NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 25, NO.

INCREASED PRODUCTION OF VASCULAR

ENDOTHELIAL GROWTH FACTOR (VEGF) BY ANGIOTENSIN II

IN NEURO-2A CELLS

Krishna Pada Sarker, Masanori Nakata, Toshihiro Nakajima,

Isao Kitajima and Ikuro Maruyama

Department of Laboratory and Molecular Medicine, Faculty of Medicine,

Kagoshima University, Kagoshima-890, Kagoshima-shi, Japan

(accepted July 1, 1999)

Summary
Vascular endothelial growth factor (VEGF) is an endothelial cell
specific mitogen that potently stimulates vasodilatation, microvascular
hyperpermeability, and angiogenesis. VEGF is synthesized and secreted in
different cell types by various agents, including vasoconstrictor angiotensin
II (Ang II). Knowledge about Ang II-induced VEGF release is still limited to
endothelial cells (ECs) and vascular smooth muscle cells (VSMCs). Here, we
demonstrate that Ang II enhanced VEGF production in Neuro-2a cells in a
dose- and time-dependent fashion in the culture media, determined by
Enzyme-Linked Immunosorbent Assay (ELISA). The Ang II receptor
antagonist Sari-Ileu8 and heparin prevented Ang II-induced VEGF release
in the culture media. Coincubation of the Neuro-2a cells with Ang II and
thrombin increased VEGF release markedly. However, Ang II-induced VEGF
production was inhibited by the transcriptional inhibitor actinomycin D and
protein synthesis inhibitor cycloheximide. Furthermore, Ang II significantly
enhanced thrombin receptor expression. These results suggest that Ang II
receptor activation may be involved in the production of VElGF, which is
dependent on both transcription and translation.

Key words.- Neuro-2a cells, Ang II, Ang II receptor, VEGF release

0 1999 Wiley-Liss, Inc.

 NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 25, NO. 2

Introduction
Vascular endothelial growth factor (VEGF), also termed vascular
permeability factor (VPS) is a homodimeric glycoprotein. VEGF is encoded
by a single gene that finally produces four protein products, VEGF 121,
VEGF 165, VEGF 189 and VEGF 206 (based on the number of amino acids),
generated by the post-transcriptional modification. WGF is expressed in a
wide variety of cells, including neuronal cells [1, 21. Mouse VEGF is only one
amino acid shorter than human counterpart. VEGF 121 and VEGF165 are
secreted, but VEGF 189 and VEGF 206 remain in the extracellular matrix.
VEGF 206 is only present in the fetal liver. VEGF 165, VEIGF 189 and
VEGF 206 possess heparin binding sites, whereas VEGF 121 lacks heparin
binding site [3-51. It is well established that VEGF plays a pivotal role in
the normal vessel growth, development, differentiation, wound healing and

reproduction. VEGF has been shown to inhibit TNF-a -induced apoptosis of

endothelial cells [6]. In addition, it has recently been reported that ischemic
damage has been reduced by the addition of VEGF in the rat brain [7].
Induction of VEGF gene expression can be promoted by the cytokines, for
instance tumor necrosis factor-alpha [8] or mitogens, for example thrombin

PI .
Ang II is an octapeptide produced by the enzymatic cleavage of
angiotensin I by angiotensin converting enzyme (ACE), exerts a large
number of physiological effects, including vascular tone, hormone secretion,
tissue growth, and neuronal activities [lo, 111. In addition to these functions,
Ang II has been shown to induce angiogenesis in some animal models [12],
and has been demonstrated to enhance growth factors expression, such as

platelets derived growth factor (PDGF), transforming growth factor-p 1 (TGF-

pl) or basic fibroblast growth factor (bFGF) [ 13, 143. The importance of Ang

II in brain has been realized by the reveal that angiotensinogen and all the
NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 25, NO. 2

receptors of Ang II (AT I, AT II, AT III) are expressed in the nervous system.
In order to investigate whether Ang II can induce VEGF release in neuronal
cells, we used mouse neuroblastoma (Neuro-Za) cells that express Ang II
receptor subtype AT II as well [15]

Materials and Methods

Materials. Actinomycin D, cycloheximide, ethylene diamine
tetraacetic acid (EDTA) and heparin were purchased from Sigma Chemicals
Co.(St Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM),
streptomycin, penicillin, fetal bovine serum (FBS) were from GIBCO (USA).
Angiotensins (Ang I, Ang II, Ang III or Ang IV) and Sarl-Ileu8 were from
Peptide Institute, Inc. (Osaka, Japan). Thrombin was from Mochida
Pharmaceutical Co. (Tokyo, Japan). Thrombin receptor antibody was from
Teijin Chemical Co. (Tokyo, Japan).
Cell. The mouse neuroblastoma cells were grown as described [16] in
low glucose DMEM, supplemented with 10% fetal bovine serum (FBS),
penicillin 100 pg/ml, and streptomycin 100 pglml. Growth was under 5% CO2
and 95% air at 37°C. Cells were passagedwithout trypsinization, twice in a
week.
ELLSA for VEGF. VEGF levels in culture media were measured by a
newly developed calorimetric ELISA by a slight modifications of a
chemiluminescence enzyme immunoassay method as described [ 171. The
anti-VEGF/VPF IgG was used as a plate-coating and a peroxidase(POD)-
conjugated Fab-fragment of the antibody was used as a secondary antibody,
respectively. Microtiter plate (Immulon 2, dynatech USA) were coated with
anti-VEGF/VPF polyclonal antibody (100 ml, 2 mg/ml) in 0.1 M NaCl, ), 025
M carbonate buffer (pH9.0), then blocked with 1 % bovine serum albumin
(BSA), 0.2 M carbonate buffer (pH9.5), 0.1 M NaCl and 0.1% NaN3. Diluted
samples with phosphate-buffered saline (PBS) containing 1% BSA was
added to the wells and incubated for 1 hr at 22°C. After washing, POD-
conjugated Fab’ was added and incubated for 1 hr at 22°C o-
Phenylenediamine was used as substrate for the enzyme reaction and the
absorbance at 490 nm was measured after 30 min of the reaction in a plate
reader (Microplate reader M-Vmax, Molecular Devices, MA).
Fluorescence-activated cell sorting (FAGS). After treatment of the
Neuro-2a cells with Ang II for the indicated period, cells were washed with
PBS by centrifugation. Ang II-treated cells were incubated with thrombin
receptor antibody, rabbit anti-SF14 poly-clonal IgG, and control cells were
incubated with normal IgG for 1 hr at 4°C. After washing with PBS, cells
 NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 25, NO. 2
were incubated with FITC-conjugated secondary antibody for 1 hr at 4°C.
Finally, cells were washed with PBS, and fluorescence was measured by
FACS-can analyzer (EPICS, COULTER, USA).

Results and Discussion

As neurons are vulnerable, blood-brain barrier (BBB) is formed by the

tight endothelial cell junction, which prevents neurons from the contact of
proteins, proteases as well as toxic metabolites that are present in the
plasma. After brain injury, capillary endothelial cells are damaged,
resulting in the disruption of BBB. Endothelial cell growth is therefore a
prerequisite in the reconstitution of BBB. Recent study has shown that BBB
is disrupted in the Ang II-knock out mice [18]. Therefore, it is speculated
that local renin-angiotensin system may be activated following brain injury,
resulting in the generation of Ang II. Angiotensin II generated in this way
may cause angiogenesis of the brain capillary endothelial cells to repair
leaky BBB by producing VEGF.
In the present study, we demonstrate that Ang II enhanced VEGF
production dose-dependently in the conditioned media. As shown in Figure
1A Ang II, at a concentration of 500 nlM increased about 3.7-fold VEGF
release compared to control. Although, Ang II increases the message level of
VEGF in rat heart endothelial cells but only trace amount of VEGF has
been possible to detect in the conditioned media by Western blotting [19].
This contradictory result to our finding could be attributed to the difference
of assay procedure or dependency of cell type on the production of VEGF by
Ang II. Release of VEGF in the culture media of CD4la+ megakariocyte cells
induced by thrombin has been detected by using ELISA. In contrast, the
message level of VEGF is upregulated by thrombin, but no VEGF has been
detected in the culture media of CD4la- megakariocytes [9]. To know

whether other forms of Ang can enhance VEGF production, we treated the
NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 25, NO. 2 83

Neuro-2a cells with different concentrations of Ang I, Ang III and Ang IV but
neither of them increased VEGF release in the culture media (data not
shown).
Ang II-induced signaling is mediated through the seven
transmembrane G-protein coupled receptors [ZO-221. To determine Ang II-
induced VEGF release was its receptor mediated, we used Sarl-Ileu8, the
nonspecific angiotensin II receptor antagonist [15]. Sari-Ileu8 binds to Ang
II receptor (AT) and desensitizes it, resulting in inhibition of the Ang II-
mediated signaling. Figure 1B shows that Sari dose-dependently inhibited
Ang II-induced VEGF production. One hour pretreatment of the Neuro-2a
cells with Sarl-Ileu8 (1000 nlM) drastically abolished Ang II-induced VEGF
production in the conditioned media. This result suggests that Ang II-
induced VEGF production is mediated through its receptor activation.

1800 n

1600
A B * C

*
*
L s 800-
u-
” & 600-
>Q

Figure 1. Effects of Ang II on Neuro-2a cells in VEGF

production. Neuro-2a cells were plated onto 12-well dishes (1x105
/well). After a 1%hr of attachment, cells were differentiated with db-
cAMP for 3 days. (A) Cells were treated with Ang II (l-1000 nM)
for 24 hr, (B) Cells were pretreated with Sar 1 (l-1000 r&I) and heparin
(l-10 U/ml) and then coincubated with Ang II for 24 hr, (C) Following
differentiation, cells were pretreated 1 hr with actinomycin D and
cycloheximide, and further incubated in the presence or absence of
Ang 11 for 24 hr. Values are given LSD (n=6). pro.05 was considered
statistically significant compared to controlin (A), and compared to Ang
11 in (B) and (C).
 NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 25, NO. 2
Ang II exhibits different cellular activities both in a calcium-
dependent and independent manner. So, we investigated the dependency of
Ang II-evoked VEGF release on extracellular calcium. Ang II-induced VEGF
release was not blunted in the presence of 1 mM EDTA (data not shown).
However, on endothelium Ang II induces PDGF release in a calcium
dependent fashion [23]. In contrast, Ang II transduces its signal in calcium
independent manner in sympathetic neurons [Z4, 251, demonstrates that the
dependency of Ang II on calcium to induce growth factors production may
vary with cell types. However, our study does not exclude the possibility that

the Ang II receptor activation causes increase in intracellular Ca2+ level,

because Ang II receptor causes membrane phosphatydyl turnover, which in
turn, mobilizes intracellular calcium.
Figure 1B further demonstrates that the Ang II-induced VEGF
production had been attenuated by the pretreatment of heparin (10 U/ml).

Mechanism of the inhibitory pathway of heparin on Ang II-induced VEGF

production in the Neuro-2a cells is unknown. However, inhibition of VEGF
production by heparin can be explained by the two possible ways. First,
heparin binds to VEGF 165 in the culture media and simply interfere the
assay of VEGF. Second, heparin binds with its receptor and inhibits the
intracellular signaling pathway of Ang II-induced VEGF production. The
later is supported by the recent report that heparin inhibits mitogen
activated signaling pathway leading to DNA synthesis by Ang II and
thrombin [26]. Furthermore, heparin appeared to be ineffective to prevent
Ang II-induced VEGF production, when it was applied 2 hr later of Ang II
addition (data not shown), suggests that to inhibit Ang II-induced VEGF
production heparin needs to be bound with its receptor, and thereby
antagonizes Ang II signaling in VEGF production.
NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 25, NO. 2
Thrombin is a classical mitogen, which increases VEGF production in
various cells [9]. In order to observe the impact of thrombin on Ang II-induced
VEIGF release, Neuro-2a cells were coincubated with Ang II (500 nM) and
thrombin (10 U/ml). Coincubation of Ang II and thrombin markedly
increased VEGF production than Ang II or thrombin alone (Fig. lC), implies
that VEGF production has been increased by the synergistic effect of
thrombin. Next, we sought to demonstrate whether Ang II-induced VEGF
production was inhibited by RNA synthesis inhibitor or protein synthesis
inhibitor. Figure 1C further shows that pretreatment of the Neuro-2a cells

with actinomycin D (1 pglml) and cycloheximide (10 pglml) abrogated Ang II-

induced VEGF production. This result illustrates that Ang II induces VEGF
production transcriptional- and translational-dependently. Neither

actinomycin D nor cycloheximide in the above concentration caused any

cytotoxic effects.

Recently, it has been shown that thrombin receptor expression in

messenger level is increased by Ang II in VSMCs [27], and VEGF

production is also induced by thrombin in several cell types [9]. Thrombin

enhances VEGF production in the culture media of PC-12 cells (manuscript
in preparation). In order to investigate, the correlation of thrombin receptor
over expression and VEGF production by Ang II, we used fluorescence-

activated cell sorting (FACS) analysis using thrombin receptor antibody

against thrombin receptor agonist peptide (TRAP) region. Figure 2 shows
that thrombin receptor expression was increased by Ang II overwhelmedly at
18 hr as the fluorescence intensity has been shifted towards right side. Thus
it favors the concept that over expression of thrombin receptor by Ang II may
play an important role in Ang II-induced VEGF production in neuronal cells.
Upregulation of the growth factors or proteins by different agents
occurred at different time phages, for example Ang II induces PDGF
86 NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 25, NO. 2

FITC-Fluorescence Intensity

Figure 2. Effects of Ang II in thrombin

receptor expression on Neuro-2a cells. Neuro-
2a cells were plated onto 100 mm dishes (5x10” cells
/dish). Following treatment with Ang II (500 nM)
or 18 hr, thrombin receptor expression was
analyzed by FACS. Similar results were obtained
by duplicate experiments.

1000
v)
-5
E 0 800
Lrn
m
600

200

6 12 38 24 48

Time (hours)

Figure 3. Time-course effect of Ang II on Neuro-Za cells in

VEGF production. Cells were plated onto 12-well dishes (lxlO5/weU).
Following differentiation, cells were incubated in the presence or absence of
Ang II (500 nM) for (O-48 hr). Data are given +SD from three experiments
in triplicate.
NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 25, NO. 2
production relatively at a delayed phage on endothelium [Z8, 291. On the
other hand, Ang II has been shown to upregulate the message level of
thrombin receptor within 2 hr in VSMCs [29]. We therefore investigated the
time-course effects of Ang II on VEGF production. Addition of Ang II led
VEGF production within 12 hr, which dramatically increased at 24 hr later.
Ang II-induced VEGF production gradually diminished and ceased almost to
basal level at 48 hr (Fig. 3). It has been shown that the message level of
VEGF is increased by Ang II at 8 to 12 hr in VSMCs, and then reached
plateau [ 181.
In conclusion, this is the first evidence that Ang II causes VEGF
production in neuronal cells. Our data may explain partly the increased
vascular permeability, which is observed after infusion of Ang II in the rat
brain [30, 311. Moreover, Ang II-induced VEGF production may explain its
angiogenic and anti-apoptotic roles in neuronal cells.

References
1 Charnock,
1. J.D.S., Sharkey, A.M., Rajput, W. J., Burch, D., Schofield, J.P.,
Fountain, S.A., Boocock, C.A. and Smith, SK. (1993) Biol. Reprod. 48,
1120-1128.
2. Tischer, E., Mitchell, R., Hartman T., Silva, M., Gospodarowicz, D.,
Fiddes, J.C. and Abraham, J.A. (1991) JBiol. Chem. 266, 11947-
11954.
3. Conn, G., Soderman, D.D., Schaeffer, M.T., Wile, M., Hatcher, VJ% and
Thomas, K.A. (1990) Proc. Natl. Acad. Sci. 87, 1323-1327.
4. Park, J.E., Keller, G.A. and Ferrara, N. (1993) Mol. Biol. Cell 4
1317-1326.
5. Pertovaara, L., Kaipainen, A., Mustonen, T., Orpana A., Ferrara,
N., Saksela, 0. and Alitalo, K. (1994) J. Biol, Chem. 269, 6271-
6274.
6. Spyridopoulos, I., Brogi, E., Kearney, M., Sullivan, A-B., Cetrulo, C.,
Isner, J.M. and Losordo, D.W. (1997) J. Mol. Cell Cardiol. 29, 1321-
1330.
7. Hayashi, T., Abe, K. and Itoyama, Y. (1998) J. Cereb. Blood Flow
Metab. 18, 887-895.
8. Ryuto, M., Ono, M., Izumi, H., Yoshida, S. Weich, H.A., Kohno,
K. and Kuwano, M. (1996) J. Biol. Chem. 271, 28220-28228.
 NEUROSCIENCE RESEARCH COMMUNICATIONS, VOL. 25, NO, 2
9. Mohle, R., Green, D., Moore, M.A., Nachman, R.L. and Rafii, S. (1997)
Proc. Natl. Acad. Sci. 94, 663-668.
10. Dzau, V.J., Gibbons,G.H. and Pratt, R.E. (1991) Hypertension 18,
(4 Suppl.),II 100-105.
11. Sasaki, K., Yamano, Y., Bardhan, S., Iwai, N., Murray, J. J.,
Hasegawa, M., Matsuda, Y. and Inagami, T. (1991) Nature 351, 2300
233.
12. Le -Noble, F.A., Hekking, J.W., Van-Straaten, H.W., Slaaf, D.W. and
Struyker-Boudier, H.A.( 1991). Eur. J. Pharmacol. 195, 305306.
13. Itoh, H., Mukoyama, M., Pratt, R.E. Gibbons, G.H. and Dzau, V-J.
(1993) J. Clin. Invest. 91, 2268-2274.
14 Nafiilan, A.J., Pratt, R.E. and Dzau, V.J.(1989) J. Clin. Invest., 83,
1419-1424
15 Chaki, S. and Inagami, T. (1992) E. J. Pharmacol. 225, 355356.
16 Suidan, H.S., Stone, S.R, Hemmings, B.A. and Monard, D. (1992)
Neuron8,363-375.
17. Hanatani, M., Tanaka, Y., Kondo, S., Ohmori, I. and Suzuki, H. (1995)
Biosci. Biotechnol. Biochem. 59, 19584959.
18. Kakinuma, Y., Hama, H., Sugiyama, F., Yagami, K., Goto, K.,
Murakami, K. and Fukamizu, A. (1998) Nature Med. 4, 1078-1080.
19. Chua, C.C., Hamdy, R.C. and Chua, B.H. (1998) Biochem. Biophys.
Acta 1401, 187-194.
20. Bottari, S.P., de-Gasparo, M., Steckelings U.M. and Levens, N.R.
(1993) Front. Neuroendocrinol. 14, 123-171.
21. Phillips, M.I. (1987) Annu. Rev. Physiol. 49, 413-435.
22. Steckelings, U.M., Bottari, S.P. and Unger, T. (1992) Trends
Pharmacol. Sci. 13, 365-368.
23. Okuda, Y., Adrogue, H.J., Nakajima, T., Mizutani, M., Asano, M.,
Tachi, Y., Suzuki, S. andyamashita, K. (1996) Life Sci. 59, 1455
1461.
24. Bacal, K. and Kunze, D.L. (1994) J. Neurosci. 14, 7159-7167.
25. Shapiro, M.S., Wollmuth, L.P. andHiRe, B. (1994) Neuron 12, 1319-
1329.
26. Hedin, U., Daum, G. and Clowes, A.W. (1998) J. Vast. Surg. 27, 512-
520.
27. Fisslthaler, B., Schini-Kerth, V.B., Fleming, I., Busse, R. (1998)
Cardiovasc. Res. 38, 263-271.
28. Berk, B.C. and Rao, G.N. (1993) J. Cell Physiol. 154, 368-380.
29. Sharma, H.S., van-Heugten, H.A., Goedbloed, M.A., Verdouw,
P.D. and Lamers, J.M. (1994) Biochem. Biophys. Res. Commun. 205,
105-112.
30. Wiener, J. and Giacomelli, F. (1976) Am. J. Path. 72, 221-231.
31. Wiener, J. Lattes, R.G., Meltzer, B.G. and Spiro D. (1969) Am. J.
Pathol. 54, 187-207.