Role of hemodynamic forces in the ex vivo arterialization of human saphenous veins Xavier Berard, MD, PhD, Sébastien Déglise, MD, Florian Alonso, PhD,

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Presentation transcript:

Role of hemodynamic forces in the ex vivo arterialization of human saphenous veins Xavier Berard, MD, PhD, Sébastien Déglise, MD, Florian Alonso, PhD, François Saucy, MD, Paolo Meda, MD, Laurence Bordenave, MD, PhD, Jean-Marc Corpataux, MD, Jacques-Antoine Haefliger, PhD Journal of Vascular Surgery Volume 57, Issue 5, Pages 1371-1382 (May 2013) DOI: 10.1016/j.jvs.2012.09.041 Copyright © 2013 Society for Vascular Surgery Terms and Conditions

Fig 1 Intimal hyperplasia develops within 7 days of ex vivo pulsatile perfusion. Upper panel, Representative histological sections show the normal intima thickness (arrow) in nonperfused veins (control) and veins perfused at low pressure (arterial shear stress [SS] 7 mm Hg). In contrast, intimal hyperplasia (arrow) is evident in veins perfused at high pressure (arterial SS 70 mm Hg). L, Lumen; M, media. Bar represents 50 μm. Lower panels, Measurements of intima thickness show that intimal hyperplasia was induced by perfusion at high pressure (70 mm Hg), despite unchanged thickness of the media layer. Data represent mean ± standard error of the mean of two series of eight and nine experiments. *P < .05 and ***P < .001 vs the nonperfused segments (controls) of the same veins. Journal of Vascular Surgery 2013 57, 1371-1382DOI: (10.1016/j.jvs.2012.09.041) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

Fig 2 Human saphenous veins after 7 days of ex vivo perfusion show persistence of endothelial cells and altered distribution of smooth muscle cells (SMCs). Upper row, Hematoxylin-eosin staining (HE) reveals the lining of the lumen by nuclei of endothelial cells and the nuclei of SMCs in the media layer. Nuclei of the latter cells were also observed within the intimal hyperplasia regions (right panel). Middle rows, Immunolabeling using antibodies against von Willebrand factor (vWf) or alpha-smooth muscle actin (SMA) demonstrate the presence of endothelial cells at the luminal surface of all veins and of SMCs in the media of all vessels. SMCs were also detected in the intimal hyperplasia regions (right panels). Lower row, Immunolabeling using antibodies against KI-67 (dark blue) revealed the presence of proliferating SMCs mainly in the intimal hyperplasia regions. L, Lumen; M, media. Bars represent 100 μm in figures and 400 μm in the insets. Journal of Vascular Surgery 2013 57, 1371-1382DOI: (10.1016/j.jvs.2012.09.041) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

Fig 3 The expression of matrix metalloproteinase (MMP)-2 and MMP-9 is increased by arterial shear stress (SS). A, MMP-2 transcripts are upregulated by arterial shear stress, and this upregulation is increased by high pressure (70 mm Hg). B, Western blots showed increased levels of both MMP-2 and MMP-9 after perfusion, under both low and high hemodynamic pressure. C and D, Both pro-MMP-2 and MMP-2 protein were significantly increased by perfusion and high pressure. E and F, MMP-9 transcript and protein levels were also upregulated by perfusion. However, the MMP-9 protein did not appear to be further induced by high pressure. Data represent mean ± standard error of the mean of two series of eight and nine experiments. GADPH, Glyceraldehyde 3-phosphate dehydrogenase. *P < .05; **P < .01; ***P < .001 vs nonperfused veins and veins exposed to low pressure. Journal of Vascular Surgery 2013 57, 1371-1382DOI: (10.1016/j.jvs.2012.09.041) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

Fig 4 The lytic activity of matrix metalloproteinase (MMP)-2 and MMP-9 is increased by arterial shear stress (SS) and blood pressure. A, Zymographic analysis distinguished the gelatin lysis induced by pro-MMP-2, MMP-2, and MMP-9. B-D, Quantitative analysis showed that the activities of these enzymes increased in veins exposed to arterial shear stress. However, only MMP-2 and MMP-9 were further enhanced by high pressure. Data represent mean ± standard error of the mean of two series of eight and nine experiments. *P < .05; **P < .01; ***P < .001 vs control veins or veins submitted to low pressure. E, Immunostaining showed that both MMP-2 (top row) and MMP-9 (bottom row) increased mostly within the media layer of the perfused veins. L, Lumen; M, media. Bars represent 100 μm. Journal of Vascular Surgery 2013 57, 1371-1382DOI: (10.1016/j.jvs.2012.09.041) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

Fig 5 The inhibitors of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 are differentially affected by arterial shear stress (SS) and pressure. A and B, Veins exposed to arterial shear stress featured increased expression of TIMP-1 mRNA but decreased expression of the TIMP-2 transcripts. C, Consistent with the transcript change, Western blots showed upregulation of the TIMP-1 protein in the presence of both low and high pressure. D, In marked contrast to the mRNA changes, the levels of the TIMP-2 protein were markedly increased only in the presence of high pressure. Data represent mean ± standard error of the mean of two series of eight and nine experiments. *P < .05; **P < .01, ***P < .001 vs control veins. E, Immunostaining confirmed that the abundance of TIMP-1-positive and TIMP-2-positive smooth muscle cells increased under arterial hemodynamic conditions. GADPH, Glyceraldehyde 3-phosphate dehydrogenase; L, lumen; M, media; PAI, plasminogen activator inhibitor. Insets show the boxed areas at higher magnification. Bars represents 100 μm in figures and 400 μm in the insets. Journal of Vascular Surgery 2013 57, 1371-1382DOI: (10.1016/j.jvs.2012.09.041) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

Fig 6 The levels of plasminogen activator inhibitor (PAI)-1 increase in human saphenous vein exposed to arterial shear stress (SS). A, PAI-1 transcripts were increased in human saphenous veins submitted to arterial shear stress. B and C, Western blot analysis and immunocytochemistry showed that PAI-1 mostly increased in the media layer of the arterializing veins. GADPH, Glyceraldehyde 3-phosphate dehydrogenase; L, lumen; M, media. Bar represents 50 μm. Data represent mean ± standard error of the mean of two series of eight and nine experiments. **P < .01; ***P < .001 vs control veins. Journal of Vascular Surgery 2013 57, 1371-1382DOI: (10.1016/j.jvs.2012.09.041) Copyright © 2013 Society for Vascular Surgery Terms and Conditions

Fig 7 The expression of Ephrin-B2 and Eph-B4 decreases in the presence of high pressure. A and B, The transcription of Ephrin-B2 and Eph-B4 decreased in veins submitted to arterial shear stress (SS). C and D, The levels of the cognate proteins were also reduced under these conditions. In vessels exposed to low pressure, only Eph-B4 was found significantly reduced. Data represent mean ± standard error of the mean of two series of eight and nine experiments. GADPH, Glyceraldehyde 3-phosphate dehydrogenase. **P < .01; ***P < .001 vs control veins. Journal of Vascular Surgery 2013 57, 1371-1382DOI: (10.1016/j.jvs.2012.09.041) Copyright © 2013 Society for Vascular Surgery Terms and Conditions