scholarly journals Chronic intrauterine pulmonary hypertension increases endothelial cell Rho kinase activity and impairs angiogenesis in vitro

2008 ◽  
Vol 295 (4) ◽  
pp. L680-L687 ◽  
Author(s):  
Jason Gien ◽  
Gregory J. Seedorf ◽  
Vivek Balasubramaniam ◽  
Nancy Tseng ◽  
Neil Markham ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Kinase Activity ◽  
Ductus Arteriosus ◽  
Rho Kinase ◽  
Tube Formation ◽  
Normal Lung ◽  
No Production ◽  
Fetal Sheep ◽  
Enos Protein

Persistent pulmonary hypertension of the newborn (PPHN) is characterized by endothelial dysfunction and decreased vascular growth. The role of Rho kinase activity in modulating endothelial function and regulating angiogenesis during normal lung development and in PPHN is unknown. We hypothesized that PPHN increases Rho kinase activity in fetal pulmonary artery endothelial cells (PAECs) and impairs angiogenesis in vitro. Proximal PAECs were harvested from fetal sheep with partial ligation of the ductus arteriosus in utero (PPHN) and age-matched controls. Rho kinase activity was measured by RhoA, Rho GTP, and phosphorylated MYPT-1 protein content. The effects of Rho kinase activity on angiogenesis, endothelial nitric oxide (NO) synthase (eNOS) protein expression, and NO production were determined in normal and PPHN PAECs. Angiogenesis was assessed by tube formation in vitro with/without Y-27632 (a Rho kinase inhibitor) and calpeptin (a Rho kinase activator) in the presence/absence of N-nitro-l-arginine (l-NA, an NOS inhibitor). RhoA, Rho GTP, and phosphorylated MYPT-1 protein were increased in PPHN PAECs. Tube formation was reduced 29% in PPHN PAECs ( P < 0.001) and increased with Y-27632 treatment in normal and PPHN PAECs, with PPHN PAECs achieving levels similar to those of normal PAECs. l-NA inhibited the Y-27632-induced increase in tube formation in normal, but not PPHN, PAECs. Calpeptin reduced tube formation in normal and PPHN PAECs. eNOS expression was reduced 42% in PPHN PAECs ( P < 0.01). Y-27632 increased eNOS protein and NO production in normal and PPHN PAECs. Calpeptin decreased eNOS protein only in normal PAECs but reduced NO production in normal and PPHN PAECs. We conclude that Rho kinase activity is increased in PPHN PAECs and impairs angiogenesis and downregulates eNOS protein and NO production in vitro.

scholarly journals Endothelin-1 decreases endothelial PPARγ signaling and impairs angiogenesis after chronic intrauterine pulmonary hypertension

2014 ◽  
Vol 306 (4) ◽  
pp. L361-L371 ◽  
Author(s):  
David Wolf ◽  
Nancy Tseng ◽  
Gregory Seedorf ◽  
Gates Roe ◽  
Steven H. Abman ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Endothelial Cell ◽  
Ductus Arteriosus ◽  
Tube Formation ◽  
No Production ◽  
Fetal Sheep ◽  
Endothelial Cell Function ◽  
Endothelin 1 ◽  
Pparγ Agonists ◽  
Enos Protein

Increased endothelin-1 (ET-1) disrupts angiogenesis in persistent pulmonary hypertension of the newborn (PPHN), but pathogenic mechanisms are unclear. Peroxisome proliferator activated receptor γ (PPARγ) is decreased in adult pulmonary hypertension, but whether ET-1-PPARγ interactions impair endothelial cell function and angiogenesis in PPHN remains unknown. We hypothesized that increased PPHN pulmonary artery endothelial cell (PAEC) ET-1 production decreases PPARγ signaling and impairs tube formation in vitro. Proximal PAECs were harvested from fetal sheep after partial ligation of the ductus arteriosus in utero (PPHN) and controls. PPARγ and phospho-PPARγ protein were compared between normal and PPHN PAECs ± ET-1 and bosentan (ETA/ETB receptor blocker). Tube formation was assessed in response to PPARγ agonists ± ET-1, N-nitro-l-arginine (LNA) (NOS inhibitor), and PPARγ siRNA. Endothelial NO synthase (eNOS), phospho-eNOS, and NO production were measured after exposure to PPARγ agonists and PPARγ siRNA. At baseline, PPHN PAECs demonstrate decreased tube formation and PPARγ protein expression and activity. PPARγ agonists restored PPHN tube formation to normal. ET-1 decreased normal and PPHN PAEC tube formation, which was rescued by PPARγ agonists. ET-1 decreased PPARγ protein and activity, which was prevented by bosentan. PPARγ agonists increased eNOS protein and activity and NO production in normal and PPHN PAECs. LNA inhibited the effect of PPARγ agonists on tube formation. PPARγ siRNA decreased eNOS protein and tube formation in normal PAECs. We conclude that ET-1 decreases PPARγ signaling and contributes to PAEC dysfunction and impaired angiogenesis in PPHN. We speculate that therapies aimed at decreasing ET-1 production will restore PPARγ signaling, preserve endothelial function, and improve angiogenesis in PPHN.

scholarly journals Endothelin-1 impairs angiogenesis in vitro through Rho-kinase activation after chronic intrauterine pulmonary hypertension in fetal sheep

2012 ◽  
Vol 73 (3) ◽  
pp. 252-262 ◽  
Author(s):  
Jason Gien ◽  
Nancy Tseng ◽  
Gregory Seedorf ◽  
Gates Roe ◽  
Steven H. Abman
Keyword(s):  
Pulmonary Hypertension ◽  
Rho Kinase ◽  
Fetal Sheep ◽  
Kinase Activation ◽  
Endothelin 1

scholarly journals Cinaciguat, a soluble guanylate cyclase activator, augments cGMP after oxidative stress and causes pulmonary vasodilation in neonatal pulmonary hypertension

2011 ◽  
Vol 301 (5) ◽  
pp. L755-L764 ◽  
Author(s):  
Marc Chester ◽  
Gregory Seedorf ◽  
Pierre Tourneux ◽  
Jason Gien ◽  
Nancy Tseng ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Ductus Arteriosus ◽  
No Donor ◽  
Fetal Sheep ◽  
Cgmp Production ◽  
Pulmonary Vasodilation

Although inhaled NO (iNO) therapy is often effective in treating infants with persistent pulmonary hypertension of the newborn (PPHN), up to 40% of patients fail to respond, which may be partly due to abnormal expression and function of soluble guanylate cyclase (sGC). To determine whether altered sGC expression or activity due to oxidized sGC contributes to high pulmonary vascular resistance (PVR) and poor NO responsiveness, we studied the effects of cinaciguat (BAY 58-2667), an sGC activator, on pulmonary artery smooth muscle cells (PASMC) from normal fetal sheep and sheep exposed to chronic intrauterine pulmonary hypertension (i.e., PPHN). We found increased sGC α1- and β1-subunit protein expression but lower basal cGMP levels in PPHN PASMC compared with normal PASMC. To determine the effects of cinaciguat and NO after sGC oxidation in vitro, we measured cGMP production by normal and PPHN PASMC treated with cinaciguat and the NO donor, sodium nitroprusside (SNP), before and after exposure to 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, an sGC oxidizer), hyperoxia (fraction of inspired oxygen 0.50), or hydrogen peroxide (H2O2). After treatment with ODQ, SNP-induced cGMP generation was markedly reduced but the effects of cinaciguat were increased by 14- and 64-fold in PPHN fetal PASMC, respectively ( P < 0.01 vs. controls). Hyperoxia or H2O2enhanced cGMP production by cinaciguat but not SNP in PASMC. To determine the hemodynamic effects of cinaciguat in vivo, we compared serial responses to cinaciguat and ACh in fetal lambs after ductus arteriosus ligation. In contrast with the impaired vasodilator response to ACh, cinaciguat-induced pulmonary vasodilation was significantly increased. After birth, cinaciguat caused a significantly greater fall in PVR than either 100% oxygen, iNO, or ACh. We conclude that cinaciguat causes more potent pulmonary vasodilation than iNO in experimental PPHN. We speculate that increased NO-insensitive sGC may contribute to the pathogenesis of PPHN, and cinaciguat may provide a novel treatment of severe pulmonary hypertension.

Pulmonary hypertension and vascular remodeling in fetal sheep

1994 ◽  
Vol 266 (6) ◽  
pp. H2303-H2309 ◽  
Author(s):  
J. Belik ◽  
F. W. Keeley ◽  
F. Baldwin ◽  
M. Rabinovitch
Keyword(s):  
Pulmonary Hypertension ◽  
Vascular Remodeling ◽  
Ductus Arteriosus ◽  
Weight Ratio ◽  
Wall Stress ◽  
Progressive Increase ◽  
Left Ventricular ◽  
Fetal Sheep ◽  
In Vitro Synthesis

Vascular remodeling is commonly associated with pulmonary hypertension (PH) postnatally, but little is known about its presence in fetuses. In sixteen fetal sheep (126 wk gestation), the ductus arteriosus (DA) was ligated, and the animals were studied at 4, 8, and 14 days after surgery. The uninstrumented twins served as controls. Four days post-DA ligation PH resulted in an increase in the right and left ventricular free wall weight ratio (1.2 +/- 0.1 vs. 1.0 +/- 0.1 in the controls; P < 0.01), with a further progressive increase at 8 (1.4 +/- 0.1 vs. 1.0 +/- 0.1; P < 0.01) and 14 days (1.5 +/- 0.2 vs. 1.0 +/- 0.1; P < 0.01). An increase in vascular percent medial thickness was observed after 4 days of DA ligation and was restricted to small vessels. The large arteries collagen and elastin contents were 28.9 +/- 3.4 and 27.1 +/- 3.4 micrograms/micrograms of DNA, respectively, and were not significantly different from control values even after 14 days DA ligation. We further compared elastin synthesis in fetal and neonatal arteries in vitro. Synthesis in the fetus was greater than the newborn (10.6 +/- 1.4 vs. 4.6 +/- 0.7 cpm.mg wet wt-1.h-1; P < 0.01). Vessel endothelium denudation reduced synthesis to 60 +/- 8% of controls in the fetus, whereas no change was seen in the newborn. After an increase in wall stress, synthesis increased in the fetus (194 +/- 28% of control P < 0.01) and newborn (173 +/- 25%; P < 0.01). Removal of the endothelium abolished the response.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

A novel vasculo-angiogenic effect of cilostazol mediated by cross-talk between multiple signalling pathways including the ERK/p38 MAPK signalling transduction cascade

2012 ◽  
Vol 123 (3) ◽  
pp. 147-159 ◽  
Author(s):  
Ting-Hsing Chao ◽  
Shih-Ya Tseng ◽  
Yi-Heng Li ◽  
Ping-Yen Liu ◽  
Chung-Lung Cho ◽  
...  
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Umbilical Vein ◽  
Tube Formation ◽  
Mitogen Activated Protein Kinase ◽  
Signalling Pathways ◽  
No Production ◽  
Angiogenic Effect

Cilostazol is an anti-platelet agent with vasodilatory activity that acts by increasing intracellular concentrations of cAMP. Recent reports have suggested that cilostazol may promote angiogenesis. In the present study, we have investigated the effect of cilostazol in promoting angiogenesis and vasculogenesis in a hindlimb ischaemia model and have also examined its potential mechanism of action in vitro and in vivo. We found that cilostazol treatment significantly increased colony formation by human early EPCs (endothelial progenitor cells) through a mechanism involving the activation of cAMP/PKA (protein kinase A), PI3K (phosphoinositide 3-kinase)/Akt/eNOS (endothelial NO synthase) and ERK (extracellular-signal-regulated kinase)/p38 MAPK (mitogen-activated protein kinase) signalling pathways. Cilostazol also enhanced proliferation, chemotaxis, NO production and vascular tube formation in HUVECs (human umbilical vein endothelial cells) through activation of multiple signalling pathways downstream of PI3K/Akt/eNOS. Cilostazol up-regulated VEGF (vascular endothelial growth factor)-A165 expression and secretion of VEGF-A in HUVECs through activation of the PI3K/Akt/eNOS pathway. In a mouse hindlimb ischaemia model, recovery of blood flow ratio (ipsilateral/contralateral) 14 days after surgery was significantly improved in cilostazol-treated mice (10 mg/kg of body weight) compared with vehicle-treated controls (0.63±0.07 and 0.43±0.05 respectively, P<0.05). Circulating CD34+ cells were also increased in cilostazol-treated mice (3614±670 compared with 2151±608 cells/ml, P<0.05). Expression of VEGF and phosphorylation of PI3K/Akt/eNOS and ERK/p38 MAPK in ischaemic muscles were significantly enhanced by cilostazol. Our data suggest that cilostazol produces a vasculo-angiogenic effect by up-regulating a broad signalling network that includes the ERK/p38 MAPK, VEGF-A165, PI3K/Akt/eNOS and cAMP/PKA pathways.

scholarly journals Serotonin contributes to high pulmonary vascular tone in a sheep model of persistent pulmonary hypertension of the newborn

2013 ◽  
Vol 304 (12) ◽  
pp. L894-L901 ◽  
Author(s):  
Cassidy Delaney ◽  
Jason Gien ◽  
Gates Roe ◽  
Nicole Isenberg ◽  
Jenai Kailey ◽  
...  
Keyword(s):  
Pulmonary Hypertension ◽  
Pulmonary Artery ◽  
Tryptophan Hydroxylase ◽  
Late Gestation ◽  
Persistent Pulmonary Hypertension ◽  
Sheep Model ◽  
Pulmonary Hemodynamics ◽  
Fetal Sheep ◽  
Pulmonary Arterial

Although past studies demonstrate that altered serotonin (5-HT) signaling is present in adults with idiopathic pulmonary arterial hypertension, whether serotonin contributes to the pathogenesis of persistent pulmonary hypertension of the newborn (PPHN) is unknown. We hypothesized that 5-HT contributes to increased pulmonary vascular resistance (PVR) in a sheep model of PPHN and that selective 5-HT reuptake inhibitor (SSRI) treatment increases PVR in this model. We studied the hemodynamic effects of 5-HT, ketanserin (5-HT2A receptor antagonist), and sertraline, an SSRI, on pulmonary hemodynamics of the late gestation fetal sheep with PPHN caused by prolonged constriction of the ductus arteriosis. Brief intrapulmonary infusions of 5-HT increased PVR from 1.0 ± 0.07 (baseline) to 1.4 ± 0.22 mmHg/ml per minute of treatment ( P < 0.05). Ketanserin decreased PVR from 1.1 ± 0.15 (baseline) to 0.82 ± 0.09 mmHg/ml per minute of treatment ( P < 0.05). Sertraline increased PVR from 1.1 ± 0.17 (baseline) to 1.4 ± 0.17 mmHg/ml per minute of treatment ( P = 0.01). In addition, we studied 5-HT production and activity in vitro in experimental PPHN. Compared with controls, pulmonary artery endothelial cells from fetal sheep with PPHN exhibited increased expression of tryptophan hydroxylase 1 and 5-HT production by twofold and 56%, respectively. Compared with controls, 5-HT2A R expression was increased in lung homogenates and pulmonary artery smooth muscle cell lysates by 35% and 32%, respectively. We concluded that increased 5-HT contributes to high PVR in experimental PPHN through activation of the 5-HT2A receptor and that SSRI infusion further increases PVR in this model.

scholarly journals Intrauterine growth restriction decreases pulmonary alveolar and vessel growth and causes pulmonary artery endothelial cell dysfunction in vitro in fetal sheep

2011 ◽  
Vol 301 (6) ◽  
pp. L860-L871 ◽  
Author(s):  
Paul J. Rozance ◽  
Gregory J. Seedorf ◽  
Alicia Brown ◽  
Gates Roe ◽  
Meghan C. O'Meara ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Cell Growth ◽  
Pulmonary Artery ◽  
Intrauterine Growth Restriction ◽  
Tube Formation ◽  
No Production ◽  
Vascular Growth ◽  
Intrauterine Growth ◽  
Pulmonary Artery Endothelial Cell

Intrauterine growth restriction (IUGR) increases the risk for bronchopulmonary dysplasia (BPD). Abnormal lung structure has been noted in animal models of IUGR, but whether IUGR adversely impacts fetal pulmonary vascular development and pulmonary artery endothelial cell (PAEC) function is unknown. We hypothesized that IUGR would decrease fetal pulmonary alveolarization, vascular growth, and in vitro PAEC function. Studies were performed in an established model of severe placental insufficiency and IUGR induced by exposing pregnant sheep to elevated temperatures. Alveolarization, quantified by radial alveolar counts, was decreased 20% ( P < 0.005) in IUGR fetuses. Pulmonary vessel density was decreased 44% ( P < 0.01) in IUGR fetuses. In vitro, insulin increased control PAEC migration, tube formation, and nitric oxide (NO) production. This response was absent in IUGR PAECs. VEGFA stimulated tube formation, and NO production also was absent. In control PAECs, insulin increased cell growth by 68% ( P < 0.0001). Cell growth was reduced in IUGR PAECs by 29% at baseline ( P < 0.01), and the response to insulin was attenuated ( P < 0.005). Despite increased basal and insulin-stimulated Akt phosphorylation in IUGR PAECs, endothelial NO synthase (eNOS) protein expression as well as basal and insulin-stimulated eNOS phosphorylation were decreased in IUGR PAECs. Both VEGFA and VEGFR2 also were decreased in IUGR PAECs. We conclude that fetuses with IUGR are characterized by decreased alveolar and vascular growth and PAEC dysfunction in vitro. This may contribute to the increased risk for adverse respiratory outcomes and BPD in infants with IUGR.

Disruption of cGMP production in pulmonary arteries isolated from fetal lambs with pulmonary hypertension

1995 ◽  
Vol 268 (4) ◽  
pp. H1483-H1489 ◽  
Author(s):  
R. H. Steinhorn ◽  
J. A. Russell ◽  
F. C. Morin
Keyword(s):  
Pulmonary Hypertension ◽  
Soluble Guanylate Cyclase ◽  
Ductus Arteriosus ◽  
Pulmonary Veins ◽  
Pulmonary Arteries ◽  
Fourth Generation ◽  
Response Curves ◽  
Fetal Sheep ◽  
A 23187 ◽  
Arteries And Veins

Ligation of the ductus arteriosus of the fetal sheep produces severe pulmonary hypertension at birth. Standard tissue bath techniques were used to study third- and fourth-generation pulmonary arteries and veins isolated from fetal sheep with pulmonary hypertension created by ligation of the ductus arteriosus 11–12 days before birth as well as from age-matched control sheep. Vessels pretreated with indomethacin and propranolol were submaximally preconstricted with norepinephrine before exposure to A-23187 (10(-8) to 3 x 10(-7) M), sodium nitroprusside (SNP; 10(-9) to 10(-5) M), and nitric oxide (NO) gas (1-973 ppm). Pulmonary veins in both control and ligated animals relaxed similarly and completely to A-23187, SNP, and NO. Control pulmonary arteries relaxed by 16 +/- 2% to A-23187 and relaxed completely to SNP and NO, with concentration-response curves shifted rightward of those observed in pulmonary veins. Pulmonary arteries from ligated animals did not relax at all to A-23187. SNP relaxations in ligated arteries were shifted rightward of control. Ligated arteries relaxed by only 11 +/- 5% to the highest dose of NO. However, control and ligated pulmonary arteries relaxed similarly to 8-bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP; 10(-5) to 10(-3) M) and atrial natriuretic peptide (10(-9) to 10(-7) M). These data are most simply explained by decreased arterial vascular smooth muscle sensitivity to NO at the level of soluble guanylate cyclase.

Pulmonary endothelial NO synthase gene expression is decreased in fetal lambs with pulmonary hypertension

1997 ◽  
Vol 272 (5) ◽  
pp. L1005-L1012 ◽  
Author(s):  
P. W. Shaul ◽  
I. S. Yuhanna ◽  
Z. German ◽  
Z. Chen ◽  
R. H. Steinhorn ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pulmonary Hypertension ◽  
Protein Expression ◽  
Enzymatic Activity ◽  
Ductus Arteriosus ◽  
Muscle Growth ◽  
No Synthase ◽  
Enos Gene ◽  
Enos Expression ◽  
Enos Protein

Nitric oxide (NO), produced by endothelial (e) NO synthase (NOS), is critically involved in the cardiopulmonary transition from fetal to neonatal life. We have previously shown that NO-dependent relaxation is attenuated in intrapulmonary arteries from fetal lambs with pulmonary hypertension (PHT) created by prenatal ligation of the ductus arteriosus. In the present study, we determined whether this is due to altered pulmonary eNOS expression. eNOS and neuronal NOS (nNOS) protein expression were assessed in lungs from near-term control lambs and PHT lambs that underwent ductal ligation 10 days earlier. eNOS protein expression was decreased 49% in PHT lung. In contrast, nNOS protein abundance was unchanged. NOS enzymatic activity was also diminished in PHT vs. control lung (60 +/- 3 vs. 110 +/- 7 fmol.mg protein-1.min-1, respectively). Paralleling the declines in eNOS protein and NOS enzymatic activity, eNOS mRNA abundance was decreased 64% in PHT lung. Thus pulmonary eNOS gene expression is attenuated in the lamb model of fetal PHT. Because NO modulates both vasodilation and vascular smooth muscle growth, diminished eNOS expression may contribute to both the abnormal vasoreactivity and the excessive muscularization of the pulmonary circulation in fetal PHT.

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