Volume 20, Issue 12, Pages 2212-2221 (December 2012) AAV9-mediated VEGF-B Gene Transfer Improves Systolic Function in Progressive Left Ventricular Hypertrophy  Jenni Huusko, Line Lottonen, Mari Merentie, Erika Gurzeler, Andrey Anisimov, Atsushi Miyanohara, Kari Alitalo, Pasi Tavi, Seppo Ylä- Herttuala  Molecular Therapy  Volume 20, Issue 12, Pages 2212-2221 (December 2012) DOI: 10.1038/mt.2012.145 Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Progression of left ventricular hypertrophy. Myocardial function measured by (a) ejection fraction (EF) and (b) fractional shortening (FS) was preserved 2 weeks after TAC operation but significantly deteriorated 4 and 10 weeks after the operation. Compensatory hypertrophy was shown as (d) increased left ventricle anterior wall thickness during diastole (LVAWd) and (e) decreased LV internal diameter during diastole (LVIDd). In heart failure, 4 and 10 weeks after TAC operation, (c) LV mass (LV mass), (d) LVAWd, and (e) LVIDd were increased. (f) LV volume did not change significantly. Echocardiographic measurements were done on the day of killing. Results were obtained from parasternal short axis M-mode projections and each time point is compared with the SHAM-operated group of the same time point by Student's t-test. Data in different time points is from individual set of animals and is not therefore compared over time. Mean ± SD, n = 10/group, *P < 0.05, **P < 0.01, ***P < 0.001. TAC, transversal aortic constriction. Molecular Therapy 2012 20, 2212-2221DOI: (10.1038/mt.2012.145) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Angiogenic and cardiomyocyte responses to progressive left ventricular hypertrophy (LVH). (a,e–h) Progressive LVH triggered endogenous angiogenesis seen as enlarged capillaries. (b) Ten weeks after TAC operation, the number of capillaries per myocyte was significantly increased. Progressive LVH was seen as (c) decreased number of capillaries per mm2 2 weeks after the operation and (d) cardiomyocytes per mm2 in all time points. Quantification was done from five endothelium-stained microscopic fields from each animal at ×400 magnification. Mean ± SEM, n = 6/group (in a–d), statistical analyses with one-way analysis of variance and Bonferroni's multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001. Representative pictures from endothelial stainings with Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin I, bars 50 µm (in e–h). TAC, transversal aortic constriction. Molecular Therapy 2012 20, 2212-2221DOI: (10.1038/mt.2012.145) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Fibrosis in progressive left ventricular hypertrophy. (a–i) The amount of fibrosis increased progressively in TAC-operated animals while there was no fibrosis seen in intact animals. Quantification was done in a blinded fashion by three observers screening collagen-stained microscopic sections at ×12.5 magnification using the following grading criteria: 1, minor or no fibrosis; 2, moderate fibrosis; and 3, severe fibrosis. Mean ± SEM, statistical analyses with one-way analysis of variance and Bonferroni's multiple comparison test (n = 6/group), *P < 0.05, **P < 0.01, ***P < 0.001, (b,d,f,h) hematoxylin-eosin (HE) staining and (c,e,g,i) Masson trichrome staining for collagen, bars 100 µm. TAC, transversal aortic constriction. Molecular Therapy 2012 20, 2212-2221DOI: (10.1038/mt.2012.145) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Relative mRNA expression of VEGFs, their receptors (VEGFRs, NRP-1) and mitochodrional genes (PGC-1α, CytB) in progressive LVH. (a) Relative expression of VEGF-A mRNA did not change 1 day, 2 or 4 weeks after TAC operation. (b) Relative expression of VEGF-B mRNA was decreased 4 weeks after TAC operation and (c,d,g) those of VEGF-C, VEGF-D, and VEGFR-3 mRNAs were increased 2 weeks after TAC operation. (e,f,h) The expressions of mRNA encoding VEGFR-1, VEGFR-2 or NRP-1 were not changed. (i,j) The expressions of PGC-1α and CytB mRNAs were decreased 4 weeks after TAC operation. mRNA expression of each gene was measured by RT-PCR and normalized to 18S ribosomal RNA as SHAM-operated group of each time point set to one. The relative expression of each gene in TAC-operated animals is compared with that in the SHAM group of the same time point by Student's t-test. Mean ± SEM, n = 6/group, *P < 0.05, **P < 0.01. CytB, cytochrome B; LVH, left ventricular hypertrophy; NRP-1, neuropilin-1; RT-PCR, reverse transcription-PCR; TAC, transversal aortic constriction; VEGFR, vascular endothelial growth factor receptor. Molecular Therapy 2012 20, 2212-2221DOI: (10.1038/mt.2012.145) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Progression of LVH after AAV9-VEGF-B186 gene transfer. AAV9-VEGF-B186 gene transfer (1 × 1010 viral genomes in 10 µl) sustained the systolic function ((a) EF and (b) FS) of TAC-operated mice when compared with AAV9-LacZ treated controls. (c) Left ventricle (LV) mass did not increase in VEGF-B186 treated group similarly than in LacZ control group (d) while LVAWd remained unchanged in both groups. (e) LVIDd and (f) LV volume stayed at the normal level in AAV9-VEGF-B186 treated animals, while both increased in AAV9-LacZ control animals. Echocardiographic measurements were done before the gene transfer and on the day of killing. The results were obtained from parasternal short axis M-mode projections. Mean ± SD, statistical analyses with repeated measurements two-way analysis of variance (n = 8/group), *P < 0.05. AAV, adeno-associated virus; EF, ejection fraction; FS, fractional shortening; LVAWd, left ventricle anterior wall thickness during diastole; LVH, left ventricular hypertrophy; LVIDd, left ventricle internal diameter during diastole; VEGF, vascular endothelial growth factor. Molecular Therapy 2012 20, 2212-2221DOI: (10.1038/mt.2012.145) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 The angiogenic response to AAV9-VEGF-B186 gene therapy. (a,f,g) AAV9-VEGF-B186 gene therapy significantly increased mean capillary area when compared with AAV9-LacZ group and the response was accelerated compared with the endogenous response of TAC-operated animals. Quantification was done from five endothelium-stained microscopic fields from each animal at ×400 magnification. (b–e) Gene therapy was done by direct ultrasound-guided injection and caused only a minor needle mark visible in hematoxylin-eosin (HE) staining. Mean ± SEM, statistical analyses with one-way analysis of variance and Bonferroni's multiple comparison test, n = 4/group, *P < 0.05, **P < 0.01, ***P < 0.001 (as shown in a), bars 1 mm (in b,c) and 50 µm (in d–g). HE staining (in b–e) and endothelium staining with Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin I (in f and g). AAV, adeno-associated virus; TAC, transversal aortic constriction; VEGF, vascular endothelial growth factor. Molecular Therapy 2012 20, 2212-2221DOI: (10.1038/mt.2012.145) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 The effect of AAV9-VEGF-B186 gene therapy on cell proliferation and apoptosis. AAV9-VEGF-B186 gene therapy (a,c,d) increased the number of proliferating cardiomyocytes and (b,e–h) decreased the number of apoptotic cardiomyocytes. Quantifications in a and b were done from five Ki-67 and cleaved caspase-3 microscopic fields, respectively, from each animal at ×400 magnification. Mean ± SEM, statistical analyses with Student's t-test, n = 4/group, *P < 0.05 (as shown in a,b), bars 25 µm (in c–f) and 50 µm (in g,h), Ki-67 staining for proliferating cells (c,d), and cleaved caspase-3 staining for apoptotic cells (e–h). AAV, adeno-associated virus; VEGF, vascular endothelial growth factor. Molecular Therapy 2012 20, 2212-2221DOI: (10.1038/mt.2012.145) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Relative mRNA expression of PGC-1α, mVEGF-B, and ANP after AAV9-VEGF-B186 gene therapy. AAV9-VEGF-B gene therapy increased the relative expression levels of (a) PGC-1α and (b) mVEGF-B and decreased the relative expression of (c) ANP mRNA compared with LacZ control group. mRNA expression was measured by quantitative RT-PCR and normalized to 18S ribosomal RNA. Mean ± SEM, statistical analyses with Student's t-test, n = 4/group, *P < 0.05, **P < 0.01. (d,e) ANP staining, bars 25 µm. AAV, adeno-associated virus; ANP, atrial natriuretic peptide; VEGF, vascular endothelial growth factor. Molecular Therapy 2012 20, 2212-2221DOI: (10.1038/mt.2012.145) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions