Functional and gene expression analysis of hTERT overexpressed endothelial cells

Abstract

Haruna Takano1, Satoshi Murasawa1,2, Takayuki Asahara1,2,31Institute of Biomedical Research and Innovation, Kobe, Japan; 2RIKEN Center for Developmental Biology, Kobe 650-0047, Japan; 3Tokai University of School of Medicine, Tokai, JapanAbstract: Telomerase dysfunction contributes to cellular senescence. Recent advances indicate the importance of senescence in maintaining vascular cell function in vitro. Human telomerase reverse transcriptase (hTERT) overexpression is thought to lead to resistance to apoptosis and oxidative stress. However, the mechanism in endothelial lineage cells is unclear. We tried to generate an immortal endothelial cell line from human umbilical vein endothelial cells using a no-virus system and examine the functional mechanisms of hTERT overexpressed endothelial cell senescence in vitro. High levels of hTERT genes and endothelial cell-specific markers were expressed during long-term culture. Also, angiogenic responses were observed in hTERT overexpressed endothelial cell. These cells showed a delay in senescence and appeared more resistant to stressed conditions. PI3K/Akt-related gene levels were enhanced in hTERT overexpressed endothelial cells. An up-regulated PI3K/Akt pathway caused by hTERT overexpression might contribute to anti-apoptosis and survival effects in endothelial lineage cells.Keywords: endothelial, telomerase, senescence, oxidative stress, anti-apoptosis, PI3K/Akt pathway

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© 2008 Takano et al, publisher and licensee Dove Medical Press Ltd. This is an Open Access article
which permits unrestricted noncommercial use, provided the original work is properly cited.
Biologics: Targets & Theraphy 2008:2(3) 547–554
547
O R I G I N A L R E S E A R C H
Functional and gene expression analysis
of hTERT overexpressed endothelial cells
Haruna Takano1
Satoshi Murasawa1,2
Takayuki Asahara1,2,3
1Institute of Biomedical Research
and Innovation, Kobe, Japan; 2RIKEN
Center for Developmental Biology,
Kobe 650-0047, Japan; 3Tokai
University of School of Medicine,
Tokai, Japan
Correspondence: Takayuki Asahara
Institute of Biomedical Research
and Innovation, 2-2
Minatojima-Minamimachi,
Chuo-ku, Kobe 650-0047, Japan
Tel +81 78 304 5772
Fax +81 78 304 5263
Email asa777@aol.com
Abstract: Telomerase dysfunction contributes to cellular senescence. Recent advances indicate
the importance of senescence in maintaining vascular cell function in vitro. Human telomerase
reverse transcriptase (hTERT) overexpression is thought to lead to resistance to apoptosis and
oxidative stress. However, the mechanism in endothelial lineage cells is unclear. We tried to
generate an immortal endothelial cell line from human umbilical vein endothelial cells using a
no-virus system and examine the functional mechanisms of hTERT overexpressed endothelial
cell senescence in vitro. High levels of hTERT genes and endothelial cell-specifi c markers were
expressed during long-term culture. Also, angiogenic responses were observed in hTERT over-
expressed endothelial cell. These cells showed a delay in senescence and appeared more resistant
to stressed conditions. PI3K/Akt-related gene levels were enhanced in hTERT overexpressed
endothelial cells. An up-regulated PI3K/Akt pathway caused by hTERT overexpression might
contribute to anti-apoptosis and survival effects in endothelial lineage cells.
Keywords: endothelial, telomerase, senescence, oxidative stress, anti-apoptosis, PI3K/Akt
pathway
In vitro, normal somatic cells undergo a non-dividing state termed cellular replicative
senescence (Harley et al 1990; Hastie et al 1990; Wright and Shay 1992). The erosion
of telomeres, a reverse transcriptase synthesize telomeric DNA, has been suggested to
contribute to cellular replicative senescence (Greider 1990; Nakamura et al 1997). It
is necessary to increase the number of human endothelial progenitor cells (EPCs) in
vitro to obtain large numbers for research and cell transplantation. A potential obstacle
to this increase, however, is the fact that EPCs, as well as other somatic cells, have a
limited replication lifespan.
Human telomerase reverse transcriptase (hTERT)-transduced differentiated endo-
thelial cell (ECs) exhibit neither evidence of malignant transformation nor loss of
functional and morphologic characteristics of parental cells (Yang et al 1999). These
fi ndings support the stability of endothelial lineage cells after hTERT overexpression.
We previously reported on the therapeutic potential of adenoviral hTERT overex-
pressed endothelial lineage cells in an ischemic hindlimb mouse model (Murasawa
et al 2002). However, ectopic hTERT expression by adenovirus transduction was
limited to 4 weeks and did not lead to immortal EPCs. We had expected that the
enhanced regenerative activity of EPCs by hTERT transduction would provide a
novel therapeutic strategy for potential neovascularization in patients with severe
ischemic disease. However, the mechanism by which hTERT-transduced EC lines
appear more resistant to programmed cell-death and exhibit a survival advantage
beyond replicative senescence is elusive. Thus, further investigation of the possible
mechanistic pathways is needed.
Here we perform gene transfer with a no-virus system to transduce hTERT genes
into human umbilical vein endothelial cells (HUVECs), one of the endothelial lineage

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Takano et al
cell models, to investigate the relation between senescence
and telomerase in vitro, determine the possible mechanistic
pathways, and investigate the characteristics of these cells
for future clinical application.
Materials and methods
Reagents
Recombinant human VEGF165 and bFGF were purchased
from R&D Systems Inc. (Minneapolis, MN, USA). Hoechst
33342 solution was purchased from Dojin Chemical Labora-
tory (Kumamoto, Japan).
Cell culture and gene transfer
HUVECs were grown in endothelial cell growth media
EGM-2 Bullet kit (CAMBREX, Walkersville, MD, USA)
supplemented with 2% fetal bovine serum (CAMBREX).
The cDNA encoding hTERT was cloned downstream of the
human cytomegalovirus (CMV) promoter in pcDNA3.1(+)
(Invitrogen, Carlsbad, CA, USA) with LipofectamineTM
2000 (Invitrogen). Forty-eight hours after transfection, the
transfected-cell populations were selected with 600 μg/mL
of G418 (GIBCO, Grand Island, NY). Cells transfected with
empty vectors were used as controls. Population doublings
(PD) were calculated as follows: PD = log (number of cells
obtained/initial number of cells)/log 2.
RT-PCR for telomerase transcripts
Total RNA was isolated with an RNA extraction kit (Ambion,
Austin, TX, USA). DNase digestion was performed after
RNA extraction. cDNA was synthesized by AMV First-
strand Synthesis System (Takara Bio, Shiga, Japan). PCR
reaction of hTERT and endothelial markers were performed
by a system according to the manufacture (BD Biosciences,
San Diego, CA and Applied Biosystems, Foster City, CA,
USA). The primers for RT-PCR are shown in Table 1.
Senescence-associated (SA)-
Galactosidase (Gal) Staining
4 x 104 cells were plated on 12-well plates pre-coated with
fi bronectin and cultivated in growth medium for 24 hours.
Senescence was investigated by a SA-Gal activity assay
system according to the manufacture (CALBIOCHEM, San
Diego, CA, USA). Quantifi cation of SA-Gal–positive cells
was obtained by counting fi ve random fi elds per dish and
assessing the percentage.
Proliferation assay
3.5 x 103 cells grown in 96-well plates pre-coated with fi bro-
nectin were cultivated in growth medium for 24 hours then in
basal medium for another 24 hours. Then the culture medium
was changed to basal medium in the presence of angiogenic
cytokines for 24 hours. Proliferative activity was evaluated
using the MTS assay (Promega, Madison, WI, USA).
Apoptosis assay
1 x 105 cells grown in 12-well plates pre-coated with fi bro-
nectin were cultivated in growth medium for 24 hours then in
basal medium for another 24 hours. To detect the frequency of
cellular apoptosis, fl uorescence-labeled Annexin-V-FLUOS
staining of transduced HUVECs was performed according to
manufacture’s instructions (Roche Molecular Biochemicals,
Mannheim, Germany). Quantifi cation of Annexin-V positive
cells was obtained by counting 5 random fi elds per dish and
assessing the percentage.
Oxidative stress detection assay
1 x 105 cells were grown in 12-well plates pre-coated with
fi bronectin cultivated in growth medium. After 24 hours’
incubation, the cells were incubated with 1mM H2O2 in
HEPES buffer for 1 hour. Then the medium was changed to
growth medium for another 3 hours. Detection of oxidative
damage in live cells was performed using the Image-iTTM
LIVE Green Reactive Oxygen Species (ROS) Detection
Kit (Molecular Probes, Eugene, OR, USA). Quantifi cation
of ROS detected cells was obtained by counting 5 random
fi elds per dish and assessing the percentage.
Gene array analysis
Targeted cDNA arrays designed to analyze endothelial cell
biology, angiogenesis, apoptosis, cell cycle, and PI3K/Akt
pathway (GEArray HS-036, HS-009, HS-603, and HS-058)
Table 1 List of primer sequences for PCR
Molecule Sense (5’–3’) Antiense (5’–3’)
hTERT
hCD31
hKDR
heNOS
hGAPDH
CACCTCACCTCACCCACGCGAAA
AGGACATCCATGTTCCGAGA
CCCTGCCGTGTTGAAGAGTT
TTACCATGGCAACCAACGTC
GCCCCAGCAAGAGCACAAGA
CCAAAGAGTTTGCGACGCATGTT
TGAACCGTGTCTTCAGGTTG
GGACAGGGGGAAGAACAAAA
AAAAGCTCTGGGTGCGTATG
TAGGCCCCTCCCCTCTTCAA

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Biologics: Targets & Therapy 2008:2(3)
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hTERT overexpressed endothelial cells
were purchased from SuperArray Bioscience Corporation
(Frederick, MD, USA). Hybridization was quantifi ed using
a Luminescent Image Analyzer LAS-3000 (Fujifi lm, Tokyo,
Japan) and data analysis was performed using Science Lab
2001 Image Gauge (Fujifi lm) software. The background was
subtracted from each representing dot signal, and values were
normalized according to the GAPDH signal.
Statistical analysis
All values are expressed as mean ± SD. Comparisons of
results between different groups were performed using
Student’s t test.
Results
hTERT transduced HUVECs expressed
telomerase gene during long term culture
hTERT transduced HUVECs (Td/TERTs) were isolated
from several donors cell sources. RT-PCR analysis showed
that telomerase gene expression was maintained during
long term culture, and the level achieved in Td/TERTs was
comparable to that expressed by the 293 human embryonic
kidney tumor cell lines (Figure 1A). By contrast, mock
transduced HUVECs (Td/mocks) did not express endogenous
telomerase at early cultures (data not shown). Further, the
transduced HUVECs expressed endothelial markers such as
CD31, KDR, and eNOS during long term culture (Figure 1B).
Sequences of PCR primers are shown in Table 1.
Transduced HUVEC lines maintained
endothelial characteristics
Phenotypes of these cell lines displayed serum-dependence
for growth (Figure 2A) and normal contact inhibition
A
B
Passage 3
day 92
day 176
Td/TERT Td/mock HUVEC
CD31
KDR
eNOS
GAPDH
day 176 day 92
293
Td/TERT Td/mock
hTERT
GAPDH
day 176
day 176day 176
day 92
day 92day 92
293
293293
Td/TERT
Td/TERTTd/TERT
Td/mock
Td/mockTd/mock
Passage 3
day 92
day 176
Td/TERT Td/mock HUVEC
Passage 3
day 92
day 176
Td/TERT Td/mock HUVEC
Passage 3
day 92
day 176
Td/TERT Td/mock HUVEC
Figure 1 hTERT transduced HUVECs expressed telomerase genes during long-term
culture. RNA samples from transduced HUVECs were analyzed by RT-PCR for gene
expression. (A) Expression of hTERT gene in 293 cells served as the positive standard.
(B) Expression of endothelial markers.
A
PD
days
0
2
4
6
1020304050
Td/mock
Td/TERT
B
C
Td/mock day 25Td/TERT day 96
Td/TERT
day 124
Td/mock
day 159
0
0.2
0.4
0.6
0.8
1
*****
**********
Absorbance (490 nm)
2%serum medium
10% serum medium
Figure 2 Transduced HUVEC lines maintained endothelial characteristics. (A) Growth
of transduced HUVECs, Td/mock (day 159) and Td/TERT (day 124), was inhibited in
low serum (*p ? 0.001, ** p ? 0.05). (B) Growth curves of transduced HUVEC. About
in a month, most transduced HUVECs showed almost a typical senescent endothelial
morphology and stopped continuous growth. However, hTERT overexpression allowed
the maintanance of a relatively normal endothelial morphology during the aging process.
(C) Phase microscopy demonstrated endothelial morphology of Td/TERTs (day 96)
and Td/mocks (day 25). Original magnifi cation x100.

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Biologics: Targets & Therapy 2008:2(3)
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Takano et al
(data not shown). Thus, the transduced HUVEC lines
showed no signs of tumorigenic transformation. About in a
month, transduced HUVECs ordinary entered the growth-
arrested state (Figure 2B), and these phenotypes developed
an enlarged and fl attened morphology (Figure 2C, Td/mocks
[day 25]). In contrast, the Td/TERTs (day 96) maintained a
relatively normal endothelial morphology during the aging
process (Figure 2C). However, some Td/mocks survived
more than a month and maintained the same normal endo-
thelial morphology as the Td/TERTs (data not shown).
Transduced HUVEC senescence
evaluated by SA-Gal staining
The cytochemical senescent phenotype was also examined
using SA-Gal activity. As expected, hTERT expression
facilitated the delay in senescence (Figure 3A, B). During
the aging process, transduced HUVECs revealed SA-Gal
activity. However, the ratio to total cells in Td/mocks (day 32)
was signifi cantly increased compared with that in Td/TERTs
(day 105) (41% ± 13% vs 7% ± 4%, p ? 0.001). These results
indicated that hTERT overexpression delayed the cells from
entering their senescent state.
hTERT overexpression exceeded
mitogenic potential.
Endothelial cell lines have a mitogenic response to angio-
genic cytokines. To determine whether Td/TERTs produced
angiogenic responses on stimulation by growth factors,
we performed angiogenic proliferation assays by treating
transduced HUVECs with different cytokines in VEGF165
and bFGF while applying serum-depleted basal EBM-2
medium. As shown in Figure 3C, D, when treated with
VEGF165 (Figure 3C) and bFGF (Figure 3D), MTS assay
showed signifi cantly increased proliferation in Td/TERTs
Figure 3 Senescence in transduced HUVECs was evaluated by SA-Gal staining. (A) Quantifi cation of SA-Gal–positive cells in transduced HUVECs obtained by counting
5 random fi elds per dish (p ? 0.001 vs Td/mocks). (B) Representative photomicrographs show SA-Gal–positive cells (blue) in Td/TERTs (day 105) and Td/mocks (day 32).
Original magnifi cation x100. hTERT overexpression exceeded mitogenic potential. (C) Proliferative activity assay response to VEGF165. (D) Proliferative activity assay response
to bFGF. The increase in mitogenic response to angiogenic cytokines of Td/TERTs (day 73) was statistically signifi cant compared with Td/mocks (day 59) (VEGF: p ? 0.001;
bFGF: p ? 0.001 vs Td/mocks).
BA
Td/TERT
day 105
Td/mock
day 32
Td/TERT
day 105
Td/mock
day 32
0
% of total
60
20
40
* * * *
DC
0
0.2
0.4
0.6
*
Td/TERT
day 73
Td/mock
day 59
Td/mock
day 59
Td/TERT
day 73
0
0.2
0.4
0.6
0.8
Absorbance (490 nm)
Absorbance (490 nm)
*
control
VEGF (100 ng/ml)
control
bFGF(100 ng/ml)
* * * *
* **

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Biologics: Targets & Therapy 2008:2(3)
551
hTERT overexpressed endothelial cells
(at day 73) than that in Td/mocks (at day 59) (VEGF; 0.506
nm ± 0.061 nm vs 0.239 nm ± 0.013 nm, p ? 0.001, bFGF;
0.697 nm ± 0.067 nm vs 0.261 nm ± 0.027 nm, p ? 0.001).
These data suggest that Td/TERTs generate more remark-
able angiogenic responses to angiogenic cytokines, perhaps
as part of their general endothelial function, than that of
Td/mocks.
hTERT transduction contributes to cell
survival
To confi rm whether hTERT modulates cell survival, cell
apoptosis and oxidative damage were evaluated in transduced
HUVECs. Strong Annexin-V (green) staining, corresponding
to apoptotic cells, was observed in Td/mocks (Figure 4A). The
staining indicated that serum starvation-induced apoptosis
was markedly reduced in Td/TERTs (Figure 4, B)
(4.9% ± 1.5% vs 54.9% ± 11.0%, p ? 0.001). Furthermore,
strong carboxy-H2DCFDA (green) staining, corresponding
to oxidized by ROS, was observed in Td/mocks (Figure 4C).
The oxidatively damaged cells signifi cantly increased in
Td/mocks (10.3% ± 2.2% vs 42.1% ± 6.7%, p ? 0.001)
(Figure 4, D). Staining in blue indicated nuclei by Hoechst
33342 (Figure 4A, C) Taken together, these results suggest
that hTERT overexpression in HUVECs contributes to
resisting apoptosis and oxidative damage.
Gene expression up-regulated in hTERT
HUVEC
Finally we examined gene levels of transduced HUVECs. To
explore the genes involved in hTERT-induced cell survival,
A
Td/TERT
day 51
Td/mock
day 51
B
0
20
40
60
80
Td/TERT
day 51
Td/mock
day 51
% of total
* * *
Td/TERT
day 59
Td/mock
day 55
C
D
Td/TERT
day 59
Td/mock
day 55
0
10
20
30
40
50
60
% of total
* * * *
Figure 4 hTERT transduction contributes to cell survival. (A) Representative photomicrographs show Annexin-V (green), propidium iodide (red), and nuclei were stained
with blue-fl uorescent Hoechst 33342. Original magnifi cation x100. (B) Quantifi cation of apoptosis-induced. The number of apoptotic cells was signifi cantly increased in
Td/mocks (p ? 0.001) vs Td/TERTs. (C) Oxidative stress measurement in live cells. Representative photomicrographs show the cells stained with carboxy-H2DCFDA (green),
corresponding to oxidized cells by ROS, and nuclei were stained with blue-fl uorescent Hoechst 33342. Original magnifi cation x100. (D) Quantifi cation of oxidatively damaged
cells. The oxidatively damaged cells signifi cantly increased in Td/mocks (p ? 0.001) vs Td/TERTs.