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Background: Aging-associated left ventricular dysfunction promotes cardiopulmonary fibrogenic remodeling, Group 2 pulmonary hypertension (PH), and right ventricular failure. At the time of diagnosis, cardiac function has declined, and cardiopulmonary fibrosis has often developed. Here, we sought to develop a molecular positron emission tomography (PET)-magnetic resonance imaging (MRI) protocol to detect both cardiopulmonary fibrosis and fibrotic disease activity in a left ventricular dysfunction model., Methods and Results: Left ventricular dysfunction was induced by transverse aortic constriction (TAC) in 6-month-old senescence-accelerated prone mice, a subset of mice that received sham surgery. Three weeks after surgery, mice underwent simultaneous PET-MRI at 4.7 T. Collagen-targeted PET and fibrogenesis magnetic resonance (MR) probes were intravenously administered. PET signal was computed as myocardium- or lung-to-muscle ratio. Percent signal intensity increase and Δ lung-to-muscle ratio were computed from the pre-/postinjection magnetic resonance images. Elevated allysine in the heart ( P =0.02) and lungs ( P =0.17) of TAC mice corresponded to an increase in myocardial magnetic resonance imaging percent signal intensity increase ( P <0.0001) and Δlung-to-muscle ratio ( P <0.0001). Hydroxyproline in the heart ( P <0.0001) and lungs ( P <0.01) were elevated in TAC mice, which corresponded to an increase in heart (myocardium-to-muscle ratio, P =0.02) and lung (lung-to-muscle ratio, P <0.001) PET measurements. Pressure-volume loop and echocardiography demonstrated adverse left ventricular remodeling, function, and increased right ventricular systolic pressure in TAC mice., Conclusions: Administration of collagen-targeted PET and allysine-targeted MR probes led to elevated PET-magnetic resonance imaging signals in the myocardium and lungs of TAC mice. The study demonstrates the potential to detect fibrosis and fibrogenesis in cardiopulmonary disease through a dual molecular PET-magnetic resonance imaging protocol.

Pulmonary hypertension (PH) pathogenesis is driven by inflammatory and metabolic derangements as well as glycolytic reprogramming. Induction of both interleukin 6 (IL6) and transglutaminase 2 (TG2) expression participates in human and experimental cardiovascular diseases. However, little is known about the role of TG2 in these pathologic processes. The current study aimed to investigate the molecular interactions between TG2 and IL6 in mediation of tissue remodeling in PH. A lung-specific IL6 over-expressing transgenic mouse strain showed elevated right ventricular (RV) systolic pressure as well as increased wet and dry tissue weights and tissue fibrosis in both lungs and RVs compared to age-matched wild-type littermates. In addition, IL6 over-expression induced the glycolytic and fibrogenic markers, hypoxia-inducible factor 1α, pyruvate kinase M2 (PKM2), and TG2. Consistent with these findings, IL6 induced the expression of both glycolytic and pro-fibrogenic markers in cultured lung fibroblasts. IL6 also induced TG2 activation and the accumulation of TG2 in the extracellular matrix. Pharmacologic inhibition of the glycolytic enzyme, PKM2 significantly attenuated IL6-induced TG2 activity and fibrogenesis. Thus, we conclude that IL6-induced TG2 activity and cardiopulmonary remodeling associated with tissue fibrosis are under regulatory control of the glycolytic enzyme, PKM2., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Penumatsa, Sharma, Warburton, Singhal, Toksoz, Bhedi, Qi, Preston, Anderlind, Hill and Fanburg.)

Rho kinase (ROCK) is implicated in the development of pulmonary arterial hypertension (PAH) in which abnormal pulmonary vascular smooth muscle (VSM) contractility and remodeling lead to right heart failure. Pharmacologic ROCK inhibitors block experimental pulmonary hypertension (PH) development in rodents but can have off-target effects and do not distinguish between the two ROCK forms, ROCK1 and ROCK2, encoded by separate genes. An earlier study using gene knock out (KO) in mice indicated that VSM ROCK2 is required for experimental PH development, but the role of ROCK1 is not well understood. Here we investigated the in vivo role of ROCK1 in PH development by generating a VSM-targeted homozygous ROCK1 gene KO mouse strain. Adult control mice exposed to Sugen5416 (Su)/hypoxia treatment to induce PH had significantly increased right ventricular systolic pressures (RVSP) and RV hypertrophy versus normoxic controls. In contrast, Su/hypoxia-exposed VSM ROCK1 KO mice did not exhibit significant RVSP elevation, and RV hypertrophy was blunted. Su/hypoxia-induced pulmonary small vessel muscularization was similarly elevated in both control and VSM ROCK1 KO animals. siRNA-mediated ROCK1 knock-down (KD) in human PAH pulmonary arterial SM cells (PASMC) did not affect cell growth. However, ROCK1 KD led to reduced AKT and MYPT1 signaling in serotonin-treated PAH PASMC. The findings suggest that like VSM ROCK2, VSM ROCK1 actively contributes to PH development, but in distinction acts via nonproliferative pathways to promote hypoxemia, and thus may be a distinct therapeutic target in PH., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Abnormalities that characterize pulmonary arterial hypertension include impairment in the structure and function of pulmonary vascular endothelial and smooth muscle cells. Aldosterone levels are elevated in human pulmonary arterial hypertension and in experimental pulmonary hypertension, while inhibition of the aldosterone-binding mineralocorticoid receptor attenuates pulmonary hypertension in multiple animal models. We explored the role of mineralocorticoid receptor in endothelial and smooth muscle cells in using cell-specific mineralocorticoid receptor knockout mice exposed to sugen/hypoxia-induced pulmonary hypertension. Treatment with the mineralocorticoid receptor inhibitor spironolactone significantly reduced right ventricular systolic pressure. However, this is not reproduced by selective mineralocorticoid receptor deletion in smooth muscle cells or endothelial cells. Similarly, spironolactone attenuated the increase in right ventricular cardiomyocyte area independent of vascular mineralocorticoid receptor with no effect on right ventricular weight or interstitial fibrosis. Right ventricular perivascular fibrosis was significantly decreased by spironolactone and this was reproduced by specific deletion of mineralocorticoid receptor from endothelial cells. Endothelial cell-mineralocorticoid receptor deletion attenuated the sugen/hypoxia-induced increase in the leukocyte-adhesion molecule, E-selectin, and collagen IIIA1 in the right ventricle. Spironolactone also significantly reduced pulmonary arteriolar muscularization, independent of endothelial cell-mineralocorticoid receptor or smooth muscle cell-mineralocorticoid receptor. Finally, the degree of pulmonary perivascular inflammation was attenuated by mineralocorticoid receptor antagonism and was fully reproduced by smooth muscle cell-specific mineralocorticoid receptor deletion. These studies demonstrate that in the sugen/hypoxia pulmonary hypertension model, systemic-mineralocorticoid receptor blockade significantly attenuates the disease and that mineralocorticoid receptor has cell-specific effects, with endothelial cell-mineralocorticoid receptor contributing to right ventricular perivascular fibrosis and smooth muscle cell-mineralocorticoid receptor participating in pulmonary vascular inflammation. As mineralocorticoid receptor antagonists are being investigated to treat pulmonary arterial hypertension, these findings support novel mechanisms and potential mineralocorticoid receptor targets that mediate therapeutic benefits in patients., (© The Author(s) 2021.)

The pathophysiology of pulmonary hypertension (PH) and heart failure (HF) includes fibrogenic remodeling associated with the loss of pulmonary arterial (PA) and cardiac compliance. We and others have previously identified transglutaminase 2 (TG2) as a participant in adverse fibrogenic remodeling. However, little is known about the biologic mechanisms that regulate TG2 function. We examined physiological mouse models of experimental PH, HF, and type 1 diabetes that are associated with altered glucose metabolism/glycolysis and report here that TG2 expression and activity are elevated in pulmonary and cardiac tissues under all these conditions. We additionally used PA adventitial fibroblasts to test the hypothesis that TG2 is an intermediary between enhanced tissue glycolysis and fibrogenesis. Our in vitro results show that glycolytic enzymes and TG2 are upregulated in fibroblasts exposed to high glucose, which stimulates cellular glycolysis as measured by Seahorse analysis. We examined the relationship of TG2 to a terminal glycolytic enzyme, pyruvate kinase M2 (PKM2), and found that PKM2 regulates glucose-induced TG2 expression and activity as well as fibrogenesis. Our studies further show that TG2 inhibition blocks glucose-induced fibrogenesis and cell proliferation. Our findings support a novel role for glycolysis-mediated TG2 induction and tissue fibrosis associated with experimental PH, HF, and hyperglycemia., (© 2019 Federation of American Societies for Experimental Biology.)

One of the major contributors to sickle cell disease (SCD) pathobiology is the hemolysis of sickle red blood cells (RBCs), which release free hemoglobin and platelet agonists including adenosine 5'-diphosphate (ADP) into the plasma. While platelet activation/aggregation may promote tissue ischemia and pulmonary hypertension in SCD, modulation of sickle platelet dysfunction remains poorly understood. Calpain-1, a ubiquitous calcium-activated cysteine protease expressed in hematopoietic cells, mediates aggregation of platelets in healthy mice. We generated calpain-1 knockout Townes sickle (SSCKO) mice to investigate the role of calpain-1 in steady state and hypoxia/reoxygenation (H/R)-induced sickle platelet activation and aggregation, clot retraction, and pulmonary arterial hypertension. Using multi-electrode aggregometry, which measures platelet adhesion and aggregation in whole blood, we determined that steady state SSCKO mice exhibit significantly impaired PAR4-TRAP-stimulated platelet aggregation as compared to Townes sickle (SS) and humanized control (AA) mice. Interestingly, the H/R injury induced platelet hyperactivity in SS and SSCKO, but not AA mice, and partially rescued the aggregation defect in SSCKO mice. The PAR4-TRAP-stimulated GPIIb-IIIa (α IIb β 3 ) integrin activation was normal in SSCKO platelets suggesting that an alternate mechanism mediates the impaired platelet aggregation in steady state SSCKO mice. Taken together, we provide the first evidence that calpain-1 regulates platelet hyperactivity in sickle mice, and may offer a viable pharmacological target to reduce platelet hyperactivity in SCD., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Tissue matrix remodeling and fibrosis leading to loss of pulmonary arterial and right ventricular compliance are important features of both experimental and clinical pulmonary hypertension (PH). We have previously reported that transglutaminase 2 (TG2) is involved in PH development while others have shown it to be a cross-linking enzyme that participates in remodeling of extracellular matrix in fibrotic diseases in general. In the present studies, we used a mouse model of experimental PH (Sugen 5416 and hypoxia; SuHypoxia) and cultured primary human cardiac and pulmonary artery adventitial fibroblasts to evaluate the relationship of TG2 to the processes of fibrosis, protein cross-linking, extracellular matrix collagen accumulation, and fibroblast-to-myofibroblast transformation. We report here that TG2 expression and activity as measured by serotonylated fibronectin and protein cross-linking activity along with fibrogenic markers are significantly elevated in lungs and right ventricles of SuHypoxic mice with PH. Similarly, TG2 expression and activity, protein cross-linking activity, and fibrogenic markers are significantly increased in cultured cardiac and pulmonary artery adventitial fibroblasts in response to hypoxia exposure. Pharmacological inhibition of TG2 activity with ERW1041E significantly reduced hypoxia-induced cross-linking activity and synthesis of collagen 1 and α-smooth muscle actin in both the in vivo and in vitro studies. TG2 short interfering RNA had a similar effect in vitro. Our results suggest that TG2 plays an important role in hypoxia-induced pulmonary and right ventricular tissue matrix remodeling in the development of PH., (Copyright © 2017 the American Physiological Society.)

This study aimed to characterize alterations in select eicosanoids in experimental and human pulmonary arterial hypertension (PAH) and to assess their potential utility as predictors of outcome. Using liquid chromatography-mass spectrometry, we performed targeted lipidomic analyses of the lungs and right ventricles (RVs) of chronically hypoxic rats and plasma of consecutive PAH patients and healthy controls. In rat lungs, chronic hypoxia was associated with significantly decreased lung prostacyclin (PGI2)/thromboxane B2 (TXB2) ratio and elevated lung 8-hydroxyeicosanoid (HETE) acid concentrations. RV eicosanoids did not exhibit any changes with chronic hypoxia. PAH treatment-naïve patients had significantly increased plasma concentrations of TXB2 and 5-, 8-, 12-, and 15-HETE. The PGI2/TXB2 ratio was lower in PAH patients than in controls, especially in the treatment-naïve cohort (median: 2.1, 0.3, and 1.3 in controls, treatment-naïve, and treated patients, respectively, P = 0.001). Survival was significantly worse in PAH patients with 12-HETEhigh (≥57 pg/mL) and 15-HETEhigh (≥256 pg/mL) in unadjusted and adjusted analyses (hazard ratio [HR]: 2.8 [95% confidence interval (CI): 1.1-7.3], P = 0.04 and HR: 4.3 [95% CI: 1.6-11.8], P = 0.004, respectively; adjustment was performed with the REVEAL [Registry to Evaluate Early and Long-Term PAH Disease Management] risk score). We demonstrate significant alterations in eicosanoid pathways in experimental and human PAH. We found that 12- and 15-HETE were independent predictors of survival in human PAH, even after adjusting for the REVEAL score, suggesting their potential role as novel biomarkers.

Anthrax is associated with severe vascular leak, which is caused by the bacterial lethal toxin (LeTx). Pleural effusions and pulmonary edema that occur in anthrax are believed to reflect endothelial injury caused by the anthrax toxin. Since vascular leak can also be observed consistently in rats injected intravenously with LeTx, the latter might present a simple physiologically relevant animal model of acute lung injury (ALI). Such a model could be utilized in evaluating and developing better treatment for ALI or acute respiratory distress syndrome (ARDS), as other available rodent models do not consistently produce the endothelial permeability that is a major component of ARDS. The biological activity of LeTx resides in the lethal factor metalloprotease that specifically degrades MAP kinase kinases (MKKs). Recently, we showed that LeTx inactivation of p38 MAP kinase signaling via degradation of MKK3 in pulmonary vascular endothelial cells can be linked to compromise of the endothelial permeability barrier. LeTx effects were linked specifically to blocking activation of p38 substrate and MAP kinase-activated protein kinase 2 (MAPKAPK2 or MK2) and phosphorylation of the latter's substrate, heat shock protein 27 (HSP27). We have now designed a peptide that directly and specifically activates MK2, causing HSP27 phosphorylation in cells and in vivo. The MK2-activating peptide (MK2-AP) also blocks the effects of LeTx on endothelial barriers in cultured cells and reduces LeTx-induced pulmonary vascular leak in rats. Hence, MK2-AP has the therapeutic potential to counteract anthrax or pulmonary edema and vascular leak due to other causes., (Copyright © 2015 the American Physiological Society.)

Despite high expression levels, the role of Tsc1 in cardiovascular tissue is ill defined. We launched this study to examine the role of Tsc1 in cardiac physiology and pathology. Mice in which Tsc1 was deleted in cardiac tissue and vascular smooth muscle (Tsc1c/cSM22cre(+/-)), developed progressive cardiomegaly and hypertension and died early. Hearts of Tsc1c/cSM22cre(+/-) mice displayed a progressive increase in cardiomyocyte number, and to a lesser extent, size between the ages of 1 and 6 weeks. In addition, compared to control hearts, proliferation markers (phospho-histone 3 and PCNA) were elevated in Tsc1c/cSM22cre(+/-) cardiomyocytes at 0-4 weeks, suggesting that cardiomyocyte proliferation was the predominant mechanism underlying cardiomegaly in Tsc1c/cSM22cre(+/-) mice. To examine the contribution of Tsc1 deletion in peripheral vascular smooth muscle to the cardiac phenotype, Tsc1c/cSM22cre(+/-) mice were treated with the antihypertensive, hydralazine. Prevention of hypertension had no effect on survival, cardiac size, or cardiomyocyte number in these mice. We furthermore generated mice in which Tsc1 was deleted only in vascular smooth muscle but not in cardiac tissue (Tsc1c/cSMAcre-ER(T2+/-)). The Tsc1c/cSMAcre-ER(T2+/-) mice also developed hypertension. However, their survival was normal and no cardiac abnormalities were observed. Our results suggest that loss of Tsc1 in the heart causes cardiomegaly, which is driven by increased cardiomyocyte proliferation that also appears to confer relative resistance to afterload reduction. These findings support a critical role for the Tsc1 gene as gatekeeper in the protection against uncontrolled cardiac growth., (Copyright © 2014 Elsevier Inc. All rights reserved.)

We previously reported that transglutaminase 2 (TG2) activity is markedly elevated in lungs of hypoxia-exposed rodent models of pulmonary hypertension (PH). Since vascular remodeling of pulmonary artery smooth muscle cells (PASMCs) is important in PH, we undertook the present study to determine whether TG2 activity is altered in PASMCs with exposure to hypoxia and whether that alteration participates in their proliferative response to hypoxia. Cultured distal bovine (b) and proximal human (h) PASMCs were exposed to hypoxia (3% O2) or normoxia (21% O2). mRNA and protein expression were determined by PCR and Western blot analyses. TG2 activity and function were visualized and determined by fluorescent labeled 5-pentylamine biotin incorporation and immunoblotting of serotonylated fibronectin. Cell proliferation was assessed by [(3)H]thymidine incorporation assay. At 24 h, both TG2 expression and activity were stimulated by hypoxia in bPASMCs. Activation of TG2 by hypoxia was blocked by inhibition of the extracellular calcium-sensing receptor or the transient receptor potential channel V4. In contrast, TG2 expression was blocked by inhibition of the transcription factor hypoxia-inducible factor-1α, supporting the presence of separate mechanisms for stimulation of activity and expression of TG2. Pulmonary arterial hypertension patient-derived hPASMCs were found to proliferate significantly more rapidly and respond to hypoxia more strongly than control-derived hPASMCs. Similar to bovine cells, hypoxia-induced proliferation of patient-derived cells was blocked by inhibition of TG2 activity. Our results suggest an important role for TG2, mediated by intracellular calcium fluxes and HIF-1α, in hypoxia-induced PASMC proliferation and possibly in vascular remodeling in PH., (Copyright © 2014 the American Physiological Society.)

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.

Serotonin (5-HT) and fibronectin (FN) have been associated with pulmonary hypertension (PH). We previously reported that FN is posttranslationally modified by tissue transglutaminase (TGase) to form serotonylated FN (s-FN) in pulmonary artery smooth muscle cells and that serotonylation stimulates their proliferation and migration, hallmarks of PH. We hypothesized that s-FN and its binding to TGase are elevated in human and experimental PH. To assess this hypothesis, FN isolation and electrophoretic, immunoblotting, and densitometric techniques were used. Mean ratio of serum s-FN to total FN level (s-FN/FN) was elevated in 19 consecutive pulmonary arterial hypertension (PAH) patients compared with 25 controls (0.3 ± 0.18 vs. 0.05 ± 0.07, P < 0.001). s-FN/FN also was increased in lungs of mice and rats with hypoxia-induced PH and in rats with monocrotaline-induced PH. In mice, the increase was detected at 1 wk of hypoxia, preceding the development of PH. Hypoxic rats had elevated serum s-FN/FN. Enhanced binding of TGase to its substrate FN occurred in serum from patients with PAH (mean 0.50 ± 0.51 vs. 0.063 ± 0.11, P = 0.002) and s-FN/FN and TGase-bound FN were highly correlated (R(2) = 0.77). TGase-bound FN also was increased in experimental PH. We conclude that increased serotonylation of FN occurs in human and experimental PH and may provide a biomarker for the disease.

Exposure to anthrax causes life-threatening disease through the action of the toxin produced by the Bacillus anthracis bacteria. Lethal factor (LF), an anthrax toxin component which causes severe vascular leak and edema, is a protease which specifically degrades MAP kinase kinases (MKK). We have recently shown that p38 MAP kinase activation leading to HSP27 phosphorylation augments the endothelial permeability barrier. We now show that treatment of rat pulmonary microvascular endothelial cells with anthrax lethal toxin (LeTx), which is composed of LF and the protective antigen, increases endothelial barrier permeability and gap formation between endothelial cells through disrupting p38 signaling. LeTx treatment increases MKK3b degradation and in turn decreases p38 activity at baseline as well as after activation of p38 signaling. Consequently, LeTx treatment decreases activation of the p38 substrate kinase, MK2, and the phosphorylation of the latter's substrate, HSP27. LeTx treatment disrupts other signaling pathways leading to suppression of Erk-mediated signaling, but these effects do not correlate with LeTx-induced barrier compromise. Overexpressing phosphomimicking (pm)HSP27, which protects the endothelial permeability barrier against LeTx, blocks LeTx inactivation of p38 and MK2, but it does not block MKK3b degradation or Erk inactivation. Our results suggest that LeTx might cause vascular leak through inactivating p38-MK2-HSP27 signaling and that activating HSP27 phosphorylation specifically restores p38 signaling and blocks anthrax LeTx toxicity. The fact that barrier integrity could be restored by pmHSP27 overexpression without affecting degradation of MKK3b, or inactivation of Erk, suggests a specific and central role for p38-MK2-HSP27 in endothelial barrier permeability regulation., (Copyright © 2011 Wiley Periodicals, Inc.)

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.

This manuscript describes how the permeability of pulmonary artery microvascular endothelial cell (RPMEC) monolayer is elevated by hypoxia and the role played by HSP27 phosphorylation. p38 MAP kinase activation leading to HSP27 phosphorylation was previously shown by our laboratory to alter the actin cytoskeleton and tethering properties of RPMEC. This effect was independent of hypoxia-induced contractility which was ROCK-dependent rather than HSP27-dependent. Results described here show that increased HSP27 phosphorylation not only does not underlie hypoxia-induced permeability, but may actually augment the endothelial barrier. Hypoxia causes gap formation between RPMEC and increases MLC2 phosphorylation. The phosphorylation of MYPT1, which inhibits MLC2 phosphatase, is also increased in hypoxia. In addition, FAK phosphorylation, which alters focal adhesion signaling, is increased in hypoxia. Overexpressing phosphomimicking HSP27 (pmHSP27), which induces significant actin stress fiber formation, surprisingly renders RPMEC resistant to hypoxia- or TGFbeta-induced permeability. siRNA against pmHSP27 reverses the increased actin stress fiber formation in pmHSP27-overexpressing cells, and disrupting actin stress fibers in pmHSP27-overexpressing RPMEC renders them more susceptible to hypoxia. Finally, hypoxia-induced gap formation, as well as phosphorylation of MLC2, MYPT1 and FAK are almost abolished by overexpressing pmHSP27 in RPMEC. These effects of pmHSP27 overexpression might represent decreased cytoskeletal plasticity and increased tethering which counteracts permeability-inducing contractility. Thus hypoxia activates two pathways one leading to contractility and increased permeability, the other leading to actin stress fibers, stronger adhesion, and reduced permeability. Altering HSP27 phosphorylation, which tips the balance towards decreased permeability, might be targeted in managing endothelial barrier dysfunction.

There have been reports of marrow cells converting into pulmonary epithelial cells after marrow transplantation in irradiated mice. We evaluated the impact of whole bone marrow (WBM) infusion in mice, with or without total body irradiation (TBI), treated with saline or monocrotaline (MCT), which induces pulmonary hypertension (PH). C57BL/6 mice were injected with MCT or saline weekly for 4 weeks. Cohorts were then infused with saline vehicle (vehicle) or WBM from C57BL/-Tg(UBC-GFP)30Scha/J mice, with or without previous TBI (WBM or WBM/TBI). Four weeks later, right ventricular peak pressures (RVPP), right ventricular free wall-to-body weight ratios (RV/BW), and pulmonary vessel wall thickness-to-blood vessel diameter ratios (PVWT/D) were determined. WBM infusion and WBM following TBI induced increases in RVPP and RV/BW in saline-treated mice, while only TBI-exposed mice showed additional increases in PVWT/D. MCT increased RVPP, RV/BW, and PVWT/D in mice given vehicle or WBM alone, but not in mice given WBM/TBI. RVPP and RV/BW were not significantly lower in MCT mice given WBM/TBI than in MCT mice treated with vehicle, but MCT-treated mice given WBM or TBI/WBM had significantly lower PVWT/D compared to MCT-treated mice given saline vehicle. No donor WBM-derived pulmonary vascular cells were detected, suggesting that the observed effects of WBM infusion may be due to paracrine effects separate from cell conversions. The observation of PH after marrow infusion suggests an additional mechanism for lung toxicity seen in marrow transplantation. In conclusion, WBM alone appears to increase RVPP and RV/BW in normal mice but the combination of WBM and TBI attenuates MCT-induced PH.

Fibroblasts play a major role in tissue repair and remodeling. Their differentiation into myofibroblasts, marked by increased expression of smooth muscle-specific alpha-actin (alpha-SMA), is believed to be important in wound healing and fibrosis. We have recently described a role for MK2 in this phenotypic differentiation in culture. In this article, we demonstrate that MK2 also regulates myofibroblasts in vivo. Disruption of MK2 in mice prevented myofibroblast formation in a model of pulmonary fibrosis. However, MK2 disruption and consequent lack of myofibroblast formation exacerbated fibrosis rather than ameliorated it as previously postulated. When mice lacking MK2 (MK2-/-) were exposed to bleomycin, more collagen accumulated and more fibroblasts populated fibrotic regions in their lungs than in similarly treated wild-type mice. While there were many vimentin-positive cells in the bleomycin-treated MK2-/- mouse lungs, few alpha-SMA-positive cells were observed in these lungs compared with wild-type mouse lungs. siRNA against MK2 reduced alpha-SMA expression in wild-type mouse embryonic fibroblasts (MEF), consistent with its suppression in MK2-/- MEF. On the other hand expressing constitutively active MK2 in MK2-/- MEF significantly increased alpha-SMA expression. MK2-/-MEF proliferated at a faster rate and produced more collagen; however, they migrated at a slower rate than wild-type MEF. Overexpressing phosphomimicking HSP27, a target of MK2, did not reverse the effect of MK2 disruption on fibroblast migration. MK2 disruption did not affect Smad2 activation by transforming growth factor-beta. Thus, MK2 appears to mediate myofibroblast differentiation, and inhibiting that differentiation might contribute to fibrosis rather than protect against it.

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 PDGFRbeta through the 5-HTT in pulmonary artery (PA) SMCs. Inhibition of PDGFR kinase with imatinib or AG1296 blocks 5-HT-stimulated PDGFRbeta phosphorylation. 5-HTT inhibitors and the Na+/K+-ATPase inhibitor ouabain, but not 5-HT2 and 5-HT1B/1D receptor inhibitors, block PDGFRbeta activation by 5-HT. Notably, 5-HTT binds the PDGFRbeta upon 5-HT stimulation and the 5-HTT inhibitor fluoxetine blocks both the binding and PDGDRbeta activation. Activation of PDGFRbeta may occur through oxidation of a catalytic cysteine of tyrosine phosphatase. 5-HT-activated PDGFRbeta phosphorylation is blocked by the antioxidant N-acetyl-L-cysteine and the NADPH oxidase inhibitor, DPI. Inhibition of PDGFR kinase with imatinib or AG1296 significantly inhibits SMC proliferation and migration induced by 5-HT in vitro. Infusion of 5-HT by miniosmotic pumps enhances PDGFRbeta activation in mouse lung in vivo. In summary, these results demonstrate that 5-HT transactivates PDGFRbeta in PASMCs leading to SMC proliferation and migration, and may be an important signaling pathway in the production of PH in vivo.

The 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism stimulates cell growth and metastasis of various cancer cells and the 12-LO metabolite, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], enhances proliferation of aortic smooth muscle cells (SMCs). However, pulmonary vascular effects of 12-LO have not been previously studied. We sought evidence for a role of 12-LO and 12(S)-HETE in the development of hypoxia-induced pulmonary hypertension. We found that 12-LO gene and protein expression is elevated in lung homogenates of rats exposed to chronic hypoxia. Immunohistochemical staining with a 12-LO antibody revealed intense staining in endothelial cells of large pulmonary arteries, SMCs (and possibly endothelial cells) of medium and small-size pulmonary arteries and in alveolar walls of hypoxic lungs. 12-LO protein expression was increased in hypoxic cultured rat pulmonary artery SMCs. 12(S)-HETE at concentrations as low as 10(-5) microM stimulated proliferation of pulmonary artery SMCs. 12(S)-HETE induced ERK 1/ERK 2 phosphorylation but had no effect on p38 kinase expression as assessed by Western blotting. 12(S)-HETE-stimulated SMC proliferation was blocked by the MEK inhibitor PD-98059, but not by the p38 MAPK inhibitor SB-202190. Hypoxia (3%)-stimulated pulmonary artery SMC proliferation was blocked by both U0126, a MEK inhibitor, and baicalein, an inhibitor of 12-LO. We conclude that 12-LO and its product, 12(S)-HETE, are important intermediates in hypoxia-induced pulmonary artery SMC proliferation and may participate in hypoxia-induced 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 (r(2) = 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.

Targeted disruption of the gene for natriuretic peptide receptor-A (NPR-A) worsens pulmonary hypertension and right ventricular hypertrophy during hypoxia, but its effect on left ventricular mass and systemic pressures is not known. We examined the effect of 3 wk of hypobaric hypoxia (0.5 atm) on right and left ventricular pressure and mass in mice with 2 (wild type), 1, or 0 copies of Npr1, the gene that encodes for NPR-A in mice. Under normoxic conditions, right ventricular peak pressure (RVPP) was greater in 0 than in 2 copy mice, but there were no genotype-related differences in carotid artery PP (CAPP). The left ventricular free wall weight-to-body weight (LV/body wt) ratio was greater in 0 than in 2 copy mice and there was a trend toward a greater right ventricular weight-to-body weight (RV/body wt) ratio. Three weeks of hypoxia increased RVPP and RV/body wt in all genotypes. The increase in RVPP was similar in all genotypes (11-14 mmHg), but the hypoxia-induced increase in RV/body wt was more than twice as great in 0 copy mice than in 2 copy mice (1.11 +/- 0.06 to 2.65 +/- 0.46 vs. 0.96 +/- 0.04 to 1.4 +/- 0.09, P < 0.05). Chronic hypoxia had no effect on CAPP in any genotype and did not effect LV/body wt in 1 or 2 copy mice, but increased LV/body wt 41% in 0 copy mice. We conclude that absent expression of NPR-A worsens right ventricular hypertrophy and causes left ventricular hypertrophy during exposure to chronic hypoxia without increasing pulmonary or systemic arterial pressure responses.

Intact Madison (M) rats have greater pulmonary pressor responses to acute hypoxia than Hilltop (H) rats. We tested the hypothesis that the difference in pressor response is intrinsic to pulmonary arteries and that endothelium contributes to the difference. Pulmonary arteries precontracted with phenylephrine (10(-7) M) from M rats had greater constrictor responses [hypoxic pulmonary vasoconstriction (HPV)] to acute hypoxia (0% O(2)) than those from H rats: 473 +/- 30 vs. 394 +/- 29 mg (P < 0.05). Removal of the endothelium or inhibition of nitric oxide (NO) synthase by N(omega)-nitro-L-arginine (L-NA, 10(-3) M) significantly blunted HPV in both strains. Inhibition of cyclooxygenase by meclofenamate (10(-5) M) or blockade of endothelin type A and B receptors by BQ-610 (10(-5) M) + BQ-788 (10(-5) M), respectively, had no effect on HPV. Constrictor responses to phenylephrine, endothelin-1, and prostaglandin F(2alpha) were similar in pulmonary arteries from both strains. The relaxation response to ACh, an NO synthase stimulator, was significantly greater in M than in H rats (80 +/- 3 vs. 62 +/- 4%, P < 0.01), but there was no difference in response to sodium nitroprusside, an NO donor. L-NA potentiated phenylephrine-induced contraction to a greater extent in pulmonary arteries from M than from H rats. These findings indicate that at least part of the strain-related difference in acute HPV is attributable to differences in endothelial function, possibly related to differences in NO production.

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.

Atrial and brain natriuretic peptides (ANP and BNP, respectively) are potent pulmonary vasodilators that are upregulated in hypoxia-adapted rats and may protect against hypoxic pulmonary hypertension. To test the hypothesis that C-type natriuretic peptide (CNP) also modulates pulmonary vascular responses to hypoxia, we compared the vasodilator effect of CNP with that of ANP on pulmonary arterial rings, thoracic aortic rings, and isolated perfused lungs obtained from normoxic and hypoxia-adapted rats. We also measured CNP and ANP levels in heart, lung, brain, and plasma in normoxic and hypoxia-adapted rats. Steady-state CNP mRNA levels were quantified in the same organs by relative RT-PCR. CNP was a less potent vasodilator than ANP in preconstricted thoracic aortic and pulmonary arterial rings and in isolated lungs from normoxic and hypoxia-adapted rats. Chronic hypoxia increased plasma CNP (15 +/- 2 vs. 6 +/- 1 pg/ml; P < 0.05) and decreased CNP in the right atrium (35 +/- 14 vs. 65 +/- 17 pg/mg protein; P < 0.05) and in the lung (3 +/- 1 vs. 14 +/- 3 pg/mg protein; P < 0.05) but had no effect on CNP in brain or right ventricle. Chronic hypoxia increased ANP levels fivefold in the right ventricle (49 +/- 5 vs. 11 +/- 2 pg/mg protein; P < 0.05) but had no effect on ANP in lung or brain. There was a trend toward decreased ANP levels in the right atrium (2,009 +/- 323 vs. 2,934 +/- 397 pg/mg protein; P = not significant). No differences in CNP transcript levels were observed between the two groups of rats except that the right atrial CNP mRNA levels were lower in hypoxia-adapted rats. We conclude that CNP is a less potent pulmonary vasodilator than ANP in normoxic and hypoxia-adapted rats and that hypoxia raises circulating CNP levels without increasing cardiopulmonary CNP expression. These findings suggest that CNP may be less important than ANP or BNP in protecting against hypoxic pulmonary hypertension in rats.

Brain natriuretic peptide (BNP) is a pulmonary vasodilator that is elevated in the right heart and plasma of hypoxia-adapted rats. To test the hypothesis that BNP protects against hypoxic pulmonary hypertension, we measured right ventricular systolic pressure (RVSP), right ventricle (RV) weight-to-body weight (BW) ratio (RV/BW), and percent muscularization of peripheral pulmonary vessels (%MPPV) in rats given an intravenous infusion of BNP, atrial natriuretic peptide (ANP), or saline alone after 2 wk of normoxia or hypobaric hypoxia (0.5 atm). Hypoxia-adapted rats had higher hematocrits, RVSP, RV/BW, and %MPPV than did normoxic controls. Under normoxic conditions, BNP infusion (0.2 and 1.4 micro g/h) increased plasma BNP but had no effect on RVSP, RV/BW, or %MPPV. Under hypoxic conditions, low-rate BNP infusion (0.2 micro g/h) had no effect on plasma BNP or on severity of pulmonary hypertension. However, high-rate BNP infusion (1.4 micro g/h) increased plasma BNP (69 +/- 8 vs. 35 +/- 4 pg/ml, P < 0.05), lowered RV/BW (0.87 +/- 0.05 vs. 1.02 +/- 0.04, P < 0.05), and decreased %MPPV (60 vs. 74%, P < 0.05). There was also a trend toward lower RVSP (55 +/- 3 vs. 64 +/- 2, P = not significant). Infusion of ANP at 1.4 micro g/h increased plasma ANP in hypoxic rats (759 +/- 153 vs. 393 +/- 54 pg/ml, P < 0.05) but had no effect on RVSP, RV/BW, or %MPPV. We conclude that BNP may regulate pulmonary vascular responses to hypoxia and, at the doses used in this study, is more effective than ANP at blunting pulmonary hypertension during the first 2 wk of hypoxia.

Endothelin-1 (ET-1), a potent vasoactive and mitogenic peptide, has been implicated in the pathogenesis of several forms of pulmonary hypertension. We hypothesized that nonspecific blockade of ET receptors would blunt the development of monocrotaline (MCT)-induced pulmonary hypertension in rats. A single dose of the nonspecific ET blocker bosentan (100 mg/kg) given to intact rats by gavage completely blocked the pulmonary vasoconstrictor actions of Big ET-1 and partially blunted hypoxic pulmonary vasoconstriction. After 3 wk, MCT-injected (105 mg/kg sc) rats gavaged once daily with bosentan (200 mg/kg) had lower right ventricular (RV) systolic pressure (RVSP), RV-to-body weight (RV/BW) and RV-to-left ventricular (LV) plus septal (S) weight [RV/(LV+S)] ratios and less percent medial thickness of small pulmonary arteries than control MCT-injected rats. Lower dose bosentan (100 mg/kg) had no effect on these parameters after MCT or saline injection. Bosentan raised plasma ET-1 levels but had no effect on lung ET-1 levels. Bosentan (200 mg/kg) also had no effect on wet-to-dry lung weight ratios 6 days after MCT injection. When given during the last 10 days, but not the first 11 days of a 3-wk period after MCT injection, bosentan reduced RV/(LV+S) compared with MCT-injected controls. We conclude that ET-1 contributes to the pathogenesis of MCT-induced pulmonary hypertension and acts mainly during the later inflammatory rather than the acute injury phase after injection.

Mechanisms that regulate atrial natriuretic peptide (ANP) expression during hypoxia are not well defined. We hypothesized that plasma immunoreactive ANP (irANP) and right heart irANP and ANP mRNA levels would be greater in a strain of Sprague-Dawley rats that develops more severe hypoxic pulmonary hypertension (H rats) than another strain (M rats). After 3 wk of hypoxia (0.5 atm), right ventricular systolic pressure (RVSP) and the right ventricle (RV) weight-to-left ventricle plus septum (LV (+) S) weight ratio [RV/(LV+S)] were greater in H rats than in M rats (70 +/- 4 vs. 40 +/- 2 mmHg and 0.59 +/- 0.02 vs. 0.50 +/- 0.02, respectively; P < 0.05 for both), but plasma ANP increased twofold and RV irANP and ANP mRNA increased fivefold in both rat strains. After 3 days of normoxic recovery from chronic hypoxia, RVSP, RV/(LV+S), and RV irANP and ANP mRNA levels decreased in M rats but not in H rats. Plasma irANP decreased to baseline levels in both rat strains. We conclude that, in addition to changes in RV pressure and hypertrophy, hypoxia acts through other mechanisms to modulate RV ANP synthesis and circulating ANP levels in hypoxia-adapted rats.

We previously found that augmentation of polycythemia by exogenous human recombinant erythropoietin (EPO) failed to worsen the severity of hypoxic pulmonary hypertension in rats. We asked whether this unexpected finding was related to reductions in cardiac output, left ventricular end-diastolic pressure, pulmonary vascular resistance, or some combination of these factors. Four groups of Sprague-Dawley rats were studied over a 3-wk period: hypoxic (0.5 ATM) and normoxic animals each injected with EPO (500 U/kg sc thrice weekly) or saline (control animals). As observed previously, we found that pulmonary arterial (PA) pressures and right ventricular hypertrophy were not increased in EPO-treated rats despite significant increases in hematocrit and blood viscosity. Cardiac outputs, blood volumes, and left ventricular end-diastolic pressures were similar in EPO-treated and control rats. Acute PA pressure responses to acute normoxia in hypoxic rats and to acute hypoxia in normoxic rats were similar, suggesting no differences in vasoreactivity. However, lungs isolated from EPO-treated hypoxic rats had lower pulmonary vascular resistance than saline-treated hypoxic rats when perfused with blood from normocythemic donor rats. PA medial thickness and the percentage of muscularized small PAs were significantly lower in EPO-treated hypoxic rats. These results indicate that augmented polycythemia fails to worsen hypoxic pulmonary hypertension in rats because of a decrease in the severity of structural remodeling.

We hypothesized that a downregulation in pulmonary atrial natriuretic peptide (ANP) receptors helps raise plasma ANP levels during chronic hypoxia. We measured in vivo pulmonary uptake and plasma clearance of 125I-ANP and in vitro pulmonary binding kinetics of 125I-ANP in normoxic and chronically hypoxic rats. Exposure to 21 days of hypobaric (0.5 atm) hypoxia did not decrease specific binding of 125I-ANP in the kidney, but pulmonary binding decreased 35 and 75% after 1 and 3 days of hypoxia, respectively, and increased 200% after 3 days of normoxic recovery from 21 days of hypoxia. The total binding capacity for ANP to lung membrane fractions from normoxic rats, chronically hypoxic rats, and rats that had recovered from hypoxia was 488 +/- 59, 109 +/- 17, and 338 +/- 48 fmol/mg, respectively (P < 0.05 for hypoxic vs. normoxic or recovered lung membranes). The area under the 125I-ANP plasma concentration curve for normoxic and hypoxic rats and normoxic rats that were infused with the ANP C-receptor ligand C-ANF-(4-23) was 3,292 +/- 216, 5,022 +/- 466, and 8,205 +/- 1,059 disintegrations.min-1.ml-1, respectively [P < 0.05 for hypoxic vs. normoxic or C-ANF-(4-23)-infused rats]. We conclude that pulmonary ANP clearance is reduced during chronic hypoxia secondary to a downregulation in pulmonary ANP clearance receptors. Reduced pulmonary clearance of ANP may represent an adaptation that contributes to increased plasma ANP levels during chronic hypoxia.

To test the hypothesis that brain natriuretic peptide (BNP) plays a role similar to that of atrial natriuretic peptide (ANP) in modulating pulmonary vascular responses to hypoxia, we measured the vasodilator potency of ANP and BNP in rat pulmonary artery (PA) and thoracic aorta (TA) rings and in isolated rat lungs. We also measured the effect of chronic hypoxia on plasma levels and cardiac gene expression of both peptides. BNP had a vasorelaxant effect equipotent to that of ANP on preconstricted TA and PA rings, but was less potent than ANP in relaxing the vasoconstrictor response to hypoxia in isolated lungs [mean 50% inhibitory concentration (IC50) 10(-7) vs. 10(-6) M for ANP and BNP, respectively]. Plasma BNP levels were 30-fold lower than ANP, but both peptides increased approximately 70% during chronic hypoxia. In the right atrium, hypoxia lowered BNP mRNA slightly, but had no effect on ANP mRNA or tissue levels of either peptide. However, hypoxia increased right ventricular content and mRNA levels of both peptides by three- to fourfold. We conclude that BNP and ANP have similar pulmonary vasodilator effects and are upregulated proportionally during chronic hypoxia. These results support a role for BNP in modulating the pulmonary hypertensive response to chronic hypoxia.

Neutral endopeptidase (NEP) inhibition is thought to blunt hypoxic pulmonary hypertension by reducing atrial natriuretic peptide (ANP) metabolism, but this hypothesis has not been confirmed. We measured NEP activity, guanosine 3',5'-cyclic monophosphate (cGMP) production, plasma ANP levels, and cardiac ANP synthesis in rats given an orally active NEP inhibitor (SCH-34826) during 3 wk of hypoxia. Under normoxic conditions, SCH-34826 had no effect on plasma ANP levels but reduced pulmonary and renal NEP activity by 50% and increased urinary cGMP levels (60 +/- 6 vs. 22 +/- 4 pg/mg creatinine; P < 0.05). Under hypoxic conditions, SCH-34826-treated rats had lower plasma ANP levels (1,259 +/- 361 vs. 2,101 +/- 278 pg/ml; P < 0.05), lower right ventricular systolic pressure (53 +/- 5 vs. 73 +/- 2 mmHg; P < 0.05), lower right ventricle weight-to-left ventricle+septum weight ratio (0.47 +/- 0.04 vs. 0.53 +/- 0.03; P < 0.05), and less muscularization and percent medial wall thickness of peripheral pulmonary arteries (22 +/- 5 vs. 45 +/- 8% and 17 +/- 1 vs. 25 +/- 1%, respectively; P < 0.05 for all values) than did rats treated with vehicle alone. These values were not affected by SCH-34826 under normoxic conditions. SCH-34826 decreased right ventricular ANP tissue levels in hypoxic rats (27 +/- 10 vs. 8 +/- 1 ng/mg protein; P < 0.05) but did not affect steady-state ANP mRNA levels. We conclude that NEP inhibition blunts pulmonary hypertension without increasing plasma ANP levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated plasma atrial natriuretic peptide (ANP) levels have been shown to blunt pulmonary hemodynamic responses to chronic hypoxia, but whether elevated circulating ANP levels negatively feedback on cardiac expression of the ANP gene is unknown. Using a recently developed strain of transgenic mouse (TTR-ANF) that expresses a transthyretin promoter-ANP fusion gene in the liver, we studied the effect of chronically elevated plasma ANP levels on cardiac hypertrophic and pulmonary hemodynamic responses and expression of the endogenous cardiac ANP gene during chronic hypoxia. Plasma ANP levels were 10-fold higher in TTR-ANF mice than in their non-transgenic littermates. After 3 wk of hypobaric hypoxia (0.5 atm), right ventricular hypertrophy and pulmonary hypertension had developed in both groups of mice, but TTR-ANF mice had lower right ventricle-to-left ventricle plus septum weight ratios (0.39 +/- 0.01 vs. 0.45 +/- 0.02), right ventricular systolic pressures (25 +/- 2 vs. 29 +/- 2 mmHg), and lung dry weight-to-body weight ratios (0.48 +/- 0.03 vs. 0.57 +/- 0.01 mg/g) and less muscularization of peripheral pulmonary vessels (8.3 +/- 1.4 vs. 17.4 +/- 2.5%) than nontransgenic controls. Right atrial and ventricular steady-state ANP mRNA levels were the same in both groups of mice under normoxic and hypoxic conditions despite much higher plasma ANP levels and less pulmonary hypertension in TTR-ANF mice. We conclude that chronically elevated plasma ANP levels attenuate the development of hypoxic pulmonary hypertension in mice but do not suppress cardiac expression of the endogenous ANP gene under normoxic conditions nor blunt the upregulation of right ventricular ANP expression during chronic hypoxia.

In two rat strains (H and M) with differing susceptibilities to chronic hypoxia we examined the role of polycythemia in the differing hypoxic pulmonary hemodynamic responses. We hypothesized that augmentation of hematocrit (Hct) during hypoxia in the resistant M strain would render cardiopulmonary responses similar to those obtained in the susceptible H strain. Administration of human recombinant erythropoietin (EPO) in doses of 100, 250 and 500 U.kg-1 s.c. thrice weekly for three weeks raised Hct similarly in both strains indicating that normoxic rats had similar sensitivities to EPO. In rats exposed to hypobaric hypoxia (0.5 atm) for 21 days, EPO (500 U.kg-1 thrice weekly) significantly increased Hct and whole blood viscosity as expected. Surprisingly, right ventricular (RV) to body weight (BW) ratio as an index of right ventricular hypertrophy (RVH) and RV peak systolic pressure did not increase in EPO-injected rats of either strain compared to hypoxic controls. Among hypoxic animals, Hct correlated highly with viscosity but not with RV/BW. We conclude, contrary to our hypothesis, that polycythemia does not appear to be responsible for the strain difference in RVH and pulmonary hypertension.

We have previously described the development of greater right ventricular hypertrophy after 7 days of hypoxia in the altitude-susceptible H strain compared to the resistant M strain of Sprague-Dawley rat. Greater polycythemia also occurs in the H strain after 2-3 weeks of hypoxia and is characterized by increased mean red cell volume (MCV), reticulocyte count (Retic), and blood viscosity after 4 weeks of hypoxia. In the present study, we determined the time course of development of these hematologic responses, whether differences in MCV are associated with differences in red cell deformability, and whether the hematologic differences might contribute to the early cardiopulmonary differences between the strains. We found that although hematocrit (Hct) did not differ between the strains until 21 days of hypoxia, MCV and Retic were greater in the H strain after only 3 days and whole blood viscosity was greater after 7 days. However, no differences in the viscosity or deformability of reconstituted red cells (Hcts 10% and 25%) were apparent at any time during hypoxic exposure. Furthermore, pressure-flow curves obtained using blood and lungs isolated from 7-day hypoxic rats suggested that the largest component of pressure elevation in the H rats was related to pulmonary vascular rather than hematologic factors. We conclude that although H rats have exaggerated hematologic responses to hypoxia, differences in pulmonary vascular structure and tone are more likely to be responsible for the strain differences in cardiopulmonary responses occurring after 7 days of hypoxia.

Pulmonary clearance of atrial natriuretic peptide (ANP) was measured by indicator dilution technique in isolated perfused rat lungs with and without ANP clearance receptor (C-receptor) blockade. Approximately 50% of a bolus injection of 125I-ANP was removed during a single pass through the lungs compared with the intravascular marker 14C-dextran. Pulmonary clearance of 125I-ANP was suppressed in a dose-dependent fashion by unlabeled ANP. C-receptor blockade suppressed pulmonary clearance of 125I-ANP to the same degree as unlabeled ANP. High-performance liquid chromatography analysis of the pulmonary venous effluent from lungs treated with C-receptor ligand demonstrated intact 125I-ANP. We conclude that virtually all of the pulmonary vascular uptake of 125I-ANP during a single pass through isolated lungs is secondary to removal by ANP C-receptors.