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(Chest. 2005;128:633S-642S.)
© 2005 American College of Chest Physicians

Role of Angiopoietin-1 in Experimental and Human Pulmonary Arterial Hypertension*

Lakshmi Kugathasan, MSc; André E. Dutly, MD; Yidan D. Zhao, MD; Yupu Deng, MD; Malcolm J. Robb, MD; Shaf Keshavjee, FCCP and Duncan J. Stewart, MD

* From the Terrence Donnelly Heart Centre (Mr. Kugathasan and Drs. Zhao, Deng, Robb, and Stewart), Division of Cardiology, St. Michael’s Hospital, Toronto, ON, Canada; and the Division of Thoracic Surgery (Drs. Dutly and Keshavjee), Toronto General Hospital, Toronto, ON, Canada.

Correspondence to: Duncan J. Stewart, Dexter Hung-Cho Man Chair and Director of the Division of Cardiology, St. Michael’s Hospital, 30 Bond St, Room 6–050K, Queen Wing, Toronto, ON, Canada, M5B 1W8; e-mail: stewartd{at}smh.toronto.on.ca


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Introduction: The pulmonary microvasculature, consisting mainly of an endothelial cell (EC) monolayer and scant matrix support, is incompletely muscularized. Thus, the distal pulmonary arterioles may be predisposed to regression on exposure to environmental stresses (ie, hypoxia) and may be dependent on EC survival factors, like angiopoietin (Ang) 1, to attenuate the development of pulmonary arterial hypertension (PAH). In order to clarify the link between Ang1 expression and the development of PAH in patients, we also studied messenger RNA and protein expression in lung samples from healthy control subjects and patients with idiopathic PAH (IPAH) or PAH associated with other diseases (APAH).

Methods: Ang/Tie2 gene expression was assessed in rats that had been exposed to hypoxia (ie, 10% O2) for 1, 3, or 7 days. In a separate experiment, the cell-based gene transfer of Ang1/Ang2 was performed, and the effects were evaluated in rats with hypoxia-induced PAH.

Results: Hypoxia induced significant early increases in right ventricular systolic pressure (RVSP) and right ventricle/left ventricle-plus-septum mass ratio (RV/[LV + S]), with a significant decrease in Tie2 expression. Hypoxic rats receiving Ang1 demonstrated significant improvements in RVSP and RV/(LV + S), with a partial normalization in Tie2 protein levels. Robust Ang1 expression was observed in healthy human lungs. Furthermore, there were no significant changes in the levels of Ang1 or Ang2 in IPAH or APAH samples vs those in control subjects.

Conclusions: Decreased activity of the Tie2 pathway with hypoxia may contribute to PAH, possibly by loss of EC survival signaling, which can be overcome by Ang1 gene transfer.


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pulmonary arterial hypertension (PAH) is a progressive, debilitating disease that is characterized by a persistent elevation in pulmonary arterial resistance, ultimately resulting in right ventricular (RV) failure and death.1 Clinically, PAH is defined as a mean pulmonary arterial pressure of > 25 mm Hg at rest or > 30 mm Hg during exercise.2 In the normal setting, the pulmonary vascular bed functions as a high-flow, low-pressure system, with the capacity to recruit normally nonperfused parallel vessels to accommodate increases in cardiac output34 with little change in pulmonary vascular resistance even at peak exercise. In patients with PAH, however, these unique hemodynamic features of the pulmonary circulation are compromised due to the obliteration and remodeling of the pulmonary microvessels, leading to the progressive increase in pulmonary arterial pressure.5

While much of the etiologic mechanism underlying PAH remains elusive, it is widely held to be instigated by a perturbation of endothelial cell (EC) homeostasis26 and may be particularly important at the level of intraacinar arterioles, consisting primarily of ECs and scant matrix support. Furthermore, the paucity in surrounding smooth muscle cells (SMCs) may predispose the "fragile" distal pulmonary arterioles to regression on exposure to various environmental stresses, such as hypoxia.1 We have previously suggested that EC apoptosis may be an initiating mechanism for PAH, leading to the regression of pulmonary microvessels, resulting in increased vascular resistance due to the rarefaction of distal pulmonary arterioles.7 Accordingly, we hypothesize that the maintenance of endothelial integrity and survival may be essential in preventing the onset and progression of PAH, and this may be dependent on the action of EC-specific survival factors.

Tie2 is an EC-specific receptor belonging to the family of receptor tyrosine kinase and is essential for angiogenesis during embryonic development.8 The two main ligands identified for Tie2 are the angiopoietin (Ang) 1 and Ang2 ligands, both of which bind to Tie2 with equal affinity but result in distinct effects: Ang1 induces the autophosphorylation of Tie2 while Ang2 is capable of competitively inhibiting this kinase activation. Through activation of the Tie2 receptor and stimulation of the Akt/survivin pathway, Ang1, the agonist to the Tie2 receptor, protects against EC apoptosis910 and also functions to recruit and sustain periendothelial support cells, allowing ECs to stabilize the structure and modulate the function of blood vessels. Ang2, on the other hand, although first identified as the endogenous antagonist to Tie2 is now recognized to have "context-dependent" agonistic properties.11121314 Therefore, we propose that Ang1 will be protective in rats with hypoxia-induced PAH, possibly by promoting EC survival and maintaining microvascular homeostasis.

A variety of nonpulmonary EC lines have been used to study the effects of the varying durations and degrees of hypoxia on Ang-Tie2 expression1516171819; however, given the fundamental difference in the response between these two circulations to hypoxia, it is important to examine the hypoxic effects on the Ang-Tie2 system in pulmonary vascular ECs. Similarly, while various in vivo studies have evaluated the regulation of Ang-Tie2 following myocardial, brain, or retinal ischemia, only one study20 thus far has examined this system in the hypoxic lung, albeit following acute exposure to hypoxia. In this report, a significant decline in Ang1 and Tie2 messenger RNA, and Tie2 protein and activation were observed, which was most pronounced in the lungs compared to that in other organs. Based on these observations, we undertook a study, first, to understand the role of the Ang-Tie2 system under conditions of chronic hypoxia and, second, to determine whether Ang1 overexpression through cell-based gene transfer would elicit a protective response and attenuate the development of hypoxia-induced PAH. Finally, we also explored whether similar changes in the Ang-Tie2 pathway could be identified in individuals with idiopathic PAH (IPAH) or PAH associated with other diseases (APAH) by assessing the levels of transcript and protein in lung biopsy samples obtained from patients undergoing lung transplantation for end-stage disease.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture, Transfection, and Delivery
All animal procedures were approved by the Animal Care Committee of St. Michael’s Hospital. Fibroblast cells were obtained from the abdominal region of syngeneic 21 day-old Fisher-344 rats (Charles River Laboratories; Wilmington, MA), and were cultured in Dulbecco modified Eagle medium with 10% fetal bovine serum and 2% penicillin/streptomycin in a humidified environment with 95% O2 and 5% CO2 at 37°C. All cells between the fifth and seventh passages were transfected with human plasmid Ang1 (Dr. I. Kim; Woosuk University; Chonju, South Korea), human pAng2, (Dr. R. Stewart; St. Michael’s Hospital; Toronto, ON, Canada), or the insert-deficient vector pFLAG (control) using a transfection reagent (Superfect; Qiagen; Valencia, CA), as described previously.7 Following transfection, cells were allowed to recover for 24 h, and then were trypsinized and divided into aliquots of 1 x 106 cells/mL saline solution per rat for delivery into the pulmonary circulation via the left external jugular vein.

Hypoxia Chamber
Groups of three Fisher-344 rats were placed in sealed plastic (Plexiglas; Degussa; Dusseldorf, Germany) normobaric chambers (12 x 5 x 7 inches) for 1, 3, or 7 days for the timecourse experiments, and for 7 days for the gene-therapy experiments. The oxygen concentration in the chamber was maintained at 8 to 10% by controlling the inflow rates from tanks containing mixed air and nitrogen. The O2 concentration in the chamber was measured daily using an O2 analyzer (Engineered Systems and Designs; Wilmington, DE). Control rats were maintained in room air. Normal rat chow and water were provided ad libitum.

Hemodynamic Measurements and Tissue Harvest
Following each end point of hypoxic exposure, the rats were isolated from their chambers individually, anesthetized, and a 3F microtip catheter (Millar Instruments; Houston, TX) was inserted into the exposed right external jugular vein, and was advanced through the superior vena cava level, right atrium, and finally, into the RV. The pattern of pressure tracing was used to confirm the correct location of the catheter, and the average RV systolic pressure (RVSP) measurement was recorded from 2 min of readings, once the pressure was deemed to have stabilized. The catheter was then slowly withdrawn, and the vein was tied off. The rats were then sacrificed, and the right lung lobes were dissected and snap-frozen in liquid nitrogen for RNA and protein extractions and analyses. The heart was dissected free from the atria, the aorta, and the pulmonary trunk, and the RV was separated from the left ventricle and the ventricular septum. The RV/left ventricle-plus-septum mass ratio (RV/LV + S) was determined as an indicator of the RV hypertrophic response to pulmonary hypertension.

Collection and Processing of Human Lung Tissue
All patients signed an informed consent form, and the study was approved by the ethics boards of the University Health Network and St. Michael’s Hospital. Control lung samples were obtained through a small wedge resection that was performed on brain-dead lung donors, prior to the flush with lung preservation solution. Lung sections were obtained from patients with IPAH and APAH who were undergoing lung transplantation. Samples were snap-frozen and stored at –80°C until the time for RNA or protein extraction. Control and patient characteristics are presented in Table 1 .


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  		Table 1.. Control Subject and Patient Characteristics*

 
Reverse-Transcription and Quantitative Polymerase Chain Reaction
Total RNA was extracted from lungs using a reagent (TRIzol; Invitrogen; Carlsbad, CA). Extracted RNA was then reverse-transcribed (Omniscript Reverse Transcriptase; Qiagen) using random hexamers (Promega; Madison, WI). Primers for rats and human genes of interest were designed, and their sequences and expected product sizes are presented in Tables 2 and 3 . Quantitative polymerase chain reaction (PCR) was performed using a sequence detection system (Prism 7700; ABI; Foster City, CA). The reaction conditions were as follows: initial activation of hot-start SYBR green at 95°C for 10 min; followed by 40 cycles consisting of 15 s at 95°C for denaturing and 1 min at 62°C for annealing and extension. Relative quantification of messenger RNA expression was calculated using a relative expression software tool (REST; provided by Pfaffl et al21).


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  		Table 2.. Rat Gene Primer Sequences and PCR Product Size*

 

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  		Table 3.. Human Gene Primer Sequences and PCR Product Size*

 
Enzyme-Linked Immunosorbent Assay
The enzyme-linked immunosorbent assay (ELISA) system was based on a sandwich noncompetitive ELISA, consisting of an antihuman Ang1 or Ang2 capture antibody (R&D Systems; Minneapolis, MN) and a biotinylated goat antihuman Ang1 or Ang2 detection antibody. Initially, 96-welled microtiter immunoassay plates were coated with 100 µL per well of 1 µg/mL mouse antihuman Ang1 or Ang2 overnight at room temperature. Plates were then washed three times with a washing solution (0.05% Tween 20 in phosphate-buffered saline solution [PBS], pH 7.2). Blocking was performed for a minimum of 1 h at room temperature with PBS containing bovine serum albumin (300 µL per well). Recombinant human Ang1 or Ang2 standards and protein samples were added in duplicate (100 µL per well), and were incubated for 2 h at room temperature and washed three times with a wash solution. Following this, 100 µL per well of biotinylated detection antibody was then added for a further 2 h of incubation at room temperature and was washed three times with the wash solution. A 1:200 dilution of streptavidin-horse-radish perioxidase (100 µL per well) was then added for 20 min at room temperature. The plates were then washed again with the wash solution before color development using tetramethyl benzidine substrate (100 µL per well). The reaction was finally stopped with the addition of 2N H2SO4 (50 µL per well), and the absorbance was read at 450 nm and corrected at 570 nm.

Western Blot Analyses (Tie2, Activated Caspase-3, and Phosphorylated Tie2)
Frozen tissues were homogenized in a modified radioimmunoprecipitation assay lysis buffer, and 30 µg of proteins were subjected to electrophoresis. Membranes were incubated overnight at 4°C with either polyclonal rabbit anti-Tie2 primary antibody (1:500 dilution) [Santa Cruz Biotechnology; Santa Cruz, CA] or polyclonal rabbit anti-caspase-3 primary antibody (1:1000 dilution) [Cell Signaling Technology; Beverly, MA]. Equivalent protein loading was demonstrated by probing the same blot with monoclonal anti-ß-actin primary antibody (1:5,000) [Santa Cruz Biotechnology]. For the evaluation of phosphorylated tyrosine levels of Tie2, 500 µg of tissue homogenate was immunoprecipitated with polyclonal rabbit anti-Tie2 primary antibody prior to electrophoresis. Membranes were then probed with monoclonal antiphosphotyrosine primary antibody (1:4,000) [4G10; Upstate Biotechnology; Lake Placid, NY], followed by horseradish peroxidase-conjugated antimouse secondary antibody (1:4,000). Blots were scanned with an imaging densitometer, and the optical densities of the protein bands were quantified. Predetermined molecular mass standards were used as markers (Invitrogen).

Immunohistochemistry
Lung tissue from healthy subjects and IPAH patients were fixed in freshly prepared 4% paraformaldehyde in PBS at 4°C overnight. The samples were then processed for paraffin embedding and cut into 5-µm sections. The avidin-biotin complex method was used for immunostaining (Vector; Burlingame, CA). Sections were incubated with rabbit antimouse Ang1 (1:100) [Alpha Diagnostic International; San Antonio, TX] at 4°C overnight followed by incubation with biotinylated goat antirabbit IgG as a secondary antibody (1:200) [Alpha Diagnostic International] for 1 h at room temperature. Negative controls were incubated only with a secondary antibody.

Statistical Analysis
All data are presented as the mean ± SEM. Statistical analysis was performed using a statistical software package (GraphPad InStat software, version 3.00 for Windows 95; GraphPad Software; San Diego, CA). Either one-way analysis of variance followed by Dunnett posttest analysis or unpaired t test was performed.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Hypoxia on Expression of the Angiogenic Growth Factors in the Lung
A significant elevation in RVSP (Fig 1 , top left, A) and RV hypertrophy (Fig 1, bottom left, B) was observed in rats over the first 3 days of hypoxic exposure, and this was sustained at 7 days of exposure. In parallel with the hemodynamic abnormalities induced by hypoxia, pulmonary Ang2 messenger RNA levels, assessed using quantitative PCR, were significantly increased even after a day of hypoxia associated with a significant decline in Tie2 transcript levels after 3 days, whereas Ang1 levels were not significantly altered (Fig 1, top right, C). Vascular endothelial growth factor (VEGF) expression was also reduced, but only after 7 days of hypoxia. Western blot analysis confirmed the decrease in Tie2 protein expression over the course of hypoxic exposure (Fig 1, bottom right, D). Levels of activated caspase-3 in lung tissue increased on exposure to hypoxia, peaking after 3 days (Fig 1, far right, E), but subsequently decreasing to levels below baseline by 7 days.



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  		Figure 1.. The effect of hypoxic exposure on hemodynamics and levels of angiogenic factors. Increasing (top left, A) RVSP measurements and (bottom left, B) RV hypertrophy following prolonged exposure to hypoxia. Top right, C: quantitative PCR analysis demonstrating a transient but no significant increase in Ang1, an early and significant elevation in Ang2, and a significant down-regulation of Tie2 and VEGF (normal samples set a 1.0). Bottom right, D: protein analysis of Tie2 receptors demonstrating a significant decrease in expression following hypoxic exposure. Far right, E: protein analysis of activated caspase-3 levels peaking at 3 days of hypoxia (n = 6 to 9). * = p < 0.05, appears in Fig 1C above Ang2 (3 days); ** = p < 0.01; {dagger} = p < 0.001, appears in Fig 1C, above Ang2 (1 day) (compared to normoxia).

 
Effect of Ang Gene-Transfer on Hypoxic PAH
Ang1 or Ang2 was overexpressed within the lung using a cell-based method for selective gene transfer to the distal (precapillary) arteriole, which has been carefully validated in previous reports.72223 Rats that received Ang1 prior to being exposed to a week of hypoxia were significantly protected against PAH (Fig 2 , top left, A) and RV hypertrophy (Fig 2, bottom left, B). Ang2-treated rats, on the other hand, exhibited neither improved nor worsened PAH. The decrease in Tie2 levels was also significantly attenuated (Fig 2, top right, C), which is consistent with a protective role being mediated through Ang1, while there was no change in the overall activity of the receptors (Fig 2, bottom right, D). Furthermore, activated levels of caspase-3 were significantly reduced in the Ang1-treated group compared to the hypoxic control or Ang2-treated rats at 7 days (Fig 2, far right, E).



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  		Figure 2.. Effect of Ang gene transfer on hemodynamics and levels of angiogenic factors. Protective effects of pAng1 gene transfer against elevations in (top left, A) RVSP and (bottom left, B) RV hypertrophy are shown; however, pAng2 gene transfer had no significant effects (n = 13–15). Protein analyses demonstrating an increase in (top right, C) Tie2 receptor levels but without an increase in (bottom right, D) overall activity following pAng1 gene transfer (n = 6). Far right, E: protein analysis indicating a significant decrease in activated caspase-3 levels following pAng1 gene transfer (n = 6). ** = p < 0.01 (compared to hypoxic controls); {dagger} = p < 0.001 (compared to respective normoxic rats); {ddagger} = p < 0.01 (compared to pAng1 normoxic rats).

 
Expression of Ang in Patients With PAH
In order to clarify the link between Ang1 expression and the development of PAH in patients, we also studied messenger RNA and protein expression in lung samples from healthy control subjects and from patients with IPAH or APAH. The relative expression of Ang1, Ang2, and Tie2 messenger RNA was determined by quantitative PCR in the various groups (Fig 3 , top left, A). In agreement with the rat studies, there were no significant changes in Ang1 messenger RNA expression in patients with PAH, and, although there was a trend toward increased Ang2 messenger RNA expression for the IPAH group, this was not significant. Similarly, the ELISA for Ang1 (Fig 3, bottom left, B) and Ang2 (Fig 3, top left, C) showed only a weak trend toward increased Ang2 protein levels. In contrast, Tie2 transcript levels were significantly increased in the lungs of patients with IPAH but not in those of patients with APAH; however, no differences were observed in Tie2 activity in tissue samples between the groups (Fig 3, bottom right, D). In addition, immunohistochemistry results demonstrated robust Ang1 staining that was localized to vascular SMCs of small and medium-sized pulmonary arteries in the lungs of healthy subjects and IPAH patients (Fig 3, far right, E).



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  		Figure 3.. Angiogenic factors in normal, IPAH, and APAH human lung samples. Top left, A: quantitative PCR analysis demonstrating a significant increase in Tie2 messenger RNA in IPAH samples compared to normal lung samples (normalized to 1.0; n = 6–8; * = p < 0.05). ELISAs demonstrating no significant differences in (bottom left, B) Ang1 levels and (top right, C) Ang2 levels from samples of patients with IPAH and APAH compared to those from healthy subjects (Normal). Bottom right, D: protein analysis demonstrating no change in the overall activity of Tie2 receptors in normal, IPAH, and APAH lung samples. Far right, E: immunohistochemistry demonstrating prominent Ang1 staining localized mainly to the vascular SMCs of small-sized and medium-sized pulmonary arteries (insert, negative control).

 

   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report, we explored the expression and activity of the Ang system under basal conditions and described the changes in response to hypoxia, which are consistent with a protective role for Ang-Tie2 signaling against the development of PAH. This was confirmed in gene-transfer experiments, which demonstrated the ability of Ang1 overexpression to reduce hypoxic PAH. Finally, we now show that this system is robustly expressed in the healthy human lung and that only minor changes can be demonstrated in the lungs of patients with severe PAH compared to those from healthy donors.

At the very least, the present data do not support a causal role for Ang1 in the development of PAH, as had been suggested previously by Du et al.24 Du et al24 hypothesized that Ang1 was involved in promoting the proliferation of SMCs of small pulmonary arteries and terminal arterioles, leading to increased pulmonary vascular resistance. This hypothesis was based on their observation that Ang1 expression and Tie2 activation were undetectable in the healthy human lung but were highly elevated in patients with pulmonary hypertension.2425 Also, Du et al24 reported a strong positive correlation between Ang1 protein expression and the degree of pulmonary vascular resistance, leading them to conclude that Ang1 expression is a marker for the severity of pulmonary hypertension.

These observations, however, challenge findings previously established in the literature. Perhaps the most striking contradiction lies with the lack of basal Ang1 expression and Tie2 activity in the healthy human and rat lung,2426 in contradistinction to a number of earlier reports of rodent and human lungs.7202728 Furthermore, as outlined in the commentary by Rudge et al,29 in studies with Ang1 overexpression or overactivation of Tie2, SMC hyperplasia was not observed within the vascular beds examined,30313233 suggesting that the excessive activation of the Ang-Tie2 pathway need not lead to SMC hyperplasia.

The apparent differences in Ang1 expression in the healthy human lung may reflect the use of different controls between our study and that of Du et al.24 Unlike the surgical controls used by Du et al,24 the samples obtained in our study were from organ donors who were undergoing lung transplantation. Contrary to the findings by Du et al24 and Thistlethwaite et al,25 we were able to detect robust levels of both Ang1 messenger RNA and protein, and phosphorylated levels of Tie2 in the healthy lung samples. Additionally, while Du et al24 reported bone morphogenetic protein receptor (BMPR)-1 levels to be inversely correlated to Ang1 levels with there being a down-regulation in BMPR1 in their pulmonary hypertension samples, we did not detect any difference between our control and PH samples (data not shown). These findings in both rats and humans are consistent with a role for Ang1 in the maintenance of vascular homeostasis in the healthy lung. Furthermore, there were no significant differences observed in messenger RNA and protein levels of Ang1 compared to samples from IPAH and APAH patients. Interestingly, only Tie2 messenger RNA levels were significantly elevated in samples from IPAH patients compared to samples from healthy subjects, and not in those patients with PAH secondary to various other pathologies (ie, APAH), which made up the majority of patients included in the previous report. Since the human samples examined were obtained from patients in the late stages of pulmonary hypertension, it is difficult to determine mechanistically the role of the Ang-Tie2 system from these results. Nevertheless, it is clear that Ang1 is indeed expressed in the healthy lung and is not significantly elevated in patients with either primary or secondary PH.

The hypoxia-induced changes in the expression of components of the Ang system in the rat lung are in keeping with the results of a number of in vitro and in vivo studies17343536 examining the response of systemic cells and tissues exposed to hypoxia. Although in the study by Abdulmalek et al,20 Ang2 levels in the lungs were not elevated following 12 and 48 h of hypoxia, levels of another Tie2 antagonist, Ang3, were elevated.20 This discrepancy may be explained by differences in the analytic methods since a semiquantitative technique for reverse-transcription PCR was used in the earlier study, while in our study a more sensitive method of quantitative PCR was utilized. Furthermore, in agreement with the findings by Abdulmalek et al,20 Tie2 protein levels were significantly decreased in the lungs after a day of hypoxia and were further sustained with long-term exposure to hypoxia. This is an important finding that points to the reduced overall influence of the Ang-Tie2 pathway in the pulmonary circulation of rats with long-term exposure to hypoxia. Although the mechanism of decreased receptor expression was not explored in either study, the following two main possibilities exist: first, that this represents a true down-regulation of expression at the level of the individual EC; and, second, that there is no change in cellular Tie2 expression but an overall reduction in the mass of ECs and, therefore, in microcirculation. In this context, the observed initial unchanged levels and the subsequent down-regulation in VEGF messenger RNA expression in this study, which is also consistent with the findings of Partovian et al,37 Pfeifer et al,38 and Sandner et al,39 is intriguing since this creates the conditions that are thought to favor EC apoptosis and vascular degeneration.40

According to this paradigm, the consequences of increased Ang2 expression will depend critically on the expression of VEGF. In conditions of high VEGF production, Ang2 is thought to play a critical role in releasing ECs from the tonic inhibitory effects of Ang1 and in facilitating their activation in response to the angiogenic stimulus. However, in the presence of reduced VEGF activity, the principle effect of Ang2 is to block Ang1 survival signaling and, thus, to induce EC apoptosis. Therefore, these findings raise the possibility that the increase in Ang2 with a concomitant decrease in VEGF may predispose ECs to apoptosis and subsequent microvascular regression. In agreement with this interpretation, levels of active caspase-3 increased initially on exposure to hypoxia, corresponding to the peak reciprocal changes in Ang2 and Tie2 expression. Thus, EC apoptosis induced by environmental triggers such as hypoxia may play an important role in mediating pulmonary microvascular rarefaction, which may be an underrecognized precipitant of PAH. Interestingly, activated caspase-3 levels were reduced once the presence of chronic PAH had been established, which is consistent with reports41 suggesting that decreased SMC apoptosis is a mechanism of pulmonary arteriolar remodeling contributing to abnormal arteriolar muscularization, as seen in the established disease.

It follows, therefore, that strategies to decrease EC apoptosis in response to hypoxia may impede the development of PAH. Ang1, being an EC-selective survival factor, is an ideal candidate gene for this experiment. Therefore, based on these observations, a second series of experiments was performed to evaluate the effect of Ang1 or Ang2 cell-based gene transfer on hypoxia-induced PAH. Ang1 gene transfer was effective in reducing both hypoxia-induced PAH and caspase activation, whereas Ang2 had no significant effect in this model. These results are consistent with the effect of Ang1 gene therapy in a monocrotaline-treated rat model 7 and suggest that the beneficial effects of Ang1 gene therapy may be in part dependent on its ability to decrease apoptosis. Although the spatial distribution of apoptosis was not determined in this study, it is unlikely that Ang1 therapy could directly affect apoptosis in non-ECs since the expression of the Tie2 receptor is largely restricted to ECs. Although it is also possible that the increased Tie2 levels, and hence increased activity, may have resulted in improved EC function and thereby protection against PAH by other mechanisms, it is well-established that Ang-1 induces potent direct survival signaling.

However, our gene transfer experiments again are in conflict with those of Thistlethwaite et al.25 These authors also conducted studies2642 in rats in which Ang1 was overexpressed using adenoviral and adeno-associated viral vectors. The delivery of Ang1 into the pulmonary circulation resulted in a spontaneous and significant elevation in pulmonary artery pressures. Histologic analysis of the lungs of Ang1-treated rats also exhibited severe muscular hyperplasia and hypertrophy in the medial layer of small pulmonary arteries and arterioles, which is consistent with the pathologic changes observed in those with advanced pulmonary hypertension. It is difficult to fully reconcile these data with our own; however, it is possible that the effect of Ang1 gene transfer critically depends on the level of overexpression or on the specific cell types that are transfected. Our system produces a low overall mass of gene transfer, which is highly localized to the distal arteriolar bed, whereas the intratracheal administration of viral vectors results in a massive transfection of many different cell types, predominantly the airway epithelium.

In conclusion, our studies demonstrate that the cell-based gene transfer of Ang1 to the pulmonary microvasculature effectively protects against hypoxia-induced PAH, while reducing the down-regulation of Tie2 expression and hypoxia-induced apoptosis. These results further suggest that the Ang-Tie2 system may be important in the protection of the pulmonary endothelium against hypoxia-induced injury and that Ang1 gene therapy may be useful in the prevention of PAH.


   Acknowledgements
 
The authors thank Drs. Gerald A Proteau and Robin Han for their technical assistance.


   Footnotes
 
This study was funded by Northern Therapeutics. Dr. Dutly is a recipient of a grant from the Swiss National Scientific Foundation (grant 81ZH-068485).

Abbreviations: Ang = angiopoietin; APAH = pulmonary arterial hypertension associated with other diseases; BMPR = bone morphogenetic protein receptor; EC = endothelial cell; ELISA = enyme-linked immunosorbent assay; IPAH = idiopathic pulmonary arterial hypertension; PAH = pulmonary arterial hypertension; PBS = phosphate-buffered saline solution; PCR = polymerase chain reaction; RV = right ventricle ventricular; RV/ = right ventricle/left ventricle; RVSP = right ventricular systolic pressure; SMC = smooth muscle cell; VEGF = vascular endothelial growth factor


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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