Antonio Rapacciuolo

Inhibition of cellular ras prevents smooth muscle cell proliferation after vascular injury in vivo

Proliferation of smooth muscle cells of the arterial wall in response to local injury is an important aetiologic factor of vascular proliferative disorders such as atherosclerosis and restenosis after angioplasty. Ras proteins are key transducers of mitogenic signals from membrane to nucleus in many cell types. We investigated the role of ras proteins in the vascular response to arterial injury by inactivating cellular ras of rats in which the common carotid artery was subjected to balloon injury. DNA vectors expressing ras transdominant negative mutants, which interfere with ras function, reduced neointimal formation after injury. Our results indicate a key role for ras in smooth muscle cell proliferation and show that the local delivery of transdominant negative mutants of ras in vivo might prevent some of the acute vascular injury caused by balloon injury.

Smooth Muscle Cell Proliferation Is Proportional to the Degree of Balloon Injury in a Rat Model of Angioplasty

BACKGROUND A variable degree of smooth muscle cell (SMC) proliferation after balloon injury has been reported in previous rat studies. It is unknown whether balloon injury induces c-fos expression and whether it is related to the degree of vascular injury in vivo. Therefore, we tested the hypothesis that proportional increases in neointimal formation and c-fos expression might be present after different degrees of balloon dilation. METHODS AND RESULTS Angioplasty of the carotid artery was performed with a balloon catheter. Vascular injury was evaluated at 0, 0.5, 1.0, 1.5, and 2 atm (n = 6 for all). In 40 additional rats, total RNA dot blots were performed to assess the effect of various degrees of balloon injury on c-fos expression. SMC proliferation proportional to the increases of inflation pressure was found between 0 and 2 atm with neointimal areas of 0.002 +/- 0.002, 0.069 +/- 0.014, 0.128 +/- 0.043, 0.190 +/- 0.010, and 0.255 +/- 0.041 mm2, respectively. When the degree of SMC proliferation (neointima and neointima/media ratio) was plotted against balloon inflation pressure, a linear relation was observed (r = .733, P < .001 and r = .755, P < .001, respectively). An increase in c-fos expression proportional to the degree of injury was found 30 minutes after injury. CONCLUSIONS Neointimal proliferation produced by balloon injury is related to balloon inflation pressure, supporting the concept of an SMC proliferative response proportional to the degree of injury. The increase in SMC proliferation is associated with a proportional increase in the early expression of the c-fos nuclear proto-oncogene.

Cardiac Overexpression of a Gq Inhibitor Blocks Induction of Extracellular Signal–Regulated Kinase and c-Jun NH2-Terminal Kinase Activity in In Vivo Pressure Overload

Background —Understanding the cellular signals that initiate cardiac hypertrophy is of critical importance in identifying the pathways that mediate heart failure. The family of mitogen-activated protein kinases (MAPKs), including the extracellular signal–regulated kinases (ERKs), c-Jun NH2-terminal kinase (JNK), and p38 MAPKs, may play specific roles in myocardial growth and function. Methods and Results —To determine the mechanism of activation of MAPK pathways during the development of cardiac hypertrophy, we evaluated the induction of MAPK activity after aortic constriction in wild-type and in 2 types of cardiac gene-targeted mice: one overexpressing a carboxyl-terminal peptide of G&agr;q that inhibits Gq-mediated signaling (TG GqI mouse) and another overexpressing a carboxyl-terminal peptide of &bgr;-adrenergic receptor kinase-1 that inhibits G&bgr;&ggr; signaling (TG &bgr;ARKct mouse). Wild-type mice with pressure overload showed an acute induction of JNK, followed by the induction of p38/p38&bgr; at 3 days and ERK at 7 days. Both JNK and p38 activity remained elevated at 7 days after banding. In TG GqI mice, hypertrophy was significantly attenuated, and induction of ERK and JNK activity was abolished, whereas the induction of p38 and p38&bgr; was robust, but delayed. By contrast, all 3 MAPK pathways were activated by aortic constriction in the TG &bgr;ARKct hearts, suggesting a role for G&agr;q, but not G&bgr;&ggr;. Conclusions —Taken together, these data show that the induction of ERK and JNK activity in in vivo pressure-overload hypertrophy is mediated through the stimulation of Gq-coupled receptors and that non–Gq-mediated pathways are recruited to activate p38 and p38&bgr;.

Defective beta-adrenergic receptor signaling precedes the development of dilated cardiomyopathy in transgenic mice with calsequestrin overexpression.

Calsequestrin is a high capacity Ca2+-binding protein in the junctional sarcoplasmic reticulum that forms a quaternary complex with junctin, triadin, and the ryanodine receptor. Transgenic mice with cardiac-targeted calsequestrin overexpression show marked suppression of Ca2+-induced Ca2+ release, myocyte hypertrophy, and premature death by 16 weeks of age (Jones, L. R., Suzuki, Y. J., Wang, W., Kobayashi, Y. M., Ramesh, V., Franzini-Armstrong, C., Cleemann, L., and Morad, M. (1998)J. Clin. Invest. 101, 1385–1393). To investigate whether alterations in intracellular Ca2+ trigger changes in the β-adrenergic receptor pathway, we studied calsequestrin overexpressing transgenic mice at 7 and 14 weeks of age. As assessed by echocardiography, calsequestrin mice at 7 weeks showed mild left ventricular enlargement, mild decreased fractional shortening with increased wall thickness. By 14 weeks, the phenotype progressed to marked left ventricular enlargement and severely depressed systolic function. Cardiac catheterization in calsequestrin mice revealed markedly impaired β-adrenergic receptor responsiveness in both 7- and 14- week mice. Biochemical analysis in 7- and 14-week mice showed a significant decrease in total β-adrenergic receptor density, adenylyl cyclase activity, and the percent high affinity agonist binding, which was associated with increased β-adrenergic receptor kinase 1 levels. Taken together, these data indicate that alterations in β-adrenergic receptor signaling precede the development of overt heart failure in this mouse model of progressive cardiomyopathy.

Effects of Balloon Injury on Neointimal Hyperplasia in Streptozotocin-Induced Diabetes and in Hyperinsulinemic Nondiabetic Pancreatic Islet–Transplanted Rats

Background—The mechanisms of increased neointimal hyperplasia after coronary interventions in diabetic patients are still unknown. Methods and Results—Glucose and insulin effects on in vitro vascular smooth muscle cell (VSMC) proliferation and migration were assessed. The effect of balloon injury on neointimal hyperplasia was studied in streptozotocin-induced diabetic rats with or without adjunct insulin therapy. To study the effect of balloon injury in nondiabetic rats with hyperinsulinemia, pancreatic islets were transplanted under the kidney capsule in normal rats. Glucose did not increase VSMC proliferation and migration in vitro. In contrast, insulin induced a significant increase in VSMC proliferation and migration in cell cultures. Furthermore, in VSMC culture, insulin increased MAPK activation. A reduction in neointimal hyperplasia was consistently documented after vascular injury in hyperglycemic streptozotocin-induced diabetic rats. Insulin therapy significantly increased neointimal hyperplasia in these rats. This effect of hyperinsulinemia was totally abolished by transfection on the arterial wall of the N17H-ras–negative mutant gene. Finally, after experimental balloon angioplasty in hyperinsulinemic nondiabetic islet-transplanted rats, a significant increase in neointimal hyperplasia was observed. Conclusions—In rats with streptozotocin-induced diabetes, balloon injury was not associated with an increase in neointimal formation. Exogenous insulin administration in diabetic rats and islet transplantation in nondiabetic rats increased both blood insulin levels and neointimal hyperplasia after balloon injury. Hyperinsulinemia through activation of the ras/MAPK pathway, rather than hyperglycemia per se, seems to be of crucial importance in determining the exaggerated neointimal hyperplasia after balloon angioplasty in diabetic animals.

Important role of endogenous norepinephrine and epinephrine in the development of in vivo pressure-overload cardiac hypertrophy.

OBJECTIVES We sought to define the role of norepinephrine and epinephrine in the development of cardiac hypertrophy and to determine whether the absence of circulating catecholamines alters the activation of downstream myocardial signaling pathways. BACKGROUND Cardiac hypertrophy is associated with elevated plasma catecholamine levels and an increase in cardiac morbidity and mortality. Although considerable evidence suggests that G-protein-coupled receptors are involved in the hypertrophic response, it remains controversial whether catecholamines are required for the development of in vivo cardiac hypertrophy. METHODS We performed transverse aortic constriction (TAC) in dopamine beta-hydroxylase knockout mice (Dbh(-/-), genetically altered mice that are completely devoid of endogenous norepinephrine and epinephrine) and littermate control mice. After induction of cardiac hypertrophy, the mitogen-activated protein kinase (MAPK) signaling pathways were measured in pressure-overloaded/wild-type and Dbh(-/-) hearts. RESULTS Compared with the control animals, cardiac hypertrophy was significantly blunted in Dbh(-/-) mice, which was not associated with altered cardiac function, as assessed by transthoracic echocardiography in conscious mice. The extracellularly regulated kinase (ERK 1/2), c-jun-NH(2)-terminal kinase (JNK) and p38 MAPK pathways were all activated by two- to threefold after TAC in the control animals. In contrast, induction of the three pathways (ERK 1/2, JNK and p38) was completely abolished in Dbh(-/-) mice. CONCLUSIONS These data demonstrate a nearly complete requirement of endogenous norepinephrine and epinephrine for the induction of in vivo pressure-overload cardiac hypertrophy and for the activation of hypertrophic signaling pathways.

Regulation of myocardial βARK1 expression in catecholamine-induced cardiac hypertrophy in transgenic mice overexpressing α1B-adrenergic receptors

OBJECTIVES Using a transgenic mouse model of myocardial-targeted overexpression of the wild-type α1Badrenergic receptor (AR) (Tgα43), we studied the role of the βAR kinase (βARK1) in the evolution of myocardial hypertrophy and its transition to heart failure (HF). BACKGROUND Increased myocardial expression of βARK1 has been shown to be associated with HF and certain models of hypertrophy. METHODS Tgα43 mice and their nontransgenic littermate controls were treated with the α1AR agonist phenylephrine (PE) for 3, 7 or 14 days to characterize the cardiac consequences. RESULTS Nontransgenic littermate control mice treated for 14 days with PE display cardiac hypertrophy with no increase in βARK1 expression. However, Tgα43 animals show a reduced tolerance to 14-day PE treatment, demonstrated by reduced survival and severe cardiac hypertrophy. Moreover, PE treatment for three and seven days in Tgα43 mice resulted in an exaggerated hypertrophic response accompanied by significant cardiac biochemical abnormalities that are normally associated with HF, including fetal gene expression, reduced βAR density and enhanced βARK1 expression. We also found reduced myocardial stores of the sympathetic neurotransmitter neuropeptide Y. CONCLUSIONS These data suggest that PE-treated Tgα43 mice have chronic activation of the cardiac sympathetic nervous system, which may be responsible for the appearance of apparent maladaptive hypertrophy with an evolution towards HF and sudden death. Thus, the cardiac phenotypes found in these mice are not the direct result of enhanced α1BAR signaling and suggest that βARK1 is a key molecule in the transition of myocardial hypertrophy to HF.

In vivo gene transfer: prevention of neointima formation by inhibition of mitogen-activated protein kinase kinase

BackgroundThe mitogenactivated protein kinase kinase (MAPKK) is a protein downstream ras which is rapidly activated in cells stimulated with various extracellular signals. These proteins are believed to play a pivotal role in integrating and transmitting transmembrane signals required for cell growth.Methods and ResultsTo study the effect of inhibition of MAPKK on smooth muscle cell (SMC) proliferationin vivo after vascular injury, we performed experimental balloon angioplasty using the standard Clowes technique in male Wistar rats 14-weeks old. The animals did not receive any treatment after vascular injury (N=6) or were randomly assigned to receive, after balloon injury, a 30% (w/v) pluronic gel solution applied to the injured carotid artery, containing respectively: 1) no plasmid DNA (n=10); 2) RSV-lacZ (encoding the β-galactosidase gene) as control gene without effects on SMC proliferation (n=10); 3) Tg-CAT (encoding cloramphenicol acetyl-transferase gene under the control of thyreoglobulin promoter) as an additional control gene without effects on SMC proliferation (n=7); 4) a negative mutant of Mitogen-Activated Protein Kinase Kinase (MAPKK) (n=13). Fourteen days after vascular injury, carotid arteries were removed and cross sections were cut and stained with hematoxylin/eosin. Morphometric analysis demonstrated, in the MAPKK-treated rats, a significant reduction of both neointima (0.096±0.018 mm2 vs. 0.184±0.019 mm2, p<0.01) and neointima/media ratio (0.603±0.103 vs. 1.147±0.161, p<0.01) compared to control DNA.ConclusionsThe inhibition of MAPKK, by a dominant inhibitor mutant gene, prevents the SMC proliferation after vascular injuryin vivo.

Multicentre experience with MGuard net protective stent in ST-elevation myocardial infarction: safety, feasibility, and impact on myocardial reperfusion.

Objective: To report, for the first time, angiographic and ECG results as well as in‐hospital and 1‐month clinical follow‐up, after MGuard net protective stent (Inspire‐MD, Tel‐Aviv, Israel—MGS) implantation in consecutive, not randomized, STEMI patients undergoing primary or rescue PCI. Background: Distal embolization may decrease coronary and myocardial reperfusion after percutaneous coronary intervention (PCI), in ST‐elevation myocardial infarction (STEMI) setting. Methods: One‐hundred consecutive patients underwent PCI, with MGS deployment for STEMI, in five different high‐volume PCI centres. Sixteen patients presented cardiogenic shock at admission. Results: All patients underwent successful procedures: mean TIMI flow grade and mean corrected TIMI frame count—cTFC(n)—improved from baseline values to 2.85 ± 0.40 and to 17.20 ± 10.51, respectively, with a mean difference in cTFC(n) between baseline and postprocedure of 46.88 ± 31.86. High‐myocardial blush grade (90% MBG 3; 10% MBG 2) was also achieved in all patients. Sixty minutes post‐PCI, a high rate (90%) of complete (≥70%) ST‐segment resolution was achieved. At in‐hospital follow‐up, seven deaths occurred: noteworthy, 5 of 16 patients with cardiogenic shock at admission died. After hospital discharge, no Major Adverse Cardiac Events have been reported up to 30‐day follow‐up. Conclusions: MGS might represent a safe and feasible option for PCI in STEMI patients, providing high perfusional and ECG improvement. Further randomized trials comparing this strategy with the conventional one are needed in the near future to assess the impact on clinical practice of this strategy.

Selective Inhibition of Heterotrimeric Gs Signaling: Targeting the Receptor-G Protein Interface Using a Peptide Minigene Encoding the Gαs Carboxyl Terminus.

The blockade of heptahelical receptor coupling to heterotrimeric G proteins by the expression of peptides derived from G protein Gα subunits represents a novel means of simultaneously inhibiting signals arising from multiple receptors that share a common G protein pool. Here we examined the mechanism of action and functional consequences of expression of an 83-amino acid polypeptide derived from the carboxyl terminus of Gαs(GsCT). In membranes prepared from GsCT-expressing cells, the peptide blocked high affinity agonist binding to β2adrenergic receptors (AR) and inhibited β2AR-induced [35S]GTPγS loading of Gαs. GsCT expression inhibited β2AR- and dopamine D1Areceptor-mediated cAMP production, without affecting the cellular response to cholera toxin or forskolin, indicating that the peptide inhibited receptor-Gs coupling without impairing G protein or adenylyl cyclase function. [35S]GTPγS loading of Gαq/11 by α1BARs and Gαi by α2AARs and Gq/11- or Gi-mediated phosphatidylinositol hydrolysis was unaffected, indicating that the inhibitory effects of GsCT were selective for Gs. We next employed the GsCT construct to examine the complex role of Gs in regulation of the ERK mitogen-activated protein kinase cascade, where activation of the cAMP-dependent protein kinase (PKA) pathway reportedly produces both stimulatory and inhibitory effects on heptahelical receptor-mediated ERK activation. For the β2AR in HEK-293 cells, where PKA activity is required for ERK activation, expression of GsCT caused a net inhibition of ERK activation. In contrast, α2AAR-mediated ERK activation in COS-7 cells was enhanced by GsCT expression, consistent with the relief of a downstream inhibitory effect of PKA. ERK activation by the Gq/11-coupled α1BAR was unaffected by GsCT. These findings suggest that peptide G protein inhibitors can provide insights into the complex interplay between G protein pools in cellular regulation.