Departments of 1 Pediatrics and 2 Molecular Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611-3008; 3 Cardiovascular Research Center, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; and 4 Department of Pediatrics and 5 Cardiovascular Research Institute, University of California, San Francisco, California 94143-0106
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ABSTRACT |
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Utilizing aortopulmonary vascular graft placement, we established a lamb model of pulmonary hypertension that mimics congenital heart disease with increased pulmonary blood flow. We previously demonstrated that endothelial nitric oxide synthase (eNOS) is increased in lambs at age 4 wk. However, these lambs display a selective impairment of endothelium-dependent pulmonary vasodilation that is suggestive of a derangement downstream of NO release. Thus our objective was to characterize potential alterations in the expression and activity of soluble guanylate cyclase (sGC) and phosphodiesterase type 5 (PDE5) induced by increased pulmonary blood flow and pulmonary hypertension. Late-gestational fetal lambs (n = 10) underwent in utero placement of an aortopulmonary vascular graft (shunt). Western blotting analysis on lung tissue from 4-wk-old shunted lambs and age-matched controls showed that protein for both subunits of sGC was increased in shunted lamb lungs compared with age-matched controls. Similarly, cGMP levels were increased in shunted lamb lungs compared with age-matched controls. However, PDE5 expression and activity were also increased in shunted lambs. Thus although cGMP generation was increased, concomitant upregulation of PDE5 expression and activity may have (at least partially) limited and accounted for the impairment of endothelium-dependent pulmonary vasodilation in shunted lambs.
phosphodiesterase type 5; soluble guanylate cyclase; circulation
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INTRODUCTION |
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RECENT EVIDENCE
SUGGESTS that normal pulmonary vascular tone is regulated by a
complex interaction of vasoactive substances that are produced locally
by the vascular endothelium (7, 15, 28). Nitric oxide (NO)
is an endothelium-derived relaxing factor synthesized by the
oxidation of the guanidino nitrogen moiety of L-arginine
after activation of NO synthase (NOS) (19). Once released
from endothelial cells, NO diffuses into vascular smooth muscle cells
(SMCs) and activates soluble guanylate cyclase (sGC), a heterodimer
with 1- and
1-subunits, which catalyzes
the production of cGMP from GTP. cGMP induces vascular SMC relaxation
through activation of a cGMP-dependent protein kinase (PKG)
(9). Although the exact mechanism remains unclear, it has
recently been shown that PKG-mediated relaxation in response to NO and
cGMP is via a leucine zipper interaction with the myosin-binding
subunit (MBS) of myosin phosphatase, which binds to smooth muscle
contractile apparatus (25). Cyclic nucleotide
phosphodiesterases (PDEs) regulate intracellular levels of cGMP by
catalyzing cGMP to GMP (2). PDE type 5 (PDE5) is the
predominant cGMP-metabolizing PDE of pulmonary tissues and is
selectively inhibited by zaprinast (23).
Evidence that the NO-cGMP cascade mediates normal pulmonary vascular tone has led to the hypothesis that endothelial injury induced by congenital heart disease with increased pulmonary blood flow disrupts these regulatory mechanisms and participates in the development of pulmonary hypertension and its associated altered vascular reactivity (8, 14, 26). For example, adults with advanced pulmonary hypertension have impaired endothelium-dependent pulmonary vasodilation and decreased endothelial NOS (eNOS) gene expression within pulmonary vascular endothelial cells (10, 13). However, because most patients who undergo histological evaluation have advanced pulmonary hypertension, it has been difficult to investigate early aberrations in the NO-cGMP cascade and their potential role in the development of pulmonary hypertension secondary to increased pulmonary blood flow.
Utilizing aortopulmonary shunt placement in the late-gestational fetal
lamb, we recently established a unique animal model of pulmonary
hypertension that mimics congenital heart disease with increased
pulmonary blood flow (20). We have previously shown that
these lambs have physiological alterations in the NO-cGMP cascade as
early as 4 wk of age. These alterations include a selective impairment
of endothelium-dependent pulmonary vasodilation that is suggestive of
decreased NO activity in that the pulmonary vasodilating effects of
acetylcholine and ATP are attenuated compared with control lambs
(20). However, shunted lambs also had an augmented increase in pulmonary vascular resistance in response to NO inhibition (induced by N-nitro-L-arginine)
and increased expression of eNOS, which is suggestive of increased
basal NO production (4, 20, 21). Therefore, the mechanism
responsible for the impaired endothelium-dependent pulmonary
vasodilation remains unclear.
The purpose of the present study was to characterize at the molecular level the alterations in the downstream effectors of NO in lambs with increased pulmonary blood flow. Using Western blotting and immunohistochemistry, we compared sGC and PDE5 protein in 4-wk-old lambs with increased pulmonary blood flow (shunted lambs) to age-matched controls (control lambs). In addition, changes in lung tissue cGMP concentrations and PDE5 activity were determined.
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METHODS |
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Surgical preparation and care.
Ten mixed-breed, pregnant Western ewes (137-141 days gestation,
term = 145 days) were operated on under sterile conditions as
previously described (20). After spontaneous delivery of the lambs, antibiotics (106 U of penicillin G potassium and
25 mg of gentamicin sulfate im) were administered for 2 days. The lambs
were weighed daily, and respiratory rate and heart rate were measured.
Furosemide (1 mg/kg im) was administered daily, and elemental iron (50 mg im) was given weekly. At 4 wk of age, the lambs were anesthetized
with intravenous infusions of ketamine hydrochloride (~1
mg · kg1 · min
1) and
diazepam (0.002 mg · kg
1 · h
1), intubated
with a 5.5-mm-OD endotracheal tube, and mechanically ventilated with a
pediatric, time-cycled, pressure-limited ventilator (Healthdyne,
Marietta, GA). Ventilation with 21% O2 was adjusted to
maintain an arterial PCO2
(PaCO2) between 35 and 45 Torr. Via a midsternotomy
incision, the lambs were instrumented to measure pulmonary and systemic
arterial pressure, right and left atrial pressure, heart rate, and left
pulmonary blood flow as previously described (20, 21).
After 60 min of recovery, baseline hemodynamic variables and
O2 saturation values were obtained. The lambs were then
euthanized with an intravenous injection of pentobarbital sodium
(Euthanasia CII, Central City Medical, Union City, CA) and were
subjected to bilateral thoracotomy. An autopsy was performed to confirm
patency of the vascular graft. The lungs were removed and prepared for
Western blot analysis, immunohistochemistry, and measurements of cGMP
concentration and PDE5 activity. All procedures and protocols were
approved by the Committee on Animal Research of the University of
California, San Francisco.
Tissue preparation.
The heart and lungs were removed en bloc. The lungs were dissected with
care to preserve the integrity of the vascular endothelium. Sections
(2-3 g) from each lobe of the lung were removed. These tissues
were snap-frozen in liquid N2 and stored at 70°C until analysis.
Measurement of cGMP. For cGMP content measurements, the snap-frozen peripheral lung tissue was homogenized in cold (4°C) 6% trichloroacetic acid (10% wet wt/vol) and centrifuged for 15 min at 4°C. cGMP was measured using an 125I radioimmunoassay kit with reagents provided according to the manufacturer's instructions (cGMP 125I assay system RPA 525, Amersham International, Arlington Heights, IL). The supernatant was washed 4 times with 5 volumes of diethyl ether. The remaining aqueous extract was dried under a stream of air and then resuspended in 1 ml of assay buffer. A 500-µl sample and two cGMP standards (128 fmol/tube) were acetylated with 25 µl of triethylamine-acetic anhydride (a 2:1 dilution). To a 100-µl aliquot, 100 µl of antiserum were added and incubated for 1 h at room temperature; then 100 µl of 125I-cGMP were added and incubated for 18 h at 4°C. Next, 500 µl of Amerlex-M second antibody reagent were added and incubated for 10 min at room temperature. The antibody-bound fraction was separated by centrifugation and was counted using a gamma scintillation counter.
Measurement of cGMP PDE activity. Peripheral lung extracts prepared from 4-wk-old shunted and control lambs were assayed for both total cGMP PDE activity and zaprinast-inhibited PDE activity. The difference between cGMP hydrolytic activity in the presence and absence of 2 µM zaprinast was used as an estimate of PDE5 activity as we have detailed previously (23).
Western blot analysis.
Western blot analysis was performed as previously described
(6). Protein extracts (100 µg) prepared from control and
shunted lungs were separated on 10% SDS-polyacrylamide gel and
electrophoretically transferred to Hybond polyvinylidene difluoride
membranes (Amersham). The membranes were blocked with 5% nonfat dry
milk in Tris-buffered saline (TBS) containing 0.1% Tween. Once
blocked, the membranes were incubated at room temperature with the
appropriate dilution of the antiserum of interest [1:1,000 dilution
for the 1-subunit of sGC (sGC-
1),
1:10,000 dilution for the
1-subunit of sGC
(sGC-
1; antisera for sGC-
1 and sGC-
1
were a gift from Dr. Peter Yuen), or 1:2,000 dilution for PDE5 (a gift
from Dr. Stefan Janssens, University Hospital Gasthuisberg, Lueven,
Belgium)], washed with TBS containing 0.1% Tween, and then incubated
with the appropriate species anti-IgG-horseradish peroxidase conjugate.
After membranes were washed, chemiluminescence (Pierce Laboratories)
was used to detect the protein bands of interest.
Immunohistochemistry.
Immunohistochemistry was performed as previously described
(4). Studies were performed on serial sections of control
and shunted ovine lungs using polyclonal antisera to either the
sGC-1 protein or PDE5. The tissue sections were
incubated in the appropriate antiserum (a 1:1,000 dilution for
sGC-
1 and a 1:500 dilution for PDE5) for 12-18 h at
4°C. Appropriate biotin-conjugated secondary antibodies were used and
detected with an anti-rabbit IgG conjugated to FITC. Sections were
viewed under a fluorescence microscope (Olympus BX40) and imaged with a
digital camera (Pixera DiRactor).
Data analysis. Quantitation of chemiluminescent studies was performed by scanning (Scan Jet IICX, Hewlett Packard, Palo Alto, CA) the bands of interest into an image-editing software program (Adobe Photoshop, Adobe Systems, Mountain View, CA). Band intensities from Western blot analysis were analyzed densitometrically on a Macintosh computer (model 9500, Apple Computer, Cupertino, CA) using the public domain NIH Image program (developed at National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). To ensure equal protein loading for Western blot analysis, duplicate polyacrylamide gels were run and one was stained with Coomassie blue. Results from control lungs were assigned the value of 1 (relative protein of interest). Means ± SD were calculated for the relative protein of interest from control and shunted lungs and were compared by the unpaired t-test. A P < 0.05 was considered statistically significant.
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RESULTS |
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Shunted lambs had a mean pulmonary arterial pressure of 44.8 ± 11.7 mmHg, which represented 76% of systemic values. In comparison, control lambs had a mean pulmonary arterial pressure of 16.5 ± 3.7 mmHg, which represents 22% of systemic values (P < 0.05). Although systolic systemic arterial pressure was similar in both groups, diastolic systemic pressure was lower in shunted lambs as a result of the runoff across the graft (40.3 ± 10.6 vs. 63.3 ± 17.1 mmHg; P < 0.05). Therefore, mean systemic arterial pressure was lower in shunted lambs (61.7 ± 11.7 vs. 77.8 ± 14.0 mmHg; P < 0.05). In addition, shunted lambs had a pulmonary-to-systemic blood flow ratio of 2.3 ± 0.9 as determined by the Fick equation.
There were changes in the expression of the sGC subunits after in utero
insertion of an aortopulmonary vascular graft. We found that shunted
lambs had a 2.53-fold increase in sGC-1
(P < 0.05; Fig. 1) and a
3.41-fold increase in sGC-
1 (P < 0.05; Fig. 2). Additional analysis using
immunohistochemistry also demonstrated increased expression of
sGC-
1 in shunted versus control vessels (Fig.
3). sGC-
1 expression was
also observed in the airway SMC layer. Tissue cGMP levels were also
found to be increased by 2.13-fold in the lungs of shunted lambs
(P < 0.05; Fig. 4),
which indicates an increase in both NO production and sGC activity.
Similarly, we found that shunted lambs had a 2.1-fold increase in PDE5
protein expression (Fig. 5). This change
in PDE5 protein expression was shown to be present in the vessels of
the shunted lambs (Fig. 6). However, as
with sGC-
1, expression of PDE5 was detected in the
airway SMC layer. We were also able to demonstrate a 1.92-fold increase
in overall PDE activity (Fig.
7A). Zaprinast-inhibited cGMP
hydrolytic activity (an estimate of PDE5 activity) was also found to be
increased by 2.1-fold in shunted lambs (Fig. 7B).
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DISCUSSION |
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Children with early, reversible pulmonary hypertension that is
secondary to increased pulmonary blood flow suffer morbidity from
enhanced pulmonary vascular reactivity (8, 14, 26). The
NO-cGMP cascade is an important modulator of pulmonary vascular reactivity. We have previously developed a novel model of pulmonary hypertension with increased pulmonary blood flow in the lamb after in
utero placement of an aorta-to-pulmonary shunt. At 4 wk of age, these
lambs have a selective impairment of endothelium-dependent pulmonary
vasodilation but evidence of increased basal NOS activity and gene
expression (4, 20, 21). Taken together, these results
suggest that shunted lambs have an alteration in the ability to
transduce increased NOS activity into SMC relaxation. This could be
caused by insufficient cGMP production or increased capacity of shunted
lambs to degrade cGMP into the biologically inactive GMP. To examine
these possibilities, we determined the expression and activity of the
two subunits of sGC (the enzyme responsible for cGMP biosynthesis) and
PDE5 (the enzyme responsible for cGMP degradation). In the present
study, we demonstrate that sGC-1, sGC-
1,
and PDE5 are upregulated in 4-wk-old shunted lambs. However, the net
changes are associated with an increase in tissue cGMP levels, which
suggests that the altered pulmonary vascular reactivity noted in
shunted lambs is not due to a decrease in either sGC expression or activity.
It should be noted that cGMP production and accumulation in the lung
are regulated not only by sGC activity but also by the action of
natriuretic peptides acting on particulate guanylate cyclase (pGC).
Levels of natriuretic peptides such as atrial natriuretic peptide (ANP)
have been shown to be increased in congestive heart failure (16,
17). Thus at least part of the increased cGMP accumulation that
we observed could be due to the activity of pGC. However, because the
increases in cGMP accumulation closely mirror the changes in
1- and
1-protein expression, it is likely that the increased cGMP is predominantly from the sGC.
Factors responsible for the regulation of sGC expression in lambs with
increased pulmonary blood flow remain to be elucidated. However, we
have demonstrated that sGC expression and activity increase in
correlation with the increases in eNOS expression and activity that we
have previously observed (4). Similarly, we have
previously demonstrated such a coordinated regulation between eNOS and
sGC expression in another model of pulmonary hypertension produced by
in utero ductal ligation (5). However, in this case, both
systems were downregulated (5). This suggests that factors
such as NOS levels, NO production, and cGMP accumulation may all
contribute to the regulation of sGC expression. These interactions are
likely to be complex; recent studies have suggested that either
increased eNOS expression or exogenous NO can reduce sGC expression
(12, 18). The latter study, using cultured vascular SMCs,
suggested that the mechanism of action is via a reduction in the
stability of the mRNAs for both sGC-1 and
sGC-
1 (12). However, the exact relationship
between endogenous NO release and sGC expression is far from clear. A
more recent report found that sGC expression was unchanged in mice that
have one, two, or no copies of a functional eNOS gene, although sGC
activity was decreased (27). Thus additional studies are
needed to determine the exact relationship between NO production and
sGC expression in shunted lambs. It is interesting to note that both
eNOS and sGC are downregulated in the ductal ligation model, which has a stimulus of increased pressure and decreased flow, whereas both are
upregulated in the shunt model, which has a stimulus of increased pressure and increased flow. We speculate that the upregulation of the
NO-cGMP cascade may represent an early adaptive response of the
pulmonary circulation to maintain a low pulmonary vascular resistance
in response to increased pulmonary blood flow.
Intracellular cGMP concentrations are not simply determined by the accumulation of cGMP but rather by a balance between synthesis and degradation. Cyclic nucleotide PDEs are the enzymes responsible for cGMP degradation (2). In the mammalian lung, there are a number of PDEs, but the cGMP-specific PDE, namely PDE5, is prevalent, especially early in development (23). In this study, we have demonstrated that the expression and activity of PDE5 is increased in shunted lambs compared with age-matched controls. Because cGMP concentrations are elevated in lungs from shunted lambs, this increased cGMP hydrolytic activity does not appear to be sufficient to completely abrogate the increased cGMP accumulation afforded by the increase in sGC activity. Shunted lambs display a selective impairment of endothelium-dependent pulmonary vasodilation (21). It is possible that the increased PDE5 activity present in lungs from shunted lambs may be sufficient to limit cGMP accumulation to a suboptimal level after our acute stimulus, thereby reducing SMC relaxation and producing the altered pulmonary vascular reactivity (21).
As with sGC, little is known about the regulation of PDE5 gene expression. Recent studies in lambs with chronic fetal pulmonary hypertension secondary to in utero ductal ligation suggest that sGC activity is decreased but PDE5 activity is increased (5, 22, 24). We have previously shown that in the ductal ligation model, vasodilator gene expression was suppressed although vasoconstrictor gene expression was enhanced (5). This apparent coordinated regulation does not seem to occur in shunted lambs (3, 4). However, in both cases of pulmonary hypertension, PDE5 gene expression and activity were increased. Because in utero ductal ligation produces fetal pulmonary hypertension and low pulmonary blood flow as opposed to high pulmonary blood flow in the postnatal model that we used, it is unclear whether the PDE5 gene is regulated in a similar manner in both models. In fact, it is possible that different regulatory mechanisms are involved in these two types of pulmonary hypertension. Again, additional studies will be necessary to determine the factors responsible for the upregulation of PDE5 gene expression in shunted lambs.
In conclusion, we report that lambs with pulmonary hypertension secondary to increased pulmonary blood flow exhibit increased expression and activity for the cell systems responsible for both increased synthesis and degradation of cGMP. The net result of these changes in expression is an increase in tissue cGMP levels, which may be limited in part by increased PDE5 activity. We speculate that the upregulated PDE5 system may participate in the enhanced pulmonary vascular reactivity noted in shunted lambs. Recent studies demonstrate that PDE inhibitors produce potent pulmonary vasodilation in animals and children with pulmonary hypertension (1, 11, 29). The findings of this current study may partially explain these beneficial effects. Additional studies into the alterations and the mechanisms of the NO-cGMP cascade in pulmonary hypertension may continue to lead to important treatment and prevention strategies.
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ACKNOWLEDGEMENTS |
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This research was supported in part by National Heart, Lung, and Blood Institute Grants HL-60190 (to S. M. Black), HL-61284 (to J. R. Fineman), and HL-54705 (to R. H. Steinhorn); National Institute of Child Health and Human Development Grant HD-398110 (to S. M. Black); March of Dimes Grants FY00-98 (to S. M. Black) and FY99-421 (to J. R. Fineman); and American Heart Association, Midwest Affiliate Grant 0051409Z (to S. M. Black).
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FOOTNOTES |
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S. M. Black is a member of the Feinberg Cardiovascular Research Institute.
Address for reprint requests and other correspondence: S. M. Black, Northwestern Univ. Medical School, Ward 12-191, 303 E. Chicago Ave., Chicago, IL 60611-3008 (E-mail: steveblack{at}northwestern.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.
Received 16 December 2000; accepted in final form 20 July 2001.
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