Endothelial actions of atrial natriuretic peptide prevent pulmonary hypertension in mice.

The cardiac hormone atrial natriuretic peptide (ANP) regulates systemic and pulmonary arterial blood pressure by activation of its cyclic GMP-producing guanylyl cyclase-A (GC-A) receptor. In the lung, these hypotensive effects were mainly attributed to smooth muscle-mediated vasodilatation. It is unknown whether pulmonary endothelial cells participate in the homeostatic actions of ANP. Therefore, we analyzed GC-A/cGMP signalling in lung endothelial cells and the cause and functional impact of lung endothelial GC-A dysfunction. Western blot and cGMP determinations showed that cultured human and murine pulmonary endothelial cells exhibit prominent GC-A expression and activity which were markedly blunted by hypoxia, a condition known to trigger pulmonary hypertension (PH). To elucidate the consequences of impaired endothelial ANP signalling, we studied mice with genetic endothelial cell-restricted ablation of the GC-A receptor (EC GC-A KO). Notably, EC GC-A KO mice exhibit PH already under resting, normoxic conditions, with enhanced muscularization of small arteries and perivascular infiltration of inflammatory cells. These alterations were aggravated on exposure of mice to chronic hypoxia. Lung endothelial GC-A dysfunction was associated with enhanced expression of angiotensin converting enzyme (ACE) and increased pulmonary levels of Angiotensin II. Angiotensin II/AT1-blockade with losartan reversed pulmonary vascular remodelling and perivascular inflammation of EC GC-A KO mice, and prevented their increment by chronic hypoxia. This experimental study indicates that endothelial effects of ANP are critical to prevent pulmonary vascular remodelling and PH. Chronic endothelial ANP/GC-A dysfunction, e.g. provoked by hypoxia, is associated with activation of the ACE-angiotensin pathway in the lung and PH.

Mast cells and fibroblasts work in concert to aggravate pulmonary fibrosis: role of transmembrane SCF and the PAR-2/PKC-α/Raf-1/p44/42 signaling pathway.

Mast cell (MC) accumulation has been demonstrated in the lungs of idiopathic pulmonary fibrosis (IPF) patients. Mediators released from MCs may regulate tissue remodeling processes, thereby contributing to IPF pathogenesis. We investigated the role of MC-fibroblast interaction in the progression of lung fibrosis. Increased numbers of activated MCs, in close proximity to fibroblast foci and alveolar type II cells, were observed in IPF lungs. Correspondingly elevated tryptase levels were detected in IPF lung tissue samples. Coculture of human lung MCs with human lung fibroblasts (HLFs) induced MC activation, as evinced by tryptase release, and stimulated HLF proliferation; IPF HLFs exhibited a significantly higher growth rate, compared with control. Tryptase stimulated HLF growth in a PAR-2/PKC-α/Raf-1/p44/42-dependent manner and potentiated extracellular matrix production, but independent of PKC-α, Raf-1, and p44/42 activities. Proproliferative properties of tryptase were attenuated by knockdown or pharmacological inhibition of PAR-2, PKC-α, Raf-1, or p44/42. Expression of transmembrane SCF, but not soluble SCF, was elevated in IPF lung tissue and in fibroblasts isolated from IPF lungs. Coculture of IPF HLFs with MCs enhanced MC survival and proliferation. These effects were cell-contact dependent and could be inhibited by application of anti-SCF antibody or CD117 inhibitor. Thus, fibroblasts and MCs appear to work in concert to perpetuate fibrotic processes and so contribute to lung fibrosis progression.

Effects of multikinase inhibitors on pressure overload-induced right ventricular remodeling.

BACKGROUND: Little is known about the effects of current PAH therapies and receptor tyrosine kinase inhibitors on heart remodeling. We sought to investigate the effects of the multikinase inhibitors sunitinib (PDGFR-, VEGFR- and KIT-inhibitor) and sorafenib (raf1/b-, VEGFR-, PDGFR-inhibitor) on pressure overload induced right ventricular (RV) remodeling. METHODS: We investigated the effects of the kinase inhibitors on hemodynamics and remodeling in rats subjected either to monocrotaline (MCT)-induced PH or to surgical pulmonary artery banding (PAB). MCT rats were treated from days 21 to 35 with either vehicle, sunitinib (1mg/kg, 5mg/kg and 10mg/kg/day) or sorafenib (10mg/kg/day). PAB rats were treated with vehicle, sunitinib (10mg/kg/day) or sorafenib (10mg/kg/day) from days 7 to 21. RV function and remodeling were determined using echocardiography, invasive hemodynamic measurement and histomorphometry. RESULTS: Treatment with both sorafenib and sunitinib decreased right ventricular systolic pressure, pulmonary vascular remodeling, RV hypertrophy and fibrosis in MCT rats. This was associated with an improvement of RV function. Importantly, after PAB, both compounds reversed RV chamber and cellular hypertrophy, reduced RV interstitial and perivascular fibrosis, and improved RV function. CONCLUSION: We demonstrated that sunitinib and sorafenib reversed RV remodeling and significantly improved RV function measured via a range of invasive and non-invasive cardiopulmonary endpoints in experimental models of RV hypertrophy.

The peroxisome proliferator-activated receptor ß/δ agonist GW0742 has direct protective effects on right heart hypertrophy.

Pulmonary hypertension is a debilitating disease with no cure. We have previously shown that peroxisome proliferator-activated receptor (PPAR) ß/δ agonists protect the right heart in hypoxia-driven pulmonary hypertension without affecting vascular remodeling. PPARß/δ is an important receptor in lipid metabolism, athletic performance, and the sensing of prostacyclin. Treatment of right heart hypertrophy and failure in pulmonary hypertension is an emerging target for future therapy. Here we have investigated the potential of GW0742, a PPARß agonist, to act directly on the right heart in vivo and what transcriptomic signatures are associated with its actions. Right heart hypertrophy and failure was induced in mice using a pulmonary artery banding (PAB) model. GW0742 was administered throughout the study. Cardiovascular parameters were measured using echocardiography and pressure monitoring. Fibrosis and cellular changes were measured using immunohistochemistry. Transcriptomics were measured using the Illumina MouseRef-8v3 BeadChip array and analyzed using GeneSpring GX (ver. 11.0). PAB resulted in right heart hypertrophy and failure and in increased fibrosis. GW0742 reduced or prevented the effects of PAB on all parameters measured. GW0742 altered a number of genes in the transcriptome, with Angptl4 emerging as the top gene altered (increased) in animals with PAB. In conclusion, the PPARß/δ agonist GW0742 has direct protective effects on the right heart in vivo. These observations identify PPARß/δ as a viable therapeutic target to treat pulmonary hypertension that may complement current and future vasodilator drugs.

Immune and inflammatory cell involvement in the pathology of idiopathic pulmonary arterial hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is characterized by vasoconstriction and vascular remodeling. Recent studies have revealed that immune and inflammatory responses play a crucial role in pathogenesis of idiopathic PAH. OBJECTIVES: To systematically evaluate the number and cross-sectional distribution of inflammatory cells in different sizes of pulmonary arteries from explanted lungs of patients with idiopathic PAH versus healthy donor lungs and to demonstrate functional relevance by blocking stromal-derived factor-1 by the Spiegelmer NOX-A12 in monocrotaline-induced pulmonary hypertension in rats. METHODS: Immunohistochemistry was performed on lung tissue sections from patients with idiopathic PAH and healthy donors. All positively stained cells in whole-lung tissue sections, surrounding the vessels, and in the different compartments of the vessels were counted. To study the effects of blocking SDF-1, rats with monocrotaline-induced pulmonary hypertension were treated with NOX-A12 from Day 21 to Day 35 after monocrotaline administration. MEASUREMENTS AND MAIN RESULTS: We found a significant increase of the perivascular number of macrophages (CD68(+)), macrophages/monocytes (CD14(+)), mast cells (toluidine blue(+)), dendritic cells (CD209(+)), T cells (CD3(+)), cytotoxic T cells (CD8(+)), and helper T cells (CD4(+)) in vessels of idiopathic PAH lungs compared with control subjects. FoxP3(+) mononuclear cells were significantly decreased. In the monocrotaline model, the NOX-A12-induced reduction of mast cells, CD68(+) macrophages, and CD3(+) T cells was associated with improvement of hemodynamics and pulmonary vascular remodeling. CONCLUSIONS: Our findings reveal altered perivascular inflammatory cell infiltration in pulmonary vascular lesions of patients with idiopathic pulmonary arterial hypertension. Targeting attraction of inflammatory cells by blocking stromal-derived factor-1 may be a novel approach for treatment of PAH.

Inhibition of microRNA-17 improves lung and heart function in experimental pulmonary hypertension.

RATIONALE: MicroRNAs (miRs) control various cellular processes in tissue homeostasis and disease by regulating gene expression on the posttranscriptional level. Recently, it was demonstrated that the expression of miR-21 and members of the miR-17-92 cluster was significantly altered in experimental pulmonary hypertension (PH). OBJECTIVES: To evaluate the therapeutic efficacy and antiremodeling potential of miR inhibitors in the pathogenesis of PH. METHODS: We first tested the effects of miR inhibitors (antagomirs), which were specifically designed to block miR-17 (A-17), miR-21 (A-21), and miR-92a (A-92a) in chronic hypoxia-induced PH in mice and A-17 in monocrotaline-induced PH in rats. Moreover, biological function of miR-17 was analyzed in cultured pulmonary artery smooth muscle cells. MEASUREMENTS AND MAIN RESULTS: In the PH mouse model, A-17 and A-21 reduced right ventricular systolic pressure, and all antagomirs decreased pulmonary arterial muscularization. However, only A-17 reduced hypoxia-induced right ventricular hypertrophy and improved pulmonary artery acceleration time. In the monocrotaline-induced PH rat model, A-17 treatment significantly decreased right ventricular systolic pressure and total pulmonary vascular resistance index, increased pulmonary artery acceleration time, normalized cardiac output, and decreased pulmonary vascular remodeling. Among the tested miR-17 targets, the cyclin-dependent kinase inhibitor 1A (p21) was up-regulated in lungs undergoing A-17 treatment. Likewise, in human pulmonary artery smooth muscle cells, A-17 increased p21. Overexpression of miR-17 significantly reduced p21 expression and increased proliferation of smooth muscle cells. CONCLUSIONS: Our data demonstrate that A-17 improves heart and lung function in experimental PH by interfering with lung vascular and right ventricular remodeling. The beneficial effects may be related to the up-regulation of p21. Thus, inhibition of miR-17 may represent a novel therapeutic concept to ameliorate disease state in PH.

Hypoxic pulmonary hypertension in mice with constitutively active platelet-derived growth factor receptor-ß.

Platelet-derived growth factor (PDGF) has been implicated in the pathobiology of vascular remodeling. The multikinase inhibitor imatinib that targets PDGF receptor (PDGFR), c-kit and Abl kinases, shows therapeutic efficacy against experimental pulmonary hypertension (PH); however, the role of PDGFR-b in experimental PH has not been examined by genetic approach. We investigated the chronic hypoxia-induced PH in mice carrying an activating point mutation of PDGFR-ß (D849N) and evaluated the therapeutic efficacy of imatinib. In addition, we studied pulmonary global gene expression and confirmed the expression of identified genes by immunohistochemistry. Chronically hypoxic D849N mice developed PH and strong pulmonary vascular remodeling that was improved by imatinib (100 mg/kg/day) as evident from the significantly reduced right ventricular systolic pressure, right ventricular hypertrophy and muscularization of peripheral pulmonary arteries. Global gene expression analysis revealed that stromal cell derived factor SDF)-1α was significantly upregulated, which was confirmed by immunohistochemistry. Moreover, an enhanced immunoreactivity for SDF-1α, PDGFR-ß and CXCR4, the receptor for SDF-1α was localized to the α-smooth muscle cell (SMC) actin positive pulmonary vascular cells in hypoxic mice and patients with idiopathic pulmonary arterial hypertension (IPAH). In conclusion, our findings substantiate the major role of PDGFR activation in pulmonary vascular remodeling by a genetic approach. Immunohistochemistry findings suggest a role for SDF-1α/CXCR4 axis in pulmonary vascular remodeling and point to a potential interaction between the chemokine SDF-1 and the growth factor PDGF signaling. Future studies designed to elucidate an interaction between the chemokine SDF-1 and the PDGF system may uncover novel therapeutic targets.

Therapeutic efficacy of TBC3711 in monocrotaline-induced pulmonary hypertension.

BACKGROUND: Endothelin-1 signalling plays an important role in pathogenesis of pulmonary hypertension. Although different endothelin-A receptor antagonists are developed, a novel therapeutic option to cure the disease is still needed. This study aims to investigate the therapeutic efficacy of the selective endothelin-A receptor antagonist TBC3711 in monocrotaline-induced pulmonary hypertension in rats. METHODS: Monocrotaline-injected male Sprague-Dawley rats were randomized and treated orally from day 21 to 35 either with TBC3711 (Dose: 30 mg/kg body weight/day) or placebo. Echocardiographic measurements of different hemodynamic and right-heart hypertrophy parameters were performed. After day 35, rats were sacrificed for invasive hemodynamic and right-heart hypertrophy measurements. Additionally, histologic assessment of pulmonary vascular and right-heart remodelling was performed. RESULTS: The novel endothelin-A receptor antagonist TBC3711 significantly attenuated monocrotaline-induced pulmonary hypertension, as evident from improved hemodynamics and right-heart hypertrophy in comparison with placebo group. In addition, muscularization and medial wall thickness of distal pulmonary vessels were ameliorated. The histologic evaluation of the right ventricle showed a significant reduction in fibrosis and cardiomyocyte size, suggesting an improvement in right-heart remodelling. CONCLUSION: The results of this study suggest that the selective endothelin-A receptor antagonist TBC3711 demonstrates therapeutic benefit in rats with established pulmonary hypertension, thus representing a useful therapeutic approach for treatment of pulmonary hypertension.

Involvement of mast cells in monocrotaline-induced pulmonary hypertension in rats.

BACKGROUND: Mast cells (MCs) are implicated in inflammation and tissue remodeling. Accumulation of lung MCs is described in pulmonary hypertension (PH); however, whether MC degranulation and c-kit, a tyrosine kinase receptor critically involved in MC biology, contribute to the pathogenesis and progression of PH has not been fully explored. METHODS: Pulmonary MCs of idiopathic pulmonary arterial hypertension (IPAH) patients and monocrotaline-injected rats (MCT-rats) were examined by histochemistry and morphometry. Effects of the specific c-kit inhibitor PLX and MC stabilizer cromolyn sodium salt (CSS) were investigated in MCT-rats both by the preventive and therapeutic approaches. Hemodynamic and right ventricular hypertrophy measurements, pulmonary vascular morphometry and analysis of pulmonary MC localization/counts/activation were performed in animal model studies. RESULTS: There was a prevalence of pulmonary MCs in IPAH patients and MCT-rats as compared to the donors and healthy rats, respectively. Notably, the perivascular MCs were increased and a majority of them were degranulated in lungs of IPAH patients and MCT-rats (p < 0.05 versus donor and control, respectively). In MCT-rats, the pharmacological inhibitions of MC degranulation and c-kit with CSS and PLX, respectively by a preventive approach (treatment from day 1 to 21 of MCT-injection) significantly attenuated right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH). Moreover, vascular remodeling, as evident from the significantly decreased muscularization and medial wall thickness of distal pulmonary vessels, was improved. However, treatments with CSS and PLX by a therapeutic approach (from day 21 to 35 of MCT-injection) neither improved hemodynamics and RVH nor vascular remodeling. CONCLUSIONS: The accumulation and activation of perivascular MCs in the lungs are the histopathological features present in clinical (IPAH patients) and experimental (MCT-rats) PH. Moreover, the accumulation and activation of MCs in the lungs contribute to the development of PH in MCT-rats. Our findings reveal an important pathophysiological insight into the role of MCs in the pathogenesis of PH in MCT-rats.

Hypoxia enhances platelet-derived growth factor signaling in the pulmonary vasculature by down-regulation of protein tyrosine phosphatases.

RATIONALE: Platelet-derived growth factor (PDGF) plays a pivotal role in the pathobiology of pulmonary hypertension (PH) because it promotes pulmonary vascular remodeling. PH is frequently associated with pulmonary hypoxia. OBJECTIVES: To investigate whether hypoxia alters PDGF ß receptor (ßPDGFR) signaling in the pulmonary vasculature. METHODS: The impact of chronic hypoxia on signal transduction by the ßPDGFR was measured in human pulmonary arterial smooth muscle cells (hPASMC) in vitro, and in mice with hypoxia-induced PH in vivo. MEASUREMENTS AND MAIN RESULTS: Chronic hypoxia significantly enhanced PDGF-BB-dependent proliferation and chemotaxis of hPASMC. Pharmacologic inhibition of PI3 kinase (PI3K) and PLCγ abrogated these events under both normoxia and hypoxia. Although hypoxia did not affect ßPDGFR expression, it increased the ligand-induced tyrosine phosphorylation of the receptor, particularly at binding sites for PI3K (Y751) and PLCγ (Y1021). The activated ßPDGFR is dephosphorylated by protein tyrosine phosphatases (PTPs). Interestingly, hypoxia decreased expression of numerous PTPs (T cell PTP, density-enhanced phosphatase-1, PTP1B, and SH2 domain-containing phosphatase-2), resulting in reduced PTP activity. Hypoxia-inducible factor (HIF)-1α is involved in this regulation of gene expression, because hypoxia-induced ßPDGFR hyperphosphorylation and PTP down-regulation were abolished by HIF-1α siRNA and by the HIF-1α inhibitor 2-methoxyestradiol. ßPDGFR hyperphosphorylation and PTP down-regulation were also present in vivo in mice with chronic hypoxia-induced PH. CONCLUSIONS: Hypoxia reduces expression and activity of ßPDGFR-antagonizing PTPs in a HIF-1α-dependent manner, thereby enhancing receptor activation and proliferation and chemotaxis of hPASMC. Because hyperphosphorylation of the ßPDGFR and down-regulation of PTPs occur in vivo, this mechanism likely has significant impact on the development and progression of PH and other hypoxia-associated diseases.

Nebulization of the acidified sodium nitrite formulation attenuates acute hypoxic pulmonary vasoconstriction.

BACKGROUND: Generalized hypoxic pulmonary vasoconstriction (HPV) occurring during exposure to hypoxia is a detrimental process resulting in an increase in lung vascular resistance. Nebulization of sodium nitrite has been shown to inhibit HPV. The aim of this project was to investigate and compare the effects of nebulization of nitrite and different formulations of acidified sodium nitrite on acute HPV. METHODS: Ex vivo isolated rabbit lungs perfused with erythrocytes in Krebs-Henseleit buffer (adjusted to 10% hematocrit) and in vivo anesthetized catheterized rabbits were challenged with periods of hypoxic ventilation alternating with periods of normoxic ventilation. After baseline hypoxic challenges, vehicle, sodium nitrite or acidified sodium nitrite was delivered via nebulization. In the ex vivo model, pulmonary arterial pressure and nitric oxide concentrations in exhaled gas were monitored. Nitrite and nitrite/nitrate were measured in samples of perfusion buffer. Pulmonary arterial pressure, systemic arterial pressure, cardiac output and blood gases were monitored in the in vivo model. RESULTS: In the ex vivo model, nitrite nebulization attenuated HPV and increased nitric oxide concentrations in exhaled gas and nitrite concentrations in the perfusate. The acidified forms of sodium nitrite induced higher levels of nitric oxide in exhaled gas and had longer vasodilating effects compared to nitrite alone. All nitrite formulations increased concentrations of circulating nitrite to the same degree. In the in vivo model, inhaled nitrite inhibited HPV, while pulmonary arterial pressure, cardiac output and blood gases were not affected. All nitrite formulations had similar potency to inhibit HPV. The tested concentration of appeared tolerable. CONCLUSION: Nitrite alone and in acidified forms effectively and similarly attenuates HPV. However, acidified nitrite formulations induce a more pronounced increase in nitric oxide exhalation.

Macrophage galactose-type C-type lectins as novel markers for alternatively activated macrophages elicited by parasitic infections and allergic airway inflammation.

Molecular markers, especially surface markers associated with type II, cytokine-dependent, alternatively activated macrophages (aaMF), remain scarce. Besides the earlier documented markers, macrophage mannose receptor and arginase 1, we demonstrated recently that murine aaMF are characterized by increased expression of found in inflammatory zone 1 (FIZZ1) and the secretory lectin Ym. We now document that expression of the two members of the mouse macrophage galactose-type C-type lectin gene family (mMGL1 and mMGL2) is induced in diverse populations of aaMF, including peritoneal macrophages elicited during infection with the protozoan Trypanosoma brucei brucei or the Helminth Taenia crassiceps and alveolar macrophages elicited in a mouse model of allergic asthma. In addition, we demonstrate that in vitro, interleukin-4 (IL-4) and IL-13 up-regulate mMGL1 and mMGL2 expression and that in vivo, induction of mMGL1 and mMGL2 is dependent on IL-4 receptor signaling. Moreover, we show that expression of MGL on human monocytes is also up-regulated by IL-4. Hence, macrophage galactose-type C-type lectins represent novel surface markers for murine and human aaMF.