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<b>31</b>
P Magnetic Resonance Spectroscopy of Endothelial Cells Grown in
Three-Dimensional Matrigel Construct as an Enabling Platform Technology: I.
The Effect of Glial Cells and Valproic Acid on Phosphometabolite Levels
M. Sterin a; I. Ringel a; S. Lecht a; P. I. Lelkes b; P. Lazarovici a
a
Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine,
The Hebrew University of Jerusalem, Jerusalem, Israel b Laboratory of Cellular Tissue Engineering, School of
Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, Pennsylvania, USA

Online Publication Date: 01 September 2008

To cite this Article Sterin, M., Ringel, I., Lecht, S., Lelkes, P. I. and Lazarovici, P.(2008)'<b>31</b>P Magnetic Resonance Spectroscopy of
Endothelial Cells Grown in Three-Dimensional Matrigel Construct as an Enabling Platform Technology: I. The Effect of Glial Cells and
Valproic Acid on Phosphometabolite Levels',Endothelium,15:5,288 — 298
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 Endothelium, 15:288–298, 2008
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ISSN: 1062-3329 print/1543-5261 online
DOI: 10.1080/10623320802487841

31
P Magnetic Resonance Spectroscopy of Endothelial Cells
Grown in Three-Dimensional Matrigel Construct as an
Enabling Platform Technology: I. The Effect of Glial Cells
and Valproic Acid on Phosphometabolite Levels

M. Sterin, I. Ringel, and S. Lecht

Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine,
The Hebrew University of Jerusalem, Jerusalem, Israel

P. I. Lelkes
Laboratory of Cellular Tissue Engineering, School of Biomedical Engineering, Science and Health
Systems, Drexel University, Philadelphia, Pennsylvania, USA

P. Lazarovici
Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine,
Downloaded By: [Lazarovici, P.] At: 07:07 23 December 2008

The Hebrew University of Jerusalem, Jerusalem, Israel

such as glycerophosphocholine. This endothelial model will be use-

Very few studies describe endothelial cell (EC) properties under
three-dimensional (3D) conditions using 31 P magnetic resonance
spectroscopy (MRS). The authors developed a model in which liv-
ing ECs growing in Matrigel threads (3D conditions) for 5 days
are monitored by 31 P MRS, providing the fingerprint of the major
EC phosphometabolites. Organic extracts of membranal phospho-
lipids were also analyzed by 31 P MRS. For comparison and as a
model for two-dimensional (2D) tissue culture conditions, 31 P MRS
spectra of aqueous extracts of EC phosphometabolites grown un-
der 2D conditions were also evaluated. The phosphometabolites
fingerprint of the cells cultured under 3D was significantly differ-
ent from that of ECs maintained under 2D. Moreover, the pattern
of phosphometabolites was affected by coculture with C6-glioma
cells and upon treatment with valproic acid, which is under clinical
investigation as an antioangiogenic anticancer drug. The major ef-
fects were modulation of (i) energy metabolism intermediates such
as phosphocreatine, (ii) precursors of phospholipids such as phos-
phomonoesters, and (iii) degradation products of phospholipids
Received 9 July 2008; accepted 7 September 2008.
The generous financial support from the Pharmalogica Consortium,
MAGNET Program of the Israeli Ministry of Trade and Industry, is
gratefully acknowledged (I.R. and P.L.). This work was also supported
at Drexel University in part by a grant-in-aid form the Stein Family
Foundations (P.L. and P.I.L.). I.R. and P.L. are affiliated and supported
at Drexel University in part by David R. Bloom Center for Pharmacy,
School of Pharmacy, The Hebrew University of Jerusalem. S.L. is
supported by “Eshkol Fellowship” from the Israeli Ministry of Science,
Culture and Sport.
Address correspondence to Prof. Philip Lazarovici, Department of
Pharmacology and Experimental Therapeutics, School of Pharmacy,
Faculty of Medicine, The Hebrew University, Jerusalem 91120, Israel.
E-mail: philipl@ekmd.huji.ac.il
full as an enabling platform technology for tissue engineering.
31
Keywords 3D, Endothelium, Matrigel, P MRS, Phospholipids,
Phosphometabolites
Endothelial cells (ECs) represent valuable cellular models
for studying hemostasis, vasomotor control, angiogenesis, and
inflammation (Kirkpatrick et al. 1997) and for blood vessel
tissue engineering (Nomi et al. 2006). ECs are the key compo-
nents of the tumor microvasculature and play a major role in
tumor progression and metastasis (McDonald and Foss 2000).
Furthermore, ECs can be maintained in three-dimensional (3D)
conditions in bioreactors to study cell-cell and cell-biomaterial
interactions (Isenberg et al. 2006; Williams and Wick 2004).
Natural extracellular matrix–derived biomaterials, such as
Matrigel (MG), can serve as a template for endothelial cells
attachment, growth, and differentiation (Schnaper et al. 1993).
MG, isolated from the murine Engelbreth-Holm-Swarm sar-
coma, is comprised of a complex mixture of basement mem-
brane proteins, containing 60% to 85% laminin, 5 to 30% type
IV collagen, 1% to 5% nidogen, 1% to 10% heparin sulfate
proteoglycan, and 1% to 5% entactin, as well as some essen-
tial growth factors and cytokines, such as transforming growth
factor-β (TGFβ), epidermal growth factor (EGF), insulin-like
growth factor-1 (IGF-1), basic fibroblast growth factor (FGF-
2), and platelet-derived growth factor (PDGF; Vukicevic et al.
1992). Due to its unique composition and viscoelastic properties,
MG provides a permissive environment that induces organotypic

288
 ENDOTHELIAL PHOSPHOMETABOLITES IN 3D
differentiation of diverse cell cultures (Kleinman and Martin
2005). MG is widely used for a variety of in vitro and in vivo
angiogenic assays to study endothelial functionality (Auerbach
et al. 2003). Furthermore, by freezing and lyophilization and/or
electrospinning, MG can be manufactured into biologically ac-
tive, 3D scaffolds capable of supporting growth and differentia-
tion of a number of different cell types (Lazarovici et al. 2007).
In the past, we established an immortalized endothe-
lial cell line that was generated as a result of spontaneous
somatic hybridization in a coculture between rat adrenal
medullary pheochromocytoma cell line (PC12) and bovine
adrenal medullary endothelial cells (Lelkes et al. 1992). The
result of hybridization between a neuronal cell line and pri-
mary endothelial cells was a novel, stable, cross-species, and
highly tumorigenic hybrid cell line, named PC12EN. Under
conventional two-dimensional (2D) culture conditions, these
endothelial cells attain a histiotypic cobblestone/spindle-like
morphology, form a tubular network on MG, take up acetylated
low-density lipoprotein, and express an endothelial-specific an-
Downloaded By: [Lazarovici, P.] At: 07:07 23 December 2008
giotensin converting enzyme isoform (Rasouly et al. 1996). We
utilized this endothelial model to develop an in vitro blood-
brain barrier (BBB) model upon coculture with C6-glioma cells
(Lecht et al. 2003). In situ, glial cells are amongst the major
cellular contributors to BBB properties. Among the different
critical effects of glial cells on endothelial cells is ability of
glial cells to tighten the endothelial monolayer (Megard et al.
2002). C6-glioma, a cell line isolated from a rat glioma tumor,
has been frequently used in 2D coculture with a wide variety of
endothelial cells (Tan et al. 2001).
In recent years, it has become evident that the transition
from 2D to 3D cell culture models provides a more realistic
approach to study in vitro cellular functions and morphogenetic
processes, such as cell-cell interactions, which are pivotal for
successfully engineering tissues for diagnosis and implantation
(Mueller-Klieser 1997; Kunz-Schughart et al. 2004). Further-
more, 3D tissue culture models play an invaluable role in tumor
biology research, providing important insights into tumorogenic
cell transformation and as models for tumor therapy (Friedrich
et al. 2007). By increasing our understanding of homeostasis,
cellular differentiation, and tissue organization, these 3D mod-
els provide a well-defined environment for research, in contrast
to the complex environment of an in vivo model and the sim-
plicity of 2D cultures. 3D homotypic and heterotypic cultures
are currently being developed to understand some of the com-
plexities of tumor biology (Sung and Chung 2002; Kim 2005;
Gottfried et al. 2006). Characterization and optimization of the
performance of the diverse cell types interacting with each other
in these 3D tumor models requires novel experimental strategies
to evaluate, e.g., cell viability, cell metabolism, tissue formation,
histological properties, and interactions with drugs.
Nuclear magnetic resonance spectroscopy (MRS) is among
the few noninvasive analytical techniques that yield such infor-
mation from living cells. In the past decade, MRS has become a
useful technique to study the development of engineered tissue
289
both in vitro and in vivo (Neves et al. 2003; Neves and Brindle
2005; Stabler et al. 2005) and tumor biology (Griffin and Kaup-
pinen 2007). Although MRS of cells grown under 2D conditions
can be performed only with organic or aqueous extracts of cell
extracts, MRS of cells grown under 3D conditions enables the
online acquisition of MRS signals in intact living cells in a
noninvasive fashion and without the requirement for prior ex-
traction. Living cells maintained under appropriate conditions in
the MRS instrument are metabolically stable and proliferating
and represent a suitable cellular model to study phospholipid
metabolism (Sterin et al. 2001, 2004).
Being a versatile technique, MRS is able to detect a wide
variety of elements that are found in cells, such as 31 P. 31 P
MRS is able to identify compounds belonging to several groups:
(i) molecules involved in generation of energy, such as nu-
cleotide triphosphates (NTPs), which give rise to three sig-
nals representing the α, β, and γ location of the phosphates
in the NTP molecule, phosphocreatine (PCr), and inorganic
phosphate (Pi); (ii) compounds involved in the metabolism
of phospholipids such as phosphocholine (PC) and phospho-
ethanolamine (PE), which are phospholipids precursors and
represent the major components of the phosphomonoester
(PM), glycerophosphoethanolamine (GPE), and glycerophos-
phocholine (GPC) signals, which are products of phospholipid
degradation; (iii) phosphorylated sugars, such as uridinediphos-
phosugars (UDPS), generate two signals that represent the dif-
ferent locations of the two phosphates in the (UDPS) which
generate molecule.
To date, very few studies have attempted to characterize
endothelial phospholipid metabolism and adenosine triphos-
phate (ATP) content by MRS (Culic et al. 1997; Gorodetsky
et al. 1999; Mori et al. 2003). The purpose of this study was to
compare the phosphometabolite fingerprints in an endothelial
cell model cultured under 3D conditions to that of ECs cultured
in 2D. The usefulness of 31 P MRS in endothelial research, and
more generally in studying 3D tissue assemblies, is emphasized
by our results, which show distinct and significant changes
in the phosphometabolite fingerprints upon coculture of the
endothelial cells with glial cells in 3D and upon treatment with
a common, BBB-permeating drug, valproic acid.
MATERIALS AND METHODS
Materials
Matrigel was prepared according to an Institutional Animal
Care and Use Committee (IACUC)-approved protocol and fol-
lowing published procedures (Kleinman et al. 1986). Dulbecco
modified Eagle’s medium (DMEM), fetal calf serum (FCS),
horse serum (HS), L-glutamine, penicillin and streptomycin,
trypsin, and other culture medium constituents were purchased
from Biological Industries (Beit Haemek, Afula, Israel). Val-
proic Acid (VPA) sodium salt (>98% pure) and all other chem-
icals were purchased from Sigma Chemical (St. Louis, MO),
unless otherwise stated. A stock solution of VPA was dissolved
 290 M. STERIN ET AL.
in dimethyl sulfoxide (DMSO); the VPA final concentration in
the MG threads did not exceed 0.1% v/v. The control cells were
treated with 0.1% DMSO.
Cell Culture
Endothelial cells (PC12EN) were maintained in DMEM sup-
plemented with 7.5% FCS and 7.5% HS, as previously described
(Lelkes et al. 1992). Glial cells (C6-glioma) were maintained in
DMEM supplemented with 10% FCS and 2 mM L-glutamine,
both media containing 100 µg/ml streptomycin and 100 U/ml
penicillin (Lecht et al. 2003). The cultures were maintained in
T-175 flasks (Nunc, Rochester, NY) in a humidified incubator at
37◦ C in an atmosphere of 95% air/5% CO2 . The growth medium
was replaced twice weekly. The cultures were passaged once a
week at a 1:10 ratio after treatment with 0.25% trypsin. For 2D
experiments, monolayers of endothelial or glioma cells were
dissociated with 0.25% trypsin, centrifuged and washed several
times with fresh medium, carefully resuspended to prevent ag-
gregate formation, and plated in three culture dishes (15 cm in
Downloaded By: [Lazarovici, P.] At: 07:07 23 December 2008
diameter) in a dilution that would bring the culture to ∼90%
confluence after 5 days (2 × 107 cells per dish). The medium
was changed after 3 days of culture. To generate a 2D coculture,
endothelial and glioma cells were mixed at 1:1 ratio before cul-
turing, After 5 days the ratio of the two cell types, which exhibit
similar population doubling times, had remained unchanged.
For 3D experiments, the cells were grown as above and, upon
harvesting, introduced individually or as 1:1 mixture into the
MG threads (see below).
Preparation of Matrigel Threads and Treatment with
Valproic Acid
To ensure adequate VPA penetration into the MG threads,
the cell suspensions were preloaded with drug by incubation
with a 0.5 mM VPA for 1 h. The cell suspension was mixed
every 10 min to prevent attachment. To generate MG threads,
the cells were centrifuged at 4◦ C, as above. The precooled cell
pellet was mixed with liquid ice-cold MG at a ratio of ∼1:3
(v/v). For EC–glial cell cocultures, the cells were mixed at
a ratio of 1:1 prior to admixing them to the MG. The cold
cell/MG mixtures were poured into a plastic syringe by an
attached syringe (Figure 1A) and left to partially gelate at
room temperature for 15 to 60 s. To generate the threads,
the gel was extruded into the growth medium containing the
same VPA concentration and maintained for 2 days to ensure
VPA effect on the cells. Each thread was 60 cm long and 0.5
mm in diameter. Threads were transferred to the sterilized
MRS perfusion system. As a control, a similar procedure was
performed using a solution of 0.1% DMSO in culture medium.
All procedures were carried out under aseptic conditions.
Perfusion System
The perfusion system (Figure 1B) was used as previously
described (Daly et al. 1988; Sterin et al. 2001). Briefly, the cells
were maintained in the MRS tube and perfused continuously
with fresh medium containing or lacking VPA at 37◦ C at a flow
rate of 0.5 ml/min using a peristaltic pump. The medium was
gently bubbled with a mixture of 5% CO2 and 95% O2 from a
gas tank to ensure sufficient oxygenation and to maintain a pH
7.4. The perfusate medium flowing through the MG construct
in the MRS instrument tube was collected in a waste beaker.
The MRS probe containing the MG threads was maintained
at 37◦ C.
Cell Growth in Matrigel
Cell viability in the MG threads prior to the MRS experiments
was tested in several threads using the 3-(4,5-Dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Mosmann
1983). Viable cells were stained blue, thus enabling both light
microscopy visualization as well as spectrophotometric estima-
tion of cell numbers (Mosmann 1983).
Phosphometabolites
Phosphometabolites were extracted using perchloric acid
(PCA) as previously described (Sterin et al. 2001). Briefly, the
cell cultures were harvested as above, washed with ice-cold
saline, trypsinized, and pelleted. Ice-cold PCA (0.5 M), 1.5 ml,
was added and mixed followed by sonication for 5 min at 4◦ C.
The extracts were neutralized with about 0.5 ml 5 M KOH and
centrifuged for 10 min at 10,000 rpm. The supernatants were
treated with 50 mg/ml Chelex-100 for 1 h to remove param-
agnetic ions. Thereafter, the Chelex was removed by filtration,
the pH was adjusted to 8.0, and the water was removed by
lyophilization. The dried solid material was stored at −70◦ C.
Prior to MRS analysis, the samples were dissolved in 250 µl
D2 O and 250 µl of 20 mM EDTA. Before extracting phospho-
metabolites from the cells loaded in the MG threads, the cells
were isolated by treatment of the MG threads with Matrisperse
for 1 h on ice. Thereafter, the phosphometabolites were extracted
from the cells as above.
Phospholipids
Phospholipids were extracted as previously described (Sterin
et al. 2004). Briefly, cell cultures from three culture dishes of
15 cm diameter were washed with ice-cold saline, scraped and
collected into 20 ml of ice-cold methanol. Each sample was
supplemented with two volumes of chloroform (40 ml) fol-
lowed by shaking and filtering into separation funnel. The ex-
tract was washed with 12 ml of 1 M KCl and the phases were
left overnight at 4◦ C for separation. The lower phase (chloro-
form) was collected, evaporated using a rotatory evaporator to
remove the chloroform, and stored at −20◦ C under nitrogen.
Before the MRS analysis, the samples were dissolved in 1.2
ml chloroform and 0.6 ml of a mixture of methanol and 40
mM of K4 EDTA at a ratio of 1:4 v/v, respectively. To calculate
the level of membranal phospholipids, the area of each sig-
nal was divided by the total areas of all signals and multiplied
 ENDOTHELIAL PHOSPHOMETABOLITES IN 3D 291
Downloaded By: [Lazarovici, P.] At: 07:07 23 December 2008

FIG. 1. Characterization of an MRS experimental system to study endothelial cells phosphometabolites in 3D. (A) Preparation of MG threads by injecting
semi-solid MG-containing cells through a plastic tube into a Petri dish. The presence of the cells in the MG thread is visualized by the MTT blue color staining
of the living cells (magnification 200×, right figure). (B) The perfusion system used during collection of MRS spectra of perfused cells. (a) 95% O2 , 5% CO2
tank; (b) container of growth medium to be perfused in the MRS tube; (c) peristaltic pump; (d) MRS Varian instrument; (e) tube containing the endothelial cells
embedded in MG threads; (f) container collecting the medium from the tube. (C) A typical 31 P MRS spectrum representing the three phosphates at α, β, and
γ positions in ATP molecule, the inorganigc phosphate (Pi), and typical phosphometabolites: 1 = phosphomonoesters; 2 = glycerophosphoethanolamine; 3 =
glycerophosphocholine; 4 = phosphocreatine; 5 and 5* = uridinediphosphosugars.

by 100. Therefore, the results represent the percentage of each
phospholipid normalized to the total level of phospholipids in the
samples.
31
P Magnetic Resonance Spectroscopy
31
P MRS of cells embedded in MG threads superfused with
culture medium were recorded on a Varian Inova500 instrument
with a 10-mm probe operating at 121 MHz with the following
characteristics: delay time of 2 s; acquisition time of 0.5 s; spec-
tral width and number of points of 8000; flip angle of 60◦ . Broad-
band Waltz proton decoupling was used. For each spectrum 1000
scans were accumulated over a 1-h period and processed using
10-Hz line broadening. Chemical shifts were referenced to the
GPC signal at 0.49 ppm as internal standard. Peak areas were
normalized to the β-ATP signal. We also compared individual
 292 M. STERIN ET AL.
peak area to total phosphate (sum of all signals excluding Pi),
which originates mostly from the perfusion medium and the re-
sults were qualitatively the same. The time delay and flip angle
were sufficient for full relaxation of all signals, except for the
Pi signal, between scans. This was confirmed by using a 10-s
delay.
As described previously (Sterin et al. 2001) and shown here
in Figure 1C, 31 P MRS of cells is able to identify compounds
belonging to several groups: (i) molecules involved in gener-
ation of energy such as nucleotide triphosphates (NTPs), gen-
erating three signals that represent the α (α-ATP), β (β-ATP),
and γ (γ -ATP) location of the phosphates in the NTP molecule,
phosphocreatine (PCr, peak 4), and inorganic phosphate (Pi);
(ii) compounds involved in the metabolism of phospholipids
such as phosphomonoesters (PM, peak 1), glycerophospho-
ethanolamine (GPE, peak 2), and glycerophosphocholine (GPC,
peak 3); (iii) phosphorylated sugars such as uridinediphospho-
sugars (UDPS, peaks 5 and 5*; for quantitative calculations of
UDPS concentration, only the well-defined down-field 31 P sig-
Downloaded By: [Lazarovici, P.] At: 07:07 23 December 2008
nal [peak 5] was used). The phosphometabolites average level of
each signal (relative to β-ATP) was calculated from several inde-
pendent experiments. For comparative purposes, the calculated
additive contribution of the individual cell type cultures is pre-
sented in addition to the experimentally measured signals level
of coculture, which represent combined contribution of each cell
type.
Statistical Analysis of the Data
Each experiment was performed at least three times. The
results are presented as the mean ± SD. Statistical comparisons
between experimental groups were made using InStat program
and included the analysis of variance (ANOVA) followed by
Dunnet’s multiple comparison test; p < .05 was considered
significant.
RESULTS
Pattern of Phosphometabolites of Endothelial Cells
Cultured under 3D Compared to 2D Conditions
To assess the influence of a 3D environment on endothelial
cell function, some recent studies compared their behavior under
2D and 3D culture conditions (Matsumoto et al. 2007; Sun et al.
2006; Martins and Kolega 2006). Several studies described the
use of MRS to evaluate EC function in vitro (Culic et al. 1997)
and in vivo (Barber et al. 2004). The data presented in Figure 2
show a comparison between the phosphometabolites of ECs cul-
tured under 2D (aqueous extracts) versus 3D (living cells) con-
ditions. As expected, 31 P MRS of all endothelial cells indicated
the presence of several compounds involved in generation of cell
energy ATP, PCr (signal number 4); phospholipid metabolites,
PC and PE, which are precursors of phospholipids, are main
components of PM signal (signal number 1). GPE (signal num-
ber 2) and GPC (signal number 3) are products of phospholipid
degradation. Activated sugar forms, such as UDPS, are denoted
as signal number 5 (Sterin et al. 2001). The phosphometabolite
data from three independent measurements for endothelial cells
cultured under 2D and 3D conditions are summarized in Figure
2B. It is evident that the level of PM, GPC, and PCr (signal num-
bers 1, 3, and 4, respectively) are significantly increased in 3D
compared to 2D by six-, nine-, and sevenfold, respectively. In
contrast, the levels of GPE (signal number 2) and UDPS (signal
number 5) were not affected by growing the cells in 3D versus
2D.
Characterization of Phosphometabolites of Endothelial
Cells in the Presence or Absence of Glial Cells under 3D
and 2D Conditions
Endothelial cells are a key component of the tumor vascu-
lature and hence play a major role in spreading metastases.
Of particular interest in the development and progression of
gliomas in the brain is the influence of the tumor microenviron-
ment on endothelial pathophysiology (Frontczak-Baniewicz et
al. 2008), as manifested, for example, by the increased leakiness
of the blood-tumor barrier, which can be measured by magnetic
resonance imaging (Cao et al. 2005). To evaluate such an influ-
ence on phosphometabolites, we performed a series of experi-
ments with ECs under 3D and 2D conditions, in the presence
or absence of C6-glioma cells. By comparing the global (total)
phosphometabolite signature measured in a well-defined cocul-
ture of these two cell types to that of the signals originating from
the individual cell types, we might get an insight into the cell-
cell interactions as inferred from differences in the levels of the
various phosphometabolites. Figure 3A shows the signature of
phosphometabolites in endothelial and glioma cells cultured in
3D either alone or upon coculturing at a ratio 1:1. The averaged
phosphometabolite data from 31 P MRS of three independent
measurements are summarized in the Figure 3B. A compari-
son of the signals in the 3D coculture to the calculated additive
values (based on the individual contribution of each cell type)
might indicate differential effects of the coculture on specific
phosphometabolites. The only statistically significant change
observed was on the level of PM (signal number 1), which was
increased in coculture by fourfold compared to the twofold ad-
ditive level of the endothelial and glial monocultures signal.
Therefore, the increased change in the level of PM in coculture
represents a synergistic effect of the coculture conditions on this
phosphometabolite.
Because 31 P MRS of aqueous extracts (2D) is more sensitive
than MRS of living cells (3D; Pianet et al. 1991), we evalu-
ated the effect of the coculture on phosphometabolite pattern
in 2D conditions (Figure 4). It is evident that the levels of PM
(signal number 1), GPE (signal number 2), GPC (signal num-
ber 3), and PCr (signal number 4) were significantly higher
than the additive contribution of the respective signals of each
cell type. These findings emphasize the sensitivity of 31 P MRS
with aqueous extracts and further support the synergistic effect
 ENDOTHELIAL PHOSPHOMETABOLITES IN 3D 293
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FIG. 2. Typical 31 P MRS spectra and signal intensities of endothelial cells grown under 3D (living cells) and 2D (aqueous extracts). For the 2D conditions, the
cells were grown in tissue culture dishes for 48 h, harvested, and phosphometabolites were extracted by PCA, partially purified by Chelex 100, and submitted for
MRS analysis, as described in Materials and Methods. For 3D conditions, the cells were grown in MG threads for 48 h, transferred to the NMR tube, and perfused,
as detailed in Materials and Methods. (A) 31 P MRS spectra; (B) averages of phosphometabolite data from 31 P MRS spectra; Signal areas are normalized to that of
β-ATP. Gray bars, 2D conditions; black bars, 3D conditions. The values represent the mean ± SD of three independent experiments. *p < .01. Assignments of
the signals are 1 = phosphomonoesters; 2 = glycerophosphoethanolamine; 3 = glycerophosphocholine; 4 = phosphocreatine; 5 = uridinediphosphosugars; PC
= phosphocholine; Pi = inorganic phosphate.

measured at the level of PM with living cells under 3D conditions
(Figure 3). Interestingly, the level of PC decreased by 2.3-fold
under 2D coculture conditions. Because PC levels were signifi-
cantly lower in the glioma cells than in the ECs, the low level of
expression in the coculture suggests a drastic decline in the PC
levels in the ECs under these conditions. We note that the levels
of UDPS were not changed significantly under our coculture
conditions.
The Effects of Valproic Acid on Phosphometabolites and
Phospholipids of Endothelial Cells under 3D and 2D
Conditions
In this study we used VPA, an endothelium/BBB-permeating
drug (Fischer et al. 2008) to paradigmatically investigate its pos-
sible effects on 31 P MRS phosphometabolites in the above 3D
cell culture systems (Figure 5). The phosphometabolite data
from 31 P MRS, such as shown in Figure 5A, are summarized in
Figure 5B. These results indicate that VPA induced a significant
decrease in PM, GPC, and UDPS levels in ECs cultured in 3D.
Furthermore, VPA also had a distinct effect on the level of ex-
pression of some of the membranal phospholipids. Specifically,
the levels of two negatively charged species were differentially
regulated: cardiolipin levels were decreased, whereas those of
phosphatidylserine were elevated. Interestingly, VPA-treated
ECs had also increased levels of plasmenyl phosphatidylcholine
(Table 1), suggesting that the increased cytoplasmic GPC may
be derived from the mebranal phosphotidylcholine pool. From
the summary of the data in Figure 5B it is evident that for some,
but not all, phosphometabolites, the VPA-induced effects in 3D
were opposite to those observed in 2D. For example, VPA sig-
nificantly increased the level of PM (signal number 1) and PCr
(signal number 4) in 2D cultures, whereas the levels of the same
 294 M. STERIN ET AL.
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FIG. 3. 31 P MRS spectra and signal intensities of living endothelial and glial cells and their coculture at a ratio 1:1. The cells were grown under 3D conditions
in MG threads for 48 h, transferred to the NMR tube, perfused, and the level of cellular phosphometabolites was monitored by NMR measurements. The
signals shown are typical for three independent experiments. (A) 31 P MRS; (B) averages of phosphometabolite data from 31 P MRS spectra. Signal areas are
normalized to that of β-ATP. The values represent the mean ± SD of three independent experiments. *p < .05. White bars, endothelial signal; gray bars, glial
signal; striped bars, calculated sum of endothelial and glial signals; black bars, coculture signal. Assignments of the signals are 1 = phosphomonoesters; 2 =
glycerophosphoethanolamine; 3 = glycerophosphocholine; 4 = phosphocreatine; 5 = uridinediphosphosugars; β-ATP.

phosphometabolites were decreased in ECs cultured in 3D in
the presence of VPA.
DISCUSSION
Using 31 P MRS, we evaluated the pattern of the phospho-
metabolites in a novel model of endothelial cells growing in 3D
inside MG threads. We found significant changes of phospho-
metabolites in ECs growing in 3D versus 2D growth conditions,
as well as upon coculture with glial cells and treatment with
the drug VPA. The analysis of the phosphometabolites focused
on species related to energy metabolism and species related to
phospholipid metabolism.
 ENDOTHELIAL PHOSPHOMETABOLITES IN 3D 295
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FIG. 4. 31 P MRS spectra and signal intensities of aqueous extracts of phosphometabolites from 2D cultures of endothelial cells, glial cells, and their coculture at
a ratio 1:1. The cells were grown under 2D conditions in tissue culture dishes for 48 h and harvested. Phosphometabolites were extracted by PCA, partially purified
by Chelex 100, and submitted for MRS analysis. Signal areas are normalized to the β-ATP (100%). (A) 31 P MRS. (B) The phosphometabolite data from 31 P MRS
of three independent measurements. The values represent the mean ± SD. *p < .05; **p < .01. White bars, endothelial signal; gray bars, glial signal; striped
bars, sum of endothelial and glial signals; black bars, coculture signal. Assignments of the signals are 1 = phosphomonoesters; 2 = glycerophosphoethanolamine;
3 = glycerophosphocholine; 4 = phosphocreatine; 5 = uridinediphosphosugars; PC = phosphocholine; β-ATP; γ -ATP.

The phosphometabolite fingerprint of the endothelial cells
in 3D versus 2D revealed increased levels of the high energy
metabolism intermediate PCr, which is responsible for the nu-
cleoside triphosphate synthesis, as also reflected by a higher
PCr/ATP ratio in the 3D cultures (Figure 2). This increased
metabolism in 3D versus 2D indirectly supports our fnding of
high mitochondrial activity of the cells in the MG threads (Figure
1C), as evident from the MTT staining of the cells (the chro-
mogenic conversion of MTT reflects mitochondrial oxidation).
Phosphorylated metabolic intermediates denoting precursors
and products of membrane phospholipid metabolism represent
the majority of the signals measured by 31 P MRS. These signals
can be further classified according to the phosphorus head group
into PM, including predominantly phosphorylethanolamine and
phosphodiesters, such as GPE and GPC. The significant in-
crease in PM under 3D versus 2D conditions in ECs (Figure 2)
most probably reflects alterations in the plasma membrane lipid
packing related to the surface pressure in 3D conditions, activat-
ing a phospholipase A2 (PLA2 )-based mechanosensor (Ko and
McCulloch 2000). In addition, forces applied to the ECs under
3D conditions may be transduced from the plasma membrane
to the cytoskeleton, affecting the microfilament-microtubule
 296 M. STERIN ET AL.
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FIG. 5. 31 P MRS spectra and signal intensities of endothelial cells grown under 3D and 2D upon treatment with VPA. The cells were grown under 3D (white
bars) conditions in MG threads or under 2D (gray bars) conditions in Petri dishes for 48 h in the presence or absence of 0.5 mM VPA. Cellular phosphometabolites
were assessed as above. (A) 31 P MRS of control (black spectra) and VPA-treated (gray spectra) cultures. Assignments of the signals are 1 = phosphomonoesters;
2 = glycerophosphoethanolamine; 3 = glycerophosphocholine; 4 = phosphocreatine; 5 = uridinediphosphosugars. (B) The phosphometabolite data from 31 P
MRS of three independent measurements. Signal areas are normalized to the β-ATP. −, absence of drug; +, presence of drug. The values represent mean ± SD.
*p < .05.

interaction (Ko and McCulloch 2000). These interactions in
turn may change the phospholipid metabolism, in particular
GPC level, as evident from studies in which cell treatment with
antimicrotubule drugs induced a profound elevation in intracel-
lular GPC that was not associated with the phospholipid com-
position of cell membrane (Sterin et al. 2001). Therefore, it is
tempting to hypothesize that the increase in the GPC signal upon
culturing ECs in 3D conditions (Figure 2) may reflect EC cy-
toskeletal dynamics. Furthermore, GPC and GPE were found to
act as competitive inhibitors of lysophospholipase activity, thus
reducing lysophospholipid hydrolysis in the cells and lowering
the rate of membrane phospholipid degradation (Fallbrook et
al. 1999). Therefore, we may also suggest that the increased
level of GPC under 3D in ECs may reflect a membranal pro-
tective effect in the plasma membrane. The profound elevation
of GPC was also reported in human umbilical vein endothe-
lial cells treated with cyclooxygenase inhibitor indomethacin
and monitored by 1 H nuclear magnetic resonance (NMR; Mori
et al. 2003). This effect may be explained by a selective induction
of actin-cytoskeleton reorganization induced by the drug (Jain et
al. 2004) or by shifting the arachidonic acid from the cyclooxy-
genase pathway to the PLA2 /lysophospholipase pathway.
In another approach, we used 31 P MRS as an enabling plat-
form technology to assess the levels of phosphometabolites upon
interaction of ECs with glial cells cocultured in the MG threads.
According to the 31 P MRS data, these coculture conditions
 ENDOTHELIAL PHOSPHOMETABOLITES IN 3D
TABLE 1
The levels of membranal phospholipids extracts of endothelial
cells in 3D and treated with VPA
Control VPA
Phospholipid (% of total) (mean ± SD) (mean ± SD)
Phosphatidic acid 3.48 ± 0.28 3.56 ± 0.23
Cardiolipin 4.17 ± 0.27 2.95 ± 0.27*
Plasmenyl 11.95 ± 1.89 11.71 ± 1.01
phosphatidylethanolamine
Phosphatidylethanolamine 10.81 ± 0.3 11.08 ± 0.25
Phosphatidylserine 4.72 ± 0.47 6.16 ± 0.5*
Sphingomyelin 6.02 ± 0.14 6.27 ± 0.46
Phosphotidylinosine 7.11 ± 0.37 6.63 ± 0.24
Plasmenyl 2.57 ± 0.26 3.34 ± 0.25*
phosphatidylcholine
Phosphatidylcholine 48.69 ± 1.3 49.25 ± 2.2
∗
The cells were grown under 3D conditions in MG threads for 48 h,
Downloaded By: [Lazarovici, P.] At: 07:07 23 December 2008
transferred to the MRS tube, and perfused. The 31 P MRS spectra are
presented in Figure 5. Following the perfusion, the cellular phospho-
lipids were extracted from the cells upon release from the threads
using Matrisperse treatment and assessed by NMR spectroscopy. The
levels of each phospholipid species were calculated. The values, as
percentage of the total phospholipid content, represent the mean ±
SD of three independent experiments. *p < .05 compared to control
(untreated) cultures.
yielded an increase in the PM level with no change in the high-
energy metabolism intermediates. Although we cannot attribute
in the coculture system the 31 P MRS fingerprint to a partic-
ular cell type, we conclude that a 3D coculture system does
not interfere with the viability of either cell type. It also may
be possible that the 3D condition favored enhanced secretion
of growth factors by ECs such as vascular endothelial growth
factor (VEGF), as found with osteogenic progenitors (Jarrahy
et al. 2005), causing a change in the phospholipid metabolism
(Bernatchez et al. 2001) and resulting with increased GPC level.
Finally, we used the 31 P MRS enabling platform technology
to assess the levels of phosphometabolites upon treatment of ECs
with VPA, a widely used antiepileptic drug that can cross the
BBB and is undergoing clinical evaluation for anticancer and an-
tiangiogenic therapeutic indications (Michaelis et al. 2004; Isen-
berg et al. 2007). VPA treatment of ECs in 3D conditions (Figure
5), in contrast to 2D, indicated a slight decrease in energy and
phospholipid metabolism intermediates compared to untreated
3D cultures. Presently, it is unclear whether these changes repre-
sent a direct effect of VPA on phospholipid metabolic enzymes
or whether this is a reflection of the antiangiogenic effect of
VPA, which was previously observed in the in vitro MG angio-
genesis assay and is probably mediated by inhibition of histone
deacetylase (Michaelis et al. 2004).
In summary, 31 P MRS of ECs cultured in 3D in MG threads
offers a several unique advantages over 2D cultures; for exam-
297
ple, in experiments designed for tissue engineering purposes.
This technique will be useful for noninvasively monitoring the
proliferation and energy levels of the cells, the interactions of
multiple cell types in the same constructs, and for analyzing the
effects of drugs. Thus, this system represents a novel enabling
technology for more realistic evaluations of the ECs organized
in engineered tissue-like constructs.
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