Pulmonary arterial hypertension (PAH) is a progressive and fatal disease without a cure. The exact pathogenic mechanisms of PAH are complex and poorly understood, yet a number of abnormally expressed genes and regulatory pathways contribute to sustained vasoconstriction and vascular remodeling of the distal pulmonary arteries. Mammalian target of rapamycin (mTOR) is one of the major signaling pathways implicated in regulating cell proliferation, migration, differentiation, and protein synthesis. Here we will describe the canonical mTOR pathway, structural and functional differences between mTOR complexes 1 and 2, as well as the crosstalk with other important signaling cascades in the development of PAH. The pathogenic role of mTOR in pulmonary vascular remodeling and sustained vasoconstriction due to its contribution to proliferation, migration, phenotypic transition, and gene regulation in pulmonary artery smooth muscle and endothelial cells will be discussed. Despite the progress in our elucidation of the etiology and pathogenesis of PAH over the two last decades, there is a lack of effective therapeutic agents to treat PAH patients representing a significant unmet clinical need. In this review, we will explore the possibility and therapeutic potential to use inhibitors of mTOR signaling cascade to treat PAH.

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.

Using RNAseq, we identified a 61 gene-based circulating transcriptomic profile most correlated with four indices of pulmonary arterial hypertension severity. In an independent dataset, 13/61 (21%) genes were differentially expressed in lung tissues of pulmonary arterial hypertension cases versus controls, highlighting potentially novel candidate genes involved in pulmonary arterial hypertension development.

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⁺ (K_v ) 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 K_v 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.

Pulmonary arterial hypertension is a progressive and fatal disease and rodents with experimental pulmonary hypertension (PH) are often used to study pathogenic mechanisms, identify therapeutic targets, and develop novel drugs for treatment. Here we describe a hands-on set of experimental approaches including ex vivo lung angiography and histology and in vivo right heart catheterization (RHC) to phenotypically characterize pulmonary hemodynamics and lung vascular structure in normal mice and mice with experimental PH. We utilized Microfil polymer as contrast in our ex vivo lung angiogram to quantitatively examine pulmonary vascular remodeling in mice with experimental PH, and lung histology to estimate pulmonary artery wall thickness. The peripheral lung vascular images were selected to determine the total length of lung vascular branches, the number of branches and the number of junctions in a given area (mm^-2). We found that the three parameters determined by angiogram were not significantly different among the apical, middle, and basal regions of the mouse lung from normal mice, and were not influenced by gender (no significant difference between female and male mice). We conducted RHC in mice to measure right ventricular systolic pressure, a surrogate measure for pulmonary artery systolic pressure and right ventricle (RV) contractility (RV ± dP/dt_max) to estimate RV function. RHC, a short time (4-6 min) procedure, did not alter the lung angiography measurements. In summary, utilizing ex vivo angiogram to determine peripheral vascular structure and density in the mouse lung and utilizing in vivo RHC to measure pulmonary hemodynamics are reliable readouts to phenotype normal mice and mice with experimental PH. Lung angiogram and RHC are also reliable approaches to examine pharmacological effects of new drugs on pulmonary vascular remodeling and hemodynamics.

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 β 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 β in pulmonary arterial smooth muscle cell, as determined by phosphorylation of platelet-derived growth factor receptor α and platelet-derived growth factor receptor β. The platelet-derived growth factor-mediated activation of platelet-derived growth factor receptor α/platelet-derived growth factor receptor β 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.

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.