Fig 2: 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 show individual values and mean ± SEM. n = 5–8 mice per group. *P < 0.05, ***P < 0.001 vs. vehicle; # P < 0.05, ### P < 0.001 vs. WT. (H) Immunohistochemistry (scale bars: 100 µm) of BPIFB4, SDF-1 and myosin heavy chain isoform alpha (MyHC-a) expression (all in green), a-sarcomeric actin in red, and DAPI blue. Triangles indicate cardiomyocytes co-expressing the three proteins.

Fig 3: 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) Dose–response curves to nitroglycerine (10-9 M to 10-5 M). n = 5 experiments. All data are given as means ± SEM.

Fig 4: 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 a-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.

Fig 5: 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 a-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-a). 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-a and increased ratio between MyHC-a and MyHC-ß. 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 a-smooth muscle actin (red) positive smooth muscle cells, with nuclei identified by DAPI (blue). Scale bars: 50 µm. Graph showing the relative abundance of capillaries (n = 7 to 10 mice per group). Individual values and mean ± SEM. **P < 0.01 vs. vehicle, ## P < 0.01 vs. WT. (E) Lipid accumulation. Representative images of Oil red O staining and graph showing morphometric data of lipid accumulation expressed as percentage of the area section. Scale bars: 5 µm. Individual values and mean ± SEM. n = 4 to 5 mice per group. **P < 0.01 vs. vehicle, ## P < 0.01 vs. WT. (F,G) Interstitial (F) and perivascular (G) fibrosis (triangles point to blood vessels). Representative images of picrosirius red staining and graph showing morphometric data of fibrosis. Scale bars: 50 µm. Individual values and mean ± SEM; n = 5 mice per group. *P < 0.05 and **P < 0.01 vs. vehicle, ## P < 0.01 vs. WT. (H) Cardiac Col1a1 mRNA individual values and mean ± SEM; n = 6 per group. (I) Western blot of cardiac atrial natriuretic peptide (ANP). Individual values and mean ± SEM; n = 6 per group. (J) Immunoreactive ANP levels in peripheral blood. Individual values and mean ± SEM; n = 8 to 10 per group.