(Circulation. 1999;100:1909-1916.)
© 1999 American Heart Association, Inc.
Basic Science Reports |
Downregulation of Protein Kinase C
Activity Enhances Endothelial Cell Adaptation to Hypoxia
From the Cardiovascular Division, Department of Medicine, and the Department of Molecular Pharmacology, Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, NY.
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Abstract |
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Background—Although protein kinase C (PKC) has been implicated in ischemic cell death, the role of individual PKC isoenzymes in the response of endothelial cells (ECs) to hypoxia is unknown.
Methods and Results—To test the effect of hypoxia on the
activity of individual PKC isoenzymes, human ECs were exposed to 95%
N2 with 5% CO2 for 24 hours. This severe
hypoxia reduced PKC
specific activity in both human
umbilical vein ECs (HUVECs) and a HUVEC-derived EC line (ECVs)
significantly (80.5±5.7% and 55.5±8.6% of normoxia controls,
respectively); the activities of PKC
and PKC
were unchanged. The
protein levels of PKC, PKC, and PKC were unchanged by
hypoxia. To determine whether PKC downregulation by
hypoxia was linked to EC function, ECVs in which PKC was
stably overexpressed (PKC-ECs) were exposed to hypoxia. A
significant increase in cell death was observed in PKC-ECs compared
with controls (5.8±0.6% versus 2.3±0.4% at 24 hours, 13.2±1.2%
versus 4.1±0.4% at 48 hours, P<0.05) during
hypoxia. Neither the DNA laddering assay nor TUNEL staining
revealed an increase in apoptosis of PKC-ECs exposed to
hypoxia, suggesting a hypoxia-induced increase in
nonapoptotic cell death of PKC-ECs. Inhibition of NO
synthase with
NG-monomethyl-L-arginine
(L-NMMA) affected neither the decline in PKC activity nor the EC
death induced by hypoxia.
Conclusions—PKC activity is decreased by hypoxia by a
mechanism that does not involve NO synthase; this downregulation
appears to enhance EC survival during hypoxia by decreasing
nonapoptotic cell death.
Key Words: endothelium • hypoxia • cell death • apoptosis • protein kinase C
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Subjecting the endothelium to hypoxia increases vascular permeability, thrombogenicity, leukocyte adhesion, and the production of proinflammatory cytokines and impairs the control of vasomotor tone.1 2 3 These effects of hypoxia are sustained in vivo even after reoxygenation, a phenomenon that may be explained by decreased NO and cAMP levels.3 Hypoxia induces cell proliferation under certain experimental conditions,4 5 but severe hypoxia results in cell death.6 7 8
In various cell types, the protein kinase C (PKC) family members have
been found to be important mediators of hypoxia-induced
changes.4 5 9 Previously, PKC has been reported to be
involved in hypoxia-mediated cell proliferation in both
mesangial cells4 and smooth muscle cells
derived from the pulmonary artery5 and in enhanced
monocyte migration into endothelial cells
(ECs).9 Increased release of platelet-activating
factor during hypoxia may upregulate PKC activity, which in
turn might increase phosphorylation of platelet and
endothelial cell adhesion molecule in ECs and thus
enhance monocyte migration.9 PKC also mediates various
aspects of EC function; however, whether PKC mediates or protects
against hypoxia-induced EC death is not understood. Only
limited information is available about the role of individual PKC
isoenzymes in hypoxia; in the majority of studies,
activators or inhibitors of PKC with
nonselective, or at best unknown, effects on specific isoenzymes were
used to investigate the role of PKC in hypoxia. In
cardiomyocytes, PKC and PKC translocate from soluble
to particulate fractions of the cell with hypoxia; conversely,
PKC dissociates from the particulate to the soluble portions under
similar conditions, although the total amount of intracellular protein
corresponding to all 3 individual PKC isoenzymes remains
constant.7 PKC may prevent hypoxia-induced cell
death by triggering a preconditioning mechanism6 in which
a hypoxic injury can be attenuated by a preceding short period of
exposure to reduced oxygen tension.10 11 Whether
hypoxia alters the activity of selective PKC isoenzymes in ECs
is unknown.
In this study, we found that the activity of PKC was
specifically suppressed by hypoxia, whereas that of PKC and
PKC were not. To assess whether PKC suppression was required for
ECs to survive during hypoxia, we tested the effects of
hypoxia on PKC-overexpressing ECs. We also investigated the
role of NO and heat-shock proteins (HSPs), which protect various cell
types from hypoxia-induced injuries.12 13
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Cell Cultures, Hypoxia, and Other Materials
Human umbilical vein ECs (HUVECs) were cultured as previously described.14 Immortalized human ECs (ECVs, obtained from American Type Culture Collection) were cultured in M-199 (Gibco BRL Products) supplemented with 10% FBS (Gemini Bio Products Inc) and antibiotics (100 U/mL penicillin, 100 g/mL streptomycin) on 100-mm dishes. To expose ECs to hypoxia, subconfluent ECs in a 100-mm dish were incubated in either 5 mL of reduced-serum medium containing 5% newborn calf serum without endothelial cell growth supplements (for HUVECs) or 4 mL of serum-free medium (for ECVs) and placed in an airtight culture chamber. The chamber was ventilated with 95% N2 and 5% CO2 for 15 minutes, then switched to a closed circuit to maintain the N2 gas mixture in the chamber and incubated at 37°C for 24 to 48 hours. ECs incubated in the same amount of serum-free medium in normal oxygen conditions served as controls for each experiment. For experiments in which an NO synthase inhibitor was used, NG-monomethyl-L-arginine (L-NMMA, Sigma Chemical Co) was added to the medium at a final concentration of 250 µmol/L to 1 mmol/L. The full-length cDNA of PKC
was cloned into the pcDNA3 mammalian expression vector
(Invitrogen) by use of the EcoRI restriction
endonucleotide enzyme site. The construct was transfected
into ECs by the lipofectin method (Life Technologies Inc). ECs that
stably expressed the empty vector or PKC were selected by resistance
to neomycin.
Immunoblot Analysis and Translocation of
PKC Isoenzymes
Cell lysates were prepared by addition of 2 mL of lysis buffer
(PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 57.4
mol/L phenylmethylsulfonyl fluoride, 2 µg/mL aprotinin, 4.2
mol/L leupeptin) per 1x107 cells.
Immunoblots were performed as previously
described.15 Polyclonal antibodies were used to
immunoblot PKC, PKC, and HSP27 (Santa Cruz
Biotechnology Inc), and mouse monoclonal antibodies were used to
immunoblot PKC, HSP70, and HSP90 (Transduction
Laboratories). To determine intracellular distribution of PKC
isoenzymes, the particulate and cytosolic fractions of ECs were
separately collected by differential
centrifugation,16 and the percentages of
individual PKC isoenzymes in each fraction were calculated as
previously reported.16
Kinase Activity Assay
Subconfluent monolayers of ECs (in 100-mm plates) were treated
with trypsin, counted, and then washed with PBS. ECs
(4.0x106 for HUVECs or
2.0x106 for ECVs) were resuspended in 1 mL of
ice-cold lysis buffer for 10 minutes and then were
homogenized by repeated aspiration through a 21-gauge
needle. Cell debris was removed by centrifugation at
3500 rpm at 4°C for 15 minutes.
To determine the specific activity of PKC, PKC, and PKC, each
individual PKC isoenzyme was immunoprecipitated from the whole-cell
lysate with the same anti-PKC specific antibody as used for
immunoblottings, and the kinase assay was carried out
according to methods described previously.15 In the
experiments that assessed PKC and PKC, the reaction mixture did
not contain additional calcium acetate. The presence of individual PKC
isoenzymes was confirmed by immunoblotting with an
anti-PKC isoenzyme specific antibody. PKC activity was normalized to
the cell number and expressed as the percentage of
simultaneously measured PKC activity of ECs cultured under
normoxic conditions.
Cell Proliferation Analysis
EC growth was determined by counting the cells with a
hemocytometer under x50 magnification. Subconfluent cells were seeded
at 2.0x105 per 16-mm plate in 1 mL of medium. At
24 hours after seeding, the ECs were exposed to either hypoxia
or normoxia with 0.3 mL serum-free medium. After a 24- to 48-hour
incubation period, ECs were harvested for counting.
Trypan Blue Exclusion Analysis
After exposure to hypoxia, ECs in 100-mm dishes were
gently treated with trypsin, suspended, and mixed with the same volume
of 0.4% trypan blue solution (Gibco BRL Products). Percentages of
viable cells were evaluated under the field of a light microscope and
normalized to the total cell number in the field.
DNA Ladder Detection Assay
ECs were harvested, washed with PBS, digested with 1 mL of lysis
buffer [10 mmol/L Tris (pH 8.0), 100 mmol/L NaCl, 25
mmol/L EDTA, 0.5% SDS, 0.1 mg/mL protease K (Gibco BRL Products)]
overnight at 37°C. Genomic DNA was precipitated with isopropanol
after phenol chloroform precipitation. Equal quantities of each sample
(20 to 30 µg) were subjected to electrophoresis on 1.25% agarose
gels containing 0.5 µg/mL ethidium bromide.
Terminal
Deoxynucleotidyltransferase
Nick-End Labeling
Subconfluent ECs in 100-mm dishes were treated with trypsin and
washed with PBS. Approximately 1x106 ECs were
fixed with 1 mL of 4% paraformaldehyde in PBS for 10
minutes, then centrifuged and resuspended with 80% ethanol for
24 hours. ECs were placed on slides and air-dried overnight. Terminal
deoxynucleotidyltransferase (TdT)
nick-end labeling (TUNEL) assay was performed on slides with a Trevigen
TACS 2 TdT (TBL) kit (Trevigen). For each slide, the number of
TUNEL-positive EC nuclei in a 0.0625-mm2 area was
scored in 12 randomly chosen high-power fields with a grid (x400). The
number of TUNEL-positive nuclei was normalized to the number of total
cells counted.
Statistical Analysis
All data are presented as mean±SEM. The effects of
different time points and cell lines were compared by ANOVA. Multigroup
comparison was carried out with Bonferroni-modified t tests.
The comparisons between normoxia and hypoxia in the same cell
lines were performed with an unpaired Student's t test.
Probability values <0.05 were accepted as statistically
significant.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Individual PKC Isoenzyme Activity of ECs in Hypoxia
Figure 1
shows individual PKC
activity normalized to normoxic ECs under similar conditions after 24
hours of hypoxia in HUVECs (A) and ECVs (B). The activity of
PKC did not change significantly with hypoxia; however,
PKC specific activity markedly decreased to 80.5±5.7% and
55.5±8.6% of that in normoxic ECs in HUVECs (n=5, P<0.05
versus normoxic controls) and HUVEC-derived ECVs (n=8,
P<0.01 versus normoxic controls), respectively. Although
PKC expression was detected in both HUVECs and ECVs by
immunoblotting, its activity could be measured only in
ECVs, in which PKC activity was unchanged by 24 hours of
hypoxia.
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For comparison, we also assessed activation of individual PKC
isoenzymes by their ability to translocate as detected by cell
fractionation and immunoblotting. The expression of
PKC in cytosolic fractions increased significantly with 24 hours of
hypoxia in both HUVECs (Figure 2A) and ECVs (Figure 2B) compared
with normoxic controls. Neither PKC nor PKC translocated
significantly. These changes could be correlated with the changes in
enzymatic activity of individual PKC isoenzymes seen in hypoxia
(Figure 1
). Conversely, the total protein levels of PKC,
PKC, and PKC did not change over a 24-hour period of
hypoxia by immunoblotting (Figure 3). Therefore, the decrease in PKC
activity in hypoxia resulted from posttranslational mechanisms,
including translocation of intracellular distribution.
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Mechanism of Hypoxia-Induced EC Death in HUVECs and
ECVs
We examined whether hypoxia injures ECs in a similar
fashion in both HUVECs and ECVs. Forty-eight hours of hypoxia
induced total cell death in 11.8±0.4% of HUVECs (n=4) and 5.9±1.0%
of ECVs (n=6). This degree of hypoxia also provoked
apoptotic ECs in 2.7±0.3% of HUVECs (n=5) and 1.1±0.2% of
ECVs (n=5). The magnitude of EC death by hypoxia in ECVs was
half that of HUVECs; however, we found that the large proportion of EC
death was due to nonapoptotic cell death in both HUVECs and
ECVs. Furthermore, HUVECs and ECVs both demonstrated a similar ratio of
apoptotic ECs to total EC death (23% in HUVECs and 19% in
ECVs). Thus, although it is a cell line, ECVs demonstrated
characteristics of EC death by hypoxia that were similar to
those of HUVECs.
EC Proliferation in Hypoxia
We examined the effect of hypoxia on ECV proliferation.
The number of ECVs incubated in serum-free medium increased over a
2-day period in normoxia in wild-type and vector controls and also in
PKC-overexpressing ECVs (PKC-ECs), but the number of ECVs
subjected to 2 days of hypoxia dropped significantly compared
with normoxic ECVs. This decrease was more severe in PKC-ECs, as
shown in Figure 4. This suggests that
PKC-ECs are more susceptible to hypoxia-induced cell
death.
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Effect of PKC Overexpression on Hypoxia-Induced EC
Death
To examine whether PKC suppression by hypoxia affected
ECV survival during hypoxia, we used trypan blue staining to
determine the number of ECVs that died after 2 days of hypoxia.
PKC-ECs demonstrated a significant increase of EC death compared
with vector controls at 24 and 48 hours of hypoxia
(5.8±1.8% versus 2.3±1.2%, P<0.05, at 24 hours of
hypoxia and 13.2±1.2% versus 4.1±0.4%, P<0.05,
at 48 hours of hypoxia, Figure 5A). PKC-ECs showed a somewhat higher
baseline cell death compared with vector controls at 24 hours of
serum-free normoxia; however, baseline cell death was comparable to
that seen at 48 hours of normoxia (2.7±0.4% versus 2.7±0.3%,
P=NS). Our results suggest that PKC suppression during
hypoxia may contribute to EC survival.
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Effect of PKC Overexpression on Hypoxia-Induced EC
Apoptosis
To investigate whether this increase of cell death in PKC-ECs
during hypoxia was due to apoptotic cell death, we
assessed the number of TUNEL-positive cells after 24 and 48 hours of
hypoxia. A small population of ECVs underwent apoptosis
at either 24 or 48 hours of hypoxia (Figure 5B), as
previously discussed. PKC-ECs did not demonstrate an increase in
apoptotic cell death above that seen with vector controls at
either 24 or 48 hours of hypoxia (0.7±0.1% versus 1.1±0.2%
and 1.6±0.3% versus 1.1±0.1%, respectively). To confirm these
findings, a DNA laddering assay was performed in ECVs subjected to up
to 48 hours of hypoxia. As shown in Figure 6, no laddering was seen in either
normoxia- or hypoxia-treated ECVs in either wild-type cells,
vector controls, or PKC-ECs. Although it is possible that neither
assay is sufficiently sensitive to detect apoptosis, these
results suggest that PKC suppression by hypoxia enhances
cell survival mainly by preventing nonapoptotic cell death
rather than apoptosis.
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Effect of NO Synthase Inhibition and HSP Expression on
Hypoxia-Induced PKC Specific Activity and
Hypoxia-Induced EC Death
Because we have reported that PKC activity can be downregulated
by activation of NO synthase in ECs stimulated by vascular
endothelial growth factor,17 we tested
whether the hypoxia-induced decrease of PKC activity was
mediated by NO synthase. In such a case, NO synthase inhibition might
exacerbate hypoxia-induced EC death by reversing PKC
suppression. No reversal of hypoxia-induced PKC suppression
was seen, however, in the presence of L-NMMA in concentrations ranging
from 250 µmol/L to 1 mmol/L [58.9±2.4% of normoxic
controls at 250 µmol/L (n=4), 41.1±10.0% at 500 µmol/L
(n=4), and 56.4±10.3% at 1 mmol/L (n=6)]. Thus, our data
suggest that the hypoxia-induced decrease in PKC activity is
mediated through a pathway that does not involve NO synthase. To
determine whether NO inhibition might affect ECV death during
hypoxia by a mechanism independent from PKC activity, the NO
inhibitor L-NMMA (500 µmol/L) was added. Such an
addition did not further increase ECV death assessed by trypan blue
exclusion, nor did it enhance apoptotic cell death in wild-type
ECVs at either 24 or 48 hours of hypoxia (Figure 7). This suggests that inhibition of NO
does not increase ECV death during hypoxia by a mechanism
independent from that resulting from sustained expression of PKC.
Similarly, immunoblots of HSP27, HSP70, and HSP90, the
latter of which, in particular, has recently been identified as a
coactivator of NO synthase,18 revealed that
these proteins were not upregulated by 24 hours of hypoxia in
either wild-type or vector controls or in PKC-ECs, although 60
minutes of 42°C heat exposure to ECVs increased expression of these
HSPs in these ECs (Figure 8).
Furthermore, HSP27, HSP70, and HSP90 were not upregulated in HUVECs by
24 hours of hypoxia (data not shown). Thus, induction of these
HSPs is not an essential function of EC adaptation to
hypoxia.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The major finding of our study is that the specific activity of PKC
is decreased by hypoxia in human ECs in both primary
cultured human ECs (HUVECs) and human EC lines (ECVs). After we
confirmed that ECVs preserved the characteristic EC injury pattern of
HUVECs by hypoxia, we overexpressed PKC in ECVs to
investigate further the physiological role of
decreased PKC activity in ECs. The enhancement of
hypoxia-induced EC death by overexpression of PKC activity,
which prevents the hypoxia-induced decrease in its activity,
suggests that the decrease in PKC activity favors EC survival during
hypoxia by preventing nonapoptotic cell death. The fact
that PKC and PKC activities were not altered by hypoxia
as determined by either enzymatic assay or translocation study suggests
that these effects are specific for PKC.
The time course of the decrease in PKC isoenzyme activity in hypoxic
ECs is consistent with the time course of the loss of the
association of this isoenzyme with the membrane of
cardiomyocytes subjected to hypoxia.7
In that experiment, as in ours, the protein levels of each PKC
isoenzyme were not altered by 24 hours of hypoxia. Thus, the
decrease of PKC activity as a consequence of hypoxia appears
to result from a posttranslational process. Our translocation studies
further support this finding. Thus, it seems that the individual PKC
isoenzymes can be altered in the physiological or
pathological setting by posttranslational mechanisms.
This study also demonstrates that overexpression of PKC in ECs
increases nonapoptotic cell death but is not associated with an
increase in hypoxia-induced apoptosis of ECs over that
seen in control ECs. The observation is in contrast to that seen in
human myeloid cancer cells,19 in which PKC activation
is required for ionizing irradiation to induce apoptosis. The
increase in nonapoptotic cell death is seen predominantly in
the presence of hypoxia; only a minimal increase is seen in the
PKC-ECs not exposed to hypoxia. Thus, it is likely that the
decrease in PKC by hypoxia is a defense mechanism that
preserves ECs by preventing nonapoptotic cell death.
The mechanism of how PKC overexpression increases
nonapoptotic cell death in the presence of hypoxia is
not clear. PKC activates Ras-dependent signals, including
AP1/Jun and MAP kinase.20 Thus, in wild-type ECs,
suppression of PKC by hypoxia may be beneficial by
decreasing the activation of the MAP cascade, which in turn decreases
fos and jun,21 which may
directly elicit changes in the expression of other genes that promote
cell death. Also, increased PKC activity both causes ECs to enter
the S phase inappropriately and slows the exit of ECs from the S
phase.22 It is possible that growth signals induced by
hypoxia,4 5 together with the accumulation of ECs
in the S phase, exert an additive or synergistic effect to initiate
cell death pathways, even though neither insult is itself sufficient to
cause cell death. Neither the decrease in PKC activity nor the
increase in EC death by hypoxia was mediated by NO synthase. NO
can decrease activity after other stimuli17 and thus might
be expected to enhance EC survival in hypoxia via suppression
of PKC. NO can also downregulate the oxygen demand of
cells23 and thus may be beneficial for cell survival
during hypoxia. In our study, however, we revealed that PKC
suppression by hypoxia is not mediated by NO synthase, nor does
inhibition of NO further increase EC death or apoptotic cell
death, suggesting that NO is not crucial for ECs to survive under
hypoxia.
HSPs have been reported to protect against
ischemia- or hypoxia-induced injury in various cell
types.12 13 The potential functions of HSPs include
suppression of proinflammatory cytokines, reduction of the
oxidative burst, NO-mediated prevention of apoptosis, and
collagen synthesis.24 Among HSPs, HSP70, HSP90, and HSP27
exert a "molecular chaperone" function that is proposed to assist
in the assembly or repair of newly synthesized or damaged proteins.
These functions may protect cells from ischemia- or
hypoxia-induced injury. We assessed whether these proteins were
involved in the protective effect of decreased PKC activity on
hypoxia-induced injury. The protein levels of HSP70 and HSP90
do not seem to be determinants of whether PKC suppression enhances
EC survival during hypoxia in our study. It has been reported
previously that hypoxia induces new categories of proteins,
called "hypoxia-associated proteins," in ECs.1
These proteins differ from HSPs, which are more commonly induced by
hypoxia in other types of cells. Recently, 1 of these proteins
was identified as a glycolytic enzyme,
glyceraldehyde-3-phosphate dehydrogenase
(GAPDH).1 In ECs, these proteins may be more important in
the cellular adaptation to hypoxia than are HSPs. The roles of
HSPs other than HSP70, HSP90, and HSP27 and that of such
hypoxia-associated proteins in hypoxia-induced EC death
remain undefined.
In conclusion, hypoxia decreases PKC specific activity in
ECs. This decrease in PKC activity may enhance EC survival during
hypoxia by preventing nonapoptotic cell death. Because
the activities of PKC and PKC do not change in ECs exposed to
hypoxia, our investigation raises the possibility that a
selective inhibitor of PKC may prevent
hypoxia-induced EC injury.
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Acknowledgments |
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This study was supported by grants HL-51043 and HL-47032 from the National Heart, Lung, and Blood Institute.
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Footnotes |
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Reprint requests to J. Anthony Ware, MD, Albert Einstein College of Medicine, Forchheimer Building, G-46, 1300 Morris Park Ave, Bronx, NY 10461.
Received April 7, 1999; revision received June 9, 1999; accepted June 17, 1999.
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Y. Shizukuda, M. E. Reyland, and P. M. Buttrick Protein kinase C-delta modulates apoptosis induced by hyperglycemia in adult ventricular myocytes Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1625 - H1634. [Abstract] [Full Text] [PDF]
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A. Wang, M. Nomura, S. Patan, and J. A. Ware Inhibition of Protein Kinase C{alpha} Prevents Endothelial Cell Migration and Vascular Tube Formation In Vitro and Myocardial Neovascularization In Vivo Circ. Res., March 22, 2002; 90(5): 609 - 616. [Abstract] [Full Text] [PDF]
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