Disrupted PI3K subunit p110α signaling protects against pulmonary hypertension and reverses established disease in rodents
Eva M. Berghausen, … , Ralph T. Schermuly, Stephan Rosenkranz
Eva M. Berghausen, … , Ralph T. Schermuly, Stephan Rosenkranz
Published October 1, 2021
Citation Information: J Clin Invest. 2021;131(19):e136939. https://doi.org/10.1172/JCI136939.
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Research Article Cell biology Vascular biology

Disrupted PI3K subunit p110α signaling protects against pulmonary hypertension and reverses established disease in rodents

Abstract

Enhanced signaling via RTKs in pulmonary hypertension (PH) impedes current treatment options because it perpetuates proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells (PASMCs). Here, we demonstrated hyperphosphorylation of multiple RTKs in diseased human vessels and increased activation of their common downstream effector phosphatidylinositol 3′-kinase (PI3K), which thus emerged as an attractive therapeutic target. Systematic characterization of class IA catalytic PI3K isoforms identified p110α as the key regulator of pathogenic signaling pathways and PASMC responses (proliferation, migration, survival) downstream of multiple RTKs. Smooth muscle cell–specific genetic ablation or pharmacological inhibition of p110α prevented onset and progression of pulmonary hypertension (PH) as well as right heart hypertrophy in vivo and even reversed established vascular remodeling and PH in various animal models. These effects were attributable to both inhibition of vascular proliferation and induction of apoptosis. Since this pathway is abundantly activated in human disease, p110α represents a central target in PH.

Authors

Eva M. Berghausen, Wiebke Janssen, Marius Vantler, Leoni L. Gnatzy-Feik, Max Krause, Arnica Behringer, Christine Joseph, Mario Zierden, Henrik ten Freyhaus, Anna Klinke, Stephan Baldus, Miguel A. Alcazar, Rajkumar Savai, Soni Savai Pullamsetti, Dickson W.L. Wong, Peter Boor, Jean J. Zhao, Ralph T. Schermuly, Stephan Rosenkranz

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Figure 3

The class IA PI3K isoform p110α mediates growth factor–dependent responses in PASMCs.

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The class IA PI3K isoform p110α mediates growth factor–dependent respons...
(A) Western blot analysis demonstrating the expression of the catalytic class IA PI3K subunits in hPASMCs. Blots were probed with isoform-specific p110 antibodies; α-actin shown as a loading control (3 repetitions). (B) Expression of p110 subunits in human lung tissue from healthy donors and patients with IPAH (n = 9 each). Shown is the ratio of p110 mRNA to the internal control (18S) as assessed by TaqMan probes. (C) p110α expression in human (upper; n = 7 each) and mouse lung tissue (lower; n = 5 each). (D) Real-time PCR demonstrating the mRNA expression of PDGFRβ (upper), PDGF-B (middle), and p110α (lower) in PASMCs from healthy donors or patients with IPAH (n = 3 each). (E and F) PDGF-BB–dependent (30 ng/mL) proliferation (E) (BrdU-incorporation; n = 11, 11, 3, 5, 5, 5, 3, 3, 3, 3, 4) and chemotaxis (F) (modified Boyden chambers; n = 15, 15, 4, 4, 6, 9, 2, 4, 3, 3, 3) of hPASMCs in the presence of PI3K isoform-specific inhibitors of p110α (PIK75), p110β (TGX221), and p110δ (IC87114) in the indicated concentrations. (G and H) Proliferation (n = 5, 5, 5, 5, 5, 4, 4, 3, 3, 3, 4) (F) and chemotaxis (n = 15, 15, 3, 3, 3, 3, 3, 3, 3, 3, 3) (H) of GFM-stimulated hPASMCs in the absence or presence of PI3K inhibitors. (I) Impact of PI3K inhibitors against p110α (left) and p110β or p110δ (right) on GFM-induced AKT phosphorylation (Thr 308) in PASMCs (n = 3 each). RasGAP shown as loading control and quantification performed by densitometric analysis. Data shown as fold-increase normalized to quiescent controls. (J) Downstream signaling in hPASMCs preincubated with PIK75 (300 nM) and stimulated with GFM for either 5 minutes (top) or 16 hours (bottom). RasGAP shown as loading control. (K) Apoptosis of starved (24 hours) hPASMCs stimulated with GFM in absence or presence of PIK75 (0.1 and 0.3 μM) (n = 7, 7, 2, 5). (L and M) Proliferation (n = 12) and apoptosis (n = 12) of hPASMCs from patients with PAH stimulated with GFM in absence or presence of PIK75 (at indicated concentrations). Data expressed as percentage of quiescent control (apoptosis) or GFM (proliferation). Data in D–I and K–M represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 as assessed by (E–I and K–M) 1-way ANOVA with Dunnett’s test and (D) 2-tailed Student’s t test.