2-Amino-phenoxazine-3-one attenuates glucose-induced augmentation of embryonic form of myosin heavy chain, endothelin-1 and plasminogen activator inhibitor-1 in human umbilical vein endothelial cells

Article abstract of DOI:10.1248/bpb.28.797

The aim of this study was to investigate the changes in mRNA level of embryonic form of myosin heavy chain (SMemb), endothelin-1 (ET-1) and plasminogen activator inhibitor-1 (PAI-1), which are considered to be involved in the angiogenesis and atherosclerosis in diabetic blood vessels, in human umbilical vein endothelial cells (HUVECs) caused by high ambient glucose, and the effects of 2-aminophenoxazine-3-one (Phx-3), which was produced by the reaction of bovine hemoglobin with o-aminophenol, on them. The mRNA level of SMemb, ET-1 and PAI-1 and the level of SMemb protein were extensively upregulated in HUVECs treated with high concentration of glucose (15 mM), compared with those in the cells with normal concentration of glucose (5 mM). The migration activity of HUVECs evaluated by the cell migration assay was accelerated by 15 mM glucose. When 10 μM Phx-3, at the concentration of which the proliferation of HUVECs was not affected, was administered to HUVECs with 15 mM glucose, the mRNA level of SMemb, ET-1 and PAI-1 and the level of SMemb protein were significantly downregulated to the normal levels in the cells. However, when 10 μM Phx-3 was administered to HUVECs with 5 mM of glucose, the mRNA level of SMemb, ET-1 and PAI-1 and the level of SMemb protein were not affected. The migration activity of HUVECs, which was accelerated by high glucose, was reversed by 10 μM Phx-3. The present results suggest that Phx-3 may be a drug to prevent the high glucose-associated endothelial damage, vascular angiogenesis in diabetic patients, by inhibiting the expression of angiogenic factors, such as SMemb, ET-1 and PAI-1, in the endothelial cells.

Full text of DOI:10.1248/bpb.28.797

May 2005
Biol. Pharm. Bull. 28(5) 797—801 (2005)
797
2-Amino-phenoxazine-3-one Attenuates Glucose-Induced Augmentation of
Embryonic Form of Myosin Heavy Chain, Endothelin-1 and Plasminogen
Activator Inhibitor-1 in Human Umbilical Vein Endothelial Cells
Gen FUKUDA,a Noriko YOSHITAKE,a Zia Ali KHAN,b Masao KANAZAWA,c Yo ko N OTOYA,c
d
d
Xiao-Fang CHE, Shin-ichi AKIYAMA, Akio TOMODA, ,d Subrata CHAKRABARTI,b and
*
a
Masato ODAWARA
b
a Third Department of Internal Medicine, Tokyo Medical University; Tokyo 160–0023, Japan: Department of Pathology,
c
University of Western Ontario; London, ON N6A 5C1, Canada: Health Care Liaison Center, Department of Molecular
Oncology, Field of Oncology, Course of Advanced Therapeutics, Tokyo Medical University; Tokyo 160–0023, Japan:
d Graduate School of Medical and Dental Sciences, Kagoshima University; Kagoshima 890–8544, Japan: and
e Department of Biochemistry and Intractable Immune System Disease Research Center, Tokyo Medical University; Tokyo
160–0022, Japan. Received October 5, 2004; accepted February 10, 2005
The aim of this study was to investigate the changes in mRNA level of embryonic form of myosin heavy
chain (SMemb), endothelin-1 (ET-1) and plasminogen activator inhibitor-1 (PAI-1), which are considered to be
involved in the angiogenesis and atherosclerosis in diabetic blood vessels, in human umbilical vein endothelial
cells (HUVECs) caused by high ambient glucose, and the effects of 2-aminophenoxazine-3-one (Phx-3), which
was produced by the reaction of bovine hemoglobin with o-aminophenol, on them. The mRNA level of SMemb,
ET-1 and PAI-1 and the level of SMemb protein were extensively upregulated in HUVECs treated with high con-
centration of glucose (15 mM), compared with those in the cells with normal concentration of glucose (5 mM). The
migration activity of HUVECs evaluated by the cell migration assay was accelerated by 15 mM glucose. When
10 mM Phx-3, at the concentration of which the proliferation of HUVECs was not affected, was administered to
HUVECs with 15 mM glucose, the mRNA level of SMemb, ET-1 and PAI-1 and the level of SMemb protein were
significantly downregulated to the normal levels in the cells. However, when 10 mM Phx-3 was administered to
HUVECs with 5 mM of glucose, the mRNA level of SMemb, ET-1 and PAI-1 and the level of SMemb protein were
not affected. The migration activity of HUVECs, which was accelerated by high glucose, was reversed by 10 mM
Phx-3. The present results suggest that Phx-3 may be a drug to prevent the high glucose-associated endothelial
damage, vascular angiogenesis in diabetic patients, by inhibiting the expression of angiogenic factors, such as
SMemb, ET-1 and PAI-1, in the endothelial cells.
Key words human umbilical vein endothelial cell; high glucose; 2-aminophenoxazine-3-one
Endothelial migration, proliferation and differentiation and whether the expression of SMemb may be changed
have been recognized to be associated with diabetes induced under hyperglycemic conditions.
angiogenesis and atherosclerosis,1—3) inflammation,2,4,5) and
The development of drugs to attenuate the expression of
angiogenesis in malignancies,2,6) where a number of cy- endothelial factors in the vascular endothelial cells are criti-
tokines and growth factors released by the endothelial cells cal for preventing the angiogenesis and atherosclerosis in the
and the smooth muscle cells are involved. In diabetes, the diabetic patients. Lohray et al.14) indicated that a phenox-
vascular damage caused by hyperglycemia may induce dys- azine analogue of phenyl propanoic acid activated peroxi-
function of endothelial cells and the activation of smooth some proliferator-activator inhibitor gamma (PPARg), which
muscle cells in the vascular wall, initiating the events in an- is highly expressed in adipocytes,15) and is involved in the
giogenesis and atherosclerosis.1—3) Therefore, it may be im- improvement of type 2 diabetes through insulin sensitizing
portant to study the expression of the factors in these cells mechanism.16) Chemically synthesized phenoxazines are gen-
under hyperglycemic conditions. Especially, expression of erally insoluble in water, and have been shown to exert lim-
endothelin-1 (ET-1), a vasoconstrictive peptide, plasminogen ited biological effects including anticancer activity.17,18)
activator inhibitor-1 (PAI-1), a prothrombic protein, and Hence, phenoxazine compounds have not been studied in
ICAM-1, VCAM-1 and ECAM-1, a group of adhesion pro- terms of biological activities, except for actinomycin D hav-
teins, are of significant interest in this context.7—12) Chen et ing strong anticancer effects but causing adverse effects.19) In
al.7,8) reported that the upregulated expression of endothelin- the 1990s, Tomoda et al.20—22) reported that novel and rela-
1 was shown in human umbilical vein endothelial cells (HU- tively water-soluble phenoxazines such as 2-amino-4,4a-
VECs) under hyperglycemic conditions, however, the behav- dihydro-4a,7-dimethyl-3H-phenoxazine-3-one (Phx-1), 3-
ior of PAI-1 in the cells was not studied under such condi- amino-1,4a-dihydro-4a,8-dimethyl-2H-phenoxazine-2-one
tions, hitherto.
(Phx-2), and 2-amino-phenoxazine-3-one (Phx-3) were pro-
On the other hand, the phenotypic switch of smooth mus- duced, when o-aminophenol and its derivatives (2-amino-5-
cle cells from contractile to synthetic type manifested by ex- methylphenol, 2-amino-4-methylphenol) were reacted with
pression of an embryonic form of myosin heavy chain human or bovine hemoglobin. Among these phenoxazines,
(SMemb) was demonstrated to be relevant to the atheroscle- the biological effects of Phx-1 have been exclusively studied,
rosis in diabetes.1,13) However, it was not sufficiently investi- in terms of antitumor effects in vivo and in vitro,23,24) im-
gated whether SMemb may be expressed in the HUVECs munosuppressive effects,25,26) anti-proliferating effects on
∗ To whom correspondence should be addressed. e-mail: tomoda@tokyo-med.ac.jp
© 2005 Pharmaceutical Society of Japan

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Vol. 28, No. 5
polio virus transfected in Vero cells.27) However, the biologi-
cal activity of Phx-3 remained obscure, though only anti-my-
cobacterial activity of Phx-3 was indicated by Anzai et al.28)
and Shimizu et al.22) Therefore, it will be tempting to study
the effects the effects of novel phenoxazines on high glucose-
induced changes in the endothelial factors that may be in-
volved in the diadetes induced angiogenic and atherogenic
processes.
Present manuscript deals with the augmentation of
SMemb, ET-1 and PAI-1 in human umbilical endothelial
cells (HUVECs) under hyperglycemic conditions, and the at-
tenuating effects of Phx-3 on high glucose augmented ex-
pression of these endothelial factors in HUVECs. The possi-
bility of Phx-3 to treat the atherosclerotic lesions in type 2
diabetes was suggested.
termined by calculating the ratio of absorbance at 260 nm
and 280 nm.
Four micrograms of total RNA was used for cDNA synthe-
sis using the Superscrpt IITM system (Invitrogen Japan).
Briefly, RNA was added to Oligo (dT) primers and denatured
at 70 °C for 10 min. Reverse transcription was carried out by
addition of MMLV reverse transcriptase and incubation of
samples at 42 °C for 55 min. The resulting cDNA products
were stored at ꢁ20 °C.
Real Time PCR Real time polymerase chain reaction
(RT-PCR) was carried out in the Opticon2TM (MJ Japan,
Tokyo) using SYBR·Green 1.30) PCR reactions were per-
formed in 96-well multiplateTM (MJ Japan) with a final vol-
ume of 20 ml. The reaction mixture consisted of 10 ml of
2ꢀDyNAmoTM SYBR·Green qPCR kit (MJ Japan), 0.5 ml of
each forward and reverse 10 mmol/l primers, 8 ml H2O and
1 ml cDNA template. Primers for SMemb, ET-1, PAI-1 and
b-actin were designed by use of the Primer 3 software
(Whitehead Institute, Cambridge, MA, U.S.A.). The gene
specific forward and reverse primer sequences are 5ꢂ-AGAG-
GAGGCAATACAGTGG-3ꢂ, 5ꢂ-TGGAGAGCCTTAAGAC-
ACAG-3ꢂ for SMemb mRNA with the expected 117 bp PCR
product, 5ꢂ-TCCTCTGCTGGTTCCTGACT-3ꢂ, 5ꢂ-CAGAA-
ACTCCACCCCTGTGT-3ꢂ for ET-1 mRNA with the ex-
pected 243 bp PCR product, 5ꢂ-CCCACTTCTTCAGGCT-
GTTC-3ꢂ, 5ꢂ-CCGTTGAAGTAGAGGGCATT-3ꢂ for PAI-1
mRNA with the expected 187 bp PCR product.
The following PCR program was performed on the Opti-
con2: an initial denaturation at 95 °C for 10 min to activate
DNA polymerase, followed by a 39 cycle program consisting
of heating at 20 °C/s to 95 °C with 10 s hold, cooling at 59 °C
with a 30 s hold for annealing and heating at 20 °C/s to 72 °C
with 30 s hold for extension. PCR was immediately followed
by a melting curve analysis to determine the melting point of
the double stranded PCR product produced. mRNA levels
were quantified using standard curve method. Standard
curves were constructed using serially diluted standard tem-
plate. Ct value was used to compute mRNA levels from the
standard curve. mRNA levels were normalized to b-actin,
whose primers were 5ꢂ-CCTCTATGCCAACACAGTGC-3ꢂ
(forward), and 5ꢂ-CATCGTACTCCTGCTTGCTG-3ꢂ (re-
verse), to account for differences in reverse transcription effi-
ciencies and amount of cDNA in reaction mixtures.
Immunoblotting Analysis of SMemb Protein HU-
VECs (1ꢀ106 cells) were grown on poly-L-lysine coated
dishes, at 37 °C, for 24 h. Then, the cells were treated with
5 mM glucose alone, 5 mM glucose plus 10 mM Phx-3, 15 mM
glucose alone or 15 mM glucose plus 10 mM Phx-3, and were
incubated at 37 °C for 8 h. The cells in each dish were rinsed
twice with chilled PBS and lyzed with the solution composed
of RIPA buffer (150 mM NaCl, 50 mM Tris–HCl (pH 7.5), 1%
Triton X-100, 1% deoxycholate), and a complete tablet pro-
tease inhibitor cocktail (Roche, Indianaplis, IN), for 30 min
at 4 °C. The whole cell extracts were sedimented at 15000
rpm for 15 min to remove the insoluble material. Total pro-
tein concentration in the supernatants was determined using
the Bio-Rad Protein Assay® reagent (Life Science, Tokyo).
Namely, the extracts containing 50 mg proteins were heated
to 100 °C for 5 min, and subjected to SDS-PAGE elec-
trophoresis. The proteins were tranferred to PVDF mem-
branes using XCell II SureLock Mini-Cell system (Invitoro-
MATERIALS AND METHODS
Phx-3 Phx-3 was prepared by the reactions of bovine he-
moglobin solution and o-aminophenol hydrochloride (Wako
Pure Chemicals., Tokyo), and was purified by using a column
of Sephadex LH20 (4 cmꢀ50 cm), previously equilibrated
with 50% ethanol, and eluted with 50% ethanol.22) Purified
Phx-3 was identified by measuring UV and visible spectra,
and by a thin layer chromatography.22) The chemical structure
is shown in Fig. 1.
Cell Culture Methods and Cell Proliferation Assay
HUVECs were seeded on a 96-well microplate (Asahi
Technoglass, Tokyo) at a density of 5000 cells per well in
EGM-2 culture medium (Takara Bio Japan, Tokyo) supple-
mented with 2% fetal bovine serum (FBS, Invitorogen Japan,
Tokyo) and 10 mg/l human recombinant epidermal growth
factor, and were cultured at 37 °C, in 5% CO2/air, for 24 h.
Appropriate concentrations of glucose (final concentrations:
5 mM, as normal glucose concentration or 15 mM, as high
glucose concentration) were added to the medium, when the
cells were 80% confluent. Ten mM Phx-3 dissolved in di-
methyl sulfoxide (DMSO) was diluted with EGM-2 and
added to the culture medium containing HUVECs to make a
final concentration of 10 mM. Cell proliferation was evaluated
by the microculture tetrazolium assay using WST-8 (Dojindo
Molecular Technologies, Kumamoto, Japan).29)
Isolation of RNA and Synthesis of cDNA HUVECs
(1ꢀ106 cells) were grown on poly-L-lysine coated dishes, at
37 °C, for 24 h. Then, the cells were treated with 5 mM glu-
cose alone, 5 mM glucoe plus 10 mM Phx-3, 15 mM glucose
alone or 15 mM glucose plus 10 mM Phx-3, and were incu-
bated at 37 °C for 8 h. Then, total RNA was isolated from
HUVECs using TRIzolTM reagent (Invitrogen Japan).8) After
addition of chloroform, samples were centrifuged to separate
aqueous and organic phases. RNA was recovered from the
aqueous phase by isopropyol alcohol precipitation and sus-
pended in DEPC-treated water. RNA was quantified by mea-
suring UV absorbance at 260 nm. Purity of samples was de-
Fig. 1. Chemical Structure of Phx-3

May 2005
799
gen Japan, Tokyo). The membranes were blocked with 5%
non-fat milk prior to immunoblot analysis reacted with a
mouse anti-human SMemb IgG antibody (dilution 1 : 3000,
Yamasa Co. Ltd., Tokyo) and a horseradish peroxidase-con-
jugated goat anti-mouse secondary antibody (dilution
1 : 2000, Amarsham Biocsiences, Tokyo). Finally, the blots
were visualized using the enhanced chmiluminescence sys-
tem (ECL Plus western Blotting Detection System, Amer-
sham Biosciences).
Cell Migration Assay of HUVECs Measurement of mi-
grating ability of HUVECs was performed according to the
method of Yamakawa et al.31) HUVECs were cultured in en-
dothelial basal medium (EBM, Sigma-Aldrich Japan, Tokyo,
Japan) supplemented with 2% FBS under a humidified at-
mosphere of 5% CO2 in air. The cells (1ꢀ106 cells/dish) in
EBM were fluorescence-labeled by incubation with 3 mM 5-
carboxyfluorescein, diacetoxy-methylester (DCECF-AM,
Dojindo Laboratories, Kumamoto, Japan) for 30 min at
37 °C. After the labeled cells had been washed with EBM,
5ꢀ104 of them in 300 ml medium were added into culture in-
sert with 8 mm-pore HTS FluoroBlokTM Inserts membrane
(Becton Dickinson, Franklin Lakes, NJ, U.S.A.) precoated
with MatrigelTM matrix (Becton Dickinson) with a density of
25 mg/insert. Then, 3 ml of 1 mM Phx-2 was added to this
Fig. 2. Effects of Phx-3 on the Proliferation of HUVECs Treated with
Normal Concentrations or High Concentrations of Glucose
(ꢀ): normal glucose (5 mM); (ꢁ): normal glucoseꢅ10 mM Phx-3; (ꢂ): normal glu-
coseꢅ30 mM Phx-3; (ꢃ): normal glucoseꢅ100 mM Phx-3; (ꢅ): normal glucoseꢅ
300 mM Phx-3; (ꢄ): high glucose (15 mM); (ꢅ): high glucoseꢅ10 mM Phx-3; (ꢆ) high
ꢅ
glucoseꢅ30 mM Phx-3; (ꢇ) high glucoseꢅ100 mM Phx-3; ( ): high glucoseꢅ300 mM
Phx-3. Each point shows the mean values (nꢆ5).
medium. The culture insert was set into the well of a 24-well tors and the Effects of Phx-3 on the Expression We stud-
plate which contained 700 ml of EBM with 10% FBS as a ied the mRNA level of SMemb, ET-1 and PAI-1 in HUVECs
chemoattractant. The serum was used without heat inactiva- treated with either normal concentrations of glucose alone
tion. The appropriate amount of recombinant human vascular (5 mM), normal concentrations of glucose (5 mM) plus 10 mM
endothelial growth factor protein (rhVEGF, Becton Dickin- Phx-3, high concentrations of glucose alone (15 mM) or high
son) was added to the both side of medium and incubated concentrations of glucose (15 mM) plus 10 mM Phx-3 for 8 h.
at 37 °C for 8 h in a CO2 incubator. Following incubation, As shown in Fig. 3A, the level of SMemb mRNA of the HU-
FluoroBlokTM Inserts membrane was examined under a fluo- VECs with normal concentrations of glucose (5 mM) was not
rescence microscope and fluorescence of invaded cells was affected by the addition of 10 mM Phx-3. The levels of
read directly in a fluorescence plate reader with bottom SMemb mRNA of the HUVECs treated with high concentra-
reading capabilities at excitation/emission wavelengths of tions of glucose increased to 200% of the control (5 mM glu-
530/590 nm every 1 h without further manipulation. Only cose alone), while those of HUVECs treated with high con-
those labeled cells that have invaded the MatrigelTM matrix centrations of glucose and 10 mM Phx-3 decreased exten-
and passed through the pores of the FluoroBlokTM membrane sively (by 50% of HUVECs with high concentrations of glu-
will be detected.
cose alone). The mRNA level of ET-1 (Fig. 3B) and PAI-1
Statistics The data were expressed as meanꢃS.E.M. and (Fig. 3C) in HUVECs was not altered in the HUVECs treated
were analyzed by ANOVA followed by post-hoc test. Differ- with 5 mM glucose and 10 mM Phx-3, compared with the cells
ences were considered significant at values of pꢄ0.05.
treated with 5 mM glucose alone. The mRNA level of ET-1
and PAI-1 was upregulated by 15 mM glucose (by 210% and
220%, respectively), but these were reversed to the levels of
normal control (5 mM glucose alone) by the addition of
RESULTS
Effect of Phx-3 on Endothelial Proliferation Prolifera- 10 mM Phx-3.
tion of HUVECs in the presence of normal (5 mM) glucose or
Figure 4 shows the representative immunoblotting patterns
high (15 mM) glucose, and the effects of various concentra- of SMemb protein in each cells treated with 5 mM glucose
tions of Phx-3 (10 to 300 mM) for 24 h was studied by WST-8 alone, 15 mM glucose alone, 5 mM glucose plus 10 mM Phx-3
assay (Fig. 2). The cell counts were increased gradually dur- or 15 mM glucose plus 10 mM Phx-3. Consequently, it was
ing 8 h incubation of HUVECs in the presence of 5 mM or found that the amounts of SMemb protein were not altered in
15 mM glucose, 5 mM glucose plus 10 mM Phx-3 or 15 mM the HUVECs treated with 5 mM glucose and 10 mM Phx-3,
glucose plus 10 mM Phx-3. The proliferation curve of these compared with the cells treated with 5 mM glucose alone.
groups increased at the similar rates after 8 h. However, the These were extensively increased in HUVECs treated with
cell counts were much suppressed in the presence of 5 mM high glucose (15 mM) alone for 8 h, compared with those
glucose plus Phx-3 over 30 mM, or 15 mM glucose plus Phx-3 treated with 5 mM glucose alone. Furthermore, the additional
over 30 mM, dependent on the dose of Phx-3 added, showing treatment of 10 mM Phx-3 reversed the high glucose effect.
that Phx-3 suppresses the proliferation of HUVECs at higher These results are consistent with those for the mRNA level of
concentrations over 30 mM, but does not affect it at the con- SMemb as shown in Fig. 3.
centration of 10 mM.
Effect of Phx-3 on Glucose Augmented Endothelial
Glucose Augmented Expression of the Vasoactive Fac- Cell Migration Since the migration of endothelial cells to

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Vol. 28, No. 5
Fig. 5. Changes in Counts of Migrating Cells after Treatment of HUVECs
with Normal Glucose, Normal Glucose plus 10 mM Phx-3, High Concentra-
tions of Glucose or High Concentrations of Glucose plus 10 mM Phx-3
Data was expressed as the mean percent migration derived from the mean fluorescent
units of cell migration through the Matrigel coated FluoroBlok inserts. (ꢀ): normal
concentrations of glucose (5 mM); (ꢁ) normal glucose (5mM)ꢅ10 mM Phx-3; (ꢄ): high
concentrations of glucose (15 mM); (ꢅ) high concentrations of glucose (15 mM)ꢅ10 mM
Phx-3. Each point shows the mean values (nꢆ5).
(Fig. 5). Consequently, it was found that the counts of mi-
grating HUVECs were significantly increased in the cells
treated with high glucose, compared with the cells treated
with normal concentration of glucose at each point examined
for 8 h. On the contrary, when HUVECs were treated with
both high glucose and 10 mM Phx-3, the counts of migrating
HUVECs were reduced more markedly than those of the
cells with high glucose alone, at each time examined for 8 h.
At 8 h, the counts of migrating HUVECs treated with 15 mM
glucose and 10 mM Phx-3 were about 67% of those treated
with 15 mM glucose alone. However, the counts of migrating
HUVECs were not affected when HUVECs with 5 mM glu-
cose was treated with 10 mM Phx-3. These results show that
the migration activity of HUVECs was accelerated under hy-
perglycemic conditions, but was reduced by the treatment
with 10 mM Phx-3.
Fig. 3. Changes in Expressed Amounts of mRNA of SMemb, ET-1 and
PAI-1 in HUVECs Treated with 10 mM Phx-3 (nꢆ4)
DISCUSSION
(A) The amounts of SMemb mRNA; (B) the amounts of ET-1 mRNA; (C) the
amounts of PAI-1 mRNA. NG, HUVECs with normal glucose concentrations (5 mM);
HG, HUVECs with high glucose concentrations (15 mM); NGꢅPhx3, HUVECs with
normal glucose concentrations (5 mM) and 10 mM Phx-3; HGꢅPhx3, HUVECs with
high glucose concentrations (15 mM) and 10 mM Phx-3. The amounts of each mRNA
were based on the mRNA amounts of b-actin, as a house-keeping gene transcript.
Since diabetes mellitus leads to endothelial dysfunction
and development of atherosclerotic vascular diseases,1,2,7) it
is critical to identify alteration of vasoactive factors involved
in such events in the endothelial cells to identify potential
targets of newer therapy as well as novel disease biomarker.
It has recently been shown that various intracellular, extracel-
lular and intercellular factors of the endothelial cells and
smooth muscle cells, such as SMemb, ET-1, PAI-1, ICAM-1,
VCAM-1, ECAM-1, and fibronectin etc. are involved in the
atherosclerotic process in diabetes.1,9—12)
NG
HG
NG + Phx-3
HG + Phx-3
SMemb was originally found in embryonic smooth muscle
cells,32) and shown to be increased at the mRNA and protein
levels in diabetic meningial cells.13) SMemb has been charac-
terized to cause vascular smooth muscle cells to switch from
contractile to synthetic type, possibly leading to atheroscle-
rotic lesions.13,32) However, SMemb was not quantified at the
mRNA level in HUVECs. ET-1, a potent vasoconstrictor, is
implicated as a cause of the micro- and macrovascular com-
plications of diabetes mellitus,7,8) and is detected in large
quantities in HUVECs treated with high glucose.33) PAI-1, on
the other hand accelerates the proliferation of HUVECs
through VEGF activation, promoting the angiogenesis34) and
is detectable in HUVECs.10)
Fig. 4. Immunoblotting Patterns of SMemb in HUVECs Treated with
10 mM Phx-3
NG, HUVECs with normal glucose concentrations (5 mM); HG, HUVECs with high
glucose concentrations (15 mM); NGꢅPhx-3, HUVECS with normal glucose concen-
trations (5 mM) and 10 mM Phx-3; HGꢅPhx-3, HUVECs with high glucose concentra-
tions (15 mM) and 10 mM Phx-3.
the extracellular matrix has been considered as a marker of
atherosclerotic process in diabetes, we studied the migration
ability of HUVECs which were treated with normal concen-
tration of glucose (5 mM) or high concentration of glucose
(15 mM) in the presence or absence of 10 mM Phx-3 for 8 h

May 2005
801
397—405 (1998).
5) Reape T. J., Groot P. H., Atherosclerosis, 147, 213—225 (1999).
Present results demonstrated that the mRNA level of these
factors tightly relevant to angiogenesis and atherosclerosis in
diabetes were upregulated in HUVECs treated with high am-
bient glucose (Fig. 3). Namely, we found that SMemb was
expressed at the mRNA level in HUVECs, and that high glu-
cose resulted in the increased mRNA level of SMemb (Fig.
3) and the level of SMemb protein (Fig. 4) in the cells, for
the first time. It remains unclarified whether such increased
SMemb in HUVECs may be directly linked to the intensified
invasion activity of the cells as shown in Fig. 4 or not. We
found that mRNA of PAI-1 was also expressed in HUVECs
and was upregulated in the cells with high ambient glucose
(Fig. 3). We confirmed the increased expression of ET-1 at
the mRNA level in HUVECs with high ambient glucose (Fig.
3), as has been shown by Keynan et al.33) These results
strongly support the views that the changes in the factors
such as SMemb, ET-1 and PAI-1 in the endothelial cells may
be associated with the angiogenesis and atherosclerosis
under hyperglycemic conditions.
Furthermore, we found that Phx-3, which is equivalent to
questiomycin A, whose actions have not been clarified except
for the anti-mycobacterial activity,22,28) attenuated the in-
creased expression of SMemb, ET-1 and PAI-1 in HUVECs
caused by high ambient glucose (Fig. 3), and inhibited the in-
creased invasion activity of the cells with high ambient glu-
cose (Fig. 5). These attenuating effects of Phx-3 on HUVECs
with high ambient glucose may be beneficial for inhibiting
the progress of diabetic angiogenesis and atherosclerosis ac-
companying the migration of the activated endothelial cells
into the extracellular matrix. However, the mechanism for at-
tenuating action of Phx-3 on the expression of intracellular
factors in HUVECs with high ambient glucose is currently
obscure. Present results suggest Phx-3, which has been
shown to have little adverse effects on mice (our unpublished
data) may be a useful drug to treat diabetes, atherosclerosis
and cancer accompanying blood vessel proliferations, which
awaits further investigation.
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Acknowledgements The present research was supported
by research funds from the Tokyo Medical University Cancer
Research Foundation (2004), from High-Tech Research Pro-
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