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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Houston VA Medical Center and the Departments
of Medicine and Pharmacology, Baylor College of Medicine, Houston, TX.
Vascular smooth muscle cells (SMCs) generate carbon monoxide (CO)
via the catabolism of heme by the enzyme heme oxygenase (HO). In the
present study, we found that serum stimulated a time- and
concentration-dependent increase in the levels of HO-1 messenger RNA
(mRNA) and protein in vascular SMCs. The induction of HO-1 expression
by serum was inhibited by actinomycin D or cycloheximide. In addition,
serum stimulated HO activity, as reflected by an increase in the
concentration of bilirubin in the culture media. Treatment of vascular
SMCs with serum stimulated DNA synthesis and this was potentiated by
the HO inhibitors, zinc and tin protoporphyrin-IX as well as by the CO
scavenger, hemoglobin. The iron chelator desferrioxamine had no effect
on DNA synthesis. However, exposure of vascular SMCs to exogenous CO
inhibited serum-stimulated SMC proliferation and the phosphorylation of
retinoblastoma protein. In addition, CO arrested SMCs at the
G1/S transition phase of the cell cycle and selectively
blocked the serum-stimulated expression of cyclin A mRNA and protein
without affecting the expression of cyclin D1 and E. CO also inhibited
the serum-stimulated activation of cyclin A-associated kinase activity
and cyclin-dependent kinase 2 activity. These results demonstrate that
serum stimulates HO-1 gene expression and CO synthesis. Furthermore,
they show that CO acts in a negative feedback fashion to inhibit
vascular SMC growth by regulating specific components of the cell cycle
machinery. The capacity of vascular mitogens to induce CO
synthesis may provide a novel mechanism by which these agents modulate
cell growth.
(Blood. 2002;99:4443-4448) Heme oxygenase (HO) catalyzes the initial and
rate-limiting step in heme metabolism. It oxidatively degrades heme
into equimolar amounts of carbon monoxide (CO), free iron, and
biliverdin.1 Biliverdin is subsequently metabolized to the
potent antioxidant bilirubin by biliverdin reductase.1
Three distinct isoforms of HO have been identified and
cloned.2-4 These isozymes are products of different genes
and differ markedly in their tissue distribution. Both HO-2 and HO-3
are constitutively expressed isoforms that are present in high
concentration in selected mammalian tissues.3,4 In
contrast, the HO-1 isoform is ubiquitously distributed and is strongly
induced by a variety of physiologic and pathophysiologic stimuli,
including heme, heavy metals, inflammatory cytokines, endotoxin, nitric
oxide, and hemodynamic forces.3,5-7 All 3 isoforms of HO
are inhibited by various metalloprotoporphyrins, such as zinc (Zn) and
tin (Sn) protoporphryin-IX (ZnPP, SnPP)8. In addition, the
HO-1 metabolites iron and CO can be inactivated by the iron chelator
desferrioxamine and by the CO scavenger hemoglobin (Hb),
respectively.9,10
Recent studies indicate that HO-1 plays an important role in the
circulation. The induction of HO-1 by endotoxin contributes to the
severe hypotension associated with septic shock.6 In addition, the expression of HO-1 in the blood vessel impairs
contractile function and causes a marked decrease in blood pressure in
spontaneously hypertensive rats.11,12 In contrast, HO
inhibitors increase blood pressure and peripheral resistance,
suggesting a critical vasoregulatory role for HO-1.13 HO-1
also modulates platelet-vessel wall interactions. Using a
platelet-vascular smooth muscle cell (SMC) coincubation system, we
found that the induction of HO-1 in SMCs inhibits platelet aggregation,
indicating a potentially important antithrombotic role for this
enzyme.7 These HO-1-mediated effects on cell function are
mediated via the release of CO.7,12-14 Finally, HO-1 also
confers significant cytoprotective effects on vascular cells. The
induction of HO-1 in vascular cells leads to an increased resistance to
oxidative stress, whereas HO-1 deficiency results in enhanced cell
injury.15-17 The cytoprotective effect of HO-1 is
dependent on the generation of bilirubin.15,16
Interestingly, we and others have demonstrated that growth
factors are potent inducers of HO-1.10,18,19 These
findings raise the possibility that HO-1 may also affect cell growth.
Accordingly, the present study examined whether HO-1 affects the
proliferative response of vascular SMCs. We now report that serum
stimulates HO-1 gene expression and that the HO-1-catalyzed release of
CO functions in an autocrine manner to limit vascular SMC
proliferation. The ability of growth factors to induce CO synthesis may
represent a novel mechanism by which vascular mitogens regulate SMC growth.
Materials
Cell culture
Messenger RNA analysis HO-1 messenger RNA (mRNA) levels were determined by ribonuclease protection analysis using a commercially available kit (Ambion). Total RNA (10 µg) was hybridized with approximately 1 × 106 cpm of [32P]UTP-labeled antisense HO-1 (284-base pair [bp]) and GAPDH (316-bp) riboprobes. The HO-1 antisense RNA probe was prepared as described earlier.5 Protected RNA was analyzed by electrophoresis using 6% acrylamide/8 mM urea gel. Gels were exposed overnight to x-ray film at 70°C in the presence of
intensifying screens. The size of the predicted nucleotide-protected
fragments was confirmed by using a 32P-labeled RNA ladder.
Cyclin A mRNA levels were determined by Northern blotting. Total RNA
(30 µg) was loaded on 1.2% agarose gels and fractionated by
electrophoresis. RNA was blot transferred to Gene Screen Plus membranes
and prehybridized for 4 hours at 68°C in hybridization buffer
(rapid-hyb buffer, Amersham). Membranes were hybridized overnight at
68°C in hybridization buffer containing [32P]DNA probes
(1 × 108 cpm) for cyclin A and 18 S ribosomal RNA. DNA
probes were labeled with Protein analysis The SMCs were lysed in electrophoresis buffer (125 mM Tris, pH 6.8, 12.5% glycerol, 2% SDS, and trace bromophenol blue) and boiled for 10 minutes. SDS-polyacrylamide gel electrophoresis (PAGE) was performed on gels with 20 µg protein using the buffer system of Laemmli.21 The separated blots were electrophoretically transferred to nitrocellulose membranes and blocked for one hour in phosphate-buffered saline (PBS) containing 0.1% Tween-20 and 3% nonfat milk. Blots were incubated with antibodies directed against HO-1 (1:500 dilution), pRb (1:750 dilution), cyclin D1 (1 µg/mL), cyclin E (2.5 µg/mL), cyclin A (0.7 µg/mL), cdk2 (1 µg/mL), or the housekeeping protein -tubulin (5 µg/mL) in PBS containing Tween-20
(0.1%) for 1 hour. Membranes were then washed in PBS and incubated for
1 hour with horseradish peroxidase-conjugated goat antirabbit or goat
antimouse antibody. After further washing with PBS, blots were
incubated in commercial chemoluminescence reagents (Amersham) and
exposed to photographic film.
Cyclin A-associated kinase and cdk2 assay The SMCs were collected in lysis buffer (150 mM Tris, pH 7.5, 200 mM NaCl, 2.0 mM EDTA, 2.0 mM EGTA, 10% glycerol, 0.1% Tween-20, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 1 mM PMSF, 1 mM Na3VO4, 50 mM NaF, and 1 mM DTT), and sonicated twice on ice at 30% for 10 seconds. Protein (300-500 µg) was immunoprecipitated using protein A agarose beads (40 µL; Santa Cruz Biotechnology), cyclin A (2 µg), or cdk2 (1 µg) antibody, and lysis buffer (400 µL) overnight at 4°C. Agarose beads were washed 3 times with lysis buffer, 2 times with kinase buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 1 mM DTT, 1 mM Na3VO4, and 1 mM DTT), and all wash buffer removed. For kinase assays, kinase buffer (40 µL), histone H1 (2.0 µg; Roche, Indianapolis, IN), ATP (50 µM), and -[32P]ATP (6 µCi [.222
MBq]) were added to the agarose beads for 20 minutes at
30°C. Reactions were stopped by adding Laemmli buffer and samples
boiled for 5 minutes. Proteins were separated by SDS-PAGE, fixed, and
exposed to x-ray film.
HO activity The activity of HO was measured spectrophotometrically by quantifying the release of bilirubin into the culture media.22 Culture media (500 µL) were collected and combined with barium chloride (250 mg) and benzene (750 µL). After vortexing, the benzene phase containing the extracted bilirubin was separated from the aqueous phase by centrifugation at 13 000g for 30 minutes. Bilirubin was determined spectrophotometrically as a difference in absorbance between 450 and 600 nm using an excitation coefficient of 27.3 mM 1cm1.
SMC proliferation The SMCs were seeded at a density of 5 × 104 cells/well in 12-well plates in serum (10%)-containing media. After 24 hours, culture media were exchanged for serum-free media, and SMCs were incubated for an additional 48 hours. SMCs were then treated with serum in the presence or absence of CO. Media with appropriate additions were replenished every second day. Cell number determinations were performed after 4 days of treatment by dissociating cells with trypsin (0.025%)/EDTA (1 mM) and counting cells in a calibrated Coulter Counter (model ZF, Coulter Electronics, Hialeah, FL).DNA synthesis Quiescent SMCs were treated with serum in the presence or absence of CO for 20 hours. Then [3H]thymidine (1 µCi/mL [0.037 MBq]) was added and cells incubated for an additional 4 hours. For [3H]thymidine incorporation, SMCs were washed 3 times with ice-cold PBS, fixed with 10% trichloroacetic acid for 30 minutes at 4°C, and DNA extracted with 0.2% SDS/0.2 N NaOH. Radioactivity was determined by scintillation spectrophotometry (Tricarb liquid scintillation analyzer, model 1900, Packard, Meriden, CT). In some experiments the uptake of [3H]thymidine by SMC was monitored. For these experiments, SMCs were incubated with [3H]thymidine (1 µCi/mL for 4 hours [0.037 MBq]), lysed in 0.2% SDS/0.2 N NaOH, and radioactivity measured by scintillation counting.Cell cycle analysis Quiescent SMCs were treated with serum in the presence and absence of CO for 24 hours. Cells were then dispersed with trypsin (0.025%)/EDTA (1 mM), suspended in PBS, and fixed in 70% ethanol for 30 minutes. The ethanol was removed and SMCs were incubated in PBS containing RNase (0.5 mg/mL) for 30 minutes at 37°C, and then stained with 0.005% propidium iodide. DNA fluorescence was measured on a Dickinson FACScan flow cytometer (Franklin Lakes, NJ). The area versus the width of the fluorescent signal was analyzed to gate out cellular multiplets. Histograms of DNA content were analyzed using Modfit Lt V1.01 (Verity Software House, Topsham, ME) to determine fractions of the population in each phase of the cell cycle.CO exposure The SMCs were exposed to CO via a previously described environmental chamber.23,24 CO at a concentration of 1% in air was combined with air containing 5% CO2 in a stainless steel mixing cylinder prior to delivery into the humidified 37°C environmental chamber. Flow into the chamber was at 1 L/min and CO levels in the chamber were continuously monitored by electrochemical detection using a CO analyzer (Interscan, Chatsworth, CA).Statistics Results are expressed as the means ± SEM. Statistical analysis was performed with the use of a Student 2-tailed t test, and an analysis of variance when more than 2 treatment regimens were compared. P < .05 was considered statistically significant.
Treatment of vascular SMCs with serum (10%) resulted in a
time-dependent increase in HO-1 mRNA and protein. An increase in HO-1
message was first detected after 4 hours of exposure, reached a maximum
at 8 hours, and then declined to basal levels by 24 hours (Figure
1A). Serum (10%) also induced a marked
increase in HO-1 protein that was first detected after 4 hours of
exposure; however, this increase was sustained for 24 hours (Figure
1B). Serum-mediated increases in HO-1 mRNA and protein were
concentration-dependent (Figure 2) and
were specific for the HO-1 isoform (data not shown). Incubation of SMCs
with the protein synthesis inhibitor cycloheximide (5 µg/mL) or with
the transcriptional inhibitor actinomycin D (2 µg/mL) blocked the
serum-mediated increase in HO-1 mRNA and protein (Figure
3). In addition, serum had no effect on
the stability of HO-1 mRNA. Incubation of SMCs with actinomycin D (2 µg/mL) resulted in the decay of HO-1 message with a half-life of
about 2 hours, and this remained unchanged in the presence of
serum (data not shown). Finally, because PDGF and TGF-
In subsequent experiments, HO-1 activity was measured by monitoring the
release of bilirubin by vascular SMCs. In the absence of serum,
bilirubin was not detected in culture media (Figure 4). However, serum (10%) induced a
marked rise in bilirubin concentration that was dependent on HO
activity because it was blocked by ZnPP (Figure 4).
Treatment of vascular SMCs with serum stimulated over a 3-fold increase
in DNA synthesis that was potentiated by the HO inhibitors, ZnPP (20 µM) and SnPP (20 µM; Figure 5). In
addition, the CO scavenger Hb (50 µM) further increased
serum-stimulated DNA synthesis (Figure 5). In contrast, the iron
chelator desferrioxamine (100 µM) had no effect on DNA synthesis
(Figure 5). In the absence of serum, the HO inhibitors or Hb minimally
affected DNA synthesis (data not shown).
To confirm that CO exerts an antiproliferative effect, vascular
SMCs were treated with SnPP (20 µM), to prevent the endogenous formation of CO, placed in an environmental chamber, and exposed to
CO.23,24 Exposure of vascular SMCs to CO (100 ppm)
significantly attenuated serum-stimulated SMC proliferation and
thymidine incorporation (Figure 6A,B). In
addition, the inhibition of thymidine incorporation by CO (0-200 ppm)
was concentration dependent (Figure 6C). Control experiments indicated
that CO had no effect on the uptake of thymidine by SMCs (data not
shown).
The CO-mediated inhibition of SMC growth was associated with a marked
reduction in serum-mediated phosphorylation of pRb (Figure 7). In addition, flow cytometric data
indicated that CO arrested SMCs in the G1/S transition
phase of the cell cycle (Figure 8A). Treatment of vascular SMC with serum also stimulated the expression of
cyclin A, cyclin D1, and cyclin E protein (Figure 8B). However, CO
exposure selectively attenuated the serum-stimulated expression of
cyclin A protein without affecting the expression of cyclin D1 or E
(Figure 8B). CO also blocked the induction of cyclin A mRNA expression
by serum (Figure 8C). Because cyclin A is an activating subunit of
cdk2, we also examined the effect of CO on cdk activity.25 CO potently inhibited the serum-mediated increase in both cyclin A-associated kinase and cdk2 activity (Figure
9A). However, CO had no effect on the
expression of cdk2 protein (Figure 9B). Finally, the administration of
CO did not induce cell necrosis or apoptosis (data not shown).
The present study demonstrates that serum induces the expression of the HO-1 gene and that HO-1 acts in a negative feedback manner to limit vascular SMC proliferation. Serum stimulates HO-1 mRNA and protein in a time- and concentration-dependent manner. The serum-mediated up-regulation of HO-1 gene expression is dependent on de novo RNA and protein synthesis and likely involves the transcriptional activation of the gene because serum does not alter the stability of HO-1 mRNA. In addition, the induction of HO-1 by serum is associated with a marked increase in HO activity. The ability of serum to stimulate HO-1 gene expression is also observed
after the administration of PDGF, TGF- We also found that CO interacts with various components of the cell cycle machinery. CO inhibits the phosphorylation of pRb, a critical event required for S-phase entry and DNA synthesis.27 The inhibition of pRb phosphorylation was demonstrated by a mobility shift assay where CO blocked the serum-mediated formation of the slower-migrating hyperphosphorylated form of pRb. Moreover, Western blotting using a phosphorylation-specific antibody against pRb directly confirmed the inhibitory effect of CO on pRb phosphorylation. In addition, CO arrests SMCs at the G1/S transition of the cell cycle and selectively inhibits the expression of cyclin A while having no effect on the expression of cyclin D1 and E. Suppression of cyclin A expression by CO also results in the inhibition of cyclin A-associated kinase activity and cdk2 activity, independent of any changes in the level of cdk2 protein. Because cdk2 is a key regulator of both G1- and S-phase cell cycle progression,25 the ability of CO to block cdk2 activity may provide a potent mechanism by which CO inhibits SMC proliferation. Interestingly, the HO-1-catalyzed release of CO also up-regulates the expression of the cdk inhibitor, p21, suggesting that CO may block cdk2 activity via multiple mechanisms.26 In addition to directly blocking SMC growth, CO may also indirectly
influence cell growth by regulating the release of growth factors. In
this respect, Morita and Kourembanas28 have demonstrated in a coculture system that SMC-derived CO decreases the endothelial synthesis of the vascular mitogens endothelin-1 and PDGF. Moreover, studies in our laboratory and others found that CO inhibits platelet activation, thereby preventing the release of growth factors from platelet The capacity of growth factors to induce HO-1 and CO synthesis may be of pathophysiologic importance. After local injury of the blood vessel wall, growth factors are released from vascular cells and from activated platelets, and they stimulate the development of a neointima. However, the simultaneous induction of HO-1 and formation of CO by growth factors may limit the extent of SMC proliferation. In support of this proposal, a recent study found that inhibition of HO-1 activity by SnPP exacerbates intimal thickening following balloon injury.30 Alternatively, the induction of HO-1 by gene transfer or hemin administration blocks neointimal formation.26,30-33 In addition to regulating the growth of SMCs, CO may also preserve blood flow at sites of vascular damage by relaxing blood vessels and blocking platelet aggregation.7,11-14 Thus, the HO-1-catalyzed release of CO may provide an important adaptive mechanism to maintain homeostasis at sites of vascular injury. In conclusion, the present study demonstrates that serum induces HO-1 gene expression in vascular SMC and that HO-1-catalyzed CO formation inhibits SMC growth. In addition, they show that the antiproliferative effect of CO is associated with a decrease in cyclin A expression and cdk2 activity. The ability of growth factors to induce CO synthesis may serve an important negative feedback role to limit SMC proliferation and the development of vascular disease. The HO-1/CO system represents a potentially new therapeutic target for modifying the vascular response to injury.
Submitted June 12, 2001; accepted February 1, 2002.
Supported in part by the National Heart, Lung, and Blood Institute grants HL59976, HL36045, and HL62467, and a grant from the American Heart Association. H.W. is an awardee of the Junior Faculty Scholar Award from the American Society of Hematology. W.D. is an Established Investigator of the American Heart Association.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: William Durante, Houston VA Medical Center, Bldg 109, Rm 130, 2002 Holcombe Blvd, Houston, TX 77030; e-mail: wdurante{at}bcm.tmc.edu.
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A. Uc and B. E. Britigan Does Heme Oxygenase-1 Have a Role in Caco-2 Cell Cycle Progression? Experimental Biology and Medicine, May 1, 2003; 228(5): 590 - 595. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-mercaptoethanol,
cycloheximide, actinomycin, EDTA, Tris, Tes, HEPES, Tween 20, urea,
bilirubin, biliverdin, benzene, barium chloride, Hb, acrylamide, trichloroacetic acid, aprotinin, leupeptin, phenylmethylsulfonyl fluoride (PMSF), NaF, dithiothreitol (DTT), guanidine isothiocyanate, CsCl, desferrioxamine, and minimum essential medium were purchased from
Sigma Chemical (St Louis, MO). Penicillin, streptomycin, neomycin,
elastase, trypsin, and calf serum were from Gibco BRL (Rockville, MD);
RNase and propidium iodide were from Boehringer Mannheim (Indianapolis,
IN); and GAPDH complementary DNA (cDNA) and molecular markers were from
Ambion (Austin, TX). A polyclonal HO-1 antibody was from Stressgen
(Victoria, BC, Canada); a monoclonal 
