Activation of the mammalian target of rapamycin complex 1 (mTORC1) subunit Raptor induces cell growth and is a downstream target of Akt. Elevated levels of aldosterone activate Akt, and, in pulmonary arterial hypertension (PAH), correlate with pulmonary arteriole thickening, which suggests that mTORC1 regulation by aldosterone may mediate adverse pulmonary vascular remodeling. We hypothesized that aldosterone-Raptor signaling induces abnormal pulmonary artery smooth muscle cell (PASMC) survival patterns to promote PAH. Remodeled pulmonary arterioles from SU-5416/hypoxia-PAH rats and monocrotaline-PAH rats with hyperaldosteronism expressed increased levels of the Raptor target, p70S6K, which provided a basis for investigating aldosterone-Raptor signaling in human PASMCs. Aldosterone (10(-9) to 10(-7) M) increased Akt/mTOR/Raptor to activate p70S6K and increase proliferation, viability, and apoptosis resistance in PASMCs. In PASMCs transfected with Raptor-small interfering RNA or treated with spironolactone/eplerenone, aldosterone or pulmonary arterial plasma from patients with PAH failed to increase p70S6K activation or to induce cell survival in vitro Optimal inhibition of pulmonary arteriole Raptor was achieved by treatment with Staramine-monomethoxy polyethylene glycol that was formulated with Raptor-small interfering RNA plus spironolactone in vivo, which decreased arteriole muscularization and pulmonary hypertension in 2 experimental animal models of PAH in vivo Up-regulation of mTORC1 by aldosterone is a critical pathobiologic mechanism that controls PASMC survival to promote hypertrophic vascular remodeling and PAH.-Aghamohammadzadeh, R., Zhang, Y.-Y., Stephens, T. E., Arons, E., Zaman, P., Polach, K. J., Matar, M., Yung, L.-M., Yu, P. B., Bowman, F. P., Opotowsky, A. R., Waxman, A. B., Loscalzo, J., Leopold, J. A., Maron, B. A. Up-regulation of the mammalian target of rapamycin complex 1 subunit Raptor by aldosterone induces abnormal pulmonary artery smooth muscle cell survival patterns to promote pulmonary arterial hypertension.
In pulmonary arterial hypertension (PAH), elevated levels of aldosterone (ALDO) induce a vasculopathy that is characterized by dysregulated pulmonary artery smooth muscle cell (PSMC) growth. Upregulation of the mammalian target of rapamycin complex 1 subunit Raptor induces PSMC growth; however, the factors regulating Raptor in PAH are not known. We hypothesized that Raptor activation by ALDO induces PSMC proliferation, hypertrophic vascular remodeling, and PAH. To test this hypothesis, PSMCs were exposed to vehicle (V) control or ALDO (10 -7 mol/l) for 1 h. Compared to V-treated cells, ALDO increased expression of P-Raptor(Ser792) and the Raptor target P-p70S6K(Thr389) by 68% (P<0.01) and 50% (P<0.01), respectively, without affecting total Raptor/p70S6K levels. Raptor upregulation in ALDO-treated cells also increased PSMC proliferation by 19% (P<0.01), assessed by BrdU incorporation. To explore the relevance of ALDO-Raptor signaling in vivo , we studied the monocrotaline (MCT) model of PAH, which is characterized by hyperALDO. In a prevention protocol, MCT-PAH rats were treated with spironolactone (SP)(25 mg/kg/d) in the drinking water to inhibit ALDO and/or intravenous Staramine-mPEG labeled with Raptor-siRNA (2 mg/kg dose x4)(si-Raptor). Anti-Raptor and anti-α-SM actin immunohistochemistry showed that compared to controls (N=4), Raptor expression and vessel thickness was increased in MCT-PAH rats (N=5) by 87% (P<0.01) and 5.8-fold (P<0.01), respectively, and pulmonary artery systolic pressure (PASP) was increased significantly (25 ± 4 vs. 77 ± 12 mmHg, P<0.01) as assessed by catheterization. Compared to MCT-PAH, MCT-si-Raptor (N=5) decreased Raptor levels by 31% (P<0.05), arteriole thickness by 41% (P<0.05), and PASP (77 ± 12 vs. 46 ± 5 mmHg, P<0.05) without affecting central blood pressure significantly. A further benefit was observed in MCT-si-Raptor+SP rats (N=5) for arterial thickness (-57%, P<0.01) and PASP (46 ± 5 vs. 39 ± 5 mmHg, P<0.05) compared to MCT-si-Raptor. Taken together, Raptor activation by ALDO induces PSMC proliferation in vitro to promote PAH in vivo . Identifying ALDO-Raptor signaling as a pathobiological mechanism underlying pulmonary vascular remodeling may have novel therapeutic implications for PAH patients.
Dextran sulfate (DS) and chitosan (CS) nanoparticles (NPs) have a glycan matrix that may be useful for in vivo delivery of proteins. Current procedures for the formation of DS‐CS NPs with incorporated proteins is to first combine DS or CS with the protein of interest and then proceed to particle formation. In general, this process requires a significant amount of protein for particle formation, and after the reaction the unincorporated protein cannot be easily recovered for future incorporation. Considering the high cost and limited availability of various recombinant therapeutic proteins, in this study we investigated whether proteins can be incorporated efficiently in pre‐formed DS‐CS NPs. To do so, DS‐CS NPs were formulated, prepared on a large scale, and lyophilized. The reconstituted DS‐CS NPs were sized 333±16 nm, with a polydispersity of 0.119±0.026 and zeta potential of ‐32.4±1.4 mV. Stromal cell‐derived factor‐1α (SDF‐1α) was loaded onto the DS‐CS NPs at various concentrations. At the ratio of SDF‐1α to DS of 0.02:1, 85±8 % of the loaded SDF‐1α was incorporated in the NPs. The resulted particles (SDFNPs) had a diameter of 393±17 nm, a polydispersity of 0.147±0.032, and a zeta potential of ‐28.6±1.9 mV. The incorporated SDF‐1α had the same chemotactic activity as that of free SDF‐1α. Administration of aerosolized SDFNP to the lungs of rats showed that ~ 30% SDF‐1α remained in the tissue at 72 hr after the delivery, while free SDF‐1α was mostly cleared from the lung (~97%) after 16 hr. This study shows that pre‐formed DS‐CS NPs incorporated SDF‐1α with high efficiency, and can be a useful vehicle for delivery of recombinant therapeutic proteins to the lung parenchyma.
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