All animals in the studies were handled according to the National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of Guangzhou Medical University. For the MCT model, male Sprague-Dawley (SD) rats (200–250 g) were studied after a single intraperitoneal monocrotaline (MCT) injection (50 mg/kg, #HY-N0750, MCE, United States). For the hypoxia-SU5416 model, SD rats (200–250 g) placed under hypoxic condition (FiO₂ 10%) for 3 weeks after given single dose of SU5416 (20 mg/kg, #HY-10374, MCE, United States). They were transitioned to normoxia for 2 weeks. Rats were randomly assigned to four groups: rapamycin alone (2 mg/kg/d, #HY-10219, MCE, United States), imatinib alone (10 mg/kg/d, #S1026, Selleck, United States), rapamycin (2 mg/kg/d) combined with imatinib (10 mg/kg/d), or vehicle only. All drugs were given once daily by intraperitoneal injection for the last 2 weeks of each model. Terminal measurements were recorded. Right ventricular systolic pressure (RVSP) was measured as previously published (Tang et al., 2018b) with a pressure transducer catheter (Millar Instruments) and used as a surrogate for pulmonary arterial pressure. Hearts were excised and dissected to determine the ratio of the RV weight to the left ventricle (LV) and septum (S) weight ratio.

Human PASMCs (hPASMCs) were obtained from Sciencell (#3110) and Promocell (#399Z003.1). Cells were maintained in SMC medium (#1101, Sciencell, United States of America) with 2% fetal bovine serum and SMC growth supplement (#1152, Sciencell, United States). For migration assay, hPASMCs suspension was diluted to 2 × 10⁵/ml. 70 ul of cell suspension was seeded into culture-inserts of 2-wells in 35 mm dish (#81176, IBIDI, Germany). After cell attachment, inserts were removed using sterilized forceps. Cells were cultured in serum-free medium containing rapamycin (100 nM), imatinib (5 μM) or vehicle and pictures recorded every 4 h. The number of cells migrating out of the wound edge was scored using Image J image analysis software. Cell viability was assessed by CCK8 kit (#96992, Sigma-Aldrich, United States). hPASMCs were seeded into 96-well plates at a density of 5,000 cells per well with six replicates per group. Cells were cultured in 2% serum-containing medium exposed to either rapamycin (100 nM), imatinib (5 μM), or vehicle. After addition of 10ul CCK8 per well, the plate was cultured for 4 h at 37°C. The absorbance value of each well was measured and collected at 450 nm. Cell proliferation assays were performed using BrdU incorporation kit (#QIA58, Merck Millipore, United States). hPASMCs were seeded into 96-well plates at a density of 5,000 cells per well with four replicates per group. Cells were cultured in 2% serum-containing medium exposed to either rapamycin (100 nM), imatinib (5 μM), or vehicle. BrdU assay was conducted according to the standard protocol of the manufacturer and BrdU uptake was measured in each well at dual wavelengths of 450–540 nm.

Total cellular and isolated pulmonary arterial (PA) proteins were extracted using RIPA lysis buffer containing proteinase and phosphatase inhibitor. Western blotting was performed using the following antibodies: anti-mTOR (#2983), anti-Rictor (#5379) anti-phospho-PDGFRα/β (#3170), anti-PDGFRα (#3174), anti-PDGFRβ (#3169), anti-phospho-AKT (T308) (#13038), anti-phospho-AKT (S473) (#4060), anti-AKT (Pan) (#4691), anti-phospho-p70S6k (#9204), anti-p70S6k (9202), anti-phospho-S6 (#4858), and anti-S6 (#2317) (Cell Signaling Technology, United States). For Co-immunoprecipitation, cell protein was extracted in the non-deformed protein lysate. Non-specific proteins were removed by adding protein A/G-beads (#sc-2003, Santa Cruz Biotechnzazology, United States). An antibody targeting mTOR was incubated at room temperature to bind the antibody to the target antigen. The antigen-antibody complex was incubated overnight at 4°C and then, boiled for 5 mins. SDS-polyacrylamide gel electrophoresis was performed by adding the sample buffer on the protein (Sarbassov et al., 2006).

For immunofluorescence staining, lung tissue sections were blocked in 2% BSA for 30 min and incubated overnight at 4°C with alpha-smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA). After extensive washes with PBS for 30 min, lung tissue was incubated with Alexa 488 anti-rabbit secondary or Alexa 594 anti-mouse secondary antibodies at a 1:500 dilution for 1 h at room temperature. Nuclei were stained with DAPI (4, 6-diamidino-2-phenylindole).

Data were presented as means ± standard error (SE) and analyzed by GraphPad Prism software. Statistical differences among multiple experimental groups were tested by one-way analysis of variance (ANOVA) and two-way ANOVA. p < 0.05 was considered as statistically significant.

While rapamycin is well-established as an mTORC1 inhibitor, its effects on mTORC2 has not been well studied. Thus, we initially investigated the effects of longer rapamycin exposure on mTORC2 in hPASMCs. As shown in Figure 1A, rapamycin treatment reduced phosphorylated p70S6k (p-p70S6k) and increased total p70S6k levels across all time points in hPASMCs, validating rapamycin-mediated inhibition of mTORC1. In contrast, phosphorylated AKT (p-AKT S473) levels were reduced only with longer exposures of rapamycin, suggesting reductions of mTORC2 activity with chronic exposure. Specifically, inhibition of mTORC1 by rapamycin can result in increased availability of free mTOR generating increases in mTORC2 activity (Hussain et al., 2013). To test this possibility, hPASMCs treated with insulin to activate mTORC2 and found that p-AKT S473 was not increased after treatment with rapamycin for 6 h (Figure 1B).

Rapamycin works through a gain-of-function mechanism in which it binds to the intracellular protein FKBP12 to generate a drug-receptor complex that binds to and inhibits mTORC1 (Annett et al., 2020). To determine whether rapamycin inhibits mTORC2 activity via a similar mechanism, we evaluated the effect of rapamycin on the interaction between Rictor and mTOR in hPASMCs using immunoprecipitation analysis. As shown in Figure 1C, rapamycin caused a time-dependent reduction in the binding between Rictor and mTOR. Expression levels of mTOR and Rictor remained unchanged in whole cell lysates.

Previously, we found elevated expression of PDGFRs in isolated pulmonary arteries (PA) from smooth muscle-specific Rictor knockout mice (Rictor ^SM-/-) (Tang et al., 2018a). Base on rapamycin reducing mTORC2 activity, the effects of long-term rapamycin treatment on both phosphorylated and total PDGFRs was investigated. As shown in Figure 2A, phosphorylated PDGFR levels were increased in hPASMCs after treatment with rapamycin at 48 and 72 h. Impact of combination therapy on mTOR and PDGFR signaling pathways also was detected. Consistent with previous results, exposure to rapamycin alone significantly reduced p-S473AKT and increased phosphorylation of p-T308AKT (Figure 2B). In parallel, rapamycin increased p-PDGFRα (T849)/β (T857) in hPASMCs. Imatinib alone inhibited the phosphorylation of PDGFR. Imatinib use alone also mildly inhibited mTOR activation. Rapamycin combined with imatinib robustly inhibited the activation of mTORC1 and mTORC2 along with the phosphorylation of PDGFRα/β.

Since hPASMCs proliferation and migration drives obliterative pulmonary arterial vascular remodeling in PAH, we investigated the impact of rapamycin and imatinib on each process. We first detected impact of rapamycin and imatinib on cell viability. Result showed that both rapamycin and imatinib alone significantly inhibited hPASMCs viability. Importantly, the inhibitory effects of rapamycin combined with imatinib was more significant than that of either drug alone (Figure 3A). Both drugs alone significantly inhibited the migration of hPASMCs using a cell scratch assay, while the inhibitory effect of the combined regimen was more potent than either drug alone (Figure 3B). To further confirm the inhibitory effects of rapamycin combined with imatinib on PASMCs proliferation, a BrdU assay was performed. As shown in Figure 3C, hypoxia induced hPASMCs proliferation at 48 h, validating our prior observations (Tang et al., 2015; Tang et al., 2016). Both rapamycin and imatinib alone significantly inhibited hPASMCs proliferation (Figure 3D) despite hypoxic exposure. The inhibitory effect of the combined regimen was more potent than either drug alone.

We evaluated combination therapy with rapamycin and imatinib in MCT and Hypoxia/Sugen rat models of PAH. Rats develop PH in both MCT and Hypoxia/Sugen treated (Figures 4A, 6A), as evidenced by significant increases in RVSP, [RV/(LV + S)] and RV/BW (Figures 4B–F, 6B–E) compared to normal control animals. In the MCT model, as shown in Figures 4B–F, neither rapamycin or low dose imatinib (10 mg/kg) alone not attenuated the development of PH in MCT-rats with no significant reductions in RVSP, the Fulton index and RV/BW, compared to controls. However, combination therapy significantly decreased RVSP and Fulton index, but no effect on RV/BW. In the Hypoxia/Sugen rat model, as shown in Figures 6B–E, rapamycin exposure alone resulted in significant decreases in RVSP and the Fulton index but not RV/BW. Imatinib alone decreased the Fulton index but had no effect on RVSP and RV/BW. However, combination therapy resulted in significantly decreased RVSP, Fulton index and RV/BW, compared to controls.

We next evaluated the effects of combination therapy on the extensive vascular remodeling and muscularized vessels in both rat models. For the MCT rodent model (Figures 5A,B), H&E staining showed that rapamycin or imatinib alone had no significant effect on pulmonary vascular wall thickening or the number of muscularized vessels. However, combination therapy significantly inhibited the thickening of the vascular wall and the number of muscularized vessels. Moreover, rapamycin or imatinib alone had no effect on the percentage of proliferating cell nuclear antigen (PCNA)-positive PASMCs in the lung. The combination strategy significantly reduced the percentage of PCNA-positive PASMCs in small pulmonary arterioles. Similarly, in the Hypoxia/Sugen model (Figures 7A,B), imatinib alone had no significant effect on pulmonary vascular thickening or the number of muscularized vessel. Rapamycin alone significantly inhibited the thickening of pulmonary vascular thickening with diameter less than 50 μm but had no effect on the number of muscularized vessels. Combination therapy of rapamycin with imatinib significantly inhibited both of vascular wall thickening and the number of muscularized vessels. Additionally, PCNA staining revealed significantly decreased cell proliferation within small pulmonary arterioles after combination strategy.

We evaluated the effects of combination therapy on mTOR and PDGFR signaling pathways in isolated PAs from rat lungs. As shown in Figure 8A, expression of p-PDGFR, p-S6 and pS473AKT was increased in PAs isolated from MCT-exposed rats. Confirming in vitro data, rapamycin inhibited the activity of both, mTORC1 and mTORC2. Imatinib alone inhibited the phosphorylation of PDGFR. Rapamycin combined with imatinib inhibited mTORC1, mTORC2 and PDGFR signaling mediators.