| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Department of Medicine, University of Minnesota Health Service Center, Minneapolis, Minnesota 55455
Submitted 31 December 2002 ; accepted in final form 14 May 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
(IL-1)-activated
inducible nitric oxide (NO) synthase (iNOS) expression and that NO production
can trigger cardiac fibroblast (CFb) apoptosis. Here, we provide evidence that
angiotensin II (ANG II) significantly attenuated IL-1-induced iNOS
expression and NO production in CFbs while simultaneously decreasing apoptotic
frequency. The anti-apoptotic effect of ANG II was abolished when cells were
pretreated with the specific ANG II type 1 receptor (AT1) antagonist losartan,
but not by the AT2 antagonist DP-123319. Furthermore, ANG II also protected
CFbs from apoptosis induced by the NO donor diethylenetriamine NONOate and
this effect was associated with phosphorylation of Akt/protein kinase B at
Ser473. The effects of ANG II on Akt phosphorylation and NO
donor-induced CFb apoptosis were abrogated when cells were preincubated with
the specific phosphatidylinositol 3-kinase inhibitors wortmannin or LY-294002.
These data demonstrate that ANG II protection of CFbs from IL-1-induced
apoptosis is associated with downregulation of iNOS expression and requires an
intact phosphatidylinositol 3-kinase-Akt survival signal pathway. The findings
suggest that ANG II and NO may play a role in regulating the cell population
size by their countervailing influences on cardiac fibroblast viability.
inducible nitric oxide synthase; donor; losartan
ANG II has been shown to stimulate cell proliferation and ECM production by CFbs during the development of pathological ventricular hypertrophy caused by ischemic injury or hemodynamic overload (22, 24, 31, 36, 37). Current evidence indicates that ANG II can indirectly promote proliferation of CFbs by stimulating the synthesis of growth factors such as the platelet-derived growth factor (2, 5, 24, 33) because there is no evidence that ANG II can directly trigger cell division through activation of the cyclin-dependent pathway (2, 24). The size of a cell population is governed by the balance between cell proliferation and cell death, but little is known about the effect of ANG II on fibroblast viability in the heart.
We previously demonstrated that interleukin-1 (IL-1)-induced
inducible NO synthase (iNOS) expression and NO production can trigger CFb
apoptosis (31), identifying NO
as a candidate for regulation of CFb viability. It is unknown whether ANG II
can modulate NO-induced CFb apoptosis, although ANG II has been shown to
attenuate NO-induced vascular smooth muscle cell (SMC) apoptosis
(30). Consequently, this study
was performed to examine the impact of ANG II on endogenous and exogenous
NO-induced CFb apoptosis. We found that ANG II significantly attenuated
IL-1 and NO donor-induced CFb apoptosis through AT1 receptor-mediated
mechanisms. This anti-apoptotic effect of ANG II is associated with
downregulation of IL-1-induced iNOS expression and requires a
phosphatidylinositol 3-kinase (PI3-kinase)-Akt/protein kinase B
(PKB)-dependent survival signal.
|
METHODS AND MATERIALS |
|---|
|
TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
was purchased from R&D
System (Minneapolis, MN). The antibody against iNOS was from Santa Cruz
Biotechnology (Santa Cruz, CA). The antibodies against phosphorylated Akt were
from Cell Signaling (Beverly, MA). The NO donor diethylenetriamine (DETA)
NONOate was purchased from Cayman Chemical (Ann Arbor, MI). All other reagents
were purchased from Sigma (St. Louis, MO). Primary culture of cardiac fibroblasts. All animal procedures were conducted in accordance with the National Institutes of Health Guide for the Care of Laboratory Animals, and were approved by the University of Minnesota Animal Care Committee. Cardiac fibroblasts were isolated from adult male Sprague-Dawley rats (180–200 g) as previously described with minor modification (35). Rats were anesthetized with pentobarbital sodium (25 mg/kg), and the hearts were removed. The left ventricles were minced and washed in Hanks' balanced salt solution. Cells were released by digesting the tissue with a mixture of 0.1% trypsin (GIBCO-BRL Life Technologies; Grand Island, NY) and 100 U/ml of collagenase (Type IV) for 10 min per cycle at 37°C. Cells from the second to fifth digestion cycle were collected and cultured in flasks containing DMEM plus 20% FCS at 37°C, 10% CO2-90% O2 for 2 h. The attached cells (>95% are cardiac fibroblasts) were allowed to grow in DMEM containing 10% FCS at 37°C, 5% CO2-95% O2 until they were confluent. The cells were subcultured one or two more times before use. Fibroblasts were distinguished from other myocardial cell types by the presence of the fibroblast marker vimentin (catalog no. V-5255, Sigma), and absence of the endothelial cell marker von Willebrand factor (catalog no. F-3520, Sigma), or the muscle cell marker desmin (catalog no. D-1033, Sigma). The presence of these various markers was examined by immunofluorescent staining with appropriate secondary antibodies as previously described (35).
Induction of iNOS expression. Induction of iNOS expression was
performed as previously described
(35). Cardiac fibroblasts were
subcultured in dishes containing 10% FCS (at 37°C, 5% CO2-95%
O2) for 16 h. Cells were washed with prewarmed 1x PBS and
switched to DMEM with low serum (0.1% FCS) and incubated for 24 h. All
subsequent experiments were performed in low-serum DMEM. Cells were treated
with IL-1 or the NO donor DETA NONOate in the presence or absence of the
designated agents for the indicated time intervals. The concentrations of
agents used in this study were chosen on the basis of our previous studies
(35). The medium from the
treated and nontreated cells was collected and stored at –70°C until
use. Cells were lysed and lysate protein was subjected to immunoblot analysis
for iNOS protein expression. Another parallel set of cells were fixed with
precooled methanol and subjected to immunostaining for morphological
analysis.
NO measurement. Assessment of NO production in the culture medium was performed with the use of the Griess reagent that measures NO2, the major NO metabolite in the cell culture system (35). One hundred microliters of sample or standard (sodium NO2 served as the standard) was added into each well of the 96-well plate and 100 µl of Griess reagent, a mixture of one part of Griess reagent A containing 0.1 g of N-[-1-naphythyl]-ethylenediamine hydrochloride in 100 ml of water and one part of Griess reagent B containing 1 g of sulfanilamide in 100 ml of 3 N HCl was added. After incubation at room temperature for 15 min, the samples were read in a spectrophotometer at a 550-nm wavelength. The amount of NO2 was calculated with the use of a standard curve calibrated with NaNO2 at concentrations of 0.1–80 µM. Assessment of apoptosis. Apoptotic cells were identified by direct staining of the condensed nuclei or fragmented DNA in cells with bisbenzimide (Hoechst 33258) or TdT-mediated dUTP nick end labeling (TUNEL)-based staining. For Hoechst 33258 staining, the bisbenzimide stock solution was added directly into the culture medium (at a final concentration of 0.02%) and incubated with the cells for 20 min at 37°C. The cells were subjected to microscopic analysis with the use of an inverted phase-contrast fluorescence microscope (model MDIRB/E, Leica; Deerfield, IL). TUNEL-based staining was performed on methanol-fixed CFbs with the use of the TUNEL staining kit by following the manufacturer's instructions (Roche; Indianapolis, IN). Image processing was performed with the use of NIH Image software, Adobe Photoshop, and a Fuji Pictography 3000 color printer. For quantitative assessment of apoptosis, TUNEL-positive cells were counted in 500 cells per sample and expressed as the percentage of apoptotic cells.
Western blot analysis. Western blot analysis for iNOS expression
was performed as previously described
(34,
35) with minor modification.
Cells were harvested and lysed with lysis buffer (50 mM Tris · HCl, pH
7.5, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, and 0.5% Nonidet P-40) containing a
protease inhibitor cocktail (Boehringer-Mannheim; Indianapolis, IN). The
lysate was clarified by centrifugation at 16,000 g for 15 min at
4°C. Equal amounts of total protein were subjected to 8% SDS-PAGE and
electrophoretically transferred to a High-Bond nitrocellulose membrane
(Amersham Life Science; Arlington Heights, IL). After being blocked with 20 mM
Tris · HCl, pH 7.6, 137 mM NaCl, and 0.05% Tween 20 (TTBS) containing
5% nonfat milk for 1 h, the membrane was incubated for 1 h at room temperature
with appropriate primary antibodies (anti-iNOS antibody was diluted at 1:500
and anti-phospho-Akt antibody was diluted at 1:1,000) in blotting buffer (TTBS
with 5% nonfat milk). After being washed three times for 10 min each in TTBS,
the membrane was incubated with an appropriately diluted
horseradish-peroxidase-labeled secondary antibody (1:2,000) in blotting buffer
for 1 h at room temperature. The membrane was washed three times, reacted with
enhanced chemiluminescence reagent (Amersham Life Science) and subjected to
autoradiography. The strength of the signal was analyzed using densitometry
and the results expressed as arbitrary units. Protein levels were standardized
by comparison with anti--tubulin antibody.
Statistical analysis. Each experiment was repeated at least three times. Data are presented as means ± SE. Comparison between groups was performed by two-way ANOVA. Significance was considered as P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
-induced CFb apoptosis. To
examine the effect of ANG II on IL-1-induced apoptosis, CFbs were seeded
in culture dishes or chamber slides and treated with IL-1 alone or
combined with other reagents for 16 h, followed by assessment of apoptosis by
immunostaining and TUNEL assay. In control CFbs, no apoptosis was detected
(Fig. 1A,A). In accord
with our earlier study (35),
in IL-1 (5 ng/ml)-treated CFbs apoptosis was apparent as evidenced by
nuclear condensation and cell shrinkage
(Fig. 1A,B). The
apoptotic frequency was markedly attenuated when ANG II was added immediately
before the addition of IL-1 (10–6 M,
Fig. 1A,C). The
anti-apoptotic effect of ANG II was abolished by preincubating the cells for 3
h with the ANG type 1 (AT1)-selective receptor blocker losartan (3 x
10–5 M, Fig.
1A,D), but not by preincubating for 3 h with the
selective AT2 receptor blocker P-123319 (3 x
10–5 M; Fig.
1A,E). CFb apoptosis was quantitated with the use of a
TUNEL-based assay (Fig.
1B). TUNEL-positive cells constituted <2% of the
control group, whereas the frequency of TUNEL-positive cells was increased to
35% after exposure to IL-1. The frequency of TUNEL-positive cells was
decreased to 7.5% by treatment with ANG II. The antiapoptotic effect of ANG II
was abolished when the cells were preincubated for 3 h with the selective AT1
receptor blocker losartan, whereas preincubation for 3 h with the selective
AT2 receptor blocker had no effect on the antiapoptotic effect of ANG II.
These data imply that ANG II is capable of rescuing CFbs from
IL-1-induced apoptosis through AT1 receptor-mediated mechanism(s).
|
ANG II decreases IL-1-induced iNOS expression and NO
production. Because IL-1-induced apoptosis in CFbs has been shown
to be mediated, at least in part, by NO production, studies were performed to
determine whether ANG II has an effect on IL-1-induced iNOS expression.
CFbs were treated with IL-1 alone or combined with other reagents for 16
h, followed by Western blot analysis for iNOS protein expression. iNOS protein
expression was induced in CFbs treated with IL-1 (5 ng/ml), and this
increase was significantly reduced when ANG II (1 x
10–6 M) was added immediately before IL-1
(Fig. 2A). The
decrease in IL-1-induced iNOS expression produced by ANG II was reversed
by preincubation with losartan (3 x 10–5 M)
for 3 h before the addition of ANG II, but not by preincubation with DP-123319
(3 x 10–5 M) for 3 h, implying that the ANG
II receptor on iNOS expression is mediated via a type 1 ANG II receptor. ANG
II alone had no effect on iNOS expression
(Fig. 2A). Parallel
changes were observed in the NO concentration in the medium collected from
these CFbs (Fig. 2B).
These data demonstrate that ANG II can rescue CFbs from endogenous NO-induced
apoptosis, at least in part by downregulating iNOS expression induced by
IL-1.
|
ANG II rescues cells from NO donor-induced CFb apoptosis. Although
the anti-apoptotic effect of ANG II on IL-1-induced CFb apoptosis
appeared to be at least in part due to downregulation of iNOS protein
expression, we also examined whether ANG II is capable of protecting CFbs from
apoptosis induced by exogenous NO. In these experiments, CFbs were exposed to
the NO donor DETA NONOate (3 x 10–5 M) alone
or combined with ANG II (10–6 M; ANG II was added
5 min before the addition of DETA NONOate) for 8 h, followed by analysis of
nuclei by Hoechst 33258 staining. Compared with the control CFbs
(Fig. 3A,A), apoptotic
frequency in CFbs treated with DETA NONOate was significantly increased
(Fig. 3A,B).
Preincubation of the cells with ANG II for 5 min markedly attenuated DETA
NONOate-induced apoptotic frequency (Fig.
3A,C). The antiapoptotic effect of ANG II was abolished
when CFbs were preincubated with the selective AT1 inhibitor losartan (3
x 10–5 M) for 5 min before exposure to ANG
II and DETA NONOate (Fig.
3A,D). Apoptosis quantified by TUNEL-based staining
(Fig. 3B) demonstrated
that ANG II inhibited CFb apoptosis induced by exogenous NO.
|
ANG II stimulates Akt/PKB activity via PI3-kinase-dependent pathway. Because ANG II was able to rescue CFbs from exogenous NO-induced apoptosis, we next addressed the question of mechanism. Because phosphorylation of Akt/PKB on Ser473 has been implicated as a universal survival mediator in many cell types, we examined the effect of ANG II on Akt/PKB phosphorylation. We found that ANG II rapidly phosphorylated Akt on Ser473 in a dose- and time-dependent fashion (Fig. 4, A and B), implying that phosphorylation of Akt by ANG II may activate an Akt-mediated survival signaling pathway. ANG II stimulated phophorylation of Akt Ser473 was detected as early as 2.5 min, peaked between 5 and 10 min, and returned to the basal level within 30 min (Fig. 4A). The effect of ANG II on Akt phosphorylation at Ser473 was completely abolished by preincubation for 3 h with the PI3-kinase inhibitors wortmannin (20 nM) or LY-294002 (20 µM) (Fig. 4C). Furthermore, the antiapoptotic effect of ANG II on DETA NONOate-induced apoptosis was abolished when the cells were preincubated for 3 h with wortmannin or LY-294002 (Fig. 5). The apoptotic frequency in control CFbs was <2% and was increased to 45% after exposure to DETA NONOate. The apoptotic frequency was reduced to 6.5% in cells preincubated with ANG II for 5 min before exposure to DETA NONOate. The antiapoptotic effect of ANG II was abolished when the cells were preincubated for 3 h with either wortmannin (47%) or LY-294002 (46%) before treatment with DETA and ANG II. These data provide evidence that ANG II can rescue CFbs from NO-induced apoptosis by activation of a PI3-kinase-Akt/PKB-mediated survival-signaling pathway.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
. Second, when NO was supplied from an exogenous source, ANG II
provided protection against apoptosis by activation of a
PI3-kinase-Akt/PKB-mediated survival signaling pathway. Our data establish a
role for both ANG II and NO in regulating the size of CFb population by their
opposing influences on the viability of cardiac fibroblasts. Accumulating evidence suggests that elevated levels of ANG II play an important role in regulation of cardiac remodeling after myocardial infarction or in response to hemodynamic overload (31, 36, 37). ANG II exerts several relevant biological effects on cardiac remodeling. Chronic infusion of low levels of ANG II can induce cardiac hypertrophy and myocardial fibrosis (14), and overexpression of AT1 receptors in transgenic mice induces cardiomyocte hypertrophy and collagen deposition (28). In vitro studies have shown that ANG II potently stimulates the synthesis of a variety of ECM proteins by CFbs, including types I and III collagen (2, 22). In addition, ANG II causes proliferation of CFbs by stimulating autocrine production of growth factors such as platelet-derived growth factor (5, 33). In agreement with these findings, inhibition of the reninangiotensin system with angiotensin-converting enzyme inhibitors or AT1 blockers has been shown to prevent or cause regression of excessive deposition of collagen and CFb hyperplasia both in patients with hypertrophic cardiomyopathy and in experimental animal models of cardiac overload (32, 36, 37). Finally, the present study provides evidence that ANG II enhances CFb survival by activating a PI3-kinase Akt/PKB-mediated survival signal. Together, these data support the concept that ANG II can enhance CFb survival and function by its influence on proliferation and viability.
The influence of ANG II on viability is cell type and cell context specific. Several investigators (18, 30) have reported that ANG II can rescue SMC and cardiac myocytes from apoptosis. In contrast, ANG II has been reported to induce apoptosis in alveolar epithelial cells, endothelial cells, and vascular SMC (1, 10, 11, 20, 27). The mechanism(s) of ANG II-induced apoptosis in these cells is not well understood, although it has been shown that both AT1 and AT2 receptors were involved in ANG II-induced apoptosis (7, 9, 11, 13, 19, 27). The involvement of the AT2 receptor in ANG II-induced apoptosis is not unexpected because AT2 receptor overexpression has been reported to mediate ANG II-induced apoptosis, whereas deletion of the AT2 receptor antagonizes ANG II-induced apoptosis (15, 22, 38). However, involvement of the AT1 receptor in ANG II-induced apoptosis was unexpected because ANG II has been shown to promote proliferation or hypertrophy of several cell types (2, 9, 16, 33). In the present study, we found that ANG II prevented both endogenous and exogenous NO-induced CFb apoptosis through an AT1 receptor-mediated mechanism. It is worth noting that we did not observe detectable levels of AT2 receptor in cultured adult CFbs by immunostaining or by Western blot analysis (data not shown), indicating that lack of response to the AT2 blocker may have been due to extremely low expression of this receptor in adult rat CFbs. Nevertheless, our data support the hypothesis that ANG II can regulate CFb viability by AT1 receptor-mediated activation of PI3-kinase/Akt-dependent survival signaling.
The PI3-kinase/Akt/PKB signaling pathway has been demonstrated to elaborate
important survival signals in many cell types
(3,
6). ANG II can activate Akt/PKB
in a PI3-kinase-dependent manner in several types of cells
(12), but it had been unclear
whether activation of the PI3-kinase-Akt/PKB pathway is involved in ANG
II-mediated survival signaling in CFbs. We previously demonstrated that
phosphorylation of Ser473 of Akt by antibody ligation of
1-integrin can prevent human lung fibroblast apoptosis
induced by collagen contraction, suggesting that the PI3-kinase-Akt/PKB signal
pathway can regulate lung fibroblast viability
(34). In the present study, we
have demonstrated that ANG II is capable of reversing NO-induced CFb apoptosis
by providing a PI3-kinase-Akt/PKB-mediated survival signal. In an attempt to
confirm the involvement of Akt phosphorylation in the ANG II activated
survival signaling pathway, we transiently transfected CFbs with truncated Akt
(lacking the catalytic domain) to determine whether this would interrupt the
ability of ANG II to rescue the cells from NO-induced apoptosis. However, we
were unable to perform this experiment because, on reducing or withdrawing
serum in anticipation of adding the NO donor, we found that the majority of
cells spontaneously underwent apoptosis (data not shown). Nevertheless, this
indicates that Akt is important for CFb survival, because loss of Akt function
itself induced CFb apoptosis during low-serum conditions. Because ANG II is
involved in multiple signaling pathways, such as activation of protein kinase
C, ANG II may also activate other signaling pathways having antiapoptotic
effects (8). Moreover, much
evidence suggests that Akt/PKB mediates a universal survival pathway, which
regulates the viability of many types of mammalian cells
(6); >10 different
downstream effectors of Akt have been identified, reflecting the complexity of
this signaling pathway (3,
6). Additional studies will be
needed to determine which downstream factors are involved in the ANG
II-induced PI3-kinase-Akt/PKB survival-signaling pathway in CFbs.
NO has been reported to trigger apoptosis or protect cells from apoptotic stimuli, depending on the concentration of NO as well as the cell type (26). The relatively low physiological concentrations of NO generated by eNOS or nNOS can act as an antiapoptotic effector in several cell systems, including endothelial cells (26). In contrast, iNOS expression associated with pathological processes, such as inflammation, wound repair, or heart failure results in production of high NO levels that can trigger apoptosis in several cell types, including macrophages and SMCs (1, 23). Our previous study (35) demonstrated that both endogenous NO produced by iNOS or exogenous NO can induce CFb apoptosis in vitro, suggesting that NO may participate in regulation of the population size of cardiac fibroblasts.
In summary, the present data provide evidence that ANG II can modulate
NO-induced CFb apoptosis by downregulating iNOS expression induced by
IL-1 and by activating a PI3-kinase-Akt/PKB-mediated survival signal
that opposes NO-stimulated apoptosis. Our data support the hypothesis that ANG
II and NO may play a role in balancing the population size of cardiac
fibroblasts by their countervailing influences on cell viability.
|
DISCLOSURES |
|---|
|
TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
|
FOOTNOTES |
|---|
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.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODS AND MATERIALSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
1 integrin regulates fibroblast
viability during collagen matrix contraction through a phosphatidylinositol
3-kinase/Akt/protein kinase B signaling pathway. J Biol
Chem 277:
24667–24675, 2002.This article has been cited by other articles:
|
|
 |
|
  L. Pattacini, B. Casali, L. Boiardi, N. Pipitone, L. Albertazzi, and C. Salvarani Angiotensin II protects fibroblast-like synoviocytes from apoptosis via the AT1-NF-{kappa}B pathway Rheumatology, August 1, 2007; 46(8): 1252 - 1257. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. R. Olson, J. E. Naugle, X. Zhang, J. A. Bomser, and J. G. Meszaros Inhibition of cardiac fibroblast proliferation and myofibroblast differentiation by resveratrol Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1131 - H1138. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Schiffrin and R. M. Touyz From bedside to bench to bedside: role of renin-angiotensin-aldosterone system in remodeling of resistance arteries in hypertension Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H435 - H446. [Full Text] [PDF]
|
|
|
|
 |
|
 F. Andreozzi, E. Laratta, A. Sciacqua, F. Perticone, and G. Sesti Angiotensin II Impairs the Insulin Signaling Pathway Promoting Production of Nitric Oxide by Inducing Phosphorylation of Insulin Receptor Substrate-1 on Ser312 and Ser616 in Human Umbilical Vein Endothelial Cells Circ. Res., May 14, 2004; 94(9): 1211 - 1218. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
