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Ann Thorac Surg 2004;77:449-456
© 2004 The Society of Thoracic Surgeons
a Division of Cardiothoracic Surgery, University of California, San Diego, California, USA
b Department of Pathology, Veterans Administration Medical Center, La Jolla, California, USA
c Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, California, USA
Accepted for publication April 28, 2003.
* Address reprint requests to Dr Thistlethwaite, Division of Cardiothoracic Surgery, University of California, San Diego, 200 West Arbor Dr, San Diego, CA 92103-8892, USA
e-mail: pthistlethwaite{at}ucsd.edu
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Abstract |
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METHODS: We injected 2 x 1010 genomic particles of adeno-associated virus-angiopoietin-1 (AAV-Ang-1) into the right ventricular outflow tract of 30 Fischer rats while using adeno-associated virus-lacZ (AAV-lacZ) injected rats and carrier-injected rats as our control groups. All animals underwent survival surgery and were sacrificed at serial timepoints postgene delivery. At each timepoint, pulmonary artery pressures were measured and pulmonary angiography using the Microfil polymer perfusion technique was performed. The lungs were harvested for pathologic analysis, mRNA analysis, Western blot assays, and in situ RNA hybridization to localize gene expression.
RESULTS: Pulmonary artery pressures of AAV-Ang-1 injected rats were significantly increased compared with the control groups (p < 0.01) at all timepoints. Pathologic analysis of AAV-Ang-1 lung specimens demonstrated increased smooth muscle cell proliferation within the medial layer of arterioles with obliteration of small vessels similar to that seen in human pulmonary hypertension. Angiograms of AAV-Ang-1 injected lungs showed blunting of small peripheral arterioles consistent with advanced pulmonary hypertension. In situ RNA hybridization localized angiopoietin-1 expression to the vascular wall of small-caliber pulmonary vessels. Protein and mRNA assays confirmed persistent angiopoietin-1 expression in the lung for up to 60 days postgene delivery.
CONCLUSIONS: Overexpression of angiopoietin-1 using an adeno-associated virus vector causes pulmonary hypertension in rats. These data provide a novel physiologic animal model for pulmonary hypertension.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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In embryonic development, the establishment and remodeling of blood vessels are controlled by paracrine signals between endothelial cells and smooth muscle cells [3]. Angiopoietin-1 (Ang-1), a molecule secreted by smooth muscle cells, plays an essential role in the formation of arteries and arterioles in utero and has been implicated in pathologic angiogenesis [4]. As a muscle-secreted ligand, Ang-1 signals vascular endothelial cells through the endothelial-specific receptor, TIE2, to recruit and stimulate the proliferation of smooth muscle cells around nascent endothelial tubes to create mature arterial structures [5]. Ang-1 has also been shown to act synergistically with vascular endothelial growth factor (VEGF) to potentiate vascular network maturation in vivo [6]. Knockout mice deficient in Ang-1 die in utero from defects in embryonic vascular development including severe vascular malformation of the lungs [7]. Endothelial cells of these animals were arranged into tubular structures and were present in normal numbers but the vessels in these genetically engineered mice had no smooth muscle cell encasement and lacked branch networks. These results suggested that Ang-1 plays an essential role in arterial vascular formation and remodeling.
Because Ang-1 plays a role in the muscularization of arteries in utero we hypothesized that its aberrant expression in the adult lung could be a key molecular step in the genesis of pulmonary hypertension. In this study, we sought to determine if constitutive Ang-1 expression in the adult lung causes pulmonary hypertension and to create a reproducible animal model for this disease. We have previously shown that Ang-1 is overexpressed in lung tissues of humans with different forms of pulmonary hypertension [8] and that the level of Ang-1 expression is directly proportional to the severity of the disease [9]. Our earlier results suggested that Ang-1 correlated with the pulmonary hypertension phenotype but did not prove it played a causal role in the disease process. In order to establish whether up-regulation of the Ang-1 gene product causes pulmonary hypertension we created an animal model whereby Ang-1 was constitutively expressed in the adult rodent lung. Our results demonstrate that Ang-1 induces pulmonary hypertensive vascular pathology and clinical development of pulmonary hypertension leading to right ventricular failure and death. This is the first reproducible model of this disease based on tissue-specific alteration of the expression of a single gene.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Large scale recombinant serotype 2 AAV containing the Ang-1 gene (AAV-Ang-1) and AAV containing the lacZ gene (AAV-lacZ) were produced and purified by cotransfecting 293T cells. Triple cotransfections with pXX2, pXX6, and pAAV-Ang-1/pAAV-lacZ plasmids were accomplished with Polyfect Transfection Reagent (Qiagen, Valencia, CA). Cells were collected and resuspended in media and underwent two freeze/thaw cycles to lyse their membranes. Benzonase (Sigma-Aldrich, St. Louis, MO) was added to the mixture to eliminate unpackaged DNA. Cellular debris was separated from the cleared cell lysate containing AAV via centrifugation.
AAV vector high-grade purification was modified from the single-step gravity-flow column technique described by Auricchio and associates [11]. Type I heparin columns (Sigma-Aldrich, St. Louis, MO) were equilibrated with phosphate-buffered saline (PBS)/1 mmol/L MgCl2 using an Econo-Pump (Bio-Rad, Hercules, CA). Crude AAV vector was added directly to the column with a flow rate of 0.2 ml/min and subsequently washed with PBS/1 mmol/L MgCl2/0.1 mol/L NaCl. The virus was eluted with PBS/1 mmol/L MgCl2/0.4 mol/L NaCl and further dialyzed against PBS/1 mmol/L MgCl2 gradient. The concentration of the vectors was determined by real-time polymerase chain reaction (PCR) (7700 Sequence Detector, Applied Biosystems, Foster City, CA) with SYBR Green detection kit (Applied Biosystems). All viral preparations had at least 1 x 1011 genomic particles/ml. The purified vectors were then retested for transgene expression by infecting 293 cells and nucleic acid sequenced to confirm transgene integrity.
Gene transfer protocol into rat lungs in vivo
Twelve week-old pathogen-free Fischer rats (Harlan, San Diego, CA) housed in micro-isolator boxes (2–3 animals per box) were anesthetized with intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg). The animals were intubated and ventilated using a Harvard rodent ventilator Model 683 (Harvard Apparatus, Holliston, MA). The heart was exposed via a left antero-lateral thoracotomy at the fourth intercostal space. 2 x 1010 genomic particles of AAV-Ang-1 in 200 µl of PBS/1 mmol/L MgCl2 were injected directly into the right ventricular outflow tract (just beneath the pulmonic valve) of the hearts of 30 rats in our test group whereas the same amount of AAV-lacZ was injected in 30 rats in our first control group. Thirty sham animals forming our second control group were injected with 200 µl of the carrier solution, PBS/1 mmol/L MgCl2 alone. Rats from each group were sacrificed at 1-month and 2-months postgene delivery timepoints and organs/blood collected for tissue and molecular analysis. Four animals in each group were followed for one year or until death (if earlier) in order to observe the natural history of their pulmonary disease. All animals received care in accordance with the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (N.I.H. publication 85 to 23, revised 1985). This study was approved by the University of California, San Diego Animal Subjects Program.
Hemodynamic measurements and pulmonary angiography
At two serial timepoints after the gene delivery operation pulmonary artery systolic and diastolic pressures as well as heart rate and systemic blood pressures were measured just before the sacrifice of the animals. To accomplish this a sternotomy was performed after anesthetic induction and intubation in order to expose the heart. A 22-gauge angiocatheter was inserted directly into the right ventricular outflow tract and advanced into the pulmonary artery. The pulmonary artery pressures were measured using a standardized pressure transducer (SpaceLabs, Inc., Issaquah, WA) whereas systemic blood pressures were measured by direct aortic transduction. Afterwards the animals were sacrificed by exsanguination through the abdominal aorta. For 5 animals in each group at each timepoint the pulmonary vasculature was flushed with saline and perfused with Microfil (FlowTech, Inc., Carver, MA), a liquid silicon polymer, at 0.25 ml per minute for 4 min using a syringe infusion pump (Harvard Apparatus, Holliston, MA). After 12 h of 4° storage the lungs were sequentially bathed in increasing concentrations of ethanol, placed in methyl-salicylate, and photographed with a digital camera (DSC-S30, Sony Corporation, Japan) through a dissecting microscope (WILD M651, Leica, Switzerland).
Protein and mRNA assays
Rat lung protein extracts and Western blot analyses were performed according to standard procedures using 100 mg of whole tissue extracts. Both the polyclonal goat Ang-1 and actin antibodies were obtained from Santa Cruz Biotechnology. Total RNA was isolated using RNAzol B reagent (Tel-Test, Friendswood, TX). For all PCR analyses, 2 µg of total RNA was digested with DNase I and reverse-transcribed according to the manufacturer's instructions (Superscript II kit, Invitrogen, Carlsbad, CA). PCR amplifications were performed under standard conditions and each PCR product was electrophoresed on 1% SeaKem LE (BioWhittaker Molecular Applications, Walkersville, MD) agarose gels stained with ethidium bromide. Each gene was analyzed using specific primer pairs: viral-specific Ang-1: 5'- GGTACCCGAATGACAGTTTTCC-3' 5'-CTCCATTTCTAAGATTTTGTGCTC-3' rodent Ang-1: 5'-GAGAAGCAACTTCTCCAACAG-3' 5'- CTCGTTCCCGAGCCAATATTC-3' glyceraldehyde-3-phosphate dehydrogenase (GAPDH): 5'-CATCATCTCTGCCCCCTCTG-3' and 5'-CCTGCTTCACCACCTTCTTG-3'. The viral-specific Ang-1 primer sequence overlapped with the multiple cloning site sequence in pAAV-shuttle vector and therefore only recognized the Ang-1 gene sequence delivered by AAV.
Immunohistochemistry and in situ hybridization
At each timepoint, The lungs from 5 rodents per group were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 µm thickness. After hematoxylin and eosin staining the specimens were analyzed by a pulmonary pathologist in blinded fashion. In situ hybridizations were performed using RNA probes subcloned in our lab specific to the SV40 Poly-A domain of the pAAV-shuttle vector expression cassette. The SV40 Poly-A domain was inserted into pBluescript II SK(+) (Stratagene, LA Jolla, CA) and served as a template for the synthesis of digoxigenin-labeled antisense RNA probes using T3 polymerase and the DIG RNA labeling mix (Roche, Indianapolis, IN). Sense probes were made using T7 polymerase and served as controls. Hybridization was carried out at 50°C with 50% formamide/0.3 mol/L sodium chloride, 0.03 mol/L sodium citrate (2xSSC). The digoxigenin (DIG)-label was detected by an anti-DIG Fab (Roche) coupled to alkaline phosphatase using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (Roche).
Statistical analysis
Values for the pulmonary artery systolic/diastolic pressure were expressed as the mean ± standard error of the mean. Continuous variables were compared with a Student's t test. A p value of less than 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed that vector-specific Ang-1 transcripts were present in the lungs of animals at 1 and 2 months after injection with AAV-Ang-1 (Fig 1A). The 561 base pair PCR product representing wild-type endogenous Ang-1 was present in small amounts in all rat lung samples tested. Because our AAV-Ang-1 injected rodents expressed not only wild-type but also virally delivered Ang-1 this group of animals had greater overall steady-state levels of Ang-1 mRNA in their lung tissue compared with control animals. We found that levels of GAPDH mRNA, our control for RNA amount in each lane, was consistently equal among the samples studied. Western blot protein analysis using an antibody against the carboxy-terminus of murine Ang-1 demonstrated increased overall steady-state levels of Ang-1 protein expression in lungs of animals injected with AAV-Ang-1 compared with control animals injected with AAV-lacZ (Fig 1B). Equal amounts of the internal control, actin filament protein, confirmed that all samples studied by Western blot analysis had equivalent amounts of protein present in each lane.
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High levels of Ang-1 in the lung induces pulmonary arteriolar muscular hyperplasia and hypertrophy
Animals constitutively expressing high levels of Ang-1 protein in the lung from injection of our AAV-Ang-1 vector showed diffuse pulmonary pathologic changes consistent with advanced pulmonary hypertension. By histologic analysis these lungs demonstrated severe muscular hyperplasia and hypertrophy in the medial layer of small pulmonary arteries and arterioles measuring 
500 µm in diameter (Fig 2A).
This small vessel medial hypertrophy/hyperplasia was present in more than 80% of the arterioles examined at 1 and 2 months post AAV-Ang-1 gene injection and occurred throughout all lobes of both lungs. By micrometer analysis on review of ten fields/lung slide at 40x magnification we found that small pulmonary vessels on average increased their vessel wall diameter by 400% (range 210%–630%) and increased the number of myocytes per vessel wall area by 300% (range 250%–430%) 1 month after gene transfer. One fourth of all pulmonary arterioles examined in animals constitutively expressing the virally delivered Ang-1 gene were occluded from severe medial hyperplasia/hypertrophy at each time point. There was no lymphocytic infiltration in or around the pulmonary vessels with this pathology as has been shown for adenovirally transduced genes. In contrast to the human form of this disease few ( < 1%) stenosed vessels in the AAV-Ang-1 treated animals manifest plexiform pathology at 1 and 2 month intervals after gene injection. Rats injected in the right ventricular outflow tract with either AAV-lacZ or an equal volume of PBS/1 mmol/L MgCl2 demonstrated normal lung histology (less than 2% of pulmonary arterioles 500 µm being muscularized) without evidence of small vessel muscle cell hypertrophy or hyperplasia.
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500 µm in diameter (or greater than four branch divisions from the main pulmonary artery) were nearly occluded. A similar angiographic pattern is seen in the human form of this disease.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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The aim of our study was to understand the molecular basis by which normal pulmonary vessels develop smooth muscle hyperplasia and hypertrophy which eventually causes clinical manifestations of pulmonary hypertension. In this study we show that high levels of Ang-1 protein induced by viral injection of transgene into the pulmonary circulation leads to the development of clinical and pathologic pulmonary hypertension in rodents. This pathology persisted after transgene expression had ceased. The phenotype observed with gene transfer was not due to the viral vector, method of surgical delivery, nor carrier solution used. Our findings suggest that in the pulmonary vasculature, supranormal levels of Ang-1 facilitate the development of profuse muscular hyperplasia of small pulmonary blood vessels which ultimately leads to the development of pulmonary hypertension. From a mechanistic point of view we believe that derangement in expression of a single gene, Ang-1, may be the molecular trigger for this organ-specific vasculopathy.
Using sustained gene transfer into the lung we have created a novel physiologic animal model to study the mechanisms responsible for pulmonary hypertension. Although several animal models have been used in the past to study pulmonary hypertension none of them accurately mimics the human form of the disease. In one model, the subcutaneous injection of a pyrrolizidine alkaloid, monocrotaline, in rodents results in global inflammation of the lung with thickening of arterioles and venules and the eventual sporadic development of lung adenocarcinomas [20, 21]. The inflammatory component induced by this chemical damage in the lung is not seen in our animal model or in the human form of pulmonary hypertension. Other pulmonary hypertensive animal models rely on rodents with both systemic and pulmonary hypertension or on placing rodents into hypoxic or high altitude conditions mimicking hypoxia [22, 23]. Although low partial pressure of oxygen will induce the vasoreactive component of this disease in humans, the majority of patients with pulmonary hypertension develop this disease under normoxic conditions. Supranormal expression of Ang-1 in our rodent lung model induced elevated pulmonary artery pressures and increased muscularization of distal pulmonary vessels similar to the human disease without chemically induced lung inflammation or pulmonary hypoxia. We found that the viral vector, AAV, itself introduced into the pulmonary circulation did not stimulate perivascular lymphocytic infiltration or inflammatory reaction in lung tissue as has been seen with gene delivery systems to the lung [24].
Clues as to why Ang-1 plays a role in pulmonary hypertension comes from earlier studies of its function in angiogenesis and vasculogenesis. Ang-1 is a vascular smooth muscle cell growth factor in the developing lung and its expression is shut-off once development is complete [7]. Constitutive Ang-1 expression in the adult lung leads to an exaggerated and organ-specific vascular smooth muscle cell proliferative response. Ang-1 is known to bind the endothelial-restricted promoter, TIE2, resulting in tyrosine phosphorylation and activation of this receptor into an active tyrosine kinase [6]. TIE2 intracellular signaling involves tyrosine phosphorylation of several intracellular proteins [25] although the final downstream effect of this signal transduction in the lung is unknown. We suspect that Ang-1 signaling through the endothelial-specific TIE2 receptor results in the release of a paracrine muscle-specific growth factor by vascular endothelial cells. In the normal adult lung where Ang-1 expression is minimal vascular smooth muscle cells are quiescent, whereas in the adult pulmonary hypertensive lung where Ang-1 expression is de-regulated vascular smooth muscle cells are proliferative. Recent evidence has suggested that haploid mutations in the bone morphogenetic receptor type 2 (BMPR2) are seen in patients with the rare form of inherited primary pulmonary hypertension [26, 27]. The molecular mechanism by which Ang-1 causes pulmonary hypertension may be based on co-signaling with this membrane bound receptor.
Several limitations of this study deserve comment. First our animal experiments are based on the transduction of a specific gene product (Ang-1) for only 60 days postgene delivery. This is an inherent limitation of gene transfer experiments and does not necessarily reflect the natural time course of the human disease. Second although overexpression of Ang-1 in the lung resulted in smooth muscle cell hyperplasia and hypertrophy, it rarely resulted in the development of plexiform lesions in the lung. We recognize that the end-stage pathology of human pulmonary hypertension includes a complex array of stenosed, occluded, and plexogenic vessels. End-stage disease in our model system was limited to vessel stenosis and occlusion by smooth muscle cell proliferation and hypertrophy. Despite these limitations we show that constitutive expression of Ang-1 in the rodent lung induces pulmonary hypertension and that this vasculopathy is similar to that seen in the human form of this disease. Our results suggest that Ang-1 may be a molecular signal for vascular muscle cell hyperplasia in the lung. Future inhibition of the expression or effect of Ang-1 in the adult pulmonary vasculature may be a useful strategy to prevent and treat pulmonary hypertension in humans.
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