Beneficial effects of vascular endothelial growth factor B gene transfer in the aged heart

Figures

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  Figure 1. Vegfb gene expression is declined with age. (A, B) Published transcriptomic data of hearts derived from 2-month-old (young) vs. 18- to 20-month-old (old) mice28 (n = 4). Panel (A) shows Vegfa and panel (B) shows Vegfb expression. (C–F) Vegfb expression detected by RT-qPCR on whole heart tissues from 3-month-old (young) vs. 18- to 20-month-old (old) mice (C, n = 11), in isolated cardiac endothelial cells from 3-month-old (young) vs. 18− to 20-month-old (old) mice (D, n = 7), in isolated fibroblasts (FB, E, n = 3) and cardiomyocytes (CM) from 3-month-old (young) vs. 24-month-old (old) mice (F, n = 4 vs. n = 3). (G, H) Vegfb expression data derived from published bulk RNA sequencing data from isolated cardiac endothelial cells (G, n = 6)24 and cardiac fibroblasts (H, n = 4 vs. n = 3).29 (I) VEGFB expression in human cardiac endothelial cells, fibroblasts, and cardiomyocytes. Data are derived from published human cardiac snRNA sequencing data of LV samples from individuals being below or above 55 years old.30 Data are shown as mean and error bars indicate SEM. Data are Gaussian distributed and P-value was assessed by two-tailed, unpaired Student’s t-test (A–H). To calculate P-values…

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  Figure 2. Vegfb overexpression attenuates fibrosis and denervation. (A, B) Eighteen-month-old mice were treated with AAV9 to overexpress Vegfb (AAV9 Vegfb). Empty vectors served as control (AAV9 Control). Vegfb induction was determined in heart samples by RT-qPCR against Vegfb186 and Vegfb167 (panel A, n = 5) and by ELISA (panel B, n = 5). (C) Picrosirius red staining in hearts of aged mice treated with AAV9-Vegfb vs. control (n = 5). (D) Human cardiac fibroblasts treated for 72 h with recombinant TGF-β2, VEGFB, and both in combination, immunostained with Phalloidin, Collagen 1a1, aSMA, and DAPI (n = 3). (E, F) Histological staining for Isolectin B4 (IB4, green), WGA, and DAPI in hearts of aged mice treated with AAV9-Vegfb vs. control (n = 5) to assess capillary count and perimeter. Panel (F) shows the quantification of capillary count and perimeter (μm) in the endocardium and epicardium (n = 5). (G) Assessment of endothelial proliferation (yellow arrow) using phosphor histone 3 (PH3), isolectin B4 (IB4), and DAPI in hearts of aged mice treated with AAV9-Vegfb vs. control (n = 5). (H) Angiogenic sprouting was determined by aortic ring assay applied on aortic rings from aged mice treated…

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  Figure 3. AAV9-mediated overexpression of vegfb rescues age-related cardiac denervation. (A–C) Histological assessment of left ventricular innervation in aged mice after AAV9-control vs. AAV9-Vegfb treatment. Axons were marked in green with TUJ1 (A, n = 5 vs. n = 4) or tyrosine hydroxylase-TH (B, n = 5). Sensory fibres were marked in red with calcitonin gene-related peptide—CGRP (C, n = 5). DAPI served as counterstain. (D) Neurons were isolated from the stellate ganglia (SG) from aged mice 8 weeks after AAV9-control vs. AAV9-Vegfb treatment. Neurons were cultured for 72 h and axon outgrowth was determined by TUJ1 and DAPI immunostaining (n = 4 vs. n = 5). (E) Mouse cortical neurons were cultured in the presence and absence of recombinant VEGFB. Axon outgrowth was determined by MAP-2 staining and apoptosis was determined by cleaved Caspase 3 staining (n = 3). (F) Mouse cortical neurons were cultured in supernatants from long-passaged (P15) human umbilical vein endothelial cells. The supernatant was supplemented with recombinant VEGFB. Axon outgrowth was determined by MAP-2 staining (n = 6). (G) Cardiac senescence was determined in hearts from aged mice after AAV9-Control vs. AAV9-Vegfb…

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  Figure 4. AAV9-mediated overexpression of vegfb delays age-related diastolic dysfunction but induces cardiac hypertrophy in aging heart. (A) Schematic representation showing the experimental design. (B–F) Echocardiography of 18-month-old mice before (baseline) and 4 and 8 weeks after AAV9-control/AAV9-Vegfb injection (n = 10). (B) Systolic function represented as ejection fraction [EF (%)]. (C) Diastolic function represented as MV (E/E′−1). (D) Left ventricular mass. (E, F) Left ventricular wall thickness at systole and diastole (n = 5). (G) Heart weight as determined by heart weight vs. tibia length or body weight in aged mice 8 weeks after AAV9 injection (n = 10). (H–J) Histological assessment of cardiac hypertrophy in the sub-endocardial and sub-epicardial area by WGA staining. IB4 and DAPI served as counter stain (n = 5). (K) Immunostaining of the cardiomyocytes against phosphor histone 3 (PH3), WGA, and DAPI (n = 5). (L) Neonatal rat cardiomyocytes cultured in the presence and absence of phenylephrine (PE), recombinant VEGFB, and PE + VEGFB (n = 3). Data are shown as mean and error bars indicate SEM. Data are Gaussian distributed and P-value was assessed by two-tailed, unpaired…

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  Figure 5. STAT3 contributes to VEGFB-induced cardiac hypertrophy. Single-nuclei RNA sequencing of hearts from aged mice after 8 weeks after AAV9-Control vs. AAV9-Vegbf injection (n = 3). (A) UMAP plot representing the distinct cell types of the heart in all conditions. (B) UMAP plots showing different cell type markers for cardiomyocytes (Tnnt2), endothelial cells (Cdh5), fibroblasts (Pdgfra), immune cells (CD74), epicardial cells (Krt19), and pericytes (Rgs5). (C) PROGENy pathway analysis of differentially regulated genes in the cardiomyocyte of AAV9-Vegfb vs. AAV9-control treated aged mice. (D) Dot plot displaying JAK-STAT-related genes that are dysregulated in the cardiomyocyte cluster. (E, F) STAT3 target expression (E) and STAT family expression levels (F) displayed as mean UMI in the cardiomyocyte cluster of AAV9-Vegfb vs. AAV9-Control treated aged mice (n = 3). (G) Western blot of phosphor-STAT3 and total STAT3 vs. a-tubulin in whole hearts of AAV9-Vegfb vs. AAV9-Control treated aged mice (n = 5). (H, I) VEGF receptor expression levels displayed as mean UMI in the cardiomyocyte (H) and endothelial cells cluster (I) of AAV9-Vegfb vs. AAV9-Control treated aged mice (n = 3). (J)…

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  Figure 6. VEGFB induces cardiac hypertrophy in aged mice. (A) Length-width ratio of cardiomyocytes after TAC surgery (n = 3). (B) Shown is the length-width ratio of cardiomyocytes from young vs. aged mice (n = 5) and from aged mice 2 months after AAV9-control or AAV9-Vegfb treatment (n = 5). (C) Length-width ratio of cardiomyocytes from young mice with and without AAV9-Vegfb treatment (n = 3, 4 weeks of treatment). (D) Length-width ratio of cardiomyocytes from young mice after 2 months of voluntary exercise (n = 5). (E–G) Expression of Nppa and Nppb in sham vs. TAC-operated mouse hearts (E, n = 3), in hearts from aged AAV9-control vs. AAV9-Vegfb treated mice (F, n = 3) and in young vs. old whole mouse tissue (G, n = 4). (H) Expression of miR-222-3p in hearts from aged AAV9-Control vs. AAV9-Vegfb treated mice (n = 10). (I, J) Immunolabelling of CD45 + leucocytes (I) and CD68 + macrophages (J) in hearts from aged mice 2 month after AAV9-control or AAV9-Vegfb treatment (n = 5). (K) Comparison of Nfkb, Il6, Tnfa, Il10 transcript levels in hearts from aged AAV9-control vs. AAV9-Vegfb treated mice (n = 5). P-values were calculated using two-sided t-test (A–J), Mann–Whitney test (K) or…