Abstract Increased fluid shear stress (FSS) is a key initiating stimulus for arteriogenesis, the outward remodeling of collateral arterioles in response to upstream occlusion. Placental growth factor (PLGF) is an important arteriogenic mediator. We previously showed that elevated FSS increases PLGF in a reactive oxygen species (ROS)-dependent fashion both in vitro and ex vivo . Heme oxygenase 1 (HO-1) is a cytoprotective enzyme that is upregulated by stress and has arteriogenic effects. In the current study, we used isolated murine mesentery arterioles and cocultures of human coronary artery endothelial cells (EC) and smooth muscle cells (SMC) to test the hypothesis that HO-1 mediates the effects of FSS on PLGF. HO-1 mRNA was increased by conditions of increased flow and shear stress in both cocultures and vessels. Both inhibition of HO-1 with zinc protoporphyrin and HO-1 knockdown abolished the effect of FSS on PLGF. Conversely, induction of HO-1 activity increased PLGF. To determine which HO-1 product upregulates PLGF, cocultures were treated with a CO donor (CORM-A1), biliverdin, ferric ammonium citrate (FAC), or iron-nitrilotriacetic acid (iron-NTA). Of these FAC and iron-NTA induced an increase PLGF expression. This study demonstrates that FSS acts through iron to induce pro-arteriogenic PLGF, suggesting iron supplementation as a novel potential treatment for revascularization.
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.
Glutathione is a low molecular weight thiol that is present at high levels in the cell. The high levels of glutathione in the cell make it one of the most abundant antioxidants contributing to cellular redox homeostasis. As a general rule, throughout cardiovascular disease and progression there is an imbalance in redox homeostasis characterized by reactive oxygen species overproduction and glutathione underproduction. As research into these imbalances continues, glutathione concentrations are increasingly being observed to drive various physiological and pathological signaling responses. Interestingly in addition to acting directly as an antioxidant, glutathione is capable of post translational modifications (S-glutathionylation) of proteins through both chemical interactions and enzyme mediated events. This review will discuss both the chemical and enzyme-based S-glutathionylation of proteins involved in cardiovascular pathologies and angiogenesis.
Atherosclerosis is the leading cause of coronary artery disease (CAD) and stroke which are the primary contributors to cardiovascular disease related deaths in the world. This is an inflammatory disease characterized by the accumulation of lipid and fibrous elements in large arteries resulting in the development of a plaque. Progression of the disease leads to the accumulation of apoptotic cells and formation of a necrotic core making the plaque unstable and prone to rupture. Rupture can lead to artery occlusion resulting in stroke or myocardial infarction (MI). One of the main contributors to a large necrotic core is macrophages that are unable to clear the accumulated apoptotic cells and as a result undergo secondary necrosis. This clearance process, referred to as efferocytosis, is impaired in advanced atherosclerotic lesions. It has been shown that the process that leads to a thin fibrous cap (characteristic of an unstable plaque) and a large necrotic core are accelerated by a decrease in glutathione (GSH) availability due to a deletion on the modifier subunit of Glutamate Cysteine Ligase (GCLM). The objective of this study is to identify the role and mechanism of GSH during the formation of the necrotic core in atherosclerosis. We hypothesize that availability of GSH is important in macrophages for adequate efferocytosis of apoptotic cells during atherosclerosis progression.Triglycerides, total cholesterol, and HDL levels were measured on PCSK9 virus driven atherosclerosis in wild type and heterozygous GCLM mice. Sections of the aortic root from these mice were stained with Movat Pentachrome to measure plaque area and percent necrosis. An efferocytic index was measured using PHK26 labelled apoptotic cells (AC). AC were added to cultured bone marrow derived macrophages (BMDM) allowing for their uptake. Percent positive macrophages containing a fluorescently labelled AC were quantified. BMDM from WT, Het, and KO GCLM mice were collected for total protein, surface receptor expression, and mRNA analysis.Efferocytosis was affected in KO mice as compared to WT and Het BMDM. RNA analysis has showed a decrease in efferocytosis receptors such as MerTK, confirmed by cell surface expression.These results suggest that a loss of GCLM and GSH during efferocytosis impairs adequate efferocytosis.
Atherosclerosis (ASVD) is characterized by the buildup of lipids and fibrous elements in large arteries creating a plaque. As it progresses, apoptotic cells (AC) accumulate forming a necrotic core making the plaque unstable and prone to rupture. Rupture can lead to artery occlusion and moreover to stroke or myocardial infarction (MI). A main contributor to a large necrotic core is the inability of macrophages to clear accumulated AC and as a result undergo secondary necrosis. This clearance process, referred to as efferocytosis, is impaired in advanced atherosclerotic lesions. It has been shown that the process that leads to a thin fibrous cap and a large necrotic core are accelerated by a decrease in glutathione (GSH) availability by a deletion of the modifier subunit of Glutamate Cysteine Ligase (GCLM) in mice. Additionally, it has also been shown that a single nucleotide polymorphism (SNP) in the promoter region of human GCLM (-588 C/T) has an increased risk for MI. Our objective is to identify the role and mechanism of the GCLM SNP during the formation of the necrotic core in atherosclerosis. We hypothesize that proper function of GCLM and GSH is important in macrophages for adequate function and clearance of AC in ASVD. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from blood donors, genotyped to test for the presence of SNP, and used for RNA sequencing. Efferocytic index was measured using labelled AC in GCLM WT, Het, and KO murine bone marrow derived macrophages (BMDM) and PBMCs. Percent positive macrophages containing fluorescently labelled AC were quantified for each sample. Cells were collected for total protein, surface receptor expression, and mRNA analysis. Results: Efferocytosis was affected in macrophages with defective GCLM as compared to control. RNA analysis has showed a decrease in efferocytosis receptors such as MerTK, confirmed by cell surface expression. Conclusion: These results suggest that loss of GCLM and GSH in macrophages impairs adequate efferocytosis.
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.
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
Abstract Supraphysiological levels of the osteoblast‐enriched mineralization regulator ectonucleotide pyrophosphatase or phosphodiesterase‐1 (NPP1) is associated with type 2 diabetes mellitus. We determined the impact of osteoblast‐specific Enpp1 ablation on skeletal structure and metabolic phenotype in mice. Female, but not male, 6‐week‐old mice lacking osteoblast NPP1 expression (osteoblast‐specific knockout [KO]) exhibited increased femoral bone volume or total volume (17.50% vs. 11.67%; p < .01), and reduced trabecular spacing (0.187 vs. 0.157 mm; p < .01) compared with floxed (control) mice. Furthermore, an enhanced ability of isolated osteoblasts from the osteoblast‐specific KO to calcify their matrix in vitro compared to fl/fl osteoblasts was observed ( p < .05). Male osteoblast‐specific KO and fl/fl mice showed comparable glucose and insulin tolerance despite increased levels of insulin–sensitizing under‐carboxylated osteocalcin (195% increase; p < .05). However, following high‐fat‐diet challenge, osteoblast‐specific KO mice showed impaired glucose and insulin tolerance compared with fl/fl mice. These data highlight a crucial local role for osteoblast NPP1 in skeletal development and a secondary metabolic impact that predominantly maintains insulin sensitivity.
Abstract Placental growth factor (PLGF) and vascular endothelial growth factor-A (VEGF-A) are important regulators of both physiological and pathological vascular remodeling. We previously reported that in monoculture human coronary EC primarily expresses PLGF, while human coronary smooth muscle cells (SMC) primarily express VEGF-A. However, in the vasculature, EC and SMC are in close proximity. Thus, in this study we sought to 1) determine whether this cell-specific expression pattern is maintained when EC and SMC are cocultured, and 2) test the hypothesis that EC and SMC co-regulate the expression of VEGF-A and PLGF. EC and SMC were cultured on either side of a porous Transwell insert and media PLGF and VEGF-A levels were measured by ELISA. We confirmed that the cell type-specific expression of PLGF and VEGF-A we observed in monocultures remains evident in the coculture model. Interestingly, coculture of EC and SMC increased media PLGF relative to EC monoculture, but decreased media VEGF-A compared to SMC monoculture. Coculture conditions also increased VEGFR2 levels on the surface of EC but decreased VEGFR1 levels on the surface of SMC. Inhibition of VEGFR2 tyrosine kinase activity decreased PLGF and increased VEGF in both EC and cocultures but not in SMC monocultures. We conclude that PLGF and VEGF-A exert both paracrine and autocrine regulatory effects on each other and that these effects are mediated by VEGFR2 (in EC) and VEGFR1 (in SMC). Based on these results, we recommend that researchers investigating VEGFR signaling consider the use of coculture models when designing studies.
The process of arterial calcification shares many similarities to skeletal mineralisation, and involves the deposition of hydroxyapatite in the arteries. However, the cellular mechanisms responsible have yet to be fully elucidated. Accumulating evidence suggests that aerobic glycolysis (the Warburg effect), plays a critical role in meeting the demand for energy and biosynthetic precursors during proliferation and differentiation in numerous cell types. Therefore we addressed the hypothesis that vascular smooth muscle cell (VSMC) calcification requires aerobic glycolysis to produce energy and the necessary biosynthetic precursors.
Methods
Calcification of murine aortic VSMCs was induced by 3 mM Pi for 7 days. Calcium deposition was determined using alizarin red staining and a modified o-cresolphthalein method. VSMCs were cultured with the fluorescent glucose analogue 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)−2-Deoxyglucose (2-NBDG) to determine changes in glucose uptake. Gene expression was analysed by qRT-PCR.
Results
Calcium deposition was significantly increased in VSMCs cultured in 3 mM Pi versus control conditions (124%, p<0.001). Calcified VSMCs also showed increased mRNA expression of Runx2, Phospho1, Ocn and PiT-1 (p<0.001), recognised osteogenic markers of arterial calcification. Furthermore 3 mM Pi treatment increased glucose uptake (98%, p<0.05) and Glut-1 mRNA expression (1.47 fold, p<0.001). Glycolysis converts glucose to pyruvate which is subsequently converted to either (i) acetyl-CoA by the pyruvate dehydrogenase complex (PDH) or (ii) lactate by lactate dehydrogenase (LDH). Notably, decreased VSMC calcification was observed in cells treated with sodium dicholoroacetate, an inducer of PDH activity (1 mM; 40%; p<0.01) and citric acid, synthesised in the mitochondria from acetyl CoA (1 mM; 72%, p<0.001). Treatment with the LDH inhibitor sodium oxamate (20 mM) or sodium lactate (50 mM) to induce pyruvate production also inhibited VSMC calcification (68% and 53% respectively, p<0.05). Activation of the Wnt pathway – an established regulator of Warburg metabolism – using the selective GSK3 inhibitor CHIR99021 (1 nM) significantly increased VSMC calcification (417%, p<0.001). However, co-?treatment with sodium oxamate (20 mM) significantly blunted the pro-calcification effect of CHIR99021 (69%, p<0.01).
Conclusion
Together these data suggest that arterial calcification requires glucose metabolism through a mechanism involving Wnt signalling. Interruption of the glycolysis pathway may therefore represent a novel therapeutic target for clinical intervention.
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