Research ArticlePulmonary Arterial Hypertension

PPARγ agonist pioglitazone reverses pulmonary hypertension and prevents right heart failure via fatty acid oxidation

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Science Translational Medicine  25 Apr 2018:
Vol. 10, Issue 438, eaao0303
DOI: 10.1126/scitranslmed.aao0303
  • Fig. 1 Targeted deletion of PPARγ in cardiomyocytes leads to biventricular systolic dysfunction in mice.

    RVEDV and LVEDV (A and E), RV end-systolic volume (RVESV) and LVESV (B and F), and RVEF and LVEF (C and G) as measures of ventricular dilation and systolic function, as well as RV and LV mass over body weight (BW) (D and H), assessed by cardiac MRI in 12- to 16-week-old male and female cmPPARγ−/− mice (α-MHC Cre Pparγflox/flox) and littermate controls. (I) PAAT as a surrogate for pulmonary artery pressure was not significant between cmPPARγ−/− and control mice. ECHO, echocardiography. (J to L) Invasive hemodynamic measurements in cmPPARγ−/− mice and controls. No difference observed. Means ± SEM, n = 4 to 6, two-tailed t test, *P < 0.05, **P < 0.01. CATH, cardiac catheterization. (M) In vivo cardiac magnetic resonance single voxel 1H spectroscopy (MRS) analysis of IMCL accumulation in the RV anterior wall of the cmPPARγ−/− mice versus controls. Average MR spectra of four mice per group are shown. Black line for controls and red for cmPPARγ−/− mice. TMA, trimethylamine; Tau, taurine; ppm, parts per million. (N) Relative mRNA expression of the FAO-related genes Cpt1b and Fabp4 in the RV and LV of cmPPARγ−/− and control mice. Means ± SEM, n = 6, Student’s two-tailed t test, *P < 0.05, **P < 0.01.

  • Fig. 2 PPARγ agonist pioglitazone fully reverses PAH and prevents RV failure in the SuHx rat model.

    (A) Experimental design. Four age-matched groups: (i) ConNx; (ii) ConHx [injected once subcutaneously with vehicle (DMSO; v/v), and then exposed to chronic hypoxia (FiO2, 0.1) for 3 weeks, followed by a 6-week period in room air (FiO2, 0.21)]; (iii) SuHx [injected with SU5416 (20 mg/kg per dose, sc), and then exposed to hypoxia (3 weeks), followed by 6 weeks of room air]; (iv) SuHx + Pio [treated orally with Pio (20 mg/kg per day) for 5 weeks]. po, per os. (B to D) Invasive hemodynamic measurements performed 6 weeks after the end of hypoxia to assess the RVSP, RVEDP, and systolic blood pressure (SAP). (E to G) PAAT as a surrogate of mean PA pressure and PVR, end-diastolic diameter of the RV free wall (RVAWD), and tricuspid annular peak systolic excursion (TAPSE) as a measure of longitudinal systolic RV function, assessed via ECHO. (H to M) RVEDV, RVESV, and RVEF as a measure of RV dilation and systolic function, RV mass, LVEDV, and LVEF were assessed by cardiac MRI. Means ± SEM, n = 3 to 12, analysis of variance (ANOVA)–Bonferroni post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

  • Fig. 3 PPARγ agonist pioglitazone fully prevents (normalizes) RVH, glucose uptake, vessel loss, and cardiac fibrosis in hearts of SuHx-exposed rats.

    (A) Representative images of explanted rat hearts and mass ratio of RV over LV plus septum (Fulton’s index, RV/LV + S). (B) FDG-PET/CT images of glucose uptake in rat hearts. SUV, standardized uptake value. (C) Representative hematoxylin and eosin (H&E) staining of the hearts at the papillary muscle level and NT-proBNP plasma concentrations as a marker of heart failure [bar graph in (C)]. (D) H&E images of RV anterior wall cardiomyocyte size as cross-sectional area (CSA) (μm2). (E) Tomato lectin staining of capillaries in RV tissue. (F) Picrosirius red staining of interstitial collagen in RV tissue. Means ± SEM, n = 3 to 7 individual animals, ANOVA-Bonferroni post hoc-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Detailed statistics in the Supplementary Materials. (D to F) Scale bars, 50 μm. For experimental design, see Fig. 2A.

  • Fig. 5 PPARγ agonist pioglitazone reverses obliterative pulmonary vascular remodeling in the SuHx rat model of PAH.

    (A and B) Representative images of small, peripheral pulmonary arteries in H&E and Masson’s trichrome (M. trichrome) staining, respectively. Scale bars, 50 μm. Bar graph (A): Plasma adiponectin, measured by radioimmunosorbent assay, as an indicator of an appropriate Pio delivery, resorption, and bioavailability. Means ± SEM, n = 4 to 7, ANOVA-Bonferroni post hoc test, *P < 0.05, **P < 0.01. (C) Representative images of α-SMA staining of small, peripheral pulmonary artery muscularization. Scale bars, 25 μm. Means ± SEM, ANOVA-Bonferroni post hoc test, n = 119 to 265 vessels, ****P < 0.0001. (D) Representative images of immunofluorescence staining for vWF and quantification of small vessel loss in pulmonary hypertensive SuHx rats. Scale bars, 100 μm. Means ± SEM, n = 7 to 10 individual animals, 563 to 790 fields calculated, ANOVA-Bonferroni post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001; ****P < 0.0001. (E to G) Representative images of stainings for vWF, CD45, and CD3, respectively. Scale bars, 50 μm. For experimental design, see Fig. 2A.

  • Fig. 4 Mitochondrial disarray, disturbed T-tubule/SR structure, and increased IMCL content in failing SuHx hearts are prevented by pioglitazone treatment.

    (A) Representative 2D electron micrographs of RV cardiomyocytes from ConNx, ConHx, SuHx, and SuHx + Pio hearts. Green arrowheads indicate T-tubule/SR couplons; red arrowheads, abnormal T-tubule/SR couplon morphology; and yellow arrowheads, autophagosome (see also fig. S4A). n = 2-3 animals per group. Scale bars, 1 μm. (B) Representative dual-axis electron tomographic slices showing mitochondrial clustering and size heterogeneity (quantification in fig. S4B). n = 2 to 3 animals/12 to 16 dual-axis tomograms per group. Scale bars, 500 nm. (C) 3D segmented models based on (B). (D) Representative electron tomographic slices of T-tubule/SR remodeling in RV cardiomyocytes. Green arrowheads indicate T-tubule/SR couplons, and red arrowheads indicate abnormal T-tubule/SR couplon morphology. n = 2 to 3 animals/12 to 16 dual-axis tomograms per group. Scale bars, 500 nm. (E) In vivo cardiac MRS of IMCL content in rat hearts. Averaged 1H spectra with chemical shift selective (CHESS) water suppression for all groups. IMCL as area under the curve, normalized to water peak. The peak of non-IMCL lipids [“other lipid” (OL) content] is also shown. Average MR spectra of 3 to 5 rats per group. For experimental design, see Fig. 2A.

  • Fig. 6 Differential miRNA expression profiles in hearts of SuHx rats with PAH and RV failure are recapitulated in human end-stage PAH cardiac tissue and regulated by PPARγ agonist pioglitazone.

    (A) Results of microRNA profiling of rat RVs using 384-well array cards. Relative to global mean expression profiles of four novel miRNAs in the SuHx model. Means ± SEM, n = 3 individual animals, two-tailed, two-sample, unequal variance t test, *P < 0.05, **P < 0.01. (B) Expression profiles of three novel miRNAs in human explanted hearts (RV and LV, IPAH, and healthy donors). Means ± SEM, n = 6 to 7, unpaired two-tailed t test, *P < 0.05, **P < 0.01. (C) Representative images of in situ hybridization (ISH) of miR-197 in rat RV tissue. Scale bars, 200 μm. The inset was captured at ×400 magnification. (D) Expression profiles of novel miRNAs in human whole-lung tissue (IPAH versus downsizing lung). Means ± SEM, n = 7 to 10, unpaired two-tailed t test. PA-control (Con), PA-IPAH, PL-IPAH (n = 5). Expression profiles of novel miRNAs in laser-captured microdissected PLs (PL-IPAH) of IPAH patients, microdissected pulmonary arteries of IPAH patients <500-μm inner diameter, characterized by concentric medial hypertrophy but no PLs (PA-IPAH), and pulmonary arteries <500 μm of downsizing lungs (PA-control). PA-control, PA-IPAH, and PL-IPAH (n = 5). Means ± SEM, ANOVA-Bonferroni post hoc test, **P < 0.01, ****P < 0.0001. For experimental design, see Fig. 2A.

  • Fig. 7 Differential cardiac mRNA expression in the RV and LV of controls, SuHx-exposed rats with RV failure, and SuHx rats treated with pioglitazone (RNA-seq).

    (A) All differentially expressed genes in the SuHx versus ConHx group comparison in RVs, ranging from the most up-regulated (red) to the most down-regulated (blue), and their counterparts in the other comparison groups. Each group comparison contains two columns: Log2 of fold change (FC) and the corresponding FDR-adjusted P values (q values) represented as −log10(q) for up-regulated genes and log10(q) for down-regulated genes. (B) All differentially expressed genes in the RVs of SuHx-exposed rats (SuHx + Pio) treated orally with Pio versus SuHx group and their counterparts in the other comparison groups. (C) Volcano plot of the SuHx versus ConHx comparison in RV (q values are capped at 10). (D) Volcano plot of the SuHx + Pio versus SuHx comparison in RV (q values are capped at 15). n = 3 individual animals in each group. For experimental design, see Fig. 2A.

  • Fig. 8 PPARγ agonist pioglitazone induces FAO, ATP production, and FAO-driving genes in cardiomyocytes, whereas FAO genes are repressed in human IPAH.

    (A and B) Oxygen consumption rates (OCR) in the setting of FAO of RV and LV NRCMs, respectively, with and without 10 μM Pio. Number of biological replicates, n = 3; number of technical replicates, n = 5; number of each measurement, n = 3; unpaired t test, ****P < 0.0001. (C) Bar graph showing percentage of dependency (reliance to maintain baseline respiration) and flexibility (ability to increase oxidation of the substrate to compensate for inhibition of alternative substrate pathways) of NRCMs to oxidize FAs (as a percentage of oxidation of glucose, FAs, and glutamate). Number of biological replicates, n = 3; number of technical replicates, n = 5; number of each measurement, n = 3; unpaired t tests, **P < 0.01. GLC, glucose; GLN, glutamine. (D) Relative extracellular ATP production of NRCMs induced by Pio. Number of biological replicates, n = 3; number of technical replicates, n = 16; means ± SEM, unpaired two-tailed t test, **P < 0.01. (E) Relative expression of FAO-related genes in NRCMs with and without 10 μM Pio. Means ± SEM, unpaired two-tailed t tests. Number of biological replicates, n = 3; number of technical replicates, n = 6; *P < 0.05, **P < 0.01, ****P < 0.0001. (F) Relative expression of FAO-related genes in human explanted hearts (RV, IPAH, and healthy donors). Means ± SEM; control RV, n = 6, IPAH RV, n = 7, unpaired two-tailed t-test, *P < 0.05, **P < 0.01. (G and H) Relative expression of FAO-related genes in NRCMs after 72 hours of preincubation with pre-miR-197 or pre-miR-146b (each 30 μM), respectively. Number of biological replicates, n = 3; number of technical replicates, n = 6; means ± SEM, unpaired two-tailed t tests, *P < 0.05, **P < 0.01, *** P < 0.001. Means ± SEM, n = 6.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/438/eaao0303/DC1

    Materials and Methods

    Fig. S1. Mice with targeted deletion of PPARγ in cardiomyocytes (cmPPARγ−/−), in the absence of PAH, do not develop cardiac hypertrophy or fibrosis at the age of 12 to 16 weeks.

    Fig. S2. Schematic depicting the vicious cycle of RV failure in PAH.

    Fig. S3. PAH but no RV failure is evident 1 week after the end of hypoxia (3 + 1 weeks), and RV failure develops by week 6 after the end of hypoxia in SuHx-exposed rats.

    Fig. S4. RV glucose uptake increases with chronic RV pressure afterload and correlates with RV systolic function 6 weeks after the end of hypoxia in SuHx-exposed rats.

    Fig. S5. Decrease in minimal mitochondrial diameter, presence of autophagosomes and cytoplasmic vacuoles, and collagen deposits indicating RV failure and fibrosis are present in SuHx RVs but not SuHx + Pio RVs.

    Fig. S6. In silico predicted miRNA/mRNA pairing of miR-491 with the mRNA of monoacylglycerol lipase (MGLL).

    Fig. S7. Expression of miRNAs that are altered in rat and human RV failure is not changed by hypoxia in the RV of FVB mice.

    Fig. S8. mRNA/miRNA expression signatures and networks in the failing RV (SuHx, on the left) and the PPARγ-mediated effects in the RV of the SuHx rat PAH model (SuHx + Pio, on the right).

    Fig. S9. Important molecular interactions based on differential gene expression (mRNA) analysis in the RV of SuHx + Pio versus SuHx rats.

    Fig. S10. Pioglitazone has no negative effects on survival, GO, or FAO in human PAECs and no impact on cardiomyocyte survival.

    Fig. S11. Neither VEGFR2 blockade nor oral pioglitazone treatment cause any significant changes in blood glucose in rats.

    Table S1. Cardiac MRI, ECHO, and cardiac catheterization hemodynamic and morphological data obtained in cmPPARγ−/− mice and littermate controls at 12 to 16 weeks of age.

    Table S2. Echocardiographic measurements in rats 1 week after the end of hypoxia (=SuHx rat with PAH but no RV failure yet, 3 + 1 weeks).

    Table S3A. Invasive hemodynamic and echocardiographic measurements in rats at 3 + 6 weeks (SuHx rat PAH/RV failure model).

    Table S3B. Cardiac MRI, 18FDG-PET/CT, and heart weight measurements in rats of the SuHx study at 3 + 6 weeks.

    Table S3C. Complete blood count, plasma NT-proBNP, and plasma APN in rats of the SuHx study at 3 + 6 weeks.

    Table S4. Human heart and lung tissue specimens used for laser capture microdissection or whole-tissue gene expression assays.

    Table S5. List of significantly differentially expressed genes (FDR < 5%) based on inversion of RV mRNA expression patterns in the SuHx PAH/RV failure model (RNA-seq) with PPARγ agonist pioglitazone, first up- or down-regulated in PAH/RV failure (SuHx) and inversely regulated with pioglitazone treatment (SuHx + Pio).

    Table S6. List of mRNA transcripts related to angiogenesis identified by RNA-seq in the RV of control rats (ConHx), rats with PAH and RV failure (SuHx), and Pio-treated rats (SuHx + Pio).

    Table S7. Fresh human lung tissue specimens obtained during lung transplantation.

    Movie S1. Cardiac MRI ConNx.

    Movie S2. Cardiac MRI ConHx.

    Movie S3. Cardiac MRI SuHx.

    Movie S4. Cardiac MRI SuHx + Pio.

    References (6681)

  • Supplementary Material for:

    PPARγ agonist pioglitazone reverses pulmonary hypertension and prevents right heart failure via fatty acid oxidation

    Ekaterina Legchenko, Philippe Chouvarine, Paul Borchert, Angeles Fernandez-Gonzalez, Erin Snay, Martin Meier, Lavinia Maegel, S. Alex Mitsialis, Eva A. Rog-Zielinska, Stella Kourembanas, Danny Jonigk, Georg Hansmann*

    *Corresponding author. Email: georg.hansmann{at}gmail.com

    Published 25 April 2018, Sci. Transl. Med. 10, eaao0303 (2018)
    DOI: 10.1126/scitranslmed.aao0303

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Mice with targeted deletion of PPARγ in cardiomyocytes (cmPPARγ−/−), in the absence of PAH, do not develop cardiac hypertrophy or fibrosis at the age of 12 to 16 weeks.
    • Fig. S2. Schematic depicting the vicious cycle of RV failure in PAH.
    • Fig. S3. PAH but no RV failure is evident 1 week after the end of hypoxia (3 + 1 weeks), and RV failure develops by week 6 after the end of hypoxia in SuHx-exposed rats.
    • Fig. S4. RV glucose uptake increases with chronic RV pressure afterload and correlates with RV systolic function 6 weeks after the end of hypoxia in SuHx-exposed rats.
    • Fig. S5. Decrease in minimal mitochondrial diameter, presence of autophagosomes and cytoplasmic vacuoles, and collagen deposits indicating RV failure and fibrosis are present in SuHx RVs but not SuHx + Pio RVs.
    • Fig. S6. In silico predicted miRNA/mRNA pairing of miR-491 with the mRNA of monoacylglycerol lipase (MGLL).
    • Fig. S7. Expression of miRNAs that are altered in rat and human RV failure is not changed by hypoxia in the RV of FVB mice.
    • Fig. S8. mRNA/miRNA expression signatures and networks in the failing RV (SuHx, on the left) and the PPARγ-mediated effects in the RV of the SuHx rat PAH model (SuHx + Pio, on the right).
    • Fig. S9. Important molecular interactions based on differential gene expression (mRNA) analysis in the RV of SuHx + Pio versus SuHx rats.
    • Fig. S10. Pioglitazone has no negative effects on survival, GO, or FAO in human PAECs and no impact on cardiomyocyte survival.
    • Fig. S11. Neither VEGFR2 blockade nor oral pioglitazone treatment cause any significant changes in blood glucose in rats.
    • Table S1. Cardiac MRI, ECHO, and cardiac catheterization hemodynamic and morphological data obtained in cmPPARγ−/− mice and littermate controls at 12 to 16 weeks of age.
    • Table S2. Echocardiographic measurements in rats 1 week after the end of hypoxia (=SuHx rat with PAH but no RV failure yet, 3 + 1 weeks).
    • Table S3A. Invasive hemodynamic and echocardiographic measurements in rats at 3 + 6 weeks (SuHx rat PAH/RV failure model).
    • Table S3B. Cardiac MRI, 18FDG-PET/CT, and heart weight measurements in rats of the SuHx study at 3 + 6 weeks.
    • Table S3C. Complete blood count, plasma NT-proBNP, and plasma APN in rats of the SuHx study at 3 + 6 weeks.
    • Table S4. Human heart and lung tissue specimens used for laser capture microdissection or whole-tissue gene expression assays.
    • Table S5. List of significantly differentially expressed genes (FDR < 5%) based on inversion of RV mRNA expression patterns in the SuHx PAH/RV failure model (RNA-seq) with PPARγ agonist pioglitazone, first up- or down-regulated in PAH/RV failure (SuHx) and inversely regulated with pioglitazone treatment (SuHx + Pio).
    • Table S6. List of mRNA transcripts related to angiogenesis identified by RNA-seq in the RV of control rats (ConHx), rats with PAH and RV failure (SuHx), and Pio-treated rats (SuHx + Pio).
    • Table S7. Fresh human lung tissue specimens obtained during lung transplantation.
    • Legends for movies S1 to S4
    • References (6681)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). Cardiac MRI ConNx.
    • Movie S2 (.mov format). Cardiac MRI ConHx.
    • Movie S3 (.mov format). Cardiac MRI SuHx.
    • Movie S4 (.mov format). Cardiac MRI SuHx + Pio.

    [Download Movies S1 to S4]

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