In silico model of atherosclerosis with individual patient calibration to enable precision medicine for cardiovascular disease.

OBJECTIVE: Guidance for preventing myocardial infarction and ischemic stroke by tailoring treatment for individual patients with atherosclerosis is an unmet need. Such development may be possible with computational modeling. Given the multifactorial biology of atherosclerosis, modeling must be based on complete biological networks that capture protein-protein interactions estimated to drive disease progression. Here, we aimed to develop a clinically relevant scale model of atherosclerosis, calibrate it with individual patient data, and use it to simulate optimized pharmacotherapy for individual patients. APPROACH AND RESULTS: The study used a uniquely constituted plaque proteomic dataset to create a comprehensive systems biology disease model for simulating individualized responses to pharmacotherapy. Plaque tissue was collected from 18 patients with 6735 proteins at two locations per patient. 113 pathways were identified and included in the systems biology model of endothelial cells, vascular smooth muscle cells, macrophages, lymphocytes, and the integrated intima, altogether spanning 4411 proteins, demonstrating a range of 39-96% plaque instability. After calibrating the systems biology models for individual patients, we simulated intensive lipid-lowering, anti-inflammatory, and anti-diabetic drugs. We also simulated a combination therapy. Drug response was evaluated as the degree of change in plaque stability, where an improvement was defined as a reduction of plaque instability. In patients with initially unstable lesions, simulated responses varied from high (20%, on combination therapy) to marginal improvement, whereas patients with initially stable plaques showed generally less improvement. CONCLUSION: In this pilot study, proteomics-based system biology modeling was shown to simulate drug response based on atherosclerotic plaque instability with a power of 90%, providing a potential strategy for improved personalized management of patients with cardiovascular disease.

Mast cells participate in smooth muscle cell reprogramming and atherosclerotic plaque calcification.

BACKGROUND: Calcification, a key feature of advanced human atherosclerosis, is positively associated with vascular disease burden and adverse events. We showed that macrocalcification can be a stabilizing factor for carotid plaque molecular biology, due to inverse association with immune processes. Mast cells (MCs) are important contributors to plaque instability, but their relationship with macrocalcification is unexplored. With a hypothesis that MC activation negatively associates with carotid plaque macrocalcification, we aimed to investigate the link between MCs and carotid plaque vulnerability, and study MC role in plaque calcification via smooth muscle cells (SMCs). METHODS: Pre-operative computed tomography angiographies of patients (n = 40) undergoing surgery for carotid stenosis were used to characterize plaque morphology. Plaque microarrays (n = 40 and n = 126) were used for bioinformatic deconvolution of immune cell populations. Tissue microarrays (n = 103) were used to histologically validate the contribution of activated and resting MCs in plaques. RESULTS: Activated MCs and their typical markers were negatively correlated with macrocalcification. The ratio of activated vs. resting MCs was increased in low-calcified plaques from symptomatic patients. There was no modulating effect of medication on MC ratios. In vitro experiments showed that SMC calcification attenuated MC activation, while both active and resting MCs stimulated SMC calcification and induced dedifferentiation towards a pro-inflammatory-, osteochondrocyte-like phenotype, without modulating their migro-proliferative function. CONCLUSIONS: Integrative analyses from human plaques showed that MC activation is inversely associated with macrocalcification and positively with parameters of plaque vulnerability. Mechanistically, MCs induce SMC osteogenic reprograming, while matrix calcification in turn attenuates MC activation, offering new therapeutic avenues for exploration.

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.

Osteomodulin attenuates smooth muscle cell osteogenic transition in vascular calcification.

RATIONALE: Vascular calcification is a prominent feature of late-stage diabetes, renal and cardiovascular disease (CVD), and has been linked to adverse events. Recent studies in patients reported that plasma levels of osteomodulin (OMD), a proteoglycan involved in bone mineralisation, associate with diabetes and CVD. We hypothesised that OMD could be implicated in these diseases via vascular calcification as a common underlying factor and aimed to investigate its role in this context. METHODS AND RESULTS: In patients with chronic kidney disease, plasma OMD levels correlated with markers of inflammation and bone turnover, with the protein present in calcified arterial media. Plasma OMD also associated with cardiac calcification and the protein was detected in calcified valve leaflets by immunohistochemistry. In patients with carotid atherosclerosis, circulating OMD was increased in association with plaque calcification as assessed by computed tomography. Transcriptomic and proteomic data showed that OMD was upregulated in atherosclerotic compared to control arteries, particularly in calcified plaques, where OMD expression correlated positively with markers of smooth muscle cells (SMCs), osteoblasts and glycoproteins. Immunostaining confirmed that OMD was abundantly present in calcified plaques, localised to extracellular matrix and regions rich in α-SMA+ cells. In vivo, OMD was enriched in SMCs around calcified nodules in aortic media of nephrectomised rats and in plaques from ApoE-/- mice on warfarin. In vitro experiments revealed that OMD mRNA was upregulated in SMCs stimulated with IFNγ, BMP2, TGFß1, phosphate and ß-glycerophosphate, and by administration of recombinant human OMD protein (rhOMD). Mechanistically, addition of rhOMD repressed the calcification process of SMCs treated with phosphate by maintaining their contractile phenotype along with enriched matrix organisation, thereby attenuating SMC osteoblastic transformation. Mechanistically, the role of OMD is exerted likely through its link with SMAD3 and TGFB1 signalling, and interplay with BMP2 in vascular tissues. CONCLUSION: We report a consistent association of both circulating and tissue OMD levels with cardiovascular calcification, highlighting the potential of OMD as a clinical biomarker. OMD was localised in medial and intimal α-SMA+ regions of calcified cardiovascular tissues, induced by pro-inflammatory and pro-osteogenic stimuli, while the presence of OMD in extracellular environment attenuated SMC calcification.

Integrative Multi-Omics Analysis in Calcific Aortic Valve Disease Reveals a Link to the Formation of Amyloid-Like Deposits.

Calcific aortic valve disease (CAVD) is the most prevalent valvular heart disease in the developed world, yet no pharmacological therapy exists. Here, we hypothesize that the integration of multiple omic data represents an approach towards unveiling novel molecular networks in CAVD. Databases were searched for CAVD omic studies. Differentially expressed molecules from calcified and control samples were retrieved, identifying 32 micro RNAs (miRNA), 596 mRNAs and 80 proteins. Over-representation pathway analysis revealed platelet degranulation and complement/coagulation cascade as dysregulated pathways. Multi-omics integration of overlapping proteome/transcriptome molecules, with the miRNAs, identified a CAVD protein-protein interaction network containing seven seed genes (apolipoprotein A1 (APOA1), hemoglobin subunit ß (HBB), transferrin (TF), α-2-macroglobulin (A2M), transforming growth factor ß-induced protein (TGFBI), serpin family A member 1 (SERPINA1), lipopolysaccharide binding protein (LBP), inter-α-trypsin inhibitor heavy chain 3 (ITIH3) and immunoglobulin κ constant (IGKC)), four input miRNAs (miR-335-5p, miR-3663-3p, miR-21-5p, miR-93-5p) and two connector genes (amyloid beta precursor protein (APP) and transthyretin (TTR)). In a metabolite-gene-disease network, Alzheimer's disease exhibited the highest degree of betweenness. To further strengthen the associations based on the multi-omics approach, we validated the presence of APP and TTR in calcified valves from CAVD patients by immunohistochemistry. Our study suggests a novel molecular CAVD network potentially linked to the formation of amyloid-like structures. Further investigations on the associated mechanisms and therapeutic potential of targeting amyloid-like deposits in CAVD may offer significant health benefits.