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

Potus F, Malenfant S, Graydon C, Mainguy V, Tremblay E, Breulis-Bonnet S, et al. American Journal of Respiratory and Critical Care Medicine 2014;190(3):318-328

Skeletal muscle defects are seen in patients with pulmonary arterial hypertension (PAH), which have been linked to reduced exercise tolerance. These defects are thought to be due to reduced contact between capillaries and skeletal myocytes, perhaps due to impaired angiogenesis.  MicroRNAs (miRs) are single-stranded non-coding RNAs that regulate gene expression by binding to a target mRNA.  miR-126 is an important regulator of angiogenesis that is expressed primarily in endothelial cells (ECs).  miR-126 targets SPRED-1, which represses the VEGF pathway by targeting downstream effectors RAF-1 and PI3K.  The authors hypothesized that miR-126 down-regulation accounts for the decrease in peripheral muscle microcirculation and contributes to exercise intolerance in PAH.

Methods and Results

Twenty patients with idiopathic PAH (n=16) or heritable PAH (n=4) [functional class II-III] and 20 sedentary healthy matched controls had quadriceps biopsies. Eleven PAH and 9 control subjects also performed a cardiopulmonary exercise test to determine peak VO2. Skeletal muscle of PAH patients had lower % of CD31+ cells (as a marker of microcirculation), and there was a strong correlation between % CD31+ cells and peak VO2 (r=0.84). There was no difference in VEGF levels between PAH and control subjects, but there was a 60% decrease in miR-126 expression in the quadriceps of PAH subjects. SPRED-1 was up-regulated, and the protein activity of RAF and ERK were significantly decreased in PAH.  Skeletal muscle miR-126 expression was significantly correlated to the proportion of CD31+ quadriceps cells (r=0.48) and also with peak VO2 (r=0.52).

To substantiate these findings, CD31+ cells were isolated from the quadriceps of 2 subjects with idiopathic PAH, 2 with heritable PAH, and 3 controls. Cells were transfected with a miR-126 mimic or an antagomir, anti-miR-126.  ECs from PAH skeletal muscle had decreased miR-126 expression and impairments in angiogenic potential. Ectopic over-expression of miR-126 increased the number of branch points between ECs, to a similar degree seen in control quadriceps.  Downregulation of miR-126 by the antagomir in control ECs reduced angiogenic potential to the level seen in PAH patients.  ECs derived from PAH patients quadriceps had decreased proliferation and increased apoptosis, and both of these parameters normalized when treated with the miR-126 mimic. Lastly, antagomirs were injected into rat quadriceps, and this led to decreased microvessel density and impairments in exercise capacity.

Reductions in quadriceps microcirculation in patients with PAH were strongly associated with worse maximal exercise capacity.  It appears that this loss of microcirculation occurs downstream in the VEGF pathway and is mediated by reductions in miR-126 expression.  In in vitro experiments, angiogenesis could be restored by application of a miR-126 mimic. This work adds further understanding to the pathogenesis of muscle dysfunction in PAH and identifies a potential therapeutic target, which deserves further study.

Expert Commentary

This article reports the novel and important observation that there is rarefaction of the skeletal muscle microcirculation in patients with PAH that is associated with exercise intolerance and may further contribute to inactivity.  The mechanism is investigated and low miR-126 is identified, in association with elevation of its target SPRED-1 accounting for reduced p-RAF and p-ERK and impaired angiogenesis.    Moreover, there is convincing data that the miR-126 mimic can improve angiogenesis in PAH endothelial cells associated with skeletal muscle and that the antagomir can repress the angiogenesis response in endothelial cells from control muscle.   There is also evidence in animals that skeletal muscle angiogenesis can be improved by miR-126 particularly in a rat monocrotaline PH model.   This new avenue of research leads to many new questions.  It is not known how this pathway affects the pulmonary arterial endothelial cells.  Although it is presumed that inflammation contributes to the down-regulation of miR-126, we do not know whether the authors could identify more inflammation associated with skeletal muscle endothelial cells, although high circulating cytokine levels previously described in PAH patients could certainly support this possibility.   While much attention is focused on miR-126 and the VEGF pathway, it is almost a footnote that pVEGFR2 down-regulation is affecting endothelial migration by a separate pathway related to migration.  It would be important to know about pVEGFR2 levels that affect this pathway and also to assess the migratory response of the endothelial cells. Since the downstream effectors are not selective to VEGF signaling, a broad discussion and exploration of other pro-angiogenic pathways that could be affected such as the BMP pathway would be of interest particularly since this pathway is repressed in PAH.   

Article summary by: Matthew Lammi, MD; Assistant Professor of Medicine, Louisiana State University Health Sciences Center

Expert commentary by: Marlene Rabinovitch, MD; Dwight and Vera Dunlevie Professor of Pediatrics, Stanford University School of Medicine

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