Inhibition of acid sphingomyelinase‐mediated autophagic flux contributes to amitriptyline‐induced perturbation of tube formation in murine endothelial cells

Abstract

Amitriptyline is an inhibitor of lysosomal acid sphingomyelinase (gene symbol Smpd1) and a well‐known tricyclic anti‐depressant with cardiovascular side effects. We previously reported that loss of acid sphingomyelinase function leads to defective autophagic flux and enhanced dedifferentiation in vascular smooth muscle cells. However, it remains unknown whether amitriptyline has any cardiovascular side effects on the angiogenic capability of vascular endothelial cells and whether such effect is associated with impaired autophagy signaling pathways. Using an aortic ring assay, we demonstrated that the wild‐type aortas treated with amitriptyline or Smpd1+/− aortas exhibited decreased endothelial tube outgrowth from small slices of aorta embedded in Matrigel indicating attenuated ex vivo angiogenesis. Similarly, amitriptyline or Smpd1 gene silencing perturbed the tube formation capability of cultured mouse microvascular endothelial cells (MVECs). The inhibitory effect of amitriptyline on angiogenesis in MVECs was correlated with decreased activation of angiogenic signaling (phosphorylation of eNOS, Akt, and Erk1/2) and reduced endothelial cell proliferation. Mechanistically, amitriptyline alone had no effect on autophagy induction in MVECs but inhibited autophagic flux as shown by increased expression of LC3B and p62. Blockade of autophagic flux by lysosome function inhibitor bafilomycin or chloroquine mimics the effects of amitriptyline on angiogenic signaling, proliferation, and tube formation in MVECs. Collectively, these data suggest that defective acid sphingomyelinase‐mediated autophagic flux contributes to the cardiovascular side effects and toxicity of amitriptyline on proliferation and angiogenic capability of vascular endothelial cells.Support or Funding InformationNational Heart, Lung, and Blood Institute grants (HL122769, HL122937)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.