Semaphorin3A Exacerbates Cardiac Microvascular Rarefaction in Pressure Overload‐Induced Heart Disease

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  Scheme 1. Schematic diagram of this study This schematic presentation depicts the mechanism by which the Sema3A‐positive sEV/NRP1 axis meditates microvascular rarefaction. The mechanism of impaired angiogenesis involves Sema3A‐positive sEVs competing with VEGF‐A to bind to NRP1, thus inhibiting the adaptive angiogenic response. In addition, communication between MiVECs and fibroblasts via Sema3A‐containing sEVs causes myofibroblast activation and promotes the development and progression of cardiac fibrosis.

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  Figure 1. Angiogenic capacity and transcriptome alterations of MiVECs in cardiac hypertrophy. A) Schematic diagram of angiotensin II (Ang II)‐induced cardiac diastolic dysfunction in mice and its detection at the corresponding time points. B) Capillaries were stained with the endothelial cell marker CD31 (red), myocardial tissue was stained with cTnt (green), and nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. n = 6 mice per group. C) Overview of the experimental design for isolation and detection of MiVECs in vitro. D) MiVEC proliferation evaluation performed using the EdU assay. n = 6 mice per group. E) Representative images of migrated MiVECs by the transwell assay. Scale bar = 200 µm. n = 6 mice per group. F) Representative images and quantification of MiVECs in the tube formation experiment, Scale bar = 100 µm. n = 6 mice per group. G) Heatmap of RNA‐sequencing analysis showed that gene expression was significantly upregulated in MiVECs isolated from the hearts of the Ang II‐induced group compared with the saline control group. For all statistical plots, the data are presented as the mean ± SEM. *p < 0.05 between the two indicated groups by 2‐tailed Student's t‐test.…

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  Figure 2. Kinetics of Sema3A expression in cardiac pressure overload‐induced cardiac hypertrophy. A) Representative images of immunoblotting analysis of Sema3A protein expression in Ang II‐infused mouse hearts after 4 weeks. The protein expression was quantified and normalized to that of β‐actin. n = 6 mice per group. B) Representative immunoblotting of Sema3A levels after 4 weeks in serum collected by cardiac puncture from an Ang II‐infused mouse heart. Blots stained with Coomassie brilliant blue served as loading controls (n = 6 mice per group). C) Sema3A serum concentration (pg mL−1) measured using ELISA; n = 6 mice per group. D) Double immunofluorescence staining of Sema3A (green) and CD31 (red) in Ang II‐infused mouse hearts. The nuclei were stained with DAPI (blue). Immunoglobulin G (IgG) antibodies were used as negative controls. Scale bar = 20 µm. E) Immunoblotting analysis of the expression of Sema3A in cardiac fibroblasts (Fibrob.), cardiomyocytes (Cardiom.), white blood cells (WBC), and MiVECs isolated directly from the heart. n = 5 mice per group. F) Representative double immunofluorescence staining images of Sema3A (red) and the vascular endothelial cell marker CD31 (green) in…

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  Figure 3. Loss of Sema3A attenuated the Ang II‐Induced dysfunction of MiVECs. A,B) Representative immunoblotting and quantification of Sema3A protein expression after lentivirus carrying Sema3A shRNA (shSema3A) and control shRNA (shCtrl) transfection in the presence or absence of Ang II treatment. n = 3 samples per group. C) Representative images of EdU incorporation assays (left panel) and quantification of EdU‐positive cells (right panel). Scale bar = 100 µm. n = 6 samples per group. D) Representative images of transwell migration assays and quantification of the migrated number of MiVECs after Sema3A knockdown. Scale bar = 100 µm. n = 5 samples per group. E) Representative images and quantification of MiVECs in the tube formation experiment after Sema3A knockdown. Scale bar = 100 µm. n = 5 samples per group. *p < 0.05, versus shCtrl group; n = 5. All data are presented as mean ± SEM, *p < 0.05. ns indicates no significant difference between the two indicated groups by one‐way analysis ANOVA. WCL, whole cell lysate. CBB, Coomassie brilliant blue staining; EdU, 5‐ethynyl‐2′‐deoxyuridine.

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  Figure 4. SRF promotes Sema3A expression in MiVECs. A) Venn diagrams of the four transcription factors (TFs) screened from databases and RNA sequencing analysis. B) Quantitative RT‐PCR (RT‐qPCR) analysis of relative mRNA expression (n = 3 samples per group). C) Immunoblotting detection of SRF protein expression in total cell lysates and nuclear fractions of MiVECs treated with or without Ang II (1 µM for 24 h). β‐actin and Histone H3 were loading controls for the total cell lysate and nuclear extracts, respectively. *p < 0.05, compared to the control group; n = 3 samples per group. D) Representative immunoblotting images (left panel) and quantification (right panel) of SRF and Sema3A protein expression after transfection with shSRF or shSema3A after Ang II treatment. *p < 0.05, compared with the control shRNA group; # p < 0.05, versus the control shRNA group; n = 3 samples per group. E) SRF binding sequence in the Sema3A promoter region predicted by JASPAR software; the predicted potential binding sequence and score are shown. F) Schematic diagram of the Sema3A promoter and deletion mutants. G) The original plasmid and digested DNA fragments were analyzed by agarose gel electrophoresis. P…

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  Figure 5. SRF knockdown attenuated the Ang II‐Induced dysfunction of MiVECs. A,B) Representative Immunoblotting showing the knockdown efficiency of SRF shRNA (shSRF) and control shRNA (shCtrl) in with or without Ang II treatment. n = 3 samples per group. C) Effect of SRF knockdown tested by shSRF on cell proliferation. Scale bar = 100 µm. n = 6 samples per group. D) Representative images of transwell migration assays and quantification of the number of migrated MiVECs after SRF knockdown. Scale bar = 100 µm. n = 5 samples per group. E) Representative images and quantification of MiVECs in tube formation experiments after SRF knockdown. Scale bar = 100 µm. n = 5 samples per group. All data are presented as mean ± SEM, *p < 0.05. ns indicates no significant difference between the two indicated groups by one‐way analysis ANOVA.

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  Figure 6. Sema3A impaired angiogenic potential by competitively binding to NRP1 with VEGFA. A) Computational prediction of Sema3A protein partner network visualization on the STRING website. B) General domain structure of NRP1. C) Interaction between endogenous Sema3A and NRP1 detected by co‐immunoprecipitation (co‐IP) experiments in MiVECs treated with Ang II. D) Representative co‐IP analysis of NRP1, Sema3A, and VEGFA in PBS‐treated or Ang II‐treated MiVECs. E) Decreased combination of Sema3A with VEGFA in MiVECs transfected with shNRP1. F) Co‐IP analysis of NRP1, Sema3A, and VEGFA in MiVECs infected with adenovirus (Ad)‐Sema3A or Ad‐VEGFA. G) Schematic representation of proximity ligation assay (PLA) detection and analysis of NRP1 interaction (protein‐protein interaction) with Sema3A in MiVECs treated with or without Ang II. I) Representative images and H) quantification of proximity ligation assay (PLA) analysis showing the interaction of Sema3A with NRP1 in MiVECs. PLA‐positive spots are shown in green. Nuclei were counterstained with DAPI (blue). No antibodies: PLA was performed in the absence of primary antibodies. Scale bar = 25 µm. n = 12 samples per group. J) Schematic of PLA…

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  Figure 7. sEV‐mediated secretion of Sema3A impaired angiogenic potential. A) Electronic microscopy analysis of sEVs stained by immunogold for Sema3A. The high‐density dots indicated by the red triangles represent the immunogold labelling of the Sema3A protein. Scale bar = 50 nm. B) Schematic workflow of sEV immunofluorescence staining. C) Immunofluorescence of sEVs from cell supernatants of MiVECs and mouse plasma samples stained with CD63‐Alexa Fluor 647 and Sema3A‐Alexa Fluor 488 antibodies. The yellow spots represent sEVs that contain both CD63 and Sema3A proteins. Scale bar = 30 µm; Ab indicates the antibody. D) Immunoblotting (left panel) and densitometric analysis (right panel) of Sema3A, NRP1, and VEGFA in sEVs obtained from MiVECs with or without treatment with Ang II. *p < 0.05, compared to the control group. n = 4 per group. E) Bivariate dot plots of FITC fluorescence versus SS‐A for sEVs preparations from MiVECs. sEVs were fluorescently labelled with FITC‐conjugated mAbs specific to Sema3A. F,G) Rab27a, a key gene involved in extracellular vesicle biogenesis, was silenced by shRNA to block sEVs secretion. F) Immunoblotting images and G) quantification of NRP1, SRF, Sema3A,…

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  Figure 8. Sema3A deficiency attenuated pressure overload‐induced cardiac dysfunction and capillary rarefaction. A,B) Schematic illustration of the injection of AAV9‐ENT‐ICAM2 in vivo: Control (shCtrl) or AAV‐ENT‐ICAM2‐shSema3A (shSema3A) into mouse hearts. AAV‐ENT‐ICAM2: A serotype targeting vascular endothelial cells adeno‐associated virus with endothelial‐specific promoter ICAM2. C) Graphs showing the time course of the E/e' ratio (upper panel) and E/A ratio (lower panel). D) Gross morphology (upper panel, Scale bar = 2 mm) and histopathological sections stained with hematoxylin and eosin (H&E) (lower panel, Scale bar = 1 mm) of the shCtrl and shSema3A hearts after saline or Ang II infusion. E) Quantification of capillary density based on CD31 staining: red, CD31; green, cTnT; and blue, nuclei. Scale bar = 50 µm. n = 6 mice per group. F) Cardiomyocyte apoptosis assessed by TUNEL staining (red), cTnT marks cardiomyocytes (green), and DAPI marks nuclei (blue). Scale bar = 50 µm. n = 6 mice per group. G) Immunoblotting analysis of fibrosis‐related proteins (Collagen I, Collagen III, and α‐SMA) in AAV‐CON or AAV‐shSema3A hearts after infusion with saline control or Ang II (n = 6 mice per…