gms | German Medical Science

24. Jahrestagung der Deutschen Gesellschaft für Arterioskleroseforschung

Deutsche Gesellschaft für Arterioskleroseforschung

18.03. - 20.03.2010, Blaubeuren

The VEGF/VEGF receptor system modulates pro-apoptotic endonucleases and their chaperones in the vascular endothelium

Meeting Contribution

  • corresponding author N. Al-Fakhri - Department of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, Philipps-University, Marburg, Germany
  • L. Mey - Department of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, Philipps-University, Marburg, Germany
  • M. Hörmann - Department of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, Philipps-University, Marburg, Germany; Clinic for Vascular Surgery, University Hospital of Cologne, Germany
  • Z. Kharip - Department of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, Philipps-University, Marburg, Germany
  • K. Nemeth - Department of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, Philipps-University, Marburg, Germany
  • A. Hildenberg - Department of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, Philipps-University, Marburg, Germany
  • C. Löwer - Department of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, Philipps-University, Marburg, Germany
  • M. Heidt - Department of Cardiology, Justus Liebig University, Giessen, Germany
  • H. Renz - Department of Laboratory Medicine and Pathobiochemistry, Molecular Diagnostics, 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. Doc10dgaf06

doi: 10.3205/10dgaf06, urn:nbn:de:0183-10dgaf065

Published: March 23, 2011

© 2011 Al-Fakhri et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( You are free: to Share – to copy, distribute and transmit the work, provided the original author and source are credited.


Vascular endothelial growth factor (VEGF) has the most potent anti-apoptotic effect on vascular endothelial cells (EC). Apoptosis is executed by endonucleases, but the influence of VEGF on endonucleases and their inhibitors, such as caspase-activated deoxyribonuclease (CAD) and inhibitor of caspase-activated deoxyribonuclease (ICAD), has not been investigated yet. Therefore, the effects of VEGF on CAD and ICAD were investigated in EC cultures, in an organ culture model and in human arteries. Incubation of EC with VEGF reduced the sensitivity to apoptosis induction and increased expression of ICAD leading to a reduced endonuclease activity of CAD. The effect on endothelial apoptosis was demonstrated to be ICAD specific by mRNA knockdown experiments. VEGF signaling involved VEGFR-2 and neuropilin-1. The relevance of the described VEGF effects on ICAD and CAD were demonstrated in an organ culture model and in human arteriosclerotic compared to normal arteries. Thus, VEGF exerts part of its anti-apoptotic effect by regulation of the endonuclease inhibitor ICAD. VEGF influences endothelial survival by interfering with the terminal stage of apoptosis execution through the inhibition of endonuclease activity. This offers a new potent target for the modulation of VEGF-driven vascular processes.


Vascular endothelial growth factor (VEGF) is the most potent anti-apoptotic factor for endothelial cells (EC). VEGF induces the resistance of vascular EC towards pro-apoptotic stimuli by activation of JNK and Akt dependent signaling pathways and by induction of PARP production, as recently demonstrated [1]. Endonucleases are the main factors determining the end-phase of apoptosis execution. The effect of VEGF on endonucleases and their chaperones that act as nuclease inhibitors has not been investigated previously.

The main endonuclease of human cells is caspase-activated deoxyribonuclease (CAD), syn. DNA-fragmentation factor-40 (DFF-40), that exerts a DNAse I-like action leading to internucleosomal DNA-fragmentation and the typical apoptotic DNA-fragmentation pattern. Its chaperone is inhibitor of caspase-activated deoxyribonuclease (ICAD). ICAD has two isoforms, the S-(35kD) and L-(45kD) isoform (syn. DFF-45 and -35) [2]. Caspase 3 is the primary inactivator of ICAD leading to liberation and activation of CAD [3]. However, ICAD may also inhibit the caspase cascade and is thus an anti-apoptotic intracellular protein [2].

The aim of the present study was to analyze the influence of VEGF and its receptor system on the endothelial endonuclease CAD and the inhibitor ICAD in cultivated EC and human arteries.


Human EC, primary cells (HUVEC) and EC lines (EA.hy.926, hCMEC/D3) were incubated with VEGF-A(165) 100 pg/ml – 1 µg/ml. Apoptosis was induced by cRGDfV (alpha v-integrin inhibitor cyclo-[Arg-Gly-Asp-D-Phe-Val]) 5 µg/ml. The incubation period was dependent on the experimental setting: for analysis of mRNA 12 h, ICAD protein and CAD nuclease activity 24 h – 6 d (48 h intervals), flow cytometry 20 h, caspase activity 1–20 h, signal transduction 15–120 min. Apoptosis was analyzed by flow cytometric measurement of the apoptosis rate via annexin V / propidium iodide staining, and quantification of caspase 3/7 activity and of CAD endonuclease activities. ICAD protein was analyzed by means of Western blot and quantified by automated densitometry relative to the house-keeping protein beta-actin. ICAD mRNA was quantified by real-time PCR and normalized to the house-keeping gene GAPDH. The functional relevance of ICAD was investigated by longterm mRNA knockdown experiments using ICAD siRNA directed against both ICAD isoforms (Qiagen). The impact of VEGF receptors (VEGFR) VEGFR-2 and neuropilin-1 (NP-1) on VEGF signaling related to ICAD regulation was demonstrated by incubation with the specific receptor inhibitors SU5416 and A7R. Signal transduction was investigated by Western blot of phospho and total MAPK p38, ERK-1/2, JNK, and Akt and by inhibition with specific signaling inhibitors (triciribine, Akti-1/2, SP600125). VEGFR-1, VEGFR-2 and NP-1 were demonstrated on the surface of EC by immunofluorescence microscopy. Nuclear translocation of CAD was analyzed by CAD immunofluorescence and DAPI counterstaining. Human arteriosclerotic (n=25) and normal (n=10) arteries and a vascular organ culture model (n=9) were investigated by immunohistochemistry and by immunofluorescence microscopy for the expression of ICAD, CAD, VEGF, VEGFR-1, VEGFR-2 and NP-1. The vascular organ culture model was performed by incubating 2 mm rings of internal mammary artery (IMA) and saphenous vein (SV) for 6 d with or without VEGF 10 ng/ml.


VEGF induced a time and dose-dependent increase in apoptosis resistance of EC. Apoptosis induction by adhesion inhibition through cRGDfV lead to the activation of the caspase cascade and to an increase in endonuclease activity. Following caspase activation, ICAD was degraded and CAD was translocated to the nucleus. Preincubation of EC with VEGF for 24 h or longer (up to 6 d) resulted in a reduced capacity of CAD endonuclease activation. VEGF induced the expression of ICAD, the chaperone of CAD, wich was shown on the mRNA and protein level (Figure 1 [Fig. 1]) and wich was paralleled by an inhibition of CAD activity (Figure 2 [Fig. 2]).

mRNA knockdown of ICAD reduced ICAD protein production only after repeated transfection for 4 weeks or longer, since ICAD is present in EC in great abundance and in a stoichiometric excess to CAD [2]. ICAD turnover in the cell may also be low, since mRNA knockdown was demonstrated already after 48 h, thus making longterm gene knockdown necessary. Repeated ICAD siRNA incubation for 4 weeks partly reversed apoptosis inhibition by VEGF, thus confirming that the effect of VEGF was mediated by ICAD proving the specificity of the VEGF effect on ICAD production.

VEGF signaling was transduced by VEGFR-2 and NP-1, as demonstrated by incubation with specific receptor inhibitors, and intracellularly by JNK and Akt dependent pathways. VEGFR-2 and NP-1 were shown to be colocalized on the EC surface, whereas VEGFR-1 was not detectable.

ICAD could be induced in the endothelium of an arterial organ culture model by incubation with VEGF 10 ng/ml over 2–6 d. VEGF preincubation also reduced CAD translocation to the nucleus of EC in the culture model. In arteriosclerotic arteries, ICAD expression of the endothelium was increased as compared to the endothelium of normal arteries. Also, CAD expression and translocation was increased in arteriosclerosis implying an increased requirement of endonuclease inhibitors because of increased endonuclease activation in the endothelium of arteriosclerotic arteries.


VEGF exerts its anti-apoptotic properties through regulation of anti-apoptotic proteins and signaling pathways on different cellular levels. Here, we show that VEGF influences the terminal processes of apoptosis execution. By increasing ICAD expression, VEGF inhibits the activation of endonucleases, mainly of CAD, the most abundant nuclease in human cells.

Persistent and strong stimulation of proliferation or cell replication, such as in virus-infected cells, has previously been shown to induce ICAD gene expression and promote ICAD protein expression [4]. The total cellular content of ICAD was thus increased and protected nuclear structures from the initiation of apoptotic cell death [4]. ICAD binds CAD and an increased level of the protein confers increased resistance to caspase cleavage. ICAD can be induced independently from CAD expression, changing the normal 1:1 ratio between the endonuclease CAD and its chaperone ICAD, thereby increasing the apoptotic threshold [4]. Overexpression of ICAD inhibited staurosporine- or Fas-induced DNA-fragmentation in other experimental models [5]. ICAD is therefore a potent target for the anti-apoptotic action of VEGF. Through this VEGF protects the endothelium from apoptosis and shields the vasculature from endothelial cell death induced by pro-apoptotic stimuli.


Hörmann M, Mey L, Kharip Z, Hildenberg A, Nemeth K, Heidt M, Renz H, Al-Fakhri N. Vascular endothelial growth factor promotes endothelial survival by induction of poly(ADP-ribose)-polymerase expression in the human vasculature. J Thromb Haemost. 2010 (in revision)
Widlak P, Garrard WT. Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G. J Cell Biochem. 2005;94:1078-87. DOI: 10.1002/jcb.20409 External link
Wolf BB, Schuler M, Echeverri F, Green DR. Caspase-3 is the primary activator of apoptotic DNA fragmentation via DNA fragmentation factor-45/inhibitor of caspase-activated DNase inactivation. J Biol Chem. 1999;274:30651-6. DOI: 10.1074/jbc.274.43.30651 External link
Sacco R, Tsutsumi T, Suzuki R, Otsuka M, Aizaki H, Sakamoto S, Matsuda M, Seki N, Matsuura Y, Miyamura T, Suzuki T. Antiapoptotic regulation by hepatitis C virus core protein through up-regulation of inhibitor of caspase-activated DNase. Virology. 2003;317(1):24-35. DOI: 10.1016/j.virol.2003.08.028 External link
Sakahira H, Enari M, Nagata S. Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature. 1998;391:96-9. DOI: 10.1038/34214 External link