Impairments in carbohydrate, lipid, and amino acid metabolism drive features of plaque instability. However, where these impairments occur within the atheroma remains largely unknown. Therefore, we sought to characterize the spatial distribution of metabolites within stable and unstable atherosclerosis in both the fibrous cap and necrotic core. Atherosclerotic tissue specimens were scored based on the Stary classification scale and subdivided into stable and unstable atheromas. After performing mass spectrometry imaging (MSI) on these samples, we identified over 850 metabolite-related peaks. Using MetaboScape, METASPACE, and HMDB, we confidently annotated 170 of these metabolites and found over 60 of these were different between stable and unstable atheromas. We then integrated these results with an RNA-sequencing dataset comparing stable and unstable human atherosclerosis. Upon integrating our MSI results with the RNA-seq dataset, we discovered that pathways related to lipid metabolism and long-chain fatty acids were enriched in stable plaques, whereas reactive oxygen species, aromatic amino acid, and tryptophan metabolism were increased in unstable plaques. Acylcarnitines and acylglycines were increased in stable plaques whereas tryptophan metabolites were enriched in unstable plaques. Evaluating spatial differences in stable plaques revealed lactic acid in the necrotic core, whereas pyruvic acid was elevated in the fibrous cap. In unstable plaques, 5-hydroxyindole-acetic acid was enriched in the fibrous cap. Our work herein represents the first step to defining an atlas of metabolic pathways involved in plaque destabilization in human atherosclerosis. We anticipate this will be a valuable resource and open new avenues of research in cardiovascular disease.
Cardiovascular disease continues to be a leading cause of death in the United States. A major underlying cause of cardiovascular events such as stroke and myocardial infarction is atherosclerosis. Notable mediators in the pathogenesis of atherosclerosis include vascular smooth muscle cells (VSMCs) and inflammation. During atherogenesis, the SMCs undergo phenotypic change from a quiescent state to a synthetic, proliferative state. The synthetic SMCs drive neointima formation which influences the stenosis characteristic of atherosclerosis. Signaling of a receptor tyrosine kinase called EphA2 has been previously determined to have a role in SMC phenotypic change. It was found that a global and SMC specific knockdown (KD) of EphA2 resulted in decreased plaque formation, suggesting the role of EphA2 expression in promoting SMC proliferation. Inflammation is another important factor that contributes to plaque formation. Oxidized low-density lipoproteins (LDLs) have been found to also cause VSMCs to undergo change to a synthetic phenotype, one that resembles macrophages. Cytokines released by macrophages orchestrate the pro-inflammatory response that affects the development and stability of atherosclerotic plaques. Considering the important role inflammation also serves in the progression of plaque formation, we investigated the relationship between EphA2 and VSMC-mediated inflammatory response. Cre-flox breeding was performed to achieve EphA2 knockout (KO) specifically in VSMCs of 8-10-wk old ApoE -/- mice. Successful KO was confirmed with PCR and Western blot. Si-RNA was transfected to achieve KD in human SMCs. The cells were then treated with cytokines, including IL-1 beta, TNF-alpha, IL-6, IL-33, and interferon-gamma, followed by RT-qPCR analysis to measure expression of inflammatory genes. To assess cytokine secretion, we utilized a cytokine array to measure presence of the cytokines of interest. Protein analysis to quantify expression of inflammatory proteins in KO vs control cells was done via Western blot. We observed that the loss of EphA2 causes significant reductions in the production of several proinflammatory cytokines and chemokines, indicating its role in mediating VSMC inflammatory responses.
The Nck family of signaling adaptors, including both Nck1 and Nck2, classically regulate cell motility during vascular development and postnatal angiogenesis. While Nck1 and Nck2 play redundant roles in this regard with deletion of both isoforms required to affect tissue remodeling, we demonstrated that global Nck1 deletion was sufficient to reduce atherosclerotic plaque formation and selective Nck1 inhibition reduces proinflammatory endothelial activation in response to oscillatory shear stress. Therefore, we sought to determine the role of endothelial Nck1 in atherosclerosis and ischemic angiogenesis using novel endothelial-specific Nck1 knockout mice. Endothelial-specific Nck1 inhibition significantly reduced Western dietinduced atherosclerotic plaque formation at multiple sites compared to mice expressing endothelial-specific Cre alone or to endothelial-specific Nck2 knockout mice. This reduced plaque formation was characterized by diminished macrophage and smooth muscle incorporation despite enhanced weight gain in endothelial Nck1 knockout mice. RNA Sequencing analysis of endothelial cells in culture showed that Nck1 depletion reduces the expression of a variety of proinflammatory pathways but enhances the expression of genes involved in central carbon metabolism and proliferation. In addition, mass spectrometry analysis of Nck1 interacting proteins showed overrepresentation in binding partners involved in cell metabolism, consistent with published GWAS associations between Nck1 and body mass index and circulating plasma lipids. In contrast, Nck1 inhibition did not affect angiogenesis or limb perfusion in the femoral artery ligation model of hind limb ischemia. Taken together, endothelial Nck1 inhibition reduces atherosclerotic plaque formation associated with diminished proinflammatory responses without limiting ischemic angiogenesis, suggesting that Nck1 inhibition could be utilized to reduce atherosclerosis without preventing ischemic angiogenesis in the affected tissue.
Despite significant advances in diagnosing and treating cardiovascular disease, atherosclerosis remains the leading cause of death worldwide. While most atherosclerotic plaques remain asymptomatic, a subset can lead to myocardial infarction, stroke, or sudden death. A plethora of evidence has demonstrated that rupture-prone atheromas contains a large necrotic core owed to defective efferocytosis, the process by which apoptotic cells (ACs) are engulfed and cleared by macrophages. The efficient clearance of apoptotic cells blocks post-apoptotic necrosis and prevents the release of tissue-degrading enzymes, immunogenic epitopes, and proinflammatory mediators. As atherosclerosis advances, substantial remodeling of the extracellular matrix (ECM) occurs whereby transitional ECM proteins, notably fibronectin (FN), deposit into the subendothelial matrix normally dominated by collagen IV and laminins. However, whether macrophage interactions with the ECM alters efferocytosis, inflammation resolution, and atherosclerosis remains unknown. Surprisingly, we discovered that macrophage interactions with FN enhance efferocytosis compared to their interactions with basement membrane proteins. Furthermore, we demonstrate that the primary FN receptor, integrin α5β1, is required for FN-mediated efferocytosis and TIM3 expression, (encoded by Havcr2 ), a known phosphatidylserine receptor that binds to ACs. Moreover, treating mice with established atherosclerosis with the integrin α5β1 antagonistic peptide ATN-161 reduced TIM3 expression in macrophages and expanded necrotic core formation. We also found lesional macrophages from human unstable atheromas displayed significantly lower TIM3 expression compared to macrophages within stable plaques. In a mouse model of atherosclerosis regression, where atherogenic stimuli dissipate, efferocytosis is restored, and FN remains, lesional macrophages showed a substantial increase in TIM3 expression. Thus, while FN is traditionally considered atherogenic in endothelial and smooth muscle cells in the initial stages of atherosclerosis, our data uncovers that macrophage adhesion to FN upregulates TIM3 expression, mediating efferocytosis and promoting features of plaque stability.
ABSTRACT Impairments in carbohydrate, lipid, and amino acid metabolism drive features of plaque instability. However, where these impairments occur within the atheroma remains largely unknown. Therefore, we sought to characterize the spatial distribution of metabolites within stable and unstable atherosclerosis in both the fibrous cap and necrotic core. Atherosclerotic tissue specimens were scored based on the Stary classification scale and subdivided into stable and unstable atheromas. After performing mass spectrometry imaging (MSI) on these samples, we identified over 850 metabolite-related peaks. Using MetaboScape, METASPACE, and HMDB, we confidently annotated 170 of these metabolites and found over 60 of these were different between stable and unstable atheroma. We then integrated these results with an RNA-sequencing dataset comparing stable and unstable human atherosclerosis. Upon integrating our MSI results with the RNA-seq dataset, we discovered that pathways related to lipid metabolism and long-chain fatty acids were enriched in stable plaques, whereas reactive oxygen species, aromatic amino acid, and tryptophan metabolism were increased in unstable plaques. Acylcarnitines and acylglycines were increased in stable plaques whereas tryptophan metabolites were enriched in unstable plaques. Evaluating spatial differences in stable plaques revealed lactic acid in the necrotic core, whereas pyruvic acid was elevated in the fibrous cap. In unstable plaques, 5-hydroxyindole-acetic acid was enriched in the fibrous cap. Our work herein represents the first step to defining an atlas of metabolic pathways involved in plaque destabilization in human atherosclerosis. We anticipate this will be a valuable resource and open new avenues of research in cardiovascular disease. GRAPHICAL ABSTRACT
Background: Nonalcoholic fatty liver disease (NAFLD), the leading cause of chronic liver disease worldwide, results from diet-induced hepatic steatosis that can result in hepatocyte ballooning, inflammation and fibrosis driving nonalcoholic steatohepatitis (NASH). The Eph receptors, the largest family of receptor tyrosine kinases in the mammalian genome, affect inflammation and fibrosis in other model systems, and we have shown that EphA2 deletion reduces atherosclerosis despite increasing plasma cholesterol levels. While a GWAS study has linked EphA2 to NAFLD, the potential role of EphA2 in NAFLD has never been investigated. Methods: EphA2 expression and signaling was assessed in mice fed a high fat diet (HFD) for 8 and 24 weeks, in human liver samples from NASH patients, and in HuH7 cells treated with palmitic acid (FA), low density lipoprotein (LDL), and/or fructose. Global EphA2 KO mice were fed a HFD for 12 weeks followed by histological and molecular analyses. Results: Mice with early-stage non-alcoholic fatty liver disease (NAFLD) showed elevated EphA2 expression along with markers of EphA2 ligand-dependent signaling (EphA2 tyrosine phosphorylation). Consistently, HuH7 cells treated with FA, LDL, and/or fructose displayed a similar enhancement of EphA2 expression upon treatment. However, a mouse model of late-stage NASH exhibited reduced expression of the EphA2 ligand ephrinA1 and elevated markers of ligand-independent signaling (Ser897 phosphorylation). Similarly, human liver samples from NASH patients show reduced ephrinA1 expression and elevated markers of EphA2 ligand independent signaling. Consistent with a role for EphA2 in NAFLD/NASH, global EphA2 KO mice show significantly lower indices of NAFLD following high fat diet feeding, including reduced liver-to-body weight ratios, hepatic steatosis, and inflammation. Conclusion: Taken together, our data demonstrate that EphA2 expression and signaling are altered during NAFLD/NASH pathogenesis and suggest that EphA2 inhibition may reduce NAFLD/NASH disease progression.
Impairments in carbohydrate, lipid, and amino acid metabolism drive features of plaque instability. However, where these impairments occur within the atheroma remains largely unknown. Therefore, we sought to characterize the spatial distribution of metabolites within stable and unstable atherosclerosis in both the fibrous cap and necrotic core.
Despite significant advancements in therapeutics, atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of death worldwide. While the role of lipids in the pathogenesis of ASCVD is well-known, the contribution of amino acids is becoming increasingly recognized. Lower circulating glycine levels are consistently reported in patients with ASCVD. Previous studies from our group and others demonstrated that patients with significant coronary artery disease exhibit a reduced glycine:serine ratio compared with healthy individuals. Primarily expressed in the liver, mitochondrial serine hydroxymethyl transferase (SHMT2) is thought to be responsible for the bulk of glycine biosynthesis. Nevertheless, the role of hepatic SHMT2 in regulating serine and glycine metabolism in the context of atherosclerosis and cardiometabolic diseases has not been studied. Here, we describe novel Shmt2 -floxed mice crossed with Alb -cre mice, yielding hepatocyte-specific loss of SHMT2 ( Shmt2 HKO ). We further crossed our Shmt2 HKO mice with athero-prone ( Apoe -/- ) mice to study the role of hepatocyte SHMT2 in the pathogenesis of atherosclerosis. While loss of hepatocyte SHMT2 was expected to limit glycine biosynthesis, our targeted metabolomics analysis uncovered a significant increase in circulating glycine with no significant changes in serine, but a marked increase in the glycine:serine ratio, indicating an unexpected reverse SHMT2 activity. Unbiased transcriptomics of livers from Shmt2 HKO / Apoe -/- mice fed a high-fat, high-cholesterol, high-sucrose Western diet revealed an upregulation of pathways known to play protective roles in atherosclerosis and cardiometabolic diseases, including glutathione metabolism, AMPK, and PPAR signaling, while proinflammatory pathways were significantly downregulated. Consistently, we noted a significant reduction in the liver-to-body weight ratio, an indicator of reduced liver damage under Western diet feeding. Our findings uncover an unexpected reverse activity of hepatic SHMT2 in mice with atherosclerosis and fatty liver. Our ongoing studies aim to elucidate a potential causative role of hepatic SHMT2 in atherosclerosis, nonalcoholic fatty liver disease, and other cardiometabolic diseases.
