Sphingosine 1­phosphate induced by hypoxia increases the expression of PAI­1 in HepG2 cells via HIF­1α.

Our group has recently reported that in the immortal human HepG2 liver cell line, sphingosine 1­phosphate (S1P) increases transcription of plasminogen activator inhibitor type­1 (PAI­1), the major physiological inhibitor of fibrinolysis, within 4 h. The present study aimed to elucidate the molecular mechanisms underlying this effect. PAI­1 expression was measured by reverse transcription­quantitative polymerase chain reaction and immunoblotting. It was demonstrated that S1P increased PAI­1 promoter activity but did not increase the activity of promoters lacking the hypoxia responsive element (HRE) 2. In addition, S1P transiently increased the concentration of hypoxia inducible factor (HIF)­1α, a transcription factor capable of binding to HRE. When HIF­1α was knocked down, the induction of transcription of PAI­1 by S1P was no longer observed. Sphingosine kinase (SPHK) activity is increased by hypoxia. It was demonstrated that increases in the concentration of the HIF­1α protein induced by hypoxia were prevented by treatment with SPHK inhibitor or S1P receptor antagonists. Thus, modification of the induction of HIF­1α by S1P, leading to increased transcription of PAI­1, may be an attractive therapeutic target for thrombosis and consequent inhibition of fibrinolysis associated with hypoxia.

Differential effects of insulin sensitization and insulin provision treatment strategies on concentrations of circulating adipokines in patients with diabetes and coronary artery disease in the BARI 2D trial.

AIMS: To determine the effects of insulin sensitization (IS) and insulin provision (IP) treatment strategies on adipokines associated with cardiovascular disease in patients with type 2 diabetes mellitus and coronary artery disease in the Bypass Angioplasty Revascularization Investigation 2 Diabetes trial (BARI 2D). METHODS AND RESULTS: Changes in adipokine levels were compared in patients with type 2 diabetes mellitus and coronary artery disease randomized to IS (n = 1037) versus IP (n = 1019) treatment strategies in BARI 2D. Circulating concentrations of leptin, adiponectin, monocyte chemoattractant protein-1, tumor necrosis factor-alpha, interleukin 6 and C-reactive protein were evaluated at baseline and one year. IS and IP treatment strategies exerted significant (p < 0.0001) differential effects on: leptin (IS: 0.02% decrease, p = 0.01; IP: 13% increase, p < 0.0001); adiponectin (IS: 73% increase, p < 0.0001; IP: no change, p = 0.52); interleukin 6 (IS: 14% decrease, p < 0.0001; IP: no change, p = 0.68). Changes in monocyte chemoattractant protein-1 and tumor necrosis factor-alpha were not statistically different between groups. C-reactive protein decreased, but the effect was significantly greater in the IS group (-32%, p < 0.0001) than in the IP group (-5%, p = 0.0005). CONCLUSION: The IS and IP treatment strategies exerted divergent effects on adipokine and inflammatory profile in patients with type 2 diabetes mellitus and coronary artery disease. The IS treatment strategy-induced changes may be more favorable than the IP treatment strategy regarding cardiovascular pathophysiology.

Human epicardial cell-conditioned medium contains HGF/IgG complexes that phosphorylate RYK and protect against vascular injury.

AIMS: The aim of this study was to evaluate the paracrine activity of human epicardial-derived cells (hEPDCs) to screen for secreted vasoprotective factors and develop therapeutics to treat vascular reperfusion injury. METHODS AND RESULTS: Epicardial cells support cardiac development, repair, and remodelling after injury in part, through paracrine activity. We hypothesized that secreted ligands from hEPDCs would protect vascular integrity after myocardial infarction (MI) with reperfusion. During simulated ischaemia in culture (24-48 h), concentrated hEPDC-conditioned medium (EPI CdM) increased survival of primary cardiac endothelial cells. In a rat MI model, EPI CdM treatment reduced vascular injury in vivo after reperfusion. By phospho-receptor tyrosine kinase (RTK) arrays, ELISA, and neutralizing antibody screens, we identified hepatocyte growth factor (HGF) as a key vasoprotective factor in EPI CdM. Unexpectedly, we observed that some of the HGF in EPI CdM formed complexes with polyclonal IgG. Following reperfusion, preparations of HGF/IgG complexes provided greater vascular protection than free HGF with IgG. HGF/IgG complexes localized to blood vessels in vivo and increased HGF retention time after administration. In subsequent screens, we found that 'related to tyrosine kinase' (RYK) receptor was phosphorylated after exposure of cardiac endothelial cells to HGF/IgG complexes, but not to free HGF with IgG. The enhanced protection conferred by HGF/IgG complexes was lost after antibody blockade of RYK. Notably, the HGF/IgG complex is the first 'ligand' shown to promote phosphorylation of RYK. CONCLUSION: Early treatment with HGF/IgG complexes after myocardial ischaemia with reperfusion may rescue tissue through vasoprotection conferred by c-Met and RYK signalling.

Priming with ligands secreted by human stromal progenitor cells promotes grafts of cardiac stem/progenitor cells after myocardial infarction.

Transplantation of culture-expanded adult stem/progenitor cells often results in poor cellular engraftment, survival, and migration into sites of tissue injury. Mesenchymal cells including fibroblasts and stromal cells secrete factors that protect injured tissues, promote tissue repair, and support many types of stem/progenitor cells in culture. We hypothesized that secreted factors in conditioned medium (CdM) from adult bone marrow-derived multipotent stromal cells (MSCs) could be used to prime adult cardiac stem/progenitor cells (CSCs/CPCs) and improve graft success after myocardial infarction (MI). Incubation of adult rat CPCs in CdM from human MSCs isolated by plastic adherence or by magnetic sorting against CD271 (a.k.a., p75 low-affinity nerve growth factor receptor; p75MSCs) induced phosphorylation of STAT3 and Akt in CPCs, supporting their proliferation under normoxic conditions and survival under hypoxic conditions (1% oxygen). Priming CSCs with 30× p75MSC CdM for 30 minutes prior to transplantation into subepicardial tissue 1 day after MI markedly increased engraftment compared with vehicle priming. Screening CdM with neutralizing/blocking antibodies identified connective tissue growth factor (CTGF) and Insulin as key factors in p75MSC CdM that protected CPCs. Human CTGF peptide (CTGF-D4) and Insulin synergistically promoted CPC survival during hypoxia in culture. Similar to CdM priming, priming of CSCs with CTGF-D4 and Insulin for 30 minutes prior to transplantation promoted robust engraftment, survival, and migration of CSC derivatives at 1 week and 1 month after MI. Our results indicate that short-term priming of human CSCs with CTGF-D4 and Insulin may improve graft success and cardiac regeneration in patients with MI.

Improved spatial resolution and electrogram wave direction independence with the use of an orthogonal electrode configuration.

To improve spatial resolution in recordings of intra-cardiac electrograms we characterized the utility of a novel configuration of two recording electrodes arranged perpendicularly to the endocardial surface. We hypothesized that this configuration denoted as orthogonal close unipolar (OCU) would combine advantages of conventional unipolar and contact bipolar (CBP) configurations. Electrical excitation was simulated in a computational model as arising from dipole current or from multi-wavelet reentry sources. Recordings were calculated for electrode tips 1 mm above the plane of the heart. Analogous recordings were obtained from swine hearts. Electrograms recorded with CBP showed strong dependence on orientation of the electrode pair with respect to the direction of spread of tissue excitation. By contrast, OCU recordings exhibited no directional dependence. OCU was significantly superior to CBP with respect to avoidance of far-field confounding of local tissue activity; the average far-field/near-field ratios for CBP and OCU were 0.09 and 0.05, respectively, for the simulated dipole current sources. In the swine hearts the ratios of ventricular to atrial signals for CBP and OCU were 0.15 ± 0.07 and 0.08 ± 0.09, respectively (p < 0.001). The difference between the actual dominant frequency in the tissue and that recorded by the electrodes was 0.44 ± 0.33 Hz for OCU, 0.58 ± 0.40 Hz for unipolar, and 0.62 ± 0.46 Hz for CBP. OCU confers improved spatial resolution compared with both unipolar and CBP electrode configurations. Unlike the case with CBP, OCU recordings are independent of excitation wave-front direction.

Increased plasma sphingosine-1-phosphate in obese individuals and its capacity to increase the expression of plasminogen activator inhibitor-1 in adipocytes.

OBJECTIVES: Concentrations of plasminogen activator inhibitor-1 (PAI-1) are increased in obese individuals. One source of PAI-1 is adipocytes. Hypoxia develops within adipose tissue as it expands, presumably contributing to increased levels of sphingosine-1-phosphate (S1P). S1P is a breakdown product of sphingosine, ubiquitous in cell membranes. We have shown previously that S1P increases the expression of PAI-1 in human liver-derived cell line. In the present study, we aimed to determine whether hypoxia induces S1P in adipocytes, thereby potentially contributing to an increase in PAI-1 and hence constraints on fibrinolysis associated with obesity. MATERIALS AND METHODS: Mouse 3T3-L1 adipocytes were exposed to CoCl2 to simulate hypoxia. Assays were performed for PAI-1 mRNA (quantitative PCR) and S1P (high-performance liquid chromatography). RESULTS: The physiologic concentration of S1P increased PAI-1 mRNA expression. The S1P2 receptor antagonist attenuated the increase in PAI-1. Adipocytes expressed sphingosine kinase 1/2 (SPHK1/2) and S1P lyase, key enzymes involved in S1P production and degradation. Hypoxia increased SPHK activity and decreased S1P lyase mRNA. Hypoxia reduced cytosolic sphingosine and increased S1P release into conditioned medium. Inhibitors of ABCA1 and ABCC1 reduced the release of S1P into conditioned media. In obese patients with uncomplicated dyslipidemia and hypertension, plasma S1P was increased compared with that in nonobese and lean individuals. CONCLUSION: Hypoxia in adipose tissue of obesity can promote elaboration of S1P that binds to S1P2 receptors in an autocrine or a paracrine manner. S1P potentially contributes toward increased expression of PAI-1 and consequent constraints on fibrinolysis. S1P production and extracellular transport provide an attractive target for therapy to attenuate impaired fibrinolysis associated with obesity.

The efficacy and tolerability of azilsartan in obese insulin-resistant mice with left ventricular pressure overload.

Angiotensin II receptor blockers (ARBs) are used widely for the treatment of heart failure. However, their use in obese and insulin-resistant patients remains controversial. To clarify their potential efficacy in these conditions, we administered azilsartan medoxomil (azilsartan), a prodrug of an angiotensin II receptor blocker to mice fed a high-fat diet (HFD) with left ventricular (LV) pressure overload (aortic banding). LV fibrosis (hydroxyproline), cardiac plasminogen activator inhibitor-1 (PAI-1; a marker of profibrosis), and creatine kinase (a marker of myocardial viability and energetics) were assessed. LV wall thickness and cardiac function were assessed echocardiographically. Mice given a HFD were obese and insulin resistant. Their LV hypertrophy was accompanied by greater LV PAI-1 and reduced LV creatine kinase compared with normal diet controls. Drug treatment reduced LV wall thickness, hypertrophy, and PAI-1 and increased cardiac output after aortic banding compared with results in HFD vehicle controls. Thus, azilsartan exerted favorable biological effects on the hearts of obese insulin-resistant mice subjected to LV pressure overload consistent with its potential utility in patients with analogous conditions.

The efficacy and tolerability of azilsartan in mice with left ventricular pressure overload or acute myocardial infarction.

Angiotensin II receptor blockers (ARBs) are used for the treatment of patients with heart failure and hypertension. Yet their safety has been questioned by some who observed delayed cardiac healing and scar thinning after myocardial infarction (MI). To clarify potential efficacy and safety of ARBs, we administered Azilsartan medoxomil, a prodrug of an ARB (Takeda Pharmaceutical Company Limited), assessed cardiac fibrosis (hydroxyproline content), left ventricular (LV) wall thickness (premortem echocardiography and caliper measurement at necropsy), and LV mass and cardiac function with high-resolution ultrasound in mice with either surgically induced LV pressure overload (aortic banding) or acute MI. Drug-treated aortic-banded mice exhibited less LV wall thickness, hypertrophy, and dilation compared with that exhibited by controls. Survival in drug-treated MI mice was greater though not significantly. Drug-treated mice with acute MI exhibited less cardiomyocyte injury reflected by LV creatine kinase content and less LV hypertrophy and dilation. Thus, Azilsartan exerted favorable biological effects on the hearts of mice subjected to LV pressure overload or MI without compromising survival consistent with its potential utility and tolerability in patients with analogous conditions.

Attenuation of cardiac vascular rhexis: a promising therapeutic target.

BACKGROUND: We have previously identified a phenomenon that we called vascular rhexis (VR) after coronary occlusion in mice. To explore its potential pathogenetic impact on the destruction of vessels described, its potential universality, and its characterization as a scaffolding for evaluating amelioration, we studied Sprague-Dawley rats from which multiple blood samples and robust tissue samples can be obtained. METHODS: Rats were subjected to nonsustained coronary occlusion for 15 min, 1 h, 90 min, 3 h, or 4 h, followed by reperfusion. Soluble fractions of left ventricular (LV) homogenates were obtained 48 h later and assayed by western blotting for quantification of α-smooth muscle actin, diminution of which has been immunohistochemically shown to reflect delineation of VR. The functional integrity of vessel walls was assessed 24 h after induction of ischemia for selected intervals, followed by reperfusion using fluorescein isothiocyanate-tagged albumin injected through the tail vein 2 h before harvesting tissue. RESULTS: Nonsustained coronary occlusion for 1 h initiated VR, evident 47 h after reperfusion; however, more brief ischemic insults did not. Loss of functional integrity was evident as judged from increases in extravasated fluorescein isothiocyanate-conjugated albumin in LV walls. CONCLUSION: Because VR occurs early and may be ameliorated by interventions that can be initiated soon after the onset of nonsustained ischemia before myocardial cell death is substantial, its amelioration is an attractive target for diminution of late negative LV remodeling associated with the 'no reflow' phenomenon to which it can contribute.

Posttranscriptional regulation of expression of plasminogen activator inhibitor type-1 by sphingosine 1-phosphate in HepG2 liver cells.

Altered expression of plasminogen activator inhibitor type-1 (PAI-1), a major physiologic inhibitor of fibrinolysis, is implicated in the progression of atherosclerosis. Sphingosine 1-phosphate (S1P) regulates expression of diverse genes and alters expression of PAI-1 in several types of cells. However, the nature of posttranscriptional regulation of expression of PAI-1 by S1P has not yet been thoroughly elucidated. The present study was undertaken to determine whether S1P has important effects on the posttranscriptional regulation of PAI-1 expression. To evaluate this possibility, we determined promoter activity, mRNA levels, 3'-untranslated region (UTR) activity, and protein levels of PAI-1 in HepG2 cells. S1P increased PAI-1 promoter activity and the expression of PAI-1 mRNA within 4h of exposure. It decreased the expression of PAI-1 mRNA and the accumulation of PAI-1 protein into the media in 24h. Human PAI-1 mRNA exists in two subspecies (3.2 and 2.2kb). S1P decreased the baseline luciferase activity of the 1kb fragment of the 3' terminus (+2177 to 3176nt) of the 3'-UTR of the 3.2kb PAI-1 mRNA [3'-UTR (+2177-3176)]. S1P decreased expression of PAI-1 protein, presumably by regulating PAI-1 expression at the posttranscriptional level thereby affecting mRNA stability. SERPINE1 mRNA binding protein (SERBP1) and ARE3 in the 3'-UTR were involved in the posttranscriptional regulation by S1P. Our data suggest that S1P can destabilize 3.2kb PAI-1 mRNA through specific effects on the 3'-UTR. These effects appear to involve SERBP1 leading to decreased expression of PAI-1 protein.

Toward a comprehensive approach to pharmacoinvasive therapy for patients with ST segment elevation acute myocardial infarction.

What exactly is "pharmacoinvasive therapy" for treatment of patients with ST segment elevation myocardial infarction (STEMI)? When this term was introduced in 2003, it addressed the need for clinical trials besides those comparing fibrinolysis with primary percutaneous coronary intervention (PCI). Primary PCI is recognized as the best strategy for treatment of patients for whom it is applicable. However, use of fibrinolytic drugs initially is necessary in many patients for logistic reasons. Studies of pharmacoinvasive therapy addressed the question of what should be done after initial fibrinolysis. Confusion of the terms pharmacoinvasive therapy, facilitated PCI, rescue PCI, and delayed invasive approaches has obscured the principles that have emerged from such studies. In our view, a uniform conceptualization of pharmacoinvasive therapy emerges on the basis of three key considerations--transfer time, initial pharmacologic therapy, and time to PCI. We propose the following definition: Pharmacoinvasive therapy is the treatment of choice for patients with STEMI who require greater than a 60 min transfer time to a PCI center. It entails immediate use of full doses of fibrinolytic agents followed by prompt transfer to a PCI center and a plan to implement PCI within 2-12 h of the time of onset of initial therapy.

Modulators of induction of plasminogen activator inhibitor type-1 in HepG2 cells by transforming growth factor-ß.

OBJECTIVE: An increased expression of plasminogen activator inhibitor type-1 (PAI-1) has been implicated in accelerating atherogenesis and coronary artery disease in patients with type 2 diabetes. Transforming growth factor (TGF)-ß increases its expression. An increased PAI-1 appears to predispose also to augmented fibrosis potentially contributing to negative left ventricular remodeling and heart failure after myocardial infarction. Diabetes is well known to induce oxidative stress. To elucidate molecular mechanisms underlying an increased PAI-1 production, the effects of TGF-ß and oxidative stress implicated as agonists of PAI-1 synthesis were characterized with the use of human liver-derived HepG2 cells. METHODS: PAI-1 mRNA was assayed by real-time PCR, and PAI-1 protein was assayed by western blotting. PAI-1 promoter (-825 -+42 bp) activity was assessed with the luciferase assay. The role of the 3'-untranslated region was delineated with the use of luciferase constructs containing the 3'-untranslated region. Oxidative stress was measured after loading carboxy-2,7-dichlorodihydrofluorescein into cells. RESULTS: TGF-ß increased oxidative stress, which was accompanied by increases in NADPH oxidase 3 mRNA and membrane translocation of Rac proteins. TGF-ß-inducible increases in the PAI-1 promoter activity involved Smad-binding elements and a nuclear factor-κB-binding site. TGF-ß did not increase the activity of the PAI-1 mRNA 3'-untranslated region. TGF-ß-inducible PAI-1 expression was attenuated by simvastatin and curcumin, a natural polyphenol. CONCLUSION: TGF-ß can increase the expression of PAI-1 through multiple mechanisms involving Smad and nuclear factor-κB pathways and oxidative stress. As both oxidative stress and PAI-1 production were reduced by simvastatin and curcumin, modulation of oxidative stress and PAI-1 production are attractive targets for pharmacotherapy of cardiovascular disorders associated with an increased PAI-1 including type 2 diabetes and its associated consequences including accelerated coronary artery disease and an increased fibrosis that may exacerbate adverse left ventricular remodeling after myocardial infarction.

The impact of pharmacologic sympathetic and parasympathetic blockade on atrial electrogram characteristics in patients with atrial fibrillation.

BACKGROUND: Ablation of atrial autonomic inputs exerts antifibrillatory effects. However, because ablation destroys both myocardium and nerve cells, the effect of autonomic withdrawal alone remains unclear. We therefore examined the effects of pharmacologic autonomic blockade (PAB) on frequency and fractionation in patients with atrial fibrillation (AF). METHODS: Esmolol and atropine were administered and electrograms were recorded simultaneously from both atria and the coronary sinus. In 17 patients, AF was recorded for 5 minutes and dominant frequency (DF) and continuous activity (CA) were compared before and during PAB. RESULTS: Examination of the pooled data (537 sites, 17 patients) revealed a statistically significant decrease in mean DF (5.61­5.43Hz, P < 0.001) during PAB. Site-by-site analysis showed that 67% of sites slowed (0.45 ± 0.59 Hz), whereas 32% accelerated (0.49 ± 0.59Hz). Fractionation was reduced: median CA decreased from 31% to 26% (P < 0.001). In patient-by-patient analysis, mean DF/median CA decreased in 13 of 17 patients and increased in four. The spatial heterogeneity of DF decreased in nine of 17 patients (spatial coefficient of variation of DF at "nondriver sites" decreased by a mean of 2%). CONCLUSION: PAB decreases DF and CA in the majority of sites. Given the complexity of interactions between atrial cells during AF, the effects of PAB on DF and fractionation are more heterogeneous than the effects of PAB on isolated cells.

Posttranscriptional regulation of expression of plasminogen activator inhibitor type-1 by cAMP in HepG2 liver cells.

Altered expression of plasminogen activator inhibitor type-1 (PAI-1), a physiologic fibrinolysis inhibitor, is implicated in atherosclerosis. Cyclic adenosine monophosphate (cAMP) alters PAI-1 expression in several cells. Nevertheless, posttranscriptional regulation of PAI-1 has not been elucidated. To determine whether cAMP affects PAI-1 expression at posttranscriptional level, we determined promoter activity, mRNA levels, 3'-untranslated region (UTR) activity and protein levels of PAI-1 using HepG2 cells. cAMP decreased PAI-1 promoter activity at 24 h and mRNA expression at 4 h while it increased mRNA expression and accumulation of PAI-1 protein into media at 24 h. Human PAI-1 mRNA exists in two subspecies (3.2 and 2.2 kb), and cAMP increased baseline luciferase activity of 3'-UTR of the 3.2 kb PAI-1 mRNA [3'-UTR (+1358-3176)] and 1 kb fragment of 3'-terminus of 3'-UTR of 3.2 kb mRNA [3'-UTR (+2177-3176)]. cAMP increased PAI-1 protein expression despite decrease in promoter activity, presumably by regulating PAI-1 expression at the posttranscriptional level and thereby affecting mRNA stability. The 53-nt fragment in 3'-UTR (+2591 to +2643 nt) was involved in posttranscriptional regulation by cAMP. Thus, cAMP can stabilize 3.2 kb PAI-1 mRNA mediated by specific effects on 3'-UTR, and these effects are associated with increased expression of PAI-1 protein.

Absence of altered autophagy after myocardial ischemia in diabetic compared with nondiabetic mice.

OBJECTIVES: The extent of autophagy in myocardium following persistent ischemia and the effects of insulin resistance and diabetes on cardiac autophagy following myocardial infarction (MI) have not been well elucidated. It is generally thought that autophagy reflects the nutritional status of cells, presumably alterable by diabetes. It has been conjectured that diminution of autophagy early after the onset of MI may preserve jeopardized myocardium thereby improving prognosis. METHODS: Ten-week-old nondiabetic C57BL6 mice, 20-week-old diabetic and nondiabetic C57BL6 mice were subjected to MI for 4 weeks. Hearts from these mice were harvested and assayed for markers of autophagy. RESULT: Hearts of 10-week-old C57BL6 mice subjected to 4 weeks of MI had similar levels of LC3-II, a protein indicator of autophagy, as measured by western blotting compared with hearts from sham operated controls. In 20-week-old C57BL6 mice rendered diabetic by feeding a high-fat diet, the amounts of autophagy were comparable to those in 20-week-old nondiabetic C57BL6 mice on a normal diet. CONCLUSION: The magnitude of autophagy in the heart after infarction is of very modest extent and is not modulated by diabetes. Thus, diminution of autophagy is not likely to reduce infarct size or attenuate late negative remodeling after MI in patients with diabetes.