Myeloid PHD2 Conditional Knockout Improves Intraplaque Angiogenesis and Vascular Remodeling in a Murine Model of Venous Bypass Grafting.

BACKGROUND: Intraplaque angiogenesis occurs in response to atherosclerotic plaque hypoxia, which is driven mainly by highly metabolically active macrophages. Improving plaque oxygenation by increasing macrophage hypoxic signaling, thus stimulating intraplaque angiogenesis, could restore cellular function and neovessel maturation, and decrease plaque formation. Prolyl hydroxylases (PHDs) regulate cellular responses to hypoxia. We therefore aimed to elucidate the role of myeloid PHD2, the dominant PHD isoform, on intraplaque angiogenesis in a murine model for venous bypass grafting. METHODS AND RESULTS: Myeloid PHD2 conditional knockout (PHD2cko) and PHD2 wild type mice on an Ldlr-/- background underwent vein graft surgery (n=11-15/group) by interpositioning donor caval veins into the carotid artery of genotype-matched mice. At postoperative day 28, vein grafts were harvested for morphometric and compositional analysis, and blood was collected for flow cytometry. Myeloid PHD2cko induced and improved intraplaque angiogenesis by improving neovessel maturation, which reduced intraplaque hemorrhage. Intima/media ratio was decreased in myeloid PHD2cko vein grafts. In addition, PHD2 deficiency prevented dissection of vein grafts and resulted in an increase in vessel wall collagen content. Moreover, the macrophage proinflammatory phenotype in the vein graft wall was attenuated in myeloid PHD2cko mice. In vitro cultured PHD2cko bone marrow-derived macrophages exhibited an increased proangiogenic phenotype compared with control. CONCLUSIONS: Myeloid PHD2cko reduces vein graft disease and ameliorates vein graft lesion stability by improving intraplaque angiogenesis.

Unlocking Deprescribing Potential in Nursing Homes: Insights from a Focus Group Study with Healthcare Professionals.

BACKGROUND: The nursing home population is characterized by multimorbidity and disabilities, which often result in extensive prescription of medication and subsequent polypharmacy. Deprescribing, a planned and supervised process of dose reduction or total cessation of medication, is a solution to combat this. OBJECTIVE: This study aimed to identify barriers and enablers of deprescribing as experienced by nursing home physicians (NHPs) and collaborating pharmacists in the specific nursing home setting. METHODS: This qualitative study utilized a semi-structured interview format with two focus groups consisting of a mix of NHPs and pharmacists. Directed content analysis was performed based on the Theoretical Domains Framework, a validated framework for understanding determinants of behavior change among health care professionals. RESULTS: Sixteen health care professionals participated in two focus groups, including 13 NHPs and three pharmacists. The participating NHPs and pharmacists believed that deprescribing is a valuable process with enablers, such as multidisciplinary collaboration, good communication with patients and family, and involvement of the nursing staff. NHPs and pharmacists view deprescribing as a core task and feel assured in their ability to carry it out successfully. However, they also noted barriers: deprescribing is time-consuming; communication with residents, their relatives or medical specialists is difficult; and electronic patient systems often do not adequately support it. CONCLUSIONS: This study provides insight into the various barriers and enablers faced by NHPs and pharmacists when deprescribing in nursing homes. Specific for this population, deprescribing barriers focus on communication (with residents and their relatives, and also with medical specialists) and resources, while knowledge and expertise are mentioned as enablers.

Protective role of chaperone-mediated autophagy against atherosclerosis.

Chaperone-mediated autophagy (CMA) contributes to regulation of energy homeostasis by timely degradation of enzymes involved in glucose and lipid metabolism. Here, we report reduced CMA activity in vascular smooth muscle cells and macrophages in murine and human arteries in response to atherosclerotic challenges. We show that in vivo genetic blockage of CMA worsens atherosclerotic pathology through both systemic and cell-autonomous changes in vascular smooth muscle cells and macrophages, the two main cell types involved in atherogenesis. CMA deficiency promotes dedifferentiation of vascular smooth muscle cells and a proinflammatory state in macrophages. Conversely, a genetic mouse model with up-regulated CMA shows lower vulnerability to proatherosclerotic challenges. We propose that CMA could be an attractive therapeutic target against cardiovascular diseases.

Deficiency of myeloid PHD proteins aggravates atherogenesis via macrophage apoptosis and paracrine fibrotic signalling.

AIMS: Atherosclerotic plaque hypoxia is detrimental for macrophage function. Prolyl hydroxylases (PHDs) initiate cellular hypoxic responses, possibly influencing macrophage function in plaque hypoxia. Thus, we aimed to elucidate the role of myeloid PHDs in atherosclerosis. METHODS AND RESULTS: Myeloid-specific PHD knockout (PHDko) mice were obtained via bone marrow transplantation (PHD1ko, PHD3ko) or conditional knockdown through lysozyme M-driven Cre recombinase (PHD2cko). Mice were fed high cholesterol diet for 6-12 weeks to induce atherosclerosis. Aortic root plaque size was significantly augmented 2.6-fold in PHD2cko, and 1.4-fold in PHD3ko compared to controls but was unchanged in PHD1ko mice. Macrophage apoptosis was promoted in PHD2cko and PHD3ko mice in vitro and in vivo, via the hypoxia-inducible factor (HIF) 1α/BNIP3 axis. Bulk and single-cell RNA data of PHD2cko bone marrow-derived macrophages (BMDMs) and plaque macrophages, respectively, showed enhanced HIF1α/BNIP3 signalling, which was validated in vitro by siRNA silencing. Human plaque BNIP3 mRNA was positively associated with plaque necrotic core size, suggesting similar pro-apoptotic effects in human. Furthermore, PHD2cko plaques displayed enhanced fibrosis, while macrophage collagen breakdown by matrix metalloproteinases, collagen production, and proliferation were unaltered. Instead, PHD2cko BMDMs enhanced fibroblast collagen secretion in a paracrine manner. In silico analysis of macrophage-fibroblast communication predicted SPP1 (osteopontin) signalling as regulator, which was corroborated by enhanced plaque SPP1 protein in vivo. Increased SPP1 mRNA expression upon PHD2cko was preferentially observed in foamy plaque macrophages expressing 'triggering receptor expressed on myeloid cells-2' (TREM2hi) evidenced by single-cell RNA, but not in neutrophils. This confirmed enhanced fibrotic signalling by PHD2cko macrophages to fibroblasts, in vitro as well as in vivo. CONCLUSION: Myeloid PHD2cko and PHD3ko enhanced atherosclerotic plaque growth and macrophage apoptosis, while PHD2cko macrophages further activated collagen secretion by fibroblasts in vitro, likely via paracrine SPP1 signalling through TREM2hi macrophages.

Partial Inhibition of the 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase-3 (PFKFB3) Enzyme in Myeloid Cells Does Not Affect Atherosclerosis.

BACKGROUND: The protein 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) is a key stimulator of glycolytic flux. Systemic, partial PFKFB3 inhibition previously decreased total plaque burden and increased plaque stability. However, it is unclear which cell type conferred these positive effects. Myeloid cells play an important role in atherogenesis, and mainly rely on glycolysis for energy supply. Thus, we studied whether myeloid inhibition of PFKFB3-mediated glycolysis in Ldlr-/-LysMCre+/-Pfkfb3 fl/fl (Pfkfb3 fl/fl ) mice confers beneficial effects on plaque stability and alleviates cardiovascular disease burden compared to Ldlr-/-LysMCre+/-Pfkfb3 wt/wt control mice (Pfkfb3 wt/wt ). METHODS AND RESULTS: Analysis of atherosclerotic human and murine single-cell populations confirmed PFKFB3/Pfkfb3 expression in myeloid cells, but also in lymphocytes, endothelial cells, fibroblasts and smooth muscle cells. Pfkfb3 wt/wt and Pfkfb3 fl/fl mice were fed a 0.25% cholesterol diet for 12 weeks. Pfkfb3 fl/fl bone marrow-derived macrophages (BMDMs) showed 50% knockdown of Pfkfb3 mRNA. As expected based on partial glycolysis inhibition, extracellular acidification rate as a measure of glycolysis was partially reduced in Pfkfb3 fl/fl compared to Pfkfb3 wt/wt BMDMs. Unexpectedly, plaque and necrotic core size, as well as macrophage (MAC3), neutrophil (Ly6G) and collagen (Sirius Red) content were unchanged in advanced Pfkfb3 fl/fl lesions. Similarly, early lesion plaque and necrotic core size and total plaque burden were unaffected. CONCLUSION: Partial myeloid knockdown of PFKFB3 did not affect atherosclerosis development in advanced or early lesions. Previously reported positive effects of systemic, partial PFKFB3 inhibition on lesion stabilization, do not seem conferred by monocytes, macrophages or neutrophils. Instead, other Pfkfb3-expressing cells in atherosclerosis might be responsible, such as DCs, smooth muscle cells or fibroblasts.

A Switch from Cell-Associated to Soluble PDGF-B Protects against Atherosclerosis, despite Driving Extramedullary Hematopoiesis.

Platelet-derived growth factor B (PDGF-B) is a mitogenic, migratory and survival factor. Cell-associated PDGF-B recruits stabilizing pericytes towards blood vessels through retention in extracellular matrix. We hypothesized that the genetic ablation of cell-associated PDGF-B by retention motif deletion would reduce the local availability of PDGF-B, resulting in microvascular pericyte loss, microvascular permeability and exacerbated atherosclerosis. Therefore, Ldlr-/-Pdgfbret/ret mice were fed a high cholesterol diet. Although plaque size was increased in the aortic root of Pdgfbret/ret mice, microvessel density and intraplaque hemorrhage were unexpectedly unaffected. Plaque macrophage content was reduced, which is likely attributable to increased apoptosis, as judged by increased TUNEL+ cells in Pdgfbret/ret plaques (2.1-fold) and increased Pdgfbret/ret macrophage apoptosis upon 7-ketocholesterol or oxidized LDL incubation in vitro. Moreover, Pdgfbret/ret plaque collagen content increased independent of mesenchymal cell density. The decreased macrophage matrix metalloproteinase activity could partly explain Pdgfbret/ret collagen content. In addition to the beneficial vascular effects, we observed reduced body weight gain related to smaller fat deposition in Pdgfbret/ret liver and adipose tissue. While dampening plaque inflammation, Pdgfbret/ret paradoxically induced systemic leukocytosis. The increased incorporation of 5-ethynyl-2'-deoxyuridine indicated increased extramedullary hematopoiesis and the increased proliferation of circulating leukocytes. We concluded that Pdgfbret/ret confers vascular and metabolic effects, which appeared to be protective against diet-induced cardiovascular burden. These effects were unrelated to arterial mesenchymal cell content or adventitial microvessel density and leakage. In contrast, the deletion drives splenic hematopoiesis and subsequent leukocytosis in hypercholesterolemia.

CARMN Loss Regulates Smooth Muscle Cells and Accelerates Atherosclerosis in Mice.

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Comprehensive autophagy evaluation in cardiac disease models.

Autophagy is a highly conserved recycling mechanism essential for maintaining cellular homeostasis. The pathophysiological role of autophagy has been explored since its discovery 50 years ago, but interest in autophagy has grown exponentially over the last years. Many researchers around the globe have found that autophagy is a critical pathway involved in the pathogenesis of cardiac diseases. Several groups have created novel and powerful tools for gaining deeper insights into the role of autophagy in the aetiology and development of pathologies affecting the heart. Here, we discuss how established and emerging methods to study autophagy can be used to unravel the precise function of this central recycling mechanism in the cardiac system.