The impact of PARPs and ADP-ribosylation on inflammation and host-pathogen interactions.

Poly-adenosine diphosphate-ribose polymerases (PARPs) promote ADP-ribosylation, a highly conserved, fundamental posttranslational modification (PTM). PARP catalytic domains transfer the ADP-ribose moiety from NAD+ to amino acid residues of target proteins, leading to mono- or poly-ADP-ribosylation (MARylation or PARylation). This PTM regulates various key biological and pathological processes. In this review, we focus on the roles of the PARP family members in inflammation and host-pathogen interactions. Here we give an overview the current understanding of the mechanisms by which PARPs promote or suppress proinflammatory activation of macrophages, and various roles PARPs play in virus infections. We also demonstrate how innovative technologies, such as proteomics and systems biology, help to advance this research field and describe unanswered questions.

Directional Endothelial Communication by Polarized Extracellular Vesicle Release.

BACKGROUND: Extracellular vesicles (EVs) contain bioactive cargo including miRNAs and proteins that are released by cells during cell-cell communication. Endothelial cells (ECs) form the innermost lining of all blood vessels, interfacing with cells in the circulation and vascular wall. It is unknown whether ECs release EVs capable of governing recipient cells within these 2 separate compartments. Given their boundary location, we propose ECs use bidirectional release of distinct EV cargo in quiescent (healthy) and activated (atheroprone) states to communicate with cells within the circulation and blood vessel wall. METHODS: EVs were isolated from primary human aortic ECs (plate and transwell grown; ±IL [interleukin]-1ß activation), quantified, visualized, and analyzed by miRNA transcriptomics and proteomics. Apical and basolateral EC-EV release was determined by miRNA transfer, total internal reflection fluorescence and electron microscopy. Vascular reprogramming (RNA sequencing) and functional assays were performed on primary human monocytes or smooth muscle cells±EC-EVs. RESULTS: Activated ECs increased EV release, with miRNA and protein cargo related to atherosclerosis. EV-treated monocytes and smooth muscle cells revealed activated EC-EV altered pathways that were proinflammatory and atherogenic. ECs released more EVs apically, which increased with activation. Apical and basolateral EV cargo contained distinct transcriptomes and proteomes that were altered by EC activation. Notably, activated basolateral EC-EVs displayed greater changes in the EV secretome, with pathways specific to atherosclerosis. In silico analysis determined compartment-specific cargo released by the apical and basolateral surfaces of ECs can reprogram monocytes and smooth muscle cells, respectively, with functional assays and in vivo imaging supporting this concept. CONCLUSIONS: Demonstrating that ECs are capable of polarized EV cargo loading and directional EV secretion reveals a novel paradigm for endothelial communication, which may ultimately enhance the design of endothelial-based therapeutics for cardiovascular diseases such as atherosclerosis where ECs are persistently activated.

Major Facilitator Superfamily Domain Containing 5 Inhibition Reduces Lipoprotein(a) Uptake and Calcification in Valvular Heart Disease.

BACKGROUND: High circulating levels of Lp(a) (lipoprotein[a]) increase the risk of atherosclerosis and calcific aortic valve disease, affecting millions of patients worldwide. Although atherosclerosis is commonly treated with low-density lipoprotein-targeting therapies, these do not reduce Lp(a) or risk of calcific aortic valve disease, which has no available drug therapies. Targeting Lp(a) production and catabolism may provide therapeutic benefit, but little is known about Lp(a) cellular uptake. METHODS: Here, unbiased ligand-receptor capture mass spectrometry was used to identify MFSD5 (major facilitator superfamily domain containing 5) as a novel receptor/cofactor involved in Lp(a) uptake. RESULTS: Reducing MFSD5 expression by a computationally identified small molecule or small interfering RNA suppressed Lp(a) uptake and calcification in primary human valvular endothelial and interstitial cells. MFSD5 variants were associated with aortic stenosis (P=0.027 after multiple hypothesis testing) with evidence suggestive of an interaction with plasma Lp(a) levels. CONCLUSIONS: MFSD5 knockdown suppressing human valvular cell Lp(a) uptake and calcification, along with meta-analysis of MFSD5 variants associating with aortic stenosis, supports further preclinical assessment of MFSD5 in cardiovascular diseases, the leading cause of death worldwide.

Multimodal Analytical Tools to Enhance Mechanistic Understanding of Aortic Valve Calcification.

This review focuses on technologies at the core of calcific aortic valve disease (CAVD) and drug target research advancement, including transcriptomics, proteomics, and molecular imaging. We examine how bulk RNA sequencing and single-cell RNA sequencing have engendered organismal genomes and transcriptomes, promoting the analysis of tissue gene expression profiles and cell subpopulations, respectively. We bring into focus how the field is also largely influenced by increasingly accessible proteome profiling techniques. In unison, global transcriptional and protein expression analyses allow for increased understanding of cellular behavior and pathogenic pathways under pathologic stimuli including stress, inflammation, low-density lipoprotein accumulation, increased calcium and phosphate levels, and vascular injury. We also look at how direct investigation of protein signatures paves the way for identification of targetable pathways for pharmacologic intervention. Here, we note that imaging techniques, once a clinical diagnostic tool for late-stage CAVD, have since been refined to address a clinical need to identify microcalcifications using positron emission tomography/computed tomography and even detect in vivo cellular events indicative of early stage CAVD and map the expression of identified proteins in animal models. Together, these techniques generate a holistic approach to CAVD investigation, with the potential to identify additional novel regulatory pathways.

Understanding HDL Metabolism and Biology Through In Vivo Tracer Kinetics.

HDL (high-density lipoprotein), owing to its high protein content and small size, is the densest circulating lipoprotein. In contrast to lipid-laden VLDL (very-low-density lipoprotein) and LDL (low-density lipoprotein) that promote atherosclerosis, HDL is hypothesized to mitigate atherosclerosis via reverse cholesterol transport, a process that entails the uptake and clearance of excess cholesterol from peripheral tissues. This process is mediated by APOA1 (apolipoprotein A-I), the primary structural protein of HDL, as well as by the activities of additional HDL proteins. Tracer-dependent kinetic studies are an invaluable tool to study HDL-mediated reverse cholesterol transport and overall HDL metabolism in humans when a cardiovascular disease therapy is investigated. Unfortunately, HDL cholesterol-raising therapies have not been successful at reducing cardiovascular events suggesting an incomplete picture of HDL biology. However, as HDL tracer studies have evolved from radioactive isotope- to stable isotope-based strategies that in turn are reliant on mass spectrometry technologies, the complexity of the HDL proteome and its metabolism can be more readily addressed. In this review, we outline the motivations, timelines, advantages, and disadvantages of the various tracer kinetics strategies. We also feature the metabolic properties of select HDL proteins known to regulate reverse cholesterol transport, which in turn underscore that HDL lipoproteins comprise a heterogeneous particle population whose distinct protein constituents and kinetics likely determine its function and potential contribution to cholesterol clearance.

FAM222A, Part of the BET-Regulated Basal Endothelial Transcriptome, Is a Novel Determinant of Endothelial Biology and Angiogenesis-Brief Report.

BACKGROUND: BETs (bromodomain and extraterminal domain-containing epigenetic reader proteins), including BRD4 (bromodomain-containing protein 4), orchestrate transcriptional programs induced by pathogenic stimuli, as intensively studied in cardiovascular disease and elsewhere. In endothelial cells (ECs), BRD4 directs induced proinflammatory, proatherosclerotic transcriptional responses; BET inhibitors, like JQ1, repress these effects and decrease atherosclerosis. While BET effects in pathogenic conditions have prompted therapeutic BET inhibitor development, BET action under basal conditions, including ECs, has remained understudied. To understand BET action in basal endothelial transcriptional programs, we first analyzed EC RNA-Seq data in the absence versus presence of JQ1 before using BET regulation to identify novel determinants of EC biology and function. METHODS: RNA-Seq datasets of human umbilical vein ECs without and with JQ1 treatment were analyzed. After identifying C12orf34, also known as FAM222A (family with sequence similarity 222 member A), as a previously unreported, basally expressed, potently JQ1-induced EC gene, FAM222A was studied in endothelial and angiogenic responses in vitro using small-interference RNA silencing and lentiviral overexpression, in vitro, ex vivo and in vivo, including aortic sprouting, matrigel plug assays, and murine neonatal oxygen-induced retinopathy. RESULTS: Resting EC RNA-Seq data indicate BETs direct transcriptional programs underlying core endothelial properties including migration, proliferation, and angiogenesis. BET inhibition in resting ECs also significantly induced a subset of mRNAs, including FAM222A-a unique BRD4-regulated gene with no reported EC role. Silencing endothelial FAM222A significantly decreased cellular proliferation, migration, network formation, aorta sprouting, and Matrigel plug vascularization through coordinated modulation of VEGF (vascular endothelial growth factor) and NOTCH mediator expression in vitro, ex vivo, in vivo; lentiviral FAM222A overexpression had opposite effects. In vivo, siFAM222A significantly repressed retinal revascularization in neonatal murine oxygen-induced retinopathy through similar angiogenic signaling modulation. CONCLUSIONS: BET control over the basal endothelial transcriptome includes FAM222A, a novel, BRD4-regulated, key determinant of endothelial biology and angiogenesis.

Effects of Recombinant Human Lecithin Cholesterol Acyltransferase on Lipoprotein Metabolism in Humans.

BACKGROUND: LCAT (lecithin cholesterol acyl transferase) catalyzes the conversion of unesterified, or free cholesterol, to cholesteryl ester, which moves from the surface of HDL (high-density lipoprotein) into the neutral lipid core. As this iterative process continues, nascent lipid-poor HDL is converted to a series of larger, spherical cholesteryl ester-enriched HDL particles that can be cleared by the liver in a process that has been termed reverse cholesterol transport. METHODS: We conducted a randomized, placebocontrolled, crossover study in 5 volunteers with atherosclerotic cardiovascular disease, to examine the effects of an acute increase of recombinant human (rh) LCAT via intravenous administration (300-mg loading dose followed by 150 mg at 48 hours) on the in vivo metabolism of HDL APO (apolipoprotein)A1 and APOA2, and the APOB100-lipoproteins, very low density, intermediate density, and low-density lipoproteins. RESULTS: As expected, recombinant human LCAT treatment significantly increased HDL-cholesterol (34.9 mg/dL; P≤0.001), and this was mostly due to the increase in cholesteryl ester content (33.0 mg/dL; P=0.014). This change did not affect the fractional clearance or production rates of HDL-APOA1 and HDL-APOA2. There were also no significant changes in the metabolism of APOB100-lipoproteins. CONCLUSIONS: Our results suggest that an acute increase in LCAT activity drives greater flux of cholesteryl ester through the reverse cholesterol transport pathway without significantly altering the clearance and production of the main HDL proteins and without affecting the metabolism of APOB100-lipoproteins. Long-term elevations of LCAT might, therefore, have beneficial effects on total body cholesterol balance and atherogenesis.

Multiomics of Tissue Extracellular Vesicles Identifies Unique Modulators of Atherosclerosis and Calcific Aortic Valve Stenosis.

BACKGROUND: Fewer than 50% of patients who develop aortic valve calcification have concomitant atherosclerosis, implying differential pathogenesis. Although circulating extracellular vesicles (EVs) act as biomarkers of cardiovascular diseases, tissue-entrapped EVs are associated with early mineralization, but their cargoes, functions, and contributions to disease remain unknown. METHODS: Disease stage-specific proteomics was performed on human carotid endarterectomy specimens (n=16) and stenotic aortic valves (n=18). Tissue EVs were isolated from human carotid arteries (normal, n=6; diseased, n=4) and aortic valves (normal, n=6; diseased, n=4) by enzymatic digestion, (ultra)centrifugation, and a 15-fraction density gradient validated by proteomics, CD63-immunogold electron microscopy, and nanoparticle tracking analysis. Vesiculomics, comprising vesicular proteomics and small RNA-sequencing, was conducted on tissue EVs. TargetScan identified microRNA targets. Pathway network analyses prioritized genes for validation in primary human carotid artery smooth muscle cells and aortic valvular interstitial cells. RESULTS: Disease progression drove significant convergence (P<0.0001) of carotid artery plaque and calcified aortic valve proteomes (2318 proteins). Each tissue also retained a unique subset of differentially enriched proteins (381 in plaques; 226 in valves; q<0.05). Vesicular gene ontology terms increased 2.9-fold (P<0.0001) among proteins modulated by disease in both tissues. Proteomics identified 22 EV markers in tissue digest fractions. Networks of proteins and microRNA targets changed by disease progression in both artery and valve EVs revealed shared involvement in intracellular signaling and cell cycle regulation. Vesiculomics identified 773 proteins and 80 microRNAs differentially enriched by disease exclusively in artery or valve EVs (q<0.05); multiomics integration found tissue-specific EV cargoes associated with procalcific Notch and Wnt signaling in carotid arteries and aortic valves, respectively. Knockdown of tissue-specific EV-derived molecules FGFR2, PPP2CA, and ADAM17 in human carotid artery smooth muscle cells and WNT5A, APP, and APC in human aortic valvular interstitial cells significantly modulated calcification. CONCLUSIONS: The first comparative proteomics study of human carotid artery plaques and calcified aortic valves identifies unique drivers of atherosclerosis versus aortic valve stenosis and implicates EVs in advanced cardiovascular calcification. We delineate a vesiculomics strategy to isolate, purify, and study protein and RNA cargoes from EVs entrapped in fibrocalcific tissues. Integration of vesicular proteomics and transcriptomics by network approaches revealed novel roles for tissue EVs in modulating cardiovascular disease.

Cellular Heterogeneity of Activated Primary Human Macrophages and Associated Drug-Gene Networks: From Biology to Precision Therapeutics.

BACKGROUND: Interferon-γ (IFNγ) signaling plays a complex role in atherogenesis. IFNγ stimulation of macrophages permits in vitro exploration of proinflammatory mechanisms and the development of novel immune therapies. We hypothesized that the study of macrophage subpopulations could lead to anti-inflammatory interventions. METHODS: Primary human macrophages activated by IFNγ (M(IFNγ)) underwent analyses by single-cell RNA sequencing, time-course cell-cluster proteomics, metabolite consumption, immunoassays, and functional tests (phagocytic, efferocytotic, and chemotactic). RNA-sequencing data were analyzed in LINCS (Library of Integrated Network-Based Cellular Signatures) to identify compounds targeting M(IFNγ) subpopulations. The effect of compound BI-2536 was tested in human macrophages in vitro and in a murine model of atherosclerosis. RESULTS: Single-cell RNA sequencing identified 2 major clusters in M(IFNγ): inflammatory (M(IFNγ)i) and phagocytic (M(IFNγ)p). M(IFNγ)i had elevated expression of inflammatory chemokines and higher amino acid consumption compared with M(IFNγ)p. M(IFNγ)p were more phagocytotic and chemotactic with higher Krebs cycle activity and less glycolysis than M(IFNγ)i. Human carotid atherosclerotic plaques contained 2 such macrophage clusters. Bioinformatic LINCS analysis using our RNA-sequencing data identified BI-2536 as a potential compound to decrease the M(IFNγ)i subpopulation. BI-2536 in vitro decreased inflammatory chemokine expression and secretion in M(IFNγ) by shrinking the M(IFNγ)i subpopulation while expanding the M(IFNγ)p subpopulation. BI-2536 in vivo shifted the phenotype of macrophages, modulated inflammation, and decreased atherosclerosis and calcification. CONCLUSIONS: We characterized 2 clusters of macrophages in atherosclerosis and combined our cellular data with a cell-signature drug library to identify a novel compound that targets a subset of macrophages in atherosclerosis. Our approach is a precision medicine strategy to identify new drugs that target atherosclerosis and other inflammatory diseases.

Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Promotes Macrophage Activation via LDL Receptor-Independent Mechanisms.

BACKGROUND: Activated macrophages contribute to the pathogenesis of vascular disease. Vein graft failure is a major clinical problem with limited therapeutic options. PCSK9 (proprotein convertase subtilisin/kexin 9) increases low-density lipoprotein (LDL)-cholesterol levels via LDL receptor (LDLR) degradation. The role of PCSK9 in macrophage activation and vein graft failure is largely unknown, especially through LDLR-independent mechanisms. This study aimed to explore a novel mechanism of macrophage activation and vein graft disease induced by circulating PCSK9 in an LDLR-independent fashion. METHODS: We used Ldlr-/- mice to examine the LDLR-independent roles of circulating PCSK9 in experimental vein grafts. Adeno-associated virus (AAV) vector encoding a gain-of-function mutant of PCSK9 (rAAV8/D377Y-mPCSK9) induced hepatic PCSK9 overproduction. To explore novel inflammatory targets of PCSK9, we used systems biology in Ldlr-/- mouse macrophages. RESULTS: In Ldlr-/- mice, AAV-PCSK9 increased circulating PCSK9, but did not change serum cholesterol and triglyceride levels. AAV-PCSK9 promoted vein graft lesion development when compared with control AAV. In vivo molecular imaging revealed that AAV-PCSK9 increased macrophage accumulation and matrix metalloproteinase activity associated with decreased fibrillar collagen, a molecular determinant of atherosclerotic plaque stability. AAV-PCSK9 induced mRNA expression of the pro-inflammatory mediators IL-1ß (interleukin-1 beta), TNFα (tumor necrosis factor alpha), and MCP-1 (monocyte chemoattractant protein-1) in peritoneal macrophages underpinned by an in vitro analysis of Ldlr-/- mouse macrophages stimulated with endotoxin-free recombinant PCSK9. A combination of unbiased global transcriptomics and new network-based hyperedge entanglement prediction analysis identified the NF-κB (nuclear factor-kappa B) signaling molecules, lectin-like oxidized LOX-1 (LDL receptor-1), and SDC4 (syndecan-4) as potential PCSK9 targets mediating pro-inflammatory responses in macrophages. CONCLUSIONS: Circulating PCSK9 induces macrophage activation and vein graft lesion development via LDLR-independent mechanisms. PCSK9 may be a potential target for pharmacologic treatment for this unmet medical need.

A Novel Spectral Annotation Strategy Streamlines Reporting of Mono-ADP-ribosylated Peptides Derived from Mouse Liver and Spleen in Response to IFN-γ.

Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.

Sortilin enhances fibrosis and calcification in aortic valve disease by inducing interstitial cell heterogeneity.

AIMS: Calcific aortic valve disease (CAVD) is the most common valve disease, which consists of a chronic interplay of inflammation, fibrosis, and calcification. In this study, sortilin (SORT1) was identified as a novel key player in the pathophysiology of CAVD, and its role in the transformation of valvular interstitial cells (VICs) into pathological phenotypes is explored. METHODS AND RESULTS: An aortic valve (AV) wire injury (AVWI) mouse model with sortilin deficiency was used to determine the effects of sortilin on AV stenosis, fibrosis, and calcification. In vitro experiments employed human primary VICs cultured in osteogenic conditions for 7, 14, and 21 days; and processed for imaging, proteomics, and transcriptomics including single-cell RNA-sequencing (scRNA-seq). The AVWI mouse model showed reduced AV fibrosis, calcification, and stenosis in sortilin-deficient mice vs. littermate controls. Protein studies identified the transition of human VICs into a myofibroblast-like phenotype mediated by sortilin. Sortilin loss-of-function decreased in vitro VIC calcification. ScRNA-seq identified 12 differentially expressed cell clusters in human VIC samples, where a novel combined inflammatory myofibroblastic-osteogenic VIC (IMO-VIC) phenotype was detected with increased expression of SORT1, COL1A1, WNT5A, IL-6, and serum amyloid A1. VICs sequenced with sortilin deficiency showed decreased IMO-VIC phenotype. CONCLUSION: Sortilin promotes CAVD by mediating valvular fibrosis and calcification, and a newly identified phenotype (IMO-VIC). This is the first study to examine the role of sortilin in valvular calcification and it may render it a therapeutic target to inhibit IMO-VIC emergence by simultaneously reducing inflammation, fibrosis, and calcification, the three key pathological processes underlying CAVD.

Second Heart Field-Derived Cells Contribute to Angiotensin II-Mediated Ascending Aortopathies.

BACKGROUND: The ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies. METHODS: Ascending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry-assisted proteomics and molecular features of SHF-derived cells were determined by single-cell transcriptomic analyses. Genetic deletion of either Lrp1 (low-density lipoprotein receptor-related protein 1) or Tgfbr2 (transforming growth factor-ß receptor type 2) in SHF-derived cells was conducted to examine the effect of SHF-derived cells on vascular integrity. RESULTS: Pathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas after AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion before overt pathology occurred evoked downregulation of smooth muscle cell proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single-cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single-cell transcriptomics results identified PAI1 (plasminogen activator inhibitor 1) as the most increased protein in SHF-derived smooth muscle cells and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies. CONCLUSIONS: SHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and transforming growth factor-ß signaling associated with increases of aortic PAI1.

Prothymosin Alpha: A Novel Contributor to Estradiol Receptor Alpha-Mediated CD8+ T-Cell Pathogenic Responses and Recognition of Type 1 Collagen in Rheumatic Heart Valve Disease.

BACKGROUND: Rheumatic heart valve disease (RHVD) is a leading cause of cardiovascular death in low- and middle-income countries and affects predominantly women. The underlying mechanisms of chronic valvular damage remain unexplored and regulators of sex predisposition are unknown. METHODS: Proteomics analysis of human heart valves (nondiseased aortic valves, nondiseased mitral valves [NDMVs], valves from patients with rheumatic aortic valve disease, and valves from patients with rheumatic mitral valve disease; n=30) followed by system biology analysis identified ProTα (prothymosin alpha) as a protein associated with RHVD. Histology, multiparameter flow cytometry, and enzyme-linked immunosorbent assay confirmed the expression of ProTα. In vitro experiments using peripheral mononuclear cells and valvular interstitial cells were performed using multiparameter flow cytometry and quantitative polymerase chain reaction. In silico analysis of the RHVD and Streptococcuspyogenes proteomes were used to identify mimic epitopes. RESULTS: A comparison of NDMV and nondiseased aortic valve proteomes established the baseline differences between nondiseased aortic and mitral valves. Thirteen unique proteins were enriched in NDMVs. Comparison of NDMVs versus valves from patients with rheumatic mitral valve disease and nondiseased aortic valves versus valves from patients with rheumatic aortic valve disease identified 213 proteins enriched in rheumatic valves. The expression of the 13 NDMV-enriched proteins was evaluated across the 213 proteins enriched in diseased valves, resulting in the discovery of ProTα common to valves from patients with rheumatic mitral valve disease and valves from patients with rheumatic aortic valve disease. ProTα plasma levels were significantly higher in patients with RHVD than in healthy individuals. Immunoreactive ProTα colocalized with CD8+ T cells in RHVD. Expression of ProTα and estrogen receptor alpha correlated strongly in circulating CD8+ T cells from patients with RHVD. Recombinant ProTα induced expression of the lytic proteins perforin and granzyme B by CD8+ T cells as well as higher estrogen receptor alpha expression. In addition, recombinant ProTα increased human leukocyte antigen class I levels in valvular interstitial cells. Treatment of CD8+ T cells with specific estrogen receptor alpha antagonist reduced the cytotoxic potential promoted by ProTα. In silico analysis of RHVD and Spyogenes proteomes revealed molecular mimicry between human type 1 collagen epitope and bacterial collagen-like protein, which induced CD8+ T-cell activation in vitro. CONCLUSIONS: ProTα-dependent CD8+ T-cell cytotoxicity was associated with estrogen receptor alpha activity, implicating ProTα as a potential regulator of sex predisposition in RHVD. ProTα facilitated recognition of type 1 collagen mimic epitopes by CD8+ T cells, suggesting mechanisms provoking autoimmunity.

ApoC-III is a novel inducer of calcification in human aortic valves.

Calcific aortic valve disease (CAVD) occurs when subpopulations of valve cells undergo specific differentiation pathways, promoting tissue fibrosis and calcification. Lipoprotein particles carry oxidized lipids that promote valvular disease, but low-density lipoprotein-lowering therapies have failed in clinical trials, and there are currently no pharmacological interventions available for this disease. Apolipoproteins are known promoters of atherosclerosis, but whether they possess pathogenic properties in CAVD is less clear. To search for a possible link, we assessed 12 apolipoproteins in nonfibrotic/noncalcific and fibrotic/calcific aortic valve tissues by proteomics and immunohistochemistry to understand if they were enriched in calcified areas. Eight apolipoproteins (apoA-I, apoA-II, apoA-IV, apoB, apoC-III, apoD, apoL-I, and apoM) were enriched in the calcific versus nonfibrotic/noncalcific tissues. Apo(a), apoB, apoC-III, apoE, and apoJ localized within the disease-prone fibrosa and colocalized with calcific regions as detected by immunohistochemistry. Circulating apoC-III on lipoprotein(a) is a potential biomarker of aortic stenosis incidence and progression, but whether apoC-III also induces aortic valve calcification is unknown. We found that apoC-III was increased in fibrotic and calcific tissues and observed within the calcification-prone fibrosa layer as well as around calcification. In addition, we showed that apoC-III induced calcification in primary human valvular cell cultures via a mitochondrial dysfunction/inflammation-mediated pathway. This study provides a first assessment of a broad array of apolipoproteins in CAVD tissues, demonstrates that specific apolipoproteins associate with valvular calcification, and implicates apoC-III as an active and modifiable driver of CAVD beyond its potential role as a biomarker.

Systems Approach to Discovery of Therapeutic Targets for Vein Graft Disease: PPARα Pivotally Regulates Metabolism, Activation, and Heterogeneity of Macrophages and Lesion Development.

BACKGROUND: Vein graft failure remains a common clinical challenge. We applied a systems approach in mouse experiments to discover therapeutic targets for vein graft failure. METHODS: Global proteomics and high-dimensional clustering on multiple vein graft tissues were used to identify potential pathogenic mechanisms. The PPARs (peroxisome proliferator-activated receptors) pathway served as an example to substantiate our discovery platform. In vivo mouse experiments with macrophage-targeted PPARα small interfering RNA, or the novel, selective activator pemafibrate demonstrate the role of PPARα in the development and inflammation of vein graft lesions. In vitro experiments further included metabolomic profiling, quantitative polymerase chain reaction, flow cytometry, metabolic assays, and single-cell RNA sequencing on primary human and mouse macrophages. RESULTS: We identified changes in the vein graft proteome associated with immune responses, lipid metabolism regulated by the PPARs, fatty acid metabolism, matrix remodeling, and hematopoietic cell mobilization. PPARα agonism by pemafibrate retarded the development and inflammation of vein graft lesions in mice, whereas gene silencing worsened plaque formation. Pemafibrate also suppressed arteriovenous fistula lesion development. Metabolomics/lipidomics, functional metabolic assays, and single-cell analysis of cultured human macrophages revealed that PPARα modulates macrophage glycolysis, citrate metabolism, mitochondrial membrane sphingolipid metabolism, and heterogeneity. CONCLUSIONS: This study explored potential drivers of vein graft inflammation and identified PPARα as a novel potential pharmacological treatment for this unmet medical need.

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

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

S100A9-RAGE Axis Accelerates Formation of Macrophage-Mediated Extracellular Vesicle Microcalcification in Diabetes Mellitus.

OBJECTIVE: Vascular calcification is a cardiovascular risk factor and accelerated in diabetes mellitus. Previous work has established a role for calcification-prone extracellular vesicles in promoting vascular calcification. However, the mechanisms by which diabetes mellitus provokes cardiovascular events remain incompletely understood. Our goal was to identify that increased S100A9 promotes the release of calcification-prone extracellular vesicles from human macrophages in diabetes mellitus. Approach and Results: Human primary macrophages exposed to high glucose (25 mmol/L) increased S100A9 secretion and the expression of receptor for advanced glycation end products (RAGE) protein. Recombinant S100A9 induced the expression of proinflammatory and osteogenic factors, as well as the number of extracellular vesicles with high calcific potential (alkaline phosphatase activity, P<0.001) in macrophages. Treatment with a RAGE antagonist or silencing with S100A9 siRNA in macrophages abolished these responses, suggesting that stimulation of the S100A9-RAGE axis by hyperglycemia favors a procalcific environment. We further showed that an imbalance between Nrf-2 (nuclear factor 2 erythroid related factor 2) and NF-κB (nuclear factor-κB) pathways contributes to macrophage activation and promotes a procalcific environment. In addition, streptozotocin-induced diabetic Apoe-/-S100a9-/- mice and mice treated with S100a9 siRNA encapsulated in macrophage-targeted lipid nanoparticles showed decreased inflammation and microcalcification in atherosclerotic plaques, as gauged by molecular imaging and comprehensive histological analysis. In human carotid plaques, comparative proteomics in patients with diabetes mellitus and histological analysis showed that the S100A9-RAGE axis associates with osteogenic activity and the formation of microcalcification. CONCLUSIONS: Under hyperglycemic conditions, macrophages release calcific extracellular vesicles through mechanisms involving the S100A9-RAGE axis, thus contributing to the formation of microcalcification within atherosclerotic plaques.

Multiorgan Systems Study Reveals Igfbp7 as a Suppressor of Gluconeogenesis after Gastric Bypass Surgery.

Roux-en-Y gastric bypass (RYGB) surgery reduces weight in obese patients. A marked decrease in blood glucose levels occurs before weight loss; however, key molecules that improve the glycemic profile remain largely unknown. Using a murine RYGB surgery model, we performed multiorgan proteomics and bioinformatics to monitor the proteins and molecular pathways that change in this early glycemic response. Multiplexed proteomic kinetics data analysis revealed that the Roux limb, biliopancreatic limb, liver, and pancreas each exhibited unique temporal and molecular responses to the RYGB surgery. In addition, protein-protein network analysis indicated that the changes to the microbial environment in the intestine may play a crucial role in the beneficial effects of RYGB surgery. Furthermore, insulin-like growth factor binding protein 7 (Igfbp7) was identified as an early induced protein in the Roux limb. Known secretory properties of Igfbp7 prompted us to further investigate its role as a remote organ regulator of glucose metabolism. Igfbp7 overexpression decreased blood glucose levels in diet-induced obese mice and attenuated gluconeogenic gene expression in the liver. Secreted Igfbp7 appeared to mediate these beneficial effects. These results demonstrate that organs responded differentially to RYGB surgery and indicate that Igfbp7 may play an important role in improving blood glucose levels.

Uremic Toxin Indoxyl Sulfate Promotes Proinflammatory Macrophage Activation Via the Interplay of OATP2B1 and Dll4-Notch Signaling.

BACKGROUND: Chronic kidney disease (CKD) increases cardiovascular risk. Underlying mechanisms, however, remain obscure. The uremic toxin indoxyl sulfate is an independent cardiovascular risk factor in CKD. We explored the potential impact of indoxyl sulfate on proinflammatory activation of macrophages and its underlying mechanisms. METHODS: We examined in vitro the effects of clinically relevant concentrations of indoxyl sulfate on proinflammatory responses of macrophages and the roles of organic anion transporters and organic anion transporting polypeptides (OATPs). A systems approach, involving unbiased global proteomics, bioinformatics, and network analysis, then explored potential key pathways. To address the role of Delta-like 4 (Dll4) in indoxyl sulfate-induced macrophage activation and atherogenesis in CKD in vivo, we used 5/6 nephrectomy and Dll4 antibody in low-density lipoprotein receptor-deficient (Ldlr-/-) mice. To further determine the relative contribution of OATP2B1 or Dll4 to proinflammatory activation of macrophages and atherogenesis in vivo, we used siRNA delivered by macrophage-targeted lipid nanoparticles in mice. RESULTS: We found that indoxyl sulfate-induced proinflammatory macrophage activation is mediated by its uptake through transporters, including OATP2B1, encoded by the SLCO2B1 gene. The global proteomics identified potential mechanisms, including Notch signaling and the ubiquitin-proteasome pathway, that mediate indoxyl sulfate-triggered proinflammatory macrophage activation. We chose the Notch pathway as an example of key candidates for validation of our target discovery platform and for further mechanistic studies. As predicted computationally, indoxyl sulfate triggered Notch signaling, which was preceded by the rapid induction of Dll4 protein. Dll4 induction may result from inhibition of the ubiquitin-proteasome pathway, via the deubiquitinating enzyme USP5. In mice, macrophage-targeted OATP2B1/Slco2b1 silencing and Dll4 antibody inhibited proinflammatory activation of peritoneal macrophages induced by indoxyl sulfate. In low-density lipoprotein receptor-deficient mice, Dll4 antibody abolished atherosclerotic lesion development accelerated in Ldlr-/- mice. Moreover, coadministration of indoxyl sulfate and OATP2B1/Slco2b1 or Dll4 siRNA encapsulated in macrophage-targeted lipid nanoparticles in Ldlr-/- mice suppressed lesion development. CONCLUSIONS: These results suggest that novel crosstalk between OATP2B1 and Dll4-Notch signaling in macrophages mediates indoxyl sulfate-induced vascular inflammation in CKD.