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. 2017 Dec 19;136(25):2451-2467.
doi: 10.1161/CIRCULATIONAHA.117.028034. Epub 2017 Sep 26.

Identification of MicroRNA-124 as a Major Regulator of Enhanced Endothelial Cell Glycolysis in Pulmonary Arterial Hypertension via PTBP1 (Polypyrimidine Tract Binding Protein) and Pyruvate Kinase M2

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Free PMC article

Identification of MicroRNA-124 as a Major Regulator of Enhanced Endothelial Cell Glycolysis in Pulmonary Arterial Hypertension via PTBP1 (Polypyrimidine Tract Binding Protein) and Pyruvate Kinase M2

Paola Caruso et al. Circulation. .
Free PMC article

Abstract

Background: Pulmonary arterial hypertension (PAH) is characterized by abnormal growth and enhanced glycolysis of pulmonary artery endothelial cells. However, the mechanisms underlying alterations in energy production have not been identified.

Methods: Here, we examined the miRNA and proteomic profiles of blood outgrowth endothelial cells (BOECs) from patients with heritable PAH caused by mutations in the bone morphogenetic protein receptor type 2 (BMPR2) gene and patients with idiopathic PAH to determine mechanisms underlying abnormal endothelial glycolysis. We hypothesized that in BOECs from patients with PAH, the downregulation of microRNA-124 (miR-124), determined with a tiered systems biology approach, is responsible for increased expression of the splicing factor PTBP1 (polypyrimidine tract binding protein), resulting in alternative splicing of pyruvate kinase muscle isoforms 1 and 2 (PKM1 and 2) and consequently increased PKM2 expression. We questioned whether this alternative regulation plays a critical role in the hyperglycolytic phenotype of PAH endothelial cells.

Results: Heritable PAH and idiopathic PAH BOECs recapitulated the metabolic abnormalities observed in pulmonary artery endothelial cells from patients with idiopathic PAH, confirming a switch from oxidative phosphorylation to aerobic glycolysis. Overexpression of miR-124 or siRNA silencing of PTPB1 restored normal proliferation and glycolysis in heritable PAH BOECs, corrected the dysregulation of glycolytic genes and lactate production, and partially restored mitochondrial respiration. BMPR2 knockdown in control BOECs reduced the expression of miR-124, increased PTPB1, and enhanced glycolysis. Moreover, we observed reduced miR-124, increased PTPB1 and PKM2 expression, and significant dysregulation of glycolytic genes in the rat SUGEN-hypoxia model of severe PAH, characterized by reduced BMPR2 expression and endothelial hyperproliferation, supporting the relevance of this mechanism in vivo.

Conclusions: Pulmonary vascular and circulating progenitor endothelial cells isolated from patients with PAH demonstrate downregulation of miR-124, leading to the metabolic and proliferative abnormalities in PAH ECs via PTPB1 and PKM1/PKM2. Therefore, the manipulation of this miRNA or its targets could represent a novel therapeutic approach for the treatment of PAH.

Keywords: endothelial cells; endothelial progenitor cells; glycolysis; hypertension, pulmonary; metabolism; microRNAs.

Figures

Figure 1
Figure 1. Glycolysis is increased in BOECs from HPAH/IPAH patients and in siBMPR2-treated control ECs
(A–B) Glycolytic flux in BMPR2 mutant HPAH BOECs (A, n=4) and IPAH BOECs (B, n=4) compared to control BOECs (n=3). (C–D) Lactate production was measured in HPAH BOECs (C) or IPAH BOECs (D) compared to control BOECs. (E–F) Glycolytic flux in control BOECs (E) or PAECs (F) transfected with siBMPR2 compared to transfection reagent alone (CTR) or scrambled siRNA. Sample were tested in triplicate. Data are presented as the mean ± S.E.M. of four biological replicates and analyzed using an unpaired t-test (A–D), or a 1-way ANOVA followed by Bonferroni post hoc test (E–F) (***p<0.001, **p<0.01, *p<0.05).
Figure 2
Figure 2. miR-124 expression is reduced in PAH BOECs and in control cells following BMPR2 silencing
1066 miRNAs were screened by quantitative PCR-array in BOECs from 4 HPAH, 3 IPAH and 4 control subjects. (A) Volcano plot showing 17 miRNAs significantly altered (unadjusted p-value<0.05) between HPAH patients and control subjects. (B) Heat-map showing relative expression patterns for the miRNAs altered in HPAH and IPAH patients, and the hierarchical clustering across all subjects. (C) 4 miRNAs were concordantly dysregulated in both groups of patients. Unadjusted p-values, adjusted p-values (false discovery rate (FDR) adjusted p-value) and fold change (FC) are shown. Importantly, it should be noted that the miRNA candidates identified in this screen did not pass typical false discovery rate thresholds, and therefore were prioritized based on p-values unadjusted for multiple comparisons. miR-124 expression was validated using 7 HPAH, 5 IPAH and 8 control BOEC lines (D–E) to confirm the observations in the quantitative PCR-array. (F) Control BOECs were transfected with a siBMPR2 and tested for miR-124 expression 24, 48 and 72 hours post-transfection compared to transfection reagent alone (CTR) or scrambled siRNA (n=3). Data are presented as the mean ± S.E.M. Samples were tested in triplicate. Data were analyzed using an unpaired t-test (D–E) or a 1-way ANOVA followed by Bonferroni post-hoc test (F). (***p<0.001, *p<0.05).
Figure 3
Figure 3. PTBP1 expression is increased in PAH BOECs and in siBMPR2-treated control cells
(A, D) qPCR analysis of PTBP1 was conducted in HPAH BOECs (A, n=7) or IPAH BOECs (D, n=5) versus control cells (n=8). (**p<0.01, *p<0.05). (B, E) PTBP1 protein expression was determined in whole cell lysates from 6 HPAH (B) and 6 IPAH (E) samples compared with 6 controls and normalized to β-actin expression. (C, F) Quantitative densitometry analysis of the immunoblots shown in B and E, respectively (**p<0.01). (G) PTBP1 localisation was confirmed by immunostaining in lung tissue from HPAH patients and non-affected controls. Scale bars: 100 μm. (H) PTBP1 gene expression was assessed by qPCR in control BOECs transfected with siBMPR2 or scrambled siRNA. Cells treated with the transfection reagent alone were used as a negative control (CTR). Total RNA was isolated 24, 48 and 72 hours post-transfection. Data were analyzed using a 1-way ANOVA followed by Bonferroni post hoc test. (n=3, ***p<0.001). (I–J) Immunoblot analysis of PTBP1 expression 72h hours after transfection with a siBMPR2 or scrambled siRNA. Non-transfected cells were used as a negative control (CTR). Expression was normalized to β-actin. (J) Quantitative densitometry analysis of the immunoblot (*p<0.05). Data were analyzed using an unpaired t-test.
Figure 4
Figure 4. PKM2 levels are elevated in PAH BOECs and in siBMPR2 control BOECs
(A–B) The expression of PKM2 was assessed by qPCR in BOECs isolated from HPAH (A, n=7) or IPAH (B, n=5) patients versus controls (n=8). Data were analyzed using an unpaired t-test (***p<0.001, **p<0.01). (C) PKM2 localisation and expression was confirmed by immunofluorescence labeling of PKM2 in HPAH BOECs and control samples (n=3). PKM2 is stained in green; actin was counterstained using phalloidin (red) and nuclei counterstained using DAPI (blue). Scale bars: 25 μm. (D) PKM2 gene expression was assessed in control BOECs (n=3) transfected with siBMPR2 or scrambled siRNA control. Non-transfected cells were used as a negative control (CTR). Data were analyzed using a 1-way ANOVA followed by Bonferroni post hoc test (*p<0.05). (E, F, H, I) Glycolytic factors MCT1 and LDHA were analysed by qPCR in BOECs isolated from HPAH (E, H, n=7) and IPAH (F, I, n=5) versus controls (n=8). Data were analyzed using an unpaired t-test (***p<0.001, **p<0.01, *p<0.05). (G, J) siBMPR2-transfected control BOECs were analysed for MCT1 (G) and LDHA (J) expression levels compared to scrambled control. Cells treated with the transfection reagent alone were used as a negative control (CTR). Data were analyzed using a 1-way ANOVA followed by Bonferroni post hoc test (n=3, *p<0.05). Data are presented as the mean ± S.E.M. Samples were tested in triplicate.
Figure 5
Figure 5. miR-124, PTBP1 and PKM2 are dysregulated in IPAH PAECs
(A) miR-124 expression was analysed by qPCR in IPAH versus control PAECs (n=4, *p<0.05). Data are presented as the mean ± S.E.M. Sample were tested in triplicate. Data were analysed using an unpaired t-test. (B) Immunofluorescence staining demonstrated high expression levels of PTBP1 (red) in endothelial and adventitial cells (EC and Adv., respectively. M = media) in the pulmonary artery of IPAH patients, whereas it is undetectable in normal donor arteries. Scale bars: 100 μm. (C) Immunoblotting for PTBP1 and PKM2 protein expression in PAEC. (D–E) Quantitative densitometry analysis of PTBP1 (D) and PKM2 (E) protein expression. Immunoblotting was conducted upon whole cell lysates from PAECs from 3 IPAH patients compared with 3 controls and normalized to β-actin expression. Data were analysed using an unpaired t-test (***p<0.001, *p<0.05).
Figure 6
Figure 6. Upregulation of miR-124 restores the expression of glycolytic genes to control levels
(A–G) HPAH BOECs (n=3) were transfected with a miR-124 mimic or with scrambled control. Cells treated with the transfection reagent alone were used as a negative control (CTR). Total RNA was extracted 48 hours post-transfection and analyzed by qPCR for the expression of PTBP1 (A), PKM2 (B), PKM1 (C) LDHA (D) MCT1 (E), PDK1 (F) and PDK2 (G). Gene expression was also assessed in control BOECs (n=3) to establish the basal level of expression in unaffected subjects. Data are presented as the mean ± S.E.M. Samples were tested in triplicate. Data were analyzed using a 1-way ANOVA followed by Bonferroni post hoc test. (***p<0.001, **p<0.01, *p<0.05).
Figure 7
Figure 7. Upregulation of miR-124 or downregulation of PTBP1 restores glycolysis, proliferation and mitochondrial activity to basal levels
(A, C) Control (n=3; black bars) and HPAH (A, n=3; white bars) or IPAH BOECS (C, n=3; white bars) were transfected with a miR-124 mimic or a siPTBP1 and glycolytic flux was measured after 48 hours and compared with untreated (CTR) or siScramble-treated cells. (B, D) Lactate production was assessed in control and HPAH (B) or IPAH BOECs (D) treated in the same way than (A) and (C) respectively. (E–F) Glycolysis was assessed in control BOECs (E, n=3) and PAECs (F, n=3) transfected with siBMPR2, alone or in presence of miR-124 mimic or siPTBP1 and compared to untreated (CTR) or siScramble-treated cells. (G) Control BOECs (n=4) were transfected with a siScramble (red line) and HPAH cells were transfected with a miR-124 mimic (purple line), siPTBP1 (green line) or siScramble as negative control (blue line). After 48 hours, cells were serum and growth factor starved for 4 hours. Cells were counted at day 0, 3, 5 and 7. (H) TCA cycle activity was measured in HPAH BOECs untreated (CTR) or transfected with a miR-124 mimic, siPTBP1 or siScramble. Basal TCA cycle activity was also measured in control BOECs as a reference (n=4). Data are presented as the mean ± S.E.M. Every sample was tested in triplicate. Data were analyzed using a 1-way ANOVA or 2-way ANOVA (for section 7G) followed by Bonferroni post hoc test. (***p<0.001, **p<0.01, *p<0.05).
Figure 8
Figure 8. Dysregulation of miR-124 and glycolytic genes is conserved in SUGEN-hypoxia rats
To induce the development of PAH, 12 weeks old Sprague–Dawley rats received a single injection of SUGEN-5416 on day 1 (20mg/kg) and were maintained in 10% O2 for 3 weeks. Thereafter, rats were returned to normoxia for 5 weeks. At the 8-week time point, right ventricular systolic pressure (A, RVSP) was measured and right ventricular hypertrophy was assessed (B, Fulton index, RV/(LV+Sep)) to confirm the development of PAH. (C–E) miR-124 (C), Ptbp1 (D) Pkm2 (E) gene expression was analysed by qPCR. Data were analyzed using an unpaired t-test (**p<0.01, *p<0.05). (F) Immunoblot analysis of PTBP1 protein expression in whole lung lysates extracted from 3 SUGEN-hypoxia and 3 control rats and quantitative densitometry analysis of the immunoblots. Data were analyzed using an unpaired t-test (*p<0.05). (E) PTBP1 localisation was confirmed by PTBP1 immunostaining in lung tissue from SUGEN-hypoxia and control rats. Scale bars: 100 μm. (H–J) The same samples used in C, D and E were analysed by qPCR to assess the expression levels of Mct1 (H), Ldha (I) and Bmpr2 (J). Data are presented as the mean ± S.E.M. Every sample was tested in triplicate. Data were analyzed using an unpaired t-test (***p<0.001, **p<0.01, *p<0.05).

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