Azelnidipine, Not Amlodipine, Induces Secretion of Vascular Endothelial Growth Factor From Smooth Muscle Cells and Promotes Endothelial Tube Formation

Abstract

Background: We previously reported that the calcium channel blocker (CCB) nifedipine-induced secretion of vascular endothelial growth factor (VEGF) from human coronary smooth muscle cells (HCSMCs) promoted human coronary endothelial cell (HCEC) tube formation. Therefore, we analyzed whether other CCBs, azelnidipine and amlodipine, also induced the secretion of VEGF and promoted HCEC tube formation, and the underlying molecular mechanisms.

Methods: To evaluate the tube formation, HCECs were grown on Matrigel for 18 hours in the supernatants from HCSMCs that had been treated with different kinds of reagents. Concentrations of VEGF in cultured HCSMCs were determined by specific enzyme immunoassays. Nuclear extracts from HCSMCs were prepared, and nuclear factor-kappa B (NF-κB) activation was measured by EZ-Detect^TM Transcription Factor Kits for NF-κB p50 or p65.

Results: Although azelnidipine dose-dependently stimulated the significant secretion of VEGF from HCSMCs and this stimulation was abolished by a protein kinase C inhibitor, amlodipine-induced secretion of VEGF was significantly lower than that induced by azelnidipine. The medium derived from azelnidipine (at up to 2 μM)-treated HCSMCs led to HCEC tube formation, whereas that obtained with amlodipine did not. Azelnidipine-induced tube formation was blocked by an inhibitor of kinase insert domain-containing receptor/fetal liver kinase-1 tyrosine kinase. Azelnidipine at up to 2 μM induced NF-κB activation.

Conclusions: Azelnidipine, but not amlodipine, stimulated the secretion of VEGF from HCSMCs and induced HCEC tube formation. This secretion is mediated at least in part via the activation of NF-κB. Azelnidipine may have a novel beneficial effect in improving coronary microvascular blood flow in addition to its main effect of lowering blood pressure.

Keywords: Calcium channel blocker; Endothelial cell tube formation; Nuclear factor-kappa B; Smooth muscle cell; Vascular endothelial growth factor.

Figure 1

Cell proliferation in HCSMCs or HCECs with or without the indicated concentrations of azelnidipine (A) and amlodipine (B) for 18 h as assessed by an MTS assay. Open and closed circles indicate HCECs and HCSMCs, respectively. Graph shows the % cell proliferation compared with that in the untreated control sample (100%). *P < 0.05 vs. control.

Figure 2

(A) VEGF secretion from HCSMCs with or without the indicated concentrations of azelnidipine (closed circle) and amlodipine (open circles). *P < 0.05 vs. no treatment. #P < 0.05 vs. amlodipine. (B) VEGF secretion from HCSMCs with or without bradykinin, azelnidipine or Go6983. *P < 0.05 vs. no treatment.

Figure 3

NF-κB activation using azelnidipine. *P < 0.05 vs. 0 μM azelnidipine.

Figure 4

HCEC tube formation with or without the indicated concentrations of azelnidipine (A) and amlodipine (B). Representative pictures of HCECs plated on Matrigel are shown. Data show the % tube formation compared with that under no treatment (100%). *P < 0.05 vs. no treatment.

Figure 5

HCEC tube formation with or without 2 μM azelnidipine or 5 μM Tki. Representative pictures of HCECs plated on Matrigel are shown. Data show the % tube formation compared with that under no treatment (100%). *P < 0.05 vs. no treatment.

Figure 6

Proposed mechanisms of the effects of azelnidipine on endothelial tube formation.