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Fenofibrate Reverses Dysfunction of EPCs Caused by Chronic Heart Failure

  • Wen-Pin Huang
  • Wei-Hsian Yin
  • Jia-Shiong Chen
  • Po-Hsun HuangEmail author
  • Jaw-Wen Chen
  • Shing-Jong LinEmail author
Original Article
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Abstract

The enhanced activity of endothelial progenitor cells (EPCs) by AMP-activated protein kinase (AMPK) agonists might explain the reversal of chronic heart failure (CHF)–mediated endothelial dysfunction. We studied baseline circulating EPC numbers in patients with heart failure and clarified the effect of fenofibrate on both circulating angiogenic cell (CAC) and late EPC activity. The numbers of circulating EPCs in CHF patients were quantified by flow cytometry. Blood-derived mononuclear cells were cultured, and CAC and late EPC functions, including fibronectin adhesion, tube formation, and migration, were evaluated. We focused on the effect of fenofibrate, an AMPK agonist, on EPC function and Akt/eNOS cascade activation in vitro. The number of circulating EPCs (CD34+/KDR+) was significantly lower in CHF patients (ischemic cardiomyopathy (ICMP): 0.07%, dilated cardiomyopathy (DCMP): 0.068%; p < 0.05) than in healthy subjects (0.102% of the gating region). In CACs, fibronectin adhesion function was reversed by fenofibrate treatment (p < 0.05). Similar results were also found for tube formation and migration in late EPCs, which were significantly improved by fenofibrate in an AMPK-dependent manner (p < 0.05), suggesting that fenofibrate reversed CACs and late EPC dysfunction in CHF patients. The present findings reveal the potential application of the AMPK agonist fenofibrate to reverse endothelial dysfunction in CHF patients.

Keywords

Chronic heart failure Endothelial progenitor cell eNOS AMPK Fenofibrate 

Abbreviations

EPCs

Endothelial progenitor cells

CACs

Circulating angiogenesis cells

CHF

Chronic heart failure

PBMCs

Peripheral blood mononuclear cells

ICMP

Ischemic cardiomyopathy

DCMP

Dilated cardiomyopathy

VEGFR-2

Vascular endothelial growth factor receptor 2

PPARs

Peroxisome proliferator–activated receptors

LVEF

Left ventricular ejection fraction

FITC

Fluorescein isothiocyanate

PerCP

Peridinin chlorophyll

APC

Allophycocyanin

AMPK

AMP-dependent protein kinase

AICAR

AICA-riboside

Compound C

Dorsomorphin

Notes

Funding Information

This study was supported, in part, by research grants from the Ministry of Science and Technology of Taiwan (MOST 104-2314-B-075-047), the Novel Bioengineering and Technological Approaches to Solve Two Major Health Problems in Taiwan sponsored by the Taiwan Ministry of Science and Technology Academic Excellence Program (MOST 108-2633-B-009-001), the Ministry of Health and Welfare (MOHW106-TDU-B-211-113001), and Taipei Veterans General Hospital (V105C-0207, V106C-045). The funders had no role in the study design, data collection, data analysis, decision to publish, or preparation of the manuscript.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Human Subjects/Informed Consent Statement

The Human Investigation Committee of the Cheng-Hsin Rehabilitation Medical Center approved the study protocol (CHGH-IRB: (136) 97-17-2). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000(5). Informed consent was obtained from all patients included in the study.

Supplementary material

12265_2019_9889_MOESM1_ESM.pptx (1.3 mb)
Supplementary Fig. 1 Characterization of circulating progenitor cells and circulating endothelial progenitor cells from peripheral blood. Gating strategy used to detect circulation-derived progenitor cells and endothelial progenitor cells (EPCs) by flow cytometry; one representative plot is shown. A first gate is set on the total cell region in a forward (FSC) and sideways scatter (SSC) plot. A second gate identifies the CD45-positive and CD45-negative cells in an SSC peak plot. Gating N identifies the percentage of CD34+/KDR+ progenitor cells measured within this population and is expressed as positive events/106 total events. Isotype-matched controls and unstained cell samples are processed as negative controls. (B-G) Quantification of the percentage of circulation-derived progenitor and EPCs in HF patients and healthy controls. Data are presented as mean ± SD; ICMP, N = 57; DCMP, N = 71; Normal, N = 17. (PPTX 1362 kb)
12265_2019_9889_MOESM2_ESM.pptx (901 kb)
Supplementary Fig. 2 Morphology and characterization of early and late endothelial progenitor cells from peripheral blood. (A) MNCs were isolated and plated on fibronectin-coated culture dishes on the first day. (B) Four days after plating, adherent circulating angiogenic cells (CACs) with spindle shapes were observed. (C) Fourteen days after plating, late EPCs with cobblestone-like morphology were selected and reseeded. Immunofluorescence detected EPC morphology (D), DiI-acLDL uptake (E), and lectin binding (F) in CACs. Late EPCs were shown to simultaneously endocytose DiI-acLDL (red). Phenotyping of the endothelial characteristics of EPCs. Immunofluorescence detected (green) CD31 (G), VE-cadherin (H), lectin binding (I), KDR (J), CD34 (K), and AC133 (L) in late EPCs. Scale bar: 50 μm. (PPTX 901 kb)
12265_2019_9889_MOESM3_ESM.pptx (1.3 mb)
Supplementary Fig. 3 Effects of fenofibrate on late endothelial progenitor cells migration and tube formation derived from heart failure patients and healthy volunteers. (A) An in vitro angiogenesis assay was performed with late endothelial progenitor cells (EPCs) in an ECMatrix gel. Representative photomicrographs demonstrating tube formation in late EPCs derived from heart failure (HF) patients and healthy volunteers. (B) An in vitro angiogenesis assay was performed with late EPCs in an ECMatrix gel. Representative photomicrographs demonstrating tube formation in healthy volunteers-derived late EPCs in response to fenofibrate. (C) A modified Boyden chamber assay was used with VEGF as the chemoattractant to measure the migration of late EPCs derived from HF patients and healthy volunteers. Representative photos are shown; the small dots are holes in the barrier membrane. The migrated cells were stained with hematoxylin and counted under a microscope. (D) An in vitro angiogenesis assay was performed with late EPCs in an ECMatrix gel. Representative photomicrographs demonstrating tube formation in late EPCs in response to fenofibrate with or without L-NAME. (PPTX 1299 kb)
12265_2019_9889_MOESM4_ESM.pptx (1.1 mb)
Supplementary Fig. 4 The effect of fenofibrate on circulating angiogenic cells (CACs) adhesion and proliferation. The incubation of CACs with fenofibrate (10 μmol/L) for 24 h increased p-Akt protein levels as assessed by western blot (Fig. 1Cfull gel). (PPTX 1093 kb)
12265_2019_9889_MOESM5_ESM.pptx (2.6 mb)
Supplementary Fig. 5 Effects of fenofibrate on eNOS, Akt phosphorylation, and nitric oxide production in late EPCs. (A) Representative immunoblots indicating the levels of eNOS and β-actin in late EPCs in response to the indicated concentration of fenofibrate for 24 hours. (Fg. 3A full gel). (B) After the incubation of EPCs with 50 μmol/L fenofibrate for the indicated time, the levels of eNOS and Akt protein phosphorylation were analyzed. (Fg. 3B full gel). (C) After the incubation of EPCs with fenofibrate at the indicated concentration for 2 h, the levels of eNOS and Akt protein phosphorylation were analyzed. (Fg. 3C full gel). (PPTX 2680 kb)
12265_2019_9889_MOESM6_ESM.pptx (782 kb)
Supplementary Figure 6 The Akt and eNOS activation pathway mediates fenofibrate–induced cell migration in late endothelial progenitor cells. A representative immunoblot indicating the levels of eNOS and β-actin in late endothelial progenitor cells (EPCs) in response to treatment with the indicated concentration of eNOS siRNA and fenofibrate for 24 hours. (Fg. 4B full gel). (PPTX 782 kb)
12265_2019_9889_MOESM7_ESM.pptx (745 kb)
Supplementary Figure 7 The AMPK/Akt pathway mediates fenofibrate–induced cell tube formation in late endothelial progenitor cells. (B) After the incubation of endothelial progenitor cells (EPCs) with the indicated concentration of fenofibrate, compound C, or AICAR (AMPK activator) for 2 h, Akt protein phosphorylation levels were analyzed. (Fg. 5B full gel). (C) After the incubation of EPCs with the indicated concentration of fenofibrate for 24 h, eNOS protein accumulation levels were analyzed. (Fg. 5C full gel) (PPTX 744 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Wen-Pin Huang
    • 1
    • 2
  • Wei-Hsian Yin
    • 2
    • 3
  • Jia-Shiong Chen
    • 1
  • Po-Hsun Huang
    • 1
    • 4
    • 5
    • 3
    Email author
  • Jaw-Wen Chen
    • 4
    • 6
    • 7
    • 3
  • Shing-Jong Lin
    • 1
    • 4
    • 8
    • 3
    Email author
  1. 1.Institute of Clinical MedicineNational Yang-Ming UniversityTaipeiTaiwan
  2. 2.Division of CardiologyCheng-Hsin Rehabilitation Medical CentreTaipeiTaiwan
  3. 3.Cardiovascular Research CenterNational Yang-Ming UniversityTaipeiTaiwan
  4. 4.Division of CardiologyTaipei-Veterans General HospitalTaipeiTaiwan
  5. 5.Department of Critical Care MedicineTaipei-Veterans General HospitalTaipeiTaiwan
  6. 6.Department of Medical ResearchTaipei Veterans General HospitalTaipeiTaiwan
  7. 7.Institute of PharmacologyNational Yang-Ming UniversityTaipeiTaiwan
  8. 8.Healthcare and Management CenterTaipei Veterans General HospitalTaipeiTaiwan

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