Mitogen-activated protein kinases activation in T lymphocytes of patients with acute coronary syndromes.

Current available biomarkers cannot identify myocardial ischemia without necrosis. To overcome this issue and to increase diagnostic power, we evaluated the activation of the three MAPK pathways, ERK1/2, JNK and p38, in T lymphocytes of patients with acute coronary syndromes (ACS). We included sixty consecutive patients affected by either unstable angina (UA, N = 22), Non- ST-segment elevation MI (NSTEMI, N = 19) or ST-segment elevation MI (STEMI, N = 19). Two separate groups of patients were matched as controls: healthy subjects (CTRL, N = 20) and patients with stable coronary artery disease (CAD, N = 21). MAPK activation in T lymphocytes, measured by phospho-ERK1/2, phospho-JNK and phospho-p38 levels, was assessed by flow cytometry analysis which revealed significantly increased phosphorylated levels of ERK1/2 in patients with UA, compared to controls. In UA patients no significant changes were detected for phospho-JNK compared to both control groups. NSTEMI and STEMI groups showed a statistically significant increase in both phospho-ERK1/2 and phospho-JNK, compared to control groups. All ACS groups demonstrated significantly increased phosphorylation of p38 compared to CTRL, but not CAD. ROC curves showed that a cut-off value of 22.5 intensity of fluorescence for phospho-ERK1/2 was able to significantly discriminate UA patients from patients with stable angina with 78% sensitivity and 90% specificity. Therefore, a differential MAPK activation in T lymphocytes denotes patients with ACS. Indeed, patients with unstable angina are identified with high specificity by activated ERK1/2 and normal JNK levels. These data could represent a valuable new molecular signature to be used as specific biomarkers for the diagnosis of unstable angina within ACS.

Differential regulation of vascular smooth muscle and endothelial cell proliferation in vitro and in vivo by cAMP/PKA-activated p85alphaPI3K.

cAMP inhibits proliferation in most cell types, triggering different and sometimes opposing molecular pathways. p85alpha (phosphatidylinositol 3-kinase regulatory subunit) is phosphorylated by cAMP/PKA in certain cell lineages, but its effects on vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) are unknown. In the present study, we evaluated 1) the role of p85alpha in the integration of cAMP/PKA-dependent signaling on the regulation of VSMC and EC growth in vitro; and 2) the effects of PKA-modified p85alpha on neointimal hyperplasia and endothelial healing after balloon injury in vivo. Plasmid constructs carrying wild-type and PKA-modified p85alpha were employed in VSMCs and ECs in vitro and after balloon injury in rat carotid arteries in vivo. cAMP/PKA reduced VSMC proliferation through p85alpha phosphorylation. Transfected PKA-activated p85alpha binds p21ras, reducing ERK1/2 activation and VSMC proliferation in vitro. In contrast, EC proliferation inhibition by cAMP is independent from PKA modification of p85alpha and ERK1/2 inhibition; indeed, PKA-activated p85alpha did not inhibit per se ERK1/2 activation and proliferation in ECs in vitro. Interestingly, cAMP reduced both VSMC and EC apoptotic death through p85alpha phosphorylation. Accordingly, PKA-activated p85alpha triggered Akt activation, reducing both VSMC and EC apoptosis in vitro. Finally, compared with controls, vascular gene transfer of PKA-activated p85alpha significantly reduced neointimal formation after balloon injury in rats, without inhibiting endothelial regeneration of the injured arterial segment. In conclusions, PKA-activated p85alpha integrates cAMP/PKA signaling differently in VSMCs and ECs. By reducing neointimal hyperplasia without inhibiting endothelial regeneration, it exerts a protective effect against restenosis after balloon injury.

Proteomics reveals high levels of vitamin D binding protein in myocardial infarction.

The pathogenic mechanisms underlying cardiovascular diseases involve significant alterations in myocardial gene and protein expression. Proteomics analysis can define new protein and peptide changes associated with cardiac pathology, including myocardial infarction. The aim of the present study was to analyze serum proteome of patients with ST-Elevation myocardial infarction (STEMI). Serum samples were collected from STEMI patients (age 65.0+/-10.3) at 5.3+/-2.7 hours after the onset of typical chest pain and before initiating standard therapy. Ten age- and sex-matched donors were used as controls. The samples were albumin- and IgG-depleted. Isotope-coded affinity tag method was employed to label cysteine residues and liquid chromatography-Tandem Mass Spectrometry analysis was performed to measure the labeled proteins. Our proteomic approach identified increased levels of vitamin D-binding protein precursor (VDB) in the serum of STEMI patients when compared to control donors. Western blot analysis confirmed the increase in VDB protein in STEMI patients. Moreover, fresh thrombotic plaques, obtained during primary angioplasty, showed high expression of VDB protein. Mechanistically, VDB protein reduces platelet aggregation and prolongs coagulation time ex vivo.

Fludarabine prevents smooth muscle proliferation in vitro and neointimal hyperplasia in vivo through specific inhibition of STAT-1 activation.

Drug-eluting stents are increasingly used to reduce in-stent restenosis and adverse cardiac events after percutaneous coronary interventions. However, the race for the ideal drug-eluting stent is still on, with special regard to the best stent-coating system and the most effective and less toxic drug. Fludarabine, a nucleoside analog, has both anti-inflammatory and antiproliferative cellular effects. The aim of the present study was to assess the cellular and molecular effects of fludarabine on vascular smooth muscle cell (VSMC) growth in vitro and in vivo and the feasibility and efficacy of a fludarabine-eluting stent. To study the biomolecular effects of fludarabine on VSMC proliferation in vitro, rat VSMCs were grown in the presence of 50 microM fludarabine or in the absence of the same. To evaluate the in vivo effect of this drug, male Wistar rats underwent balloon injury of the carotid artery, and fludarabine was locally delivered at the time of injury. Finally, fludarabine-eluting stents were in-laboratory manufactured and tested in a rabbit model of in-stent restenosis. Fludarabine markedly inhibited VSMC proliferation in cell culture. Furthermore, fludarabine reduced neointimal formation after balloon angioplasty in a dose-dependent manner, and fludarabine-eluting stents reduced neointimal hyperplasia by approximately 50%. These in vitro and in vivo cellular effects were specifically associated with the molecular switch-off of signal transducer and activator of transcription (STAT)-1 activation, without affecting other STAT proteins. Fludarabine abolishes VSMC proliferation in vitro and reduces neointimal formation after balloon injury in vivo through specific inhibition of STAT-1 activation. Fludarabine-eluting stents are feasible and effective in reducing in-stent restenosis in rabbits.

Acute beta-adrenergic overload produces myocyte damage through calcium leakage from the ryanodine receptor 2 but spares cardiac stem cells.

A hyperadrenergic state is a seminal aspect of chronic heart failure. Also, "Takotsubo stress cardiomyopathy," is associated with increased plasma catecholamine levels. The mechanisms of myocyte damage secondary to excess catecholamine exposure as well as the consequence of this neurohumoral burst on cardiac stem cells (CSCs) are unknown. Cardiomyocytes and CSCs were exposed to high doses of isoproterenol (ISO), in vivo and in vitro. Male Wistar rats received a single injection of ISO (5 mg kg-1) and were sacrificed 1, 3, and 6 days later. In comparison with controls, LV function was impaired in rats 1 day after ISO and started to improve at 3 days. The fraction of dead myocytes peaked 1 day after ISO and decreased thereafter. ISO administration resulted in significant ryanodine receptor 2 (RyR2) hyperphosphorylation and RyR2-calstabin dissociation. JTV519, a RyR2 stabilizer, prevented the ISO-induced death of adult myocytes in vitro. In contrast, CSCs were resistant to the acute neurohumoral overload. Indeed, CSCs expressed a decreased and inverted complement of beta1/beta2-adrenoreceptors and absence of RyR2, which may explain their survival to ISO insult. Thus, a single injection of ISO causes diffuse myocyte death through Ca2+ leakage secondary to the acutely dysfunctional RyR2. CSCs are resistant to the noxious effects of an acute hyperadrenergic state and through their activation participate in the response to the ISO-induced myocardial injury. The latter could contribute to the ability of the myocardium to rapidly recover from acute hyperadrenergic damage.