures de mise en culture; les cellules endotheliales adherent au collagene et forment des rosettes. En se multipliant; elles colonisent peu a peu l'ensemble de la surface de la boite de culture pour former a confluence un tapis pavimenteux relativement homogene. A confluence; la proliferation des cellules endotheliales est inhibee par le contact et elle presente un aspect polygonal. Nos resultats montrent que les cellules cultivees presentent un intense marquage au Dil-Ac-LDL. Conclusion : Les cellules endotheliales de l'artere coronaire de porc en culture constituent un bon outil pour l'etude des fonctions endotheliales dans des conditions physiologiques et pathologiques

Nifedipine increases cytochrome P4502C expression and endothelium-derived hyperpolarizing factor-mediated responses in coronary arteries.

In addition to NO and prostacyclin, endothelial cells release a factor that elicits vasodilatation by hyperpolarizing the underlying vascular smooth muscle cells. In some vascular beds, this so-called endothelium-derived hyperpolarizing factor (EDHF) displays the characteristics of a cytochrome P450 (CYP)-derived arachidonic acid metabolite, such as an epoxyeicosatrienoic acid. Native porcine and cultured human coronary artery endothelial cells were screened for CYP epoxygenases, and CYP2B, CYP2C, and CYP2J were detected with reverse transcription-polymerase chain reaction. The CYP inducer beta-naphthoflavone and the Ca(2+) antagonist nifedipine significantly increased CYP2C mRNA but did not change the expression of CYP2J or CYP2B. To determine the relationship between CYP2C expression and EDHF production in native endothelial cells, we incubated porcine coronary arteries with nifedipine. Nifedipine enhanced endothelial CYP2C protein expression, as well as the generation of 11,12-epoxyeicosatrienoic acid. In organ bath experiments, pretreatment with nifedipine enhanced bradykinin-induced, EDHF-mediated relaxations as well as the concomitant hyperpolarization of smooth muscle cells. The specific CYP2C9 inhibitor sulfaphenazole, on the other hand, significantly attenuated EDHF-mediated hyperpolarization and relaxation. These results demonstrate that in porcine coronary arteries, the elevated expression of a CYP epoxygenase, homologous to CYP2C8/9, is associated with enhanced EDHF-mediated hyperpolarization in response to bradykinin. Therefore, we propose that an isozyme of CYP2C is the most likely candidate for the CYP-dependent EDHF synthase in porcine coronary arteries.

3-Morpholinosydnonimine (SIN-1) and K(+) channels in smooth muscle cells of the rabbit and guinea pig carotid arteries.

Experiments were designed to determine the subtype of K(+) channels activated by the nitrovasodilator 3-morpholinosydnonimine (SIN-1) in smooth muscle cells of the rabbit and guinea pig carotid arteries. Membrane potential was recorded in isolated segments with intracellular microelectrode and K(+) currents in freshly dissociated smooth muscle cells, with the patch-clamp technique. In the guinea pig carotid artery, SIN-1 caused a glibenclamide-sensitive hyperpolarization. The nitrovasodilator did not affect the whole-cell K(+) current, but activated a glibenclamide-sensitive K(+) current. In the rabbit carotid artery, SIN-1 induced only an iberiotoxin-sensitive repolarization in phenylephrine-depolarized tissue and in isolated cells, enhanced the activity of an iberiotoxin-sensitive K(+) current. These findings demonstrate that the population of K(+) channels activated by nitric oxide (NO) is species-dependent and support the conclusion that, in the guinea pig carotid artery, in contrast to the rabbit carotid artery, the release of NO cannot account for the responses attributed to endothelium-derived hyperpolarizing factor (EDHF).

Acetylcholine-induced relaxation in blood vessels from endothelial nitric oxide synthase knockout mice.

1. Isometric tension was recorded in isolated rings of aorta, carotid, coronary and mesenteric arteries taken from endothelial nitric oxide synthase knockout mice (eNOS(-/-) mice) and the corresponding wild-type strain (eNOS(+/+) mice). The membrane potential of smooth muscle cells was measured in coronary arteries with intracellular microelectrodes. 2. In the isolated aorta, carotid and coronary arteries from the eNOS(+/+) mice, acetylcholine induced an endothelium-dependent relaxation which was inhibited by N(omega)-L-nitro-arginine. In contrast, in the mesenteric arteries, the inhibition of the cholinergic relaxation required the combination of N(omega)-L-nitro-arginine and indomethacin. 3. The isolated aorta, carotid and coronary arteries from the eNOS(-/-) mice did not relax in response to acetylcholine. However, acetylcholine produced an indomethacin-sensitive relaxation in the mesenteric artery from eNOS(-/-) mice. 4. The resting membrane potential of smooth muscle cells from isolated coronary arteries was significantly less negative in the eNOS(-/-) mice (-64.8 +/- 1.8 mV, n = 20 and -58.4 +/- 1.9 mV, n = 17, for eNOS(+/+) and eNOS(-/-) mice, respectively). In both strains, acetylcholine, bradykinin and substance P did not induce endothelium-dependent hyperpolarizations whereas cromakalim consistently produced hyperpolarizations (- 7.9 +/- 1.1 mV, n = 8 and -13.8 +/- 2.6 mV, n = 4, for eNOS(+/+) and eNOS(-/-) mice, respectively). 5. These findings demonstrate that in the blood vessels studied: (1) in the eNOS(+/+) mice, the endothelium-dependent relaxations to acetylcholine involve either NO or the combination of NO plus a product of cyclo-oxygenase but not EDHF; (2) in the eNOS(-/-) mice, NO-dependent responses and EDHF-like responses were not observed. In the mesenteric arteries acetylcholine releases a cyclo-oxygenase derivative.

Cannabinoid CB1 receptor and endothelium-dependent hyperpolarization in guinea-pig carotid, rat mesenteric and porcine coronary arteries.

1. The purpose of these experiments was to determine whether or not the endothelium-dependent hyperpolarizations of the vascular smooth muscle cells (observed in the presence of inhibitors of nitric oxide synthase and cyclo-oxygenase) can be attributed to the production of an endogenous cannabinoid. 2. Membrane potential was recorded in the guinea-pig carotid, rat mesenteric and porcine coronary arteries by intracellular microelectrodes. 3. In the rat mesenteric artery, the cannabinoid receptor antagonist, SR 141716 (1 microM), did not modify either the resting membrane potential of smooth muscle cells or the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (17.3 +/- 1.8 mV, n = 4 and 17.8 +/- 2.6 mV, n = 4, in control and presence of SR 141716, respectively). Anandamide (30 microM) induced a hyperpolarization of the smooth muscle cells (12.6 +/- 1.4 mV, n = 13 and 2.0 +/- 3.0 mV, n = 6 in vessels with and without endothelium, respectively) which could not be repeated in the same tissue, whereas acetylcholine was still able to hyperpolarize the preparation. The hyperpolarization induced by anandamide was not significantly influenced by SR 141716 (1 microM). HU-210 (30 microM), a synthetic CB1 receptor agonist, and palmitoylethanolamide (30 microM), a CB2 receptor agonist, did not influence the membrane potential of the vascular smooth muscle cells. 4. In the rat mesenteric artery, the endothelium-dependent hyperpolarization induced by acetylcholine (1 microM) (19.0 +/- 1.7 mV, n = 6) was not altered by glibenclamide (1 microM; 17.7 +/- 2.3 mV, n = 3). However, the combination of charybdotoxin (0.1 microM) plus apamin (0.5 microM) abolished the acetylcholine-induced hyperpolarization and under these conditions, acetylcholine evoked a depolarization (7.7 +/- 2.7 mV, n = 3). The hyperpolarization induced by anandamide (30 microM) (12.6 +/- 1.4 mV, n = 13) was significantly inhibited by glibenclamide (4.0 +/- 0.4 mV, n = 4) but not significantly affected by the combination of charybdotoxin plus apamin (17.3 +/- 2.3 mV, n = 4). 5. In the guinea-pig carotid artery, acetylcholine (1 microM) evoked endothelium-dependent hyperpolarization (18.8 +/- 0.7 mV, n = 15). SR 141716 (10 nM to 10 microM), caused a direct, concentration-dependent hyperpolarization (up to 10 mV at 10 microM) and a significant inhibition of the acetylcholine-induced hyperpolarization. Anandamide (0.1 to 3 microM) did not influence the membrane potential. At a concentration of 30 microM, the cannabinoid agonist induced a non-reproducible hyperpolarization (5.6 +/- 1.3 mV, n = 10) with a slow onset. SR 141716 (1 microM) did not affect the hyperpolarization induced by 30 microM anandamide (5.3 +/- 1.5 mV, n = 3). 6. In the porcine coronary artery, anandamide up to 30 microM did not hyperpolarize or relax the smooth muscle cells. The endothelium-dependent hyperpolarization and relaxation induced by bradykinin were not influenced by SR 141716 (1 microM). 7. These results indicate that the endothelium-dependent hyperpolarizations, observed in the guinea-pig carotid, rat mesenteric and porcine coronary arteries, are not related to the activation of cannabinoid CB1 receptors.

Epoxyeicosatrienoic acids, potassium channel blockers and endothelium-dependent hyperpolarization in the guinea-pig carotid artery.

1. Using intracellular microelectrodes, we investigated the effects of 17-octadecynoic acid (17-ODYA) on the endothelium-dependent hyperpolarization induced by acetylcholine in the guinea-pig isolated internal carotid artery with endothelium. 2. In the presence of Nomega-nitro-L-arginine (L-NOARG, 100 microM) and indomethacin (5 microM) to inhibit nitric oxide synthase and cyclo-oxygenase, acetylcholine (1 microM) evoked an endothelium-dependent hyperpolarization which averaged -16.4 mV starting from a resting membrane potential of -56.8 mV. There was a negative correlation between the amplitude of the hyperpolarization and the absolute values of the resting membrane potential. 3. The acetylcholine-induced endothelium-dependent hyperpolarization was not altered by charybdotoxin (0.1 microM) or iberiotoxin (30 nM). It was partially but significantly reduced by apamin (0.5 microM) to -12.8+/-1.2 mV (n=10) or the combination of apamin plus iberiotoxin (-14.3+/-3.4mV, n=4). However, the combination of charybdotoxin and apamin abolished the hyperpolarization and under these conditions, acetylcholine evoked a depolarization (+ 7.1+/-3.7 mV, n = 8). 4. 17-ODYA (10 microM) produced a significant hyperpolarization of the resting membrane potential which averaged -59.6 mV and a partial but significant inhibition of the acetylcholine-induced endothelium-dependent hyperpolarization (-10.9 mV). 5. Apamin did not modify the effects of 17-ODYA but in the presence of charybdotoxin or iberiotoxin, 17-ODYA no longer influenced the resting membrane potential or the acetylcholine-induced hyperpolarization. 6. When compared to solvent (ethanol, 1% v/v), epoxyeicosatrienoic acids (EpETrEs) (5,6-, 8,9-, 11,12- and 14,15-EpETrE, 3 microM) did not affect the cell membrane potential and did not relax the guinea-pig isolated internal carotid artery. 7. These results indicate that, in the guinea-pig internal carotid artery, the involvement of metabolites of arachidonic acid through the cytochrome P450 pathway in endothelium-dependent hyperpolarization is unlikely. Furthermore, the hyperpolarization mediated by the endothelium-derived hyperpolarizing factor (EDHF) is probably not due to the opening of BK(Ca) channels.