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.

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.

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.

Profibrinolytic, antithrombotic, and antiinflammatory effects of an insulin-sensitizing strategy in patients in the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trial.

BACKGROUND: Effects were compared in patients in the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D) trial of 2 mechanistically different strategies for treatment of hyperglycemia, insulin-sensitizing and insulin-providing strategies, on biomarker profiles reflecting the balance between fibrinolysis and thrombosis and the intensity of inflammation implicated in diabetic vasculopathy. METHODS AND RESULTS: A total of 2368 patients with type 2 diabetes mellitus and clinically stable, angiographically documented coronary artery disease were randomized to treatment with 1 of the 2 strategies and followed for an average of 5 years. Plasminogen activator inhibitor type 1 antigen and activity, tissue plasminogen activator antigen, fibrinogen, D-dimer, C-reactive protein, insulin, and hemoglobin A(1c) were assayed in blood samples acquired at baseline and at 12 regular intervals throughout the follow-up interval. Higher baseline D-dimer, fibrinogen, and C-reactive protein portended a poor prognosis in patients in both groups. In contrast to the insulin-providing strategy, the insulin-sensitizing strategy led to (1) lower plasma insulin; (2) lower plasminogen activator inhibitor type 1 antigen and activity and lower tissue plasminogen activator antigen (known to track with plasminogen activator inhibitor type 1); and (3) lower C-reactive protein and fibrinogen at all intervals after baseline (P<0.001 for each). CONCLUSIONS: The insulin-sensitizing treatment strategy led to changes in biomarker profiles indicative of decreased insulin resistance, an altered balance between thrombosis and fibrinolysis favoring fibrinolysis, and diminished intensity of the systemic inflammatory state, factors that have been associated with cardiovascular risk. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov. Unique identifier: NCT00006305.

A randomized trial of therapies for type 2 diabetes and coronary artery disease.

BACKGROUND: Optimal treatment for patients with both type 2 diabetes mellitus and stable ischemic heart disease has not been established. METHODS: We randomly assigned 2368 patients with both type 2 diabetes and heart disease to undergo either prompt revascularization with intensive medical therapy or intensive medical therapy alone and to undergo either insulin-sensitization or insulin-provision therapy. Primary end points were the rate of death and a composite of death, myocardial infarction, or stroke (major cardiovascular events). Randomization was stratified according to the choice of percutaneous coronary intervention (PCI) or coronary-artery bypass grafting (CABG) as the more appropriate intervention. RESULTS: At 5 years, rates of survival did not differ significantly between the revascularization group (88.3%) and the medical-therapy group (87.8%, P=0.97) or between the insulin-sensitization group (88.2%) and the insulin-provision group (87.9%, P=0.89). The rates of freedom from major cardiovascular events also did not differ significantly among the groups: 77.2% in the revascularization group and 75.9% in the medical-treatment group (P=0.70) and 77.7% in the insulin-sensitization group and 75.4% in the insulin-provision group (P=0.13). In the PCI stratum, there was no significant difference in primary end points between the revascularization group and the medical-therapy group. In the CABG stratum, the rate of major cardiovascular events was significantly lower in the revascularization group (22.4%) than in the medical-therapy group (30.5%, P=0.01; P=0.002 for interaction between stratum and study group). Adverse events and serious adverse events were generally similar among the groups, although severe hypoglycemia was more frequent in the insulin-provision group (9.2%) than in the insulin-sensitization group (5.9%, P=0.003). CONCLUSIONS: Overall, there was no significant difference in the rates of death and major cardiovascular events between patients undergoing prompt revascularization and those undergoing medical therapy or between strategies of insulin sensitization and insulin provision. (ClinicalTrials.gov number, NCT00006305.)

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.

The angiotensin receptor blocker, azilsartan medoxomil (TAK-491), suppresses vascular wall expression of plasminogen activator inhibitor type-I protein potentially facilitating the stabilization of atherosclerotic plaques.

Increased expression of plasminogen activator inhibitor type-I (PAI-1) in vessel walls seems to accelerate atherosclerosis. Angiotensin II can increase the synthesis of PAI-1. Inhibition of this process may facilitate migration of vascular smooth muscle cells (VSMCs) stabilizing atherosclerotic plaques. To determine whether the inhibition of the angiotensin II type 1 receptor can blunt the expression of PAI-1 protein in the aortic wall, we administered azilsartan medoxomil (AZL-M), a prodrug of an angiotensin II type 1 receptor blocker developed by the Takeda Pharmaceutical Company Limited, for 16 weeks to ApoE knockout mice on a high fat diet rendered overexpressors of PAI-1 in VSMCs. Homogenates of the pooled aortas from each group were assayed for PAI-1 by enzyme-linked immunosorbent assay. Cellularity of atherosclerotic lesions was assessed by 4',6-diamidino-2-phenylindole staining in sections of aortic lesions, and collagen content in the lesions was quantified by immunohistochemistry. Aortic wall PAI-1 was decreased by each of the 3 dosage regimens of AZL-M (0.1-10 mg/kg). Cellularity and collagen were increased in lesions from mice given AZL-M, consistent with the development of more stable plaques. Accordingly, the suppression of PAI-1 expression by AZL-M may attenuate the evolution of atherosclerotic plaques vulnerable to rupture.

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.

Autophagy in myocardium of murine hearts subjected to ischemia followed by reperfusion.

Autophagy in myocardium has been thought to be cardioprotective, but its extent after transient or prolonged myocardial ischemia remains unclear. Accordingly, we characterized its magnitude in myocardium of murine hearts subjected to ischemia with or without reperfusion. Ten-week-old transgenic GFP-LC3 mice and C57Bl6 mice were subjected to coronary ligation for 1 or 4 h followed by 24 h of reperfusion (1HTL, 4HTL) or to 24 h of persistent ligation (24HPL). Their hearts were analyzed by fluorescence microscopy, electron microscopy, and by Western blotting. Fluorescent GFP-LC3 dots indicative of autophagy were absent in infarct zones and reduced markedly in the peri-infarct zones compared with dots in sham controls (p ≤ 0.05). The LC3-II/LC3-I ratio indicative of autophagy did not increase in LV homogenates from hearts following ischemia. Phosphorylation of ribosomal protein S6 increased in LV homogenates in hearts from mice subjected to 4HTL and 24HPL (p ≤ 0.05). Virtually no autophagic cells recognizable by electron microscopy were evident in infarct or peri-infarct zones. Autophagy is virtually absent within 24 h in the center of zones of infarction and is decreased significantly in the peri-infarct zones compared with that in normal hearts.

The magnitude and temporal dependence of apoptosis early after myocardial ischemia with or without reperfusion.

In view of the conventional wisdom in the cardiology literature that apoptosis is extensive early after myocardial ischemia, predicated largely from results with the TUNEL assay known to be nonspecific, this study was performed to delineate its extent with multiple assays and at multiple intervals. Coronary occlusion with and without subsequent revascularization was induced in 10-wk-old C57BL6 mice subjected to 1 or 4 h of transient ligation followed by 24 h of reperfusion, or 24 h persistent ligation. Apoptosis was quantified throughout the left ventricle immunohistochemically by assay of TUNEL, single-stranded DNA (ssDNA), and cleaved caspase 3; electron microscopy (EM); and activity assays of caspase 3 and 8. TUNEL staining was marked, but ssDNA and cleaved caspase 3 staining were significantly less (P<0.001 compared with TUNEL), and apoptosis defined by EM was virtually absent in all groups. Caspase 3 and caspase 8 activities per milligram protein were not significantly different from those in normal hearts. Only rare, potentially apoptotic cells were seen by EM in hearts from any group. Thus, the results with TUNEL were not specific, and the extent of apoptosis was markedly less than that predicated on the results with the TUNEL procedure. Apoptosis is de minimus early after transitory or persistent ischemia, though it is overestimated by TUNEL assays. Thus, antiapoptotic interventions per se are not likely to preserve substantial amounts of myocardium early after ischemic insults.

Inhibition by fibrates of plasminogen activator inhibitor type-1 expression in human adipocytes and preadipocytes.

Plasminogen activator inhibitor type-1 (PAI-1), an established marker and mediator of cardiovascular risk, is produced extensively in adipose tissue. Fibrates are hypolipidemic peroxisome proliferator activated receptor-alpha (PPARalpha) agonists. Recent laboratory and clinical observations indicate that they are also anti-atherosclerotic. Mechanisms responsible, however, remain to be fully understood. The present study was designed to elucidate modulation of PAI-1 expression in adipose cells by fibrates as a potential mechanism. Expression of PPARalpha was verified by PCR, immunohistochemistry, and Western blotting. In cultured preadipocytes and adipocytes gemfibrozil and fenofibrate significantly reduced PAI-1 protein expression by up to 55 +/- 5% and 34 +/- 4% under basal conditions and up to 56 +/- 6% and 31 +/- 6% under conditions of stimulation of the cells with 40 pM transforming growth factor (TGF)beta, respectively. Quantification of mRNA showed that the gemfibrozil-induced effect was at least in part regulated at the transcriptional level. Incubations with non-fibrate PPARalpha agonists showed similar reductions in PAI-1 expression. The decrease in PAI-1 expression induced by gemfibrozil was inhibited by MK886, a PPARalpha inhibitor. Furthermore, preadipocytes isolated from PPARalpha-deficient mice produced significantly more PAI-1 than those from wild-type mice upon stimulation with TGFbeta. Finally, fenofibrate reduced PAI-1 expression both in plasma and adipose tissue of hyperlipidemic mice. Our data support the view that PPARalpha activation down-regulates PAI-expression in adipose cells that may contribute in part to the reduction in cardiovascular mortality seen with fibrates in clinical trials.

A profibrotic effect of plasminogen activator inhibitor type-1 (PAI-1) in the heart.

Increased expression of PAI-1 is profibrotic in several organs. However, its potentially profibrotic effects in the heart subjected to infarction have not been elucidated. Accordingly, we induced coronary occlusion in 10-week-old mice congenic on a C57BL6 background and in mice overexpressing PAI-1 (PTG) in multiple tissues. Compared with C57BL6 control mice without myocardial infarction (MI), PTG mice exhibited consistently elevated PAI-1 in plasma at 16 weeks of age but virtually identical PAI-1 content in left ventricular (LV) myocardium. However, they exhibited a 2-fold increase in LV PAI-1 content 6 weeks after induction of MI (4.21 +/- 1.0 ng/ml tissue protein) compared with that in C57BL6 mice (2.04 +/- 0.5, P < 0.05). In 16-week-old mice, ultrasonically delineated LV fractional shortening (FS) was comparable in normal PTG and normal C57BL6 controls. However, 6 weeks after MI, PTG (n = 21) compared with C57BL6 (n = 14) mice exhibited markedly thinner LV posterior walls in both diastole (C57BL6 0.79 +/- 0.05 mm, PTG 0.55 +/- 0.06, P < 0.05) and systole (0.97 +/- 0.05 mm, 0.75 +/- 0.06, P < 0.05); increased end systolic LV dimensions (4.54 +/- 0.2 mm, 5.17 +/- 0.2, P < 0.05); and significantly depressed FS, more impaired LV segmental function, and greater mitral E wave amplitude. Compared with fibrosis assessed by Masson staining of sections from apex to base in C57BL6 mice (10.85 +/- 0.43% LV area), PTG mice exhibited 33% more LV fibrosis after MI (P < 0.05). Thus, PAI-1 is profibrotic in the heart subjected to infarction. Accordingly, overexpression of PAI-1 is a promising target for attenuation of heart failure after MI that may be exacerbated by fibrosis.

Lack of early augmentation of platelet reactivity after coronary intervention in patients treated with bivalirudin.

To determine whether a regimen of aspirin pretreatment, bivalirudin during the procedure and clopidogrel (600 mg) immediately after percutaneous coronary intervention (PCI) was associated with platelet activation during and shortly after (1 and 2 h) PCI, we characterized platelet function in 10 patients with the use of flow cytometry in the absence of agonist and in response to thrombin (10 nM), ADP (1 microM), the collagen-mimetic convulxin (5 ng/ml), and platelet activating factor (10 nM). Activation of platelets in peripheral blood was rare (<0.5% of platelets) before, during and after PCI. Platelet reactivity in response to each of the agonists was lower after the PCI compared with that in blood taken before the PCI. Accordingly, platelet activation and platelet reactivity were not increased after elective PCI when patients were treated during the procedure with aspirin and bivalirudin and immediately after the procedure with a 600 mg loading dose of clopidogrel.

The effect of plasminogen activator inhibitor type 1 on apoptosis.

Plasminogen activator inhibitor type-1 (PAI-1), an inhibitor of plasminogen activators, inhibits formation of plasmin and plasmin-mediated proteolysis. Apoptosis, or programmed cell death, is a potentially important phenomenon in mediating overall cell death. This review focuses on the influence of PAI-1 on apoptosis. Greater expression of PAI-1 has been associated with increased survival of cells and resistance to apoptosis. PAI-1 appears to influence apoptosis by decreasing cell adhesion (anoikis) as well as its effect on intracellular signaling. Mechanisms by which PAI-1 may render a cell resistant to apoptosis include its ability to inhibit generation of plasmin, its ability to inhibit caspase 3, and its ability to inhibit cell adhesion mediated by vitronectin. Inhibition of caspase 3 by PAI-1 may divert intracellular signalling from induction of apoptosis to induction of proliferation.

Contributions of young platelets and of previously activated platelets to platelet reactivity in patients with coronary artery disease.

INTRODUCTION: We sought to determine why some patients with coronary artery disease have more platelets that do not activate (express P-selectin on their surface) despite exposure in vitro to high concentrations of agonists and whether this finding is associated with altered platelet reactivity. METHODS: We assessed platelet activation and the proportion of young platelets with the use of flow cytometry in blood from 50 patients with coronary artery disease undergoing cardiac catheterization. Ultrastructural characteristics of platelets isolated with the use of a fluorescence activated cell sorter were assessed with the use of electron microscopy. RESULTS: Ultrastructural characteristics of platelets that did not exhibit surface expression of P-selectin in response to 50 nM thrombin delineated 2 groups; resting platelets devoid of glycogen stores and activated platelets. Because the activated platelets had shed their surface P-selectin we refer to them as 'previously activated' platelets. To estimate the proportion of 'previously activated' platelets we quantified the percentage of platelets that bound annexin-V but did not express P-selectin after exposure to 50 nM thrombin. The fraction of 'previously activated' platelets ranged from 0.3%-4.8% and correlated modestly though positively with the fraction of young platelets (r=0.41, p=0.003). Patients with more young platelets tended to have more 'previously activated' platelets and exhibited greater platelet reactivity. CONCLUSIONS: A greater proportion of 'previously activated' platelets is associated with a greater fraction of young platelets and increased platelet reactivity. The presence of 'previously activated' platelets in circulating blood may be a marker of micro or macro thrombosis.

Progress in pharmacogenomics and its promise for medicine.

Pharmacogenomics addresses the impacts of diverse and multiple genes in populations as determinants of responses of individual patients to drugs. The field has its roots in basic science, and is pivotal in drug development, elucidation of therapeutic efficacy, and constraining the risks of adverse drug reactions. Regulatory agencies are relying increasingly on pharmacogenomics for identification of patients who are particularly likely to benefit from treatment with specific agents and exclusion of those at risk of adverse drug reactions. Practical applications of pharmacogenomics already abound particularly in the use of drugs acting on the central nervous system and on the cardiovascular system. The Society for Experimental Biology and Medicine (SEBM) is proud and pleased to have devoted its 2008 symposium, presented at the annual Experimental Biology meeting in San Diego on April 6, 2008, to advances in pharmacogenomics with emphasis on drug development, regulatory agency considerations, and clinical applications.

Impact of statins on modulation by insulin of expression of plasminogen activator inhibitor type-1.

OBJECTIVE: Plasminogen activator inhibitor type-1 (PAI-1) is a physiologic inhibitor of fibrinolysis and a known determinant of cardiovascular risk. That its expression is stimulated by insulin in HepG2 human hepatoma cells has been shown earlier. Others have found that increased expression of PAI-1 is a risk factor for acute myocardial infarction. Pleiotropic (noncholesterol lowering) effects of statins seem to reduce cardiovascular risk. This study was designed to determine whether insulin stimulation of PAI-1 expression is attenuated by statins, and if so to explore mechanisms that are responsible for the attenuation. METHODS: PAI-1 protein in the conditioned media was assayed by western blotting, and PAI-1 mRNA expression was measured by real-time reverse transcriptase polymerase chain reactions. RESULTS: Insulin (0.001-10 micromol/l) increased accumulation of PAI-1 protein in the conditioned media and PAI-1 mRNA expression in HepG2 cells. A transient transfection assay of the human PAI-1 promoter-luciferase construct demonstrated that insulin increased PAI-1 promoter activity. Increased PAI-1 mRNA was attenuated significantly by U0126 and PD98059, specific inhibitors of mitogen-activated protein kinase. In contrast, GF109203X, an inhibitor of the protein kinase C pathway, and LY294002, an inhibitor of phosphatidylinositol 3-kinase, exerted no effects. Simvastatin (10 micromol/l), known to translocate membrane-bound sterol regulatory element-binding protein to nuclei, attenuated PAI-1 mRNA expression induced by insulin. It did not affect baseline PAI-1 mRNA expression. Intraperitoneal injection of insulin (0.1 IU/kg) increased concentrations of PAI-1 antigen in plasma in mice within 3 h, correlating with hepatic PAI-1 mRNA expression. CONCLUSION: Insulin-mediated augmented expression of PAI-1 may be amenable to suppression attributable to pleiotropic effects of statins, potentially diminishing cardiovascular risk in patients with insulin resistance.

Attenuation of apoptosis and the eye of the beholder.

OBJECTIVES: We consider the conundrum suggested by myocardial hibernation and late restoration of function despite the absence of a substantial lateral peri-infarction border zone with respect to oxygenation, and suggest a pivotal role for apoptosis and its attenuation in salvaging jeopardized myocardium. METHODS: Selective pertinent literature is reviewed, and some recent observations indicating difficulties in identifying and quantifying apoptosis microscopically are summarized. RESULTS: Apoptosis seems to occur primarily after reperfusion following ischemia rather than persistent ischemia leading to necrosis. Refinements of markers of its presence are needed in vitro for use ultimately in vivo and should be pivotal in defining the extent to which tissue-protective interventions can salvage myocardium in the context of a fixed magnitude and duration of ischemia. CONCLUSION: Apoptosis is strongly implicated in the overall demise of jeopardized myocardium. Its attenuation seems likely to be potentially beneficial. Validation of this hypothesis will require progress in identification, delineation, and assessment of the extent of apoptosis in the threatened heart.