Journal of Molecular and Cellular Cardiology Lack of haptoglobin results in unbalanced VEGFα/angiopoietin-1 expression, intramural hemorrhage and impaired wound healing after myocardial infarction  Fatih Arslan, Mirjam B. Smeets, Brigitta Buttari, Elisabetta Profumo, Rachele Riganò, Lars Akeroyd, Emrah Kara, Leo Timmers, Joost P. Sluijter, Ben van Middelaar, Krista den Ouden, Gerard Pasterkamp, Sai Kiang Lim, Dominique P.V. de Kleijn  Journal of Molecular and Cellular Cardiology  Volume 56, Pages 116-128 (March 2013) DOI: 10.1016/j.yjmcc.2012.12.012 Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 1 Haptoglobin deficiency worsens survival and causes intramural hemorrhage after MI. (A) Haptoglobin protein levels in the heart during infarct development. Representative western blots are shown; *p=0.037 compared to baseline levels; n=3/time point (B) Infarct size (IS) as a percentage of the left ventricle (LV) 2days after infarction. Representative cross-sections after TTC staining are shown below corresponding bars; n=5/group. (C) Cardiac rupture before and after removal of thrombus. Arrow indicates site of rupture, note the massive hemorrhage area within the infarct (pale area), asterisk indicates the LCA ligation. Each bar represents Mean±SEM. Journal of Molecular and Cellular Cardiology 2013 56, 116-128DOI: (10.1016/j.yjmcc.2012.12.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 2 Hp mediates wound healing, scar formation and remodeling. (A) Representative images of hematoxyllin–eosin stained hearts. Note the excessive red blood cell deposition and disorganized granulation in the Hp−/− hearts. (B) Quantification of total red blood cell positive area as a percentage of the infarcted area(10× magnification); *p=0.026 and †p=0.029 compared to WT animals. (C) Quantification of residual necrotic area (% of infarct area) 7days after infarction; *p=0.019 compared to WT animals. (D) Total collagen content (determined by total gray value of light-converted polarized cross-sections stained with picrosirius red) and collagen density within the infarct area. Representative images of 28days post-infarct hearts are shown using polarized light and 3D confocal microscopy. Note the dense and organized collagen fiber structure in WT heart in contrast to Hp−/− heart; *p=0.046 and †p=0.031 compared to WT animals. (E) Representative images in the end-diastolic phase are shown. Each bar represents Mean±SEM, MRI study: n=10 in WT, n=20 in Hp−/−, n=6/sham group; histology n=7/group. Journal of Molecular and Cellular Cardiology 2013 56, 116-128DOI: (10.1016/j.yjmcc.2012.12.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 2 Hp mediates wound healing, scar formation and remodeling. (A) Representative images of hematoxyllin–eosin stained hearts. Note the excessive red blood cell deposition and disorganized granulation in the Hp−/− hearts. (B) Quantification of total red blood cell positive area as a percentage of the infarcted area(10× magnification); *p=0.026 and †p=0.029 compared to WT animals. (C) Quantification of residual necrotic area (% of infarct area) 7days after infarction; *p=0.019 compared to WT animals. (D) Total collagen content (determined by total gray value of light-converted polarized cross-sections stained with picrosirius red) and collagen density within the infarct area. Representative images of 28days post-infarct hearts are shown using polarized light and 3D confocal microscopy. Note the dense and organized collagen fiber structure in WT heart in contrast to Hp−/− heart; *p=0.046 and †p=0.031 compared to WT animals. (E) Representative images in the end-diastolic phase are shown. Each bar represents Mean±SEM, MRI study: n=10 in WT, n=20 in Hp−/−, n=6/sham group; histology n=7/group. Journal of Molecular and Cellular Cardiology 2013 56, 116-128DOI: (10.1016/j.yjmcc.2012.12.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 2 Hp mediates wound healing, scar formation and remodeling. (A) Representative images of hematoxyllin–eosin stained hearts. Note the excessive red blood cell deposition and disorganized granulation in the Hp−/− hearts. (B) Quantification of total red blood cell positive area as a percentage of the infarcted area(10× magnification); *p=0.026 and †p=0.029 compared to WT animals. (C) Quantification of residual necrotic area (% of infarct area) 7days after infarction; *p=0.019 compared to WT animals. (D) Total collagen content (determined by total gray value of light-converted polarized cross-sections stained with picrosirius red) and collagen density within the infarct area. Representative images of 28days post-infarct hearts are shown using polarized light and 3D confocal microscopy. Note the dense and organized collagen fiber structure in WT heart in contrast to Hp−/− heart; *p=0.046 and †p=0.031 compared to WT animals. (E) Representative images in the end-diastolic phase are shown. Each bar represents Mean±SEM, MRI study: n=10 in WT, n=20 in Hp−/−, n=6/sham group; histology n=7/group. Journal of Molecular and Cellular Cardiology 2013 56, 116-128DOI: (10.1016/j.yjmcc.2012.12.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 3 Hp mediates oxidative stress and microvascular permeability. (A) Vascular permeability in the infarcted ventricle 3days after MI; *p<0.014 compared to WT hearts (n=4/group). Representative images are shown of infarcted hearts 1h after Evans' blue dye injection and subsequent flushing with saline. Note the massive extravasation of the blue dye in Hp−/− mice. The arrows (yellow) point red areas of hemorrhage. (B) Plasma Evans' blue dye concentration. (C) Oxidative stress in cardiac protein extracts 3days after infarction; *p=0.043 (n=4/group). PAI-1 activity in heart (D) and plasma (E) 3days after MI; *p=0.022, †p=0.036 compared to WT animals (n=7/group). (F) Capillary density after MI. Each bar represents Mean±SEM. Journal of Molecular and Cellular Cardiology 2013 56, 116-128DOI: (10.1016/j.yjmcc.2012.12.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 3 Hp mediates oxidative stress and microvascular permeability. (A) Vascular permeability in the infarcted ventricle 3days after MI; *p<0.014 compared to WT hearts (n=4/group). Representative images are shown of infarcted hearts 1h after Evans' blue dye injection and subsequent flushing with saline. Note the massive extravasation of the blue dye in Hp−/− mice. The arrows (yellow) point red areas of hemorrhage. (B) Plasma Evans' blue dye concentration. (C) Oxidative stress in cardiac protein extracts 3days after infarction; *p=0.043 (n=4/group). PAI-1 activity in heart (D) and plasma (E) 3days after MI; *p=0.022, †p=0.036 compared to WT animals (n=7/group). (F) Capillary density after MI. Each bar represents Mean±SEM. Journal of Molecular and Cellular Cardiology 2013 56, 116-128DOI: (10.1016/j.yjmcc.2012.12.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 4 Hp deficiency is associated with unbalanced VEGFα/Ang-1 expression. Tissue levels of (A) VEGFα and (B) Ang-1 mRNA after infarction; for VEGFα *p=0.005, †p=0.002, ‡p=0.001; for Ang-1 *p=0.011; for (C) VEGFα/Ang-1 ratio *p=0.021, †p=0.002, ‡p=0.028 compared to WT animals. Each bar represents Mean±SEM, n=7/group/time point. Journal of Molecular and Cellular Cardiology 2013 56, 116-128DOI: (10.1016/j.yjmcc.2012.12.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 5 HUVECs exposed to oxidative stress with and without Hp. (A) Oxidative stress; *p=0.006, (B) VEGFα; *p=0.024, (C) Ang-1; *p=0.012 and (D) VEGFα/Ang-1 expression ratios in protein lysates from stimulated HUVECs; *p=0.039 compared to hemoglobin-stimulated HUVECs. Representative western blot images are shown. Each bar represents Mean±SEM, n=4/group for VEGF/Ang-1 western blots; n=2 for protein oxidation assay; US=unstimulated, Hb=hemoglobin, Hb/Hp=Hb with haptoglobin. Journal of Molecular and Cellular Cardiology 2013 56, 116-128DOI: (10.1016/j.yjmcc.2012.12.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 6 Hp deficiency and leukocyte influx after MI. (A) Neutrophil (Ly6-Gpos) and (B) macrophage (MAC3pos) influx in infarct area; *p=0.016 compared to WT animals. Representative cross-sections of hearts positive for neutrophils and macrophages are shown. (C) CD163pos cell count in infarct area. Representative images are shown; *p=0.03 and †p=0.024 compared to WT animals. Each bar represents Mean±SEM, n=7/group. Journal of Molecular and Cellular Cardiology 2013 56, 116-128DOI: (10.1016/j.yjmcc.2012.12.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Fig. 6 Hp deficiency and leukocyte influx after MI. (A) Neutrophil (Ly6-Gpos) and (B) macrophage (MAC3pos) influx in infarct area; *p=0.016 compared to WT animals. Representative cross-sections of hearts positive for neutrophils and macrophages are shown. (C) CD163pos cell count in infarct area. Representative images are shown; *p=0.03 and †p=0.024 compared to WT animals. Each bar represents Mean±SEM, n=7/group. Journal of Molecular and Cellular Cardiology 2013 56, 116-128DOI: (10.1016/j.yjmcc.2012.12.012) Copyright © 2012 Elsevier Ltd Terms and Conditions