Abstract: Integrins are heterodimeric receptors that play a critical role in cell-cell and cell-matrix adhesion processes. Among them, αVβ3 integrin, that recognizes the aminoacidic RGD triad, is reported to be involved in angiogenesis, tissue repair and tumor growth. We have recently synthesized a new and selective ligand of αVβ3 receptor, referred to as RGDechiHCit, that contains a cyclic RGD motif and two echistatin moieties.
Abstract: Adrenergic stimulation of the heart engages cAMP and phosphoinositide second messenger signaling cascades. Cardiac phosphoinositide 3-kinase p110 gamma participates in these processes by sustaining beta-adrenergic receptor internalization through its catalytic function and by controlling phosphodiesterase 3B (PDE3B) activity via an unknown kinase-independent mechanism. We have discovered that p110 gamma anchors protein kinase A (PKA) through a site in its N-terminal region. Anchored PKA activates PDE3B to enhance cAMP degradation and phosphorylates p110 gamma to inhibit PIP(3) production. This provides local feedback control of PIP(3) and cAMP signaling events. In congestive heart failure, p110 gamma is upregulated and escapes PKA-mediated inhibition, contributing to a reduction in beta-adrenergic receptor density. Pharmacological inhibition of p110 gamma normalizes beta-adrenergic receptor density and improves contractility in failing hearts.
Abstract: Lymphocyte G protein coupled receptor kinase 2 (GRK2) levels are increased in patients with chronic heart failure, and in this condition, they correlate with cardiac function. The aim of this study was to assess the prognostic role of GRK2 during acute cardiac dysfunction in humans. A study was designed to investigate the role of GRK2 levels in patients with acute coronary syndromes. Lymphocyte GRK2 levels were examined at admission and after 24 and 48 hours in 42 patients with acute coronary syndromes, 32 with ST-segment elevation myocardial infarction and 10 with unstable angina as a control group. Echocardiographic parameters of systolic and diastolic function and left ventricular remodeling were evaluated at admission and after 2 years. GRK2 levels increased during ST-segment elevation myocardial infarction and were associated with worse systolic and diastolic function. This association held at 2-year follow-up, when GRK2 was correlated with the ejection fraction and end-systolic volume, indicating a prognostic value for GRK2 levels during acute ST-segment elevation myocardial infarction. In conclusion, lymphocyte GRK2 levels increase during acute myocardial infarction and are associated with worse cardiac function. Taken together, these data indicate that GRK2 could be predictive of ventricular remodeling after myocardial infarction and could facilitate the tailoring of appropriate therapy for high-risk patients. (C) 2011 Elsevier Inc. All rights reserved. (Am J Cardiol 2011;107:1125-1130)
Abstract: Background: Integrins are heterodimeric receptors that play a critical role in cell-cell and cell-matrix adhesion processes. Among them, alpha(V)beta(3) integrin, that recognizes the aminoacidic RGD triad, is reported to be involved in angiogenesis, tissue repair and tumor growth. We have recently synthesized a new and selective ligand of alpha(V)beta(3) receptor, referred to as RGDechiHCit, that contains a cyclic RGD motif and two echistatin moieties. xD;Methods: The aim of this study is to evaluate in vitro and in vivo the effects of RGDechiHCit. Therefore, we assessed its properties in cellular (endothelial cells [EC], and vascular smooth muscle cells [VSMC]) and animal models (Wistar Kyoto rats and c57Bl/6 mice) of angiogenesis. xD;Results: In EC, but not VSMC, RGDechiHCit inhibits intracellular mitogenic signaling and cell proliferation. Furthermore, RGDechiHCit blocks the ability of EC to form tubes on Matrigel. In vivo, wound healing is delayed in presence of RGDechiHCit. Similarly, Matrigel plugs demonstrate an antiangiogenic effect of RGDechiHCit. xD;Conclusions: Our data indicate the importance of RGDechiHCit in the selective inhibition of endothelial alpha(V)beta(3) integrin in vitro and in vivo. Such inhibition opens new fields of investigation on the mechanisms of angiogenesis, offering clinical implications for treatment of pathophysiological conditions such as cancer, proliferative retinopathy and inflammatory disease.
Abstract: There is much evidence supporting the role of β₂-adrenoceptors (β₂AR) in angiogenesis but the mechanisms underlying their effects have not been elucidated. Hence, we studied post-ischaemic angiogenesis in the hindlimb (HL) of β₂AR knock-out mice (β₂AR-/-) in vivo and explored possible molecular mechanisms in vitro.
Abstract: BACKGROUND AND PURPOSE xD;There is much evidence supporting the role of beta(2)-adrenoceptors (beta(2)AR) in angiogenesis but the mechanisms underlying their effects have not been elucidated. Hence, we studied post-ischaemic angiogenesis in the hindlimb (HL) of beta(2)AR knock-out mice (beta(2)AR-/-) in vivo and explored possible molecular mechanisms in vitro. xD;EXPERIMENTAL APPROACH xD;Femoral artery resection (FAR) was performed in wild-type and beta(2)AR-/- mice and adaptive responses to chronic HL ischaemia were explored; blood flow was measured by ultrasound and perfusion of dyed beads, bone rarefaction, muscle fibrosis and skin thickness were evaluated by immunoflourescence and morphometric analysis. Intrafemoral delivery of an adenovirus encoding the human beta(2)AR (AD beta(2)AR) was used to reinstate beta(2)ARs in beta(2)AR-/- mice. Molecular mechanisms were investigated in mouse-derived aortic endothelial cells (EC) in vitro, focusing on NF kappa B activation and transcriptional activity. xD;RESULTS xD;Angiogenesis was severely impaired in beta(2)AR-/- mice subjected to FAR, but was restored by gene therapy with AD beta(2)AR. The proangiogenic responses to a variety of stimuli were impaired in beta(2)AR-/- EC in vitro. Moreover, removal of beta(2)ARs impaired the activation of NF kappa B, a transcription factor that promotes angiogenesis; neither isoprenaline (stimulates beta ARs) nor TNF alpha induced NF kappa B activation in beta(2)AR-/- EC. Interestingly, cAMP response element binding protein (CREB), a transcription factor that counter regulates NF kappa B, was constitutively increased in beta(2)AR-/- ECs. AD beta(2)AR administration restored beta(2)AR membrane density, reduced CREB activity and reinstated the NF kappa B response to isoprenaline and TNF alpha. xD;CONCLUSIONS AND IMPLICATIONS xD;Our results suggest that beta(2)ARs control angiogenesis through the tight regulation of nuclear transcriptional activity.
Abstract: The clinical impact of PlA2 polymorphism has been investigated in several diseases, but the definition of its specific role on thrombotic cardiovascular complications has been challenging. We aimed to explore the effect of PlA2 polymorphism on outcome in patients with atherosclerosis.
Abstract: Several studies underline the role of the transcription factor NF-kappa B in the development of left cardiac hypertrophy (LVH). We have demonstrated recently that the RGS homology domain within the amino terminus of GRK5 (GRK5-NT) is able to inhibit NF-kappa B transcription activity and its associated phenotypes. The aim of this study was to evaluate the ability of GRK5-NT to regulate LVH through the inhibition of NF-kappa B both in vitro and in vivo. In cardiomyoblasts, GRK5-NT inhibits phenylephrine-induced transcription of both NF-kappa B and atrial natriuretic factor promoters, assessed by luciferase assay, thus confirming a role for this protein in the regulation of cardiomyocyte hypertrophy. In vivo, we explored 2 rat models of LVH, the spontaneously hypertensive rat and the normotensive Wistar Kyoto rat exposed to chronic administration of phenylephrine. Intracardiac injection of an adenovirus encoding for GRK5-NT reduces cardiac mass in spontaneously hypertensive rats and prevents the development of phenylephrine-induced LVH in Wistar Kyoto rats. This associates with inhibition of NF-kappa B signaling (assessed by NF-kappa B levels), transcriptional activity and phenotypes (fibrosis and apoptosis). Such phenomenon is independent from hemodynamic changes, because adenovirus encoding for GRK5-NT did not reduce blood pressure levels in spontaneously hypertensive rats or in Wistar Kyoto rats. In conclusion, our study supports the regulation of LVH based on the GRK5-NT inhibition of the NF-kappa B transduction signaling. (Hypertension. 2010;56:696-704.)
Abstract: Background: The clinical impact of PIA2 polymorphism has been investigated in several diseases, but the definition of its specific role on thrombotic cardiovascular complications has been challenging. We aimed to explore the effect of PIA2 polymorphism on outcome in patients with atherosclerosis. xD;Methods: We studied 400 consecutive patients with coronary artery disease (CAD) undergoing percutaneous coronary intervention. A replication study was conducted in 74 hypertensive patients with cerebrovascular events while a group of 100 healthy subjects was included as control population. PIA genotype was determined by PCR-RFLP on genomic DNA from peripheral blood cells. Major adverse cardiac events (MACE), were considered as end points, and recorded at a mean follow up of 24 +/- 4.3 months. xD;Results: The frequencies of PIA2 polymorphism was similar between groups and genotype distribution was in Hardy-Weinberg equilibrium. In patients with CAD, the presence of PIA2 allele was associated with higher incidence of cardiac death (13.1% vs. 1.5%, p = 0.0001), myocardial infarction (10.7% vs. 2.6%, p = 0.004) and needs of new revascularization (34.8% vs. 17.7%, p = 0.010). Accordingly, the Kaplan-Meier analysis for event free survival in patients harboring the PIA2 allele showed worse long-term outcome for these patients (p = 0.015). Cox regression analysis identified the presence of PlA2 as an independent predictor of cardiac death (OR: 9.594, 95% CI: 2.6 to 35.3, p = 0.002) and overall MACE (OR: 1.829, 95% CI: 1.054 to 3.176, p = 0.032). In the replication study, the PlA2 polymorphism increased the risk of stroke (OR: 4.1, 95% CI: 1.63-12.4, p = 0.02) over TIA and was identified as an independent risk factor for stroke (B:-1.39; Wald: 7.15; p = 0.001). xD;Conclusions: Our study demonstrates that in patients with severe atherosclerosis the presence of PlA2 allele is associated with thrombotic cardiovascular complications.
Abstract: We observed a patient with syncope, who implanted a pacemaker with advanced algorithms such as "atrial-tachy response" and "dynamic atrio-ventricular delay". After one year, conventional ECG Holter showed pacemaker malfunction, wrongly attributed to exposure to electromagnetic field. In fact, telemetry revealed an inappropriate programming and solved our case. Holter monitoring is commonly performed in the evaluation of pacemaker malfunction, albeit it remains a quite shallow diagnostic method especially to detect electromagnetic interferences. New algorithms seem important, but it is reasonable to obtain more suitable analytical tools, too. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
Abstract: Several studies underline the role of the transcription factor NF-κB in the development of left cardiac hypertrophy (LVH). We have demonstrated recently that the RGS homology domain within the amino terminus of GRK5 (GRK5-NT) is able to inhibit NF-κB transcription activity and its associated phenotypes. The aim of this study was to evaluate the ability of GRK5-NT to regulate LVH through the inhibition of NF-κB both in vitro and in vivo. In cardiomyoblasts, GRK5-NT inhibits phenylephrine-induced transcription of both NF-κB and atrial natriuretic factor promoters, assessed by luciferase assay, thus confirming a role for this protein in the regulation of cardiomyocyte hypertrophy. In vivo, we explored 2 rat models of LVH, the spontaneously hypertensive rat and the normotensive Wistar Kyoto rat exposed to chronic administration of phenylephrine. Intracardiac injection of an adenovirus encoding for GRK5-NT reduces cardiac mass in spontaneously hypertensive rats and prevents the development of phenylephrine-induced LVH in Wistar Kyoto rats. This associates with inhibition of NF-κB signaling (assessed by NF-κB levels), transcriptional activity and phenotypes (fibrosis and apoptosis). Such phenomenon is independent from hemodynamic changes, because adenovirus encoding for GRK5-NT did not reduce blood pressure levels in spontaneously hypertensive rats or in Wistar Kyoto rats. In conclusion, our study supports the regulation of LVH based on the GRK5-NT inhibition of the NF-κB transduction signaling.
Abstract: Overproduction of phosphoprotein enriched in diabetes (PED, also known as phosphoprotein enriched in astrocytes-15 [PEA-15]) is a common feature of type 2 diabetes and impairs insulin action in cultured cells and in mice. Nevertheless, the potential role of PED in diabetic complications is still unknown. xD;We studied the effect of PED overproduction and depletion on kidney function in animal and cellular models. xD;Transgenic mice overexpressing PED (PEDTg) featured age-dependent increases of plasma creatinine levels and urinary volume, accompanied by expansion of the mesangial area, compared with wild-type littermates. Serum and kidney levels of TGF-beta 1 were also higher in 6- and 9-month-old PEDTg. Overexpression of PED in human kidney 2 cells significantly increased TGF-beta 1 levels, SMAD family members (SMAD)2/3 phosphorylation and fibronectin production. Opposite results were obtained following genetic silencing of PED in human kidney 2 cells by antisense oligonucleotides. Inhibition of phospholipase D and protein kinase C-beta by 2-butanol and LY373196 respectively reduced TGF-beta 1, SMAD2/3 phosphorylation and fibronectin production. Moreover, inhibition of TGF-beta 1 receptor activity and SMAD2/3 production by SB431542 and antisense oligonucleotides respectively reduced fibronectin secretion by about 50%. TGF-beta 1 circulating levels were significantly reduced in Ped knockout mice and positively correlated with PED content in peripheral blood leucocytes of type 2 diabetic patients. xD;These data indicate that PED regulates fibronectin production via phospholipase D/protein kinase C-beta and TGF-beta 1/SMAD pathways in kidney cells. Raised PED levels may therefore contribute to the abnormal accumulation of extracellular matrix and renal dysfunction in diabetes.
Abstract: The main regulator of neovascularization is Vascular Endothelial Growth Factor (VEGF). We recently demonstrated that QK, a de novo engineered VEGF mimicking peptide, shares in vitro the same biological properties of VEGF, inducing capillary formation and organization. On these grounds, the aim of this study is to evaluate in vivo the effects of this small peptide. Therefore, on Wistar Kyoto rats, we evaluated vasomotor responses to VEGF and QK in common carotid rings. Also, we assessed the effects of QK in three different models of angiogenesis: ischemic hindlimb, wound healing and Matrigel plugs. QK and VEGF present similar endothelium-dependent vasodilatation. Moreover, the ability of QK to induce neovascularization was confirmed us by digital angiographies, dyed beads dilution and histological analysis in the ischemic hindlimb as well as by histology in wounds and Matrigel plugs. Our findings show the proangiogenic properties of QK, suggesting that also in vivo this peptide resembles the full VEGF protein. These data open to new fields of investigation on the mechanisms of activation of VEGF receptors, offering clinical implications for treatment of pathophysiological conditions such as chronic ischemia.
Abstract: Insulin (Ins) resistance (IRES) associates to increased cardiovascular risk as observed in metabolic syndrome. Chronic stimulation of beta-adrenergic receptors (beta AR) due to exaggerated sympathetic nervous system activity is involved in the pathogenesis of IRES. The cellular levels of G protein coupled receptor kinase 2 (GRK2) increase during chronic beta AR stimulation, leading to beta AR desensitization. We tested the hypothesis that GRK2 plays a role in beta AR-induced IRES. xD;We evaluated Ins-induced glucose uptake and signalling responses in vitro in cell overexpressing the beta(2)AR, the GRK2, or the catalytically dead mutant GRK2-DN. In a model of increased adrenergic activity, IRES and elevated cellular GRK2 levels, the spontaneously hypertensive rats (SHR) we performed the intravenous glucose tolerance test load. To inhibit GRK2, we synthesized a peptide based on the catalytical sequence of GRK2 conjugated with the antennapedia internalization sequence (Ant-124). Ins in human kidney embryonic (HEK-293) cells causes rapid accumulation of GRK2, tyrosine phosphorylation of Ins receptor substrate 1 (IRS1) and induces glucose uptake. In the same cell type, transgenic beta(2)AR overexpression causes GRK2 accumulation associated with significant deficit of IRS1 activation and glucose uptake by Ins. Similarly, transgenic GRK2 overexpression prevents Ins-induced tyrosine phosphorylation of IRS1 and glucose uptake, whereas GRK2-DN ameliorates glucose extraction. By immunoprecipitation, GRK2 binds IRS1 but not the Ins receptor in an Ins-dependent fashion, which is lost in HEK-GRK2 cells. Ant-124 improves Ins-induced glucose uptake in HEK-293 and HEK-GRK2 cells, but does not prevent GRK2/IRS1 interaction. In SHR, Ant-124 infusion for 30 days ameliorates IRES and IRS1 tyrosine phosphorylation. xD;Our results suggest that GRK2 mediates adrenergic IRES and that inhibition of GRK2 activity leads to increased Ins sensitivity both in cells and in animal model of IRES.
Abstract: Background: Based on its role in angiogenesis and apoptosis, the inhibition of NF kappa B activity is considered an effective treatment for cancer, hampered by the lack of selective and safe inhibitors. We recently demonstrated that the RH domain of GRK5 (GRK5-RH) inhibits NF kappa B, thus we evaluated its effects on cancer growth. xD;Methods: The role of GRK5-RH on tumor growth was assessed in a human cancer cell line (KAT-4). RH overexpression was induced by adenovirus mediated gene transfer; alternatively we administered a synthetic protein reproducing the RH domain of GRK5 (TAT-RH), actively transported into the cells. xD;Results: In vitro, adenovirus mediated GRK5-RH overexpression (AdGRK5-NT) in human tumor cells (KAT-4) induces I kappa B accumulation and inhibits NF kappa B transcriptional activity leading to apoptotic events. In BALB/c nude mice harboring KAT-4 induced neoplasias, intra-tumor delivery of AdGRK5-NT reduces in a dose-dependent fashion tumor growth, with the highest doses completely inhibiting it. This phenomenon is paralleled by a decrease of NF kappa B activity, an increase of I kappa B levels and apoptotic events. To move towards a pharmacological setup, we synthesized the TAT-RH protein. In cultured KAT-4 cells, different dosages of TAT-RH reduced cell survival and increased apoptosis. In BALB/c mice, the anti-proliferative effects of TAT-RH appear to be dose-dependent and highest dose completely inhibits tumor growth. xD;Conclusion: Our data suggest that GRK5-RH inhibition of NF kappa B is a novel and effective anti-tumoral strategy and TAT-RH could be an useful tool in the fighting of cancer.
Abstract: Insulin (Ins) resistance (IRES) associates to increased cardiovascular risk as observed in metabolic syndrome. Chronic stimulation of beta-adrenergic receptors (betaAR) due to exaggerated sympathetic nervous system activity is involved in the pathogenesis of IRES. The cellular levels of G protein coupled receptor kinase 2 (GRK2) increase during chronic betaAR stimulation, leading to betaAR desensitization. We tested the hypothesis that GRK2 plays a role in betaAR-induced IRES.
Abstract: Overproduction of phosphoprotein enriched in diabetes (PED, also known as phosphoprotein enriched in astrocytes-15 [PEA-15]) is a common feature of type 2 diabetes and impairs insulin action in cultured cells and in mice. Nevertheless, the potential role of PED in diabetic complications is still unknown.
Abstract: Based on its role in angiogenesis and apoptosis, the inhibition of NFkappaB activity is considered an effective treatment for cancer, hampered by the lack of selective and safe inhibitors. We recently demonstrated that the RH domain of GRK5 (GRK5-RH) inhibits NFkappaB, thus we evaluated its effects on cancer growth.
Abstract: The alpha(v) integrins are key receptors involved in mediating cell migration and angiogenesis. In age-related macular degeneration (AMD) and diabetic retinopathy, angiogenesis plays a critical role in the loss of vision. These ocular vasculopathies might be treatable with a suitable alpha(v) antagonist, and an oral drug would offer a distinct advantage over current therapies. (3,S,beta,S)-1,2,3,4-Tetrahydro-beta-[[1-[1-oxo-3-(1,5,6,7-tetrahydro-1,8-naphthyridin-2-yl) propyl]-4-piperidinyl]methyl]-3-quinolinepropanoic acid (JNJ-26076713) is a potent, orally bioavailable, nonpeptide alpha(v) antagonist derived from the arginine-glycine-asparagine binding motif in the matrix protein ligands (e.g., vitronectin). This compound inhibits alpha(v)beta(3) and alpha(v)beta(5) binding to vitronectin in the low nanomolar range, it has excellent selectivity over integrins alpha(11b)beta(3) and alpha(5)beta(1), and it prevents adhesion to human, rat, and mouse endothelial cells. JNJ-26076713 blocks cell migration induced by vascular endothelial growth factor, fibroblast growth factor (FGF), and serum, and angiogenesis induced by FGF in the chick chorioallantoic membrane model. JNJ-26076713 is the first alpha(V) antagonist reported to inhibit retinal neovascularization in an oxygen-induced model of retinopathy of prematurity after oral administration. In diabetic rats, orally administered JNJ-26076713 markedly inhibits retinal vascular permeability, a key early event in diabetic macular edema and AMD. Given this profile, JNJ-26076713 represents a potential therapeutic candidate for the treatment of age-related macular degeneration, macular edema, and proliferative diabetic retinopathy.
Abstract: Background and purpose: Intact endothelium plays a pivotal role in post-ischaemic angiogenesis. It is a phenomenon finely tuned by activation and inhibition of several endothelial receptors. The presence of alpha(1)-adrenoceptors on the endothelium suggests that these receptors may participate in regenerative phenomena by regulating the responses of endothelial cells involved in neo-angiogenesis. xD;Experimental approach: We evaluated the expression of the subtypes of the alpha(1)-adrenoceptor in isolated endothelial cells harvested from Wistar-Kyoto (WKY) rats. We explored the possibility these alpha(1)-adrenoceptors may influence the pro-angiogenic phenotype of endothelial cells in vitro. In vivo, we used a model of hindlimb ischaemia in WKY rats, to assess the effects of alpha(1) adrenoceptor agonist or antagonist on angiogenesis in the ischaemic hindlimb by laser Doppler blood flow measurements, digital angiographies, hindlimb perfusion with dyed beads and histological evaluation. xD;Key results: In vitro, pharmacological antagonism of alpha(1)-adrenoceptors in endothelial cells from WKY rats by doxazosin enhanced, while stimulation of these adrenoceptors with phenylephrine, inhibited endothelial cell proliferation and DNA synthesis, ERK and retinoblastoma protein (Rb) phosphorylation, cell migration and tubule formation. In vivo, we found increased alpha(1)-adrenoceptor density in the ischaemic hindlimb, compared to non-ischaemic hindlimb, suggesting an enhanced alpha(1)-adrenoceptor tone in the ischaemic tissue. Treatment with doxazosin (0.06mg kg(-1) day(-1) for 14 days) did not alter systemic blood pressure but enhanced neo-angiogenesis in the ischaemic hindlimb, as measured by all our assays. xD;Conclusions: Our findings support the hypothesis that the alpha(1)-adrenoceptors in endothelial cells provide a negative regulation of angiogenesis.
Abstract: Increased levels of G protein coupled receptor kinase GRK2 appear to participate in hypertension presumably through the desensitization of beta adrenergic receptors (beta ARs) that mediate vasodilatation. There are contrasting data on the occurrence of beta AR desensitization in the vasculature, we therefore investigated beta AR vasodilatation and desensitization in normotensives and in hypertensive humans. In blood lymphocytes, we assessed beta AR signaling and GRK2 expression and found beta AR signaling alterations and consistent with desensitization, increased GRK2 levels in hypertensives. We studied in vivo vasodilatation to the beta AR agonist isoproterenol (ISO) injected in the brachial artery in control conditions and during the concomitant infusion of heparin, a known in vitro nonspecific GRK inhibitor. ISO induced a dose-dependent vasorelaxation that was attenuated in hypertensives indicating a loss of beta AR signaling. Intra-arterial infusion of heparin inhibited lymphocyte GRK2 activity and prevented desensitization of beta AR vasodilatation in normotensives. In hypertensives, heparin restored vasodilatation to ISO, to levels observed in normotensives. Our results suggest that beta AR desensitization does indeed occur at the vascular levels in vivo, and that heparin by acting as a GRK inhibitor prevents this in normotensives and restores impaired beta AR vasodilation in hypertensives. We conclude that desensitization participates to impaired beta AR vasodilation in hypertension.
Abstract: G-protein-coupled receptor (GPCR) kinases, GPCR, are known as serine/threonine kinases that regulate GPCR signaling, but recent findings propose functions for these kinases besides receptor desensitization. Indeed, GRK5 can translocate to the nucleus by means of a nuclear localization sequence, suggesting that this kinase regulates transcription events in the nucleus. To evaluate the effect of GRK5-I kappa b alpha interaction on NF kappa B signaling, we induced the overexpression and the knockdown of GRK5 in cell cultures. GRK5 overexpression causes nuclear accumulation of I kappa B alpha, leading to the inhibition of NF kappa B transcriptional activity. Opposite results are achieved by GRK5 knockdown through siRNA. A physical interaction between GRK5 and I kappa B alpha, rather than phosphorylative events, appears as the underlying mechanism. We identify the regulator of gene protein signaling homology domain of GRK5 (RH) and the IN-terminal domain Of I kappa B alpha as the regions involved in such interaction. To confirm the biological relevance of this mechanism of regulation for NF kappa B, we evaluated the effects of GRK5-RH on NF kappa B-dependent phenotypes. In particular, GRK5-RH overexpression impairs apoptosis protection and cytokine production in vitro and inflammation and tissue regeneration in vivo. Our results reveal an unexpected role for GRK5 in the regulation of NF kappa B transcription activity. Placing these findings in perspective, this mechanism may represent a therapeutic target for all those conditions involving excessive NF kappa B activity.
Abstract: We have recently demonstrated that endothelial beta(2) adrenergic receptors beta(2)AR regulate eNOS activity and consequently vascular tone, through means of PKB/AKT. In this work we explored the signal transduction pathway leading to AKT/eNOS activation in endothelial cells (EC). Using pharmacological and molecular inhibitors both in cultured EC cells and in ex vivo rat carotid preparations, we found that G(i) coupling of the beta 2AR is needed for AKT activation and vasorelaxation. Since endothelial activation is sensitive to pertussis toxin but not to G(i beta gamma), inhibition by beta ARKct, we conclude that G(alpha i) mediates beta AR induced AKT activation. Downstream, beta AR signalling requires the soluble tyrosine kinase SRC, as both in cultured EC and rat carotid, the mutant dominant negative of SRC prevent beta(2)AR induced endothelial activation and vasodilation. In EC, G(alpha i) directly interacts with SRC and this interaction leads to SRC activation and phosphorylation in a manner that is regulated by Balk stimulation. We propose a novel signal transduction pathway for beta(2)AR stimulation trough G(alpha i) and SRC, leading to activation of AKT. (c) 2007 Elsevier Inc. All rights reserved.
Abstract: Objective: The platelet GPIIIa plays a pivotal role in platelet aggregation. Previous studies showed an association between the GPIIIa PIA1/A2 polymorphism and coronary thrombosis, while there is only contrasting evidence about its role in stroke. We explored the possibility that this polymorphism represents a risk factor for stroke in hypertensive patients. xD;Methods: We studied two populations. In loco, we genotyped 140 hypertensive control individuals and 28 hypertensive patients with ischemic stroke. Furthermore, we performed an analysis of previously published data of 451 Sardinian hypertensive patients, already characterized and genotyped. xD;Results: Association analysis revealed that the PIA2 distribution was similar between hypertensive patients with and without stroke, but when considering a more homogeneous population of high-risk hypertensive patients, defined according to ESH/ESC 2003 guidelines, we observed that the frequency of the PIA2 allele was higher among stroke versus nonstroke patients (stroke, 46.4%; nonstroke, 22.6%; P = 0.01). The multiple regression analysis taking into account this polymorphism among other factors known to contribute to ischemic stroke confirmed the PIA2 allele as an additive risk factor for stroke (B = 0.986, Wald = 4.943, P < 0.03), increasing the risk of stroke by 2.9 (95% confidence interval = 1.23-6.85, P < 0.02). Similar results were obtained in the Sardinian population: in hypertensive patients with three or more risk factors, PIA2 increases the risk (odds ratio = 2.8, 95% confidence interval = 1.3-6.0, P < 0.001) and is an additive risk factor for stroke (B = 1.073, Wald =6.920, P < 0.01). xD;Conclusions: Our data suggest that the PIA2 polymorphism is a genetic determinant of ischemic stroke in a selected high-risk hypertensive population.
Abstract: beta(2)-Adrenergic receptors (beta(2)ARs) are widely expressed, although their physiological relevance in many tissues is not yet fully understood. In vascular endothelial cells, they regulate NO release and vessel tone. Here we provide novel evidence that beta(2)ARs can regulate neoangiogenesis in response to chronic ischemia. We used in vivo adenoviral-mediated gene transfer of the human beta(2)AR to the endothelium of the rat femoral artery and increased beta(2)AR signaling resulting in ameliorated angiographic blood flow and hindlimb perfusion after chronic ischemia. Histological analysis confirmed that beta(2)AR overexpression also produced benefits on capillary density. The same maneuver partially rescued impaired angiogenesis in spontaneously hypertensive rats (SHR), whereas gene delivery of the G-protein coupling defective mutant Ile164 beta(2)AR failed to provide ameliorations. Stimulation of endogenous and overexpressed beta(2)AR on endothelial cells in vitro was found to regulate cell number by inducing proliferation and [H-3]-thymidine incorporation through means of extracellular receptor-activated kinase and vascular endothelial growth factor. The beta(2)AR also has novel effects on endothelial cell number through stimulation of proapoptosis and antiapoptosis pathways involving p38 mitogen-activated protein kinase and PI3-kinase/Akt activation. Therefore, beta(2)ARs play a critical role in endothelial cell proliferation and function including revascularization, suggesting a novel and physiologically relevant role in neoangiogenesis in response to ischemia.
Abstract: Background - In hypertension, reduced nitric oxide production and blunted endothelial vasorelaxation are observed. It was recently reported that AKT phosphorylates and activates endothelial nitric oxide synthase and that impaired kinase activity may be involved in endothelial dysfunction. xD;Methods and Results - To identify the physiological role of the kinase in normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR), we used adenoviral vectors to transfer the human AKT1 gene selectively to the common carotid endothelium. In vitro, endothelial vasorelaxations to acetylcholine, isoproterenol, and insulin were blunted in control carotids from SHR compared with WKY rats, and human AKT1 overexpression corrected these responses. Similarly, blood flow assessed in vivo by Doppler ultrasound was reduced in SHR compared with WKY carotids and normalized after AKT1 gene transfer. In primary cultured endothelial cells, we evaluated AKT phosphorylation, activity, and compartmentalization and observed a mislocalization of the kinase in SHR. xD;Conclusions - We conclude that AKT participates in the settings of endothelial dysfunction in SHR rats by impaired membrane localization. Our data suggest that AKT is involved in endothelium dysfunction in hypertension.
