Departments of 1 Medicine and 3 Pharmacology, Duke University Medical Center, Durham 27710; and 2 Division of Human Studies, Environmental Protection Agency, Chapel Hill, North Carolina 27514
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ABSTRACT |
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CO is a biologically active gas
that produces cellular effects by multiple mechanisms. Because cellular
binding of CO by heme proteins is increased in hypoxia, we tested the
hypothesis that CO interferes with hypoxic pulmonary vascular
remodeling in vivo. Rats were exposed to inspired CO (50 parts/million)
at sea level or 18,000 ft of altitude [hypobaric hypoxia (HH)], and
changes in vessel morphometry and pulmonary pressure-flow relationships were compared with controls. Vascular cell single strand DNA
(ssDNA) and proliferating cell nuclear antigen (PCNA) were assessed,
and changes in gene and protein expression of smooth muscle -actin (sm-
-actin),
-actin, and heme oxygenase-1 (HO-1) were evaluated by Western analysis, RT-PCR, and immunohistochemistry. After 21 days of
HH, vascular pressure at constant flow and vessel wall thickness
increased and lumen diameter of small arteries decreased significantly.
The presence of CO, however, further increased both pulmonary vascular
resistance (PVR) and the number of small muscular vessels compared with
HH alone. CO + HH also increased vascular PCNA and nuclear
ssDNA expression compared with hypoxia, suggesting accelerated cell
turnover. CO in hypoxia downregulated sm-
-actin and strongly
upregulated
-actin. CO also increased lung HO activity and HO-1 mRNA
and protein expression in small pulmonary arteries during hypoxia.
These data indicate an overall propensity of CO in HH to promote
vascular remodeling and increase PVR in vivo.
pulmonary hypertension; hypoxic pulmonary vasoconstriction; actin; heme oxygenase
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INTRODUCTION |
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CHRONIC HYPOXIA ALTERS the structure of pulmonary arterial vessels by inducing proliferation and hypertrophy of smooth muscle in larger arteries and growth of smooth muscle in small arteries that are normally nonmuscular (14, 20, 24). Pulmonary hypertension gradually becomes irreversible, accompanied by right heart hypertrophy and failure. These responses occur in chronic lung and cyanotic heart diseases and in natives and sojourners to high altitude (12). Among the complex cellular and molecular mechanisms regulating the vascular responses to hypoxia is increased expression of heme oxygenase-1 (HO-1) (11), which produces carbon monoxide (CO) endogenously during heme degradation (22). CO, like nitric oxide (NO), can activate guanylate cyclase (15), resulting in smooth muscle relaxation, and the molecule may have other regulatory effects (26). HO-1 gene expression in vascular smooth muscle cells (VSMC) can be activated by hypoxia inducible factor-1 (HIF-1) (11), which regulates transcription of several hypoxia-responsive proteins, including erythropoietin and vascular endothelial growth factor (9, 10). Thus regulation of HO-1 gene expression by HIF-1 suggests a unique function for the enzyme in hypoxia, which could be mediated by CO production and its effects on heme proteins (Hp).
The effects of CO on the pulmonary vessels in hypoxia are unclear. A previous study has shown that inhaled CO does not attenuate hypoxic pulmonary vasoconstriction in isolated rat lungs after chronic hypoxia (3). Exogenous CO inhibits in vitro VSMC growth (16) and HO-1 knockout mice develop more right ventricular hypertrophy in chronic hypoxia than wild-type mice (28), implying HO-1 activity inhibits pulmonary vascular responses to hypoxia. The latter changes are difficult to attribute solely to endogenous CO due to the multifaceted consequences of failing to express HO-1 including increased tissue heme and iron content.
Pulmonary vascular phenotype changes in response to chronic hypoxia
differently among species, but in the rat, new smooth muscle in small
arteries correlates with increased medial thickness after 3 wk of
hypoxia (14), and previously nonmuscular arterioles become
muscular (20). Subpopulations of pulmonary VSMC also have
been described that have multiple differentiation profiles in hypoxia
(1, 19). The VSMC express different contractile and
structural elements in hypoxia. For example, expression of smooth
muscle--actin (sm-
-actin), a structural protein specific to
smooth muscle (19), decreases in proliferating neonatal
lung vascular cells after hypoxia but is maintained in mature muscle cells (7).
In the rat lung, HO-1 protein and mRNA increase in the first week
of hypoxia, return to control values by 2 wk, followed by a second
increase in HO-1 mRNA at 3 wk (4). Because this bimodal change in HO-1 in hypoxic lung makes it difficult to assess the effects
of endogenous CO production in specific phases of hypoxia, we studied
the effects of exogenous CO on pulmonary vascular structure and
function. Based on observations that CO interferes with VSMC proliferation in hypoxia in vitro (16) and the known
effects of CO binding on Hp function, we hypothesized that sustained CO exposure would influence pulmonary vascular remodeling in hypoxia by
altering vascular metabolism. These effects of CO could either inhibit
or promote vessel hypertrophy by its affects on cell turnover rate. We
tested the hypothesis in rats exposed to hypoxia or hypoxia plus
inspired CO [50 parts/million (ppm)] for 3 wk and found CO accentuated the hypoxic increases in vascular resistance and promoted the expected structural changes in small pulmonary arteries. The structural changes correlated with changes in cell proliferation, apoptosis, sm--actin,
-actin, and HO-1 gene and protein
expression in the lungs and pulmonary vasculature.
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METHODS |
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Materials. All materials were obtained from Sigma (St. Louis, MO) unless otherwise stated.
Animal exposures. Rats were exposed to hypobaric hypoxia (HH) using an altitude chamber simulating altitude of 18,000 ft (380 Torr) for 1, 3, 7, 14, or 21 days (n = 8 animals per group) on a protocol approved by the Duke University Animal Care and Use Committee. The chamber was returned briefly to sea level once daily to clean the cages and replenish the food and water supply for the animals. CO exposures were performed at 50 ppm in rats either in air (21% O2) or at altitude as above (Air + CO and HH + CO). CO levels were monitored continuously using a Toxilog gas detector (Biosystems, Rockfall, CT). After the exposures, the animals were anesthetized with pentobarbital (50 mg/kg ip), an arterial catheter was placed, and blood samples drawn at the altitude to which the rats were exposed. Arterial blood gases, pH, hemoglobin, and carboxyhemoglobin (COHb) were measured on a calibrated blood gas analyzer (Instrumentation Laboratories model 1640) and Co-oximeter (Instrumentation Laboratories model 480).
Tissue preparation.
The thorax was opened, and the lungs were flushed gently with 0.9%
NaCl through the right ventricle, excised from the hilar structures,
snap-frozen in liquid nitrogen, and stored at 80°C. Frozen lungs
were later used for Western blot and RT-PCR analysis and to measure HO
enzyme activity. The heart was excised and immediately dissected, and
the wet hearts were weighed to determine right ventricle to left
ventricle plus septum (R/L heart) ratio as a measure of right heart
hypertrophy. Lungs from three rats exposed to air, CO + air,
hypoxia, or CO + hypoxia were inflation and perfusion fixed with
4% paraformaldehyde for morphometry studies according to the method of
Meyrick and Reid (14). Lungs from additional rats in the
14- and 21-day exposure groups were inflation fixed for
immunohistochemistry to localize sm-
-actin and HO-1 and assess
apoptosis and cell proliferation by staining for single strand
DNA (ssDNA) and proliferating cell nuclear antigen (PCNA). Control lung
tissue came from animals exposed to air at sea level or air containing
50 ppm CO for 1-21 days.
Pulmonary vessel morphometry. Oriented lung sections were cut from both upper and lower lobes in a plane perpendicular to the hilum. Sections were paraffin embedded, cut into 6-µm-thick sections, and stained with hematoxylin and eosin. We avoided large central vessels (>200 µM) by analyzing only vessels in the peripheral 2 mm of lung parenchyma. In addition, the measurements were limited to vessels with a complete circumferential smooth muscle layer. Digital image analysis was used to measure the area of the lung sections that were sampled. Using an ocular grid on a Nikon Optiphot-2 light microscope, an observer unaware of the exposure conditions measured external diameter and wall thickness for each vessel. Eyepiece grid sampling dimensions were determined using a micrometer calibration slide. The grid was used as a systematic guide to ensure 100% sampling of selected tissue areas. The slides were scanned using an EPSON Expression 800, and scanned images were processed and printed with Adobe Photoshop 5.5. Printed images were digitized and analyzed with a commercial software package (Quickmeasure 1.6; Tally Systems, San Diego, CA), and the surface area was calculated for each tissue sample.
Muscular vessels were counted and grouped according to diameter into three categories: small, <50 µm; medium, 50-100 µm; and large, >100 µm. Vessel wall thickness as percent diameter [(2 × measured wall thickness/diameter) × 100] was determined from the measurements for small and medium arteries. Lumen diameter was calculated from the measurements of vessel diameter and wall thickness.Measurement of pulmonary pressure-flow curves. Pulmonary pressure-flow measurements were used to estimate fixed vascular resistance ex vivo in air control rats, after 21 days of Air + CO (50 ppm) or 21 days of HH or HH + CO (50 ppm). Animals were removed from the exposure chambers to room air and anesthetized with halothane, and tracheal catheters were placed. The thorax was opened, and the lungs were inflated with 5 ml of air. The main pulmonary artery (PA) was cannulated through an incision in the right ventricle with fluid-filled PE-70 tubing connected to a calibrated pressure transducer and a calibrated infusion pump. The left atrium was opened, and the lungs were perfused with Ringer lactate at constant flow until PA pressure stabilized. Then, pulmonary pressure-flow curves were recorded at different flows for both increasing and decreasing flow rates. The average of the two values was taken for the pressure at each flow rate.
Western blot analysis.
Tissue was homogenized on ice in cold lysis buffer [150 mM NaCl, 50 mM
Tris, pH 7.6, 1% SDS (Bio-Rad, Carpenteria, CA), 3% Nonidet P-40, 5 mM EDTA, 1 mM MgCl2, 2 mM 1,3-dichloroisocoumarin, 2 mM
1,10-phenanthroline, and 0.5 mM E-64]. The homogenate was centrifuged at 10,000 g for 10 min. The supernatant was
decanted, and an aliquot was stored at 20°C for measurement of
protein concentration. The remaining supernatant was mixed with an
equal volume of double-strength Laemmli sample buffer [250 mM
Tris · HCl (Bio-Rad), pH 6.8, 4% SDS, 10% glycerol, 0.006%
bromphenol blue, 2%
-mercaptoethanol], divided into aliquots, and
stored at
80°C.
HO activity assay.
HO activity was measured as reported (5, 21) with
modification from the method of Tenhunen et al. (22).
Briefly, lungs were homogenized on ice in one volume of 100 mM
phosphate buffer with 2 mM MgCl2. The homogenates were
sonicated and centrifuged, and HO activity in the supernatant was
measured using liver cytosol as a source of biliverdin reductase. The
reaction mixture also contained 20 mM hemin, 0.8 mM NADPH, 2 mM glucose
6-phosphate, and 0.0016 U/µl of glucose-6-phosphate dehydrogenase. An
NADPH-free reaction mixture provided a background sample.
Bilirubin was extracted with chloroform and measured on a
spectrophotometer based on optical density (OD) at 464 nm minus 530 nm
using an extinction coefficient of 40 mM1 · cm
1.
Immunohistochemistry for sm--actin and HO-1.
Inflation-fixed lungs were paraffin embedded and cut into 6-µm
sections. Before labeling, the tissue sections were deparaffinized in
xylene and rehydrated in graded alcohol solutions. The sections were
blocked in a solution of 5% nonfat dry milk, 1% BSA, 5% goat serum
in 0.01 M PBS, and 0.1% Triton X-100 before overnight incubation at
4°C with a monoclonal antibody to sm-
actin (Sigma) and HO-1 (Stress-Gen) in 1% milk and 1% BSA in 0.01 M PBS and 0.1% Triton X-100 using a 1:200 dilution. The sections were washed three times with
PBS with 0.1% Triton X-100 (5 min each) and incubated with the
secondary antibody, biotinylated goat anti-mouse IgG (Jackson Laboratories), at a dilution of 1:1,000 in 1% milk in 0.01 M PBS and
0.1% Triton X-100 at room temperature for 1 h. The signal was
detected with peroxidase-conjugated avidin and diaminobenzidine. The
slides were counterstained with 1% hematoxylin. Negative control sections were processed as above except primary incubations were performed with nonimmune rabbit serum (Jackson Laboratories) instead of
primary antibody.
Detection of ssDNA and PCNA. Paraffin-embedded fixed lung tissue was cut into 6-µm sections and placed on slides for labeling PCNA and ssDNA. Immunohistochemical labeling for PCNA was performed using a commercial protocol and antibody to PCNA (Boehringer Mannheim, Indianapolis, IN). The ssDNA method, a specific and sensitive cellular marker for detection of apoptosis before frank internucleosomal DNA fragmentation, required sections to be incubated in 60°C hot formamide (50%) for 30 min and washed in PBS before being stained with monoclonal antibody to ssDNA, diluted 1:100 (Chemicon International, Temecula, CA) using our standard immunohistochemical protocol (see above).
RT-PCR.
Lung tissue was homogenized (1 g/5 ml) with 4 M guanidine thiocyanate
(Boehringer Mannheim), 50 mM sodium citrate, 0.5% sarkosyl, and 0.01 M
dithiothreitol. RNA was pelleted by ultracentrifugation through cesium
chloride (Boehringer Mannheim) and 0.1 M EDTA. Total RNA (100 ng) was
reverse transcribed (M-MLV Reverse transcriptase; Life Technologies),
and the resultant cDNA was amplified for 27 and 37 cycles for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and HO-1,
respectively, in separate reactions using gene-specific primers.
Oligonucleotide sequences were synthesized using an Applied Biosystems
391 DNA synthesizer (Foster City, CA) based on sequences published in
the GenBank DNA database. The following sense and anti-sense sequences
were employed: GAPDH: 5'-CCA TGG AGA AGG CTG GGG-3' and -5' CAA AGT TGT
CAT GGA TGA CC-3'; HO-1: 5'-ATT GGA GGC TGG AGC TAT TCT G-3' and 5'-
CCT TCG GTG CAG CTC CTC AG-5'; sm--actin: 5'-CGA TAG AAC ACG GCA TCA
TC-3' and -5' CAT CAG GCA GTT CGT AGC TC-3';
-actin: 5'-CCT TCC TGG
GCA TGG AGT CCT G-3' and 5'-GGA GCA ATG ATC TTG ATC TTC-3'.
Statistical analysis. Experimental data from each group were expressed as means ± SE. Statistical analyses were performed with two-way ANOVA (factorial design) with a post hoc comparison test (Fisher's exact test) using commercially available software (Statview 4.0, Calabasas, CA). A P value of 0.05 or less was accepted as significant.
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RESULTS |
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Physiological responses.
The altitude exposures in this study produced a stable range of
arterial PO2 values of 40-46 mmHg both
without and with added CO. The responses in hematocrit and R/L heart
ratios for rats at each point of study during HH and HH + CO are
shown Fig. 1, A and
B. In rats exposed to HH alone, the hematocrit increased steadily for 21 days. In rats exposed to HH + CO, hematocrit was significantly lower after 7 and 14 days than HH alone (50.6 ± 2 and 56.2 ± 1.2, respectively, in HH + CO animals, and
57.6 ± 1.7 and 61 ± 1.7, respectively, in hypoxia,
P 0.05). By 21 days, however, hematocrit in HH + CO-exposed rats approached the values of the HH animals. R/L heart
ratios increased similarly in both HH and HH + CO groups at all
time points. COHb levels measured in arterial blood at the end of each
study (Fig. 1C), used as a marker of exposure, indicate
consistent CO exposures. In the HH animals, COHb averaged 1.5 ± 0.14%
after 3 days and was stable until 21 days, when COHb reached 2.8 ± 0.1%. This was significantly higher than the 14-day values
(P
0.05). In rats exposed to HH + CO, the mean COHb
level remained stable between 3.5% and 3.9% during the entire
experiment.
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Pulmonary vascular morphometry.
Changes in pulmonary vascular structure were measured to assess the
effect of inspired CO on hypoxic vascular remodeling in the lung.
Quantitative measurements were made in four groups of rats including
air control, 21-day air + CO (50 ppm), 21-day HH, and 21-day
HH + CO (50 ppm). Figure 2 shows
summarized data for small (<50 µm; A-C) and
medium-sized (50-100 µm; D-F) muscular vessels
including wall thickness, lumen diameter, and number of muscular
arteries after 21 days of HH + CO or 21 days of HH + CO.
Vessel wall thickness as a percentage of total diameter is shown in
Fig. 2, A and D. After 21 days of HH + CO,
the mean wall thickness of small, muscular arteries had increased
significantly compared with air control animals (Fig. 3A,
22.3% vs. 14.9%, P < 0.01). When CO was present
during hypoxia, small vessel wall thickness averaged ~20% after 3 wk, which was greater than control, but significantly less than HH
animals (P < 0.05). CO alone had no significant effect
on small-vessel wall thickness. In medium-sized vessels (Fig.
3D), no differences were found in wall thickness after
exposure to HH or HH + CO, whereas air + CO resulted in a
slight decrease in wall thickness.
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Measurement of pulmonary pressure-flow curves.
Pressure-flow relationships for the pulmonary vasculature were
determined ex vivo in lungs of rats after 21 days of CO exposure in
air, HH, and HH + CO, and compared with air control rat lungs. PA
pressure was measured during perfusion at four controlled flow rates,
and mean pressure values are plotted for each of the four groups in
Fig. 4. The pulmonary vascular resistance
(PVR) was estimated from the ratios of pressure to flow on the curves.
Chronic HH significantly increased vascular resistance over air control animals (P < 0.01), whereas 21 days of air + CO
exposure had no effect. The administration of CO during 21 days of HH
increased PVR significantly more than HH alone (P < 0.01).
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Immunohistochemistry for ssDNA and PCNA.
To assess cell turnover in the vessels of the lung,
immunohistochemistry was performed for ssDNA (as an early marker of
apoptosis) and PCNA (as a marker of cell proliferation).
Representative immunohistochemistry stains for ssDNA are shown in Fig.
5, from control lungs and after 21 days
of HH and HH + CO. In control lung, minimal staining for ssDNA was
present (Fig. 5A). After 21 days of hypoxia, nuclear staining for ssDNA was detected in alveolar macrophages and occasional vascular cells (Fig. 5B). After HH + CO (Fig.
5C), ssDNA staining was prominent in both macrophages and
vascular cells. Figure 5C, inset, shows strong nuclear
staining for ssDNA was present in some cells while neighboring cells
were undergoing mitosis.
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Lung sm- actin expression.
The sm-
-actin, a major structural and functional protein in vascular
smooth muscle, was measured on Western blots of the lung after HH and
HH + CO. The sm-
-actin mRNA content of the lungs was measured
semiquantitatively by RT-PCR and compared with control; sm-
-actin
was localized by immunohistochemistry to determine whether the measured
changes occurred in vascular smooth muscle. Figure
7A shows a representative
Western blot for sm-
-actin in the lungs of rats exposed to air,
air + CO, HH, and HH + CO. Control rat lung expresses
sm-
-actin, which appears as a single strong band at 42 kDa, and the
signal is not affected by exposure to 50 ppm CO in air. After hypoxia,
sm-
-actin expression increased slightly in the lung by 14 and 21 days. In animals exposed to HH + CO, however, sm-
-actin was
markedly decreased in the lung after 14 and 21 days. Thus HH + CO
significantly decreased sm-
-actin expression in the lung after
14-21 days, whereas after HH alone, sm-
-actin content of the
lung increased over control. After air + CO, sm-
-actin in the
lung was similar to control and hypoxia-exposed animals.
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Lung HO-1 expression.
HO-1 protein, activity, and mRNA were measured in the lungs after HH
and HH + CO and compared with control lungs. In Fig. 8A, a Western blot for HO-1
shows representative results in the lungs of one control animal and one
from each time point after HH and HH + CO. HO-1 protein expression
increased in the lungs after hypoxia over the first 7 days, and, after
HH + CO, HO-1 expression remained elevated for 21 days. In the
lungs of rats breathing air + CO, minimal or no increase in HO-1
was seen over 21 days of exposure (data not shown).
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Immunohistochemistry for sm--actin and HO-1.
Figure 9 shows representative
photomicrographs of immunohistochemistry in lungs of an air-exposed rat
and after 14 days of exposure to HH or HH + CO. Figure 9,
A-C, shows immunohistochemistry for sm-
-actin, and
immunohistochemistry for HO-1 is shown in Fig. 9, D-F.
The sm-
-actin staining in control lung was limited to bronchial and
vascular smooth muscle. In control lung, sm-
-actin is present in the
thin smooth muscle layer of a small arteriole (Fig. 9A).
After 14 days of HH, sm-
-actin staining increased significantly in
walls of small blood vessels, reflecting the thickened smooth muscle
layer (Fig. 9B). In addition, sm-
-actin staining was
present in the alveolar region, likely representing new smooth muscle
in small vessels. In contrast, staining for sm-
-actin after 14 days
of HH + CO was attenuated in the vessel walls and alveolar region.
In control lungs (Fig. 9D), HO-1 was present primarily in
lung macrophages. After 14 days of HH, HO-1 was present diffusely in
macrophages, in the alveolar region, and in the walls of thickened
blood vessels (Fig. 9E). After 14 days of HH + CO, HO-1
was present throughout the alveolar region, in the macrophages, and in
blood vessel walls (Fig. 9F).
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DISCUSSION |
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The main finding of this study is that continuous exposure of rats
to low concentrations of CO alters vascular remodeling during HH in
vivo. This result was somewhat unanticipated, but it correlates with a
greater increase in the lung's pressure-flow curve in chronic HH with
CO than without it. This effect was independent of right heart
hypertrophy and differences in erythrocyte response between the groups.
The CO-related changes in vascular structure and PVR were associated
with decreased expression of sm--actin in small pulmonary arteries
during hypoxia. Because CO in air at sea level did not produce similar
effects, the combination of CO and hypoxia appears to be critical and
implicates an Hp-based mechanism for the interaction.
The lack of difference in morphometric and pressure-flow data between CO control and air control animals confirms that the amount of CO used in these experiments was not sufficient to cause significant hypoxia in the lung. Therefore, the finding that the presence of CO accelerates hypoxic vascular remodeling cannot be attributed to an exaggerated vascular response to hypoxia. Higher concentrations of CO in air do result in significant tissue hypoxia, and previous studies report that neonatal rats exposed to 500 ppm CO in air develop right heart remodeling and polycythemia but not increased PVR (13, 17, 25). COHb levels of 3-4% in the current experiments are too low to cause significant changes in lung tissue PO2 that would promote more rapid remodeling of the lung to chronic HH. Although the accuracy of optical measurements of COHb in the 2% range is a problem, the low values do indicate that lung cell concentrations of CO during HH were in the range of those attained by increases in endogenous CO production in vivo (14).
In an earlier study, CO was found to decrease smooth muscle cell growth in vitro at a concentration 1,000 times greater than used in this study (16). The living rat lung, however, shows evidence of a higher degree of vascular cell turnover when CO is persistently present during long-term hypoxia. This interpretation is suggested by increases in both ssDNA consistent with apoptosis and nuclear PCNA consistent with cell proliferation. If true, the biochemical linkage of CO to the regulation of growth responses is quite complex. Such complex effects could occur by a variety of biochemical mechanisms, e.g., when CO interactions with reduced cellular hemoproteins increase during hypoxia.
Some of our findings suggest that CO increases muscular pulmonary
vessels during hypoxia by stimulating smooth muscle cell proliferation.
Although mature contractile VSMC contain sm--actin as a major
structural and functional protein, proliferating VSMC show low
expression of sm-
-actin and other mature muscle proteins after
mechanical injury or during hypoxic vascular remodeling (2,
7). A less well-differentiated phenotype of proliferating smooth
muscle cells possibly results from either a phenotypic shift or the
expansion of a less-differentiated subpopulation of smooth muscle cells
(7). The relative decrease in sm-
-actin mRNA with CO in
hypoxia suggests that its lack of expression is due to decreased gene
transcription or altered message stability. The twofold increase in
-actin mRNA transcript in the lungs after hypoxia + CO confirms
the switch in actin isoform expression, as reported in proliferating
smooth muscle cells (2). This raises several intriguing
possibilities for future investigation of how CO modifies actin message
expression, translation, and/or mRNA or protein degradation in hypoxia.
The mechanisms responsible for the effects of CO during hypoxia
potentially involve HO enzyme activity in the lung. It is known that
initial expression of many hypoxia-responsive genes is regulated by
HIF-1, including HO-1. Although HIF-1 DNA binding activity was not
measured, prolonged expression of HO-1 in the lung after CO in hypoxia
may be associated with early HIF-1 activation, whereas HO-1 expression
at later time points may be regulated by either HIF-1 or other
factors. For example, hypoxia in cells leads to induction of
c-Fos and appearance of c-Jun/c-Fos activator protein-1 heterodimer,
promoting gene expression of proteins involved in cell proliferation,
including HO-1 (8). Recent studies have found that
hypoxia alters expression of cytokines such as interleukin-6 and
stimulates cellular reactive oxygen species (ROS) associated with
nuclear factor-B activation (6, 27). Such mechanisms may allow CO to augment the effect of hypoxia through HIF-1-independent pathways.
In this study, CO increased HO-1 expression in the lung in vivo only during HH; however, in endothelial cells in vitro, CO (at 100 ppm) also increased HO-1 expression in normoxia (23), where, it was proposed, to increase cellular NO metabolites, which then serve as toxic or signaling mediators. However, we detected minimal to no overall increase in HO-1 expression in air control lung exposed to CO at 50 ppm for up to 3 wk. The effects of HO-1 induction by CO in hypoxic lung are not clear, and multiple biochemical mechanisms are possible for enhanced HO-1 expression by a metabolic product like CO. Enzyme inhibition by CO substrate binding leading to HO-1 induction by heme is the only such mechanism demonstrated to date (5).
A unifying mechanism for the vascular effects of CO in hypoxia would have to explain apparent increases in apoptosis, cell proliferation, and HO-1 induction. Perhaps such responses could be triggered by enhanced generation of reactive species of oxygen or nitrogen. For instance, high concentrations of CO actually increase oxidative stress in the brain (18), where ROS generation by mitochondria has been demonstrated (29). In the present study, however, such oxidative mechanisms have not been explored and remain for investigation.
The increase in smooth muscle content of pulmonary arteries in hypoxia has important functional consequences for the processes that influence vascular resistance. Although CO in HH increased resistance, our technique did not independently assess the contribution of small arteries. However, the morphometry provides additional perspective because the histograms show a greater number of muscular vessels in the lungs after CO + hypoxia than hypoxia alone. These vessels may represent previously nonmuscular arteries not accounted for in hypoxia but which contribute to an apparent increase in PVR after CO. On the other hand, total lumen cross-sectional area of muscular vessels in rats exposed to CO in hypoxia increase relative to air control animals (~25%), whereas lumen area of muscular vessels decreased in hypoxia relative to control (~20%). The extent to which any of this increase in lumen area by CO represented vasculogenesis would tend to counteract an increase in PVR in HH. Alternatively, CO may have slowed smooth muscle growth in one size of vessel but stimulated vessels of another size, which contributed more to an overall increase in PVR.
In conclusion, we find that exogenous CO can promote vascular
remodeling and the increase in vascular resistance in HH in the lung.
These responses are associated with complex cellular changes comprising
evidence for both apoptosis and increased proliferation of
resident cell populations suggestive of increased cell turnover. Structural changes induced by CO in hypoxia correlate with changes in
sm--actin and HO-1 expression in small pulmonary arteries. The effects of CO require hypoxia, as they are absent after the same CO
exposure in air, strongly implicating
PO2-dependent Hp-based mechanisms in the
responses. These new findings may have direct relevance to
environmental exposures and human lung disease, for example, in
smokers, where alveolar CO levels reach hundreds of parts per million,
and hypoxic lung cells may take up and retain CO for long periods of time.
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ACKNOWLEDGEMENTS |
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Funding for this work was provided by an American Lung Association Research Grant and National Heart, Lung, and Blood Institute Grant HL-4-2444.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. A. Piantadosi, Div. of Pulmonary & Critical Care Medicine, P.O. Box 3315, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: piant001{at}mc.duke.edu).
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.
10.1152/ajplung.00211.2001
Received 8 June 2001; accepted in final form 23 October 2001.
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