MicroRNA-34a regulation of endothelial senescence

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Abstract

Endothelial senescence is thought to play a role in cardiovascular diseases such as atherosclerosis. We hypothesized that endothelial microRNAs (miRNAs) regulate endothelial survival and senescence. We found that miR-34a is highly expressed in primary endothelial cells. We observed that miR-34a expression increases in senescent human umbilical cord vein endothelial cells (HUVEC) and in heart and spleen of older mice. MiR-34a over-expression induces endothelial cell senescence and also suppresses cell proliferation by inhibiting cell cycle progression. Searching for how miR-34a affects senescence, we discovered that SIRT1 is a target of miR-34a. Over-expressing miR-34a inhibits SIRT1 protein expression, and knocking down miR-34a enhances SIRT1 expression. MiR-34a triggers endothelial senescence in part through SIRT1, since forced expression of SIRT1 blocks the ability of miR-34a to induce senescence. Our data suggest that miR-34a contributes to endothelial senescence through suppression of SIRT1.

Research highlights

► MicroRNA-34a (miR-34a) regulates senescence and cell cycle progression in endothelial cells. ► MiR-34a expression increases during endothelial cell senescence and in older mice. ► SIRT1 is a miR-34a target gene in endothelial cells. ► SIRT1 mediates the effects of miR-34a upon cell senescence in endothelial cells.

Introduction

Senescence of endothelial cells may play an important role in cardiovascular diseases such as atherosclerosis and thrombosis [1], [2], [3], [4]. Changes in many transcription factors are associated with aging [5], [6], [7]. Sirtuin 1 (SIRT1), the mammalian homolog of Sir2, regulates cell cycle, cellular senescence, and metabolism by deacetylating p53, Forkhead box subfamily O (Foxo), Ku70 and peroxisome proliferator-activated receptor gamma (PPAR-gamma) [8], [9], [10], [11], [12], [13], [14]. Over-expression of Sir2 can extend the life span of yeast, worms, and flies [15], [16], [17]. In human fibroblasts, increased expression of SIRT1 can delay cellular senescence and extend cellular life span [18], [19]. SIRT1 also has a role in vascular homeostasis by controlling endothelial cell senescence and angiogenesis [20], [21]. However, the mechanisms regulating senescence of endothelial cells mostly remain unknown.

MicroRNA (miRNA) are small non-coding RNAs that inhibits gene expression by binding to complementary sequences of 3′UTR in their target mRNAs [22], [23], [24], [25]. MiRNAs modulate a variety of biological functions such as cell development, cell differentiation, and apoptosis [26], [27], [28]. MiRNAs regulate endothelial biology, since knockdown of Dicer or Drosha in human endothelial cells decrease angiogenesis in vitro [29], [30]. Several miRNAs involved in endothelial cell functions have been identified. MiR-126, miR-92, and miR-221/222 control the growth of new blood vessels [31], [32], [33], [34], [35]. A recent report showed that miR-217 regulates senescence in endothelial cells [36], but the role of other miRNAs that regulate senescence in endothelial cells are not fully clarified.

MiR-34a has been shown to regulate genes involved in cell cycle regulation and apoptosis in a p53 dependent or independent manner in cancer cells [37], [38], [39], [40], [41], [42], [43], [44]. However, the function of miR-34a in endothelial cells is not known. We now show that miR-34a is abundantly expressed in primary endothelial cells. We investigated the role of miR-34a in endothelial cells and found that miR-34a regulates SIRT1 expression and controls cell senescence. Aging endothelial cells expressed high levels of miR-34a and low levels of SIRT1 protein. Over-expression of miR-34a decreased SIRT1 protein level and increased acetylated p53 level in endothelial cells. Our data suggest that miR-34a regulates endothelial senescence in part through SIRT1.

Section snippets

Cell culture and transfection

Pooled HUVECs (population doubling level; PDL 7–45), HAEC, and HMVEC were cultured in endothelial basal medium (EBM2) supplemented with growth factors (Lonza). Reagents were obtained from Sigma (St. Louis, MO). For primary endothelial cell culture, serial passage was performed when the cells got 80% confluence. The number of PDL was calculated as described previously [45]. We used cells in PDL < 10 unless we mentioned in the manuscript. Pre-miRNAs and AS-miRNAs were obtained from Applied

MiR-34a is expressed in endothelial cells

We previously performed microarray for miRNAs in human umbilical cord endothelial cell (HUVEC) [33]. Since we found that miR-34a is moderately expressed in HUVEC, we analyzed miR-34a levels in several types of primary endothelial cells, including HUVEC, human aortic endothelial cell (HAEC) and human dermal microvascular endothelial cell (HMVEC) by qRT-PCR. These primary endothelial cells (within passage 5) expressed miR-34a at levels higher than HCT116 colon cancer cells and HeLa epithelial

Discussion

The major findings of this study are that endothelial cells express miR-34a, expression of miR-34a increases during senescence, and miR-34a regulates levels of SIRT1. Since SIRT1 is linked to cellular aging, our study suggests that a specific miRNA may regulate aging pathways.

Acknowledgments

The author is grateful to Dr. Charles Lowenstein for helpful discussion and critical reading of the manuscript. This work was supported by an SDG grant from American Heart Association (0835446N).

References (48)

  • D.P. Bartel

    MicroRNAs: target recognition and regulatory functions

    Cell

    (2009)
  • S. Wang et al.

    The endothelial-specific microRNA miR-126 governs vascular integrity and angiogenesis

    Dev. Cell

    (2008)
  • L. Poliseno et al.

    MicroRNAs modulate the angiogenic properties of HUVECs

    Blood

    (2006)
  • T.C. Chang et al.

    Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis

    Mol. Cell

    (2007)
  • N. Raver-Shapira et al.

    Transcriptional activation of miR-34a contributes to p53-mediated apoptosis

    Mol. Cell

    (2007)
  • N. Morisaki et al.

    New indices of ischemic heart disease and aging: studies on the serum levels of soluble intercellular adhesion molecule-1 (ICAM-1) and soluble vascular cell adhesion molecule-1 (VCAM-1) in patients with hypercholesterolemia and ischemic heart disease

    Atherosclerosis

    (1997)
  • T. Kumazaki et al.

    Expression of endothelin, fibronectin, and mortalin as aging and mortality markers

    Exp. Gerontol.

    (1997)
  • R.P. Brandes et al.

    Endothelial aging

    Cardiovasc. Res.

    (2005)
  • T. Minamino et al.

    Vascular cell senescence: contribution to atherosclerosis

    Circ. Res.

    (2007)
  • A. Orlandi et al.

    Aging influences development and progression of early aortic atherosclerotic lesions in cholesterol-fed rabbits

    Arterioscler. Thromb. Vasc. Biol.

    (2000)
  • M.Y. Lee et al.

    Senescence of cultured porcine coronary arterial endothelial cells is associated with accelerated oxidative stress and activation of NFkappaB

    J. Vasc. Res.

    (2009)
  • H.L. Hu et al.

    Antioxidants may contribute in the fight against ageing: an in vitro model

    Mech. Ageing Dev.

    (2000)
  • I. van Leeuwen et al.

    Sirtuins and p53

    Adv. Cancer Res.

    (2009)
  • S. Pillarisetti

    A review of Sirt1 and Sirt1 modulators in cardiovascular and metabolic diseases

    Recent Pat. Cardiovasc. Drug Discov.

    (2008)
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