Cystamine Treatment Fails to Prevent the Development of Pulmonary Hypertension in Chronic Hypoxic Rats.

INTRODUCTION: Pulmonary hypertension is characterized by vasoconstriction and remodeling of pulmonary arteries, leading to right ventricular hypertrophy and failure. We have previously found upregulation of transglutaminase 2 (TG2) in the right ventricle of chronic hypoxic rats. The hypothesis of the present study was that treatment with the transglutaminase inhibitor, cystamine, would inhibit the development of pulmonary arterial remodeling, pulmonary hypertension, and right ventricular hypertrophy. METHODS: Effect of cystamine on transamidase activity was investigated in tissue homogenates. Wistar rats were exposed to chronic hypoxia and treated with vehicle, cystamine (40 mg/kg/day in mini-osmotic pumps), sildenafil (25 mg/kg/day), or the combination for 2 weeks. RESULTS: Cystamine concentration-dependently inhibited TG2 transamidase activity in liver and lung homogenates. In contrast to cystamine, sildenafil reduced right ventricular systolic pressure and hypertrophy and decreased pulmonary vascular resistance and muscularization in chronic hypoxic rats. Fibrosis in the lung tissue decreased in chronic hypoxic rats treated with cystamine. TG2 expression was similar in the right ventricle and lung tissue of drug and vehicle-treated hypoxic rats. DISCUSSION/CONCLUSIONS: Cystamine inhibited TG2 transamidase activity, but cystamine failed to prevent pulmonary hypertension, right ventricular hypertrophy, and pulmonary arterial muscularization in the chronic hypoxic rat.

Soluble guanylate cyclase stimulator, trans-4-methoxy-ß-nitrostyrene, has a beneficial effect in monocrotaline-induced pulmonary arterial hypertension in rats.

The soluble guanylate cyclase (sGC)/GMPc pathway plays an important role in controlling pulmonary arterial hypertension (PAH). We investigated whether the novel sGC stimulator trans-4-methoxy-ß-nitrostyrene (T4MN), ameliorates monocrotaline (MCT)-induced PAH. At Day 0, rats were injected with MCT (60 mg/kg, s. c.). Control (CNT) rats received an equal volume of monocrotaline vehicle only (s.c.). Four weeks later, MCT-treated rats were orally treated for 14 days with T4MN (75 mg/kg/day) (MCT-T4MN group) or its vehicle (MCT-V group), and with sildenafil (SIL; 50 mg/kg) (MCT-SIL group). Compared to the CNT group, MCT treatment induced a significant increase in both the Fulton index and RV systolic pressure but significantly reduced the maximum relaxation induced by acetylcholine. Indeed, MCT treatment increased the wall thickness of small and larger pulmonary arterioles. Oral treatment with T4MN and SIL reduced the Fulton index and RV systolic pressure compared to the MCT-V group. Maximum relaxation induced by acetylcholine was significantly enhanced in MCT-SIL group. Both T4MN and SIL significantly reduced the enhanced wall thickness of small and larger pulmonary arterioles. Treatment with T4MN has a beneficial effect on PAH by reducing RV systolic pressure and consequently right ventricular hypertrophy, and by reducing pulmonary artery remodeling. T4MN may represent a new therapeutic or complementary approach for the treatment of PAH.

Nox2 Upregulation and p38α MAPK Activation in Right Ventricular Hypertrophy of Rats Exposed to Long-Term Chronic Intermittent Hypobaric Hypoxia.

One of the consequences of high altitude (hypobaric hypoxia) exposure is the development of right ventricular hypertrophy (RVH). One particular type of exposure is long-term chronic intermittent hypobaric hypoxia (CIH); the molecular alterations in RVH in this particular condition are less known. Studies show an important role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex-induced oxidative stress and protein kinase activation in different models of cardiac hypertrophy. The aim was to determine the oxidative level, NADPH oxidase expression and MAPK activation in rats with RVH induced by CIH. Male Wistar rats were randomly subjected to CIH (2 days hypoxia/2 days normoxia; n = 10) and normoxia (NX; n = 10) for 30 days. Hypoxia was simulated with a hypobaric chamber. Measurements in the RV included the following: hypertrophy, Nox2, Nox4, p22phox, LOX-1 and HIF-1α expression, lipid peroxidation and H2O2 concentration, and p38α and Akt activation. All CIH rats developed RVH and showed an upregulation of LOX-1, Nox2 and p22phox and an increase in lipid peroxidation, HIF-1α stabilization and p38α activation. Rats with long-term CIH-induced RVH clearly showed Nox2, p22phox and LOX-1 upregulation and increased lipid peroxidation, HIF-1α stabilization and p38α activation. Therefore, these molecules may be considered new targets in CIH-induced RVH.

Taurine prevents cardiomyocyte apoptosis by inhibiting the calpain-1/cytochrome c pathway during RVH in broilers.

The calpain-1-activated apoptotic pathway plays a key role in right ventricular hypertrophy (RVH). Taurine has been shown to attenuate apoptosis by inhibiting calpain activity. This experiment aimed to determine whether taurine could prevent RVH by inhibiting the calpain-1/cytochrome c apoptotic pathway. The broilers were given 1% taurine dissolved in drinking water and were raised at 10 °C ~ 12 °C from day 21 to day 42. At 21 d, 28 d, 35 d and 42 d, the right ventricular (RV) tissues were collected. Increased RVH index, angiotensin II, norepinephrine and atrial natriuretic peptide mRNA expression were reduced by taurine in the broiler RVs. Taurine obviously inhibited cardiomyocyte apoptosis via maintaining the mitochondrial membrane potential and decreased the activation of caspase-9 and caspase-3 in the broiler RVs. The antioxidant assay demonstrated that taurine enhanced the activities of superoxide dismutase, total antioxidant capacity and glutathione peroxidase and the glutathione/glutathione disulfide ratio. Western blot results revealed that taurine also downregulated the expression of calpain-1 and cytosolic cytochrome c while upregulating the expression of Bcl-2/Bax and mitochondrial cytochrome c in broiler cardiomyocytes during RVH. In summary, we found that taurine could enhance cardiomyocyte antioxidant ability and further prevented cardiomyocyte apoptosis by inhibiting the calpain-1/cytochrome c pathway during RVH in broilers.

Altered proteasome function in right ventricular hypertrophy.

AIMS: In patients with pulmonary hypertension, right ventricular hypertrophy (RVH) is a detrimental condition that ultimately results in right heart failure and death. The ubiquitin proteasome system has been identified as a major protein degradation system to regulate cardiac remodelling in the left heart. Its role in right heart hypertrophy, however, is still ambiguous. METHODS AND RESULTS: RVH was induced in mice by pulmonary artery banding (PAB). Both, expression and activity of the proteasome was found to be up-regulated in the hypertrophied right ventricle (RV) compared to healthy controls. Catalytic inhibition of the proteasome by the two proteasome inhibitors Bortezomib (BTZ) and ONX-0912 partially improved RVH both in preventive and therapeutic applications. Native gel analysis revealed that specifically the 26S proteasome complexes were activated in experimental RVH. Increased assembly of 26S proteasomes was accompanied by elevated expression of Rpn6, a rate-limiting subunit of 26S proteasome assembly, in hypertrophied cardiomyocytes of the right heart. Intriguingly, patients with RVH also showed increased expression of Rpn6 in hypertrophied cardiomyocytes of the RV as identified by immunohistochemical staining. CONCLUSION: Our data demonstrate that alterations in expression and activity of proteasomal subunits play a critical role in the development of RVH. Moreover, this study provides an improved understanding on the selective activation of the 26S proteasome in RVH that might be driven by the rate-limiting subunit Rpn6. In RVH, Rpn6 therefore represents a more specific target to interfere with proteasome function than the commonly used catalytic proteasome inhibitors.

Effects of toceranib compared with sorafenib on monocrotaline-induced pulmonary arterial hypertension and cardiopulmonary remodeling in rats.

Sorafenib reverses pulmonary arterial hypertension (PAH) and cardiopulmonary remodeling (CPR), but the effects of toceranib are unknown. This study investigated anti-remodeling effects and determined optimal doses of toceranib and sorafenib on monocrotaline (MCT)-induced PAH and CPR in rats. MCT-treated rats were orally treated with a 14-day course of sorafenib (10, 30, or 100 mg/kg), toceranib (1, 3, or 10 mg/kg), or water. Both sorafenib and toceranib significantly reversed the right ventricular (RV) hypertrophy at 10 mg/kg, but only sorafenib significantly improved the RV systolic and mean pressures. Sorafenib significantly normalized the B-type natriuretic peptide mRNA level of the RV and increased the non-muscularized pulmonary artery percentage. However, these effects were only observed at the highest toceranib dose, and neither toceranib dose reduced the fully muscularized pulmonary artery percentage. Further, the inhibition on vascular endothelial growth factor (VEGF) signaling was stronger in sorafenib than in toceranib. Besides the stronger inhibition on mitogen-activated protein kinase signaling, the greater reversal ability of sorafenib may be also due to the simultaneous blockade on the C-X-C chemokine receptor type 4 and autophagy induction. Toceranib insignificantly reversed CPR, and a high-dose therapy did not improve the RV hemodynamic outcomes. Sorafenib significantly reversed CPR, and a low-dose sorafenib therapy may be a suitable therapeutic agent for PAH.

Protein kinase C isozyme expression in right ventricular hypertrophy induced by pulmonary hypertension in chronically hypoxic rats.

In chronic hypoxia, pulmonary hypertension (PH) induces right ventricular hypertrophy (RVH). Evidence indicates that protein kinase C (PKC) serves a crucial role in hypoxia­induced RVH. The present study investigated PKC isoform-specific expression and its involvement in RVH. Rats were exposed to normobaric hypoxia for a number of days to induce PH. PKC isoform­specific membrane translocation and protein expression in the myocardium were evaluated by western blotting and immunostaining. A total of six isoforms of conventional PKC (cPKC; α, ßI and ßII) and of novel PKC (nPKC; δ, ε and η), were detected in the rat myocardium. Hypoxic exposure (1­21 days) induced PH with RVH and vascular remodeling. nPKCδ membrane translocation at 3­7 days and cPKCßI expression at 1­21 days in the RV following hypoxic exposure were significantly decreased as compared with the normoxia control group. Membrane translocation of cPKCßII at 14­21 days and of nPKCη at 7­21 days in the left ventricle following hypoxic exposure was significantly increased when compared with the control. The results of the present study suggested that the alterations in membrane translocation, and nPKCδ and cPKCßI expression, are associated with RVH following PH, and the upregulation of cPKCßII membrane translocation is involved in left­sided heart failure.

p38 MAPK Inhibition Improves Heart Function in Pressure-Loaded Right Ventricular Hypertrophy.

Although p38 mitogen-activated protein kinase (MAPK) is known to have a role in ischemic heart disease and many other diseases, its contribution to the pathobiology of right ventricular (RV) hypertrophy and failure is unclear. Therefore, we sought to investigate the role of p38 MAPK in the pathophysiology of pressure overload-induced RV hypertrophy and failure. The effects of the p38 MAPK inhibitor PH797804 were investigated in mice with RV hypertrophy/failure caused by exposure to hypoxia or pulmonary artery banding. In addition, the effects of p38 MAPK inhibition or depletion (by small interfering RNA) were studied in isolated mouse RV fibroblasts. Echocardiography, invasive hemodynamic measurements, immunohistochemistry, collagen assays, immunofluorescence staining, and Western blotting were performed. Expression of phosphorylated p38 MAPK was markedly increased in mouse and human hypertrophied/failed RVs. In mice, PH797804 improved RV function and inhibited cardiac fibrosis compared with placebo. In isolated RV fibroblasts, p38 MAPK inhibition reduced transforming growth factor (TGF)-ß-induced collagen production as well as stress fiber formation. Moreover, p38 MAPK inhibition/depletion suppressed TGF-ß-induced SMAD2/3 phosphorylation and myocardin-related transcription factor A (MRTF-A) nuclear translocation, and prevented TGF-ß-induced cardiac fibroblast transdifferentiation. Moreover, p38 MAPK inhibition in mice exposed to pulmonary artery banding led to diminished nuclear levels of MRTF-A and phosphorylated SMAD3 in RV fibroblasts. Together, our data indicate that p38 MAPK inhibition significantly improves RV function and inhibits RV fibrosis. Inhibition of p38 MAPK in RV cardiac fibroblasts, resulting in coordinated attenuation of MRTF-A cytoplasmic-nuclear translocation and SMAD3 deactivation, indicates that p38 MAPK signaling contributes to distinct disease-causing mechanisms.

Histone deacetylase adaptation in single ventricle heart disease and a young animal model of right ventricular hypertrophy.

BackgroundHistone deacetylase (HDAC) inhibitors are promising therapeutics for various forms of cardiac diseases. The purpose of this study was to assess cardiac HDAC catalytic activity and expression in children with single ventricle (SV) heart disease of right ventricular morphology, as well as in a rodent model of right ventricular hypertrophy (RVH).MethodsHomogenates of right ventricle (RV) explants from non-failing controls and children born with a SV were assayed for HDAC catalytic activity and HDAC isoform expression. Postnatal 1-day-old rat pups were placed in hypoxic conditions, and echocardiographic analysis, gene expression, HDAC catalytic activity, and isoform expression studies of the RV were performed.ResultsClass I, IIa, and IIb HDAC catalytic activity and protein expression were elevated in the hearts of children born with a SV. Hypoxic neonatal rats demonstrated RVH, abnormal gene expression, elevated class I and class IIb HDAC catalytic activity, and protein expression in the RV compared with those in the control.ConclusionsThese data suggest that myocardial HDAC adaptations occur in the SV heart and could represent a novel therapeutic target. Although further characterization of the hypoxic neonatal rat is needed, this animal model may be suitable for preclinical investigations of pediatric RV disease and could serve as a useful model for future mechanistic studies.

Activation of the nicotinamide N-methyltransferase (NNMT)-1-methylnicotinamide (MNA) pathway in pulmonary hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is associated with inflammatory response but it is unknown whether it is associated with alterations in NNMT activity and MNA plasma concentration. Here we examined changes in NNMT-MNA pathway in PAH in rats and humans. METHODS: PAH in rats was induced by a single subcutaneous injection of MCT (60 mg/kg). Changes in NNMT activity in the lungs and liver (assessed as the rate of conversion of nicotinamide (NA) to MNA), changes in plasma concentration of MNA and its metabolites (analyzed by LC/MS) were analyzed in relation to PAH progression. PAH was characterized by right ventricular hypertrophy (gross morphology), cardiac dysfunction (by MRI), lung histopathology, lung ultrastructure, and ET-1 concentration in plasma. NO-dependent and PGI2-dependent function in isolated lungs was analyzed. In naive patients with idiopathic pulmonary hypertension (IPAH) characterized by hemodynamic and biochemical parameters MNA and its metabolites in plasma were also measured. RESULTS: MCT-injected rats developed hypertrophy and functional impairment of the right ventricle, hypertrophy of the pulmonary arteries, endothelial ultrastructural defects and a progressive increase in ET-1 plasma concentration-findings all consistent with PAH development. In isolated lung, NO-dependent regulation of hypoxic pulmonary vasoconstriction was impaired, while PGI2 production (6-keto-PGF1α) was increased. NNMT activity increased progressively in the liver and in the lungs following MCT injection, and NNMT response was associated with an increase in MNA and 6-keto-PGF1α concentration in plasma. In IPAH patients plasma concentration of MNA was elevated as compared with healthy controls. CONCLUSIONS: Progression of pulmonary hypertension is associated with the activation of the NNMT-MNA pathway in rats and humans. Given the vasoprotective activity of exogenous MNA, which was previously ascribed to PGI2 release, the activation of the endogenous NNMT-MNA pathway may play a compensatory role in PAH.

Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to the development of pulmonary arterial hypertension.

AIMS: Pulmonary arterial hypertension [1] is a proliferative disorder associated with enhanced proliferation and suppressed apoptosis of pulmonary artery smooth muscle cells (PASMCs). Reactive oxygen species (ROS) is implicated in the development of PAH and regulates the vascular tone and functions. However, which cellular signaling mechanisms are triggered by ROS in PAH is still unknown. Hence, here we wished to characterize the signaling mechanisms triggered by ROS. METHODS AND RESULTS: By Western blots, we showed that increased intracellular ROS caused inhibition of the glycolytic pyruvate kinase M2 (PKM2) activity through promoting the phosphorylation of PKM2. Monocrotaline (MCT)-induced rats developed severe PAH and right ventricular hypertrophy, with a significant increase in the P-PKM2 and decrease in pyruvate kinase activity which could be attenuated with the treatments of PKM2 activators, FBP and l-serine. The antioxidant NAC, apocynin and MnTBAP had the similar protective effects in the development of PAH. In vitro assays confirmed that inhibition of PKM2 activity could modulate the flux of glycolytic intermediates in support of cell proliferation through the increased pentose phosphate pathway (PPP). Increased ROS and decreased PKM2 activity also promoted the Cav1.2 expression and intracellular calcium. CONCLUSION: Our data provide new evidence that PKM2 makes a critical regulatory contribution to the PAHs for the first time. Decreased pyruvate kinase M2 activity confers additional advantages to rat PASMCs by allowing them to sustain anti-oxidant responses and thereby support cell survival in PAH. It may become a novel treatment strategy in PAH by using of PKM2 activators.

Aberrant gene expression of heparanase in ventricular hypertrophy induced by monocrotaline in rats.

The gene expression levels of heparanase, matrix metalloproteinase 2 (MMP2) and MMP9 were examined in ventricles after treatment with monocrotaline (MCT) to induce cardiac hypertrophy in rats. Rats received a single intraperitoneal injection of MCT (60 mg/kg) or saline. Twenty-five days after the injection, the right ventricle and lung wet weights were increased in MCT-treated rats compared with the control. Histological analysis revealed cardiomyocyte hypertrophy in the right ventricle of MCT-treated rats. Northern blot hybridization showed that heparanase and MMP2 expression increased significantly in the right and left ventricles of MCT-treated rats, whereas MMP9 was not induced. These findings indicate that heparanase and MMP2 might play an important role in the development of MCT-induced cardiac hypertrophy.

Tetrandrine prevents monocrotaline-induced pulmonary arterial hypertension in rats through regulation of the protein expression of inducible nitric oxide synthase and cyclic guanosine monophosphate-dependent protein kinase type 1.

OBJECTIVE: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by a persistent elevation of pulmonary artery pressure and ventricular hypertrophy. Tetrandrine is a bisbenzylisoquinoline alkaloid that can decrease blood pressure, inhibit the proliferation of vascular smooth muscle cells, and block cardiac hypertrophy, but whether it has a therapeutic effect on PAH remains poorly defined. This study was undertaken to investigate the efficacy of tetrandrine on PAH. METHODS: Forty-eight male Sprague-Dawley rats were randomly and equally divided into four groups. The control group was injected with normal saline; the others were injected with monocrotaline (MCT) to induce PAH, then treated with saline, tetrandrine, and vardenafil, respectively, from day 21 to day 42. On day 43, we measured the mean pulmonary artery pressure under general anesthesia, dissected the rat, and calculated the right ventricular hypertrophy index [right ventricle/(left ventricle plus septum)]. Later we observed the changes in the pulmonary vascular wall; measured the expression of cyclic guanosine monophosphate-dependent protein kinase type 1 and inducible nitric oxide synthase; measured the levels of superoxide dismutase, glutathione, malondialdehyde, and catalase; and then compared the results among groups. RESULTS: Compared with the MCT group, rats treated with tetrandrine had attenuated mean pulmonary artery pressure (20.48 ± 1.49 vs 30.07 ± 1.51; P < .01) and right ventricular hypertrophy index (49.19 ± 2.45 vs 68.50 ± 1.95; P < .01), inhibited proliferation of pulmonary artery smooth muscle cells, and improved endothelial function. Tetrandrine also upregulated the expression of protein kinase type 1 (90.86 ± 1.95 vs 67.34 ± 1.50; P < .01); downregulated the expression of inducible nitric oxide synthase (74.76 ± 1.48 vs 80.19 ± 0.28; P < .01); increased levels of superoxide dismutase (245.54 ± 12.98 vs 166.16 ± 21.42; P < .01), glutathione (0.699 ± 0.032 vs 0.514 ± 0.056; P < .01), and catalase (32.13 ± 2.33 vs 27.19 ± 2.72; P < .01); and decreased malondialdehyde (1.027 ± 0.039 vs 1.462 ± 0.055; P < .01). CONCLUSIONS: Tetrandrine alleviated MCT-induced PAH through regulation of nitric oxide signaling pathway and antioxidant and antiproliferation effects.

Systems pharmacology-based approach for dissecting the active ingredients and potential targets of the Chinese herbal Bufei Jianpi formula for the treatment of COPD.

BACKGROUND: The Chinese herbal Bufei Jianpi formula (BJF) provides an effective treatment option for chronic obstructive pulmonary disease (COPD). However, the systems-level mechanism underlying the clinical effects of BJF on COPD remains unknown. METHODS: In this study, a systems pharmacology model based on absorption filtering, network targeting, and systems analyses was applied specifically to clarify the active compounds and therapeutic mechanisms of BJF. Then, a rat model of cigarette smoke- and bacterial infection-induced COPD was used to investigate the therapeutic mechanisms of BJF on COPD and its comorbidity. RESULTS: The pharmacological system successfully identified 145 bioactive ingredients from BJF and revealed 175 potential targets. There was a significant target overlap between the herbal constituents of BJF. These results suggested that each herb of BJF connected with similar multitargets, indicating potential synergistic effects among them. The integrated target-disease network showed that BJF probably was efficient for the treatment of not only respiratory tract diseases but also other diseases, such as nervous system and cardiovascular diseases. The possible mechanisms of action of BJF were related to activation of inflammatory response, immune responses, and matrix metalloproteinases, among others. Furthermore, we demonstrated that BJF treatment could effectively prevent COPD and its comorbidities, such as ventricular hypertrophy, by inhibition of inflammatory cytokine production, matrix metalloproteinases expression, and other cytokine production in vivo. CONCLUSION: This study using the systems pharmacology method, in combination with in vivo experiments, helped us successfully dissect the molecular mechanism of BJF for the treatment of COPD and predict the potential targets of the multicomponent BJF, which provides a new approach to illustrate the synergetic mechanism of the complex prescription and discover more effective drugs against COPD.

Lack of EC-SOD worsens alveolar and vascular development in a neonatal mouse model of bleomycin-induced bronchopulmonary dysplasia and pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) worsens clinical outcomes in former preterm infants with bronchopulmonary dysplasia (BPD). Oxidant stress disrupts alveolar and vascular development in models of BPD. Bleomycin causes oxidative stress and induces BPD and PAH in neonatal rats. Disruption in the vascular endothelial growth factor (VEGF) and nitric oxide signaling pathways contributes to BPD. We hypothesized that loss of EC-SOD would worsen PAH associated with BPD in a neonatal mouse model of bleomycin-induced BPD by disrupting the VEGF/NO signaling pathway. METHODS: Neonatal wild-type mice (WT), and mice lacking EC-SOD (EC-SOD KO) received intraperitoneal bleomycin (2 units/kg) or phosphate-buffered saline (PBS) three times weekly and were evaluated at weeks 3 or 4. RESULTS: Lack of EC-SOD impaired alveolar development and resulted in PH (elevated right ventricular systolic pressures, right ventricular hypertrophy (RVH)), decreased vessel density, and increased small vessel muscularization. Exposure to bleomycin further impaired alveolar development, worsened RVH and vascular remodeling. Lack of EC-SOD and bleomycin treatment decreased lung total and phosphorylated VEGFR2 and eNOS protein expression. CONCLUSION: EC-SOD is critical in preserving normal lung development and loss of EC-SOD results in disrupted alveolar development, PAH and vascular remodeling at baseline, which is further worsened with bleomycin and associated with decreased activation of VEGFR2.

Chymase: a multifunctional player in pulmonary hypertension associated with lung fibrosis.

Limited literature sources implicate mast-cell mediator chymase in the pathologies of pulmonary hypertension and pulmonary fibrosis. However, there is no evidence on the contribution of chymase to the development of pulmonary hypertension associated with lung fibrosis, which is an important medical condition linked with increased mortality of patients who already suffer from a life-threatening interstitial lung disease.The aim of this study was to investigate the role of chymase in this particular pulmonary hypertension form, by using a bleomycin-induced pulmonary hypertension model.Chymase inhibition resulted in attenuation of pulmonary hypertension and pulmonary fibrosis, as evident from improved haemodynamics, decreased right ventricular remodelling/hypertrophy, pulmonary vascular remodelling and lung fibrosis. These beneficial effects were associated with a strong tendency of reduction in mast cell number and activity, and significantly diminished chymase expression levels. Mechanistically, chymase inhibition led to attenuation of transforming growth factor ß1 and matrix-metalloproteinase-2 contents in the lungs. Furthermore, chymase inhibition prevented big endothelin-1-induced vasoconstriction of the pulmonary arteries.Therefore, chymase plays a role in the pathogenesis of pulmonary hypertension associated with pulmonary fibrosis and may represent a promising therapeutic target. In addition, this study may provide valuable insights on the contribution of chymase in the pulmonary hypertension context, in general, regardless of the pulmonary hypertension form.

Mouse lung development and NOX1 induction during hyperoxia are developmentally regulated and mitochondrial ROS dependent.

Animal models demonstrate that exposure to supraphysiological oxygen during the neonatal period compromises both lung and pulmonary vascular development, resulting in a phenotype comparable to bronchopulmonary dysplasia (BPD). Our prior work in murine models identified postnatal maturation of antioxidant enzyme capacities as well as developmental regulation of mitochondrial oxidative stress in hyperoxia. We hypothesize that consequences of hyperoxia may also be developmentally regulated and mitochondrial reactive oxygen species (ROS) dependent. To determine whether age of exposure impacts the effect of hyperoxia, neonatal mice were placed in 75% oxygen for 72 h at either postnatal day 0 (early postnatal) or day 4 (late postnatal). Mice exposed to early, but not late, postnatal hyperoxia demonstrated decreased alveolarization and septation, increased muscularization of resistance pulmonary arteries, and right ventricular hypertrophy (RVH) compared with normoxic controls. Treatment with a mitochondria-specific antioxidant, (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (mitoTEMPO), during early postnatal hyperoxia protected against compromised alveolarization and RVH. In addition, early, but not late, postnatal hyperoxia resulted in induction of NOX1 expression that was mitochondrial ROS dependent. Because early, but not late, exposure resulted in compromised lung and cardiovascular development, we conclude that the consequences of hyperoxia are developmentally regulated and decrease with age. Attenuated disease in mitoTEMPO-treated mice implicates mitochondrial ROS in the pathophysiology of neonatal hyperoxic lung injury, with potential for amplification of ROS signaling through NOX1 induction. Furthermore, it suggests a potential role for targeted antioxidant therapy in the prevention or treatment of BPD.

PKC δ and ßII regulate angiotensin II-mediated fibrosis through p38: a mechanism of RV fibrosis in pulmonary hypertension.

Pulmonary hypertension (PH) eventually leads to right ventricular (RV) fibrosis and dysfunction that is associated with increased morbidity and mortality. Although angiotensin II plays an important role in RV remodeling associated with hypoxic PH, the molecular mechanisms underlying RV fibrosis in PH largely remain unresolved. We hypothesized that PKC-p38 signaling is involved in RV collagen accumulation in PH and in response to angiotensin II stimulation. Adult male Sprague-Dawley rats were exposed to 3 wk of normoxia or hypoxia (10% FiO2 ) as a model of PH. Hypoxic rats developed RV hypertrophy and fibrosis associated with an increase in PKC ßII and δ protein expression and p38 dephosphorylation in freshly isolated RV cardiac fibroblasts. Further mechanistic studies were performed in cultured primary cardiac fibroblasts stimulated with angiotensin II, a key activator of ventricular fibrosis in PH. Angiotensin II induced a reduction in p38 phosphorylation that was attenuated following chemical inhibition of PKC ßII and δ. Molecular and chemical inhibition of PKC ßII and δ abrogated angiotensin II-induced cardiac fibroblast proliferation and collagen deposition in vitro. The effects of PKC inhibition on proliferation and fibrosis were reversed by chemical inhibition of p38. Conversely, constitutive activation of p38 attenuated angiotensin II-induced increase of cardiac fibroblast proliferation and collagen accumulation. PKC ßII- and δ-dependent inactivation of p38 regulates cardiac fibroblast proliferation and collagen deposition in response to angiotensin II, which suggests that the PKC-p38 signaling in cardiac fibroblasts may be involved and important in the pathophysiology of RV fibrosis in PH.

Acetyl-lysine erasers and readers in the control of pulmonary hypertension and right ventricular hypertrophy.

Acetylation of lysine residues within nucleosomal histone tails provides a crucial mechanism for epigenetic control of gene expression. Acetyl groups are coupled to lysine residues by histone acetyltransferases (HATs) and removed by histone deacetylases (HDACs), which are also commonly referred to as "writers" and "erasers", respectively. In addition to altering the electrostatic properties of histones, lysine acetylation often creates docking sites for bromodomain-containing "reader" proteins. This review focuses on epigenetic control of pulmonary hypertension (PH) and associated right ventricular (RV) cardiac hypertrophy and failure. Effects of small molecule HDAC inhibitors in pre-clinical models of PH are highlighted. Furthermore, we describe the recently discovered role of bromodomain and extraterminal (BET) reader proteins in the control of cardiac hypertrophy, and provide evidence suggesting that one member of this family, BRD4, contributes to the pathogenesis of RV failure. Together, the data suggest intriguing potential for pharmacological epigenetic therapies for the treatment of PH and right-sided heart failure.