gms | German Medical Science

24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung

Deutsche Gesellschaft für Arterioskleroseforschung

18.03. - 20.03.2010, Blaubeuren

Effect of Growth Differentiation Factor-15 (GDF-15) on the expression of apoptosis-relevant genes in a mouse-model of atherosclerosis

Meeting Contribution

  • corresponding author J. Thogersen - Ruprecht-Karls-University Heidelberg, Germany
  • S. Zügel - Philipps-University Marburg, Germany
  • G. A. Bonaterra - Philipps-University Marburg, Germany
  • G. Bendner - Ruprecht-Karls-University Heidelberg, Germany
  • S. Vorwald - Ruprecht-Karls-University Heidelberg, Germany
  • J. Fey - Ruprecht-Karls-University Heidelberg, Germany
  • U. Traut - Ruprecht-Karls-University Heidelberg, Germany
  • J. Strelau - Ruprecht-Karls-University Heidelberg, Germany
  • R. Kinscherf - Philipps-University Marburg, Germany

Deutsche Gesellschaft für Arterioskleroseforschung e.V.. 24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung. Blaubeuren, 18.-20.03.2010. Düsseldorf: German Medical Science GMS Publishing House; 2011. Doc10dgaf13

DOI: 10.3205/10dgaf13, URN: urn:nbn:de:0183-10dgaf135

Published: March 23, 2011

© 2011 Thogersen et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.en). You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Abstract

Growth differentiation factor-15 (GDF-15) is a distant and divergent member of the TGF-β super family and is expressed in macrophages after stimulation by several biological mediators. Interactions between peripheral blood mononuclear cells (PBMCs) and those within plaques are suggested to be pathophysiologically relevant to lipid-induced atherosclerosis. We therefore postulated that the expression of GDF-15 is associated with the development and progression of atherosclerotic plaque, possibly through the regulation of apoptotic processes.


Introduction

Atherosclerosis is a common disorder that specifically affects middle and large arteries [1], [2] and is a slow, complex disease that typically starts in childhood and often progresses when people grow older [1], [2]. Atherosclerosis is characterized by the accumulation on the inner wall of lipids together with complex carbohydrates and fibrous tissue that form the atherosclerotic plaque (atheroma) [1]. Furthermore, the activation of monocytes to macrophages (MФ) plays a central role in the pathophysiological process [1]. Additionally, immune- and inflammatory reactions are involved in the development of the disease, which are caused by the ability of MФ to phagocyte injurious material and to release biologically active molecules, like cytokines [2].

GDF-15 is widely distributed in adult tissue, being most strongly expressed in choroid plexus epithelium, Schwann cells and in cardiomyocytes, microglia and macrophages after stress exposure [3]. Furthermore, GDF-15 is a new biomarker of cardiac vascular dysfunction and an independent predictor of coronary heart disease (CHD) mortality across the broad spectrum of patients with stable and unstable CHD [4]. Moreover, GDF-15 is expressed in MФ after stimulation by several biological mediators, including tumor necrosis factor-a (TNFα), interleukin 1, macrophage-colony stimulating factor (M-CSF), oxidized low-density lipoprotein (oxLDL), ceramide or hydrogen peroxide, but is not expressed in resting MФ [5]. Activation of PBMCs and their interaction with those within plaques are suggested to be pathophysiologically relevant for lipid-induced atherosclerosis [6].


Methods

Animals: To analyze the physiological functions of GDF-15, we generated GDF-15 LacZ-knockin (GDF15-/-) mice and crossbred them with apoE-deficient (apoE-/-) mice to yield apoE-/-/GDF-15-/- mice. After 10 weeks, the offspring were fed for 12 weeks with a cholesterol-enriched (western) diet.

Immunohistomorphometrical analyses of CD11b, CD68 (macrophages) and α-actin (smooth muscle cells) were performed on cryostat cross-sections of the innominate artery (brachiocephalic trunk). Lumen stenosis, cell density and apoptosis (TUNEL) were also measured histomorphometrically.

Peritoneal macrophages were isolated from apoE-/-/GDF15+/+ as well as from apoE-/-/GDF15-/- mice after intraperitoneal injection of pristane (2,6,10,14 tetramethylpentadecane). Thereafter, they were incubated with 100 µg/ml native (nLDL) or oxidized LDL (oxLDL) for 24 h. The mRNA was isolated, tested for quality and analyzed by real-time PCR (qRT-PCR).

qRT-PCR: The mRNA expressions of Bcl-2-associated death promoter (BAD), caspase-3 (Casp-3), TNF receptor super family member 6 (Fas-R, CD95) and CD40 antigen (CD40) were measured by qRT-PCR. The qRT-PCR data were analyzed using the Mx3005P™ QPCR System (Stratagene). For comparison of stability, six reference candidate genes (housekeeping genes) were used: glucuronidase-beta (Gusb); hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1); cytosolic heat shock protein 90 alpha; class B member 1 (Hsp90ab1); glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin-beta (ACTB).

The software Best Keeper (BK), version 1 was employed according to manufacturer's recommendations to calculate a BK normalization factor. Expression of mRNA was normalized using the BK factor.

Statistical analyses: Results are presented as mean + SEM. Depending on the mode of distribution, statistical procedures were performed by the Mann-Whitney or by the Student’s t-test for unpaired data. A p-value of 0.05 or less was chosen for statistical significance.


Results

Figure 1 [Fig. 1] shows a significant (p<0.04) increase of the percentage of the CD68 immunoreactive MΦ in atherosclerotic lesions of apoE-/-/GDF-15-/- compared to apoE-/-/GDF-15+/+ mice (Figure 1A [Fig. 1]), whereas the percentage of CD11b (Figure 1B [Fig. 1]) immunoreactive cells was decreased (p<0.05), but percentage of α-actin immunoreactive smooth muscle cells was similar in both groups (Figure 1C [Fig. 1]). Furthermore, in atherosclerotic lesions of apoE-/-/GDF-15-/- mice, we found a significant (p<0.004) increase of the cell density (Figure 1D [Fig. 1]) as well as a significant (p<0.04) decrease of the lumen stenosis (Figure 1E [Fig. 1]), whereas the percentage of apoptotic cells (TUNEL positive) was marginally decreased compared to apoE-/-/GDF-15+/+ mice (Figure 1F [Fig. 1]).

Treatment of cultured peritoneal MФ of apoE-/-/GDF-15-/- mice with ox-LDL, compared to treatment of native LDL, revealed changes in the expression of several apoptosis-relevant genes (e. g. Atf5, Bag3, Bak1, Bid, Casp3, Casp7 and TNF) (Figure 2 [Fig. 2]).


Discussion

Atherosclerosis is an inflammatory disease [1] involving several cytokines and growth factors. It has been suggested that in atherosclerotic lesions, apoptosis is triggered by inflammatory processes, both via cell-cell contact and by cytokines and oxidized lipids [7]. Interestingly, pro-inflammatory and pro-apoptotic markers (which might be interpreted also as markers of anti-inflammatory processes) have been shown to be in part co-localized in cells of human atherosclerotic lesions [5], [8]. Now, interest is increasing about intracellular interactions between the cell death program (anti-/pro-apoptotic) and the inflammatory process (anti-/pro-inflammatory), which are key processes in MФ of atherosclerotic plaques. In activated, human MФ a participation of GDF-15 in the oxLDL induced expression of cytokines and apoptosis was likewise found [5], thus it can be hypothesized that GDF-15 is apparently responsible for an early mediator of damage, which could adjust inflammation [1] survival, proliferation or apoptosis during illness processes [9]. Our studies show that after a 12-week western-diet the increased serum cholesterol level in apoE-/-/GDF-15-/- in comparison with apoE-/-/GDF-15+/+ mice (results not shown) were not different between both groups. However, an increase of weight and adipositas was observed in apoE-/-/GDF-15-/- mice, possibly being an indicator that GDF-15 is associated with a regulation of appetite and the intake of food. However, we found a reduction of the lumen stenosis in the innominate artery of apoE-/-/GDF-15-/- mice that seems to be independent from food intake. Possible explanations for these findings might be the decreased percentage of CD11b cells in atherosclerotic lesions of the innominate artery of apoE-/-/GDF-15-/- mice and unchanged amount of CD68 positive cells and proliferation status of smooth muscle cells (α-actin). Simultaneously, the reduction of the cell density may be an evidence that GDF-15 expression is related to the plaque stability/instability status. Previous publications from our group [9] show pro-apoptotic effects of oxLDL on human MФ [5] and that GDF-15 is inducible by oxLDL and its mediators in vitro [9]. Treatment of peritoneal MФ from apoE-/-/GDF-15-/- mice with oxLDL, compared to treatment with native LDL, revealed changes in the expression of several apoptosis-relevant genes (e. g. Atf5, Bag3, Bak1, Bid, Casp3, Casp7 and TNFα). Therefore, we conclude from our results a possible relation between GDF-15 and apoptosis as well as atherosclerosis regulation, both in vivo and in vitro. Complementary experiments must be performed to confirm this hypothesis.


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