Increased MAO-A Activity Promotes Progression of Pulmonary Arterial Hypertension.

Monoamine oxidases (MAOs), a class of enzymes bound to the outer mitochondrial membrane, are important sources of reactive oxygen species. Increased MAO-A activity in endothelial cells and cardiomyocytes contributes to vascular dysfunction and progression of left heart failure. We hypothesized that inhibition of MAO-A can be used to treat pulmonary arterial hypertension (PAH) and right ventricular (RV) failure. MAO-A levels in lung and RV samples from patients with PAH were compared with levels in samples from donors without PAH. Experimental PAH was induced in male Sprague-Dawley rats by using Sugen 5416 and hypoxia (SuHx), and RV failure was induced in male Wistar rats by using pulmonary trunk banding (PTB). Animals were randomized to receive either saline or the MAO-A inhibitor clorgyline at 10 mg/kg. Echocardiography and RV catheterization were performed, and heart and lung tissues were collected for further analysis. We found increased MAO-A expression in the pulmonary vasculature of patients with PAH and in experimental experimental PAH induced by SuHx. Cardiac MAO-A expression and activity was increased in SuHx- and PTB-induced RV failure. Clorgyline treatment reduced RV afterload and pulmonary vascular remodeling in SuHx rats through reduced pulmonary vascular proliferation and oxidative stress. Moreover, clorgyline improved RV stiffness and relaxation and reversed RV hypertrophy in SuHx rats. In PTB rats, clorgyline had no direct clorgyline had no direct effect on the right ventricle effect. Our study reveals the role of MAO-A in the progression of PAH. Collectively, these findings indicated that MAO-A may be involved in pulmonary vascular remodeling and consecutive RV failure.

Contribution of Impaired Parasympathetic Activity to Right Ventricular Dysfunction and Pulmonary Vascular Remodeling in Pulmonary Arterial Hypertension.

BACKGROUND: The beneficial effects of parasympathetic stimulation have been reported in left heart failure, but whether it would be beneficial for pulmonary arterial hypertension (PAH) remains to be explored. Here, we investigated the relationship between parasympathetic activity and right ventricular (RV) function in patients with PAH, and the potential therapeutic effects of pyridostigmine (PYR), an oral drug stimulating the parasympathetic activity through acetylcholinesterase inhibition, in experimental pulmonary hypertension (PH). METHODS: Heart rate recovery after a maximal cardiopulmonary exercise test was used as a surrogate for parasympathetic activity. RV ejection fraction was assessed in 112 patients with PAH. Expression of nicotinic (α-7 nicotinic acetylcholine receptor) and muscarinic (muscarinic acetylcholine type 2 receptor) receptors, and acetylcholinesterase activity were evaluated in RV (n=11) and lungs (n=7) from patients with PAH undergoing heart/lung transplantation and compared with tissue obtained from controls. In addition, we investigated the effects of PYR (40 mg/kg per day) in experimental PH. PH was induced in male rats by SU5416 (25 mg/kg subcutaneously) injection followed by 4 weeks of hypoxia. In a subgroup, sympathetic/parasympathetic modulation was assessed by power spectral analysis. At week 6, PH status was confirmed by echocardiography, and rats were randomly assigned to vehicle or treatment (both n=12). At the end of the study, echocardiography was repeated, with additional RV pressure-volume measurements, along with lung, RV histological, and protein analyses. RESULTS: Patients with PAH with lower RV ejection fraction (<41%) had a significantly reduced heart rate recovery in comparison with patients with higher RV ejection fraction. In PAH RV samples, α-7 nicotinic acetylcholine receptor was increased and acetylcholinesterase activity was reduced versus controls. No difference in muscarinic acetylcholine type 2 receptor expression was observed. Chronic PYR treatment in PH rats normalized the cardiovascular autonomic function, demonstrated by an increase in parasympathetic activity and baroreflex sensitivity. PYR improved survival, increased RV contractility, and reduced RV stiffness, RV hypertrophy, RV fibrosis, RV inflammation, and RV α-7 nicotinic acetylcholine receptor and muscarinic acetylcholine type 2 receptor expression, as well. Furthermore, PYR reduced pulmonary vascular resistance, RV afterload, and pulmonary vascular remodeling, which was associated with reduced local and systemic inflammation. CONCLUSIONS: RV dysfunction is associated with reduced systemic parasympathetic activity in patients with PAH, with an inadequate adaptive response of the cholinergic system in the RV. Enhancing parasympathetic activity by PYR improved survival, RV function, and pulmonary vascular remodeling in experimental PH.

Prevention of progression of pulmonary hypertension by the Nur77 agonist 6-mercaptopurine: role of BMP signalling.

Pulmonary arterial hypertension (PAH) is a progressive fatal disease characterised by abnormal remodelling of pulmonary vessels, leading to increased vascular resistance and right ventricle failure. This abnormal vascular remodelling is associated with endothelial cell dysfunction, increased proliferation of smooth muscle cells, inflammation and impaired bone morphogenetic protein (BMP) signalling. Orphan nuclear receptor Nur77 is a key regulator of proliferation and inflammation in vascular cells, but its role in impaired BMP signalling and vascular remodelling in PAH is unknown.We hypothesised that activation of Nur77 by 6-mercaptopurine (6-MP) would improve PAH by inhibiting endothelial cell dysfunction and vascular remodelling.Nur77 expression is decreased in cultured pulmonary microvascular endothelial cells (MVECs) and lungs of PAH patients. Nur77 significantly increased BMP signalling and strongly decreased proliferation and inflammation in MVECs. In addition, conditioned medium from PAH MVECs overexpressing Nur77 inhibited the growth of healthy smooth muscle cells. Pharmacological activation of Nur77 by 6-MP markedly restored MVEC function by normalising proliferation, inflammation and BMP signalling. Finally, 6-MP prevented and reversed abnormal vascular remodelling and right ventricle hypertrophy in the Sugen/hypoxia rat model of severe angioproliferative PAH.Our data demonstrate that Nur77 is a critical modulator in PAH by inhibiting vascular remodelling and increasing BMP signalling, and activation of Nur77 could be a promising option for the treatment of PAH.

Reduced exercise capacity occurs before intrinsic skeletal muscle dysfunction in experimental rat models of pulmonary hypertension.

Reduced exercise capacity in pulmonary hypertension (PH) significantly impacts quality of life. However, the cause of reduced exercise capacity in PH remains unclear. The objective of this study was to investigate whether intrinsic skeletal muscle changes are causative in reduced exercise capacity in PH using preclinical PH rat models with different PH severity. PH was induced in adult Sprague-Dawley (SD) or Fischer (CDF) rats with one dose of SU5416 (20 mg/kg) injection, followed by 3 weeks of hypoxia and additional 0-4 weeks of normoxia exposure. Control s rats were injected with vehicle and housed in normoxia. Echocardiography was performed to assess cardiac function. Exercise capacity was assessed by VO2 max. Skeletal muscle structural changes (atrophy, fiber type switching, and capillary density), mitochondrial function, isometric force, and fatigue profile were assessed. In SD rats, right ventricular systolic dysfunction is associated with reduced exercise capacity in PH rats at 7-week timepoint in comparison to control rats, while no changes were observed in skeletal muscle structure, mitochondrial function, isometric force, or fatigue profile. CDF rats at 4-week timepoint developed a more severe PH and, in addition to right ventricular dysfunction, the reduced exercise capacity in these rats is associated with skeletal muscle atrophy; however, mitochondrial function, isometric force, and fatigue profile in skeletal muscle remain unchanged. Our data suggest that cardiopulmonary impairments in PH are the primary cause of reduced exercise capacity, which occurs before intrinsic skeletal muscle dysfunction.

Right Ventricular Architectural Remodeling and Functional Adaptation in Pulmonary Hypertension.

BACKGROUND: Global indices of right ventricle (RV) function provide limited insights into mechanisms underlying RV remodeling in pulmonary hypertension (PH). While RV myocardial architectural remodeling has been observed in PH, its effect on RV adaptation is poorly understood. METHODS: Hemodynamic assessments were performed in 2 rodent models of PH. RV free wall myoarchitecture was quantified using generalized Q-space imaging and tractography analyses. Computational models were developed to predict RV wall strains. Data from animal studies were analyzed to determine the correlations between hemodynamic measurements, RV strains, and structural measures. RESULTS: In contrast to the PH rats with severe RV maladaptation, PH rats with mild RV maladaptation showed a decrease in helical range of fiber orientation in the RV free wall (139º versus 97º; P=0.029), preserved global circumferential strain, and exhibited less reduction in right ventricular-pulmonary arterial coupling (0.029 versus 0.017 mm/mm Hg; P=0.037). Helical range correlated positively with coupling (P=0.036) and stroke volume index (P<0.01). Coupling correlated with global circumferential strain (P<0.01) and global radial strain (P<0.01) but not global longitudinal strain. CONCLUSIONS: Data analysis suggests that adaptive RV architectural remodeling could improve RV function in PH. Our findings suggest the need to assess RV architecture within routine screenings of PH patients to improve our understanding of its prognostic and therapeutic significance in PH.

Geranylgeranylacetone reduces cardiomyocyte stiffness and attenuates diastolic dysfunction in a rat model of cardiometabolic syndrome.

Titin-dependent stiffening of cardiomyocytes is a significant contributor to left ventricular (LV) diastolic dysfunction in heart failure with preserved LV ejection fraction (HFpEF). Small heat shock proteins (HSPs), such as HSPB5 and HSPB1, protect titin and administration of HSPB5 in vitro lowers cardiomyocyte stiffness in pressure-overload hypertrophy. In humans, oral treatment with geranylgeranylacetone (GGA) increases myocardial HSP expression, but the functional implications are unknown. Our objective was to investigate whether oral GGA treatment lowers cardiomyocyte stiffness and attenuates LV diastolic dysfunction in a rat model of the cardiometabolic syndrome. Twenty-one-week-old male lean (n = 10) and obese (n = 20) ZSF1 rats were studied, and obese rats were randomized to receive GGA (200 mg/kg/day) or vehicle by oral gavage for 4 weeks. Echocardiography and cardiac catheterization were performed before sacrifice at 25 weeks of age. Titin-based stiffness (Fpassive ) was determined by force measurements in relaxing solution with 100 nM [Ca2+ ] in permeabilized cardiomyocytes at sarcomere lengths (SL) ranging from 1.8 to 2.4 µm. In obese ZSF1 rats, GGA reduced isovolumic relaxation time of the LV without affecting blood pressure, EF or LV weight. In cardiomyocytes, GGA increased myofilament-bound HSPB5 and HSPB1 expression. Vehicle-treated obese rats exhibited higher cardiomyocyte stiffness at all SLs compared to lean rats, while GGA reduced stiffness at SL 2.0 µm. In obese ZSF1 rats, oral GGA treatment improves cardiomyocyte stiffness by increasing myofilament-bound HSPB1 and HSPB5. GGA could represent a potential novel therapy for the early stage of diastolic dysfunction in the cardiometabolic syndrome.

α7 Nicotinic acetylcholine receptor mediates right ventricular fibrosis and diastolic dysfunction in pulmonary hypertension.

Right ventricular (RV) fibrosis is a key feature of maladaptive RV hypertrophy and dysfunction and is associated with poor outcomes in pulmonary hypertension (PH). However, mechanisms and therapeutic strategies to mitigate RV fibrosis remain unrealized. Previously, we identified that cardiac fibroblast α7 nicotinic acetylcholine receptor (α7 nAChR) drives smoking-induced RV fibrosis. Here, we sought to define the role of α7 nAChR in RV dysfunction and fibrosis in the settings of RV pressure overload as seen in PH. We show that RV tissue from PH patients has increased collagen content and ACh expression. Using an experimental rat model of PH, we demonstrate that RV fibrosis and dysfunction are associated with increases in ACh and α7 nAChR expression in the RV but not in the left ventricle (LV). In vitro studies show that α7 nAChR activation leads to an increase in adult ventricular fibroblast proliferation and collagen content mediated by a Ca2+/epidermal growth factor receptor (EGFR) signaling mechanism. Pharmacological antagonism of nAChR decreases RV collagen content and improves RV function in the PH model. Furthermore, mice lacking α7 nAChR exhibit improved RV diastolic function and have lower RV collagen content in response to persistently increased RV afterload, compared with WT controls. These finding indicate that enhanced α7 nAChR signaling is an important mechanism underlying RV fibrosis and dysfunction, and targeted inhibition of α7 nAChR is a potentially novel therapeutic strategy in the setting of increased RV afterload.

The BMP Receptor 2 in Pulmonary Arterial Hypertension: When and Where the Animal Model Matches the Patient.

Background: Mutations in bone morphogenetic protein receptor type II (BMPR2) are leading to the development of hereditary pulmonary arterial hypertension (PAH). In non-hereditary forms of PAH, perturbations in the transforming growth factor-ß (TGF-ß)/BMP-axis are believed to cause deficient BMPR2 signaling by changes in receptor expression, the activity of the receptor and/or downstream signaling. To date, BMPR2 expression and its activity in the lungs of patients with non-hereditary PAH is poorly characterized. In recent decades, different animal models have been used to understand the role of BMPR2 signaling in PAH pathophysiology. Specifically, the monocrotaline (MCT) and Sugen-Hypoxia (SuHx) models are extensively used in interventional studies to examine if restoring BMPR2 signaling results in PAH disease reversal. While PAH is assumed to develop in patients over months or years, pulmonary hypertension in experimental animal models develops in days or weeks. It is therefore likely that modifications in BMP and TGF-ß signaling in these models do not fully recapitulate those in patients. In order to determine the translational potential of the MCT and SuHx models, we analyzed the BMPR2 expression and activity in the lungs of rats with experimentally induced PAH and compared this to the BMPR2 expression and activity in the lungs of PAH patients. Methods: the BMPR2 expression was analyzed by Western blot analysis and immunofluorescence (IF) microscopy to determine the quantity and localization of the receptor in the lung tissue from normal control subjects and patients with hereditary or idiopathic PAH, as well as in the lungs of control rats and rats with MCT or SuHx-induced PAH. The activation of the BMP pathway was analyzed by determining the level and localization of phosphorylated Smad1/5/8 (pSmad 1/5/8), a downstream mediator of canonical BMPR2 signaling. Results: While BMPR2 and pSmad 1/5/8 expression levels were unaltered in whole lung lysates/homogenates from patients with hereditary and idiopathic PAH, IF analysis showed that BMPR2 and pSmad 1/5/8 levels were markedly decreased in the pulmonary vessels of both PAH patient groups. Whole lung BMPR2 expression was variable in the two PAH rat models, while in both experimental models the expression of BMPR2 in the lung vasculature was increased. However, in the human PAH lungs, the expression of pSmad 1/5/8 was downregulated in the lung vasculature of both experimental models. Conclusion: BMPR2 receptor expression and downstream signaling is reduced in the lung vasculature of patients with idiopathic and hereditary PAH, which cannot be appreciated when using human whole lung lysates. Despite increased BMPR2 expression in the lung vasculature, the MCT and SuHx rat models did develop PAH and impaired downstream BMPR2-Smad signaling similar to our findings in the human lung.

Nintedanib improves cardiac fibrosis but leaves pulmonary vascular remodelling unaltered in experimental pulmonary hypertension.

Aims: Pulmonary arterial hypertension (PAH) is associated with increased levels of circulating growth factors and corresponding receptors such as platelet derived growth factor, fibroblast growth factor and vascular endothelial growth factor. Nintedanib, a tyrosine kinase inhibitor targeting primarily these receptors, is approved for the treatment of patients with idiopathic pulmonary fibrosis. Our objective was to examine the effect of nintedanib on proliferation of human pulmonary microvascular endothelial cells (MVEC) and assess its effects in rats with advanced experimental pulmonary hypertension (PH). Methods and results: Proliferation was assessed in control and PAH MVEC exposed to nintedanib. PH was induced in rats by subcutaneous injection of Sugen (SU5416) and subsequent exposure to 10% hypoxia for 4 weeks (SuHx model). Four weeks after re-exposure to normoxia, nintedanib was administered once daily for 3 weeks. Effects of the treatment were assessed with echocardiography, right heart catheterization, and histological analysis of the heart and lungs. Changes in extracellular matrix production was assessed in human cardiac fibroblasts stimulated with nintedanib. Decreased proliferation with nintedanib was observed in control MVEC, but not in PAH patient derived MVEC. Nintedanib treatment did not affect right ventricular (RV) systolic pressure or total pulmonary resistance index in SuHx rats and had no effects on pulmonary vascular remodelling. However, despite unaltered pressure overload, the right ventricle showed less dilatation and decreased fibrosis, hypertrophy, and collagen type III with nintedanib treatment. This could be explained by less fibronectin production by cardiac fibroblasts exposed to nintedanib. Conclusion: Nintedanib inhibits proliferation of pulmonary MVECs from controls, but not from PAH patients. While in rats with experimental PH nintedanib has no effects on the pulmonary vascular pathology, it has favourable effects on RV remodelling.

Renal Denervation Reduces Pulmonary Vascular Remodeling and Right Ventricular Diastolic Stiffness in Experimental Pulmonary Hypertension.

Neurohormonal overactivation plays an important role in pulmonary hypertension (PH). In this context, renal denervation, which aims to inhibit the neurohormonal systems, may be a promising adjunct therapy in PH. In this proof-of-concept study, we have demonstrated in 2 experimental models of PH that renal denervation delayed disease progression, reduced pulmonary vascular remodeling, lowered right ventricular afterload, and decreased right ventricular diastolic stiffness, most likely by suppression of the renin-angiotensin-aldosterone system.

Right Ventricular Myocardial Stiffness in Experimental Pulmonary Arterial Hypertension: Relative Contribution of Fibrosis and Myofibril Stiffness.

BACKGROUND: The purpose of this study was to determine the relative contribution of fibrosis-mediated and myofibril-mediated stiffness in rats with mild and severe right ventricular (RV) dysfunction. METHODS AND RESULTS: By performing pulmonary artery banding of different diameters for 7 weeks, mild RV dysfunction (Ø=0.6 mm) and severe RV dysfunction (Ø=0.5 mm) were induced in rats. The relative contribution of fibrosis- and myofibril-mediated RV stiffness was determined in RV trabecular strips. Total myocardial stiffness was increased in trabeculae from both mild and severe RV dysfunction in comparison to controls. In severe RV dysfunction, increased RV myocardial stiffness was explained by both increased fibrosis-mediated stiffness and increased myofibril-mediated stiffness, whereas in mild RV dysfunction, only myofibril-mediated stiffness was increased in comparison to control. Histological analyses revealed that RV fibrosis gradually increased with severity of RV dysfunction, whereas the ratio of collagen I/III expression was only elevated in severe RV dysfunction. Stiffness measurements in single membrane-permeabilized RV cardiomyocytes demonstrated a gradual increase in RV myofibril stiffness, which was partially restored by protein kinase A in both mild and severe RV dysfunction. Increased expression of compliant titin isoforms was observed only in mild RV dysfunction, whereas titin phosphorylation was reduced in both mild and severe RV dysfunction. CONCLUSIONS: RV myocardial stiffness is increased in rats with mild and severe RV dysfunction. In mild RV dysfunction, stiffness is mainly determined by increased myofibril stiffness. In severe RV dysfunction, both myofibril- and fibrosis-mediated stiffness contribute to increased RV myocardial stiffness.