Published online before print July 31, 2008, doi: 10.1161/ATVBAHA.108.170571
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© 2008 American Heart Association, Inc.
Integrative Physiology/Experimental Medicine |
Adenovirus-Mediated VEGF Gene Therapy Enhances Venous Thrombus Recanalization and Resolution
From King’s College London, Academic Dept. of Surgery (B.M., J.H., J.A.G., M.W., K.G.B., A.W., A.S.) and Dept. of Cardiology (G.S.K.) Cardiovascular Division, st. Thomas’ Hospital, London, UK; and the Kennedy Institute of Rheumatology (A.A., E.P.), Imperial College London, UK.
Correspondence to Dr Alberto Smith, Academic Department of Surgery, St Thomas’ Hospital, 1st Floor, North Wing, London SE1 7EH, United Kingdom. E-mail alberto.smith{at}kcl.ac.uk
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Abstract |
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Objective— Rapid thrombus recanalization reduces the incidence of post–thrombotic complications. This study aimed to discover whether adenovirus-mediated transfection of the vascular endothelial growth factor gene (ad.VEGF) enhanced thrombus recanalization and resolution.
Methods and Results— In rats, thrombi were directly injected with either ad.VEGF (n=40) or ad.GFP (n=37). Thrombi in SCID mice (n=12) were injected with human macrophages transfected with ad.VEGF or ad.GFP. Thrombi were analyzed at 1 to 14 days. GFP was found mainly in the vein wall and adventitia by 3 days, but was predominantly found in cells within the body of thrombus by day 7. VEGF levels peaked at 4 days (376±299 pg/mg protein). Ad.VEGF treatment reduced thrombus size by >50% (47.7±5.1 mm2 to 22.0±4.0 mm2, P=0.0003) and increased recanalization by >3-fold (3.9±0.69% to 13.6±4.1%, P=0.024) compared with controls. Ad.VEGF treatment increased macrophage recruitment into the thrombus by more than 50% (P=0.002). Ad.VEGF-transfected macrophages reduced thrombus size by 30% compared with controls (12.3±0.89 mm2 to 8.7±1.4 mm2, P=0.04) and enhanced vein lumen recanalization (3.39±0.34% to 5.07±0.57%, P=0.02).
Conclusion— Treatment with ad.VEGF enhanced thrombus recanalization and resolution, probably as a consequence of an increase in macrophage recruitment.
Key Words: thrombosis • angiogenesis • gene therapy
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Introduction |
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Deep vein thrombosis (DVT), which develops in the deep veins of the calf muscles, is associated with outflow restriction, valvular damage, valvular reflux, and ambulatory venous hypertension. This can lead to the postthrombotic syndrome, which occurs in approximately a third of affected patients and is characterized by leg swelling, pain, and chronic ulceration.1,2
The majority of patients who develop a DVT are treated by anticoagulation with low-molecular-weight heparin or unfractionated heparin, and warfarin.3 Such treatment prevents thrombus extension, death by pulmonary embolism, and recurrence of venous thromboembolism, but does not accelerate thrombus resolution, and is associated with a risk of bleeding.4 Rapid restoration of vein patency using thrombolytic agents may reduce the incidence of postthrombotic syndrome5 but has a greater risk of bleeding, and patients must be treated within a few days of thrombus development. Rapid natural recanalization is associated with improved valve preservation and a lower incidence of reflux, while patients with shorter recanalization times are reported to have a better clinical outcome.6,7 Valve damage and subsequent incompetence is therefore not an inevitable consequence of thrombosis, and it is reasonable to expect that enhancing natural thrombus resolution might reduce the incidence of postthrombotic complications.
Thrombus resolution occurs by a process of organization that is similar to that found in wound healing.8,9 Thrombus organization begins immediately and is accompanied by the formation of vascular channels,8,10,11 which appear within and around the thrombus as resolution proceeds. The development of the recanalizing channels may be regulated by the recruitment of a variety of cells, including endothelial cells, macrophages, and bone marrow–derived progenitors.8,12,13 The invading proangiogenic cells express growth and chemotactic factors such as vascular endothelial growth factor (VEGF), which may promote the formation of the vascular channels within the thrombus.8,14,15
VEGF is a potent angiogenic cytokine and has been delivered as recombinant protein, using plasmids or viral gene constructs to encourage blood vessel formation in ischemic tissues.16,17 We have previously shown that VEGF is expressed in a temporal pattern by cells recruited into the thrombus during natural resolution,15 and that treating thrombi with recombinant VEGF protein or plasmid containing the VEGF gene accelerates thrombus recanalization and resolution.18,19
This study aimed to determine whether more efficient transfer of the VEGF gene using an adenoviral vector would have a greater effect on thrombus recanalization and resolution. The mechanisms by which VEGF enhanced thrombus resolution were also investigated.
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Materials and Methods |
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Adenoviral Vectors
Delivery of VEGF was achieved using an adenovirus vector containing cDNA for the 165 splice variant of human VEGF (ad.VEGF).20 Virus containing the gene for green fluorescent protein (ad.GFP) was used as a control. (See supplemental material for details of construct growth and purification, available online at http://atvb.ahajournals.org).
Adenovirus-Mediated VEGF Gene Transfer In Vitro
Production of VEGF by ad.VEGF-transfected HEK293 cells was assessed by ELISA (R&D Systems). Ad.GFP-infected cells were used as a control.
The biological activity of the VEGF produced by ad.VEGF-transfected HEK293 cells was assessed by measuring human umbilical vein endothelial cell proliferation21 (see online supplemental material).
Human monocytes were isolated from whole blood using Optiprep (Accurate Chemical, USA).22 Recovered monocytes were transfected with ad.VEGF or ad.GFP at 500 MOI (multiplicity of infection) after stimulation with MCSF (see online supplemental materials). VEGF was measured in cell-conditioned-medium by ELISA.
Rat and Mouse Models of Thrombosis
Laminated thrombus was produced in the inferior vena cava (IVC) of rats and mice using a flow model of thrombosis previously described (See online supplemental material).12,18
Adenovirus-Mediated Gene Transfer Into Thrombus: Tracking Studies
Thrombus, formed 24 hours earlier in adult male Wistar rats (n=9), was injected with 108 PFU ad.GFP (10 µL). Three animals were euthanized at 3, 7, and 14-days after gene transfer to assess viral transfection and GFP protein expression. These were the only group of animals in which the thrombus was perfuse-fixed with 4%-paraformaldehyde. Sections (10 µm), taken at intervals through each thrombus, were mounted with DAPI (Vectashield, Vector Laboratories). Fluorescent images were captured and processed as shown in online supplemental material.
VEGF Production in Thrombus Transfected With ad.VEGF
Thrombi were induced in 28 adult male Wistar rats. Thrombi were injected with 10 µL sterile PBS containing108 pfu ad.VEGF (n=20, 5 per group) or 108 pfu ad.GFP (n=8, 2 per group) 24 hours after induction. Animals were killed 1, 2, 4, and 7 days later. Blood was collected and the IVC harvested from the ligature to the confluence of the iliac veins and snap frozen in liquid nitrogen (LN2). Human VEGF protein concentration was measured in tissue homogenates and plasmas by ELISA (Quantikine, R&D Systems).
Thrombus Resolution After VEGF Gene Therapy
Treatment With Naked ad.VEGF
Thrombi were induced in 48 adult male Wistar rats (supplemental Figure I). Thrombi were injected with 10 µL of sterile PBS containing 108 pfu (n=20) or 109 pfu (n=8) ad.VEGF or 109 pfu ad.GFP (n=20) 24 hours after induction.
Treatment With ad.VEGF Transfected Macrophages
Human macrophages (106/mL) were transfected with ad.VEGF or ad.GFP (500-MOI) after MCSF stimulation. Thrombi were formed in 22 mice with severe combined immunodeficiency (SCID) and injected 24 hours later with 105 (10 µL) macrophages transfected in vitro with either ad.VEGF (n=11) or ad.GFP (n=11, supplemental Figure II). Animals were killed 7 days after gene transfer, and IVC, from the ligature to the confluence of the iliac veins, harvested and processed for paraffin sectioning. Sections were stained with Martius Scarlet Blue (MSB). Confirmation of endothelial cell phenotype of cells lining channels was carried out by immunohistochemistry (IHC) with VEGF (A-20, Santa Cruz) and VEGF receptor-2 (VEGFR2, A3, Santa Cruz).
Six thrombi were randomly chosen from each of the 108 pfu ad.VEGF and ad.GFP-treated animals and analyzed for macrophage and endothelial cell content. Cells were located in the thrombus using IHC with a monoclonal mouse antirat macrophage antibody (ED1, Serotec) and polyclonal rabbit anti VEGFR2 antibody (A3), respectively.
Image Analysis
Thrombus area was expressed as a percentage of the total vein lumen area, while the areas of recanalization and macrophage and endothelial cell staining were calculated as a percentage of the area of the vein lumen and thrombus, respectively.12,18 Full details of this analysis are given in the online supplemental material.
The Effect of ad.VEGF Treatment on Circulating Cell Numbers
Thrombus was formed in a cohort of 20 8-week-old FVB/N mice (supplemental Figure III). A sham-operated group (n=10) and untreated animals (n=10) were used as controls. The thrombi were injected after 24 hours with either 108 pfu ad.VEGF (n=10) or with 108 pfu ad.GFP (n=10). The sham operated group underwent relaparotomy and closure, without an injection. Blood was analyzed for expression of haemtopoietic (CD4523), progenitor (ScaI24), and endothelial (VEGFR225) markers at 3 days (see online supplemental material). Expression of VEGFR2 distinguished endothelial progenitors from the ScaI+/CD45+ circulating stem cell population.26
Statistics
Results are expressed as mean±SE, or median and range for small sample sizes. Statistical significance was determined by using parametric or nonparametric tests as appropriate.
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Results |
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Adenovirus-Mediated VEGF Gene Transfer In Vitro
Conditioned-medium from ad.VEGF-infected HEK293 cells contained more than 5-fold more VEGF protein than cells infected with ad.GFP (500pg/mL [450 to 600] versus 96 pg/mL [60 to 105]). Macrophages transfected with ad.VEGF produced 1445 pg/mL (1350 to 1602) of VEGF protein compared with no detectable VEGF in the conditioned-media from ad.GFP transfected cells. Incubation with neat and a 1/10 dilution of conditioned medium from ad.VEGF-transfected HEK293 cells increased HUVEC proliferation by
75% and 15%, respectively, compared with 2% induced by basal-medium (P<0.001 ANOVA) and 30% by VEGF standard (5 µg/mL, P<0.01; supplemental Figure IV).
Adenovirus-Mediated Gene Transfer Into Thrombus
GFP expression was predominantly seen in the innermost aspect of the vein wall at 3 days, with a small number of transfected cells present both inside the thrombus and in the periphery of the wall (Figure 1a). More GFP-expressing cells were seen within the thrombus at day 7 (Figure 1b and 1c). The pattern of transfection at 2 weeks was similar to that seen at 1 week, albeit with fewer GFP-expressing cells in the thrombus.
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Human VEGF was detected in ad.VEGF-treated thrombi at all time points with the highest amounts after 2 days (300±125 pg/mg protein) and 4 days (376±299 pg/mg protein). VEGF was not detectable in ad.GFP transfected thrombi (Figure 2). Human VEGF was not detected at any time point in the plasma from animals treated with either ad.VEGF or ad.GFP.
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Thrombus Resolution After Treatment With Naked ad.VEGF
All animals developed thrombi. Treatment with 108 pfu ad.VEGF resulted in a greater than 50% reduction in thrombus size (47.7±5.1 mm2 versus 22.0±4.0 mm2, P=0.0003; Figure 3a) and more than 3-fold increase in recanalization area (3.9±0.69% versus 13.6±4.1%, P=0.024; Figure 3b) compared with thrombi injected with ad.GFP (high-powered micrographs of recanalizing channels are shown in supplemental Figure V). The amount of thrombus, expressed as a percentage of the vein lumen, was significantly reduced in animals treated with 108 pfu ad.VEGF (96.1±0.69% versus 86.4±4.1%, P=0.024; Figure 3c). Representative sections of thrombi from treated and control animals are shown in Figure 4 (and supplemental Figure VI). Thrombi treated with the 109 pfu ad.VEGF dose were also smaller and better recanalized than those treated with ad.GFP, but the 109 pfu did not confer any advantage compared with 108 pfu ad.VEGF.
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Macrophage and VEGFR2+ Cell Content of ad.VEGF-Treated Thrombi
Thrombi injected with ad.VEGF at a 108 pfu dose had a higher macrophage cell content than thrombi treated with ad.GFP (19.9±1.6% versus 12.8±1.17%, P=0.002; Figure 5a, staining with ED1 is shown in Figure 5c through 5e). There was no difference between the number of VEGFR2+ cells in sections of thrombus from ad.GFP and ad.VEGF treated groups (Figure 5b).
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Thrombi Treated With ad.VEGF-Transfected Macrophages
Mouse thrombi injected with macrophages transfected with ad.VEGF were 30% smaller than thrombi treated with ad.GFP transfected macrophages (12.3±0.89 mm2 to 8.7±1.4 mm2, P=0.04; Figure 6a). Lumen recanalization was also enhanced by more than 30% after treatment with ad.VEGF-transfected macrophages (3.39±0.34% to 5.07±0.57%, P=0.02; Figure 6b). There was a small but significant reduction in thrombus size expressed as a percentage of vein lumen in ad.VEGF-transfected macrophage-treated animals (96.6±0.34% versus 94.9±0.57%, P=0.02; Figure 6c).
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The Effect of VEGF Gene Transfer on Circulating Cells
There were no significant differences in the number of circulating CD45+/ScaI+/VEGFR2+ cells between any of the groups of animals studied (supplemental Figure VIIa). There were, however, twice as many ScaI+ cells after ad.VEGF-treatment compared with no intervention (75230±11696 versus 34888±7235, P=0.014), or the sham-operated group (75230±11696 versus 36795±6266, P=0.0096; supplemental Figure VIIb). The mean number of circulating ScaI+ cells in the ad.VEGF-treated group was 33% higher than that of the ad.GFP-treated mice (75230±11696 versus 56617±8067), but this difference did not reach statistical significance (P>0.05). There was 41% more ScaI+ cells in Ad.GFP-treated animals compared with sham controls, but this also did not reach statistical significance (P>0.05). No difference was found in circulating ScaI+ cell numbers between sham controls and animals having no intervention (P>0.1).
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Discussion |
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The resolution of experimental thrombi was markedly enhanced within 7 days of treatment with ad.VEGF. Thrombus size was halved, and vein lumen recanalization was increased by more than 3-fold. This was a more potent effect than the differences observed 14 days after delivery of either recombinant human VEGF protein or a plasmid expressing human VEGF in our previous studies.18,19 This was not unexpected as adenoviral vectors are more efficient transfectants of both dividing and nondividing cells than expression plasmids.27 The VEGF concentrations in the thrombus were more than 20-fold higher than that obtained after VEGF-plasmid transfection.19 This transient elevation of VEGF expression is in keeping with other studies that have used adenovirus-mediated VEGF gene delivery and reported increased revascularization.28,29 Our study supports these findings, showing that an increase in VEGF in the first 4 days after transfection was sufficient to enhance resolution by day 7. It appears that early transient VEGF expression is sufficient to drive the remodeling processes needed for resolution and that these processes continue after the VEGF levels have decreased. The mechanism for the effect of VEGF on thrombus resolution requires elucidation.
Angiogenic compounds have previously been used to enhance resolution in experimental thrombi, with modest results. Administration of basic fibroblast growth factor (bFGF) protein increased recanalization of thrombi but had no effect on thrombus volume or organization.30 Injection of interleukin (IL)-8 and monocyte chemotactic protein 1(MCP1), proteins with known angiogenic properties, has been shown to improve blood flow through resolving experimental thrombi and accelerated their dissolution.14,31 VEGF has been used extensively to promote neovascularization in ischemic tissues. VEGF has been delivered either as recombinant protein, or using plasmid and viral gene constructs, to human subjects in an attempt to improve lower limb perfusion.16,32 So called "therapeutic angiogenesis" has also been used to treat ischemic heart disease.33,34
It is difficult to determine whether the channels that developed around the thrombus were purely associated with angiogenesis or whether there is a contribution from thrombus retraction. Our previous studies suggest that the small vascular channels that develop between the thrombus and the vessel wall may influence the formation of large vascular spaces that are important for the patency of the vein lumen.11 Thrombus appears to resolve through mechanisms similar to those found during wound healing, and it is likely that a combination of neovascularization and thrombus retraction is important in this process.
A variety of cells including monocytes, endothelial cells, and bone marrow–derived progenitor cells infiltrate the thrombus during its resolution.8,12,13 Monocytes are naturally recruited into resolving thrombi over time,9 and thrombus resolution is inhibited when their recruitment is restricted.12 VEGF has a number of properties that affect the recruitment, migration, and proliferation of many cells, including the monocytes.35,36 VEGF also activates macrophages to produce a number of proteases (including plasminogen activators and matrix metalloproteinases), growth factors, and chemokines that regulate recruitment of cells and turnover of extracellular matrix.37 In the present study, treatment with ad.VEGF increased the numbers of thrombus macrophages (ED1+ cells). The ad.VEGF could, therefore, have enhanced thrombus recanalization and resolution by stimulating the recruitment and activation of macrophages.38
VEGF mobilizes and recruits EPCs from the bone marrow and may, therefore, have contributed to de novo formation of vascular spaces within and around the thrombus (vasculogenesis).39 The number of circulating EPCs (VEGFR2+/ScaI+/CD45+) was, however, similar in all groups of animals, suggesting that EPC mobilization was not the mechanism by which ad.VEGF affected thrombus recanalization. The total number of progenitors (ScaI+ cells) was elevated in the ad.VEGF-treated animals. This may have been a consequence of the inflammatory response induced in the host by the adenovirus rather a direct effect of VEGF.
It was hoped that treating the thrombus with ad.VEGF-transfected macrophages would be as effective as injecting the naked construct, as these cells are normally recruited into the thrombus in large numbers. This would have given the impetus for the development of a less invasive treatment, whereby ad.VEGF-transfected macrophages are injected peripherally; or alternatively the ad.VEGF construct could be targeted to circulating monocytes.40 The increase in thrombus organization and recanalization that occurred after direct injection of transfected monocytes was, however, more modest than that observed with injection of naked construct. This may have been for a number of reasons. The direct injection of naked ad.VEGF into thrombus probably resulted in the transfection of a variety of cells (endothelial, macrophage, smooth muscle cells, and neutrophils) found within the resolving thrombus. In contrast, ad.VEGF "packaged" in a relatively small number (105) of injected cells, all of which may not have been retained in the thrombus and may have limited the total amount of VEGF protein expressed. Another plausible explanation is that differences between the speed of natural resolution/recanalization in the rat and mouse models may have accounted for this discrepancy. The use of human macrophages may have been another confounding factor, as their migration and activity in the thrombus may not have had the same impact on resolution as native macrophages. Targeting rat or mouse monocytes with adenovirus-gene construct in vivo40 could address these problems.
The acceleration in experimental thrombus resolution obtained in the present study was substantial. Similar benefits have, however, been reported in experimental models of tissue ischemia, which were not borne out in the double blind, randomized, controlled clinical trials.33,41 The lack of therapeutic benefit in man is thought to be because the blood vessels that grow after short-term treatment with proangiogenic agents are unstable and of small caliber. The processes that regulate resolution in young healthy animals are also radically different from those of older human populations. Thrombus resolution may, however, only require a short-term stimulus for the rapid formation of cell-lined vascular spaces within and around the fresh thrombus to establish a patent lumen. Although thrombus resolution in the rodent vena cava is a more rapid and almost complete process compared with human thrombi,42 the recanalization process and cellular changes that occur are remarkably similar.
The long-term aim of our work is to develop treatments that will enhance natural resolution, and in the context of the human literature this should reduce postthrombotic complications. Our models of thrombus resolution clearly do not develop postthrombotic syndrome (PTS), and our outcome measures are therefore restricted to recanalization and organization. It will have to wait for clinical studies before outcome measures regarding PTS can be assessed.
Adenoviral delivery of VEGF was chosen for this proof of concept study as it provided a greater sustained production of VEGF than the expression plasmid or VEGF protein used in our previous studies.18,19 Administration of adenovirus gene constructs given locally is reported to be relatively safe.43 There are, however, problems, such as activation of the immune system, which limit the use of adenoviruses as gene delivery vehicles. These may be overcome through advances in gene therapy, whereas the development of cell specific or tissue targeting techniques could also provide a means of directing gene transfer and improve the efficacy of VEGF gene delivery.44 There are also methods of sustained or targeted VEGF protein and naked gene delivery that could potentially be used to circumvent many of the real and perceived problems associated with the use of viral constructs and are worthy of further investigation. These include encapsulation of VEGF protein in biodegradable microspheres,45 which could be administered into the thrombus by image-guided (eg, ultrasound) injection; or encapsulation in microbubbles, delivered systemically and targeted to thrombus by ultrasound-induced release.46,47
Although thrombolysis is a therapeutic option for the rapid removal of a venous thrombus when it is relatively fresh, it does not remove organizing thrombus and there is also a significant risk (up to 16%) of pathological bleeding. It is clear, therefore, that the development of alternative strategies that enhance the natural process of resolution are worth pursuing. Increasing VEGF levels in the thrombus might provide a useful adjunct to current conventional anticoagulation.
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Acknowledgments |
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Disclosures
This work was funded by a Clinical PhD studentship grant to B.M.
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Footnotes |
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Adenovirus-Mediated VEGF Gene Therapy Enhances Venous Thrombus Recanalization and Resolution: Correction
In the article that appeared on page 1753 of the 28.10 issue, author A. Wadoodi’s name should be moved prior to G.S. Kanaganayagam in the author byline. Additionally, K.G. Burnand should move to the third place in the byline, prior to J.A. Gossage. A correction to the article has been published in the 29.12 issue of the journal.
Reference
Modarai B, Humphries J, Gossage JA, Waltham M, Burnand KG, Kanaganayagam GS, Afuwape A, Paleolog E, Smith A, and Wadoodi A. Adenovirus-mediated VEGF gene therapy enhances venous thrombus recanalization and resolution. Arterioscler Thromb Vasc Biol 2008;28:1753–1759.
Original received March 7, 2006; final version accepted June 27, 2008.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Strandness DE Jr, Langlois Y, Cramer M, Randlett A, Thiele BL. Long-term sequelae of acute venous thrombosis. JAMA. 1983; 250: 1289–1292.
2. Kearon C. Natural history of venous thromboembolism. Circulation. 2003; 107: I22–I30.[Medline] [Order article via Infotrieve]
3. Hirsh J, Lee AY. How we diagnose and treat deep vein thrombosis. Blood. 2002; 99: 3102–3110.
4. Kearon C. Long-term management of patients after venous thromboembolism. Circulation. 2004; 110: I10–I18.[CrossRef][Medline] [Order article via Infotrieve]
5. Watson LI, Armon MP Thrombolysis for acute deep vein thrombosis. Cochrane Database Syst Rev. 2004; CD002783.
6. Meissner MH, Caps MT, Zierler BK, Polissar N, Bergelin RO, Manzo RA, Strandness DE Jr. Determinants of chronic venous disease after acute deep venous thrombosis. J Vasc Surg. 1998; 28: 826–833.[CrossRef][Medline] [Order article via Infotrieve]
7. Wakefield TW. Treatment options for venous thrombosis. J Vasc Surg. 2000; 31: 613–620.[CrossRef][Medline] [Order article via Infotrieve]
8. Wakefield TW, Linn MJ, Henke PK, Kadell AM, Wilke CA, Wrobleski SK, Sarkar M, Burdick MD, Myers DD, Strieter RM. Neovascularization during venous thrombosis organization: a preliminary study. J Vasc Surg. 1999; 30: 885–892.[CrossRef][Medline] [Order article via Infotrieve]
9. McGuinness CL, Humphries J, Waltham M, Burnand KG, Collins M, Smith A. Recruitment of labelled monocytes by experimental venous thrombi. Thromb Haemost. 2001; 85: 1018–1024.[Medline] [Order article via Infotrieve]
10. Sevitt S Organic canalisation and vascularisation of deep vein thrombi studied with dyed-micropaque injected at necropsy. J Pathol. 1970; 100: i.
11. Modarai B, Burnand KG, Humphries J, Waltham M, Smith A. The role of neovascularisation in the resolution of venous thrombus. Thromb Haemost. 2005; 93: 801–809.[Medline] [Order article via Infotrieve]
12. Singh I, Burnand KG, Collins M, Luttun A, Collen D, Boelhouwer B, Smith A. Failure of thrombus to resolve in urokinase-type plasminogen activator gene-knockout mice: rescue by normal bone marrow–derived cells. Circulation. 2003; 107: 869–875.
13. Modarai B, Burnand KG, Sawyer B, Smith A. Endothelial progenitor cells are recruited into resolving venous thrombi. Circulation. 2005; 111: 2645–2653.
14. Humphries J, McGuinness CL, Smith A, Waltham M, Poston R, Burnand KG. Monocyte chemotactic protein-1 (MCP-1) accelerates the organization and resolution of venous thrombi. J Vasc Surg. 1999; 30: 894–899.[CrossRef][Medline] [Order article via Infotrieve]
15. Waltham M, Burnand KG, Collins M, Smith A. Vascular endothelial growth factor and basic fibroblast growth factor are found in resolving venous thrombi. J Vasc Surg. 2000; 32: 988–996.[CrossRef][Medline] [Order article via Infotrieve]
16. Isner JM, Baumgartner I, Rauh G, Schainfeld R, Blair R, Manor O, Razvi S, Symes JF. Treatment of thromboangiitis obliterans (Buerger’s disease) by intramuscular gene transfer of vascular endothelial growth factor: preliminary clinical results. J Vasc Surg. 1998; 28: 964–973.[CrossRef][Medline] [Order article via Infotrieve]
17. Whitlock PR, Hackett NR, Leopold PL, Rosengart TK, Crystal RG. Adenovirus-mediated transfer of a minigene expressing multiple isoforms of VEGF is more effective at inducing angiogenesis than comparable vectors expressing individual VEGF cDNAs. Mol Ther. 2004; 9: 67–75.[Medline] [Order article via Infotrieve]
18. Waltham M, Burnand KG, Collins M, McGuinness CL, Singh I, Smith A. Vascular endothelial growth factor enhances venous thrombus recanalisation and organisation. Thromb Haemost. 2003; 89: 169–176.[Medline] [Order article via Infotrieve]
19. Waltham M, Burnand K, Fenske C, Modarai B, Humphries J, Smith A. Vascular endothelial growth factor naked DNA gene transfer enhances thrombus recanalization and resolution. J Vasc Surg. 2005; 42: 1183–1189.[CrossRef][Medline] [Order article via Infotrieve]
20. Mack CA, Magovern CJ, Budenbender KT, Patel SR, Schwarz EA, Zanzonico P, Ferris B, Sanborn T, Isom P, Ferris B, Sanborn T, Isom OW, Crystal RG, Rosengart TK. Salvage angiogenesis induced by adenovirus-mediated gene transfer of vascular endothelial growth factor protects against ischemic vascular occlusion. J Vasc Surg. 1998; 27: 699–709.[CrossRef][Medline] [Order article via Infotrieve]
21. Roehm NW, Rodgers GH, Hatfield SM, Glasebrook AL. An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. J Immunol Methods. 1991; 142: 257–265.[CrossRef][Medline] [Order article via Infotrieve]
22. Graziani-Bowering GM, Graham JM, Filion LG. A quick, easy and inexpensive method for the isolation of human peripheral blood monocytes. J Immunol Methods. 1997; 207: 157–168.[CrossRef][Medline] [Order article via Infotrieve]
23. Lagasse E, Connors H, Al Dhalimy M, Reitsma M, Dohse M, Osborne L, Wang X, Finegold M, Weissman IL, Grompe M. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med. 2000; 6: 1229–1234.[CrossRef][Medline] [Order article via Infotrieve]
24. Yonemura Y, Ku H, Lyman SD, Ogawa M. In vitro expansion of hematopoietic progenitors and maintenance of stem cells: comparison between FLT3/FLK-2 ligand and KIT ligand. Blood. 1997; 89: 1915–1921.
25. Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med. 2003; 9: 702–712.[CrossRef][Medline] [Order article via Infotrieve]
26. Ishizawa K, Kubo H, Yamada M, Kobayashi S, Suzuki T, Mizuno S, Nakamura T, Sasaki H. Hepatocyte growth factor induces angiogenesis in injured lungs through mobilizing endothelial progenitor cells. Biochem Biophys Res Commun. 2004; 324: 276–280.[CrossRef][Medline] [Order article via Infotrieve]
27. Baker AH. Designing gene delivery vectors for cardiovascular gene therapy. Prog Biophys Mol Biol. 2004; 84: 279–299.[CrossRef][Medline] [Order article via Infotrieve]
28. Perrin LA, June JE, Rosebury W, Robertson A, Kovesdi I, Bruder JT, Kessler PD, Keiser JA, Gordon D. Increased revascularization efficacy after administration of an adenovirus encoding VEGF(121). Gene Ther. 2004; 11: 512–521.[CrossRef][Medline] [Order article via Infotrieve]
29. Gounis MJ, Spiga MG, Graham RM, Wilson A, Haliko S, Lieber BB, Wakhloo AK, Webster KA. Angiogenesis is confined to the transient period of VEGF expression that follows adenoviral gene delivery to ischemic muscle. Gene Ther. 2005; 12: 762–771.[CrossRef][Medline] [Order article via Infotrieve]
30. Varma MR, Moaveni DM, Dewyer NA, Varga AJ, Deatrick KB, Kunkel SL, Upchurch GR Jr, Wakefield TW, Henke PK. Deep vein thrombosis resolution is not accelerated with increased neovascularization. J Vasc Surg. 2004; 40: 536–542.[CrossRef][Medline] [Order article via Infotrieve]
31. Henke PK, Wakefield TW, Kadell AM, Linn MJ, Varma MR, Sarkar M, Hawley A, Fowlkes JB, Strieter RM. Interleukin-8 administration enhances venous thrombosis resolution in a rat model. J Surg Res. 2001; 99: 84–91.[CrossRef][Medline] [Order article via Infotrieve]
32. Makinen K, Manninen H, Hedman M, Matsi P, Mussalo H, Alhava E, Yla-Herttuala S. Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study. Mol Ther. 2002; 6: 127–133.[CrossRef][Medline] [Order article via Infotrieve]
33. Henry TD, Annex BH, McKendall GR, Azrin MA, Lopez JJ, Giordano FJ, Shah PK, Willerson JT, Benza RL, Berman DS, Gibson CM, Bajamonde A, Rundle AC, Fine J, McCluskey ER. The VIVA trial: Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis. Circulation. 2003; 107: 1359–1365.
34. Losordo DW, Dimmeler S. Therapeutic angiogenesis and vasculogenesis for ischemic disease. Part I: angiogenic cytokines. Circulation. 2004; 109: 2487–2491.
35. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science. 1989; 246: 1306–1309.
36. Helmlinger G, Endo M, Ferrara N, Hlatky L, Jain RK. Formation of endothelial cell networks. Nature. 2000; 405: 139–141.
37. Polverini PJ, DiPietro LA. Role of the macrophage in the regulation of physiological and pathological angiogenesis. In: Maragoudakis ME, Gullino P, Lelkes PI, eds. Angiogenesis in health and disease. New York: Plenum Press; 1992: 43–53.
38. Ohtani K, Egashira K, Hiasa K, Zhao Q, Kitamoto S, Ishibashi M, Usui M, Inoue S, Yonemitsu Y, Sueishi K, Sata M, Shibuya M, Sunagawa K. Blockade of vascular endothelial growth factor suppresses experimental restenosis after intraluminal injury by inhibiting recruitment of monocyte lineage cells. Circulation. 2004; 110: 2444–2452.
39. Asahara T, Takahashi T, Masuda H, Kalka C, Chen D, Iwaguro H, Inai Y, Silver M, Isner JM. VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells. EMBO J. 1999; 18: 3964–3972.[CrossRef][Medline] [Order article via Infotrieve]
40. Pearce OM, Fisher KD, Humphries J, Seymour LW, Smith A, Davis BG. Glycoviruses: chemical glycosylation retargets adenoviral gene transfer. Angew Chem Int Ed Engl. 2005; 44: 1057–1061.[CrossRef]
41. Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, Dauerman H, Udelson JE, Gervino EV, Pike M, Whitehouse MJ, Moon T, Chronos NA. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial. Circulation. 2002; 105: 788–793.
42. Holmstrom M, Lindmarker P, Granqvist S, Johnsson H, Lockner D. A 6-month venographic follow-up in 164 patients with acute deep vein thrombosis. Thromb Haemost. 1997; 78: 803–807.[Medline] [Order article via Infotrieve]
43. Crystal RG, Harvey BG, Wisnivesky JP, O'Donoghue KA, Chu KW, Maroni J, Muscat JC, Pippo AL, Wright CE, Kaner RJ, Leopold PL, Kessler PD, Rasmussen HS, Rosengart TK, Hollmann C. Analysis of risk factors for local delivery of low- and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of comorbid conditions. Hum Gene Ther. 2002; 13: 65–100.[CrossRef][Medline] [Order article via Infotrieve]
44. White K, Nicklin SA, Baker AH. Novel vectors for in vivo gene delivery to vascular tissue. Expert Opin Biol Ther. 2007; 7: 809–821.[CrossRef][Medline] [Order article via Infotrieve]
45. Faranesh AZ, Nastley MT, Perez dlC, Haller MF, Laquerriere P, Leong KW, McVeigh ER. In vitro release of vascular endothelial growth factor from gadolinium-doped biodegradable microspheres. Magn Reson Med. 2004; 51: 1265–1271.[CrossRef][Medline] [Order article via Infotrieve]
46. Hernot S, Klibanov AL. Microbubbles in ultrasound-triggered drug and gene delivery. Adv Drug Deliv Rev. 2008; 60: 1153–1166.[CrossRef][Medline] [Order article via Infotrieve]
47. Leong-Poi H, Kuliszewski MA, Lekas M, Sibbald M, Teichert-Kuliszewska K, Klibanov AL, Stewart DJ, Lindner JR. Therapeutic arteriogenesis by ultrasound-mediated VEGF165 plasmid gene delivery to chronically ischemic skeletal muscle. Circ Res. 2007; 101: 295–303.
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