Sex-dependent Remodeling of Right-Ventricular Function in a Rat Model of Pulmonary Arterial Hypertension.

Right-ventricular (RV) function is an important prognostic indicator for pulmonary arterial hypertension (PAH), a vasculopathy that primarily and disproportionally affects women with distinct pre- and post-menopausal clinical outcomes. However, most animal studies have overlooked the impact of sex and ovarian hormones on RV remodeling in PAH. Here, we combined invasive measurements of RV hemodynamics and morphology with computational models of RV biomechanics in sugen-hypoxia (SuHx) treated male, ovary-intact female, and ovariectomized female rats. Despite similar pressure overload levels, SuHx induced increases in end-diastolic elastance and passive myocardial stiffening, notably in male SuHx animals, corresponding to elevated diastolic intracellular calcium. Increases in end-systolic chamber elastance were largely explained by myocardial hypertrophy in male and ovary-intact female rats, whereas ovariectomized females exhibited contractility recruitment via calcium transient augmentation. Ovary-intact female rats primarily responded with hypertrophy, showing fewer myocardial mechanical alterations and less stiffening. These findings highlight sex-related RV remodeling differences in rats, affecting systolic and diastolic RV function in PAH.

Sphingosine Kinase 1 Deficiency in Smooth Muscle Cells Protects against Hypoxia-Mediated Pulmonary Hypertension via YAP1 Signaling.

Sphingosine kinase 1 (SPHK1) and the sphingosine-1-phosphate (S1P) signaling pathway have been shown to play a role in pulmonary arterial hypertension (PAH). S1P is an important stimulus for pulmonary artery smooth muscle cell (PASMC) proliferation and pulmonary vascular remodeling. We aimed to examine the specific roles of SPHK1 in PASMCs during pulmonary hypertension (PH) progression. We generated smooth muscle cell-specific, Sphk1-deficient (Sphk1f/f TaglnCre+) mice and isolated Sphk1-deficient PASMCs from SPHK1 knockout mice. We demonstrated that Sphk1f/f TaglnCre+ mice are protected from hypoxia or hypoxia/Sugen-mediated PH, and pulmonary vascular remodeling and that Sphk1-deficient PASMCs are less proliferative compared with ones isolated from wild-type (WT) siblings. S1P or hypoxia activated yes-associated protein 1 (YAP1) signaling by enhancing its translocation to the nucleus, which was dependent on SPHK1 enzymatic activity. Further, verteporfin, a pharmacologic YAP1 inhibitor, attenuated the S1P-mediated proliferation of hPASMCs, hypoxia-mediated PH, and pulmonary vascular remodeling in mice and hypoxia/Sugen-mediated severe PH in rats. Smooth muscle cell-specific SPHK1 plays an essential role in PH via YAP1 signaling, and YAP1 inhibition may have therapeutic potential in treating PH.

Endothelial upregulation of mechanosensitive channel Piezo1 in pulmonary hypertension.

Piezo is a mechanosensitive cation channel responsible for stretch-mediated Ca2+ and Na+ influx in multiple types of cells. Little is known about the functional role of Piezo1 in the lung vasculature and its potential pathogenic role in pulmonary arterial hypertension (PAH). Pulmonary arterial endothelial cells (PAECs) are constantly under mechanic stretch and shear stress that are sufficient to activate Piezo channels. Here, we report that Piezo1 is significantly upregulated in PAECs from patients with idiopathic PAH and animals with experimental pulmonary hypertension (PH) compared with normal controls. Membrane stretch by decreasing extracellular osmotic pressure or by cyclic stretch (18% CS) increases Ca2+-dependent phosphorylation (p) of AKT and ERK, and subsequently upregulates expression of Notch ligands, Jagged1/2 (Jag-1 and Jag-2), and Delta like-4 (DLL4) in PAECs. siRNA-mediated downregulation of Piezo1 significantly inhibited the stretch-mediated pAKT increase and Jag-1 upregulation, whereas downregulation of AKT by siRNA markedly attenuated the stretch-mediated Jag-1 upregulation in human PAECs. Furthermore, the mRNA and protein expression level of Piezo1 in the isolated pulmonary artery, which mainly contains pulmonary arterial smooth muscle cells (PASMCs), from animals with severe PH was also significantly higher than that from control animals. Intraperitoneal injection of a Piezo1 channel blocker, GsMTx4, ameliorated experimental PH in mice. Taken together, our study suggests that membrane stretch-mediated Ca2+ influx through Piezo1 is an important trigger for pAKT-mediated upregulation of Jag-1 in PAECs. Upregulation of the mechanosensitive channel Piezo1 and the resultant increase in the Notch ligands (Jag-1/2 and DLL4) in PAECs may play a critical pathogenic role in the development of pulmonary vascular remodeling in PAH and PH.

TRPC6, a therapeutic target for pulmonary hypertension.

Idiopathic pulmonary arterial hypertension (PAH) is a fatal and progressive disease. Sustained vasoconstriction due to pulmonary arterial smooth muscle cell (PASMC) contraction and concentric arterial remodeling due partially to PASMC proliferation are the major causes for increased pulmonary vascular resistance and increased pulmonary arterial pressure in patients with precapillary pulmonary hypertension (PH) including PAH and PH due to respiratory diseases or hypoxemia. We and others observed upregulation of TRPC6 channels in PASMCs from patients with PAH. A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in PASMC triggers PASMC contraction and vasoconstriction, while Ca2+-dependent activation of PI3K/AKT/mTOR pathway is a pivotal signaling cascade for cell proliferation and gene expression. Despite evidence supporting a pathological role of TRPC6, no selective and orally bioavailable TRPC6 antagonist has yet been developed and tested for treatment of PAH or PH. In this study, we sought to investigate whether block of receptor-operated Ca2+ channels using a nonselective blocker of cation channels, 2-aminoethyl diphenylborinate (2-APB, administered intraperitoneally) and a selective blocker of TRPC6, BI-749327 (administered orally) can reverse established PH in mice. The results from the study show that intrapulmonary application of 2-APB (40 µM) or BI-749327 (3-10 µM) significantly and reversibly inhibited acute alveolar hypoxia-induced pulmonary vasoconstriction. Intraperitoneal injection of 2-APB (1 mg/kg per day) significantly attenuated the development of PH and partially reversed established PH in mice. Oral gavage of BI-749327 (30 mg/kg, every day, for 2 wk) reversed established PH by ∼50% via regression of pulmonary vascular remodeling. Furthermore, 2-APB and BI-749327 both significantly inhibited PDGF- and serum-mediated phosphorylation of AKT and mTOR in PASMC. In summary, the receptor-operated and mechanosensitive TRPC6 channel is a good target for developing novel treatment for PAH/PH. BI-749327, a selective TRPC6 blocker, is potentially a novel and effective drug for treating PAH and PH due to respiratory diseases or hypoxemia.

Distinct time courses and mechanics of right ventricular hypertrophy and diastolic stiffening in a male rat model of pulmonary arterial hypertension.

Although pulmonary arterial hypertension (PAH) leads to right ventricle (RV) hypertrophy and structural remodeling, the relative contributions of changes in myocardial geometric and mechanical properties to systolic and diastolic chamber dysfunction and their time courses remain unknown. Using measurements of RV hemodynamic and morphological changes over 10 wk in a male rat model of PAH and a mathematical model of RV mechanics, we discriminated the contributions of RV geometric remodeling and alterations of myocardial material properties to changes in systolic and diastolic chamber function. Significant and rapid RV hypertrophic wall thickening was sufficient to stabilize ejection fraction in response to increased pulmonary arterial pressure by week 4 without significant changes in systolic myofilament activation. After week 4, RV end-diastolic pressure increased significantly with no corresponding changes in end-diastolic volume. Significant RV diastolic chamber stiffening by week 5 was not explained by RV hypertrophy. Instead, model analysis showed that the increases in RV end-diastolic chamber stiffness were entirely attributable to increased resting myocardial material stiffness that was not associated with significant myocardial fibrosis or changes in myocardial collagen content or type. These findings suggest that whereas systolic volume in this model of RV pressure overload is stabilized by early RV hypertrophy, diastolic dilation is prevented by subsequent resting myocardial stiffening.NEW & NOTEWORTHY Using a novel combination of hemodynamic and morphological measurements over 10 wk in a male rat model of PAH and a mathematical model of RV mechanics, we found that compensated systolic function was almost entirely explained by RV hypertrophy, but subsequently altered RV end-diastolic mechanics were primarily explained by passive myocardial stiffening that was not associated with significant collagen extracellular matrix accumulation.

Quantification of uncertainty in a new network model of pulmonary arterial adventitial fibroblast pro-fibrotic signalling.

Here, we present a novel network model of the pulmonary arterial adventitial fibroblast (PAAF) that represents seven signalling pathways, confirmed to be important in pulmonary arterial fibrosis, as 92 reactions and 64 state variables. Without optimizing parameters, the model correctly predicted 80% of 39 results of input-output and inhibition experiments reported in 20 independent papers not used to formulate the original network. Parameter uncertainty quantification (UQ) showed that this measure of model accuracy is robust to changes in input weights and half-maximal activation levels (EC50), but is more affected by uncertainty in the Hill coefficient (n), which governs the biochemical cooperativity or steepness of the sigmoidal activation function of each state variable. Epistemic uncertainty in model structure, due to the reliance of some network components and interactions on experiments using non-PAAF cell types, suggested that this source of uncertainty had a smaller impact on model accuracy than the alternative of reducing the network to only those interactions reported in PAAFs. UQ highlighted model parameters that can be optimized to improve prediction accuracy and network modules where there is the greatest need for new experiments. This article is part of the theme issue 'Uncertainty quantification in cardiac and cardiovascular modelling and simulation'.

Quantification of model and data uncertainty in a network analysis of cardiac myocyte mechanosignalling.

Cardiac myocytes transduce changes in mechanical loading into cellular responses via interacting cell signalling pathways. We previously reported a logic-based ordinary differential equation model of the myocyte mechanosignalling network that correctly predicts 78% of independent experimental results not used to formulate the original model. Here, we use Monte Carlo and polynomial chaos expansion simulations to examine the effects of uncertainty in parameter values, model logic and experimental validation data on the assessed accuracy of that model. The prediction accuracy of the model was robust to parameter changes over a wide range being least sensitive to uncertainty in time constants and most affected by uncertainty in reaction weights. Quantifying epistemic uncertainty in the reaction logic of the model showed that while replacing 'OR' with 'AND' reactions greatly reduced model accuracy, replacing 'AND' with 'OR' reactions was more likely to maintain or even improve accuracy. Finally, data uncertainty had a modest effect on assessment of model accuracy. This article is part of the theme issue 'Uncertainty quantification in cardiac and cardiovascular modelling and simulation'.

Relative Contributions of Matrix and Myocytes to Biaxial Mechanics of the Right Ventricle in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) commonly leads to right ventricular (RV) hypertrophy and fibrosis that affect the mechanical properties of the RV myocardium (MYO). To investigate the effects of PAH on the mechanics of the RV MYO and extracellular matrix (ECM), we compared RV wall samples, isolated from rats in which PAH was induced using the SuHx protocol, with samples from control animals before and after the tissues were decellularized. Planar biaxial mechanical testing, a technique first adapted to living soft biological tissues by Fung, was performed on intact and decellularized samples. Fung's anisotropic exponential strain energy function fitted the full range of biaxial test results with high fidelity in control and PAH samples both before and after they were decellularized. Mean RV myocardial apex-to-outflow tract and circumferential stresses during equibiaxial strain were significantly greater in PAH than control samples. Mean RV ECM circumferential but not apex-to-outflow tract stresses during equibiaxial strain were significantly greater in the PAH than control group. The ratio of ECM to myocardial stresses at matched strains did not change significantly between groups. Circumferential stresses were significantly higher than apex-to-outflow tract stresses for all groups. These findings confirm the predictions of a mathematical model based on changes in RV hemodynamics and morphology in rat PAH, and may provide a foundation for a new constitutive analysis of the contributions of ECM remodeling to changes in RV filling properties during PAH.

Nicotinamide Phosphoribosyltransferase Promotes Pulmonary Vascular Remodeling and Is a Therapeutic Target in Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension is a severe and progressive disease, a hallmark of which is pulmonary vascular remodeling. Nicotinamide phosphoribosyltransferase (NAMPT) is a cytozyme that regulates intracellular nicotinamide adenine dinucleotide levels and cellular redox state, regulates histone deacetylases, promotes cell proliferation, and inhibits apoptosis. We hypothesized that NAMPT promotes pulmonary vascular remodeling and that inhibition of NAMPT could attenuate pulmonary hypertension. METHODS: Plasma, mRNA, and protein levels of NAMPT were measured in the lungs and isolated pulmonary artery endothelial cells from patients with pulmonary arterial hypertension and in the lungs of rodent models of pulmonary hypertension. Nampt+/- mice were exposed to 10% hypoxia and room air for 4 weeks, and the preventive and therapeutic effects of NAMPT inhibition were tested in the monocrotaline and Sugen hypoxia models of pulmonary hypertension. The effects of NAMPT activity on proliferation, migration, apoptosis, and calcium signaling were tested in human pulmonary artery smooth muscle cells. RESULTS: Plasma and mRNA and protein levels of NAMPT were increased in the lungs and isolated pulmonary artery endothelial cells from patients with pulmonary arterial hypertension, as well as in lungs of rodent models of pulmonary hypertension. Nampt+/- mice were protected from hypoxia-mediated pulmonary hypertension. NAMPT activity promoted human pulmonary artery smooth muscle cell proliferation via a paracrine effect. In addition, recombinant NAMPT stimulated human pulmonary artery smooth muscle cell proliferation via enhancement of store-operated calcium entry by enhancing expression of Orai2 and STIM2. Last, inhibition of NAMPT activity attenuated monocrotaline and Sugen hypoxia-induced pulmonary hypertension in rats. CONCLUSIONS: Our data provide evidence that NAMPT plays a role in pulmonary vascular remodeling and that its inhibition could be a potential therapeutic target for pulmonary arterial hypertension.

Biaxial Properties of the Left and Right Pulmonary Arteries in a Monocrotaline Rat Animal Model of Pulmonary Arterial Hypertension.

In a monocrotaline (MCT) induced-pulmonary arterial hypertension (PAH) rat animal model, the dynamic stress-strain relation was investigated in the circumferential and axial directions using a linear elastic response model within the quasi-linear viscoelasticity theory framework. Right and left pulmonary arterial segments (RPA and LPA) were mechanically tested in a tubular biaxial device at the early stage (1 week post-MCT treatment) and at the advanced stage of the disease (4 weeks post-MCT treatment). The vessels were tested circumferentially at the in vivo axial length with matching in vivo measured pressure ranges. Subsequently, the vessels were tested axially at the mean pulmonary arterial pressure by stretching them from in vivo plus 5% of their length. Parameter estimation showed that the LPA and RPA remodel at different rates: axially, both vessels decreased in Young's modulus at the early stage of the disease, and increased at the advanced disease stage. Circumferentially, the Young's modulus increased in advanced PAH, but it was only significant in the RPA. The damping properties also changed in PAH; in the LPA relaxation times decreased continuously as the disease progressed, while in the RPA they initially increased and then decreased. Our modeling efforts were corroborated by the restructuring organization of the fibers imaged under multiphoton microscopy, where the collagen fibers become strongly aligned to the 45 deg angle in the RPA from an uncrimped and randomly organized state. Additionally, collagen content increased almost 10% in the RPA from the placebo to advanced PAH.

A computational study of right ventricular mechanics in a rat model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) presents a significant challenge to right ventricular (RV) function due to progressive pressure overload, necessitating adaptive remodeling in the form of increased wall thickness, enhanced myocardial contractility and stiffness to maintain cardiac performance. However, the impact of these remodeling mechanisms on RV mechanics in not clearly understood. In addition, there is a lack of quantitative understanding of how each mechanism individually influences RV mechanics. Utilizing experimental data from a rat model of PAH at three distinct time points, we developed biventricular finite element models to investigate how RV stress and strain evolved with PAH progression. The finite element models were fitted to hemodynamic and morphological data to represent different disease stages and used to analyze the impact of RV remodeling as well as the altered RV pressure. Furthermore, we performed a number of theoretical simulation studies with different combinations of morphological and physiological remodeling, to assess and quantify their individual impact on overall RV load and function. Our findings revealed a substantial 4-fold increase in RV stiffness and a transient 2-fold rise in contractility, which returned to baseline by week 12. These changes in RV material properties in addition to the 2-fold increase in wall thickness significantly mitigated the increase in wall stress and strain caused by the progressive increase in RV afterload. Despite the PAH-induced cases showing increased wall stress and strain at end-diastole and end-systole compared to the control, our simulations suggest that without the observed remodeling mechanisms, the increase in stress and strain would have been much more pronounced. Our model analysis also indicated that while changes in the RV's material properties-particularly increased RV stiffness - have a notable effect on its mechanics, the primary compensatory factor limiting the stress and strain increase in the early stages of PAH was the significant increase in wall thickness. These findings underscore the importance of RV remodeling in managing the mechanical burden on the right ventricle due to pressure overload.

A murine experimental model for the mechanical behaviour of viable right-ventricular myocardium.

Although right-ventricular function is an important determinant of cardio-pulmonary performance in health and disease, right ventricular myocardium mechanical behaviour has received relatively little attention. We present a novel experimental method for quantifying the mechanical behaviour of transmurally intact, viable right-ventricular myocardium. Seven murine right ventricular free wall (RVFW) specimens were isolated and biaxial mechanical behaviour measured, along with quantification of the local transmural myofibre and collagen fibre architecture. We developed a complementary strain energy function based method to capture the average biomechanical response. Overall, murine RVFW revealed distinct mechanical anisotropy. The preferential alignment of the myofibres and collagen fibres to the apex-to-outflow-tract direction was consistent with this also being the mechanically stiffer axis. We also observed that the myofibre and collagen fibre orientations were remarkably uniform throughout the entire RVFW thickness. Thus, our findings indicate a close correspondence between the tissue microstructure and biomechanical behaviour of the RVFW myocardium, and are a first step towards elucidating the structure­function of non-contracted murine RVFW myocardium in health and disease.

Computational models of ventricular mechanics and adaptation in response to right-ventricular pressure overload.

Pulmonary arterial hypertension (PAH) is associated with substantial remodeling of the right ventricle (RV), which may at first be compensatory but at a later stage becomes detrimental to RV function and patient survival. Unlike the left ventricle (LV), the RV remains understudied, and with its thin-walled crescent shape, it is often modeled simply as an appendage of the LV. Furthermore, PAH diagnosis is challenging because it often leaves the LV and systemic circulation largely unaffected. Several treatment strategies such as atrial septostomy, right ventricular assist devices (RVADs) or RV resynchronization therapy have been shown to improve RV function and the quality of life in patients with PAH. However, evidence of their long-term efficacy is limited and lung transplantation is still the most effective and curative treatment option. As such, the clinical need for improved diagnosis and treatment of PAH drives a strong need for increased understanding of drivers and mechanisms of RV growth and remodeling (G&R), and more generally for targeted research into RV mechanics pathology. Computational models stand out as a valuable supplement to experimental research, offering detailed analysis of the drivers and consequences of G&R, as well as a virtual test bench for exploring and refining hypotheses of growth mechanisms. In this review we summarize the current efforts towards understanding RV G&R processes using computational approaches such as reduced-order models, three dimensional (3D) finite element (FE) models, and G&R models. In addition to an overview of the relevant literature of RV computational models, we discuss how the models have contributed to increased scientific understanding and to potential clinical treatment of PAH patients.

Cellular mechanosignaling in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a vasculopathy characterized by sustained elevated pulmonary arterial pressures in which the pulmonary vasculature undergoes significant structural and functional remodeling. To better understand disease mechanisms, in this review article we highlight recent insights into the regulation of pulmonary arterial cells by mechanical cues associated with PAH. Specifically, the mechanobiology of pulmonary arterial endothelial cells (PAECs), smooth muscle cells (PASMCs) and adventitial fibroblasts (PAAFs) has been investigated in vivo, in vitro, and in silico. Increased pulmonary arterial pressure increases vessel wall stress and strain and endothelial fluid shear stress. These mechanical cues promote vasoconstriction and fibrosis that contribute further to hypertension and alter the mechanical milieu and regulation of pulmonary arterial cells.

Substrate Stiffness and Stretch Regulate Profibrotic Mechanosignaling in Pulmonary Arterial Adventitial Fibroblasts.

Pulmonary arterial adventitial fibroblasts (PAAFs) are important regulators of fibrotic vascular remodeling during the progression of pulmonary arterial hypertension (PAH), a disease that currently has no effective anti-fibrotic treatments. We conducted in-vitro experiments in PAAFs cultured on hydrogels attached to custom-made equibiaxial stretchers at 10% stretch and substrate stiffnesses representing the mechanical conditions of mild and severe stages of PAH. The expression of collagens α(1)I and α(1)III and elastin messenger RNAs (Col1a1, Col3a1, Eln) were upregulated by increased stretch and substrate stiffness, while lysyl oxidase-like 1 and α-smooth muscle actin messenger RNAs (Loxl1, Acta2) were only significantly upregulated when the cells were grown on matrices with an elevated stiffness representative of mild PAH but not on a stiffness representative of severe PAH. Fibronectin messenger RNA (Fn1) levels were significantly induced by increased substrate stiffness and transiently upregulated by stretch at 4 h, but was not significantly altered by stretch at 24 h. We modified our published computational network model of the signaling pathways that regulate profibrotic gene expression in PAAFs to allow for differential regulation of mechanically-sensitive nodes by stretch and stiffness. When the model was modified so that stiffness activated integrin ß3, the Macrophage Stimulating 1 or 2 (MST1\2) kinases, angiotensin II (Ang II), transforming growth factor-ß (TGF-ß), and syndecan-4, and stretch-regulated integrin ß3, MST1\2, Ang II, and the transient receptor potential (TRP) channel, the model correctly predicted the upregulation of all six genes by increased stiffness and the observed responses to stretch in five out of six genes, although it could not replicate the non-monotonic effects of stiffness on Loxl1 and Acta2 expression. Blocking Ang II Receptor Type 1 (AT1R) with losartan in-vitro uncovered an interaction between the effects of stretch and stiffness and angiotensin-independent activation of Fn1 expression by stretch in PAAFs grown on 3-kPa matrices. This novel combination of in-vitro and in-silico models of PAAF profibrotic cell signaling in response to altered mechanical conditions may help identify regulators of vascular adventitial remodeling due to changes in stretch and matrix stiffness that occur during the progression of PAH in-vivo.

Upregulation of Calcium Homeostasis Modulators in Contractile-To-Proliferative Phenotypical Transition of Pulmonary Arterial Smooth Muscle Cells.

Excessive pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and migration are implicated in the development of pathogenic pulmonary vascular remodeling characterized by concentric arterial wall thickening and arteriole muscularization in patients with pulmonary arterial hypertension (PAH). Pulmonary artery smooth muscle cell contractile-to-proliferative phenotypical transition is a process that promotes pulmonary vascular remodeling. A rise in cytosolic Ca2+ concentration [(Ca2+) cyt ] in PASMCs is a trigger for pulmonary vasoconstriction and a stimulus for pulmonary vascular remodeling. Here, we report that the calcium homeostasis modulator (CALHM), a Ca2+ (and ATP) channel that is allosterically regulated by voltage and extracellular Ca2+, is upregulated during the PASMC contractile-to-proliferative phenotypical transition. Protein expression of CALHM1/2 in primary cultured PASMCs in media containing serum and growth factors (proliferative PASMC) was significantly greater than in freshly isolated PA (contractile PASMC) from the same rat. Upregulated CALHM1/2 in proliferative PASMCs were associated with an increased ratio of pAKT/AKT and pmTOR/mTOR and an increased expression of the cell proliferation marker PCNA, whereas serum starvation and rapamycin significantly downregulated CALHM1/2. Furthermore, CALHM1/2 were upregulated in freshly isolated PA from rats with monocrotaline (MCT)-induced PH and in primary cultured PASMC from patients with PAH in comparison to normal controls. Intraperitoneal injection of CGP 37157 (0.6 mg/kg, q8H), a non-selective blocker of CALHM channels, partially reversed established experimental PH. These data suggest that CALHM upregulation is involved in PASMC contractile-to-proliferative phenotypical transition. Ca2+ influx through upregulated CALHM1/2 may play an important role in the transition of sustained vasoconstriction to excessive vascular remodeling in PAH or precapillary PH. Calcium homeostasis modulator could potentially be a target to develop novel therapies for PAH.

Halofuginone, a promising drug for treatment of pulmonary hypertension.

BACKGROUND AND PURPOSE: Halofuginone is a febrifugine derivative originally isolated from Chinese traditional herb Chang Shan that exhibits anti-hypertrophic, anti-fibrotic and anti-proliferative effects. We sought to investigate whether halofuginone induced pulmonary vasodilation and attenuates chronic hypoxia-induced pulmonary hypertension (HPH). EXPERIMENTAL APPROACH: Patch-clamp experiments were conducted to examine the activity of voltage-dependent Ca2+ channels (VDCCs) in pulmonary artery smooth muscle cells (PASMCs). Digital fluorescence microscopy was used to measure intracellular Ca2+ concentration in PASMCs. Isolated perfused and ventilated mouse lungs were used to measure pulmonary artery pressure (PAP). Mice exposed to hypoxia (10% O2 ) for 4 weeks were used as model of HPH for in vivo experiments. KEY RESULTS: Halofuginone increased voltage-gated K+ (Kv ) currents in PASMCs and K+ currents through KCNA5 channels in HEK cells transfected with KCNA5 gene. HF (0.03-1 µM) inhibited receptor-operated Ca2+ entry in HEK cells transfected with calcium-sensing receptor gene and attenuated store-operated Ca2+ entry in PASMCs. Acute (3-5 min) intrapulmonary application of halofuginone significantly and reversibly inhibited alveolar hypoxia-induced pulmonary vasoconstriction dose-dependently (0.1-10 µM). Intraperitoneal administration of halofuginone (0.3 mg·kg-1 , for 2 weeks) partly reversed established PH in mice. CONCLUSION AND IMPLICATIONS: Halofuginone is a potent pulmonary vasodilator by activating Kv channels and blocking VDCC and receptor-operated and store-operated Ca2+ channels in PASMCs. The therapeutic effect of halofuginone on experimental PH is probably due to combination of its vasodilator effects, via inhibition of excitation-contraction coupling and anti-proliferative effects, via inhibition of the PI3K/Akt/mTOR signalling pathway.

Revisiting the mechanism of hypoxic pulmonary vasoconstriction using isolated perfused/ventilated mouse lung.

Hypoxic Pulmonary Vasoconstriction (HPV) is an important physiological mechanism of the lungs that matches perfusion to ventilation thus maximizing O2 saturation of the venous blood within the lungs. This study emphasizes on principal pathways in the initiation and modulation of hypoxic pulmonary vasoconstriction with a primary focus on the role of Ca2+ signaling and Ca2+ influx pathways in hypoxic pulmonary vasoconstriction. We used an ex vivo model, isolated perfused/ventilated mouse lung to evaluate hypoxic pulmonary vasoconstriction. Alveolar hypoxia (utilizing a mini ventilator) rapidly and reversibly increased pulmonary arterial pressure due to hypoxic pulmonary vasoconstriction in the isolated perfused/ventilated lung. By applying specific inhibitors for different membrane receptors and ion channels through intrapulmonary perfusion solution in isolated lung, we were able to define the targeted receptors and channels that regulate hypoxic pulmonary vasoconstriction. We show that extracellular Ca2+ or Ca2+ influx through various Ca2+-permeable channels in the plasma membrane is required for hypoxic pulmonary vasoconstriction. Removal of extracellular Ca2+ abolished hypoxic pulmonary vasoconstriction, while blockade of L-type voltage-dependent Ca2+ channels (with nifedipine), non-selective cation channels (with 30 µM SKF-96365), and TRPC6/TRPV1 channels (with 1 µM SAR-7334 and 30 µM capsazepine, respectively) significantly and reversibly inhibited hypoxic pulmonary vasoconstriction. Furthermore, blockers of Ca2+-sensing receptors (by 30 µM NPS2143, an allosteric Ca2+-sensing receptors inhibitor) and Notch (by 30 µM DAPT, a γ-secretase inhibitor) also attenuated hypoxic pulmonary vasoconstriction. These data indicate that Ca2+ influx in pulmonary arterial smooth muscle cells through voltage-dependent, receptor-operated, and store-operated Ca2+ entry pathways all contribute to initiation of hypoxic pulmonary vasoconstriction. The extracellular Ca2+-mediated activation of Ca2+-sensing receptors and the cell-cell interaction via Notch ligands and receptors contribute to the regulation of hypoxic pulmonary vasoconstriction.

Endothelial platelet-derived growth factor-mediated activation of smooth muscle platelet-derived growth factor receptors in pulmonary arterial hypertension.

Platelet-derived growth factor is one of the major growth factors found in human and mammalian serum and tissues. Abnormal activation of platelet-derived growth factor signaling pathway through platelet-derived growth factor receptors may contribute to the development and progression of pulmonary vascular remodeling and obliterative vascular lesions in patients with pulmonary arterial hypertension. In this study, we examined the expression of platelet-derived growth factor receptor isoforms in pulmonary arterial smooth muscle and pulmonary arterial endothelial cells and investigated whether platelet-derived growth factor secreted from pulmonary arterial smooth muscle cell or pulmonary arterial endothelial cell promotes pulmonary arterial smooth muscle cell proliferation. Our results showed that the protein expression of platelet-derived growth factor receptor α and platelet-derived growth factor receptor ß in pulmonary arterial smooth muscle cell was upregulated in patients with idiopathic pulmonary arterial hypertension compared to normal subjects. Platelet-derived growth factor activated platelet-derived growth factor receptor α and platelet-derived growth factor receptor ß in pulmonary arterial smooth muscle cell, as determined by phosphorylation of platelet-derived growth factor receptor α and platelet-derived growth factor receptor ß. The platelet-derived growth factor-mediated activation of platelet-derived growth factor receptor α/platelet-derived growth factor receptor ß was enhanced in idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell compared to normal cells. Expression level of platelet-derived growth factor-AA and platelet-derived growth factor-BB was greater in the conditioned media collected from idiopathic pulmonary arterial hypertension-pulmonary arterial endothelial cell than from normal pulmonary arterial endothelial cell. Furthermore, incubation of idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell with conditioned culture media from normal pulmonary arterial endothelial cell induced more platelet-derived growth factor receptor α activation than in normal pulmonary arterial smooth muscle cell. Accordingly, the conditioned media from idiopathic pulmonary arterial hypertension-pulmonary arterial endothelial cell resulted in more pulmonary arterial smooth muscle cell proliferation than the media from normal pulmonary arterial endothelial cell. These data indicate that (a) the expression and activity of platelet-derived growth factor receptor are increased in idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell compared to normal pulmonary arterial smooth muscle cell, and (b) pulmonary arterial endothelial cell from idiopathic pulmonary arterial hypertension patients secretes higher level of platelet-derived growth factor than pulmonary arterial endothelial cell from normal subjects. The enhanced secretion (and production) of platelet-derived growth factor from idiopathic pulmonary arterial hypertension-pulmonary arterial endothelial cell and upregulated platelet-derived growth factor receptor expression (and function) in idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell may contribute to enhancing platelet-derived growth factor/platelet-derived growth factor receptor-associated pulmonary vascular remodeling in pulmonary arterial hypertension.