PKM2-dependent glycolysis promotes the proliferation and migration of vascular smooth muscle cells during atherosclerosis.

Increased glycolysis is involved in the proliferation and migration of vascular smooth muscle cells (VSMCs). Pyruvate kinase isoform M2 (PKM2), a key rate-limiting enzyme in glycolysis, accelerates the proliferation and migration of tumor cells. Although the intracellular mechanisms associated with oxidized low-density lipoprotein (oxLDL)-stimulated VSMC proliferation and migration have been extensively explored, it is still unclear whether oxLDL promotes the proliferation and migration of VSMCs by enhancing PKM2-dependent glycolysis. In the present study, we detected PKM2 expression and pyruvate kinase activity in oxLDL-treated VSMCs and explored the regulation of PKM2 in oxLDL-treated VSMCs and apoE-/- mice. The results showed that PKM2 expression in VSMCs was higher in the intima than in the media in plaques from atherosclerotic rabbits. Moreover, PKM2 level in VSMCs was increased during atherosclerosis progression in apoE-/- mice. Both PKM2 expression and pyruvate kinase activity were found to be upregulated by oxLDL stimulation in VSMCs. Shikonin (SKN), a specific inhibitor of PKM2, was found to inhibit the oxLDL-induced proliferation and migration in VSMCs, in addition to delaying the atherosclerosis progression in apoE-/- mice. More importantly, oxLDL increased glucose uptake, ATP and lactate production, and the extracellular acidification rate in VSMCs, which could be reversed by SKN. Meanwhile, oxygen consumption rate was unchanged after oxLDL stimulation, suggesting that glycolysis is the main contributor to the energy supply in oxLDL-treated VSMCs. Our results suggest that oxLDL induces VSMC proliferation and migration by upregulating PKM2-dependent glycolysis, thereby contributing to the atherosclerosis progression. Thus, targeting PKM2-dependent glycolysis might provide a novel therapeutic approach for the treatment of atherosclerosis.

Trimethylamine N-oxide as a risk marker for ischemic stroke in patients with atrial fibrillation.

Trimethylamine N-oxide (TMAO) is an independent risk factor of cardiovascular disease. Our objective was to explore the relation between TMAO and ischemic stroke (IS) in patients with atrial fibrillation (AF). A total of 68 patients with AF with IS and 111 ones without IS were enrolled. The plasma levels of TMAO remarkably increased in IS-AF patients (8.25 ± 1.58 µM) compared with patients with AF (2.22 ± 0.09 µM, P < 0.01). The receiver operating characteristic analysis revealed that the best cutoff value of TMAO to predict IS in patients with AF was 3.53 µM with 75.0% sensitivity and 92.8% specificity (area under the curve: 0.917, 95% confidence intervals: 0.877-0.957). Univariate and multivariate logistic regression analysis showed that TMAO was an independent predictor in IS. The level of TMAO was correlated with the CHA2DS2-VASc score. In conclusion, TMAO was an independent predictor of IS, which could potentially refine stroke stratification in patients with AF.

5-Aminolevulinic Acid-Mediated Sonodynamic Therapy Alleviates Atherosclerosis via Enhancing Efferocytosis and Facilitating a Shift in the Th1/Th2 Balance Toward Th2 Polarization.

BACKGROUND/AIMS: We and other groups have demonstrated that 5-aminolevulinic acid (ALA)-mediated sonodynamic therapy (ALA-SDT) induces macrophage and foam cell apoptosis and stabilizes atherosclerosis (AS) plaques in animal models. Lymphocytes also play vital roles in the development of AS. The primary purpose of the present study was to investigate the effects of ALA-SDT on T helper (Th) cell fate and function, Th subset differentiation, and atherosclerotic lesion stability. METHODS: We utilized ALA-SDT on Western diet-fed apoE-/-mice in vivo and human Jurkat cells in vitro. Hematoxylin and eosin staining and TUNEL assays were used to evaluate the atherosclerotic plaque size and apoptosis within the atheroma. ALA induced cytotoxicity on cultured Jurkat cells was determined with CCK-8 assay. To address the mechanisms, levels of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and mitochondrial permeability transition pore (MPTP) opening were evaluated by staining with fluorescent probes. Western blot analysis and confocal microscopy were used to analyze the protein levels of caspases, Bax and cytochrome c and the release of cytochrome c. Cell apoptosis and necrosis and phagocytosis were examined by flow cytometry. ELISAs and immunofluorescent staining were used to assess the corresponding cytokine levels and Th subset cell numbers within the atheroma. RESULTS: Our studies revealed that ALA-SDT significantly enhanced CD4+ cell apoptosis and macrophage-mediated phagocytosis and hence reduced the necrotic core size. ALA-SDT activated the mitochondrial apoptotic signaling pathway with minimal necrosis in Jurkat cells. ALA-SDT inhibited the Th1 response and enhanced the Th2 response. These effects of ALA-SDT were mediated primarily through the generation of ROS. CONCLUSION: ALA-SDT alleviates AS by enhancing cytotoxic effects on Th cells, subsequently stimulating efferocytosis and facilitating a shift in the Th1/Th2 balance toward Th2 cells, a discovery that might help elucidate the mechanism underlying SDT as a potential treatment to prevent atherothrombotic events.

(-)-Epicatechin rescues the As2 O3 -induced HERG K+ channel deficiency possibly through upregulating transcription factor SP1 expression.

(-)-Epicatechin (EPI) has beneficial effects on the cardiovascular disease. The human ether-a-go-go-related gene (HERG) potassium channel is crucial for repolarization of cardiac action potential. Dysfunction of the HERG channel can cause long QT syndrome type 2 (LQT2). Arsenic trioxide (As2 O3 ) has shown efficacy in the treatment of acute promyelocytic leukemia. However, As2 O3 can induce the deficiency of HERG channel and cause LQT2. In this study, we examined whether EPI could rescue the As2 O3 -induced HERG channel deficiency. We found that 3 µM EPI obviously increased protein expression and current of HERG channel. EPI was able to recover the protein expression and current of HERG channel disrupted by As2 O3 . EPI was able to increase the expression of SP1 protein and recover the expression of SP1 protein disrupted by As2 O3 . In addition, EPI significantly shortened action potential duration prolonged by As2 O3 . Our data suggest that EPI rescues As2 O3 -induced HERG channel deficiency through upregulating SP1 expression.

Trimethylamine N-Oxide is Associated with Heart Failure Risk in Patients with Preserved Ejection Fraction.

BACKGROUND: Trimethylamine N-oxide (TMAO) has been considered to be an independent risk factor of heart failure (HF). OBJECTIVES: To further determine the plasma levels of TMAO in patients who have HF with preserved ejection fraction (HFpEF), and to analyze the relationship between TMAO and HFpEF risk. METHODS: A total of 57 control participants and 61 patients with HFpEF were recruited. We measured and analyzed plasma levels of TMAO and performed biochemical examination of all patients. RESULTS: The mean (SD) plasma levels of TMAO in patients with HFpEF (6.84 [1.12] µmol/L) were significantly higher than in controls (1.63 [0.08] µmol/L; P <.01). The area under the curve (AUC) of TMAO and N-terminal pro b-type natriuretic peptide (NT-proBNP) was 0.817 and 0.924, respectively, which were determined by receiver operating characteristic (ROC) analysis. TMAO was an independent risk factor in patients with HFpEF, as revealed by univariate and multivariate logistic regression analysis. The level of TMAO was correlated with blood urea nitrogen (BUN), creatinine, and NT-proBNP. CONCLUSIONS: TMAO level was highly associated with HFpEF risk.

Non-lethal sonodynamic therapy facilitates the M1-to-M2 transition in advanced atherosclerotic plaques via activating the ROS-AMPK-mTORC1-autophagy pathway.

Emerging evidence indicates that macrophage functional polarization is critically involved in the development of atherosclerosis (AS). Here, we examined the role of 5-aminolaevulinic acid (ALA)-mediated non-lethal sonodynamic therapy (NL-SDT) in macrophage-subset polarization and atherosclerotic lesion stability and explored the potential underlying mechanisms. Using Western diet-fed apolipoprotein E (apoE)-/- and green fluorescent protein (GFP)-positive bone marrow (BM) chimeric mouse models, we demonstrated that NL-SDT promoted phenotypic switching of both BM-derived and resident macrophages from M1 to M2 and significantly inhibited AS progression. Further mechanistic studies indicated that NL-SDT enhanced macrophage differentiation toward the M2 phenotype by activating the reactive oxygen species (ROS)-5' AMP-activated protein kinase (AMPK)-mammalian target of rapamycin complex 1 (mTORC1)-autophagy signaling pathway in murine BM-derived M1 macrophages (BMDM1s). Moreover, NL-SDT drastically reduced lipid droplets, mainly by promoting apoAI-mediated cholesterol efflux in vitro. Specifically, administration of pharmacological inhibitors to the animal model showed a reciprocal effect on NL-SDT-induced macrophage polarization. These findings indicate that NL-SDT engages a virtuous cycle that enhances M1-to-M2 polarization, cholesterol efflux, and anti-inflammatory reactions in advanced plaque in vivo and in BMDM1s in vitro by activating the ROS-AMPK-mTORC1-autophagy pathway. This discovery might help elucidate the mechanism underlying NL-SDT as a potential treatment to prevent atherothrombotic events.

Early modulation of macrophage ROS-PPARγ-NF-κB signalling by sonodynamic therapy attenuates neointimal hyperplasia in rabbits.

Disruption of re-endothelialization and haemodynamic balance remains a critical side effect of drug-eluting stents (DES) for preventing intimal hyperplasia. Previously, we found that 5-aminolevulinic acid-mediated sonodynamic therapy (ALA-SDT) suppressed macrophage-mediated inflammation in atherosclerotic plaques. However, the effects on intimal hyperplasia and re-endothelialization remain unknown. In this study, 56 rabbits were randomly assigned to control, ultrasound, ALA and ALA-SDT groups, and each group was divided into two subgroups (n = 7) on day 3 after right femoral artery balloon denudation combined with a hypercholesterolemic diet. Histopathological analysis revealed that ALA-SDT enhanced macrophage apoptosis and ameliorated inflammation from day 1. ALA-SDT inhibited neointima formation without affecting re-endothelialization, increased blood perfusion, decreased the content of macrophages, proliferating smooth muscle cells (SMCs) and collagen but increased elastin by day 28. In vitro, ALA-SDT induced macrophage apoptosis and reduced TNF-α, IL-6 and IL-1ß via the ROS-PPARγ-NF-κB signalling pathway, which indirectly inhibited human umbilical artery smooth muscle cell (HUASMC) proliferation, migration and IL-6 production. ALA-SDT effectively inhibits intimal hyperplasia without affecting re-endothelialization. Hence, its clinical application combined with bare-metal stent (BMS) implantation presents a potential strategy to decrease bleeding risk caused by prolonged dual-antiplatelet regimen after DES deployment.

Membrane-permeabilized sonodynamic therapy enhances drug delivery into macrophages.

Macrophages play a pivotal role in the formation and development of atherosclerosis as a predominant inflammatory cell type present within atherosclerotic plaque. Promoting anti-atherosclerotic drug delivery into macrophages may provide a therapeutic potential on atherosclerotic plaque. In this study, we investigated whether membrane-permeabilized sonodynamic therapy (MP-SDT) enhances drug delivery into THP-1 macrophages. Images of confocal microscopy confirmed that the optimal plasma distribution of the sonosensitizer protoporphyrin IX (PpIX) was at 1 hour incubation. The non-lethal parameter of MP-SDT was determined by cell viability as measured by a CCK-8 assay. Bright field microscopy demonstrated plasma membrane deformation in response to MP-SDT. Using SYTOX Green, a model drug for cellular uptake, we found that MP-SDT significantly induced membrane permeabilization dependent on ultrasound intensity and exposure time. Using Fluo-3 AM, intracellular calcium elevation during MP-SDT was confirmed as a result of membrane permeabilization. Membrane perforation of MP-SDT-treated cells was observed by scanning electron microscopy and transmission electron microscopy. Moreover, MP-SDT-induced membrane permeabilization and perforation were remarkably prevented by scavenging reactive oxygen species (ROS) during MP-SDT. Furthermore, we assessed the therapeutic effect of MP-SDT in combination with anti-atherosclerotic drug atorvastatin. Our results showed that MP-SDT increased the therapeutic effect of atorvastatin on lipid-laden THP-1-derived foam cells, including decreasing lipid droplets, increasing the cholesterol efflux and the expression of PPARγ and ABCG1. In conclusion, MP-SDT might become a promising approach to facilitating the delivery of anti-atherosclerotic drugs into macrophages via membrane permeabilization.

The Association between Plasma Levels of Trimethylamine N-Oxide and the Risk of Coronary Heart Disease in Chinese Patients with or without Type 2 Diabetes Mellitus.

AIM: Trimethylamine N-oxide (TMAO) has been demonstrated as an independent risk factor for cardiovascular disease. Our objective was to determine the plasma levels of TMAO in Chinese coronary heart disease (CHD) patients with or without type 2 diabetes mellitus (T2DM). METHODS: A total of 132 control participants, 243 CHD patients, and 175 CHD patients with T2DM were enrolled. Plasma levels of TMAO in all patients were measured and analyzed. RESULTS: The plasma levels of TMAO were significantly higher in CHD patients than in control subjects (3.08 ± 0.13 µM versus 1.49 ± 0.05 µM; P < 0.01). In addition, plasma levels of TMAO were remarkably increased in CHD patients with T2DM compared with CHD patients (7.63 ± 0.97 µM versus 3.08 ± 0.13 µM; P < 0.01). The receiver operating characteristic analysis revealed that the area under the curve of TMAO was 0.794 and 0.927 to predict CHD or CHD-T2DM patients (P < 0.01). Univariate and multivariate logistic regression analysis showed that TMAO was an independent predictor in CHD patients with or without T2DM. The level of TMAO was correlated with high-sensitive troponin I (hs-TnI) and creatine kinase MB (CKMB). CONCLUSIONS: TMAO was an independent predictor of CHD in Chinese patients; moreover, the TMAO levels were highly associated with diabetes in CHD patients.