Abstract
Byline: Filipe F Stoyell Conti, Univ of Miami, Pembroke Pnes, FL; Miguel Rojas, Surgery, Univ of Miami; Zachary Zigmond, Surgery, Univ of Miami; Mohdahmar Rauf, Univ of Miami, FL; Laisel Martinez, UNIVERSITY OF MIAMI, Miami, FL; Roberto I Vazquez-Padron, UNIVERSITY OF MIAMI, Miami, FL Aim: Dissect the mechanisms by which LOX increases atherogenesis and plaque calcification in hyperlipidemic mice. Methods: LOX conditional knockout [LOXf/fMyh11-CreERT2 ApoE -/-] and control littermate [LOXwt/wtMyh11-CreERT2 ApoE -/-] mice were injected with tamoxifen before high fat diet (HFD) feeding for atherosclerosis and calcification assessment and molecular analyses. Results: scRNA sequence showed full Lox gene inactivation in 72.5% of the aortic SMCs without any significant Loxl compensatory regulation. Gene deletion in SMCs decreased global arterial LOX deposition by ~3-fold in relation to control mice. Surprisingly, the loss of vascular LOX neither promoted aneurysm nor affected the aorta's stiffness. However, it reduced atherosclerosis burden (WT: 23Ø1 vs. KO: 13Ø2 %, p ëñ0.01) and calcium deposition (11.8Ø3 versus 5Ø0.4%, pëñ 0.05) within the plaque after 16 weeks of high fat diet (HFD). Using bulk RNA sequencing, we further demonstrated that Lox gene inactivation leads to a marked decrease in Frizzled Related Protein-3 (FRZB) gene expression (127-fold and pëñ0.0001), a biphasic modulator of the proatherogenic Wnt signaling that facilitates the association of ð-catenin with TCF4 in the nucleus. The compromised FRZB /ð-catenin/TCF4 signaling axis reduced the downstream production of Bone Morphogenetic Protein 2 (BMP2), which is responsible for the osteogenic transition of SMCs. Further scRNAseq analysis of atherosclerotic aortas from conditional KO and control mice also showed a reduction of BMP2 in SMCs. Interestingly, genetic inactivation of LOX increased the number of contractile SMCs (KO: 40 vs. WT: 7 %), characterized by the high expression of ACTA2 and TAGLN, in the aorta of HFD fed mice when compared to the control group. BAPN, an irreversible inhibitor of LOX activity, was not able to reduce BMP2 expression and protein levels nor reduce calcium deposition in vitro, suggesting that the pro-calcification effect of LOX is not activity dependent. Conclusions: Vascular gene inactivation of Lox protects mice against atherosclerosis by reducing ð-catenin/TCF4/BMP2 proatherogenic signaling.