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CELLULAR AND MOLECULAR
Departments of Pharmacology (K.Y.K., K.W.H.) and Internal Medicine (Y.W.S.), College of Medicine, and Research Institute of Genetic Engineering (K.W.H.), Pusan National University, Busan, Korea; Dongbu Hannong Chemical Co. (S.O.K., H.L.), Daejon, Korea; and Research Institute of Chemical Technology (S.-E.Y.), Daejon, Korea
Received January 6, 2003; accepted March 3, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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). Vascular endothelial growth factor (VEGF), as an
endothelial cell-specific mitogen, promotes angiogenesis by stimulation of
endothelial cell proliferation and inhibition of endothelial cell apoptosis
(Kuzuya et al., 1999).
Apoptosis, a programmed cell death, is critical during embryonal development
and in tissue renewal (Cohen,
1993). Induction of endothelial cell apoptosis has recently
attracted considerable interest as a potential target for antitumor therapy
(Sgonc et al., 1998).
Phosphatase and tensin homolog deleted from chromosome 10 (PTEN) has dual
specificity protein phosphatase (toward phospho-serine/threonine and
phospho-tyrosine) and phosphoinositide 3-phosphatase activities
(Myers et al., 1997;
Maehama and Dixon, 1998) and
antagonizes the phosphatidylinositol 3-kinase (PI3-K) pathway by catalyzing
degradation of the phosphatidylinositol (3,4,5)-triphosphate
[PI(3,4,5)P3], which is generated by PI3-K, thereby leading to a
control role in converting PI(3,4,5)P3 to phosphatidylinositol
(3,4)-diphosphate, an inactive state
(Stambolic et al., 1998;
Cantley and Neel, 1999).
Reportedly, PTEN-induced-down-regulation of Bcl-2 protein requires reduction
in serine/threonine kinase (Akt)/cAMP response element-binding protein
signaling (Huang et al.,
2001).
On the other hand, the existence of a mitochondrial ATP-sensitive potassium
channels (mitoKATP) that are reversibly inactivated by ATP and
inhibited by glibenclamide has been first demonstrated within the inner
mitochondrial membrane by Inoue et al.
(1991). Mitochondrial channels
have higher sensitivity to opening by diazoxide, which exceeds the sensitivity
of sarcolemmal channels by 2000-fold
(Garlid et al., 1996).
Glibenclamide, a potent and nonselective KATP channel blocker has
been demonstrated to inhibit the mitoKATP in the heart and brain
(Jaburek et al., 1998;
Bajgar et al., 2001).
5-Hydroxydecanoic acid (5-HD) blocks the mitoKATP reconstituted in
liposomes and isolated mitochondria, but it does not block cardiac type
sarcolemmal KATP channels
(Jaburek et al., 1998). There
is, however, little information on the relationships between
mitoKATP activation and regulation of PTEN phosphorylation in the
angiogenesis in relation with apoptosis.
In the preliminary study, KR-31372 showed very weak vasodilator action
despite having a benzopyran moiety in its structure, which is a striking
contrast to levcromakalim, and showed a glibenclamide-inhibitable opening of
KATP channel in isolated rat ventricular myocytes, suggestive of
the KATP channel opener. Recently, KR-31372 exerted an inhibitory
effect on the oxidized low-density lipoprotein-stimulated syntheses of
[3H]thymidine incorporation and migrations of the cultured rat
aortic smooth muscle cells (Kim et al.,
2000). The antiangiogenic effect of KR-31372 was first
demonstrated in rat sponge implant model, in that oral administration of
KR-31372 (1 mg/kg for 7 days) significantly suppressed the neovascularization
induced by angiotensin II and VEGF165
(Kim et al., 2001).
In the present study, we designed to clarify the signal transduction mechanism by which KR-31372 exerts an inhibitory action on the angiogenesis in relation with apoptosis in the human umbilical vein endothelial cells (HUVECs) under treatment with and without 5-HD, a mitoKATP blocker. The results were compared with those of diazoxide, a specific mitoKATP opener. We examined the effect of KR-31372 against VEGF165-induced increase in phosphorylated PTEN in the HUVECs and U87-MG cells, a PTEN-null glioblastoma cell line, transfected with expression vectors for sense PTEN in the absence and presence of 5-HD. To clarify whether KR-31372 increased the PTEN phosphorylation specifically in HUVECs, we used U-373 MG, a human brain glioblastoma cells for comparison with HUVECs.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Cell Proliferation Assay. HUVECs, seeded at a density of 1 x 104 cells/well in 96-well plates, were incubated in growth media and allowed to attach for 24 h. Thereafter, cells were incubated for 3 h in Kaighn's F-12K medium containing 1% fetal bovine serum. Cells were exposed to KR-31372 (10-8–10-4 M) or diazoxide (10-8–10-4 M) for 3 h, and then were stimulated by VEGF165 (10 ng/ml) for 48 h. After addition of 5-bromo-2'-deoxyuridine, cells were reincubated for 2 h for incorporation of 5-bromo-2'-deoxyuridine into the DNA of proliferating cells. The reaction product was quantified by measuring the absorbance at the 450 nm (reference wavelength 690 nm) using the ELISA reader (Bio-Tek Instruments, Winooski, VT).
Basal Tubular Formation Assays. The incubation of HUVECs were performed on 24-well plates that have been coated with 250 µl of growth factor-reduced Matrigel (10 mg of protein/ml) per well and polymerized for 30 min at 37°C. Cells were detached with 0.05% trypsin-EDTA solution, suspended in F-12K with 1% fetal bovine serum, and plated onto a layer of Matrigel at a density of 1 x 105 cells/well. 5-HD was given to the cells 30 min before and after seeding. After 18 h, the culture plate was photographed (200x). The picture of the tubules was scanned and network area was determined using the GS-710 calibrated imaging densitometer (Bio-Rad, Hercules, CA).
Matrigel Plug Assay in Mice. Mice (C57BL/6) were injected subcutaneously with 0.5 ml of Matrigel containing VEGF165 (5 ng/ml) with KR-31372 (10-6 M) or diazoxide (10-5 M). The injected Matrigel rapidly formed a single, solid gel plug. After 5 days, mice were sacrificed, and Matrigel plug was recovered, fixed with 10% formaldehyde/phosphate-buffered saline (pH 7.4), and embedded in paraffin and examined with hematoxylin and eosin stain. To quantify the formation of new blood vessel, the amount of hemoglobin was measured using the total hemoglobin kit (Sigma-Aldrich, St. Louis, MO). Four mice were used for one group, and the experiment was repeated twice.
DNA Fragmentation Assays. Cells were lysed in 1 ml of lysis buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1% sodium dodecyl sulfate, and 0.5 mg/ml proteinase K). Digestion was continued for 1 to 3 h at 55°C, followed by addition of RNase A to 0.1 mg/ml and running dye (10 mM EDTA, 0.25% bromophenol blue, and 50% glycerol). Equivalent amounts of DNA (15–20 µg) were loaded into wells of 1.6% agarose gel and electrophoresed in 0.5x TAE buffer (40 mM Tris-acetate and 1 mM EDTA) for 2 h at 6 V/cm. DNA was visualized by ethidium bromide staining. Gel pictures were taken by UV transillumination with the Polaroid camera. Bands were quantified by the Molecular Analyst software using the Bio-Rad's Image Analysis System (Bio-Rad).
Western Blot Analyses. Cells were incubated for 24 h in the presence of 10 ng/ml VEGF165. KR-31372 and diazoxide were applied 3 h, and 5-HD 30 min before experiment. For determination of Bcl-2, Bax protein, PTEN, phosphorylated PTEN, Akt, and phosphorylated Akt levels, cells were grown in 100-mm tissue culture dishes and treated with the indicated compounds. After washing, the cells were lysed in lysis buffer containing 50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 100 µg/ml phenylmethylsulflonyl fluoride, 1 µg/ml aprotinin, and 1% Triton X-100. After centrifugation at 12,000 rpm, 50 µg of total protein of each sample was loaded into 12% SDS-polyacrylamide gel electrophoresis gel, and transferred to nitrocellulose membrane (Amersham Biosciences, Inc., Piscataway, NJ). The blocked membranes were then incubated with the antibody of Bcl-2, Bax (Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), PTEN and phospho-PTEN (Ser380/Thr382/383), Akt, and phospho-Akt (Cell Signaling Technology, Inc., Beverly, MA).
Mitochondrial cytochrome c was prepared via following procedures. After washing cells (12 x 106) with ice-cold phosphate-buffered saline, cell pellets were suspended in buffer (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM Na-EDTA, 1 mM Na-EGTA, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride) containing 250 mM sucrose. The cells were homogenized and then centrifuged twice at 750g for 10 min at 4°C. The harvested supernatants were centrifuged at 10,000g for 10 min at 4°C, and the resulting mitochondrial pellets were dissolved in 1x SDS sample buffer. Western blots were performed with the antibody of cytochrome c (Santa Cruz Biotechnology, Inc.). The immunoreactive bands were visualized using chemiluminescent reagent of the Super-Signal west dura extended duration substrate kit (Pierce Chemical, Rockford, IL). The signals of the bands were quantified using the calibrated imaging densitometer (GS-710; Bio-Rad). The protein concentration of the lysate was determined using the Bio-Rad DC assay kit (Bio-Rad).
Plasmid Construction. The expression of plasmid encoding the human PTEN protein was cloned by reverse transcription-polymerase chain reaction using the total RNA of SK-N-SH cells. Sequence analysis was performed to confirm the nucleotide sequences. The following sequences of oligodeoxynucleotides were used as primers containing linker recognizable by XhoI as underlined: 5'-GCGCTCGAGATGACAGCCATCAAAG-3'. Amplified 1264-bp fragments containing the human PTEN coding region were ligated into the XhoI site of pcDNA3.1 HisC (Invitrogen, Carlsbad, CA). pcDNA3.1-sPTEN is transcripted sense nucleotides.
DNA Transfection. U87-MG cells were seeded for 24 h before transfection in tissue culture dishes. At 50 to 70% confluence, the dishes were washed twice with Opti-MEM medium to remove the fetal bovine serum and a transfection cocktail containing 10 µg DNA and 10 µl of LipofectAMINE reagent (Invitrogen) per 100-mm dish was added. The medium was removed and then 7 ml of MEM medium containing 10% fetal bovine serum was added to each dish.
Drugs. VEGF165 was purchased from the R & D Systems (Minneapolis, MN). (2R,3R,4S)-N''-Cyano-N-(6-nitro-3,4-dihydro-hydroxy-2-methyl-2-dimethoxymethyl-2H-1-benzopyran-4yl)-N'-benzyl guanidine (KR-31372) was donated from The Korea Research Institute of Chemical Technology (Daejon, Korea). 5-Hydroxydecanoic acid (sodium salt), endothelial cell growth supplement, and total hemoglobin kit were purchased from the Sigma-Aldrich (St. Louis, MO). 5-Bromo-2'-deoxyuridine kit was from the Roche Diagnostics (Mannheim, Germany). Matrigel was from the BD Biosciences Discovery Labware (Bedford, MA). KR-31372 and diazoxide were dissolved in dimethyl sulfoxide as a 10-1 M stock solution. 5-Hydroxydecanoic acid was dissolved in distilled water as a 10-1 M stock solution.
Statistics. The results are expressed as means ± S.E.M. Two-way repeated measures analysis of variance was used for the comparison of concentration-dependent changes in cell proliferation in response to agonists between inhibitor-treated and untreated groups. Statistical differences between groups were determined by paired or unpaired Student's t test or analysis of variance. P < 0.05 was considered significant.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Matrigel Plug Assay in Mice. Five days after implantation, histological examination and hemoglobin content were estimated. As shown in Fig. 1, C and D, the neomicrovessel formation significantly increased in the Matrigel containing VEGF165 (5 ng/ml), whereas it was not evident in the Matrigel without VEGF165. Matrigel containing either KR-31372 (10-6 M) or diazoxide (10-5 M) significantly reduced the formation of VEGF165-stimulated neomicrovessels, which was antagonized by 5-HD (10-5 M). The neovascular formation was further confirmed by measurement of the hemoglobin content in the Matrigel. The hemoglobin content was elevated in the Matrigel containing 5 ng/ml VEGF165 to 5.2 ± 0.6 g/dl (P < 0.001), which was markedly suppressed by treatment with 10-6 M KR-31372 (1.6 ± 0.3 g/dl, P < 0.001) and 10-5 M diazoxide (2.2 ± 0.4 g/dl, P < 0.05). Pretreatment with 5-HD (10-5 M) significantly reversed both KR-31372- and diazoxide-induced reduction in hemoglobin content (Fig. 1D), indicating that KR-31372 as well as diazoxide elicit antiangiogenic activities in the in vivo experiment.
Cell Proliferation. VEGF165 (1–20 ng/ml) concentration dependently increased DNA synthesis. After 48-h incubation, 10 ng/ml VEGF165 increased DNA synthesis to 185.3 ± 7.7% of control cells (Fig. 2, inset). Cell proliferation was concentration dependently suppressed by simultaneous incubation with either KR-31372 or diazoxide (10-8–10-4 M, each), respectively. 5-HD (10-5 M), when applied 30 min before KR-31372 or diazoxide treatment, significantly reversed the suppressed DNA synthesis induced by either KR-31372 or diazoxide (Fig. 2). After application of 5-HD (10-5 M) in the absence of VEGF165, the cells showed little effect on the proliferation of HUVECs (102.9 ± 3.7%).
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Apoptotic Effect. Effect of KR-31372 was compared with diazoxide on the laddered feature of DNA fragmentation, which was pretreated with 10 ng/ml VEGF165 in the HUVECs. Exposure of HUVECs to KR-31372 (10-5 and 10-4 M) and diazoxide (10-5 M) induced prominent oligonucleosomal DNA fragmentation. Pretreatment with 5-HD (10-5 M) strongly suppressed the DNA laddering induced by KR-31372 (10-5 M) and diazoxide (10-5 M), respectively (Fig. 3).
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On the other hand, the effect of KR-31372 on the DNA fragmentation was identified in the U-373MG cells, naive U87-MG cells, and U87-MG cells of sPTEN. The cells were exposed to KR-31372 (10-6–10-4 M) for 3 h in the presence of VEGF165 (10 ng/ml). KR-31372 (10-6–10-4 M) induced prominent oligonucleosomal DNA fragmentation in the U-373MG and U87-MG cells of sPTEN, but did not show DNA fragmentation in U87-MG lacking a wild-type PTEN (Fig. 4).
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PTEN and Akt Phosphorylation. PTEN phosphorylation was concentration dependently increased by KR-31372 [1.82 ± 0.14-fold (P < 0.05) by 10-6 M and 2.78 ± 0.31-fold (P < 0.01) by 10-5 M KR-31372] when applied 3 h before VEGF165 (10 ng/ml) in the HUVECs. Pretreatment with 5-HD (10-5 M, P < 0.01) significantly suppressed the increased PTEN phosphorylation induced by KR-31372 (10-5 M) to 0.88 ± 0.09-fold (P < 0.01). Diazoxide (10-5 M) and 5-HD (10-5 M) showed similar interactions as shown with KR-31372 and 5-HD (Fig. 5A). The similar results were also evident in the U-373MG cells, human brain glioblastoma cells (Fig. 5B). However, the PTEN protein expression was not altered by these agents.
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Otherwise, PTEN protein was expressed in the HUVECs and U-373MG cells, but not in the naive U87-MG cells, whereas the Akt expression was well identified in the three cells. Introduction of PTEN cDNA (sense oligodeoxynucleotide) into the U87-MG cells, lacking a wild-type PTEN, caused a large increase in PTEN expression (Fig. 6A). In the U87-MG cells of sPTEN, both KR-31372 and diazoxide significantly increased the PTEN phosphorylation as shown in the HUVECs and U-373MG cells, and the phosphorylated Akt levels were, in contrast, significantly decreased by these agonists (Fig. 6B). The alterations induced by KR-31372 and diazoxide were well antagonized by 10-5 M of 5-HD (Fig. 6, C and D). The levels of Akt and Akt phosphorylation were not altered by KR-31372 (10-6–10-4 M) and diazoxide (10-5 M) in the U87-MG cells lacking a wild-type PTEN (data not shown).
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Western Blot for Bcl-2, Bax, and Cytochrome c. Under treatment with VEGF165 (10 ng/ml for 24 h), the Bcl-2 protein expression was markedly increased to 2.60 ± 0.54 relative density, which was strongly suppressed by KR-31372 (10-6–10-4 M) in a concentration-dependent manner (Fig. 7A). Diazoxide (10-5 M) also significantly decreased the Bcl-2 protein level. In the presence of 5-HD (10-5 M), both KR-31372- and diazoxide-induced inhibitions of Bcl-2 protein levels were significantly reversed (Fig. 7B).
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In the presence of VEGF165 (10 ng/ml), the basal levels of Bax protein and cytochrome c release from mitochondria were 0.77 ± 0.06 and 0.97 ± 0.08 relative densities, respectively. In contrast, both were largely elevated by pretreatment with KR-31372 (10-6, 10-5, and 10-4 M) and diazoxide (10-5 M). 5-HD (10-5 M) significantly suppressed the increased Bax protein and cytochrome c release induced by either KR-31372 or diazoxide (Fig. 8, A and B).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The angiogenesis requires the triad processes: endothelial cell
proliferation, migration, and local protease activity such as matrix
metalloproteinases (Hanahan and Folkman,
1996). Overexpression of VEGF and its receptors is associated with
chronic inflammation, tumor growth, and diabetic retinopathy
(Williams, 1998). Of the
various VEGF species, VEGF165 is well characterized
(Neufeld et al., 1999), and
the expression of cell surface receptors for VEGF165 was
demonstrated in the endothelial cells
(Millauer et al., 1993).
Binding of VEGF to VEGF receptor-2 leads to the receptor phosphorylation and
subsequent activation of PI3-K, phospholipase C-
1, and Src family
tyrosine kinases (Thakker et al.,
1999).
PTEN was originally identified as a tumor suppressor gene based on its high
frequency of mutation in a variety of tumors
(Li et al., 1997).
3-Phosphoinositides are important substrates for PTEN both in vitro
(Maehama and Dixon, 1998) and
in vivo experiments (Sun et al.,
1999). PTEN potently modulates VEGF-mediated signaling and
function. Most recently, Huang and Kontos
(2002) have provided evidence
that adenovirus-mediated overexpression of a dominant negative PTEN mutant
enhances VEGF-mediated endothelial cell proliferation and migration, whereas
overexpression of wildtype PTEN, in contrast, shows inhibition of cell
proliferation and chemotactic effects of VEGF. Our data showed for the first
time that both KR-31372 and diazoxide exerted concentration-dependent increase
in PTEN phosphorylation in the HUVECs, which was suppressed by 5-HD
(10-5 M, P < 0.01). PTEN has the property to
restrain the PI3-K pathway by catalyzing degradation of the
PI(3,4,5)P3, which is generated by PI3-K
(Stambolic et al., 1998;
Cantley and Neel, 1999;
Huang et al., 2001). It is
known that overexpression of PI3-K and its downstream effector Akt mediate
growth factor-induced neuronal survival, and Akt in turn up-regulates Bcl-2
promoter activity in association with Bcl-2 protein expression through
enhanced cAMP response element-binding protein activation
(Walton et al., 1999;
Pugazhenthi et al., 2000).
Therefore, it is speculated that activation of PTEN in the HUVECs may provide
a signaling event that may induce a decrease in phosphorylated Akt and Bcl-2
expression followed by increased Bax protein and cytochrome c release
from mitochondria, thereby enhancing cell apoptosis.
In our results, when the U87-MG cells, a glioblastoma cell line that lacks
expression of wild type PTEN (Haas-Kogan
et al., 1998), were transfected with expression vectors for sense
PTEN, they exerted increased PTEN protein expression. These transfected cells
also showed high phosphorylated PTEN accompanied by decreased Akt
phosphorylation under treatment with KR-31372 and diazoxide, which was fully
reversed by mitoKATP blocker 5-HD. Based on these facts, it is
likely that KR-31372 has a role for regulation of PTEN phosphorylation as a
mitoKATP opener.
In the previous results, glibenclamide reversed the KR-31372-induced
suppression of the VEGF165-stimulated chemotactic motility of
HUVECs (Kim et al., 2003). At
that time, they did not identify whether the action site of glibenclamide is
on the sarcolemmal KATP channels or the mitoKATP
channels. In the light of the present study, it seemed that the action of
glibenclamide was ascribed to the inhibition of the mitoKATP
(Jaburek et al., 1998;
Bajgar et al., 2001). Tamura et
al. (1998) have emphasized the
importance of PTEN in the cell migration, in that overexpression of PTEN
dephosphorylates FAK in vivo and in vitro, and reduces its tyrosine
phosphorylation and inhibits integrin-mediated cell spreading, whereas
antisense PTEN enhanced migration. Most recently, Kim et al.
(2003) have demonstrated that
KR-31372 significantly inhibited the KDR/Flk-1 tyrosine phosphorylation-linked
extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein
kinase, and p125FAK tyrosine phosphorylation, which were inhibited
by glibenclamide, a KATP channel blocker. Considering that KR-31372
significantly suppressed the VEGF-stimulated triad of angiogenesis (DNA
synthesis, migration, and MMP-2 release; data not shown) in HUVECs, it is
likely predictable that KR-31372 might suppress the tube formation in HUVECs
and Matrigel-induced neovascularization in mice via mediation of the
mitoKATP opening, thereby leading to the antiangiogenesis.
Accumulating reports have shown that the mitoKATP is the
receptor for cardioprotection by KATP channel openers and for 5-HD
blockade and that 5-HD inhibits the mitoKATP, but not the
sarcolemmal KATP channel
(McCullough et al., 1991;
Grover and Garlid, 2000).
Garlid et al. (1996,
1997) showed that diazoxide was
1000 to 2000 times more potent in opening mitoKATP than in opening
sarcolemmal KATP channels. Thus, it is intriguing that the
mitoKATP openers, including diazoxide and KR-31372, increased the
expression of PTEN phosphorylation and induced 5-HD-inhibitable apoptosis in
the HUVECs, despite a number of studies demonstrating a cardioprotection by
diazoxide and cromakalim as openers of mitoKATP
(Grover and Garlid, 2000).
Currently, it is not possible to reconcile the disparate results of diazoxide
and KR-31372 between cardiac myocytes and HUVECs. In contrast to other cells,
the endothelial cells possessed the unique properties. The electrochemical
gradient or driving force for Ca2+ entry into
endothelial cells is characteristically influenced by the membrane potential
(Lückhoff and Busse,
1990), such that depolarization decreases, whereas
hyperpolarization increases, Ca2+ entry. Resting
membrane potentials in endothelial cells are thought to be controlled
primarily by K+ channels; in particular, inwardly rectifying
K+ channels (Nilius et al.,
1997).
Although the data are not shown, mitochondrial membrane potential and intramitochondrial calcium levels were decreased by both KR-31372 and diazoxide in a concentration-dependent manner. These values were significantly reversed by treatment with 5-HD. Moreover, these reductions were accompanied by increase in Bax protein and increased cytochrome c release, indicating that KR-31372 and diazoxide augmented the apoptotic action via mitoKATP opening. Presently, it remains to be clarified how the mitoKATP opening triggers to increase the PTEN phosphorylation.
Apoptosis of endothelial cells occurs during the vascular regression in the
process of scar formation (Desmouliere et
al., 1995), atherosclerosis
(Dimmeler et al., 1998), and
progressive glomerulonephritis (Shimizu et
al., 1997). The expression of Bcl-2 protein in the mitochondrial
outer membrane prevents the association of the proapoptotic Bax protein with
permeability transition pore and its pore forming activity, and then acts to
inhibit cytochrome c release from mitochondria to cytosol
(Kluck et al., 1997;
Shimizu and Tsujimoto, 2000).
The consequence of mitoKATP activation in the isolated rat heart
mitochondria was emphasized by Holmuhamedov et al.
(1998), in that
KATP channel openers (pinacidil, cromakalim, and levcromakalim)
induced mitochondrial membrane depolarization with an increase in the rate of
mitochondrial respiration and consequently a decrease in ATP synthesis, these
cascades resulting in enhanced release of cytochrome c from
mitochondria.
Together, these findings provide a strong evidence to support that the proapoptotic effect of KR-31372 in the HUVECs is closely linked to 5-HD-sensitive mitoKATP opening and the up-regulation of PTEN phosphorylation and down-regulation of Akt phosphorylation and Bcl-2 protein expression, thereby leading to antiangiogenesis in the HUVECs.
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Acknowledgements |
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Footnotes |
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This study was supported by funds from the Critical National Technology Program of the Korea Science and Engineering Foundation and Research Institute of Genetic Engineering, Pusan National University.
ABBREVIATIONS:VEGF, vascular endothelial growth factor; PTEN, phosphatase and tensin homolog deleted from chromosome 10; Akt, serine/threonine kinase; PI3-K, phosphatidylinositol 3-kinase; PI(3,4,5)P3, phosphatidylinositol (3,4,5)-triphosphate; mitoKATP, mitochondrial ATP-sensitive potassium channel; KATP, ATP-sensitive potassium channel; 5-HD, 5-hydroxydecanoic acid; HUVEC, human umbilical vein endothelial cell; MEM, minimal essential medium; bp, base pair(s); sPTEN, sense phosphatase and tensin homolog deleted from chromosome 10.
Address correspondence to: Dr. Ki Whan Hong, Department of Pharmacology, College of Medicine, Pusan National University, 10 Ami-Dong, 1-Ga, Seo-Gu Busan 602-739, Korea. E-mail: kwhong{at}pusan.ac.kr
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, P < 0.05
versus KR-31372 or diazoxide alone.
