MMP-10 rs17435959 Polymorphism is Associated with the Formation and Stability of Carotid Atherosclerosis Plaque: A Case-Control Study.

BACKGROUND: Matrix metalloproteinase 10 (MMP-10) has a close relationship with carotid atherosclerosis (CAS) and cerebral infarction. The MMP-10 rs17435959 polymorphism causes a leucine to valine transition at codon 4 in exon 1 of the MMP-10 gene and may have functional effects. OBJECTIVES: To investigate the relationship between the MMP-10 rs17435959 polymorphism and the formation and stability of CAS plaques. MATERIALS AND METHODS: The present case-control study contains 738 visitors who came to our health examination center for the first time. According to the carotid ultrasound examinations, visitors were classified into the vulnerable plaque group (41-86 years old, 141 male, 105 female), the stable plaque group (41-86 years old, 141 male, 105 female) and the no plaque group (41-85 years old, 141 male, 105 female). All visitors in the three groups were sex- and- age-matched, and cardiovascular and cerebrovascular diseases were absent. The polymorphism was genotyped by real-time polymerase chain reaction- restriction. RESULTS: Compared to the GG genotype, the frequency of the CC and CG genotypes was significantly more common in the vulnerable plaque group than in the no plaque group (18.7% vs. 7.7%, unadjusted P = 0.002). Moreover, compared to the G allele, the frequency of the C allele was significantly more common in the vulnerable plaque group than in the no plaque group (10.4% vs. 3.9%, unadjusted P = 0.000) and in the vulnerable plaque group than in the stable plaque group (10.4% vs. 5.1%, unadjusted P = 0.008). Binary logistic regression showed that the CC and CG genotype was independent risk factor for the formation (P = 0.019, OR = 1.961, 95% CI [1.117, 3.444]) and vulnerability (P = 0.035, OR = 1.842, 95% CI [1.045, 3.247]) of CAS plaques. Moreover, individuals who have the C allele showed a higher level of fibrinogen, which was an independent risk factor for the formation of CAS plaques (P = 0.000, OR = 2.425, 95% CI [1.475, 3.985]). CONCLUSIONS: The rs17435959 polymorphism was associated with the formation and vulnerability of CAS plaques. Individuals who had variant-type MMP-10 showed higher levels of fibrinogen, which promoted the formation of CAS plaques.

Chitinase 3 like 1 is a regulator of smooth muscle cell physiology and atherosclerotic lesion stability.

AIMS: Atherosclerotic cerebrovascular disease underlies the majority of ischaemic strokes and is a major cause of death and disability. While plaque burden is a predictor of adverse outcomes, plaque vulnerability is increasingly recognized as a driver of lesion rupture and risk for clinical events. Defining the molecular regulators of carotid instability could inform the development of new biomarkers and/or translational targets for at-risk individuals. METHODS AND RESULTS: Using two independent human endarterectomy biobanks, we found that the understudied glycoprotein, chitinase 3 like 1 (CHI3L1), is up-regulated in patients with carotid disease compared to healthy controls. Further, CHI3L1 levels were found to stratify individuals based on symptomatology and histopathological evidence of an unstable fibrous cap. Gain- and loss-of-function studies in cultured human carotid artery smooth muscle cells (SMCs) showed that CHI3L1 prevents a number of maladaptive changes in that cell type, including phenotype switching towards a synthetic and hyperproliferative state. Using two murine models of carotid remodelling and lesion vulnerability, we found that knockdown of Chil1 resulted in larger neointimal lesions comprised by de-differentiated SMCs that failed to invest within and stabilize the fibrous cap. Exploratory mechanistic studies identified alterations in potential downstream regulatory genes, including large tumour suppressor kinase 2 (LATS2), which mediates macrophage marker and inflammatory cytokine expression on SMCs, and may explain how CHI3L1 modulates cellular plasticity. CONCLUSION: CHI3L1 is up-regulated in humans with carotid artery disease and appears to be a strong mediator of plaque vulnerability. Mechanistic studies suggest this change may be a context-dependent adaptive response meant to maintain vascular SMCs in a differentiated state and to prevent rupture of the fibrous cap. Part of this effect may be mediated through downstream suppression of LATS2. Future studies should determine how these changes occur at the molecular level, and whether this gene can be targeted as a novel translational therapy for subjects at risk of stroke.

N6-methyladenosine in RNA of atherosclerotic plaques: An epitranscriptomic signature of human carotid atherosclerosis.

BACKGROUND: More than 170 post-transcriptional RNA modifications regulate the localization, processing and function of cellular RNAs, and aberrant RNA modifications have been linked to a range of human diseases. The RNA modification landscape in atherosclerosis, the main underlying cause of cardiovascular diseases, is still largely unknown. METHODS: We used mass spectrometry to analyse a selection of RNA-modifying enzymes and the N6-methyladenosine (m6A) in carotid atherosclerotic lesion samples representing early and advanced stages of atherosclerosis as compared to non-atherosclerotic arteries from healthy controls. FINDINGS: (i) the detection of different levels of several enzymes involved in methylations occurring in rRNA and mRNA; (ii) these findings included changes in the levels of methyltransferases ('writers'), binding proteins ('readers') and demethylases ('erasers') during atherosclerosis as compared to non-atherosclerotic control arteries, with generally the most prominent differences in samples from early atherosclerotic lesions; and (iii) these changes were accompanied by a marked downregulation of m6A in rRNA, the most abundant and well-studied modification in mRNA with a wide range of effects on cell biology. INTERPRETATION: We show for the first time that RNA-modifying enzymes and the well-studied RNA modification m6A are differentially regulated in atherosclerotic lesions, which potentially could help creating new prognostic and treatment strategies.

Nck1, But Not Nck2, Mediates Disturbed Flow-Induced p21-Activated Kinase Activation and Endothelial Permeability.

Background Alteration in hemodynamic shear stress at atheroprone sites promotes endothelial paracellular pore formation and permeability. The molecular mechanism remains unknown. Methods and Results We show that Nck (noncatalytic region of tyrosine kinase) deletion significantly ameliorates disturbed flow-induced permeability, and selective isoform depletion suggests distinct signaling mechanisms. Only Nck1 deletion significantly reduces disturbed flow-induced paracellular pore formation and permeability, whereas Nck2 depletion has no significant effects. Additionally, Nck1 re-expression, but not Nck2, restores disturbed flow-induced permeability in Nck1/2 knockout cells, confirming the noncompensating roles. In vivo, using the partial carotid ligation model of disturbed flow, Nck1 knockout prevented the increase in vascular permeability, as assessed by Evans blue and fluorescein isothiocyanate dextran extravasations and leakage of plasma fibrinogen into the vessel wall. Domain swap experiments mixing SH2 (phosphotyrosine binding) and SH3 (proline-rich binding) domains between Nck1 and Nck2 showed a dispensable role for SH2 domains but a critical role for the Nck1 SH3 domains in rescuing disturbed flow-induced endothelial permeability. Consistent with this, both Nck1 and Nck2 bind to platelet endothelial adhesion molecule-1 (SH2 dependent) in response to shear stress, but only Nck1 ablation interferes with shear stress-induced PAK2 (p21-activated kinase) membrane translocation and activation. A single point mutation into individual Nck1 SH3 domains suggests a role for the first domain of Nck1 in PAK recruitment to platelet endothelial cell adhesion molecule-1 and activation in response to shear stress. Conclusions This work provides the first evidence that Nck1 but not the highly similar Nck2 plays a distinct role in disturbed flow-induced vascular permeability by selective p21-activated kinase activation.

Association between rs2107595 HDAC9 gene polymorphism and advanced carotid atherosclerosis in the Slovenian cohort.

BACKGROUND: Histone deacetylase 9 (HDAC9) plays an important role in transcriptional regulation, cell cycle progression and developmental events; moreover, it has been investigated as a candidate gene in a number of conditions, including the onset and progression of atherosclerosis. We hypothesized that the rs2107595 HDAC9 gene polymorphism may be associated with advanced carotid artery disease in a Slovenian cohort. We also investigated the effect of this polymorphism on HDAC9 receptor expression in the internal carotid artery (ICA) specimens obtained by endarterectomy. METHODS: This case-control study enrolled 619 unrelated Slovenian patients: 311 patients with ICA stenosis > 75% as the study group and 308 patients with ICA stenosis < 50% as the control group. Patient laboratory and clinical data were obtained from the medical records. The rs2107595 polymorphisms were genotyped using TaqMan SNP Genotyping assay. HDAC9 expression was assessed by immunohistochemistry in 30 ICA specimens from patients with ICA atherosclerosis > 75%, and the numerical areal density of HDAC9 positive cells was calculated. RESULTS: The occurrence of advanced ICA atherosclerosis in the Slovenian cohort was 3.81 times higher in the codominant genetic model (OR = 3.81, 95%CI = 1.06-13.77, p = 0.04), and 3.10 times higher in the recessive genetic model (OR = 3.10, 95%CI = 1.16-8.27, p = 0.02). In addition, the A allele of rs2107595 was associated with increased HDAC9 expression in the ICA specimens obtained by endarterectomy. CONCLUSIONS: We observed a significant association between the AA genotype of rs2107595 with the advanced carotid artery disease in our Slovenian cohort, indicating that this polymorphism may be a genetic risk factor for ICA atherosclerosis.

Effects of exercise training and detraining on atheromatous matrix metalloproteinase activity in mice.

BACKGROUND AND AIMS: Exercise training (ET) helps treat atherosclerosis. However, many patients stop regular ET for various reasons. The effect of detraining on atherosclerosis is not well studied. We examined the effects of ET vs. short-term detraining on atheromatous matrix-metalloproteinase (MMP) activity in preexisting plaque and circulating cytokines/lipids. METHODS AND RESULTS: Eighteen-week-old apolipoprotein-E-/- mice (n = 56) on a Western diet underwent: 1) ET for 6-weeks (ET5+1), 2) ET for 5-weeks and detraining for 1-week (ET5+0), 3) ET for the last 1-week (ET0+1), or 4) no treadmill ET at all for 6-weeks (ET0+0). Atheromatous MMP-activity was visualized using molecular imaging with an MMP-2/9-activatable near-infrared-fluorescent probe. Compared with no ET (ET0+0), regular ET (ET5+1) decreased carotid atheromatous MMP activity, but this protective effect was significantly blunted by short-term detraining (ET5+0). Short-term detraining after longer-term ET showed a reduction in MMP-activity similar to short-term ET (ET0+1). Blood levels of lipids and cytokines paralleled the molecular imaging results: exercise caused higher levels of high-density lipoprotein, adiponectin, and interleukin-10 and lower levels of vascular cell adhesion molecule, monocyte chemoattractant protein-1, interleukin-1ß, and low-density lipoprotein. However, this beneficial effect was short-lived, with the ET5+0 group being similar to the ET0+0 group, and the ET0+1 group being similar to the ET5+1 group. The effect of exercise can be modeled with an exponential-decay of the protective factor of about 15%/day. CONCLUSIONS: Even short-term detraining reduces atheroprotective effects, and tips the balance towards atherosclerosis. This suggests that ET, to be effective, needs to be prolonged and regular, and that detraining should be avoided.

Sickle Cell Anemia Mediates Carotid Artery Expansive Remodeling That Can Be Prevented by Inhibition of JNK (c-Jun N-Terminal Kinase).

OBJECTIVE: Sickle cell anemia (SCA) causes chronic inflammation and multiorgan damage. Less understood are the arterial complications, most evident by increased strokes among children. Proteolytic mechanisms, biomechanical consequences, and pharmaceutical inhibitory strategies were studied in a mouse model to provide a platform for mechanistic and intervention studies of large artery damage due to sickle cell disease. Approach and Results: Townes humanized transgenic mouse model of SCA was used to test the hypothesis that elastic lamina and structural damage in carotid arteries increased with age and was accelerated in mice homozygous for SCA (sickle cell anemia homozygous genotype [SS]) due to inflammatory signaling pathways activating proteolytic enzymes. Elastic lamina fragmentation observed by 1 month in SS mice compared with heterozygous littermate controls (sickle cell trait heterozygous genotype [AS]). Positive immunostaining for cathepsin K, a powerful collagenase and elastase, confirmed accelerated proteolytic activity in SS carotids. Larger cross-sectional areas were quantified by magnetic resonance angiography and increased arterial compliance in SS carotids were also measured. Inhibiting JNK (c-jun N-terminal kinase) signaling with SP600125 significantly reduced cathepsin K expression, elastin fragmentation, and carotid artery perimeters in SS mice. By 5 months of age, continued medial thinning and collagen degradation was mitigated by treatment of SS mice with JNK inhibitor. CONCLUSIONS: Arterial remodeling due to SCA is mediated by JNK signaling, cathepsin proteolytic upregulation, and degradation of elastin and collagen. Demonstration in Townes mice establishes their utility for mechanistic studies of arterial vasculopathy, related complications, and therapeutic interventions for large artery damage due to SCA.

Regulatory T cells suppress the expression of COX-2 in vulnerable plaque.

COX-2 contributes to local inflammation in atherosclerotic lesions. Regulatory T cells (Tregs) enhance the stability of atherosclerotic plaques. The aim of this study was to detect the potential relationship between Tregs and COX-2 in vulnerable plaques. Thirty ApoE -/- mice were fed a high-fat diet, and a silastic perivascular collar was placed around the right common carotid artery to induce vulnerable plaques. Eight weeks after collar placement, the mice were divided randomly into three groups: control, PBS, and Treg groups. Four weeks later, the right common carotid arteries were collected to detect the expression of COX-2. The results showed that Tregs significantly suppressed the expression of COX-2 in vulnerable plaques. In an in vitro experiment, RAW264.7 cells were divided randomly into three groups, which were precultured without T cells or with CD4 + CD25- T cells or Tregs for 48 h with an anti-CD3 antibody; then the cells were stimulated with LPS for 24 h. The RAW264.7 cells were harvested for RT-PCR and western blot assays and the results showed that Tregs downregulated COX-2 expression in RAW264.7 cells. Therefore, Tregs inhibited the expression of COX-2 in vulnerable plaques and macrophages, and COX-2 inhibition may be an important effect of Tregs that results in atherosclerotic plaque stabilization.

Cigarette Smoke Extract Activates Tartrate-Resistant Acid Phosphatase-Positive Macrophage.

BACKGROUND: It has been reported that smoking is one of the strongest positive risk factors for abdominal aortic aneurysms (AAAs). Although many studies have been directed to decipher the effect of smoking on AAA, its effect on macrophage activation has not yet been explored. OBJECTIVES: We have reported the importance of osteoclastogenesis (OCG) in aneurysm formation. Therefore, we examined the effect of cigarette smoking on OCG and arterial aneurysmal formation by using cigarette smoke extract (CSE) in this study. METHODS: Macrophage cell lines were stimulated with CSE, and their activation and differentiation were examined in vitro. Since macrophages activated through the OCG pathway are identified by tartrate-resistant acid phosphatase (TRAP) expression, these cells are referred to as TRAP-positive macrophages (TPMs) in this study. We also applied CSE-contained PBS in the calcium chloride-induced mouse carotid aneurysm model in vivo. RESULTS: Macrophages stimulated with CSE expressed significantly higher levels of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), TRAP, cathepsin K, matrix metalloproteinase-9 and membrane-type metalloproteinase (MT1-MMP). CSE-treated mouse aneurysms showed increased aneurysm size with increased TPM infiltration and protease expression compared to non-CSE-treated mouse aneurysms. CONCLUSIONS: These results suggest that CSE intensifies OCG in macrophages and promotes arterial aneurysmal progression.

Association of serum proprotein convertase subtilisin/kexin type 9 with early atherosclerosis in newly diagnosed type 2 diabetes mellitus.

BACKGROUND AND AIM: Proprotein convertase subtilisin/kexin type 9 (PCSK9) is rapidly gaining attention as a potential risk of developing atherosclerosis due to its crucial role in the regulation of low-density lipoprotein cholesterol (LDL-C) metabolism. The present study investigated the relationship between serum PCSK9 levels and early atherosclerosis as assessed by carotid intimal-medial thickness (CIMT) and brachial-ankle pulse wave velocity (ba-PWV) in newly diagnosed type 2 diabetes mellitus (T2DM). METHODS AND RESULTS: A total of 100 newly diagnosed T2DM were enrolled and further divided into the thickened CIMT group (n = 41) and the non-thickened CIMT group (n = 59) according to the results of color Doppler ultrasonography. Serum PCSK9 levels, CIMT, ba-PWV, and metabolic parameters were measured. Patients in the thickened CIMT group had higher serum PCSK9 levels than patients in the non-thickened CIMT group (all P < 0.05). CIMT and ba-PWV were both positively correlated to serum PCSK9 levels, while serum PCSK9 levels were positively correlated to white blood cell count, neutrophil, lymphocyte, and high-sensitivity C-reactive protein (P < 0.05). Multiple linear regression indicated that serum PCSK9 level was an independent predictor of CIMT (ß = 0.637, P < 0.001) and ba-PWV (ß = 0.600, P < 0.001). Binary logistic regression analysis showed that serum PCSK9 levels were independent risk factors of thickened CIMT [OR = 1.120, 95%CI (1.041-1.204), P = 0.002]. CONCLUSION: Serum PCSK9 levels are significantly correlated with CIMT and ba-PWV, independent of CAD risk factors. Therefore, serum PCSK9 level may have the potential to serve as a prescriptive biomarker for early arteriosclerosis in newly diagnosed T2DM.

Paraoxonase-1: Characteristics and Role in Atherosclerosis and Carotid Artery Disease.

Paraoxonase-1 (PON-1) is a calcium-dependent enzyme that is synthesized in the liver and then secreted in blood where it is bound to high density lipoprotein (HDL). PON-1 is a hydrolase with a wide range of substrates, including lipid peroxides. It is considered responsible for many of the antiatherogenic properties of HDL. PON-1 prevents low density lipoprotein (LDL) oxidation, a process that is considered to contribute to the initiation and development of atherosclerosis. PON-1 activity and levels are influenced by gene polymorphisms; of the 2 common variants, one is in position 192 (Q192R) and one in position 55 (M55L). Also, many drugs affect PON-1 activity. The role of PON-1 in carotid atherosclerosis is inconsistent. Some studies show an association of PON-1 polymorphisms with carotid plaque formation, whereas others do not. The aim of this review is to summarize the characteristics of PON-1, its interactions with drugs and its role in atherosclerosis and especially its relationship with carotid artery disease.

Cystathionine γ Lyase Sulfhydrates the RNA Binding Protein Human Antigen R to Preserve Endothelial Cell Function and Delay Atherogenesis.

BACKGROUND: Hydrogen sulfide (H2S), generated by cystathionine γ lyase (CSE), is an important endogenous regulator of vascular function. The aim of the present study was to investigate the control and consequences of CSE activity in endothelial cells under physiological and proatherogenic conditions. METHODS: Endothelial cell CSE knockout mice were generated, and lung endothelial cells were studied in vitro (gene expression, protein sulfhydration, and monocyte adhesion). Mice were crossed onto the apolipoprotein E-deficient background, and atherogenesis (partial carotid artery ligation) was monitored over 21 days. CSE expression, H2S bioavailability, and amino acid profiling were also performed with human material. RESULTS: The endothelial cell-specific deletion of CSE selectively increased the expression of CD62E and elevated monocyte adherence in the absence of an inflammatory stimulus. Mechanistically, CD62E mRNA was more stable in endothelial cells from CSE-deficient mice, an effect attributed to the attenuated sulfhydration and dimerization of the RNA-binding protein human antigen R. CSE expression was upregulated in mice after partial carotid artery ligation and in atheromas from human subjects. Despite the increase in CSE protein, circulating and intraplaque H2S levels were reduced, a phenomenon that could be attributed to the serine phosphorylation (on Ser377) and inhibition of the enzyme, most likely resulting from increased interleukin-1ß. Consistent with the loss of H2S, human antigen R sulfhydration was attenuated in atherosclerosis and resulted in the stabilization of human antigen R-target mRNAs, for example, CD62E and cathepsin S, both of which are linked to endothelial cell activation and atherosclerosis. The deletion of CSE from endothelial cells was associated with the accelerated development of endothelial dysfunction and atherosclerosis, effects that were reversed on treatment with a polysulfide donor. Finally, in mice and humans, plasma levels of the CSE substrate l-cystathionine negatively correlated with vascular reactivity and H2S levels, indicating its potential use as a biomarker for vascular disease. CONCLUSIONS: The constitutive S-sulfhydration of human antigen R (on Cys13) by CSE-derived H2S prevents its homodimerization and activity, which attenuates the expression of target proteins such as CD62E and cathepsin S. However, as a consequence of vascular inflammation, the beneficial actions of CSE-derived H2S are lost owing to the phosphorylation and inhibition of the enzyme.

FUNDC2 regulates platelet activation through AKT/GSK-3ß/cGMP axis.

AIMS: AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. METHODS AND RESULTS: We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5'-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3ß in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3ß/cGMP axis also regulates clot retraction of platelet-rich plasma. CONCLUSION: FUNDC2 positively regulates platelet functions via AKT/GSK-3ß/cGMP signalling pathways, which provides new insight for platelet-related diseases.

Human mast cell neutral proteases generate modified LDL particles with increased proteoglycan binding.

BACKGROUND AND AIMS: Subendothelial interaction of LDL with extracellular matrix drives atherogenesis. This interaction can be strengthened by proteolytic modification of LDL. Mast cells (MCs) are present in atherosclerotic lesions, and upon activation, they degranulate and release a variety of neutral proteases. Here we studied the ability of MC proteases to cleave apoB-100 of LDL and affect the binding of LDL to proteoglycans. METHODS: Mature human MCs were differentiated from human peripheral blood-derived CD34+ progenitors in vitro and activated with calcium ionophore to generate MC-conditioned medium. LDL was incubated in the MC-conditioned medium or with individual MC proteases, and the binding of native and modified LDL to isolated human aortic proteoglycans or to human atherosclerotic plaques ex vivo was determined. MC proteases in atherosclerotic human coronary artery lesions were detected by immunofluorescence and qPCR. RESULTS: Activated human MCs released the neutral proteases tryptase, chymase, carboxypeptidase A3, cathepsin G, and granzyme B. Of these, cathepsin G degraded most efficiently apoB-100, induced LDL fusion, and enhanced binding of LDL to isolated human aortic proteoglycans and human atherosclerotic lesions ex vivo. Double immunofluoresence staining of human atherosclerotic coronary arteries for tryptase and cathepsin G indicated that lesional MCs contain cathepsin G. In the lesions, expression of cathepsin G correlated with the expression of tryptase and chymase, but not with that of neutrophil proteinase 3. CONCLUSIONS: The present study suggests that cathepsin G in human atherosclerotic lesions is largely derived from MCs and that activated MCs may contribute to atherogenesis by enhancing LDL retention.

Noncanonical Matrix Metalloprotease 1-Protease-Activated Receptor 1 Signaling Drives Progression of Atherosclerosis.

OBJECTIVE: Protease-activated receptor-1 (PAR1) is classically activated by thrombin and is critical in controlling the balance of hemostasis and thrombosis. More recently, it has been shown that noncanonical activation of PAR1 by matrix metalloprotease-1 (MMP1) contributes to arterial thrombosis. However, the role of PAR1 in long-term development of atherosclerosis is unknown, regardless of the protease agonist. APPROACH AND RESULTS: We found that plasma MMP1 was significantly correlated (R=0.33; P=0.0015) with coronary atherosclerotic burden as determined by angiography in 91 patients with coronary artery disease and acute coronary syndrome undergoing cardiac catheterization or percutaneous coronary intervention. A cell-penetrating PAR1 pepducin, PZ-128, currently being tested as an antithrombotic agent in the acute setting in the TRIP-PCI study (Thrombin Receptor Inhibitory Pepducin-Percutaneous Coronary Intervention), caused a significant decrease in total atherosclerotic burden by 58% to 70% (P<0.05) and reduced plaque macrophage content by 54% (P<0.05) in apolipoprotein E-deficient mice. An MMP1 inhibitor gave similar beneficial effects, in contrast to the thrombin inhibitor bivalirudin that gave no improvement on atherosclerosis end points. Mechanistic studies revealed that inflammatory signaling mediated by MMP1-PAR1 plays a critical role in amplifying tumor necrosis factor α signaling in endothelial cells. CONCLUSIONS: These data suggest that targeting the MMP1-PAR1 system may be effective in tamping down chronic inflammatory signaling in plaques and halting the progression of atherosclerosis.

Loss of Sirt3 accelerates arterial thrombosis by increasing formation of neutrophil extracellular traps and plasma tissue factor activity.

Aims: Sirtuin 3 (Sirt3) is a mitochondrial, nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase that reduces oxidative stress by activation of superoxide dismutase 2 (SOD2). Oxidative stress enhances arterial thrombosis. This study investigated the effects of genetic Sirt3 deletion on arterial thrombosis in mice in an inflammatory setting and assessed the clinical relevance of these findings in patients with ST-elevation myocardial infarction (STEMI). Methods and results: Using a laser-induced carotid thrombosis model with lipopolysaccharide (LPS) challenge, in vivo time to thrombotic occlusion in Sirt3-/- mice (n = 6) was reduced by half compared to Sirt3+/+ wild-type (n = 8, P < 0.01) controls. Ex vivo analyses of whole blood using rotational thromboelastometry revealed accelerated clot formation and increased clot stability in Sirt3-/- compared to wild-type blood. rotational thromboelastometry of cell-depleted plasma showed accelerated clotting initiation in Sirt3-/- mice, whereas overall clot formation and firmness remained unaffected. Ex vivo LPS-induced neutrophil extracellular trap formation was increased in Sirt3-/- bone marrow-derived neutrophils. Plasma tissue factor (TF) levels and activity were elevated in Sirt3-/- mice, whereas plasma levels of other coagulation factors and TF expression in arterial walls remained unchanged. SOD2 expression in bone marrow -derived Sirt3-/- neutrophils was reduced. In STEMI patients, transcriptional levels of Sirt3 and its target SOD2 were lower in CD14+ leukocytes compared with healthy donors (n = 10 each, P < 0.01). Conclusions: Sirt3 loss-of-function enhances experimental thrombosis in vivo via an increase of neutrophil extracellular traps and elevation of TF suggesting thrombo-protective effects of endogenous Sirt3. Acute coronary thrombosis in STEMI patients is associated with lower expression levels of SIRT3 and SOD2 in CD14+ leukocytes. Therefore, enhancing SIRT3 activity by pan-sirtuin activating NAD+-boosters may provide a novel therapeutic target to prevent or treat thrombotic arterial occlusion in myocardial infarction or stroke.

Evaluation of the oxidative stress-related genes ALOX5, ALOX5AP, GPX1, GPX3 and MPO for contribution to the risk of type 2 diabetes mellitus in the Han Chinese population.

AIM: Type 2 diabetes mellitus is a polygenic metabolic disorder resulting from oxidative stress, the root cause of insulin resistance, ß-cell dysfunction and impaired glucose tolerance. The aim of this study was to investigate the role of oxidative stress-related genes ALOX5, ALOX5AP, GPX1, GPX3 and MPO in type 2 diabetes mellitus susceptibility in the Chinese Han population. METHODS: A total of 396 type 2 diabetes mellitus patients and 678 controls were recruited. The ALOX5 rs10900213, ALOX5AP rs4293222, GPX1 rs1050450, GPX3 rs3828599 and MPO rs2107545 gene polymorphisms were genotyped. RESULTS: We found one single-nucleotide polymorphism in the MPO gene was associated with type 2 diabetes mellitus susceptibility [rs2107545: odds ratio = 1.563 (1.166-2.096); p = 0.003], after adjusting for covariates. Furthermore, we also considered the likely complexity of effects of genetic and conventional risk factors in type 2 diabetes mellitus-related vascular complications, such as carotid plaques. Our analysis revealed that the GPX1 rs1050450 and MPO rs2107545 were significantly associated with increased risk of carotid plaques in type 2 diabetes mellitus patients. CONCLUSION: Our study presents novel evidence for main effects of MPO gene on type 2 diabetes mellitus susceptibility. Furthermore, our study supported the association between variants of oxidative stress-related genes ( GPX1 and MPO) and carotid plaques in type 2 diabetes mellitus patients, which indicated a modulation of type 2 diabetes mellitus-related vascular complication susceptibility by genetic predisposition.

Impaired mitochondrial respiration in human carotid plaque atherosclerosis: A potential role for Pink1 in vascular smooth muscle cell energetics.

BACKGROUND AND AIMS: DNA damage and mitochondrial dysfunction are thought to play an essential role in ageing and the energetic decline of vascular smooth muscle cells (VSMCs) essential for maintaining plaque integrity. We aimed to better understand VSMCs and identify potentially useful compensatory pathways that could extend their lifespan. Moreover, we wanted to assess if defects in mitochondrial respiration exist in human atherosclerotic plaques and to identify the appropriate markers that may reflect a switch in VSMC energy metabolism. METHODS: Human plaque tissue and cells were assessed for composition and evidence of DNA damage, repair capacity and mitochondrial dysfunction. Fresh plaque tissue was evaluated using high resolution oxygen respirometry to assess oxidative metabolism. Recruitment and processing of the mitochondrial regulator of autophagy Pink1 kinase was investigated in combination with transcriptional and protein markers associated with a potential switch to a more glycolytic metabolism. RESULTS: Human VSMC have increased nuclear (nDNA) and mitochondrial (mtDNA) damage and reduced repair capacity. A subset of VSMCs within plaque cap had decreased oxidative phosphorylation and expression of Pink1 kinase. Plaque cells demonstrated increased glycolytic activity in response to loss of mitochondrial function. A potential compensatory glycolytic program may act as energetic switch via AMP kinase (AMPK) and hexokinase 2 (Hex2). CONCLUSIONS: We have identified a subset of plaque VSMCs required for plaque stability that have increased mitochondrial dysfunction and decreased oxidative phosphorylation. Pink1 kinase may initiate a cellular response to promote a compensatory glycolytic program associated with upregulation of AMPK and Hex2.

Relationship between ADAMTS4 and carotid atherosclerotic plaque vulnerability in humans.

BACKGROUND: Rupture of atherosclerotic plaques and the resulting thrombosis are vital causes of clinical ischemic events. Recent studies have shown that ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) is a pathogenic factor of plaque vulnerability in mice. However, the relationship between ADAMTS4 and carotid atherosclerotic vulnerable plaques in humans remains unclear. METHODS: Forty-eight carotid atherosclerotic plaque specimens were obtained from 48 carotid artery stenosis inpatients undergoing carotid endarterectomy. We performed hematoxylin and eosin and Movat pentachrome staining for histologic characteristics; immunohistochemical staining for ADAMTS4, versican, and macrophages; and serologic tests for ADAMTS4. Patients were divided into stable and vulnerable groups on the basis of histologic characterization according to the classification criteria of the American Heart Association. Comparison between the groups was carried out using SPSS 17.0 (SPSS Inc, Chicago, Ill). RESULTS: Expression of ADAMTS4 in the plaque and its serum concentration were significantly higher in the vulnerable group compared with the stable one (P = .004 and P = .021, respectively), whereas the expression of versican was lower in the vulnerable group than in the stable group (P = .015). Univariate analysis revealed that the incidence of symptomatic cerebral ischemic events and ADAMTS4 serum levels were statistically higher in the vulnerable group compared with the stable group (P = .021 and P = .029, respectively). Multivariate analysis showed that ADAMTS4 was an independent risk factor (odds ratio, 1.14; P = .038). CONCLUSIONS: Our study revealed that ADAMTS4 expression was upregulated during carotid atherosclerotic plaque development. Serum levels of ADAMTS4 were associated with increased plaque vulnerability in both symptomatic and asymptomatic patients with carotid artery stenosis. ADAMTS4 may be a potential biomarker for plaque vulnerability.

Peroxisome proliferator-activated receptor gamma agonists for preventing recurrent stroke and other vascular events in people with stroke or transient ischaemic attack.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists are insulin-sensitising drugs used for the treatment of insulin resistance. In addition to lowering glucose in diabetes, these drugs may also protect against hyperlipidaemia and arteriosclerosis, which are risk factors for stroke. This is an update of a review first published in January 2014 and subsequently updated in October 2015. OBJECTIVES: To assess the efficacy and safety of PPAR-γ agonists in the secondary prevention of stroke and related vascular events for people with stroke or transient ischaemic attack (TIA). SEARCH METHODS: We searched the Cochrane Stroke Group Trials Register (16 May 2017), the Cochrane Central Register of Controlled Trials (CENTRAL; 2017, Issue 5), MEDLINE (1949 to 16 May 2017), Embase (1980 to 16 May 2017), CINAHL (1982 to 16 May 2017), AMED (1985 to 16 May 2017), and 11 Chinese databases (16 May 2017). In an effort to identify further published, unpublished, and ongoing trials, we searched ongoing trials registers, reference lists, and relevant conference proceedings, and contacted authors and pharmaceutical companies. We did not impose any language restrictions. SELECTION CRITERIA: We included randomised controlled trials (RCTs) evaluating PPAR-γ agonists versus placebo for the secondary prevention of stroke and related vascular events in people with stroke or TIA, with the outcomes of recurrent stroke, vascular events, and adverse events. DATA COLLECTION AND ANALYSIS: Two review authors independently screened the titles and abstracts of identified records, selected studies for inclusion, extracted eligible data, cross-checked the data for accuracy, and assessed methodological quality and risk of bias. We evaluated the quality of evidence for each outcome using the GRADE approach. MAIN RESULTS: We identified five RCTs with 5039 participants; two studies had a low risk of bias for all domains. Four studies evaluated the drug pioglitazone, and one study evaluated rosiglitazone. The participants in different studies were heterogeneous.Recurrent strokeThree studies evaluated the number of participants with recurrent stroke (4979 participants, a single study contributing 3876 of these). Peroxisome proliferator-activated receptor gamma agonists probably reduce the recurrence of stroke compared with placebo (risk ratio (RR) 0.66, 95% confidence interval (CI) 0.44 to 0.99; moderate-quality evidence).Adverse eventsEvidence that adverse events occurred more frequently in participants treated with PPAR-γ agonists when compared with placebo was uncertain due to wide confidence interval and high levels of statistical heterogeneity: risk difference 10%, 95% CI -8% to 28%; low-quality evidence).Data were available on additional composite outcomes reflecting serious vascular events (all-cause death and other major vascular events; all-cause mortality, non-fatal myocardial infarction or non-fatal stroke) from one study in 984 people. This study provided low-quality evidence that PPAR-γ agonists led to fewer events (data not meta-analysed).Vascular eventsPeroxisome proliferator-activated receptor gamma agonists given over a mean duration of 34.5 months in a single trial of 984 participants may reduce serious vascular events expressed as a composite outcome of total events of cardiovascular death, non-fatal myocardial infarction or non-fatal stroke (RR 0.73, 95% CI 0.54 to 0.99; low-quality evidence).Other outcomesOne study in 20 people measured insulin sensitivity, and one study in 40 people measured the ubiquitin-proteasome activity in carotid plaques. Our confidence in the improvements observed with PPAR-γ agonists were limited by small sample sizes and risk of bias. None of the studies reported the number of participants with disability due to vascular events or improvement in quality of life. AUTHORS' CONCLUSIONS: Peroxisome proliferator-activated receptor gamma agonists probably reduce recurrent stroke and total events of cardiovascular death, non-fatal myocardial infarction or non-fatal stroke, and may improve insulin sensitivity and the stabilisation of carotid plaques. Their effects on adverse events are uncertain. Our conclusions should be interpreted with caution considering the small number and the quality of the included studies. Further well-designed, double-blind RCTs with large samples are required to assess the efficacy and safety of PPAR-γ agonists in the secondary prevention of stroke and related vascular events in people with stroke or TIA.