Vascular inflammation inhibits gene transfer to the pulmonary circulation in vivo

Robert C. Tyler1, Karen A. Fagan2, Robert C. Unfer1, Cornelia Gorman3, Molly McClarrion3, Clayton Bullock3, and David M. Rodman1,2,4

1 Cardiovascular Pulmonary Research Laboratory, 2 Division of Pulmonary Sciences and Critical Care Medicine, and 4 Department of Physiology, University of Colorado Health Sciences Center, Denver, Colorado 80262; and 3 Valentis, Burlingame, California 94010


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We report gene transfer to the normal and injured murine pulmonary circulation via systemic (intravascular) and airway (intratracheal) delivery of novel polycationic liposomes (imidazolium chloride, imidazolinium chloride-cholesterol, and ethyl phosphocholine). With use of the reporter genes chloramphenicol acetyltransferase (CAT) or human placental alkaline phosphatase (hpAP), intravascular injection of lipid-DNA complexes resulted in gene expression primarily in the lung, with lesser expression in the heart (11% of lung, P < 0.05) and spleen (8% of lung, P < 0.05). Histochemical staining for the hpAP reporter gene showed localized transgene expression in the microvascular endothelium. Monocrotaline (80 mg/kg body wt sc) treatment produced endovascular inflammation and reduced lung CAT activity (2 days postintravascular transfection) by 75 ± 8 and 86 ± 6% at 7 and 21 days, respectively, after monocrotaline (P < 0.05). Despite the apparent decrease in functional CAT protein, Southern blot analysis suggested maintained plasmid delivery, whereas quantitative PCR (TaqMan) showed decreased CAT mRNA levels in monocrotaline mice. In contrast, intratracheal delivery of lipid-DNA complexes showed enhanced CAT expression in monocrotaline mice. Transfection in alternate pulmonary vascular disorders was studied by inducing hypoxic pulmonary hypertension (4 wk at barometric pressure of 410 mmHg). Efficiency and duration of gene transfer, assessed by CAT activity, were similar in pulmonary hypertensive and normal lungs. We conclude that imidazolinium-derived polycationic liposomes provide a means of relatively selective and efficient gene transfer to the normal and injured murine microvascular circulation, although translation of transgene mRNA may be reduced by preexisting endothelial injury.

vascular injury; polycationic liposomes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SUCCESSFUL GENE TRANSFER to the normal pulmonary circulation has been reported by several groups including our own (8, 13, 14, 17). This technique holds promise as both an investigational and a therapeutic tool. Targeting genes to the injured pulmonary vasculature has the potential of enabling one to selectively modulate the injury. This could have relevance to many pulmonary vascular diseases including pulmonary vasculitis, primary pulmonary hypertension, and acute respiratory distress syndrome. Whereas gene transfer to the normal pulmonary circulation can be accomplished using either viral or nonviral vectors (1, 8, 13, 17), the suitability of these vectors in the abnormal pulmonary circulation has not been established.

A well-characterized model of pulmonary vascular injury is treatment with the plant alkaloid monocrotaline (5, 7, 10). Monocrotaline induces selective endothelial damage, leading to mild pulmonary vascular inflammation and vessel wall remodeling. In rats, this injury results in a loss of capillary surface area, medial arterial hypertrophy, and pulmonary hypertension (10). In mice, the degree of damage and remodeling is less severe, although endovascular inflammation is still apparent (12).

Using monocrotaline treatment as our model, we questioned whether the success of intravenous gene delivery would be affected by endovascular injury. International cancer research (ICR) mice, either control or monocrotaline treated, were transfected through a tail vein using cationic lipid-DNA complexes encoding either the chloramphenicol acetyltransferase (CAT) or human placental alkaline phosphatase (hpAP) reporter gene. Expression of functional protein was evaluated by CAT activity assay, and tissue distribution was determined by alkaline phosphatase staining. Plasmid delivery and mRNA transcription were evaluated by Southern blotting and quantitative PCR, respectively. The selectivity of monocrotaline injury to vascular gene delivery was assessed by comparing intravascular with intratracheal gene delivery. To ascertain if reduced transgene expression was due to endovascular inflammation, the effect of chronic hypoxic exposure, which produces pulmonary vascular remodeling without inflammation (3), was also tested.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmids. Expression plasmids were prepared using standard methods. Two plasmids were used, coding for either Escherichia coli CAT [plasmid human cytomegalovirus (hCMV)-CAT, 4,752 bp] or hpAP (plasmid hCMV/hpAP, 5,836 bp). Each plasmid was under transcriptional control of the CMV immediate-early promoter-enhancer and a chimeric intron (from human preproinsulin) preceding the coding region of the plasmid. Transcription termination and polyadenylation signals were supplied by the simian virus 40 polyadenylation sequence.

Assembly of lipid-DNA complexes for in vivo transfection. The cationic lipid 1-[2-(acyloxy)ethyl]-2-alkyl(alkenyl)-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM) was used for intravenous transfection and 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EDMPC) chloride salt for intratracheal transfections. The synthesis of DOTIM (15) and EDMPC (2) have been previously described and were generously provided by Valentis (Burlingame, CA). Aqueous suspensions of 1:1 molar ratio of DOTIM or EDMPC with cholesterol were prepared under sterile conditions using 5% dextrose in water as the diluent. In vivo gene delivery utilized the previously determined optimum DNA-lipid ratio of 1:6 (DOTIM) and 1:3 (EDMPC). Lipids were prepared from sonicated lipid suspensions containing 1:1 cationic lipid-cholesterol mixed with appropriate amounts of plasmid DNA.

Animals. Three groups of female ICR (25-32 g) mice were used for CAT transfections. Pulmonary vascular inflammation was induced by subcutaneously injecting monocrotaline (80 mg/kg body wt) (12). Saline-treated ICR mice were used as control groups and for hpAP transfections and immunohistochemistry. Chronic hypobaric hypoxic exposure (barometric pressure 410 mmHg) for 4 wk was used to produce noninflammatory pulmonary hypertension as previously described (3, 11).

Transfection protocols. Mice were injected intravenously with a single dose of 125 µg DNA (hpAP or CAT)/200 µl complexes. The diluent, 5% dextrose in water, was injected as a control. Pulmonary inflammation was induced with subcutaneous injection of monocrotaline on day 0. Control mice received saline subcutaneously. On either day 5, 12, or 19 after monocrotaline, mice were injected intravenously with hCMV-CAT containing complexes and killed 2 days later (i.e., day 7, 14, or 21). In a second series of experiments, mice were first transfected and then treated 2 days later with monocrotaline and killed 7, 14, or 21 days after transfection. In addition, mice from the control and monocrotaline groups were killed on days 7, 14, and 21, and lung tissue was isolated and fixed as described for subsequent hematoxylin and eosin staining. To determine whether monocrotaline treatment interfered with either plasmid delivery or transcription, Southern blots and competitive PCR were performed using DNA and RNA extracted from 7-day postmonocrotaline-treated, 2-day post-CAT-transfected mice as described below.

For intratracheal transfection, mice were anesthetized (methoxyflurane) and had a 20-gauge feeding tube directed into the left lung. EDMPC (62.5 µg DNA/100 µl) was then injected into the left lung airways. The diluent, 5% dextrose in water, was injected as a control. Monocrotaline-induced pulmonary inflammation was induced, and airway transfections were performed 5 days after monocrotaline treatment. Tissues were harvested on day 7 and assayed for CAT protein by ELISA (Boehringer Mannheim).

CAT assay. Quick-frozen tissues were homogenized in lysis buffer (0.25 M Tris, 5 mM EDTA, and 5 mM phenylmethylsulfonyl fluoride with 10 mg/ml aprotinin) on ice with a hand-held homogenizer (Biospec Products, model 985-370 Tissue Tearor). Soluble CAT enzyme was separated from other tissue components by microcentrifugation for 5 min; 25 ml of supernatant were reserved for total soluble protein assay and the rest was heat inactivated at 65°C for 10 min. The supernatants were then diluted in TE-CAT buffer (0.25 M Tris-5 mM EDTA plus 2 mg/ml BSA) to 55 µl total, and 50 µl of 1:10 diluted 14C-labeled chloramphenicol (DuPont NEN) in TE-CAT buffer and 25 µl of 25 mg/ml n-butyryl coenzyme A (Sigma) were added. After a 2-h incubation at 37°C, the reactions were extracted once with 300 µl of mixed xylenes (Aldrich). The mixed xylenes were then extracted once with 750 µl of homogenization buffer, and 200 µl of extracted xylenes were counted on a Beckman LS-6500 scintillation counter for 1 min. When necessary, tissues were assayed at higher dilutions to obtain values in the linear range. Values were also corrected for total soluble protein using BCA Protein Assay Reagent (Pierce) in a 96-well format, with BSA as a standard.

Alkaline phosphatase staining. For tissue harvest, mice were anesthetized with ketamine and rompin (0.03 and 0.02 mg, respectively). The trachea was cannulated, the chest was opened, and the pulmonary artery was cannulated and flushed with PBS, after which the pulmonary circulation was perfused with 1% paraformaldehyde at 25°C. The airways were filled with 1% agarose at 55°C to improve the preservation of lung architecture. Lungs were then immersed in 1% paraformaldehyde borate buffer for 16 h, placed in 70% ethyl alcohol, and paraffin embedded. Paraffin blocks were sectioned at a thickness of 6 µm onto poly-L-lysine-coated slides, deparaffinized in three changes of xylene, dehydrated in 100, 95, and 75% ethyl alcohol, and incubated in PBS at 65°C for 60 min to inactivate endogenous alkaline phosphatase activity. Specimens were then incubated in a chromogenic substrate of 5-bromo-4-chloro-3-indolylphosphate p-toluidine (1 mg/ml, GIBCO BRL) and nitro blue tetrazolium chloride (1 mg/ml, GIBCO BRL) for 19 h. In the presence of alkaline phosphatase, this substrate yields dark-blue staining. Sections were rinsed with PBS and counterstained with hematoxylin.

Morphological assessment of vascular inflammation in monocrotaline-treated mice. Lung tissue sections were mounted, deparaffinized, and hydrated as described in Alkaline phosphatase staining. Standard methods of hematoxylin and eosin staining were used. Sections were then reviewed in a blinded fashion, and vascular inflammation and endothelial morphology were evaluated. Four lung sections from three animals in each experimental group were examined. The number of sections demonstrating any vessels with perivascular inflammation was counted and compared.

Southern blots. Lung tissue was harvested, snap-frozen in liquid nitrogen, and stored at -70°C. DNA was isolated from 25 mg of lung tissue using a Wizard Genomic DNA preparation (Promega), and stored at 2-8°C. The concentration per sample was determined by spectrophotometry. The efficiency of DNA extraction did not differ between groups (saline 0.55 ± 0.15 µg/µl, monocrotaline 0.50 ± 0.15 µg/µl). DNA (5 µg) was cut with a BamH I digest and run on a 0.8% agarose gel. The bands were then transferred to nitrocellulose and hybridized with a CAT 32P-labeled probe. The Southern blot DNA probe was constructed by cutting the CAT cDNA from plasmids with restriction enzymes BamH I and Spe I (GIBCO BRL), which yielded two fragments (3.3 and 1.8 kb). The 1.8-kb fragment was purified with Geneclean procedure (Bio 101) and random prime labeled with 32P (Amersham Life Science). The nitrocellulose was placed on autoradiographic film and exposed for 48 h. Densitometry on the autoradiograph was used to determine DNA levels.

Quantitative PCR. Poly(A)+ RNA was isolated from snap-frozen lung tissue using standard techniques. CAT transgene mRNA was quantified using the TaqMan PCR technique (Perkin-Elmer), which takes advantage of the exonuclease activity of Taq DNA polymerase described by Holland et al. (6). Briefly, we incorporated a unique DNA sequence into the transcribed, untranslated 5' sequence of our CAT expression plasmid. A complementary DNA sequence, which hybridizes with this unique sequence, was synthesized and complexed with a fluorophore on the 3'-end and a quencher on the 5'-end and included into the PCR mix. A similar construct designed to hybridize with alpha -actin was used as a loading control in a second reaction. Fluorescence was detected with the ABI Prism Sequence Detection System (PE Applied Biosystems). Increase in fluorescence resulting specifically from amplification of the target sequence was detected real time so that the number of PCR cycles and thus the number of molecules of transgene mRNA could be expressed as a percentage of alpha -actin mRNA.

Statistical methods. All values are expressed as means ± SE. For normally distributed dependent variables, differences between means were compared by either one- or two-way analysis of variance (ANOVA) and Fisher's protected least squares difference post hoc testing. For measurement of transgene mRNA, which was not normally distributed, Mann-Whitney two-group comparison was used. Differences were considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Intravenous transfection with lipid-DNA complexes encoding the CAT reporter gene resulted primarily in lung expression, with lesser amounts in heart and spleen (Fig. 1). No expression was detected in liver, kidney, ear, brain, skeletal muscle, or gonads (no difference from background level of 700 counts · min-1 · mg protein-1). Transfection with the hpAP reporter gene revealed lung expression to be localized to microvascular endothelium (Fig. 2C). Mice treated with monocrotaline developed mild endovascular inflammation (Fig. 2, A and B). Whereas only 1 vessel in 12 sections examined in the saline group showed detectable inflammation, 5 of 12 sections in monocrotaline-treated animals had from 1 to 3 vessels with detectable inflammation. We also measured right ventricular pressure in lightly anesthetized mice and found that monocrotaline-treated mice had only a small, nonstatistically significant increase in right ventricle systolic pressure 21 days after monocrotaline (monocrotaline 33.1 ± 1.4 vs. control 28.4 ± 0.07 mmHg, P = 0.09).


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Fig. 1.   Chloramphenicol acetyltransferase (CAT) activity in lung tissue 2 days after intravenous transfection (n = 6-8 mice/group). Heart and spleen had 11 and 8%, respectively, of lung CAT expression. CPM, counts/min. * Significantly less gene expression than lung (P < 0.05).



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Fig. 2.   Lung inflammation from monocrotaline treatment is evident from increased number of leukocytes dispersed throughout lung (×10; A) and localized around small arteries (a; ×40; B) compared with saline-treated controls (×10; D and ×40; E). Intravenous transfection of human placental alkaline phosphatase (hpAP) shows expression in alveolar capillary endothelium (×60; C), whereas intratracheal transfection shows expression in airway epithelium and alveolar wall but not in vasculature (×4; F) (arrows).

We injected lipid-hCMV-CAT complexes 5, 12, and 19 days after monocrotaline injection and measured CAT activity 2 days later (i.e., 7, 14, and 21 days after monocrotaline treatment). Thus in this protocol CAT activity was assayed at the anticipated peak expression from the transfected plasmid. Results indicated that CAT activity at the three time points was reduced by 86.0 ± 6, 75 ± 8, and 84 ± 5%, respectively, compared with control mice treated with saline rather than monocrotaline (Fig. 3). A comparable reduction in CAT expression was also seen in heart and spleen (heart 84.1 ± 5%, spleen 80.8 ± 6%). In a separate series of experiments, CAT complexes or saline were injected on day 0, and monocrotaline was injected 2 days later. Lungs were then harvested either on day 2 before monocrotaline or on day 7, 14, or 21 posttransfection. These lungs were then assayed for CAT activity. As Fig. 4 shows and as expected, CAT activity at all three later time points was significantly lower than peak activity 2 days after transfection. However, in contrast to results of the first protocol, CAT activity did not significantly differ between monocrotaline- and saline-treated groups.


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Fig. 3.   CAT activity 2 days after intravenous transfection in mice (n = 5/group) 7, 14, and 21 days after monocrotaline treatment. CAT activity was significantly reduced at 3 time points compared with subcutaneously saline-treated controls. Controls (subcutaneous saline) from each time point were not different so they were pooled into 1 control group. * Significantly different from control (P < 0.05).



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Fig. 4.   CAT activity in mice transfected on day 0 and then treated with monocrotaline (MC trt) 2 days later. Typical decline in CAT activity over 3 wk is shown, with no differences between control (saline) vs. monocrotaline-treated animals (n = 5/group).

To determine if the defect in CAT expression was due to impaired delivery of lipid-DNA complexes to the lung, Southern blots using DNA isolated from lung tissue harvested from CAT-transfected mice 7 days after monocrotaline treatment were performed. As Fig. 5 demonstrates, DNA delivery did not appear to be impaired in monocrotaline-treated mice and, in fact, tended to be increased. To test if the defect in CAT expression was due to impaired transcription of plasmid DNA, we performed quantitative RT-PCR using mRNA from similar groups. Using the TaqMan technique, we detected CAT mRNA in all seven dextrose in water-treated control animals (mean 12.4 ± 6.6 copies/1,000 copies alpha -actin), whereas in monocrotaline-treated animals, CAT mRNA was detected in only one of six animals (mean 0.8 ± 0.8 copies/1,000 copies alpha -actin, P = 0.012 vs. control).


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Fig. 5.   Southern blot of CAT DNA 2 days after intravenous transfection in mice that received either control (saline) or monocrotaline treatment 7 days earlier. Autoradiograph densitometry showed trend for greater DNA levels in monocrotaline- vs. saline-treated mice (n = 7-8/group).

Because the mechanism of action of monocrotaline is mediated via pulmonary endothelial injury, we questioned whether monocrotaline caused selective impairment of vascular gene transfer. Therefore, we tested the effect of monocrotaline treatment on transgene expression in airway after intratracheal delivery of lipid-DNA complexes. As Fig. 2F demonstrates, intratracheal delivery of lipid-DNA complexes resulted in transgene expression in airway epithelium as well as in the alveolar wall but not in vessels. In contrast to intravenous delivery, after airway delivery of the transgene, CAT activity was increased in monocrotaline-treated vs. saline control mice (Fig. 6).


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Fig. 6.   Left lung CAT expression 2 days after intratracheal transfection in mice (n = 5/group) receiving either control (saline) or monocrotaline treatment 7 days earlier. Monocrotaline-treated mice had greater CAT protein levels than controls. * Significantly greater than control (P < 0.01).

We questioned whether the defect in pulmonary vascular transgene expression was present in another model of pulmonary vascular disease, chronic hypobaric hypoxia. Therefore, we tested intravenous CAT gene delivery in mice exposed to hypobaric hypoxia for 4 wk. This exposure produces vascular injury characterized by secondary pulmonary hypertension without morphological evidence of endothelial damage or vascular inflammation. As Fig. 7 demonstrates, in contrast to the effect of monocrotaline, CAT activity after intravenous delivery of lipid-DNA complexes was not reduced in chronic hypoxic pulmonary hypertension.


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Fig. 7.   CAT activity in mice intravenously transfected and immediately either placed in hypobaric hypoxia (barometric pressure 410 mmHg, 17,000 ft) or kept at Denver's altitude (barometric pressure 630 mmHg, 5,280 ft). Mice were killed at 2, 7, 14, 28 (n = 7-8/group), and 60 (n = 3-4) days posttransfection. Typical decline in CAT expression was observed, with ~20% of CAT expression present at 60 days posttransfection. No differences were observed between groups.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Intravenous delivery of DOTIM-choline-DNA complexes resulted in relatively selective lung gene expression. This relative lung selectivity is a prominent feature of imidazolinium-based lipid vectors, although most cationic lipids produce some expression in lung. The reason for the relative lung selectivity of DOTIM is uncertain, although complexes with higher cationic charge and higher DNA content appear to preferentially target the lung endothelium (16). Staining for the hpAP reporter gene demonstrated that expression was restricted to endothelium, with no transgene detected in medial smooth muscle or airway epithelial cells. Treatment with monocrotaline produced modest vascular inflammation. Whereas lung architecture and vascular morphology were nearly normal, a subtle increase in lung cellularity was appreciated in monocrotaline-treated mice. This is consistent with a prior report of the effect of monocrotaline in mice (12). Also consistent with a prior report (12), we found only a minimal increase in right ventricular pressure in monocrotaline-treated mice.

Monocrotaline treatment resulted in a significant reduction in lung CAT activity detected 48 h after intravenous transfection, suggesting that endovascular injury reduced the efficiency of cationic lipid-mediated gene transfer to the pulmonary circulation. Gene transfer with nonviral vectors requires a sequential cascade of events if transgene expression is to be successful. These events include 1) membrane fusion and endocytosis, 2) release of DNA and translocation to the nucleus, and 3) episomal DNA transcription and RNA translation. Vascular injury could interfere with any or all of the steps necessary for successful transgene expression, thus limiting the ability to express new proteins in vivo.

To address the first step, we performed Southern analysis to determine if monocrotaline reduced the delivery of plasmid DNA to the lung. We found that there was no reduction in plasmid DNA detected in monocrotaline-treated mice. This suggests that complex delivery and endocytosis were unaffected by monocrotaline. We therefore tested if transcription was affected by monocrotaline using quantitative RT-PCR. CAT mRNA was detected in all saline-treated control animals. In contrast, CAT mRNA was detected in only one of six monocrotaline-treated mice. This difference was most likely due to reduced transcriptional activity in the pulmonary endothelium of monocrotaline-treated mice, although we cannot exclude the possibility of selective destabilization of transgene mRNA.

We wondered whether reduced transgene transcription was a nonspecific effect of monocrotaline or a selective effect due to endothelial injury and/or inflammation. We therefore tested whether monocrotaline treatment would affect transgene expression after airway transfection. Using hpAP staining, we confirmed that intratracheal delivery of lipid-DNA complexes resulted in expression in epithelial but not endothelial cells. In contrast to results in intravascular transfected mice, we found that monocrotaline treatment increased CAT activity after airway transfection. This result suggests that the defect produced by monocrotaline was selective to endothelial gene expression. Whereas the reason for the increase in CAT expression after airway transfection is uncertain, it is possible that the inflammatory response resulted in augmented transgene expression either in epithelial cells or in retained inflammatory cells such as macrophages.

To further establish the role of endothelial inflammation, we tested the effect of chronic hypoxic pulmonary hypertension on transgene expression. In contrast to the effect of monocrotaline, we found that chronic hypoxia had no effect on CAT activity after transfection. Because endothelial damage and inflammation are largely absent in this model (3), we conclude that the endothelial and/or inflammatory effects of monocrotaline are the likely mechanisms of impaired transgene expression in our studies.

In all experimental groups, a significant decrease in transgene expression was seen with time. There was a bimodal pattern of decay in transgene expression, in that initial peak expression at 48 h rapidly declined by day 7, followed by a much slower phase of decline over the next 2 wk. The initial rapid decline in maximum expression, which precedes any immunological response to transgene, has been attributed by other investigators to promoter downregulation (4), although the specific mechanisms remain unknown. Interestingly, we found that the effect of monocrotaline was predominantly seen by 48 h, before significant promoter downregulation occurs. In contrast, when transgene expression was measured 1-3 wk after transfection, monocrotaline treatment produced no further downregulation. This suggests that the mechanism of action of monocrotaline on transcriptional activity of the CMV promoter may be similar to endogenous, time-dependent promoter downregulation, which occurs in normal endothelial cells. Although the determination of this mechanism of promoter downregulation is beyond the scope of the current project, it is possible that monocrotaline interferes with the activity of nuclear factor-kappa B or other transcription factors that are essential to activation of the CMV promoter (9).

In summary, we found that monocrotaline-induced endovascular inflammation markedly reduced the efficacy of lipid-mediated pulmonary vascular gene transfer. Whereas DNA delivery appeared normal, reduced steady-state transgene mRNA was found. Endothelial dysfunction or inflammation rather than pulmonary hypertension per se appears to be primarily responsible because chronically hypoxic mice demonstrated normal transfection efficiency. The defect was restricted to the vascular compartment because lipid-mediated airway transfection was not impaired by monocrotaline. We conclude that whereas cationic lipids are suitable vectors for gene delivery to the pulmonary circulation, preexisting endothelial inflammation and/or injury may greatly reduce transgene expression by interfering with mRNA synthesis and/or stability. As transfection efficiency does not appear to be affected, it is likely that alternative promoters may be required to achieve adequate gene expression in the inflamed pulmonary circulation.


    ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute (NHLBI) Program Project Grant HL-14985.


    FOOTNOTES

R. C. Tyler was supported by NHLBI National Research Service Award Grant HL-07670 and an American Heart Association of Colorado Fellowship Award. D. M. Rodman was supported by a Clinical-Scientist Award from the American Heart Association. C. Gorman was supported by NHLBI Small Business Innovation Research Grant HL-56479.

This work was presented in part at the NHLBI Workshop Mechanisms of Proliferative and Obliterative Vascular Disease, September 2-3, 1997.

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: D. M. Rodman, Univ. of Colorado Health Sciences Center, CVP Research Laboratory, B-133, 4200 9th Ave., Denver, CO 80262 (E-mail: david.rodman{at}uchsc.edu).

Received 18 March 1999; accepted in final form 27 July 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 277(6):L1199-L1204
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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