Publications by authors named "Ljubica Matic"

31 Publications

Integrative multiomics analysis of human atherosclerosis reveals a serum response factor-driven network associated with intraplaque hemorrhage.

Clin Transl Med 2021 Jun;11(6):e458

Department of Pathology, School for Cardiovascular Diseases (CARIM), Maastricht UMC+, Maastricht, The Netherlands.

Background: While single-omics analyses on human atherosclerotic plaque have been very useful to map stage- or disease-related differences in expression, they only partly capture the array of changes in this tissue and suffer from scale-intrinsic limitations. In order to better identify processes associated with intraplaque hemorrhage and plaque instability, we therefore combined multiple omics into an integrated model.

Methods: In this study, we compared protein and gene makeup of low- versus high-risk atherosclerotic lesion segments from carotid endarterectomy patients, as judged from the absence or presence of intraplaque hemorrhage, respectively. Transcriptomic, proteomic, and peptidomic data of this plaque cohort were aggregated and analyzed by DIABLO, an integrative multivariate classification and feature selection method.

Results: We identified a protein-gene associated multiomics model able to segregate stable, nonhemorrhaged from vulnerable, hemorrhaged lesions at high predictive performance (AUC >0.95). The dominant component of this model correlated with αSMA PDGFRα fibroblast-like cell content (p = 2.4E-05) and Arg1 macrophage content (p = 2.2E-04) and was driven by serum response factor (SRF), possibly in a megakaryoblastic leukemia-1/2 (MKL1/2) dependent manner. Gene set overrepresentation analysis on the selected key features of this model pointed to a clear cardiovascular disease signature, with overrepresentation of extracellular matrix synthesis and organization, focal adhesion, and cholesterol metabolism terms, suggestive of the model's relevance for the plaque vulnerability. Finally, we were able to corroborate the predictive power of the selected features in several independent mRNA and proteomic plaque cohorts.

Conclusions: In conclusion, our integrative omics study has identified an intraplaque hemorrhage-associated cardiovascular signature that provides excellent stratification of low- from high-risk carotid artery plaques in several independent cohorts. Further study revealed suppression of an SRF-regulated disease network, controlling lesion stability, in vulnerable plaque, which can serve as a scaffold for the design of targeted intervention in plaque destabilization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ctm2.458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8236116PMC
June 2021

Proteoglycan 4 Modulates Osteogenic Smooth Muscle Cell Differentiation during Vascular Remodeling and Intimal Calcification.

Cells 2021 May 21;10(6). Epub 2021 May 21.

Vascular Surgery, Department of Molecular Medicine and Surgery, Karolinska Institutet, 17164 Stockholm, Sweden.

Calcification is a prominent feature of late-stage atherosclerosis, but the mechanisms driving this process are unclear. Using a biobank of carotid endarterectomies, we recently showed that Proteoglycan 4 (PRG4) is a key molecular signature of calcified plaques, expressed in smooth muscle cell (SMC) rich regions. Here, we aimed to unravel the PRG4 role in vascular remodeling and intimal calcification. expression in human carotid endarterectomies correlated with calcification assessed by preoperative computed tomographies. PRG4 localized to SMCs in early intimal thickening, while in advanced lesions it was found in the extracellular matrix, surrounding macro-calcifications. In experimental models, was upregulated in SMCs from partially ligated mice and rat carotid intimal hyperplasia, correlating with osteogenic markers and . Furthermore, PRG4 was enriched in cells positive for chondrogenic marker SOX9 and around plaque calcifications in mice on warfarin. In vitro, was induced in SMCs by IFNg, TGFb1 and calcifying medium, while SMC markers were repressed under calcifying conditions. Silencing experiments showed that expression was driven by transcription factors and Functionally, the addition of recombinant human PRG4 increased ectopic SMC calcification, while arresting cell migration and proliferation. Mechanistically, it suppressed endogenous , and , and restored SMC markers' expression. PRG4 modulates SMC function and osteogenic phenotype during intimal remodeling and macro-calcification in response to TGFb1 signaling, SMAD3 and SOX9 activation. The effects of PRG4 on SMC phenotype and calcification suggest its role in atherosclerotic plaque stability, warranting further investigations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells10061276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8224064PMC
May 2021

AMPA-Type Glutamate Receptors Associated With Vascular Smooth Muscle Cell Subpopulations in Atherosclerosis and Vascular Injury.

Front Cardiovasc Med 2021 20;8:655869. Epub 2021 Apr 20.

Laboratory of Immunobiology, Center for Bioelectronic Medicine, Department of Medicine, Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden.

Vascular smooth muscle cells (VSMCs) are key constituents of both normal arteries and atherosclerotic plaques. They have an ability to adapt to changes in the local environment by undergoing phenotypic modulation. An improved understanding of the mechanisms that regulate VSMC phenotypic changes may provide insights that suggest new therapeutic targets in treatment of cardiovascular disease (CVD). The amino-acid glutamate has been associated with CVD risk and VSMCs metabolism in experimental models, and glutamate receptors regulate VSMC biology and promote pulmonary vascular remodeling. However, glutamate-signaling in human atherosclerosis has not been explored. We identified glutamate receptors and glutamate metabolism-related enzymes in VSMCs from human atherosclerotic lesions, as determined by single cell RNA sequencing and microarray analysis. Expression of the receptor subunits glutamate receptor, ionotropic, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA)-type subunit 1 (GRIA1) and 2 (GRIA2) was restricted to cells of mesenchymal origin, primarily VSMCs, as confirmed by immunostaining. In a rat model of arterial injury and repair, changes of GRIA1 and GRIA2 mRNA level were most pronounced at time points associated with VSMC proliferation, migration, and phenotypic modulation. , human carotid artery SMCs expressed GRIA1, and selective AMPA-type receptor blocking inhibited expression of typical contractile markers and promoted pathways associated with VSMC phenotypic modulation. In our biobank of human carotid endarterectomies, low expression of AMPA-type receptor subunits was associated with higher content of inflammatory cells and a higher frequency of adverse clinical events such as stroke. AMPA-type glutamate receptors are expressed in VSMCs and are associated with phenotypic modulation. Patients suffering from adverse clinical events showed significantly lower mRNA level of GRIA1 and GRIA2 in their atherosclerotic lesions compared to asymptomatic patients. These results warrant further mapping of neurotransmitter signaling in the pathogenesis of human atherosclerosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcvm.2021.655869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8093397PMC
April 2021

Long Non-coding RNA Aerrie Controls DNA Damage Repair via YBX1 to Maintain Endothelial Cell Function.

Front Cell Dev Biol 2020 11;8:619079. Epub 2021 Jan 11.

Department of Physiology, Amsterdam UMC, Vrije Universiteit Amsterdam, Amsterdam, Netherlands.

Aging is accompanied by many physiological changes. These changes can progressively lead to many types of cardiovascular diseases. During this process blood vessels lose their ability to maintain vascular homeostasis, ultimately resulting in hypertension, stroke, or myocardial infarction. Increase in DNA damage is one of the hallmarks of aging and can be repaired by the DNA signaling and repair system. In our study we show that long non-coding RNA Aerrie (linc01013) contributes to the DNA signaling and repair mechanism. Silencing of Aerrie in endothelial cells impairs angiogenesis, migration, and barrier function. Aerrie associates with YBX1 and together they act as important factors in DNA damage signaling and repair. This study identifies Aerrie as a novel factor in genomic stability and as a binding partner of YBX1 in responding to DNA damage.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcell.2020.619079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829583PMC
January 2021

Chitinase 3 like 1 (CHI3L1) is a regulator of smooth muscle cell physiology and atherosclerotic lesion stability.

Cardiovasc Res 2021 Jan 20. Epub 2021 Jan 20.

Division of Vascular Surgery, Stanford University School of Medicine, Stanford, USA.

Aims: Atherosclerotic cerebrovascular disease underlies the majority of ischemic strokes and is a major cause of death and disability. While plaque burden is a predictor of adverse outcomes, plaque vulnerability is increasingly recognized as a driver of lesion rupture and risk for clinical events. Defining the molecular regulators of carotid instability could inform the development of new biomarkers and/or translational targets for at-risk individuals.

Methods And Results: Using two independent human endarterectomy biobanks, we found that the understudied glycoprotein, Chitinase 3 like 1 (CHI3L1), is upregulated in patients with carotid disease compared to healthy controls. Further, CHI3L1 levels were found to stratify individuals based on symptomatology and histopathological evidence of an unstable fibrous cap. Gain- and loss-of-function studies in cultured human carotid artery smooth muscle cells (SMCs) showed that CHI3L1 prevents a number of maladaptive changes in that cell type, including phenotype switching towards a synthetic and hyperproliferative state. Using two murine models of carotid remodelling and lesion vulnerability, we found that knockdown of Chil1 resulted in larger neointimal lesions comprised by de-differentiated SMCs that failed to invest within and stabilize the fibrous cap. Exploratory mechanistic studies identified alterations in potential downstream regulatory genes, including large tumor suppressor kinase 2 (LATS2), which mediates macrophage marker and inflammatory cytokine expression on SMCs, and may explain how CHI3L1 modulates cellular plasticity.

Conclusion: CHI3L1 is upregulated in humans with carotid artery disease and appears to be a strong mediator of plaque vulnerability. Mechanistic studies suggest this change may be a context-dependent adaptive response meant to maintain vascular SMCs in a differentiated state and to prevent rupture of the fibrous cap. Part of this effect may be mediated through downstream suppression of LATS2. Future studies should determine how these changes occur at the molecular level, and whether this gene can be targeted as a novel translational therapy for subjects at risk of stroke.

Translational Perspective: Taken together, CHI3L1 has the potential to become a new translational target for cardiovascular disease. With further studies to understand its full causal relationship to inflammatory pathways, it could have a role in the diagnosis and management of patients with cerebrovascular disease at risk for stroke.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/cvr/cvab014DOI Listing
January 2021

Expression of Interleukin 6 signaling receptors in carotid atherosclerosis.

Vasc Med 2021 02 22;26(1):3-10. Epub 2020 Dec 22.

Cardiovascular Medicine Unit, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.

Interleukin (IL) 6 contributes to atherosclerotic plaque development through IL6 membrane-bound (IL6R and gp130) and soluble (sIL6R and sgp130) receptors. We investigated IL6 receptor expression in carotid plaques and its correlation with circulating IL6 and soluble receptor levels. Plasma samples and carotid plaques were obtained from 78 patients in the Biobank of Karolinska Endarterectomies study. IL6, sIL6R, and sgp130 were measured in plasma and , and s (s) gene expression assessed in carotid plaques. Correlations between plaque IL6 signaling gene expression and plasma levels were determined by Spearman's correlation. Differences in plasma and gene expression levels between patients with ( = 53) and without ( = 25) a history of a cerebral event and statin-treated ( = 65) and non-treated ( = 11), were estimated by Kruskal-Wallis. and its receptors were all expressed in carotid plaques. There was a positive, borderline significant, moderate correlation between plasma IL6 and sIL6R and the respective gene expression levels (rho 0.23 and 0.22, both = 0.05). expression was higher in patients with a history of a cerebrovascular event compared to those without ( = 0.007). Statin-treated had higher , and s expression levels and plasma sIL6R compared to non-treated patients (all < 0.05). In conclusion, all components of the IL6 signaling pathways are expressed in carotid artery plaques and IL6 and sIL6R plasma levels correlate moderately with and . Our data suggest that IL6 signaling in the circulation might mirror the system activity in the plaque, thus adding novel perspectives to the role of IL6 signaling in atherosclerosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/1358863X20977662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7879223PMC
February 2021

Expression Associates With Inflammation in Early Atherosclerosis in Humans and Can Be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice.

Circulation 2021 Jan 23;143(2):163-177. Epub 2020 Nov 23.

University of Ottawa Heart Institute, Canada (D.K., M.-A.N., M.G., Z.L., H.W., J.W.K., R.J., A.M., A.R., P.L., K.J.R.).

Background: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. We previously showed that macrophages in the atherogenic plaque undergo RIPK3 (receptor-interacting serine/threonine-protein kinase 3)-MLKL (mixed lineage kinase domain-like protein)-dependent programmed necroptosis in response to sterile ligands such as oxidized low-density lipoprotein and damage-associated molecular patterns and that necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1 (receptor-interacting serine/threonine-protein kinase 1), which acts as a master switch that controls whether the cell undergoes NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells)-dependent inflammation, caspase-dependent apoptosis, or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is driven largely by NF-κB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NF-κB-dependent inflammation in early atherogenic lesions, and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis.

Methods: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and used loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 antisense oligonucleotides to mice fed a cholesterol-rich (Western) diet for 8 weeks.

Results: We find that RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 antisense oligonucleotides led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, <0.01) and plasma inflammatory cytokines (IL-1α [interleukin 1α], IL-17A [interleukin 17A], <0.05) in comparison with controls. knockdown in macrophages decreased inflammatory genes (NF-κB, TNFα [tumor necrosis factor α], IL-1α) and in vivo lipopolysaccharide- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of , , and monocyte attachment.

Conclusions: We identify RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.118.038379DOI Listing
January 2021

A neutralizing antibody against DKK1 does not reduce plaque formation in classical murine models of atherosclerosis: Is the therapeutic potential lost in translation?

Atherosclerosis 2020 12 6;314:1-9. Epub 2020 Oct 6.

Global Drug Discovery, Novo Nordisk A/S, Maaloev, Denmark. Electronic address:

Background And Aims: Clinical interventions targeting nonlipid risk factors are needed given the high residual risk of atherothrombotic events despite effective control of dyslipidemia. Dickkopf-1 (DKK1) plays a lipid-independent role in vascular pathophysiology but its involvement in atherosclerosis development and its therapeutic attractiveness remain to be established.

Methods: Patient data, in vitro studies and pharmacological intervention in murine models of atherosclerosis were utilized.

Results: In patients' material (n = 127 late stage plaque specimens and n = 10 control vessels), DKK1 mRNA was found to be higher in atherosclerotic plaques versus control arteries. DKK1 protein was detected in the luminal intimal area and in the necrotic core of plaques. DKK1 was released from isolated primary human platelets (~12 - 21-fold) and endothelial cells (~1.4-2.5-fold) upon stimulation with different pathophysiological stimuli. In ApoE and Ldlr mice, plasma DKK1 concentrations were similar to those observed in humans, whereas DKK1 expression in different atheroprone arterial segments was very low/absent. Chronic treatment with a neutralizing DKK1 antibody effectively reduced plasma concentrations, however, plaque lesion area was not reduced in ApoE and Ldlr mice fed a western diet for 14 and 16 weeks. Anti-DKK1 treatment increased bone volume and bone mineral content.

Conclusions: Functional inhibition of DKK1 with an antibody does not alter atherosclerosis progression in classical murine models. This may reflect the absence of DKK1 expression in plaques and more advanced animal disease models could be needed to evaluate the role and therapeutic attractiveness of DKK1 in late stage complications such as plaque destabilization, calcification, rupture and thrombosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.atherosclerosis.2020.10.001DOI Listing
December 2020

Genomic and drug target evaluation of 90 cardiovascular proteins in 30,931 individuals.

Nat Metab 2020 10 16;2(10):1135-1148. Epub 2020 Oct 16.

SCALLOP consortium.

Circulating proteins are vital in human health and disease and are frequently used as biomarkers for clinical decision-making or as targets for pharmacological intervention. Here, we map and replicate protein quantitative trait loci (pQTL) for 90 cardiovascular proteins in over 30,000 individuals, resulting in 451 pQTLs for 85 proteins. For each protein, we further perform pathway mapping to obtain trans-pQTL gene and regulatory designations. We substantiate these regulatory findings with orthogonal evidence for trans-pQTLs using mouse knockdown experiments (ABCA1 and TRIB1) and clinical trial results (chemokine receptors CCR2 and CCR5), with consistent regulation. Finally, we evaluate known drug targets, and suggest new target candidates or repositioning opportunities using Mendelian randomization. This identifies 11 proteins with causal evidence of involvement in human disease that have not previously been targeted, including EGF, IL-16, PAPPA, SPON1, F3, ADM, CASP-8, CHI3L1, CXCL16, GDF15 and MMP-12. Taken together, these findings demonstrate the utility of large-scale mapping of the genetics of the proteome and provide a resource for future precision studies of circulating proteins in human health.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42255-020-00287-2DOI Listing
October 2020

Integrative Multi-Omics Analysis in Calcific Aortic Valve Disease Reveals a Link to the Formation of Amyloid-Like Deposits.

Cells 2020 09 24;9(10). Epub 2020 Sep 24.

Department of Internal Medicine I-Cardiology, Medical Faculty, RWTH Aachen University, 52074 Aachen, Germany.

Calcific aortic valve disease (CAVD) is the most prevalent valvular heart disease in the developed world, yet no pharmacological therapy exists. Here, we hypothesize that the integration of multiple omic data represents an approach towards unveiling novel molecular networks in CAVD. Databases were searched for CAVD omic studies. Differentially expressed molecules from calcified and control samples were retrieved, identifying 32 micro RNAs (miRNA), 596 mRNAs and 80 proteins. Over-representation pathway analysis revealed platelet degranulation and complement/coagulation cascade as dysregulated pathways. Multi-omics integration of overlapping proteome/transcriptome molecules, with the miRNAs, identified a CAVD protein-protein interaction network containing seven seed genes (apolipoprotein A1 (APOA1), hemoglobin subunit β (HBB), transferrin (TF), α-2-macroglobulin (A2M), transforming growth factor β-induced protein (TGFBI), serpin family A member 1 (SERPINA1), lipopolysaccharide binding protein (LBP), inter-α-trypsin inhibitor heavy chain 3 (ITIH3) and immunoglobulin κ constant (IGKC)), four input miRNAs (miR-335-5p, miR-3663-3p, miR-21-5p, miR-93-5p) and two connector genes (amyloid beta precursor protein (APP) and transthyretin (TTR)). In a metabolite-gene-disease network, Alzheimer's disease exhibited the highest degree of betweenness. To further strengthen the associations based on the multi-omics approach, we validated the presence of APP and TTR in calcified valves from CAVD patients by immunohistochemistry. Our study suggests a novel molecular CAVD network potentially linked to the formation of amyloid-like structures. Further investigations on the associated mechanisms and therapeutic potential of targeting amyloid-like deposits in CAVD may offer significant health benefits.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9102164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600313PMC
September 2020

Induction of the Coxsackievirus and Adenovirus Receptor in Macrophages During the Formation of Atherosclerotic Plaques.

J Infect Dis 2020 11;222(12):2041-2051

Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet and Karolinska University Hospital Huddinge, Stockholm, Sweden.

Multiple viruses are implicated in atherosclerosis, but the mechanisms by which they infect cells and contribute to plaque formation in arterial walls are not well understood. Based on reports showing the presence of enterovirus in atherosclerotic plaques we hypothesized that the coxsackievirus and adenovirus receptor (CXADR/CAR), although absent in normal arteries, could be induced during plaque formation. Large-scale microarray and mass spectrometric analyses revealed significant up-regulation of CXADR messenger RNA and protein levels in plaque-invested carotid arteries compared with control arteries. Macrophages were identified as a previously unknown cellular source of CXADR in human plaques and plaques from Ldr-/-Apob100/100 mice. CXADR was specifically associated with M1-polarized macrophages and foam cells and was experimentally induced during macrophage differentiation. Furthermore, it was significantly correlated with receptors for other viruses linked to atherosclerosis. The results show that CXADR is induced in macrophages during plaque formation, suggesting a mechanism by which enterovirus infect cells in atherosclerotic plaques.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/infdis/jiaa418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7661765PMC
November 2020

Lack of PCSK6 Increases Flow-Mediated Outward Arterial Remodeling in Mice.

Cells 2020 04 18;9(4). Epub 2020 Apr 18.

Department of Molecular Medicine and Surgery, Karolinska Institutet, 171 76 Stockholm, Sweden.

Proprotein convertases (PCSKs) process matrix metalloproteases and cytokines, but their function in the vasculature is largely unknown. Previously, we demonstrated upregulation of PCSK6 in atherosclerotic plaques from symptomatic patients, localization to smooth muscle cells (SMCs) in the fibrous cap and positive correlations with inflammation, extracellular matrix remodeling and cytokines. Here, we hypothesize that PCSK6 could be involved in flow-mediated vascular remodeling and aim to evaluate its role in the physiology of this process using knockout mice. Pcsk6 and wild type mice were randomized into control and increased blood flow groups and induced in the right common carotid artery (CCA) by ligation of the left CCA. The animals underwent repeated ultrasound biomicroscopy (UBM) examinations followed by euthanization with subsequent evaluation using wire myography, transmission electron microscopy or histology. The Pcsk6 mice displayed a flow-mediated increase in lumen circumference over time, assessed with UBM. Wire myography revealed differences in the flow-mediated remodeling response detected as an increase in lumen circumference at optimal stretch with concomitant reduction in active tension. Furthermore, a flow-mediated reduction in expression of SMC contractile markers SMA, MYH11 and LMOD1 was seen in the Pcsk6 media. Absence of PCSK6 increases outward remodeling and reduces medial contractility in response to increased blood flow.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9041009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7225991PMC
April 2020

Proteoglycan 4 is Increased in Human Calcified Aortic Valves and Enhances Valvular Interstitial Cell Calcification.

Cells 2020 03 11;9(3). Epub 2020 Mar 11.

Department of Medicine, Karolinska Institutet, 17177 Stockholm, Sweden.

Aortic valve stenosis (AVS), a consequence of increased fibrosis and calcification of the aortic valve leaflets, causes progressive narrowing of the aortic valve. Proteoglycans, structural components of the aortic valve, accumulate in regions with fibrosis and moderate calcification. Particularly, proteoglycan 4 (PRG4) has been identified in fibrotic parts of aortic valves. However, the role of PRG4 in the context of AVS and aortic valve calcification has not yet been determined. Here, transcriptomics, histology, and immunohistochemistry were performed in human aortic valves from patients undergoing aortic valve replacement. Human valve interstitial cells (VICs) were used for calcification experiments and RNA expression analysis. PRG4 was significantly upregulated in thickened and calcified regions of aortic valves compared with healthy regions. In addition, mRNA levels of PRG4 positively associated with mRNA for proteins involved in cardiovascular calcification. Treatment of VICs with recombinant human PRG4 enhanced phosphate-induced calcification and increased the mRNA expression of bone morphogenetic protein 2 and the runt-related transcription factor 2. In summary, PRG4 was upregulated in the development of AVS and promoted VIC osteogenic differentiation and calcification. These results suggest that an altered valve leaflet proteoglycan composition may play a role in the progression of AVS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cells9030684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140654PMC
March 2020

Antagonistic relationship between the unfolded protein response and myocardin-driven transcription in smooth muscle.

J Cell Physiol 2020 10 10;235(10):7370-7382. Epub 2020 Feb 10.

Department of Experimental Medical Science, Lund, Sweden.

Smooth muscle cells (SMCs) are characterized by a high degree of phenotypic plasticity. Contractile differentiation is governed by myocardin-related transcription factors (MRTFs), in particular myocardin (MYOCD), and when their drive is lost, the cells become proliferative and synthetic with an expanded endoplasmic reticulum (ER). ER is responsible for assembly and folding of secreted proteins. When the load on the ER surpasses its capacity, three stress sensors (activating transcription factor 6 [ATF6], inositol-requiring enzyme 1α [IRE1α]/X-box binding protein 1 [XBP1], and PERK/ATF4) are activated to expand the ER and increase its folding capacity. This is referred to as the unfolded protein response (UPR). Here, we hypothesized that there is a reciprocal relationship between SMC differentiation and the UPR. Tight negative correlations between SMC markers (MYH11, MYOCD, KCNMB1, SYNPO2) and UPR markers (SDF2L1, CALR, MANF, PDIA4) were seen in microarray data sets from carotid arterial injury, partial bladder outlet obstruction, and bladder denervation, respectively. The UPR activators dithiothreitol (DTT) and tunicamycin (TN) activated the UPR and reduced MYOCD along with SMC markers in vitro. The IRE1α inhibitor 4μ8C counteracted the effect of DTT and TN on SMC markers and MYOCD expression. Transfection of active XBP1s was sufficient to reduce both MYOCD and the SMC markers. MRTFs also antagonized the UPR as indicated by reduced TN and DTT-mediated induction of CRELD2, MANF, PDIA4, and SDF2L1 following overexpression of MRTFs. The latter effect did not involve the newly identified MYOCD/SRF target MSRB3, or reduced production of either XBP1s or cleaved ATF6. The UPR thus counteracts SMC differentiation via the IRE1α/XBP1 arm of the UPR and MYOCD repression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jcp.29637DOI Listing
October 2020

PCSK6 Is a Key Protease in the Control of Smooth Muscle Cell Function in Vascular Remodeling.

Circ Res 2020 02 2;126(5):571-585. Epub 2020 Jan 2.

From the Department of Molecular Medicine and Surgery, Karolinska Institute and Karolinska University Hospital Solna, Stockholm, Sweden (U.R., B.E.S., S.R., A.R., M.L., M.K., U.H., L.M.).

Rationale: PCSKs (Proprotein convertase subtilisins/kexins) are a protease family with unknown functions in vasculature. Previously, we demonstrated PCSK6 upregulation in human atherosclerotic plaques associated with smooth muscle cells (SMCs), inflammation, extracellular matrix remodeling, and mitogens.

Objective: Here, we applied a systems biology approach to gain deeper insights into the PCSK6 role in normal and diseased vessel wall.

Methods And Results: Genetic analyses revealed association of intronic variant rs1531817 with maximum internal carotid intima-media thickness progression in high-cardiovascular risk subjects. This variant was linked with PCSK6 mRNA expression in healthy aortas and plaques but also with overall plaque SMA+ cell content and pericyte fraction. Increased PCSK6 expression was found in several independent human cohorts comparing atherosclerotic lesions versus healthy arteries, using transcriptomic and proteomic datasets. By immunohistochemistry, PCSK6 was localized to fibrous cap SMA+ cells and neovessels in plaques. In human, rat, and mouse intimal hyperplasia, PCSK6 was expressed by proliferating SMA+ cells and upregulated after 5 days in rat carotid balloon injury model, with positive correlation to PDGFB (platelet-derived growth factor subunit B) and MMP (matrix metalloprotease) 2/MMP14. Here, PCSK6 was shown to colocalize and cointeract with MMP2/MMP14 by in situ proximity ligation assay. Microarrays of carotid arteries from Pcsk6 versus control mice revealed suppression of contractile SMC markers, extracellular matrix remodeling enzymes, and cytokines/receptors. Pcsk6 mice showed reduced intimal hyperplasia response upon carotid ligation in vivo, accompanied by decreased MMP14 activation and impaired SMC outgrowth from aortic rings ex vivo. PCSK6 silencing in human SMCs in vitro leads to downregulation of contractile markers and increase in MMP2 expression. Conversely, PCSK6 overexpression increased PDGFBB (platelet-derived growth factor BB)-induced cell proliferation and particularly migration.

Conclusions: PCSK6 is a novel protease that induces SMC migration in response to PDGFB, mechanistically via modulation of contractile markers and MMP14 activation. This study establishes PCSK6 as a key regulator of SMC function in vascular remodeling. Visual Overview: An online visual overview is available for this article.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCRESAHA.119.316063DOI Listing
February 2020

Endothelin-1 increases expression and activity of arginase 2 via ETB receptors and is co-expressed with arginase 2 in human atherosclerotic plaques.

Atherosclerosis 2020 01 1;292:215-223. Epub 2019 Oct 1.

From the Department of Medicine, Division of Cardiology, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden.

Background And Aims: Endothelin-1 (ET-1) and arginase are both suggested to be involved in the inflammatory processes and development of endothelial dysfunction in atherosclerosis. However, information regarding the roles of ET-1 and arginase, as well as the interactions between the two in human atherosclerosis, is scarce. We investigated the expression of ET-1 and its receptors, ET and ET, as well as arginase in human carotid atherosclerotic plaques and determined the functional interactions between ET-1 and arginase in endothelial cells and THP-1-derived macrophages.

Methods: Carotid plaques and blood samples were retreived from patients undergoing surgery for symptomatic or asymptomatic carotid stenosis. Plaque gene and protein expression was determined and related to clinical characteristics. Functional interactions between ET-1 and arginase were investigated in endothelial cells and THP-1 cells.

Results: Expression of ET-1 and ET receptors was increased in plaques from patients with symptomatic carotid artery disease. ET-1 was co-localized with arginase 1 and arginase 2 in the necrotic core, together with macrophage markers CD163 and CD68. Arginase 2, ET-1 and ET receptors were expressed in endothelial cells as well as in smooth muscle cells in the fibrous cap. ET-1 increased arginase 2 mRNA expression and arginase activity in endothelial cells and arginase activity in macrophages. Moreover, ET-1 stimulated formation of reactive oxygen species (ROS) in THP-1-derived macrophages via an arginase-dependent mechanism.

Conclusions: This is the first study that demonstrates co-localization of ET-1 and arginase 2 in human atherosclerotic plaques. ET-1 stimulated arginase 2 expression and activity in endothelial cells, as well as arginase activity and ROS formation in macrophages via an arginase-dependent mechanism. These results indicate an important interaction between the ET pathway and arginase in human atherosclerotic plaques.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.atherosclerosis.2019.09.020DOI Listing
January 2020

Oncostatin M reduces atherosclerosis development in APOE*3Leiden.CETP mice and is associated with increased survival probability in humans.

PLoS One 2019 28;14(8):e0221477. Epub 2019 Aug 28.

Laboratory of Experimental Cardiology, University Medical Center Utrecht, Utrecht, The Netherlands.

Objective: Previous studies indicate a role for Oncostatin M (OSM) in atherosclerosis and other chronic inflammatory diseases for which inhibitory antibodies are in development. However, to date no intervention studies with OSM have been performed, and its relation to coronary heart disease (CHD) has not been studied.

Approach And Results: Gene expression analysis on human normal arteries (n = 10) and late stage/advanced carotid atherosclerotic arteries (n = 127) and in situ hybridization on early human plaques (n = 9) showed that OSM, and its receptors, OSM receptor (OSMR) and Leukemia Inhibitory Factor Receptor (LIFR) are expressed in normal arteries and atherosclerotic plaques. Chronic OSM administration in APOE*3Leiden.CETP mice (n = 15/group) increased plasma E-selectin levels and monocyte adhesion to the activated endothelium independently of cholesterol but reduced the amount of inflammatory Ly-6CHigh monocytes and atherosclerotic lesion size and severity. Using aptamer-based proteomics profiling assays high circulating OSM levels were shown to correlate with post incident CHD survival probability in the AGES-Reykjavik study (n = 5457).

Conclusions: Chronic OSM administration in APOE*3Leiden.CETP mice reduced atherosclerosis development. In line, higher serum OSM levels were correlated with improved post incident CHD survival probability in patients, suggesting a protective cardiovascular effect.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0221477PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713386PMC
March 2020

Identification of the intermediate filament protein synemin/SYNM as a target of myocardin family coactivators.

Am J Physiol Cell Physiol 2019 12 28;317(6):C1128-C1142. Epub 2019 Aug 28.

Department of Experimental Medical Science, Lund, Sweden.

Myocardin (MYOCD) is a critical regulator of smooth muscle cell (SMC) differentiation, but its transcriptional targets remain to be exhaustively characterized, especially at the protein level. Here we leveraged human RNA and protein expression data to identify novel potential MYOCD targets. Using correlation analyses we found several targets that we could confirm at the protein level, including SORBS1, SLMAP, SYNM, and MCAM. We focused on , which encodes the intermediate filament protein synemin. rivalled smooth muscle myosin () for SMC specificity and was controlled at the mRNA and protein levels by all myocardin-related transcription factors (MRTFs: MYOCD, MRTF-A/MKL1, and MRTF-B/MKL2). MRTF activity is regulated by the ratio of filamentous to globular actin, and was accordingly reduced by interventions that depolymerize actin, such as latrunculin treatment and overexpression of constitutively active cofilin. Many MRTF target genes depend on serum response factor (SRF), but lacked SRF-binding motifs in its proximal promoter, which was not directly regulated by MYOCD. Furthermore, resisted SRF silencing, yet the time course of induction closely paralleled that of the SRF-dependent target gene . was repressed by the ternary complex factor (TCF) FLI1 and was increased in mouse embryonic fibroblasts lacking three classical TCFs (ELK1, ELK3, and ELK4). Imaging showed colocalization of SYNM with the intermediate filament proteins desmin and vimentin, and MRTF-A/MKL1 increased SYNM-containing intermediate filaments in SMCs. These studies identify SYNM as a novel SRF-independent target of myocardin that is abundantly expressed in all SMCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpcell.00047.2019DOI Listing
December 2019

The long noncoding RNA CHROME regulates cholesterol homeostasis in primate.

Nat Metab 2019 01 3;1(1):98-110. Epub 2018 Dec 3.

Department of Medicine, Leon H. Charney Division of Cardiology, New York University School of Medicine, New York, New York, USA.

The human genome encodes thousands of long non-coding RNAs (lncRNAs), the majority of which are poorly conserved and uncharacterized. Here we identify a primate-specific lncRNA (), elevated in the plasma and atherosclerotic plaques of individuals with coronary artery disease, that regulates cellular and systemic cholesterol homeostasis. LncRNA expression is influenced by dietary and cellular cholesterol via the sterol-activated liver X receptor transcription factors, which control genes mediating responses to cholesterol overload. Using gain- and loss-of-function approaches, we show that promotes cholesterol efflux and HDL biogenesis by curbing the actions of a set of functionally related microRNAs that repress genes in those pathways. knockdown in human hepatocytes and macrophages increases levels of miR-27b, miR-33a, miR-33b and miR-128, thereby reducing expression of their overlapping target gene networks and associated biologic functions. In particular, cells lacking show reduced expression of ABCA1, which regulates cholesterol efflux and nascent HDL particle formation. Collectively, our findings identify as a central component of the non-coding RNA circuitry controlling cholesterol homeostasis in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42255-018-0004-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691505PMC
January 2019

Regulation of expression in human vascular endothelial cells by a neighboring divergently transcribed long noncoding RNA.

Proc Natl Acad Sci U S A 2019 08 26;116(33):16410-16419. Epub 2019 Jul 26.

Toronto General Hospital Research Institute, University Health Network, Toronto, ON M5G 2C4, Canada;

Atherosclerosis is a chronic inflammatory disease that is driven, in part, by activation of vascular endothelial cells (ECs). In response to inflammatory stimuli, the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway orchestrates the expression of a network of EC genes that contribute to monocyte recruitment and diapedesis across the endothelium. Although many long noncoding RNAs (lncRNAs) are dysregulated in atherosclerosis, they remain poorly characterized, especially in the context of human vascular inflammation. Prior studies have illustrated that lncRNAs can regulate their neighboring protein-coding genes via interaction with protein complexes. We therefore identified and characterized neighboring interleukin-1β (IL-1β)-regulated messenger RNA (mRNA)-lncRNA pairs in ECs. We found these pairs to be highly correlated in expression, especially when located within the same chromatin territory. Additionally, these pairs were predominantly divergently transcribed and shared common gene regulatory elements, characterized by active histone marks and NF-κB binding. Further analysis was performed on , which is transcribed divergently to the gene, , encoding a proatherosclerotic chemokine. and showed coordinate up-regulation in response to inflammatory stimuli, and their expression was correlated in unstable symptomatic human atherosclerotic plaques. Knock-down experiments revealed that positively regulated mRNA levels in multiple primary ECs and EC cell lines. This regulation appeared to involve the interaction of with RNA binding proteins, including HNRNPU and IGF2BP2. Hence, our approach has uncovered a network of neighboring mRNA-lncRNA pairs in the setting of inflammation and identified the function of an lncRNA, , which may contribute to atherogenesis in humans.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1904108116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6697820PMC
August 2019

Correlation of computed tomography with carotid plaque transcriptomes associates calcification with lesion-stabilization.

Atherosclerosis 2019 09 11;288:175-185. Epub 2019 May 11.

Department of Vascular Surgery, Karolinska University Hospital, Stockholm, Sweden; Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden. Electronic address:

Background And Aims: Unstable carotid atherosclerosis causes stroke, but methods to identify patients and lesions at risk are lacking. We recently found enrichment of genes associated with calcification in carotid plaques from asymptomatic patients. Here, we hypothesized that calcification represents a stabilising feature of plaques and investigated how macro-calcification, as estimated by computed tomography (CT), correlates with gene expression profiles in lesions.

Methods: Plaque calcification was measured in pre-operative CT angiographies. Plaques were sorted into high- and low-calcified, profiled with microarrays, followed by bioinformatic analyses. Immunohistochemistry and qPCR were performed to evaluate the findings in plaques and arteries with medial calcification from chronic kidney disease patients.

Results: Smooth muscle cell (SMC) markers were upregulated in high-calcified plaques and calcified plaques from symptomatic patients, whereas macrophage markers were downregulated. The most enriched processes in high-calcified plaques were related to SMCs and extracellular matrix (ECM) organization, while inflammation, lipid transport and chemokine signaling were repressed. These findings were confirmed in arteries with high medial calcification. Proteoglycan 4 (PRG4) was identified as the most upregulated gene in association with plaque calcification and found in the ECM, SMA+ and CD68+/TRAP + cells.

Conclusions: Macro-calcification in carotid lesions correlated with a transcriptional profile typical for stable plaques, with altered SMC phenotype and ECM composition and repressed inflammation. PRG4, previously not described in atherosclerosis, was enriched in the calcified ECM and localized to activated macrophages and smooth muscle-like cells. This study strengthens the notion that assessment of calcification may aid evaluation of plaque phenotype and stroke risk.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.atherosclerosis.2019.05.005DOI Listing
September 2019

Dual roles of heparanase in human carotid plaque calcification.

Atherosclerosis 2019 04 5;283:127-136. Epub 2019 Jan 5.

Department of Molecular Medicine and Surgery, Bioclinicum J8:20, Karolinska Institutet, 171 64, Solna, Sweden.

Background And Aims: Calcification is a hallmark of advanced atherosclerosis and an active process akin to bone remodeling. Heparanase (HPSE) is an endo-β-glucuronidase, which cleaves glycosaminoglycan chains of heparan sulfate proteoglycans. The role of HPSE is controversial in osteogenesis and bone remodeling while it is unexplored in vascular calcification. Previously, we reported upregulation of HPSE in human carotid endarterectomies from symptomatic patients and showed correlation of HPSE expression with markers of inflammation and increased thrombogenicity. The present aim is to investigate HPSE expression in relation to genes associated with osteogenesis and osteolysis and the effect of elevated HPSE expression on calcification and osteolysis in vitro.

Methods: Transcriptomic and immunohistochemical analyses were performed using the Biobank of Karolinska Endarterectomies (BiKE). In vitro calcification and osteolysis were analysed in human carotid smooth muscle cells overexpressing HPSE and bone marrow-derived osteoclasts from HPSE-transgenic mice respectively.

Results: HPSE expression correlated primarily with genes coupled to osteoclast differentiation and function in human carotid atheromas. HPSE was expressed in osteoclast-like cells in atherosclerotic lesions, and HPSE-transgenic bone marrow-derived osteoclasts displayed a higher osteolytic activity compared to wild-type cells. Contrarily, human carotid SMCs with an elevated HPSE expression demonstrated markedly increased mineralization upon osteogenic differentiation.

Conclusions: We suggest that HPSE may have dual functions in vascular calcification, depending on the stage of the disease and presence of inflammatory cells. While HPSE plausibly enhances mineralization and osteogenic differentiation of vascular smooth muscle cells, it is associated with inflammation-induced osteoclast differentiation and activity in advanced atherosclerotic plaques.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.atherosclerosis.2018.12.027DOI Listing
April 2019

Recent advances in therapeutic targeting of inflammation in atherosclerosis.

J Vasc Surg 2019 03 24;69(3):944-951. Epub 2018 Dec 24.

Departments of Vascular Surgery and Molecular Medicine and Surgery, Karolinska University Hospital and the Karolinska Institute, Stockholm, Sweden.

Objective: Current prevention of peripheral vascular disease (PVD) focuses on blood pressure control, lipid lowering, and platelet inhibition with statins and aspirin. A critical role for inflammation in the pathophysiology of atherosclerosis has been established for decades and, although both statins and aspirin have anti-inflammatory properties, the management of inflammation is becoming increasingly recognized. Here, we summarize recent clinical and translational discoveries that outline how inflammation may become targeted in PVD in the future.

Methods: A PubMed search using a combination of the following MeSH terms-inflammation, pathophysiology, atherosclerosis, cancer, auto immune disease, therapy, and clinical trial-was performed and literature selected with a focus on basic pathophysiology of inflammation and clinical investigations targeting inflammation in cardiovascular disease, cancer, and autoimmune diseases.

Results: Based on this literature overview, we summarized the common features of inflammation in these different diseases and how inflammation may also translate into common therapeutic strategies. Finally, the results of recent clinical and translational investigations highlighting inflammation in cardiovascular disease are reviewed with a focus on hematopoietic mutations that generate more active immune cells and increase cardiovascular risk, treatment with anti-inflammatory biological pharmaceuticals that reduce cardiovascular risk, and translational studies demonstrating how the treatment of defective immune-mediated clearance of dying cells in lesions may prevent disease progression.

Conclusions: Progress in clinical and translational atherosclerosis research has now brought inflammation in clinical focus, because recent discoveries with respect to cardiovascular risk prediction and pharmacotherapy targeting inflammation have shown the potential to improve future care of patients with PVD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jvs.2018.10.051DOI Listing
March 2019

Deficiency of the T cell regulator Casitas B-cell lymphoma-B aggravates atherosclerosis by inducing CD8+ T cell-mediated macrophage death.

Eur Heart J 2019 01;40(4):372-382

Department of Medical Biochemistry, Amsterdam Cardiovascular Sciences (ACS), Amsterdam University Medical Centers, University of Amsterdam, Room K1-110, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

Aims: The E3-ligase CBL-B (Casitas B-cell lymphoma-B) is an important negative regulator of T cell activation that is also expressed in macrophages. T cells and macrophages mediate atherosclerosis, but their regulation in this disease remains largely unknown; thus, we studied the function of CBL-B in atherogenesis.

Methods And Results: The expression of CBL-B in human atherosclerotic plaques was lower in advanced lesions compared with initial lesions and correlated inversely with necrotic core area. Twenty weeks old Cblb-/-Apoe-/- mice showed a significant increase in plaque area in the aortic arch, where initial plaques were present. In the aortic root, a site containing advanced plaques, lesion area rose by 40%, accompanied by a dramatic change in plaque phenotype. Plaques contained fewer macrophages due to increased apoptosis, larger necrotic cores, and more CD8+ T cells. Cblb-/-Apoe-/- macrophages exhibited enhanced migration and increased cytokine production and lipid uptake. Casitas B-cell lymphoma-B deficiency increased CD8+ T cell numbers, which were protected against apoptosis and regulatory T cell-mediated suppression. IFNγ and granzyme B production was enhanced in Cblb-/-Apoe-/- CD8+ T cells, which provoked macrophage killing. Depletion of CD8+ T cells in Cblb-/-Apoe-/- bone marrow chimeras rescued the phenotype, indicating that CBL-B controls atherosclerosis mainly through its function in CD8+ T cells.

Conclusion: Casitas B-cell lymphoma-B expression in human plaques decreases during the progression of atherosclerosis. As an important regulator of immune responses in experimental atherosclerosis, CBL-B hampers macrophage recruitment and activation during initial atherosclerosis and limits CD8+ T cell activation and CD8+ T cell-mediated macrophage death in advanced atherosclerosis, thereby preventing the progression towards high-risk plaques.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/eurheartj/ehy714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340101PMC
January 2019

Functional regulatory mechanism of smooth muscle cell-restricted LMOD1 coronary artery disease locus.

PLoS Genet 2018 11 16;14(11):e1007755. Epub 2018 Nov 16.

Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, California, United States of America.

Recent genome-wide association studies (GWAS) have identified multiple new loci which appear to alter coronary artery disease (CAD) risk via arterial wall-specific mechanisms. One of the annotated genes encodes LMOD1 (Leiomodin 1), a member of the actin filament nucleator family that is highly enriched in smooth muscle-containing tissues such as the artery wall. However, it is still unknown whether LMOD1 is the causal gene at this locus and also how the associated variants alter LMOD1 expression/function and CAD risk. Using epigenomic profiling we recently identified a non-coding regulatory variant, rs34091558, which is in tight linkage disequilibrium (LD) with the lead CAD GWAS variant, rs2820315. Herein we demonstrate through expression quantitative trait loci (eQTL) and statistical fine-mapping in GTEx, STARNET, and human coronary artery smooth muscle cell (HCASMC) datasets, rs34091558 is the top regulatory variant for LMOD1 in vascular tissues. Position weight matrix (PWM) analyses identify the protective allele rs34091558-TA to form a conserved Forkhead box O3 (FOXO3) binding motif, which is disrupted by the risk allele rs34091558-A. FOXO3 chromatin immunoprecipitation and reporter assays show reduced FOXO3 binding and LMOD1 transcriptional activity by the risk allele, consistent with effects of FOXO3 downregulation on LMOD1. LMOD1 knockdown results in increased proliferation and migration and decreased cell contraction in HCASMC, and immunostaining in atherosclerotic lesions in the SMC lineage tracing reporter mouse support a key role for LMOD1 in maintaining the differentiated SMC phenotype. These results provide compelling functional evidence that genetic variation is associated with dysregulated LMOD1 expression/function in SMCs, together contributing to the heritable risk for CAD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1007755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6268002PMC
November 2018

Novel Multiomics Profiling of Human Carotid Atherosclerotic Plaques and Plasma Reveals Biliverdin Reductase B as a Marker of Intraplaque Hemorrhage.

JACC Basic Transl Sci 2018 Aug 1;3(4):464-480. Epub 2018 Aug 1.

Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, Sweden.

Clinical tools to identify individuals with unstable atherosclerotic lesions are required to improve prevention of myocardial infarction and ischemic stroke. Here, a systems-based analysis of atherosclerotic plaques and plasma from patients undergoing carotid endarterectomy for stroke prevention was used to identify molecular signatures with a causal relationship to disease. Local plasma collected in the lesion proximity following clamping prior to arteriotomy was profiled together with matched peripheral plasma. This translational workflow identified biliverdin reductase B as a novel marker of intraplaque hemorrhage and unstable carotid atherosclerosis, which should be investigated as a potential predictive biomarker for cardiovascular events in larger cohorts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jacbts.2018.04.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115646PMC
August 2018

Integrated Human Evaluation of the Lysophosphatidic Acid Pathway as a Novel Therapeutic Target in Atherosclerosis.

Mol Ther Methods Clin Dev 2018 Sep 27;10:17-28. Epub 2018 Jun 27.

Translational Sciences, Cardiovascular, Renal and Metabolism, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.

Variants in the gene encoding for lipid phosphate phosphohydrolase 3 have been associated with susceptibility to atherosclerosis independently of classical risk factors. PLPP3 inactivates lysophosphatidic acid (LPA), a pro-inflammatory, pro-thrombotic product of phospholipase activity. Here we performed the first exploratory analysis of PLPP3, LPA, and LPA receptors (LPARs 1-6) in human atherosclerosis. PLPP3 transcript and protein were repressed when comparing plaques versus normal arteries and plaques from symptomatic versus asymptomatic patients, and they were negatively associated with risk of adverse cardiovascular events. PLPP3 localized to macrophages, smooth muscle, and endothelial cells (ECs) in plaques. LPAR 2, 5, and especially 6 showed increased expression in plaques, with LPAR6 localized in ECs and positively correlated to PLPP3. Utilizing mass spectrometry imaging, LPA and its precursors were found in the plaque fibrous cap, co-localizing with PLPP3 and LPAR6. , PLPP3 silencing in ECs under LPA stimulation resulted in increased expression of adhesion molecules and cytokines. LPAR6 silencing inhibited LPA-induced cell activation, but not when PLPP3 was silenced simultaneously. Our results show that repression of PLPP3 plays a key role in atherosclerosis by promoting EC activation. Altogether, the PLPP3 pathway represents a suitable target for investigations into novel therapeutic approaches to ameliorate atherosclerosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.omtm.2018.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039967PMC
September 2018

Local Delivery of miR-21 Stabilizes Fibrous Caps in Vulnerable Atherosclerotic Lesions.

Mol Ther 2018 04 31;26(4):1040-1055. Epub 2018 Jan 31.

Department of Medicine, Karolinska Institute, Stockholm, Sweden; Department of Vascular and Endovascular Surgery, Technical University Munich, Munich, Germany; German Center for Cardiovascular Research (DZHK), Munich, Germany. Electronic address:

miRNAs are potential regulators of carotid artery stenosis and concordant vulnerable atherosclerotic plaques. Hence, we analyzed miRNA expression in laser captured micro-dissected fibrous caps of either ruptured or stable plaques (n = 10 each), discovering that miR-21 was significantly downregulated in unstable lesions. To functionally evaluate miR-21 in plaque vulnerability, miR-21 and miR-21/apolipoprotein-E double-deficient mice (ApoemiR-21) were assessed. miR-21 mice lacked sufficient smooth muscle cell proliferation in response to carotid ligation injury. When exposing ApoemiR-21 mice to an inducible plaque rupture model, they presented with more atherothrombotic events (93%) compared with miR-21Apoe mice (57%). We discovered that smooth muscle cell fate in experimentally induced advanced lesions is steered via a REST-miR-21-REST feedback signaling pathway. Furthermore, ApoemiR-21 mice presented with more pronounced atherosclerotic lesions, greater foam cell formation, and substantially higher levels of arterial macrophage infiltration. Local delivery of a miR-21 mimic using ultrasound-targeted microbubbles into carotid plaques rescued the vulnerable plaque rupture phenotype. In the present study, we identify miR-21 as a key modulator of pathologic processes in advanced atherosclerosis. Targeted, lesion site-specific overexpression of miR-21 can stabilize vulnerable plaques.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ymthe.2018.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6080193PMC
April 2018

TCF21 and the environmental sensor aryl-hydrocarbon receptor cooperate to activate a pro-inflammatory gene expression program in coronary artery smooth muscle cells.

PLoS Genet 2017 May 8;13(5):e1006750. Epub 2017 May 8.

Division of Cardiovascular Medicine, Stanford University, Stanford, California, United States of America.

Both environmental factors and genetic loci have been associated with coronary artery disease (CAD), however gene-gene and gene-environment interactions that might identify molecular mechanisms of risk are not easily studied by human genetic approaches. We have previously identified the transcription factor TCF21 as the causal CAD gene at 6q23.2 and characterized its downstream transcriptional network that is enriched for CAD GWAS genes. Here we investigate the hypothesis that TCF21 interacts with a downstream target gene, the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that mediates the cellular response to environmental contaminants, including dioxin and polycyclic aromatic hydrocarbons (e.g., tobacco smoke). Perturbation of TCF21 expression in human coronary artery smooth muscle cells (HCASMC) revealed that TCF21 promotes expression of AHR, its heterodimerization partner ARNT, and cooperates with these factors to upregulate a number of inflammatory downstream disease related genes including IL1A, MMP1, and CYP1A1. TCF21 was shown to bind in AHR, ARNT and downstream target gene loci, and co-localization was noted for AHR-ARNT and TCF21 binding sites genome-wide in regions of HCASMC open chromatin. These regions of co-localization were found to be enriched for GWAS signals associated with cardio-metabolic as well as chronic inflammatory disease phenotypes. Finally, we show that similar to TCF21, AHR gene expression is increased in atherosclerotic lesions in mice in vivo using laser capture microdissection, and AHR protein is localized in human carotid atherosclerotic lesions where it is associated with protein kinases with a critical role in innate immune response. These data suggest that TCF21 can cooperate with AHR to activate an inflammatory gene expression program that is exacerbated by environmental stimuli, and may contribute to the overall risk for CAD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1006750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5439967PMC
May 2017

Extracellular matrix proteomics identifies molecular signature of symptomatic carotid plaques.

J Clin Invest 2017 Apr 20;127(4):1546-1560. Epub 2017 Mar 20.

Background: The identification of patients with high-risk atherosclerotic plaques prior to the manifestation of clinical events remains challenging. Recent findings question histology- and imaging-based definitions of the "vulnerable plaque," necessitating an improved approach for predicting onset of symptoms.

Methods: We performed a proteomics comparison of the vascular extracellular matrix and associated molecules in human carotid endarterectomy specimens from 6 symptomatic versus 6 asymptomatic patients to identify a protein signature for high-risk atherosclerotic plaques. Proteomics data were integrated with gene expression profiling of 121 carotid endarterectomies and an analysis of protein secretion by lipid-loaded human vascular smooth muscle cells. Finally, epidemiological validation of candidate biomarkers was performed in two community-based studies.

Results: Proteomics and at least one of the other two approaches identified a molecular signature of plaques from symptomatic patients that comprised matrix metalloproteinase 9, chitinase 3-like-1, S100 calcium binding protein A8 (S100A8), S100A9, cathepsin B, fibronectin, and galectin-3-binding protein. Biomarker candidates measured in 685 subjects in the Bruneck study were associated with progression to advanced atherosclerosis and incidence of cardiovascular disease over a 10-year follow-up period. A 4-biomarker signature (matrix metalloproteinase 9, S100A8/S100A9, cathepsin D, and galectin-3-binding protein) improved risk prediction and was successfully replicated in an independent cohort, the SAPHIR study.

Conclusion: The identified 4-biomarker signature may improve risk prediction and diagnostics for the management of cardiovascular disease. Further, our study highlights the strength of tissue-based proteomics for biomarker discovery.

Funding: UK: British Heart Foundation (BHF); King's BHF Center; and the National Institute for Health Research Biomedical Research Center based at Guy's and St Thomas' NHS Foundation Trust and King's College London in partnership with King's College Hospital. Austria: Federal Ministry for Transport, Innovation and Technology (BMVIT); Federal Ministry of Science, Research and Economy (BMWFW); Wirtschaftsagentur Wien; and Standortagentur Tirol.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI86924DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373893PMC
April 2017
-->