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Endothelial-mesenchymal transition (EndMT) is associated with various cardiovascular diseases and in particular with atherosclerosis and plaque instability. However, the molecular pathways that govern EndMT are poorly defined. Specifically, the role of epigenetic factors and histone deacetylases (HDACs) in controlling EndMT and the atherosclerotic plaque phenotype remains unclear. Here, we identified histone deacetylation, specifically that mediated by HDAC9 (a class IIa HDAC), as playing an important role in both EndMT and atherosclerosis. Using in vitro models, we found class IIa HDAC inhibition sustained the expression of endothelial proteins and mitigated the increase in mesenchymal proteins, effectively blocking EndMT. Similarly, ex vivo genetic knockout of Hdac9 in endothelial cells prevented EndMT and preserved a more endothelial-like phenotype. In vivo, atherosclerosis-prone mice with endothelial-specific Hdac9 knockout showed reduced EndMT and significantly reduced plaque area. Furthermore, these mice displayed a more favorable plaque phenotype, with reduced plaque lipid content and increased fibrous cap thickness. Together, these findings indicate that HDAC9 contributes to vascular pathology by promoting EndMT. Our study provides evidence for a pathological link among EndMT, HDAC9, and atherosclerosis and suggests that targeting of HDAC9 may be beneficial for plaque stabilization or slowing the progression of atherosclerotic disease.

BACKGROUND: Arterial 18fluorodeoxyglucose (FDG) positron emission tomography (PET) is considered a measure of atherosclerotic plaque macrophages and is used for quantification of disease activity in clinical trials, but the distribution profile of FDG across macrophages and other arterial cells has not been fully clarified. OBJECTIVES: The purpose of this study was to analyze FDG uptake in different arterial tissues and their contribution to PET signal in normal and atherosclerotic arteries. METHODS: Wild-type and D374Y-PCSK9 transgenic Yucatan minipigs were fed a high-fat, high-cholesterol diet to induce atherosclerosis and subjected to a clinical FDG-PET and computed tomography scan protocol. Volumes of arterial media, intima/lesion, macrophage-rich, and hypoxic tissues were measured in serial histological sections. Distributions of FDG in macrophages and other arterial tissues were quantified using modeling of the in vivo PET signal. In separate transgenic minipigs, the intra-arterial localization of FDG was determined directly by autoradiography. RESULTS: Arterial FDG-PET signal appearance and intensity were similar to human imaging. The modeling approach showed high accuracy in describing the FDG-PET signal and revealed comparable FDG accumulation in macrophages and other arterial tissues, including medial smooth muscle cells. These findings were verified directly by autoradiography of normal and atherosclerotic arteries. CONCLUSIONS: FDG is taken up comparably in macrophage-rich and -poor arterial tissues in minipigs. This offers a mechanistic explanation to a growing number of observations in clinical imaging studies that have been difficult to reconcile with macrophage-selective FDG uptake. This study was supported by the Danish Council for Independent Research/Medical Sciences, Lundbeck Foundation, Danish Heart Foundation, and Aarhus University Research Foundation (AU IDEAS). The CNIC is supported by the Ministerio de Ciencia, Innovación y Universidades, and the Pro CNIC Foundation; and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Dr. Bentzon has served as a consultant for Novo Nordisk A/S; and has within the last 5 years received an investigator-initiated preclinical research grant from Regeneron Pharmaceuticals Sí

AIMS/HYPOTHESIS: Diabetes is associated with an increased risk of atherosclerotic cardiovascular disease, but whether there is a direct and independent role for impaired glucose control in atherogenesis remains uncertain. We investigated whether diabetes with poor glycaemic control would accelerate atherogenesis in a novel pig model of atherosclerosis, the D374Y-PCSK9 (+) transgenic minipig.METHODS: Nineteen minipigs were fed a cholesterol-enriched, high-fat diet; ten of these pigs were injected with streptozotocin to generate a model of diabetes. Restricted feeding was implemented to control the pigs' weight gain and cholesterol intake. After 49 weeks of high-fat feeding, the major arteries were harvested for a detailed analysis of the plaque burden and histological plaque type.RESULTS: Stable hyperglycaemia was achieved in the diabetic minipigs, while the plasma total and LDL-cholesterol and creatinine levels were unaffected. Diabetes failed to increase atherosclerosis in any of the vessels examined. The plaque burden in the aorta and right coronary artery was comparable between the groups, and was even reduced in the left anterior descending (LAD) coronary and iliofemoral arteries in the diabetic pigs compared with the controls. The distribution of plaque types and the collagen and macrophage contents were similar between the groups, except for a reduced infiltration of macrophages in the LAD arteries of the diabetic pigs.CONCLUSIONS/INTERPRETATION: Poorly controlled diabetes with no alterations in plasma cholesterol or creatinine concentrations did not augment the plaque burden or promote the development of more advanced lesions in this large-animal model of human-like atherosclerosis. This is consistent with clinical studies in patients with type 1 diabetes, indicating that hyperglycaemia per se is not an independent promoter of atherosclerotic disease, but that other diabetes-associated risk factors are important.

Background— Although disturbed flow is thought to play a central role in the development of advanced coronary atherosclerotic plaques, no causal relationship has been established. We evaluated whether inducing disturbed flow would cause the development of advanced coronary plaques, including thin cap fibroatheroma. Methods and Results— D374Y -PCSK9 hypercholesterolemic minipigs (n=5) were instrumented with an intracoronary shear-modifying stent (SMS). Frequency-domain optical coherence tomography was obtained at baseline, immediately poststent, 19 weeks, and 34 weeks, and used to compute shear stress metrics of disturbed flow. At 34 weeks, plaque type was assessed within serially collected histological sections and coregistered to the distribution of each shear metric. The SMS caused a flow-limiting stenosis, and blood flow exiting the SMS caused regions of increased shear stress on the outer curvature and large regions of low and multidirectional shear stress on the inner curvature of the vessel. As a result, plaque burden was ≈3-fold higher downstream of the SMS than both upstream of the SMS and in the control artery ( P P P P Conclusions— These data support a causal role for lowered and multidirectional shear stress in the initiation of advanced coronary atherosclerotic plaques. Persistently lowered shear stress appears to be the principal flow disturbance needed for the formation of thin cap fibroatheroma.