- Email: [email protected]
GRK2 induces cardiac hypertrophy through the regulation of NFκB activity
Abstracts
p b 0.001) an effect which was partially prevented by MMP and EGFR inhibition and abolished by PP2 and other inhibitors. Ape reduced IS from 64 ± 4% to 30 ± 3% (p b 0.001). This effect was abolished by all inhibitors. As shown by preliminary data, Ape increased EGFR phosphorylation (p b 0.05). This increase was prevented by inhibition of Src and EGFR, but not by prevention of MMP shedding of EGFlike ligands. Conclusions: The results show that EGFR mediates Ape-induced protection. The protection is abolished by inhibition of the various steps from PI3K activation to the opening of K + mito via NO–cGMP pathway, a cascade which is also involved in IPost. However, while the limitation of IS and of LVDP increase requires both liganddependent and -independent transactivations of EGFR, the recovery of developed LVP is mainly mediated by the ligand-independent pathway. It may be argued the ligand-independent pathway plays a critical role in the overall cardioprotection. doi:10.1016/j.vph.2015.11.034
Integrative approach to the investigation of the performance–perfusion interaction in skeletal muscles A. Folinoa,, A. Messerea, S. De Angelisb, S. Roattaa a Department of Neuroscience, University of Turin, Turin, Italy b Città della Salute e della scienza, Turin, Italy Objectives: How does exercise affects muscle blood flow? To what extent actual muscle perfusion affects muscle performance? What is the interplay between metabolic and mechanical factors in the hyperemic response at the beginning of exercise? And at the onset of fatigue? May a long-lasting perfusion metabolism mismatch underlie the development of muscle disorders? Many questions about the control of muscle blood flow in health and disease remain to be clarified. We present here an experimental set-up designed for the investigation of these issues. Materials and methods: The system is based on a wrist actuator servo-controlled (WAS) for accurate control of passive and active wrist exercise and a rapid cuff inflator for providing controlled compressive stimuli to the muscles under study and it integrates continuous assessment of blood flow (eco-Doppler), blood volume and oxygenation with near infrared spectroscopy (NIRS), electric activity with a surface electromyography (EMG) and mechanical variables. Different types of dynamic exercise can be performed with the WAS, ranging from the standard isometric contraction to active exercise against different type of load (elastic, viscous and inertial), calibrate on maximal voluntary contraction (MVC) of the subject. Results: Preliminary experiments allowed us to detect the fast hemodynamic and oxygenation transients occurring at the beginning of exercise. In particular, depending on the type of exercise, it is possible to detect a transient increase in tissue oxygenation, indicative of a feedforward control of muscle blood flow. Moreover it is possible to follow the functional hyperemia in long lasting muscle activity and correlate the progressive increase in blood flow with myoelectric and mechanical evidence of muscle fatigue. Discussion: This particular set up takes advantage of a multidisciplinary approach: it can be employed to investigate the muscle function under a variety of working conditions in order to explain physiological question still under debate. The preliminary results outlined above may have implications in different disciplines ranging from basic sciences to sport sciences and occupational medicine. doi:10.1016/j.vph.2015.11.035
53
Estrogen receptor β is involved in 17β-estradiol-mediated Notch1 activation and angiogenesis enhancement in human endothelial cells F. Fortinia,, C. Calicetia, M. Pannellab, G. Aquilaa, M.B. Morellia, A. Pannutic,e, L. Mielec,e, P. Rizzod, R. Ferraria a Department of Medical Sciences, University of Ferrara, Ferrara, Italy b GoldyneSavad Institute of Gene Therapy, Hadassah-Hebrew University Medical Center, 91120 Jerusalem, Israel c Stanley Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA, USA d Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy e Louisiana Cancer Research Consortium, New Orleans, LA, USA Objectives: Many studies have shown that estrogens play a protective role in the cardiovascular system. The mechanisms of action are still poorly understood, although a role for estrogens in stimulation of angiogenesis has been suggested. The Notch pathway modulates sprouting angiogenesis and Notch inhibition, by treatment with DAPT or with pro-inflammatory cytokine TNFα, enhances new sprouts formation. We have previously demonstrated that 17β-estradiol (E2) enhances the VEGFA-Delta-like ligand 4 (Dll4)mediated activation of Notch signaling in human umbilical vein endothelial cells (HUVECs) and that E2 counteracts the increase in endothelial cells sprouting caused by the inhibition of Notch with DAPT. Estrogen receptors (ERs) antagonist ICI 182.780 partially inhibits E2-induced Notch1 activation, suggesting that the action of E2 is mediated by ERs (α and/or β). The aims of this study were: 1) to determine whether the effects of E2 on endothelial cells network formation and Notch signaling are mediated by one or both ERs; 2) to establish whether E2 could counteract Notch inhibition induced by TNFα; and 3) to investigate whether E2 regulation of Notch is specific to HUVECs or it is present also in artery endothelial cells. Materials and methods: Human endothelial cells sprouting was determined by Matrigel assay. HUVECs, HCAECs (human coronary artery endothelial cells) and HAECs (human aortic endothelial cells) were hormones-deprived and treated with E2, ERβ specific agonist (DPN) and ERα specific agonist (PPT), in the presence or absence of TNFα. Notch signaling was assessed by measuring Notch1 protein and mRNA levels (Western Blot and qRT-PCR). Results: Human endothelial cells tube formation assay in Matrigel showed that: i) ICI 182.780 partially reverses the effects of E2 on DAPT-mediated increase in sprouting of endothelial cells; and ii) DPN counteracts the increase in endothelial cells sprouting caused by DAPT. In HUVECs, HCAECs and HAECs, treatment with DPN but not with PPT induced activation of Notch1. Notch1 mRNA levels were not affected. E2 and DPN treatment antagonized TNFα-induced decreased of cleaved Notch1 in HUVEC and HAECs. Conclusions: Our data confirm and expand our previous observations showing activation of Notch1 by E2 and suggest that ERβ might be implicated in E2-mediated activation of Notch1 processing in artery and vein endothelial cells as well as Notch regulation of sprouting angiogenesis. Furthermore, we found that DPN treatment partially counteracted Notch1 inhibition induced by TNFα treatment. It remains to be established whether DPN is able to counteract sprouting angiogenesis promoted by TNFα.
doi:10.1016/j.vph.2015.11.036
GRK2 induces cardiac hypertrophy through the regulation of NFκB activity A. Francoa,, D. Sorrientob, M. Ciccarellic, G. Santullid, E. Cipollettac, J. Gambardellac, B. Trimarcoa, G. Iaccarinoc
54
Abstracts
a Department of Advanced Biomedical Science, “Federico II” University, Naples, Italy b Institute of Biostructure and Bioimaging (IBB) of the Italian National Research Council (CNR), Naples, Italy c Department of Medicine and Surgery, University of Salerno, Salerno, Italy d Columbia University Medical Center, College of Physicians & Surgeons, New York Presbyterian Hospital, Manhattan, NY, USA
Objectives: G protein coupled receptors kinases (GRKs) belong to a family of serine/threonine kinases involved in phosphorylation and desensitization of agonist occupied GPCRs. In prenatal life GRK2 is pivotal for heart development. Less known is the role of GRK2 in cardiac adaptative responses in adult life. The aim of this study was to assess the effect of GRK2 on the regulation of cardiac hypertrophy and its related genes, such as ANF. Materials and methods: In vitro study was performed in cardiac myoblasts (H9C2). Hypertrophy was induced in vitro by chronic stimulation with phenylephrine. In vivo study was performed in two different animal models: spontaneously hypertensive rats (SHR) and normotensive rats (WKY) in which hypertrophy was induced through means of subcutaneously implantation of miniosmotic pumps releasing PE. The inhibition of GRK2 activity was induced in vivo by intra-cardiac injections of Ant-124 (10-4 M) once a week for three weeks. Results: In H9C2 cells, GRK2 overexpression increased ANF activity (+332 ± 15% vs control) and enhanced phenylephrine (PE)-induced ANF response (+118 ± 12% vs PE). The overexpression of GRK2-DN, an inactive mutant of GRK2, reduced ANF promoter activity in basal condition and after stimulation with PE (− 76 ± 1.8% vs control; −67 ± 4.5% vs PE). Moreover, we used a specific inhibitor of the catalytic activity of GRK2, Ant-124, designed on HJ loop domain of GRK2 and conjugated with Antennapoedia sequence. Ant-124 inhibited ANF promoter activity (Ant-124: 57 ± 1.1%) and reduced the response to both GRK2 (Ant-124: −43 ± 0.8) and PE (Ant-124: 60 ± 0.9%). These data suggest that the catalytic activity of GRK2 is necessary to regulate the expression of cardiac genes. Several studies have demonstrated that the activation of the transcription factor NFkappaB has a key role in the induction of hypertrophy by modulation of specific genes. Thus, we evaluated the effect of GRK2 on NFkappaB activity. GRK2 increased NFkappaB transcriptional activity and the overexpression of a NFkappaB inhibitor prevented ANF activation induced by GRK2, suggesting that GRK2 regulates hypertrophy through upregulation of NFkappaB activity. In two in vivo models of left ventricle hypertrophy (LVH), SHR and PE-treated WKY, the selective inhibition of GRK2 catalytic activity by Ant-124 has counteracted development of cardiac hypertrophy. The same result was obtained in a mouse model of GRK2 knock out. Conclusions: Our results suggest a novel role of GRK2 to regulate cardiac hypertrophic responses both in vitro and in vivo and propose GRK2 as potential therapeutic target for limiting LV hypertrophy.
doi:10.1016/j.vph.2015.11.037
Arachidonic acid stimulates endothelial progenitor cell proliferation through an increase in Ca2 + concentration and nitric oxide production E. Zuccoloa,, V. Polettob, G. Guerrac, V. Rostic, F. Mocciaa a Laboratory of General Physiology, Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Pavia, Italy b Center for the Study of Myelofibrosis, IRCCS Policlinico S Matteo Foundation, Pavia, Italy c Department of Medicine and Health Sciences “V. Tiberio”, University of Molise, Campobasso, Italy
Objectives: Endothelial progenitor cells (EPCs) are released from bone marrow (BM) into circulation to replace injured/senescent endothelial cells and promote revascularization of ischemic tissues. Additionally, they may sustain the angiogenic switch in a growing number of solid tumors. BM stromal cells release arachidonic acid (AA), which stimulates angiogenesis in vascular endothelial cells (ECs) through an increase in intracellular Ca2 + concentration ([Ca2 +]i). AA may also stimulate vasculogenesis and EPC proliferation, but the underlying mechanism is still unknown. Nitric oxide (NO) is a gasotransmitter which has long been known to mediate the proangiogenic effect Ca2 + signaling both in ECs and in human EPCs (hEPCs). Therefore, herein we sought to elucidate whether AA promotes hEPC proliferation through an increase in [Ca2 +]i and the following activation of the endothelial NO synthase (eNOS). Materials and methods: Changes in [Ca2 +]i were monitored from hEPCs loaded with the Ca2 +-sensitive dye, Fura-2/AM (2 μM, 30 min), by using a CCD camera. NO production was evaluated by loading the cells with DAF-FM (4 μM, 1 h). Among the many EPC subtypes described in literature, we focused on the endothelial colony forming cells (ECFCs), which are the only EPC population truly belonging to the endothelial phenotype. Results: AA induced a dose-dependent elevation in [Ca2 +]i which was significantly reduced by pharmacologically inhibiting cytochrome P450 monooxygenase and lipoxygenase metabolic pathways, but not cycloxygenase activity. AA-evoked Ca2 + signals were triggered by intracellular Ca2 + release and maintained by external Ca2 + inflow. AA-induced Ca2 + release was suppressed by preventing endogenous Ca2 + release from ryanodine receptors (RyRs), but not from inositol1,4,5-trisphosphate receptors. Finally, AA-evoked Ca2 + entry mediated Transient Receptor Potential Vanilloid 4 (TRPV4) channels, but did not involve store-operated Ca2 + entry. Moreover, AA caused an increase in NO levels which was blocked by preventing the concomitant increase in [Ca2 +]i and by pretreating the cells with L-NAME, which interferes with eNOS activity. Finally, the pharmacological blockade of AA-induced Ca2 + and NO signals prevented AA-dependent hEPC proliferation. Conclusions: These results indicate that AA-induced elevation in [Ca2 +]i Ca2 + requires RyRs-dependent Ca2 + release and TRPV4 activation in hEPCs. AA-evoked Ca2 + signals, in turn, promote NO production and hEPC growth.
doi:10.1016/j.vph.2015.11.038
Different methods for assessing hemodynamic congestion using inferior vena cava echocardiographic indexes. A retrospective study C. Arianoa,, R. De Vecchisa, F. Piemonteb a Cardiology Unit, Presidio Sanitario Intermedio “Elena d'Aosta”, ASL Napoli 1 Centro, Napoli, Italy b Cardiology Division, “San Giovanni di Dio” Hospital, ASL Napoli 2 Nord, Frattamaggiore, NA, Italy Objectives: Among the indices able to replace invasive central venous pressure (CVP) measurement for patients with acute decompensated heart failure (ADHF) the diameters of the inferior vena cava (IVC) and their respiratory fluctuations, the so-called IVC collapsibility index (IVCCI), measured by echocardiography, have recently gained ground as a quite reliable proxy of CVP. The aims of our study were to compare three different ways of evaluating cardiac overload by using the IVC diameters and/or their respiratory fluctuations and by calculating the inter-method agreement. Materials and methods: Medical records of patients hospitalized for right or bi-ventricular acute decompensated heart failure from January to December 2014 were retrospectively evaluated. The predictive significance of the IVC expiratory diameter and IVC collapsibility index (IVCCI) was analyzed using three different methods, namely a) the
p b 0.001) an effect which was partially prevented by MMP and EGFR inhibition and abolished by PP2 and other inhibitors. Ape reduced IS from 64 ± 4% to 30 ± 3% (p b 0.001). This effect was abolished by all inhibitors. As shown by preliminary data, Ape increased EGFR phosphorylation (p b 0.05). This increase was prevented by inhibition of Src and EGFR, but not by prevention of MMP shedding of EGFlike ligands. Conclusions: The results show that EGFR mediates Ape-induced protection. The protection is abolished by inhibition of the various steps from PI3K activation to the opening of K + mito via NO–cGMP pathway, a cascade which is also involved in IPost. However, while the limitation of IS and of LVDP increase requires both liganddependent and -independent transactivations of EGFR, the recovery of developed LVP is mainly mediated by the ligand-independent pathway. It may be argued the ligand-independent pathway plays a critical role in the overall cardioprotection. doi:10.1016/j.vph.2015.11.034
Integrative approach to the investigation of the performance–perfusion interaction in skeletal muscles A. Folinoa,, A. Messerea, S. De Angelisb, S. Roattaa a Department of Neuroscience, University of Turin, Turin, Italy b Città della Salute e della scienza, Turin, Italy Objectives: How does exercise affects muscle blood flow? To what extent actual muscle perfusion affects muscle performance? What is the interplay between metabolic and mechanical factors in the hyperemic response at the beginning of exercise? And at the onset of fatigue? May a long-lasting perfusion metabolism mismatch underlie the development of muscle disorders? Many questions about the control of muscle blood flow in health and disease remain to be clarified. We present here an experimental set-up designed for the investigation of these issues. Materials and methods: The system is based on a wrist actuator servo-controlled (WAS) for accurate control of passive and active wrist exercise and a rapid cuff inflator for providing controlled compressive stimuli to the muscles under study and it integrates continuous assessment of blood flow (eco-Doppler), blood volume and oxygenation with near infrared spectroscopy (NIRS), electric activity with a surface electromyography (EMG) and mechanical variables. Different types of dynamic exercise can be performed with the WAS, ranging from the standard isometric contraction to active exercise against different type of load (elastic, viscous and inertial), calibrate on maximal voluntary contraction (MVC) of the subject. Results: Preliminary experiments allowed us to detect the fast hemodynamic and oxygenation transients occurring at the beginning of exercise. In particular, depending on the type of exercise, it is possible to detect a transient increase in tissue oxygenation, indicative of a feedforward control of muscle blood flow. Moreover it is possible to follow the functional hyperemia in long lasting muscle activity and correlate the progressive increase in blood flow with myoelectric and mechanical evidence of muscle fatigue. Discussion: This particular set up takes advantage of a multidisciplinary approach: it can be employed to investigate the muscle function under a variety of working conditions in order to explain physiological question still under debate. The preliminary results outlined above may have implications in different disciplines ranging from basic sciences to sport sciences and occupational medicine. doi:10.1016/j.vph.2015.11.035
53
Estrogen receptor β is involved in 17β-estradiol-mediated Notch1 activation and angiogenesis enhancement in human endothelial cells F. Fortinia,, C. Calicetia, M. Pannellab, G. Aquilaa, M.B. Morellia, A. Pannutic,e, L. Mielec,e, P. Rizzod, R. Ferraria a Department of Medical Sciences, University of Ferrara, Ferrara, Italy b GoldyneSavad Institute of Gene Therapy, Hadassah-Hebrew University Medical Center, 91120 Jerusalem, Israel c Stanley Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA, USA d Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy e Louisiana Cancer Research Consortium, New Orleans, LA, USA Objectives: Many studies have shown that estrogens play a protective role in the cardiovascular system. The mechanisms of action are still poorly understood, although a role for estrogens in stimulation of angiogenesis has been suggested. The Notch pathway modulates sprouting angiogenesis and Notch inhibition, by treatment with DAPT or with pro-inflammatory cytokine TNFα, enhances new sprouts formation. We have previously demonstrated that 17β-estradiol (E2) enhances the VEGFA-Delta-like ligand 4 (Dll4)mediated activation of Notch signaling in human umbilical vein endothelial cells (HUVECs) and that E2 counteracts the increase in endothelial cells sprouting caused by the inhibition of Notch with DAPT. Estrogen receptors (ERs) antagonist ICI 182.780 partially inhibits E2-induced Notch1 activation, suggesting that the action of E2 is mediated by ERs (α and/or β). The aims of this study were: 1) to determine whether the effects of E2 on endothelial cells network formation and Notch signaling are mediated by one or both ERs; 2) to establish whether E2 could counteract Notch inhibition induced by TNFα; and 3) to investigate whether E2 regulation of Notch is specific to HUVECs or it is present also in artery endothelial cells. Materials and methods: Human endothelial cells sprouting was determined by Matrigel assay. HUVECs, HCAECs (human coronary artery endothelial cells) and HAECs (human aortic endothelial cells) were hormones-deprived and treated with E2, ERβ specific agonist (DPN) and ERα specific agonist (PPT), in the presence or absence of TNFα. Notch signaling was assessed by measuring Notch1 protein and mRNA levels (Western Blot and qRT-PCR). Results: Human endothelial cells tube formation assay in Matrigel showed that: i) ICI 182.780 partially reverses the effects of E2 on DAPT-mediated increase in sprouting of endothelial cells; and ii) DPN counteracts the increase in endothelial cells sprouting caused by DAPT. In HUVECs, HCAECs and HAECs, treatment with DPN but not with PPT induced activation of Notch1. Notch1 mRNA levels were not affected. E2 and DPN treatment antagonized TNFα-induced decreased of cleaved Notch1 in HUVEC and HAECs. Conclusions: Our data confirm and expand our previous observations showing activation of Notch1 by E2 and suggest that ERβ might be implicated in E2-mediated activation of Notch1 processing in artery and vein endothelial cells as well as Notch regulation of sprouting angiogenesis. Furthermore, we found that DPN treatment partially counteracted Notch1 inhibition induced by TNFα treatment. It remains to be established whether DPN is able to counteract sprouting angiogenesis promoted by TNFα.
doi:10.1016/j.vph.2015.11.036
GRK2 induces cardiac hypertrophy through the regulation of NFκB activity A. Francoa,, D. Sorrientob, M. Ciccarellic, G. Santullid, E. Cipollettac, J. Gambardellac, B. Trimarcoa, G. Iaccarinoc
54
Abstracts
a Department of Advanced Biomedical Science, “Federico II” University, Naples, Italy b Institute of Biostructure and Bioimaging (IBB) of the Italian National Research Council (CNR), Naples, Italy c Department of Medicine and Surgery, University of Salerno, Salerno, Italy d Columbia University Medical Center, College of Physicians & Surgeons, New York Presbyterian Hospital, Manhattan, NY, USA
Objectives: G protein coupled receptors kinases (GRKs) belong to a family of serine/threonine kinases involved in phosphorylation and desensitization of agonist occupied GPCRs. In prenatal life GRK2 is pivotal for heart development. Less known is the role of GRK2 in cardiac adaptative responses in adult life. The aim of this study was to assess the effect of GRK2 on the regulation of cardiac hypertrophy and its related genes, such as ANF. Materials and methods: In vitro study was performed in cardiac myoblasts (H9C2). Hypertrophy was induced in vitro by chronic stimulation with phenylephrine. In vivo study was performed in two different animal models: spontaneously hypertensive rats (SHR) and normotensive rats (WKY) in which hypertrophy was induced through means of subcutaneously implantation of miniosmotic pumps releasing PE. The inhibition of GRK2 activity was induced in vivo by intra-cardiac injections of Ant-124 (10-4 M) once a week for three weeks. Results: In H9C2 cells, GRK2 overexpression increased ANF activity (+332 ± 15% vs control) and enhanced phenylephrine (PE)-induced ANF response (+118 ± 12% vs PE). The overexpression of GRK2-DN, an inactive mutant of GRK2, reduced ANF promoter activity in basal condition and after stimulation with PE (− 76 ± 1.8% vs control; −67 ± 4.5% vs PE). Moreover, we used a specific inhibitor of the catalytic activity of GRK2, Ant-124, designed on HJ loop domain of GRK2 and conjugated with Antennapoedia sequence. Ant-124 inhibited ANF promoter activity (Ant-124: 57 ± 1.1%) and reduced the response to both GRK2 (Ant-124: −43 ± 0.8) and PE (Ant-124: 60 ± 0.9%). These data suggest that the catalytic activity of GRK2 is necessary to regulate the expression of cardiac genes. Several studies have demonstrated that the activation of the transcription factor NFkappaB has a key role in the induction of hypertrophy by modulation of specific genes. Thus, we evaluated the effect of GRK2 on NFkappaB activity. GRK2 increased NFkappaB transcriptional activity and the overexpression of a NFkappaB inhibitor prevented ANF activation induced by GRK2, suggesting that GRK2 regulates hypertrophy through upregulation of NFkappaB activity. In two in vivo models of left ventricle hypertrophy (LVH), SHR and PE-treated WKY, the selective inhibition of GRK2 catalytic activity by Ant-124 has counteracted development of cardiac hypertrophy. The same result was obtained in a mouse model of GRK2 knock out. Conclusions: Our results suggest a novel role of GRK2 to regulate cardiac hypertrophic responses both in vitro and in vivo and propose GRK2 as potential therapeutic target for limiting LV hypertrophy.
doi:10.1016/j.vph.2015.11.037
Arachidonic acid stimulates endothelial progenitor cell proliferation through an increase in Ca2 + concentration and nitric oxide production E. Zuccoloa,, V. Polettob, G. Guerrac, V. Rostic, F. Mocciaa a Laboratory of General Physiology, Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, Pavia, Italy b Center for the Study of Myelofibrosis, IRCCS Policlinico S Matteo Foundation, Pavia, Italy c Department of Medicine and Health Sciences “V. Tiberio”, University of Molise, Campobasso, Italy
Objectives: Endothelial progenitor cells (EPCs) are released from bone marrow (BM) into circulation to replace injured/senescent endothelial cells and promote revascularization of ischemic tissues. Additionally, they may sustain the angiogenic switch in a growing number of solid tumors. BM stromal cells release arachidonic acid (AA), which stimulates angiogenesis in vascular endothelial cells (ECs) through an increase in intracellular Ca2 + concentration ([Ca2 +]i). AA may also stimulate vasculogenesis and EPC proliferation, but the underlying mechanism is still unknown. Nitric oxide (NO) is a gasotransmitter which has long been known to mediate the proangiogenic effect Ca2 + signaling both in ECs and in human EPCs (hEPCs). Therefore, herein we sought to elucidate whether AA promotes hEPC proliferation through an increase in [Ca2 +]i and the following activation of the endothelial NO synthase (eNOS). Materials and methods: Changes in [Ca2 +]i were monitored from hEPCs loaded with the Ca2 +-sensitive dye, Fura-2/AM (2 μM, 30 min), by using a CCD camera. NO production was evaluated by loading the cells with DAF-FM (4 μM, 1 h). Among the many EPC subtypes described in literature, we focused on the endothelial colony forming cells (ECFCs), which are the only EPC population truly belonging to the endothelial phenotype. Results: AA induced a dose-dependent elevation in [Ca2 +]i which was significantly reduced by pharmacologically inhibiting cytochrome P450 monooxygenase and lipoxygenase metabolic pathways, but not cycloxygenase activity. AA-evoked Ca2 + signals were triggered by intracellular Ca2 + release and maintained by external Ca2 + inflow. AA-induced Ca2 + release was suppressed by preventing endogenous Ca2 + release from ryanodine receptors (RyRs), but not from inositol1,4,5-trisphosphate receptors. Finally, AA-evoked Ca2 + entry mediated Transient Receptor Potential Vanilloid 4 (TRPV4) channels, but did not involve store-operated Ca2 + entry. Moreover, AA caused an increase in NO levels which was blocked by preventing the concomitant increase in [Ca2 +]i and by pretreating the cells with L-NAME, which interferes with eNOS activity. Finally, the pharmacological blockade of AA-induced Ca2 + and NO signals prevented AA-dependent hEPC proliferation. Conclusions: These results indicate that AA-induced elevation in [Ca2 +]i Ca2 + requires RyRs-dependent Ca2 + release and TRPV4 activation in hEPCs. AA-evoked Ca2 + signals, in turn, promote NO production and hEPC growth.
doi:10.1016/j.vph.2015.11.038
Different methods for assessing hemodynamic congestion using inferior vena cava echocardiographic indexes. A retrospective study C. Arianoa,, R. De Vecchisa, F. Piemonteb a Cardiology Unit, Presidio Sanitario Intermedio “Elena d'Aosta”, ASL Napoli 1 Centro, Napoli, Italy b Cardiology Division, “San Giovanni di Dio” Hospital, ASL Napoli 2 Nord, Frattamaggiore, NA, Italy Objectives: Among the indices able to replace invasive central venous pressure (CVP) measurement for patients with acute decompensated heart failure (ADHF) the diameters of the inferior vena cava (IVC) and their respiratory fluctuations, the so-called IVC collapsibility index (IVCCI), measured by echocardiography, have recently gained ground as a quite reliable proxy of CVP. The aims of our study were to compare three different ways of evaluating cardiac overload by using the IVC diameters and/or their respiratory fluctuations and by calculating the inter-method agreement. Materials and methods: Medical records of patients hospitalized for right or bi-ventricular acute decompensated heart failure from January to December 2014 were retrospectively evaluated. The predictive significance of the IVC expiratory diameter and IVC collapsibility index (IVCCI) was analyzed using three different methods, namely a) the