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We investigated the effect of As(2)O(3), an anti-cancer drug, on endothelial gap junctions. Human aortic endothelial cells (HAEC) were treated with As(2)O(3) at 1, 10, 100, and 1000 ng/ml and the cells were examined to evaluate the expression of connexin43 (Cx43) and to assess gap-junction communication. Endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) were measured to assess for endothelial dysfunction. Male Sprague-Dawley rats were given intravenous As(2)O(3) (200 mug/kg/day) or saline for 4 weeks, after which aortic endothelial gap junctions, eNOS, and circulating NO levels were evaluated. We found that HAEC Cx43 transcripts and gap junctions were reduced and gap-junction communication was attenuated by As(2)O(3). This decrease of Cx43 gap junctions was prevented by the addition of protease inhibitors. At a dose of 100 ng/ml of As(2)O(3), eNOS was reduced at 48 h, but NO was markedly reduced by 1 h. In animals treated with As(2)O(3), endothelial gap junctions comprising Cx37, Cx40, or Cx43 were all reduced in amount, while eNOS and circulating NO levels remained unchanged. In both in vitro and in vivo rat experiments, endothelial gap junctions were consistently reduced by As(2)O(3), unlike the response of eNOS and NO, which were decreased in cells but not in the rat aortic endothelium. The reduction in Cx43 involved both down-regulation at the transcriptional level and increased degradation. These findings indicate that gap-junction communication in the vascular endothelium is inhibited by treatment with As(2)O(3).

We examined the effect of 2',5'-dihydroxychalcone on connexin43 (Cx43) expression and gap-junctional communication in human umbilical vein endothelial cells (HUVEC). The result showed that expression of Cx43 is rapidly reduced by 2',5'-dihydroxychalcone in a dose-dependent manner, Concomitantly, the communication function, determined by fluorescence recovery after photobleaching (FRAP), is decreased. We further investigated whether the mitogen-activated protein (MAP) kinase and the degradation pathway of gap junctions are involved in these processes. Although the change of Cx43 is not affected by the level of fetal calf serum (FCS) used in the medium, activation of MAP kinase varies, depending on the FCS level. At a low level (0.5%), the chalcone inhibits the activation, like PD98059, a specific inhibitor of MAP kinase kinase. However, at a high level (20%), MAP kinase is activated. On the other hand, the chalcone's down-regulating effect on Cx43, while is totally blocked by protease inhibitors leupeptin and N-acetyl-leucyl-norleucinal (ALLN), persists in the presence of PD98059, We concluded that 2',5'-dihydroxychalcone down-regulates Cx43 expression and gap-junctional communication in the HUVEC via enhancement of the proteolysis pathway, and this compound possesses dual effects on MAP kinase activation.

Generally, inflammatory response to the metal stents contributes to the formation of in-stent restenosis. Howerver, recent development of biodegradable stents made of magnesium alloys has a potential to overcome this drawback. Nevertheless, the degradation profile of such stents and the influence on the endothelial cells remained unclear.

We compared the behavior of endothelial cells grown on stent materials. Human umbilical vein endothelial cells (HUVEC) were seeded (200 or 800 cells/mm(2)) onto different metallic sheets, including 316 stainless steel (low carbon; 316LSS), nitinol, and 316LSS coated with TiN or TiO(2). Cells seeded onto tissue culture-treated polystyrene dish coated with gelatin were used as controls. Forty-eight hours later, the cells were examined by Western blotting, immunofluorescence microscopy, and scanning electron microscopy (SEM). The results showed that for either seeding values, the levels of cellularity on TiN and TiO(2) are comparable or higher, and those on 316LSS and nitinol are lower compared to the controls (p < 0.05). SEM demonstrated that cells are well-attached on the metallic surface with various amount of cellular processes. In metals seeded with 800 cells/mm(2), Western blotting showed that the overlying cells expressed less amounts of endothelial nitric oxide synthase (eNOS), Von Willebrand factor (VWF), and connexin43 protein compared to the controls (p < 0.05). Immunofluorescence microscopy confirmed the results of immunoblotting. In conclusion, stent materials affect HUVEC's growth and protein expression profile. Down-regulation of eNOS, VWF, and connexin43 gap junctions is a common phenomenon in the cells growing on the examined metallic materials, suggesting the existence of endothelial dysfunction in the arterial segments containing the stents made of such materials.

The aim of this study was to clarify the response of endothelial connexins to hypertension and antihypertensive drugs.Rats remained normotensive (group 1, N = 10) or were made hypertensive using N(omega)-nitro-L-arginine methyl ester (L-NAME) (groups 2 to 4, N = 10/group). Seven weeks later groups 3 and 4 were respectively fed atenolol or carvedilol daily for 7 days and the aortic endothelial gap junctions were analyzed. In parallel the effects of atenolol, carvedilol, labetalol, vitamin C, and vitamin E on connexin43 (Cx43) in human umbilical vein endothelial cells (HUVEC) were compared.Endothelial Cx43 was reduced by 35% and Cx37 by 59% in hypertensive rats (P.001). The reduction was recovered fully by carvedilol but only partially by atenolol (P.05), although both drugs lowered the blood pressure to similar levels. Cx40 remained stationary in all groups. In HUVEC, carvedilol (10 microg/mL) increased Cx43 protein expression by70% (P.01), whereas other drugs had minimal effects. The upregulation by carvedilol was associated with increased transcripts and decreased proteolysis of Cx43.The study showed that L-NAME-induced hypertension has differential effects on endothelial connexins, which respond variously to carvedilol and atenolol. In HUVEC carvedilol directly upregulates endothelial Cx43 and the effect is independent of its antioxidant activity.

We investigated the effect of nicotine on connexin43 (Cx43) expression and gap-junctional communication in human umbilical vein endothelial cells (HUVEC). We also evaluated whether the effect requires activation of acetyl choline receptors sensitive to nicotine (nAChRs) and is altered by statins. The results showed that expression of Cx43 protein is reduced by nicotine in a dose-dependent manner (6 x 10(-4) M nicotine vs control, 33% reduction, p0.01), though Cx43 mRNA is up-regulated (6 x 10(-4) M nicotine vs control, 36% increase, p0.01). Concomitantly, the communication function, determined by fluorescence recovery after photobleaching, is decreased (6 x 10(-4) M nicotine vs control, 38% reduction, p0.05). Such a down-regulation of Cx43 gap junctions by nicotine disappears in the presence of the nAChRs antagonist, dihydro-beta-erythroidine, and protease inhibitors leupeptin plus N-acetyl-Leu-Leu-Norleu-al (ALLN). Similarly, the effect of nicotine is attenuated by statins, including fluvastatin, lovastatin, pravastatin, and simvastatin, even at the presence of mevalonate. We concluded that i) nicotine down-regulates Cx43 expression and gap-junctional communication in HUVEC via post-transcriptional modification, which involves enhancement of Cx43 proteolysis; ii) the effect of nicotine is mediated via activation of nAChRs; and iii) the effect of nicotine is attenuated by statins through mechanisms outside the hypolipidemic pathway.