Transfer of a Human Gene Variant Associated with Exceptional Longevity Improves Cardiac Function in Obese Type 2 Diabetic Mice Through Induction of the SDF-1/CXCR4 Signalling Pathway

Figures

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  Figure 1. BPIFB4 expression in human cardiac samples. Samples were obtained from control hearts (A,E, control) and patients with ischaemic heart failure (IHF) with diabetes (B,F) or without (C,G). (A–D) BPIFB4 expression in cardiomyocytes. Fluorescence microscopy images show BPIFB4 in red fluorescence, cardiomyocytes α‐sarcomeric actin in green fluorescence, nuclei in blue (DAPI). (E–H) BPIFB4 expression in endothelial cells. BPIFB4 is in red fluorescence, CD34+ endothelial cells are shown with cyan pseudocolour, nuclei blue. All scale bars = 10 μm. The BPIFB4 channel (red) is provided also separately with the microphotographs labelled as (ii) for a better observation of the positive staining. In (D,H) data indicate the expression of BPIFB4 in cardiomyocytes (D) and endothelial cells (H) calculated as the positive volume fraction (percent of BPIFB4 positive staining in either cardiomyocytes or endothelial cells). In (D): n = 10 control, 14 diabetic, and 26 non‐diabetic. In (H): n = 10 control, 14 diabetic, and 26 non‐diabetic. Data are shown as individual values and mean. *P < 0.05 vs. controls. Red symbols correspond to carriers of the LAV‐BPIFB4 variant.

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  Figure 2. LAV‐BPIFB4 gene therapy improves cardiac function in type 2 diabetic mice. (A) Diagram of mice randomisation (n = 8 mice per group). (B) Immunohistochemistry of BPIFB4 (green fluorescence) in the whole left ventricle (bar 1 mm) and a magnified section (bar 100 μm). Bar graph showing individual values and mean ± SEM of positive staining in the three groups. **P < 0.01, ***P < 0.001 vs. the vehicle group (V). (C) Body weight assessed at 8 and 13 weeks. Individual and mean values. **P < 0.01 vs. 8 weeks. (D–E) Peripheral blood levels of C‐peptide and insulin (mean ± SEM). (F) Representative images and bar graph (mean ± SEM) of 18F‐fluorodeoxyglucose (FDG) positron emission tomography/computed tomography imaging [values expressed as percent of injected dose (ID) per gram of tissue; n = 3 per group]. (G–M) Graphs showing results of echocardiography assessments at 8 and 13 weeks. Representative images of E and A waves measured using pulsed‐wave Doppler (G) and M‐mode traces (H). Graphs showing the improvement induced by AAV‐LAV‐BPIFB4 (LAV) on indices of diastolic and systolic function: (I) deceleration time (DT), (J) fractional shortening (FS), (K) stroke volume (SV), (L) left…

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  Figure 3. LAV‐BPIFB4 promotes cardiomyocyte expression of contractile protein and prevents capillary rarefaction, lipid accumulation and fibrosis. (A) Representative fluorescence images showing areas of transversally sectioned myocardium. Cardiomyocytes identified by α‐sarcomeric actin staining (white pseudocolour) and nuclei labelled by DAPI. Scale bars: 50 μm. Right panels are higher magnifications of the areas delimited by a green outline. Graph shows morphometric data of cardiomyocyte cross‐sectional area (CSA) from individual mice (n = 5 to 6 per group) together with mean ± SEM. (B) Representative fluorescence images showing the expression of myosin heavy chain isoform alpha (MyHC‐α). Scale bar: 100 μm. Graph with individual values (n = 6 per group) together with mean ± SEM. **P < 0.01 vs. vehicle, # P < 0.05 vs. WT. (C) Western blot analysis confirming the induction of MyHC‐α and increased ratio between MyHC‐α and MyHC‐β. Individual values (n = 6 per group) with mean ± SEM. *P < 0.05 vs. vehicle, # P < 0.05 vs. WT. (D) Myocardial microvasculature. Representative images of isolectin B4 (green) positive endothelial cells and α‐smooth muscle actin (red) positive smooth muscle cells,…

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  Figure 4. LAV‐BPIFB4 induces stromal cell‐derived factor‐1 (SDF‐1) in human monocytes in vitro and in peripheral blood and hearts of diabetic mice in vivo. (A–D) Cytofluorimetric analysis of the peripheral blood mononuclear cells (PBMCs) from type 2 diabetic patients incubated with WT‐BPIFB4 or LAV‐BPIFB4 protein or vehicle for 48 h. LPS was used as a positive control stimulation. At the end of the cell culture treatment, PBMCs were recovered, stained for the different antigens and anti‐intracellular SDF‐1, and analysed by flow cytometry. (A) T cells, NK cells, neutrophils, and B cells. (B) Monocytes. (C) Frequency of SDF‐1 positive events within subclasses of monocytes. (D) Relative fluorescence intensity of SDF‐1 in the intermediate monocyte population. Individual values and mean ± SEM. n = 4 biological replicates. *P < 0.05. (E) Immunoreactive SDF‐1 levels in murine peripheral blood. Bar graphs show individual values and mean ± SEM. n = 11–13 mice per group. **P < 0.001 between longevity‐associated variant (LAV) and other groups. (F) Immunohistochemistry (SDF‐1 in green, DAPI in blue, scale bars: 100 μm) and (G) western blot analyses of SDF‐1 expression in the mice heart. Bar graphs…

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  Figure 5. Inhibition of the effects of LAV‐BPIFB4 gene therapy by an antagonist of the CXCR4 receptor. (A) Experimental protocol: seven mice entered the study but only six in each antagonist group were considered because compliant in taking the medicated jelly. All data shown as individual values and means ± SEM. (B–F) Results of echocardiography. The antagonist inhibits the effect of LAV‐BPIFB4 gene therapy on deceleration time (DT) (B), end‐systolic volume (ESV) (C), fractional shortening (FS) (D), left ventricular ejection fraction (LVEF) (E), and cardiac output (CO) (F). *P < 0.05 and **P < 0.01 vs. GFP + DMSO, # P < 0.05 and ## P < 0.01 in comparison with changes between LAV + DMSO and GFP + DMSO. (G) Capillary density (scale bars: 50 μm). Isolectin B4 (green) identifies endothelial cells, α‐smooth muscle actin (red), and DAPI nuclei (blue). Bar graph showing the results. **P < 0.01 vs. GFP + DMSO, ## P < 0.01 in comparison with changes between LAV + DMSO and GFP + DMSO. (H,I) Cardiac interstitial (H) and perivascular (I) fibrosis (blue) analysed by Azan Mallory staining (scale bars: 50 μm). ***P < 0.001 vs. GFP + DMSO, ## P < 0.01 in comparison with changes between LAV + DMSO and GFP…

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  Figure 6. LAV‐BPIFB4 gene therapy benefits vascular function in diabetic mice (study 3). (A) Systolic blood pressure (SBP) in non‐diabetic (ND) mice and obese diabetic mice treated with AAV9‐LAV‐BPIFB4 or AAV9‐GFP (n = 5 to 6 per group). Arrow indicates gene therapy delivery time point. *P < 0.05 diabetic vs. diabetic given AAV9‐GFP; # P < 0.05 ND vs. ND given AAV9‐GFP. (B) Heart rate data in the same animals. (C,D) Graphs show blood glucose levels (C) and body mass (D) before and after treatment with AAV9‐LAV‐BPIFB4 or AAV9‐GFP. *P < 0.05 vs. diabetic given AAV9‐GFP and # P < 0.05 vs. diabetic given AAV9‐LAV‐BPIFB4. (E–H) Assays performed on mesenteric arteries excised 4 weeks after gene therapy. (E) Graphs show the dose–response curves to acetylcholine (ACh, 10−9 M to 10−5 M), n = 5 experiments. *P < 0.05 and **P < 0.01 vs. ND given AAV9‐GFP; § P < 0.05 and §§ P < 0.01 vs. ND given AAV9‐GFP; # P < 0.05 and ## P < 0.01 vs. all. (F) Representative western blot of mesenteric arteries obtained from ND and diabetic mice treated with AAV9‐LAV‐BPIFB4 or AAV9‐GFP. (G) Bar graphs show optical density quantification. n = 3 experiments. Individual values and means ± SEM. *P < 0.05. (H)…