Single-Cell RNA-Seq Reveals a Crosstalk between Hyaluronan Receptor LYVE-1-Expressing Macrophages and Vascular Smooth Muscle Cells.

Background: Atherosclerosis is a chronic inflammatory disease where macrophages participate in the progression of the disease. However, the role of resident-like macrophages (res-like) in the atherosclerotic aorta is not completely understood. Methods: A single-cell RNA sequencing analysis of CD45+ leukocytes in the atherosclerotic aorta of apolipoprotein E-deficient (Apoe-/-) mice on a normal cholesterol diet (NCD) or a high cholesterol diet (HCD), respecting the side-to-specific predisposition to atherosclerosis, was performed. A population of res-like macrophages expressing hyaluronan receptor LYVE-1 was investigated via flow cytometry, co-culture experiments, and immunofluorescence in human atherosclerotic plaques from carotid artery disease patients (CAD). Results: We identified 12 principal leukocyte clusters with distinct atherosclerosis disease-relevant gene expression signatures. LYVE-1+ res-like macrophages, expressing a high level of CC motif chemokine ligand 24 (CCL24, eotaxin-2), expanded under hypercholesteremia in Apoe-/- mice and promoted VSMC phenotypic modulation to osteoblast/chondrocyte-like cells, ex vivo, in a CCL24-dependent manner. Moreover, the abundance of LYVE-1+CCL24+ macrophages and elevated systemic levels of CCL24 were associated with vascular calcification and CAD events. Conclusions: LYVE-1 res-like macrophages, via the secretion of CCL24, promote the transdifferentiation of VSMC to osteogenic-like cells with a possible role in vascular calcification and likely a detrimental role in atherosclerotic plaque destabilization.

NLRP3 Inflammasome Activation Controls Vascular Smooth Muscle Cells Phenotypic Switch in Atherosclerosis.

(1) Background: Monocytes and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome orchestrate lipid-driven amplification of vascular inflammation promoting the disruption of the fibrous cap. The components of the NLRP3 inflammasome are expressed in macrophages and foam cells within human carotid atherosclerotic plaques and VSMCs in hypertension. Whether monocytes and NLRP3 inflammasome activation are direct triggers of VSMC phenotypic switch and plaque disruption need to be investigated. (2) Methods: The direct effect of oxLDL-activated monocytes in VSMCs co-cultured system was demonstrated via flow cytometry, qPCR, ELISA, caspase 1, and pyroptosis assay. Aortic roots of VSMCs lineage tracing mice fed normal or high cholesterol diet and human atherosclerotic plaques were used for immunofluorescence quantification of NLRP3 inflammasome activation/VSMCs phenotypic switch. (3) Results: OxLDL-activated monocytes reduced α-SMA, SM22α, Oct-4, and upregulation of KLF-4 and macrophage markers MAC2, F4/80 and CD68 expression as well as caspase 1 activation, IL-1ß secretion, and pyroptosis in VSMCs. Increased caspase 1 and IL-1ß in phenotypically modified VSMCs was detected in the aortic roots of VSMCs lineage tracing mice fed high cholesterol diet and in human atherosclerotic plaques from carotid artery disease patients who experienced a stroke. (4) Conclusions: Taken together, these results provide evidence that monocyte promote VSMC phenotypic switch through VSMC NLRP3 inflammasome activation with a likely detrimental role in atherosclerotic plaque stability in human atherosclerosis.

Cardiomyocyte-Specific JunD Overexpression Increases Infarct Size following Ischemia/Reperfusion Cardiac Injury by Downregulating Sirt3.

Ischemia/reperfusion (I/R) injury in acute myocardial infarction activates several deleterious molecular mechanisms. The transcription factor JunD regulates pathways involved in oxidative stress as well as in cellular proliferation, differentiation, and death. The present study investigated the potential role of JunD as a modulator of myocardial injury pathways in a mouse model of cardiac I/R injury. Infarct size, systemic and local inflammation, and production of reactive oxygen species, as well as cytosolic and mitochondrial apoptotic pathways were investigated in adult males after myocardial I/R. In wild-type (WT) mice, 30 minutes after ischemia and up to 24 hours following reperfusion, cardiac JunD messenger ribonucleic acid expression was reduced while JunB increased. Cardiac-specific JunD overexpressing mice (JunDTg/0 ) displayed larger infarcts compared with WT. However, postischemic inflammatory or oxidative responses did not differ. JunD overexpression reduced Sirt3 transcription by binding to its promoter, thus leading to mitochondrial dysfunction, myocardial cell death, and increased infarct size. On the other hand, JunD silencing reduced, while Sirt3 silencing increased infarct size. In human myocardial autopsy specimens, JunD-positive areas within the infarcted left ventricle staining corresponded to undetectable Sirt3 areas in consecutive sections of the same heart. Cardiac-specific JunD overexpression increases myocardial infarct size following I/R. These effects are mediated via Sirt3 transcriptional repression, mitochondrial swelling, and increased apoptosis, suggesting that JunD is a key regulator of myocardial I/R injury. The present data set the stage for further investigation of the potential role of Sirt3 activation as a novel target for the treatment of acute myocardial infarction.

The Reduction of Visceral Adipose Tissue after Roux-en-Y Gastric Bypass Is more Pronounced in Patients with Impaired Glucose Metabolism.

PURPOSE: Visceral adipose tissue (VAT) is associated with cardiometabolic risk factors and insulin resistance. The physiological mechanisms underlying the benefits of Roux-en-Y gastric bypass surgery (RYGB) on glucose metabolism remain incompletely understood. The impact of RYGB on VAT was assessed among three groups of patients stratified by their glucose tolerance before surgery. METHODS: Forty-four obese women were categorized into normoglycemia (n = 21), impaired glucose tolerance (IGT, n = 18) and diabetes (n = 5) before surgery. Body composition measured by dual-energy X-ray absorptiometry (DXA) was performed before surgery, 6 months and 12 months after. RESULTS: The three groups had comparable mean age (mean 38.6 ± SD 9.9) and BMI at baseline (41.9 ± 4.3 kg/m2). After 12 months, total weight loss (mean 35.1% ± 7.5) and excess weight loss (91.1% ± 25.1) were similar between groups. Pre-surgery mean VAT was significantly higher in diabetes (mean 2495 ± 616 g) than in normoglycemia (1750 ± 617 g, p = 0.02). The percentage of VAT to total body fat was significantly higher in diabetes (mean 4.4% ± 0.9) compared to normoglycemia (2.9% ± 0.8, p = 0.003). Twelve months after surgery, VAT loss was significantly greater among patients with diabetes (mean 1927 ± 413 g) compared to normoglycemia (1202 ± 450, p = 0.009). CONCLUSIONS: RYGB leads to important VAT loss, and this loss is greater in patients with diabetes prior to surgery. As VAT is associated with insulin resistance, this reduction may account for the profound impact of this surgery on glucose metabolism.

Intraplaque Expression of C-Reactive Protein Predicts Cardiovascular Events in Patients with Severe Atherosclerotic Carotid Artery Stenosis.

Serum c-reactive protein (CRP) was suggested for the assessment of intermediate cardiovascular (CV) risk. Here, systemic or intraplaque CRP levels were investigated as predictors of major adverse cardiovascular events (MACEs) in patients with severe carotid stenosis. CRP levels were assessed in the serum and within different portions (upstream and downstream) of carotid plaques of 217 patients undergoing endarterectomy. The association between CRP and intraplaque lipids, collagen, neutrophils, smooth muscle cells (SMC), and macrophage subsets was determined. No correlation between serum CRP and intraplaque biomarkers was observed. In upstream portions, CRP content was directly correlated with intraplaque neutrophils, total macrophages, and M1 macrophages and inversely correlated with SMC content. In downstream portions, intraplaque CRP correlated with M1 and M2 macrophages. According to the cut-off point (CRP > 2.9%) identified by ROC analysis in upstream portions, Kaplan-Meier analysis showed that patients with high CRP levels had a greater rate of MACEs. This risk of MACEs increased independently of age, male gender, serum CRP, and statin use. In conclusion, in patients with severe carotid artery stenosis, high CRP levels within upstream portions of carotid plaques directly and positively correlate with intraplaque inflammatory cells and predict MACEs at an 18-month follow-up period.

Vitamin D receptor is expressed within human carotid plaques and correlates with pro-inflammatory M1 macrophages.

The role of Vitamin D system in cardiovascular diseases remains controversial. Here, we investigated whether intraplaque levels of vitamin D receptor (VDR) predicted major adverse cardiovascular events (MACEs) at 18month-follow-up and correlated with macrophage subsets in 164 patients undergoing endarterectomy for carotid stenosis. In human carotid plaque portions upstream and downstream the blood flow, VDR, lipid, collagen, as well as macrophage subsets were determined. Human primary monocytes were then differentiated in vitro to M1 and M2 macrophages and treated with 1,25(OH)2D3. Intraplaque VDR positively correlated with total and M1 macrophages. According to the result of ROC curve analysis, downstream portions of plaques having high VDR expression were characterized by increased M1 macrophages. Kaplan-Meier analysis showed that the risk of MACEs was greater in patients having low downstream VDR levels (8.2% vs. 1.3%; p=0.005). Cox proportional hazard regression analyses confirmed that MACE risk decreased with increasing downstream VDR (adjusted HR 0.78 [95% CI 0.62-0.98]; p=0.032). In vitro, VDR expression was prevalent in M1, but not M2. Incubation of M1 macrophages with 1,25(OH)2D3, increased VDR expression and suppressed toll-like receptor 4 expression. These results suggest that low intraplaque VDR expression predict MACEs in patients with carotid stenosis potentially involving M1 macrophages.

Anti-ApoA-1 IgG serum levels predict worse poststroke outcomes.

BACKGROUND: Autoantibodies to apolipoprotein A-1 (anti-ApoA-1 IgG) were shown to predict major adverse cardiovascular events and promote atherogenesis. However, their potential relationship with clinical disability and ischaemic lesion volume after acute ischaemic stroke (AIS) remains unexplored. MATERIALS AND METHODS: We included n = 76 patients admitted for AIS and we investigated whether baseline serum anti-ApoA-1 IgG levels could predict (i) AIS-induced clinical disability [assessed by the modified Rankin Scale (mRS)], and (ii) AIS-related ischaemic lesion volume [assessed by Computed Tomography (CT)]. We also evaluated the possible pro-apoptotic and pro-necrotic effects of anti-ApoA-1 IgG on human astrocytoma cell line (U251) using flow cytometry. RESULTS: High levels of anti-ApoA-1 IgG were retrieved in 15·8% (12/76) of patients. Increased baseline levels of anti-ApoA-1 IgG were independently correlated with worse mRS [ß = 0·364; P = 0·002; adjusted odds ratio (OR): 1·05 (95% CI 1·01-1·09); P = 0·017] and CT-assessed ischaemic lesion volume [ß = 0·333; P < 0·001; adjusted OR: 1·06 (95% CI 1·01-1·12); P = 0·048] at 3 months. No difference in baseline clinical, biochemical and radiological characteristics was observed between patients with high vs. low levels of anti-ApoA-1 IgG. Incubating human astrocytoma cells with anti-ApoA-1 IgG dose dependently induced necrosis and apoptosis of U251 cells in vitro. CONCLUSION: Anti-ApoA-1 IgG serum levels at AIS onset are associated with poorer clinical recovery and worse brain lesion volume 3 months after AIS. These observations could be partly explained by the deleterious effect of anti-ApoA-1 IgG on human brain cell survival in vitro and may have clinical implication in the prediction of poor outcome in AIS.

Anti-apolipoprotein A-1 auto-antibodies as active modulators of atherothrombosis.

Humoral autoimmune-mediated inflammation plays a role in atherogenesis, and potentially in arterial thrombosis. Anti-apolipoprotein A-1 (apoA-1) IgG have been reported to represent emergent mediators of atherogenesis through Toll-like receptors (TLR) 2, 4 and CD14 signalling. We investigated the role of anti-apoA-1 IgG on tissue factor (TF) expression and activation, a key coagulation regulator underlying atherothrombosis. Atherothrombosis features were determined by immunohistochemical TF staining of human carotid biopsies derived from patients with severe carotid stenosis undergoing elective surgery (n=176), and on aortic roots of different genetic backgrounds mice (ApoE-/-; TLR2-/-ApoE-/- and TLR4-/-ApoE-/-) exposed to passive immunisation with anti-apoA-1 IgG. Human serum levels of anti-apoA-1 IgG were measured by ELISA. In vitro, on human-monocyte-derived-macrophages (HMDM) the anti-apoA-1 IgG increased TF expression and activity were analysed by FACS and chromogenic assays in presence of different pharmacological inhibitors. Human serum anti-apoA-1 IgG levels significantly correlated to intraplaque TF expression in carotid biopsies (r=0.31, p<0.001), which was predictive of clinically symptomatic lesions. On HMDM, anti-apoA-1 IgG induced a TLR2, 4 and CD14-dependent increase in TF expression and activity, involving NF-kappaB and a c-Jun N-terminal kinase-dependent AP-1 transcription factors. In ApoE-/- mice, anti-apoA-1 IgG passive immunisation significantly enhanced intraplaque TF expression when compared to control IgG. This effect was lost in both TLR2-/-ApoE-/- and TLR4-/-ApoE-/- mice. These results demonstrate that anti-apoA-1 IgG are associated with TF expression in human atherosclerotic plaques, induce TF expression in vitro and in vivo through TLR2 and 4 signalling, supporting a possible causal relationship between anti-apoA-1 IgG and atherothrombosis.

Treatment with anti-RANKL antibody reduces infarct size and attenuates dysfunction impacting on neutrophil-mediated injury.

Selective pharmacological treatments targeting reperfusion injury produced modest protective effects and might be associated with immunosuppression. In order to identify novel and better-tolerated approaches, we focused on the neutralization of receptor activator of nuclear factor kappa-B ligand [RANKL], a cytokine recently shown to activate inflammatory cells (i.e. neutrophils) orchestrating post-infarction injury and repair. Myocardial ischemia (60min) and reperfusion injury was surgically induced in C57Bl/6 mice. In hearts and serum, RANKL was early upregulated during reperfusion. A "one-shot" injection with neutralizing anti-RANKL IgG during ischemia ameliorated myocardial infarct size and function, but not adverse remodeling (determined by Magnetic Resonance Imaging [MRI]) as compared to Vehicle or control IgG. These beneficial effects were accompanied in vivo by reduction in cardiac neutrophil infiltration, reactive oxygen species (ROS) and MMP-9 release. Anti-RANKL IgG treatment suppressed sudden peak of neutrophil granule products in mouse serum early after reperfusion onset. In vitro, RANK mRNA expression was detected in isolated mouse neutrophils. Co-incubation with neutralizing anti-RANKL IgG abrogated RANKL-induced mouse neutrophil degranulation and migration, suggesting a critical role of RANKL in neutrophil-mediated injury. Conversely, anti-RANKL IgG did not affect salvage pathways in cardiac cells (i.e. ERK p42/p44, Akt and STAT-3) or macrophage cardiac infiltration. Finally, treatment with anti-RANKL IgG showed no effect on B and T lymphocyte polarization (in serum, spleen and infarcted myocardium) and circulating chemokines as compared with Vehicle or control IgG. In conclusion, acute treatment with anti-RANKL IgG improved cardiac infarct size and function by potentially impacting on neutrophil-mediated injury and repair.

Treatment with KLEPTOSE® CRYSMEB reduces mouse atherogenesis by impacting on lipid profile and Th1 lymphocyte response.

The ability of pharmacological agents to target both "classical" risk factors and inflammation may be key for successful outcomes in the prevention and treatment of atherogenesis. Among the promising drugs interfering with cholesterol metabolism, we investigated whether methyl beta-cyclodextrin (KLEPTOSE® CRYSMEB) could positively impact on atherogenesis, lipid profile and atherosclerotic plaque inflammation in ApoE-/- mice. Eleven-week old ApoE-/- mice were fed either a normal diet (N.D.) or a high-cholesterol diet (H.D.), resulting in different levels of hypercholesterolemia. KLEPTOSE® CRYSMEB (40mg/kg) or vehicle was intraperitoneally administrated 3 times per week in the last 16weeks before euthanasia in mice under N.D. and in the last 11weeks under H.D. Treatment with KLEPTOSE® CRYSMEB reduced triglyceride serum levels in both atherogenesis mouse models. In H.D. mice, treatment with KLEPTOSE® CRYSMEB increased HDL-cholesterol levels and reduced free fatty acids and spleen weight. In both mouse models, treatment with KLEPTOSE® CRYSMEB reduced atherosclerotic plaque size in thoraco-abdominal aortas and intraplaque T lymphocyte content, but did not induce relevant improvements in other histological parameters of vulnerability (macrophage, neutrophil, MMP-9 and collagen content). Conversely and more markedly in H.D. mice, treatment with KLEPTOSE® CRYSMEB was associated with a reduction in genetic markers of Th1-mediated immune response. In vitro, KLEPTOSE® CRYSMEB dose-dependently abrogated Th1 proliferation and IFNγ release. In conclusion, treatment with KLEPTOSE® CRYSMEB reduced atherosclerotic plaque size by improving triglyceride serum levels and Th1-mediated response. These results indicate this drug as a potential tool for blocking atheroprogression associated with different severity degrees of hypercholesterolemia.