This paper was presented at the 2nd Micro and Nano Flows Conference (MNF2009), which was held at Brunel University, West London, UK. The conference was organised by Brunel University and supported by the Institution of Mechanical Engineers, IPEM, the Italian Union of Thermofluid dynamics, the Process Intensification Network, HEXAG - the Heat Exchange Action Group and the Institute of Mathematics and its Applications.Angiogenesis, formation of new blood vessels, is a complex but critical phenomenon. In particular, it is regulated by different angiogenic factors. Nitric oxide (NO) is also a very well known biological mediator involved in vascular physiology. This study focuses on relationships between the effect of angiogenin, a major angiogenic factor, and extracellular NO release. NO concentration was sensed electrochemically using a fibronectin coated multiple microelectrode array. Angiogenin was shown to increase NO levels, thus triggering nitric oxide synthase (NOS) activity. Angiogenin reactive pathway being very complex, we have used various selective inhibitors of angiogenin to investigate the mechanism leading to NO production. Neomycin, an antibiotic blocking nuclear translocation, inhibited angiogenin effect on NOS. This result demonstrates that angiogenin activates NOS by interacting with the cell nucleus.This study is funded by Medermica Ltd; the DIUS;  KICOS (K20602000681-08B0100-02210); the Korea Science and Engineering Foundation (M10749000231-08N4900-23110); and the
Korea Biotech R&D Group of Next-Generation Growth Engine Project (F104AB010004-08A0201-00410)

2nd Micro and Nano Flows Conference (MNF2009)

Chang, SI

Chung, HM

Kang, DK

O'Hare, D

Trouillon, R

English

Brunel University Research Archive

2nd Micro and Nano Flows ConferenceWest London, UK, 1-2 September 2009Electrochemical sensing of angiogenin induced endothelial nitric oxide synthase activityRaphaël TROUILLON 1,*, Dong-Ku KANG 2, Soo-Ik CHANG 2, Hyung-Min CHUNG3, Danny O’HARE 1*Corresponding author: Tel.: ++44 (0)20 7594 6498; Fax: +44 (0)207 594 5177; Email: raphael.trouillon06@imperial.ac.uk1 Department of Bioengineering, Imperial College London, UK2 Department of Biochemistry, Chungbuk National University, Republic of Korea3 Graduate School of Life Science, CHA Stem Cell Institute, Pochon CHA University, Republic of KoreaAbstract: Angiogenesis,  formation  of  new  blood  vessels,  is  a  complex  but  critical  phenomenon.  In particular,  it  is  regulated by different  angiogenic  factors.  Nitric  oxide  (NO) is  also a  very well  known biological mediator involved in vascular physiology. This study focuses on relationships between the effect of  angiogenin,  a  major  angiogenic  factor,  and  extracellular  NO  release.  NO concentration  was  sensed electrochemically  using  a  fibronectin  coated  multiple  microelectrode  array.  Angiogenin  was  shown  to increase NO levels, thus triggering nitric oxide synthase (NOS) activity. Angiogenin reactive pathway being very complex, we have used various selective inhibitors of angiogenin to investigate the mechanism leading to NO production. Neomycin, an antibiotic blocking nuclear translocation, inhibited angiogenin effect on NOS. This result demonstrates that angiogenin activates NOS by interacting with the cell nucleus.Keywords: angiogenesis,  electrochemical  sensing,  nitric  oxide  synthase,  biomeasurements,  stem cells, angiogeninIntroductionAngiogenesis,  formation  of  new  blood vessels,  is  critical  factor  in  much pathology like  chronic  wounds  (Harding  et  al.,  2002) and  rheumatoid  arthritis  (Walsh,  1999).  In particular,  it  has  been  proven  that  tumours release angiogenic factors in order to support their  growth,  carcinogenesis  being  strongly dependent on blood supply (Carmeliet et al., 2000).  However,  angiogenesis  is  a  rather complex  biological  phenomenon  that  is dependent on the extra cellular matrix (Clark, 1995),  mechanical  or  shear  stress  (Baum et al.,  2004)  and  biochemical  factors.  For example,  some  growth  factors  like  vascular endothelial  growth  factors  are  known  to promote endothelial growth and angiogenesis (Ferrara, 2001). This biological phenomenon involves the activation of blood vessel cells to an  altered  state  as  a  response to  a  complex balance of competing stimuli. Investigating its biochemistry  therefore  seems  the  most obvious  way  to  understand  and  modulate angiogenesis.In  this  study,  we  are  focusing  on  an angiogenic protein, angiogenin. Angiogenin is a 123 amino acids long single-chain protein of about 14,400 kDa (Gao and Xu, 2008). This molecule  is  a  requirement  for  cell proliferation,  even  for  angiogenesis  induced by  other  angiogenic  factors,  like  fibroblast growth factors or endothelial  growth factors (Kishimoto et al., 2005). In particular, it has been  reported  that  nuclear  translocation  of angiogenin,  e.g.  angiogenin  entering  cell nucleus, is a critical step for the biochemical cascade triggering angiogenesis (Kishimoto et al., 2005). Furthermore, angiogenin is known to have several effects on cells (Gao and Xu, 2008): (i) it  can  bind  to  the  actin  receptors and cleave the extracellular matrix, thus allowing cell migration (Hu et al., 1994), (ii) it has an RNase activity (Russo et al., 1994), (iii) it  can  bind  to  cell  membrane receptors  and  trigger  an - 1 -2nd Micro and Nano Flows ConferenceWest London, UK, 1-2 September 2009intracellular  biochemical  cascade (probably  via  signal-associated kinases ERK1/2 (Liu et al., 2001) and  B/Akt  (Kim et  al.,  2008)  or via  the  phosphorylation  of  stress associated kinase SAPK/JNK (Xu et al., 2001)),(iv) it  can  enter  the  cell  nucleus  and interact  with  ribonucleotides (nuclear  translocation)  (Xu et  al., 2001). Because of these numerous  sites and modes of  action,  the  physiology  of  angiogenin  is quite unclear. NO  is  produced  by  nitric  oxide  synthase (NOS,  in  our  case  the  endothelial  NOS, eNOS) from L-arginine (Palmer et al. 1988). NO is  a  biological  transmitter  mediating  in particular  vasodilation  and  angiogenesis (Fukumura et  al.,  1998).  NO  is  therefore implied in a wide range of heart and vascular diseases. It has been demonstrated that nitric oxide  (NO)  plays  a  central  role  in angiogenesis  (Fukumura et  al.,  1998)  by triggering  the  proliferation  of  angiogenic growth  factors  and  the  differentiation  of endothelial  cells  into  vascular  tube  cells (Babaei  et  al.,  1998).  Ziche et  al.  have also shown that  NO can  lead  to  DNA synthesis and migration of coronary endothelial cells as many other  angiogenic  factors  (Ziche  et  al., 1993  and  1994).  In  addition,  NO may  also promote angiogenesis through its endothelial antiapoptotic  properties  (Chavakis  et  al., 2002):  angiogenesis  is  mainly  based  on proliferation of endothelial cells and therefore on inhibition of their apoptosis. As  a  consequence,  NO  is  a  key  factor  in angiogenesis. The purpose of this study is to investigate  relationships  between angiogenin and extracellular NO release. Measurement of NO was performed electrochemically, using a multiple  microelectrode  array  (MMA) developed at Imperial College London (UK) (Patel  et  al.,  2008).  Electrochemical  sensors are  attractive  devices  for  biomeasurements because  of  robustness,  linear  response  to concentration,  ease  of  production  and miniaturization.  We  have  already demonstrated the utility of the MMA for NO sensing in fibroblast cells (Patel et al., 2008). To  improve  biocompatibility,  the  surface  of the  MMA  was  coated  with  fibronectin,  an extra  cellular  matrix  protein.  Fibronectin  is known to promote adequate cell adhesion and growth, and we have shown that it  does not interfere  with  electrochemical  measurements (Trouillon et al., 2009). Firstly, (i)  human  umbilical  vascular endothelila cells (HUVEC) and (ii) embryonic stem  cells  (ESC)  derived  endothelial  cells were  grown  directly  on  the  sensor  (2x104 cells.cm-1 to promote angiogenesis (Hu et al., 1997)), angiogenin was added and NO levels were monitored. Then, several inhibitors were used  to  study  more  deeply  the  effect  of angiogenin  on  NO:  L-NAME,  RNase inhibitor  and  neomycin.  L-NAME,  an analogue of L-arginine, the eNOS substrate, is a competitive eNOS inhibitor, and blocks NO production  (Rees  et  al.,  1990).  RNase inhibitor  (RI)  and  neomycin  inhibit selectively  different  reaction  pathways  of angiogenin. RI binds to the angiogenin RNase site with very high affinity (Ki ~ 10-13 – 10-16 M) (Chen et al., 1997). In addition, neomycin is an antibiotic used in cancer treatment. It is known  to  block  angiogenin  nuclear translocation (Hu, 1998).Examining the release of NO in the presence of these inhibitors should therefore elucidate the relationships between NO and angiogenin and identify the mode and site of action.Materials and methodsMaterialsAll  the  chemicals  used  in  this  study  were purchased  from  Sigma,  unless  stated otherwise,  and  were  used  without  further purification. Deionized water purified through a  Millipore  system was  used  throughout  all the experiments.  Angiogenin was purified as previously  described  (Jang  et  al.,  2004). RI was purchased from Intron Biotechnology. Characterization of sensorsThe  MMA was  calibrated  using  aliquots  of authentic  solution of NO. This solution was - 2 -2nd Micro and Nano Flows ConferenceWest London, UK, 1-2 September 2009prepared  according  to  Feelisch  and  has already been described elsewhere (Patel et al., 2008). Background current was measured in degassed phosphate buffered saline (PBS, pH =  7.4)  by  differential  pulse  voltammetry (DPV),  versus  the  MMA  pseudo-gold reference. NO was then added to reach a final concentration of 75 μM. DPV was performed again to identify the peak potential.Cell cultureHUVEC were purchased from Clonetics (San Diego,  USA)  and  cultivated  in  EBM-2 medium (Clonetics)  on gelatin coated 75 ml flasks, at 37°C, 95% O2, 5% CO2. Cells from passage 6 to 9 were used.ESC-derived endothelial  cells  were prepared by mechanical isolation and cell sorting (Cho et al., 2007).Modification  and  preparation  of  the sensorsThe  MMA  were  modified  using  a  sylgard (Dow Corning) custom-made reaction cell to allow deposition of a 500 μl volume on the sensor. A lid made from a Petri dish was also fitted on the cell to minimise evaporation.Prior to any experiment, biological debris was removed using trypsin solution. Trypsin was deposited  into  the  cell  and  incubated  it  at 37°C  for  one  hour.  They  were  then  rinsed with  70%  ethanol  followed  by  water.  The gold  electrodes  were  electrochemically cleaned by performing cyclic voltammograms between 1 V and -0.8 V at 0.5 V.s-1 in 0.1 M sulphuric  acid  until  stability  of  the  graphs. The electrodes potential was then held at –0.6 V to reduce gold oxides.The  sensor  was  then  sterilized  with  70% ethanol  and  UV  light  and  rinsed  with  PBS (pH = 7.4). 100 μl of human fibronectin (20 μg.ml-1,  in  DMEM)  was  deposited  on  the sensor  and  let  to  dry.  The  excess  of fibronectin was rinsed with PBS.Electrochemical measurementsCells  were  harvested  using  trypsin  and counted  with  a  cytometer  and  Trypan  blue. 1000  cells  were  suspended  in  500  μl  of EBM-2 medium and deposited on the sensor. The  chip  was  then  incubated  for  at  least  2 hours.A DPV was then recorded between 0 V and 1.5 V vs Ag/AgCl.2  μl  of  bovine  angiogenin  (5.26  mg.ml-1  in deionized water) were added with the relevant inhibitor  if  required  (protocol  described below)  to  reach  a  final  concentration  of  21 μg.ml-1.  The  MMA  was  placed  in  the incubator for one hour and another DPV was recorded.InhibitorsFor  the  L-NAME  study,  the  cells  were maintained in culture medium containing 100 μM L-NAME during at least one hour before any measurement.Similarly,  in the neomycin  setup, cells  were cultivated  for  at  least  one  hour  in  medium containing  50  μM  neomycin  before  any measurement.The  RNase inhibitor  was mixed  with angiogenin (1:2 mass ratio) and incubated at 37°C  for  one  hour.  This mixture  was then added  to  the  cell  medium  to  reach  a  final angiogenin  concentration  of  21  μg.ml-1 and incubated for 1 hour.Data processingThe  DPV  results  were  processed  by normalizing  the  peak  current  obtained  after angiogenin  injection  by  the  peak  current measured before injection. This normalization guaranteed consistency between the different measurements  by  minimizing  the  effect  of variations  in  size,  background  species, heterogeneity  in  cell  population. The results obtained  for  each  experimental  condition were then averaged, and the means compared using Student’s t-tests.ResultsMeasurement  of  nitric  oxide  and  other nitrous compoundsFig.1  shows  curves  obtained  for  various dissolved  NO  solutions.  We  notice  a  sharp peak  at  0.6V.  This  peak  is  due  to  NO oxidation  and  increases  with  NO concentration.- 3 -2nd Micro and Nano Flows ConferenceWest London, UK, 1-2 September 2009Figure  1:  Differential  pulse voltammograms in PBS, pH = 7.4, with increasing concentration of nitric oxideFigure  2:  Differential  pulse  voltammograms  in EBM-2  medium  in  presence  of  HUVEC,  with angiogenin,  in presence (  )  or absence of L-NAME (),  and  control  without  angiogenin  and  L-NAME (∙∙∙∙∙)  (top);  Results  for  the  differential  pulse voltammograms  performed  in  EBM-2  medium  in presence of HUVEC, with angiogenin, in presence or absence of L-NAME, and control without angiogenin and L-NAME ( n ≥ 17, ***: p <  0.001) (bottom)Effect of angiogenin Fig.2 displays  typical  voltammograms obtained from HUVEC measurements.Results for control setup and after angiogenin addition, in presence or absence of L-NAME, are  also presented.  We see similar  peaks  to the ones obtained for pure NO solutions.  In particular, we see that if L-NAME is present, the  response  is  very  similar  to  the  one measured in the control experiment. However, if only angiogenin is added to the medium, we notice an increase in peak magnitude. These results  are  summarized  on  Fig.2,  which presents the ratio of the peak magnitude after and before addition of angiogenin. There is a significant  increase  in  peak  height  if  only angiogenin is present (+ 21%, p < 0.0001, n ≥ 17). However, if the HUVEC were previously incubated 100 μM L-NAME, we do not see any significant change in peak magnitude (p > 0.8, n ≥ 17). As a control, the same protocol was applied to stem cell derived endothelial cells (Fig.3). We obtained  exactly  similar  results,  angiogenin leading to an increase in the peak current (+22 %,  p<0.0001,  n  ≥  18),  and  L-NAME inhibiting this increase.Figure  3:  :  Results  for  the  differential  pulse voltammograms  performed  in  EBM-2  medium  in presence  of  ESC derived  endothelial  cells,  with angiogenin,  in presence or absence of L-NAME, and controls  without  angiogenin  and  L-NAME,  and  with L-NAME only ( n ≥ 18, ***: p <  0.001)Effect of inhibitorsTo understand the physiological pathway of angiogenin,  several  selective  inhibitors  have been used in the same kind of experiment - 4 -2nd Micro and Nano Flows ConferenceWest London, UK, 1-2 September 2009a) RNase inhibitorFig.4 shows the statistical results obtained for addition of angiogenin in presence or absence of RNase inhibitor (RI). Angiogenin alone led to an increase in magnitude of 22% (p<0.001, n ≥ 10) and addition of a mixture of RI and angiogenin increased the peak magnitude by 17%  (p<0.001,  n  ≥  10).  The  increases observed  for  both  treatments  are  not statistically significantly different (p > 0.45, n ≥10). Figure  4:  Results  for  the  differential  pulse voltammograms  performed  in  EBM-2  medium  in presence of HUVEC, with angiogenin, in presence or absence of RI, and control without angiogenin and with RI ( n ≥ 10, ***: p <  0.001)Figure  5:  Results  for  the  differential  pulse voltammograms  performed  in  EBM-2  medium  in presence of HUVEC, with angiogenin, in presence or absence of neomycin, and control without angiogenin and with neomycin ( n ≥ 10, ***: p <  0.001)b) NeomycinFig.5 shows  the  results  obtained  for  this experiment.  Again,  if  we add angiogenin  to the cell medium, we see an increase in current peak (+ 27%, p < 0.001, n ≥ 10). However, if the  cells  were  previously  incubated  with neomycin, no significant increase is measured (p  <  0.55,  n  ≥  10).  Neomycin  inhibits  the angiogenin induced increase in peak currentDiscussionEffect  of  angiogenin  on  nitric  oxide synthase activityWe have shown that adding angiogenin to cell medium  led  to  a  higher  current  peak.  In addition, this peak is very similar to the one obtained previously in pure NO solutions and is  expected  to be due to  an increase  in  NO concentration.  This  NO  is  expected  to  be produced by the HUVEC, NOS activity being probably  triggered  by  angiogenin.  This assertion  was  confirmed  by  the  L-NAME tests. As an analogue of L-arginine, L-NAME is  known to  inhibit  competitively  NOS and therefore  NO  production.  By  adding  L-NAME to our setup, NOS in our population of  HUVEC  is  inhibited  and  no  current increase  is  observed.  This  shows  that  this increase  in  peak  current  was  due  to  NOS activity  and  that  the  peaks  we  measure  are due  to  NO  oxidation.  Furthermore,  these experiments  show  that  angiogenin  triggers NOS  activity  and  leads  to  increases  in extracellular NO.The  same  results  were  obtained  when  the same experiment was carried out in stem cell derived endothelial cells as a control showing that angiogenin induced NOS activation is a general feature of endothelial cells. Figure 4: A hypothetical scheme for the mechanism of action of angiogenin modified from Kim et al. (2008)- 5 -2nd Micro and Nano Flows ConferenceWest London, UK, 1-2 September 2009Physiological pathway of angiogeninThe  physiological  pathway  leading  to  NOS activation  was  studied  using  2 angiogenin inhibitors:  RI and neomycin.  We do not see any effect  of  RI,  indicating  that  angiogenin induced  eNOS  activity  does  not  depend  on the  RNase activity. However,  neomycin completely  inhibits  the  NO  increase. Neomycin blocking nuclear translocation, this result  shows that eNOS is activated through angiogenin  interactions  with cell  nucleus. A modification  of  hypothetical  scheme for  the mechanism of action of angiogenin is shown in Fig. 6. This conclusion has to be related to the  fact  that  nuclear  translocation  of angiogenin  is  a  general  requirement  for angiogenesis (Kishimoto et al., 2005).ConclusionWe have demonstrated that angiogenin leads to  enhanced  release  of  NO.  Interaction  of angiogenin with the cell nucleus is a critical requirement  for  this  angiogenin  induced eNOS  activity,  which  tends  to  prove  that nuclear translocation of angiogenin is a major event  in  angiogenesis.  Selective  inhibitors demonstrate  that  nuclear  translocation  is required for angiogenin mediated NO release.AcknowledgementsRT  gratefully  acknowledges Medermica Ltd. for funding. This work was supported by the DIUS  and  KICOS (K20602000681-08B0100-02210)  funded UK/Korea  Focal  Point  Programme  on  Life Science,  the Korea Science and Engineering Foundation (M10749000231-08N4900-23110)  and  the Korea  Biotech  R&D  Group  of  Next-Generation  Growth  Engine  Project (F104AB010004-08A0201-00410).ReferencesBabaei, S. et al. Circ. Res., 1998, 82, 1007-1015Baum, O. et al.  Am. J. Physiol.  Heart Circ.  Physiol., 2004, 287, 2300-2308Carmeliet,  P.  et  al.  Nature,  2000,  407, 249-257 Chavakis, E et al. Arterioscler. Thromb. Vasc. Biol., 2002, 22, 887-893Chen, C.Z. et al. Proc. Natl. Acad. Sci. USA, 1997, 94, 1761-1766Cho, S-W; Moon, S-H et al. Circulation, 2007, 116, 2409-2419Clark, R.A. Science, 1995, 268, 233-239Ferrara,  N.  Am.  J.  Physiol.  cell  Physiol., 2001, 280, 1358-1366Fukumura,  D.  et  al.  Canc.and  Metas.  Rev., 1998, 17, 77-89Gao,  X.;  Xu,  Z.  Acta  Biochim Biophys  Sin, 2008, 40, 619-624Harding, K.G. et al. Brit. Med. Journal, 2002, 324, 160-163Hu, G. et al. Proc. Natl. Acd. Sci USA, 1994, 91, 12096-12100Hu,  G.  et  al.  Proc.  Natl.  Acad.  Sci.  USA, 1997, 94, 2204-2209Hu, G. Proc. Natl.  Acad. Sci. USA, 1998, 95, 9791-9795Jang, S-H; Kang, D-K et al. Biotechnology  letters, 2004, 26, 1501-1504Kim,  H.M.  et  al.  Biochem.  Biophys.  Res.  Commun., 2008, 352, 509-513Kishimoto, K.; Liu, S. et al. Oncogene, 2005, 24, 445-456Liu, S.  et  al.  Biochem.  Biophys.  Res.  Commun., 2001, 287, 305-310Palmer,  R.M.J.  et  al.  Nature,  1988,  333, 664-666Patel, B.A. et al. Anal. Bioanal. Chem., 2008, 390, 1379-1387Rees, D. et al.  Br. J. Pharmacol., 1990,  101, 746-752Russo,  N.  et  al.  Proc.  Natl.  Acd.  Sci  USA, 1994, 91, 2920-2924Trouillon,  R.  et  al.  The Analyst,  2009,  134, 784-793 Walsh,  D.A.  Rheumatology,  1999,  38, 103-112Xu,  Z.  et  al.  Biochem.  Biophys.  Res.  Commun., 2001, 285, 909-914Ziche, M. et al. Biochem. Biophys. Res.  Commun., 1993, 192, 1198-1203Ziche,  M.  et  al.  J.  Clin.  Invest., 1994,  94, 2036-2044- 6 -

Acta Biochim Biophys Sin,

Electrochemical sensing of angiogenin induced endothelial nitric oxide synthase activity

Electrochemical sensing of angiogenin induced endothelial nitric oxide synthase activity

Abstract

Similar works

Full text

Available Versions

Brunel University Research Archive