Effect of the calcium channel blockers amlodipine and diltiazem on erythropoietin-mediated angiogenesis.

VITTORI DANIELA; SCHIAPPACASSE AGUSTINA; NESSE ALCIRA; MALTANERI ROMINA EUGENIA; NESSE ALCIRA; MALTANERI ROMINA EUGENIA; CHAMORRO MARÍA EUGENIA; CHAMORRO MARÍA EUGENIA; VITTORI DANIELA; SCHIAPPACASSE AGUSTINA

CABA

Congreso; LXII Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica; 2017

SAIC

Calcium (Ca) is an important factor involved in cellmigration. Our group has previously demonstrated its participation in thepromigratory effect of erythropoietin (Epo) on endothelial cells. Stemming fromthis, we hypothesized that Ca channel blockers (CCBs) could negatively affectangiogenesis. This is an important issue since many of these compounds areclinically used as antihypertensives.  Amlodipine (Aml), a dihydropyridine, and thebenzothiazepine diltiazem (Dil) are both able to block voltage-dependent Cachannels (VDCCs) in smooth muscle cells. With an aim to determine the abilityof these compounds to block Ca channels in the endothelium, we used theEA.hy926 cell line as an experimental model. In accordance to previous reports,we found that both, Aml and Dil prevented staurosporin-induced apoptosis inthis cell line (Western blotting, detection of caspase-cleaved PARP-1).However, only Aml decreased the intracellular accumulation of ROS induced byTNF-α (flow cytometry with the ROS probe DCFH-DA). In inhibition assays, cellswere preincubated with Aml or Dil before exposing them to Epo. Epo (200 ng/mL)provoked a significant transient rise in intracellular Ca (<30 s, flow cytometrywith Fluo-4AM), which was abrogated by both channel blockers. Wound healingassays (15 h) revealed an inhibitory effect of Aml (1 µM) on Epo-induced cell migration(Control: 25.6±1.6% *Epo: 34.8±2.0%, #Epo+Aml:22.4±2.7%; *P<0.05 vs. Control, #P<0.01 vs Epo, KruskalWallis-Dunn, n=7) while Dil (5 µM) showed no activity (Control: 25.0±1.6%,*Epo: 32.4±1.5%, Epo+Dil: 33.4±2.6%; *P<0.05 vs. Control, Kruskal Wallis-Dunn,n=7). Accordingly, Aml, but not Dil, was able to inhibit Epo-induced tubeformation in EA.hy926 cells.These results not only support the much-debatedpresence of VDCCs in endothelial cells, but also suggest a differential effectof amlodipine and diltiazem on angiogenesis, with possible implications in theircoadministration with a proangiogenic therapy.