Genome-wide chromatin contacts of super-enhancer-associated lncRNA identify LINC01013 as a regulator of fibrosis in the aortic valve.

Calcific aortic valve disease (CAVD) is characterized by a fibrocalcific process. The regulatory mechanisms that drive the fibrotic response in the aortic valve (AV) are poorly understood. Long noncoding RNAs derived from super-enhancers (lncRNA-SE) control gene expression and cell fate. Herein, multidimensional profiling including chromatin immunoprecipitation and sequencing, transposase-accessible chromatin sequencing, genome-wide 3D chromatin contacts of enhancer-promoter identified LINC01013 as an overexpressed lncRNA-SE during CAVD. LINC01013 is within a loop anchor, which has contact with the promoter of CCN2 (CTGF) located at ~180 kb upstream. Investigation showed that LINC01013 acts as a decoy factor for the negative transcription elongation factor E (NELF-E), whereby it controls the expression of CCN2. LINC01013-CCN2 is part of a transforming growth factor beta 1 (TGFB1) network and exerts a control over fibrogenesis. These findings illustrate a novel mechanism whereby a dysregulated lncRNA-SE controls, through a looping process, the expression of CCN2 and fibrogenesis of the AV.

Oxyphospholipids in Cardiovascular Calcification.

Mineralization of cardiovascular structures including blood vessels and heart valves is a common feature. We postulate that ectopic mineralization is a response-to-injury in which signals delivered to cells trigger a chain of events to restore and repair tissues. Maladaptive response to external or internal signals promote the expression of danger-associated molecular patterns, which, in turn, promote, when expressed chronically, a procalcifying gene program. Growing evidence suggest that danger-associated molecular patterns such as oxyphospholipids and small lipid mediators, generated by enzyme activity, are involved in the transition of vascular smooth muscle cells and valve interstitial cells to an osteoblast-like phenotype. Understanding the regulation and the molecular processes underpinning the mineralization of atherosclerotic plaques and cardiac valves are providing valuable mechanistic insights, which could lead to the development of novel therapies. Herein, we provide a focus account on the role oxyphospholipids and their mediators in the development of mineralization in plaques and calcific aortic valve disease.

Enhancer-mediated enrichment of interacting JMJD3-DDX21 to ENPP2 locus prevents R-loop formation and promotes transcription.

ENPP2, which encodes for the enzyme autotaxin (ATX), is overexpressed during chronic inflammatory diseases and various cancers. However, the molecular mechanism involved in the ENPP2 transcription remains elusive. Here, in HEK 293T cells, we demonstrated that lipopolysaccharide (LPS) increased the transcription process at ENPP2 locus through a NF-кB pathway and a reduction of H3K27me3 level, a histone repressive mark, by the demethylase UTX. Simultaneously, the H3K27me3 demethylase JMJD3/KDM6B was recruited to the transcription start site (TSS), within the gene body and controlled the expression of ENPP2 in a non-enzymatic manner. Mass spectrometry data revealed a novel interaction for JMJD3 with DDX21, a RNA helicase that unwinds R-loops created by nascent transcript and DNA template. Upon LPS treatment, JMJD3 is necessary for DDX21 recruitment at ENPP2 locus allowing the resolution of aberrant R-loops. CRISPR-Cas9-mediated deletion of a distant-acting enhancer decreased the expression of ENPP2 and lowered the recruitment of JMJD3-DDX21 complex at TSS and its progression through the gene body. Taken together, these findings revealed that enhancer-mediated enrichment of novel JMJD3-DDX21 interaction at ENPP2 locus is necessary for nascent transcript synthesis via the resolution of aberrant R-loops formation in response to inflammatory stimulus.

Activated platelets promote an osteogenic programme and the progression of calcific aortic valve stenosis.

AIMS: Calcific aortic valve stenosis (CAVS) is characterized by a fibrocalcific process. Studies have shown an association between CAVS and the activation of platelets. It is believed that shear stress associated with CAVS promotes the activation of platelets. However, whether platelets actively participate to the mineralization of the aortic valve (AV) and the progression of CAVS is presently unknown. To identify the role of platelets into the pathobiology of CAVS. METHODS AND RESULTS: Explanted control non-mineralized and mineralized AVs were examined by scanning electron microscope (SEM) for the presence of activated platelets. In-depth functional assays were carried out with isolated human valve interstitial cells (VICs) and platelets as well as in LDLR-/- apoB100/100 IGFII (IGFII) mice. Scanning electron microscope and immunogold markings for glycoprotein IIb/IIIa (GPIIb/IIIa) revealed the presence of platelet aggregates with fibrin in endothelium-denuded areas of CAVS. In isolated VICs, collagen-activated platelets induced an osteogenic programme. Platelet-derived adenosine diphosphate induced the release of autotaxin (ATX) by VICs. The binding of ATX to GPIIb/IIIa of platelets generated lysophosphatidic acid (LysoPA) with pro-osteogenic properties. In IGFII mice with CAVS, platelet aggregates were found at the surface of AVs. Administration of activated platelets to IGFII mice accelerated the development of CAVS by 2.1-fold, whereas a treatment with Ki16425, an antagonist of LysoPA receptors, prevented platelet-induced mineralization of the AV and the progression of CAVS. CONCLUSIONS: These findings suggest a novel role for platelets in the progression of CAVS.

Altered DNA Methylation of Long Noncoding RNA H19 in Calcific Aortic Valve Disease Promotes Mineralization by Silencing NOTCH1.

BACKGROUND: Calcific aortic valve disease is characterized by an abnormal mineralization of the aortic valve. Osteogenic activity in the aortic valve is under the control of NOTCH1, which regulates the expression of key pro-osteogenic genes such as RUNX2 and BMP2. Long noncoding RNAs (lncRNAs) may reprogram cells by altering the gene expression pattern. METHODS: Multidimensional genomic profiling was performed in human aortic valves to document the expression of lncRNAs and the DNA methylation pattern in calcific aortic valve disease. In-depth functional assays were carried out to document the impact of lncRNA on the mineralization of the aortic valve. RESULTS: We documented that lncRNA H19 (H19) was increased in calcific aortic valve disease. Hypomethylation of the promoter region was observed in mineralized aortic valves and was inversely associated with H19 expression. Knockdown and overexpression experiments showed that H19 induces a strong osteogenic phenotype by altering the NOTCH1 pathway. Gene promoter analyses showed that H19 silenced NOTCH1 by preventing the recruitment of p53 to its promoter. A knockdown of H19 in valve interstitial cells (VICs) increased the expression of NOTCH1 and decreased the level of RUNX2 and BMP2, 2 downstream targets repressed by NOTCH1. In rescue experiments, the transfection of a vector encoding for the active Notch intracellular domain prevented H19-induced mineralization of valve interstitial cells. CONCLUSIONS: These findings indicate that a dysregulation of DNA methylation in the promoter of H19 during calcific aortic valve disease is associated with a higher expression of this lncRNA, which promotes an osteogenic program by interfering with the expression of NOTCH1.

Autotaxin Derived From Lipoprotein(a) and Valve Interstitial Cells Promotes Inflammation and Mineralization of the Aortic Valve.

BACKGROUND: Mendelian randomization studies have highlighted that lipoprotein(a) [Lp(a)] was associated with calcific aortic valve disease (CAVD). Lp(a) transports oxidized phospholipids with a high content in lysophosphatidylcholine. Autotaxin (ATX) transforms lysophosphatidylcholine into lysophosphatidic acid. We hypothesized that ATX-lysophosphatidic acid could promote inflammation/mineralization of the aortic valve. METHODS AND RESULTS: We have documented the expression of ATX in control and mineralized aortic valves. By using different approaches, we have also investigated the role of ATX-lysophosphatidic acid in the mineralization of isolated valve interstitial cells and in a mouse model of CAVD. Enzyme-specific ATX activity was elevated by 60% in mineralized aortic valves in comparison with control valves. Immunohistochemistry studies showed a high level of ATX in mineralized aortic valves, which colocalized with oxidized phospholipids and apolipoprotein(a). We detected a high level of ATX activity in the Lp(a) fraction in circulation. Interaction between ATX and Lp(a) was confirmed by in situ proximity ligation assay. Moreover, we documented that valve interstitial cells also expressed ATX in CAVD. We showed that ATX-lysophosphatidic acid promotes the mineralization of the aortic valve through a nuclear factor κB/interleukin 6/bone morphogenetic protein pathway. In LDLR(-/-)/ApoB(100/100)/IGFII mice, ATX is overexpressed and lysophosphatidic acid promotes a strong deposition of hydroxyapatite of calcium in aortic valve leaflets and accelerates the development of CAVD. CONCLUSIONS: ATX is transported in the aortic valve by Lp(a) and is also secreted by valve interstitial cells. ATX-lysophosphatidic acid promotes inflammation and mineralization of the aortic valve and thus could represent a novel therapeutic target in CAVD.

A functional interplay between the small GTPase Rab11a and mitochondria-shaping proteins regulates mitochondrial positioning and polarization of the actin cytoskeleton downstream of Src family kinases.

It is believed that mitochondrial dynamics is coordinated with endosomal traffic rates during cytoskeletal remodeling, but the mechanisms involved are largely unknown. The adenovirus early region 4 ORF4 protein (E4orf4) subverts signaling by Src family kinases (SFK) to perturb cellular morphology, membrane traffic, and organellar dynamics and to trigger cell death. Using E4orf4 as a model, we uncovered a functional connection between mitochondria-shaping proteins and the small GTPase Rab11a, a key regulator of polarized transport via recycling endosomes. We found that E4orf4 induced dramatic changes in the morphology of mitochondria along with their mobilization at the vicinity of a polarized actin network typifying E4orf4 action, in a manner controlled by SFK and Rab11a. Mitochondrial remodeling was associated with increased proximity between Rab11a and mitochondrial membranes, changes in fusion-fission dynamics, and mitochondrial relocalization of the fission factor dynamin-related protein 1 (Drp1), which was regulated by the Rab11a effector protein FIP1/RCP. Knockdown of FIP1/RCP or inhibition of Drp1 markedly impaired mitochondrial remodeling and actin assembly, involving Rab11a-mediated mitochondrial dynamics in E4orf4-induced signaling. A similar mobilization of mitochondria near actin-rich structures was mediated by Rab11 and Drp1 in viral Src-transformed cells and contributed to the biogenesis of podosome rosettes. These findings suggest a role for Rab11a in the trafficking of Drp1 to mitochondria upon SFK activation and unravel a novel functional interplay between Rab11a and mitochondria during reshaping of the cell cytoskeleton, which would facilitate mitochondria redistribution near energy-requiring actin-rich structures.

Early development of calcific aortic valve disease and left ventricular hypertrophy in a mouse model of combined dyslipidemia and type 2 diabetes mellitus.

OBJECTIVE: This study aimed to determine the potential impact of type 2 diabetes mellitus on left ventricular dysfunction and the development of calcified aortic valve disease using a dyslipidemic mouse model prone to developing type 2 diabetes mellitus. APPROACH AND RESULTS: When compared with nondiabetic LDLr(-/-)/ApoB(100/100), diabetic LDLr(-/-)/ApoB(100/100)/IGF-II mice exhibited similar dyslipidemia and obesity but developed type 2 diabetes mellitus when fed a high-fat/sucrose/cholesterol diet for 6 months. LDLr(-/-)/ApoB(100/100)/IGF-II mice showed left ventricular hypertrophy versus C57BL6 but not LDLr(-/-)/ApoB(100/100) mice. Transthoracic echocardiography revealed significant reductions in both left ventricular systolic fractional shortening and diastolic function in high-fat/sucrose/cholesterol fed LDLr(-/-)/ApoB(100/100)/IGF-II mice when compared with LDLr(-/-)/ApoB(100/100). Importantly, we found that peak aortic jet velocity was significantly increased in LDLr(-/-)/ApoB(100/100)/IGF-II mice versus LDLr(-/-)/ApoB(100/100) animals on the high-fat/sucrose/cholesterol diet. Microtomography scans and Alizarin red staining indicated calcification in the aortic valves, whereas electron microscopy and energy dispersive x-ray spectroscopy further revealed mineralization of the aortic leaflets and the presence of inflammatory infiltrates in diabetic mice. Studies showed upregulation of hypertrophic genes (anp, bnp, b-mhc) in myocardial tissues and of osteogenic genes (spp1, bglap, runx2) in aortic tissues of diabetic mice. CONCLUSIONS: We have established the diabetes mellitus -prone LDLr(-/-)/ApoB(100/100)/IGF-II mouse as a new model of calcified aortic valve disease. Our results are consistent with the growing body of clinical evidence that the dysmetabolic state of type 2 diabetes mellitus contributes to early mineralization of the aortic valve and calcified aortic valve disease pathogenesis.

Mechanical strain induces the production of spheroid mineralized microparticles in the aortic valve through a RhoA/ROCK-dependent mechanism.

Calcific aortic valve disease (CAVD) is a chronic disorder characterized by an abnormal mineralization of the leaflets, which is accelerated in bicuspid aortic valve (BAV). It is suspected that mechanical strain may promote/enhance mineralization of the aortic valve. However, the effect of mechanical strain and the involved pathways during mineralization of the aortic valve remains largely unknown. Valve interstitial cells (VICs) were isolated and studied under strain conditions. Human bicuspid aortic valves were examined as a model relevant to increase mechanical strain. Cyclic strain increased mineralization of VICs by several-fold. Scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analyses revealed that mechanical strain promoted the formation of mineralized spheroid microparticles, which coalesced into larger structure at the surface of apoptotic VICs. Apoptosis and mineralization were closely associated with expression of ENPP1. Inhibition of ENPP1 greatly reduced mineralization of VIC cultures. Through several lines of evidence we showed that mechanical strain promoted the export of ENPP1-containing vesicles to the plasma membrane through a RhoA/ROCK pathway. Studies conducted in human BAV revealed the presence of spheroid mineralized structures along with the expression of ENPP1 in areas of high mechanical strain. Mechanical strain promotes the production and accumulation of spheroid mineralized microparticles by VICs, which may represent one important underlying mechanism involved in aortic valve mineralization. RhoA/ROCK-mediated export of ENPP1 to the plasma membrane promotes strain-induced mineralization of VICs.

P2Y2 receptor represses IL-6 expression by valve interstitial cells through Akt: implication for calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is a disorder characterized by an abnormal mineralization, which may have intricate links with inflammation. Interleukin-6 (IL-6) and its cognate cytokines are widely expressed and exert pleiotropic effects on different tissues. In this study, we examined the expression of the IL-6 family of cytokines in human CAVD by using a transcriptomic approach and we performed in-depth functional assays with valve interstitial cells (VICs) to unravel the process regulating IL-6 expression and its role during the mineralization of the aortic valve. We documented by both microarray and q-PCR analyses an elevated expression of IL-6 in human CAVD, which was correlated with the remodeling process. IL-6 was highly expressed by VICs. We found that following treatment with a phosphate-containing medium the level of IL-6 expressed by VICs increased by several-fold. Phosphate-induced expression of IL-6 relied on reduced PI3K/Akt signaling downstream of the P2Y2 receptor (P2Y2R). In this regard, we found by using transfection experiments that Akt-1 is a negative regulator of the NF-κB pathway. In addition, by using a siRNA targeting IL-6 we found that phosphate-induced mineralization was largely dependent on IL-6 expression. A transfection of Akt-1 rescued the hypermineralizing phenotype of P2Y2R(-/-) mouse VICS (MVICs). Hence, we documented a novel mechanism whereby P2Y2R and Akt modulate the NF-κB pathway and its downstream target IL-6, which is a strong promoter of the mineralization of VICs.

Arginine methylation by PRMT1 regulates nuclear-cytoplasmic localization and toxicity of FUS/TLS harbouring ALS-linked mutations.

Mutations in FUS/TLS (fused in sarcoma/translated in liposarcoma) cause an inheritable form of amyotrophic lateral sclerosis (ALS6). In contrast to FUS(WT), which is concentrated in the nucleus, these mutants are abnormally distributed in the cytoplasm where they form inclusions and associate with stress granules. The data reported herein demonstrate the importance of protein arginine methylation in nuclear-cytoplasmic shuttling of FUS and abnormalities of ALS-causing mutants. Depletion of protein arginine methyltransferase 1 (PRMT1; the enzyme that methylates FUS) in mouse embryonic fibroblasts by gene knockout, or in human HEK293 cells by siRNA knockdown, diminished the ability of ALS-linked FUS mutants to localize to the cytoplasm and form inclusions. To examine properties of FUS mutants in the context of neurons vulnerable to the disease, FUS(WT) and ALS-linked FUS mutants were expressed in motor neurons of dissociated murine spinal cord cultures. In motor neurons, shRNA-mediated PRMT1 knockdown concomitant with the expression of FUS actually accentuated the shift in distribution of ALS-linked FUS mutants from the nucleus to the cytoplasm. However, when PRMT1 was inhibited prior to expression of ALS-linked FUS mutants, by pretreatment with a global methyltransferase inhibitor, ALS-linked FUS mutants were sequestered in the nucleus and cytoplasmic inclusions were reduced, as in the cell lines. Mitochondria were significantly shorter in neurons with cytoplasmic ALS-linked FUS mutants, a factor that could contribute to toxicity. We propose that arginine methylation by PRMT1 participates in the nuclear-cytoplasmic shuttling of FUS, particularly of ALS6-associated mutants, and thus contributes to the toxic gain of function conferred by these disease-causing mutations.

Lp-PLA2 is associated with structural valve degeneration of bioprostheses.

OBJECTIVES: In this study, we sought to determine the metabolic markers associated with structural valve degeneration (SVD). BACKGROUND: Structural valve degeneration (SVD) is the major cause of bioprosthetic valve failure leading to bioprostheses (BPs) stenosis or regurgitation. We hypothesized that lipoprotein-associated phospholipase A2 (Lp-PLA2) is involved in the SVD of BPs. METHODS: We included 197 patients who underwent aortic valve replacement with a bioprosthetic valve and had echocardiographic follow-up to evaluate valve function. Moreover, explanted BPs (n = 39) were analysed by immunohistochemistry for the expression of Lp-PLA2. RESULTS: After a mean follow-up of 7·9 ±0·2 years, forty-one patients (21%) were identified as developing SVD. Patients with SVD had significantly higher plasma level of Lp-PLA2 mass (151·8 ± 9·2 ng/mL vs. 133·2 ± 3·4 ng/mL, P = 0·03) and activity (27·6 ± 0·9 nmol/min/mL vs. 25·0 ± 0·4 nmol/min/mL, P = 0·005). Multivariate analysis revealed that Lp-PLA2 activity (OR: 1·09, 95% CI: 1·01-1·18; P = 0·03) was the strongest independent predictor of SVD. Immunohistochemistry studies of explanted BP showed that 77% of explanted BPs had the expression of Lp-PLA2, which correlated with the density of macrophages (CD68), and ox-LDL levels in bioprosthetic tissues. CONCLUSIONS: Increased blood plasma activity of Lp-PLA2 is associated with higher prevalence of SVD. These findings open new avenues for the identification of patients at risk for SVD and for the development of pharmacotherapy aiming at the prevention of SVD.

Ectopic visceral fat: a clinical and molecular perspective on the cardiometabolic risk.

Worldwide, cardiovascular diseases (CVDs) are a leading cause of mortality. While in many westernized societies there has been a decrease prevalence of smoking and that a special emphasis has been put on the urgency to control the, so called, classical risk factors, it is more and more recognized that there remains a residual risk, which contributes to the development of CVDs. Imaging studies conducted over two decades have highlighted that the accumulation of ectopic visceral fat is associated with a plethora of metabolic dysfunctions, which have complex and intertwined interactions and participate to the development/progression/events of many cardiovascular disorders. The contribution of visceral ectopic fat to the development of coronary artery disease (CAD) is now well established, while in the last several years emerging evidence has pointed out that accumulation of harmful ectopic fat is associated with other cardiovascular disorders such as calcific aortic valve disease (CAVD), atrial fibrillation and left ventricular dysfunction. We review herein the key molecular processes linking the accumulation of ectopic fat to the development of CVDs. We have attempted, whenever possible, to use a translational approach whereby the pathobiology processes are linked to clinical observations.

Angiotensin receptor blockers are associated with reduced fibrosis and interleukin-6 expression in calcific aortic valve disease.

BACKGROUND: Calcific aortic valve disease (CAVD) is a chronic disorder characterized by the mineralization of the aortic valve and involving fibrosis. OBJECTIVES: In this work we sought to determine if the fibrotic component of the remodeling process of CAVD was related to the use of angiotensin-converting enzyme inhibitors (ACEi) and/or angiotensin receptor blockers (ARBs). METHODS: In 477 patients with CAVD, the aortic valve was examined by histology. A semiquantitative score of fibrosis was generated and associations with clinical/cardiometabolic variables examined. In a subset of 103 patients the aortic valve was available to study the infiltration by inflammatory cells and expression of interleukin-6 (IL-6) by quantitative real-time PCR. RESULTS: The fibrosis score of the aortic valve was independently related to the hemodynamic severity of CAVD measured by echocardiography. The fibrotic score of the aortic valve was also related to the expression of IL-6. The use of ARBs but not of ACEi was associated with a lower fibrosis score of the aortic valve even after correction for covariates. In addition, patients under ARBs had lower aortic valve inflammation and expression of IL-6. CONCLUSIONS: These findings suggest that ARBs may alter the fibrotic process of the aortic valve in CAVD, possibly by lowering tissue inflammation.

Lipoprotein lipase in aortic valve stenosis is associated with lipid retention and remodelling.

BACKGROUND: Calcific aortic valve disease (CAVD) is a chronic disorder characterized by a fibrocalcific remodelling. It is suspected that lipid retention within the aortic valve may be one important mechanism participating to aortic valve remodelling. Lipoprotein lipase (LPL) is implicated in lipid metabolism and may play a role in lipid retention within the aortic valve. METHODS: In 57 patients, CAVD were analysed for the expression of LPL by q-PCR and immunohistochemistry. Expression of oxidized-LDL (ox-LDL) and decorin was also documented. In addition, a complete blood profile, including the size of LDL and high-density lipoprotein (HDL) particles, were performed to find associations between the blood lipid profile and expression of ox-LDL and LPL within CAVD. RESULTS: Immunohistochemistry studies revealed that LPL was expressed in stenotic aortic valves as a diffuse staining and also in dense cellular areas where macrophages were abundant. Expression of LPL co-localized with decorin and ox-LDL. In turn, valves with higher amount of ox-LDL had elevated number of LPL transcripts. In addition, we documented that the small, dense HDL phenotype was associated with an elevated amount of ox-LDL and LPL transcripts within CAVD. Furthermore, expression of LPL was associated with several indices of fibrocalcific remodelling of the aortic valve. CONCLUSION: Expression of LPL within CAVD is related to the amount of ox-LDL, which is, in turn, associated with the small, dense HDL phenotype. Lipid retention associated with smaller HDL particles may participate in the expression of LPL, whereby a fibrocalcific remodelling of the aortic valve is promoted.

Performance of an RNA-Based Next-Generation Sequencing Assay for Combined Detection of Clinically Actionable Fusions and Hotspot Mutations in NSCLC.

INTRODUCTION: With its expanding list of approved and emerging therapeutic indications, NSCLC is the exemplar tumor type requiring upfront assessment of several biomarkers to guide clinical management. Next-generation sequencing allows identification of different types of molecular alterations, each with specific analytical challenges. Library preparation using parallel DNA and RNA workflows can overcome most of them, but it increases complexity of laboratory operations, turnaround time, and costs. We describe the performance characteristics of a 15-gene RNA panel on the basis of anchored multiplex polymerase chain reaction for combined detection of clinically relevant oncogenic fusion transcripts and hotspot small variants. METHODS: Formalin-fixed, paraffin-embedded NSCLC clinical samples (N = 58) were used along cell lines and commercial controls to validate the assay's analytical performance, followed by an exploratory prospective cohort (N = 87). RESULTS: The raw assay sensitivity for hotspot mutations and fusions was 83% and 93%, respectively, reaching 100% after filtering for key assay metrics. Those include quantity and quality of input of nucleic acid and sequencing metric from primers on housekeeping genes included in the assay. In the prospective cohort, driver alterations were identified in most cases (≥58%). CONCLUSIONS: This ultrafocused RNA-next-generation sequencing assay offers an advantageous option with single unified workflow for simultaneous detection of clinically relevant hotspot mutations and fusions in NSCLC, focusing on actionable gene targets.

System Genetics Including Causal Inference Identify Immune Targets for Coronary Artery Disease and the Lifespan.

BACKGROUND: Randomized clinical trials indicate that the immune response plays a significant role in coronary artery disease (CAD), a disorder impacting the lifespan potential. However, the identification of targets critical to the immune response in atheroma is still hampered by a lack of solid inference. METHODS: Herein, we implemented a system genetics approach to identify causally associated immune targets implicated in atheroma. We leveraged genome-wide association studies to perform mapping and Mendelian randomization to assess causal associations between gene expression in blood cells with CAD and the lifespan. Expressed genes (eGenes) were prioritized in network and in single-cell expression derived from plaque immune cells. RESULTS: Among 840 CAD-associated blood eGenes, 37 were predicted causally associated with CAD and 6 were also associated with the parental lifespan in Mendelian randomization. In multivariable Mendelian randomization, the impact of eGenes on the lifespan potential was mediated by the CAD risk. Predicted causal eGenes were central in network. FLT1 and CCR5 were identified as targets of approved drugs, whereas 22 eGenes were deemed tractable for the development of small molecules and antibodies. Analyses of plaque immune single-cell expression identified predicted causal eGenes enriched in macrophages (GPX1, C4orf3) and involved in ligand-receptor interactions (CCR5). CONCLUSIONS: We identified 37 blood eGenes predicted causally associated with CAD. The predicted expression for 6 eGenes impacted the lifespan potential through the risk of CAD. Prioritization based on network, annotations, and single-cell expression identified targets deemed tractable for the development of drugs and for drug repurposing.

Enhancer-associated aortic valve stenosis risk locus 1p21.2 alters NFATC2 binding site and promotes fibrogenesis.

Genome-wide association studies for calcific aortic valve stenosis (CAVS) previously reported strong signal for noncoding variants at 1p21.2. Previous study using Mendelian randomization suggested that the locus controls the expression of PALMD encoding Palmdelphin (PALMD). However, the molecular regulation at the locus and the impact of PALMD on the biology of the aortic valve is presently unknown. 3D genetic mapping and CRISPR activation identified rs6702619 as being located in a distant-acting enhancer, which controls the expression of PALMD. DNA-binding assay showed that the risk variant modified the DNA shape, which prevented the recruitment of NFATC2 and lowered the expression of PALMD. In co-expression network analysis, a module encompassing PALMD was enriched in actin-based process. Mass spectrometry and functional assessment showed that PALMD is a regulator of actin polymerization. In turn, lower level of PALMD promoted the activation of myocardin-related transcription factor and fibrosis, a key pathobiological process underpinning CAVS.

Proteomic analysis of Src family kinases signaling complexes in Golgi/endosomal fractions using a site-selective anti-phosphotyrosine antibody: identification of LRP1-insulin receptor complexes.

A role for Src Family Kinases (SFKs) in the dynamics of endocytic and secretory pathways has previously been reported. Identification of low-abundance compartmentalized complexes still remains challenging, highlighting the need for novel tools. Here we describe analysis of SFK-signaling complexes of hepatic Golgi/endosomes (G/E) fractions by sequential affinity enrichment of proteins. Mouse G/E permeabilized membranes were first validated in terms of electron microscopy, 1-D electrophoresis (1-DE), insulin-mediated endocytosis and protein content. With the use of quantitative N-terminal labeling of tryptic peptides (iTRAQ), 1-DE and IEF tryptic peptides separation methods, a total of 666 proteins were identified, including the SFK Lyn. Following insulin injection, a series of proteins were recognized by an anti-phosphotyrosine antibody (alpha P42-2) raised against the residue most frequently phosphorylated by SFK on the adenoviral protein E4orf4 and that cross-reacts with endosomal SFK targets. By using affinity chromatography coupled with mass spectrometry, we identified 16 proteins classified as (1) recycling receptors, (2) vesicular trafficking proteins, (3) actin network proteins, (4) metabolism proteins, or (5) signaling proteins. One of these proteins, low density lipoprotein-related protein 1 (LRP1), which is a known SFK substrate, was found to associate with the internalized insulin receptor (IR), suggesting the presence of a co-internalization process. The identification of these proteomes should, thus, contribute to a better understanding of the molecular mechanisms that regulate trafficking events and insulin clearance.