Vascular Endothelial Growth Factor-A and Platelet-Derived Growth Factor-B Combination Gene Therapy Prolongs Angiogenic Effects via Recruitment of Interstitial Mononuclear Cells and Paracrine Effects Rather Than Improved Pericyte Coverage of Angiogenic Vessels
Vessel stabilization and the inhibition of side effects such as tissue edema are essential in angiogenic gene therapy. Thus, combination gene transfers stimulating both endothelial cell and pericyte proliferation have become of interest. However, there is currently little data to support combination gene transfer in large animal models. In this study, we evaluated the potential advantages of such a strategy by combining the transfer of adenoviral (Ad) vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF)-B into rabbit hindlimb skeletal muscle. AdLacZ alone or in combination with AdVEGF-A were used as controls. Contrast-enhanced ultrasound, modified Miles assay, and immunohistology were used to quantify perfusion, vascular permeability, and capillary size, respectively. Confocal microscopy was used in the assessment of pericyte-coverage. The transfer of AdPDGF-B alone and in combination with AdVEGF-A induced prominent proliferation of α-smooth muscle actin–, CD31-, RAM11-, HAM56-, and VEGF- positive cells. Although, pericyte recruitment to angiogenic vessels was not improved, combination gene transfer induced a longer-lasting increase in perfusion in both intact and ischemic muscles than AdVEGF-A gene transfer alone. In conclusion, intramuscular delivery of AdVEGF-A and AdPDGF-B, combined, resulted in a prolonged angiogenic response. However, the effects were most likely mediated via paracrine mechanisms rather than an increase in vascular pericyte coverage.
The stabilization of angiogenic vessels through pericyte recruitment is regarded to be essential for the maintenance of blood flow after angiogenic gene therapy of ischemic diseases.1 Thus, a gene transfer (GT) that combines vascular endothelial growth factors (VEGFs) and platelet-derived growth factors (PDGFs) and stimulates both endothelial cells and pericytes could be more effective than application of single therapies.
VEGF-A is a strong endothelial mitogen that can induce efficient vascular growth and perfusion.2 PDGF-B mediates pericyte proliferation and migration and is, thus, associated with vessel stabilization.3 Interestingly, our previous studies have shown that pericyte proliferation can also be induced after transduction with adenoviral (Ad) VEGFs alone, likely through indirect mechanisms, including increased capillary pressure, shear stress, and upregulation of other growth factors.2,4 Thus, actual benefits of the combination GT on pericyte proliferation and vascular recruitment associated to therapeutic angiogenesis are unclear.
Currently, very little data are available on combination GT in large animal models. Intramuscular injection of adenoviruses is currently the most efficient method for gene delivery in large animals and holds promise for clinical trials. We compared the effects of intramuscular GT of AdVEGF-A or AdPDGF-B alone or in combination on pericyte activation and the stability of angiogenic vessels in normoxic and ischemic rabbit hindlimbs. We found that the combination GT prolonged angiogenic effects, although pericyte coverage of the neovessels was not enhanced. Rather, AdPDGF-B GT alone or in combination with AdVEGF-A induced recruitment of mononuclear, interstitial cells expressing endogenous VEGF.
Materials and Methods
Ischemia Operation and Gene Transfers
New Zealand White rabbits (mean weight, 2.5 to 3 kg; total n=141) received intramuscular injections of adenoviruses (1011 viral particles [vp]) encoding human VEGF-A165 or human PDGF-B. A total dose of 2×1011 vp was used for the AdVEGF-A+AdPDGF-B combination GT. β-Galactosidase marker gene (LacZ) alone or in combination with AdVEGF-A was used as a control. Human clinical grade, first generation, serotype 5, replication-deficient adenoviruses produced under GMP conditions and analyzed to be free from contaminants were used.4 Intramuscular GTs were performed into the semimembranosus muscle of the thigh using a 1-mL syringe and a 25-gauge needle (1011 vp/mL divided into 10 separate 0.1-mL injections) during medetomidine (Domitor, 0.3 mg/kg, Orion) and ketamine (Ketalar, 20 mg/kg, Pfizer) anesthesia. Ligation of the profound femoral artery was performed in a subgroup of animals (n=58) before GTs as previously described.2 Animals were euthanized at 6, 14, or 28 days after GT. To reduce the number of animals, the AdVEGF+AdLacZ control was not used in ischemic animals because the results did not statistically differ from transfer of AdVEGF alone to normoxic muscle. All animal experiments were approved by the Experimental Animal Committee at the University of Kuopio.
Contrast-Enhanced Ultrasound Imaging of Perfusion
Perfusion in transduced and contralateral intact rabbit semimembranosus muscles was quantitatively measured with Acuson Sequoia 512 and 15L8 transducer (Siemens), using the power Doppler mode and the administration of a contrast agent 6, 14, or 28 days after GT.2 Two consecutive longitudinal plane video clips of 10 seconds (power Doppler at 8.5 MHz; dynamic range, 10 dB; power, −18 dB; mechanical index, 0.6; gain, 40; depth, 20 mm) were captured starting immediately on a bolus injection containing 0.3 mL of second generation contrast agent (sulfur hexafluoride in a phospholipid shell, ≈2×108 bubbles per milliliter; mean diameter, 2.5 μm; Sonovue, Bracco) into the ear vein. The perfusion ratio was calculated with Datapro 2.13 (Noesis), using the maximum signal intensities of the transduced and contralateral intact limbs.2,5 Ultrasound measurements and the analysis of data were performed in a blinded manner.
The Modified Miles Assay for the Evaluation of Tissue Edema
The modified Miles assay was used for the evaluation of tissue edema at death. Evans Blue dye (30 mg/kg, Sigma) was injected intravenously 30 minutes before euthanasia. After euthanasia, the animals were perfusion-fixed with 1 L of 1% paraformaldehyde in 0.05 mol/L citrate buffer (pH 3.5) via the left ventricle. Extravasated Evans blue dye bound to plasma proteins (mostly albumin) was extracted from transduced and contralateral intact semimembranosus muscle samples by incubation in formamide at 60°C for 48 hours. The amount of extravasated Evans blue dye was determined on the basis of absorbance at 610 nm.4 The results are represented as absorbance ratios between the transduced and contralateral intact muscles. The absorbances were normalized to the weight of the muscle sample.
The Avidin-biotin-HRP system (Vector Laboratories) with 3,prime]-5,prime]-diaminobenzidine (Zymed) color substrate or fluorescein isothiocyanate (FITC) (Zymed) fluorescent dye was used for immunocytochemistry on 7-μm-thick paraffin-embedded sections fixed in 4% paraformaldehyde/15% sucrose for 4 hours. Intraarterially injected rhodamine-labeled Ricinus communis lectin (1 mg in 2 mL of saline administered into the profound femoral artery, Vector) and FITC conjugates were used for immunocytochemistry on 50-μm-thick frozen sections. The endothelium was immunostained using a mouse monoclonal antibody (mAb) against CD31 (DAKO, dilution 1:50). Pericytes and SMCs were stained with an α-smooth muscle actin (α-SMA) mAb (Sigma, 1:250), macrophages were stained with a mAb against rabbit macrophages (RAM11, DAKO, 1:200) and mAb against human macrophages and monocytes (HAM56, DAKO, 1:500) with a trypsin pretreatment. Fibroblasts were identified with desmin and vimentin (Sigma, 1:100 and 1:50, respectively). Protein expressions were studied using a hVEGF antibody (Santa Cruz Biotechnology, 1:500) and a PDGF antibody (R&D, 1:500) with a citrate buffer boiling treatment. Receptor stainings were performed using VEGFR-1 (Santa Cruz Biotechnology, 1:250), VEGFR-2 (RDI, 1:250), and PDGFR-β (Santa Cruz Biotechnology, 1:200) antibodies. General histology and cell morphology were studied using hematoxylin/eosin stainings. Double immunostainings comprising Avidin-biotin-HRP system with FITC (x) and anti–mouse-Alexa 546 (x) were used for the detection of VEGF and HAM56, respectively.
Photographs of the 7-μm-thick histological sections were taken with an Olympus AX70 microscope (Olympus Optical) and analySIS software (Soft Imaging System). Fluorescent images in Figure 4 were taken using an Olympus U-RFL-T burner. Confocal images of the 50-μm-thick sections were taken with an Olympus IX81 microscope and a Fluoview-1000 confocal setup. Reconstructions of the confocal images were performed with an open source software package, BioImageXD.6 Images were further processed for publication with Adobe Photoshop 7.0 (Adobe).4
Blood Vessel Measurements
The mean capillary area (micrometers squared) was measured at ×200 magnification from CD31 immunostained sections of semimembranosus muscles obtained from areas covered entirely by skeletal muscle tissue.4 All measurements were performed in a blinded manner from 10 fields representing maximal angiogenic effects of each muscle section using analySIS software (Soft Imaging System). To avoid ambiguous data caused from trauma effects of the needle injection, the analysis was made outside the needle track area. Means of the measurements are reported. Total area of arteries and veins (percentage of the total muscle area) was quantified from α-SMA–stained sections of semimembranosus muscles at ×40 magnification covering the entire muscle.
Measurements of α-SMA–Positive Cells
The percentage of α-SMA–positive pericytes, SMCs, and myofibroblasts (percentage of the skeletal muscle area) were measured by immunofluorescence (FITC) of α-SMA–stained sections of semimembranosus muscles at ×200 magnification. All measurements were performed using analySIS software (Soft Imaging System) in a blinded manner from 5 fields that represented maximal α-SMA immunofluorescence of each muscle section. Measurements were taken from areas that did not contain large arteries or veins because their SMC layer could affect the results.
Quantification of VEGF-A and PDGF-B Protein Expressions
Muscle samples taken at the time of death were frozen in liquid nitrogen and stored at −70°C. T-Per buffer (Thermo Scientific) with 1× Halt protease inhibitor (Thermo Scientific) was used for protein extraction from homogenized muscle samples. The amount of protein in each sample was quantified with hVEGF-A and hPDGF-B ELISA (R&D Systems) and further normalized to the amount of total protein in each protein extract. The amount of total protein in each sample was quantified with BCA protein assay kit (Thermo Scientific).
The results are expressed as mean+SEM. Statistical significance was evaluated using the Kruskal–Wallis test, followed by the Mann–Whitney U test where appropriate. P<0.05 was considered statistically significant.
Preparation of Supplemental Video Files
Three-dimensional reconstructions of the confocal images were prepared with Imaris-software (Bitplane). See the video files in the online data supplement at http://circres.ahajournals.org.
AdPDGF-B Induces Recruitment of Interstitial Cells Six Days After Gene Transfer
The efficacy of the GTs was confirmed using protein expression analysis of the muscle samples (see Figure I in the online data supplement for results). The effect of AdPDGF-B overexpression was first studied in normoxic muscles (Figure 1). AdLacZ-transduced control muscles displayed normal skeletal muscle morphology with small capillaries (Figure 1a, red arrowheads) and the occasional α-SMA–positive pericytes surrounding the capillaries (Figure 1a, black arrowheads). The main response to AdPDGF-B transduction was the instead of angiogenesis, proliferation of cells in the muscle interstitium (Figure 1b, black arrows). Some enlarged capillaries (Figure 1b, red arrowheads), with increased pericyte coverage (Figure 1b, black arrowheads), were also detected. AdVEGF-A induced abundant capillary enlargement (Figure 1c, red arrowheads) and recruited pericytes around the angiogenic capillaries (Figure 1c, black arrowheads). The AdVEGF-A+AdLacZ controls manifested similar histology to that of AdVEGF-A alone; only a minor recruitment of inflammatory cells, attributable to increased viral dosage, was observed (Figure 1d). In contrast, both the enlargement of capillaries and strong proliferation of interstitial cells (Figure 1e, black arrows) were visible after AdVEGF-A+AdPDGF-B GT. However, recruitment of pericytes to angiogenic vessels was impaired compared to AdVEGF-A or AdVEGF-A+AdLacZ (Figure 1c through 1e). Many α-SMA–positive cells (Figure 1e, asterisk) and some CD31-positive cells (Figure 1e, red arrows) could be seen in the interstitium of AdVEGF-A+AdPDGF-B–transduced muscles. For AdVEGF-A+dPDGF-B combination GT, 2 doses were tested: 2×1011 and 1011 vp. However, both doses yielded similar results (see also supplemental Figure II for the comparison of results obtained from each dose).
The results for normoxic and ischemic animals were very similar when quantified (see also supplemental Figure III for histology from the ischemic muscles). AdPDGF-B alone could not induce significant changes in capillary size (Figure 2a) but moderately increased perfusion in normoxic conditions (Figure 2b). In both normoxic and ischemic muscles, AdVEGF-A, AdVEGF-A+AdLacZ, or AdVEGF-A+AdPDGF-B significantly increased both capillary size and perfusion when compared to AdLacZ alone (Figure 2a and 2b). AdVEGF-A+AdPDGF-B induced more moderate changes when compared to AdVEGF-A alone or combined with AdLacZ, which is probably explained by the moderately lower VEGF expression levels after the combination GT (see supplemental Figure I). AdPDGF-B could not reduce AdVEGF-A–induced edema formation, but, rather, AdPDGF-B even enhanced it in the ischemic muscles (Figure 2c).
Angiogenesis Induced by AdPDGF-B or AdVEGF-A+AdPDGF-B Persists Longer Than That Stimulated by AdVEGF-A Alone
Normal skeletal muscle perfusion was detected with contrast-enhanced ultrasound in AdLacZ-transduced normoxic muscles 6 days after GT (Figure 3a). There was a large increase in perfusion after AdVEGF-A, AdVEGF-A+AdLacZ, and AdVEGF-A+AdPDGF-B GTs (Figure 3c through 3e) but only a small increase after AdPDGF-B GT (Figure 3b). Tissue edema was observed between semimembranosus and gracilis muscles in all AdVEGF-A, AdVEGF-A+AdLacZ and AdVEGF-A+AdPDGF-B (Figure 3c through 3e, asterisks). Fourteen days after GT, perfusion increases induced by AdVEGF-A and AdVEGF-A+AdLacZ were decreased to baseline (Figure 3h and 3i). However, perfusion increases induced by AdPDGF-B and AdVEGF-A+AdPDGF-B were still visible (Figure 3g and 3j; see also supplemental Video 1). Tissue edema was also still detectable in AdVEGF-A+ AdPDGF-B 14 days after GT (Figure 3h, asterisk).
In addition to the ultrasound findings, the analysis of CD31-stained muscle sections 14 days after GT (Figure 3k through 3o and 3q) revealed that histological changes induced by AdPDGF-B alone or in combination had also persisted. For AdVEGF-A or AdVEGF-A+AdLacZ most vessels had regressed, although there were some regressing vascular structures still visible (Figure 3m and 3n, arrowheads). However, in AdPDGF-B and AdVEGF-A+AdPDGF-B–transduced muscles, large arteries and veins, and, to some extent capillaries, were still enlarged (Figure 3l and 3o, arrows). Also, cell density was still increased in the muscle interstitium and often localized to persistent angiogenic vessels (Figure 3l and 3o, asterisks).
Quantification of the ultrasound data displayed dramatic changes in skeletal muscle perfusion induced by the GTs (Figure 3p). Whereas perfusion in AdVEGF-A and AdVEGF-A+AdLacZ–transduced muscles quickly decreased after 6 days, a statistically significant increase in perfusion was still observed 14 days after AdVEGF-A+AdPDGF-B GT in both intact and ischemic muscles (Figure 3p). AdPDGF-B–transduced muscles showed very little attenuation of the effect, and ischemic muscles had increased perfusion even 28 days after GT. Ultrasound findings on day 14 were supported by quantification of the histological findings on the same time point, yielding significant increases in vascularity in AdPDGF-B and AdVEGF-A+AdPDGF-B–transduced muscles (Figure 3q).
AdPDGF-B Induces Proliferation of α-SMA– Positive Pericytes and Fibroblasts and Induces Recruitment of CD31-, HAM56-, and VEGF-Positive Cells
As expected, AdPDGF-B induced a significant proliferation of α-SMA–positive cells in rabbit skeletal muscle 6 days after GT (Figure 4a). However, not all cells in the muscle interstitium were positive for α-SMA. Thus, a series of immunostainings were performed to investigate which cells accumulated in AdPDGF-B–transduced muscles. Cell proliferation was confirmed using Ki67 staining (Figure 4b). Hematoxylin/eosin staining allowed the identification of fibroblasts (Figure 4c, arrow) and some granulocytes (Figure 4c, arrowhead). Desmin and vimentin stainings were also performed to confirm the presence of fibroblasts (data not shown). The occasional presence of macrophages was detected with RAM11 staining (Figure 4d, arrowhead). HAM56 staining showed more positivity because it also stained monocytes, in addition to macrophages (Figure 4e). CD34 only stained the vascular endothelium of large arteries in the samples (Figure 4f). However, CD31 also stained some extravascular cells in the muscle interstitium (Figure 4g, arrows), in addition to endothelial cells (Figure 4g, arrowheads). The expression of VEGF was detected in the blood vessel endothelium (Figure 4h, arrows), fibroblasts, and several mononuclear cells (Figure 4h, arrowheads) of AdPDGF-B–transduced muscles. Whereas vascular structures only displayed a weak PDGF-B expression (Figure 4i, red arrows), strong expression could be found in individual cells in the interstitium (Figure 4i, arrowheads) and in the extracellular matrix (Figure 4i, black arrow). A double staining for VEGF and HAM56 confirmed that VEGF was expressed by HAM56 positive macrophages and monocytes (Figure 4j through 4l, arrows display colocalized staining). VEGFR-1 expression was found in the vascular endothelium (Figure 4m, arrowheads) and in the interstitial cells (Figure 4m, arrows). Additionally, VEGFR-2 and PDGFR-β expression was found in the interstitial cells (Figure 4n and 4o, arrows).
The Recruitment of α-SMA–Positive Pericytes to Vessels Is Impaired in AdVEGF-A+AdPDGF-B– Transduced Muscles
Quantification of α-SMA–positive cells from histological samples revealed that AdPDGF-B induced a small but significant proliferation of α-SMA–positive cells in normoxic muscles compared to the AdLacZ control 6 days after GT (Figure 5a). In contrast, AdVEGF-A, AdVEGF-A+AdLacZ and AdVEGF-A+AdPDGF-B showed highly increased numbers of α-SMA–positive cells in the muscle. No significant difference was observed between AdVEGF-A, AdVEGF-A+AdLacZ and AdVEGF-A+AdPDGF-B (Figure 5a). However, confocal images of the transduced muscles with rhodamine lectin (endothelium in red) infusion and α-SMA immunostaining (pericytes in green) demonstrated that the recruitment of pericytes to angiogenic vessels differed between the 2 groups. In AdVEGF-A–transduced muscles, α-SMA–positive pericytes were closely associated with enlarged capillaries (Figure 5b; see also supplemental Video 2). After AdVEGF-A+AdPDGF-B GT, large numbers of α-SMA–positive cells were not associated with vessels but were scattered in the muscle interstitium (Figure 5c, asterisks; see also Figure 1e). These cells also possessed long extensions projecting away from the vessels (Figure 5c, arrowheads; see also supplemental Video 3).
Adenoviral delivery of VEGFs is a potentially useful way to increase perfusion in ischemic muscles. The main problems of AdVEGF-A GT include increased plasma protein extravasation, leading to tissue edema and the instability of the newly formed vessels.1,2,4 PDGFs induce pericyte recruitment and migration and are thus proposed to stabilize vessels and decrease edema.7–9 In this study, we tested the effect of intramuscular AdPDGF-B in combination with AdVEGF-A on vessel stability and edema formation in intact and ischemic rabbit skeletal muscles.
Rather than inducing angiogenesis, AdPDGF-B GT induced the proliferation of α-SMA–positive pericytes and fibroblasts and the accumulation of interstitial cells, including inflammatory cells such as monocytes, in both normoxic and ischemic muscles, 6 days after GT. Occasional enlargement of capillaries was detected near the interstitial cells in AdPDGF-B–transduced muscles, possibly indicating a role for the interstitial cells in the angiogenic process as previously described.10 Capillary enlargement after AdPDGF-B GT was more visible in ischemic animals. However, because capillary size was also increased in the ischemic AdLacZ controls, the increase was most likely mediated by hypoxia regulated endogenous growth factors. In contrast, AdVEGF-A induced efficient angiogenesis and recruitment of pericytes around growing vessels at 6 days. When quantified, AdVEGF-A alone was even found to increase the amount of α-SMA–positive cells significantly more than AdPDGF-B alone. However, as expected, extensive tissue edema accompanied rapid changes in vascular growth. When the 2 growth factors were combined, both angiogenesis and massive proliferation of interstitial cells were observed. The mean capillary area and perfusion were smaller in AdVEGF-A+AdPDGF-B–transduced muscles compared to AdVEGF-A alone or AdVEGF-A+AdLacZ GT in both normoxic and ischemic conditions. However, edema was rather increased than decreased in the AdVEGF-A+AdPDGF-B combination group, especially in ischemic muscles. Thus, at 6 days, the combination GT had no significant improvement of angiogenesis and failed to reduce acute edema.
Combination GT was expected to decrease angiogenesis associated edema via stabilization of angiogenic vessels. However, further analysis of the vascular structures in AdVEGF-A and AdVEGF-A+AdPDGF-B–transduced muscles showed that the recruitment of pericytes to vascular structures was impaired in the combination group compared to AdVEGF-A alone. Although the number of α-SMA–positive cells did not differ between the groups, pericytes in the AdVEGF-A+AdPDGF-B group had projections directing away from the vascular structures. An explanation of this might be the site of transgene expression in the target tissue. In tumor studies, it has been reported that PDGF secreted by tumor cells leads to abnormal attachment of pericytes to vessels.11 Also, endothelial PDGF-B retention has been shown to be crucial for proper pericyte investment on the vessels during vascular growth.12 We showed that following intramuscular GT, transgene production took place in several cell types, including monocytes, fibroblasts, and vascular cells. Our histological analysis also showed deposition of PDGF-B protein in the extracellular matrix. It is possible that overexpression of the transgene outside the vascular wall results in the lack of PDGF gradients from endothelial cells and leads to improper pericyte guidance. AdVEGF-A expression alone can induce relatively efficient pericyte recruitment, even if the transgene is not expressed in the vessel wall as a result of blood flow– and shear stress–mediated mechanisms.2,4,13 In fact, the addition of exogenous PDGF-B appears to cause pericyte detachment from the vessels, as shown in the confocal images of AdVEGF-A+AdPDGF-B–transduced muscles. Thus, the intramuscular combination GT seems to activate pericyte migration away from the vessel wall rather than toward it. These results address the importance of proper PDGF-B gradients in target tissues to induce efficient pericyte recruitment on vessels.
Fourteen days after GT, the effects of AdVEGF-A returned to baseline levels as the expression of transgene attenuated. However, in AdVEGF-A+AdPDGF-B–transduced muscles, perfusion and interstitial cell density were still increased. Also, tissue edema was still observed at 14 days after AdVEGF-A+AdPDGF-B GT. Thus, although the combination GT was unable to decrease edema, it was able to induce longer lasting increases in perfusion, compared to AdVEGF-A GT alone, in both normoxic and ischemic muscles. Interestingly, AdPDGF-B was found to induce significant recruitment of inflammatory cells and CD31-positive nonendothelial cells in the transduced muscles 6 and 14 days after GT. Additionally, the angiogenic changes at both time points were often found near sites of cell accumulation. Importantly, many of the interstitial cells in AdPDGF-B–transduced muscle, such as monocytes and macrophages, expressed endogenous VEGF. Additionally, CD31-positive nonendothelial cells have been previously described to have angiogenic potential.14 Thus, strengthening of angiogenesis via paracrine secretion of growth factors seems a plausible mechanism for the improved net effect of the combination GT and AdPDGF-B. The role of inflammatory cells in arteriogenesis10,15 and bone marrow–derived cells mediating PDGF-CC–induced revascularization16 have been suggested previously and is in line with our findings. However, the risk of fibrosis caused by the accumulation of inflammatory cells17 and the immediate effect of AdPDGF-B on fibroblasts18 needs to be considered against the therapeutic potential.
In summary, this study proposes that AdPDGF-B in combination with AdVEGF-A prolongs angiogenic effects via paracrine growth factors secreted from recruited cells. Additionally, this study displays the importance of proper transgene expression in target tissues to induce proper pericyte investment on vessels.
Technicians in the group of Molecular Medicine and at the National Experimental Animal Center of Kuopio University are acknowledged for their expertise and technical help. We thank Dr Roseanne Girnary, PhD, for linguistic revision of this manuscript and Dr Henna Parviainen for help in data reanalysis.
Sources of Funding
This study was supported by grants from the Finnish Academy, European Union (Lymphangiogenomics LSHG-CT-2004-503573), Sigrid Juselius Foundation, the Finnish Cultural Foundation, Aarne and Aili Turunen Foundation, Paavo Nurmi Foundation, and Emil Aaltonen Foundation. This research was also supported in part by NIH grants HL24136 and HL59157 from the National Heart, Lung, and Blood Institute and CA82923 from the National Cancer Institute (to D.M.M.).
Original received April 28, 2008; resubmission received June 27, 2008; revised resubmission received August 29, 2008; accepted September 22, 2008.
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