Abstract
Rationale: Enhanced proliferation and distal migration of human pulmonary arterial smooth muscle cells (hPASMCs) both contribute to the progressive increases in pulmonary vascular remodeling and resistance in pulmonary arterial hypertension (PAH). Our previous studies revealed that Rictor deletion, to disrupt mTOR Complex 2 (mTORC2), over longer periods result in a paradoxical rise in platelet-derived growth factor receptor (PDGFR) expression in PASMCs. Thus, the purpose of this study was to evaluate the role of combination therapy targeting both mTOR signaling with PDGFR inhibition to attenuate the development and progression of PAH. Methods and Results: Immunoblotting analyses revealed that short-term exposure to rapamycin (6h) significantly reduced phosphorylation of p70S6K (mTORC1-specific) in hPASMCs but had no effect on the phosphorylation of AKT (p-AKT S473, considered mTORC2-specific). In contrast, longer rapamycin exposure (>24 h), resulted in differential AKT (T308) and AKT (S473) phosphorylation with increases in phosphorylation of AKT at T308 and decreased phosphorylation at S473. Phosphorylation of both PDGFRα and PDGFRβ was increased in hPASMCs after treatment with rapamycin for 48 and 72 h. Based on co-immunoprecipitation studies, longer exposure to rapamycin (24–72 h) significantly inhibited the binding of mTOR to Rictor, mechanistically suggesting mTORC2 inhibition by rapamycin. Combined exposure of rapamycin with the PDGFR inhibitor, imatinib significantly reduced the proliferation and migration of hPASMCs compared to either agent alone. Pre-clinical studies validated increased therapeutic efficacy of rapamycin combined with imatinib in attenuating PAH over either drug alone. Specifically, combination therapy further attenuated the development of monocrotaline (MCT)- or Hypoxia/Sugen-induced pulmonary hypertension (PH) in rats as demonstrated by further reductions in the Fulton index, right ventricular systolic pressure (RVSP), pulmonary vascular wall thickness and vessel muscularization, and decreased proliferating cell nuclear antigen (PCNA) staining in PASMCs. Conclusion: Prolonged rapamycin treatment activates PDGFR signaling, in part, via mTORC2 inhibition. Combination therapy with rapamycin and imatinib may be a more effective strategy for the treatment of PAH.
Highlights
Pulmonary arterial hypertension (PAH) is characterized by progressive increases in pulmonary vascular resistance (PVR) and pressure, which can lead to deterioration of right ventricular function (Voelkel et al, 2012; Humbert et al, 2019)
While rapamycin is well-established as an mTORC1 inhibitor, its effects on mTORC2 has not been well studied
We initially investigated the effects of longer rapamycin exposure on mTORC2 in Human PASMCs (hPASMCs)
Summary
Pulmonary arterial hypertension (PAH) is characterized by progressive increases in pulmonary vascular resistance (PVR) and pressure, which can lead to deterioration of right ventricular function (Voelkel et al, 2012; Humbert et al, 2019). Deletion of tuberous sclerosis complex 1⁄2 (TSC 1/2), which is an upstream inhibitor of mTORC1 signaling, inhibits expression of PDGFRs in a rapamycin-sensitive manner (Zhang et al, 2007). In contrast to these data, smooth muscle cell-specific ablation of mTORC2 over prolonged periods results in spontaneous murine PAH, possibly due to unexpected increases in PDGFR expression (Tang et al, 2015; Tang et al, 2018a).
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