P1938In vitro characterization of a novel PI 3-kinase inhibitor for growth factor-induced effects in pulmonary artery smooth muscle cells

Abstract

Abstract Rationale Pulmonary arterial hypertension (PAH) is characterized by vascular remodeling in pulmonary arterioles. Increased activation of receptor tyrosine kinase (RTK)-mediated signaling pathways lead to increased proliferation and chemotaxis of pulmonary arterial smooth muscle cells (PASMCs). In previous studies, we identified phosphatidylinositol-3-kinase (PI3K) as a key enzyme for these processes. Since systemic PI3K inhibition is potentially associated with undesired side effects, we here for the first time tested the efficacy of the novel PI3K inhibitor CL27c to impede growth factor-mediated cellular effects in human PASMCs. CL27c is topically applicable and as a prodrug it is intracellularly activated and thus offers optimal efficacy in target cells while at the same time minimizing undesired effects in non-target cells. Methods Using Western blotting and immunohistochemistry, activation of the central PI3K effector AKT in human and murine lung tissue was investigated. Quantification of cell proliferation was performed by 5-bromo-2-deoxyuridine (BrdU) incorporation assay. PASMCs were incubated with CL27c [0.1; 0.3; 1; 3; 10μM] and stimulated either with PDGF-BB [30ng/ml] or with a growth factor combination (GFC) [PDGF-BB 30ng/ml, FGF 2ng/ml, EGF 0.5ng/ml, insulin 0.5μg/ml, FCS 5%]. Chemotaxis was quantified using modified Boyden chambers. The signal transduction cascade was investigated by Western blotting. Results Analysis of AKT activation revealed hyperphosphorylation in both the vascular wall and complete lung homogenates from PAH patients and hypoxia-treated mice. Examination of the mitogenic signal transduction revealed a markedly reduced phosphorylation of AKT and GSK3β in CL27c pretreated PASMCs. In addition, central cell cycle events, such as expression of cyclin D1, were reduced. This was in line with a CL27c concentration-dependent significant reduction of PDGF- and GFC-induced proliferation (control: 2.53±0.26 vs. CL27c: 0.73±0.14 and control: 5.27±0.61 vs. CL27c: 2.80±0.71 both at 10μM, p<0.001). Furthermore, PDGF- and GFC-induced chemotaxis were as well significantly reduced (100% ± 19 vs. CL27c: 24.43% ± 8.58 and 100% ± 5.76 vs CL27c: 25.23% ± 10.57 at 10μM p<0.001). Conclusion These results indicate that CL27c reduces both proliferation and chemotaxis of PASMCs. As these processes play a key role in the development and progression of PAH, this approach is currently being investigated in preclinical models for its therapeutic potential. This “prodrug” approach could avoid non-specific effects and systemic side effects of Pan-PI3K inhibition, especially since the substance is suitable for topical application (inhalation). Thus, CL27c represents a promising treatment option.