Down-regulation of microRNA-342-5p or Up-regulation of Wnt3a Inhibits Angiogenesis and Maintains Atherosclerotic Plaque Stability in Atherosclerosis Mice

Evidence has demonstrated that microRNA-342-5p (miR-342-5p) is implicated in atherosclerosis (AS), but little is known regarding its intrinsic regulatory mechanisms. Here, we aimed to explore the effect of miR-342-5p targeting Wnt3a on formation of vulnerable plaques and angiogenesis of AS. ApoE−/− mice were fed with high-fat feed for 16 w to replicate the AS vulnerable plaque model. miR-342-5p and Wnt3a expression in aortic tissues of AS were detected. The target relationship between miR-342-5p and Wnt3a was verified. Moreover, ApoE−/− mice were injected with miR-342-5p antagomir and overexpression-Wnt3a vector to test their functions in serum lipid levels, inflammatory and oxidative stress-related cytokines, aortic plaque stability and angiogenesis in plaque of AS mice. miR-342-5p expression was enhanced and Wnt3a expression was degraded in aortic tissues of AS mice and miR-342-5p directly targeted Wnt3a. Up-regulating Wnt3a or down-regulating miR-342-5p reduced blood lipid content, inflammatory and oxidative stress levels, the vulnerability of aortic tissue plaque and inhibited angiogenesis in aortic plaque of AS mice. Functional studies show that depleting miR-342-5p can stabilize aortic tissue plaque and reduce angiogenesis in plaque in AS mice via restoring Wnt3a.

Weak UVB Irradiation Promotes Macrophage M2 Polarization and Stabilizes Atherosclerosis

Atherosclerosis (AS) is a chronic cardiovascular disease endangering human health and is one of the most common causes of myocardial infarction and stroke. Macrophage polarization plays a vital role in regulating plaque stability. As an important component of sunlight, ultraviolet B (UVB) has been proven to promote vitamin D and nitric oxide synthesis. This research used an AS model in ApoE−/− mice to study the effects of UVB on macrophage polarization and atherosclerotic plaque stability. In vitro, UVB irradiation increased arginase-I (Arg-I, M2 macrophage) and macrophage mannose receptor (CD206) expression, while the expression of inducible nitric oxide synthase (iNOS) (M1 macrophage) and CD86 was decreased. UVB promoted Akt phosphorylation in vitro. In vivo, UVB irradiation promoted the stabilization of atherosclerotic lesion plaques, while the phenotype of M2 macrophages increased. Our research provides new evidence for UVB in preventing and treating atherosclerosis.

SMC Derived Hyaluronan Modulates Vascular SMC-Phenotype in Murine Atherosclerosis

Rationale: Plaque instability remains poorly understood and new therapeutic approaches to reduce plaque rupture and subsequent clinical events are of great interest. Recent studies revealed an important role of phenotypic switching of smooth muscle cells (SMC) in controlling plaque stability, including extracellular matrix (ECM) deposition. Objective: The aim of this study was to elucidate the role of hyaluronan (HA) derived from SMC-HA synthase 3 (Has3), in phenotypic switching and plaque stability in an animal model of atherosclerosis. Methods and Results: A mouse line with SMC-specific deletion of Has3 and simultaneous SMC lineage tracing (eYFP) on an Apoe-/- background was used. Lineage tracing of SMC with eYFP revealed that SMC-specific deletion of Has3 significantly increased the number of galectin-3 (LGALS3+) "transition-state" SMC and decreased alpha-smooth muscle actin (ACTA2+) SMC. Notably, SMC-Has3 deletion led to significantly increased collagen deposition and maturation within the fibrous cap (FC) and the whole lesion, as evidenced by Picrosirius red staining and LC-PolScope analysis. Single-cell RNA sequencing (scRNA-seq) of brachiocephalic artery (BCA) lesions demonstrated that the loss of SMC-Has3 enhanced the transition of SMC to an Lgals3+, ECM-producing phenotype with elevated acute-phase response gene expression. Experiments using cultured murine aortic SMC revealed that blocking cluster of differentiation-44 (CD44), an important HA binding receptor, recapitulated the enhanced acute-phase response and synthesis of fibrous ECM. Conclusions: These studies provide evidence that the deletion of SMC-Has3 results in an ECM-producing "transition state" SMC phenotype (characterized by LGALS3+ expression), likely via reduced CD44 signaling, resulting in increased collagen formation and maturation, an index consistent with increased plaque stability.

Huang-Lian-Jie-Du Decoction Attenuates Atherosclerosis and Increases Plaque Stability in High-Fat Diet-Induced ApoE-/- Mice by Inhibiting M1 Macrophage Polarization and Promoting M2 Macrophage Polarization

Macrophage polarization plays a vital impact in triggering atherosclerosis (AS) progression and regression. Huang-Lian-Jie-Du Decoction (HLJDD), a famous traditional Chinese decoction, displays notable anti-inflammatory and lipid-lowering effects in different animal models. However, its effects and mechanisms on AS have not been clearly defined. We determined whether HLJDD attenuated atherosclerosis and plaques vulnerability by regulating macrophage polarization in ApoE−/− mice induced by high-fat diet (HFD). Furthermore, we investigated the effects of HLJDD on macrophage polarization in oxidized low-density lipoprotein (ox-LDL) induced RAW264.7 cells. For in vivo assay, compared with the model group, HLJDD ameliorated lipid metabolism, with significantly decreased levels of serum triglyceride, total cholesterol (CHOL), and lipid density lipoprotein. HLJDD suppressed serum tumor necrosis factor α (TNF-α) and IL-1β levels with increased serum IL-10 level, and inhibited mRNA level of NLRP3 inflammasome in carotid tissues. HLJDD enhanced carotid lesion stability by decreasing macrophage infiltration together with increased expression of collagen fibers and α-SMA. Moreover, HLJDD inhibited M1 macrophage polarization, which decreased the expression and mRNA levels of M1 markers [inducible nitric oxide synthase (iNOS) and CD86]. HLJDD enhanced alternatively activated macrophage (M2) activation, which increased the expression and mRNA levels of M2 markers (Arg-1 and CD163). For in vitro assay, HLJDD inhibited foam cell formation in RAW264.7 macrophages disturbed by ox-LDL. Besides, groups with ox-LDL plus HLJDD drug had a lower expression of CD86 and mRNA levels of iNOS, CD86, and IL-1β, but higher expression of CD163 and mRNA levels of Arg-1, CD163, and IL-10 than ox-LDL group. Collectively, our results revealed that HLJDD alleviated atherosclerosis and promoted plaque stability by suppressing M1 polarization and enhancing M2 polarization.

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

BackgroundThe 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+/–Pfkfb3fl/fl (Pfkfb3fl/fl) mice confers beneficial effects on plaque stability and alleviates cardiovascular disease burden compared to Ldlr–/–LysMCre+/–Pfkfb3wt/wt control mice (Pfkfb3wt/wt).Methods and ResultsAnalysis 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. Pfkfb3wt/wt and Pfkfb3fl/fl mice were fed a 0.25% cholesterol diet for 12 weeks. Pfkfb3fl/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 Pfkfb3fl/fl compared to Pfkfb3wt/wt BMDMs. Unexpectedly, plaque and necrotic core size, as well as macrophage (MAC3), neutrophil (Ly6G) and collagen (Sirius Red) content were unchanged in advanced Pfkfb3fl/fl lesions. Similarly, early lesion plaque and necrotic core size and total plaque burden were unaffected.ConclusionPartial 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.

Understanding the Clinical Implications of Intracranial Arterial Calcification Using Brain CT and Vessel Wall Imaging

Background and Purpose: Intracranial arterial calcification (IAC) has been the focus of much attention by clinicians and researchers as an indicator of intracranial atherosclerosis, but correlations of IAC patterns (intimal or medial) with the presence of atherosclerotic plaques and plaque stability are still a matter of debate. Our study aimed to assess the associations of IAC patterns identified on computed tomography (CT) with the presence of plaque detected on vessel wall magnetic resonance imaging and plaque stability.Materials and Methods: Patients with stroke or transient ischemic attack and intracranial artery stenosis were recruited. IAC was detected and localized (intima or media) on non-contrast CT images. Intracranial atherosclerotic plaques were identified using vessel wall magnetic resonance imaging and matched to corresponding CT images. Associations between IAC patterns and culprit atherosclerotic plaques were assessed by using multivariate regression.Results: Seventy-five patients (mean age, 63.4 ± 11.6 years; males, 46) were included. Two hundred and twenty-one segments with IAC were identified on CT in 66 patients, including 86 (38.9%) predominantly intimal calcifications and 135 (61.1%) predominantly medial calcifications. A total of 72.0% of intimal calcifications coexisted with atherosclerotic plaques, whereas only 10.2% of medial calcifications coexisted with plaques. Intimal calcification was more commonly shown in non-culprit plaques than culprit plaques (25.9 vs. 9.4%, P = 0.008). The multivariate mixed logistic regression adjusted for the degree of stenosis showed that intimal calcification was significantly associated with non-culprit plaques (OR, 2.971; 95% CI, 1.036–8.517; P = 0.043).Conclusion: Our findings suggest that intimal calcification may indicate the existence of a stable form of atherosclerotic plaque, but plaques can exist in the absence of intimal calcification especially in the middle cerebral artery.