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.

The pro-fibrotic and anti-inflammatory foam cell macrophage paradox.

The formation of foamy macrophages by sequestering extracellular modified lipids is a key event in atherosclerosis. However, there is controversy about the effects of lipid loading on macrophage phenotype, with in vitro evidence suggesting either pro- or anti-inflammatory consequences. To investigate this in vivo we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in experimental subcutaneous granulomas in fat-fed ApoE null mice or normal chow-fed wild-type mice, respectively. Consistent with previous studies in peritoneal macrophages from LDL receptor null mice (Spann et al., 2012 [1]), we found that anti-inflammatory LXR/RXR pathway genes were over-represented in the foamy macrophages, but there was no change in M1 or M2 phenotypic markers. Quite unexpectedly, however, we found that genes related to the induction of fibrosis had also been up-regulated (Thomas et al., 2015 [2]). The progression of the foamy macrophages along anti-inflammatory and pro-fibrotic pathways was confirmed using immunohistochemistry (described fully in our primary research article (Thomas et al., 2015 [2]). Here we provide additional details on production of the macrophages and their transcriptomic comparison, with the raw and processed microarray data deposited in GEO (accession number GSE70126). Our observations on these cells are indeed paradoxical, because foamy macrophages have long been implicated in promoting inflammation, extracellular matrix degradation and atherosclerotic plaque rupture, which must be provoked by additional local mediators. Our findings probably explain how very early macrophage-rich lesions maintain their structural integrity.

Foam Cell Formation In Vivo Converts Macrophages to a Pro-Fibrotic Phenotype.

Formation of foam cell macrophages, which sequester extracellular modified lipids, is a key event in atherosclerosis. How lipid loading affects macrophage phenotype is controversial, with evidence suggesting either pro- or anti-inflammatory consequences. To investigate this further, we compared the transcriptomes of foamy and non-foamy macrophages that accumulate in the subcutaneous granulomas of fed-fat ApoE null mice and normal chow fed wild-type mice in vivo. Consistent with previous studies, LXR/RXR pathway genes were significantly over-represented among the genes up-regulated in foam cell macrophages. Unexpectedly, the hepatic fibrosis pathway, associated with platelet derived growth factor and transforming growth factor-ß action, was also over-represented. Several collagen polypeptides and proteoglycan core proteins as well as connective tissue growth factor and fibrosis-related FOS and JUN transcription factors were up-regulated in foam cell macrophages. Increased expression of several of these genes was confirmed at the protein level in foam cell macrophages from subcutaneous granulomas and in atherosclerotic plaques. Moreover, phosphorylation and nuclear translocation of SMAD2, which is downstream of several transforming growth factor-ß family members, was also detected in foam cell macrophages. We conclude that foam cell formation in vivo leads to a pro-fibrotic macrophage phenotype, which could contribute to plaque stability, especially in early lesions that have few vascular smooth muscle cells.

Myeloid type I interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to lesions.

Inflammatory cytokines are well-recognized mediators of atherosclerosis. Depending on the pathological context, type I interferons (IFNs; IFNalpha and IFNbeta) exert either pro- or anti-inflammatory immune functions, but their exact role in atherogenesis has not been clarified. Here, we demonstrate that IFNbeta enhances macrophage-endothelial cell adhesion and promotes leukocyte attraction to atherosclerosis-prone sites in mice in a chemokine-dependent manner. Moreover, IFNbeta treatment accelerates lesion formation in two different mouse models of atherosclerosis and increases macrophage accumulation in the plaques. Concomitantly, absence of endogenous type I IFN signaling in myeloid cells inhibits lesion development, protects against lesional accumulation of macrophages, and prevents necrotic core formation. Finally, we show that type I IFN signaling is upregulated in ruptured human atherosclerotic plaques. Hereby, we identify type I IFNs as proatherosclerotic cytokines that may serve as additional targets for prevention or treatment.

Equivalence testing in microarray analysis: similarities in the transcriptome of human atherosclerotic and nonatherosclerotic macrophages.

We focus on similarities in the transcriptome of human Kupffer cells and alveolar, splenic, and atherosclerotic plaque-residing macrophages. We hypothesized that these macrophages share a common expression signature. We performed microarray analysis on mRNA from these subsets (4 patients) and developed a novel statistical method to identify genes with significantly similar expression levels. Phenotypic and functional diversity between macrophage subpopulations reflects their plasticity to respond to microenvironmental signals. Apart from detecting differences in expression profiles, the comparison of the transcriptomes of different macrophage populations may also allow the definition of molecular similarities between these subsets. This new method calculates the maximum difference in gene expression level, based on the estimated confidence interval on that gene's expression variance. We listed the genes by equivalence ranking relative to expression level. FDR estimation was used to determine significance. We identified 500 genes with significantly equivalent expression levels in the macrophage subsets at 5.5% FDR using a confidence level of α = 0.05 for equivalence. Among these are the established macrophage marker CD68, IL1 receptor antagonist, and MHC-related CD1C. These 500 genes were submitted to IPA and GO clustering using DAVID. Additionally, hierarchical clustering of these genes in the Novartis human gene expression atlas revealed a subset of 200 genes specifically expressed in macrophages. Equivalently expressed genes, identified by this new method, may not only help to dissect common molecular mechanisms, but also to identify cell- or condition-specific sets of marker genes that can be used for drug targeting and molecular imaging.

The multi-functionality of CD40L and its receptor CD40 in atherosclerosis.

Disrupting the CD40-CD40L co-stimulatory pathway reduces atherosclerosis and induces a stable atherosclerotic plaque phenotype that is low in inflammation and high in fibrosis. Therefore, inhibition of the CD40-CD40L pathway is an attractive therapeutic target to reduce clinical complications of atherosclerosis. The CD40-CD40L dyad is known to interact with other co-stimulatory molecules, to activate antigen-presenting cells (APC) and to contribute to T-cell priming and B-cell isotype switching. Besides their presence on T-cells and APCs, CD40 and CD40L are also present on macrophages, endothelial cells and vascular smooth muscle cells in the plaque, where they can exert pro-atherogenic functions. Moreover, recent progress indicates the involvement of neutrophil CD40, platelet CD40L and dendritic cell CD40 in atherogenesis. Since systemic CD40-CD40L modulation compromises host defense, more targeted interventions are needed to develop superior treatment strategies for atherosclerosis. We believe that by unravelling the cell-cell CD40-CD40L interactions, inhibition of cell-type specific (signalling components of) CD40(L) that do not compromise the patient's immune system, will become possible. In this review, we highlight the cell-type specific multi-functionality of CD40-CD40L signalling in atherosclerosis.

The vulnerable patient: refocusing on the plaque?

The term 'vulnerable plaque' is used to refer to the lesions that are prone to rupture and may cause life-threatening events like acute coronary syndrome or stroke. The study of the vulnerable plaque phenotype and its detection has attracted increasing interest over the past decades. During this time, there have been some remarkable transitions in the paradigm on methods to identify patients at risk or patients to treat. Whereas formerly, the key factors used to determine an individual's risk were primarily population-based traditional risk factors such as age, sex, body mass index, hypertension etc., new approaches are based on conditional risk factors that represent an individual's current risk of suffering a cardiovascular event. These population based risk factors fall short in predicting near-future events in a high-risk individual. In the early 2000s, the focus of research into surrogate markers for cardiovascular event prediction shifted from the vulnerable plaque to the identification of the vulnerable patient. This new paradigm stimulated a number of new initiatives that aimed to identify vulnerable patients by testing systemic biomarkers that could identify patients at high risk for cardiovascular events. A second research paradigm is refocusing on the plaque by searching for plaque-derived biomarkers and non-invasive imaging modalities to assess characteristics of a plaque that determine its vulnerability. Although both concepts are attractive, they still need proper validation in large multicenter cohorts, while cost-effectiveness arguments also need to be assessed.

Towards high-throughput functional target discovery in angiogenesis research.

Angiogenesis is a hallmark of malignancies and other proliferative diseases, and inhibition of this process is considered to be a promising treatment strategy. Classical gene-expression analyses performed during the past decade have generated vast lists of genes associated with disease but have so far yielded only limited novel therapeutic targets for clinical applications. Recently, the focus has shifted from target identification, based on gene-expression analysis, to identification of genes, based on the function of the encoded protein. Disease-target genes can now be identified in a high-throughput fashion based on functional properties that are directly related to the disease phenotype. This new approach significantly shortens the time span for the development of therapeutic applications from the laboratory bench to the hospital bedside.