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Right ventricular (RV) function is the predominant determinant of survival in patients with pulmonary arterial hypertension (PAH). In preclinical models, pharmacological activation of BMP (bone morphogenetic protein) signaling with FK506 (tacrolimus) improved RV function by decreasing RV afterload. FK506 therapy further stabilized three patients with end-stage PAH. Whether FK506 has direct effects on the pressure-overloaded right ventricle is yet unknown. We hypothesized that increasing cardiac BMP signaling with FK506 improves RV structure and function in a model of fixed RV afterload after pulmonary artery banding (PAB). Direct cardiac effects of FK506 on the microvasculature and RV fibrosis were studied after surgical PAB in wild-type and heterozygous Bmpr2 mutant mice. RV function and strain were assessed longitudinally via cardiac magnetic resonance imaging during continuous FK506 infusion. Genetic lineage tracing of endothelial cells (ECs) was performed to assess the contribution of ECs to fibrosis. Molecular mechanistic studies were performed in human cardiac fibroblasts and ECs. In mice, low BMP signaling in the right ventricle exaggerated PAB-induced RV fibrosis. FK506 therapy restored cardiac BMP signaling, reduced RV fibrosis in a BMP-dependent manner independent from its immunosuppressive effect, preserved RV capillarization, and improved RV function and strain over the time course of disease. Endothelial mesenchymal transition was a rare event and did not significantly contribute to cardiac fibrosis after PAB. Mechanistically, FK506 required ALK1 in human cardiac fibroblasts as a BMPR2 co-receptor to reduce TGFβ1-induced proliferation and collagen production. Our study demonstrates that increasing cardiac BMP signaling with FK506 improves RV structure and function independent from its previously described beneficial effects on pulmonary vascular remodeling.

In pulmonary arterial hypertension (PAH), progressive structural remodeling accounts for the pulmonary vasculopathy including the obliteration of the lung vasculature that causes an increase in vascular resistance and mean blood pressure in the pulmonary arteries ultimately leading to right heart failure-mediated death. Deciphering the molecular details of aberrant signaling of pulmonary vascular cells in PAH is fundamental for the development of new therapeutic strategies. We aimed to identify kinases as new potential drug targets that are dysregulated in PAH by means of a peptide-based kinase activity assay. We performed a tyrosine kinase-dependent phosphorylation assay using 144 selected microarrayed substrate peptides. The differential signature of phosphopeptides was used to predict alterations in tyrosine kinase activities in human pulmonary arterial smooth muscle cells (HPASMCs) from patients with idiopathic PAH (IPAH) compared with healthy control cells. Thereby, we observed an overactivation and an increased expression of Jak2 (Janus kinase 2) in HPASMCs from patients with IPAH as compared with controls.

Pulmonary hypertension (PH) is a progressive pulmonary vasculopathy that causes chronic right ventricular pressure overload and often leads to right ventricular failure. Various kinase inhibitors have been studied in the setting of PH and either improved or worsened the disease, highlighting the importance of understanding the specific role of the respective kinases in a spatiotemporal cellular context. In this review, we will summarize the knowledge on the role of kinases in PH and focus on druggable targets for which certain criteria are met: (a) deregulation of the kinase in PH; (b) small-molecule inhibitors are available (e.g. from the oncology field); (c) preclinical studies have shown their efficacy in PH models; and (d) when available, therapeutic exploitation in human PH has been initiated. Along this line, clinical considerations such as personalized medicine approaches to predict therapy response and adverse side events such as cardiotoxicity together with their clinical management are discussed. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.1/issuetoc.

Aims The temporal sequence of events underlying functional right ventricular (RV) recovery after improvement of pulmonary hypertension-associated pressure overload is unknown. We sought to establish a novel mouse model of gradual RV recovery from pressure overload and use it to delineate RV reverse-remodelling events. Methods and results Surgical pulmonary artery banding (PAB) around a 26-G needle induced RV dysfunction with increased RV pressures, reduced exercise capacity and caused liver congestion, hypertrophic, fibrotic, and vascular myocardial remodelling within 5 weeks of chronic RV pressure overload in mice. Gradual reduction of the afterload burden through PA band absorption (de-PAB)—after RV dysfunction and structural remodelling were established—initiated recovery of RV function (cardiac output and exercise capacity) along with rapid normalization in RV hypertrophy (RV/left ventricular + S and cardiomyocyte area) and RV pressures (right ventricular systolic pressure). RV fibrotic (collagen, elastic fibres, and vimentin+ fibroblasts) and vascular (capillary density) remodelling were equally reversible; however, reversal occurred at a later timepoint after de-PAB, when RV function was already completely restored. Microarray gene expression (ClariomS, Thermo Fisher Scientific, Waltham, MA, USA) along with gene ontology analyses in RV tissues revealed growth factors, immune modulators, and apoptosis mediators as major cellular components underlying functional RV recovery. Conclusion We established a novel gradual de-PAB mouse model and used it to demonstrate that established pulmonary hypertension-associated RV dysfunction is fully reversible. Mechanistically, we link functional RV improvement to hypertrophic normalization that precedes fibrotic and vascular reverse-remodelling events.

Rationale: Pulmonary arterial hypertension (PAH) is characterized by progressive narrowing of pulmonary arteries, resulting in right heart failure and death. BMPR2 (bone morphogenetic protein receptor type 2) mutations account for most familial PAH forms whereas reduced BMPR2 is present in many idiopathic PAH forms, suggesting dysfunctional BMPR2 signaling to be a key feature of PAH. Modulating BMPR2 signaling is therapeutically promising, yet how BMPR2 is downregulated in PAH is unclear. Objectives: We intended to identify and pharmaceutically target BMPR2 modifier genes to improve PAH. Methods: We combined siRNA high-throughput screening of >20,000 genes with a multicohort analysis of publicly available PAH RNA expression data to identify clinically relevant BMPR2 modifiers. After confirming gene dysregulation in tissue from patients with PAH, we determined the functional roles of BMPR2 modifiers in vitro and tested the repurposed drug enzastaurin for its propensity to improve experimental pulmonary hypertension (PH). Measurements and Main Results: We discovered FHIT (fragile histidine triad) as a novel BMPR2 modifier. BMPR2 and FHIT expression were reduced in patients with PAH. FHIT reductions were associated with endothelial and smooth muscle cell dysfunction, rescued by enzastaurin through a dual mechanism: upregulation of FHIT as well as miR17-5 repression. Fhit(−/−) mice had exaggerated hypoxic PH and failed to recover in normoxia. Enzastaurin reversed PH in the Sugen5416/hypoxia/normoxia rat model, by improving right ventricular systolic pressure, right ventricular hypertrophy, cardiac fibrosis, and vascular remodeling. Conclusions: This study highlights the importance of the novel BMPR2 modifier FHIT in PH and the clinical value of the repurposed drug enzastaurin as a potential novel therapeutic strategy to improve PAH.

Background The role of interventricular mechanics in pediatric pulmonary arterial hypertension (PAH) and its relation to right ventricular (RV) dysfunction has been largely overlooked. Here, we characterize the impact of maintained pressure overload in the RV–pulmonary artery (PA) axis on myocardial strain and left ventricular (LV) mechanics in pediatric PAH patients in comparison to a preclinical PA-banding (PAB) mouse model. We hypothesize that the PAB mouse model mimics important aspects of interventricular mechanics of pediatric PAH and may be beneficial as a surrogate model for some longitudinal and interventional studies not possible in children. Methods Balanced steady-state free precession (bSSFP) cardiovascular magnetic resonance (CMR) images of 18 PAH and 17 healthy (control) pediatric subjects were retrospectively analyzed using CMR feature-tracking (FT) software to compute measurements of myocardial strain. Furthermore, myocardial tagged-CMR images were also analyzed for each subject using harmonic phase flow analysis to derive LV torsion rate. Within 48 h of CMR, PAH patients underwent right heart catheterization (RHC) for measurement of PA/RV pressures, and to compute RV end-systolic elastance (RV_Ees, a measure of load-independent contractility). Surgical PAB was performed on mice to induce RV pressure overload and myocardial remodeling. bSSFP-CMR, tagged CMR, and intra-cardiac catheterization were performed on 12 PAB and 9 control mice (Sham) 7 weeks after surgery with identical post-processing as in the aforementioned patient studies. RV_Ees was assessed via the single beat method. Results LV torsion rate was significantly reduced under hypertensive conditions in both PAB mice (p = 0.004) and pediatric PAH patients (p es in PAB (r = 0.91, p = 0.05) and PAH subjects (r = 0.51, p = 0.04). In order to compare combined metrics of LV torsion rate and strain parameters principal component analysis (PCA) was used. PCA revealed grouping of PAH patients with PAB mice and control subjects with Sham mice. Similar to LV torsion rate, LV global peak circumferential, radial, and longitudinal strain were significantly (p Conclusions The PAB mouse model resembles PAH-associated myocardial mechanics and may provide a potential model to study mechanisms of RV/LV interdependency.

Background: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by profound vascular remodeling in which pulmonary arteries narrow because of medial thickening and occlusion by neointimal lesions, resulting in elevated pulmonary vascular resistance and right heart failure. Therapies targeting the neointima would represent a significant advance in PAH treatment; however, our understanding of the cellular events driving neointima formation, and the molecular pathways that control them, remains limited. Methods: We comprehensively map the stepwise remodeling of pulmonary arteries in a robust, chronic inflammatory mouse model of pulmonary hypertension. This model demonstrates pathological features of the human disease, including increased right ventricular pressures, medial thickening, neointimal lesion formation, elastin breakdown, increased anastomosis within the bronchial circulation, and perivascular inflammation. Using genetic lineage tracing, clonal analysis, multiplexed in situ hybridization, immunostaining, deep confocal imaging, and staged pharmacological inhibition, we define the cell behaviors underlying each stage of vascular remodeling and identify a pathway required for neointima formation. Results: Neointima arises from smooth muscle cells (SMCs) and not endothelium. Medial SMCs proliferate broadly to thicken the media, after which a small number of SMCs are selected to establish the neointima. These neointimal founder cells subsequently undergoing massive clonal expansion to form occlusive neointimal lesions. The normal pulmonary artery SMC population is heterogeneous, and we identify a Notch3-marked minority subset of SMCs as the major neointimal cell of origin. Notch signaling is specifically required for the selection of neointimal founder cells, and Notch inhibition significantly improves pulmonary artery pressure in animals with pulmonary hypertension. Conclusions: This work describes the first nongenetically driven murine model of pulmonary hypertension (PH) that generates robust and diffuse occlusive neointimal lesions across the pulmonary vascular bed and does so in a stereotyped timeframe. We uncover distinct cellular and molecular mechanisms underlying medial thickening and neointima formation and highlight novel transcriptional, behavioral, and pathogenic heterogeneity within pulmonary artery SMCs. In this model, inflammation is sufficient to generate characteristic vascular pathologies and physiological measures of human PAH. We hope that identifying the molecular cues regulating each stage of vascular remodeling will open new avenues for therapeutic advancements in the treatment of PAH.

Pulmonary hypertension (PH) results in right ventricular (RV) pressure overload and eventual failure. Current research efforts have focused on the RV while overlooking the left ventricle (LV), which is responsible for mechanically assisting the RV during contraction. The objective of this study is to evaluate the biomechanical and gene expression changes occurring in the LV due to RV pressure overload in a mouse model. Nine male mice were divided into two groups: (a) pulmonary arterial banding (PAB, N = 4) and (b) sham surgery (Sham, N = 5). Tagged and steady‐state free precision cardiac MRI was performed on each mouse at 1, 4, and 7 weeks after surgery. At/week7, the mice were euthanized following right/left heart catheterization with RV/LV tissue harvested for histology and gene expression (using RT‐PCR) studies. Compared to Sham mice, the PAB group revealed a significantly decreased LV and RV ejection fraction, and LV maximum torsion and torsion rate, within the first week after banding. In the PAB group, there was also a slight but significant increase in LV perivascular fibrosis, which suggests elevated myocardial stress. LV fibrosis was also accompanied with changes in gene expression in the hypertensive group, which was correlated with LV contractile mechanics. In fact, principal component (PC) analysis of LV gene expression effectively separated Sham and PAB mice along PC2. Changes in LV contractile mechanics were also significantly correlated with unfavorable changes in RV contractile mechanics, but a direct causal relationship was not established. In conclusion, a purely biomechanical insult of RV pressure overload resulted in biomechanical and transcriptional changes in both the RV and LV. Given that the RV relies on the LV for contractile energy assistance, considering the LV could provide prognostic and therapeutic targets for treating RV failure in PH., Right ventricular (RV) failure is a common endpoint in pulmonary hypertension. While most clinical and research efforts are focused on the RV, our research shows that the left ventricle undergoes bio‐mechanical and gene‐expression changes in response to RV pressure overload.

Progression of pulmonary arterial hypertension (PAH) is associated with pathological remodeling of the pulmonary vasculature and the right ventricle (RV). Oxidative stress drives the remodeling process through activation of MAPKs (mitogen-activated protein kinases), which stimulate apoptosis, inflammation, and fibrosis.We investigated whether pharmacological inhibition of the redox-sensitive apical MAPK, ASK1 (apoptosis signal-regulating kinase 1), can halt the progression of pulmonary vascular and RV remodeling.A selective, orally available ASK1 inhibitor, GS-444217, was administered to two preclinical rat models of PAH (monocrotaline and Sugen/hypoxia), a murine model of RV pressure overload induced by pulmonary artery banding, and cellular models.Oral administration of GS-444217 dose dependently reduced pulmonary arterial pressure and reduced RV hypertrophy in PAH models. The therapeutic efficacy of GS-444217 was associated with reduced ASK1 phosphorylation, reduced muscularization of the pulmonary arteries, and reduced fibrotic gene expression in the RV. Importantly, efficacy was observed when GS-444217 was administered to animals with established disease and also directly reduced cardiac fibrosis and improved cardiac function in a model of isolated RV pressure overload. In cellular models, GS-444217 reduced phosphorylation of p38 and JNK (c-Jun N-terminal kinase) induced by adenoviral overexpression of ASK1 in rat cardiomyocytes and reduced activation/migration of primary mouse cardiac fibroblasts and human pulmonary adventitial fibroblasts derived from patients with PAH.ASK1 inhibition reduced pathological remodeling of the pulmonary vasculature and the right ventricle and halted progression of pulmonary hypertension in rodent models. These preclinical data inform the first description of a causal role of ASK1 in PAH disease pathogenesis.

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)-b-induced collagen production as well as stress fiber formation. Moreover, p38 MAPK inhibition/depletion suppressed TGF-b-induced SMAD2/3 phosphorylation and myocardin-related transcription factor A (MRTF-A) nuclear translocation, and prevented TGF-b-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.

Assessment of right ventricular (RV) function in rodents is a challenge due to the complex RV anatomy and structure. Subsequently, the best characterization of RV function is achieved by accurate cardiovascular phenotyping, involving a combination of non-invasive imaging and intra-cardiac pressure-volume measurements. We sought to investigate the feasibility of two complementary phenotyping techniques for the evaluation of RV function in an experimental mouse model of sustained RV pressure overload. Mice underwent either Sham surgery (n = 5) or pulmonary artery banding (PAB) (n = 8) to induce isolated RV pressure overload. After three weeks indices of RV function were assessed by echocardiography (Vevo2100) and closed chest-derived invasive pressure-volume measurements (PVR-1030). PAB resulted in RV hypertrophy and dilatation accompanied by systolic and diastolic dysfunction. Invasive RV hemodynamic measurements demonstrate an increased end-systolic as well as arterial elastance after PAB as compared to sham, resulting in ventricular-arterial decoupling. Regression analysis revealed that TAPSE is rather correlated with ventricular-arterial coupling (r² = 0.77, P = 0.002) than RV contractility (r² = -0.61, P = 0.07). Furthermore, IVRT/RR and E/E' correlate well with RV end-diastolic pressure (r² = 0.87, P = 0.0001 and r² = 0.82, P = 0.0009; respectively). Commonly used indices of systolic RV function are associated with RV-arterial coupling rather than contractility, while diastolic indices are interrelated with end-diastolic pressure where there is maintained pressure overload. This article is protected by copyright. All rights reserved.

The ability of the right ventricle to compensate pressure overload determines survival in pulmonary arterial hypertension (PAH). Nitric oxide (NO) reduces the right ventricular afterload through pulmonary vasodilation, but excessive NO amounts cause oxidative stress. Oxidative stress drives remodeling of pulmonary arteries and the right ventricle. In the present study, we hypothesized that nitric oxide synthase 2 (NOS2) induction leads to excessive NO amounts that contribute to oxidative stress and impair right ventricular adaptation to PAH. We used a surgical pulmonary artery banding (PAB) mouse model in which right ventricular dysfunction and remodeling occur independently of changes in the pulmonary vasculature. Three weeks after PAB, NOS2 expression was increased twofold in the hypertrophied right ventricle on transcript and protein levels together with increased NO production. Histomorphology localized NOS2 in interstitial and perivascular cardiac fibroblasts after PAB, which was confirmed by cell isolation experiments. In the hypertrophied right ventricle, NOS2 induction was accompanied by an increased formation of reactive oxidants blocked by ex vivo NOS inhibition. We show that reactive oxidant formation in the hypertrophied right ventricle is in part NOS2 dependent (in NOS2-deficient mice [NOS2