Volume 21, Issue 10, Pages 1841-1851 (October 2013) Transcriptional Profiling of Hmgb1-Induced Myocardial Repair Identifies a Key Role for Notch Signaling  Federica Limana, Grazia Esposito, Pasquale Fasanaro, Eleonora Foglio, Diego Arcelli, Christine Voellenkle, Anna Di Carlo, Daniele Avitabile, Fabio Martelli, Matteo Antonio Russo, Giulio Pompilio, Antonia Germani, Maurizio C Capogrossi  Molecular Therapy  Volume 21, Issue 10, Pages 1841-1851 (October 2013) DOI: 10.1038/mt.2013.137 Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Gene epression analysis in control and infarcted hearts. (a) Hierarchical clustering of differentially expressed genes selected by significance analysis of microarrays (SAM) in the border zone (BZ) and in the infarcted area (IA) of 3 day infarcted hearts compared to sham operated hearts. Each row represents the expression of a single gene and columns 1 and 2 correspond to a sample pool of 3 hearts. Expression levels are represented by a color tag, with red representing the highest levels and green the lowest levels of expression. (b) The Venn diagrams shows the number of differentially up and downregulated transcripts, as obtained by SAM, and the number of overlapping transcripts between the bz and the IA. Molecular Therapy 2013 21, 1841-1851DOI: (10.1038/mt.2013.137) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Comparison of biological functions and pathways between the border zone and the infarcted area. Ingenuity pathway analysis showing (a) selected biological functions and (b) Pathways in the border zone and in the infarcted area compared with the sham group. Bars, P values evaluated by exact Fisher test expressed in logarithmic scale. Threshold (evidenced by the red line) indicates the minimally accepted significance level of P < 0.05. Molecular Therapy 2013 21, 1841-1851DOI: (10.1038/mt.2013.137) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Gene expression analysis in untreated and HMGB1-treated infarcted hearts. (a) Hierarchical clustering of differentially expressed genes selected by SAM in the BZ and in the IA 3 days following MI and HMGB1 treatment. Each row represents the expression of a single gene and columns 1 and 2 correspond to a sample pool of infarcted hearts from three animals in each column. Expression levels are represented by a color tag, with red representing the highest levels and green the lowest levels of expression. (b) The Venn diagrams shows the number of differentially up and downregulated transcripts, as obtained by SAM in the BZ and in the IA. No overlapping transcripts between BZ and IA were detected. Molecular Therapy 2013 21, 1841-1851DOI: (10.1038/mt.2013.137) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Comparison of biological functions and pathways between the border zone and the infarcted area of HMGB1-treated infarcted hearts. Ingenuity pathway analysis showing (a) selected biological functions and (b) pathways in BZ and in the IA 3 days following MI and HMGB1 treatment. Bars, P values evaluated by exact Fisher test expressed in logarithmic scale. Threshold indicates the minimally accepted significance level of P < 0.05. Molecular Therapy 2013 21, 1841-1851DOI: (10.1038/mt.2013.137) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 HMGB1 enhances Notch expression in the border zone of HMGB1-treated infarcted hearts. (a) HMGB1 enhances Notch activation in cardiac tissue. Left panel: Average number of myocytes expressing NICD1 in the border zone of 3 day infarcted hearts treated either with HMGB1 (+) or its denatured form (-) (n = 6/each group). Right panel: representative immunohistochemical analysis showing NICD1 in myocyte nuclei (arrow). (b) qRT-PCR analysis of Hes1 and Hey1 in the IA of 3-day HMGB1-treated hearts. IA from hearts treated with denatured HMGB1 was used as control (-). (c,d) HMGB1 enhances Notch activation in CPCs in vivo. CPCs were isolated from infarcted hearts treated either with HMGB1 (+) or its denatured form (-) 3 days following infarction. (c) Average number of CPCs coexpressing c-kit and NICD1 (n = 10/each group). Lower panel: representative image of double immunofluorescence showing freshly isolated CPCs from HMGB1-treated hearts stained for c-kit (red fluorescence) and NICD1 (green fluorescence). Hoechst staining of nuclei in blue. (d) qRT-PCR analysis of Notch1, Hes1 and Hey 1 in isolated CPCs (n = 3, *P < 0.05). (e,f) HMGB1 enhances Notch activation in vitro. CPCs were isolated from non-infarcted hearts, in vitro expanded and grown for 1 and 3 days either in the absence (-) or in the presence of 100 ng HMGB1 (e) Bar graph shows the average number of cells coexpressing c-kit and NICD1 (n = 8, *P < 0.05). (f) Western blot analysis showing Hes1 and NICD1 accumulation in 1 day HMGB1-treated CPCs. The same filter was probed with anti-α-tubulin mAb to show the equal loading. Molecular Therapy 2013 21, 1841-1851DOI: (10.1038/mt.2013.137) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Notch signaling modulates in vivo CPC number and proliferation. The γ-secreatse inhibitor DAPT reduces Notch activation in infarcted hearts (a,b) and in CPCs isolated from HMGB1-treated infarcted hearts (c,d). DAPT was administered to mice 1 day before, 1 and 2 days after MI. The zone bordering the infarct was collected from six different mouse hearts and three of them were used for Western blot analysis while the others were subjected to qRT-PCR. (a) Western blot analysis was performed on heart extracts and on control brain samples (positive control), to detect NICD1. The same filter was probed with anti-Notch1 antibody. DAPT reduced NICD levels. (b) DAPT inhibited the expression of Notch-regulated genes, that is, hes, hey and jagged1 (n = 3, *P < 0.05 vs. each control). CPCs expressing c-kit (c) and c-kit/Ki67 (d) were isolated from infarcted hearts in the absence (-) and in presence (+) of DAPT, 3 days following MI and HMGB1 administration (n = 16/each group). Lower panel: representative image of double immunofluorescence showing freshly isolated CPCs from HMGB1-treated hearts stained for c-kit (red fluorescence) and Ki67 (green fluorescence). Hoechst staining of nuclei in blue. Molecular Therapy 2013 21, 1841-1851DOI: (10.1038/mt.2013.137) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Downregulation of Notch signaling inhibits CPC cardiovascular differentiation. CPCs were isolated from infarcted hearts in the absence (-) and in the presence (+) of DAPT treatment, 3 days following MI and HMGB1 administration. qRT-PCR was performed to detect the expression of myocardial (Tbx5), endothelial (Tie2) and smooth muscle (smMHC) cell markers. n = 3, *,†P < 0.05 versus each control. Molecular Therapy 2013 21, 1841-1851DOI: (10.1038/mt.2013.137) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 8 Schematic representation of HMGB1-mediated activation of Notch1 signaling. HMGB1 activates Notch1 receptor through an undefined mechanism that could involve NF-kB. Notch1 is cleaved by the γ-secretase complex and the Notch1 intracellular domain (NICD1) translocates to the nucleus, forms a transcription activation complex with RBP-jk and activates the expression of target genes Hes1, Hey1, Wnt11. Hes1/Hey1 could further modulate p53 expression which in turn may increase Notch1 expression. The activation of Nkx2.5 by Notch1 could drive commitment of CPCs to the myocyte lineage. Finally, via the modulation of miR-301a, HMGB1 could control HIF1α levels. Molecular Therapy 2013 21, 1841-1851DOI: (10.1038/mt.2013.137) Copyright © 2013 The American Society of Gene & Cell Therapy Terms and Conditions