Effects of Interferons a/b on the Proliferation
of Human Micro- and Macrovascular Endothelial Cells
Joris Erdmann,1,2Giovanni Vitale,2–4Peter M. van Koetsveld,2Ed Croze,5Diane M. Sprij-Mooij,2
Leo J. Hofland,2and Casper H.J. van Eijck1
Synthetic interferons (IFNs) are used in the treatment of several types of cancer. In addition to an antitumor effect,
IFNs show antiangiogenic activity. The aim of this study was to investigate the effects of IFN-a and IFN-b
on human micro- and macrovascular endothelial cells in vitro [human micro vascular lung endothelial cells
(HMVEC-L) and human umbilical cord endothelial cells (HUVEC)]. By immunohistochemical staining and
quantitative reverse transcriptase (RT)-polymerase chain reaction, we studied expression of type I IFN receptors.
We evaluated the effects of IFN-a and IFN-b on the proliferation (DNA content), apoptosis (DNA fragmentation by
enzyme-linked immunosorbent assay), and cell cycle distribution (flow-cytometric analysis) of endothelial cells.
HUVEC and HMVEC-L cells show comparable expression level of the distinct IFN receptor subtypes. Proliferation
of HMVEC-L and HUVEC was inhibited by IFN-b (the half maximal inhibitory concentration [IC50]¼60 and
90IU/mL, respectively), but not by IFN-a at a dose up to 1,000IU/mL. An interesting and unexpected observation
was an inhibition of apoptosis by IFN-b. After 72h of treatment with IFN-b. Cell cycle inhibition occurs in late S-
phase in both cell lines. In conclusion, only IFN-b, not IFN-a (10–1,000IU/mL), has an inhibitory activity on
endothelial cell proliferation. Surprisingly, apoptosis was decreased by IFN treatment, whereas inhibition of
proliferation is caused by cell cycle arrest in late S-phase.
1957) as cytokines released by virus-infected cells inducing
endonucleases and inhibitors of protein synthesis in neigh-
bouring cells creating an antiviral state (Samuel 2001). Syn-
thetic IFNs are currently used in the treatment of several types
of cancer, multiple sclerosis, and viral infections such as hep-
atitis B/C and severe acute respiratory syndrome. IFNs are
highly species specific and consist of 3 families of glycopro-
teins: type 1–3 IFNs. The type 1 family includes the IFN-a
(leukocyte IFN), IFN-b (fibroblast IFN), IFN-o, and IFN-t
subtypes. Type 2 (IFN-g) and the recently discovered type 3
IFNs (IFN-l) (Kotenko and others 2003) will not be further
discussed in this article. Thirteen forms of IFN-a and only one
of IFN-b are known to exist (Roberts and others 1998). The
type 1–3 IFNs act through separate membrane receptors that
have distinct properties and induce different responses. The
type 1 receptor consists of 2 subunits, AR1 and AR2. AR2 is
known to have 3 splice variants: long, short, and soluble forms.
nterferons (IFNs) were first discovered in 1956 by
Alick Isaacs and Jean Lindemann (Isaacs and Lindenmann
Only one form of AR1 has been identified (Novick and others
1994). Binding of IFNs to their receptor causes hetero-
dimerization of the 2 receptor subunits, AR1 and AR2c, the
long form splice variant (Croze and others 1996). One of the 2
membrane-bound variants of AR2, the short form AR2b, is
able to bind IFNs but does not couple to signal transduction
because it lacks the signal transducing tail of AR2c (Novick
and others 1994, 1995). This soluble AR2a may act as regula-
and others 2004). After heterodimerization, Tyk 2 and Jak 1
associate with the receptor complex and form a docking site
for STATS 1 and 2. These STATS then translocate to the nu-
cleus were they activate transcription of IFN-stimulated genes
(Caraglia and others 1997, 2005; Jonasch and Haluska 2001).
The list of IFN-stimulated genes is still increasing and currently
over 300 genes have been identified (de Veer and others 2001).
IFN-a and IFN-b induce different subsets of genes in fibrosar-
coma cells (Der and others 1998); however, no differences were
found in endothelial cells (da Silva and others 2002).
IFNs have known antitumor effects (Jonasch and Haluska
2001) and we recently demonstrated a more potent in vitro
1Department of Surgery, Erasmus MC, Rotterdam, The Netherlands.
2Department of Internal Medicine, Division Endocrinology, Erasmus MC, Rotterdam, The Netherlands.
3Chair of Endocrinology, Department of Medical Sciences, Faculty of Medicine, University of Milan, Milan, Italy.
4IRCCS, Instituto Auxologico Italiano, Milan, Italy.
5Department of Immunology, Berlex Bioscience, Inc., Richmond, California.
JOURNAL OF INTERFERON & CYTOKINE RESEARCH
Volume 31, Number 5, 2011
ª Mary Ann Liebert, Inc.
antitumor activity of IFN-b compared to IFN-a in adrenal,
pancreatic, and neuroendocrine tumors (van Koetsveld and
others 2006; Vitale and others 2006, 2007).
In vivo studies show growth inhibition of several types of
cancer by IFNs. Inability to respond to endogenous IFN-a
and IFN-b in type 1 receptor-deficient mice accelerated de-
velopment, tumorigenity, and metastasis of cutaneous neo-
plasms (McCarty and others 2002). In hepatocellular
carcinoma grown in nude mice, IFN-a inhibits metastasis
and recurrence after curative resection by inhibition of an-
giogenesis mediated through downregulation of vascular
endothelial growth factor (Wang and others 2003). Gene-
therapy-delivered IFN-b is able to reduce invasiveness and
angiogenic potential of prostate and renal cell cancer in vivo
(Dong and others 1999; Nakanishi and others 2003).
It is clear that endothelial cells play a critical role in tumor
growth, since the size to which a tumor can grow is limited
to the diffussional capacity of the tissue for oxygen and nu-
trients. An angiogenic switch (Folkman 2002) is necessary to
expand beyond this point. Proliferation of endothelial cells is
thought to be an essential step in this process of angiogen-
esis; therefore, inhibition of proliferation may result in anti-
angiogenic effects (Carmeliet 2003; Tonini and others 2003;
Taraboletti and Giavazzi 2004). In vitro both stimulatory
(Cozzolino and others 1993; Mintzer and others 1998; Gomez
and Reich 2003) and inhibitory (Heyns and others 1985;
Hicks and others 1989; Ruszczak and others 1990; Yoshida
and others 1996; Hong and others 2000; Minischetti and
others 2000; da Silva and others 2002; Pammer and others
2006) effects of type 1 IFNs on endothelial proliferation have
been reported. Remarkably, IFN-b, potentially the most po-
tent subtype (van Koetsveld and others 2006; Vitale and
others 2006, 2007), has not been studied extensively. There-
fore, in the current study a comparison is made between the
anti-proliferative effects of IFN-a and IFN-b on micro- and
macro vascular endothelial cells in vitro.
Materials and Methods
Human umbilical cord endothelial cells (HUVEC) and
human microvascular lung endothelial cells (HMVEC-L)
were supplied by Clonetics-Cambrex Biosciencea ¨ (Walkers-
ville), and cultured in EBM-2MV?medium supplied by the
same company. All supplements (human epidermal growth
factor [hEGF], Hydrocortisone, vascular endothelial growth
factor, human fibroblast growth factor [hFGF-B], Long R3-
IGF-1, Ascorbic Acid, GA-1000 [antibiotics], and fetal bovine
serum 5%) were added to the medium. The cultures were
grown at 378C and 5% CO2. Standard culture instructions
supplied by Clonetics were applied. Cells were passaged by
Grand Island, Canada). Before plating, the cells were coun-
ted microscopically using a standard heamocytometer. Try-
pan Blue staining was used to assess cell viability, which
always exceeded 95%. HMVEC-L were used only from
passages 3–6 and HUVEC, passages 3–7.
Drugs and reagents
Intron-A (IFN-a-2b), REBIF (Recombinant IFN-b 1a), and
Poly I:C were obtained from Schering-Plough Corporation
(Utrecht, The Netherlands), Serono (Rockland, MA), and
Sigma-Aldrich (Leiden, The Netherlands), respectively. All
compounds were stored at ?208C, and the stock solution
was constituted in distilled water according to the manu-
Quantitative reverse transcriptase-polymerase
Expression of receptors (AR1, AR2 total, the short-form
AR2b, and the long-form AR2c) and housekeeping gene
was evaluated by quantitative reverse transcriptase (RT)-
polymerase chain reaction (PCR) in HUVEC and HMVEC-L
cells as published previously (Vitale and others 2006). In ad-
dition, IFN-b primer and probe sequences were purchased
from Biosource (Nivelles, Belgium): forward, 50-CAGCAATTT
TCAGTGTCAGAAGCT-30; reverse, 50-TTCATCCTGTCCTT
GAGGCAG-30; probe 50-FAM TGTGGCAATTGAATGGGAG
The detection of HPRT mRNA was used for normalization
of mRNA levels. Expression of AR2a mRNA, the soluble
form of AR2 subunit, was determined indirectly by sub-
tracting AR2b and AR2c from AR2 total. Several controls
were included in the RT-PCR experiments. To exclude con-
tamination of the PCR reaction mixtures, the reactions were
also performed in the absence of DNA template in parallel
with cDNA samples. As a positive control for the PCR re-
actions of HPRT and type I IFN receptors, human cDNA
[from BON cells (Vitale and others 2006)] was amplified in
parallel with the cDNA samples.
HUVEC and HMVEC-L cells were grown on Thermanox?
coverslips (Nunc, Naperville, IL) fixed with acetone and
subsequently incubated for 30min at room temperature with
antibodies to human AR-1 (rabbit polyclonal antibody; Santa
Cruz Biotechnology, Santa Cruz, CA) or AR-2c (monoclonal
antibody, Dr E. Croze, Berlex Biosciences, Richmond, CA)
subunits. Standard streptavidin-biotinylated alkaline phos-
phatase (IL Immunologic, Duiven, The Netherlands) was
used according to the manufacturer’s recommendations to
observe the bound antibodies. Negative controls for the im-
munohistochemistry were included by omission of the pri-
After plating of cells (20,000 cells per well HMVEC-L and
10,000 cells per well HUVEC experiments) on 24-well plates
(Corning, Schiphol-Rijk, The Netherlands), the plates were
left in an incubator for 24h for the cells to adhere to the
bottom of the wells. After refreshing the medium, com-
pounds were added. The medium was refreshed at day 3
and compounds added again. After 6 days the cells were
harvested and DNA contents were measured. Total DNA
contents, representative for the number of cells, were mea-
sured using the Hoechst 33258 reagent as published previ-
ously (Hofland and others 1990).
DNA fragmentation assay
After plating 100,000 cells/well for HUVEC and 200,000 for
HMVEC-L on a 24-well plate, cells were left overnight to
452ERDMANN ET AL.
adhere. After refreshingthe medium, compounds wereadded.
After incubation for 24h DNA fragmentation (apoptosis) was
assessed using the Roche Cell Death Detection ELISA Plus?
(Penzberg, Germany). This assay is based on a quantitative
monoclonal antibodies directed against DNA and histones,
respectively. This allows the specific determination of mono-
and oligonucleosomes in the cytoplasmatic fraction of cell ly-
sates, typical for apoptosis. The standard protocol supplied by
the manufacturer was used. By both light microscopy and
measurement of DNA contents, we confirmed that cell num-
bers were not significantly affected during these experiments.
In baseline conditions IFN-b expression is low but detect-
able. To explore the possible induction of IFN-b expression,
cells were plated and refreshed as described for the apoptosis
assay. Poly I:C, double-stranded RNA, a potent activator of
IFN-b expression (Field and others 1972), was added at a
concentration of 100mg/mL and samples were collected after
2 and 4h as described in the Quantitative RT-PCR section.
Cell cycle analysis by fluorescence-activated
Cells (0.8–2?106) were plated in 75cm2flasks. After 3
days, the medium was changed with a fresh medium (con-
trol group) or with a fresh medium plus IFN-a or IFN-b at
the concentration of 1,000IU/mL. After 3 days of incubation
(with a confluence of about 60%–70%), cells were harvested
by gentle trypsinization and prepared for propidium iodide
(Sigma-Aldrich) staining as published previously (Vitale and
others 2006). The stained cells were analyzed on a FACSca-
libur flow cytometer (Becton Dickinson, Erembodegem,
Belgium) using CellQuest Pro Software.
All experiments were performed in quadruplicate, except
for RT-PCR and fluorescence-activated cell sorting (FACS),
which were performed in duplicate, and at least twice with
comparable results. For statistical analysis, GraphPad Prism?
4.0 (GraphPad Software, San Diego, CA) was used. One-way
analysis of variance, followed by multiple comparison Tukey
test, was used to determine statistical significance, defined as
P<0.05. All data are shown as mean?standard error of the
mean. Statistical significance is marked with an asterisk. Bars
represent means, and whiskers, standard error of the mean.
Expression of AR1 and all the variants of AR2 mRNA
have been evaluated by quantitative RT-PCR, as shown in
Fig. 1A. In both cell lines, receptor subtypes AR1 and AR2
a/b/c have comparable distribution; overall expression lev-
els are not significantly different between cell lines (P>0.05
for all subtypes). Immunostaining of both cell lines shows
the presence of AR1 and AR2c on endothelial cells (Fig. 1B–
G). These 2 subtypes jointly represent the active signal
transducing complex. Both cell lines show a comparable
staining and distribution of receptors: whereas AR1 staining
is diffuse and membrane bound and/or cytoplasmic, AR2
staining is granular and appears to be more cytoplasmic than
Endothelial cell proliferation
the proliferation of HUVEC and HMVEC-L, IFN-b (10–
1,000IU/mL) treatment shows a statistically significant cell
growth inhibition in both cell lines (Fig. 2A, B) in a dose-
dependent manner [HMVEC-L: the half maximal inhibitory
concentration [IC50]¼60IU/mL (95% confidence interval:38–
84); HUVEC: 90IU/mL (78–122) P>0.05]. Population dou-
blingtimesfor HUVECare41h,and81hafter IFN-b treatment
(1,000IU/mL), for HMVEC-L 81 and 384h, respectively.
Stimulation of apoptosis by IFN-b has been reported to be
one of the main mechanisms involved in the inhibition of
proliferation, particularly in cancer cells (van Koetsveld and
others 2006; Vitale and others 2006). Therefore, we measured
DNA fragmentation after 24h of IFN treatment (Fig. 2C, D).
Surprisingly, IFN-b inhibits DNA fragmentation in a dose-
dependent manner [HMVEC-L: IC50¼51IU/mL (95% con-
fidence interval: 26–97); HUVEC: 105IU/mL (39–281) at
1,000IU P<0.001 for both cell lines]. IFN-a did not have a
significant effect on apoptosis at a similar dose (P>0.05).
After 24h of incubation with IFN-a or IFN-b, there was no
change in cell number (DNA measurement; data not shown).
Figure 3 shows the IFN-b expression levels with and
without poly I:C treatment. Poly I:C binds to a toll-like re-
ceptor (TLR-3) and is a known inducer of an antiviral re-
sponse in cells (Basu and Fenton 2004). Under baseline
conditions, IFN-b expression is very low but detectable. After
2h of Poly I:C treatment there was a dramatic increase of
IFN-b mRNA (HUVEC: P<0.001 and HMVEC-L: P<0.05),
followed by a decrease after 4h (HUVEC: P<0.05 and
HMVEC-L: P>0.05). The increase of IFN-b mRNA during
stimulation was significantly higher in HUVEC than in
Cell cycle effects
The effects of treatment with IFN-a (1,000IU/mL) and
IFN-b (1,000IU/mL) on the cell cycle phase distribution have
been evaluated in HUVEC and HMVEC-L cells. While IFN-a
does not affect S-phase phase cell counts after 72h of incu-
bation, IFN-b induces a significant accumulation of both cell
lines in S phase compared to the control. The proportion of
cells in G0-G1-phase is not modified by either treatment in
HUVEC and HMVEC-L.
Endothelial cell proliferation is an essential step in an-
giogenesis. Therefore, the inhibition of this process may re-
sult in antiangiogenic effects (Carmeliet 2003; Tonini and
others 2003; Taraboletti and Giavazzi 2004). In literature
there are contradictory results about the effects of IFN-a and
IFN-b on endothelial cells, showing both inhibitory (Ruszc-
zak and others 1990; Hong and others 2000; da Silva and
EFFECTS OF INTERFERONS a/b
others 2002; Pammer and others 2006; Wada and others
2009) and stimulatory effects (Cozzolino and others 1993;
Mintzer and others 1998; Gomez and Reich 2003) on prolif-
eration. These apparent discrepancies between the different
studies may be related to the culture conditions, concentra-
tions, and the synthetic IFN subtypes used. The aim of the
present study was to evaluate the antiproliferative effects of
IFN treatment on endothelial cells, and especially the dif-
ferences between IFN-a and IFN-b subtypes. Parallel to
HUVEC, the most commonly used model for endothelial
cells, we also studied HMVEC-L because of their adult mi-
crovascular origin, possibly resembling tumor endothelial
cells more accurately. A completely different endothelial cell
model next to HUVEC adds to the biological validity of our
For the first time, type I IFN receptors are identified im-
munohistochemically on both HUVEC and HMVEC-L. Both
cell lines have similar expression of these receptors. Func-
tional activity of the receptor complex is shown by the re-
sponse to IFN treatment. Both cell lines can promptly express
IFN-b when stimulated by poly I:C. Presumably this is
similar to in vivo pathophysiological circumstances, such as
during viral infections.
We assessed growth of endothelial cells after IFN treat-
ment. Both cell lines HUVEC and HMVEC-L show a potent
inhibition of proliferation by IFN-b. IFN-a is not able to in-
hibit the growth of endothelial cells at a concentration
ranging from 10 to 1,000IU/mL. Additionally, cell cycle
analysis by FACS shows an increase in S-phase after 72h of
IFN-b treatment, for both cell lines (Fig. 4). This represents a
cell-cycle block in late S-phase, as has been reported before in
several tumor cell lines as well (van Koetsveld and others
2006; Vitale and others 2006, 2007). IFN-a does not induce
any effect on cell cycle distribution at this dose range. The
number of cells in G1/0 seems unaffected after IFN-b treat-
ment, considering that we showed that the apoptosis, mito-
AR1 and AR2a/b/c mRNA
(Copies/HPRT) (P>0.05 for
HUVEC AR1, (C) HMVEC-L
AR1, (D) HUVEC AR2c, (E)
HMVEC-L AR2c, (F) HUVEC-
negative control, (G) HMVEC-
transferase; HUVEC, human
cells; HMVEC-L, human mi-
cro vascular lung endothelial
(A) Expression of
454ERDMANN ET AL.
sis, and growth rates are decreased in the treatment group
(Fig. 2). The increase in S-phase can be compensated by a
decrease in the aforementioned phases. Possibly, only after
prolonged incubation, a decrease in G0/1 may be observed.
Since our cells were not synchronised, more and more cells
arrive at S-phase and are blocked during IFN-b treatment,
leading to accumulation in S- and decrease in G1/0 and
Both HUVEC and HMVEC-L show a dose-dependent
decrease in apoptosis after IFN-b treatment. This was unex-
pected and in contrast with our findings in tumor cells (van
Koetsveld and others 2006; Vitale and others 2006, 2007). An
analogous effect was noted by Pammer et al. (Pammer and
others 2006). They found that IFN-a could prevent apoptosis
induced by serum starvation in HUVEC, using an identical
cell death detection ELISA. They also studied endothelial cell
apoptosis under normal culture conditions, without observ-
ing a significant effect on apoptosis by IFN-a.
Wada and others (2009) recently showed a moderate in-
hibitory effect of high-dose IFN-a (10,000IU) on cell growth
of HUVEC cells. The following were observed: (1) a slight
suppression of apoptosis was reported during IFN-a treat-
ment (apoptosis in control versus IFN-a: 11 versus 7.6%); (2)
IFN-a induced accumulation of HUVEC cells in S-phase after
24h of incubation. In these experiments a less proliferative
culture medium was used possibly leading to lower dose
thresholds for IFN effects. However, a proliferative medium
may more accurately resemble a highly angiogenic tumor
environment. Inadvertently, HMVEC-L must be cultured in
EBM-2MV?medium; in a less proliferative environment
these cells do not proliferate consistently. For matters of
comparability it was necessary to culture HUVEC in this
medium as well.
As mentioned above, Wada and others (2009) reported a
moderate inhibitory effect on cell proliferation using high-
dose of IFN-a (10,000IU/mL) and only a slight effect with
lower doses. We did not observe a significant inhibitory ef-
fect on cell proliferation at 1,000IU/mL of IFN-a, but we did
Dose Interferon b b
DNA Contents (% control)
DNA Contents (% control)
on proliferation after 6 days (dotted line: HMVEC-L). (C) Effects of IFN-a and IFN-b treatment on DNA fragmentation
(apoptosis) after 24h. (D) Dose-dependent effects of IFN-b treatment on DNA fragmentation after 24h. (*P<0.05). IFN,
(A) Effects of IFN-a and IFN-b treatment on proliferation after 6 days. (B) Dose-dependent effects of IFN-b treatment
(0h), and after 2 and 4h of poly I:C treatment. In baseline
conditions, expression is low but detectable. After stimulation
by poly I:C, a vast increase can be observed in IFN-b mRNA
expression in HUVEC (*P<0.001) and HMVEC-L (*P<0.05).
From left to right: 0, 2 and 4 hours for both cell lines.
Expression of IFN-b mRNA, baseline conditions
EFFECTS OF INTERFERONS a/b
not evaluate the activity of this cytokine at higher supra-
clinical dose. In addition, we observed a potent inhibitory
effect on HUVEC and HMVEC-L cell proliferation after in-
cubation with IFN-b. This could partially be explained by the
fact that although IFN-a and IFN-b interact with the same
receptor, IFN-b has a binding affinity *10-fold higher than
IFN-a (Johns and others 1992).
Apoptosis and the cell cycle are considered to be inti-
mately coupled (Vermeulen and others 2003). Indeed both
processes share many of the controlling pathways. However,
according to the classical proliferation–survival model (Bla-
gosklonny 2003) apoptosis should increase when prolifera-
tion is inhibited. Our results show a contradictory relation. In
a study by Tanaka and others (1998) it was shown that IFNs
are key mediators of apoptosis in virus-infected cells. Heal-
thy cells could not be induced to undergo apoptosis unless
they were co-stimulated. Similar co-stimulatory effects were
seen in adhesion molecule expression (inter-cellular adhesion
molecule 1 [ICAM-1] and vascular cell adhesion molecule 1
[VCAM-1]) after IFN-b treatment; endothelial cells were only
stimulated by IFN-b to express these molecules when co-
stimulated by tumor necrosis factor-a (Kobayashi and others
2008). Takaoka and Taniguchi (2003) propose that even under
physiological circumstances, IFNs play a critical role in
to external and internal signalling. In pathophysiological cir-
cumstances, tumor and virus-infected cells have intrinsic and
extrinsic co-stimuli; this makes them susceptible to the cyto-
toxic effects of IFN-b (Tanaka and others 1998; Takaoka and
Taniguchi 2003; Takaoka and others 2003). HUVEC and
HMVEC-L may lack these co-stimuli.
In conclusion, there is clear evidence that IFN-b inhibits
proliferation of endothelial cells. The main cause of growth
inhibition is a cell-cycle block in S-phase. This may partially
explain the antiangiogenic effects as seen in vivo after IFN
treatment, although paracrine effects also appear to be in-
volved (Wang and others 2003; Wada and others 2009). This
hypothesis should be further strengthened by future in vivo or
in vitro studies (such as endothelial cell migration or tube
formation on matrigel). Furthermore, IFN treatment is not
directly cytotoxic or pro-apoptotic for endothelial cells, in
contrast to tumor cells (van Koetsveld and others 2006; Vitale
and others 2006, 2007). Currently, IFN-a is the most widely
used type 1 IFN in clinical-oncological treatment protocols
(Jonasch and Haluska 2001; Picozzi and others 2003). Since
our data suggest a more potent effect of IFN-b treatment,
further in vivo and clinical research on this point is warranted.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:
Dr. Joris Erdmann
Department of Surgery
Dr. Molewaterplein 40
3015 GD Rotterdam
Received 5 October 2009/Accepted 13 October 2010
458ERDMANN ET AL.