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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2016 Oct 31;113(46):13144–13149. doi: 10.1073/pnas.1616168113

VEGF-B gene therapy inhibits doxorubicin-induced cardiotoxicity by endothelial protection

Markus Räsänen a,b, Joni Degerman a,b, Tuuli A Nissinen c, Ilkka Miinalainen d, Risto Kerkelä d, Antti Siltanen e, Janne T Backman f, Eero Mervaala e, Juha J Hulmi c,g, Riikka Kivelä a,b,1,2, Kari Alitalo a,b,1,2
PMCID: PMC5135329  PMID: 27799559

Significance

The cardiotoxicity of anthracyclines is a major problem in cancer chemotherapy, and its alleviation would improve the life expectancy of cancer patients. This study shows that vascular endothelial growth factor-B (VEGF-B) gene therapy can be used to prevent the cardiotoxicity of doxorubicin (DOX). VEGF-B inhibited DOX-induced cardiac atrophy, protected endothelial cells from apoptosis, and preserved the myocardial capillary network. Importantly, DOX-induced whole body wasting (cachexia), which both impairs the quality of life and increases drug toxicity in patients as well as decreases their survival, was inhibited by VEGF-B treatment in the DOX-treated mice. Additional preclinical studies are needed for development of the VEGF-B gene therapy for cardiac protection in patients.

Keywords: VEGFB, heart, cancer, endothelial cell, heart failure

Abstract

Congestive heart failure is one of the leading causes of disability in long-term survivors of cancer. The anthracycline antibiotic doxorubicin (DOX) is used to treat a variety of cancers, but its utility is limited by its cumulative cardiotoxicity. As advances in cancer treatment have decreased cancer mortality, DOX-induced cardiomyopathy has become an increasing problem. However, the current means to alleviate the cardiotoxicity of DOX are limited. We considered that vascular endothelial growth factor-B (VEGF-B), which promotes coronary arteriogenesis, physiological cardiac hypertrophy, and ischemia resistance, could be an interesting candidate for prevention of DOX-induced cardiotoxicity and congestive heart failure. To study this, we administered an adeno-associated viral vector expressing VEGF-B or control vector to normal and tumor-bearing mice 1 wk before DOX treatment, using doses mimicking the concentrations used in the clinics. VEGF-B treatment completely inhibited the DOX-induced cardiac atrophy and whole-body wasting. VEGF-B also prevented capillary rarefaction in the heart and improved endothelial function in DOX-treated mice. VEGF-B also protected cultured endothelial cells from apoptosis and restored their tube formation. VEGF-B increased left ventricular volume without compromising cardiac function, reduced the expression of genes associated with pathological remodeling, and improved cardiac mitochondrial respiration. Importantly, VEGF-B did not affect serum or tissue concentrations of DOX or augment tumor growth. By inhibiting DOX-induced endothelial damage, VEGF-B could provide a novel therapeutic possibility for the prevention of chemotherapy-associated cardiotoxicity in cancer patients.

Cancer mortality has decreased during the last decades; thus, the number of patients suffering from the long-term effects of cardiotoxic antineoplastic drugs is increasing. Several agents used in cancer treatment can adversely affect the heart and lead to congestive heart failure, which is a growing problem in cardio-oncology. Reversible cardiac changes occur, e.g., during monoclonal antibody therapy with trastuzumab, whereas anthracyclines are associated with cumulative dose-limiting toxicity, which can lead to irreversible cardiac changes and ultimately to dilated cardiomyopathy with long-term heart problems (1, 2).

Doxorubicin (DOX) is an anthracycline cytostatic agent, which has been in clinical use for almost a half century. DOX and other anthracyclines arrest the cell cycle and thus inhibit the proliferation of malignant cells (3). DOX is known to be effective against a wide variety of cancers, which is in part why it has remained one of the most commonly used antineoplastic drugs. However, soon after its discovery, cumulative doses of DOX were found to be harmful also to healthy tissues, especially to the myocardium (4). Cumulative DOX doses are associated with cardiotoxicity, which leads to left ventricular dysfunction and development of heart failure. The toxic mechanisms have been suggested to include mitochondrial iron accumulation and associated redox reactions (5), abnormal protein processing, activation of innate immune responses, DNA damage, failure of cardiac repair, and decreased vasculature (6).

Weight loss in cancer cachexia affects skeletal muscle and adipose tissue and also internal organs including the liver, kidneys, lungs, and heart. Both large tumors and anticancer treatments can cause cachexia alone or in combination (7). Generally, the term “cardiac cachexia” refers to the unintentional weight loss caused by heart disease. However, in the case of cancer cachexia, cardiac cachexia describes the cardiac atrophy, remodeling, and dysfunction associated with cancer (7).

Endothelial dysfunction occurs in many diseases, but endothelial cells (ECs) are seldom considered as therapeutic targets, although many cardiac problems arise as a result of vascular defects (8). Vascular endothelial growth factor-B (VEGF-B) is the least studied member of the five known VEGFs that regulate the proliferation, sprouting, and migration of ECs (9). The highest endogenous concentrations of VEGF-B are found in cardiomyocytes (9). Exogenous VEGF-B is a potent arteriogenic growth factor that can promote physiological cardiac hypertrophy and counteract metabolic problems of obesity (10–12). Furthermore, reduced VEGF-B expression occurs in dilated cardiomyopathy and in ischemic heart disease, and VEGF-B has been suggested as a candidate for the therapy of dilated cardiomyopathy (11, 13–16). Interestingly, exogenous VEGF-B maintained mitochondrial respiratory complex 1 function after ischemia/reperfusion injury and protected the heart from ischemic damage (11).

In this study, we have addressed the protective effects of VEGF-B in anthracycline-treated mice. We treated normal and tumor-bearing mice with DOX, which induced damage to the cardiac microvasculature and cardiomyocyte atrophy. We show that these adverse effects, as well as the DOX-induced decrease in lean body mass, can be prevented by VEGF-B gene therapy. Importantly, VEGF-B treatment did not affect the therapeutic levels of DOX or augment tumor growth.

Results

DOX-Induced Cardiac Atrophy Is Inhibited by VEGF-B.

To analyze the effects of VEGF-B in DOX-treated mice, we injected adeno-associated viral vector 9 (AAV9)–control vector or AAV9–VEGF-B186 i.p. to the mice 7 d before the first DOX injection (6 mg/kg). DOX treatment was repeated every third day for four times, and the mice were terminated at 2 or 4 wk (Fig. 1A). VEGF-B transgene expression in the heart was confirmed by quantitative polymerase chain reaction (qPCR) and ELISA, and VEGF-B serum levels were measured by ELISA (Fig. S1). We also analyzed VEGF-B expression in other tissues after AAV delivery and found the highest levels in the liver, heart, and epididymal adipose tissue (Fig. S2). This is in line with our previously published findings on VEGF-B levels in different tissues after systemic AAV9 delivery (12).

Fig. 1.

Fig. 1.

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DOX-induced cardiac atrophy is inhibited by VEGF-B. (A) Schedule of the 4- and 2-wk experiments. The mice received four doses of DOX (6 mg/kg) or PBS every 3 d, as indicated. (B) Heart weight/tibial length (HW/TL) and (C) quantitation of the cardiomyocyte (CMC) size. (D) Representative images of laminin staining for cardiomyocyte size analysis. Mean ± SD. *P = 0.05; **P = 0.01; ***P = 0.001.

Fig. S1.

Fig. S1.

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Analysis of VEGF-B mRNA in the heart and VEGF-B protein in serum in AAV–VEGF-B transduced and control mice. VEGF-B mRNA quantification at termination of the (A) 2-wk, (B) 4-wk, and (C) acute 20-h experiment. The values are presented as fold increase compared with the control group (CTRL = 1). (D) VEGF-B protein levels in serum were measured with ELISA. Mean ± SD. **P = 0.01; ***P = 0.001.

Fig. S2.

Fig. S2.

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ELISA analysis of VEGF-B protein expression in different tissues and serum 3 wk after AAV–VEGF-B injection. Mean ± SD. **P = 0.01; ***P = 0.001.

DOX treatment induced significant cardiac atrophy after the cumulative 24 mg/kg dose (Fig. 1B). Importantly, the reduction in heart weight and cardiomyocyte size was completely inhibited by pretreatment with AAV–VEGF-B (Fig. 1 B–D). In addition, left ventricle (LV) posterior wall and septum thickness were decreased after DOX treatment in hearts fixed in diastole, and this was prevented by VEGF-B pretreatment (Fig. S3C). DOX treatment also resulted in whole-body cachexia, mainly due to reduced fat and muscle mass (Fig. 2). AAV–VEGF-B inhibited the loss of body mass both in the 2- and 4-wk experiments, but it did not affect body weight in normal untreated mice (Fig. 2 and Fig. S4). VEGF-B did not affect food consumption, which was decreased by 12% in the DOX group and by 10% in the DOX + AAV–VEGF-B group during DOX treatment. Dual-energy X-ray absometry (DXA) analysis of body composition showed similar loss of fat mass in both DOX and DOX + VEGF-B groups, but the lean body mass was significantly higher in the VEGF-B–treated group (Fig. 2 E–G). However, VEGF-B did not rescue the decrease in bone mineral density (BMD) or maximal running capacity of the DOX-treated mice (Fig. S5).

Fig. S3.

Fig. S3.

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Effect of VEGF-B on cardiac function and wall thickness in DOX-treated mice. Shown is the echocardiographic analysis after 24 mg/kg cumulative DOX dose. (A) Two-week experiment. (B) Four-week experiment. LV, left ventricle. (C) Posterior wall and septum thickness in hearts fixed in diastole. Mean ± SD. *P = 0.05; **P = 0.01; ***P = 0.001.

Fig. 2.

Fig. 2.

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DOX-induced whole-body wasting is inhibited by VEGF-B. (A) Body weight follow-up during the 2-wk experiment and (B) 4-wk experiment. (C and D) Body weights at the end of the experiments. (E) Fat mass analyzed by DXA at 4 wk. (F) Tibialis anterior muscle weight adjusted to tibial length at 4 wk. (G) Lean body mass analyzed by DXA at 4 wk. Arrows indicate the time points of DOX or PBS injections (VEGF-B injections were performed 7 d before the first DOX treatment). Mean ± SD. *P = 0.05; **P = 0.01; ***P = 0.001 (DOX vs. DOX + VEGF-B). #P = 0.05; ##P = 0.01; ###P = 0.001 (DOX vs. CTRL).

Fig. S4.

Fig. S4.

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VEGF-B increases heart weight but has no effect on body weight in healthy, untreated mice. (A and B) Heart weight/body weight ratio and body weight at (A) 2 wk and (B) 4 wk after AAV9–VEGF-B injection. Mean ± SD. *P = 0.05.

Fig. S5.

Fig. S5.

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Effects of DOX on bone mineral density (BMD), bone mineral content (BMC), and maximal running capacity. (A) Changes in BMD and BMC measured by DXA. (B) Maximal running capacity in seconds on a treadmill. Mean ± SD. **P = 0.01; ***P = 0.001.

VEGF-B Protects Cardiac ECs from DOX-Induced Damage.

DOX cardiotoxicity has been attributed mainly to cardiomyocyte damage, but because VEGF-B acts via binding to its receptor vascular endothelial growth factor receptor-1 (VEGFR-1), which is expressed in ECs at robustly higher levels than in cardiomyocytes (17), we analyzed the effects of DOX on cardiac microvasculature. DOX caused EC damage in coronary microvasculature and reduced cardiac capillary area significantly in both 2- and 4-wk experiments (Figs. 3A and 4). Importantly, this reduction was completely prevented by VEGF-B-treatment (Fig. 3A).

Fig. 3.

Fig. 3.

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VEGF-B protects the endothelium against DOX-induced apoptosis and capillary rarefaction. (A) Quantification of cardiac capillary area and in DOX and DOX + VEGF-B–treated mice in the 2- and 4-wk experiments compared with the control mice. (B) Human arterial ECs were pretreated with medium from AAV9–VEGF-B or AAV9–HSA-transfected 293T cells, followed by administration of DOX (0.5 µM). Apoptosis (Caspase3/7 activity) was analyzed for 48 h by fluorescent time-lapse imaging. *P = 0.05; **P = 0.01; ***P = 0.001 (DOX vs. DOX + VEGF-B). #P = 0.05; ##P = 0.01; ###P = 0.001 (DOX vs. CTRL). Mean ± SD.

Fig. 4.

Fig. 4.

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DOX-induced endothelial and cardiomyocyte damage. Electron microscopic images from hearts of mice treated with PBS (Ctrl), DOX, and VEGF-B + DOX. (A) Arrow points to damaged EC in the mouse treated with DOX. (B) Arrow points to damaged and atrophied mitochondria in mice treated with DOX. (Scale bar, 2 μm.)

To further assess if VEGF-B protects cardiac endothelium from DOX-induced apoptosis, we studied human cardiac microvascular and arterial ECs (HCMECs and HCAECs) in culture. The cells were incubated with conditioned medium from either AAV–VEGF-B or AAV–human serum albumin (HSA) transduced 293T cells, after which the ECs were exposed to DOX. Video microscopy using fluorescent Caspase3/7 activity reporter showed that DOX induced EC apoptosis, which was alleviated by VEGF-B (Fig. 3B).

Electron microscopic images of the DOX-treated hearts showed damaged endothelium and mitochondria as well as significant myofibrillar degradation (Fig. 4). The number of cardiomyocytes with disturbed myofibrillar and sarcolemmal integrity tended to decrease in the hearts of mice treated with VEGF-B (30.5 ± 11.4 vs. 19.3 ± 13.2 cells per mm2, P = 0.2) (Mann–Whitney U test).

VEGF-B pretreatment also inhibited endothelial dysfunction induced by DOX in the assay of aortic relaxation to acetylcholine (ACh) after phenylephrine-induced contraction. Fig. 5A shows significantly improved aortic relaxation in the VEGF-B–pretreated mice in comparison with the DOX-treated mice (Fig. 5A). Inhibition of the endothelium-dependent vascular relaxation by N[omega]-nitro-l-arginine methyl ester (l-NAME) abolished the differences (Fig. S6). We also observed significant up-regulation of heat shock protein 70 in the DOX-treated but not in DOX + VEGF-B–treated ECs (Fig. S7A). As another measure of endothelial dysfunction, HCAEC tube formation on Geltrex matrix was significantly inhibited by 0.5 μmol/L DOX, but this was restored by VEGF-B treatment (Fig. 5 B and C).

Fig. 5.

Fig. 5.

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VEGF-B protects against DOX-induced endothelial dysfunction both in vivo and in vitro. (A) Aortic relaxation after phenylephrine contraction and descending ACh concentrations in mice after DOX treatment. (B) HCAEC 3D tube formation in Geltrex-coated wells. Cells were pretreated with VEGF-B or HSA as a control and then exposed to DOX (0.5 µM). (C) Quantification of the number of junctions and total segment length. Mean ± SD. *P = 0.05.

Fig. S6.

Fig. S6.

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Monitoring of aortic relaxation after addition of the nitric oxide synthesis inhibitor l-NAME. Aortas were isolated from VEGF-B–overexpressing and control mice treated with DOX or PBS, as indicated. The aortas were then pretreated with l-NAME, followed by phenylephrine treatment, and treatment with the descending acetylcholine concentrations shown on the x axis.

Fig. S7.

Fig. S7.

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Analysis of Hsp70 expression in DOX-treated ECs and DNA fragmentation in cardiomyocytes and cardiac ECs. (A) Hsp70 mRNA in ECs treated with 0.5 μM DOX for 24 h. (B) Quantification of TUNEL-positive nucleae in podocalyxin-positive endothelial cells and cardiomyocytes per microscope field. Mean ± SD. *P = 0.05.

VEGF-B Restores pERK1/2 Signaling in DOX-Treated Hearts.

ERK signaling is an important mediator of angiogenesis and arteriogenesis downstream of VEGFR2 activation, and VEGF-B increases ERK1/2 phosphorylation in the heart (10, 18). Here were found that DOX treatment significantly reduced ERK1/2 phosphorylation, which was reversed by VEGF-B (Fig. 6A). Consistent with its effect on heart weight, VEGF-B also increased phosphorylation of the ribosomal protein pS6, which is one of the main regulators of the mTOR pathway necessary for cardiomyocyte growth (19) (Fig. 6B).

Fig. 6.

Fig. 6.

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VEGF-B restores DOX-induced reduction in ERK1/2 phosphorylation and increases phosphorylation of S6. (A) Western blot of p-Erk1/2 and total Erk1/2; GADPH was used as a loading control. (B) Western blot of p-S6 and total S6. (C) Quantification of p-Erk1/2/total Erk1/2 and (D) p-S6/total S6. Mean ± SD. *P = 0.05. (Scale bar, 50 µm.)

VEGF-B Decreases DOX-Induced DNA Damage and Induction of Markers of Pathological Cardiac Remodeling.

To analyze the acute cardiac effects of DOX treatment, we injected 15 mg/kg of DOX intraperitoneally and collected the hearts 20 h after the injection. Cardiac sections were stained for phosphorylated histone H2Ax (γH2Ax), which is used as a marker for the cellular response to DNA double-strand breaks (20). DOX increased the amount of γH2Ax-positive cells significantly compared with PBS, and this effect was attenuated by VEGF-B pretreatment (Fig. 7 A and B). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) showed a trend toward increased DNA fragmentation after DOX treatment in both cardiomyocytes and ECs, but this was not statistically significant (Fig. S7B).

Fig. 7.

Fig. 7.

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VEGF-B attenuates DOX-induced DNA damage in cardiomyocytes and inhibits the up-regulation of pathological cardiac transcripts. (A) Representative images of immunofluorescence stainings for γH2Ax (red), laminin (green), and DAPI (blue) in cardiac sections. (B) Quantification of the percentage of γH2Ax-positive nuclei. mRNA expression of (C) REDD1, (D) ANP, (E) BNP, and (F) β-MHC. All values are presented as a fold change compared with the control group. Mean ± SD. *P = 0.05; **P = 0.01; ***P = 0.001. (Scale bar, 300 µm.)

Microarray analysis revealed that acute DOX treatment up-regulated the expression of 46 known transcripts and down-regulated 25 known transcripts in the heart [false discovery rate (FDR) < 0.05]. VEGF-B together with DOX in turn up-regulated 1,111 and down-regulated 927 known transcripts. According to the Venn diagram, expression of 16 genes (8 up and 8 down) was significantly changed only in the DOX-treated hearts but not in the VEGF-B + DOX-treated hearts (Fig. S8). Interestingly, the DNA damage-inducible transcript 4 (Ddit4/REDD1), which mediates cardiomyocyte autophagy and apoptosis through the mTOR pathway (21), was one of the significantly increased transcripts in DOX- but not in VEGF-B + DOX-treated hearts (Fig. 7C). Furthermore, DOX significantly increased the expression of BNP, ANP, and β-MHC, which are markers of pathological cardiac remodeling, and this was significantly inhibited by VEGF-B (Fig. 7 D–F). GSEA pathway analysis indicated increased apoptotic signaling and decreased voltage-gated channel activity and cardiac contraction in DOX-treated hearts (Fig. S8). VEGF-B treatment increased cytoskeleton biogenesis, angiogenesis, and cell cycle-related transcripts compared with both control and DOX-treated hearts (Fig. S8).

Fig. S8.

Fig. S8.

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Effects of acute DOX and VEGF-B + DOX treatments on cardiac transcriptome. Shown is a Venn diagram analysis of cardiac transcripts affected by DOX and VEGF-B + DOX. Also shown is a list of 16 genes affected by DOX only and results from GSEA pathway analysis of the indicated groups. FDR, false discovery rate q value; NES, normalized enrichment score; Nom P, nominal P value.

Effect of VEGF-B on Cardiac and Mitochondrial Function in the DOX-Treated Mice.

To assess the effect of DOX and VEGF-B on cardiac function, we carried out echocardiographic analyses after DOX treatment. Ventricular ejection fraction or fractional shortening was not significantly altered in mice treated with DOX for 2 or 4 wk. VEGF-B + DOX treatment increased LV mass and systolic and diastolic LV volumes (Fig. S3) but did not have any adverse effects on cardiac function.

Intact vasculature is necessary for optimal mitochondrial function, as blood vessels provide nutrients and oxygen for cardiomyocyte mitochondria. To analyze the effect of DOX treatment on cardiac mitochondrial function, we performed high-resolution oxygraph analysis of myocardial samples from the LV 3 d after the last DOX injection.

VEGF-B increased mitochondrial oxygen consumption in the DOX-treated hearts when complex I substrates (malate, pyruvate, and glutamate) were used in ADP-stimulated state 3 respiration analysis (Fig. S9A). In addition, maximal uncoupled respiration (ETS) was significantly enhanced. To test if this is due to differences in the number of mitochondria, we analyzed cardiac mitochondrial DNA (mtDNA) content. There was a significant increase in total mtDNA in the DOX + VEGF-B group compared with DOX and control groups (Fig. S9B). However, there was no difference in expression of peroxisome proliferator-activated receptor gamma co-activator 1 (PGC-1α), which is a major regulator of mitochondrial biosynthesis (Fig. S9B).

Fig. S9.

Fig. S9.

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VEGF-B improves cardiac mitochondrial function in DOX-treated mice. (A) Mitochondrial function was measured at termination of the 2-wk experiment using the SUIT-protocol in a high-resolution oxygen analyzer. CI, complex I; CytC, cytochrome c; CII, complex II; ETS, electron transfer system (maximal uncoupled respiration). (B) Quantification of myocardial mtDNA content at 2 and 4 wk and relative levels of cardiac PGC1α RNA at 2 wk. Mean ± SD. *P = 0.05; **P = 0.01.

VEGF-B Does Not Affect Tumor Growth or Tissue Concentrations of DOX.

To mimic the use of DOX in the clinic, we implanted Lewis lung carcinoma (LLC) cells s.c. to isogenic C57Bl6 mice. AAV–VEGF-B or AAV–Control was administered on day 3 after tumor implantation, and DOX was injected on days 6 and 11. These data indicated that VEGF-B administration did not affect the growth of the LLC xenotransplants (Fig. 8 B and C). DOX treatment induced significant cardiac and whole-body atrophy in the tumor-bearing mice, which was inhibited by VEGF-B (Fig. 8 D–F).

Fig. 8.

Fig. 8.

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VEGF-B restores body weight and cardiac mass in DOX-treated LLC tumor-bearing mice but does not increase tumor growth. (A) Schedule of the experiment. (B) Tumor growth curves and (C) tumor tissue mass at termination. (D) Body weight follow-up and (E) body weight and (F) heart weight adjusted to tibial length at termination. (G) DOX concentrations in vivo in serum, heart, and tumor tissue. *P = 0.05; **P = 0.01; ***P = 0.001 (LLC + DOX vs. LLC + DOX + VEGF-B). ###P = 0.001(LLC + DOX vs. LLC + VEGF-B). §P = 0.05 (LLC vs. LLC+DOX). Mean ± SD.

To assess if VEGF-B affects DOX availability in target tissues, we measured DOX concentrations in serum, heart, and tumor tissue. These measurements did not reveal significant differences in DOX concentrations between the DOX- and DOX + VEGF-B–treated groups (Fig. 8G). Thus, the protective effects of VEGF-B were not due to reduced availability of DOX.

Discussion

Here we show that VEGF-B treatment, starting 1 wk before DOX administration, completely inhibits DOX-induced cardiac atrophy and whole-body wasting, demonstrating a promising anticachexia effect. VEGF-B also prevented capillary rarefaction and protected ECs from apoptosis, improved endothelial and mitochondrial function, and reduced DNA damage. Importantly, VEGF-B treatment did not affect tumor growth or the availability or efficacy of DOX.

Cancer cachexia-related cardiac problems begin with an early atrophy of the LV, which is followed by thinning of the septal and interventricular walls and chamber dilation (7). In later stages, systolic and diastolic dysfunction become evident, accompanied by increased fibrosis and, in the most severe cases, heart failure (7). The decreased LV mass, left ventricular wall and septum thickness, diastolic and systolic volume, and stroke volume in the DOX-treated hearts were prevented by VEGF-B pretreatment. Previous echocardiographic studies have reported a DOX-induced decrease in fractional shortening and ventricular ejection fraction (5, 6). However, our DOX dosing protocol at 24 mg/kg (equivalent to ∼72 mg/m2 in humans) was designed to mimic patient treatment with low DOX doses (30–90 mg/m2) (22). This dosing did not cause functional systolic impairment during the 2- and 4-wk follow-up. Importantly, there were no deaths in any of the DOX-treated groups in any of the experiments. In human patients, decreased ejection fraction is usually not seen until advanced heart failure, when it is a predictor of a poor prognosis.

Whole-body wasting is shown to be an independent risk factor for mortality in chronic heart failure (23), and it increases chemotherapy toxicity and decreases survival (24). Importantly, although VEGF-B inhibited DOX-induced whole-body wasting in all experiments, it did not increase body weight in healthy mice without cachexia, as we have reported previously (10, 11). Mechanistically, VEGF-B increased phosphorylation of ERK1/2, a downstream mediator of VEGF receptor signaling in the heart, and ribosomal protein S6, indicating activation of the mTORC1 pathway. The effect of VEGF-B is consistent with our previous studies, in which VEGF-B induced mild cardiac and cardiomyocyte hypertrophy that was not converted to cardiomyopathy even upon prolonged VEGF-B overexpression (10, 11). This may be because VEGF-B–induced cardiomyocyte hypertrophy is based on enhanced signaling by endogenous VEGF via endothelial VEGFR2, leading to a concomitant increase of the coronary vasculature (11).

Several mechanisms have been suggested to mediate DOX-induced toxicity to cardiomyocytes (4, 25), whereas endothelial injury has received little attention (26, 27). In our experiments, the apoptosis-associated DNA fragmentation was observed approximately to the same extent in cardiomyocytes and ECs. DOX-induced apoptosis of ECs and capillary rarefaction in mouse hearts was inhibited by VEGF-B. Furthermore, VEGF-B had a protective effect against DOX-induced endothelial dysfunction both in vivo and in vitro, which supports our hypothesis that besides its direct toxicity to cardiomyocytes, DOX induces cardiac damage via ECs by disturbing the protective endothelial–cardiomyocyte cross-talk.

DOX-induced DNA damage in cardiomyocytes, estimated by staining for phosphorylated γH2Ax, a marker of DNA double-strand breakpoints (20), was significantly inhibited by VEGF-B. Furthermore, DOX induction of another DNA damage marker, Ddit4 mRNA, was inhibited by VEGF-B. The gene expression pathway analysis indicated increased apoptosis signaling and decreased expression of genes involved in cardiac contraction in the DOX-treated hearts. Interestingly, the DOX-induced changes in the cardiac transcriptome were similar to those recently reported in human induced pluripotent stem cell-derived cardiomyocytes treated with DOX (28). Previous studies have shown that VEGF-B can protect cultured rat neonatal cardiomyocytes against reactive oxygen species or chemotherapy-induced damage (16, 17). Even though the majority of VEGFR1 in the heart is found in ECs (11), VEGF-B may thus protect both ECs and cardiomyocytes against anthracyclin-induced damage.

In agreement with previous reports (29), DOX treatment tended to decrease cardiac mitochondrial complex I activity, whereas VEGF-B treatment increased the activity. This is consistent with our previous data showing that VEGF-B protects against ischemia reperfusion-induced mitochondrial dysfunction (11). The generation of reactive oxygen species (ROS) in cardiomyocytes is known to cause myocardial damage during cancer therapy, especially when anthracyclines are used (30). Sildenafil is cardioprotective against DOX-induced myocardial injury, presumably because it activates the nitric oxide pathway, which opens up the mitochondrial ATP-sensitive potassium channels (31). We have shown that VEGF-B up-regulates endogenous VEGF–VEGFR2 signaling and endothelial nitric oxide synthase (eNOS) expression, which is likely to increase NO production by ECs (11). This is consistent with our present finding that VEGF-B protected against DOX-induced endothelial dysfunction and endothelial tube formation.

Considering the translational potential of the present findings, it was important to confirm that VEGF-B does not compromise therapeutic DOX levels or promote tumor growth. In a previous study, overexpression of VEGF-B was actually shown to inhibit tumor growth, and tumors in VEGF-B–deficient mice grew faster (32). However, we did not experiment with enforced overexpression of VEGF-B in transfected tumor cells, which has been claimed to increase metastasis of tumor grafts via a VEGFR1-independent mechanism (33). Our results, and the fact that unlike other VEGF family members VEGF-B does not promote significantly increased vascular permeability or inflammation, thus encourage further studies on the translational potential of VEGF-B.

Cardiotoxicity limits the successful use of several cytotoxic anticancer agents. Thus, prevention of cardiac atrophy and dysfunction could improve the efficacy of cancer chemotherapy and enhance the chances of patient survival. Our present study demonstrates cardioprotective effects of VEGF-B gene therapy in an anthracycline-induced cardiotoxicity model in mice. VEGF-B inhibited the loss of body and heart weight and protected against DOX-induced DNA damage and mitochondrial dysfunction but did not induce tumor growth. Thus, VEGF-B could provide a safe and effective clinical treatment to alleviate cardiac problems related to cancer treatment, which is in line with previous studies showing beneficial effects of VEGFB in different models of cardiac disease (11, 16, 34). Importantly, our results highlight the possibility of protecting the heart via the vascular endothelium and suggest that VEGF-B could be useful in the therapy of those cardiac disorders where cardiac endothelium plays a pathogenic role.

Methods

A detailed description of the materials and methods can be found in SI Methods.

Study Design.

In the 2-wk (n = 6 per group) and 4-wk (n = 7 per group) experiments to mimic chronic DOX exposure, the mice in the DOX and DOX + VEGF-B groups were injected i.p. with 6 mg/kg of DOX every 3 d, four times total; the control mice received PBS (5). To mimic the acute toxicity, we injected 15 mg/kg DOX hydrochloride (Sigma-Aldrich), diluted in 2.5 mL of PBS solution, 20 h before the mice (n = 7 per group) were killed. In all experiments, AAV9–mVEGF-B186 (2 × 1011 AAV particles) was administered 7 d before the first DOX injection. The control mice were injected with the same amount of scrambled control vector (AAV9-Ctrl) at the same time.

In the 2-wk tumor experiment, the mice were injected s.c. with 5 × 105 LLC cells at day 0. AAV9–mVEGF-B186 or control vector was injected i.p. on day 3. The mice received injections of 6 mg/kg DOX on days 6 and 11. All mouse experiments were approved by the Provincial State Office of Southern Finland and carried out in accordance with institutional guidelines. Detailed study design is described in SI Methods.

RT-qPCR and Western Blot Analysis.

RT-qPCR and Western blot analysis are detailed in SI Methods. Primers used are listed in Table S1.

Table S1.

Primers used for qPCR and their sequences

Gene Forward primer Reverse primer
Agt 5′-AAGCTGCCCACCCTTTTG-3′ 5′-TGTTCCTCCTCTCCTGCTTTG-3′
ANP 5′-GAAAAGCAAACTGAGGGCTCTG-3′ 5′-CCTACCCCCGAAGCAGCT-3′
APJ 5′-CGCCAGGCCATTCTCAAAG-3′ 5′-AGCTGCCTCTGCTGCTATCTGTC-3′
Apln 5′-CAGGCCTATTCCCAGGCTCA-3′ 5′-CAAGATCAAGGGCGCAGTCA-3′
ATM 5′-ATAAGTCATATAGGAAGCCGAG-3′ 5′-TGCATCAAAAAGCTGGTAAC-3′
BNP 5′-AGGCGAGACAAGGGAGAACA-3′ 5′-GGAGATCCATGCCGCAGA-3′
COL-1 5′-TGTTGGCCCATCTGGTAAAGA-3′ 5′-CAGGGAATCCGATGTTGCC-3′
COL-3 5′-TGGTCCTCAGGGTGTAAAGG-3′ 5′-GTCCAGCATCACCTTTTGGT-3′
Cyt-B 5′-GCTTTCCACTTCATCTTACCATTT-3′ 5′-TGTTGGGTTGTTTGATCCTG-3′
MYH-7 5′-GCCAACTATGCTGGAGCTGATGCCC-3′ 5′-GGTGCGTGGAGCGCAAGTTTGTCATAAG-3′
PGC1α 5′-CCCTGCCATTGTTAAGACC-3′ 5′-TGCTGCTGTTCCTGTTTTC-3′
PGC1α1 5′-CATGTGCAGCCAAGACTCTG-3′ 5′-ACACCACTTCAATCCACCCA-3′
PGC1α3 5′-AAGTGAGTAACCGGAGGCATTC-3′ 5′-TTCAGGAAGATCTGGGCAAAGA-3′
PGC1α4 5′-TCACACCAAACCCACAGAAA-3′ 5′-CTGGAAGATATGGCACAT-3′
PGC1β 5′-CAGGGTGGGGACTCTGGA-3′ 5′-GAAGAGCTCGGAGTCATCGG-3′
TBP 5′-GAAGCTGCGGTACAATTCCAG-3′ 5′-CCCCTTGTACCCTTCACCAAT-3′
TOPOIIβ 5′-ATCAGTGAAACCATTTCAGG-3′ 5′-TATGCTTTCTACAATGCCAC-3′
VEGF-B 5′-AGCCACCAGAAGAAAGTGGT-3′ 5′-GCTGGGCACTAGTTGTTTGA-3′
β-actin 5′-GGAAAAGAGCCTCAGGGCAT-3′ 5′-GAAGAGCTATGAGCTGCCTGA-3′
REDD1 (DDIT4) 5′-GCCTCTGGGATCGTTTCTCG-3′ 5′-GGTCAAGGCCCTCTTCTCTG-3′
HSP-70 5′-CAAGATCACCATCACCAACG-3′ 5′-GCACCTTCTTCTTGTCAGCC-3′
Neuregulin-1 5′-TCTGACCGAAGGCTCTGC-3′ 5′-GGCCTACTGCAAAACCAAGA-3′
Angiopoeitin 2 5′-ATCCTGCCTTTCTCTTTATGGTT-3′ 5′-AGCCTTTGCACTGAGTCGTCGTA-3′
STAT 3 5′-TCCAATGTCCTCTATCAGCA-3′ 5′-GGCTGTTGGGTGGTTGGCCT-3′
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Immunohistochemical Analyses.

Frozen sections 8 μm were fixed with acetone, immunostained, and analyzed as detailed in SI Methods.

Whole-Genome Microarray and Data Analysis.

RNA samples were analyzed with genome-wide Illumina MouseWG-6 v2 microarrays (Illumina Inc.). Detailed microarray data analysis is described in SI Methods. The microarray data have been submitted to the GEO database, under the series accession number GSE81448.

Statistical Analysis.

Values are indicated as mean ± SD. Statistical analysis of multiple groups was performed with one-way or two-way ANOVAs, followed by Bonferroni or Holm–Sidak post hoc tests. All statistical tests were two-tailed. Differences were considered statistically significant at P ≤ 0.05. In graphs, *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

SI Methods

Mice.

We conducted five separate in vivo experiments on 9–10-wk-old wild-type C57BI6J male mice. The mice were randomly divided into three different groups: Control, DOX, and DOX + VEGF-B.

Study Design.

In the 2-wk (n = 6 per group) and 4-wk (n = 7 per group) experiments to mimic chronic DOX exposure, the mice in DOX and DOX + VEGF-B groups were injected i.p. with 6 mg/kg of DOX every 3 d, four times total; the control mice received PBS (5). To mimic the acute toxicity, we injected 15 mg/kg DOX hydrochloride (Sigma-Aldrich), diluted in 2.5 mL of PBS solution, 20 h before the mice (n = 7 per group) were killed. In all experiments, AAV9–mVEGF-B186 (2 × 1011 AAV particles) was administered 7 d before the first DOX injection. The AAV vectors are nonpathogenic, and they efficiently transduce postmitotic cardiomyocytes in vivo. The control mice were injected with the same amount of scrambled control vector (AAV9–Ctrl) at the same time. After termination of the mice, heart, gastrognemius, and tibialis anterior muscles and epididymal fat pads were excised, weighed, and processed for further analysis. Heart and skeletal muscle weights (mg) were normalized to tibial bone length (mm).

In the 2-wk tumor experiment, the mice were injected s.c. with 5 × 105 LLC cells at day 0. AAV9–mVEGF-B186 or control vector was injected i.p. on day 3. The mice received injections of 6 mg/kg DOX on days 6 and 11. Control mice were injected with PBS at the same time. Tumor volume was measured every other day and calculated using the following formula: volume = width × height × length/2.

The amount of food consumed was measured between days 14 and 17 (immediately after DOX treatment) and between days 21 and 24 (during the follow-up period).

Western Blot Analysis.

ECs were lysed in 0.5% Nonidet P-40 and 0.5% Tx-100 solution with protein inhibitors, and cardiac muscle samples were homogenized. Total protein content was determined using the bicinchoninic acid (BCA) protein assay (Pierce, Thermo Scientific) with an automated KoneLab device (Thermo Scientific). Thirty micrograms of total protein was separated by SDS/PAGE and transferred to PVDF membrane. The membrane was then blocked in TBS with 0.1% Tween 20 containing 5% (wt/vol) nonfat dry milk and incubated overnight at 4 °C with primary antibodies against phospho-Erk1/2 (Thr202/Tyr204), Erk1/2, phospho-rpS6 (Ser240/244), or rpS6 (Cell Signaling Technology). The membrane was then incubated with horseradish peroxidase-conjugated secondary antibody, after which the proteins were visualized by enhanced chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate; Pierce, Thermo Scientific) and quantified (Band Intensity × Volume) using a ChemiDoc XRS device in combination with Quantity One software (version 4.6.3; Bio-Rad Laboratories). The membrane was then reprobed for GAPDH (ab9485, Abcam) as a loading control.

Treadmill Running Protocol.

The mice ran first at 9, 12, and 15 m/min for 5 min each, after which the velocity was increased by 2 m/min every 2 min until exhaustion. All mice were familiarized with treadmill running on a separate day before the test.

Cardiac Echography.

Cardiac function was analyzed under isoflurane anesthesia using the Vevo 2100 Ultrasound system before the start and termination of the study. Left ventricular mass, ejection fraction, fractional shortening, interventricular spectrum width (diastolic and systolic), left ventricular internal diameter (diastolic and systolic), left ventricular posterior wall width (diastolic and systolic), and left ventricular volume (diastolic and systolic) were calculated.

DXA.

DXA assays were performed under Xylazine and Ketamine anesthesia using Lunar PIXImus 2 (GE Healthcare) equipment before and at termination of the experiment. BMD, bone mineral content (BMC), total skeletal area (AREA), lean body mass (LEAN), total body tissue mass (TOTAL), total body fat mass (FAT), and total body fat percentage (%FAT) were analyzed.

Assessment of Vascular Function.

The endothelium-intact descending aorta was placed between stainless steel hooks and mounted in an organ bath chamber in physiological salt solution (pH 7.4) of the following composition (mmol/L): NaCl 11.9%, NaHCO3 25.0, glucose 11.1, CaCl2 1.6, KCl 4.7, KH2PO4 1.2, and MgSO4 1.2 aerated with 95% O2 and 5% CO2. The rings were equilibrated for 60 min at 37 °C with a resting tension of 1.0 g. The force of contraction was measured with an isometric force-displacement transducer and recorded with IOX Software 1.569 and Data-analyst V1.68 (EMKA Technologies). The descending aortic artery rings were precontracted with 1 μmol/L phenylephrine for 6 min. The presence of intact endothelium in the vascular preparations was confirmed by observing the relaxation response to descending ACh (each 2.5 min) concentrations. To analyze the role of nonendothelial components on vascular relaxation, endothelium-dependent relaxation was blocked with l-NAME (0.1 mmol/L).

VEGF-B ELISA.

Serum levels of VEGF-B were measured by in-house built ELISA using goat–anti-mouse VEGF-B (AF590 R&D) as a capture antibody and biotinylated goat–anti-mouse VEGF-B186 (BAF767, R&D) as a detection antibody.

Electron Microscopy.

Specimens from mouse hearts were fixed in 1% glutaraldehyde and 4% formaldehyde mixture in 0.1 M phosphate buffer, postfixed in 1% osmium tetroxide, dehydrated in acetone, and embedded in Epon LX 112 (Ladd Research Industries). Thin sections (70 nm) were cut with Leica Ultracut UCT ultramicrotome, stained in uranyl acetate and lead citrate, and examined in Tecnai G2 Spirit transmission electron microscope (FEI Europe). Images were captured by Quemesa CCD camera and analyzed using iTEM software (Olympus Soft Imaging Solutions GMBH).

Analysis of Mitochondrial Function.

Mitochondrial function was measured using high-resolution respirometry (Oroboros oxygraph-2K). Five milligrams of midcardiac tissue was homogenized and analyzed using the established SUIT-protocol (35). To estimate the amount of mitochondria in the heart, we conducted qPCR assays using SYBRgreen (Bio-Rad) chemistry. The amount of mtDNA was analyzed by calculating the ratio of Cytochrome-B (mitochondrial gene) to Beta-actin (nuclear gene); sequences of these primers are listed in Table S1.

Quantification of EC Apoptosis.

HCMECs/HCAECs were grown in gelatin-coated 96-well plates. Before DOX treatment, the cells were incubated for 12 h in conditioned medium from either AAV–VEGF-B or AAV–HSA-transfected cells and then exposed to 0.5 or 1.0 µM DOX. The cells were imaged every second hour for 48 h with the IncuCyte live imaging system, and apoptosis was assessed using fluorescent Caspase3/7 activity reporter (Essen Bioscience). Altogether 12 identical replicates (n = 12) were analyzed, and puromycin, a known strong apoptosis inducer, was used as a positive control.

Tube Formation.

HCAECs (20,000 cells per well) were plated in a 48-well plate coated with Geltrex Basement membrane. Cells were pretreated with VEGF-B–conditioned media for 12 h, and then DOX was given in different concentrations from 0.1 to 0.5 μmol/L. The wells were imaged and tube length and the number of junctions were quantified at 12, 24, and 48 h.

Immunohistochemical Analysis.

Heart sections (8 µm thick) were fixed in cold acetone for 10 min and blocked using donkey immuno mix solution: normal donkey serum (5%), 10% BSA (2%), and 0.3% Triton-x-100 (Fisher Scientific) in PBS for 1 h in room temperature. The primary antibodies in 1% BSA in PBS were incubated overnight at +4 °C. Antibodies against the following proteins were used: γH2ax (Abcam, AB22551), activated caspase-3 (R&D, AF835), laminin (Thermo Scientific, RB-082), and VEGFR2 (R&D, AF644). The sections were then washed with Tris NaCl Tween 20 (TNT) buffer solution, incubated with Alexa Fluor secondary antibodies, and mounted using Vectashield mounting medium with Dapi (Vector Laboratories). Stained sections were imaged using Zeiss Axioplan epifluorescence microscope equipped with Zeiss AxioCam HRm camera and analyzed using ImageJ software.

H&E Staining.

Cardiac cross-sections at the midventricle level were stained using H&E protocol. The sections were viewed and imaged with a Leica DM LB research microscope equipped with Olympus DP50 color camera. Posterior and septal wall thicknesses were quantified.

TUNEL Staining.

DNA fragmentation was detected from formalin-fixed sections of the LV free wall by using 17–141 TUNEL Apoptosis Detection Kit from Chemicon International and the manufacturer’s protocol. From each heart, a cross-section at the midventricle level was used for staining, and all TUNEL-positive cells per 40× microscope field were counted. Sections were counterstained with DAPI (4′6-diamidino-2-phenylindole) and podocalyxin as endothelial cell marker (AF 1556, R&D) to determine the total number and localization of the nuclei.

RNA Extraction, Reverse Transcription, and Quantitative Real-Time PCR.

Cells were lysed and total RNA was extracted using the Nucleospin RNA II Kit (Macherey-Nagel). Cardiac tissue was first lysed with Trisure reagent (Bioline), and RNA was purified with the Nucleospin RNA II Kit (Macherey-Nagel). cDNA synthesis was performed with the iScript cDNA synthesis kit (Bio-Rad). qPCR was performed with the iQ SYBR green supermix (Bio-Rad), and relative gene expression levels were calculated using the Eq. 2–ΔΔCt. Primers are listed in Table S1.

Whole-Genome Microarray.

The quality of RNA was determined with Bioanalyzer (Agilent Technologies) and analyzed on genome-wide Illumina Mouse WG-6 v2 Expression BeadChips (Illumina). Illumina’s GenomeStudio software was used for initial data analysis and quality control, and further data analysis was performed with Chipster software (chipster.csc.fi) (36). After quantile normalization, statistically significant differences in individual genes between the groups were tested using Empirical Bayes statistics and the Benjamini–Hochberg algorithm controlling FDR. Adjusted FDR values of P < 0.05 were considered significant. The microarray data have been submitted to the GEO database under accession number GSE81448. n = 5 in each group.

Gene Set Enrichment Analysis.

The gene expression dataset was transferred to the Gene Set Enrichment Analysis software (software.broadinstitute.org/gsea/) (37, 38), and the analysis was carried out with default parameters, except that the “exclude smaller sets” was set to 25 and gene permutation was applied. A modified list of the kegg.v3.1.symbols gene set was used (software.broadinstitute.org/gsea/msigdb).

DOX Measurement in Serum, Heart, and Tumor Tissue.

The concentrations of DOX, doxorubicinole, doxorubicinolone, and doxorubicinone were measured with an Agilent 1100 HPLC system (Agilent Technologies) coupled to an AB Sciex API 2000 tandem mass spectrometer, as previously described (39), with slight modifications. Briefly, the serum and homogenized tissue (100 µL) proteins were precipitated with 300 µL acetonitrile containing 1% formic acid and the internal standard (daunorubicine). The supernatant phospholipids were removed using HybridSPE Phospholipid cartridges (30 mg; Supelco). Chromatography was performed on a Kinetex XB C18 column (2.1 × 100 mm; Phenomenex) using gradient elution. The mobile phase consisted of 5 mM ammonium acetate or acetonitrile with 0.2% formic acid. The mass spectrometer was operated in positive multireaction monitoring mode, and the mass-to-charge (m/z) ion transition [M+H]+ monitored was 544–396.8 for DOX. The limit of quantification and coefficient of variation (CV) were 4.0 ng/mL and 6.9% in serum and 100 pg/mg and 11.9% in tissue samples (n = 5). Doxorubicinole, doxorubicinolone, and doxorubicinone were not found in the samples in measurable levels.

Statistical Analysis.

Values are indicated as mean ± SD. Statistical analysis of multiple groups was performed with one-way or two-way ANOVAs, followed by Bonferroni or Holm–Sidak post hoc tests. All statistical tests were two-tailed. Differences were considered statistically significant at P ≤ 0.05. In graphs, *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001.

Acknowledgments

We thank Dr. Ulrich Pohl, Dr. Andrey Anisimov, and Dr. Su Ngyuen for their helpful comments and Robert Leigh for proofreading the manuscript. Karthik Amudhala Hemanthakumar, Tanja Laakkonen, Maria Arrano-Kivikko, Kirsi Lintula, Tapio Tainola, Maija Atuegwu, Jennifer Paech, and Jouko Laitila are acknowledged for excellent technical assistance. We also thank the Biomedicum Imaging Unit, the AAV Gene Transfer and Cell Therapy Core Facility of Biocenter Finland, as well as the Laboratory Animal Centre of the University of Helsinki for professional services. Funding was provided by the Wihuri Foundation, Academy of Finland (including TCB-CoE Grants 271845 and 275922), Cancer Society of Finland, Sigrid Juselius Foundation, European Research Council (Grant ERC-2010-AdG-268804), and the Leducq Foundation (Grant 11CVD03). M.R. received support from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7/2007-2013 under REA Grant 317250 and M.R. and R. Kivelä from the Finnish Foundation for Cardiovascular Research.

Footnotes

The authors declare no conflict of interest.

Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE81448).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1616168113/-/DCSupplemental.

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