Department of Pathology, Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada N6A 5C1
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ABSTRACT |
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Vascular endothelial cells are constantly exposed to oxidative stress and must be protected by physiological responses. In diabetes mellitus, endothelial cell permeability is impaired and may be increased by high extracellular glucose concentrations. It has been postulated that metallothionein (MT) can protect endothelial cells from oxidative stress with its increased expression by cytokines, thrombin, and endothelin (ET)-1. In this study, we demonstrate that high glucose concentration can induce MT expression in endothelial cells through a distinct ET-dependent pathway. Exposure of human umbilical vein endothelial cells (HUVEC) to increasing concentrations of glucose resulted in a rapid dose-dependent increase in MT-2 and ET-1 mRNA expression. MT expression may be further augmented with addition of ET-1. Preincubation of the cells with the specific ETB antagonist BQ-788 blocked MT-2 mRNA expression more effectively than the ETA inhibitor TBC-11251. High glucose also increased immunoreactive MT protein expression and induced translocation of MT into the perinuclear area. Perinuclear localization of MT was related to high-glucose-induced reorganization of F-actin filaments. These results demonstrate that an increase in extracellular glucose in HUVEC can lead to a rapid dose-dependent increase in MT-2 mRNA expression and to perinuclear localization of MT protein with changes to the cytoskeleton. These effects are mediated via the ET receptor-dependent pathway.
human umbilical vein endothelial cells; TBC-11251; BQ-788
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INTRODUCTION |
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ONE OF THE MAJOR PROBLEMS in diabetes mellitus is the disruption of glucose homeostasis, which is normally maintained by a balance of hepatic glucose production, and cellular glucose uptake and metabolism (8). Sustained hyperglycemia in diabetes may alter expression of vasoactive substances in the target organs of diabetic complications through a variety of mechanisms (16).
It has been well established that the endothelium plays a vital role in the regulation of reactivity of vascular tissues by releasing the endothelium-derived factors that act on adjacent smooth muscle cells (40). One such factor produced by vascular endothelial cells is the potent vasoconstrictive peptide endothelin (ET)-1, which is regulated by a number of inflammatory and vasoactive mediators (13, 26, 29). Regulation of ET-1 secretion by these mediators is critical in the maintenance of vascular tone in certain pathophysiological conditions. Recently, several reports have shown that insulin stimulates ET-1 secretion from endothelial cells and also enhances ET-1 binding to its receptors (35). It also has been shown that plasma ET-1 levels are elevated in patients with type II diabetes mellitus with microvascular complications, suggesting that ET-1 may be involved in diabetes-related complications such as microangiopathy (24).
The 21-amino acid ET-1 peptide is derived by proteolytic processing of prepro-ET-1, a 212-amino acid precursor protein (46). ETs are a family of three oligopeptides that includes ET-1, ET-2, and ET-3 (17). These peptides interact with a group of specific receptors such as ETA, ETB, and ETC (28, 41). The mechanism of ET expression in both constitutive and induced conditions is important for elucidating the role of ET in various diseases. Changes in ET-1 expression may affect blood flow, extracellular matrix protein synthesis, and expression of other molecules of physiological importance (32).
It has been suggested (21) that ET-1 may modulate the expression of metallothionein (MT), a zinc- and copper-binding protein (19). MT genes are expressed in most tissues and organisms, and the transcription of these genes is regulated by metals, growth factors, glucocorticoids, cytokines, and stress conditions (10, 22, 23). In experimental diabetes mellitus, increased pancreatic and hepatic MT expression has been demonstrated (6, 11, 47, 50). However, the role of MT and zinc in diabetes is not well understood. Increased expression of MT in response to cadmium, cytokines, thrombin, and ET-1 has been reported in cultured vascular endothelial cells (20, 21) and bovine aortic endothelial cells (37). Moreover, there are reports that MT can antagonize ET-1-induced vasoconstriction and lower the release of ET, stimulated by angiotensin II during limb ischemia (36). In addition, the protective effect of MT on changes in membrane protein structure caused by ET has also been demonstrated in erythrocyte ghost membranes (42).
This study was designed to investigate the mechanisms of MT induction in human umbilical vein endothelial cells (HUVEC) after exposure to high glucose levels. We have investigated the role of ET-dependent pathways in MT expression. The results suggest that MT gene expression after incubation with high glucose levels is partially mediated via an ET receptor-dependent pathway.
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MATERIALS AND METHODS |
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Cell culture. HUVEC were obtained from Bio-Whittaker (San Diego, CA) and plated in complete endothelial growth medium (catalog no. CC-3024; Bio-Whittaker). They were routinely found to be free of mycoplasma. All cells used for the following investigations were at low passage number (passage 11).
Cell treatment.
Cells were plated at a density of 1 × 105 cells/ml.
They were treated with glucose or 2-deoxy-D-glucose (2-DG;
Sigma, Ontario, ON, Canada) in a concentration range from 5 to 100 mmol/l and with ETA [TBC-11251;
4-chloro-3-methyl-5-(2-(2-(6-methylbenzo[d][1,3]dioxol-5-yl) acetyl)-3-thienylsulfonamido)isoxazole;
Texas Biotechnology, Houston, TX; courtesy of Dr. R. Tilton] and
ETB antagonists (BQ-788;
d-cis-2,6-dimethylpiperidinocarbonyl-L--methylleucyl-D-1-methoxycarbonyltrptophanyl-D-Nle; American Peptide, Sunnyvale, CA) in a concentration range up to 10 µmol/l.
Assessment of cell viability. Alamar blue was used as an indicator to assess cellular metabolic activity (4, 33). The procedure was done in accordance with the manufacturer's instructions (MediCorp, Montreal, PQ, Canada).
Immunohistochemistry. Cells were incubated with either a monoclonal antibody for MT (E9; Dako Diagnostics, Mississauga, ON, Canada) or a polyclonal rabbit antibody for MT or preimmune rabbit serum. This polyclonal antibody was prepared in our laboratory against polymerized rat liver MT-2, and its cross-reactivity with both MT-1 and -2 isoforms was demonstrated previously (5). Immunohistochemical labeling was performed as described by Apostolova et al. (2).
HUVEC were stained for F-actin according to the method described by Wang et al. (43a). F-actin was detected using Alexa Fluor 568 phalloidin (Molecular Probes, Eugene, OR). Fluorescence microscopy was performed with a Zeiss LSM 410 inverted laser scan microscope equipped with fluorescein, rhodamine, and 4,6-diamidino-2-phenylindole filters.Isolation of RNA and reverse transcription. Total RNA was extracted from cells with Trizol reagent (Life Technologies, Burlington, ON, Canada) according to the methods described by Deng et al. (9).
MT protein estimation with Western blot. The cellular proteins were resolved in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and subjected to Western blot analysis (14) with MT monoclonal antibody. The signals from Western blot were obtained with the use of commercially available horseradish peroxidase-conjugated secondary anti-mouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and developed with chemiluminescent substrate (Amersham Pharmacia Biotechnology, Amersham, UK).
Preparation of oligonucleotide primers and PCR.
Oligonucleotide primers for human MT-2, ET-1, and -actin were
obtained from Life Technologies. MT-2 primer sequences were as follows:
5'-CTCTTCAGCACGCCATGGAT-3' (sense) and 5'-CGCGTTCTTTACATCTGGGA-3' (antisense). The ET-1 primer sequences were as follows:
5'-GGACATCATTTGGGTCAACACTCC-3'(sense) and
5'-CAAGCTTGGAACAGTCTTTTCCT-3' (antisense). The sequences of rat
-actin primers were as follows: 5'-CCTCTATGCCAACACAGTGC-3' (sense) and 5'-CATCGTACTCCTGCTTGCTG-3' (antisense). The predicted sizes
of the amplified products (cDNA) were 203 bp for MT-2, 269 bp for ET-1,
and 210 bp for
-actin, respectively.
Data analysis. Statistical evaluation of data was performed by one-way analysis of variance with Origin 5.0 (Microcal Software, Northampton, MA) using Bonferroni's multiple comparison, and results were considered statistically significant if P < 0.05. The results are presented as means ± SE.
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RESULTS |
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Cell viability and proliferation.
When the HUVEC were grown in increasing concentrations of either
glucose or 2-DG (25, 50, and 100 mmol/l), there was an increase in cell
growth for 64 h in the cells treated with glucose only (Fig.
1). At 72 and 96 h, there was no
change in cell numbers for cells grown in glucose, but there was a
decrease in cell viability for cells grown in 2-DG as shown in Fig. 1,
A and B.
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Effects of high concentration of glucose on MT-2 mRNA.
The changes in MT-2 mRNA levels in HUVEC with increasing concentration
of glucose or 2-DG (5-100 mmol/l) are shown in Fig. 2, A and B. There
was a dose-dependent increase in MT-2 mRNA after exposure to glucose
(25-100 mmol/l) as determined by RT-PCR. The increase was
~2-fold with 25 mmol/l glucose and >10-fold with 100 mmol/l glucose
at 64 h. No such increase in MT-2 mRNA was observed after exposure
to 2-DG (Fig. 2B).
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Effect of high concentrations of glucose on ET-1 mRNA level.
Incubation of HUVEC with increasing concentrations of glucose
(5-100 mmol/l) resulted in a dose-dependent increase in ET-1 mRNA
(Fig. 3). The mRNA transcript analyzed by
RT-PCR showed a maximum increase after treatment with 25 mmol/l glucose
for 64 h. This increase was about twofold compared with the ET-1
mRNA level for 5 mmol/l glucose. No such increase was observed when cells were incubated with 25 mmol/l 2-DG (data not shown).
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Role of ET-1 in MT expression.
To investigate the direct role of ET-1 in MT expression, we
examined the expression of MT-2 mRNA after exposure to 25 mmol/l glucose in the presence or absence of ET-1. The results
showed that addition of ET-1 to cells grown in 5 mmol/l glucose can
induce the MT-2 mRNA expression in a concentration-dependent manner
(Fig. 4A). An additive effect
of MT-2 mRNA expression was observed when the HUVEC were treated with
both ET-1 (0.25-0.75 µmol/l) and 25 mmol/l glucose (Fig.
4B). When HUVEC treated with 25 mmol/l glucose were
coincubated with TBC-11251, a specific antagonist of the ETA receptor, at three different concentrations (0.5, 1, and 10 µmol/l), a partial block in glucose-induced MT -2 mRNA
expression was observed (Fig. 4C). At concentrations >1
µmol/l, this antagonist can block both ETA and
ETB receptors (44). To distinguish the subtype
of ET receptors involved in MT-2 mRNA expression with high glucose, we
treated the HUVEC grown in 25 mmol/l glucose with 250 nmol/l BQ-788, a
specific inhibitor of ETB receptor. This treatment also
decreased the MT mRNA expression (Fig. 4D) similarly to that
observed with 10 µmol/l TBC-11251.
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Effect on MT protein induction.
The results in MT mRNA expression are supported by changes in MT
protein levels as shown by Western blot analysis with a monoclonal MT-2
antibody (Fig. 5). The basal level of MT
in HUVEC grown in 5 mmol/l glucose was increased more than threefold
after addition of ET-1 (0.5 µmol/l) or by growing the cells in 25 mmol/l glucose. This increase in MT protein was blocked after addition
of both inhibitors (10 µmol/l TBC-11251 and 250 nmol/l BQ-788) of ET
receptors. These results demonstrate that both MT mRNA and protein
expressions are increased with high glucose and ET but can be blocked
by inhibition of ET receptors.
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Effects of high glucose and ET-1 on subcellular localization of MT.
Immunofluorescence confocal microscopy showed that HUVEC cultured in
normal medium with 5 mmol/l glucose or 2-DG had immunoreactive MT
randomly distributed in the cytoplasm (Fig.
6A). The increase in MT
immunoreactivity with high glucose was concentration dependent. The
cells treated with 25 and 50 mmol/l glucose exhibited a higher intensity of immunofluorescence for MT (green) in the perinuclear area
(Fig. 6, B and C), indicating a translocation of
MT to the perinuclear area.
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Effect of high glucose on actin cytoskeleton organization.
At glucose concentrations of 5 mmol/l, HUVEC showed the typical normal
distribution of actin filaments with most of the actin filaments
arranged along the periphery of the cells and only a few filaments in
the middle (Figs. 6 and 9A).
High glucose caused major changes in the microfilament arrangement that
were characterized by reorganization of F-actin from
cortical microfilaments into transcytoplasmic stress fibers (Fig. 9,
B and C). The stress fiber formation upon
exposure to high glucose concentration was associated with the
relocalization of MT in perinuclear area (Fig. 6, B and C). Treatment of the cells with 2-DG had no effect on either
loss of membrane protrusions or distribution of F-actin and
localization of MT (Figs. 6 and 9, D-F).
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DISCUSSION |
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The results reported in this study demonstrate a novel mechanism of induction of MT, an intracellular zinc-binding protein, that can regulate the bioavailability of zinc and may influence the redox status of the cell (1). The synthesis of MT can be induced by various stress conditions, and these proteins are also considered acute phase proteins (18). In this study, we have examined the expression of MT-2 mRNA, MT-2 protein, and the subcellular localization of MT in HUVEC after incubation with high glucose (>5 mmol/l). The results demonstrate an increased expression of MT and its translocation to the perinuclear area when HUVEC are treated with high glucose. Furthermore, these changes are probably mediated by an ET receptor-dependent pathway, since they can be prevented by ET receptor blockade. The study also shows that the induced synthesis of MT with high glucose concentrations is under transcriptional control.
In cells, several biochemical changes can occur secondarily to hyperglycemia, including augmented polyol-myoinositol-related metabolic defects, nonenzymatic glycation, augmented protein kinase C (PKC) activity, and altered redox potential (25, 27). The PKC activation may affect the expression of several vasoactive factors such as ET-1 and vascular endothelial growth factor (9, 25). Endothelial damage and altered endothelium-derived factors can induce vascular complications in diabetes (27). The changes in ET-1 expression have been demonstrated in both kidneys and heart (32). Increased ET-1 expression can have several effects, including increased extracellular matrix protein gene expression. The induction of MT in HUVEC may be one of these effects.
In this study, we also found an additive effect on MT-2 mRNA and protein expression when the HUVEC were incubated with increasing concentrations of ET-1 in 25 mmol/l glucose. Because ET-1 can stimulate glucose uptake in different cell lines (38, 45), the additive effect on MT expression could be related to an increase in glucose uptake by HUVEC.
The increased MT-2 mRNA expression that results from incubation with high glucose concentrations appears to be mediated by the ETA and ETB receptor-dependent pathway. Blocking of either ETA or ETB receptors can inhibit the increase in MT-2 mRNA and protein levels (Figs. 4 and 5). This effect is probably mediated through the ETB receptor. The results show that BQ-788 is 40 times more effective than TBC-11251 in reducing glucose-induced MT-2 expression. Incubation of cells with 2-DG did not cause any changes in MT-2 expression. These results demonstrate conclusively that the induction of MT in HUVEC by high glucose is directly related to ET and its receptors.
The exact role of increased synthesis of MT or its nuclear and perinuclear localization after exposure to high glucose is not yet understood. However, these changes may be related to the observed cytoskeleton activation and reorganization (Figs. 5 and 8). Alterations in F-actin filament assembly have been observed in cultured 3T3-L1 mesangial cells and HUVEC in response to increased glucose levels (7, 43, 49). Thus the changes in MT localization in the perinuclear area can be related to decreased resistance to shear force in diabetic endothelial cells. The alterations in cell shape and permeability also occur with changes in intracellular actin pattern (43a, 49). All these metabolic changes may result in increased endothelial cell permeability, which is characteristic of diabetes mellitus (7).
However, it is unclear whether the changes in MT synthesis and localization can alter either intracellular or membrane zinc levels, which can affect membrane properties. It has already been shown that zinc can protect free radical-induced apoptosis in endothelial cells (15, 30). The induction of MT synthesis, observed in our present study, can be considered as a stress response to protect the endothelial cells from high-glucose exposure, and it is mediated by ET-1.
There are reports on endothelial dysfunction and the impairment of nitric oxide (NO) release by endothelial cells in diabetes mellitus (12). A reduction in NO-dependent vasodilation of mesenteric arteries has been demonstrated in diabetic rats (39). Studies have shown that NO production can modulate MT expression in mouse liver and brain under both normal and stress conditions (31). MT can directly react with NO, resulting in release of zinc, and may regulate NO signaling in endothelial cells (3, 34). Hence, there may be a feedback regulatory mechanism among ET-1, NO, and MT.
In summary, we demonstrate a novel mechanism on induction of MT synthesis involving ET receptors in endothelial cells when exposed to high glucose concentrations. However, further studies are required to understand the physiological significance of these metabolic changes in these cells.
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ACKNOWLEDGEMENTS |
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This study was supported by research grants from the Medical Research Council, Canada, and the Canadian Diabetes Association.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. G. Cherian, Dept. of Pathology, Faculty of Medicine and Dentistry, Univ. of Western Ontario, London, Ontario, Canada N6A 5C1 (E-mail: mcherian{at}uwo.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4 December 2000; accepted in final form 10 April 2001.
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