- Email: [email protected]
RhoA dependent membrane protrusions maintain the endothelial barrier
Abstracts
used a fluorescence-based assay to detect H2 S release from thioglycine. In this assay, fluorescence is emitted by thiobromobinane that is produced by a reaction between dibromobinane and H2S/HS−. Thioglycine and thiovaline produced ample amounts of H2S (measured as thiobromobinane) in a buffer containing sodium bicarbonate (NaHCO3), but not any measurable amounts when dissolved in water or in the absence of NaHCO3. H2S release from these compounds was confirmed using an H2S electrode. Thioglycine dose-dependently stimulated cGMP accumulation in cultured rat aortic smooth muscle cells more than the well known H2S donors NaHS and Na2S. In endothelium-denuded aortic rings pre-contracted with phenylephrine, thioglycine caused maximal relaxations at concentrations lower than 1 mM. To evaluate the ability of thioglycine to promote H2S release in vivo, this aminoacid was administered to rats. Hydrogen sulfide levels in the plasma increased after i.p injection of 5 mg/kg in a time-dependent manner; H2S plasma levels first became significantly increased after 30 min and remained elevated for 8 h. After 12 h, at a time when plasma levels had returned to normal, increased H2S levels could be found in the urine and liver. Conclusions: Thioglycine can be used as H2S donor; the H2S released from this compound is pH and NaHCO3-dependent. Thioglycine is more potent than the widely-used H2S donor NaHS. As H2S exerts blood pressure-lowing effects and cytoprotective actions in the cardiovascular system, agents that release H2S might be of therapeutic value.
doi:10.1016/j.vph.2011.08.140
P.6.7 N-methyl-2-pyridone-5-carboxamide is 1-methylnicotinamide metabolite of low cyclooxygenase-dependent vasodilating activity Tomasz Przygodzkia, Bartlomiej Grobelskia, Piotr Kazmierczaka,b, Cezary Watalaa a Department of Haemostasis and Haemostatic Disorders, Chair of Laboratory Diagnostics, Medical University of Lodz, University Clinical Hospital No 2, Lodz, Poland b Postgraduate School of Molecular Medicine, Warsaw, Poland E-mail address: [email protected] (T. Przygodzki) 1-Methylnicotinamide (MNA) is a primary metabolite of nicotinamide recently proven to cause systemic increase in PGI2 plasma levels in an unknown mechanism. Our present study was aimed at verifying whether the increased production of PGI2, a vasodilating prostanoid, in response to MNA, its metabolite N-methyl-2-pyridone5-carboxamide (Met2PY) and nicotinamide (NA) may be reproduced under in vitro conditions. Since prostacyclin is a vasodilating prostanoid we also performed the functional tests in the ex vivo model of coronary vascular bed perfusion to evaluate the vasoactive properties of those compounds. We did not observe any significant effect of the tested drugs on either PGI2 or PGE2 secretion in our in vitro model. Nicotinamide at the concentrations of 10 μmol/l and 100 μmol/l and 100 μmol/l Met2PY, slightly but significantly increased coronary flow in rat heart. This increases however, remained very low when compared to that induced by the reference compound, bradykinin (100 nmol/l). Perfusion of rat hearts with Met2PY in the presence of 50 μmol/l indomethacin resulted in decreased coronary flow, which proves that the effect is cyclooxygenase-dependent. We conclude that MNA metabolites should be more carefully addressed in reference to pro-prostacyclin activity and that systemic mechanism of MNA-induced PGI2 production needs further clarification.
doi:10.1016/j.vph.2011.08.141
357
P.6.8 Identification of septin-5 as a novel calpain substrate and its role in platelet secretion Voahanginirina Randriamboavonjya, Johann Isaaka, Amro Elgheznawya, Manuel Mayrb, Ingrid Fleminga a Institute for Vascular Signalling, Centre for Molecular Medicine, Johann Wolfgang Goethe University, Frankfurt am Mai, Germany b King's College London, London, UK E-mail address: [email protected] (V. Randriamboavonjy) The Ca2+-activated protease, calpain, plays an important role in platelet function by cleaving a broad spectrum of proteins. We have shown that an increased activity of calpain is responsible of the enhanced aggregability of platelets from patients with type 2 diabetes and that glitazone therapy prevents calpain activation. Therefore, to characterize novel calpain substrates in platelets, platelet lysates from patients with type 2 diabetes before and after treatment with the PPARγ agonist pioglitazone (8 weeks, 20 mg/kg/ day) were analyzed by 2D-DIGE followed by mass spectrometry. The SNARE-associated protein septin-5 was one of the proteins identified. In vitro stimulation of washed human platelets with the Ca2+ ionophore ionomycin in order to activate calpain led to the cleavage of septin-5. An effect inhibited by the calpain inhibitor calpeptin (10 μmol/L, 30 min) suggesting that septin-5 is a calpain substrate. We next characterized the effect of calpain on the function of septin-5 and showed that in resting platelets, septin-5 bound the SNARE protein syntaxin-4, calpain activation led to the dissociation of septin-5 from syntaxin 4 and potentiation of the thrombin-induced α-granule secretion. Interestingly, the platelet releasate obtained after calpain activation enhanced endothelial cell proliferation. Since α-granules also store chemokines, we further investigated the effect of calpain activation on the release of RANTES, an important chemokine involved in the development of vascular diseases. Stimulation of calpain in platelets potentiated the thrombin-induced increase in RANTES secretion and its subsequent cleavage to a variant possessing an increased chemotaxis activity. Indeed, the cleaved RANTES had an enhanced capacity to attract monocytes. To further investigate the role of calpain activation in platelet in vivo, diabetes was induced in mice. Diabetes induction resulted in the activation of calpain in murine platelets and a reduction in the levels of different calpain substrates including septin-5. Treatment of the animals with the calpain inhibitor A-705253 (30 mg/kg/day for 10 days) inhibited calpain activation and restored protein levels. In conclusion, calpain overactivation in platelets not only increases the release of growth factors and chemokines but also generates new chemokine variants which may contribute to the development of vascular diseases in diabetes. Finally, inhibition of calpain in vivo represents a promising way to inhibit platelet hyperactivation. doi:10.1016/j.vph.2011.08.142
P.6.9 RhoA dependent membrane protrusions maintain the endothelial barrier Robert Szulcek, Cora Beckers, Jan van Bezu, Victor W.M. van Hinsbergh, Geerten P. van Nieuw Amerongen Department of Physiology, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, Amsterdam, The Netherlands E-mail address: [email protected] (R. Szulcek) Aims: Endothelial cells (ECs) constantly equilibrate contractile and adhesive forces to maintain vascular barrier integrity. The involvement of the small GTPase RhoA and its downstream target Rho kinase in stimulated endothelial hyperpermeability through
358
Abstracts
regulation of the actin cytoskeleton is well understood. In contrast, possible barrier protective effects of RhoA remain unclear. Here the role of RhoA in maintenance of the basal EC barrier was studied. Methods: Measurements were performed on unstimulated endothelial monolayers, and after the administration of the RhoA inhibitor C3-transferase. Electrical resistance of the endothelial layer was measured with Electric Cell-Substrate Impedance Sensing (ECIS). Absolute values of the electrical resistance are a measure of barrier integrity, whereas the noise of the signal is a reflection of the motility of cells known as micromotion. Micromotion was calculated by Fourier Transformation and analysis of the resulting frequency spectrum. Differential Interference Contrast Microscopy (DIC) was applied to fallow formation and closure of inter-endothelial gaps with a high temporal resolution (interval 10 s). ECs were transfected with the biosensor Raichu-RhoA and Fluorescence Resonance Energy Transfer (FRET) was performed to visualize RhoA activity with a high spatial resolution. Results: Live cell imaging revealed that highly confluent endothelial monolayers actively maintain barrier integrity by a continual remodeling of their cell–cell contacts. This was accompanied by a rapid opening and closure of inter-endothelial gaps. Surprisingly, enhanced RhoA activity promoted gap-closure, but not gap-formation. This high RhoA activity was localized at sites where small finger-like membrane protrusions were formed, which eventually triggered gap-closure, suggesting a role of RhoA in maintenance and restoration of barrier integrity. Inhibition of RhoA with C3-transferase (1 μM for 24 h) indeed reduced the number of those protrusive activities significantly (from 11 to 5 per ROI). Loss of membrane activities was reflected by a significant decrease in micromotion. Furthermore, inhibition of RhoA resulted in a decreased number of spontaneously formed gaps (from 60 to 20 per ROI) but those which formed showed prolonged opening times (from 160 to 1800 s). This was accompanied by a drop in electrical resistance (from 8000 to 4000 Ω), indicating an overall loss of barrier integrity. Conclusion: Here we identified the RhoA-dependent formation of membrane protrusions that regulate the closure of inter-endothelial gaps, as a novel mechanism for maintenance of the endothelial barrier. doi:10.1016/j.vph.2011.08.143
P.6.10 Wnt4 is a novel promoter of VSMC proliferation and intimal thickening by the NFATc1 non-canonical and beta-catenin canonical pathways Aikaterini Tsaousi, Georgia M. Connolly, Sarah J. George The University of Bristol, Bristol Royal Infirmary Level 7, Bristol, BS2 8HW, UK E-mail address: [email protected] (A. Tsaousi) Vascular smooth muscle cell (VSMC) proliferation causes intimal thickening in atherosclerosis and restenosis. We previously demonstrated that Wnt/beta-catenin signalling stimulated VSMC proliferation in vitro via cyclin D1 up-regulation1. We have further identified that it is Wnt4 that is responsible for VSMC proliferation and the induction of intimal thickening in ligated mouse carotid arteries, at least in part via activation of the Wnt/beta-catenin signalling pathway and cyclin D1 expression2. However, Wnts can also signal independently of beta-catenin, via a Ca2+-related pathway, involving nuclear transcription factor of activated T-cells (NFAT). To investigate whether Wnt4-induced VSMC proliferation utilises this beta-catenin independent Wnt pathway, we assessed the role of NFATc1 (previously identified as the predominant isoform in VSMCs) in Wnt4-induced VSMC proliferation in vitro. Immunocytochemistry revealed that addition of 500 ng/mL of recombinant Wnt4 protein
in vitro induced nuclear translocation of transcription factor NFATc1, directly demonstrating the activation of Wnt/Ca2+ pathway by Wnt4. Moreover, treatment of VSMCs with recombinant Wnt4 protein for 6 h in vitro, significantly upregulated the mRNA levels of three NFATtarget genes; regulator of calcineurin 1 RCAN1, cyclooxygenase2 (Cox2) and Cyclin D1, as shown by Q-PCR (1.54 ± 0.28, 1.39 ± 0.24 and 1.35 ± 0.15 fold, respectively, p b 0.05). Wnt4-induced VSMC proliferation was significantly retarded by treatment with 400 nM of NFAT inhibitor (11R-VIVIT) from 46 ± 3%, to 30 ± 3% (p b 0.01, n = 4). Gene knockdown by 89 ± 1% of NFATc1 via siRNA using nucleofection resulted in a significant reduction of both Cyclin D1 and RCAN1 mRNA, as shown by Q-PCR (by 21 ± 7% and 21 ± 17% respectively, n = 3). Finally, immunohistochemistry demonstrated increased presence of NFATc1 protein in thickened intimas of ligated mouse carotid arteries compared to the media and unligated arteries. In summary, this study demonstrates that in addition to inducing proliferation via the ‘canonical’ Wnt/beta-catenin pathway, Wnt4 can induce proliferation via the ‘non-canonical’ Wnt/Ca2+ pathway, through activation of NFATc1. We conclude that Wnt4 plays an important role in the stimulation of VSMC proliferation via both betacatenin and NFATc1 axes and is an important contributor to intimal thickening. References 1. Quasnichka H, Circ. Res. 2006. 2. Tsaousi A, Circ. Res. 2011.
doi:10.1016/j.vph.2011.08.144
P.6.11 Study on the involvement of soluble guanylyl cyclase and its different isoforms in carbon monoxide and carbon monoxide releasing molecule-2 induced vasodilatation Kelly Decaluwéa, Bart Pauwelsa, Sara Verpoesta, Peter Brouckaertb, Johan Van de Voordea a Department of Pharmacology, Ghent University, Ghent, Belgium b VIB Department of Molecular Biomedical Research, Ghent University, Ghent, Belgium E-mail address: [email protected] (J. Van de Voorde) Besides nitric oxide, carbon monoxide (CO) also activates soluble guanylyl cyclase (sGC). CO as well as the CO-donor CORM-2 has been shown to possess vasodilatory properties. Whether these vasodilatory properties by CO can be attributed to sGC activation is still a matter of debate. The aim of this study was to examine the involvement of sGC and its different subunits in CO and CORM-2 induced vasodilatation within different vascular tissues. Isometric tension recordings were performed using mice isolated aortic rings, femoral artery ring segments as well as corpora cavernosa (CC). To be able to distinguish between the different sGC subunits we evaluated responses to saturated CO solutions and CORM-2 in both sGCa1−/− and sGCβ1KI/KI mice and their wild-type controls. Saturated CO solution was unable to relax mice isolated blood vessels, whereas it induced concentration-dependent relaxations in mice CC. In CC of wild-type mice, the response to CO was completely inhibited by the sGC inhibitor ODQ. The involvement of sGC in the CO-induced corporal relaxation was further confirmed by the loss of response to CO in CC isolated from sGCβ1KI/KI mice. Moreover, the vasodilatory responses of CO in the corporal tissue of sGCa1−/− mice were strongly inhibited although not completely abolished. In contrast to CO, CORM-2 was able to relax all vascular tissues examined in the present study, although ODQ only partially blocked the response to CORM-2 in the aorta. Interestingly ODQ did not affect the CORM-2 induced relaxation in the femoral arteries and the CC, indicating that sGC is
used a fluorescence-based assay to detect H2 S release from thioglycine. In this assay, fluorescence is emitted by thiobromobinane that is produced by a reaction between dibromobinane and H2S/HS−. Thioglycine and thiovaline produced ample amounts of H2S (measured as thiobromobinane) in a buffer containing sodium bicarbonate (NaHCO3), but not any measurable amounts when dissolved in water or in the absence of NaHCO3. H2S release from these compounds was confirmed using an H2S electrode. Thioglycine dose-dependently stimulated cGMP accumulation in cultured rat aortic smooth muscle cells more than the well known H2S donors NaHS and Na2S. In endothelium-denuded aortic rings pre-contracted with phenylephrine, thioglycine caused maximal relaxations at concentrations lower than 1 mM. To evaluate the ability of thioglycine to promote H2S release in vivo, this aminoacid was administered to rats. Hydrogen sulfide levels in the plasma increased after i.p injection of 5 mg/kg in a time-dependent manner; H2S plasma levels first became significantly increased after 30 min and remained elevated for 8 h. After 12 h, at a time when plasma levels had returned to normal, increased H2S levels could be found in the urine and liver. Conclusions: Thioglycine can be used as H2S donor; the H2S released from this compound is pH and NaHCO3-dependent. Thioglycine is more potent than the widely-used H2S donor NaHS. As H2S exerts blood pressure-lowing effects and cytoprotective actions in the cardiovascular system, agents that release H2S might be of therapeutic value.
doi:10.1016/j.vph.2011.08.140
P.6.7 N-methyl-2-pyridone-5-carboxamide is 1-methylnicotinamide metabolite of low cyclooxygenase-dependent vasodilating activity Tomasz Przygodzkia, Bartlomiej Grobelskia, Piotr Kazmierczaka,b, Cezary Watalaa a Department of Haemostasis and Haemostatic Disorders, Chair of Laboratory Diagnostics, Medical University of Lodz, University Clinical Hospital No 2, Lodz, Poland b Postgraduate School of Molecular Medicine, Warsaw, Poland E-mail address: [email protected] (T. Przygodzki) 1-Methylnicotinamide (MNA) is a primary metabolite of nicotinamide recently proven to cause systemic increase in PGI2 plasma levels in an unknown mechanism. Our present study was aimed at verifying whether the increased production of PGI2, a vasodilating prostanoid, in response to MNA, its metabolite N-methyl-2-pyridone5-carboxamide (Met2PY) and nicotinamide (NA) may be reproduced under in vitro conditions. Since prostacyclin is a vasodilating prostanoid we also performed the functional tests in the ex vivo model of coronary vascular bed perfusion to evaluate the vasoactive properties of those compounds. We did not observe any significant effect of the tested drugs on either PGI2 or PGE2 secretion in our in vitro model. Nicotinamide at the concentrations of 10 μmol/l and 100 μmol/l and 100 μmol/l Met2PY, slightly but significantly increased coronary flow in rat heart. This increases however, remained very low when compared to that induced by the reference compound, bradykinin (100 nmol/l). Perfusion of rat hearts with Met2PY in the presence of 50 μmol/l indomethacin resulted in decreased coronary flow, which proves that the effect is cyclooxygenase-dependent. We conclude that MNA metabolites should be more carefully addressed in reference to pro-prostacyclin activity and that systemic mechanism of MNA-induced PGI2 production needs further clarification.
doi:10.1016/j.vph.2011.08.141
357
P.6.8 Identification of septin-5 as a novel calpain substrate and its role in platelet secretion Voahanginirina Randriamboavonjya, Johann Isaaka, Amro Elgheznawya, Manuel Mayrb, Ingrid Fleminga a Institute for Vascular Signalling, Centre for Molecular Medicine, Johann Wolfgang Goethe University, Frankfurt am Mai, Germany b King's College London, London, UK E-mail address: [email protected] (V. Randriamboavonjy) The Ca2+-activated protease, calpain, plays an important role in platelet function by cleaving a broad spectrum of proteins. We have shown that an increased activity of calpain is responsible of the enhanced aggregability of platelets from patients with type 2 diabetes and that glitazone therapy prevents calpain activation. Therefore, to characterize novel calpain substrates in platelets, platelet lysates from patients with type 2 diabetes before and after treatment with the PPARγ agonist pioglitazone (8 weeks, 20 mg/kg/ day) were analyzed by 2D-DIGE followed by mass spectrometry. The SNARE-associated protein septin-5 was one of the proteins identified. In vitro stimulation of washed human platelets with the Ca2+ ionophore ionomycin in order to activate calpain led to the cleavage of septin-5. An effect inhibited by the calpain inhibitor calpeptin (10 μmol/L, 30 min) suggesting that septin-5 is a calpain substrate. We next characterized the effect of calpain on the function of septin-5 and showed that in resting platelets, septin-5 bound the SNARE protein syntaxin-4, calpain activation led to the dissociation of septin-5 from syntaxin 4 and potentiation of the thrombin-induced α-granule secretion. Interestingly, the platelet releasate obtained after calpain activation enhanced endothelial cell proliferation. Since α-granules also store chemokines, we further investigated the effect of calpain activation on the release of RANTES, an important chemokine involved in the development of vascular diseases. Stimulation of calpain in platelets potentiated the thrombin-induced increase in RANTES secretion and its subsequent cleavage to a variant possessing an increased chemotaxis activity. Indeed, the cleaved RANTES had an enhanced capacity to attract monocytes. To further investigate the role of calpain activation in platelet in vivo, diabetes was induced in mice. Diabetes induction resulted in the activation of calpain in murine platelets and a reduction in the levels of different calpain substrates including septin-5. Treatment of the animals with the calpain inhibitor A-705253 (30 mg/kg/day for 10 days) inhibited calpain activation and restored protein levels. In conclusion, calpain overactivation in platelets not only increases the release of growth factors and chemokines but also generates new chemokine variants which may contribute to the development of vascular diseases in diabetes. Finally, inhibition of calpain in vivo represents a promising way to inhibit platelet hyperactivation. doi:10.1016/j.vph.2011.08.142
P.6.9 RhoA dependent membrane protrusions maintain the endothelial barrier Robert Szulcek, Cora Beckers, Jan van Bezu, Victor W.M. van Hinsbergh, Geerten P. van Nieuw Amerongen Department of Physiology, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, Amsterdam, The Netherlands E-mail address: [email protected] (R. Szulcek) Aims: Endothelial cells (ECs) constantly equilibrate contractile and adhesive forces to maintain vascular barrier integrity. The involvement of the small GTPase RhoA and its downstream target Rho kinase in stimulated endothelial hyperpermeability through
358
Abstracts
regulation of the actin cytoskeleton is well understood. In contrast, possible barrier protective effects of RhoA remain unclear. Here the role of RhoA in maintenance of the basal EC barrier was studied. Methods: Measurements were performed on unstimulated endothelial monolayers, and after the administration of the RhoA inhibitor C3-transferase. Electrical resistance of the endothelial layer was measured with Electric Cell-Substrate Impedance Sensing (ECIS). Absolute values of the electrical resistance are a measure of barrier integrity, whereas the noise of the signal is a reflection of the motility of cells known as micromotion. Micromotion was calculated by Fourier Transformation and analysis of the resulting frequency spectrum. Differential Interference Contrast Microscopy (DIC) was applied to fallow formation and closure of inter-endothelial gaps with a high temporal resolution (interval 10 s). ECs were transfected with the biosensor Raichu-RhoA and Fluorescence Resonance Energy Transfer (FRET) was performed to visualize RhoA activity with a high spatial resolution. Results: Live cell imaging revealed that highly confluent endothelial monolayers actively maintain barrier integrity by a continual remodeling of their cell–cell contacts. This was accompanied by a rapid opening and closure of inter-endothelial gaps. Surprisingly, enhanced RhoA activity promoted gap-closure, but not gap-formation. This high RhoA activity was localized at sites where small finger-like membrane protrusions were formed, which eventually triggered gap-closure, suggesting a role of RhoA in maintenance and restoration of barrier integrity. Inhibition of RhoA with C3-transferase (1 μM for 24 h) indeed reduced the number of those protrusive activities significantly (from 11 to 5 per ROI). Loss of membrane activities was reflected by a significant decrease in micromotion. Furthermore, inhibition of RhoA resulted in a decreased number of spontaneously formed gaps (from 60 to 20 per ROI) but those which formed showed prolonged opening times (from 160 to 1800 s). This was accompanied by a drop in electrical resistance (from 8000 to 4000 Ω), indicating an overall loss of barrier integrity. Conclusion: Here we identified the RhoA-dependent formation of membrane protrusions that regulate the closure of inter-endothelial gaps, as a novel mechanism for maintenance of the endothelial barrier. doi:10.1016/j.vph.2011.08.143
P.6.10 Wnt4 is a novel promoter of VSMC proliferation and intimal thickening by the NFATc1 non-canonical and beta-catenin canonical pathways Aikaterini Tsaousi, Georgia M. Connolly, Sarah J. George The University of Bristol, Bristol Royal Infirmary Level 7, Bristol, BS2 8HW, UK E-mail address: [email protected] (A. Tsaousi) Vascular smooth muscle cell (VSMC) proliferation causes intimal thickening in atherosclerosis and restenosis. We previously demonstrated that Wnt/beta-catenin signalling stimulated VSMC proliferation in vitro via cyclin D1 up-regulation1. We have further identified that it is Wnt4 that is responsible for VSMC proliferation and the induction of intimal thickening in ligated mouse carotid arteries, at least in part via activation of the Wnt/beta-catenin signalling pathway and cyclin D1 expression2. However, Wnts can also signal independently of beta-catenin, via a Ca2+-related pathway, involving nuclear transcription factor of activated T-cells (NFAT). To investigate whether Wnt4-induced VSMC proliferation utilises this beta-catenin independent Wnt pathway, we assessed the role of NFATc1 (previously identified as the predominant isoform in VSMCs) in Wnt4-induced VSMC proliferation in vitro. Immunocytochemistry revealed that addition of 500 ng/mL of recombinant Wnt4 protein
in vitro induced nuclear translocation of transcription factor NFATc1, directly demonstrating the activation of Wnt/Ca2+ pathway by Wnt4. Moreover, treatment of VSMCs with recombinant Wnt4 protein for 6 h in vitro, significantly upregulated the mRNA levels of three NFATtarget genes; regulator of calcineurin 1 RCAN1, cyclooxygenase2 (Cox2) and Cyclin D1, as shown by Q-PCR (1.54 ± 0.28, 1.39 ± 0.24 and 1.35 ± 0.15 fold, respectively, p b 0.05). Wnt4-induced VSMC proliferation was significantly retarded by treatment with 400 nM of NFAT inhibitor (11R-VIVIT) from 46 ± 3%, to 30 ± 3% (p b 0.01, n = 4). Gene knockdown by 89 ± 1% of NFATc1 via siRNA using nucleofection resulted in a significant reduction of both Cyclin D1 and RCAN1 mRNA, as shown by Q-PCR (by 21 ± 7% and 21 ± 17% respectively, n = 3). Finally, immunohistochemistry demonstrated increased presence of NFATc1 protein in thickened intimas of ligated mouse carotid arteries compared to the media and unligated arteries. In summary, this study demonstrates that in addition to inducing proliferation via the ‘canonical’ Wnt/beta-catenin pathway, Wnt4 can induce proliferation via the ‘non-canonical’ Wnt/Ca2+ pathway, through activation of NFATc1. We conclude that Wnt4 plays an important role in the stimulation of VSMC proliferation via both betacatenin and NFATc1 axes and is an important contributor to intimal thickening. References 1. Quasnichka H, Circ. Res. 2006. 2. Tsaousi A, Circ. Res. 2011.
doi:10.1016/j.vph.2011.08.144
P.6.11 Study on the involvement of soluble guanylyl cyclase and its different isoforms in carbon monoxide and carbon monoxide releasing molecule-2 induced vasodilatation Kelly Decaluwéa, Bart Pauwelsa, Sara Verpoesta, Peter Brouckaertb, Johan Van de Voordea a Department of Pharmacology, Ghent University, Ghent, Belgium b VIB Department of Molecular Biomedical Research, Ghent University, Ghent, Belgium E-mail address: [email protected] (J. Van de Voorde) Besides nitric oxide, carbon monoxide (CO) also activates soluble guanylyl cyclase (sGC). CO as well as the CO-donor CORM-2 has been shown to possess vasodilatory properties. Whether these vasodilatory properties by CO can be attributed to sGC activation is still a matter of debate. The aim of this study was to examine the involvement of sGC and its different subunits in CO and CORM-2 induced vasodilatation within different vascular tissues. Isometric tension recordings were performed using mice isolated aortic rings, femoral artery ring segments as well as corpora cavernosa (CC). To be able to distinguish between the different sGC subunits we evaluated responses to saturated CO solutions and CORM-2 in both sGCa1−/− and sGCβ1KI/KI mice and their wild-type controls. Saturated CO solution was unable to relax mice isolated blood vessels, whereas it induced concentration-dependent relaxations in mice CC. In CC of wild-type mice, the response to CO was completely inhibited by the sGC inhibitor ODQ. The involvement of sGC in the CO-induced corporal relaxation was further confirmed by the loss of response to CO in CC isolated from sGCβ1KI/KI mice. Moreover, the vasodilatory responses of CO in the corporal tissue of sGCa1−/− mice were strongly inhibited although not completely abolished. In contrast to CO, CORM-2 was able to relax all vascular tissues examined in the present study, although ODQ only partially blocked the response to CORM-2 in the aorta. Interestingly ODQ did not affect the CORM-2 induced relaxation in the femoral arteries and the CC, indicating that sGC is