Sustained Elevation of Vascular Endothelial Growth Factor and Angiopoietin-2 Levels After Transcatheter Aortic Valve Replacement Jeremy Ben-Shoshan,

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Sustained Elevation of Vascular Endothelial Growth Factor and Angiopoietin-2 Levels After Transcatheter Aortic Valve Replacement Jeremy Ben-Shoshan, MD, PhD, Arie Steinvil, MD, Yaron Arbel, MD, Yan Topilsky, MD, Leehee Barak, MSc, Michal Entin-Meer, PhD, Ran Levy, PhD, Arie Lorin Schwartz, MD, Gad Keren, MD, Ariel Finkelstein, MD, Shmuel Banai, MD Canadian Journal of Cardiology Volume 32, Issue 12, Pages 1454-1461 (December 2016) DOI: 10.1016/j.cjca.2016.05.020 Copyright © 2016 Canadian Cardiovascular Society Terms and Conditions

Figure 1 Ang-1, Ang-2, and vascular endothelial growth factor (VEGF) levels 2 and 30 days after transcatheter aortic valve replacement (TAVR). Sera of patients undergoing TAVR were collected 2 and 30 days after the procedure and (A) angiopoietin (Ang)-1, (B) Ang-2, and (C) VEGF levels were assessed by enzyme-linked immunoassay. (D) The correlation between the change (day 2/day 0 ratio) of Ang-2 and VEGF (∗P < 0.01; ∗∗P < 0.001). Canadian Journal of Cardiology 2016 32, 1454-1461DOI: (10.1016/j.cjca.2016.05.020) Copyright © 2016 Canadian Cardiovascular Society Terms and Conditions

Figure 2 Adhesion capacity of human umbilical vein endothelial cells (HUVECs) exposed to serum of patients undergoing transcatheter aortic valve replacement (TAVR). Sera of patients undergoing TAVR were collected 2 and 30 days after the procedure and applied to HUVEC cultures. After 48 hours, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression was assessed by flow cytometry. (A) Representative cytometric measurements of a single patient are presented. Additionally, cells were allowed to adhere to fibronectin-coated plates for 30 minutes, and nonadherent cells were washed away. (B) Adherent cells were quantified by XTT colorimetry (∗P < 0.05; ∗∗P < 0.005). Canadian Journal of Cardiology 2016 32, 1454-1461DOI: (10.1016/j.cjca.2016.05.020) Copyright © 2016 Canadian Cardiovascular Society Terms and Conditions

Figure 3 Migration and network formation capacities of human umbilical vein endothelial cells (HUVECs) exposed to serum of patients undergoing transcatheter aortic valve replacement (TAVR). For migration assay, HUVECs were seeded on 8-μm Transwell inserts and allowed to migrate toward different patients' sera. Cell number was determined in 4 random fields/insert under light microscopy (×10). The number of migrating cells (A) correlated with post-TAVR (30 days) vascular endothelial growth factor (VEGF) but not angiopoietin (Ang)-2 levels and (B) diminished in the presence of anti-VEGF bevacizumab (100 ng/mL). For network formation assay, sera of patients undergoing TAVR were collected 2 and 30 days after the procedure and applied to HUVEC cultures. Alternatively, SU5416 (50 nM) was added to cultures. After 48 hours, cells were collected and seeded on Matrigel-coated plates for 24 hours. (C) Representative microscopy of a single patient. (D) The number of closed tubules as well as (E) branching points were determined in 4 random fields/slide (×40) (∗P < 0.01; ∗∗P < 0.001). Canadian Journal of Cardiology 2016 32, 1454-1461DOI: (10.1016/j.cjca.2016.05.020) Copyright © 2016 Canadian Cardiovascular Society Terms and Conditions