Vein graft neointimal hyperplasia is exacerbated by CXCR4 signaling in vein graft-extrinsic cells

Vein graft neointimal hyperplasia is exacerbated by CXCR4 signaling in vein graft-extrinsic cells Lisheng Zhang, MD, a Leigh Brian, MS, a and Neil J. Freedman, MD, a,b Durham, NC Objective: Because vein graft neointimal hyperplasia engenders vein graft failure, and because most vein graft neointimal cells derive from outside the vein graft, we sought to determine whether vein graft neointimal hyperplasia is affected by activity of the CXC chemokine receptor-4 (CXCR4), which is important for bone marrow-derived cell migration. Methods: In congenic Cxcr4 / and wild-type (WT) recipient mice, we performed interposition grafting of the common carotid artery with the inferior vena cava (IVC) of either Cxcr4 / or WT mice to create four surgically chimeric groups of mice (n > 5 each), characterized by vein graft donor/recipient: WT/WT; Cxcr4 / /WT; WT/Cxcr4 / ; and Cxcr4 / /Cxcr4 / ; vein grafts were harvested 6 weeks postoperatively. Results: The agonist for CXCR4 is expressed by cells in the arterializing vein graft. Vein graft neointimal hyperplasia was reduced by reducing CXCR4 activity in vein graft-extrinsic cells, but not in vein graft-intrinsic cells: the rank order of neointimal hyperplasia was WT/WT Cxcr4 / /WT > WT/Cxcr4 / Cxcr4 / /Cxcr4 / ; CXCR4 deficiency in graft-extrinsic cells reduced neointimal hyperplasia by 39% to 47% (P <.05). Vein graft medial area was equivalent in all grafts except Cxcr4 / /Cxcr4 /, in which the medial area was 60% 20% greater (P <.05). Vein graft reendothelialization was indistinguishable among all three vein graft groups. However, the prevalence of medial leukocytes was 40% 10% lower in Cxcr4 / /Cxcr4 / than in WT/WT vein grafts (P <.05), and the prevalence of smooth muscle actin-positive cells was 45% 20% higher (P <.05). Conclusions: We conclude that CXCR4 contributes to vein graft neointimal hyperplasia through mechanisms that alter homing to the vein graft of graft-extrinsic cells, particularly leukocytes. (J Vasc Surg 2012;56:1390-7.) Clinical Relevance: The utility of autologous vein grafts is severely reduced by neointimal hyperplasia, which accelerates subsequent graft atherosclerosis. Our study demonstrates that vein graft neointimal hyperplasia is aggravated by activity of the cell-surface CXC chemokine receptor-4 (CXCR4), which is critical for recruitment of bone marrow-derived cells to sites of inflammation. Our model for CXCR4 deficiency used mice with heterozygous deficiency of Cxcr4. Consequently, our results suggest the possibility that a CXCR4 antagonist like plerixafor, currently in clinical use could be applied to vein grafts periadventitially, and perhaps achieve beneficial effects on vein graft neointimal hyperplasia. Although saphenous vein grafts remain the most commonly used conduit for arterial bypass surgery, 1 their durability remains suboptimal: 28% fail within 1 year of surgery 2 and 75% are either occluded or atherosclerotic within 10 years of surgery. 1 A major contributor to vein graft failure is neointimal hyperplasia, a process in which medial smooth muscle cells (SMCs) and vascular progenitor cells proliferate and migrate into the layer subjacent to the endothelium. 3,4 Even when it does not compromise vein graft patency, vein graft neointimal hyperplasia seems to accelerate vein graft atherosclerosis. 5 We have found that most vein graft neointimal cells derive from cells that reside outside of the vein graft at the time of its implantation, or from graft-extrinsic cells. 4 From the Departments of Medicine (Cardiology) a and Cell Biology, b Duke University Medical Center. The study was supported in part by the National Institutes of Health grants HL77185 and HL73042 (to N.J.F.). Author conflict of interest: none. Reprint requests: Neil J. Freedman, MD, Duke University Medical Center, Department of Medicine, Box 3187, Durham, NC 27710 (e-mail: neil.freedman@duke.edu). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214/$36.00 Copyright 2012 by the Society for Vascular Surgery. http://dx.doi.org/10.1016/j.jvs.2012.03.254 1390 Moreover, many of these graft-extrinsic cells derive from the bone marrow compartment. 4 Because an arterializing vein graft constitutes an injured vessel, 3 the pathogenesis of vein graft disease involves mechanisms pertinent to tissue repair, such as recruitment of bone marrow-derived cells. An important cell signaling system in this process is that comprising the chemokine known as stromal cell-derived factor-1 (SDF-1, or CXC chemokine ligand-12 [CXCL12]), and its primary chemokine receptor known as CXCR4. 6,7 The SDF-1/CXCR4 signaling system is critical for normal vascular development 8 and mediates cell-specific effects that engender distinct inflammatory phenotypes (Fig 1). For example, CXCR4 promotes migration of both bone marrow-derived cells 9 and SMCs. 10,11 However, whereas CXCR4 signaling reduces proliferation of hematopoietic stem cells 12 and induces endothelial cell apoptosis, 13 it seems to promote proliferation of SMCs. 11 In vivo, CXCR4 activation contributes to neointimal hyperplasia associated with wire-mediated arterial injury. 14,15 Nonetheless, CXCR4 activation seems to protect against atherosclerosis by reducing atherosclerotic plaque infiltration by neutrophils. 16 How the SDF-1/CXCR4 signaling system affects inflammation in the arterializing vein graft and consequent neointimal hyperplasia remains unknown. This study tests the hypothesis that CXCR4 activity, particularly in vein graft-extrinsic cells, contributes to vein

JOURNAL OF VASCULAR SURGERY Volume 56, Number 5 Zhang et al 1391 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. graft neointimal hyperplasia. To do so, this study used a mouse vein graft model that mimics human vein graft disease, 17 in that nonocclusive, SMC-rich neointimal hyperplasia develops over 4 to 6 weeks. Moreover, in this model, vein graft neointimal hyperplasia is sensitive to effects of single-gene deletions in either the vein graft donor or the congenic vein graft recipient. 18,19 METHODS Studies of immature vein grafts. To investigate the role of CXCR4 in the recruitment of bone marrow-derived progenitor cells to vein graft lesions, we first evaluated whether its ligand SDF-1 is expressed in cells constituting the arterializing vein graft. For this purpose, we harvested carotid interposition vein grafts from wild-type (WT) mice 2 weeks postoperatively, when the vein graft wall remains incompletely arterialized (wall thickness and neointimal hyperplasia reach steady state by 4 weeks postoperatively). 17 We compared immature (2-week-old) vein grafts with ungrafted inferior vena cava (IVC) veins from congenic mice (see Histology ). In addition, we compared vein grafts (n 4/group) implanted into WT and Cxcr4 / recipients. Study design for CXCR4 experiments. To study the effects of CXCR4 physiology on vein graft disease, we used CXCR4-deficient and congenic WT, C57BL/6 mice. However, because CXCR4 deficiency is lethal in the embryonic or perinatal period, 8 we used Cxcr4 / mice as our model for CXCR4 loss of function. We implanted interposition vein grafts from WT and congenic Cxcr4 / mice into the common carotid arteries of WT or Cxcr4 / mice, and thereby created four groups of vein graft donor/ recipient mice: WT/WT, Cxcr4 / /WT, WT/Cxcr4 /, and Cxcr4 / /Cxcr4 /. With these four groups, we could test whether neointimal hyperplasia is reduced by CXCR4 loss of function (heterozygosity) in vein graftintrinsic cells, vein graft-extrinsic cells, or both cell types. Vein grafts were harvested 6 weeks postoperatively, after neointimal hyperplasia in WT grafts reaches steady state. 17 Mice. WT and congenic Cxcr4 / mice on the C57BL/6 genetic background were purchased from Jackson Laboratories (Bar Harbor, Me), and maintained as we reported. 17 All animal experiments were performed according to protocols approved by the Duke University Institutional Animal Care and Use Committee and complied with the Guide for the Care and Use of Laboratory Animals (National Research Council). Vein graft surgery. Interposition vein graft surgery was performed as described previously. 17-19 IVCs from WT or congenic Cxcr4 / donor mice were anastomosed endto-side to the right common carotid artery of WT or congenic Cxcr4 / recipient mice. After both vein graft anastomoses were secured, the intervening common carotid artery was ligated and cut. All vein graft donors and recipients were matched for gender and age (10-15 weeks old), and there were at least five independent vein graft donors/recipients for each genotypic group. All four surgical groups underwent surgery contemporaneously, and the surgeon was blinded to the genotype of the mice. At 2 or 6 weeks postoperatively, mice were euthanized and vein grafts were harvested as described, 17,18 with phosphatebuffered saline (PBS) perfusion to achieve exsanguination followed by perfusion fixation with PBS/formalin (paraffin-embedded specimens) or incubation in 30% sucrose/ PBS overnight followed by embedding in optimal cutting temperature compound (frozen sections). Histology. All specimens were sectioned at 5 m, from the distal or middle third of the vein graft specimens; all four vein graft groups were matched for section location, so as to minimize variation among grafts of identical donor/recipient genotype groups. 17 WT vein grafts implanted into WT recipient mice ( WT/WT ) developed neointimal hyperplasia characteristic of the pre-atherosclerotic stages of human vein graft disease 17 : multiple layers of SMCs that do not cause significant luminal stenosis. Morphometry was performed as described, 17 on perfusion-fixed specimens stained with a modified Masson trichrome and Verhoeff elastic tissue stain that facilitates the simultaneous identification of collagen (green), elastin (black), cytoplasm (red), and nuclei (black). 17 The neointimal/medial boundary was defined as the transition from the cytoplasm-rich, disorganized neointima to

1392 Zhang et al JOURNAL OF VASCULAR SURGERY November 2012 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 m (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. the collagen-rich media. 17 The medial/adventitial boundary was defined as the transition from the more densely organized medial collagen to the less densely organized, vasa vasorum-containing collagenous network of the adventitia. 17 The neointimal area was measured as the cross-sectional area subtended by the luminal perimeter and the neointimal/medial boundary. The medial area was measured as the cross-sectional area subtended by the neointimal/medial and medial/adventitial boundaries. All measurements were made on two sections per vein graft, by observers blinded to specimen identity. We stained immunofluorescently as described, 4,18,20 with the following immunoglobulin Gs (IgGs; or corresponding isotype negative-control IgGs): rabbit anti- SDF-1 (Santa Cruz Biotechnology, Santa Cruz, Calif), mouse anti-collagen I (Sigma-Aldrich, St. Louis, Mo), and anti-smc- -actin (Cy5-conjugated 1A4; Sigma-Aldrich) and rabbit anti-cd45 (H-230, Santa Cruz Biotechnology), for the purpose of recognizing fibrocytes 21 as well as leukocytes. Immunofluorescence microscopy with narrow band-pass filters was performed as described. 4,18,19,22 Protein immunofluorescence was normalized to DNA fluorescence in the same microscopic field, and quantitated as described. 4,18-20,22,23 Morphometric and immunofluorescence data were quantitated by observers blinded to specimen identity. Vein graft endothelialization. Work from our group 19 and others 24 has shown that during the early phase of arterialization, there is significant damage to the vein graft endothelium. To quantitate re-endothelialization, we stained vein graft cross-sections for von Willebrand factor (vwf), as described 19 ; an observer blinded to specimen identity assessed the percentage of the luminal surface that stained positive for vwf. 19 Statistical analysis. One-way analysis of variance with Tukey s post-hoc test for multiple comparisons was used to analyze morphometric and protein expression data. Data are presented as means SD in the text and SE in the figures. RESULTS Immature vein grafts express SDF-1. In our mouse model of vein grafting, immature, 17 arterializing vein grafts express SDF-1 protein, discerned by immunofluorescence microscopy of 2-week-old vein grafts (Fig 2). However, no SDF-1 could be detected in cognate IVCs that had not been subjected to vein grafting (Fig 2). Thus, like atherosclerotic 25 and mechanically injured arteries, 26 vein grafts may use SDF-1 to recruit cells. CXCR4 promotes vein graft neointimal hyperplasia. Compared with the WT/WT control vein grafts, the Cxcr4 / /WT vein grafts demonstrated equivalent

JOURNAL OF VASCULAR SURGERY Volume 56, Number 5 Zhang et al 1393 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 m (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. 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 m (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.

1394 Zhang et al JOURNAL OF VASCULAR SURGERY November 2012 neointimal hyperplasia, as assessed by neointimal area (120 20 10 3 m 2 vs 120 10 10 3 m 2, Fig 3). Thus, CXCR4 activity in vein graft-intrinsic cells did not seem to affect vein graft neointimal hyperplasia. In contrast to these vein graft findings in WT recipient mice, however, vein grafts implanted into Cxcr4 / recipient mice showed substantially less neointimal hyperplasia, whether the vein graft donor was WT or Cxcr4 / (Fig 3). Compared with neointimal area in WT/WT or Cxcr4 / /WT vein grafts, neointimal area was 39% to 47% less in vein grafts that were WT/Cxcr4 / (76 9 10 3 m 2 )orcxcr4 / / Cxcr4 / (70 20 10 3 m 2 ; P.05; Fig 3, B). Intriguingly, the medial area was 60% 20% greater (P.05) in Cxcr4 / /Cxcr4 / vein grafts (320 40 10 3 m 2 ) than in WT/WT control vein grafts (200 40 10 3 m 2 ; Fig 3). Consequently, the total vein graft wall area (media plus neointima) did not differ among the four vein graft groups (Fig 3), and neither did the luminal area (data not shown). Thus, unlike CXCR4 activity in graft-intrinsic cells, CXCR4 activity in vein graft-extrinsic cells promotes neointimal hyperplasia. CXCR4 does not affect vein graft re-endothelialization. Because the mature vein graft endothelium comprises both vein graft-intrinsic and graft-extrinsic endothelial cells 4 that can be affected by CXCR4 activity, 13 we sought to determine whether CXCR4 in graft-intrinsic or graft-extrinsic cells affected vein graft re-endothelialization. In mature vein grafts, the degree of endothelialization was indistinguishable among our four vein graft groups: WT/WT (93% 6%), Cxcr4 / /WT (92% 7%), WT/Cxcr4 / (94% 2%) and Cxcr4 / /Cxcr4 / (91% 4%; Fig 4). Thus, as with arterial injury, 15 in vein graft arterialization there seems to be no effect of CXCR4 on re-endothelialization. CXCR4 alters vein graft composition. To determine whether CXCR4 in vein graft-intrinsic or graft-extrinsic cells altered the cellular composition of the vein graft, we first determined the vein graft prevalence of -SMC actinpositive cells. Whereas SMCs were more abundant in the larger neointimas of WT/WT vein grafts (Figs 3 and 5), they were 1.6 0.2-fold more prevalent in the media of 4 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 -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, SMCactin-stained sections. In the middle panels, the dotted rectangles delimit the areas further enlarged for depiction in the lower panels. Scale bars 100 m (original magnification 110, middle panels; 440, lower panels). B, Collagen I-stained sections; scale bar 100 m (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 Volume 56, Number 5 Zhang et al 1395 vein grafts from Cxcr4 / recipients (P.05; Fig 5). Despite the higher prevalence of medial SMCs, however, vein grafts from Cxcr4 / recipients had only 55% 8% as much medial collagen I as WT/WT vein grafts (P.05; Fig 5). Nevertheless, congruently with a higher prevalence of SMCs, the media of vein grafts from Cxcr4 / recipients had a lower prevalence of leukocytes, as demonstrated by staining for the pan-leukocyte marker CD45: as compared with WT/WT or Cxcr4 / /WT vein grafts, WT/ Cxcr4 / and Cxcr4 / /Cxcr4 / vein grafts had 56% 5% and 55% 6% as many medial leukocytes, respectively (P.05; Fig 6). Thus, the media of vein grafts from Cxcr4 / recipients seems to be enriched in SMCs and poor in leukocytes, compared with vein grafts from WT recipients. DISCUSSION Using a genetic approach to reduce CXCR4 activity, our work demonstrates that vein graft neointimal hyperplasia is exacerbated by CXCR4 activity in vein graft-extrinsic cells. Our work also suggests that an SDF-1/CXCR4 signaling system promotes inflammation associated with vein graft arterialization, by demonstrating that Cxcr4 / vein graft recipients have fewer leukocytes in the vein graft media. In vein grafts, the beneficial effects attributable to CXCR4 deficiency were observed when CXCR4 expression was reduced by only 50% 27 in Cxcr4 / graft recipient mice. Consequently, these studies highlight the possibility that inhibiting CXCR4, at least in the local vein graft environment, could achieve clinically beneficial effects. The importance of the SDF-1/CXCR4 signaling system in vein graft arterialization accords with current concepts regarding the pathophysiology of vein graft disease. Consequent to the barotrauma associated with implantation in the arterial circuit, 3,24,28 defects in the vein graft endothelium trigger platelet adhesion to the vein graft s luminal surface; platelet-secreted SDF-1 29 then functions to recruit vein graft-extrinsic cells to the vein graft. During vein graft arterialization, cells of the burgeoning vein graft media and neointima also secrete SDF-1, as Fig 2 demonstrates. Reducing CXCR4 activity in both vein graft- 4 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 m (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.

1396 Zhang et al JOURNAL OF VASCULAR SURGERY November 2012 extrinsic and graft-intrinsic cells attenuates neointimal hyperplasia substantially. However, inhibiting CXCR4 activity globally does not seem to engender thinning of the vein graft wall overall because of increased medial hypertrophy (Fig 3). Thus, because they may not prevent medial or total vein graft wall thickening, vein graft therapies that inhibit CXCR4 may decrease neointimal hyperplasia without increasing vein graft wall stress (by the Law of LaPlace). 3,5,30 That the Cxcr4 / /Cxcr4 / vein grafts demonstrated larger medial areas than other vein graft types was surprising, in light of our previous finding that a large proportion of vein graft medial cells derive from the bone marrow. 4 The higher prevalence of SMC-actin-positive cells in the media of Cxcr4 / /Cxcr4 / vein grafts (Fig 5) suggested the possibility that Cxcr4 / /Cxcr4 / vein grafts, compared with WT/WT vein grafts, comprised more fibrocytes bone marrow-derived cells that differentiate from monocyte-like cells into SMC actin-expressing myofibroblasts. 21,31 However, the SMC actin-expressing cells of the Cxcr4 / /Cxcr4 / vein grafts seemed relatively deficient in two other key markers of fibrocytes 21 : collagen I and CD45 (Figs 5 and 6). Consequently, we infer that the media of Cxcr4 / /Cxcr4 / vein grafts is populated principally by SMCs, and not bone marrowderived fibrocytes. Reduction in CXCR4 activity reduced the prevalence of leukocytes in arterialized vein grafts (Fig 6), just as CXCR4 antagonism reduced macrophage density in injured mouse femoral arteries. 15 These observations accord with models of CXCR4-dependent recruitment of inflammatory cells 6 and may explain the reduction of vein graft neointimal hyperplasia we observed in Cxcr4 / vein graft recipients. By decreasing leukocyte density in arterializing vein grafts, reduction of vein graft-extrinsic cells CXCR4 activity diminishes the number of cytokine-secreting cells in the vein graft. 18 A reduction in vein graft cytokine levels, in turn, would be expected to reduce medial SMC activation, manifest as proliferation and migration into the neointimal layer of the vein graft. 3 Perhaps for these reasons, our data suggest that vein graft neointimal hyperplasia depends upon CXCR4 activity in vein graft-extrinsic cells, rather than vein graft-intrinsic cells, even though CXCR4 activation can drive SMC proliferation and migration. 11 CXCR4 deficiency had no effect on vein graft reendothelialization, just as CXCR4 antagonism failed to affect re-endothelialization of wire-injured arteries. 14,15 This result may be attributable to one or more of several factors. First, graft-extrinsic endothelial cells constitute only 10% of total vein graft endothelial cells in our model, 4 and consequently our approach may not have been sensitive enough to discern CXCR4-dependent differences in such a small fraction of the vein graft endothelial cells. Second, CXCR4-mediated endothelial progenitor cell recruitment 27 may be offset by CXCR4-promoted endothelial cell apoptosis and cytokine secretion, 13 which by recruiting inflammatory cells to the vein graft may further promote endothelial cell apoptosis. 19 Study limitations. These mouse studies were performed with interposition vein grafts and thus cannot model all of the hemodynamic parameters that are obtained in vein grafts used to bypass partially patent, atherosclerotic vessels. In addition, our genetic approach to reducing CXCR4 activity, heterozygous gene deletion, cannot be applied to humans. However, the CXCR4 antagonist plerixafor (AMD3100) is safely used in humans 32,33 and could conceivably be used to treat vein grafts focally, at the time of implantation. Because our experiments did not use plerixafor, our work cannot determine whether vein graftlocalized plerixafor treatment, perhaps in the form of a peri-adventitial gel, 34,35 could reduce vein graft neointimal hyperplasia by inhibiting CXCR4 not only on vein graftintrinsic cells but also on graft-extrinsic cells, as they endeavor to migrate into the graft. 34,35 Thus, the clinical translatability of our CXCR4 findings remains to be determined. CONCLUSIONS The present study demonstrates that the SDF-1/ CXCR4 signaling system in vein graft-extrinsic cells contributes to vein graft neointimal hyperplasia. Mechanisms underlying this phenomenon include CXCR4-mediated recruitment of inflammatory cells to the vein graft. AUTHOR CONTRIBUTIONS Conception and design: LZ, NF Analysis and interpretation: LZ, LB, NF Data collection: LZ, LB Writing the article: LZ, NF Critical revision of the article: LZ, NF Final approval of the article: LZ, LB, NF Statistical analysis: LZ, NF Obtained funding: NF Overall responsibility: NF REFERENCES 1. Sabik JF 3rd. Understanding saphenous vein graft patency. Circulation 2011;124:273-5. 2. Alexander JH, Hafley G, Harrington RA, Peterson ED, Ferguson TB Jr, Lorenz TJ, et al. Efficacy and safety of edifoligide, an E2F transcription factor decoy, for prevention of vein graft failure following coronary artery bypass graft surgery: PREVENT IV: a randomized controlled trial. JAMA 2005;294:2446-54. 3. Cai X, Freedman NJ. New therapeutic possibilities for vein graft disease in the post-edifoligide era. Future Cardiol 2006;2:493-501. 4. Zhang L, Freedman NJ, Brian L, Peppel K. Graft-extrinsic cells predominate in vein graft arterialization. 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