NMDA-Type Glutamate Receptor Activation Promotes Vascular Remodeling and Pulmonary Arterial Hypertension.

BACKGROUND: Excessive proliferation and apoptosis resistance in pulmonary vascular cells underlie vascular remodeling in pulmonary arterial hypertension (PAH). Specific treatments for PAH exist, mostly targeting endothelial dysfunction, but high pulmonary arterial pressure still causes heart failure and death. Pulmonary vascular remodeling may be driven by metabolic reprogramming of vascular cells to increase glutaminolysis and glutamate production. The N-methyl-d-aspartate receptor (NMDAR), a major neuronal glutamate receptor, is also expressed on vascular cells, but its role in PAH is unknown. METHODS: We assessed the status of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and controls through mass spectrometry imaging, Western blotting, and immunohistochemistry. We measured the glutamate release from cultured pulmonary vascular cells using enzymatic assays and analyzed NMDAR regulation/phosphorylation through Western blot experiments. The effect of NMDAR blockade on human pulmonary arterial smooth muscle cell proliferation was determined using a BrdU incorporation assay. We assessed the role of NMDARs in vascular remodeling associated to pulmonary hypertension, in both smooth muscle-specific NMDAR knockout mice exposed to chronic hypoxia and the monocrotaline rat model of pulmonary hypertension using NMDAR blockers. RESULTS: We report glutamate accumulation, upregulation of the NMDAR, and NMDAR engagement reflected by increases in GluN1-subunit phosphorylation in the pulmonary arteries of human patients with PAH. Kv channel inhibition and type A-selective endothelin receptor activation amplified calcium-dependent glutamate release from human pulmonary arterial smooth muscle cell, and type A-selective endothelin receptor and platelet-derived growth factor receptor activation led to NMDAR engagement, highlighting crosstalk between the glutamate-NMDAR axis and major PAH-associated pathways. The platelet-derived growth factor-BB-induced proliferation of human pulmonary arterial smooth muscle cells involved NMDAR activation and phosphorylated GluN1 subunit localization to cell-cell contacts, consistent with glutamatergic communication between proliferating human pulmonary arterial smooth muscle cells via NMDARs. Smooth-muscle NMDAR deficiency in mice attenuated the vascular remodeling triggered by chronic hypoxia, highlighting the role of vascular NMDARs in pulmonary hypertension. Pharmacological NMDAR blockade in the monocrotaline rat model of pulmonary hypertension had beneficial effects on cardiac and vascular remodeling, decreasing endothelial dysfunction, cell proliferation, and apoptosis resistance while disrupting the glutamate-NMDAR pathway in pulmonary arteries. CONCLUSIONS: These results reveal a dysregulation of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and identify vascular NMDARs as targets for antiremodeling treatments in PAH.

Functional interaction between PDGFß and GluN2B-containing NMDA receptors in smooth muscle cell proliferation and migration in pulmonary arterial hypertension.

In this study, we explored the complex interactions between platelet-derived growth factor (PDGF) and N-methyl-d-aspartate receptor (NMDAR) and their effect on the excessive proliferation and migration of smooth muscle cells leading to obstructed arteries in pulmonary arterial hypertension (PAH). We report lower expression of glutamate receptor NMDA-type subunit 2B (GluN2B), a subunit composing NMDARs expected to affect cell survival/proliferation of pulmonary artery smooth muscle cells (PASMCs), in PAH patient lungs. PASMC exposure to PDGF-BB stimulated immediate increased levels of phosphorylated Src family kinases (SFKs) together with increased phosphorylated GluN2B (its active form) and cell surface relocalization, suggesting a cross talk between PDGFR-recruited SFKs and NMDAR. Selective inhibition of PDGFR-ß or SFKs with imatinib or A-419259, respectively, on one hand, or with specific small-interfering RNAs (siRNAs) on the other hand, aborted PDGF-induced phosphorylation of GluN2B, thus validating the pathway. Selective inhibition of GluN2B using Rö25-6981 and silencing with specific siRNA, in the presence of PDGF-BB, significantly increased both migration and proliferation of PASMCs, thus strengthening the functional importance of the pathway. Together, these results indicate that GluN2B-type NMDAR activation may confer to PASMCs antiproliferative and antimigratory properties. The decreased levels of GluN2B observed in PAH pulmonary arteries could mediate the excessive proliferation of PASMCs, thus contributing to medial hyperplasia and PAH development.