Department of Pediatrics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235
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ABSTRACT |
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Nitric oxide (NO), produced by endothelial (e) nitric oxide synthase (NOS), is a critical mediator of vascular function and growth in the developing lung. Pulmonary eNOS expression is diminished in conditions associated with altered pulmonary vascular development, suggesting that eNOS may be modulated by changes in pulmonary artery endothelial cell (PAEC) growth. We determined the effects of cell growth on eNOS expression in cultured ovine fetal PAEC studied at varying levels of confluence. NOS enzymatic activity was sixfold greater in quiescent PAEC at 100% confluence compared with more rapidly replicating cells at 50% confluence. To determine if there is a reciprocal effect of NO on PAEC growth, studies of NOS inhibition or the provision of exogenous NO from spermine NONOate were performed. Neither intervention had a discernable effect on PAEC growth. The influence of cell growth on NOS activity was unique to pulmonary endothelium, because varying confluence did not alter NOS activity in fetal systemic endothelial cells. The effects of cell growth induced by serum stimulation were also evaluated, and NOS enzymatic activity was threefold greater in quiescent, serum-deprived cells compared with that in serum-stimulated cells. The increase in NOS activity observed at full confluence was accompanied by parallel increases in eNOS protein and mRNA expression. These findings indicate that eNOS gene expression in fetal PAEC is upregulated during cell quiescence and downregulated during rapid cell growth. Furthermore, the interaction between cell growth and NO in the PAEC is unidirectional.
polymerase chain reaction; pulmonary circulation
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INTRODUCTION |
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NITRIC OXIDE (NO) produced by the enzyme NO synthase (NOS) is a potent pulmonary vasodilator that plays a major role in the normal cardiopulmonary transition from intrauterine to extrauterine life. Studies in fetal sheep have demonstrated that the normally dramatic increase in pulmonary blood flow seen at birth is attenuated by 50% in the presence of the inhibitor of NOS activity, nitro-L-arginine (1). A variety of processes occurring at the time of transition, including increased oxygenation, ventilation, and shear stress, acutely enhance NO production by the pulmonary endothelium through the rapid activation of the endothelial isoform of NOS (eNOS; see Ref. 5).
Along with acute stimulation of the enzyme, there is evidence of modulation of pulmonary eNOS expression in the perinatal period. Animal studies indicate that the capacity for pulmonary endothelial NO production is normally maximal at term due to an increase in pulmonary eNOS gene expression during late gestation and that this is followed by a fall in pulmonary eNOS expression in the postnatal period (11, 17, 27). In contrast, there is evidence that pulmonary eNOS expression is attenuated in certain disease states such as congenital diaphragmatic hernia and fetal pulmonary hypertension (16, 23, 27), thereby potentially contributing to the pathogenesis of persistent pulmonary hypertension of the newborn. Studies using fetal pulmonary artery endothelial cells (PAEC) have also shown that pulmonary eNOS expression is downregulated by prolonged hypoxia, and this appears to be mediated at the level of gene transcription or mRNA stability (15). Because all of these conditions are associated with changes in vascular cell growth, these findings suggest that variations in cell growth may play an important role in the regulation of eNOS expression in the fetal pulmonary circulation. However, it is not yet known if cell growth directly modifies eNOS expression in the fetal pulmonary endothelium.
In an effort to better understand the regulation of NO production in the developing lung, studies were performed on isolated, early-passage ovine fetal PAEC to determine the effects of cell growth on eNOS expression. Previous studies of the relationship between cell growth and eNOS expression have been limited to mature aortic endothelial cells (2). PAEC growth was modulated by two independent mechanisms, namely by varying the degree of cell confluence and by stimulation with serum. In addition to testing the effects of growth on eNOS expression in the fetal PAEC, experiments were performed to answer the following questions. 1) Is there a reciprocal effect of NO on PAEC growth? 2) Are the effects of cell growth on eNOS expression in the PAEC unique to that vascular bed? 3) What is the mechanism(s) by which eNOS expression is altered by the state of cell growth?
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METHODS |
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Endothelial cell culture. Endothelial cells were obtained from mixed-breed fetal lambs at 125-135 days of gestation (term = 144 days) using methods that have been described previously (22). Before being killed, the ewes were housed in the Animal Resources Center at the University of Texas Southwestern Medical Center and were given standard animal chow and water ad libitum. The procedures followed in the care and euthanasia of the study animals were approved by the Institutional Review Board for Animal Research.
The ewe and fetus(es) were euthanized with pentobarbital sodium (120 mg/kg) given intravenously to the ewe, and the fetus(es) was delivered by cesarean section. The fetal lung and the mesentery containing the cephalic mesenteric arterial cascade were immediately removed and placed in ice-cold PBS (10 mM PO4 and 150 mM NaCl, pH 7.4). Further tissue preparation was performed using a sterile technique in a cold room at 4°C. The pulmonary arterial tree was rapidly dissected from the lung parenchyma, the mesenteric arterial bed was isolated from the surrounding tissue, and both were placed in fresh, ice-cold PBS.
Endothelial cells were harvested and maintained by methods modified from those of Johnson (10). Third-generation intrapulmonary arteries and mesenteric arteries of similar size (1-3 mm external diameter) were isolated, and branch vessels were tied off with silk suture. We have previously employed endothelial cells obtained from these arteries in comparisons of phenotype in fetal pulmonary and systemic endothelium (22). The arteries were placed in ice-cold medium 199 (M199) containing 2.5% iron-supplemented calf serum, 2.5% lamb serum, 1% antibiotic-antimycotic mixture, 0.15% nystatin, 0.15% gentamicin, and 0.10% tylosin. Further processing was performed at room temperature under a laminar flow hood. Remaining fatty and connective tissues were gently removed, taking care not to disrupt the endothelium, and the arteries were flushed with M199 several times to remove cellular debris. The arteries were filled with a 0.2-0.4% solution of collagenase in M199, tied off with silk suture, and incubated at 37°C for 30 min. The lumen contents were emptied into RPMI containing 12.5% iron-supplemented calf serum, 12.5% lamb serum, 1% L-glutamine, 1% antibiotic-antimycotic mixture, 0.15% nystatin, 0.15% gentamicin, and 0.10% tylosin, and the lumen was rinsed two times with M199. The cells were pelleted, resuspended, and plated in gelatin-coated culture flasks. They were further propagated in RPMI medium containing 10% iron-supplemented calf serum and 10% lamb serum in a humidified incubator with 5% CO2 in air at 37°C. The identity of the endothelial cells was confirmed by phenotype (cobblestone appearance and contact inhibition) and by the evaluation of the uptake of acetylated low-density lipoprotein. Cells were utilized at passages 4-6. Multiple primary cultures were employed in this investigation, and results in independent experiments were verified in studies with three different primary cultures.
Manipulation of cell growth. The primary approach used to manipulate cell growth involved the contact inhibition known to occur in endothelial cell culture (10). To avoid potential confounding effects produced by differences in cell density upon initial seeding, an identical number of cells was plated into T75 flasks (1 × 106), and all passages were made using a splitting ratio of 1:3. This ensured that the number of mitoses necessary to bring the cells to a given degree of confluence was similar between experiments. Levels of cell confluence of 50, 70, and 100% were estimated by visual inspection by evaluating 8-10 fields at ×40 magnification every 24 h. With the use of this approach, 50% confluence was reached 48 h after cell passage, 70% was reached at 72 h, and 100% confluence was attained at 96 h. Preliminary studies revealed that, compared with 48 h after cell passage, cell number rose 2.2-fold by 72 h and 3.8-fold by 96 h. Cells were not studied postconfluence. Contact inhibition was evident in both the PAEC and the mesenteric endothelium. Culture medium was replaced every 24 h.
In an attempt to modify endothelial cell growth in an entirely independent manner, 50% confluent cells were placed in either serum-free medium or medium containing 20% serum for 24 h. Before the addition of serum-free medium, the cells were grown under the conditions noted above, with the exception that culture medium was replaced every 48 h.
Manipulation of NO exposure. To evaluate the potential reciprocal effect of NO on PAEC growth, studies of the roles of both endogenous and exogenous NO were performed. To assess the effects of endogenous NO, cells at 50% confluence were placed in medium deficient in L-arginine (control, Endothelial-SFM Growth Media; Life Technologies, Grand Island, NY) or medium deficient in L-arginine containing the competitive inhibitor of NOS, nitro-L-arginine methyl ester (L-NAME, 2 mM), for 24 h. We have previously shown that such treatment causes complete inhibition of NOS activity in intact PAEC (12). To determine the effects of exogenous NO on PAEC growth, the cells were placed for 24 h in RPMI propagation medium containing either spermine (Sp, 0.01 or 1.0 µM) or spermine NONOate (SpNO, 0.01 or 1.0 µM). SpNO is an NO donor compound that is stable in solution at alkaline pH; it releases NO at a predictable rate when added to medium with a normal pH, and it requires no bioconversion to release NO; Sp is the parent compound (14). We have recently successfully employed Sp and SpNO to evaluate the effects of NO exposure on eNOS expression in PAEC (29). Sp and SpNO were prepared daily in PBS at pH 8.5. Preliminary studies revealed that neither Sp nor SpNO had any effect on cell viability as assessed by the method of Denizot and Lang (6).
Evaluation of cell replication. Endothelial cells were grown under the varying conditions outlined above, and cell abundance was assessed by measurements of total protein per flask using the method of Bradford (4), with BSA as the standard. This approach was employed to avoid the trypsinization that is required for accurate cell counting, thereby providing samples that are amenable to immunoblot analysis. When changes in cell abundance were evident, cell replication was evaluated using the thymidine analog 5-bromo-2'-deoxyuridine (BrdU; Amersham). After the removal of growth medium, the cells were incubated at 37°C for 1 h with diluted BrdU (1:1,000). The cells were washed three times in PBS for 3 min at room temperature and were fixed with acid-ethanol (90% ethanol-5% acetic acid-5% water) for 30 min. The cells were rehydrated with PBS, washed two times with PBS, and incubated with reconstituted nuclease/anti-BrdU antibody (1:100) for 60 min at room temperature. The cells were subsequently washed with PBS three times, incubated with peroxidase-conjugated anti-mouse IgG for 30 min, and then incubated in 3,3'-diaminobenzidine tetrahydrochloride chromagen for 20 min. The peroxidase-chromagen reaction was quenched with distilled water, and the cells were counterstained with eosin Y and hematoxylin. Fifteen to twenty fields per T75 flask magnified at ×100 were evaluated in a blinded manner for percent nuclei stained with the chromagen. Approximately 1,000-1,500 cells were counted per flask.
NOS activity in cell lysates.
Endothelial cells were washed in ice-cold PBS, scraped and pelleted,
and resuspended in ice-cold 50 mM Tris buffer (pH 7.4) containing 1.0 mM EDTA, 5 mM mercaptoethanol, 10 µg/ml pepstatin A, 10 µg/ml
leupeptin, 90 µg/ml phenylmethylsulfonyl fluoride, and 1.0 µM
tetrahydrobiopterin. The cells were disrupted by freeze-thawing two
times in liquid nitrogen, and NOS activity in the preparation was
determined by measuring the conversion of
[3H]arginine to
[3H]citrulline (21).
The cell preparation (50 µl) was added to 50 µl of buffer, yielding
final concentrations of reagents as follows: 2 mM -NADPH, 2 µM
tetrahydrobiopterin, 10 µM FAD, 10 µM flavin mononucleotide, 2.5 mM
CaCl2 in excess of EDTA, 15 nM calmodulin, 2 µM cold L-arginine, and 2.0 µCi/ml
L-[3H]arginine.
After incubation at 37°C for 1 h, the assay was terminated by the
addition of 400 µl of 40 mM HEPES buffer, pH 5.5, with 2 mM EDTA and
2 mM EGTA. The terminated reactions were applied to 1-ml columns of
Dowex AG50WX-8 (Tris form) and eluted with 1 ml of the 40 mM HEPES
buffer. [3H]citrulline
generated was collected in scintillation vials and quantified by liquid
scintillation spectroscopy. NOS activity was linear with time for up to
2 h, and the assay was performed within this linear range over 1 h. NOS
activity was fully inhibited by 2.0 mM L-NAME. The protein
content of the samples was determined by the method of Bradford (4).
Immunoblot analysis. The methods used for immunoblot analysis generally followed those we have previously reported (13). After being washed and pelleted, PAEC were resuspended in the 50 mM Tris buffer described above and were disrupted by freeze-thawing in liquid nitrogen. The protein content of the preparation was determined, SDS-PAGE was performed with 7% acrylamide, and the proteins were electrophoretically transferred to filters. The filters were blocked for 1.5 h in buffer containing 150 mM NaCl and 10 mM Tris (pH 7.5) with 0.5% Tween 20 and 5% dried milk and were incubated overnight at 4°C with a 1:2,000 dilution of primary antiserum to eNOS. The eNOS antiserum was the kind gift from Dr. Thomas Michel (Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA). After incubation with primary antiserum, the filters were washed with the 150 mM NaCl buffer with Tween 20 and incubated for 60 min with a 1:5,000 dilution of a donkey anti-rabbit Ig antibody-horseradish peroxidase conjugate (Amersham). The filters were washed in the 150 mM NaCl buffer with Tween 20, and the bands for eNOS were visualized by chemiluminescence (ECL Western Blotting Analysis System; Amersham) and quantitated densitometrically. Under the conditions employed, there was a linear relationship between the protein load and the densitometric values for eNOS signal (r = 0.96-0.98; see Ref. 13).
RT-PCR assays. A semiquantitative RT-PCR assay was used to evaluate eNOS mRNA abundance in PAEC because detection of mRNA by Northern analysis requires the use of poly(A)+ RNA from a large volume of cells. Total cellular RNA was obtained from the cells, and RT was performed using 10 µg of total RNA. Briefly, cDNA synthesis was carried out using 200 units of Moloney murine leukemia virus reverse transcriptase, 5 µM oligo(dT), 1 mM dNTPs, and 3 mM Mg2+ in a volume of 20 µl. In selected tubes, the reverse transcriptase was omitted to control for amplification from contaminating cDNA or genomic DNA. The temperature profile was 1) annealing at room temperature for 5 min, 2) extension at 42°C for 60 min, and 3) termination at 99°C for 5 min. PCR was performed on the resulting RT products using specific oligonucleotide primers for ovine eNOS (23). The sequence of the sense primer was 5'-AGCTCGAGACCCTCAGTCAGGA-3', and that of the antisense primer was 5'-GTCTCCAGTCTTGAGTTGGC-3'. The PCR contained 1.5 mM Mg2+, 1 µM primers, 200 µM dNTPs, reaction buffer, and 5 µl of cDNA in a final volume of 45 µl. To minimize nonspecific amplification, a "hot start" procedure was employed in which the PCR reaction tubes were placed in a thermal cycler (model 480; Perkin-Elmer) prewarmed to 94°C. After 2 min, each tube was opened sequentially, and 2.5 units (in 2 µl) of Taq DNA polymerase were added. The PCR temperature profile consisted of 35 cycles at 94°C for 45 s (denaturation), 60°C for 45 s (annealing), and 72°C for 1.25 min (extension) followed by an additional 5-min final extension at 72°C. The primer location, primer concentration, Mg2+ concentration, and annealing temperature were optimized to produce the greatest amount of a single PCR product.
The PCR products were size-fractionated by agarose gel electrophoresis. The identity of the PCR products was confirmed, and they were quantitated by transferring the DNA to nylon filters, probing with a 32P end-labeled internal oligonucleotide primer specific for ovine eNOS, and performing densitometric analysis of the resulting autoradiographs. PCR product identity was also confirmed by direct double-stranded sequencing (Fmol DNA Sequencing Kit: Promega, Madison, WI). To control for the RT step, RT-PCR was also done for the housekeeping gene malate dehydrogenase (MDH; see Ref. 23). The PCR temperature profile for MDH was identical to that described above except for an annealing temperature of 56°C. Experiments performed to evaluate the relationship between the quantity of total RNA subjected to RT-PCR and the amount of PCR product generated revealed high correlations between densitometry values and the quantity of RNA used for eNOS RT-PCR (r = 0.97-0.99, n = 3 experiments) and for MDH RT-PCR (r = 0.96-0.99, n = 3; see Ref. 29). We have previously employed RT-PCR assays performed in this semiquantitative manner in studies of the ontogeny of eNOS and neuronal NOS mRNA expression in fetal and newborn rat lung and in studies of changes in their expression in lungs from fetal rats with congenital diaphragmatic hernia and lungs from adult rats subjected to prolonged hypoxia (16, 17, 20). In all investigations in which we have performed Northern analyses in parallel with RT-PCR assays, identical results have been obtained with the two techniques (17, 20).
Statistical analysis. Groups were compared using the two-tailed Student's t-test or ANOVA with Newman-Keuls post hoc testing (24). Results are expressed as means ± SE. Significance was accepted at the 0.05 level of probability.
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RESULTS |
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Effect of cell growth on NOS activity.
The effect of cell growth on NOS activity in pulmonary endothelial
cells was first examined in studies of cells at varying levels of
confluence. As expected, cell abundance as reflected by total cellular
protein per T75 flask increased with advancing confluence, being 132 ± 15 µg at 50%, 289 ± 38 µg at 70%, and 525 ± 49 µg
at 100% confluence (n = 4, P < 0.05). The relative increases in
total cellular protein were comparable to the increase in changes in
cell number seen preliminarily (see METHODS).
Pulmonary endothelial cell proliferation at varying levels of
confluence is shown in Fig. 1. The rate of cell proliferation indicated by the percent of BrdU-positive nuclei was
similar at 50 and 70% confluence. In contrast, cell proliferation fell
by almost two-thirds once 100% confluence was achieved. As a result of
these observations, all further studies were performed at 50 and 100% confluence.
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The effect of varying confluence on NOS activity in pulmonary
endothelial cells is shown in Fig.
2A. NOS
activity was sixfold greater in relatively quiescent cells at 100%
confluence compared with more rapidly proliferating cells at 50%
confluence. To determine if the effect of varying confluence is unique
to the pulmonary vasculature, parallel experiments were performed using
systemic endothelial cells from the mesenteric circulation. As
anticipated, cell abundance indicated by the total protein per T75
flask of systemic endothelial cells increased with advancing
confluence, being 208 ± 13 µg at 50% and 554 ± 21 µg at
100% confluence (n = 4, P < 0.05). The effects of varying
confluence on systemic endothelial cell proliferation rate were
comparable to the findings in the pulmonary endothelium (Fig. 1), with
11 ± 0.7 and 6 ± 0.3% BrdU-positive nuclei at 50 and 100%
confluence, respectively. In contrast to the observed increase in NOS
activity in 100% confluent pulmonary endothelium, there was no effect
of varying confluence on NOS activity in systemic endothelial cells
(Fig. 2B).
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To modify pulmonary endothelial cell growth in an entirely independent
manner, cells were also studied after 24 h of exposure to serum-free or
serum-containing medium beginning at 50% confluence. As expected,
there was greater cell abundance as reflected by total cellular protein
per T75 flask after serum exposure compared with serum-free conditions
(313 ± 20 vs. 107 ± 17 µg/T75
flask, respectively, n = 4, P < 0.05). The effect of serum
exposure on pulmonary endothelial cell proliferation is depicted in
Fig. 3. Cell proliferation was 2.3-fold
greater in serum-exposed cells compared with that in cells in
serum-free conditions.
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The effect of serum exposure on NOS activity in pulmonary endothelial
cells is shown in Fig. 4. In parallel with
the observations made at varying levels of cell proliferation
manipulated by the degree of confluence, NOS activity was threefold
greater in the relatively quiescent, serum-deprived pulmonary
endothelial cells compared with that in the rapidly dividing,
serum-stimulated cells.
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Effect of NO on cell growth. The potential reciprocal effect of NO on PAEC growth was evaluated in studies in which both endogenous NO production and exogenous NO exposure were manipulated. NOS inhibition with L-NAME for 24 h, beginning at 50% confluence, did not alter cell abundance as indicated by total cellular protein per flask (263 ± 19 µg with L-NAME vs. 285 ± 35 µg for control, n = 4). Similarly, the provision of exogenous NO from SpNO for 24 h did not affect cell abundance (258 ± 20 vs. 255 ± 19 µg protein/flask with SpNO vs. Sp at 0.01 µM, n = 4, and 255 ± 30 vs. 271 ± 21 µg protein/flask with SpNO vs. Sp at 1.0 µM, n = 4).
Effect of cell growth on eNOS protein.
The effect of varying cell growth on eNOS protein expression in the
pulmonary endothelium is shown in Fig. 5.
In the representative immunoblot shown, eNOS was detected at the
expected size of 135 kDa, and there was greater protein expression in
cells at 100% confluence compared with that at 50% confluence.
Quantitative densitometry for four independent experiments revealed
that eNOS protein expression was 3.5-fold greater in the 100%
confluent PAEC in relation to 50% confluent cells. These findings
parallel the observed changes in NOS enzymatic activity.
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Effect of cell growth on eNOS mRNA. To
determine the basis for the increase in eNOS protein expression and NOS
enzymatic activity with cell quiescence, eNOS mRNA abundance was
compared by RT-PCR in cells harvested at 50 and 100% confluence. The
PCR product obtained for eNOS was at the expected size of 281 bp (Fig.
6A, top). The representative Southern
blot reveals an increase in eNOS mRNA as determined by RT-PCR in 100%
confluent cells compared with that in cells at 50% confluence,
paralleling the findings for eNOS protein and NOS enzymatic activity.
PCR was also performed for MDH to serve as a control for the RT step,
yielding a single PCR product at the expected size of 396 bp that was
comparable in abundance in the two groups (Fig.
6A,
bottom). These results were
confirmed in four independent experiments. Summary data are shown in
Fig. 6B, revealing 3.6-fold greater
eNOS mRNA expression in quiescent PAEC at 100% confluence compared
with that in 50% confluent cells.
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DISCUSSION |
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In the present study, we sought to determine whether the state of cell growth influences eNOS expression in fetal PAEC. In experiments performed in early-passage cultured cells in which the direct effects of varying cell growth can be examined, we have shown that there is a marked upregulation of eNOS in quiescent PAEC. To our knowledge, this is the first demonstration of the importance of the degree of cell growth in the regulation of eNOS in the pulmonary endothelium.
We initially performed studies in which the approach used to manipulate cell growth involved the contact inhibition known to occur in endothelial cell culture (10). As anticipated, the rate of cell replication was higher in subconfluent compared with confluent cells. We demonstrated that NOS enzymatic activity was sixfold greater in the lysates of PAEC that were relatively quiescent at 100% confluence compared with that in more actively replicating cells at 50% confluence. Because we have previously found that NOS enzymatic activity in endothelial cell lysates is a sensitive indicator of eNOS abundance (13, 15), these findings reveal that eNOS expression is greater in quiescent compared with actively replicating PAEC.
Because it is known that NO attenuates the growth of vascular smooth muscle cells (7) and the current studies revealed greater eNOS abundance in quiescent PAEC, the potential reciprocal effect of NO on PAEC growth was evaluated. Previous studies of the role of NO in the modulation of nonpulmonary endothelial cell growth have yielded variable results (8, 18, 30). Experiments were performed in subconfluent PAEC to assess the effects of manipulating either endogenous NO production or exogenous NO exposure over a period of time during which cell abundance normally doubles. NOS inhibition with L-NAME for 24 h, beginning at 50% confluence, did not alter cell abundance as assessed by measurements of total cellular protein per flask. Similarly, the provision of exogenous NO from SpNO for 24 h did not affect PAEC abundance. These findings indicate that neither endogenous nor exogenous NO has a discernable effect on PAEC growth. Thus the interaction between cell growth and NO in the PAEC is unidirectional.
To determine if the observed changes in NOS activity with varying cell growth are specific to the fetal pulmonary vasculature, parallel experiments were performed with fetal systemic endothelial cells from similarly sized mesenteric arteries. In contrast to the findings for the pulmonary endothelium, there was no effect of varying cell confluence on NOS activity in the systemic endothelium. This suggests that the effect of cell growth on NOS activity in the PAEC is unique to the pulmonary circulation.
In addition to the experiments evaluating the effects of varying cell confluence on eNOS, PAEC were also studied after 24 h of exposure to serum-free or serum-containing medium. In parallel with the findings for PAEC at differing levels of confluence, NOS activity was threefold greater in relatively quiescent PAEC exposed to serum-free conditions versus more actively replicating, serum-exposed cells. These observations reveal that when PAEC growth is manipulated by a totally different mechanism, the same effects of cell proliferation on NOS activity are demonstrated, namely that NOS activity is increased in quiescent cells compared with that in more rapidly replicating cells.
In addition to the studies of NOS enzymatic activity, we also investigated the effects of varying cell growth on eNOS protein expression by performing immunoblot analyses at varying levels of cell confluence. We demonstrated that relatively quiescent PAEC at 100% confluence express 3.5-fold greater eNOS protein than more actively replicating cells at 50% confluence. These findings confirm that the changes in NOS enzymatic activity in the PAEC with varying cell growth are indicative of alterations in eNOS protein expression.
To determine the basis for growth-mediated effects on eNOS enzyme
activity and protein expression in the fetal PAEC, we assessed eNOS
mRNA abundance in 50 and 100% confluent cells using semiquantitative RT-PCR. In parallel with observed changes in eNOS protein abundance and
NOS enzymatic activity, steady-state eNOS mRNA levels were greater in
quiescent compared with more rapidly replicating PAEC. The degree of
changes in the protein and mRNA levels with varying cell growth was
equivalent, and this is similar to the comparable inductions in eNOS
protein and mRNA that we previously observed with varying oxygen
exposure and with estradiol treatment (13, 15). The parallel changes in
protein and mRNA abundance indicate that the mechanism(s) by which cell
growth modulates eNOS expression involves processes at the level of
gene transcription or mRNA stability. With the exception of tumor
necrosis factor-, which principally alters eNOS mRNA stability (28),
studies of both humoral and physical modulators of eNOS indicate that
levels of expression are primarily under transcriptional control (3, 9). As such, it is postulated that varying cell growth modifies eNOS
gene transcription.
The present findings in the fetal PAEC are in distinct contrast to previous observations made in adult bovine aortic endothelial cells (2) in which eNOS protein and mRNA expression were three- to sixfold greater in growing compared with growth-arrested cells studied postconfluence. Because the present results in the fetal PAEC were comparable when cell growth was manipulated by two entirely different methods, it suggests that the disparity with earlier findings in aortic endothelium is not due to the use of preconfluent PAEC versus postconfluent aortic endothelial cells. Alternatively, because parallel studies of fetal PAEC and fetal systemic endothelial cells yielded markedly contrasting findings, it suggests that the differences between the present observations in PAEC and previous results in aortic endothelium are most likely related to the vascular bed of origin. Because the intrapulmonary circulation is derived from splanchnic mesenchyme (25), the intrapulmonary and nonpulmonary endothelia, either aortic or mesenteric, originate from markedly different embryologic sources. In the past, we have noted in studies of NO regulation in both freshly obtained arteries and early-passage cultured cells that there are dramatic differences in the phenotypes of the pulmonary and systemic endothelia (19, 22). Thus it may be unwise to extrapolate or to compare observations made about NO regulation in pulmonary versus systemic endothelium.
In previous studies of eNOS in whole lung, marked changes in its expression have been observed in the perinatal period. Pulmonary eNOS expression normally increases during late gestation, and this is followed by a decline in expression to adult levels in the early newborn period (11, 17, 27). In contrast, there is evidence that pulmonary eNOS expression is attenuated in certain disease states such as congenital diaphragmatic hernia and fetal pulmonary hypertension (16, 23, 27). Because these conditions are all associated with alterations in lung vascular growth (25, 26), the present investigation in the cultured cell model was prompted. With congenital diaphragmatic hernia and fetal pulmonary hypertension, there is an attenuation of pulmonary vascular development (25, 26), and the noted decline in pulmonary eNOS abundance in these conditions suggests that eNOS expression may be blunted with fetal pulmonary endothelial cell quiescence. In addition, in studies of cultured PAEC, we have found that eNOS expression is decreased by prolonged hypoxia (15), which may also reflect downregulation related to attenuated cell growth. However, in the present studies in which the direct effects of varying cell growth were examined in culture, PAEC quiescence resulted in a marked upregulation in eNOS expression. This suggests that the observations made in diaphragmatic hernia lungs and with fetal pulmonary hypertension may be related to other mechanisms besides changes in endothelial cell growth state, such as alterations in pulmonary blood flow and shear stress (3). It further suggests that the effects of hypoxia on PAEC eNOS expression are direct and unrelated to changes in cell growth (15).
A degree of caution may be warranted in extrapolating the present findings in cultured primary cells to processes in the intact lung. For example, the level of oxygenation present in the cell culture system may differ from that in the lung, and we have previously demonstrated that eNOS gene expression in this cell type is modulated by oxygen (15). In addition, cell behavior may vary under different culture conditions. However, a variety of phenotypic characteristics relevant to the regulation of pulmonary endothelial NO production are conserved in the early-passage PAEC cultured as described herein (19, 22). Furthermore, the use of the cultured cells enables us to evaluate the direct effects of single factors such as the degree of cell growth on PAEC phenotype.
Keeping these potential limitations in mind, the present observations have important implications on the function of NO in pulmonary vascular development and disease. Because NO inhibits the proliferation of vascular smooth muscle cells (7), enhanced NO production by quiescent PAEC may attenuate the growth of the underlying vascular smooth muscle, thereby serving to orchestrate the development of the intimal and medial layers. Conversely, pathological conditions leading to enhanced pulmonary intimal proliferation may result in diminished endothelial NO production, thereby contributing to the excessive medial growth that often characterizes these disorders (25, 26). Further studies of growth-related changes in eNOS gene expression in this model may enhance our understanding of the role of NO in the developing and injured lung.
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ACKNOWLEDGEMENTS |
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We are indebted to Marilyn Dixon for preparing this manuscript.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants HD-30276 and HL-53546. The project was done during the tenure of an Established Investigatorship of the American Heart Association (P. W. Shaul).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. W. Shaul, Dept. of Pediatrics, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9063 (E-mail: pshaul{at}mednet.swmed.edu).
Received 29 March 1999; accepted in final form 18 August 1999.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abman, S. H.,
B. A. Chatfield,
S. L. Hall,
and
I. F. McMurtry.
Role of endothelium-derived relaxing factor during transition of pulmonary circulation at birth.
Am. J. Physiol. Heart Circ. Physiol.
259:
H1921-H1927,
1990
2.
Arnal, J. F.,
J. Yamin,
S. Dockery,
and
D. G. Harrison.
Regulation of endothelial nitric oxide synthase mRNA, protein, and activity during cell growth.
Am. J. Physiol. Cell Physiol.
267:
C1381-C1388,
1994
3.
Awolesi, M. A.,
and
W. C. Sessa.
Cyclic strain upregulates nitric oxide synthase in cultured bovine aortic endothelial cells.
J. Clin. Invest.
96:
1449-1454,
1995[ISI][Medline].
4.
Bradford, M. M.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
248-254,
1976[ISI][Medline].
5.
Cornfield, D. N.,
B. A. Chatfield,
J. A. McQueston,
I. F. McMurtry,
and
S. H. Abman.
Effects of birth-related stimuli on L-arginine-dependent pulmonary vasodilation in ovine fetus.
Am. J. Physiol. Heart Circ. Physiol.
262:
H1474-H1481,
1992
6.
Denizot, F.,
and
R. Lang.
Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability.
J. Immunol. Methods
89:
271-277,
1986[ISI][Medline].
7.
Garg, U. C.,
and
A. Hassid.
Nitric oxide generating vasodilators and 8-bromo cyclic GMP inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells.
J. Clin. Invest.
83:
1774-1777,
1989[ISI][Medline].
8.
Ghigo, D.,
E. Aldieri,
R. Todde,
C. Costamagna,
G. Garbarino,
G. Pescarmona,
and
A. Bosia.
Chloroquine stimulates nitric oxide synthesis in murine, porcine and human endothelial cells.
J. Clin. Invest.
102:
595-605,
1998
9.
Inoue, N.,
R. C. Venema,
H. S. Sayegh,
O. Yuichi,
T. J. Murphy,
and
D. G. Harrison.
Molecular regulation of the bovine endothelial nitric oxide synthase by transforming growth factor beta.
Arterioscler. Thromb. Vasc. Biol.
15:
1255-1261,
1995
10.
Johnson, A. R.
Human pulmonary endothelial cells in culture. Activities of cells from arteries and cells from veins.
J. Clin. Invest.
65:
841-850,
1980[ISI][Medline].
11.
Kawai, N.,
D. B. Bloch,
G. Filippov,
D. Rabkina,
H. C. Suen,
P. D. Losty,
S. P. Janssens,
W. M. Zapol,
S. de la Monte,
and
K. D. Bloch.
Constitutive endothelial nitric oxide synthase is regulated during lung development.
Am. J. Physiol. Lung Cell. Mol. Physiol.
268:
L589-L595,
1995
12.
Lantin-Hermoso, R. L.,
C. R. Rosenfeld,
I. S. Yuhanna,
Z. German,
Z. Chen,
and
P. W. Shaul.
Estrogen acutely stimulates nitric oxide synthase activity in fetal pulmonary artery endothelium.
Am. J. Physiol. Lung Cell. Mol. Physiol.
273:
L119-L126,
1997
13.
MacRitchie, A. N.,
S. S. Jun,
Z. Chen,
Z. German,
I. S. Yuhanna,
T. S. Sherman,
and
P. W. Shaul.
Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium.
Circ. Res.
81:
355-362,
1997
14.
Maragos, C. M.,
D. Morley,
D. A. Wink,
T. M. Dunams,
J. E. Saavedra,
A. Hoffman,
A. A. Bove,
L. Isaac,
J. A. Hrabie,
and
L. K. Keefer.
Complexes of NO with nucleophiles as agents for the controlled biologic release of nitric oxide. Vasorelaxant effects.
J. Med. Chem.
34:
3242-3247,
1991[ISI][Medline].
15.
North, A. J.,
K. S. Lau,
T. S. Brannon,
L. C. Wu,
L. B. Wells,
Z. German,
and
P. W. Shaul.
Oxygen upregulates nitric oxide synthase gene expression in ovine fetal pulmonary artery endothelial cells.
Am. J. Physiol. Lung Cell. Mol. Physiol.
270:
L643-L649,
1996
16.
North, A. J.,
F. R. Moya,
M. R. Mysore,
V. L. Thomas,
L. B. Wells,
L. C. Wu,
and
P. W. Shaul.
Pulmonary endothelial nitric oxide synthase gene expression is decreased in a rat model of congenital diaphragmatic hernia.
Am. J. Respir. Cell Mol. Biol.
13:
676-682,
1995[Abstract].
17.
North, A. J.,
R. A. Star,
T. S. Brannon,
K. Ujiie,
L. B. Wells,
C. J. Lowenstein,
S. H. Snyder,
and
P. W. Shaul.
NOS type I and type III gene expression is developmentally regulated in rat lung.
Am. J. Physiol. Lung Cell. Mol. Physiol.
266:
L635-L641,
1994
18.
Papapetropoulos, A.,
C. Garcia-Cardena,
J. A. Madri,
and
W. C. Sessa.
Nitric oxide production contributes to the angiogenic properties of vascular endothelial growth factor in human endothelial cells.
J. Clin. Invest.
100:
3131-3139,
1997
19.
Shaul, P. W.,
M. A. Farrar,
and
T. M. Zellers.
Oxygen modulates endothelium-derived relaxing factor production in fetal pulmonary arteries.
Am. J. Physiol. Heart Circ. Physiol.
262:
H355-H364,
1992
20.
Shaul, P. W.,
A. J. North,
T. S. Brannon,
K. Ujiie,
L. B. Wells,
P. A. Nisen,
C. J. Lowenstein,
S. H. Snyder,
and
R. A. Star.
Prolonged in vivo hypoxia enhances nitric oxide synthase type I and type III gene expression in adult rat lung.
Am. J. Respir. Cell Mol. Biol.
13:
167-174,
1995[Abstract].
21.
Shaul, P. W.,
A. J. North,
L. C. Wu,
L. B. Wells,
T. S. Brannon,
K. S. Lau,
T. Michel,
L. R. Margraf,
and
R. A. Star.
Endothelial nitric oxide synthase is expressed in cultured human bronchiolar epithelium.
J. Clin. Invest.
94:
2231-2236,
1994[ISI][Medline].
22.
Shaul, P. W.,
and
L. B. Wells.
Oxygen modulates nitric oxide production selectively in fetal pulmonary endothelial cells.
Am. J. Respir. Cell Mol. Biol.
11:
432-438,
1994[Abstract].
23.
Shaul, P. W.,
I. S. Yuhanna,
Z. German,
Z. Chen,
R. H. Steinhorn,
and
F. C. Morin III.
Pulmonary endothelial NO synthase gene expression is decreased in fetal lambs with pulmonary hypertension.
Am. J. Physiol. Lung Cell. Mol. Physiol.
272:
L1005-L1012,
1997
24.
Snedecor, G. W.,
and
W. G. Cochran.
Statistical Methods. Ames, IA: Iowa State Univ, 1980.
25.
Stenmark, K. R.,
and
R. P. Mecham.
Cellular and molecular mechanisms of pulmonary vascular remodeling.
Annu. Rev. Physiol.
59:
89-144,
1997[ISI][Medline].
26.
Tenbrinck, R.,
D. Tibboel,
J. L. J. Gaillard,
D. Kluth,
A. P. Bos,
B. Lachmann,
and
J. C. Molenaar.
Experimentally induced congenital diaphragmatic hernia in rats.
J. Pediatr. Surg.
25:
426-429,
1990[ISI][Medline].
27.
Villamor, E.,
T. D. La Cras,
M. P. Horan,
A. C. Halbower,
R. M. Tuder,
and
S. H. Abman.
Chronic intrauterine pulmonary hypertension impairs endothelial nitric oxide synthase in the ovine fetus.
Am. J. Physiol. Lung Cell. Mol. Physiol.
272:
L1013-L1020,
1997
28.
Yoshizumi, M.,
M. Perella,
J. Burnett,
and
M. Lee.
Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life.
Circ. Res.
73:
205-209,
1993[Abstract].
29.
Yuhanna, I. S., A. N. MacRitchie, R. L. Lantin-Hermoso, L. B. Wells, and P. W. Shaul. Nitric oxide upregulates endothelial nitric oxide synthase
expression in fetal intrapulmonary artery endothelial cells.
Am. J. Respir. Cell Mol.
Biol. In press.
30.
Ziche, M.,
A. Parenti,
F. Ledda,
P. Dell'Era,
H. J. Granger,
C. A. Maggi,
and
M. Presta.
Nitric oxide promotes proliferation and plasminogen activator production by coronary venular endothelium through endogenous bFGF.
Circ. Res.
80:
845-852,
1997