Impaired Angiogenesis and Peripheral Muscle Microcirculation Loss Contribute to Exercise Intolerance in Pulmonary Arterial Hypertension

Rationale: Pulmonary arterial hypertension (PAH) is characterized by significant exercise intolerance, which is multifactorial and involves skeletal muscle alterations. There is growing evidence that microRNAs (miRs) are involved in PAH pathogenesis. Objectives: We hypothesized that miR-126, an endothelial-specific, proangiogenic miR, is down-regulated in the peripheral muscles of patients with PAH, which would account for skeletal muscle microcirculation loss and exercise intolerance. Measurements and Main Results: Patients with PAH displayed decreases in exercise capacity (Vo(2)max) 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.