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Departments of 1Pediatrics, 3Cell Biology and Physiology, and 4Internal Medicine, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131; and 2Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
Submitted 24 February 2004 ; accepted in final form 12 July 2004
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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pulmonary hypertension; double occlusion technique; gene therapy; pulmonary vascular resistance; isolated lungs
Gene therapy with NOS has been advocated for pulmonary vascular disorders (5), cardiovascular diseases (9, 38), erectile dysfunction (8, 17, 18) and wound healing (37). Of the three isoforms of NOS [neuronal NOS, inducible NOS (iNOS), and eNOS], the use of the iNOS isoform in gene therapy applications has advantages over the other isoforms. For example, iNOS activity is less dependant on local calcium concentrations, and it has a far greater NO output than the other NOS isoforms (11, 27, 35). Because the decrease in NO production in patients with PPHN and ARDS is transient (6), we employed an adenoviral vector in which the iNOS gene is not incorporated into the genome, thus producing transient iNOS expression. Therefore, we tested the hypothesis that adenovirus-mediated iNOS gene transduction of the lung results in time-dependent iNOS overexpression and parallel attenuation of vasoconstrictor responses. We further contrasted the efficacy of intratracheal administration of the iNOS gene construct on pulmonary arterial vs. venous vasoreactivity.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Adenovirus-human iNOS Gene Construct
The iNOS coding sequence was cloned from cytokine-stimulated human hepatocytes in tissue culture. The fragment of the iNOS cDNA that was used in the construction of adenoviral-mediated iNOS (Adeno-iNOS) included a manufactured Nco I site at the translational start site (bp 3,705) where a Not I site was added. None of the 5' untranslated region and only 40 bp of the 3' untranslated region were included in the adenoviral construct (National Center for Biotechnology Information accession no. AY046510). This iNOS coding sequence was subcloned into pcDNA3 at the EcoRV site and then reisolated using Hind III and Not I restriction enzymes. This fragment was then cloned into the pAdlox shuttle vector (20) using these restriction sites.
Recombinant Adeno-iNOS was generated by cotransfection of Sfi I-digested pAdlox-iNOS and 
5 helper virus DNA into the adenoviral packaging cell line CRE8 (20) in the presence of the NOS inhibitor L-N5-(1-iminoethyl)-ornithine dihydrochloride (1 mM). Ten days after transfection, a clarified viral lysate was tested by infection of 293 cells and assaying for NO production using the Greiss reaction to detect nitrite accumulation in the absence of L-N5-(1-iminoethyl)-ornithine dihydrochloride. Nitrite production collected over a 24-h period was >15 µM. After iNOS expression was confirmed, the clarified viral lysate was subjected to three cycles of plaque purification with 293 cells. The resulting viral lysate was subsequently purified with two rounds of cesium chloride density gradient centrifugation followed by exchange dialysis. The viral titer was determined spectrophotometrically and by plaque assay and diluted to the desired concentration with preservation buffer and aliquots made into sterile containers stored at –80°C. This construct was provided by Dr. Andrea Gambotto from the Pre-Clinical Viral Core Facility, University of Pittsburgh, Pittsburgh, PA.
Adenovirus control construct.
The adenovirus control (Adeno-Control) virus used in this study is also an E1- and E3-deleted, replication-incompetent recombinant type 5 adenovirus. It was prepared using standard 5 helper virus DNA with the adenoviral packaging cell line CRE8, as previously described by Hardy et al. (20). This construct was provided by Dr. Andrea Gambotto from the Pre-Clinical Viral Core Facility, University of Pittsburgh.
Human iNOS gene transduction. Rats were anesthetized with halothane (Halocarbon Laboratories, River Edge, NJ) followed by 8 mg/kg xylazine sq. The rats were then intubated and mechanically ventilated using a volume-controlled ventilator (Harvard Apparatus, Holleston, MA) at constant tidal volume (2.5 ml) and constant respiratory rate (55 breaths/min). Rats received a single tracheal instilled bolus of 0.75 ml of bovine calf surfactant alone (sham), or 0.75 ml of bovine calf surfactant mixed with 5 x 108 plaque-forming units (PFU) of either Adeno-iNOS or Adeno-Control. A fourth group of age-matched rats received no treatment (control). Surfactant-assisted instillation was utilized in this study because previous studies (22, 24) have demonstrated greater transgene expression and distribution compared with aersolized instillation methods. After instillation and resumption of spontaneous room air breathing, the animals were removed from ventilator support and returned to their cages.
Total lung RNA isolation and multiplex RT-PCR. We evaluated human iNOS (hiNOS) mRNA levels in lungs from rats treated with either Adeno-iNOS (n = 3) or Adeno-Control (n = 3) virus as described above. On the third day posttransduction, the lungs were rapidly harvested and snap frozen in liquid nitrogen. The frozen lungs were pulverized in a precooled mortar and pestle and homogenized in Trizol (following manufacturer's protocol) using a polytron tissue disrupter. The quantity and quality of isolated RNA was determined by comparing absorbance at 260 and 280 nm, as well as by visualizing RNA on agarose gels and noting the presence of 18S and 28S bands of expected intensity with minimal evidence of breakdown. Multiplex RT-PCR for hiNOS mRNA and the housekeeping gene rat malate dehydrogenase (rMDH) mRNA was performed. RT reactions (40 µl) contained 20 units of avian Moloney virus in 1x buffer (Promega), 2 µg of total tissue RNA, 2.5 mM oligo(dT)16, and 1 mM each deoxynucleotide triphosphate. Tissue RNA and oligo(dT)16 were held at 65°C for 5 min, then 4°C for 5 min. The remainder of the RT reaction mixture was then added and incubated at 21°C for 10 min, 42°C for 60 min, 94°C for 5 min, and then returned to 4°C and frozen at –20°C until used for PCR. PCR reactions contained 0.3 µM each oligonucleotide primer for hiNOS and rMDH. The hiNOS primers used were forward 5'-GAG CTT CTA CCT CAA GCT ATC G-3', which were hiNOS specific and did not contain complementary sequences found in rat iNOS using a BLAST search, and reverse 5'-AGC ATG TTG GCC ACT GCA GGC-3'. The rMDH primers used were forward 5'-GAA GCA TGG CGT ATA CAA CC-3' and reverse 5'-TTT CAG CTC AGG GAT GGC C-3' [modified from Ujiie et al. (40)]. Additional components of the PCR reaction included 5.0 µl of RT product, 1 mM MgCl2, 0.2 mM deoxynucleotide triphosphate, 1x PCR buffer II, and 2.5 units of Amplitaq polymerase (Applied Biosystems). PCR reactions were denatured at 94°C for 5 min and then cycled at 94°C for 45 s, 55°C for 45 s, and 72°C for 2 min for 35 cycles. Final extension was 5 min at 72°C. PCR products were separated and sized by 1.5% agarose gel electrophoresis. Gels were visualized, and digital images were made under ultraviolet light captured using a GeneGnome darkroom apparatus (Syngene Imaging, Cambridge, UK). PCR product size was the expected 656 bp for hiNOS and 512 bp for rMDH. Thirty-five cycles were within the linear range for generation of both PCR products (data not shown).
iNOS/eNOS Immunoblot Analysis
iNOS or eNOS immunoblot analysis was carried out as previously described (10, 29, 31). Lungs transduced with either Adeno-iNOS or Adeno-Control were harvested and snap frozen in liquid nitrogen and stored at –80°C until preparation for immunoblot analysis. Briefly, frozen lungs pulverized in a precooled mortar and pestle were homogenized on ice in Tris buffer [10 mM Tris·HCl (pH 7.4) containing 255 mM sucrose, 2 mM EDTA, 12 µM leupeptin, 1 µM pepstatin A, 0.3 µM aprotinin, and 1 mM phenylmethylsulfonyl fluoride (all from Sigma)]. A commercially available protein assay (Bio-Rad) was performed on spun clarified (1,500 g at 4°C for 10 min) supernatants to ensure equal gel loading. Samples (40 µg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 7.5% acrylamide, along with molecular weight standards (Bio-Rad) and an appropriate NOS standard (Transduction Laboratories). The separated proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) and blocked overnight at 4°C with 5% nonfat milk (Carnation), 3% bovine serum albumin (Sigma), and 0.05% Tween-20 (Bio-Rad) in a Tris-buffered saline solution containing 10 mM Tris·HCl and 50 mM NaCl (pH 7.5). Rinsed blots were incubated for 4 h at room temperature with a mouse monoclonal antibody raised against iNOS or eNOS (1:2,500; Transduction Laboratories) in Tris-buffered saline. Immunochemical labeling was achieved by incubation for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG (1:10,000; Bio-Rad) in Tris-buffered saline. Chemiluminescence labeling was performed per kit instructions (Amersham). Protein bands were detected by exposure to chemiluminescence-sensitive film and quantitated by densitometric analysis (Sigma Gel, Jandel Scientific). Membranes were poststained with Coomassie brilliant blue to confirm equal protein loading per lane (30).
iNOS Immunohistochemistry
To demonstrate the localization of recombinant iNOS expression, immunohistochemistry for iNOS was performed in sham, Adeno-Control, and Adeno-iNOS groups 3 days posttransduction using previously described procedures (29). Briefly, fixed/frozen sections (10 µm thick) from the left lung hilar region from each group of rats were treated with 0.33% H2O2 to inhibit endogenous peroxidases, then blocked with normal horse serum (4%), followed by incubation for 24 h at 4°C with a mouse monoclonal iNOS antibody (1:1,000; Transduction Laboratories, isotype IgG1, clone 54) in PBS containing 0.3% Triton X-100. This antibody has been used in previous studies from our laboratory to detect rat iNOS using Western blotting methods (31). Immunocytochemical labeling was demonstrated by incubation with rat adsorbed, biotinylated, horse anti-mouse IgG (1:400) (Vector Laboratories), followed by incubation with an avidin biotinylated complex (ABC Elite Kit, Vector Laboratories). Immunoprecipitation of the antigen peroxidase conjugate was achieved by treatment of sections with a diaminobenzidine peroxidase substrate labeling kit (Vector Laboratories) for 10 min. Sections were washed in deionized water, dehydrated, cleared, and mounted. Negative control sections were prepared by incubation with mouse IgG (1:1,000; Sigma) instead of primary antibody. Positive control sections were generated from separate groups of animals administered LPS (20 mg/kg ip; serotype 0127:B8, Sigma) or vehicle (sterile saline; 2 ml/kg ip) 5 h before lung removal. We have previously used this method of LPS administration to induce pulmonary iNOS in rats (29). Images were generated with a Photometrics CoolSNAPcf digital camera from a Nikon Optiphot microscope and processed with MetaMorph Imaging System software (Universal Imaging).
Intratracheal Exhaled NO Measurement
Animals were anesthetized with 50 mg/kg ip pentobarbital, intubated, and mechanically ventilated using room air and a volume-controlled ventilator (Harvard Apparatus, Holleston, MA) at constant tidal volume (2.5 ml) and constant respiratory rate (55 breaths/min). A central venous catheter was placed via the right jugular vein for subsequent L-N6-(1-iminoethyl)lysine dihydrochloride (L-NIL) administration for iNOS inhibition (25, 31). Thirty minutes after catheter placement, baseline exhaled gas was collected for 5 min into a mylar balloon attached to the ventilator exhaust port. The gas collected in the mylar balloon was analyzed using a chemiluminescence NO analyzer (Sievers, Boulder, CO). The analyzer was calibrated using a standard curve generated daily with authentic NO (1 part/million in N2, Matheson, Chicago, IL) mixed with NO-free N2 using precision flow meters to obtain concentrations ranging from 0 to 500 parts/billion (15). The NO detection limit was 0.5 parts/billion (vol/vol). After baseline gas collection, L-NIL (10 mg/kg; Calbiochem) was administered intravenously followed by repeated exhaled gas collection at 30, 60, and 90 min post-L-NIL administration.
Segmental Vascular Resistance
The procedure for lung isolation has been previously described (13, 33). Briefly, rats were anesthetized and intubated. The lungs were ventilated at a rate of 55 breaths/min using a warmed and humidified gas mixture (6% CO2 in room air) with a 9-cmH2O peak inspiratory pressure and 3-cmH2O positive end-expiratory pressure. A median sternotomy was performed, and heparin (100 U in 0.1 ml) was injected directly into the right ventricle of the heart. The pulmonary artery and left ventricle were cannulated, and the preparation was perfused using a Masterflex roller pump with a physiological saline solution containing (in mM) 129.8 NaCl, 5.4 KCl, 0.83 MgSO4, 19 NaHCO3, 1.8 CaCl2, and 5.5 glucose with 4% albumin (wt/vol) (all from Sigma). In addition, meclofenamate (10 µg/ml) was added to the perfusate before lung removal and perfusion. The heart and lungs were removed en bloc and suspended in a humidified chamber maintained at 38°C. The perfusion rate was gradually increased from 0.8 ml/min to 30 ml·min–1·kg body wt–1 and maintained at this rate for the duration of the experiment. Twenty milliliters of perfusate were washed through the lungs and discarded before recirculation was initiated with 40 ml of physiological saline solution. The lungs were maintained under zone 3 conditions throughout the study by maintaining venous pressure (Pv) at 3–4 mmHg (14). Pulmonary arterial pressure (Pa) and pulmonary Pv were continuously measured and recorded on a Gould RS 3400 chart recorder. Data were stored and processed using a computer-based data acquisition and analysis system (Dataq Instruments). Arterial and venous vascular resistances were assessed using double occlusion techniques as previously described (13, 33). Total pulmonary vascular resistance (PVR) was calculated as (Pa – Pv)/
, where was the constant perfusate flow rate. Simultaneous occlusion of both the arterial inflow and the venous outflow caused Pa and Pv to rapidly equilibrate at the double occlusion pressure (Pd), which approximates the microvascular pressure (39). Arterial resistance was calculated as (Pa – Pd)/, and venous resistance was calculated as as (Pd – Pv)/.
Experimental Protocols
Transduction and hiNOS mRNA expression. RT-PCR analysis of lung tissue was used to assess hiNOS mRNA expression in rats 3 days after tracheal instillation of Adeno-iNOS (n = 3) or Adeno-Control (n = 3).
Dose determination. Animals were either treated with Adeno-Control (dose: 1 x 108, 5 x 108, and 1 x 109 PFU) or treated with increasing Adeno-iNOS dose (n = 3 for each dose: 1 x 108, 5 x 108, and 1 x 109 PFU). After 3 days, the lungs were removed and analyzed by immunoblot analysis for iNOS and eNOS protein production.
Tissue specificity of tracheally instilled Adeno-iNOS. The tissue specificity of tracheally instilled Adeno-iNOS was determined in animals (n = 3 each group) treated with 5 x 108 PFU/animal of either Adeno-iNOS or Adeno-Control. Lungs, hearts, kidneys, and livers were harvested 3 days posttransduction, and iNOS immunoblot analysis was performed.
Time course of hiNOS protein production and correlation with exhaled NO production. The time course of protein production and exhaled NO (exNO) production was assessed in Adeno-iNOS (1, 2, 3, 4, 7, and 14 days posttreatment), and Adeno-Control (1, 3, 4, and 14 days posttreatment)-transduced animals (n = 3–8 animals/group) were anesthetized and intubated. To determine the iNOS-mediated increase in exNO production, L-NIL, an iNOS-specific inhibitor (10 mg/kg), was administered (25, 31). Exhaled gas samples were collected and analyzed before and at 30, 60, and 90 min post-L-NIL administration.
PVR. Experiments were performed to examine the effect of hiNOS transduction on segmental vasoconstrictor responses in isolated, perfused lungs. Lungs were isolated from Adeno-iNOS (n = 8), Adeno-Control (n = 8), sham (n = 7), and nontreated age-matched controls (n = 7) at 3 days posttransduction. Total, arterial, and venous vascular resistances were assessed under basal conditions and after administration of cumulative doses of the thromboxane mimetic U-46619 (50, 100, 200, 300, and 400 nM). U-46619 was used as a vasoconstrictor because it elicits both arterial and venous constriction in the lung, allowing an assessment of hiNOS transduction in each vascular segment (4). In a second group of studies, lungs from Adeno-iNOS (n = 6), Adeno-Control (n = 9), and nontreated age-matched control (n = 3) rats were perfused with physiological saline solution containing 10 µM L-NIL, and the segmental vascular response to 400 nM U-46619 was determined.
Statistical Analysis
All data are expressed as means ± SE, where n refers to the number of animals in each group. One- or two-way ANOVA with repeated measures was used to compare groups. If differences were detected by ANOVA, individual groups were compared using the Student-Newman-Keuls test. A level of P < 0.05 was accepted as statistically significant for all comparisons.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Figure 1 demonstrates multiplex RT-PCR analysis of lung tissue isolated 3 days after transduction with either 5 x 108 PFU Adeno-iNOS or the same amount of Adeno-Control. The hiNOS band was detected only in Adeno-iNOS-treated rat lungs.
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Immunoblot analysis demonstrated an Adeno-iNOS dose-dependent increase in iNOS protein expression in the lung at 3 days posttransduction (Fig. 2A). The iNOS levels found in lungs from animals treated with 5 x 108 and 1 x 109 PFU doses were greater (P < 0.05) than the iNOS protein levels found in Adeno-Control (treated with 1 x 108, 5 x 108, and 1 x 109 PFU) and the Adeno-iNOS (treated with 1 x 108 PFU) animals (Fig. 2B). The viral dose utilized for the subsequent studies described in this manuscript was 5 x 108 PFU/animal.
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In the Adeno-iNOS (n = 3: 5 x 108 PFU/dose)-transduced animals, only lung tissue demonstrated detectable levels of iNOS by immunoblot analysis at 3 days posttransduction (Fig. 3A). Similar doses of Adeno-Control (n = 3: 5 x 108 PFU/dose) did not cause detectable levels of iNOS production by immunoblot analysis in any tissue studied (Fig. 3B). eNOS immunoblot analysis of lung tissue isolated from Adeno-iNOS- or Adeno-Control-transduced animals demonstrated no change in eNOS expression (data not shown).
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Figure 4 depicts iNOS immunostaining in lung tissue from Adeno-Control (Fig. 4, A and B) and Adeno-iNOS (Fig. 4, C and D) groups 3 days posttransduction (5 x 108 PFU/dose), and in lungs from a vehicle-treated (Fig. 4E) and an LPS-treated (Fig. 4F) rat. iNOS immunoreactivity was restricted largely to the alveolar walls of Adeno-iNOS-transduced rats, with no detectable staining observed in the vasculature or bronchi. However, iNOS immunostaining was not uniformly distributed, with some areas lacking specific staining. In agreement with results from immunoblot analyses, no detectable staining was observed in the Adeno-Control or sham groups. Positive staining for iNOS was also observed in lung tissue from an LPS-treated rat, which was localized primarily to leukocytes distributed throughout the lung parenchyma. These results confirm the reactivity of this antibody with both endogenous rat iNOS and recombinant hiNOS. No staining was detected in a vehicle-treated animal.
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The time course of pulmonary iNOS protein production in the Adeno-iNOS-transduced group is shown in Fig. 5. There were easily detected iNOS protein bands 1, 2, 3, and 4 days posttransduction, there were discernable iNOS protein bands on 7 days posttransduction, and there were no detectable iNOS protein bands on 14 days posttransduction. The time course of changes in exNO concentration is shown in Fig. 6A. The Adeno-iNOS-transduced rats had 10- to 20-fold greater exNO concentrations than did Adeno-Control-transduced rats on posttransduction days 1, 3, and 4. On day 7, the exNO concentration was approximately one-third of the values on days 1–4, and by day 14 there was no difference in exNO concentration between Adeno-iNOS-transduced rats and Adeno-Control-transduced rats. The greater exNO concentration was due to iNOS activity, since the administration of L-NIL to Adeno-iNOS-transduced rats decreased exNO concentrations to levels found in Adeno-Control-transduced rats (Fig. 6B).
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The baseline segmental vascular resistances in the isolated lungs did not differ between groups. The addition of the thromboxane mimetic U-46619 resulted in a dose-dependent increase in PVR (Fig. 7A). The change in total PVR was 
75% less in the isolated lungs from the Adeno-iNOS-transduced rats than in either Adeno-Control-transduced, sham-treated, or nontreated age-matched control rats (Fig. 7A). In addition, U-46619 caused a dose-dependent increase in pulmonary arterial resistance in the isolated lung (Fig. 7B). The increase in pulmonary arterial resistance was 67% less in the lungs isolated from Adeno-iNOS-transduced lungs than in lungs isolated from Adeno-Control-transduced, sham-treated, or nontreated age-matched control rats (Fig. 7B). Finally, U-46619 elicited a small but significant increase in pulmonary venous resistance in the lungs isolated from Adeno-Control-transduced, sham-treated, or nontreated age-matched control rats but had no significant effect on pulmonary venous resistance in lungs isolated from Adeno-iNOS-transduced rats (Fig. 7C).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Tracheal rather than vascular administration was utilized to take advantage of a delivery method that is potentially applicable to clinical gene therapy. The vascular approach could result in increased hiNOS expression more selectively within the vascular bed; however, this method requires insertion of a pulmonary arterial catheter and probably requires a period of vascular occlusion, whereas tracheal hiNOS transduction may result in more wide-spread hiNOS expression within the lung. Our immunohistochemistry results demonstrate iNOS transgene expression limited mostly to the alveolar wall cells throughout the lung parenchyma. These results are similar to those reported by others utilizing a tracheal route of administration (5, 21). Our data suggest that NO produced by transduced cells within the lung parenchyma successfully diffuses to vascular smooth muscle cells in both small arteries and importantly veins to produce vasodilation.
The dose of Adeno-iNOS necessary for effective transduction and iNOS protein expression in the present investigation is similar to that observed in previous studies that utilized either iNOS or eNOS. We found that a dose of 5 x 108 PFU resulted in greater iNOS protein expression by immnuoblot than did a dose of 1 x 108 PFU and that 1 x 109 PFU resulted in no additional increase in iNOS protein expression. Similarly, Zsengeller et al. (44) used 2 x 109 PFU intratracheally in buffer to transduce mouse lungs with LacZ. Budts et al. (5) and Janssens et al. (21) used 3–5 x 109 PFU delivered by aerosol to transfect rat lungs with hiNOS or heNOS.
The use of hiNOS to transfect rat lungs was advantageous, because it allowed the transgene product to be identified with hiNOS-specific PCR primers. As expected, we found hiNOS expression only in Adeno-iNOS-treated lungs. The specificity of hiNOS was further demonstrated by immunoblot analysis. Although the immunoblot does not necessarily differentiate between hiNOS and rat iNOS, there was no detectable iNOS protein expression in the Adeno-Control-treated lungs. Furthermore, the exNO concentration was at least 100-fold greater in the Adeno-iNOS-treated rats than in the Adeno-Control-treated rats. Thus tracheal administration of Adeno-iNOS in surfactant resulted in iNOS mRNA and protein expression and increased exNO concentration that was due specifically to the transgene. Budts et al. (5) also demonstrated specific transgene expression of iNOS in transduced rat lung utilizing aerosolized recombinant adenovirus. Furthermore, expression of the hiNOS transgene was limited to the lungs, because we found no detectable iNOS expression in nonpulmonary tissue. Finally, because we found no detectable iNOS protein expression in the lungs isolated from animals treated with Adeno-Control and no detectable increase in exNO concentration in rats treated with Adeno-Control, this suggests that the adenoviral vector itself caused no appreciable endogenous iNOS upregulation in the rat lungs in our study. Similar results were reported by Budts et al. (5) in that no evidence of endogenous iNOS expression was noted in lung tissue from Adeno-Control-treated rat lungs. In contrast, Zsengeller et al. (44) reported that adenoviral transduction or adenoviral infection resulted in the upregulation of endogenous iNOS mRNA and increased NOS activity in mouse lungs (44). This discrepancy may relate to species differences or differences in assay sensitivity. Thus, although we cannot entirely exclude a small effect of adenoviral transduction on endogenous iNOS in the rat lung, the Adeno-iNOS transduction was a specific effect resulting in hiNOS mRNA expression and greater than an 100-fold increase in exNO concentration than in the lungs of Adeno-Control animals.
The expression of iNOS was greatest 1–4 days posttransduction, was much less at 7 days posttransduction, and was essentially undetectable 14 days posttransduction. Champion et al. (7) found that adenoviral-mediated transduction with the B-galactocidase gene resulted in transgene expression that peaked at 1 day and decayed over 7–14 days. In this study, we found that the exNO concentration followed a similar time course, with an 100-fold greater exNO concentration at 1–4 days, with no difference in exNO concentration at 14 days than in Adeno-Control-transduced rats. In a rat model, Budts et al. (5) found that using aerosolized delivery of an adenovirus construct containing the hiNOS gene resulted in increased exNO concentrations at 3 days, which returned to baseline values by 10 days. The time-dependent expression of iNOS protein and production of NO may be due to the stimulation of a cellular inflammatory response by adenovirus and/or normal airway epithelial cell turnover. Although the time-dependent expression of iNOS protein may not be ideal for gene therapy of chronic pulmonary hypertensive disorders, it may be an advantage for the treatment of more transient conditions, such as PPHN or ARDS.
Our results demonstrate that tracheal administration of Adeno-iNOS results in diminished pulmonary vasoconstrictor reactivity 3 days after instillation. Because U-46619 causes both arterial and venous constriction (4), we were able to assess the vasodilatory effectiveness of hiNOS transduction in each vascular segment. We found that Adeno-iNOS treatment of the rat 3 days before lung isolation and perfusion resulted in attenuated arterial and venous responses to U-46619. Furthermore, this effect was reversed by L-NIL, suggesting that an Adeno-iNOS-induced increase in lung NO production resulted in significantly attenuated pulmonary vasoconstriction. Ours is the first study to delineate the segmental profile of altered vasoreactivity after iNOS transduction. Previously, Budts et al. (5) found that aerosol delivery of adenovirus-mediated transduction of hiNOS resulted in an attenuated total pulmonary vasoconstrictor response to 10% oxygen in the rat. Interestingly, they found that aerosol delivery of a similar dose of an adenovirus construct carrying the human eNOS gene did not attenuate the pulmonary vasoconstrictor response to 10% oxygen. Champion et al. (7) found that adenoviral-mediated eNOS transduction did not attenuate the vasoconstrictor response to U-46619 (7). Taken together, these findings suggest that Adeno-iNOS attenuates pulmonary vasoconstrictor responses and that Adeno-iNOS may be better at attenuating pulmonary vasoconstrictor responses than adenoviral-mediated eNOS.
The present study focused on the effects of Adeno-iNOS transduction and overexpression of hiNOS on pulmonary vasoconstrictor responses to U-46619. Interestingly, Eguchi et al. (12) and Gunnett et al. (19) have previously demonstrated an inhibitory effect of recombinant iNOS gene expression on endothelium-dependent vasorelaxation of transduced canine basilar artery and rabbit carotid artery, respectively. We report here that Adeno-iNOS does not change baseline pulmonary artery or venous resistances, but whether Adeno-iNOS affects endothelium-dependent vasodilation in the pulmonary vascular bed remains to be determined.
The present approach of hiNOS gene transduction produces a similar attenuation of arterial and venous vasoconstriction, such as that observed with iNO therapy. For example, we previously observed that iNO results in arterial and venous vasodilation in the U-46619 preconstricted isolated perfused rat lung (32). Similarly, Roos et al. (34) found that iNO resulted in both arterial and venous dilation in endothelin-1 preconstricted isolated perfused rat lungs. These results suggest that the NO produced by the transgene in the rat lung reaches the same resistance arteries and veins as NO gas given by inhalation. It is likely that at least a portion of this similarity is due to hiNOS expression in airway epithelial cells in our studies that would mimic airway administration of NO. In this study, we used 5 x 108 PFU based on immunoblot detection of the transgene, which may be greater than needed to elicit changes in vasoconstrictor responses. Further studies are needed to assess dosing and the vasoconstrictor response.
In summary, our data suggest that the tracheal administration of Adeno-iNOS may offset pulmonary vasoconstriction and could be useful as a treatment for some forms of pulmonary hypertension. This treatment produces both arterial and venous vasodilation similar to the response to iNO; however, the transgene approach may avoid the toxicities associated with the latter method.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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