Fenofibrate regulates retinal endothelial cell survival through the AMPK signal transduction pathway

https://doi.org/10.1016/j.exer.2007.01.009Get rights and content

Abstract

Fenofibrate, a widely used hypolipidemic drug, has anti-inflammatory and anti-atherosclerotic effects in the vessel wall. In the present study, we report an anti-apoptotic property of fenofibrate in human retinal endothelial cells (HRECs) and describe an underlying molecular mechanism. Treatment with fenofibrate protected HRECs from apoptosis in response to serum deprivation in a dose-dependent manner. This inhibition of apoptosis by fenofibrate was not altered by peroxisome proliferator-activated receptor α (PPARα) antagonist MK 886, and selective agonist for PPARα, WY-14643 had no beneficial effects on serum deprivation-induced cell death. Fenofibrate potently induced a sustained activation of AMP-activated protein kinase (AMPK) and vascular endothelial growth factor (VEGF) mRNA expression. Furthermore, compound C, a specific AMPK inhibitor, almost completely blocked the fenofibrate-induced survival effect as well as VEGF mRNA expression. Taken together, these results suggest that fenofibrate prevents apoptotic cell death induced by serum deprivation through PPARα-independent, but AMPK-dependent pathway. Thus fenofibrate may have a novel therapeutic property that can control unwanted cell death found in diabetic retinopathy.

Introduction

Fibric acid derivatives, which are peroxisome proliferator-activated receptor α (PPARα) agonists, have beneficial effects on lipid profiles, including lowering triglycerides and increasing high-density lipoprotein cholesterol, and in reducing coronary vascular events in large clinical trials (Frick et al., 1997, Robins et al., 2003). Treatment with fenofibrate, one of the most widely used fibric acid derivatives, reduces the angiographic progression of coronary artery disease in type 2 diabetes (Diabetes Atherosclerosis Intervention Study Investigators, 2001). Fenofibrates also improved endothelial dysfunction in patients with type 2 diabetes with dyslipidemia (Playford et al., 2002). The mechanisms for the vascular benefit of fenofibrates are unclear, but probably involve direct anti-inflammatory effects including the modulation of the expression of several cytokines and adhesion molecules and anti-atherosclerotic effects (Israelian-Konaraki and Reaven, 2005).

AMP-activated protein kinase (AMPK) is a ubiquitously expressed enzyme whose activity is increased by adipocyte-derived hormones, including leptin and adiponectin, as well as by pathological stresses such as hypoxia, glucose deprivation, and hyperosmolarity (Hardie and Carling, 1997, Carling, 2004). Activated AMPK phosphorylates multiple target genes, including several biosynthetic enzymes such as acetyl-CoA carboxylase (ACC), hydroxymethylglutaryl-CoA reductase, glycogen synthase, and endothelial nitric oxide synthase (eNOS) (Chen et al., 1999). AMPK has an important role in the cardiovascular protection. For example, AMPK activation by α-lipoic acid prevents endothelial dysfunction in insulin-resistant rats (Lee et al., 2005). AMPK also protects against myocardial injury and apoptosis in the ischemic heart (Russell et al., 2004, Shibata et al., 2005). A recent study suggests that fenofibrate activates AMPK and increases eNOS phosphorylation in human umbilical vein endothelial cells (Murakami et al., 2006). However, the effect of fenofibrate on endothelial cell survival has not been fully investigated. The objectives of the present study were to examine whether fenofibrate activates AMPK and promotes anti-apoptotic effects in human retinal endothelial cells. We found that the AMPK signaling plays an important role in the fenofibrate-mediated endothelial cell survival.

Section snippets

Reagents

Fenofibrate and WY-14643 were purchased from Sigma Chemical Co. (St Louis, MO). MK 886 was obtained from Alexis Biochemicals (San Diego, CA). Compound C was from Calbiochem. (La Jolla, CA). Fenofibrate, WY-14643, MK 886, and Compound C were dissolved in dimethyl sulfoxide (DMSO). Antibodies against AMPK, p-AMPK-α-Thr172, ACC, p-ACC-Ser79 were from Cell Signaling Technology (Beverly, MA). Endothelial basal medium (EBM-2) and endothelial cell growth medium (EGM-2) were from Cambrex (East

Fenofibrate protects HRECs from serum deprivation-induced apoptosis

Serum deprivation caused cell death in HRECs, as evidenced by more floating and less adherent cells seen with phase-contrast microscopy (Fig. 1A). Approximately 40% of total cells undergo cell death at 36 h during serum deprivation. As shown in Fig. 1B, addition of fenofibrate (10–50 μM) reversed the serum deprivation associated cell death in a concentration-dependent manner (P < 0.01). To verify that cell death was mediated by apoptosis, cells were subjected to DAPI staining. As shown in Fig. 1C,

Discussion

In the present study, we demonstrated that in HRECs fenofibrate is anti-apoptotic and its action is mediated in part by AMPK signaling. The survival of endothelial cells is critical for the maintenance of microvascular integrity. It has been reported that retinal endothelial cells die prematurely and undergo apoptosis in both human and experimental diabetes (Mizutani et al., 1996). Acellular or nonperfused capillaries can lead to sight-threatening proliferative retinopathy. A recent report has

Acknowledgements

We thank Professor E. Dale Abel and Dr. Adam R. Wende of the School of Medicine, University of Utah for critically reading the manuscript, and Dr. Jeong-Ki Min of the Department of Biochemistry College of Sciences, Yonsei University for very helpful discussion and advice. This work was supported by the Korea Research Foundation Grant funded by the Korean Government (KRF-2005-003-E00104).

References (44)

  • K.K. Nagothu et al.

    Fibrate prevents cisplatin-induced proximal tubule cell death

    Kidney Int.

    (2005)
  • D.A. Playford et al.

    Effect of fenofibrate on brachial artery flow-mediated dilatation in type 2 diabetes mellitus

    Am. J. Cardiol.

    (2002)
  • J. Tran et al.

    Marked induction of the IAP family antiapoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells

    Biochem. Biophys. Res. Commun.

    (1999)
  • A.N. Witmer et al.

    Vascular endothelial growth factors and angiogenesis in eye disease

    Prog. Retin. Eye Res.

    (2003)
  • H. Yun et al.

    Glucose deprivation increases mRNA stability of vascular endothelial growth factor through activation of AMP-activated protein kinase in DU145 prostate carcinoma

    J. Biol. Chem.

    (2005)
  • T. Alon et al.

    Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity

    Nat. Med.

    (1995)
  • B.J. Davis et al.

    Activation of the AMP-activated kinase by antidiabetes drug metformin stimulates nitric oxide synthesis in vivo by promoting the association of heat shock protein 90 and endothelial nitric oxide synthase

    Diabetes

    (2006)
  • Diabetes Atherosclerosis Intervention Study Investigators

    Effect of fenofibrate on progression of coronary-artery disease in type 2 diabetes: the Diabetes Atherosclerosis Intervention Study, a randomised study

    Lancet

    (2001)
  • A.B. El-Remessy et al.

    Oxidative stress inactivates VEGF survival signaling in retinal endothelial cells via PI 3-kinase tyrosine nitration

    J. Cell Sci.

    (2005)
  • N. Ferrara

    Role of vascular endothelial growth factor in regulation of physiological angiogenesis

    Am. J. Physiol. Cell. Physiol.

    (2001)
  • M.H. Frick et al.

    Prevention of the angiographic progression of coronary and vein-graft atherosclerosis by gemfibrozil after coronary bypass surgery in men with low levels of HDL cholesterol. Lopid Coronary Angiography Trial (LOCAT) Study Group

    Circulation

    (1997)
  • Y. Fujio et al.

    Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart

    Circulation

    (2000)
  • Cited by (101)

    • Metronomic anti-angiogenesis: The ideal companion of pH-centered treatments

      2020, An Innovative Approach to Understanding and Treating Cancer: Targeting pH: From Etiopathogenesis to New Therapeutic Avenues
    • Fenofibrate improves vascular endothelial function in diabetic mice

      2019, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      In addition, AMPK was indicated as a drug target for diabetes mellitus, and previous experiments reported that fenofibrate can activate AMPK [54]. Activation of AMPK by fenofibrate could prevent apoptotic cell death induced by serum deprivation [55]. Although, the effect of fenofibrate on eNOS has been described in previous studies, and other studies have shown that fenofibrate normalizes endothelium-dependent relaxation in diabetic mice aortas through activating PPAR/LKB1/AMPK/eNOS signaling pathway, there has been no further verification at the cell level [56,14].

    • Fenofibrate improves vascular endothelial function and contractility in diabetic mice

      2019, Redox Biology
      Citation Excerpt :

      We found that fenofibrate administration increased the protein expression of PPARα and AMPKα phosphorylation in aortas from diabetic mice. This result was consistent with previous cell experiments reporting that fenofibrate has a role in AMPKα activation [19,20], but the mechanism by which fenofibrate activates AMPK is poorly defined. Interestingly, a potentially important observation in our study is that fenofibrate administration upregulated LKB1 by transferring from the nucleus to the cytoplasm and increased phosphorylation of LKB1, thus activating AMPK in the cytosol.

    View all citing articles on Scopus
    View full text