Circ-MBOAT2 Regulates Angiogenesis via the miR-495/NOTCH1 Axis and Associates with Myocardial Perfusion in Patients with Coronary Chronic Total Occlusion.

Revascularization of coronary chronic total occlusion (CTO) still remains controversial. The factors that impact collateral circulation and myocardial perfusion are of interest. Circular RNA (circRNA) has been shown to regulate the process of angiogenesis. However, the effects of circ-membrane-bound O-acyltransferase domain containing 2 (circ-MBOAT2) on angiogenesis in patients with CTO were unclear. In this study, we evaluated circulating circRNAs and miRNAs in patients with CTO and stable coronary artery disease using high-throughput sequencing. Another cohort of patients were selected to verify the expressions of circ-MBOAT2 and miR-495. The role and mechanism of circ-MBOAT2 in the process of angiogenesis were explored through in vitro and vivo studies. Finally, we came back to a clinical perspective and investigated whether circ-MBOAT2 and miR-495 were associated with the improvement of myocardial perfusion evaluated by single-photon emission computed tomography (SPECT). We found that the expression of circ-MBOAT2 was significantly up-regulated while miR-495 was significantly down-regulated in patients with CTO. The expression of circ-MBOAT2 was negatively correlated with miR-495 in patients with CTO. In an in vitro study, we found that circ-MBOAT2 promoted tube formation and cell migration via the miR-495/NOTCH1 axis in endothelial cells. In an in vivo study, we showed that the inhibition of miR-495 caused the increase in collateral formation in mice after hindlimb ischemia. In a human study, we showed the expressions of circ-MBOAT2 and miR-495 were associated with myocardial perfusion improvement after revascularization of CTO. In conclusion, circ-MBOAT2 regulates angiogenesis via the miR-495/NOTCH1 axis and associates with myocardial perfusion in patients with CTO. Our findings suggest that circ-MBOAT2 and miR-495 may be potential therapeutic targets and prognostic factors for patients with CTO.

SerpinG1: A Novel Biomarker Associated With Poor Coronary Collateral in Patients With Stable Coronary Disease and Chronic Total Occlusion.

Background This study aimed to explore predictive biomarkers of coronary collateralization in patients with chronic total occlusion. Methods and Results By using a microarray expression profiling program downloaded from the Gene Expression Omnibus database, weighted gene coexpression network analysis was constructed to analyze the relationship between potential modules and coronary collateralization and screen out the hub genes. Then, the hub gene was identified and validated in an independent cohort of patients (including 299 patients with good arteriogenic responders and 223 patients with poor arteriogenic responders). Weighted gene coexpression network analysis showed that SERPING1 in the light-cyan module was the only gene that was highly correlated with both the gene module and the clinical traits. Serum levels of serpinG1 were significantly higher in patients with bad arteriogenic responders than in patients with good arteriogenic responders (472.53±197.16 versus 314.80±208.92 µg/mL; P<0.001) and were negatively associated with the Rentrop score (Spearman r=-0.50; P<0.001). Receiver operating characteristic curve analysis indicated that the area under the curve was 0.77 (95% CI, 0.72-0.81; P<0.001) for serum serpinG1 in prediction of bad arteriogenic responders. After adjusting for traditional cardiovascular risk factors, serum serpinG1 levels (per SD) remained an independent risk factor for bad arteriogenic responders (odds ratio, 2.20 [95% CI, 1.76-2.74]; P<0.001). Conclusions Our findings illustrate that SERPING1 screened by weighted gene coexpression network analysis was associated with poor collateralization in patients with chronic total occlusion.

LncRNA-LUNAR1 Levels Are Closely Related to Coronary Collaterals in Patients with Chronic Total Coronary Occlusion.

Coronary collaterals can effectively improve myocardial blood supply to the area of CTO (chronic total coronary occlusion) and can, thus, reduce infarct size. LUNAR1(leukemia-induced noncoding activator RNA-1) is a specific LncRNA regulated by Notch signaling that not only can enhance the expression of IGFR-1 but also can promote angiogenesis and cell survival. Here, we investigated the relationship between LncRNA-LUNAR1 levels in peripheral plasma and the formation of coronary collaterals. In total, 172 patients with CTO were enrolled and followed up for 12 months. Coronary collaterals were scored according to the Rentrop scoring system. Preclinical tests of tube formation were used to address the mechanisms behind the association between LncRNA-LUNAR1 and development of collaterals. Clinical data and inflammatory factors, including comorbidity, CD14++CD16- monocytes, and CCL2 (chemokine motif ligand 2), were compared and analyzed. Real-time PCR was used to detect the expression of LncRNA-LUNAR1 in peripheral blood plasma. The Rentrop score was positively correlated with LncRNA-LUNAR1 levels in patients with CTO (R = 0.47, p < 0.001). Tube formation assay proved the direct association between LncRNA-LUNAR1 and development of collaterals (p = 0.011). The univariate Kaplan-Meier analysis revealed that patients with low LncRNA-LUNAR1 expression exhibited worse clinical outcomes than those with high LncRNA-LUNAR1 levels (p = 0.008). Receiver operating characteristic (ROC) curve and correlation analysis further confirmed that LncRNA-LUNAR1 expression was closely related to chronic inflammatory diseases, especially diabetes (area = 0.644, p = 0.001; 95% CI, 0.562-0.726). Furthermore, both CD14++CD16- monocytes (r = - 0.37; p < 0.001) and CCL2 levels (r = - 0.35; p < 0.001) negatively affected the expression of LncRNA-LUNAR1. LncRNA-LUNAR1 expression was positively correlated with coronary collaterals in patients with CTO. Inflammatory factors, including CD14++CD16- monocytes and CCL2, may be risk factors affecting LncRNA-LUNAR1 expression.

Frequency and clinical outcomes of CYP2C19 genotype-guided escalation and de-escalation of antiplatelet therapy in a real-world clinical setting.

PURPOSE: To evaluate the frequency and clinical impact of switches in antiplatelet therapy following implementation of CYP2C19 genotyping after percutaneous coronary intervention (PCI). METHODS: The frequency of escalation (clopidogrel switched to prasugrel/ticagrelor) and de-escalation (prasugrel/ticagrelor switched to clopidogrel) was evaluated in 1063 PCI patients who underwent CYP2C19 genotyping. Risk of major adverse cardiovascular or cerebrovascular (MACCE) and bleeding events over one year was evaluated. RESULTS: Antiplatelet therapy switches were common (19%), with escalation (101/115: 88%) and de-escalation (77/84: 92%) occurring predominantly in patients with and without a CYP2C19 nonfunctional allele, respectively. Nonfunctional allele carriers initiated and continued on clopidogrel had a significantly higher risk of experiencing either a MACCE or bleeding event compared with those escalated to prasugrel/ticagrelor (52 vs. 19 events/100 patient-years; adjusted hazard ratio [HR] 2.89 [1.44-6.13], p = 0.003). Patients without a nonfunctional allele de-escalated to clopidogrel had no difference in risk compared with those initiated and continued on prasugrel/ticagrelor (21 vs. 19 events/100 patient-years; adjusted HR 1.13 [0.51-2.34], p = 0.751). CONCLUSION: CYP2C19-guided escalation and de-escalation is common in a real-world setting. Continuation of clopidogrel in nonfunctional allele carriers is associated with adverse outcomes. De-escalation to clopidogrel in patients without a nonfunctional allele appears safe and warrants prospective study.

Ovine Models of Congenital Heart Disease and the Consequences of Hemodynamic Alterations for Pulmonary Artery Remodeling.

The natural history of pulmonary vascular disease associated with congenital heart disease (CHD) depends on associated hemodynamics. Patients exposed to increased pulmonary blood flow (PBF) and pulmonary arterial pressure (PAP) develop pulmonary vascular disease more commonly than patients exposed to increased PBF alone. To investigate the effects of these differing mechanical forces on physiologic and molecular responses, we developed two models of CHD using fetal surgical techniques: 1) left pulmonary artery (LPA) ligation primarily resulting in increased PBF and 2) aortopulmonary shunt placement resulting in increased PBF and PAP. Hemodynamic, histologic, and molecular studies were performed on control, LPA, and shunt lambs as well as pulmonary artery endothelial cells (PAECs) derived from each. Physiologically, LPA, and to a greater extent shunt, lambs demonstrated an exaggerated increase in PAP in response to vasoconstricting stimuli compared with controls. These physiologic findings correlated with a pathologic increase in medial thickening in pulmonary arteries in shunt lambs but not in control or LPA lambs. Furthermore, in the setting of acutely increased afterload, the right ventricle of control and LPA but not shunt lambs demonstrates ventricular-vascular uncoupling and adverse ventricular-ventricular interactions. RNA sequencing revealed excellent separation between groups via both principal components analysis and unsupervised hierarchical clustering. In addition, we found hyperproliferation of PAECs from LPA lambs, and to a greater extent shunt lambs, with associated increased angiogenesis and decreased apoptosis in PAECs derived from shunt lambs. A further understanding of mechanical force-specific drivers of pulmonary artery pathology will enable development of precision therapeutics for pulmonary hypertension associated with CHD.

On the Mechanism of the Cardioprotective Action of σ1 Receptor Agonist Anxiolytic Fabomotizole Hydrochloride (Afobazole).

Original translational rat model of chronic heart failure provoked by experimental anterior transmural myocardium infarction was employed to examine the preventive action of anxiolytic Afobazole (15 mg/kg/day administered intraperitoneally during the first 15 days after coronary occlusion) on the development of the heart failure assessed in 3 months after infarction. Afobazole prevented the development of pathologic remodeling of the myocardium, maintained its inotropic function, and decreased the plasma level of brain natriuretic peptide known as a biochemical marker of chronic heart failure. In the myocardium, Afobazole down-regulated overexpression of the genes induced in chronic heart failure and assessed by corresponding RNA levels, which code angiotensin (AT1A-R), vasopressin (V1A-R), and glucocorticoid (GR) receptors as well as Epac2 protein. The revealed biochemical changes are consistent with the data on cardioprotective action of Afobazole.

Monocytic microRNA profile associated with coronary collateral artery function in chronic total occlusion patients.

An expansive collateral artery network is correlated with improved survival in case of adverse cardiac episodes. We aimed to identify cellular microRNAs (miRNA; miR) important for collateral artery growth. Chronic total occlusion (CTO) patients (n = 26) were dichotomized using pressure-derived collateral flow index (CFIp) measurements; high collateral capacity (CFIp > 0.39; n = 14) and low collateral (CFIp < 0.39; n = 12) capacity. MiRNA profiling via next generation sequencing from various monocyte phenotypes (freshly isolated monocytes, monocytes cultured without stimulant, or stimulation with lipopolysaccharide, interleukin 4, transforming growth factor beta-1, or interferon gamma) revealed significantly different miRNA expression patterns between high versus low collateral capacity patients. Validation by real-time polymerase chain reaction demonstrated significantly decreased expression of miR339-5p in all stimulated monocyte phenotypes of low collateral capacity patients. MiR339-5p showed significant correlation with CFIp values in stimulated monocytes. Ingenuity pathway analysis of predicted gene targets of miR339-5p and differential gene expression data from high versus low CFIp patients (n = 20), revealed significant association with STAT3 pathway, and also suggested a possible regulatory role for this signaling pathway. These results identify a novel association between miR339-5p and coronary collateral function. Future work examining modulation of miR339-5p and downstream effects on the STAT3 pathway and subsequent collateral vessel growth are warranted.

Carboxy-terminal deletion of the HDL receptor reduces receptor levels in liver and steroidogenic tissues, induces hypercholesterolemia, and causes fatal heart disease.

The HDL receptor SR-BI mediates the transfer of cholesteryl esters from HDL to cells and controls HDL abundance and structure. Depending on the genetic background, loss of SR-BI causes hypercholesterolemia, anemia, reticulocytosis, splenomegaly, thrombocytopenia, female infertility, and fatal coronary heart disease (CHD). The carboxy terminus of SR-BI (505QEAKL509) must bind to the cytoplasmic adaptor PDZK1 for normal hepatic-but not steroidogenic cell-expression of SR-BI protein. To determine whether SR-BI's carboxy terminus is also required for normal protein levels in steroidogenic cells, we introduced into SR-BI's gene a 507Ala/STOP mutation that produces a truncated receptor (SR-BIΔCT). As expected, the dramatic reduction of hepatic receptor protein in SR-BIΔCT mice was similar to that in PDZK1 knockout (KO) mice. Unlike SR-BI KO females, SR-BIΔCT females were fertile. The severity of SR-BIΔCT mice's hypercholesterolemia was intermediate between those of SR-BI KO and PDZK1 KO mice. Substantially reduced levels of the receptor in adrenal cortical cells, ovarian cells, and testicular Leydig cells in SR-BIΔCT mice suggested that steroidogenic cells have an adaptor(s) functionally analogous to hepatic PDZK1. When SR-BIΔCT mice were crossed with apolipoprotein E KO mice (SR-BIΔCT/apoE KO), pathologies including hypercholesterolemia, macrocytic anemia, hepatic and splenic extramedullary hematopoiesis, massive splenomegaly, reticulocytosis, thrombocytopenia, and rapid-onset and fatal occlusive coronary arterial atherosclerosis and CHD (median age of death: 9 wk) were observed. These results provide new insights into the control of SR-BI in steroidogenic cells and establish SR-BIΔCT/apoE KO mice as a new animal model for the study of CHD.

Inhibition of MicroRNA-383 Ameliorates Injury After Focal Cerebral Ischemia via Targeting PPARγ.

BACKGROUND: Peroxisome proliferator-activated receptor gamma (PPARγ) plays a critical role in protecting against distinct brain damages, including ischemia. Our previous data have shown that the protein level of PPARγ is increased in the cortex after middle cerebral artery occlusion (MCAO); PPARγ up-regulation contributes to PPARγ activation and is effective in reducing ischemic damage to brain. However, the regulatory mechanism of PPARγ after focal cerebral ischemia in rats is still unclear. In this study, we evaluated the effect of microRNA on PPARγ in rats subjected to MCAO. METHODS: Focal cerebral ischemia was established by surgical middle cerebral artery occlusion; the protein level of PPARγ was detected by Western blotting; the level of microRNA-383 (miR-383) was quantified by real-time PCR; the neurological outcomes were defined by infarct volume and neurological deficits. Luciferase assay was used to identify the luciferase activities of PPARγ and miR-383. RESULTS: We showed here that miR-383 level was down-regulated in the ischemic hemisphere of rats 24h after MCAO. Overexpression of miR-383 by miR-383 agomir increased infarct volume and aggravated neurological damage. Administration of miR-383 antagomir had the opposite effects. Furthermore, we found that PPARγ protein was down-regulated by miR-383 overexpression, and up-regulated by miR-383 inhibition both in rat model of MCAO and in primary culture cells. Finally, we found that miR-383 suppressed the luciferase activity of the vector carrying the 3'UTR of PPARγ, whereas mutation of the binding sites relived the repressive effect of miR-383. CONCLUSION: Our study demonstrated that miR-383 may play a key role in focal cerebral ischemia by regulating PPARγ expression at the post-transcriptional level, and miR-383 may be a potential therapeutic target for stroke.

Impact of exercise stress testing on diagnostic gene expression in patients with obstructive and nonobstructive coronary artery disease.

A blood-based gene expression test can diagnose obstructive coronary artery disease (CAD). The test is sensitive to inflammatory and immune processes associated with atherosclerosis. Acute exercise engages short-term inflammatory pathways, and exercise stress testing may affect results of gene expression testing during the same diagnostic workup. The objective of this study was to evaluate the effect of exercise on diagnostic gene expression testing. Ten patients with obstructive CAD (≥50% stenosis) and 10 with no/minimal CAD (≤20% stenosis) were identified by angiography. Blood samples for gene expression were obtained at baseline, peak exercise, 30 to 60 minutes after testing, and 24 to 36 hours after testing. Core-lab gene expression analysis yielded raw gene expression scores (GES) for each time point. Linear models were used to estimate changes in GES, adjusting for CAD status and other covariates. GES increased during peak exercise across both genders, with no significant differences as a function of CAD status. The overall adjusted mean GES increase at peak exercise was 0.29 (95% confidence interval 0.22 to 0.36; p <0.001). GES after exercise were not significantly different from baseline. The change in gene expression levels during peak exercise may reflect a transient inflammatory response to acute exercise that may be independent of patient gender or CAD status. In conclusion, CAD GES increase at peak exercise testing and rapidly return to baseline. Such may reflect a transient inflammatory response to acute exercise independent of gender or extent of CAD.

[ABCG1 transporter gene expression in peripheral blood mononuclear cells of patients with atherosclerosis].

Accumulation of cholesterol in arterial wall macrophages is a main hallmark of atherosclerosis. The ABCG1 transporter mediates cholesterol efflux to high density lipoproteins (HDL) and plays an important role in macrophage foam cell formation. The goal of our study was to investigate the potential role of ABCG1 in atherosclerosis development in humans. ABCG1 gene expression has been examined in leukocytes, monocytes and monocyte-derived macrophages of patients with atherosclerosis and in the control group. Real time PCR and Western blotting were used to determine ABCG1 mRNA and ABCG1 protein levels. Monocyte ABCG1 mRNA level was inversely correlated with the rate of artery occlusion (r = -0.45, P = 0.016). Patients with 100% artery occlusions had decreased monocyte ABCG1 mRNA levels compared to patients who had smaller plaques and controls (P < 0.05). ABCG1 mRNA (P < 0.001) and ABCG1 protein (P < 0.05) levels in macrophages of patients with coronary artery stenosis were significantly reduced compared to the control group. No significant correlation between the ABCG1 gene expression in mononuclear cells and HDL cholesterol concentration has been found. Our study suggests that decrease in the ABCG1 gene expression in macrophages is associated with atherosclerosis.

CYP2C19 genotype has a greater effect on adverse cardiovascular outcomes following percutaneous coronary intervention and in Asian populations treated with clopidogrel: a meta-analysis.

BACKGROUND: The degree to which cytochrome P450 (CYP) 2C19 genotype influences the effectiveness of clopidogrel remains uncertain because of considerable heterogeneity in results between studies and potential publication bias. Clopidogrel indication and ethnic population have been proposed to influence the effect of CYP2C19 genotype. METHODS AND RESULTS: A systematic review was undertaken up to 14 November 2013. Meta-analysis of the CYP2C19 genotype effect was stratified by the predominant clopidogrel indication (percutaneous coronary intervention [PCI] versus non-PCI) and ethnic population (white versus Asian) of each primary study. The primary analysis was restricted to studies with ≥500 participants, which comprised 24 studies and a total of 36 076 participants. The association between carriage of ≥1 CYP2C19 loss-of-function (LoF) allele and major cardiovascular outcomes differed significantly (P<0.001) between studies of whites not undergoing PCI (relative risk 0.99 [95% confidence interval, 0.84-1.17]; n=7043), whites undergoing PCI (1.20 [1.10-1.31]; n=19,016), and Asians undergoing PCI (1.91 [1.61-2.27]; n=10,017). Similar differences were identified in secondary analyses of 2 CYP2C19 LoF alleles, stent thrombosis outcomes, and studies with ≥200 participants. Minimal heterogeneity was apparent between studies of Asian populations. CONCLUSIONS: The reported association between CYP2C19 LoF allele carriage and major cardiovascular outcomes differs based on the ethnic population of the study and, to a lesser extent, the clopidogrel indication. This is potentially of major importance given that over 50% of Asians carry ≥1 CYP2C19 LoF alleles.

Dual-contrast molecular imaging allows noninvasive characterization of myocardial ischemia/reperfusion injury after coronary vessel occlusion in mice by magnetic resonance imaging.

BACKGROUND: Inflammation and myocardial necrosis play important roles in ischemia/reperfusion injury after coronary artery occlusion and recanalization. The detection of inflammatory activity and the extent of myocardial necrosis itself are of great clinical and prognostic interest. We developed a dual, noninvasive imaging approach using molecular magnetic resonance imaging in an in vivo mouse model of myocardial ischemia and reperfusion. METHODS AND RESULTS: Ischemia/reperfusion injury was induced in 10-week-old C57BL/6N mice by temporary ligation of the left anterior descending coronary artery. Activated platelets were targeted with a contrast agent consisting of microparticles of iron oxide (MPIOs) conjugated to a single-chain antibody directed against a ligand-induced binding site (LIBS) on activated glycoprotein IIb/IIIa (LIBS-MPIOs). After injection and imaging of LIBS-MPIOs, late gadolinium enhancement was used to depict myocardial necrosis; these imaging experiments were also performed in P2Y12 (-/-) mice. All imaging results were correlated to immunohistochemistry findings. Activated platelets were detectable by magnetic resonance imaging via a significant signal effect caused by LIBS-MPIOs in the area of left anterior descending coronary artery occlusion 2 hours after reperfusion. In parallel, late gadolinium enhancement identified the extent of myocardial necrosis. Immunohistochemistry confirmed that LIBS-MPIOs bound significantly to microthrombi in reperfused myocardium. Only background binding was found in P2Y12 (-/-) mice. CONCLUSIONS: Dual molecular imaging of myocardial ischemia/reperfusion injury allows characterization of platelet-driven inflammation by LIBS-MPIOs and myocardial necrosis by late gadolinium enhancement. This noninvasive imaging strategy is of clinical interest for both diagnostic and prognostic purposes and highlights the potential of molecular magnetic resonance imaging for characterizing ischemia/reperfusion injury.

The thromboprotective effect of bortezomib is dependent on the transcription factor Kruppel-like factor 2 (KLF2).

Multiple myeloma confers a high risk for vascular thrombosis, a risk that is increased by treatment with immunomodulatory agents. Strikingly, inclusion of the proteasome inhibitor bortezomib reduces thrombotic risk, yet the molecular basis for this observation remains unknown. Here, we show that bortezomib prolongs thrombosis times in the carotid artery photochemical injury assay in normal mice. Cell-based studies show that bortezomib increases expression of the transcription factor Kruppel-like factor 2 (KLF2) in multiple cell types. Global postnatal overexpression of KLF2 (GL-K2-TG) increased time to thrombosis, and global postnatal deletion of KLF2 (GL-K2-KO) conferred an antiparallel effect. Finally, studies in GL-K2-KO mice showed that the thromboprotective effect of bortezomib is KLF2 dependent. These findings identify a transcriptional basis for the antithrombotic effects of bortezomib.

G-CSF induced arteriogenesis in humans: molecular insights into a randomized controlled trial.

AIMS: Recent data have demonstrated the feasibility of therapeutic induction of coronary collateral growth (arteriogenesis); however, mechanisms of action of such therapeutic collateral stimulation in humans are unknown. The aim of this study was to evaluate potential mechanisms, especially the involvement of arteriogenesis-relevant genes. METHODS AND RESULTS: A total of 52 patients were randomized into two groups: subcutaneous G-CSF (10 µg/kg; n=26) or placebo (n=26). Before and after this 2-week treatment, collateral-flow index (CFI) was determined by simultaneous measurement of mean aortic, distal coronary occlusive and central venous pressure. CD34+ endothelial progenitor cells (EPC) and monocytes were quantified before, during and after treatment; gene-expression analysis of monocytes was performed with real-time polymerase chain reaction (RT-PCR). G-CSF lead to a significant increase of EPC and monocytes (4.8 and 2.6 fold, p < 0.05); for both cell types, the extent of increase correlated with CFI increase (r=0.23 and 0.14, p < 0.05). G-CSF also induced a change in gene expression of pro-and anti-arteriogenic genes in monocytes. Among nine assessed genes, three were found to be differentially regulated (IL8, JAK2, and PNPLa4; p < 0.05). CONCLUSIONS: The mechanism of induction of collateral growth by G-CSF is related to an increase of EPC and of peripheral monocytes. It also leads to a change toward a pro-arteriogenic gene expression in peripheral monocytes.

Plasminogen activator inhibitor-1 5G/5G genotype is associated with early spontaneous recanalization of the infarct-related artery in patients presenting with acute ST-elevation myocardial infarction.

BACKGROUND: We aimed to examine the association between plasminogen activator inhibitor-1 (PAI-1) genetic polymorphism and early spontaneous recanalization in patients presenting with acute ST-elevation myocardial infarction. METHODS: Patients admitted to our emergency department with ST-elevation myocardial infarction in the first 6 h of symptom onset were included. An immediate primary percutaneous coronary intervention was performed. Patients were grouped according to the initial patency of the infarct-related artery (IRA) as follows: total occlusion (TO) group [Thrombolysis in Myocardial Infarction (TIMI) 0-1 flow in the IRA], partial recanalization group (TIMI 2 flow in the IRA), and complete recanalization (CR) group (TIMI 3 flow in the IRA). PAI-1 4G/5G polymorphism was detected using the real-time PCR method. RESULTS: There were 107 patients in the TO group, 30 patients in the partial recanalization group, and 45 patients in the CR group. When we evaluated degrees of patency according to the PAI-1 genotype, TO of the IRA was the highest in patients with the PAI 4G/4G genotype (PAI-1 4G/4G: 66.7%, PAI-1 4G/5G: 65.9%, PAI-1 5G/5G: 40.4%) and CR of the IRA was the highest in patients with the PAI 5G/5G genotype (PAI-1 5G/5G: 38.5%, PAI-1 4G/5G: 19.8%, PAI-1 4G/4G: 17.9%). The distribution of genotypes in different degrees of patency of IRA was statistically significant (P=0.029). In logistic regression analysis, the PAI-1 5G/5G genotype was associated independently with the spontaneous CR of the IRA (odds ratio: 2.875, 95% confidence interval [1.059-7.086], P=0.038). CONCLUSION: Patients with the PAI-1 5G/5G genotype seem to be luckier than others in terms of early spontaneous recanalization of the IRA. Further prospective studies with large patient populations are required for more precise results.

Cytochrome P450 2J2 is protective against global cerebral ischemia in transgenic mice.

Cytochrome P450 epoxygenase metabolites of arachidonic acid, EETs, have multiple cardiovascular effects, including reduction of blood pressure, protection against myocardial ischemia-reperfusion injury, and attenuation of endothelial apoptosis. This study investigated the hypothesis that transgenic mice with endothelial overexpression of CYP2J2 (Tie2-CYP2J2-Tr) would be protected against global cerebral ischemia induced by bilateral common carotid artery occlusion (BCCAO) and action mechanisms of EETs on cerebral ischemia in cultures of astrocytes exposed to oxygen-glucose deprivation (OGD). Tie2-CYP2J2-Tr mice had significantly increased CYP2J2 expression, increased 14,15-EET production, increases regional cerebral blood flow (rCBF) and microvascular density, decreased ROS production, decreased brain infarct size and apoptosis after ischemia compared to wild type mice, these were associated with increased activation of the PI3K/AKT and apoptosis-related protein in ischemic brain. Addition of exogenous EETs or CYP2J2 transfection attenuated OGD-induced apoptosis in astrocytes via activation of PI3K/AKT and anti-apoptosis pathways. However, these effects were reduced by pretreatments with inhibitor of the PI3K (LY294002) and 14,15-EET (14,15-EEZE), respectively. These results indicate that CYP2J2 overexpression exerts marked neuroprotective effects against ischemic injury by a mechanism linked to increased level of circulating EETs and increases CBF and reduction of apoptosis.

Common haplotype of interleukin-6 gene is associated with chronic total occlusions of coronary arteries.

AIM: Genetic polymorphisms in genes coding for cytokines may predispose patients to coronary artery disease and chronic total occlusion (CTO) of coronary arteries. The aim of the study was to evaluate association of common genetic variations of interleukins (IL) with CTOs. METHODS: We reviewed coronary angiograms and evaluated presence of CTOs in 684 consecutive patients with no history of revascularization. The following genetic variations were analyzed: IL-1B +3954 C>T, IL-1B -511 C>T, IL-1RN VNTR and haplotypes of IL-6 encompassing IL-6 -596 G>A, IL-6 -572 G>C, IL-6 -373 AnTn and IL-6 -174 G>C polymorphisms. RESULTS: In 254 patients (37.1%) CTO of at least one artery was found. We observed nine IL-6 haplotypes, of which five were common (>1%) and one (GG9/12G, n=8) was not previously reported in literature. The most prevalent IL-6 haplotype (AG8/12C or Hap*1, 49.5%) correlated with CTOs, that were present in 31.5%, 36.5% and 44.5% of patients with none, one and two Hap*1, respectively (OR [95%CI] 1.252 [0.844-1.856] and 1.746 [1.104-2.762] for heterozygots and homozygots, respectively). In multivariate analysis this association became non-significant (OR [95%CI] 1.202 [0.805-1.796] and 1.529 [0.955-2.450]). In subgroup analysis Hap*1 was, however, associated with CTOs in the left anterior descending artery (p for trend 0.032) and the left circumflex artery (P=0.047), but not in the right coronary artery (p=0.799). In multivariate analysis CTOs of the left coronary arteries were associated only with total cholesterol (OR [95%CI] 1.170 [1.020-1.341]) and Hap*1 (OR [95%CI] 1.469 [0.914-2.361] and 1.970 [1.151-3.372] for heterozyogots and homozygots, respectively). CONCLUSION: Our study suggests that common IL-6 haplotype (AG8/12C or Hap*1) is associated with development of CTOs in left coronary arteries.

LOX-1 abrogation reduces cardiac hypertrophy and collagen accumulation following chronic ischemia in the mouse.

We hypothesized that lectin-like oxidized LDL receptor-1 (LOX-1) deletion may inhibit oxidative stress signals, reduce collagen accumulation and attenuate cardiac remodeling after chronic ischemia. Activation of LOX-1 plays a significant role in the development of inflammation, apoptosis and collagen signals during acute ischemia. Wild-type and LOX-1 knockout (KO) mice were subjected to occlusion of left coronary artery for 3 weeks. Markers of cardiac hypertrophy, fibrosis-related signals (collagen IV, collagen-1 and fibronectin) and oxidant load (nicotinamide adenine dinucleotide phosphate oxidase expression, activity of mitogen-activated protein kinases and left ventricular (LV) tissue thiobarbituric acid reactive substances) were analyzed. In in vitro experiments, HL-1 cardiomyocytes were transfected with angiotensin II (Ang II) type 1 receptor (AT1R) or type 2 receptor (AT2R) genes to determine their role in the cardiomyocyte hypertrophy. LOX-1 KO mice had 25% improvement in survival over the 3-week period of chronic ischemia. LOX-1 deletion reduced collagen deposition and cardiomyocyte hypertrophy (∼75%) in association with a decrease in oxidant load and AT1R upregulation (all P<0.05). The LOX-1 KO mice hearts exhibited a disintegrin and metalloproteinase 10 (ADAM10) and a disintegrin and metalloproteinase 17 (ADAM17) expression and matrix metalloproteinase 2 activity, and increased AT2R expression (P<0.05). Attenuation of cardiac remodeling was associated with improved cardiac hemodynamics (LV ±dp/dt and cardiac ejection fraction). In vitro studies showed that it is AT1R, and not AT2R overexpression that induces cardiomyocyte hypertrophy. We demonstrate for the first time that LOX-1 deletion reduces oxidative stress and related intracellular signaling, which leads to attenuation of the positive feedback loop involving AT1R and LOX-1. This results in reduced chronic cardiac remodeling.

Galectin-2 expression is dependent on the rs7291467 polymorphism and acts as an inhibitor of arteriogenesis.

AIMS: In patients with obstructive coronary artery disease (CAD), the growth of collateral arteries, i.e. arteriogenesis, can preserve myocardial tissue perfusion and function. Monocytes modulate this process, supplying locally the necessary growth factors and degrading enzymes. Knowledge on factors involved in human arteriogenesis is scarce. Thus, the aim of the present study is to identify targets in monocytes that are critical for arteriogenesis in patients with CAD. METHODS AND RESULTS: A total of 50 patients with a chronic total coronary occlusion were dichotomized according to their collateral flow index. From each patient, RNA was isolated from unstimulated peripheral blood monocytes, monocytes stimulated by lipopolysaccharide (LPS) or interleukin (IL)-4, and from macrophages. Increased mRNA expression of galectin-2 was found in three out of four monocytic cell types of patients with a low capacity of the collateral circulation (P= 0.03 for unstimulated monocytes; P= 0.02 for LPS-stimulated monocytes; P= 0.20 for IL-4-stimulated monocytes; P= 0.02 for macrophages). Additionally, galectin-2 mRNA expression was significantly associated with the rs7291467 polymorphism in LGALS2 encoding galectin-2 in all four monocytic cell types. Patient with the rs7291467 CC genotype displayed highest galectin-2 expression, and also tended to have a lower arteriogenic response. To evaluate the effect of galectin-2 on arteriogenesis in vivo, we used a murine hindlimb model. Treatment with galectin-2 markedly impaired the perfusion restoration at Day 7. CONCLUSION: Collectively, these results identify galectin-2 as a novel inhibitor of arteriogenesis. Modulation of galectin-2 may constitute a new therapeutic strategy for the stimulation of arteriogenesis in patients with CAD.