Fig 2: Only preischemic and not postischemic Nrg1ß (neuregulin 1) administration protects myocardium due to loss of endothelial-ErbB2 (Erb-B2 receptor tyrosine kinase 2) during ischemia. A, To determine the protective function of Nrg1 during ischemia or reperfusion, recombinant Nrg1ß (4 µg/kg) was injected into mice before ischemia (preischemia) or after ischemia (postischemia) but before reperfusion. After ischemia/reperfusion (I/R), the heart was isolated and perfused with annexin-V-Fe complex. The infarcted tissue was excised, and tissue bound annexin-V was quantified by electron spin resonance (EPR). B, The absolute spin count was calculated from the EPR spectra of paramagnetic-Fe bound to annexin-V and plotted as a bar graph. Values are means±SD (n=5 mice). *P<0.01 vs sham; **P<0.01 vs I/R, †P<0.01 vs Nrg1 (preischemic) plus IR (ANOVA). C, Mice were subjected to sham, I/R, remote ischemic preconditioning (RIPC) or RIPC+I/R, and the infarcted tissue region was excised. Protein extract was prepared from the excised tissue and analyzed by Western blotting for ErbB2 and ErbB4 levels. D, Sections of sham and I/R mouse heart were subjected to immunofluorescence staining using anti-ErbB2 and anti-a-actinin (cardiomyocyte marker) antibodies. Isolectin-Alexa Fluor 647 conjugate was used to stain endothelial cells selectively. Fluorescent images of the stained sections were obtained using an upright Zeiss fluorescence microscope (AxioImager Z2) via 40×/1.4 NA objective. Scale bar=20 mm. E and F, Adult mouse cardiomyocytes and mouse cardiac endothelial cells were isolated from adult mouse heart, and cell lysate was prepared. An equal amount of protein from cell lysates was analyzed for ErbB2 levels by Western blotting. Blot was reprobed for a-actinin and CD31 (F). ErbB2 levels were quantified and plotted as a bar graph (n=3). *P<0.01 vs MCEC (Student t test). G, HCAECs were pretreated with preconditioned medium (PM) or Nrg1-neutralized PM, then exposed to hypoxia/reoxygenation (H/R), and cell lysates were prepared. An equal amount of protein from each sample was analyzed for ErbB2 and ErbB4 levels by Western blotting. H, HCAECs were pretreated with or without PM and exposed to H/R. At the end of treatment, cells undergoing apoptosis were labeled with annexin V-FITC conjugate, and the percentage of apoptotic cells was quantitated by fluorescence-activated cell sorting analysis using Attune NxT Flow Cytometer and plotted as a bar graph (n=3). *P<0.01 vs normoxia, **P<0.01 vs H/R (ANOVA). I, HCAECs were transfected with nontarget (NT) or ErbB4 or ErbB4 siRNA (100 nmol/L), and after recovery from transfection, they were treated with or without PM and exposed to H/R. Cells undergoing apoptosis were labeled with annexin V-FITC conjugate, and the percentage of apoptotic cells was quantitated and plotted as a bar graph (n=3). *P<0.01 vs NT siRNA normoxia, **P<0.01 vs NT siRNA H/R; †P<0.01 vs NT siRNA PM+H/R (ANOVA). ANOVA followed by Tukey post test was performed using GraphPad-Prism software (version 8).

Fig 3: Endothelial-ErbB2 (Erb-B2 receptor tyrosine kinase 2) directly associates with Src and activate eNOS (endothelial nitric oxide synthase). A, Human coronary artery endothelial cells (HCAECs) were treated with preconditioned medium (PM) for the indicated period, and cell lysates were prepared. An equal amount of protein from cell lysates was immunoprecipitated using the anti-ErbB2 antibody. The immunoprecipitates were analyzed by Western blotting for tyrosine phosphorylation of ErbB2, level of associated Src, ErbB4, and ErbB2 using their specific antibodies. B, HCAECs were treated with CM for 1 or 4 h, and proximity ligation assay (PLA) was performed using anti-ErbB2 and anti-Src antibodies. Scale bar=20 mm. C, PLA signals were counted and plotted as a bar graph (n=5). *P<0.01 vs control (ANOVA). D, To determine the role of Nrg1 (neuregulin 1) released by HMVEC in PM, PM was pretreated with neutralizing anti-Nrg1 and then incubated with HCAECs for 1 h. At the end of treatment, cell lysates were prepared and analyzed for ErbB2 tyrosine phosphorylation and its interaction with Src immunoprecipitation followed by Western blotting. E, HCAECs were incubated with PM or Nrg1 neutralized-PM for 1 h. and PLA was performed using anti-ErbB2 and anti-Src antibodies. Scale bar=20 mm. F, PLA signals were counted and plotted as a bar graph (n=5). *P<0.01 vs control (ANOVA). G, HCAECs were pretreated with Herceptin and incubated with PM for 1 h, and cell lysates were prepared. An equal amount of proteins from cell lysates were immunoprecipitated with anti-ErbB2 antibodies, and the immunocomplexes were analyzed for associated Src by Western blotting. H, HCAEC were transfected with ErbB2 siRNA, treated with PM for 1 h, and cell lysates were prepared and analyzed for eNOS tyrosine phosphorylation. I, HCAECs were pretreated with control IgG, Herceptin, or Nrg1 neutralizing antibodies and then incubated with PM for 1 h, and cell lysates were prepared. An equal amount of proteins from cell lysates were analyzed for eNOS-Tyr81 phosphorylation by Western blotting using its specific antibodies. J, HCAECs were pretreated with control IgG or Herceptin and incubated with PM for 1 h, and cell lysates were prepared. An equal amount of protein from cell lysates was analyzed for eNOS activity and plotted as a bar graph. *P<0.01 vs control IgG, **P<0.01 vs control IgG+PM (ANOVA).

Fig 4: Immunohistochemical staining of candidate genes (brown). Nuclei were stained with hematoxylin and appear in blue. All pictures show neotissue in the thickened area of the conduit wall. (A) Black arrow points to EGFR positive macrophage in granular tissue. (B) Black arrow points to ErbB4 positive macrophage in transition area between granular and fibrous tissue. (C) Black arrows point to ErbB4 positive macrophages in granular tissue. (D) Black arrows point to FLT4 positive, spindle shaped fibroblasts in fibrous tissue. (E) Overview picture of FLT4 staining. Graft material is located on the left side of the picture. A dotted line marks the border between graft and neo tissue. Neo tissue close to the graft was mostly granular, becoming more fibrous towards the lumen (right side in the picture). Here, granular tissue was generally intensively stained, whereas in fibrous tissue, staining was restricted to the fibroblasts. Square indicates the area that is zoomed in Fig. 10.1038/s41598-021-81340-2F. (F) Close up of square in Fig. 10.1038/s41598-021-81340-2E. Black arrows point to more intensively stained FLT4 positive macrophages. Scale bars: 50 µm; scale bar Figure E: 200 µm. Stainings were perfomed on n = 11 explanted conduits.