1 Department of Medicine, Kolling Institute: Renal Research Group, and 2 Department of Immunology, Royal North Shore Hospital, St Leonards, 2065; and 3 Department of Physiology, University of Sydney, New South Wales 2006, Australia
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ABSTRACT |
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Human endothelial cells were
exposed to 5 mM glucose (control), 25 mM (high) glucose, or osmotic
control for 72 h. TGF-1 production, cell growth, death, and
cell cycle progression, and the effects of TGF-
1 and TGF-
neutralization on these parameters were studied. High glucose and
hyperosmolarity increased endothelial TGF-
1 secretion
(P < 0.0001) and bioactivity (P < 0.0001). However, high glucose had a greater effect on reducing
endothelial cell number (P < 0.001) and increasing
cellular protein content (P < 0.001) than the osmotic
control. TGF-
antibody only reversed the antiproliferative and
hypertrophic effects of high glucose. High glucose altered cell cycle
progression and cyclin-dependent kinase inhibitor expression
independently of hyperosmolarity. High glucose increased endothelial
cell apoptosis (P < 0.01), whereas
hyperosmolarity induced endothelial cell necrosis (P < 0.001). TGF-
antibody did not reverse the apoptotic effects
observed with high glucose. Exogenous TGF-
1 mimicked the increased S
phase delay but not endoreduplication observed with high glucose. High glucose altered endothelial cell growth, apoptosis, and cell
cycle progression. These growth effects occurred principally via a
TGF-
1 autocrine pathway. In contrast, apoptosis and
endoreduplication occurred independently of this cytokine and hyperosmolarity.
endothelial cells; diabetes mellitus; apoptosis; endoreduplication; transforming growth factor-1
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INTRODUCTION |
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DIABETES MELLITUS contributes significantly to the morbidity and mortality within the developed world largely as a consequence of its effects on both the micro- and macrovasculature. A number of the complications of patients with diabetes are manifestations of aberrant cell growth such as the renal hypertrophy of early diabetes, the impaired wound healing from delayed capillary morphogenesis, and proliferative diabetic retinopathy. Inhibition of endothelial cell proliferation by high glucose has been documented in a number of in vivo and in vitro studies; however, the mechanisms by which these growth patterns occur has not been fully delineated (24, 42, 43).
Previous studies have demonstrated that long-term exposure (7-14
days) to high glucose leads to a reduction in endothelial cell
proliferation and a concomitant increase in apoptosis (2, 8, 9, 13, 19, 25, 26, 37). Although the precise mechanisms that
mediate these effects remain unclear, two lines of evidence suggest
that TGF-1 may mediate the effects of high glucose: 1)
TGF-
1 has been shown to induce changes in the growth of endothelial
cells similar to those observed after exposure of endothelial cells to
high glucose (30, 31, 45, 52), and 2) it has
been demonstrated in several cell types that exposure to high glucose
is a potent inducer of TGF-
1 production (21, 38).
Furthermore, increased levels of TGF-
1 are observed in both clinical
and experimental models of diabetes (39, 41), and as a
result, the deleterious effects of high glucose are attributed primarily to the autocrine action of TGF-
1. In mesangial cells, TGF-
1 has been demonstrated to induce its growth effects by causing arrest in the cell cycle by increasing the expression of the
cyclin-dependent kinase inhibitors (CDKIs) p21Cip1 and
p27Kip1 (39, 40). There is, however, little
evidence establishing a direct link between the effects of high glucose
on growth, cell cycle progression, and apoptosis and the
autocrine actions of TGF-
1 in endothelial cells. It remains
contentious whether exposure to high glucose does induce TGF-
1
production by endothelial cells, with one study showing an increase in
TGF-
1 production (34) and the other study failing to
show such an increase (5).
The role that the increase in osmolarity associated with high glucose
may play on autocrine TGF-1 secretion has not been fully defined. If
autocrine TGF-
1 does mediate endothelial cell growth effects induced
after high glucose exposure, the relative role of hyperosmolarity plays
needs to be elucidated. In murine mesangial cells, exposure to high
glucose increased TGF-
1 bioactivity and protein production, and this
was independent of any associated increase in osmolarity
(15). However, the effects of the increased osmolarity of
high glucose on endothelial cell TGF-
1 secretion, and hence
TGF-
1-mediated growth, has not previously been investigated.
Several studies demonstrate that high glucose induces hypertrophy in a
number of cell types, but whether endothelial cell hypertrophy occurs
under these conditions has not been previously demonstrated.
Furthermore, while others have shown similarities in cell growth
responses as a consequence of the minor increase in osmolarity with
high glucose and its effect on Na+/H+
antiporter activity (1), no previous study has dissociated the glucose specific growth effects from either those induced by the
increase in osmolarity itself or the changes in cytokine profile that
this minor increase in osmolarity induces. Changes in cell volume are
known to have profound effects on cellular homeostasis. Cell swelling
inhibits proteolysis and increases protein synthesis, whereas cell
shrinkage stimulates proteolysis and decreases protein synthesis
(22). These effects on protein metabolism may be partly
mediated by alterations in the activity of lysosomal enzymes as a
result of pH changes (4). Importantly, part of the
hypertrophic effect previously demonstrated to occur with TGF-1 may
be due to the activation of Na+/H+ exchange
(18), leading to cell swelling and alkalinization with
subsequent reduction in proteolysis (47). Hence, the
hypertrophy that is observed in a number of cell types after high
glucose exposure may be as a consequence of alterations in cell volume with resultant affects on TGF-
1. Cell volume changes may also influence cell death mechanisms, with cell shrinkage being a hallmark of apoptosis (22). Apoptosis has been
shown to be increased after high glucose exposure to endothelial cells
(2), but the relative contribution of TGF-
1 and
hyperosmolarity to this increase in cell death is not known.
Exposure of endothelial cells to high glucose has at least three broad
components, those attributed to the specific metabolic effects of
increased levels of glucose such as reactive oxidative stress, those
due to the hyperosmotic component of high glucose medium, and those due
to the autocrine release of cytokines such as TGF-1 production.
Furthermore, whether TGF-
1 is a common mediator for the effects of
high glucose and increased osmolarity on endothelial cell growth and
apoptosis needs clarification. Consequently, the aim of the
current study was to define the precise contribution of TGF-
1 and
hypertonicity that is induced by high glucose on endothelial cell
growth and progression through the cell cycle.
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METHODS |
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Cell Isolation and Culture Conditions
Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords of normal pregnancies by the method of Jaffe et al. (16). Ethical approval for the study was obtained from the Royal North Shore Hospital Human Research Ethics Committee. These cells were cultured on 0.2% gelatin (BDH, Poole, UK)-coated flasks in medium M199 (ICN, Aurora, OH) supplemented with 10% fetal calf serum (GIBCO BRL, Gaithersburg, MD), penicillin (100 U/ml), streptomycin (100 µg/ml; GIBCO BRL), heparin (100 mg/ml; Sigma, St. Louis, MO), and endothelial cell growth supplement. Endothelial cell growth supplement was obtained from bovine hypothalamus (27) and tested against a commercial preparation (Sigma). Cells were grown at 37°C in a humidified 5% CO2 incubator and subcultured at confluence by trypsinization with 0.05% trypsin-0.02% EDTA (GIBCO BRL). These cell cultures were confirmed to be endothelial cells on the basis of their characteristic "cobblestone" morphology under phase-contrast microscopy and by staining positive for von Willebrand factor using standard immunoperoxidase methods (DAKO, Carpinteria, CA).HUVECs are a commonly used model in the study of diabetic vascular
disease (2, 5, 9, 25, 26, 37, 51). They were studied
between passages 1 and 4 (in which both we and
others have demonstrated no phenotypic change; Ref. 10).
In all experiments the cells were grown on 0.2% gelatin-coated plates
to 90% confluence (days 6-8 after plating) and then
exposed to medium containing 5 mM D-glucose (control),
25 mM (high) D-glucose, or 20 mM mannitol with 5 mM
D-glucose (osmotic control) for 72 h. In subsequent experiments, endothelial cells were exposed to 5 mM glucose plus TGF-1 (1 ng/ml) (Sigma) or panspecific TGF-
antibody (30 µg/ml) (R&D Systems, Minneapolis, MN), with or without high glucose, for
72 h. The medium was changed every 48 h.
Total TGF-1 Production
TGF-1 Bioactivity
Growth Studies
To determine the effects of exposure to high glucose on cell growth, we determined cell number as a measure of cell proliferation and total cellular protein content as a measure of hypertrophy. Flow cytometry was used to analyze progression through the cell cycle. Endothelial cells were grown in 96-well tissue culture plates (Costar), and measurements were performed at 24-h intervals over a 72-h period. Cell number was measured by counting trypsinized cells with a hemocytometer. Total cellular protein content was determined on cell lysates using the Bio-Rad protein assay dye (Hercules, CA). Cells were washed in PBS and solubilized in 0.2 M sodium hydroxide, and protein content was determined per the manufacturer's instructions.Cell Cycle Analysis
For cell cycle analysis, endothelial cells were grown on 6-well plates and, after 72 h of exposure to the glucose-specific medium, harvested by trypsinization. Cells for pre-G1 peak analysis were fixed in 70% ethanol, whereas cells for cell cycle analysis were not fixed. The cells were then washed once in PBS and incubated for 1 h at 4°C in 1 ml of a fluorochrome solution containing propidium iodide (PI; 50 µg/ml), RNase A (1 mg/ml), and 1.5% Triton X-100 (all from Sigma) in PBS. Flow cytometry was performed on the cells using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). The PI fluorescence of individual nuclei and the forward and side scatter were all measured using identical instrument settings with a minimum of 20,000 events.p21Cip1/Waf1 and p27Kip1 Expression
Because previous investigations have demonstrated a delay in cell cycle traversal in the S phase after exposure of high glucose to endothelial cells (26), the expression of the TGF-The expression of p21Cip1/Waf1 and p27Kip1 was
measured by Western blotting using the method described for ig-h3.
The primary antibodies anti-p21Cip1/Waf1 and
anti-p27Kip1 (Upstate Biotech, Lake Placid, NY) were used
at a concentration of 1 µg/ml. Secondary antibody concentrations used
were anti-mouse antibody (1:1,000) for p21Cip1/Waf1 and
anti-rabbit antibody for p27Kip1 (Amersham Pharmacia
Biotech). The resultant films were scanned into a computer, and the
relative band intensities were measured using the NIH Image software.
Because equal volumes of sample were loaded onto each gel, analysis of
the band intensities was then adjusted to take into account the
differences in cell number observed with each treatment.
Cellular Apoptosis
All apoptotic parameters were assessed on adherent cells, and hence, the levels of apoptosis measured may underestimate the total levels of apoptosis that occur in vitro. However, because the cell supernatants would contain a number of apoptotic cells that may have undergone secondary necrosis, use of nonadherent cells would make interpretation of results difficult.The terminal stages of apoptosis were determined by morphological assessment using 4,6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI) staining (Sigma). Cells were grown on 1-cm gelatin-coated glass coverslips, exposed to glucose-specific medium for 72 h, fixed in ice-cold methanol, and then stained with DAPI (0.8 µg/ml PBS) for 1 min at room temperature. The coverslips were mounted on slides with Glycergel mounting medium (DAKO), and the DAPI-stained nuclei were visualized with a fluorescence microscope (Olympus Bx60) with a ×60 objective by using 360-nm excitation and 460-nm emission wavelengths. Apoptotic cells were defined as those clearly showing chromatin condensation, nuclear fragmentation, or the formation of apoptotic bodies. A total of 400 cells was counted per experimental treatment group, and the numbers of apoptotic cells were then expressed as percentages of the total cells. The flow cytometry results also provided simultaneous data as to the number of cells present in the pre-G1 peak, allowing determination of the number of apoptotic cells present in the population.
Levels of the proapoptotic cytosolic protein Bax were determined using confocal immunomicroscopy. Endothelial cells were grown on coverslips and exposed to glucose-specific medium for 72 h. The cells were then fixed with 4% paraformaldehyde, washed in PBS, and blocked for 90 min in 20% horse serum in 0.1 M PBS. The coverslips were incubated overnight with rabbit anti-human Bax (DAKO) at a dilution of 1:400 at 4°C and then repeatedly washed with PBS, incubated with 1:200 FITC-labeled anti-rabbit IgG (DAKO) for 90 min, and finally mounted on slides using Glycergel (DAKO). The cells were visualized with a ×63 oil-immersion objective by using an Optiscan 5009e confocal unit (Optiscan, Victoria, Australia) mounted on an Olympus Bx60 microscope. Average pixel intensities within each cell were quantified using the NIH Image software.
Cellular Necrosis
Trypan blue exclusion and lactate dehydrogenase (LDH) release were both measured as markers of cellular necrosis. Cells were grown on 96-well plates and exposed to glucose-specific medium for 72 h. The cells were trypsinized, and 50-µl aliquots of a 0.1% trypan blue solution (Searle Diagnostics) in PBS were added to the cell suspensions. After 10 min, the cells were counted with a hemocytometer, and the number of cells stained with trypan blue was expressed as a percentage of the total cell number. Levels of LDH activity were determined in supernatants collected from cells exposed to the glucose-specific medium for 72 h. The spectrophotometric assay was performed by Pacific Laboratory Medicine Services (Sydney, Australia).TGF- Neutralization
Statistical Analysis
The experiments were performed at least in triplicate on a minimum of four and a maximum of seven different endothelial cell isolates. Results are expressed as a percentage of the control value (5 mM D-glucose) except the flow cytometry data, which represent the percentage distribution of the cells within the cell cycle. Results are expressed as means ± SE. Statistical comparisons between groups were made by analysis of variance with pairwise multiple comparisons made by Fisher's protected least significant differences test. Analyses were performed using StatView (version 5.0; Abacus Concepts, Berkley, CA). P values <0.05 were considered significant. ![]() |
RESULTS |
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TGF-1 Production
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TGF-1 Bioactivity
Growth Studies
Endothelial cells grown under control conditions showed a typical proliferative response, with a linear increase in cell number at 72 h indicating a doubling time of ~54 h (Fig. 2A). The initial effect of exposure to high glucose at 24 h was a significant increase in cell number compared with control levels (P < 0.001). However, continued exposure to high glucose arrested the proliferative response, resulting in a reduction in cell number at 72 h (P < 0.001; Fig. 2A). In contrast, exposure to the osmotic control resulted in a uniform slowing of the proliferative response over the 72-h period (Fig. 2A).
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The total protein content per cell under control conditions reflected the characteristic trend of proliferating cells. At the 48 h time point, there was a significant increase in cellular protein content that reflected the premitotic increase in cell mass. However, by 72 h, the protein content per cell had returned to baseline levels (Fig. 2B). In contrast, after 72 h of exposure to high glucose, there was a significant increase in the protein content per cell (P < 0.001; Fig. 2B). The effect of the osmotic control was markedly different from that of high glucose, with an initial significant increase in protein content at 24 h, an effect that gradually returned toward control conditions over the subsequent 48 h (Fig. 2B).
To confirm that the growth and TGF-1 response of high glucose
exposure to endothelial cells occurred under conditions that were
clinically relevant and independent of hyperosmolarity, we repeated the
growth studies and TGF-
1 ELISA using 15 mM glucose.
After exposure to 25 mM glucose for 72 h, cell number was
73.5 ± 2.4% (P < 0.0001), cell protein
content was 152.6 ± 5.5% (P < 0.0001), and
total TGF-1 release was 141.5 ± 5.6% (P < 0.001) compared with control values. Similarly, exposure to 15 mM
glucose for 72 h reduced cell number to 76.3 ± 2.2%
(P < 0.0001), increased cell protein to 149.5 ± 6.9% (P < 0.0001), and increased TGF-
1 release to
138.4 ± 6.2% (P < 0.001) compared with control
levels (Fig. 3, A-C).
Because the growth and TGF-
1 release responses were consistent
between 15 and 25 mM glucose, these data provide further confirmatory
evidence that hyperosmolarity is not a significant factor in the
effects observed.
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Cell Cycle Analysis
Fluorescence-activated cell sorter (FACS) analysis was used to determine the effects of high glucose and the osmotic control on the progression of endothelial cells through the cell cycle after 72 h. A typical FACS result of cells grown under control conditions is shown in Fig. 4A. The distribution of endothelial cells throughout the phases of the cell cycle after exposure to control conditions was 63.0 ± 8.0% for the G1 phase, 8.3 ± 1.0% for the S phase, and 24.5 ± 3.1% for the G2M phase. After exposure to high glucose, no significant differences were noted in the distribution of cells in either the G1 or G2M phases. However, there was a significant increase in the number of cells in the S phase (P < 0.005; Fig. 4B). In contrast, treatment of the cells with the osmotic control had no effect on their distribution throughout the cell cycle (Fig. 4B).
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An important finding was the identification of a subpopulation of cells
that were undergoing endoreduplication (defined as those polyploid
cells that failed to enter mitosis but proceeded directly from a gap
phase to a phase of DNA synthesis) (Fig.
5A). Under control conditions,
3.7 ± 1.1% of cells were endoreduplicative, but after exposure
to high glucose for 72 h, this population increased to 6.8 ± 0.2% (P < 0.001), representing a 186.5 ± 4.3%
increase compared with control levels (Fig. 4). In contrast, exposure
to the osmotic control did not significantly alter the numbers of cells
undergoing endoreduplication (Fig. 5B).
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Cell cycle analysis was also performed on endothelial cells grown in 5 mM glucose treated with TGF-1 (1 ng/ml) for 72 h to determine
whether the effects of high glucose exposure could be mimicked by
TGF-
1. Exposure to TGF-
1 led to an increase in the number of
cells in the S phase (P < 0.05) compared with control levels (Fig. 6A), an effect
that was similar to that observed after high glucose exposure. However,
unlike high glucose, TGF-
1 had no effect on the levels of
endoreduplicative cells (Fig. 6B).
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Cyclin-Dependent Kinases and Inhibitors
p21Cip1/Waf1.
p21Cip1/Waf1 is a TGF--inducible 21-kDa protein that
inhibits the cyclin E/Cdk2 complex, which controls S phase progression
(40). It also inhibits the cyclin B/Cdc2 complex that
controls the initiation of mitosis. Hence, an isolated increase in the
activity of p21Cip1/Waf1 leads to delayed progression
through both S phase and G2M progression (40).
Exposure of endothelial cells to high glucose for 72 h resulted in
a significant increase in p21Cip1/Waf1 expression per cell
to 138.8 ± 7.9% (n = 25; P < 0.0001) of control values. In contrast, mannitol had no effect on the
levels of this CDKI (116.5 ± 6.5%; n = 25)
compared with control levels (Fig. 7A).
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p27Kip1.
p27Kip1 is a TGF--inducible 27-kDa protein that inhibits
the cyclin E/Cdk2 complex that controls S phase progression, and an increase in the level of this protein is associated with delayed progression through the S phase (40). High glucose induced
a significant increase in p27Kip1 expression per cell to
162.2 ± 12.8% (n = 32; P < 0.0001) of control values. Mannitol had no significant effect on
cellular p27Kip1 expression (118.3 ± 8.3%;
n = 32) compared with control levels. (Fig.
7B).
Apoptosis
DAPI staining of cells grown under control conditions revealed that 3.1 ± 0.4% of the cells were apoptotic after 72 h (Fig. 8). In cells exposed to high glucose there was a 152.1 ± 12.4% increase in apoptotic cells (P < 0.05). In contrast, there was no effect on the levels of apoptosis after 72 h of exposure to the osmotic control.
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Similarly, FACS analysis revealed that under control conditions, 0.58 ± 0.6% (n = 13) of cells were in the pre-G1 peak that represents apoptotic cells (Fig. 5). After exposure to high glucose for 72 h, this value increased by 141.3 ± 8.5% (P < 0.001). No change was observed in cells exposed to the osmotic control for 72 h.
Confocal analysis of cells grown under control conditions labeled with
anti-Bax antibodies showed diffuse cytosolic staining with occasional
puncta (Fig. 9A). In cells
exposed to high glucose there was an increase in the fluorescent
intensity compared with control levels and a pronounced redistribution
of the Bax within the cells, suggestive of mitochondrial translocation
(Fig. 9B). In contrast, cells grown in the osmotic control
showed no changes in distribution or intensity of expression of Bax
(Fig. 9C).
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Necrosis
In the presence of high glucose, there was no significant change in the LDH release into the medium from control values. However, in cells exposed to the osmotic control, there was an increase in amount of LDH released (P < 0.001; Fig. 10A). This is in keeping with the findings of Wu et al. (51).
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Trypan blue staining of control cells showed that 6.2 ± 0.2% of the cells were nonviable. In the presence of high glucose, this value increased to 14.6 ± 0.8% (P < 0.05). However, exposure to the osmotic control resulted in a further increase to 27.7 ± 3.8% (P < 0.0001), an effect that was significantly greater than exposure to high glucose alone (P < 0.005) (Fig. 10B).
Effects of Neutralization of TGF-1 on Endothelial Cell Growth
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We then exposed the cells to high glucose in the presence of TGF-
neutralizing antibody to determine the extent to which the effects of
high glucose were mediated by TGF-
. The bioactivity of the
neutralizing antibody was confirmed by treating the cells with high
glucose and rabbit IgG (R&D Systems), and no difference in either
growth parameter was observed compared with high glucose alone (Fig.
11, A and B). However, the addition of the
panspecific TGF-
antibody to cells exposed to high glucose over
72 h significantly reversed the reduction in endothelial cell
number seen with high glucose back to control levels (Fig.
11A). Similarly, the increase in cell protein content
observed after 72 h of exposure to high glucose was abolished in
the presence of panspecific TGF-
antibody (Fig. 11B). The
addition of panspecific TGF-
antibody did not reverse the growth
effects observed with the osmotic control (Fig. 11, A and
B). These data indicated that the growth effects observed after exposure of endothelial cells to high glucose were mediated primarily through TGF-
dependent pathways, and this was a response independent of hyperosmolarity.
Effects of Neutralization of TGF-1 on Apoptosis
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DISCUSSION |
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The current study highlights the pathogenic effects of exposure of
high glucose on the endothelial cell and defines the role of autocrine
TGF-1 in the alterations observed in cell growth, cell cycle
progression, and the levels of apoptosis and cellular necrosis.
In addition, it confirms that these effects are largely glucose
specific and independent of any potential hyperosmolar component of
elevated glucose. Furthermore, we have identified novel cell cycle
effects of high glucose that are independent of the autocrine action of
TGF-
1. Our results demonstrate that short-term exposure of
endothelial cells to high glucose produces a hypertrophic and
antiproliferative growth response, leading to a reduction in cell
number with a concomitant increase in the levels of apoptosis
and a subpopulation of cells undergoing endoreduplication. These
findings are in keeping with the pathology observed in vivo in diabetic
models (17, 24, 28, 44).
We observed an increase in the total amount of TGF-1 released per
cell as well as increased TGF-
1 bioactivity, which was confirmed in
our studies by the increased expression of the TGF-
1-inducible protein
ig-h3. This increase in levels of total TGF-
1 released per cell is consistent with the increased total TGF-
1 release and
increased TGF-
1 mRNA expression described by others
(34). The only other study to investigate the effects of
high glucose on TGF-
1 bioactivity in HUVECs used the mink lung
epithelial cells proliferation assay, and no increase in TGF-
1
activity was observed (5). Although the reasons for this
discrepancy between the two studies remains unclear, our results are
supported by the finding of increased expression of
ig-h3 in
atherosclerotic vessels, suggesting that TGF-
1 plays a significant
role in the pathogenesis of this condition of which diabetes mellitus
is a major risk factor (33). This study also showed that
the increase in TGF-
1 bioactivity observed with high glucose may be
partly a consequence of its increase in hypertonicity. We are the first to demonstrate that this minor increase in osmolarity significantly altered endothelial cell TGF-
1 bioactivity that potentially could induce secondary effects either locally or systemically. However, because panspecific TGF-
antibody did not reverse the growth effects
associated with mannitol exposure, it would appear that this local
increase in TGF-
1 was insufficient in itself to mediate the
endothelial cell growth effects observed with mannitol exposure. The
combination of high glucose with increased TGF-
1 created a milieu
suitable for autocrine TGF-
1 to mediate the growth effects observed.
Several groups have implicated a number of growth factors such as
platelet-derived growth factor and hepatocyte growth factor that act as
important cofactors in mediating the growth effects of TGF-
1 in a
number of different cell types (7, 32, 35). Hence, similar
glucose-specific effects on these cytokines combined with autocrine
TGF-
1 may be occurring in our endothelial cell model to induce the
growth effects observed. These cofactors must be unaltered with the
osmotic control, and hence, the increase in TGF-
1 observed with
mannitol does not have an autocrine effect.
Our results demonstrate that exogenous TGF-1 mimics the
antiproliferative and hypertrophic effects of high glucose on
endothelial cells in association with an increase in the number of
cells in the S phase of the cell cycle. The antiproliferative and
hypertrophic effects of high glucose are reversed by the anti-TGF-
1
antibody, confirming that these effects are mediated by autocrine
TGF-
1 production. These findings differ from those of Cagliero et
al. (5), although in the latter studies the effects of
exogenous TGF-
1 were not reversed with the blocking antibody. We
observed a reduction in endothelial cell number despite an increased
number of cells synthesizing DNA. These data are consistent with high glucose inducing a delay in S phase progression, arrest at the G2M phase, or dysregulation of the mitotic spindle
checkpoint mechanism. This current study demonstrates increased
expression of both p21Cip1/Waf1 and p27Kip1
after exposure of high glucose to endothelial cells. The elevated expression of these CDKIs delays traversal through the S phase, and
hence, the unaltered expression associated with hyperosmolar conditions
is also in keeping with the cell cycle data described. In addition,
these data highlight a role for the cell cycle in mediating the growth
effects of high glucose but not mannitol. In other cell types TGF-
1
has been shown to increase the levels of the CDKIs
p21Cip1/Waf1 and p27Kip1 (40, 48,
49). Thus inhibition of cyclin-dependent kinases by the
autocrine effects of TGF-
1 may explain the action of high glucose on
the endothelial cell cycle.
High glucose-induced endothelial cell hypertrophy has not previously
been described. This response is glucose specific, because exposure to
the osmotic control induced a different growth response with a marked
hypertrophy in the first 24 h that diminished over the subsequent
48 h but still remained above control levels. Unlike the high
glucose-induced hypertrophy observed in mesangial cells and
fibroblasts, where G1 phase arrest is present (48,
49), the endothelial cell hypertrophy demonstrated in this study
was associated with S phase delay and abnormalities in mitosis. Because the hypertrophic response observed with high glucose was mediated by
autocrine TGF-1 (as demonstrated by coexposure with the TGF-
1 antibody), and because abnormalities in mitosis (manifest as
endoreduplication) were not mediated by TGF-
1, it would appear that
the hypertrophy observed under high glucose conditions was most largely
a consequence of S phase delay.
This study showed that the endothelial cell hypertrophy mediated by
hyperosmolar conditions was not cell cycle dependent and was not
mediated by autocrine TGF-1. Hypertrophy occurring independently of
the cell cycle is a consequence of the altered balance of cell protein
synthesis and proteolysis (48); therefore, the changes in
endothelial cell volume that occur with mannitol exposure must alter
this protein homeostasis. The effects on the proteolytic and protein
synthetic mechanisms under hyperosmolar conditions were independent of
the known effects of TGF-
1 on these protein homeostatic mechanisms.
When endothelial cells were exposed to a lower but still elevated
concentration of glucose (15 mM), the antiproliferative and
hypertrophic growth response occurred. Because 15 mM glucose does not
induce a substantial increase in osmolarity, these data confirm that
these growth and TGF-1 release effects are largely specific to the
elevated levels of glucose. Furthermore, exposure to this level of
glucose bears more resemblance to the levels observed in patients with
diabetes mellitus and is consistent with the vascular pathology
observed in these patients.
Our results demonstrate that TGF-1 mediated the growth and cell
cycle effects of high glucose more than its associated increase in
osmolarity and independently of the hyperosmolarity-induced release of
this cytokine. Furthermore, the proapoptotic effects of high
glucose and the increase in the subpopulation of cells undergoing
endoreduplication occurred independently of autocrine TGF-
1 and
hyperosmolarity and were specific to the metabolic effects of high
glucose. Endoreduplication is an atypical form of progression through
the cell cycle in which rounds of DNA synthesis repeat in the absence
of intervening mitoses (12). It is recognized in a number
of human cells such as cardiomyocytes and megakaryocytes, where
increasing levels of polyploidy underlie the hypertrophic responses in
a large proportion of these cells (12, 36). In addition,
endoreduplication with hypertrophy may manifest under conditions of
stress, for example, in vascular smooth muscle cells of hypertensive
rats (3) and in diabetic rat mesenteric smooth muscle
cells (46). Multinucleated variant endothelial cells have
also been demonstrated in human atherosclerotic vessels of which
diabetes mellitus is a major risk factor. This finding suggests that
endoreduplication is an in vivo phenomenon that may contribute to the
pathogenesis of diabetic vascular disease. Endoreduplication is
associated with an increase in cell mass, altered gene expression profiles, and the potential to produce large quantities of endogenous proteins such as cytokines (36). Thus the cellular
endoreduplication demonstrated may contribute to the altered cytokine
expression implicated in many of the inflammatory and fibrotic
consequences of diabetes mellitus.
This study demonstrated increased apoptosis after exposure to
high glucose, and the overall low levels demonstrated are in keeping
both with in vivo studies (28) and in patients, where acute hyperglycemia is not associated with sequelae of increased levels
of endothelial cell apoptosis such as thrombosis. The etiology of the proapoptotic effect of high glucose on the endothelial cell
is unresolved. High glucose exposure is associated with increased endothelial cell production of reactive oxygen species (ROS), and
oxidative stress has been the mechanism most often implicated in the
increase in the associated endothelial cell apoptosis (6, 9, 37, 51). The increased ROS appears to act through the MAPKs
with downstream upregulation of caspase-3 (32). However, TGF-1 has been demonstrated to increase ROS production in
endothelial cells (35), and therefore, because TGF-
1 is
proapoptotic in endothelial cells (45), we postulated
that autocrine TGF-
1 mediated the apoptotic effects of high
glucose. Our results demonstrated that both high glucose and TGF-
1
caused a significant increase in the levels of apoptosis and
that combined treatment with TGF-
1 and high glucose had an additive
effect on the levels of apoptosis. Neutralization of TGF-
did not reverse the apoptosis induced by high glucose,
suggesting that the increased ROS associated with high glucose-induced
apoptosis occurs independently of TGF-
1 levels. Although the
proapoptotic effect of high glucose has been previously described
(2), this is the first demonstration that high
glucose-induced endothelial apoptosis is independent of
TGF-
1. The finding of distinct apoptotic mechanisms is
significant because patients with diabetes have elevations in both
serum glucose and active TGF-
1 (29) that may,
therefore, result in a deleterious additive effect on the levels of
apoptosis in vivo.
Furthermore, we present evidence that the osmotic control had no effect on the levels of apoptosis, demonstrating that the proapoptotic effects of glucose were independent of hyperosmolarity. This is in keeping with previous studies, where high glucose-induced endothelial cell apoptosis was not reproduced by exposure to mannitol (51). In contrast to high glucose, in this study the osmotic control resulted in a significant increase in cell death via necrosis. In particular, because the reduction in cell number that occurred with mannitol exposure occurred independently of the cell cycle, necrosis more than apoptosis accounted for the cell numbers observed. Previously, increased ROS production has been demonstrated to occur with HUVEC exposure to high glucose but not with exposure to mannitol (51). Wu et al. (51) have also demonstrated that whereas taurine reversed the apoptosis associated with high glucose exposure, the necrosis induced by mannitol was not reversed by the antioxidant taurine. This finding confirms that high glucose induces subtle changes in local cytokine/ROS production independently of the increase in hyperosmolarity and that these subtle changes may account for the differing growth and apoptotic effects observed between these two conditions.
In summary, we have demonstrated that high glucose, TGF-1, and
osmotic stress all have independent but partially overlapping effects
on the growth of endothelial cells. The changes in cell growth during
exposure to high glucose occur as a result of interactions between the
autocrine effects of TGF-
1 and the metabolic and hyperosmotic
effects of glucose. Importantly, this is the first demonstration that
some of the deleterious effects of high glucose may be mediated by
increases in the populations of cells undergoing apoptosis and
endoreduplication, phenomena that occur independently of TGF-
1 and hyperosmolarity.
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ACKNOWLEDGEMENTS |
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We thank Pacific Laboratory Medicine Services, Sydney, for performing the lactate dehydrogenase assay.
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FOOTNOTES |
---|
S. McGinn was supported by scholarships from Royal North Shore Hospital and the National Health & Medical Research Council, Australia. P. Poronnik is a University of Sydney 2000 fellow. This research was also supported by the Juvenile Diabetes Research Foundation.
Address for reprint requests and other correspondence: C. Pollock, Dept. of Medicine, Level 3, Wallace Freeborn, Royal North Shore Hospital, St Leonards, NSW 2065, Australia (E-mail: carpol{at}med.usyd.edu.au).
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.
First published January 22, 2003;10.1152/ajpcell.00466.2002
Received 3 October 2002; accepted in final form 11 January 2003.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ambuhl, P,
Amemiya M,
Preisig PA,
Moe OW,
and
Alpern RJ.
Chronic hyperosmolality increases NHE3 activity in OKP cells.
J Clin Invest
101:
170-177,
1998
2.
Baumgartner-Parzer, SM,
Wagner L,
Pettermann M,
Grillari J,
Gessl A,
and
Waldhausl W.
High-glucose-triggered apoptosis in cultured endothelial cells.
Diabetes
44:
1323-1327,
1995[Abstract].
3.
Berk, BC.
Vascular smooth muscle growth: autocrine growth mechanisms.
Physiol Rev
81:
999-1030,
2001
4.
Busch, GL,
Lang HJ,
and
Lang F.
Studies on the mechanism of swelling-induced lysosomal alkalinization in vascular smooth muscle cells.
Pflügers Arch
431:
690-696,
1996[ISI][Medline].
5.
Cagliero, E,
Roth T,
Taylor AW,
and
Lorenzi M.
The effects of high glucose on human endothelial cell growth and gene expression are not mediated by transforming growth factor-beta.
Lab Invest
73:
667-673,
1995[ISI][Medline].
6.
Ceriello, A,
dello Russo P,
Amatadi P,
and
Cerutti P.
High glucose induces antioxidant enzymes in human endothelial cells: evidence linking high glucose with oxidative stress.
Diabetes
45:
471-477,
1999.
7.
Di Paolo, S,
Gesualdo L,
Ranieri E,
Grandaliano G,
and
Schena FP.
High glucose concentration induces the overexpression of transforming growth factor-beta through the activation of a platelet-derived growth factor loop in human mesangial cells.
Am J Pathol
149:
2095-2106,
1996[Abstract].
8.
Doyle, JW,
Smith RM,
and
Roth TP.
The effect of hyperglycemia and insulin on the replication of cultured human microvascular endothelial cells.
Horm Metab Res
29:
43-45,
1997[ISI][Medline].
9.
Du, XL,
Sui GZ,
Stockklauser-Farber K,
Weiss J,
Zink S,
Schwippert B,
Wu QX,
Tschope D,
and
Rosen P.
Introduction of apoptosis by high proinsulin and glucose in cultured human umbilical vein endothelial cells is mediated by reactive oxygen species.
Diabetologia
41:
249-256,
1998[ISI][Medline].
10.
Garfinkel, S,
Hu X,
Prudovsky IA,
McMahon GA,
Kapnik EM,
McDowell SD,
and
Maciag T.
FGF-1-dependent proliferative and migratory responses are impaired in senescent human umbilical vein endothelial cells and correlate with the inability to signal tyrosine phosphorylation of fibroblast growth factor receptor-1 substrates.
J Cell Biol
134:
783-791,
1996[Abstract].
11.
Gilbert, RE,
Wilkinson-Berka JL,
Johnson DW,
Cox A,
Soulis T,
Wu LL,
Kelly DJ,
Jerums G,
Pollock CA,
and
Cooper ME.
Renal expression of transforming growth factor-beta inducible gene-h3 (ig-h3) in normal and diabetic rats.
Kidney Int
54:
1052-1062,
1998[ISI][Medline].
12.
Grafi, G.
Cell cycle regulation of DNA replication: the endoreduplication perspective.
Exp Cell Res
244:
372-378,
1998[ISI][Medline].
13.
Graier, WF,
Grubenthal I,
Dittrich P,
Wascher TC,
and
Kostner GM.
Intracellular mechanism of high D-glucose-induced modulation of vascular cell proliferation.
Eur J Pharmacol
294:
221-229,
1995[ISI][Medline].
14.
Han, DC,
Isono N,
Hoffman BB,
and
Ziyadeh FN.
High glucose stimulates proliferation and collagen type I synthesis in renal cortical fibroblasts: mediation by autocrine activation of TGF-beta.
J Am Soc Nephrol
10:
1891-1899,
1999
15.
Hoffman, BB,
Sharma K,
Zhu Y,
and
Ziyadeh FN.
Transcriptional activation of transforming growth factor-beta1 in mesangial cell culture by high glucose concentration.
Kidney Int
54:
1107-1116,
1998[ISI][Medline].
16.
Jaffe, EA,
Nachman RL,
Becker CG,
and
Minick CR.
Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria.
J Clin Invest
52:
2745-2756,
1973[ISI][Medline].
17.
Jakobsen, J,
Sidenius P,
Gundersen HV,
and
Osterby R.
Quantitative changes of cerebral neocortical structure in insulin treated long term streptozocin-induced diabetes in rats.
Diabetes
36:
597-601,
1987[Abstract].
18.
Johnson, DW,
Saunders HJ,
Brew BK,
Poronnik P,
Cook DI,
Field MJ,
and
Pollock CA.
TGF-beta 1 dissociates human proximal tubule cell growth and Na+H+ exchange activity.
Kidney Int
53:
1601-1607,
1998[ISI][Medline].
19.
Kamal, K,
Du W,
Mills I,
and
Sumpio BE.
Antiproliferative effect of elevated glucose in human microvascular endothelial cells.
J Cell Biochem
71:
491-501,
1998[ISI][Medline].
20.
Klintworth, GK,
Valnickova Z,
and
Enghild JJ.
Accumulation of beta ig-h3 gene product in corneas with granular dystrophy.
Am J Pathol
152:
743-748,
1998[Abstract].
21.
Kolm, V,
Sauer U,
Olgemooller B,
and
Schleicher ED.
High glucose-induced TGF-1 regulates mesangial production of heparan sulfate proteoglycan.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F812-F821,
1996
22.
Lang, F,
Ritter M,
Gamper N,
Huber S,
Fillon S,
Tanneur V,
Lepple-Wienhues A,
Szabo I,
and
Gulbins E.
Cell volume in the regulation of cell proliferation and apoptotic cell death.
Cell Physiol Biochem
10:
417-428,
2000[ISI][Medline].
23.
Langham, RA,
Egan MK,
Dowling JP,
Gilbert RE,
and
Thomson NM.
Transforming growth factor-beta and transforming growth factor-beta inducible gene-h3 (beta ig-h3) in non renal transplant cyclosporine nephropathy.
Transplantation
72:
1826-1829,
2001[ISI][Medline].
24.
Lorenzi, M,
and
Cagliero E.
Pathobiology of endothelial and other vascular cells in diabetes mellitus. Call for data.
Diabetes
40:
653-659,
1991[Abstract].
25.
Lorenzi, M,
Cagliero E,
and
Toledo S.
Glucose toxicity for human endothelial cells in culture. Delayed replication, disturbed cell cycle, and accelerated death.
Diabetes
34:
621-627,
1985[Abstract].
26.
Lorenzi, M,
Nordberg JA,
and
Toledo S.
High glucose prolongs cell-cycle traversal of cultured human endothelial cells.
Diabetes
36:
1261-1267,
1987[Abstract].
27.
Maciag, T,
Cerundolo J,
Ilsley S,
Kelley PR,
and
Forand R.
An endothelial cell growth factor from bovine hypothalamus: identification and partial characterization.
Proc Natl Acad Sci USA
76:
5674-5678,
1979[Abstract].
28.
Mizutani, M,
Kern TS,
and
Lorenzi M.
Accelerated death of retinal microvascular cells in human and experimental diabetic retinopathy.
J Clin Invest
97:
2883-2890,
1996
29.
Mogyorosi, A,
Kapoor A,
Isono M,
Kapoor S,
Sharma K,
and
Ziyadeh FN.
Utility of serum and urinary transforming growth factor-beta levels as markers of diabetic nephropathy.
Nephron
86:
234-235,
2000[ISI][Medline].
30.
Muller, G,
Behrens J,
Nussbaumer U,
Bohlen P,
and
Birchmeier W.
Inhibitory action of transforming growth factor beta on endothelial cells.
Proc Natl Acad Sci USA
84:
5600-5604,
1987[Abstract].
31.
Myoken, Y,
Kan M,
Sato GH,
McKeehan WL,
and
Sato JD.
Bifunctional effects of transforming growth factor-beta (TGF-beta) on endothelial cell growth correlate with phenotypes of TGF-beta binding sites.
Exp Cell Res
191:
299-304,
1990[ISI][Medline].
32.
Nakagami, H,
Morishita R,
Yamamoto K,
Yoshimura SI,
Taniyama Y,
Aoki M,
Matsubara H,
Kim S,
Kaneda Y,
and
Ogihara T.
Phosphorylation of MAPK downstream of bax-caspase 3 leads to cell death induced by D glucose in human endothelial cells.
Diabetes
50:
1472-1481,
2001
33.
O'Brien, ER,
Bennett KL,
Garvin MR,
Zderic TW,
Hinohara T,
Simpson JB,
Kimura T,
Nobuyoshi M,
Mizgala H,
Purchio A,
and
Schwartz SM.
Beta ig-h3, a transforming growth factor-beta-inducible gene, is overexpressed in atherosclerotic vessels.
Arterioscler Thromb Vasc Biol
16:
576-584,
1996
34.
Pascal, MM,
Forrester JV,
and
Knott RM.
Glucose-mediated regulation of transforming growth factor-beta (TGF-beta) and TGF-beta receptors in human retinal endothelial cells.
Curr Eye Res
19:
162-170,
1999[ISI][Medline].
35.
Perella, MA,
Yoshizumu M,
Fen Z,
Tsai JC,
Haiah CM,
Kourenbanea J,
and
Lee ME.
Transforming growth factor 1, but not dexamethasone, downregulates nitric oxide synthase mRNA after its induction by interleukin 1 in rat smooth muscle cells.
J Biol Chem
269:
14595-14600,
1994
36.
Ravid, K,
Lu J,
Zimmet JM,
and
Jones MR.
Roads to polyploidy: the megakaryocyte example.
J Cell Physiol
190:
7-20,
2002[ISI][Medline].
37.
Risso, A,
Mercuri F,
Quagliaro L,
Damante G,
and
Ceriello A.
Intermittent high glucose enhances apoptosis in human umbilical vein endothelial cells in culture.
Am J Physiol Endocrinol Metab
281:
E924-E930,
2001
38.
Rocco, MV,
Chen Y,
Goldfarb S,
and
Ziyadeh FN.
Elevated glucose stimulates TGF-beta gene expression and bioactivity in proximal tubule.
Kidney Int
41:
107-114,
1992[ISI][Medline].
39.
Shankland, SJ,
Scholey JW,
Ly H,
and
Thai K.
Expression of transforming growth factor-beta 1 during diabetic renal hypertrophy.
Kidney Int
46:
430-442,
1994[ISI][Medline].
40.
Shankland, SJ,
and
Wolf G.
Cell cycle regulatory proteins in renal disease: role of hypertrophy, proliferation and apoptosis.
Am J Physiol Renal Physiol
278:
F515-F529,
2000
41.
Sharma, K,
and
Ziyadeh FN.
Hyperglycemia and diabetic kidney disease. The case for transforming growth factor-beta as a key mediator.
Diabetes
44:
1139-1146,
1995[Abstract].
42.
The Diabetes Control and Complications Trial Research Group.
The effect of intensive treatment of diabetes on the development, and progression of long-term complications in insulin-dependent diabetes mellitus.
N Engl J Med
329:
977-986,
1993
43.
The UK Prospective Diabetes Study (UKPDS) Group..
Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment, and risk of complications in patients with type 2 diabetes (UKPDS 33).
Lancet
352:
837-853,
1998[ISI][Medline].
44.
Tilton, RG,
Faller AM,
Burkhardt JK,
Hoffmann PL,
Kilo C,
and
Williamson JR.
Pericyte degeneration and acellular capillaries are increased in the feet of human diabetic patients.
Diabetologia
28:
895-900,
1985[ISI][Medline].
45.
Tsukada, T,
Eguchi K,
Migita K,
Kawabe Y,
Kawakami A,
Matsuoka N,
Takashima H,
Mizokami A,
and
Nagataki S.
Transforming growth factor beta 1 induces apoptotic cell death in cultured human umbilical vein endothelial cells with down-regulated expression of bcl-2.
Biochem Biophys Res Commun
210:
1076-1082,
1995[ISI][Medline].
46.
Vranes, D,
Cooper ME,
and
Dilley RJ.
Cellular mechanisms of diabetic vascular hypertrophy.
Microvasc Res
57:
8-18,
1999[ISI][Medline].
47.
Waldegger, S,
Busch GL,
Kaba NK,
Zempel G,
Ling H,
Heidland A,
Haussinger D,
and
Lang F.
Effect of cellular hydration on protein metabolism.
Miner Electrolyte Metab
23:
201-205,
1997[ISI][Medline].
48.
Wolf, G.
Molecular mechanisms of renal hypertrophy: role of p27Kip1.
Kidney Int
56:
1262-1265,
1999[ISI][Medline].
49.
Wolf, G,
Schroeder R,
Zahner G,
Stahl RA,
and
Shankland SJ.
High glucose-induced hypertrophy of mesangial cells requires p27Kip1, an inhibitor of cyclin-dependent kinases.
Am J Pathol
158:
1091-1100,
2001
51.
Wu, QD,
Wang JH,
Fennessy F,
Redmond HP,
and
Bouchier-Hayes D.
Taurine prevents high-glucose-induced human vascular endothelial cell apoptosis.
Am J Physiol Cell Physiol
277:
C1229-C1238,
1999
52.
Yan, Q,
and
Sage EH.
Transforming growth factor-beta1 induces apoptotic cell death in cultured retinal endothelial cells but not pericytes: association with decreased expression of p21waf1/cip1.
J Cell Biochem
70:
70-83,
1998[ISI][Medline].