Lisheng Zhang, MD, Leigh Brian, MS, Neil J. Freedman, MD

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Presentation transcript:

Vein graft neointimal hyperplasia is exacerbated by CXCR4 signaling in vein graft- extrinsic cells Lisheng Zhang, MD, Leigh Brian, MS, Neil J. Freedman, MD Journal of Vascular Surgery Volume 56, Issue 5, Pages 1390-1397 (November 2012) DOI: 10.1016/j.jvs.2012.03.254 Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 1 Possible roles of the stromal-derived factor-1 (SDF-1)/CXC chemokine receptor-4 (CXCR4) signaling system in vein graft neointimal hyperplasia. Green arrows (with “+”) indicate augmentation; red lines (with “−”) indicate inhibition, or reduction. PMN, Polymorphonuclear neutrophil; SMC, smooth muscle cell. Journal of Vascular Surgery 2012 56, 1390-1397DOI: (10.1016/j.jvs.2012.03.254) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 2 Stromal-derived factor-1 (SDF-1) is expressed in arterializing vein grafts. Carotid interposition vein grafts in wild-type (WT) mice were created with syngeneic inferior venae cavae (IVCs) and harvested 2 weeks postoperatively, before the completion of arterialization. IVCs from WT mice of equivalent age were also harvested, for comparison. Serial frozen sections were stained with SDF-1-specific or nonimmune rabbit immunoglobulin G (IgG), anti-rabbit IgG/Alexa-488 (green), and Hoechst 33342 to visualize DNA (blue). Shown are sections from single specimens, representative of four independent vein grafts and IVC specimens. Scale bar = 100 μm (original magnification ×220). The lumen is indicated by an asterisk. Equivalent results were obtained with WT/WT, WT/CXC chemokine receptor-4 (Cxcr4)−/+, and Cxcr4−/+/Cxcr4−/+ vein grafts. Journal of Vascular Surgery 2012 56, 1390-1397DOI: (10.1016/j.jvs.2012.03.254) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 3 CXC chemokine receptor-4 (CXCR4) activity in vein graft-extrinsic cells contributes to vein graft neointimal hyperplasia. Carotid interposition vein grafts from the indicated mouse donors were placed into the indicated congenic recipient mice and harvested 6 weeks later, after perfusion fixation. Vein graft sections were stained with a modified connective tissue stain. A, Photomicrographs of single specimens, representative of ≥5 obtained of each type. Specimens are aligned at the neointimal/medial border; the medial/adventitial borders are indicated by the arrows and corresponding dashed lines. Scale bar = 50 μm (original magnification ×440). B, Neointimal and medial areas were measured for each specimen cross-section by an observer blinded to specimen identity, using computerized planimetry. Total Wall refers to the sum of neointimal plus medial areas. Shown are means ± SE from ≥5 independent vein graft specimens from each group. Compared with wild-type (WT)/WT control vein grafts: *P < .05. Journal of Vascular Surgery 2012 56, 1390-1397DOI: (10.1016/j.jvs.2012.03.254) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 4 CXC chemokine receptor-4 (CXCR4) does not affect vein graft re-endothelialization. Vein grafts of the indicated donor/recipient combinations were harvested 6 weeks postoperatively and fixed in formalin. A, Specimen cross-sections were fluorescently stained simultaneously for von Willebrand factor (vWF) to identify endothelial cells (green) and for DNA (blue). Shown are photomicrographs from single samples, representative of ≥5 vein grafts in each group. Scale bar = 100 μm (original magnification ×440, lumen oriented upward). B, The length of luminal border staining positively for vWF was divided by the total lumen perimeter to obtain the % of graft re-endothelialization, which is plotted as mean ± SE of ≥5 independent vein graft specimens from each group. WT, Wild-type. Journal of Vascular Surgery 2012 56, 1390-1397DOI: (10.1016/j.jvs.2012.03.254) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 5 CXC chemokine receptor-4 (CXCR4) activity affects vein graft composition. Vein grafts of the indicated donor/recipient groups were harvested 6 weeks postoperatively and stained with a modified connective tissue stain (“Masson”), from which neointimal/medial boundaries were identified. Serial sections were fluorescently stained simultaneously for α-smooth muscle cell (SMC) actin (red) and DNA (blue) (A, middle and bottom panels), or for collagen type I (red) and DNA (blue) (B, upper panel). Dotted white lines indicate the neointimal/medial boundaries. A, SMC-actin-stained sections. In the middle panels, the dotted rectangles delimit the areas further enlarged for depiction in the lower panels. Scale bars = 100 μm (original magnification ×110, middle panels; ×440, lower panels). B, Collagen I-stained sections; scale bar = 100 μm (original magnification ×440). C, SMC actin and collagen type I fluorescence in the medial layer of each vein graft were normalized to cognate DNA fluorescence intensity within each microscopic field. The resulting ratios were normalized, within each staining group, to those of wild-type (WT)/WT samples to yield % of control. Data are plotted as the means ± SE of ≥4 independent vein graft specimens from each group. Compared with WT/WT control vein grafts: *P < .05. Journal of Vascular Surgery 2012 56, 1390-1397DOI: (10.1016/j.jvs.2012.03.254) Copyright © 2012 Society for Vascular Surgery Terms and Conditions

Fig 6 CXC chemokine receptor-4 (CXCR4) activity in vein graft-extrinsic cells promotes leukocyte recruitment to vein grafts. Vein grafts with donors and recipients of the indicated genotypes were harvested 6 weeks postoperatively and were fluorescently stained simultaneously for CD45 (green) and DNA (blue). A, Dotted white lines indicate the neointimal/medial boundaries. Shown are photomicrographs from single samples, representative of ≥4 in each group. Scale bar = 100 μm (original magnification ×220, lumen oriented upward). B, CD45 fluorescence in the medial layer of each vein graft was normalized to cognate DNA fluorescence intensity; the resulting ratios were normalized within each staining group to those of wild-type (WT)/WT samples to yield % of control, plotted as the means ± SE of ≥4 independent vein graft specimens from each group. Compared with WT/WT control vein grafts: *P < .05. Journal of Vascular Surgery 2012 56, 1390-1397DOI: (10.1016/j.jvs.2012.03.254) Copyright © 2012 Society for Vascular Surgery Terms and Conditions