Quality control in cardiovascular research: a difficult task.
Explore the source record for details and available documents.
Cardiology↗
Biomedical subjects
M Chiariello
Publications and source records attributed to M Chiariello.
[Diabetes and ischemic cardiopathy].
Diabetes represents an independent risk factor for coronary artery disease (CAD), and the prognosis in terms of survival rates is worse for diabetic patients who have CAD with respect to those with CAD but no diabetes. An acute coronary event represents a cause of death in more than 30% of diabetics. Experimental studies suggested that the increased incidence of myocardial infarction in diabetics is due to an increased risk of developing atherosclerotic plaque with subsequent ulceration and intracoronary thrombus formation. Structural abnormalities of the coronary vessel wall were associated with an abnormal pattern of coronary flow and of coagulation abnormalities: all these abnormalities explain the epidemiological evidence of widespread and severe vascular atherosclerotic disease in diabetics. Due to the extreme complexity of ischemic vascular disease in patients with diabetes, an optimal therapeutic strategy is based on the correction of elevated blood glucose and lipid levels, of blood pressure, of platelet and coagulation abnormalities and of any other risk factor. Both percutaneous and surgical myocardial revascularization have been proved equally effective for CAD treatment in diabetes, even though a recent randomized trial has shown a significantly improved outcome after surgical revascularization. More recently the characterization of the advanced glycation end-product receptor opened new perspectives in the treatment of the complications of diabetes, and gave a new impact to the need of further investigations, through new randomized trials, of the best therapeutic options for diabetic patients.
Angioplasty, Balloon, Coronary↗
Importance of the MKK6/p38 pathway for interleukin-12-induced STAT4 serine phosphorylation and transcriptional activity.
Interleukin-12 (IL-12) is a key immunoregulatory cytokine that promotes Th1 differentiation and cell-mediated immune responses. The transcription factor STAT4 (signal transducer and activator of transcription 4) is an important element in mediating IL-12 signals, as evidenced by the fact that STAT4(-/-) mice display impaired responsiveness to IL-12 and deficient Th1 differentiation. STAT4 is inducibly phosphorylated on tyrosine and serine in response to IL-12, but the kinase(s) responsible for the latter event is unknown. Here we show that IL-12 induces STAT4 phosphorylation on serine 721 and that mutation of serine 721 interferes with STAT4 transcriptional activity. In addition, we show that mutation of tyrosine 693 abrogates IL-12-induced STAT4 tyrosine phosphorylation and transcriptional activity. Although the site surrounding serine 721 is an optimum consensus sequence for mitogen-activated family of protein kinases (MAPKs)-mediated phosphorylation, we demonstrate that IL-12 does not induce extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) activation in T and natural killer (NK) cells and that IL-12-induced STAT4 transcriptional activity is not affected by these kinases. Rather, we show that IL-12 induces p38 activation. Moreover, we demonstrate that p38alpha and its upstream activator, MKK6, phosphorylate STAT4 on serine 721, and are required for STAT4 full transcriptional activity induced by IL-12, establishing the MKK6/p38alpha/STAT4 pathway as an important mediator of IL-12 actions. (Blood. 2000;96:1844-1852)
3T3 Cells↗
Regulation of cyclin-dependent kinase (Cdk) 2 Thr-160 phosphorylation and activity by mitogen-activated protein kinase in late G1 phase.
Mitogen-activated protein (MAP) kinases, p42(MAPK) and p44(MAPK), are central components of growth-promoting signalling pathways. However, how stimulation of MAP kinases culminates in cell-cycle progression is still poorly understood. Here we show that mitogenic stimulation of NIH 3T3 cells causes a sustained activation of MAP kinases, which lasts until cells begin progressing through the G(1)/S boundary. Furthermore, we observed that disruption of the MAP-kinase pathway with a selective MEK (MAP kinase/extracellular-signal-regulated protein kinase kinase) inhibitor, PD98059, prevents the activation of cyclin-dependent kinase (Cdk) 2 and DNA synthesis, even when added during late G(1) phase, once the known mechanisms by which MAP kinase controls G(1) progression, accumulation of G(1) cyclins and degradation of Cdk inhibitors have already taken place. Moreover, we provide evidence indicating that MAP kinases control Cdk2 Thr-160 activating phosphorylation and function, possibly by regulating the activity of a Cdk-activating kinase, thus promoting the re-initiation of DNA synthesis. These findings suggest the existence of a novel mechanism whereby signal-transducing pathways converging on MAP kinases can affect the cell-cycle machinery and, ultimately, participate in cell-growth control.
3T3 Cells↗
Signaling from G protein-coupled receptors to ERK5/Big MAPK 1 involves Galpha q and Galpha 12/13 families of heterotrimeric G proteins. Evidence for the existence of a novel Ras AND Rho-independent pathway.
The regulation of gene expression by cell surface receptors often involves the stimulation of signaling pathways including one or more members of the MAPK superfamily of serine-threonine kinases. Upon their activation in the cytosol, MAPKs can translocate to the nucleus and affect the activity of a variety of transcription factors. Recently, it has been observed that a novel member of the MAPK superfamily, ERK5, can be potently activated by transforming G protein-coupled receptors (GPCRs) and that ERK5 participates in the regulation of c-jun expression through the activation of MEF2 transcription factors. How cell surface receptors, including GPCRs, stimulate ERK5 is still poorly understood. In this study, we have used transiently transfected COS-7 cells to begin delineating the biochemical route linking GPCRs to ERK5. We show that receptors that can couple to the G(q) and G(12/13) families of heterotrimeric G proteins, m1 and thrombin receptors, respectively, but not those coupled to G(i), such as m2 receptors, are able to regulate the activity of ERK5. To investigate which heterotrimeric G proteins signal to ERK5, we used a chimeric system by which Galpha(q)- and Galpha(13)-mediated signaling pathways can be conditionally activated upon ligand stimulation. Using this system, as well as the expression of activated forms of G protein subunits, we show that the Galpha(q) and Galpha(12/13) families of heterotrimeric G proteins, but not the Galpha(i), Galpha(s), and betagamma subunits, are able to regulate ERK5. Furthermore, we provide evidence that the stimulation of ERK5 by GPCRs involves a novel signaling pathway, which is distinct from those regulated by Ras and Rho GTPases.
Animals↗
Endogenous tissue factor pathway inhibitor modulates thrombus formation in an in vivo model of rabbit carotid artery stenosis and endothelial injury.
BACKGROUND: Tissue factor pathway inhibitor (TFPI) is the sole known inhibitor of the extrinsic coagulation pathway of physiological importance; however, its role in modulating thrombosis in vivo is still unclear. METHODS AND RESULTS: Intravascular thrombosis was initiated by placing an external constrictor around endothelially injured rabbit carotid arteries (n=10). Carotid blood flow velocity was measured by a Doppler flow probe. After placement of the constrictor, cyclic flow reductions (CFRs), due to recurrent thrombosis, developed at the site of stenosis. Transstenotic TFPI plasma activity was measured in blood samples before induction of CFRs and after 30, 60, and 180 minutes of CFRs. TFPI plasma activity distal to the site of thrombosis was significantly lower than the corresponding proximal values at 30, 60, and 180 minutes of CFRs. In addition, a progressive decrease in TFPI plasma activity was observed in both the proximal and the distal samples, indicating consumption of TFPI during thrombus formation. In 10 additional rabbits, CFRs were abolished by administration of aspirin (10 mg/kg). In the animals in which aspirin abolished CFRs, endogenous TFPI was depleted by a bolus of a polyclonal antibody against rabbit TFPI, and the effects on restoration of CFRs were monitored. In 5 of 6 animals in which aspirin abolished CFRs, depletion of endogenous TFPI activity caused full restoration of CFRs. CONCLUSIONS: The data of the present study support the involvement of endogenous TFPI in the process of thrombus formation in vivo and its active role in modulating arterial thrombosis.
Animals↗
The assessment of myocardial viability and hibernation using resting thallium imaging.
Rest-redistribution thallium-201 imaging is widely used to assess recovery of regional systolic dysfunction in patients with chronic coronary artery disease. In several studies, this technique has demonstrated very high sensitivity but reduced specificity, as reported in general for radionuclide imaging. In clinical terms, this implicates that many dysfunctional territories will not recover after revascularization despite a substantial amount of tracer uptake. Yet, the amount of tracer uptake in a given myocardial segment, although not perfect, remains the best indicator for predicting reversible dysfunction. In fact, the occurrence of redistribution after rest injection is not very common and it does not substantially contribute to the accuracy of the test. However, it is still undetermined whether the presence of redistribution is relevant for prognostic implications.
Coronary Disease↗
Prediction of improvement in global left ventricular function in patients with chronic coronary artery disease and impaired left ventricular function: rest thallium-201 SPET versus low-dose dobutamine echocardiography.
Accurate assessment of myocardial viability permits selection of patients who would benefit from myocardial revascularization. Currently, rest-redistribution thallium-201 scintigraphy and low-dose dobutamine echocardiography are among the most used techniques for the identification of viable myocardium. Thirty-one consecutive patients (all men, mean age 60 +/- 8 years) with chronic coronary artery disease and reduced left ventricular ejection fraction (31% +/- 7%) were studied. Rest 201Tl single-photon emission tomography (SPET), low-dose dobutamine echocardiography and radionuclide angiography were performed before revascularization. Radionuclide angiography and echocardiography were repeated after revascularization. An a/dyskinetic segment was considered viable on 201Tl SPET when tracer uptake was >65%, while improvement on low-dose dobutamine echocardiography was considered a marker of viability. Increase in global ejection fraction was considered significant at > or = 5%. In identifying viable segments, rest 201Tl SPET showed higher sensitivity than low-dose dobutamine echocardiography (72% vs 53%, P<0.05), while specificity was not significantly different (86% vs 88%). In 17 patients, global ejection fraction increased > or = 5% (group 1) while in 14 it did not (group 2). A higher number of a/dyskinetic segments were viable on 201Tl SPET in group 1 than in group 2 (2.6 +/- 1.9 vs 0.6 +/- 1.2, P < 0.005), while no significant differences were observed on low-dose dobutamine echocardiography (1.7 +/- 1.6 vs 1.1 +/- 1.6). A significant correlation was found between the number of a/dyskinetic segments viable on 201Tl SPET and post-revascularization changes in ejection fraction (r = 0.52, P < 0.05), but such a correlation was not observed for low-dose dobutamine echocardiography. Using as the cut-off the presence of at least one viable a/dyskinetic segment, rest 201Tl SPET had a higher sensitivity (82% vs 53%, P = 0.07) and showed a trend towards higher accuracy and specificity (77% vs 58%, and 71% vs 64%, respectively) as compared with low-dose dobutamine echocardiography. In conclusion, these findings suggest that when severely reduced global function is present, rest 201Tl SPET evaluation of viability is more accurate than low-dose dobutamine echocardiography for the identification of patients who will benefit most from revascularization.
Adrenergic beta-Agonists↗
A new rat model of small vessel stenting.
OBJECTIVES: Restenosis is the major complication of coronary angioplasty and stenting. In addition, the small vessel diameter represents a major limitation to the wide use of the technology. The aim of this study was to assess the feasibility and the vascular response of stent deployment in rat small vessels. METHODS: In 40 Wistar rats (500-550 g) a Nir stent crimped on a 1.5 mm Comet angioplasty balloon catheter was deployed at high pressure in the common carotid artery. Neointimal area, neointima/media ratio and the arterial dimension were assessed immediately and at 7, 14, 21, and 28 days after stenting. RESULTS: After stent deployment, the neointimal area and the neointima/media ratio increased progressively and peaked at 14 days (p < 0.05 vs 0 and 7 days). Alpha-actin-positive cells were found circumferentially organized on the lumen surface. At 21 and 28 days after stenting, the neointima and the neointima/media ratio were not statistically different compared with the results obtained fourteen days after stent deployment. No significant differences in the area of external elastic lamina were observed during the study period. In contrast, the internal lumen area was reduced significantly at 14, 21, and 28 days after the stent deployment. Subacute thrombosis rate after stent implantation was 26.5 %. CONCLUSIONS: The results of this study demonstrated that the balloon expandable stents can be safely placed into rat arteries and the reduction of the internal arterial lumen observed after stent deployment was only due to the neointima formation whereas remodeling did not occur.
Animals↗
8-chloro-cAMP inhibits smooth muscle cell proliferation in vitro and neointima formation induced by balloon injury in vivo.
OBJECTIVES: The aims of the present study were to assess 1) the effect of 8-C1-cAMP (cyclic-3'-5'-adenosine monophosphate) on vascular smooth muscle cell (VSMC) proliferation in vitro and 2) the efficacy of systemic administration of 8-C1-cAMP on neointimal formation after balloon injury in vivo. BACKGROUND: Neointimal formation after vascular injury is responsible for restenosis after arterial stenting. Recently, 8-C1-cAMP, a cAMP analogue that induces growth arrest, has been safely administered in phase I studies in humans. METHODS: The effect of 8-C1-cAMP on cell proliferation was first assessed on SMCs in vitro. To study the effects of cAMP in vivo, balloon injury was performed in 67 rats using a 2F Fogarty balloon catheter. RESULTS: The 8-C1-cAMP markedly inhibited VSMC proliferation in vitro, reduced protein kinase A (PKA) RIalpha subunit expression, and induced PKA RIIbeta subunit expression. In addition, 8-C1-cAMP reduced, in a dose-dependent manner, neointimal area and neointima/media ratio after balloon injury. The proliferative activity, assessed by proliferating nuclear cell antigen immunostaining, revealed a reduction of proliferative activity of VSMCs in vivo in the 8-C1-cAMP group. Moreover, the systemic administration of 8-C1-cAMP did not affect renal function, blood pressure and heart rate. CONCLUSIONS: We conclude that 8-C1-cAMP potently inhibits VSMC proliferation in vitro and reduces neointima formation by balloon injury in vivo after systemic administration. These data may have a clinical relevance in designing future strategies to prevent restenosis after arterial stenting and perhaps after percutaneous transluminal coronary angioplasty.
8-Bromo Cyclic Adenosine Monophosphate↗
Effects of hydroxymethylglutaryl coenzyme A reductase inhibitor simvastatin on smooth muscle cell proliferation in vitro and neointimal formation in vivo after vascular injury.
OBJECTIVES: We sought to evaluate the effects of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase inhibitors on vascular smooth muscle cell (VSMC) proliferation in vitro and neointimal formation in vivo after vascular injury. BACKGROUND: Neointimal hyperplasia after vascular injury is responsible for restenosis after arterial stenting, whereas arterial remodeling and neointimal formation are the causes of restenosis after percutaneous transluminal coronary angioplasty. METHODS: We assessed the effect of simvastatin on in vitro VSMC proliferation. To study the effects of simvastatin in vivo, balloon injury and stent deployment were performed in the common carotid artery of rats. Neointimal area was measured two weeks later in the balloon injury model and three weeks after stent deployment. RESULTS: Simvastatin markedly inhibits VSMC proliferation in vitro. In vivo, simvastatin reduced, in a dose-dependent manner, the neointimal area and the neointima-media ratio after balloon injury from 0.266 +/- 0.015 mm2 to 0.080 +/- 0.026 mm2 and from 1.271 +/- 0.074 to 0.436 +/- 0.158 (p < 0.001 vs. control rats) at the highest dose. Simvastatin also significantly reduced the neointimal formation and the neointima-media ratio after stenting from 0.508 +/- 0.035 mm2 to 0.362 +/- 0.047 mm2 (p < 0.05 vs. control rats) and from 2.000 +/- 0.136 to 1.374 +/- 0.180 (p < 0.05 vs. control rats). The vessel thrombosis rate after stent deployment was 30% in the control group and 11.1% in the treated group (p = NS). Moreover, the systemic administration of simvastatin did not affect hepatic and renal functions, blood pressure or heart rate. CONCLUSIONS: Simvastatin potently inhibits VSMC proliferation in vitro and reduces neointimal formation in a rat model of vascular injury.
Animals↗
Effects of recombinant active site-blocked activated factor VII in rabbit models of carotid stenosis and myocardial infarction.
We tested the effects of human recombinant active site-blocked activated factor VII (rFVIIai) in a rabbit model of carotid artery thrombosis. Cyclic flow variations (CFVs), due to recurrent thrombus formation, were obtained in stenotic rabbit carotid arteries with endothelial injury. After 30 min of CFV, the animals received rFVIIai. If CFVs were abolished, animals were observed for 30 additional minutes, after which human recombinant activated factor VII was infused into the carotid artery to determine whether it could displace rFVIIai from tissue factor (TF), thus restoring CFV. An additional group of animals received rFVIIai to determine its duration of action. Recombinant FVIIai abolished CFVs in 8 of 9 rabbits (P < 0.01). This effect was reversible, as rFVIIa administration restored CFVs in all animals. A further study was initiated to assess whether TF-dependent reductions in coronary blood flow might contribute to the occurrence of myocardial injury during postischaemic reperfusion of rabbit hearts. Recombinant FVIIai resulted in significant reductions in both infarct size and no-reflow area, while rFVIIa produced a significant increase in both infarct size and no-reflow area. These data suggest that rFVIIai might be beneficial in patients with acute myocardial infarction undergoing reperfusion therapies.
Animals↗
Multiple mitogen-activated protein kinase signaling pathways connect the cot oncoprotein to the c-jun promoter and to cellular transformation.
The serine/threonine kinase Cot is a member of the mitogen-activated protein kinase (MAPK) kinase kinase family implicated in cellular transformation. Enhanced expression of this protein has been shown to activate both the MAPK and the c-Jun N-terminal kinase (JNK) pathways and to stimulate the nuclear factor of activated T cells and NF-kappaB-dependent transcription. However, the nature of the normal functions of the Cot protein and the molecular mechanisms responsible for its oncogenic potential are still largely unknown. Here, we show that overexpression of the cot proto-oncogene is sufficient to stimulate the expression of c-jun and that, in turn, the activity of c-Jun is required for Cot-induced transformation. These observations prompted us to explore the molecular events by which Cot regulates c-jun expression. We found that Cot potently stimulates the activity of the c-jun promoter utilizing JNK-dependent and -independent pathways, the latter involving two novel members of the MAPK family, p38gamma (ERK6) and ERK5. Molecularly, this activity was found to be dependent on the ability of Cot to activate, in vivo, members of each class of the MAPK kinase superfamily, including MEK, SEK, MKK6, and MEK5. Furthermore, the use of dominant interfering molecules revealed that Cot requires JNK, p38s, and ERK5 to stimulate the c-jun promoter fully and to induce neoplastic transformation. These findings indicate that Cot represents the first example of a serine/threonine kinase acting simultaneously on all known MAPK cascades. Moreover, these observations strongly suggest that the transforming ability of Cot results from the coordinated activation of these pathways, which ultimately converge on the regulation of the expression and activity of the product of the c-jun proto-oncogene.
3T3 Cells↗
Recombinant human, active site-blocked factor VIIa reduces infarct size and no-reflow phenomenon in rabbits.
Oxygen free radicals induce de novo synthesis of tissue factor (TF), the initiator of the extrinsic pathway of coagulation, within the coronary vasculature during postischemic reperfusion. In the present study we wanted to assess whether TF expression might cause myocardial injury during postischemic reperfusion. Anesthetized rabbits underwent 30 min of coronary occlusion followed by 5.5 h of reperfusion. At reperfusion the animals received 1) saline (n = 8), 2) human recombinant, active site-blocked activated factor VII (FVIIai, 1 mg/kg, n = 8), or 3) human recombinant activated FVII (FVIIa, 1 mg/kg, n = 8). FVIIai binds to TF as native FVII, but with the active site blocked it inhibits TF procoagulant activity. The area at risk of infarction (AR), the infarct size (IS), and the no-reflow area (NR) were determined at the end of the experiment. FVIIai resulted in a significant reduction in IS and NR with respect to control animals (28.1 +/- 11.3 and 11.1 +/- 6.1% of AR vs. 59.8 +/- 12.8 and 24.4 +/- 2.7% of AR, respectively, P < 0.01), whereas FVIIa resulted in a significant increase in IS and NR to 80.1 +/- 13. 1 and 61.9 +/- 13.8% of AR, respectively (P < 0.01). In conclusion, TF-mediated activation of the extrinsic coagulation pathway makes an important contribution to myocardial injury during postischemic reperfusion.
Amino Acid Chloromethyl Ketones↗
Genetically modified animal models to study cardiovascular disorders.
Explore the source record for details and available documents.
Animals↗
Exercise capacity in hypertrophic cardiomyopathy depends on left ventricular diastolic function.
Some studies have demonstrated that left ventricular (LV) diastolic function is the principal determinant of impaired exercise capacity in hypertrophic cardiomyopathy (HC). In this study we sought the capability of echocardiographic indexes of diastolic function in predicting exercise capacity in patients with HC. We studied 52 patients with HC while they were not on drugs;12 of them had LV tract obstruction at rest. Diastolic function was assessed by M-mode and Doppler echocardiography by measuring: (1) left atrial fractional shortening, and the slope of posterior aortic wall displacement during early atrial emptying on M-mode left atrial tracing; and (2) Doppler-derived transmitral and pulmonary venous flow velocity indexes. Exercise capacity was assessed by maximum oxygen consumption by cardiopulmonary test during cycloergometer upright exercise. Maximum oxygen consumption correlated with the left atrial fractional shortening (r = 0.63, p <0.001), the slope of posterior aortic wall displacement during early atrial emptying (r = 0.55, p <0.001), age (r = -0.50; p <0.001), pulmonary venous diastolic anterograde velocity (r = 0.41, p <0.01), and the systolic filling fraction (r = -0.43; p <0.01). By stepwise multiple linear regression analysis, left atrial fractional shortening and the pulmonary venous systolic filling fraction were the only determinants of the maximum oxygen consumption (multiple r = 0.70; p <0.001). Exercise capacity did not correlate with Doppler-derived transmitral indexes. Thus, in patients with HC, exercise capacity was determined by passive LV diastolic function, as assessed by the left atrial M-mode and Doppler-derived pulmonary venous flow velocities.
Adolescent↗
Activation of the protein kinase Akt/PKB by the formation of E-cadherin-mediated cell-cell junctions. Evidence for the association of phosphatidylinositol 3-kinase with the E-cadherin adhesion complex.
E-cadherins are surface adhesion molecules localized at the level of adherens junctions, which play a major role in cell adhesiveness by mediating calcium-dependent homophylic interactions at sites of cell-cell contacts. Recently, E-cadherins have been also implicated in a number of biological processes, including cell growth and differentiation, cell recognition, and sorting during developmental morphogenesis, as well as in aggregation-dependent cell survival. As phosphatidylinositol (PI) 3-kinase and Akt play a critical role in survival pathways in response to both growth factors and extracellular stimuli, these observations prompted us to explore whether E-cadherins could affect intracellular molecules regulating the activity of the PI 3-kinase/Akt signaling cascade. Using Madin-Darby canine kidney cells as a model system, we show here that engagement of E-cadherins in homophylic calcium-dependent cell-cell interactions results in a rapid PI 3-kinase-dependent activation of Akt and the subsequent translocation of Akt to the nucleus. Moreover, we demonstrate that the activation of PI 3-kinase in response to cell-cell contact formation involves the phosphorylation of PI 3-kinase in tyrosine residues, and the concomitant recruitment of PI 3-kinase to E-cadherin-containing protein complexes. These findings indicate that E-cadherins can initiate outside-in signal transducing pathways that regulate the activity of PI 3-kinase and Akt, thus providing a novel molecular mechanism whereby the interaction among neighboring cells and their adhesion status may ultimately control the fate of epithelial cells.
Animals↗
The small GTPases Rab5a, Rab5b and Rab5c are differentially phosphorylated in vitro.
Rab GTPases play a fundamental role in the regulation of membrane traffic. Three different Rab5 isoforms have been reported but no differences in their function in endocytosis have been discovered. As the Rab5 isoforms show a conserved consensus site for Ser/Thr phosphorylation, we investigated whether this site was phosphorylated. Here, we report that the three Rab5 proteins are differentially recognized by different kinases. Rab5a is efficiently phosphorylated by extracellular-regulated kinase 1 but not by extracellular-regulated kinase 2, while cdc2 kinase preferentially phosphorylates Ser-123 of Rab5b. These findings strongly suggest that phosphorylation could be important to differentially regulate the function of the Rab5 isoforms.
Calcium-Calmodulin-Dependent Protein Kinases↗