Ulf Hedin

BACKGROUND: Cardiovascular calcification is a significant predictor and contributor to cardiovascular diseases. Vascular smooth muscle cell (SMC)–derived extracellular vesicles (EVs) play a crucial role in microcalcification formation. EVs can originate from the endosomal system, and PIKFYVE (1-phosphatidylinositol 3-phosphate 5-kinase), a lipid kinase, plays a key role in endomembrane maturation. We hypothesize that PIKFYVE inhibition will modulate EV cargo and, thereby, arterial calcification. METHODS: Human coronary artery SMCs were cultured in osteogenic media to induce calcification. PIKFYVE inhibition was achieved using the pharmacological inhibitor apilimod and siRNA. We characterized the SMC phenotype and EVs through proteomics, transcriptomics, and kinomics. Ldlr -deficient mice fed a high-fat, high-cholesterol diet received apilimod for 5 weeks. RESULTS: Calcified human arteries and SMCs exhibited increased PIKFYVE protein expression compared with controls. In calcifying SMCs, phosphatidylinositol 3-phosphate levels were reduced but restored by apilimod. Apilimod prevented matrix mineralization and collagen deposition in calcifying SMCs, accompanied by reduced procollagen 1A1 secretion. Apilimod inhibited TNAP (tissue-nonspecific alkaline phosphatase) at mRNA, protein, and activity levels. EVs released from apilimod-treated calcifying SMCs exhibited lower mineral cargo, reduced aggregation potential, and diminished TNAP cargo. Phenotypic omics analyses revealed that apilimod induced a shift toward an adipocyte-like SMC phenotype, marked by upregulation of adipogenic TFs (transcription factors), fatty acid metabolism genes, and increased fatty acid uptake. Reactivation of YAP (Yes-associated protein)-TEAD (transcriptional enhancer factor) signaling partially reversed these phenotypic changes. In vivo, apilimod reduced vascular calcification and plaque TNAP activity but increased plaque lipid accumulation. CONCLUSIONS: Pharmacological inhibition of PIKFYVE disrupts YAP-TEAD signaling, thereby reducing arterial calcification by limiting the calcification potential of EVs and suppressing osteogenic SMC programming, but also induces an adipocyte-like SMC phenotype with lipid accumulation. These findings emphasize the need to consider SMC phenotypic plasticity and potential adverse effects when developing therapeutic strategies for arterial calcification.

Ischemic stroke affects more than 15 million people annually, where atheroembolism from unstable atherosclerotic plaques in the carotid bifurcation contributes a significant and potentially preventable aetiology. Landmark carotid endarterectomy trials conducted decades ago proved that removing high-grade symptomatic stenoses outperforms medical therapy, yet accurately identifying unstable plaques, the true embolic source, remains elusive. Consequently, procedural efficacy is modest, and more personalized diagnostic methods are necessary for refined risk stratification and improved stroke prevention. Plaque instability and atheroembolism from rupture of the fibrous cap is driven by an enlargement of the lipid-rich necrotic core, inflammation, neovascularisation, intraplaque haemorrhage, and fibrous-cap thinning. Hypoxia-induced angiogenesis produces fragile neovessels that bleed, generating oxidative stress and further destabilisation. Biomolecules escaping into the circulation from such lesions could flag individuals at imminent risk, but imaging modalities such as magnetic resonance, computed tomography angiography, or ultrasound are necessary to identify lesions at risk. Using these methods, aligning imaging phenotypes with molecular signatures of stability or vulnerability can aid in the development of refined diagnostic methods. Integrating blood-based with quantitative imaging biomarkers and systems-biology modelling promises a personalised diagnostic pipeline to screen for circulating signals of instability to detect individuals at risk, together with noninvasive imaging to localise culprit lesions, creating the basis for tailored surgical or pharmacologic therapy. In this review, we discuss scientific advances in the development of such strategies that possess the potential to sharpen patient selection, reduce unnecessary procedures, and advance precision in stroke prevention.

Aims: Atherosclerosis is a major global health challenge, with limited diagnostic and therapeutic options. Macrophages drive disease progression, but their tissue-specific phenotypes and functions remain poorly defined. This study aims to elucidate macrophage-driven mechanisms by characterizing their functional diversity across key metabolic and vascular tissues. Methods and results: We used single-cell RNA sequencing (scRNA-seq) and translating ribosome affinity purification sequencing (TRAP-seq) to profile macrophage-specific gene programmes in a mouse model of atherosclerosis across the aorta, adipose tissue, and liver. Our data highlight tissue-specific macrophage gene programmes and identify markers that are shared across mouse and human plaques. First, we identified soluble Trem2 as a potential circulating biomarker for differentiating between asymptomatic and symptomatic individuals. Secondly, we leveraged the pronounced expression of Folr2 and Slc7a7 to explore the potential of folate and glutamine as positron emission tomography (PET) tracers for disease burden assessment through in vivo PET imaging. Finally, we show that knockout of Slc7a7 inhibits acetylated low-density lipoprotein uptake and dampens the gene signature linked to lipid-associated macrophages. This suggests that glutamine signalling may play a critical role in foam cell formation, a key event in atherosclerosis. Conclusion: Our findings provide novel insights into macrophage-specific gene programmes during atherosclerosis progression and identify a set of promising biomarkers that can serve as a resource for future studies. These findings could significantly contribute to improving the diagnosis, monitoring, and treatment of atherosclerosis.

Aims: Endotypes integrate individual clinical and molecular data and can be used to formulate molecular subclassifications of diseases. We previously derived four endotypes of subclinical carotid atherosclerosis in a large European cohort, c-IMT and c-IMT Progression as Predictors of Vascular Events in a High-Risk European Population (IMPROVE), identifying individuals with a specific cardiovascular (CV) risk, ranging from low (endotype 1) to very high (endotype 4). Here, we investigate the mechanisms underlying the differences in CV risk observed across these four endotypes. Methods and results: We validated the four endotypes in SCAPIS (n = 5050) and UK Biobank (n = 50 396) using carotid plaque and carotid intima-media thickness (c-IMT) as subclinical atherosclerosis measures. Endotype 4 associated with a larger number of carotid plaques and increased c-IMT measures as compared to endotype 1. We performed a meta-analysis of individual genome wide association studies in IMPROVE (n = 3711), SCAPIS and UK Biobank, and identified 12 SNPs associated with endotypes. We investigated if they regulated gene expression and circulating protein levels. We found that rs2228145A/C at Interleukin-6 Receptor (IL6R), associated with endotype 4, regulated IL6R expression and circulating levels of OncoStatin M Receptor (OSMR), Complement Factor B (CFB) and Fibrinogen Chain A (FGA). We used rs2228145A/C as an instrument in two-sample Mendelian randomization analyses and showed that a decreasing IL6R expression, associated with increasing CFB, FGA, and OSMR circulating levels. Endotype 4, IL6R, CFB, FGA, and OSMR co-localized within 250 kb surrounding rs2228145A/C. However, only OSMR was up-regulated in advanced carotid atherosclerotic plaques in the presence of the A allele and in aortic region exposed to low wall shear stress. In the UK Biobank, we observed that each additional A allele at rs2228145 increased by 1.28-times the risk of myocardial infarction (MI) in endotype 4. Conclusion: Rs2228145A/C associated with endotype 4 clinical and molecular characteristics and amplified the MI risk in individuals assigned to endotype 4. These effects appeared to be mediated by a crosstalk with OSMR.

BACKGROUND: Carotid atherosclerosis is orchestrated by cell-cell communication that drives progression along a clinical continuum (asymptomatic to symptomatic). Extracellular vesicles (EVs) are cell-derived nanoparticles representing a new paradigm in cellular communication. Little is known about their biological cargo, cellular origin/destination, and functional roles in human atherosclerotic plaque. METHODS: EVs were enriched via size exclusion chromatography from human carotid endarterectomy samples dissected into paired plaque and marginal zones (symptomatic n=16, asymptomatic n=13). EV-cargos were assessed via whole transcriptome microRNA-sequencing and mass spectrometry–based proteomics. EV multiomics was integrated with bulk and single-cell RNA-sequencing datasets to predict EV cellular origin and ligand-receptor interactions, and multimodal biological network integration of EV-cargo was completed. EV functional impact was assessed with endothelial angiogenesis assays. RESULTS: Carotid plaques contained more EVs than adjacent marginal zones, with differential enrichment for EV-microRNAs and EV-proteins in key atherogenic pathways. EV cellular origin analysis suggested that tissue EV signatures originated from endothelial cells, smooth muscle cells, and immune cells. Integrated tissue vesiculomics and single-cell RNA-sequencing indicated complex EV–vascular cell communication that changed with disease progression and plaque vulnerability (ie, symptomatic disease). Plaques from symptomatic patients, but not asymptomatic patients, were characterized by increased involvement of endothelial pathways and more complex ligand-receptor interactions, relative to their marginal zones. Plaque EVs were predicted to mediate communication with endothelial cells. Pathway enrichment analysis delineated an endothelial signature with roles in angiogenesis and neovascularization, well-known indices of plaque instability. This was validated functionally, wherein human carotid symptomatic plaque EVs induced sprouting angiogenesis in comparison to their matched marginal zones. CONCLUSIONS: Our findings indicate that EVs may drive dynamic changes in plaques through EV–vascular cell communication and effector functions that typify vulnerability to rupture, precipitating symptomatic disease. The discovery of endothelial-directed angiogenic processes mediated by EVs creates new therapeutic avenues for atherosclerosis.

The notion of clonal cell populations in human atherosclerosis has been suggested but not demonstrated. Somatic mutations are used to define cellular clones in tumors. Here, we characterized the mutational landscape of human carotid plaques through whole-exome sequencing to explore the presence of clonal cell populations. Somatic mutations were identified in 12 of 13 investigated plaques, while no mutations were detected in 11 non-atherosclerotic arteries. Mutated clones often constituted over 10% of the sample cell population, with genes related to the contractile apparatus enriched for mutations. In carriers of clonal hematopoiesis of indeterminate potential (CHIP), hematopoietic clones had infiltrated the plaque tissue and constituted substantial fractions of the plaque cell population alongside locally expanded clones. Our findings establish somatic mutations as a common feature of human atherosclerosis and demonstrate the existence of mutated clones expanding locally, as well as CHIP clones invading from the circulation. While our data do not support plaque monoclonality, we observed a pattern suggesting the coexistence of multiple mutated clones of considerable size spanning different regions of plaques. Mutated clones are likely to be relevant to disease development, and somatic mutations will serve as a convenient tool to uncover novel pathological processes of atherosclerosis in future studies.

Selecting the appropriate kidney replacement therapy (KRT) is crucial in order to secure optimal care for chronic kidney disease (CKD) patients with end-stage renal disease (ESRD). Next to renal transplantation, the choice of dialysis modality directly affects patient well-being, treatment effectiveness, and long-term outcomes. 1 Therefore, clinical decision-making must take into account a range of factors to tailor decisions to each patient's unique needs. Previously, when the dialysis population was less diverse and resources more limited, straight-forward algorithms could be effectively implemented both for selection of KRT as well as for hemodialysis (HD) vascular access (VA). Recently, the growing ESRD population with more elderly being considered for dialysis care together with improved treatment opportunities have resulted in more challenging decision-making where on-size-fits-all strategies are being replaced by more tailored and personalized strategies aimed to ensure "the right dialysis access- to the right patient- at the right time- for the right reason." In this review, recent trends enforcing a more personalized approach in the selection of VA for HD are summarized and discussed, where these concerns have become especially relevant.