CROT (Carnitine O-Octanoyltransferase) Is a Novel Contributing Factor in Vascular Calcification via Promoting Fatty Acid Metabolism and Mitochondrial Dysfunction

Abstract

Objective: Vascular calcification is a critical pathology associated with increased cardiovascular event risk, but there are no Food and Drug Administration-approved anticalcific therapies. We hypothesized and validated that an unbiased screening approach would identify novel mediators of human vascular calcification. Approach and Results: We performed an unbiased quantitative proteomics and pathway network analysis that identified increased CROT (carnitine O-octanoyltransferase) in calcifying primary human coronary artery smooth muscle cells (SMCs). Additionally, human carotid artery atherosclerotic plaques contained increased immunoreactive CROT near calcified regions. CROT siRNA reduced fibrocalcific response in calcifying SMCs. In agreement, histidine 327 to alanine point mutation inactivated human CROT fatty acid metabolism enzymatic activity and suppressed SMC calcification. CROT siRNA suppressed type 1 collagen secretion, and restored mitochondrial proteome alterations, and suppressed mitochondrial fragmentation in calcifying SMCs. Lipidomics analysis of SMCs incubated with CROT siRNA revealed increased eicosapentaenoic acid, a vascular calcification inhibitor. CRISPR/Cas9-mediated Crot deficiency in LDL (low-density lipoprotein) receptor-deficient mice reduced aortic and carotid artery calcification without altering bone density or liver and plasma cholesterol and triglyceride concentrations. Conclusions: CROT is a novel contributing factor in vascular calcification via promoting fatty acid metabolism and mitochondrial dysfunction, as such CROT inhibition has strong potential as an antifibrocalcific therapy.

Computational Screening Strategy for Drug Repurposing Identified Niclosamide as Inhibitor of Vascular Calcification

“…The remaining 13 candidates were then assessed for their ability to decrease CROT mRNA expression and suppress calcium deposition in hSMCs cultured in osteogenic media (OM). This osteogenic condition increased CROT transcriptional levels in hSMCs as previously reported ( 4 ). The maximum concentration was determined based on each compound's solubility and cell toxicity ( Supplementary Figure IIA ).…”

“…We recently reported carnitine o -octanoyltransferase (CROT) as a candidate target to suppress vascular calcification ( 4 ). Proteomics of human smooth muscle cells (hSMCs) identified elevated levels of CROT during the transition to a procalcifying phenotype.…”

“…Small interfering RNA (siRNA)-mediated suppression of CROT in hSMCs and genetic deletion of Crot in mice inhibited vascular calcification. Additional proteomics, lipidomics and network analysis studies deduced that CROT inhibition suppresses calcification potential by ameliorating mitochondrial function and fatty acid metabolism ( 4 ). Although this recent study suggests CROT as a promising target, it is only the beginning of multiple steps toward confirming CROT inhibition is indeed a viable therapy for cardiovascular calcification.…”

“…Given that many of these virtual approaches are publicly available, we sought to identify candidate compounds that can potentially reduce CROT expression, by screening a genetic perturbation resource known as L1000, using the Connectivity Map (CMap) ( 9 , 10 ). Since the reduction of CROT mRNA inhibits calcification ( 4 ), we screened for small molecules that were predicted to reduce CROT expression, with the idea that they could be candidate calcification inhibitors. We then validated them in vitro and in vivo .…”

Gut Metabolite Trimethylamine-N-Oxide in Atherosclerosis: From Mechanism to Therapy

“…Carnitine is the precursor of TMAO; interestingly, Okui et al performed an unbiased quantitative proteomics and pathway network analysis that identified increased carnitine-O-octanoyltransferase (CROT) levels in calcifying primary human coronary artery smooth muscle cells (SMCs). CROT was identified as a novel contributing factor in vascular calcification via promoting fatty acid metabolism and mitochondrial dysfunction ( 61 ). Moreover, this study also provides new potential target genes for the clinical treatment of vascular calcification.…”