Rod R. Warburton

The 5-HT transporter transactivates the PDGFβ receptor in pulmonary artery smooth muscle cells

Serotonin (5‐HT) stimulates smooth muscle cell growth through 5‐HT receptors and the 5‐HT transporter (5‐HTT), and has been associated with pulmonary hypertension (PH). Platelet‐derived growth factor receptors (PDGFR) have also been associated with PH. We present evidence for the first time that 5‐HT transactivates PDGFRβ through the 5‐HTT in pulmonary artery (PA) SMCs. Inhibition of PDGFR kinase with imatinib or AG1296 blocks 5‐HT‐stimulated PDGFRβ phosphorylation. 5‐HTT inhibitors and the Na+/K+‐ATPase inhibitor ouabain, but not 5‐HT2 and 5‐HT1B/1D receptor inhibitors, block PDGFRβ activation by 5‐HT. Notably, 5‐HTT binds the PDGFRβ upon 5‐HT stimulation and the 5‐HTT inhibitor fluoxetine blocks both the binding and PDGDRβ activation. Activation of PDGFRβ may occur through oxidation of a catalytic cysteine of tyrosine phosphatase. 5‐HT‐acti‐vated PDGFRβ phosphorylation is blocked by the anti‐oxidant N‐acetyl‐L‐cysteine and the NADPH oxidase inhibitor, DPI. Inhibition of PDGFR kinase with ima‐tinib or AG1296 significantly inhibits SMC proliferation and migration induced by 5‐HT in vitro. Infusion of 5‐HT by miniosmotic pumps enhances PDGFRβ activation in mouse lung in vivo. In summary, these results demonstrate that 5‐HT transactivates PDGFRβ in PASMCs leading to SMC proliferation and migration, and may be an important signaling pathway in the production of PH in vivo..—Liu, Y., Li, M., Warburton, R. R., Hill, N. S., Fanburg, B. L. The 5‐HT transporter transactivates the PDGFβ receptor in pulmonary artery smooth muscle cells. FASEB J. 21, 2725–2734 (2007)

Role of matrix metalloprotease-9 in hyperoxic injury in developing lung.

Matrix metalloprotease-9 (MMP-9) is increased in lung injury following hyperoxia exposure in neonatal mice, in association with impaired alveolar development. We studied the role of MMP-9 in the mechanism of hyperoxia-induced functional and histological changes in neonatal mouse lung. Reduced alveolarization with remodeling of ECM is a major morbidity component of oxidant injury in developing lung. MMP-9 mediates oxidant injury in developing lung causing altered lung remodeling. Five-day-old neonatal wild-type (WT) and MMP-9 (-/-) mice were exposed to hyperoxia for 8 days. The lungs were inflation fixed, and sections were examined for morphometry. The mean linear intercept and alveolar counts were evaluated. Immunohistochemistry for MMP-9 and elastin was performed. MMP-2, MMP-9, type I collagen, and tropoelastin were measured by Western blot analysis. Lung quasistatic compliance was studied in anaesthetized mice. MMP-2 and MMP-9 were significantly increased in lungs of WT mice exposed to hyperoxia compared with controls. Immunohistochemistry showed an increase in MMP-9 in mesenchyme and alveolar epithelium of hyperoxic lungs. The lungs of hyperoxia-exposed WT mice had less gas exchange surface area and were less compliant compared with room air-exposed WT and hyperoxia-exposed MMP-9 (-/-) mice. Type I collagen and tropoelastin were increased in hyperoxia-exposed WT with aberrant elastin staining. These changes were ameliorated in hyperoxia-exposed MMP-9 (-/-) mice. MMP-9 plays an important role in the structural changes consequent to oxygen-induced lung injury. Blocking MMP-9 activity may lead to novel therapeutic approaches in preventing bronchopulmonary dysplasia.

Genetic disruption of atrial natriuretic peptide causes pulmonary hypertension in normoxic and hypoxic mice.

To determine whether atrial natriuretic peptide (ANP) plays a physiological role in modulating pulmonary hypertensive responses, we studied mice with gene-targeted disruption of the ANP gene under normoxic and chronically hypoxic conditions. Right ventricular peak pressure (RVPP), right ventricle weight- and left ventricle plus septum weight-to-body weight ratios [RV/BW and (LV+S)/BW, respectively], and muscularization of pulmonary vessels were measured in wild-type mice (+/+) and in mice heterozygous (+/-) and homozygous (-/-) for a disrupted proANP gene after 3 wk of normoxia or hypobaric hypoxia (0.5 atm). Under normoxic conditions, homozygous mutants had higher RVPP (22 ± 2 vs. 15 ± 1 mmHg; P < 0.05) than wild-type mice and greater RV/BW (1.22 ± 0.08 vs. 0.94 ± 0.07 and 0.76 ± 0.04 mg/g; P < 0.05) and (LV+S)/BW (4.74 ± 0.42 vs. 3.53 ± 0.14 and 3.18 ± 0.18 mg/g; P < 0.05) than heterozygous or wild-type mice, respectively. Three weeks of hypoxia increased RVPP in heterozygous and wild-type mice and increased RV/BW and RV/(LV+S) in all genotypes compared with their normoxic control animals but had no effect on (LV+S)/BW. After 3 wk of hypoxia, homozygous mutants had higher RVPP (29 ± 3 vs. 23 ± 1 and 22 ± 2 mmHg; P < 0.05), RV/BW (2.03 ± 0.14 vs. 1.46 ± 0.04 and 1.33 ± 0.08 mg/g; P < 0.05), and (LV+S)/BW (4.76 ± 0.23 vs. 3.82 ± 0.09 and 3.44 ± 0.14 mg/g; P < 0.05) than heterozygous or wild-type mice, respectively. The percent muscularization of peripheral pulmonary vessels was greater in homozygous mutants than that in heterozygous or wild-type mice under both normoxic and hypoxic conditions. We conclude that endogenous ANP plays a physiological role in modulating pulmonary arterial pressure, cardiac hypertrophy, and pulmonary vascular remodeling under normoxic and hypoxic conditions.To determine whether atrial natriuretic peptide (ANP) plays a physiological role in modulating pulmonary hypertensive responses, we studied mice with gene-targeted disruption of the ANP gene under normoxic and chronically hypoxic conditions. Right ventricular peak pressure (RVPP), right ventricle weight- and left ventricle plus septum weight-to-body weight ratios [RV/BW and (LV+S)/BW, respectively], and muscularization of pulmonary vessels were measured in wild-type mice (+/+) and in mice heterozygous (+/-) and homozygous (-/-) for a disrupted proANP gene after 3 wk of normoxia or hypobaric hypoxia (0.5 atm). Under normoxic conditions, homozygous mutants had higher RVPP (22 +/- 2 vs. 15 +/- 1 mmHg; P < 0.05) than wild-type mice and greater RV/BW (1.22 +/- 0.08 vs. 0.94 +/- 0.07 and 0.76 +/- 0.04 mg/g; P < 0.05) and (LV+S)/BW (4.74 +/- 0. 42 vs. 3.53 +/- 0.14 and 3.18 +/- 0.18 mg/g; P < 0.05) than heterozygous or wild-type mice, respectively. Three weeks of hypoxia increased RVPP in heterozygous and wild-type mice and increased RV/BW and RV/(LV+S) in all genotypes compared with their normoxic control animals but had no effect on (LV+S)/BW. After 3 wk of hypoxia, homozygous mutants had higher RVPP (29 +/- 3 vs. 23 +/- 1 and 22 +/- 2 mmHg; P < 0.05), RV/BW (2.03 +/- 0.14 vs. 1.46 +/- 0.04 and 1.33 +/- 0.08 mg/g; P < 0.05), and (LV+S)/BW (4.76 +/- 0.23 vs. 3.82 +/- 0.09 and 3.44 +/- 0.14 mg/g; P < 0.05) than heterozygous or wild-type mice, respectively. The percent muscularization of peripheral pulmonary vessels was greater in homozygous mutants than that in heterozygous or wild-type mice under both normoxic and hypoxic conditions. We conclude that endogenous ANP plays a physiological role in modulating pulmonary arterial pressure, cardiac hypertrophy, and pulmonary vascular remodeling under normoxic and hypoxic conditions.

Regulation of vimentin intermediate filaments in endothelial cells by hypoxia

Hypoxia triggers responses in endothelial cells that play roles in many conditions including high-altitude pulmonary edema and tumor angiogenesis. Signaling pathways activated by hypoxia modify cytoskeletal and contractile proteins and alter the biomechanical properties of endothelial cells. Intermediate filaments are major components of the cytoskeleton whose contribution to endothelial physiology is not well understood. We have previously shown that hypoxia-activated signaling in endothelial cells alters their contractility and adhesiveness. We have also linked p38-MAP kinase signaling pathway leading to HSP27 phosphorylation and increased actin stress fiber formation to endothelial barrier augmentation. We now show that vimentin, a major intermediate filament protein in endothelial cells, is regulated by hypoxia. Our results indicate that exposure of endothelial cells to hypoxia causes vimentin filament networks to initially redistribute perinuclearly. However, by 1 hour hypoxia these networks reform and appear more continuous across cells than under normoxia. Hypoxia also causes transient changes in vimentin phosphorylation, and activation of PAK1, a kinase that regulates vimentin filament assembly. In addition, exposure to 1 hour hypoxia increases the ratio of insoluble/soluble vimentin. Overexpression of phosphomimicking mutant HSP27 (pmHSP27) causes changes in vimentin distribution that are similar to those observed in hypoxic cells. Knocking-down HSP27 destroys the vimentin filamentous network, and disrupting vimentin filaments with acrylamide increases endothelial permeability. Both hypoxia- and pmHSP27 overexpression-induced changes are reversed by inhibition of phosphatase activity. In conclusion hypoxia causes redistribution of vimentin to a more insoluble and extensive filamentous network that could play a role in endothelial barrier stabilization. Vimentin redistribution appears to be mediated through altering the phosphorylation of the protein and its interaction with HSP27.

Mineralocorticoid receptor antagonism attenuates experimental pulmonary hypertension

Mineralocorticoid receptor (MR) activation stimulates systemic vascular and left ventricular remodeling. We hypothesized that MR contributes to pulmonary vascular and right ventricular (RV) remodeling of pulmonary hypertension (PH). We evaluated the efficacy of MR antagonism by spironolactone in two experimental PH models; mouse chronic hypoxia-induced PH (prevention model) and rat monocrotaline-induced PH (prevention and treatment models). Last, the biological function of the MR was analyzed in cultured distal pulmonary artery smooth muscle cells (PASMCs). In hypoxic PH mice, spironolactone attenuated the increase in RV systolic pressure, pulmonary arterial muscularization, and RV fibrosis. In rat monocrotaline-induced PH (prevention arm), spironolactone attenuated pulmonary vascular resistance and pulmonary vascular remodeling. In the established disease (treatment arm), spironolactone decreased RV systolic pressure and pulmonary vascular resistance with no significant effect on histological measures of pulmonary vascular remodeling, or RV fibrosis. Spironolactone decreased RV cardiomyocyte size modestly with no significant effect on RV mass, systemic blood pressure, cardiac output, or body weight, suggesting a predominantly local pulmonary vascular effect. In distal PASMCs, MR was expressed and localized diffusely. Treatment with the MR agonist aldosterone, hypoxia, or platelet-derived growth factor promoted MR translocation to the nucleus, activated MR transcriptional function, and stimulated PASMC proliferation, while spironolactone blocked these effects. In summary, MR is active in distal PASMCs, and its antagonism prevents PASMC proliferation and attenuates experimental PH. These data suggest that MR is involved in the pathogenesis of PH via effects on PASMCs and that MR antagonism may represent a novel therapeutic target for this disease.

Smooth muscle protein-22-mediated deletion of Tsc1 results in cardiac hypertrophy that is mTORC1-mediated and reversed by rapamycin

Constitutive activation of mammalian target of rapamycin complex 1 (mTORC1), a key kinase complex that regulates cell size and growth, is observed with inactivating mutations of either of the tuberous sclerosis complex (TSC) genes, Tsc1 and Tsc2. Tsc1 and Tsc2 are highly expressed in cardiovascular tissue but their functional role there is unknown. We generated a tissue-specific knock-out of Tsc1, using a conditional allele of Tsc1 and a cre recombinase allele regulated by the smooth muscle protein-22 (SM22) promoter (Tsc1c/cSM22cre+/-) to constitutively activate mTOR in cardiovascular tissue. Significant gene recombination (∼80%) occurred in the heart by embryonic day (E) 15, and reduction in Tsc1 expression with increased levels of phosphorylated S6 kinase (S6K) and S6 was observed, consistent with constitutive activation of mTORC1. Cardiac hypertrophy was evident by E15 with post-natal progression to heart weights of 142 ± 24 mg in Tsc1c/cSM22cre+/- mice versus 65 ± 14 mg in controls (P < 0.01). Median survival of Tsc1c/cSM22cre+/- mice was 24 days, with none surviving beyond 6 weeks. Pathologic and echocardiographic analysis revealed severe biventricular hypertrophy without evidence of fibrosis or myocyte disarray, and significant reduction in the left ventricular end-diastolic diameter (P < 0.001) and fractional index (P < 0.001). Inhibition of mTORC1 by rapamycin resulted in prolonged survival of Tsc1c/cSM22cre+/- mice, with regression of ventricular hypertrophy. These data support a critical role for the Tsc1/Tsc2-mTORC1-S6K axis in the normal development of cardiovascular tissue and also suggest possible therapeutic potential of rapamycin in cardiac disorders where pathologic mTORC1 activation occurs.

Serotonin induces Rho/ROCK-dependent activation of Smads 1/5/8 in pulmonary artery smooth muscle cells

Serotonin (5‐HT) stimulates pulmonary artery smooth muscle cell proliferation and has been associated with pulmonary arterial hypertension (PAH). Bone morphogenetic protein receptor 2 (BMPR2) mutations similarly have been linked to PAH. However, possible crosstalk between 5‐HT and BMPR signaling remains poorly characterized. We report here that 5‐HT activates Smads 1/5/8 in bovine and human pulmonary artery smooth muscle cells (SMCs) and causes translocation of these Smads from cytoplasm to the nucleus. DN BMPR1A blocked 5‐HT activation of Smads 1/5/8 by 5‐HT and BMPR1A overexpression enhanced it. Activation of Smads by 5‐HT occurred through the 5‐HT 1B/1D receptor as it was blocked with the inhibitor GR 55562 but unaffected by inhibitors of the 5‐HT transporter and a variety of 5‐HT receptors. Activation of the Smads by 5‐HT depended on Rho/Rho kinase signaling as it was blocked by Y27632, but unaffected by inhibitors of PI3K or MAPK. Transfection of cells with BMPR1A and ligation of the BMP receptor with BMP‐2 also activated GTP‐Rho A of these SMCs, while DN BMPR1A blocked the activation. 5‐HT stimulated an increase in serine/threonine phosphorylation of BMPR1A, supporting the activation of BMPR1A by 5‐HT in SMCs. Infusion of 5‐HT into mice with miniosmotic infusion pumps caused activation of Smads 1/5/8 in lung tissue, demonstrating the effect in vivo. The studies support a unique concept that 5‐HT transactivates the serine kinase receptor, BMPR 1A, to activate Smads 1/5/8 via Rho and Rho kinase in pulmonary artery SMCs. Rho and Rho kinase also participate in the activation of Smads by BMP.—Liu, Y.,Ren, W., Warburton, R., Toksoz, D., Fanburg, B. L. Serotonin induces Rho/ROCK‐dependent activation of Smads 1/5/8 in pulmonary artery smooth muscle cells. FASEB J. 23, 2299–2306 (2009)

Synergistic Effects of ANP and Sildenafil on cGMP Levels and Amelioration of Acute Hypoxic Pulmonary Hypertension

We hypothesized that the phosphodiesterase 5 inhibitor, sildenafil, and the guanosine cyclase stimulator, atrial natriuretic peptide (ANP), would act synergistically to increase cGMP levels and blunt hypoxic pulmonary hypertension in rats, because these compounds act via different mechanisms to increase the intracellular second messenger. Acute hypoxia: Adult Sprague-Dawley rats were gavaged with sildenafil (1 mg/kg) or vehicle and exposed to acute hypoxia with and without ANP (10-8 -10-5 M). Sildenafil decreased systemic blood pressure (103 ± 10 vs. 87 ± 6 mm Hg, P < 0.001) and blunted the hypoxia-induced increase in right ventricular systolic pressure (RVSP; percent increase 73.7% ± 9.4% in sildenafil-treated rats vs. 117.2% ± 21.1% in vehicle-treated rats, P = 0.03). Also, ANP and sildenafil had synergistic effects on blunting the hypoxia-induced increase in RVSP (P < 0.001) and on rising plasma cGMP levels (P < 0.05). Chronic hypoxia: Other rats were exposed to prolonged hypoxia (3 weeks, 0.5 atm) after subcutaneous implantation of a sustained-release pellet containing lower (2.5 mg), or higher (25 mg) doses of sildenafil, or placebo. Higher-dose, but not lower-dose sildenafil blunted the chronic hypoxia-induced increase in RVSP (P = 0.006). RVSP and plasma sildenafil levels were inversely correlated in hypoxic rats (r2 = 0.68, P = 0.044). Lung cGMP levels were increased by both chronic hypoxia and sildenafil, with the greatest increase achieved by the combination. Plasma and right ventricular (RV) cGMP levels were increased by hypoxia, but sildenafil had no effect. RV hypertrophy and pulmonary artery muscularization were also unaffected by sildenafil. In conclusion, sildenafil and ANP have synergistic effects on the blunting of hypoxia-induced pulmonary vasoconstriction. During chronic hypoxia, sildenafil normalizes RVSP, but in the doses used, sildenafil has no effect on RV hypertrophy or pulmonary vascular remodeling.

Adenosine protected against pulmonary edema through transporter- and receptor A2-mediated endothelial barrier enhancement

We have previously demonstrated that adenosine plus homocysteine enhanced endothelial basal barrier function and protected against agonist-induced barrier dysfunction in vitro through attenuation of RhoA activation by inhibition of isoprenylcysteine-O-carboxyl methyltransferase. In the current study, we tested the effect of elevated adenosine on pulmonary endothelial barrier function in vitro and in vivo. We noted that adenosine alone dose dependently enhanced endothelial barrier function. While adenosine receptor A(1) or A(3) antagonists were ineffective, an adenosine transporter inhibitor, NBTI, or a combination of DPMX and MRS1754, antagonists for adenosine receptors A(2A) and A(2B), respectively, partially attenuated the barrier-enhancing effect of adenosine. Similarly, inhibition of both A(2A) and A(2B) receptors with siRNA also blunted the effect of adenosine on barrier function. Interestingly, inhibition of both transporters and A(2A)/A(2B) receptors completely abolished adenosine-induced endothelial barrier enhancement. The adenosine receptor A(2A) and A(2B) agonist, NECA, also significantly enhanced endothelial barrier function. These data suggest that both adenosine transporters and A(2A) and A(2B) receptors are necessary for exerting maximal effect of adenosine on barrier enhancement. We also found that adenosine enhanced Rac1 GTPase activity and overexpression of dominant negative Rac1 attenuated adenosine-induced increases in focal adhesion complexes. We further demonstrated that elevation of cellular adenosine by inhibition of adenosine deaminase with Pentostatin significantly enhanced endothelial basal barrier function, an effect that was also associated with enhanced Rac1 GTPase activity and with increased focal adhesion complexes and adherens junctions. Finally, using a non-inflammatory acute lung injury (ALI) model induced by alpha-naphthylthiourea, we found that administration of Pentostatin, which elevated lung adenosine level by 10-fold, not only attenuated the development of edema before ALI but also partially reversed edema after ALI. The data suggest that adenosine deaminase inhibition may be useful in treatment of pulmonary edema in settings of ALI.

Elevated transglutaminase 2 activity is associated with hypoxia-induced experimental pulmonary hypertension in mice.

Previous studies in human patients and animal models have suggested that transglutaminase 2 (TG2) is upregulated in pulmonary hypertension (PH), a phenomenon that appears to be associated with the effects of serotonin (5-hydroxytryptamine; 5-HT) in this disease. Using chemical tools to interrogate and inhibit TG2 activity in vivo, we have shown that pulmonary TG2 undergoes marked post-translational activation in a mouse model of hypoxia-induced PH. We have also identified irreversible fluorinated TG2 inhibitors that may find use as non-invasive positron emission tomography probes for diagnosis and management of this debilitating, lifelong disorder. Pharmacological inhibition of TG2 attenuated the elevated right ventricular pressure but had no effect on hypertrophy of the right ventricle of the heart. A longitudinal study of pulmonary TG2 activity in PH patients is warranted.