A Standardized Lindera obtusiloba Extract Improves Endothelial Dysfunction and Attenuates Plaque Development in Hyperlipidemic ApoE-Knockout Mice

Lindera obtusiloba extract (LOE), a traditional herbal medicine used to enhance blood circulation and to reduce inflammation, induced NO-mediated endothelium-dependent relaxation, and reduced the formation of reactive oxygen species (ROS). The study investigated whether LOE improves endothelial dysfunction and reduces plaque inflammation and progression by inhibiting ROS generation in a mouse model of atherosclerosis. Eight-week-old apolipoprotein E-deficient (apoE−/−) mice fed with a western diet (WD) were randomized into different groups by administering vehicle (0.5% carboxymethylcellulose (CMC)), LOE (100 mg/kg/day), or losartan (30 mg/kg/day) by gavage until the age of 28 weeks. Fourteen male C57BL/6 mice that were fed normal chow and treated with CMC were used as negative controls. Similar to losartan treatment, LOE treatment induced the concentration-dependent relaxation of aorta rings in WD-fed apoE−/− mice. LOE treatment significantly reduced the vascular ROS formation and expression of NADPH oxidase subunits, including p22phox and p47phox. Compared with WD-fed apoE−/− mice, mice exposed to chronic LOE treatment exhibited reductions in plaque inflammation-related fluorescence signals and atherosclerotic lesions. These effects were greater than those of losartan treatment. In conclusion, LOE treatment improves endothelial dysfunction and reduces plaque inflammation as well as lesion areas by reducing vascular NADPH oxidase-induced ROS generation in a mouse model of atherosclerosis.

Myeloid Mineralocorticoid Receptor Transcriptionally Regulates P-Selectin Glycoprotein Ligand-1 and Promotes Monocyte Trafficking and Atherosclerosis

Objective: MR (mineralocorticoid receptor) activation associates with increased risk of cardiovascular ischemia while MR inhibition reduces cardiovascular-related mortality and plaque inflammation in mouse atherosclerosis. MR in myeloid cells (My-MR) promotes inflammatory cell infiltration into injured tissues and atherosclerotic plaque inflammation by unclear mechanisms. Here, we examined the role of My-MR in leukocyte trafficking and the impact of sex. Approach and Results: We confirm in vivo that My-MR deletion (My-MR-KO) in ApoE-KO mice decreased plaque size. Flow cytometry revealed fewer plaque macrophages with My-MR-KO. By intravital microscopy, My-MR-KO significantly attenuated monocyte slow-rolling and adhesion to mesenteric vessels and decreased peritoneal infiltration of myeloid cells in response to inflammatory stimuli in male but not female mice. My-MR-KO mice had significantly less PSGL1 (P-selectin glycoprotein ligand 1) mRNA in peritoneal macrophages and surface PSGL1 protein on circulating monocytes in males. In vitro, MR activation with aldosterone significantly increased PSGL1 mRNA only in monocytes from MR-intact males. Similarly, aldosterone induced, and MR antagonist spironolactone inhibited, PSGL1 expression in human U937 monocytes. Mechanistically, aldosterone stimulated MR binding to a predicted MR response element in intron-1 of the PSGL1 gene by ChIP-qPCR. Reporter assays demonstrated that this PSGL1 MR response element is necessary and sufficient for aldosterone-activated, MR-dependent transcriptional activity. Conclusions: These data identify PSGL1 as a My-MR target gene that drives leukocyte trafficking to enhance atherosclerotic plaque inflammation. These novel and sexually dimorphic findings provide insight into increased ischemia risk with MR activation, cardiovascular protection in women, and the role of MR in atherosclerosis and tissue inflammation.

Carotid Plaque Inflammation Imaged by PET and Prediction of Recurrent Stroke at 5 Years

Objective:To determine whether carotid plaque inflammation identified by 18F-fluorodeoxyglucose (18FDG)-PET is associated with late (5-year) recurrent stroke.Methods:We did an individual-participant data pooled analysis of three prospective studies with near-identical study methods. Eligible patients had recent non-severe (modified Rankin Score ≤3) ischaemic stroke/TIA and ipsilateral carotid stenosis (50-99%). Participants underwent carotid 18FDG-PET/CT angiography ≤14 days after recruitment. 18FDG uptake was expressed as maximum standardized uptake value (SUVmax) in the axial single hottest slice of symptomatic plaque. We calculated the previously-validated Symptomatic Carotid Atheroma Inflammation Lumen-stenosis (SCAIL) score, which incorporates a measure of stenosis severity and 18FDG uptake. The primary outcome was 5-year recurrent ipsilateral ischaemic stroke after PET imaging.Results:Of 183 eligible patients, 181 patients completed follow-up (98.9%). The median duration of follow-up was 4.9 years (interquartile range 3.3-6.4, cumulative follow-up period 901.8 patient-years). After PET imaging, 17 patients had a recurrent ipsilateral ischemic strokes at 5 years (recurrence rate 9.4%, 95% CI 5.6-14.6%). Baseline plaque SUVmax independently predicted 5-year ipsilateral recurrent stroke after adjustment for age, gender, carotid revascularization, stenosis severity, NIH Stroke Scale, and diabetes mellitus (adjusted HR 1.98; 95 % CI, 1.10-3.56, p=0.02, per 1g/mL increase SUVmax). On multivariable Cox regression, SCAIL score predicted 5-year ipsilateral stroke (adjusted HR 2.73 per 1-point increase; 95% CI 1.52-4.90, p=0.001).Conclusion:Plaque inflammation-related 18FDG uptake improved identification of 5-year recurrent ipsilateral ischaemic stroke. Addition of plaque inflammation to current selection strategies may target patients most likely to have late as well as early benefit from carotid revascularization.Classification of Evidence:This study provides Class I evidence that in individuals with recent ischemic stroke/TIA and ipsilateral carotid stenosis, carotid plaque inflammation-related 18FDG uptake on PET/CT angiography was associated with 5-year recurrent ipsilateral stroke.

Dose-Dependent Association of Inflammatory Cytokines with Carotid Atherosclerosis in Transient Ischaemic Attack: Implications for Clinical Trials

<b><i>Introduction:</i></b> The 5-year recurrence risk after ischaemic stroke and transient ischaemic attack (TIA) is 25–30%. Although inflammation may be a target for prevention trials, the contribution of plaque inflammation to acute cerebrovascular events remains unclear. We investigated the association of acute inflammatory cytokines and high-sensitivity C-reactive protein (CRP) with recently symptomatic carotid atherosclerosis in a prospective cohort study. <b><i>Methods:</i></b> Blood and Imaging markers of TIA BIO-TIA) is a multicentre prospective study of imaging and inflammatory markers in patients with TIA. Exclusion criteria were infection and other co-morbid illnesses associated with inflammation. CRP and serum cytokines (interleukin [IL]-6, IL-1β, IL-8, IL-10, IL-12, interferon-γ [IFN-γ] and tumour necrosis factor-α [TNF-α]) were measured. All patients had carotid imaging. <b><i>Results:</i></b> Two hundred and thirty-eight TIA cases and 64 controls (TIA mimics) were included. Forty-nine (20.6%) cases had symptomatic internal carotid artery stenosis. Pro-inflammatory cytokine levels increased in a dose-dependent manner across controls, TIA without carotid stenosis (CS), and TIA with CS (IL-1β, <i>p</i>_trend = 0.03; IL-6, <i>p</i>_trend &#x3c; 0.0001; IL-8, <i>p</i>_trend = 0.01; interferon (IFN)-γ, <i>p</i>_trend = 0.005; TNF-α, <i>p</i>_trend = 0.003). Results were unchanged when DWI-positive cases were excluded. On multivariable linear regression, only age (<i>p</i> = 0.01) and CS (<i>p</i> = 0.04) independently predicted log-IL-6. On multivariable Cox regression, CRP was the only independent predictor of 90-day stroke recurrence (adjusted hazard ratio per 1-unit increase 1.03 [95% CI: 1.01–1.05], <i>p</i> = 0.003). <b><i>Conclusion:</i></b> Symptomatic carotid atherosclerosis was associated with elevated cytokines in TIA patients after controlling for other sources of inflammation. High-sensitivity CRP was associated with recurrent ischaemic stroke at 90 days. These findings implicate acute plaque inflammation in the pathogenesis of cerebral thromboembolism and support a rationale for randomized trials of anti-inflammatory therapy for stroke patients, who were excluded from coronary trials.

Systemic delivery of rAAV9‐IκBα stabilized atherosclerotic plaques and attenuated intra-plaque inflammation by inhibiting nuclear factor-κB activation in apolipoprotein E–deficient mice

Background: The activated nuclear factor (NF)-κB pathway in atherosclerotic plaques promotes the progression of atherosclerosis. Targeting of plaque NF-κB may provide a novel strategy for limiting chronic inflammation. This study was conducted to examine the effect of NF-κB inhibition by using recombinant adeno-associated virus 9 (AAV9) to deliver IκBα (AAV9‐IκBα) which can be overexpressed to influence atherosclerosis.Methods and Results: Systemic delivery of AAV9‐IκBα resulted in ~2-fold overexpression of IκBα protein in the atherosclerotic plaques of apolipoprotein E-/- mice fed a high-fat diet. AAV9‐IκBα delivery did not affect the body weight or lipid deposition and plaque size in the mice. During plaque formation, overexpression of IκBα suppressed intra-plaque macrophage infiltration, inhibited the expression of pro-inflammatory genes including interleukin-6, monocyte chemoattractant protein-1, tumor necrosis factor-α and matrix metalloproteinase, enhanced the contents of collagen and vascular smooth muscle cells, and decreased the plaque vulnerability index by inhibiting the phosphorylation of NF-κB subunit p65 and its nuclear translocation. Conclusions: Systemic delivery of AAV9-IκBα promotes plaque stability by inhibiting NF-κB activation and suppressing intra-plaque inflammation, suggesting that inhibition of the NF-κB pathway in atherosclerotic plaques is a promising approach for treating atherosclerosis.

Reshaping of the gastrointestinal microbiome alters atherosclerotic plaque inflammation resolution in mice

Since alterations in the intestinal microbiota may induce systemic inflammation and polarization of macrophages to the M1 state, the microbiome role in atherosclerosis, an M1-driven disease, requires evaluation. We aimed to determine if antibiotic (Abx) induced alterations to the intestinal microbiota interferes with atherosclerotic plaque inflammation resolution after lipid-lowering in mice. Hyperlipidemic Apoe−/− mice were fed a western diet to develop aortic atherosclerosis with aortas then transplanted into normolipidemic wild-type (WT) mice to model clinically aggressive lipid management and promote atherosclerosis inflammation resolution. Gut microbial composition pre and post-transplant was altered via an enteral antibiotic or not. Post aortic transplant, after Abx treatment, while plaque size did not differ, compared to Apoe−/− mice, Abx– WT recipient mice had a 32% reduction in CD68-expressing cells (p = 0.02) vs. a non-significant 12% reduction in Abx+ WT mice. A trend toward an M1 plaque CD68-expresing cell phenotype was noted in Abx+ mice. By 16S rRNA sequence analysis, the Abx+ mice had reduced alpha diversity and increased Firmicutes/Bacteroidetes relative abundance ratio with a correlation between gut Firmicutes abundance and plaque CD68-expressing cell content (p < 0.05). These results indicate that in a murine atherosclerotic plaque inflammation resolution model, antibiotic-induced microbiome perturbation may blunt the effectiveness of lipid-lowering to reduce the content of plaque inflammatory CD68-expressing cells.

miR-33 Silencing Reprograms the Immune Cell Landscape in Atherosclerotic Plaques

Rationale: MicroRNA-33 post-transcriptionally represses genes involved in lipid metabolism and energy homeostasis. Targeted inhibition of miR-33 increases plasma HDL cholesterol and promotes atherosclerosis regression, in part, by enhancing reverse cholesterol transport and dampening plaque inflammation. However, how miR-33 reshapes the immune microenvironment of plaques remains poorly understood. Objective: To define how miR-33 inhibition alters the dynamic balance and transcriptional landscape of immune cells in atherosclerotic plaques. Methods and Results: We used single cell RNA-sequencing of aortic CD45 + cells, combined with immunohistologic, morphometric and flow cytometric analyses to define the changes in plaque immune cell composition, gene expression and function following miR-33 inhibition. We report that anti-miR-33 treatment of Ldlr -/- mice with advanced atherosclerosis reduced plaque burden and altered the plaque immune cell landscape by shifting the balance of pro- and anti-atherosclerotic macrophage and T cell subsets. By quantifying the kinetic processes that determine plaque macrophage burden, we found that anti-miR-33 reduced levels of circulating monocytes and splenic myeloid progenitors, decreased macrophage proliferation and retention, and promoted macrophage attrition by apoptosis and efferocytotic clearance. scRNA-sequencing of aortic arch plaques showed that anti-miR-33 reduced the frequency of MHCIIhi "inflammatory" and Trem2hi "metabolic" macrophages, but not tissue resident macrophages. Furthermore, anti-miR-33 led to derepression of distinct miR-33 target genes in the different macrophage subsets: in resident and Trem2hi macrophages, anti-miR-33 relieved repression of miR-33 target genes involved in lipid metabolism (e.g., Abca1, Ncoa1, Ncoa2, Crot), whereas in MHCIIhi macrophages, anti-miR-33 upregulated target genes involved in chromatin remodeling and transcriptional regulation. Anti-miR-33 also reduced the accumulation of aortic CD8+ T cells and CD4+ Th1 cells, and increased levels of FoxP3+ regulatory T cells in plaques, consistent with an immune-dampening effect on plaque inflammation. Conclusions: Our results provide insight into the immune mechanisms and cellular players that execute anti-miR-33's atheroprotective actions in the plaque.