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Brief unreferenced abstractThe genetics underlying cancer and cardiovascular disease have been studied for decades. However, despite sharing numerous risk factors and pathologic features, the contribution of shared genetics in these diseases remains poorly defined. Our study sought to identify common genetic factors between these two diseases. We found a unique relationship between coronary artery disease and prostate cancer that appears to be mediated through germline genetic variation in LPA.

Venous disease is a term that broadly covers both venous thromboembolic disease and chronic venous disease. The basic pathophysiology of venous thromboembolism and chronic venous disease differ as venous thromboembolism results from an imbalance of hemostasis and thrombosis while chronic venous disease occurs in the setting of tissue damage because of prolonged venous hypertension. Both diseases are common and account for significant mortality and morbidity, respectively, and collectively make up a large health care burden. Despite both diseases having well-characterized environmental components, it has been known for decades that family history is an important risk factor, implicating a genetic element to a patient’s risk. Our understanding of the pathogenesis of these diseases has greatly benefited from an expansion of population genetic studies from pioneering familial studies to large genome-wide association studies; we now have multiple risk loci for each venous disease. In this review, we will highlight the current state of knowledge on the epidemiology and genetics of venous thromboembolism and chronic venous disease and directions for future research.

The pleiotropic benefits of statins may result from their impact on vascular inflammation. The molecular process underlying this phenomenon is not fully elucidated. Here, RNA sequencing designed to investigate gene expression patterns following CD47-SIRPα inhibition identifies a link between statins, efferocytosis, and vascular inflammation. In vivo and in vitro studies provide evidence that statins augment programmed cell removal by inhibiting the nuclear translocation of NFκB1 p50 and suppressing the expression of the critical 'don't eat me' molecule, CD47. Statins amplify the phagocytic capacity of macrophages, and thus the anti-atherosclerotic effects of CD47-SIRPα blockade, in an additive manner. Analyses of clinical biobank specimens suggest a similar link between statins and CD47 expression in humans, highlighting the potential translational implications. Taken together, our findings identify efferocytosis and CD47 as pivotal mediators of statin pleiotropy. In turn, statins amplify the anti-atherosclerotic effects of pro-phagocytic therapies independently of any lipid-lowering effect.

Supplemental Digital Content is available in the text., Background: Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. Methods: We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. Results: We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11-, Lgals3+ population with a chondrocyte-like gene signature that was markedly reduced with SMC-Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene–positive, extracellular matrix-remodeling, “pioneer” cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. Conclusions: Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3+ osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.

Aims Until recently, the pluripotency factor Octamer (ATGCAAAT)-binding transcriptional factor 4 (OCT4) was believed to be dispensable in adult somatic cells. However, our recent studies provided clear evidence that OCT4 has a critical atheroprotective role in smooth muscle cells. Here, we asked if OCT4 might play a functional role in regulating endothelial cell (EC) phenotypic modulations in atherosclerosis. Methods and results Specifically, we show that EC-specific Oct4 knockout resulted in increased lipid, LGALS3+ cell accumulation, and altered plaque characteristics consistent with decreased plaque stability. A combination of single-cell RNA sequencing and EC-lineage-tracing studies revealed increased EC activation, endothelial-to-mesenchymal transitions, plaque neovascularization, and mitochondrial dysfunction in the absence of OCT4. Furthermore, we show that the adenosine triphosphate (ATP) transporter, ATP-binding cassette (ABC) transporter G2 (ABCG2), is a direct target of OCT4 in EC and establish for the first time that the OCT4/ABCG2 axis maintains EC metabolic homeostasis by regulating intracellular heme accumulation and related reactive oxygen species production, which, in turn, contributes to atherogenesis. Conclusions These results provide the first direct evidence that OCT4 has a protective metabolic function in EC and identifies vascular OCT4 and its signalling axis as a potential target for novel therapeutics.

The stem cell pluripotency factor Oct4 serves a critical protective role during atherosclerotic plaque development by promoting smooth muscle cell (SMC) investment. Here, we show using Myh11-CreERT2 lineage-tracing with inducible SMC and pericyte (SMC-P) knockout of Oct4 that Oct4 regulates perivascular cell migration and recruitment during angiogenesis. Knockout of Oct4 in perivascular cells significantly impairs perivascular cell migration, increases perivascular cell death, delays endothelial cell migration, and promotes vascular leakage following corneal angiogenic stimulus. Knockout of Oct4 in perivascular cells also impairs perfusion recovery and decreases angiogenesis following hindlimb ischemia. Transcriptomic analyses demonstrate that expression of the migratory gene Slit3 is reduced following loss of Oct4 in cultured SMCs, and in Oct4-deficient perivascular cells in ischemic hindlimb muscle. Together, these results provide evidence that Oct4 plays an essential role within perivascular cells in injury- and hypoxia-induced angiogenesis., Perivascular cells are essential to the formation and stabilization of new blood vessels during angiogenesis. Here, Hess and Kelly-Goss et al. show that the stem cell pluripotency factor Oct4 promotes perivascular cell survival and migration required for angiogenesis in contexts of tissue injury and hypoxia.

Stable atherosclerotic plaques are characterized by a thick, extracellular matrix-rich fibrous cap populated by protective ACTA2+ myofibroblast (MF)-like cells, assumed to be almost exclusively derived from smooth muscle cells (SMCs). Herein, we show that in murine and human lesions, 20% to 40% of ACTA2+ fibrous cap cells, respectively, are derived from non-SMC sources, including endothelial cells (ECs) or macrophages that have undergone an endothelial-to-mesenchymal transition (EndoMT) or a macrophage-to-mesenchymal transition (MMT). In addition, we show that SMC-specific knockout of the Pdgfrb gene, which encodes platelet-derived growth factor receptor beta (PDGFRβ), in Apoe-/- mice fed a Western diet for 18 weeks resulted in brachiocephalic artery lesions nearly devoid of SMCs but with no changes in lesion size, remodelling or indices of stability, including the percentage of ACTA2+ fibrous cap cells. However, prolonged Western diet feeding of SMC Pdgfrb-knockout mice resulted in reduced indices of stability, indicating that EndoMT- and MMT-derived MFs cannot compensate indefinitely for loss of SMC-derived MFs. Using single-cell and bulk RNA-sequencing analyses of the brachiocephalic artery region and in vitro models, we provide evidence that SMC-to-MF transitions are induced by PDGF and transforming growth factor-β and dependent on aerobic glycolysis, while EndoMT is induced by interleukin-1β and transforming growth factor-β. Together, we provide evidence that the ACTA2+ fibrous cap originates from a tapestry of cell types, which transition to an MF-like state through distinct signalling pathways that are either dependent on or associated with extensive metabolic reprogramming.

The results of the CANTOS trial provide exciting evidence in support of the inflammatory hypothesis of atherosclerosis in humans, and is the first phase III clinical trial to show clinical benefit of a targeted anti-inflammatory therapy. However, the modest overall clinical benefit and safety concerns with increased susceptibility to fatal infections indicate that we need much more work in this critical area.

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

Despite decades of research, our understanding of the processes controlling late-stage atherosclerotic plaque stability remains poor. A prevailing hypothesis is that reducing inflammation may improve advanced plaque stability, as recently tested in the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) trial, in which post-myocardial infarction subjects were treated with an IL-1β antibody. Here, we performed intervention studies in which smooth muscle cell (SMC) lineage-tracing Apoe-/- mice with advanced atherosclerosis were treated with anti-IL-1β or IgG control antibodies. Surprisingly, we found that IL-1β antibody treatment between 18 and 26 weeks of Western diet feeding induced a marked reduction in SMC and collagen content, but increased macrophage numbers in the fibrous cap. Moreover, although IL-1β antibody treatment had no effect on lesion size, it completely inhibited beneficial outward remodeling. We also found that SMC-specific knockout of Il1r1 (encoding IL-1 receptor type 1) resulted in smaller lesions nearly devoid of SMCs and lacking a fibrous cap, whereas macrophage-selective loss of IL-1R1 had no effect on lesion size or composition. Taken together, these results show that IL-1β has multiple beneficial effects in late-stage murine atherosclerosis, including promotion of outward remodeling and formation and maintenance of an SMC- and collagen-rich fibrous cap.

The long-term adverse effects of radiotherapy on cardiovascular disease are well documented. However, the underlying mechanisms responsible for this increased risk are poorly understood. Previous studies using rigorous smooth muscle cell (SMC) lineage tracing have shown abundant SMC investment into atherosclerotic lesions, where SMCs contribute to the formation of a protective fibrous cap. Studies herein tested whether radiation impairs protective adaptive SMC responses during vascular disease. To do this, we exposed SMC lineage tracing (Myh11-ERT2Cre YFP+) mice to lethal radiation (1,200 cGy) followed by bone marrow transplantation prior to atherosclerosis development or vessel injury. Surprisingly, following irradiation, we observed a complete loss of SMC investment in 100% of brachiocephalic artery (BCA), carotid artery, and aortic arch lesions. Importantly, this was associated with a decrease in multiple indices of atherosclerotic lesion stability within the BCA. Interestingly, we observed anatomic heterogeneity, as SMCs accumulated normally into lesions of the aortic root and abdominal aorta, suggesting that SMC sensitivity to lethal irradiation occurs in blood vessels of neural crest origin. Taken together, these results reveal an undefined and unintended variable in previous studies using lethal irradiation and may help explain why patients exposed to radiation have increased risk for cardiovascular disease.

Could improving the design of preclinical murine atherosclerosis studies help increase the success rate of cardiovascular clinical trials? In this Viewpoint, the authors advocate for a change from prevention to intervention study designs and rigorous lesion analyses that they argue will enhance the translational potential of murine atherosclerosis studies. Clinical management of patients with coronary artery disease (CAD) has made great progress by reducing risk factors like low-density lipoprotein cholesterol level, hypertension, and diabetes mellitus. However, late-stage events associated with coronary atherosclerosis including myocardial infarction still account for ≈16% of worldwide mortality and are forecasted to increase in prevalence.1 These events arise from 3 main processes: plaque rupture, plaque erosion, and shedding of calcific nodules. Plaque rupture accounts for the majority of CAD and typically occurs in vulnerable atherosclerotic lesions termed thin-capped fibroatheromas,2 characterized by thin fibrous caps (

The presence of lymph node (LN)-like vasculature in tumours, characterized by expression of peripheral node addressin and chemokine CCL21, is correlated with T-cell infiltration and positive prognosis in breast cancer and melanoma patients. However, mechanisms controlling the development of LN-like vasculature and how it might contribute to a beneficial outcome for cancer patients are unknown. Here we demonstrate that LN-like vasculature is present in murine models of melanoma and lung carcinoma. It enables infiltration by naive T cells that significantly delay tumour outgrowth after intratumoral activation. Development of this vasculature is controlled by a mechanism involving effector CD8 T cells and NK cells that secrete LTα3 and IFNγ. LN-like vasculature is also associated with organized aggregates of B lymphocytes and gp38(+) fibroblasts, which resemble tertiary lymphoid organs that develop in models of chronic inflammation. These results establish LN-like vasculature as both a consequence of and key contributor to anti-tumour immunity.

Scanning near-field optical microscopy (SNOM) has been successfully employed to generate high resolution (