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Volume: 15 Issue: 2 April 2017


Vascular Endothelial Growth Factor-B Overexpressing Hearts Are Not Protected From Transplant-Associated Ischemia-Reperfusion Injury

Objectives: Cardiac vascular endothelial growth factor-B transgene limits myocardial damage in rat infarction models. We investigated whether heart transplant vascular endothelial growth factor-B overexpression protected against ischemia-reperfusion injury.

Materials and Methods: We transplanted hearts hetero­topically from Dark Agouti to Wistar Furth rats. To characterize the role of vascular endothelial growth factor-B in ischemia-reperfusion injury, we transplanted either long-term human vascular endothelial growth factor-B transgene overexpressing hearts from Wistar Furth rats or short-term adeno-associated virus 9-human vascular endothelial growth factor-B-transduced hearts from Dark Agouti rats into Wistar Furth rats. Heart transplants were subjected to 2 hours of cold and 1 hour of warm ex vivo ischemia. Samples were collected 6 hours after reperfusion.

Results: Two hours of cold and 1 hour of warm ischemia increased vascular endothelial growth factor-B mRNA levels 2-fold before transplant and 6 hours after reperfusion. Transgenic vascular endothelial growth factor-B overexpression caused mild cardiac hyper­trophy and elevated cardiac troponin T levels 6 hours after reperfusion. Laser Doppler measurements indicated impaired epicardial tissue perfusion in these transgenic transplants. Recombinant human vascular endothelial growth factor-B increased mRNA levels of cytochrome c oxidase and extracellular ATPase CD39, suggesting active oxidative phosphorylation and high ATP production. Adeno-associated virus 9-mediated vascular endothelial growth factor-B overexpression in transplanted hearts increased intragraft macro­phages 1.5-fold and proinflammatory cytokine interleukin 12 p35 mRNA 1.6-fold, without affecting recipient serum cardiac troponin T concentration.

Conclusions: Vascular endothelial growth factor-B expression in transplanted hearts is linked to ischemia and ischemia-reperfusion injury. Cardiac transgenic vascular endothelial growth factor-B overexpression failed to protect heart transplants from ischemia-reperfusion injury.

Key words : Animal models, VEGF-B, Heart transplant


Ischemia-reperfusion injury (IRI) after a heart transplant involves a sequence of inflammatory processes. The pathogenesis of IRI is characterized by the up-regulation of cytokines and growth factors, as well as microvascular barrier dysfunction and perfusion defects. This cascade of events may lead to enhanced allorecognition and concomitant activation of the adaptive immune system, leading to cardiac fibrosis and cardiac allograft vasculopathy and, ultimately, heart transplant failure. Current phar­macologic therapies target T cell-dependent allo­immune activation in the recipient; however, no specific treatment is available against IRI.1-4

Members of the vascular endothelial growth factor (VEGF) family are known for their angiogenic and lymphangiogenic effects. Vascular endothelial growth factor-B is expressed in most tissues of the body, especially in the heart,5-7 and its main receptors are VEGFR-1 and neuropilin1,8,9 Vascular endothelial growth factor-B mRNA passes through alternative splicing to generate 2 different isoforms, VEGF-B167 and VEGF-B186. Vascular endothelial growth factor-B167 contains a heparin-binding domain that mediates adhesion to heparan sulfate proteoglycans on cell secretion. In contrast, VEGF-B186 does not contain the heparin-binding domain, allowing better distribution within tissues. Although the molecular structure of VEGF-B is closely related to that of VEGF and placental growth factor, it fails to exert similar effects on angiogenesis, lymphangiogenesis, or vascular permeability.10-17

Studies of VEGF-B have resulted in contradictory data.18 The endogenously produced factor appears to be functionally inert under normal conditions. Gain-of-function experiments with transgenic mice, however, have suggested that VEGF-B may regulate the survival of vascular cells and cardiomyocytes through regulation of anti-apoptotic signals and possibly via cellular energy metabolism.19 Vascular endothelial growth factor-B knockout decreases blood vessel survival in an oxygen-induced blood vessel regression model of mouse corneal pockets and is indispensable for endothelial cell, pericyte, and smooth muscle cell survival in vitro.20

This is the first study to investigate the effects of transgenic VEGF-B overexpression in cardiac al­lografts. We measured VEGF-B mRNA in native hearts and rat cardiac allografts during prolonged cold ischemic preservation and IRI of heart transplants. In addition, we used transgenic donor hearts overexpressing VEGF-B under the control of an α-myosin heavy chain promoter (α-MHC) or donor hearts transducted with adeno-associated virus 9 (AAV9)-carrying a VEGF-B transgene and investigated whether overexpression of recombinant human VEGF-B could affect micro­vascular dysfunction and myo­cardial cell death, the hallmarks of cardiac allograft IRI.1-4,21

Materials and Methods

Permission for animal experiments
Permission for animal experiments was obtained from the State Provincial Office of Southern Finland. The animals received care in compliance with the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Academies Press (ISBN 0-309-15400-6, revised 2011).

Characterization of VEGF-B expression in the heart during cold preservation and warm ischemia
Donor hearts from specific pathogen-free male Dark Agouti rats (DA, RT1av1) were perfused in vivo with ice-cold heparinized phosphate-buffered saline (PBS) before retrieval. The hearts were subjected to 2-hour cold preservation in PBS at +4°C (n = 5). Another group of hearts was subjected to additional 1-hour warm ischemia (n = 5). Native DA hearts without cold preservation and warm ischemia served as the control group (n = 5).

Characterization of vascular endothelial growth factor-B expression in cardiac allografts during ischemia-reperfusion injury
Intra-abdominal heterotopic heart transplant procedures were performed from specific pathogen-free fully MHC-mismatched male DAs to male Wistar Furth (WF, RT1u, Scanbur) rats weighing 300 to 350 g. Dark Agouti donor hearts were perfused with heparinized ice-cold PBS, with the vena cava and pulmonary veins ligated with 6-0 silk and the pulmonary artery and aorta cut 2 to 3 mm above their origin in the heart. After removal, allografts were immediately transplanted or preserved in PBS at +4°C for 2 hours (n = 6/group). Cardiac allograft recipients were anesthetized with isoflurane (2%-5%/L O2) and received buprenorphine 0.15 mg/kg subcutaneously (Temgesic 0.3 mg/mL, Schering-Plough, Kenilworth, NJ) for peri- and postoperative analgesia. Allografts were heterotopically anastomosed to recipient abdominal vessels using microsurgical techniques. Warm ischemia was standardized to 1 hour when performing the anastomoses. Allografts were removed for analyses 6 hours after reperfusion.

Heterotopic rat heart transplants for studying the effects of transgenic vascular endothelial growth factor-B overexpression
The generation of α-MHC-human VEGF-B-transgenic pathogen-free male Wistar (HsdBrl Han: WIST, a kind gift from Professor Kari Alitalo) rats has been described previously by Bry and associates.15 Intra-abdominal heterotopic heart transplants were performed from specific pathogen-free male Wistar (HsdBrl Han) to minor MHC-mismatched Wistar Furth (RT1u) rats, as described above. Cold preservation and warm ischemia times were standardized to 2 hours and 1 hour. Serial laser Doppler measurements were performed at 1 minute, 5 minutes, and 6 hours to evaluate tissue perfusion in the myocardium after the heart transplant. The heart transplant and recipient serum were collected at 6 hours (n = 6/group) for ex vivo analyses. At 6 hours after reperfusion, infiltration of inflammatory cells was determined by immunohistochemistry, myo­cardial injury by serum cardiac troponin T, and in situ apoptosis by terminal deoxynucleotidyl trans­ferase dUTP nick-end labeling (TUNEL). Intragraft mRNA expression of anti- and proapoptotic genes was analyzed by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Genes and their respective gene bank numbers are featured in Table 1.

Heterotopic rat heart transplants for studying the effects of adeno-associated virus 9-mediated vascular endothelial growth factor-B overex­pression
Dark Agouti donor rats were injected intravenously with 150 μL each of AAV9-human VEGF-B167 (5.55 x 109 vector genomes [vg]/μL) and AAV9-human VEGF-B186 (9.98 x 109 vg/μL) (n = 6) into the penile vein 5 days before transplant. Control group DA rats were treated with an intravenous injection of AAV9-human serum albumin (1.85 × 109 vg/μL) (n = 6). Intra-abdominal heterotopic heart transplants were performed from those previously treated specific pathogen-free male DA rats to major MHC-mismatched WF rats, as described previously. Cold preservation and warm ischemia times were standardized to 2 hours and 1 hour. The transplant and recipient serum were collected at 6 hours after reperfusion for ex vivo analyses.

Laser Doppler monitoring
Cardiac allograft tissue perfusion was analyzed 1 minute, 5 minutes, and 6 hours after reperfusion by dynamic measurement with a laser Doppler monitor, Transonic BLF21-Series (Transonic Systems Inc., Ithaca, NY). The moving Doppler effect was calculated via an 18-gauge (1.2 mm) probe head receiving fiberoptic light reflected by stationary structures within the tissue as well as by moving particles (red blood cells). A low-intensity beam of monochromatic light was emitted to a 1-mm depth of a portion of the apical myocardium, and the tissue volume under the laser Doppler monitoring was ap­proximately 1 mm3. Flow signal was sampled as 200 s-1 from transmitted 19200 baud (pulses per second) and is presented as tissue perfusion units (TPUs) that are relatively proportional to mL/min/100 g of tissue.22

Measurement of cardiac troponin T
The rat serum levels of cardiac troponin T were analyzed with the fifth-generation troponin T test (Troponin T, STAT, Roche Diagnostics, Basel, Switzerland). Cardiac troponin T was measured with electrochemiluminescence immunoassay (ECLIA) on the Elecsys 2010 immunoassay analyser (Roche Diagnostics).

In situ apoptosis detection
The analysis of cardiomyocyte apoptosis was based on nick-end labeling assay (TUNEL). Parafor­maldehyde-fixed paraffin sections (4-μm thick) were stained with an in situ apoptosis detection kit (TA5353, CardioTACS, R&D Systems, Minneapolis, MN). TUNEL-positive apoptotic cardiomyocyte nuclei were counted from 4 random fields of each quadrant of a cardiac cross section with ×40 mag­nification, and the results are given as the mean number of positive cells per 1 mm2.

Cryostat sections were stained using the peroxidase ABC method (Vectastain Elite ABC Kit, Vector Laboratories, Inc., Burlingame, CA), and the reaction was revealed by 3-amino-9-ethylcarbazole (AEC, Vector Laboratories). Counterstaining was performed using Mayer’s hemalum. Antibodies and dilutions used included CD4 (5 μg/mL, 22021D), CD8 (5 μg/mL, 22071D), OX-62 (10 μg/mL, MCA 1029G), ED-1 (5 μg/mL, 22451D, BD Pharmingen, Franklin Lakes, NJ); myeloperoxidase (20 μg/mL, ab9535, Abcam, Cambridge, United Kingdom); RECA-1 (50 μg/mL, MCA97, AbD Serotec, Dusseldorf, Germany); α-SMA (1:5000, A2547, Sigma-Aldrich, St. Louis, MO); VEGF-B (0.2 μg/mL, AF751, R&D Systems); dystrophin (3.3 μg/ml, NCL-DYS2, Leica Microsystems, Newcastle, UK), NKG2D/CD314 NK-cells (1 μg/mL, Bioss, MA, USA), and Oil-Red-O (5 μg/mL, Sigma Aldrich). Inflammatory cells, RECA1-positive capillaries, and α-SMA arteries were counted from 4 random fields of each quadrant of the cardiac cross section with ×40 magnification, with results presented as mean number of positive cells and vessels per 1 mm2. The cardiomyocyte (CMC) cross-sectional area was analyzed using computer-assisted imaging and presented as an average of CMC sizes from 4 random fields of each quadrant of cardiac cross-sections stained with dystrophin antibody.

Cardiac fibrosis was determined in a blinded review by 2 observers from paraformaldehyde-fixed pa­raffin sections stained with Masson trichrome, and scored semiquantitatively (0 to 3) as follows: 0, no fibrosis; 1, mild fibrosis; 2, moderate fibrosis; and 3, severe myocardial fibrosis. Cardiac intracellular lipid droplets were visualized with Oil-Red-O staining of cardiac paraffin sections and quantitated with computer-assisted imaging (Zeiss Axiovision 4.4, Carl Zeiss International, Oberkochen, Germany). The data were presented as the total Oil-Red-O-positive area in the cardiac cross-section. Myocardial area, septum-, and left- and right-ventricular thickness were determined from allograft midline cross sections using computer-assisted image processing.

RNA isolation and reverse transcription
Total RNA was extracted from myocardial samples using RNeasy Mini Kit (Qiagen, Hilden, Germany). Reverse transcription of mRNA was carried out from 100 ng total RNA using High-RNA-to-cDNA kit (Applied Biosystems Inc., Carlsbad, CA) in a total volume of 20 μL. After reverse trans­cription, 40 μL of PCR-grade water was added to each cDNA sample. Three μL of each sample (corresponding to 5 ng total RNA) were used in each subsequent PCR reaction.

Quantitative real-time PCR
Quantitative real-time PCR reactions were carried out on a RotorGene-6000 (Corbett Research, Hilden, Germany) using 2X DyNAmo Flash SYBR Green Master mix (Finnzymes, Espoo, Finland). Measure­ment of the PCR product was performed at the end of each extension period. Amplification specificity was checked using melting curve analysis. The number of mRNA copies of each gene of interest was calculated from a corresponding standard curve using the RotorGene software (Qiagen). The results are given in relation to 18S rRNA molecule numbers (see Table 1 for GenBank numbers).

Statistical analyses
All data are means ± standard error and were analyzed using SPSS software version 15.0 (SPSS Inc, Chicago, IL). For parametric comparison of the 2 groups, the t test was applied. The Mann-Whitney U test was used for nonparametric measurements. Comparisons between multiple groups were performed using the one-way analysis of variance (ANOVA) with Dunnett post hoc test. For comparison in longitudinal studies, data were analyzed by combined Kruskal-Wallis and Friedman tests (2-way nonparametric ANOVA). P < .05 was considered statistically significant.


Ischemia-reperfusion injury elevates vascular endo­thelial growth factor-B expression in cardiac allografts.

To simulate time frames relevant to clinical heart transplant procedures, we chose 2-hour cold pre­servation and 1-hour warm ischemia time to investigate the effects of cold ischemic preservation, warm ischemia, and IRI on the mRNA expression of VEGF-B in the heart.

Quantitative real-time RT-PCR showed that 2-hour cold- and 1-hour warm ischemia resulted in 2-fold VEGF-B mRNA expression levels (P < .05; Figure 1A) in DA donor hearts compared with native DA hearts. One-hour warm ischemia did not have any additional effects when compared with 2-hour cold ischemia; however, IRI induced a nearly 3-fold increase in the expression levels of VEGF-B mRNA (P < .05; Figure 1B) 6 hours after reperfusion in cardiac allografts subjected to 2-hour cold preservation, compared with cardiac allografts without cold preservation.

Cardiomyocyte-specific human vascular endo­thelial growth factor-B overexpression leads to cardiomyocyte hypertrophy
Real-time PCR revealed that human VEGF-B mRNA was significantly increased in the VEGF-B transgenic hearts (P < .001; Figure 2A). Correspondent runs on cDNA from wild-type animals did not result in any amplification (Figure 2B), and immunofluorescence staining of cardiac allograft cross-sections confirmed human VEGF-B protein localization in cardio­myocytes.
Vascular endothelial growth factor-B has been associated with fatty acid trafficking and metabolism in tissues.6,7,23-25 Therefore, we characterized the effects of cardiomyocyte-specific human VEGF-B transgene expression on the morphology and fat content of the heart, by determining cardiac dimensions and the amount of intracellular neutral fat deposits. In this set of VEGF-B transgenic hearts, there was no significant difference in the total cardiac cross-sectional area or thickness of the right ventricular wall, left ventricular wall, or the septum (Figure 2C-D); however, cardiomyocyte size was significantly larger in the VEGF-B transgenic hearts than in the wild-type hearts (P < .05, Figure 2E). We found no difference in the amount of neutral lipid accumulation between the transgenic and wild-type hearts (Figure 2F), and the amount of fibrosis was similar between the groups (Figure 2G).

Recombinant human vascular endothelial growth factor-B transgenic donor hearts undergo enhanced ischemia-reperfusion injury after transplant
We compared the effect of IRI on VEGF-B transgenic and wild-type cardiac grafts by applying 2-hour cold preservation and 1-hour warm ischemia before reperfusion. Six hours after reperfusion, the rats transplanted with the human VEGF-B transgenic grafts had a greater than 2-fold increase in serum levels of cardiac troponin T compared with rats that received wild-type heart transplants (P < .01; Figure 3A). There was no difference in the number of ED-1-positive macrophages, MPO-positive neutrophils, and CD4-positive and CD8-positive T cells in the heart transplants between the 2 groups (Figure 3B, Figure 4). The proapoptotic Bax-to-Bcl-2 mRNA ratio and number of TUNEL-positive apoptotic cells were similar between the VEGF-B overexpressing hearts and wild-type control hearts (Figure 3C-D). We observed no effects of transgenic human VEGF-B overexpression on inflammatory cytokines and chemokines (Figure 3E).

Cardiomyocyte-specific human vascular endo­thelial growth factor-B transgenic donor hearts showed signs of altered metabolic homeostasis
Due to the previously proposed metabolic effects of VEGF-B, we measured the mRNA levels of com­ponents of glycolysis, fatty acid oxidation, and cellular aerobic and anaerobic energy metabolism pathways in VEGF-B transgenic hearts 6 hours after reperfusion.

The mRNA levels of electron transport chain enzyme complex IV subunit 1 mRNA was signi­ficantly elevated in the human VEGF-B transgenic rat hearts compared with wild-type hearts (P < .001; Figure 3F). We also found an increase in the purine ecto­nucleotidase enzyme CD39 mRNA in the human VEGF-B-overexpressing hearts (P < .05; Figure 3F).

Recombinant human vascular endothelial growth factor-B transgenic donor hearts showed impaired myocardial perfusion after transplant
Serial laser-Doppler measurements from the apex of the heart transplants 1 minute, 5 minutes, and 6 hours after reperfusion revealed impaired perfusion in the human VEGF-B transgenic heart transplants compared with wild-type controls (P < .05; Figure 5A). We analyzed VEGF-B transgenic heart trans­plants 6 hours after reperfusion. Although we observed no signs of arterialization (Figure 5B), we found a marked reduction in the number of RECA-1-positive myocardial capillaries in the human VEGF-B transgenic hearts (P < .01; Figure 5C); however, the density of RECA-1-positive capillaries was similar between the human VEGF-B transgenic and wild-type hearts (Figure 5D).

Adeno-associated virus-mediated human human vascular endothelial growth factor-B overexpression induced macrophage proinflammatory response in the allografts
To ensure sufficient cardiomyocyte transduction, wild-type cardiac allograft donor DA rats were treated with intravenous AAV9-human VEGF-B (isotypes 167 and 186) 5 days before organ pro­curement. The AAV9 was chosen due to its cardio­tropic qualities after intravenous injection and effective transgene expression kinetics.26 The hearts were exposed to 2-hour cold preservation and 1-hour warm ischemia and transplanted heterotopically into wild-type WF rats. We removed the cardiac allografts 6 hours after reperfusion, determined the serum concentration of cardiac troponin T, and analyzed the cardiac allograft tissue samples with PCR and immunohistochemical methods.

Donor treatment with intravenous AAV9-human VEGF-B resulted in efficient cardiomyocyte trans­duction, as shown by mRNA expression and immunofluorescence staining of human VEGF-B protein (P < .001; Figure 6A). The cardiac allografts weighed the same in both groups (Figure 6B), and serum troponin T concentration was similar between the groups (Figure 6C). The number of MPO-positive neutrophils, natural killer cells, OX62-positive-dendritic cells, and CD4-positive and CD8-positve T cells was similar between the groups (Figure 6D); however, the number of intragraft ED-1-positive macrophages was increased in the VEGF-B group (1.5-fold; P < .05; Figure 6D and Figure 7C). The RECA-positive capillary density was similar between the groups (Figure 6E), and no difference was observed in the average cardiomyocyte area between the groups (Figure 6F). No differences were found in Bax/Bcl-2 mRNA and various metabolic factors (Figure 7A-B); however, we observed an increase in the proinflammatory cytokine IL-12 p35 mRNA expression in the VEGF-B overexpressing groups (1.6-fold; P < .05; Figure 6G).


We observed a relation between ischemia, reperfusion, and VEGF-B production in cardiac allografts. The VEGF-B gene expression was significantly increased in hearts subjected to cold ischemia alone, with no further effect with 1 hour of warm ischemia. Furthermore, 2 hours of preoperative cold ischemia increased the VEGF-B mRNA in transplanted hearts at 6 hours after reperfusion.

Transgenic overexpression of VEGF-B induces cardiomyocyte hypertrophy but also reduces ischemia-induced cardiomyocyte damage in infarct models.11,19 In our study, VEGF-B transgenic hearts were not protected against IRI but rather released more cardiac troponin T to the circulation after ischemia and reperfusion of the cardiac grafts. However, we found no differences in markers of cardiomyocyte apoptosis or graft inflammatory response between the groups. These results are surprising, as other studies have shown that VEGF-B treatment directly decreases the expression of apoptosis-inducing genes, particularly members of the BH3 protein family, both in vitro and in pathologic conditions in vivo.10-17,27

Vascular endothelial growth factor-B gene deletion has been shown to diminish blood vessel survival in an in vivo oxygen-induced vessel regression model of mouse corneal pockets.18,20 It has been proposed as an important survival factor for a variety of cell types under pathologic circumstances.25 Also, myocardial infarction models indicate that VEGF-B treatment induces expansion of coronary vasculature and epicardial arterialization.11,15,19 Such results suggest that VEGF-B protects coronary vasculature in pathologic circumstances. Vascular endothelial growth factor-B also functions as a survival factor for vascular endothelial cells, pericytes, and smooth muscle cells in vitro.18,20 However, myocardial perfusion was impaired in the VEGF-B transgenic hearts after transplant and reperfusion. Although VEGF-B can have a protective role in acute myocardial infarct models,11,19 this was not the case in the heart transplant model.

Transgenic overexpression of VEGF-B has been shown to lead to mild cardiac hypertrophy without progression to cardiomyopathy.11,15 Although there was no statistically significant difference in the cardiac graft dimensions between the transgenic- and wild-type groups in the present study, the individual cardiomyocyte area was greater in the transgenic hearts than in the wild-type hearts. Despite the suggested involvement of VEGF-B in lipid transport from the circulation to the heart, there were no differences in the amount of intracellular lipid droplets within cardiomyocytes. The amount of extracellular ATPase CD39 was increased in VEGF-B-overexpressing hearts, suggesting a boosted ATP production.28 Kivelä and associates have shown that transgenic cardiac human VEGF-B overexpression promotes aerobic oxidative metabolism in cardio­myocytes.29 Furthermore, we have previously described how endogenous VEGF-B mRNA levels diminish along with cardiomyocyte size in cardiac allografts during long-term follow-up and unloading of the grafts.30 Combining this information with our current findings, one could hypothesize that VEGF-B may be an important factor in the maintenance of cardiomyocyte metabolic homeostasis and adap­tation to increased workload, overexpression of which leads to cardiomyocyte hypertrophy. Moreover, this translates into increased VEGF-B mRNA expression during ischemia, as the cardiac tissue is striving to overcome the lack of nutrients.

The increased myocardial damage in transgenic cardiac grafts could be explained by their higher ATP production and thus basal metabolic demand as a result of continuous high levels of VEGF-B, leading to decreased ischemia tolerance. Also, the RNA encoding cytochrome C oxidase, a component of the electron transport chain, was increased in the VEGF-B overexpressing hearts, suggesting an impaired metabolic switch from fatty acid oxidation and oxidative phosphorylation to anaerobic glycolysis during ischemic preservation of the graft. The transgenic overexpression of VEGF-B resulted in significant cardiomyocyte hypertrophy, which was the most likely cause of the increased recipient serum troponin T concentration and decreased myocardial perfusion rate. These results, however, were not mimicked after AAV9-mediated short-term VEGF-B overexpression in heart transplants, indicating that long-term VEGF-B overexpression is needed for cardiomyocyte hypertrophy.

Overexpression of AAV9-mediated VEGF-B in heart transplants was associated with a mild increase of cardiac ED-1-positive macrophages. Furthermore, of the various cytokines analyzed, IL-12 p35 mRNA expression was increased in the VEGF-B-treated heart transplants, suggesting a proinflammatory macrophage phenotype. VEGFR-1 is expressed on the surface of monocytes and macrophages and regulates the migration of these cells.31,32 To our knowledge, however, there are no previous reports on VEGF-B acting directly on macrophages. Instead, VEGF-A has been shown to induce macrophage migration and activation via VEGFR-1.32 Furthermore, VEGFR-2 signaling has been shown to increase cardiac allograft inflammation.33 A study conducted by Kivelä and associates showed that increased cardiac VEGF-B expression made more VEGF-A available for VEGFR-2 signaling, offering a possible explanation for our findings.29

Here, for the first time, we demonstrate that VEGF-B is upregulated by ischemia-reperfusion of heart transplants. An excess of VEGF-B, however, did not protect heart transplants from heart transplant-associated IRI. The cardiomyocyte damage that we observed after long-term human VEGF-B overex­pression may have been caused by a faulty switch from oxidative phosphorylation to anaerobic meta­bolism during the ischemic period and by the cardiomyocyte hypertrophy, exacerbating ischemia.


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Volume : 15
Issue : 2
Pages : 203 - 212
DOI : 10.6002/ect.2016.0181

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From the 1Transplantation Laboratory, University of Helsinki and Cardiac Surgery, Heart and Lung Center, Helsinki University Hospital, Helsinki, Finland; the 2Wihuri Research Institute; and the 3Translational Cancer Biology Program and Helsinki University Hospital, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland
Acknowledgements: Alireza Raissadati and Raimo Tuuminen contributed equally. Authors have no conflicts of interest to declare. This study was supported by grants from the Academy of Finland, the Sigrid Juselius Foundation, Helsinki University Central Hospital Research Funds, Finnish Cultural Foundation, the Finnish Foundation for Cardiovascular Research, University of Helsinki, Emil Aaltonen Foundation, Research and Science Foundation of Farmos, Aarne Koskelo Foundation, Paavo Ilmari Ahvenainen Foundation, Sirpa and Markku Jalkanen Foundation, Finnish Transplantation Society, Päivikki and Sakari Sohlberg Foundation, the Finnish Society of Angiology, the Paulo Foundation, and the Ida Montin Foundation.
We extend our gratitude to Professor Kari Alitalo for providing us with the transgenic animals and viral vectors used in this study and for the excellent support and assistance in writing the manuscript. We thank Terhi Kärpänen, PhD, from the Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, University of Helsinki, for helpful discussions and valuable advice. We also thank Seppo Sarna, PhD, Department of Public Health, University of Helsinki, for valuable advice in the statistical assessment of this study, Ms. Leena Saraste from the Transplantation Laboratory for language editing, and Ms. Tanja Laakkonen for the production of the AAV vectors.
Corresponding author: Alireza Raissadati, Transplantation Laboratory, Haartman Institute, PO Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Finland
Phone: +358 9 1912 6590