1. Volume 26, Issue 3, Pages 902-916 (March 2018)
Therapeutic Benefit and Gene Network Regulation by Combined Gene Transfer of Apelin, FGF2, and SERCA2a into Ischemic Heart 
Edith Renaud-Gabardos, Florence Tatin, Fransky Hantelys, Benoît Lebas, Denis Calise, Oksana Kunduzova, Bernard Masri, Françoise Pujol, Pierre Sicard, Philippe Valet, Jérôme Roncalli, Xavier Chaufour, Barbara Garmy-Susini, Angelo Parini, Anne-Catherine Prats 
Molecular Therapy 
Volume 26, Issue 3, Pages (March 2018)
DOI: /j.ymthe
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

2. Molecular Therapy 2018 26, 902-916DOI: (10.1016/j.ymthe.2017.11.007)
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

3. Figure 1
Validation of Lentivector Transduction into Mouse Ischemic Heart
(A) Schema of the timeline of lentivector injection into mouse ischemic heart model. Myocardial infarction (MI) was generated as described in the Materials and Methods by ligation of the left anterior descending coronary artery, and lentivectors were immediately injected at the border between the ischemic area and the healthy area of the myocardium (2–2.5 × 106 TU). The black horizontal arrow shows the time in weeks. Week 0 (W0) corresponds to surgery. Times of vector injection, VEVO echocardiography, and sacrifice for further analyses are indicated with vertical arrows. (B and C) In order to validate lentivector transduction and stable expression in ischemic heart, a lentivector carrying the luciferase gene LucF (Lenti LucF, schematized above the histogram) was injected as described in (A). Mice were sacrificed at 1 or 7 weeks. Total RNA was purified and gene expression measured by qRT-PCR using a primer couple targeting the 3′ end sequence of the lentivector cassette. Relative transgene expression is shown as mean ± standard error of the mean, *p < 0.05, **p < 0.01 (B). Luciferase was detected at 7 weeks in tissue sections of the ischemic area (see schema on the right) by immunostaining with anti-LucF antibody. Scale bar, 50 μm. Presence or absence of MI and lentivector transduction (Lenti LucF) are indicated by + or −, respectively (C). (D and E) To validate transgene expression from an IRES-based lentivector in ischemic heart, a bicistronic lentivector carrying renilla (LucR) and firefly luciferase genes separated by the FGF1 IRES (Lenti RiF, schematized above the histograms) was administrated to mice as described in (A). LucR and LucF activities were measured 7 weeks after injection from extracts of heart ischemic area. LucR activity reflects expression of the first cistron (D), whereas the LucF/LucR ratio reflects the IRES activity, thus expression of the second cistron (E). Presence or absence of MI and lentivector transduction (Lenti RiF) are indicated by + or −, respectively. The value measured in the non-transduced heart corresponds to autoluminescence always observed in blood tissue. LucR activity and LucF/LucR ratio are shown as mean ± standard error of the mean, *p < 0.05, ***p < (n = 3–7 mice per group).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

4. Figure 2
Stimulation of HUVEC Tubulogenesis by Transduced HL-1 Cell-Conditioned Media
(A and B) HL-1 cardiomyocytes were transduced by monocistronic and bicistronic lentivectors carrying apelin and/or FGF2 cDNA (see schemas). Transgene expression was measured by qRT-PCR from HL-1 total RNAs 48 hr after transduction using primer couples targeting apelin (A) or FGF2 (B). For each transduction, the name of the transgene is indicated. A, F, or AF means the monocistronic cassette apelin or FGF2 or the bicistronic cassette apelin-IRES-FGF2, respectively. Quantifications are represented as mean ± standard error of the mean, *p < 0.05, **p < 0.01, ***p < (C–F) To determine the angiogenic potential of the lentivectors carrying apelin, FGF2, or apelin-FGF2 expression cassette, conditioned media of transduced HL-1 cells were used in a tubulogenesis assay. To assess the ability of HUVECs to form vessel-like structures in culture, they were plated on Matrigel in the presence of conditioned medium from transduced HL-1 cells, as described in the Materials and Methods. The positive control (C+) corresponds to medium with 2.5% serum. Negative controls correspond to medium with 0.25% serum (C−) or to conditioned media from non-transduced HL-1 cells (−). Conditioned media of HL-1 transduced by lentivector apelin, FGF2, or apelin-FGF2 are indicated by A, F, and AF, respectively. Cultures were photographed (C), and the mean number of branch points was determined (D). Branch points were normalized by apelin (E) and FGF2 (F) mRNA amounts to determine the biological activities of conditioned media. Studies were performed in triplicates. Histograms correspond to means ± standard error of the mean, ***p < 0.001, ****p < compared to −; #p < 0.05 compared to apelin.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

5. Figure 3
Stimulation of Angiogenesis by Therapeutic Lentivectors in Mouse Ischemic Heart
(A) Mono- and bicistronic lentivectors carrying apelin or apelin-FGF2 expression cassettes, as well as the control lentivector RiF, were injected into ischemic heart according to the timeline described in Figure 1A. qRT-PCR was performed on total RNA from heart ischemic zones, using primer couples targeting either the transgene mRNA 3′ region (PTRIP3′, common to all constructs) or endogenous apelin or endogenous FGF2 (Tables S1 and S2; mean ± standard error of the mean, *p < 0.05, compared to sham [−]; n = 3–7 mice per group). (B and C) Tissue sections were immunostained 7 weeks after lentivector injection with anti-apelin antibody to visualize transgene expression (B). Apelin quantification is shown by histograms (C) (mean ± standard error of the mean, **p < 0.01, ***p < compared to RiF; n = 3 or 4 mice per group). (D–F) Capillary density was visualized with anti-CD31 antibody in peri-infarct border zones after treatment with mono- or bicistronic lentivector (D). CD31 quantification is shown by histograms (E and F). Quantification is expressed as vessel number (D) and vessel size (E) (mean ± standard error of the mean, *p < 0.05, **p < 0.01 compared to RiF; #p < 0.05, ####p < compared to A; n = 3–6 mice per group). Treatment with lentivector control, apelin, or apelin-FGF2 is indicated by RiF, A, and AF, respectively. Scale bar, 50 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

6. Figure 4
Improvement of Heart Echocardiography Parameters following Treatment by Therapeutic Lentivectors
(A) Lentivectors carrying apelin, apelin-FGF2, or apelin-FGF2-SERCA2a expression cassettes, as well as the control lentivector RiF are represented. (B) Expression of SERCA2a (green) from the tricistronic lentivector (AFS) was detected after HL-1 cell transduction using anti-SERCA2a antibody. (C) The four lentivectors were injected into ischemic heart according to the timeline described in Figure 1A, as in Figure 3. Transgene or endogenous SERCA2 expression was measured by qRT-PCR on total RNA from heart ischemic areas, using specific primer couples (PTRIP3′ or SERCA2; Tables S1 and S2; mean ± standard error of the mean, n = 3–7 mice per group). (D and E) Capillary density was measured for AF and AFS lentivectors as in Figure 3. Vessel number (D) and size (E) are presented (mean ± standard error of the mean; n = 5 mice per group). (F–I) Echocardiography was performed 7 weeks after lentivector injection. Ejection fraction (F), representative echocardiograms (G), and left ventricular dilation (H and I) are shown (mean ± standard error of the mean, *p < 0.05 compared to RiF; n = 11–18 mice per group). Presence or absence of myocardial infarction (MI) is indicated by + or −. Treatment with lentivector control, apelin, apelin-FGF2, or apelin-FGF2-SERCA2a are indicated by RiF, A, AF, and AFS, respectively.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

7. Figure 5
Prevention of Heart and Cardiomyocyte Hypertrophy following Treatment by Therapeutic Lentivectors
Lentivectors carrying apelin, apelin-FGF2, or apelin-FGF2-SERCA2a expression cassettes. as well as the control lentivector RiF, were injected into ischemic heart (see schemas, Figure 4A). (A) Heart hypertrophy was analyzed by measurement of heart weight reported to mouse weight 7 weeks after surgery (mean ± standard error of the mean, ***p < compared to RiF; #p < 0.05, compared to A; n = 5–23 mice per group). (B and C) Cardiomyocyte hypertrophy was analyzed by measurement of cardiomyocyte area into septum region using wheat germ agglutinin (WGA) staining of tissue sections (mean ± standard error of the mean, *p < 0.05, ***p < compared to RiF; n = 3 or 4 mice per group) (B). Representative pictures are shown (C). Scale bar, 50 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

8. Figure 6
Fibrosis Prevention following Treatment by Therapeutic Lentivectors
(A–C) Fibrosis was analyzed by Sirius red staining of ischemic heart tissue sections. Whole sections are presented (A) as well as enlargements of distal non-infarct and peri-infarct zones (B). Scale bar, 100 μm (B). Collagen deposition areas were quantified in distal non-infarct (C) and peri-infarct zones (D) (mean ± standard error of the mean, *p < 0.05, **p < 0.01, ****p < compared to RiF; n = 3–7 mice per group). Treatment with lentivector control, apelin, apelin-FGF2, or apelin-FGF2-SERCA2a are indicated by RiF, A, AF, and AFS, respectively. Scale bars, 1 mm (A) and 100 μm (B).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

9. Figure 7
Effect of the Treatment with Therapeutic Vectors on the Transcriptome in Ischemic Heart
(A–D) Total RNA was purified from ischemic areas after 7 weeks of therapeutic vector treatment. cDNA was synthesized and used for a Fluidigm DELTAgene Assay, which is a PCR array dedicated to genes related to angiogenesis (A), anti-angiogenesis (B), cardiac remodeling and function (C), fibrosis (D), or stress, inflammation, or signaling (Figure S1). Messenger RNA levels are presented by histograms for treatment with lentivector control (RiF, black boxes), apelin (A, blue boxes), apelin-FGF2 (AF, purple boxes), or apelin-FGF2-SERCA2a (AFS, orange boxes) as the fold-change of repression (on the left of the axis) or induction (on the right of the axis) normalized to the group without myocardial infarction (mean ± standard error of the mean, n = 3–7 mice per group). The detailed values are presented in Table S1.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions