Injection Of SU5416 Research Articles

Rimeporide, a first in class Na/H exchanger-1 inhibitor, ameliorates right ventricular dysfunction and pulmonary arterial hypertension in a SuHx model

Abstract Background Pulmonary arterial hypertension (PAH) is characterized by pulmonary vasoconstriction and vascular remodeling leading to right ventricular (RV) failure. Although RV function is the major prognostic factor in PAH, no RV-specific therapies exist. The Na+/H+ exchanger type 1 (NHE1) regulates the intra- and extracellular pH balance mainly through the 1:1 exchange of intracellular H+ with extracellular Na+. Increased NHE-1 activity is involved in several diseases, including hypoxic PAH. Inhibition of NHE-1 activity attenuates pulmonary vascular remodeling in rodents exposed to hypoxia but its potential direct contribution in RV remodeling remains unclear. Aim In the current study, we aimed to investigate the effect of NHE-1 inhibition by Rimeporide a preclinical hypoxic models of PH: Sugen5416/hypoxia. Methods Sugen/hypoxia model: PAH was induced in adult Sprague-Dawley rats with a single injection of SU5416 (20 mg/kg) followed by 3 weeks of hypoxia (10% O2; Sugen/Hypoxia model: SuHx) and then return to normoxia. Control rats were injected with equal volume of vehicle. At week 5 post-injection, control and SuHx rats were treated with rimeporide (100 mg/Kg daily in drinking water, n=10/group) or placebo (n=11/group) for 3 weeks. Echocardiography and blood sampling were performed. RV phenotype was monitored by echocardiography and hemodynamic analysis. Lung and heart were collected for histological and protein expression analysis, such as NHE-1 (target of Rimeporide). Results SuHx caused severe PAH and RV dysfunction. RV dysfunction was accompanied by an increase in RV hypertrophy and RV fibrosis and PA remodeling (increase in the arteriolar wall thickness and fibrosis). A marked increase in macrophages was found in lung and RV tissues but not in LV. Moreover, Western blot analysis showed that RV protein levels of NHE-1 was higher in SuHx rats than Normoxia rats. Treatment with Rimeporide reversed SuHx-induced PAH as well as RV dysfunction and blunted pulmonary and RV inflammation, RV NHE-1 protein expression. Conclusion Our results indicate that NHE-1 levels are increased in RV tissues from SuHx-induced PAH rats. Rimeporide, through NHE-1 inhibition, might be a potential new target for treatment of RV dysfunction and PAH.

Inhibition of Soluble Epoxide Hydrolase Does Not Promote or Aggravate Pulmonary Hypertension in Rats.

Inhibitors of soluble epoxide hydrolase (sEH), which catalyzes the hydrolysis of various natural epoxides to their corresponding diols, present an opportunity for developing oral drugs for a range of human cardiovascular and inflammatory diseases, including, among others, diabetes and neuropathic pain. However, some evidence suggests that their administration may precipitate the development of pulmonary hypertension (PH). We thus evaluated the impact of chronic oral administration of the sEH inhibitor TPPU (N-[1-(1-Oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy)phenyl]-urea) on hemodynamics, pulmonary vascular reactivity, and remodeling, as well as on right ventricular (RV) dimension and function at baseline and in the Sugen (SU5416) + hypoxia (SuHx) rat model of severe PH. Treatment with TPPU started 5 weeks after SU5416 injection for 3 weeks. No differences regarding the increase in pulmonary vascular resistance, remodeling, and inflammation, nor the abolishment of phenylephrine-induced pulmonary artery constriction, were noted in SuHx rats. In addition, TPPU did not modify the development of RV dysfunction, hypertrophy, and fibrosis in SuHx rats. Similarly, none of these parameters were affected by TPPU in normoxic rats. Complementary in vitro data demonstrated that TPPU reduced the proliferation of cultured human pulmonary artery-smooth muscle cells (PA-SMCs). This study demonstrates that inhibition of sEH does not induce nor aggravate the development of PH and RV dysfunction in SuHx rats. In contrast, a potential beneficial effect against pulmonary artery remodeling in humans is suggested.

β3 adrenergic agonism: A novel pathway which improves right ventricular-pulmonary arterial hemodynamics in pulmonary arterial hypertension.

Efficacy of therapies that target the downstream nitric oxide (NO) pathway in pulmonary arterial hypertension (PAH) depends on the bioavailability of NO. Reduced NO level in PAH is secondary to "uncoupling" of endothelial nitric oxide synthase (eNOS). Stimulation of β3 adrenergic receptors (β3 ARs) may lead to the recoupling of NOS and therefore be beneficial in PAH. We aimed to examine the efficacy of β3 AR agonism as a novel pathway in experimental PAH. In hypoxia (5 weeks) and Sugen hypoxia (hypoxia for 5 weeks + SU5416 injection) models of PAH, we examined the effects of the selective β3 AR agonist CL316243. We measured echocardiographic indices and invasive right ventricular (RV)-pulmonary arterial (PA) hemodynamics and compared CL316243 with riociguat and sildenafil. We assessed treatment effects on RV-PA remodeling, oxidative stress, and eNOS glutathionylation, an oxidative modification that uncouples eNOS. Compared with normoxic mice, RV systolic pressure was increased in the control hypoxic mice (p < 0.0001) and Sugen hypoxic mice (p < 0.0001). CL316243 reduced RV systolic pressure, to a similar degree to riociguat and sildenafil, in both hypoxia (p < 0.0001) and Sugen hypoxia models (p < 0.03). CL316243 reversed pulmonary vascular remodeling, decreased RV afterload, improved RV-PA coupling efficiency and reduced RV stiffness, hypertrophy, and fibrosis. Although all treatments decreased oxidative stress, CL316243 significantly reduced eNOS glutathionylation. β3 AR stimulation improved RV hemodynamics and led to beneficial RV-PA remodeling in experimental models of PAH. β3 AR agonists may be effective therapies in PAH.

Alterations in Genes Associated with Bioactive Lipid Pathways during Development of Pulmonary Hypertension in Rat SU5416‐Hypoxia Model of Severe Pulmonary Arterial Hypertension

BackgroundPulmonary arterial hypertension (PAH) is a disease of the pulmonary vascular system characterized by sustained elevation in resting mean pulmonary arterial pressure (&gt;25 mmHg) with presence of occlusive vascular remodeling and progressive loss of microvasculature. PAH patients have a poor five‐year survival rate of only ~50% and the primary cause of death for PAH patients is the right (‐sided) heart failure. Better understanding of pathophysiology of PAH and RHF is required to find novel therapies. In this project, using a rat model of severe PAH, we explored the alterations in expression of genes involved in bioactive lipid pathways in the lungs and the right ventricle (RV) during development of PAH.MethodsAdult male Sprague Dawley rats were administered a single injection of SU5416 (SU; 20 mg/kg, subcutaneous) and subjected to 3 weeks of hypoxia (10% O2) followed by 4 weeks of normoxia (room air), SUHx model of sever PAH. Right ventricular systolic pressure (RVSP) was measured, and heart and lung samples were collected at 1, 3, 7, 21 and 49‐days post SU injection. Histological analysis was conducted to confirm vascular remodeling in the lungs and development of RV hypertrophy (RVH; RV/LV+S). RTqPCR was performed to evaluate gene expression changes in the genes related to bioactive lipids pathways.ResultsIn response to SUHx, progressive increase in RVSP and RVH was observed. Histology of lungs and heart samples confirmed pulmonary vascular and RV remodeling at 7 weeks post SU in the SUHx model. We observed increase in expression of ALOX‐5 in the RV and lungs of SUHx rats, compared to controls. Furthermore, alterations in various cytochrome P450 (CYP) isozymes were observed in the RV and lungs of SUHx rats compared to controls.ConclusionsTogether, we have identified unique changes in bioactive lipid pathway genes in the lungs and the RV that were associated with development of PAH and RV remodeling in SUHx model. Further studies are ongoing to confirm the role of the candidate genes in pathophysiology of PAH and development of RHF.

VEGF Modulates Neurogenesis and Microvascular Remodeling in Epileptogenesis After Status Epilepticus in Immature Rats.

Neurogenesis and angiogenesis are widely recognized to occur during epileptogenesis and important in brain development. Because vascular endothelial growth factor (VEGF) is a critical neurovascular target in neurological diseases, its effect on neurogenesis, microvascular remodeling and epileptogenesis in the immature brain after lithium-pilocarpine-induced status epilepticus (SE) was investigated. The dynamic changes in and the correlation between hippocampal neurogenesis and microvascular remodeling after SE and the influence of VEGF or SU5416 injection into the lateral ventricles at different stages after SE on neurogenesis and microvascular remodeling through regulation of VEGF expression were assessed by immunofluorescence and immunohistochemistry. Western blot analysis revealed that the VEGFR2 signaling pathway promotes phosphorylated ERK and phosphorylated AKT expression. The effects of VEGF expression regulation at different stages after SE on pathological changes in hippocampal structure and spontaneous recurrent seizures (SRS) were evaluated by Nissl staining and electroencephalography (EEG). The results showed that hippocampal neurogenesis after SE is related to microvascular regeneration. VEGF promotion in the acute period and inhibition in the latent period after SE alleviates loss of hippocampal neuron, abnormal vascular regeneration and inhibits neural stem cells (NSCs) ectopic migration, which may effectively alleviate SRS severity. Interfering with VEGF via the AKT and ERK pathways in different phases after SE may be a promising strategy for treating and preventing epilepsy in children.

Upregulation of Aquaporin 1 Mediates Increased Migration and Proliferation in Pulmonary Vascular Cells From the Rat SU5416/Hypoxia Model of Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive disorder characterized by exuberant vascular remodeling leading to elevated pulmonary arterial pressure, maladaptive right ventricular remodeling, and eventual death. The factors controlling pulmonary arterial smooth muscle cell (PASMC) and endothelial cell hyperplasia and migration, hallmark features of the vascular remodeling observed in PAH, remain poorly understood. We previously demonstrated that hypoxia upregulates the expression of aquaporin 1 (AQP1), a water channel, in PASMCs, and that this upregulation was required for hypoxia-induced migration and proliferation. However, whether the same is true in a model of severe PAH and in pulmonary microvascular endothelial cells (MVECs) is unknown. In this study, we used the SU5416 plus hypoxia (SuHx) rat model of severe pulmonary hypertension, which mimics many of the features of human PAH, to determine whether AQP1 levels were altered in PASMCs and MVECs and contributed to a hyperproliferative/hypermigratory phenotype. Rats received a single injection of SU5416 (20 mg/kg) and then were placed in 10% O2 for 3 weeks, followed by a return to normoxic conditions for an additional 2 weeks. We found that AQP1 protein levels were increased in both PASMCs and MVECs from SuHx rats, even in the absence of sustained hypoxic exposure, and that in MVECs, the increase in protein expression was associated with upregulation of AQP1 mRNA levels. Silencing of AQP1 had no significant effect on PASMCs from control animals but normalized enhanced migration and proliferation observed in cells from SuHx rats. Loss of AQP1 also reduced migration and proliferation in MVECs from SuHx rats. Finally, augmenting AQP1 levels in MVECs from control rats using forced expression was sufficient to increase migration and proliferation. These results demonstrate a key role for enhanced AQP1 expression in mediating abnormal migration and proliferation in pulmonary vascular cells from a rodent model that reflects many of the features of human PAH.

Abstract 9081: Fatal Occlusive Pulmonary Vasculopathy in Rats Late After Perinatal Hypoxia: Hyperproliferative and Pro-Inflammatory Phenotypes and Related Differential Dna Methylation in Cultured Pulmonary Arterial Smooth Muscle Cells as the Potential Substrate

Background: Perinatal insults increase the risk of developing various adult-onset vascular diseases. Although we recently established a fatal SU5416/hypoxia-induced model with worsening pulmonary vascular disease (PVD) in rats late after exposure to perinatal hypoxia, the mechanisms are elusive. Hypothesis: In rats with fatal occlusive PVD late after perinatal hypoxia, cultured pulmonary arterial smooth muscle cells (PASMCs) from rats before SU5416/hypoxia treatment exhibit phenotypic and epigenetic changes. Methods: PVD was induced in 7-week-old rats exposed to perinatal hypoxia or ambient air by injecting SU5416 followed by exposure to hypoxia for 3 weeks (SU5416/hypoxia). Rats were assessed at the baseline (7 weeks of age) and 8 weeks after SU5416 injection (15 weeks of age). Proliferation, cytokine production and genome-wide DNA methylation profile were evaluated in PASMC isolated from the baseline rats with/without perinatal hypoxia. Results: In SU5416/hypoxia rats, perinatal hypoxia reduced body weight gain and survival, and increased right ventricular systolic pressure, Fulton index and occlusive PVD (p&lt;.05), which were associated with an increase in perivascular macrophages, expression of prepro-endothelin-1 mRNA and eNOS protein in the lung (p&lt;.05) but not with impaired alveolarization or worsening right ventricular fibrosis. In PASMCs isolated from the baseline rats, perinatal hypoxia enhanced PDGF-BB-induced cell proliferation and phosphorylation of extracellular signal-regulated kinase as well as TNFα-induced expression of IL-6, monocyte chemotactic protein 1 and phosphorylation of p38 (p&lt;.05). DNA methylation profiling identified 35 differentially methylated regions (DMRs). Methylation in the region encoding phosducin like protein (Pdcl) was associated with decreased PDGF-BB-induced expression of Pdcl mRNA in PASMCs with perinatal hypoxia. Functional enrichment analysis of DMRs revealed the relation to cell proliferation and inflammatory process (ie, Wnt5A and ROR1). Conclusions: In SU5416/hypoxia rats late after perinatal hypoxia, hyperproliferative and pro-inflammatory phenotypes and functionally related differential DNA methylation in PASMCs may be the substrate for the fatal occlusive PVD.

Abstract 12203: Sotatercept Analog RAP-011 is More Effective Than Sildenafil in Improving Pulmonary Hypertension and Reducing Right Ventricular Hypertrophy in a ZSF1 Rat Model of Heart Failure With Preserved Ejection Fraction

Introduction: Pulmonary hypertension (PH) due to heart failure with preserved ejection fraction (PH-HFpEF) is the most prevalent form of WHO Group 2 PH but lacks effective treatment options. We previously found that sequestration of activins and growth differentiation factors with the sotatercept analog RAP-011, an activin receptor type IIA-Fc fusion protein, reverses pathological remodeling of pulmonary arteries and the right ventricle (RV) in experimental models of pulmonary arterial hypertension (PAH, Group 1 PH). This strategy of selective multi-ligand neutralization offers a promising new approach for treating patients with PAH and is under active clinical evaluation (PULSAR, SPECTRA, and STELLAR). Here, we compared the cardiopulmonary effects of therapeutic treatment with RAP-011 to those of sildenafil in a rat model of PH-HFpEF. Methods: PH was induced in adult male obese ZSF1 rats by a single subcutaneous injection of SU5416 (100 mg/kg). To model the timeline of diagnosis and treatment for Group 2 PH patients, we initiated treatment with RAP-011 and sildenafil 8 weeks post SU5416 administration—at which point RV dysfunction is prominent—and continued treatment for 8 weeks. RV systolic pressure (RVSP), pulmonary artery acceleration time (PAAT), RV hypertrophy, and tricuspid annular plane systolic excursion (TAPSE) were evaluated by right heart catheterization and echocardiography following the 8-week treatment regimen. Results: Obese ZSF1 rats treated with SU5416 displayed significantly increased RVSP (P &lt; 0.0001) and decreased PAAT (P &lt; 0.0001). RAP-011 treatment fully reversed changes in RVSP (P &lt; 0.0001) and PAAT (P &lt; 0.0001), whereas sildenafil treatment improved RVSP by 19% (P &lt; 0.05) and PAAT by 36% (P &lt; 0.001). Treatment with RAP-011, but not sildenafil, normalized RV hypertrophy (P &lt; 0.0001) and increased TAPSE (P &lt; 0.0001). Conclusions: Therapeutic treatment with the sotatercept analog RAP-011 exerted beneficial cardiopulmonary effects in a ZSF1 rat model of PH-HFpEF that were significantly greater than those produced by sildenafil, fully normalizing PH and RV hypertrophy and improving RV function. These findings support evaluation of sotatercept in patients with PH-HFpEF (Group 2 PH).

Riociguat can ameliorate bronchopulmonary dysplasia in the SU5416 induced rat experimental model

Background Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature neonates. Classical BPD is caused by hyperoxia and high-pressure mechanical ventilation, whereas BPD in recent era is caused by impaired pulmonary angiogenesis and alveolarization in extreme prematurity. Although sildenafil was reported to be effective in a hyperoxia-induced rat BPD model, several clinical trials could not demonstrate any significant improvement in the respiratory statuses of BPD infants. Riociguat is a soluble guanylate cyclase stimulator that increases cyclic guanosine monophosphate activity in a nitric oxide independent manner. However, a beneficial effect in BPD has not been established yet. Methods and Results We established BPD model in rats by injection of SU5416 on day 1 followed by maintenance under normoxia, which resulted in oversimplified alveoli, sparse pulmonary capillary vessels, severe pulmonary hypertension, and growth retardation, which mimicked the features observed in recent clinical management of BPD. We administered riociguat from day 10, when BPD rats exhibited growth retardation. Histological analyses demonstrated that riociguat treatment significantly but partially ameliorated lung alveolarization, vascularization, and pulmonary hypertension. However, the survival rate was not significantly improved by riociguat treatment. Conclusions Riociguat could ameliorate pulmonary alveolarization, vascularization, and hypertension in the SU5416 induced BPD rat model, but could not improve the overall survival.

Fasudil Dichloroacetate Alleviates SU5416/Hypoxia-Induced Pulmonary Arterial Hypertension by Ameliorating Dysfunction of Pulmonary Arterial Smooth Muscle Cells.

BackgroundPulmonary arterial hypertension (PAH) is an incurable disease that urgently needs therapeutic approaches. Based on the therapeutic effects of fasudil and dichloroacetate (DCA) on PAH, we aimed to explore the effects and potential mechanism of a new salt, fasudil dichloroacetate (FDCA), in a SU5416 plus hypoxia (SuHx)-induced rat model of PAH.MethodsThe rat model of PAH was established by a single subcutaneous injection of SU5416 (20 mg/kg) followed by hypoxia (10% O2) exposure for 3 weeks. FDCA (15, 45, or 135 mg/kg i.g. daily) or the positive control, bosentan (100 mg/kg i.g. daily), were administered from the first day after SU5416 injection. After 3-week hypoxia, hemodynamic parameters, and histological changes of the pulmonary arterial vessels and right ventricle (RV) were assessed. Additionally, in vitro, the effects of FDCA (50 μM), compared with equimolar doses of fasudil, DCA, or fasudil+DCA, on the proliferation, migration, and contraction of human pulmonary arterial smooth muscle cell (PASMC) under hypoxia (1% O2) were evaluated.ResultsFDCA dose-dependently attenuated SuHx-induced PAH, with significant reductions in RV systolic pressure, pulmonary artery wall thickness, pulmonary vessel muscularization, perivascular fibrosis, as well as RV hypertrophy and fibrosis. In vitro, FDCA inhibited hypoxia-induced PASMC proliferation, migration, and contraction to a greater degree than fasudil or DCA alone by restoring mitochondrial function, reducing intracellular Ca2+, and inhibiting calcium/calmodulin-dependent kinase (Ca2+/CaMK) activity as well as Rho-kinase activity.ConclusionFDCA ameliorates hypoxia-induced PASMC dysfunction by inhibiting both Ca2+/CaMK and Rho-kinase signaling pathways, as well as maintaining mitochondrial homeostasis, thus alleviating SuHx-induced PAH.

Gut microbiota modification suppresses the development of pulmonary arterial hypertension in an SU5416/hypoxia rat model.

The pathogenesis of pulmonary arterial hypertension is closely associated with dysregulated inflammation. Recently, abnormal alterations in gut microbiome composition and function were reported in a pulmonary arterial hypertension experimental animal model. However, it remains unclear whether these alterations are a result or the cause of pulmonary arterial hypertension. The purpose of this study was to investigate whether alterations in the gut microbiome affected the hemodynamics in SU5416/hypoxia rats. We used the SU5416/hypoxia rat model in our study. SU5416/hypoxia rats were treated with a single SU5416 injection (30 mg/kg) and a three-week hypoxia exposure (10% O2). Three SU5416/hypoxia rats were treated with a combination of four antibiotics (SU5416/hypoxia + ABx group) for four weeks. Another group was exposed to hypoxia (10% O2) without the SU5416 treatment, and control rats received no treatment. Fecal samples were collected from each animal, and the gut microbiota composition was analyzed by 16S rRNA sequencing. The antibiotic treatment significantly suppressed the vascular remodeling, right ventricular hypertrophy, and increase in the right ventricular systolic pressure in SU5416/hypoxia rats. 16S rRNA sequencing analysis revealed gut microbiota modification in SU5416/hypoxia + ABx group. The Firmicutes-to-Bacteroidetes ratio in SU5416/hypoxia rats was significantly higher than that in control and hypoxia rats. Compared with the control microbiota, 14 bacterial genera, including Bacteroides and Akkermansia, increased, whereas seven bacteria, including Rothia and Prevotellaceae, decreased in abundance in SU5416/hypoxia rats. Antibiotic-induced modification of the gut microbiota suppresses the development of pulmonary arterial hypertension. Dysbiosis may play a causal role in the development and progression of pulmonary arterial hypertension.

Induction and Characterization of Pulmonary Hypertension in Mice using the Hypoxia/SU5416 Model.

Pulmonary Hypertension (PH) is a pathophysiological condition, defined by a mean pulmonary arterial pressure exceeding 25 mm Hg at rest, as assessed by right heartcatheterization. A broad spectrum of diseases can lead to PH, differing in their etiology, histopathology, clinical presentation, prognosis, and response totreatment. Despite significant progress in the last years, PH remains an uncured disease. Understanding the underlying mechanisms can pave the way for the development of new therapies. Animal models are important research tools to achieve this goal. Currently, there are several models available for recapitulating PH. This protocol describes a two-hit mouse PH model. The stimuli for PH development are hypoxia and the injection of SU5416, a vascular endothelial growth factor (VEGF) receptor antagonist. Three weeks after initiation of Hypoxia/SU5416, animals develop pulmonary vascular remodeling imitating the histopathological changes observed in human PH (predominantly Group 1). Vascular remodeling in the pulmonary circulation results in the remodeling of the rightventricle (RV). The procedures for measuring RV pressures (using the open chest method), the morphometrical analyses of the RV (by dissecting and weighing both cardiac ventricles) and the histological assessments of the remodeling (both pulmonary by assessing vascular remodeling and cardiac by assessing RV cardiomyocyte hypertrophy and fibrosis) are described in detail. The advantages of this protocol are the possibility of the application both in wild type and in genetically modified mice, the relatively easy and low-cost implementation, and the quick development of the disease of interest (3 weeks). Limitations of this method are that mice do not develop a severe phenotype and PH is reversible upon return to normoxia. Prevention, as well as therapy studies, can easily be implemented in this model, without the necessity of advanced skills (as opposed to surgical rodent models).

Role of Na+/H+ Exchanger 1 in Regulating Resistance to Apoptosis in Pulmonary Arterial Smooth Muscle Cells

Vascular remodeling within the pulmonary arterioles is a key component of pulmonary arterial hypertension (PAH). In a rat model of severe PAH, the SU5416‐Hypoxia (SuHx) model, pulmonary arterial smooth muscle cells (PASMCs) demonstrate resistance to apoptosis and contribute to vascular remodeling. Na+/H+ exchange (NHE) is increased in SuHx PASMCs; however, few studies have elucidated the functional role of NHE in these cells. Previous studies have shown a link between NHE and the expression of CHOP, a critical mediator of apoptosis. Therefore, we tested the hypothesis that altering NHE1 expression and or activity is sufficient to modulate apoptosis in PASMCs. PASMCs were isolated from distal pulmonary arteries dissected from vehicle‐treated, male, Wistar rats or rats exposed to hypoxia after an injection of SU5416 (SuHx rats). PASMCs were treated with vehicle, a combination of a pharmacologic inhibitor of NHE (EIPA) and hydrogen peroxide (H2O2), an apoptosis‐inducing agent, or either EIPA or H2O2 alone. Apoptosis was measured via Hoechst staining. In other experiments, control PASMCs were infected with a viral construct to overexpress wild‐type NHE1 (AdNHE1), a translocation‐disabled NHE1 mutant (AdE266I), or green fluorescent protein (AdGFP) as a control. NHE activity was measured via the ammonium‐pulse technique. SuHx PASMCs exhibited decreased CHOP expression and minimal apoptosis rates in response to H2O2 when compared to control PASMCs. EIPA had no effect on unstimulated apoptosis in control cells, but induced apoptosis in SuHx PASMCs at baseline and sensitized the cells to H2O2. AdNHE1, but not AdE266I, increased NHE1 activity. Interestingly, both AdNHE1 and AdE266I conferred a resistance to H2O2‐induced apoptosis when compared to the AdGFP‐infected cells. Overall, these findings suggest that increased NHE1 may be responsible for attenuating apoptotic responses in SuHx PASMCs and that increasing NHE1 levels in normal PASMCs is sufficient to promote increased survival via an ion‐translocation‐independent function.Support or Funding InformationSupported by NIH grants HL073859, HL126514 and HL084762

Endogenous mechanism of right ventricular repair in pulmonary hypertension

IntroductionRight‐sided heart failure is the major cause of death among patients with pulmonary hypertension (PH) as well as children who suffer from various forms of congenital heart disease. Thus, agents that repair the damaged right ventricle (RV) have therapeutic potential to treat right‐sided heart failure. The biology of RV, however, is not well understood. The present study describes an endogenous mechanism for RV repair that was discovered in an experimental animal model of PH.Methods &amp; ResultsThe treatment of Sprague‐Dawley (SD) rats with the injection of SU5416 (VEGF inhibitor) and 3 weeks of chronic hypoxia, followed by the maintenance in normoxia, resulted in progressive PH and RV remodeling. At 17 weeks after the initiation of the treatment, RV cardiomyocytes were damaged and the fibrotic region was found to be 12% of the total RV. However, at 35 weeks, RV fibrosis was significantly reduced and new cardiomyocytes were regenerated, despite RV pressure remained elevated. Before the RV repair occurs, α‐smooth muscle actin (αSMA) was found to be expressed in myofibroblasts. In addition, we noted the expression of αSMA in some of RV cardiomyocytes. Normal terminally differentiated cardiomyocytes do not express αSMA, but induced cardiomyocytes (iCMs) derived from induced pluripotent stem cells (iPSCs) do. Thus, we hypothesized that the damaged RV is repaired by activating the cellular reprogramming process that converts resident cardiac fibroblasts into iCMs perhaps through an iPSC‐like mechanism. This hypothesis was substantiated by our observations that Sox2, stem cell‐specific transcription factor, was also expressed in RV cardiomyocytes being repaired. Further, transmission electron microscopy observations revealed that extracellular matrix contains fibroblast‐line cells with myofilaments, which are organized into myofibrils and formed into the sarcomere‐like structure similar to iCMs.ConclusionsThe RV damage promoted by PAH can be repaired through a naturally occurring process that may involve cardiomyocyte regeneration through cell reprogramming. Understanding such an endogenous repair mechanism should contribute to the development of therapeutic strategies to treat right‐sided heart failure.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 17217: ActRIIA-Fc Rebalances Activin/GDF and BMP9 Signaling to Attenuate Experimental Pulmonary Hypertension

Introduction: Therapies targeting the underlying mechanisms of pulmonary arterial hypertension (PAH) might modify the natural history of disease. The genetics of heritable PAH implicate deficient BMP signaling as a pathogenetic driver. Lung tissues from experimental and human PAH exhibit deficient BMP &amp; excessive TGFβ signaling. While previous studies suggest agonism of BMP9 signaling or inhibition of TGFβ signaling may prevent experimental PH, the roles of activin and growth and differentiation factor (GDF) ligand signaling are unclear. ACTRIIA-Fc (sotatercept) is an activin- and GDF8/11-specific ligand trap currently in Phase 2 studies for anemia and multiple myeloma. Hypothesis: ACTRIIA-Fc inhibits activin and GDF8/11 signaling in vascular cells to attenuate experimental PH and pulmonary vascular remodeling. Methods: Impact of ACTRIIA-Fc on BMP &amp; TGFβ signaling was examined in human pulmonary microvascular endothelial (PMVEC) and pulmonary arterial smooth muscle cells (PASMC). ACTRIIA-Fc (15 mg/kg s.c. 2x/week), sildenafil (30 mg/kg p.o. BID) or vehicle were given prophylactically for 4 weeks to rats developing PH following a single injection of MCT (40 mg/kg s.c.), or via single injection of SU5416 (200 mg/kg s.c.) combined with hypoxia (FIO2=0.10, SU-Hx). Alternately, rats were allowed to develop PH following SU-Hx treatment for 3 weeks or MCT treatment for 4 weeks, and then treated with ACTRIIA-Fc (1, 3, or 10 mg/kg s.c. 2x/week) or isotype-matched control antibody (3 weeks under normoxia following SU-Hx, or 2 weeks following MCT). Invasive hemodynamics, RV mass and vascular remodeling were assessed. Results: In PMVEC, ACTRIIA-Fc enhanced BMP9-induced SMAD1/5/8 activation and transcriptional activity, and reversed inhibition of BMP9 signaling by GDF11. ACTRIIA-Fc inhibited GDF8-, GDF11- and activin A-mediated SMAD2 phosphorylation and myogenic differentiation of PASMC. In vivo , a standard dose of ACTRIIA-Fc prevented PH, RV hypertrophy and PV remodeling in MCT- and SU-Hx-rats, with more potency than sildenafil. All doses of ACTRIIA-Fc attenuated PH and RV hypertrophy when given in a delayed fashion after MCT- and SU-Hx-treatment, with graded effects on PV remodeling. Conclusions: ACTRIIA-Fc rebalances GDF11 vs. BMP9 signaling, with distinct effects in vascular endothelium vs. media, and potently inhibits experimental PH in several rodent models. Given its well-defined tolerability, dosing, and efficacy in previous human studies, ACTRIIA-Fc is an attractive candidate for PAH as a novel, mechanism-targeted therapy.

Targeting the Right Ventricle as a Treatment Strategy for Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by remodeling of the pulmonary artery within the lung that leads to increased pulmonary vascular resistance (PVR). This increase in PVR can result in progressive remodeling of the right ventricle (RV) and eventual right heart failure. Current therapy aims to decrease PVR via vasodilation of the PA, but this strategy does not work for some patients, and is not a cure for the disease. This study aims to uncover novel therapeutic targets by assessing the RV for expression of G protein‐coupled receptors (GPCRs) during PAH. GPCRs are the target of more than 30% of approved drugs and regulate multiple cellular processes by activation on the cell membrane. We utilized a rat model of PAH in which Sprague Dawley rats were placed in hypoxia (10% O2) for two weeks, or normoxia as a control (n=3–4/treatment). Placement in hypoxia resulted in RV remodeling evident by an increase in the RV weight/left ventricle (LV) + septum weight (hypoxia‐treatment: 0.458 ± 0.036 vs. control rats: 0.210 ± 0.020; mean ± standard deviation, Student's t‐test p&lt;0.0001). We then utilized RNA sequencing (RNA‐seq) of the RV and the left ventricle (LV) tissue coupled with differential expression analysis to identify GPCRs expressed with significantly (false discovery rate &lt;0.1) altered expression in the RV of hypoxia‐treated rats compared to controls. In the RV, 37 GPCRs were altered in expression in hypoxia‐treated rats vs. control rats compared to 7 GPCRs altered in the LV. To examine the cell‐specific expression of these targets, cardiac myocytes and fibroblasts were isolated from a mouse model of PAH (10% O2 for 3 weeks with weekly injection of SU5416). qPCR of the target GPCRs revealed that most of the differentially expressed GPCRs were altered in the cardiac myocytes but not in the fibroblasts. These results indicate that a subset of genes is differentially expressed in the RV compared to the LV both in normoxia and hypoxia, particularly in the cardiac myocytes. This raises the possibility that treatments for PAH could be directed at GPCRs selectively expressed in RV remodeling, thus providing a novel means of treating an unmet need for patients with RV hypertrophy and failure.Support or Funding InformationSupported by NIH Grant: T32HL007444‐34 and DoD Grant: W81XWH‐14‐1‐0372This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Wave reflection correlates with pulmonary vascular wall thickening in rats with pulmonary arterial hypertension

BackgroundWave reflection is enhanced in patients with pulmonary arterial hypertension (PAH), which may be derived from a mismatch of pulmonary artery (PA) impedance between proximal and distal sites of arteries. Whether enhanced wave reflection correlates with histological remodeling remains unknown, partly because lung biopsy is not clinically recommended for PAH patients due to substantial risks of mortality and morbidity. MethodsPulmonary hypertension was induced by SU5416 injection and 3-week hypoxic exposure (SuHx-PH) in rats, and hemodynamic and histological examinations were performed at 4weeks (SuHx-PH4W) and 8weeks (SuHx-PH8W) after SU5416 injection (n=7 each). Two groups of age-matched normal rats were also analyzed (n=7 each). Using an elastic tube with a 3-element Windkessel model, PA impedance was parameterized as pulmonary artery compliance (CP), peripheral resistance (RP), characteristic impedance (ZC), and transmission time (TD) in conducting arteries. Wave reflection was quantified as reflection gain at 0 Hz (Γgain) in the frequency domain, and as the ratio of peak backward pressure to peak forward pressure (KB/F) in the time domain. ResultsThe SuHx-PH groups demonstrated increased RP and ZC, and decreased CP and TD compared with normal groups. Γgain and KB/F were significantly higher in the SuHx-PH8W group than in the SuHx-PH4W group, and correlated strongly with a histological index of vascular wall thickening (R2=0.839, P<0.001 for Γgain and R2=0.775, P<0.001 for KB/F). ConclusionsEnhanced wave reflection caused by abnormal PA impedance correlates with histological remodeling, and may have a diagnostic value in clinical staging of PAH.

Natural reversal of pulmonary vascular remodeling and right ventricular remodeling in SU5416/hypoxia-treated Sprague-Dawley rats.

AimsPulmonary arterial hypertension (PAH) is a lethal disease and improved therapeutic strategies are needed. Increased pulmonary arterial pressure, due to vasoconstriction and vascular remodeling, causes right ventricle (RV) failure and death in patients. The treatment of Sprague-Dawley rats with SU5416 injection and exposure to chronic hypoxia for three weeks followed by maintenance in normoxia promote progressive and severe PAH with pathologic features that resemble human PAH. At 5–17 weeks after the SU5416 injection, PAH is developed with pulmonary vascular remodeling as well as RV hypertrophy and fibrosis. The present study investigated subsequent events that occur in these PAH animals.Methods & resultsAt 35 weeks after the SU5416 injection, rats still maintained high RV pressure, but pulmonary vascular remodeling was significantly reduced. Metabolomics analysis revealed that lungs of normal rats and rats from the 35-week time point had different metabolomics profiles. Despite the maintenance of high RV pressure, fibrosis was resolved at 35-weeks. Masson’s trichrome stain and Western blotting monitoring collagen 1 determined 12% fibrosis in the RV at 17-weeks, and this was decreased to 5% at 35-weeks. The level of myofibroblasts was elevated at 17-weeks and normalized at 35-weeks.ConclusionsThese results suggest that biological systems possess natural ways to resolve pulmonary and RV remodeling. The resolution of RV fibrosis appears to involve the reduction of myofibroblast-dependent collagen synthesis. Understanding these endogenous mechanisms should help improve therapeutic strategies to treat PAH and RV failure.

Endothelial cell-related autophagic pathways in Sugen/hypoxia-exposed pulmonary arterial hypertensive rats

Pulmonary arterial hypertension (PAH) is characterized by progressive obstructive remodeling of pulmonary arteries. However, no reports have described the causative role of the autophagic pathway in pulmonary vascular endothelial cell (EC) alterations associated with PAH. This study investigated the time-dependent role of the autophagic pathway in pulmonary vascular ECs and pulmonary vascular EC kinesis in a severe PAH rat model (Sugen/hypoxia rat) and evaluated whether timely induction of the autophagic pathway by rapamycin improves PAH. Hemodynamic and histological examinations as well as flow cytometry of pulmonary vascular EC-related autophagic pathways and pulmonary vascular EC kinetics in lung cell suspensions were performed. The time-dependent and therapeutic effects of rapamycin on the autophagic pathway were also assessed. Sugen/hypoxia rats treated with the vascular endothelial growth factor receptor blocker SU5416 showed increased right ventricular systolic pressure (RVSP) and numbers of obstructive vessels due to increased pulmonary vascular remodeling. The expression of the autophagic marker LC3 in ECs also changed in a time-dependent manner, in parallel with proliferation and apoptotic markers as assessed by flow cytometry. These results suggest the presence of cross talk between pulmonary vascular remodeling and the autophagic pathway, especially in small vascular lesions. Moreover, treatment of Sugen/hypoxia rats with rapamycin after SU5416 injection activated the autophagic pathway and improved the balance between cell proliferation and apoptosis in pulmonary vascular ECs to reduce RVSP and pulmonary vascular remodeling. These results suggested that the autophagic pathway can suppress PAH progression and that rapamycin-dependent activation of the autophagic pathway could ameliorate PAH.

Haemodynamic unloading reverses occlusive vascular lesions in severe pulmonary hypertension.

An important pathogenic mechanism in the development of idiopathic pulmonary arterial hypertension is hypothesized to be a cancer-like cellular proliferation independent of haemodynamics. However, because the vascular lesions are inseparably coupled with haemodynamic stress, the fate of the lesions is unknown when haemodynamic stress is eliminated. We applied left pulmonary artery banding to a rat model with advanced pulmonary hypertension to investigate the effects of decreased haemodynamic stress on occlusive vascular lesions. Rats were given an injection of the VEGF blocker Sugen5416 and exposed to 3 weeks of hypoxia plus an additional 7 weeks of normoxia (total 10 weeks) (SU/Hx/Nx rats). The banding surgery to reduce haemodynamic stress to the left lung was done at 1 week prior to (preventive) or 5 weeks after (reversal) the SU5416 injection. All SU/Hx/Nx-exposed rats developed severe pulmonary hypertension and right ventricular hypertrophy. Histological analyses showed that the non-banded right lungs developed occlusive lesions including plexiform lesions with marked perivascular cell accumulation. In contrast, banding the left pulmonary artery not only prevented the development of but also reversed the established occlusive lesions as well as perivascular inflammation in the left lungs. Our results indicate that haemodynamic stress is prerequisite to the development and progression of occlusive neointimal lesions in this rat model of severe pulmonary hypertension. We conclude that perivascular inflammation and occlusive neointimal arteriopathy are driven by haemodynamic stress.