Conclusions
We demonstrate for the first time that exercise intolerance in PAH is associated with skeletal muscle microcirculation loss and impaired angiogenesis secondary to miR-126 down-regulation.
Results
Patients with PAH displayed decreases in exercise capacity ([Formula: see text]o2max) and microcirculation loss on quadriceps muscle biopsy (in CD31(+) immunofluorescence experiments) compared to control subjects. Exercise capacity correlated with muscle capillarity (r = 0.84, P < 0.01). At the cellular level, vascular endothelial growth factor (VEGF) and VEGF receptor 2 expression were similar in both groups. Conversely, PAH was associated with a 60% decrease in miR-126 expression in a quantitative reverse transcriptase polymerase chain reaction experiment (P < 0.01), resulting in up-regulation of its targeted protein, Sprouty-related, EVH1 domain-containing protein 1 (SPRED-1), and a marked decrease in the downstream effectors of the VEGF pathway, p-Raf/Raf and p-ERK/ERK, as determined by immunoblot analysis. Using freshly isolated CD31(+) cells from human quadriceps biopsies, we found that the down-regulation of miR-126 in PAH triggered the activation of SPRED-1, impairing the angiogenic response (Matrigel assay). These abnormalities were reversed by treating the PAH cells with miR-126 mimic, whereas inhibition of miR-126 (antagomir) in healthy CD31(+) cells fully mimicked the PAH phenotype. Finally, miR-126 down-regulation in skeletal muscle of healthy rats decreased muscle capillarity in immunofluorescence assays (P < 0.05) and exercise tolerance in treadmill tests (P < 0.05), whereas miR-126 up-regulation increased them in monocrotaline PAH rats. Conclusions: We demonstrate for the first time that exercise intolerance in PAH is associated with skeletal muscle microcirculation loss and impaired angiogenesis secondary to miR-126 down-regulation.