The Apelin/APJ System Mediates Pravastatin ‐ but not Atorvastatin ‐ Induced HUVEC Proliferation
Authors: Lorenz Florian , Strohbach Anne , Felix Stephan B. , Dörr Marcus , Busch Raila , Bahls Martin 
Published: November 2023

IntroductionStatins are employed to prevent cardiovascular disease via a reduction in LDL cholesterol and a variety of pleiotropic effects, for example improving endothelial cell proliferation. In vascular endothelial cells the apelin/APJ system may mediate at least some of these pleiotropic effects. The aim of this investigation was to explore the influence of the apelin/APJ system on statin induced human umbilical vein endothelial cells (HUVECs) proliferation.MethodsA total of 6,000 HUVECs (passage 1 to 3) were seeded onto E‐Plates 16. Bioelectrical impedance (BI) was continuously measured for 48 h as a surrogate for HUVEC proliferation using a real‐time cell analyzer. A physiological (0.1 μM) and a supraphysiological (1 μM) concentration of Atorvastatin (Ator) and Pravastatin (Pra) were used to induce cell proliferation. The APJ‐antagonist ML221 (10 μM) alone or in combination with statins was applied to assess the role of the apelin/APJ system. The unit‐less normalized cell index (nCI) was used at 24 h and 48 h to describe relative changes in BI. Total sample size for each group was n ≥ 10. Statistical analyses were performed by one way analysis of variance. Statistical significance was considered for p < .05. HUVECs were fixed with 4% phosphate‐buffered formaldehyde solution after 48 h to confirm BI measurements by light microscopy.ResultsAfter 24 h a significant main effect for statin and the statin*ML221 interaction was observed. After 48 h significant main effects for statins and ML221 showed significant main effects. BI was not significantly different between control (Con) and both statins after 24 h. Ator augmented HUVEC proliferation compared to Con after 48 h at physiological (Con: 3.53±.11 vs. 0.1 μM: 4.27±.27 [p = .05]) but not at supraphysiological concentrations. Pra did not influence BI after 48 h. After 24 h ML221 significantly reduced HUVEC proliferation compared to Con (Con: 1.65±.074 vs. ML221: 1.19±.16 [p < .01]) and the combination of ML221 with statins increased BI compared to ML221 alone (ML221: 1.19±.16 vs. 0.1 μM Ator: 2.02±.08 [p < .01]; 1 μM Ator: 1.90±.19 [p < .01]; 0.1 μM Pra: 1.97±.11 [p < .01]; 1 μM Pra: 2.1±.13 [p < .01]). After 48 h ML221 had no significant effect on BI compared to Con, but ML221 increased HUVEC proliferation in combination with Pra compared to Pra alone (0.1 μM Pra: 3.78±.14 vs. 4.34±.20 [p = .03]; 1 μM Pra: 4.01±.15 vs 4.86±.20 [p < .01]). Light microscopy analysis confirmed differences in HUVEC proliferation.ConclusionStatins did not increase HUVEC proliferation after 24 h. After 48 h only physiological Atorvastatin increased cell proliferation. Both statins prevented the lower HUVEC proliferation due to inhibition of the apelin/APJ system. Interestingly, after 48 h when the apelin/APJ system was blocked only pravastatin independent of concentration increased HUVEC proliferation compared to pravastatin alone. Therefore, this is the first investigation to report that pravastatin but not atorvastatin induced HUVEC proliferation is influenced by the apelin/APJ system.Support or Funding InformationDZHK (German Centre for Cardiovascular Research)