(Circulation. 2000;101:923.)
© 2000 American Heart Association, Inc.
Basic Science Reports |
In Vivo Gene Transfer of Prepro-Calcitonin Gene–Related Peptide to the Lung Attenuates Chronic Hypoxia-Induced Pulmonary Hypertension in the Mouse
From the Department of Pharmacology, Tulane University School of Medicine, New Orleans, La (H.C.C., T.J.B., A.L.H., P.J.K.), and the Departments of Internal Medicine and Pharmacology, University of Iowa College of Medicine, Iowa City (K.T., D.D.H.).
Correspondence to Philip J. Kadowitz, PhD, Department of Pharmacology SL83, Tulane University School of Medicine, 1430 Tulane Ave, New Orleans, LA 70112.
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Abstract |
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Background—Calcitonin gene–related peptide (CGRP) is believed to play an important role in maintaining low pulmonary vascular resistance (PVR) and in modulating pulmonary vascular responses to chronic hypoxia; however, the effects of adenovirally mediated gene transfer of CGRP on the response to hypoxia are unknown.
Methods and Results—In the present study, an adenoviral vector encoding prepro-CGRP (AdRSVCGRP) was used to examine the effects of in vivo gene transfer of CGRP on increases in PVR, right ventricular mass (RVM), and pulmonary vascular remodeling that occur in chronic hypoxia in the mouse. Intratracheal administration of AdRSVCGRP, followed by 16 days of chronic hypoxia (FIO2 0.10), increased lung CGRP and cAMP levels. The increase in pulmonary arterial pressure (PAP), PVR, RVM, and pulmonary vascular remodeling in response to chronic hypoxia was attenuated in animals overexpressing prepro-CGRP, whereas systemic pressure was not altered while in chronically hypoxic mice, angiotensin II and endothelin-1–induced increases in PAP were reduced, whereas decreases in PAP in response to CGRP and adrenomedullin were not changed and decreases in PAP in response to a cAMP phosphodiesterase inhibitor were enhanced by AdRSVCGRP.
Conclusions—In vivo CGRP lung gene transfer attenuates the increase in PVR and RVM, pulmonary vascular remodeling, and pressor responses in chronically hypoxic mice, suggesting that CGRP gene transfer alone and with a cAMP phosphodiesterase inhibitor may be useful for the treatment of pulmonary hypertensive disorders.
Key Words: pulmonary heart disease • hypoxia • genes • peptides • molecular biology
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Introduction |
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Calcitonin gene–related peptide (CGRP) is produced by alternative splicing of the calcitonin gene and is localized in nerves and endocrine cells within the lung.1 2 3 4 CGRP receptors are highly expressed on lung vascular smooth muscle cells, and CGRP has potent pulmonary vasodilator activity.5 6 CGRP levels and bioavailability are reduced by hypoxia, and it has been hypothesized that the peptide may play a role in the regulation of the pulmonary circulation under pathophysiological conditions.4 7 8 9 10 11 In hypoxic rats, CGRP receptor blockade exacerbates the increase in pulmonary arterial pressure (PAP) and right ventricular mass (RVM), whereas chronic CGRP infusion has a beneficial effect.12 These observations suggest that CGRP may attenuate the pulmonary response to chronic hypoxia and that CGRP may be useful in the treatment of pulmonary hypertension.12
The transfer of genes for vasoactive peptides represents a new strategy for the treatment of cardiovascular disorders.13 14 15 16 17 Gene transfer to the lung can modify pulmonary vascular resistance (PVR) and responses, suggesting that gene transfer technology may be applicable to the treatment of pulmonary hypertension.14 16 Therefore, we hypothesized that overexpression of CGRP in the lung may reduce PVR and vasoconstrictor responses. To the best of our knowledge, peptides have not been transferred to the lung by use of adenoviral vectors. To test the hypothesis that peptide gene transfer may be useful in treating cardiovascular disorders, a recombinant adenovirus containing the cDNA for human prepro-CGRP, which may facilitate the release of CGRP from transfected cells, was constructed. The purpose of the present investigation was to study the effect of in vivo gene transfer of prepro-CGRP on the increase in PAP, RVM, and pulmonary vascular remodeling induced by chronic hypoxia in the mouse.
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Methods |
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Adenovirus Vectors
Two replication-deficient recombinant adenoviruses encoding nuclear-targeted ß-galactosidase (AdRSVßgal) and prepro-CGRP (AdRSVCGRP), both driven by a Rous sarcoma virus (RSV) promoter, were generated by standard methods by the University of Iowa Gene Transfer Vector Core Laboratory.16 18 Briefly, human prepro-CGRP was cloned by blunt-end ligation into pAdRSV4. The resultant plasmid and adenovirus backbone sequences restricted of E1 were transfected into HEK 293 cells, and plaques were isolated and amplified for analysis of CGRP expression. Recombinant adenoviruses were triple plaque–purified, and virus titer was determined by plaque assay on HEK-293 cells. Purified viruses were stored in PBS with 3% sucrose and kept at -80°C until use.
In Vivo Gene Delivery to the Lung
CD-1 mice (Harlan-Sprague Dawley, Indianapolis, Ind) weighing 22
to 31 g were anesthetized with thiopentobarbital (85 to 95
µg/g IP) and placed in the supine position on a thermoregulated
surgical table. Body temperature was monitored with a rectal probe
(Yellow Springs Instruments) and maintained at 37°C with a
water-jacketed heating blanket. Under sterile technique, the trachea
was approached via a midline neck incision and isolated by blunt
dissection. With a 27-gauge needle attached to a microliter syringe, 50
µL of vehicle (3% sucrose in PBS), AdRSVßgal
(1012 particles/mL), or AdRSVCGRP
(1012 particles/mL) was instilled into the
trachea by a recently described procedure.16
Hypoxic Exposure
After intratracheal administration of vehicle, AdRSVßgal, or
AdRSVCGRP, the animals were housed 12 to 15 animals per cage, exposed
to a 12:12-hour light cycle, and fed standard mouse chow and water ad
libitum. Five days after transfection, the hypoxic group was placed in
a hypobaric chamber with an air intake valve adjusted to maintain
intrachamber pressure at 380 mm Hg for 16 days. The chamber was
opened daily for cleaning and replacement of food and water. Chamber
pressure was monitored. These procedures have been described
previously.11 12 19 Normoxic control mice were kept in
identical cages under normobaric conditions.
Measurement of Systemic and Pulmonary Vascular
Responses
Twenty-one days after transfection (16 days after hypoxic
exposure was begun), mice were anesthetized with
thiopentobarbital (85 to 95 µg/g IP) and placed on a thermoregulated
surgical table. The trachea was cannulated, and the animals breathed
room air enriched with 95% O2/5%
CO2. A femoral artery was cannulated for the
measurement of systemic arterial pressure, which was
measured with a Viggo-Spectramed transducer attached to a polygraph
(Grass Instruments, model 7). Heart rate was determined from the
systolic pressure pulses with a tachometer (Grass, model
7P44A), and the left jugular vein was cannulated for the administration
of drugs.16
The animals were strapped in the supine position to a fluoroscopic table, and PAP was measured with a pressure transducer (Schneider/Namic). Mean PAP was derived electronically, and pulmonary arterial wedge pressure was determined by recently described procedures.16
Cardiac output was measured by the thermodilution technique by injection of a known volume of 0.9% NaCl solution at 23°C into the right atrium and measurement of blood temperature changes in the root of the aorta with a cardiac output computer (Cardiotherm 500, Columbus Instruments) equipped with a small-animal interface. A thermistor microprobe (Columbus Instruments, Fr-1) was inserted into the right carotid artery and advanced to the aortic arch, where changes in aortic blood temperature were measured.6 16
Lung Histology
For histological analysis, the animals
were killed as described previously, and the lung was perfused through
the right ventricle and trachea with 2%
paraformaldehyde and 0.2%
glutaraldehyde in PBS for 10 minutes. Sagittal sections
of the lower left lobe were cut, fixed in 10% formalin, embedded in
paraffin, and prepared for Masson’s trichrome stain. The
muscularization of pulmonary vessels was determined as
described previously.12 20 Pulmonary vessels with
external diameters between 25 and 100 µm landmarked to an airway
distal to the respiratory bronchiole were counted. Vessels were
considered muscularized if a muscularized media was seen around the
entire vessel. Vessels with a muscularized media that did not
completely surround the vessel were considered partly muscularized, and
those without any muscularized media were considered nonmuscularized.
Percent muscularization of pulmonary vessels for each side
examined was calculated as (number of muscularized and partly
muscularized vessels)/total number of vessels countedx100.
Lung water was determined from the weight difference between wet and dry lungs normalized to body weight. Hearts were isolated and fixed in 10% formalin for 4 hours, blotted, dissected, and weighed while moist. The weight ratio of the right ventricle (RV) to the left ventricle (LV) plus septum [RV/(LV+S)] was used to evaluate changes in RVM; the LV index, obtained by dividing the LV by body weight (LV/BW), was used to determine whether LV changes occurred that could affect the RV/(LV+S) or whether changes occurred in the RV alone.12 20
Expression of ß-Galactosidase
Twenty-one days after adenovirus administration, the mice were
anesthetized and, after administration of heparin (1000 U/kg
IV), were killed with pentobarbital (80 mg/kg IP). The lung was
perfused through the right ventricle with PBS at 15 mm Hg and
removed. Expression of ß-galactosidase was evaluated by measurement
of ß-galactosidase activity in tissue samples and by histochemical
staining using recently described methods.16
Measurement of Lung and Plasma CGRP and Lung cAMP and cGMP
Levels
Lung and plasma CGRP levels were measured 21 days after
instillation of vehicle, AdRSVßgal, or AdRSVCGRP in normoxic and
hypoxic mice. Animals were killed as previously described, and the
lungs were excised and processed immediately or quick-frozen in liquid
nitrogen. Lungs were homogenized (Polytron, Brinkmann
Instruments), tissue peptides were extracted by boiling of
homogenates in saline for 15 minutes and
centrifugation (-4°C, 1500g for 30
minutes), and supernatants were collected. Lung pellet boiling was
repeated in 0.5 mol/L acetic acid for 15 minutes, followed by
centrifugation. For each lung, the combined
supernatants were stored lyophilized until assay. Radioimmunoassays
were performed on lung extracts and left ventricular plasma
as previously described.14 Because elevated lung
water in hypoxic mice significantly increases lung weight, CGRP levels
were expressed as picomoles per whole lung instead of wet weight. In
addition, because hematocrit is elevated in hypoxic mice, peptide
levels in plasma are expressed as picomoles per liter blood to avoid
differences due to reduced plasma volume as previously
described.11 12
Twenty-one days after administration of vehicle, AdRSVßgal, or AdRSVCGRP, treated lungs were quick-frozen in liquid nitrogen and stored at -70°C. Whole-lung tissue was homogenized in 1 mL ice-cold 6% trichloroacetic acid, pH 4.0, and then centrifuged at 1500g for 10 minutes at 4°C; the supernatant was transferred to a 10-mL test tube; and the trichloroacetic acid was extracted with H2O-saturated diethyl ether. The samples were assayed for cAMP or cGMP with an enzyme immunoassay kit (Cayman Chemical). Lung cAMP and cGMP levels are expressed as picomoles cAMP or cGMP per lung.
Drugs
Endothelin-1 (ET-1), CGRP, adrenomedullin, and
angiotensin II (Sigma Chemical Co) were dissolved in 0.9%
NaCl. U46619 (9,11-dideoxy-11
,9-epoxymethano
prostaglandin F2, Upjohn) was
dissolved in 100% ethanol and diluted with 0.9% NaCl. Zaprinast
(2-o-propoxyphenyl-8-azapurin-6-one; Rhone-Poulenc) was
dissolved in 0.15N NaOH in 0.9% NaCl and diluted in 0.9% NaCl.
Rolipram (SmithKline Beecham) was dissolved in 20% dimethylsulfoxide
and diluted with normal saline. Solvents used in this study did not
alter vascular pressures or responses. Stock solutions were stored
frozen, and working solutions were prepared daily and kept on
crushed ice.
Statistics
The data expressed as mean±SEM were analyzed with a
1-way ANOVA with repeated measures and Newman-Keuls test or a paired
t test. A value of P<0.05 was used as the
criterion for statistical significance.
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Results |
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Effect of AdRSVCGRP on Baseline Parameters
Hemodynamic parameters in normoxic and hypoxic mice after intratracheal administration of AdRSVßgal or AdRSVCGRP are presented in Table 1
. Mice treated with AdRSVßgal exposed
to hypobaric hypoxia for 16 days had increased mean right
atrial pressure, mean PAP, and PVR (Table 1).
Hemodynamic parameters were similar in
animals receiving vehicle or AdRSVßgal when exposed to
hypoxia for 16 days (data not shown). The increase in right
atrial pressure, PAP, and PVR in response to chronic hypoxia
was significantly reduced in animals treated with AdRSVCGRP compared
with AdRSVßgal-treated animals (Table 1).
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Effects of AdRSVCGRP on Responses in Chronically Hypoxic
Mice
The influence of AdRSVCGRP on responses to CGRP and adrenomedullin
in chronically hypoxic mice is summarized in Figure 1. When PAP was increased to 23 to
27 mm Hg with U46619 or ET-1, intravenous injections
of CGRP or adrenomedullin decreased PAP and systemic
arterial pressure. Decreases in pulmonary and
systemic pressures were similar in vehicle control and
AdRSVßgal-treated animals (data not shown). Decreases in
pulmonary and systemic pressures in response to the peptides
were similar in mice treated with AdRSVCGRP or AdRSVßgal.
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The influence of AdRSVCGRP on responses to type IV and type V
phosphodiesterase inhibitors in chronically hypoxic mice is
summarized in Figure 2. When PAP was
increased with U46619 or ET-1, intravenous injections of
the type IV cAMP inhibitor rolipram and the type V cGMP
inhibitor zaprinast decreased PAP and systemic
arterial pressure. Decreases in pressure were similar in
vehicle control and AdRSVßgal-treated animals (data not shown). In
mice treated with AdRSVCGRP, the decrease in PAP, but not in systemic
pressure, in response to rolipram was increased compared with responses
in AdRSVßgal-treated mice (Figure 2). Decreases in pressure in
response to zaprinast were similar in animals treated with AdRSVßgal
or with AdRSVCGRP (Figure 2).
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The influence of AdRSVCGRP on pulmonary and systemic pressor
responses in chronically hypoxic mice is summarized in Figure 3. Angiotensin II increased
PAP and systemic arterial pressure. Under elevated-tone
conditions, ET-1 induced biphasic pressure changes characterized by an
initial depressor followed by a pressor response, and the pressor phase
of the response is summarized in Figure 3. In mice treated with
AdRSVCGRP, the increases in PAP in response to angiotensin
II and ET-1 were reduced compared with responses in mice treated with
AdRSVßgal. Although pulmonary pressor responses to
angiotensin II and ET-1 were reduced in animals treated
with AdRSVCGRP, the increases in systemic pressure in response to the
peptides were not altered.
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Effect of AdRSVCGRP on the Response to Acute Ventilatory
Hypoxia and L-NAME
The influence of AdRSVCGRP on increases in PAP in response to
acute ventilatory hypoxia
(FIO2 0.10) in mice exposed to
chronic hypoxia after administration of AdRSVßgal or
AdRSVCGRP was studied. When mice were ventilated with a 10%
O2 gas mixture for 3 to 5 minutes, the PAP
increase was reduced in mice treated with AdRSVCGRP compared with
responses in animals treated with AdRSVßgal (data not shown). The
influence of AdRSVCGRP on increases in PAP in response to inhibition of
nitric oxide synthase in chronically hypoxic mice was determined, and
after treatment with
NG-nitro-L-arginine
methyl ester (L-NAME, 50 mg/kg IV), the PAP increase in response to
acute hypoxia was significantly less in mice treated with AdRSVCGRP
than in mice pretreated with AdRSVßgal.
Effect of AdRSVCGRP on the Increase in RVM and Vascular Remodeling
in Chronically Hypoxic Mice
The influence of AdRSVCGRP on the increase in RVM and vascular
remodeling in chronically hypoxic mice is summarized in Tables 2 and 3.
Body weight was less in 16-day hypoxic mice than in normoxic mice and
was similar in mice exposed to hypoxia receiving AdRSVßgal or
AdRSVCGRP (Table 2). Hematocrit, ratio of lung wet to dry
weight, and ratio of lung weight to body weight were significantly
greater in mice exposed to chronic hypoxia than in normoxic
mice, and the values were similar in mice receiving AdRSVßgal or
AdRSVCGRP. RVM, as determined by RV/LV+S and RV/BW, was increased in
mice exposed to chronic hypoxia compared with normoxic mice.
RV/LV+S and RV/BW ratios were significantly lower in chronically
hypoxic mice receiving AdRSVCGRP than in those receiving AdRSVßgal.
LV+S/BW ratios were similar in normoxic mice and chronically hypoxic
mice treated with AdRSVßgal or AdRSVCGRP, suggesting that left
ventricular hypertrophy and differences in body
weight did not account for the changes in RVM (Table 2).
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Lung sections were examined to determine the effect of AdRSVCGRP on
pulmonary remodeling in chronically hypoxic mice, and compared
with normoxia, hypoxic AdRSVßgal-treated mice had a significantly
greater number of muscular peripheral pulmonary
vessels (Table 3
). Pretreatment with AdRSVCGRP attenuated the
increase in muscular peripheral pulmonary vessels
that occurred with hypoxia compared with AdRSVßgal-treated
hypoxic animals.
Effect of AdRSVCGRP on Plasma and Lung CGRP Levels and Cyclic
Nucleotide Levels
Plasma concentrations of CGRP were significantly reduced in
chronically hypoxic mice treated with AdRSVßgal or AdRSVCGRP compared
with concentrations in normoxic control mice (Figure 4). Plasma CGRP values were not increased
in mice treated with AdRSVCGRP. There was a small increase in lung and
plasma CGRP concentrations in mice exposed to chronic hypoxia.
CGRP concentrations were increased 4-fold in lungs of mice transfected
with AdRSVCGRP compared with CGRP levels in normoxic or hypoxic
AdRSVßgal-treated mice (P<0.05; Figure 4).
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significantly different from
concentrations in AdRSVßgal-treated animals.
Lung concentrations of cAMP and cGMP in normoxic mice and in
chronically hypoxic mice after administration of AdRSVßgal or
AdRSVCGRP were evaluated, and lung cAMP concentrations were reduced in
chronically hypoxic mice pretreated with AdRSVßgal compared with
levels in normoxic animals (Figure 5). In
contrast, cAMP concentrations were significantly increased in lungs of
mice transfected with AdRSVCGRP compared with levels in
AdRSVßgal-treated animals (P<0.05; Figure 6). Lung cGMP concentrations in
chronically hypoxic mice pretreated with AdRSVßgal or AdRSVCGRP were
increased compared with levels in normoxic mice (Figure 5).
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ß-Galactosidase Activity
ß-Galactosidase activity was measured by
chemiluminescence in normoxic and hypoxic lung 21 days after
intratracheal administration of vehicle (16 days of hypoxic exposure),
AdRSVßgal, and AdRSVCGRP. In lungs from vehicle- or AdRSVCGRP-treated
mice, ß-galactosidase levels were very low (0.16±0.09 and 0.13±0.1
mU/mg protein, respectively). In lungs from mice treated with
AdRSVßgal, ß-galactosidase activity was 29±6 mU/mg protein
(P<0.05).
ß-Galactosidase enzyme transduction was assessed in the lungs of mice
treated with AdRSVßgal, and 21 days after AdRSVßgal transfection,
ß-galactosidase protein was expressed diffusely in airway epithelial
cells in proximal bronchi and distal bronchioles, alveolar lining of
cells, and adventitial cells in medium-sized and small (100 to 300
µm) pulmonary arteries (Figure 6). ß-Galactosidase
staining was not observed in the lung of mice treated with vehicle or
AdRSVCGRP.
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Discussion |
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The present results demonstrate that adenoviral gene transfer of the cDNA encoding prepro-CGRP to the lung attenuates the increase in PAP, PVR, RVM, and vascular remodeling induced by 16-day hypoxic exposure without altering systemic pressure. Hematocrit values were increased to similar levels in AdRSVßgal- and AdRSVCGRP-treated mice, suggesting that the contribution of the hypoxia-induced increase in hematocrit to the observed responses is minimal. The decrease in PVR in AdRSVCGRP-treated mice was associated with an increase in lung CGRP and cAMP levels and enhanced response to the type IV cAMP phosphodiesterase inhibitor rolipram. These results show that treatment with AdRSVCGRP attenuates the increase in PAP in response to angiotensin II, ET-1, acute hypoxia, and L-NAME in chronically hypoxic mice and suggest that the CGRP transgene has significant activity in the lung and that the major actions of intratracheal AdRSVCGRP are in the lung.
CGRP has potent vasodilator activity in the lung, and immunoreactive CGRP is localized to nerve fibers within airway epithelium and around lung blood vessels.1 5 CGRP has been suggested to play a role in regulating pulmonary vascular tone under normal and pathophysiological conditions.12
The role of the endothelium in mediating responses to CGRP is uncertain, and CGRP interacts with specific vascular smooth muscle receptors and increases cAMP levels.6 21 22 23 The increased lung levels of cAMP, but not cGMP, after AdRSVCGRP transfer may suggest that CGRP activates adenylyl cyclase, and because lung but not plasma CGRP levels were increased, it is possible that CGRP in the lung did not interact with the CGRP receptors on the vascular endothelium.
In addition to reducing vascular resistance and pressor responses, CGRP gene transfer enhanced depressor responses to rolipram, suggesting that in vivo gene transfer, along with a cAMP phosphodiesterase inhibitor, may be useful in treatment of pulmonary hypertension.
Potassium channels play an important role in regulating pulmonary vascular tone, the response to hypoxia, and are inhibited in patients with pulmonary hypertension.24 25 Decreased K+ conductance results in membrane depolarization with enhanced vasoconstrictor reactivity and decreased vasodilator reactivity.24 25 CGRP-induced vasodilation in some preparations is mediated by opening of ATP-sensitive potassium channels, and it is possible that CGRP responses in the mouse lung are mediated by K+ channel opening or a cAMP-dependent mechanism.26 The observation that AdRSVCGRP treatment reduces pressor responses to angiotensin II, ET-1, acute hypoxia, and L-NAME is consistent with the hypothesis that CGRP may increase K+ conductance in the mouse lung.
The use of vasodilator agents in the management of pulmonary hypertension is limited by the systemic actions of these drugs.27 Intratracheal administration of the reporter gene coding for ß-galactosidase showed extensive gene expression in airway epithelium and interstitial cells, with less expression in the wall of small pulmonary arteries; the mechanism by which the gene gains access to small arteries is uncertain, but examination of lung sections reveals that uptake has occurred. The present data suggest that intratracheal administration of AdRSVCGRP results in CGRP delivery to perivascular and abluminal sites in the pulmonary vascular bed. It is uncertain, however, what CGRP concentrations are present, and it is possible that peptide concentration at the receptor level may be higher. The consequence of >21-day exposure to high lung CGRP levels is uncertain; however, under the present conditions, no overt physiological changes were found.
Results showing that pressor responses are decreased by AdRSVCGRP suggest that lung cell transduction with a vasodilator peptide may be sufficient to alter vascular function. Although the distribution of the peptide is unknown when CGRP synthesis is increased by gene transfer, it is possible that the peptide may diffuse throughout the lung when released into the extracellular environment. After release and diffusion, the peptide may interact with CGRP receptors at abluminal sites to induce vasodilation.
It is possible that adenovirally mediated gene transfer may produce an inflammatory response and alter vascular responses; however, no increase in CGRP or cAMP levels was observed with AdRSVßgal transfection, an adenoviral vector that does not affect vascular resistance. Moreover, baseline pulmonary pressures were similar in animals treated with vehicle and AdRSVßgal, whereas AdRSVCGRP decreased baseline pressure and responses, and no overt sign of an inflammatory response was seen with AdRSVßgal or AdRSVCGRP treatment.
The results of the present study may suggest that adenovirally mediated transfer of the CGRP gene alone or along with a cAMP phosphodiesterase inhibitor may be useful in the treatment of pulmonary hypertension.
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Acknowledgments |
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This work was supported in part by NIH grants HL-16066, HL-14388, and HL-09474. The authors thank Drs Beverly L. Davidson and Richard D. Anderson and the University of Iowa Vector Core Laboratory for the viral vectors.
Received March 3, 1999; revision received August 18, 1999; accepted August 26, 1999.
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