Dataset Information

Carnitine O-octanoyltransferase is a novel inducer of 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 FDA-approved anti-calcific 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 carnitine O-octanoyltransferase (CROT) 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 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 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 inducer of vascular calcification via promoting fatty acid metabolism and mitochondrial dysfunction, as such CROT inhibition has strong potential as an anti-fibrocalcific therapy.

INSTRUMENT(S): Orbitrap Fusion Lumos, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Smooth Muscle Cell Of The Coronary Artery, Cell Culture, Coronary Artery Smooth Muscle Cell

SUBMITTER: Sasha Singh

LAB HEAD: Elena Aikawa

PROVIDER: PXD022575 | Pride | 2021-02-26

REPOSITORIES: Pride

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Publications

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

Okui Takehito T Iwashita Masaya M Rogers Maximillian A MA Halu Arda A Atkins Samantha K SK Kuraoka Shiori S Abdelhamid Ilyes I Higashi Hideyuki H Ramsaroop Ashisha A Aikawa Masanori M Singh Sasha A SA Aikawa Elena E

Arteriosclerosis, thrombosis, and vascular biology 20201224 2

<h4>Objective</h4>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 calci ...[more]

PMID: 33356393

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Project description:Vascular calcification is a complex process and has been associated with aging, diabetes, chronic kidney disease (CKD). Although there have been several studies studying the role of miRNAs (miRs) in bone osteogenesis, little is known about the role of miRs in vascular calcification and their role in the pathogenesis of vascular abnormalities. Matrix vesicles (MV) are known to play an important role in initiating vascular smooth muscle cell (VSMC) calcification. In the present study, we performed miRNA microarray analysis to identify the dysregulated miRs between MV and VSMC derived from CKD rats to understand the role of post-transcriptional regulatory networks governed by these miRNAs in vascular calcification and to uncover the differential miRNA content of MV. The percentage of miRNA to total RNA was increased in MV compared to VSMC. Comparison of expression profiles of miRNA by microarray demonstrated 33 miRs to be differentially expressed with the majority (~ 57%) of them down-regulated. Target genes controlled by differentially expressed miRNAs were identified utilizing two different complementary computational approaches Miranda and Targetscan to understand the functions and pathways that may be affected due to the production of MV from calcifying VSMC thereby contributing to the regulation of genes by miRs. We found several processes including vascular smooth muscle contraction, response to hypoxia and regulation of muscle cell differentiation to be enriched. Signaling pathways identified included MAP-kinase and wnt signaling that have previously been shown to be important in vascular calcification. In conclusion, our results demonstrate that miRs are concentrated in MV from calcifying VSMC, and that important functions and pathways are affected by the miRs dysregulation between calcifying VSMC and the MV they produce. This suggests that miRs may play a very important regulatory role in vascular calcification in CKD by controlling an extensive network of post-transcriptional targets. Compare miRNA from matrix vesicles to miRNA from vascular smooth muscle cells that gave rise to the matrix vesicles from 3 sets of MV and VSMC derived from 3 normal and 3 CKD rats

Project description:Background: Ectopic vascular calcifications represent a major clinical problem associated with cardiovascular disease and mortality. However, the mechanisms underlying pathological vascular calcifications are largely unknown hampering the development of therapies to tackle this life threatening medical condition. Results: In order to gain insight into the genes and mechanisms driving this pathological calcification process we analyzed the transcriptional profile of calcifying vascular smooth muscle cells (C-VSMCs). These profiles were compared to differentiating osteoblasts, cells that constitute their physiological calcification counterparts in the body. Overall the transcriptional program of C-VSMC and osteoblasts did not overlap. Several genes, some of them relevant for bone formation, were distinctly modulated by C-VSMCs which did not necessarily lose their smooth muscle cell markers while calcifying. Bioinformatics gene clustering and correlation analysis disclosed limited bone-related mechanisms being shared by two cell types. Extracellular matrix (ECM) and biomineralization genes represented common denominators between pathological vascular and physiological bone calcifications. These genes constitute the strongest link between these cells and represent potential drivers for their shared end-point phenotype. Conclusions: the analyses support the hypothesis that VSMC trans-differentiate into C-VSMCs keeping their own identity while using mechanisms that osteoblasts use to mineralize. The data provide novel insights into groups of genes and biological processes shared in MSC and VSMC osteogenic differentiation. The distinct gene regulation between C-VSMC and osteoblasts might hold clues to find cell-specific pathway modulations, opening the possibility to tackle undesired vascular calcifications without disturbing physiologic bone formation and vice versa. Total RNA obtained from hMSC and hVSMC cultured in osteogenic differentiation medium supplemented with 1.8 mM Ca2+ for 0, 2, 8, 12 or 25 days respectively. For each timepoint 3 replicates were used, with exception for day 0 where 4 replicates were collected.

Project description:Smooth muscle cells (SMCs) are important in a number of physiological systems and organs, including the cardiovascular system. The hallmark property of differentiated SMCs is the ability to contract, but contractile SMCs themselves show a range of phenotypes allowing prolonged tonic contraction in vascular smooth muscle or rapid phasic contraction in tissues such as bladder. Another distinctive characteristic, in contrast with terminally differentiated striated muscle cells, is that SMCs exhibit phenotypic plasticity. Vascular SMCs are able to modulate their phenotype along a continuum between a contractile phenotype, characteristic of healthy blood vessels, and a more proliferative âsyntheticâ phenotype, so-named for the enhanced synthesis and secretion of extracellular matrix components. Synthetic phenotype cells are found in a number of pathological situations such as atherosclerosis and arterial injury. We used mouse exon-junction (MJAY) arrays to gain insights into both the global contribution of alternative splicing events in re-shaping the transcriptome of dedifferentiating mouse aorta and bladder SMCs, and into the underlying regulatory mechanisms of the alternative splicing program. Affymetrix splice junction arrays (MJAY) were used to profile changes in both alternative splicing and transcript levels during the phenotypic modulation of smooth muscle cells when placed in culture. RNA extracted from intact aorta and bladder smooth muscle tissue was used for differentiated samples. For dedifferentiated, proliferative samples smooth muscle cells were enzymatically dispersed and grown in tissue culture for a week. Triplicate RNA samples were prepared from smooth muscle tissue of mouse aorta and bladder (differentiated) and from smooth muscle cells from each tissue cultured for 7 days (proliferative). The samples allowed comparison of alternative splicing (and other transcriptome) changes between differentiated and proliferative smooth muscle cell samples from two distinct types of smooth muscle cell, as well as allowing direct comparison of aorta (tonic smooth muscle) and bladder (phasic smooth muscle).

Dataset Information

Carnitine O-octanoyltransferase is a novel inducer of vascular calcification via promoting fatty acid metabolism and mitochondrial dysfunction

Publications

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

Similar Datasets

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