Genome-scale metabolic network of human carotid plaque reveals the pivotal role of glutamine/glutamate metabolism in macrophage modulating plaque inflammation and vulnerability.

BACKGROUND: Metabolism is increasingly recognized as a key regulator of the function and phenotype of the primary cellular constituents of the atherosclerotic vascular wall, including endothelial cells, smooth muscle cells, and inflammatory cells. However, a comprehensive analysis of metabolic changes associated with the transition of plaque from a stable to a hemorrhaged phenotype is lacking. METHODS: In this study, we integrated two large mRNA expression and protein abundance datasets (BIKE, n = 126; MaasHPS, n = 43) from human atherosclerotic carotid artery plaque to reconstruct a genome-scale metabolic network (GEM). Next, the GEM findings were linked to metabolomics data from MaasHPS, providing a comprehensive overview of metabolic changes in human plaque. RESULTS: Our study identified significant changes in lipid, cholesterol, and inositol metabolism, along with altered lysosomal lytic activity and increased inflammatory activity, in unstable plaques with intraplaque hemorrhage (IPH+) compared to non-hemorrhaged (IPH-) plaques. Moreover, topological analysis of this network model revealed that the conversion of glutamine to glutamate and their flux between the cytoplasm and mitochondria were notably compromised in hemorrhaged plaques, with a significant reduction in overall glutamate levels in IPH+ plaques. Additionally, reduced glutamate availability was associated with an increased presence of macrophages and a pro-inflammatory phenotype in IPH+ plaques, suggesting an inflammation-prone microenvironment. CONCLUSIONS: This study is the first to establish a robust and comprehensive GEM for atherosclerotic plaque, providing a valuable resource for understanding plaque metabolism. The utility of this GEM was illustrated by its ability to reliably predict dysregulation in the cholesterol hydroxylation, inositol metabolism, and the glutamine/glutamate pathway in rupture-prone hemorrhaged plaques, a finding that may pave the way to new diagnostic or therapeutic measures.

The Effect of Spironolactone in Patients With Obesity at Risk for Heart Failure: Proteomic Insights from the HOMAGE Trial.

BACKGROUND: Adipose tissue influences the expression and degradation of circulating biomarkers. We aimed to identify the biomarker profile and biological meaning of biomarkers associated with obesity to assess the effect of spironolactone on the circulating biomarkers and to explore whether obesity might modify the effect of spironolactone. METHODS AND RESULTS: Protein biomarkers (n = 276) from the Olink Proseek-Multiplex cardiovascular and inflammation panels were measured in plasma collected at baseline, 1 month and 9 months from the HOMAGE randomized controlled trial participants. Of the 510 participants, 299 had obesity defined as an increased waist circumference (≥102 cm in men and ≥88 cm in women). Biomarkers at baseline reflected adipogenesis, increased vascularization, decreased fibrinolysis, and glucose intolerance in patients with obesity at baseline. Treatment with spironolactone had only minor effects on this proteomic profile. Obesity modified the effect of spironolactone on systolic blood pressure (Pinteraction = 0.001), showing a stronger decrease of blood pressure in obese patients (-14.8 mm Hg 95% confidence interval -18.45 to -11.12) compared with nonobese patients (-3.6 mm Hg 95% confidence interval -7.82 to 0.66). CONCLUSIONS: Among patients at risk for heart failure, those with obesity have a characteristic proteomic profile reflecting adipogenesis and glucose intolerance. Spironolactone had only minor effects on this obesity-related proteomic profile, but obesity significantly modified the effect of spironolactone on systolic blood pressure.

Clonal Hematopoiesis of Indeterminate Potential From a Heart Failure Specialist's Point of View.

Clonal hematopoiesis of indeterminate potential (CHIP) is a common bone marrow abnormality induced by age-related DNA mutations, which give rise to proinflammatory immune cells. These immune cells exacerbate atherosclerotic cardiovascular disease and may induce or accelerate heart failure. The mechanisms involved are complex but point toward a central role for proinflammatory macrophages and an inflammasome-dependent immune response (IL-1 [interleukin-1] and IL-6 [interleukin-6]) in the atherosclerotic plaque or directly in the myocardium. Intracardiac inflammation may decrease cardiac function and induce cardiac fibrosis, even in the absence of atherosclerotic cardiovascular disease. The pathophysiology and consequences of CHIP may differ among implicated genes as well as subgroups of patients with heart failure, based on cause (ischemic versus nonischemic) and ejection fraction (reduced ejection fraction versus preserved ejection fraction). Evidence is accumulating that CHIP is associated with cardiovascular mortality in ischemic and nonischemic heart failure with reduced ejection fraction and involved in the development of heart failure with preserved ejection fraction. CHIP and corresponding inflammatory pathways provide a highly potent therapeutic target. Randomized controlled trials in patients with well-phenotyped heart failure, where readily available anti-inflammatory therapies are used to intervene with clonal hematopoiesis, may pave the way for a new area of heart failure treatment. The first clinical trials that target CHIP are already registered.

Two-faced Janus: the dual role of macrophages in atherosclerotic calcification.

Calcification is an independent predictor of atherosclerosis-related cardiovascular events. Microcalcification is linked to inflamed, unstable lesions, in comparison to the fibrotic stable plaque phenotype generally associated with advanced calcification. This paradox relates to recognition that calcification presents in a wide spectrum of manifestations that differentially impact plaque's fate. Macrophages, the main inflammatory cells in atherosclerotic plaque, have a multifaceted role in disease progression. They crucially control the mineralization process, from microcalcification to the osteoid metaplasia of bone-like tissue. It is a bilateral interaction that weighs heavily on the overall plaque fate but remains rather unexplored. This review highlights current knowledge about macrophage phenotypic changes in relation to and interaction with the calcifying environment. On the one hand, macrophage-led inflammation kickstarts microcalcification through a multitude of interlinked mechanisms, which in turn stimulates phenotypic changes in vascular cell types to drive microcalcification. Macrophages may also modulate the expression/activity of calcification inhibitors and inducers, or eliminate hydroxyapatite nucleation points. Contrarily, direct exposure of macrophages to an early calcifying milieu impacts macrophage phenotype, with repercussions for plaque progression and/or stability. Macrophages surrounding macrocalcification deposits show a more reparative phenotype, modulating extracellular matrix, and expressing osteoclast genes. This phenotypic shift favours gradual displacement of the pro-inflammatory hubs; the lipid necrotic core, by macrocalcification. Parallels to bone metabolism may explain many of these changes to macrophage phenotype, with advanced calcification able to show homeostatic osteoid metaplasia. As the targeted treatment of vascular calcification developing in atherosclerosis is thus far severely lacking, it is crucial to better understand its mechanisms of development.

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

Calcification is a prominent feature of late-stage atherosclerosis, but the mechanisms driving this process are unclear. Using a biobank of carotid endarterectomies, we recently showed that Proteoglycan 4 (PRG4) is a key molecular signature of calcified plaques, expressed in smooth muscle cell (SMC) rich regions. Here, we aimed to unravel the PRG4 role in vascular remodeling and intimal calcification. PRG4 expression in human carotid endarterectomies correlated with calcification assessed by preoperative computed tomographies. PRG4 localized to SMCs in early intimal thickening, while in advanced lesions it was found in the extracellular matrix, surrounding macro-calcifications. In experimental models, Prg4 was upregulated in SMCs from partially ligated ApoE-/- mice and rat carotid intimal hyperplasia, correlating with osteogenic markers and TGFb1. Furthermore, PRG4 was enriched in cells positive for chondrogenic marker SOX9 and around plaque calcifications in ApoE-/- mice on warfarin. In vitro, PRG4 was induced in SMCs by IFNg, TGFb1 and calcifying medium, while SMC markers were repressed under calcifying conditions. Silencing experiments showed that PRG4 expression was driven by transcription factors SMAD3 and SOX9. Functionally, the addition of recombinant human PRG4 increased ectopic SMC calcification, while arresting cell migration and proliferation. Mechanistically, it suppressed endogenous PRG4, SMAD3 and SOX9, and restored SMC markers' expression. PRG4 modulates SMC function and osteogenic phenotype during intimal remodeling and macro-calcification in response to TGFb1 signaling, SMAD3 and SOX9 activation. The effects of PRG4 on SMC phenotype and calcification suggest its role in atherosclerotic plaque stability, warranting further investigations.

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

BACKGROUND: While single-omics analyses on human atherosclerotic plaque have been very useful to map stage- or disease-related differences in expression, they only partly capture the array of changes in this tissue and suffer from scale-intrinsic limitations. In order to better identify processes associated with intraplaque hemorrhage and plaque instability, we therefore combined multiple omics into an integrated model. METHODS: In this study, we compared protein and gene makeup of low- versus high-risk atherosclerotic lesion segments from carotid endarterectomy patients, as judged from the absence or presence of intraplaque hemorrhage, respectively. Transcriptomic, proteomic, and peptidomic data of this plaque cohort were aggregated and analyzed by DIABLO, an integrative multivariate classification and feature selection method. RESULTS: We identified a protein-gene associated multiomics model able to segregate stable, nonhemorrhaged from vulnerable, hemorrhaged lesions at high predictive performance (AUC >0.95). The dominant component of this model correlated with αSMA- PDGFRα+ fibroblast-like cell content (p = 2.4E-05) and Arg1+ macrophage content (p = 2.2E-04) and was driven by serum response factor (SRF), possibly in a megakaryoblastic leukemia-1/2 (MKL1/2) dependent manner. Gene set overrepresentation analysis on the selected key features of this model pointed to a clear cardiovascular disease signature, with overrepresentation of extracellular matrix synthesis and organization, focal adhesion, and cholesterol metabolism terms, suggestive of the model's relevance for the plaque vulnerability. Finally, we were able to corroborate the predictive power of the selected features in several independent mRNA and proteomic plaque cohorts. CONCLUSIONS: In conclusion, our integrative omics study has identified an intraplaque hemorrhage-associated cardiovascular signature that provides excellent stratification of low- from high-risk carotid artery plaques in several independent cohorts. Further study revealed suppression of an SRF-regulated disease network, controlling lesion stability, in vulnerable plaque, which can serve as a scaffold for the design of targeted intervention in plaque destabilization.

Structural topology defines protective CD8+ T cell epitopes in the HIV proteome.

Mutationally constrained epitopes of variable pathogens represent promising targets for vaccine design but are not reliably identified by sequence conservation. In this study, we employed structure-based network analysis, which applies network theory to HIV protein structure data to quantitate the topological importance of individual amino acid residues. Mutation of residues at important network positions disproportionately impaired viral replication and occurred with high frequency in epitopes presented by protective human leukocyte antigen (HLA) class I alleles. Moreover, CD8+ T cell targeting of highly networked epitopes distinguished individuals who naturally control HIV, even in the absence of protective HLA alleles. This approach thereby provides a mechanistic basis for immune control and a means to identify CD8+ T cell epitopes of topological importance for rational immunogen design, including a T cell-based HIV vaccine.