Abstract 1633: RhoA and Rho Kinase Activation in Human Pulmonary Hypertension - Role of 5-HT Signaling

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Christophe Guilluy ◽  
Saadia Eddahibi ◽  
Christian Agard ◽  
Laurent Savale ◽  
Elie Fadel ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Smooth Muscle ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Strong Increase ◽  
Kinase Signaling ◽  
Kinase Activation ◽  
Promising Target ◽  
New Treatments

Background- The complex and multifactorial pathogenesis of pulmonary hypertension (PH) involves constriction, remodeling, and in situ thrombosis of pulmonary vessels. Both serotonin (5-HT) and Rho kinase signaling may contribute to these alterations. Here, we investigated possible links between the 5-HT transporter (5-HTT) and RhoA/Rho kinase pathways, as well as their involvement in the progression of human and experimental PH. Methods and Results- Lungs, platelets, and quiescent cultured pulmonary-artery smooth-muscle cells (PA-SMCs) from patients with idiopathic PH (iPH) were characterized by marked elevations in RhoA and Rho kinase activity and by a strong increase in serotonin binding to RhoA, compared to controls. The 5-HTT inhibitor fluoxetine and the type 2 transglutaminase inhibitor monodansylcadaverin prevented 5-HT-induced RhoA serotonylation and RhoA/Rho kinase activation, as well as 5-HT-induced proliferation of PA-SMCs from iPH patients, that was also inhibited by the Rho kinase inhibitor fasudil. Increased Rho kinase activity, RhoA activation, and RhoA serotonylation were also observed in lungs from SM22–5-HTT+ mice, which overexpress 5-HTT in smooth muscle and spontaneously develop PH. Treatment of SM22–5-HTT+ mice with either fasudil or fluoxetine limited both PH progression and RhoA/Rho kinase activation. Conclusions- RhoA and Rho kinase activities are increased in iPH, as a result of transglutaminase-mediated transamidation of RhoA by 5-HT internalized via 5-HTT. Direct involvement of this pathway in 5-HT-mediated PA-SMC proliferation and platelet activation during PH progression identify RhoA/Rho kinase signaling as a promising target for new treatments against PH.

Abstract 3571: Evidence for Rho-Kinase Activation in Patients with Pulmonary Hypertension

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Zhulanqiqige Doe ◽  
Yoshihiro Fukumoto ◽  
Aya Takaki ◽  
Shunsuke Tawara ◽  
Junko Ohashi ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Heart Diseases ◽  
Pulmonary Arteries ◽  
Connective Tissue Diseases ◽  
Rho Kinase ◽  
Kinase Activation ◽  
And Gender

Pulmonary hypertension (PH) still remains a fatal disease characterized by hyperconstriction and remodeling of pulmonary arteries (PA). We and others have previously demonstrated that long-term inhibition of Rho-kinase is useful for treatment of PH in animal models, however, it remains to be examined whether Rho-kinase is actually activated in patients with PH. First, we examined whether Rho-kinase activity is enhanced in circulating neutrophils from 40 healthy age- and gender-matched controls and 40 PH patients with various etiologies, including idiopathic PAH (n=18), and PH associated with connective tissue diseases (n=8), congenital heart diseases (n=7), or chronic thromboembolism (n=7). We measured total and phosphorylated forms of myosin binding subunit (MBS), a substrate of Rho-kinase, by Western blotting, and defined the p-MBS/t-MBS ratio as an index of systemic Rho-kinaes activity. Next, we examined Rho-kinase activity by immunostaining in lung tissues from 5 controls and 5 IPAH obtained during lung surgery and transplantation, respectively. Finally, we examined vascular responses of isolated small PA from those subjects in vitro. Systemic Rho-kinase activity was significantly increased in the PAH patients compared with the controls (P<0.0001). Among the 4 subgroups of PH, Rho-kinase activity was significantly increased in all except for PH with thromboembolism (P<0.05). Significant correlations were noted between Rho-kinase activity and mean PA pressure, pulmonary vascular resistance, and duration of the disorder in the PH patients (all P<0.05). Rho-kinase expression in small PA and Rho-kinase activity in the lung tissue also were significantly increased in the PAH patients compared the controls (both P<0.0001). Endothelium-dependent relaxations were markedly impaired and serotonin-induced contractions were markedly enhanced in the PAH patients compared with the controls, and the hypercontractions were abolished in the presence of hydroxyfasudil, a specific Rho-kinase inhibitor (all P<0.01). These results provide the first direct evidence for Rho-kinase activation in patients with PH, confirming the therapeutic importance of Rho-kinase in the treatment of PH in humans.

Smooth muscle F-actin disassembly and RhoA/Rho-kinase signaling during endotoxin-induced alterations in pulmonary arterial compliance

2004 ◽  
Vol 287 (4) ◽  
pp. L649-L655 ◽  
Author(s):  
Christa Boer ◽  
Geerten P. van Nieuw Amerongen ◽  
A. B. Johan Groeneveld ◽  
Gert Jan Scheffer ◽  
Jaap J. de Lange ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Actin Cytoskeleton ◽  
Vascular Function ◽  
Kinase Inhibitor ◽  
Arterial Compliance ◽  
Pulmonary Arteries ◽  
Rho Kinase ◽  
Kinase Signaling ◽  
Arterial Mechanics ◽  
Pulmonary Arterial

Endotoxemia is associated with changed pulmonary vascular function with respect to vasoreactivity, endothelial permeability, and activation of inducible nitric oxide synthase II (NOSII). However, whether altered passive arterial wall mechanics contribute to this endotoxin-induced pulmonary vascular dysfunction is still unknown. Therefore, we investigated whether endotoxin affects the passive arterial mechanics and compliance of isolated rat pulmonary arteries. Pulmonary arteries of pentobarbital-anesthetized Wistar rats ( n = 55) were isolated and exposed to Escherichia coli endotoxin (50 μg/ml) for 20 h. Endotoxin increased pulmonary artery diameter and compliance (transmural pressure = 13 mmHg) in an endothelium-, Ca2+-, or NOSII-induced NO release-independent manner. Interestingly, the endotoxin-induced alterations in the passive arterial mechanics were accompanied by disassembly of the smooth muscle cell (SMC) F-actin cytoskeleton. Disassembly of F-actin by incubation of control arteries with the cytoskeleton-disrupting agent cytochalasin B or the Rho-kinase inhibitor Y-27632 induced a similar increase in passive arterial diameter and compliance. In contrast, RhoA activation by lysophosphatidic acid prevented the endotoxin-induced alterations in the pulmonary SMC F-actin cytoskeleton and passive mechanics. In conclusion, these findings indicate that disassembly of the SMC F-actin cytoskeleton and RhoA/Rho-kinase signaling act as mediators of endotoxin-induced changes in the pulmonary arterial mechanics. They imply the involvement of F-actin rearrangement and RhoA/Rho-kinase signaling in endotoxemia-induced vascular lung injury.

scholarly journals Myosin light chain kinase activation and calcium sensitization in smooth muscle in vivo

2008 ◽  
Vol 295 (2) ◽  
pp. C358-C364 ◽  
Author(s):  
Yusuke Mizuno ◽  
Eiji Isotani ◽  
Jian Huang ◽  
Hailei Ding ◽  
James T. Stull ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Fluorescent Proteins ◽  
Rho Kinase ◽  
Kinase Activation ◽  
Calphostin C

Ca2+/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) in smooth muscle by myosin light chain kinase (MLCK) and dephosphorylation by myosin light chain phosphatase (MLCP) are subject to modulatory cascades that influence the sensitivity of RLC phosphorylation and hence contraction to intracellular Ca2+ concentration ([Ca2+]i). We designed a CaM-sensor MLCK containing smooth muscle MLCK fused to two fluorescent proteins linked by the MLCK CaM-binding sequence to measure kinase activation in vivo and expressed it specifically in mouse smooth muscle. In phasic bladder muscle, there was greater RLC phosphorylation and force relative to MLCK activation and [Ca2+]i with carbachol (CCh) compared with KCl treatment, consistent with agonist-dependent inhibition of MLCP. The dependence of force on MLCK activity was nonlinear such that at higher concentrations of CCh, force increased with no change in the net 20% activation of MLCK. A significant but smaller amount of MLCK activation was found during the sustained contractile phase. MLCP inhibition may occur through RhoA/Rho-kinase and/or PKC with phosphorylation of myosin phosphatase targeting subunit-1 (MYPT1) and PKC-potentiated phosphatase inhibitor (CPI-17), respectively. CCh treatment, but not KCl, resulted in MYPT1 and CPI-17 phosphorylation. Both Y27632 (Rho-kinase inhibitor) and calphostin C (PKC inhibitor) reduced CCh-dependent force, RLC phosphorylation, and phosphorylation of MYPT1 (Thr694) without changing MLCK activation. Calphostin C, but not Y27632, also reduced CCh-induced phosphorylation of CPI-17. CCh concentration responses showed that phosphorylation of CPI-17 was more sensitive than MYPT1. Thus the onset of agonist-induced contraction in phasic smooth muscle results from the rapid and coordinated activation of MLCK with hierarchical inhibition of MLCP by CPI-17 and MYPT1 phosphorylation.

Involvement of RhoA/Rho kinase signaling in pulmonary hypertension of the fawn-hooded rat

2006 ◽  
Vol 100 (3) ◽  
pp. 996-1002 ◽  
Author(s):  
Tetsutaro Nagaoka ◽  
Sarah A. Gebb ◽  
Vijaya Karoor ◽  
Noriyuki Homma ◽  
Kenneth G. Morris ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Sea Level ◽  
Kinase Inhibitor ◽  
Pulmonary Arteries ◽  
Rho Kinase ◽  
Kinase Signaling ◽  
Rhoa Activity ◽  
Pulmonary Arterial ◽  
Mild Hypoxia

The fawn-hooded rat (FHR) develops severe pulmonary hypertension (PH) when raised for the first 3–4 wk of life in the mild hypoxia of Denver’s altitude (5,280 ft.). The PH is associated with sustained pulmonary vasoconstriction and pulmonary artery remodeling. Furthermore, lung alveolarization and vascularization are reduced in the Denver FHR. We have recently shown that RhoA/Rho kinase signaling is involved in both vasoconstriction and vascular remodeling in animal models of hypoxic PH. In this study, we investigated the role of RhoA/Rho kinase signaling in the PH of Denver FHR. In α-toxin permeabilized pulmonary arteries from Denver FHR, the contractile sensitivity to Ca2+was increased compared with those from sea-level FHR. RhoA activity and Rho kinase I protein expression in pulmonary arteries of Denver FHR (10-wk-old) were higher than in those of sea-level FHR. Acute inhalation of the Rho kinase inhibitor fasudil selectively reduced the elevated pulmonary arterial pressure in Denver FHR in vivo. Chronic fasudil treatment (30 mg·kg−1·day−1, from birth to 10 wk old) markedly reduced the development of PH and improved lung alveolarization and vascularization in Denver FHR. These results suggest that Rho kinase-mediated sustained vasoconstriction, through increased Ca2+sensitivity, plays an important role in the established PH and that RhoA/Rho kinase signaling contributes significantly to the development of PH and lung dysplasia in mild hypoxia-exposed FHR.

Regulation of protein kinase by vasopressin in renal medulla in situ

1977 ◽  
Vol 232 (1) ◽  
pp. F50-F57
Author(s):  
T. P. Dousa ◽  
L. D. Barnes
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Renal Medulla ◽  
Protein Kinase Activity ◽  
Kinase Activation ◽  
Heat Stable ◽  
Protein Kinase Activation ◽  
Heat Stable Protein

Results of this study demonstrate that vasopressin activates protein kinase in intact renal medullary cells as detected by measurement of the (-cyclic AMP/+cyclic AMP) protein kinase activity ratios in freshly prepared tissue extracts (40,000 X g supernates) from bovine renal medullary slices. The activation of protein kinase was specific for vasopressin since parathyroid hormone, histamine, angiotensin II, or the inactive analog of vasopressin did not activate protein kinase. There was a direct correlation between the extent of protein kinase activation and the elevation in tissue levels of cyclic AMP elicited by increasing doses of vasopressin or with an increase in incubation time. The elevation of tissue cyclic AMP level and maximum activation of protein kinase reached maximum level at a vasopressin concentration of about 2 X 10(-9) M. Incubation of slices with vasopressin caused a dose-dependent decrease in the cyclic AMP-dependent protein kinase activity in the 40,000 X g supernate of homogenate from the renal medullary slices. This effect of vasopressin was specific for protein kinase since activity of lactate dehydrogenase or a specific [3H]colchicine-binding activity was not affected, and the decrease in the protein kinase was not due to the accumulation of a heat-stable protein kinase inhibitor. There was an increase in protein kinase was not due to the accumulation of a heat-stable protein kinase inhibitor. There was an increase in protein kinase activity extracted from 40,000 X g pellets of homogenate prepared from slices exposed to vasopressin. Results thus provide evidence that cyclic AMP-mediated protein kinase activation in the intact cells is an integral part of cellular response of the mammalian renal medulla to vasopressin.

scholarly journals Pro-Nerve Growth Factor Induces Activation of RhoA Kinase and Neuronal Cell Death

2019 ◽  
Vol 9 (8) ◽  
pp. 204 ◽  
Author(s):  
Marina Sycheva ◽  
Jake Sustarich ◽  
Yuxian Zhang ◽  
Vaithinathan Selvaraju ◽  
Thangiah Geetha ◽  
...  
Keyword(s):  
Cell Death ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Neuronal Cell Death ◽  
Rho Kinase ◽  
Neuronal Cell ◽  
Nerve Growth ◽  
Rhoa Kinase

We have previously shown that the expression of pro-nerve growth factor (proNGF) was significantly increased, nerve growth factor (NGF) level was decreased, and the expression of p75NTR was enhanced in Alzheimer’s disease (AD) hippocampal samples. NGF regulates cell survival and differentiation by binding TrkA and p75NTR receptors. ProNGF is the precursor form of NGF, binds to p75NTR, and induces cell apoptosis. The objective of this study is to determine whether the increased p75NTR expression in AD is due to the accumulation of proNGF and Rho kinase activation. PC12 cells were stimulated with either proNGF or NGF. Pull-down assay was carried out to determine the RhoA kinase activity. We found the expression of p75NTR was enhanced by proNGF compared to NGF. The proNGF stimulation also increased the RhoA kinase activity leading to apoptosis. The expression of active RhoA kinase was found to be increased in human AD hippocampus compared to control. The addition of RhoA kinase inhibitor Y27632 not only blocked the RhoA kinase activity but also reduced the expression of p75NTR receptor and inhibited the activation of JNK and MAPK induced by proNGF. This suggests that overexpression of proNGF in AD enhances p75NTR expression and activation of RhoA, leading to neuronal cell death.

Abstract 641: Expression Of GLUT4 In Vascular Smooth Muscle Affects Endothelial Function In Hypertension.

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Kevin B Atkins ◽  
Jharna Saha ◽  
Frank C Brosius
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Interesting Phenomenon ◽  
Glut4 Expression ◽  
Endothelial Denudation ◽  
Rho Kinase Inhibitor ◽  
Total Body ◽  
Rats And Mice

Expression of GLUT4 is decreased in arterial smooth muscle of hypertensive rats and mice, and total body overexpression of GLUT4 in mice prevents enhanced arterial reactivity. To demonstrate that the effect on vascular response to GLUT4 overexpression is vascular rather than systemic in origin we utilized smooth muscle-specific GLUT4 transgenic mice (SMG4). GLUT4 expression in aortae of SMG4 compared to WT mice was increased 2-3 fold. Adult wild-type (WT) and SMG4 mice were made hypertensive or not through implantation of angiotensin II (AngII; 1.4mg/kg/d for 2 wks) or vehicle containing osmotic mini-pumps. Both WT and SMG4 mice AngII-treated mice exhibited significantly increased systolic blood pressure. In AngII-treated WT mice (WT-AngII) aortic GLUT4 expression was significantly decreased, whereas GLUT4 expression in aortae of AngII-treated SMG4 mice (SMG4-AngII) was maintained. The phosphorylation of ERM and MYPT1(Thr850) were significantly increased in aortae of WT-AngII compared to WT-Sham and SMG4-AngII mice. Responsiveness to the contractile agonists, phenylephrine, 5-HT, and PGF 2 was significantly increased in endothelium-intact aortic rings from WT-AngII mice, but remained normal in aortae of SMG4-AngII mice. Following pretreatment with Rho-kinase inhibitor Y-27632, relative inhibition of contractility to 5-HT was equal in aortae from WT-AngII and SMG4-AngII-treated mice. With endothelial denudation, contractility to 5-HT was equally enhanced in aortae of WT-AngII and SMG4-AngII-treated mice. Interestingly, whereas acetylcholine stimulated relaxation was significantly decreased in aortic rings of WT-AngII mice, relaxation in rings from SMG4-AngII mice was not significantly different from WT or SMG4. These results demonstrate an interesting phenomenon whereby decreased expression of GLUT4 in vascular smooth muscle leads to an endothelial dysfunction that not only impairs relaxation, but also enhances contractility.

scholarly journals Rosuvastatin intensifies the beneficial effects of rho-kinase inhibitor in reversal of monocrotaline-induced pulmonary hypertension

2016 ◽  
Vol 4 ◽  
pp. 898-905 ◽  
Author(s):  
Magdalena Jasińska-Stroschein ◽  
Jacek Owczarek ◽  
Urszula Sołtysiak ◽  
Daria Orszulak-Michalak
Keyword(s):  
Pulmonary Hypertension ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Beneficial Effects ◽  
Rho Kinase Inhibitor

Urokinase-induced migration of human vascular smooth muscle cells requires coupling of the small GTPases RhoA and Rac1 to the Tyk2/PI3-K signalling pathway

2003 ◽  
Vol 89 (05) ◽  
pp. 904-914 ◽  
Author(s):  
Natalia Tkachuk ◽  
Hermann Haller ◽  
Inna Dumler ◽  
Ioulia Kiian
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Vascular Smooth Muscle ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Small Gtpases ◽  
Janus Kinase ◽  
Immunocytochemical Staining ◽  
Boyden Chamber ◽  
Dependent Manner

SummaryUrokinase-type plasminogen activator (uPA) facilitates cell migration by localizing proteolisys on the cell surface and by inducing intracellular signalling pathways. In human vascular smooth muscle cell (VSMC) uPA stimulates migration via the uPA receptor (uPAR) signalling complex containing the Janus kinase Tyk2 and phosphatidylinositol 3-kinase (PI3-K). We report that active GTP-bound forms of small GTPases RhoA and Rac1, but not Cdc42, are directly associated with Tyk2 and PI3-K in an uPA/uPAR-dependent fashion. Endogenous RhoA, but not Rac1 or Cdc42, was significantly activated in response to uPA. RhoA activation was abolished by cell treatment with two unrelated, structurally distinct, specific inhibitors of PI3-K, wortmannin, and LY294002. Downstream of RhoA, phosphorylation of myosin light chain (MLC) was dramatically upregulated by uPA in a Rho kinase- and PI3-K-dependent manner. Thus, selective Rho kinase inhibitor Y27632 and PI3-K inhibitors wortmannin and LY294002 prevented the uPA-induced stimulation of MLC phosphorylation. Rho kinase inhibition also decreased uPA-stimulated VSMC migration as observed in a Boyden chamber. VSMC immunocytochemical staining demonstrated redistribution of RhoA and Rac1 active forms to the newly formed leading edge of migrating cell. VSMC microinjection with antibodies to either Rho or Rac1 decreased uPA-stimulated cell migration, indicating the involvement of both GTPases in the migration process. Our results provide evidence that the small GTPases RhoA and Rac1, together with Rho kinase, are necessary to mediate the uPA/uPAR-directed migration via the Tyk2/PI3-K signalling complex in human VSMC.

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