- Email: [email protected]
c-kit+ progenitors and reduces vascular restenosis in a murine vascular injury model
A depleting antibody toward sca-1 mitigates a surge of CD34þ/c-kitþ progenitors and reduces vascular restenosis in a murine vascular injury model Bryan W. Tillman, MD, PhD,a,b,c Jeremy Kelly, PhD,b,c Tara D. Richards, BS,b,c Alex F. Chen, MD, PhD,b,d Albert D. Donnenberg, PhD,c,e,f Vera S. Donnenberg, PhD,c,e,g and Edith Tzeng, MD,a,b,d Pittsburgh, Pa Objective: Vascular restenosis remains a major obstacle to long-term success after vascular intervention. Circulating progenitor cells have been implicated in restenosis, and yet it has remained unclear if these cells, particularly nonendothelial progenitors, have an active role in this pathologic process. We hypothesized that circulating CD34D/c-kitD progenitors would increase after vascular injury, mirrored by changes in the injury signal, stromal cell-derived factor 1a (sdf1a). We further postulated that an antibody-based depletion would mitigate progenitor surge and, in turn, reduce restenosis in a murine model. Methods: C57BL6 mice underwent wire injury of the femoral artery and were compared with mice with sham surgery and vessel ligation by flow cytometry as well as by sdf1a enzyme-linked immunosorbent assay of peripheral blood. Next, injured C57BL6 mice treated with a depleting antibody toward the progenitor marker sca-1 or with an isotype control were compared in terms of sdf1a as well as enumeration of progenitors. At 28 days, restenosis was quantified between sca1- and isotype-treated animals. Results: Wire injury generated an increase in sdf1a as well as a surge of CD34D/c-kitD progenitors relative to nonsurgical controls (P [ .005). Treatment with sca-1 antibody ablated the peripheral surge compared with isotype-treated, injured animals (P [ .02), and sca progenitor depletion reduced the 28-day intima to media ratio in a statistically significant fashion compared with either nontreated (P [ .04) or isotype-treated (P [ .036) animals. Conclusions: Our study has demonstrated that sca-1 antibody reduces both progenitor surge and vascular restenosis after endoluminal vascular injury in a murine model. This suggests that circulating progenitors play an active role in restenotic disease. (J Vasc Surg 2016;64:1084-92.) Clinical Relevance: Vascular restenosis is a clinically important complication leading to failure of up to half of open and endovascular interventions in under 5 years. As a result, this pathologic process affects long-term patient outcomes and increases health care costs. Progenitor cells circulating in the blood have been suggested to contribute to restenosis, but it has not been demonstrated if these cells play an active role in this pathologic process. The results of this study suggest that circulating progenitor cells may represent an important contributor to restenosis and that blockade of these cells may represent an approach to prevent this life- and limb-threatening complication.
Restenosis is a pathologic process of subintimal cellular proliferation and matrix deposition that threatens up to half of all vascular interventions in under 5 years. It affects a From the Division of Vascular Surgerya and Department of Surgery,b University of Pittsburgh Medical Center; the McGowan Institute for Regenerative Medicine, University of Pittsburghc; the Department of Veterans Affairs Medical Center, University of Pittsburghd; the University of Pittsburgh Cancer Institute, University of Pittsburghe; and the Division of Hematology/Oncologyf and Department of Cardiothoracic Surgery,g University of Pittsburgh School of Medicine. Support for this project includes the Vascular Cures Wylie Scholar Award; the American Surgical Association Fellowship Research Award; the Vascular Medicine Institute, Pilot Project Program in Hemostasis and Vascular Biology (University of Pittsburgh); and the Division of Vascular Surgery, Department of Surgery (University of Pittsburgh Medical Center). Author conflict of interest: none. Correspondence: Bryan W. Tillman, MD, PhD, Division of Vascular Surgery, University of Pittsburgh Medical Center Shadyside, 5200 Centre Ave, Ste 307, Pittsburgh, PA 15232 (e-mail: [email protected]). 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 Copyright Ó 2016 by the Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jvs.2015.05.002
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wide spectrum of interventions spanning the coronary circulation, peripheral vasculature, and even hemodialysis access.1-4 Whereas drug-eluting stents have been an important development, they are contraindicated in a variety of contexts, including anatomic points of flexion, bifurcations, and open surgical bypass. Evidence of involvement of progenitor cells in restenotic disease has been reported by several groups, including histologic studies,5,6 murine cellular transplant models,7 animal models,8,9 and even human clinical studies.10-12 The last, specifically, have revealed associations between increased circulating progenitor cells and the occurrence of restenosis after human coronary interventions. As further evidence, the study of Inoue et al12 described an increase among progenitor cells after a bare-metal coronary stent placement in humans and yet observed a reduction in progenitors after placement of a drug-eluting stent. Unfortunately, most of these studies have been observational, and skepticism has remained as to whether progenitors are active participants or merely bystanders in the pathologic process of restenosis. To examine the role of these cells, we have developed an approach in which progenitor cells can be downmodulated in an adult murine model. In addition, multiple previous
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reports have characterized the subset of progenitors known as endothelial progenitor cells (EPCs). An important distinction of the present study is that we sought to examine a more primitive progenitor cell population known as short-term hematopoietic stem cells (STHSCs).13,14 These cells expressing CD34 and c-kit are derived from sca-1þ cells and have not been previously examined after vascular injury. In targeting this more primitive progenitor, the impact of nonendothelial progenitors could be examined; this includes myeloid lineages that have become a cornerstone of other vascular disease.15-17 As one of the potential linkages between vascular injury and progenitors, stromal cell-derived growth factor a/ CXCL12 (sdf1a) has an essential role in progenitor cell release from the bone marrow to the circulation as well as in progenitor homing,18,19 with importance for disease contexts such as diabetic wounds and cancer metastasis. Ischemia is one of the traditional vascular triggers for sdf1a, which culminates in recruitment of EPCs for angiogenesis.20 We postulated that similar increases would be observed after endoluminal injury of the artery and may represent an early event related to more primitive progenitor recruitment after vascular injury. Our group has previously reported a surge of CD34 progenitors limited to macrovascular injury and also detailed an effective affinity pheresis approach for mitigating progenitor surge in an ovine model.21 We hypothesized that similar approaches to attenuate progenitor surge would reduce restenotic disease. In the present murine model, a pheresis approach was not practical, and therefore an antibody-based depletion modeled on classic T-cell depletion approaches was instead pursued.22 We selected an antibody to sca-1, a marker of murine ST-HSCs, toward the goal of depleting these cells. In summary, the goals for this study were to characterize the pattern of both the vascular injury signal sdf1a and circulating progenitor cells after vascular injury as well as to determine the impact of sca-1 depletion therapy on both ST-HSC progenitor surge and restenotic outcome. METHODS Animals. C57BL6 mice between the ages of 8 and 12 weeks were purchased from Jackson Laboratory (Bar Harbor, Me; catalog #000664). All procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Femoral wire injury. Consistent with an Institutional Animal Care and Use Committee-approved protocol, mice anesthetized with inhaled isoflurane underwent femoral artery exposure with or without femoral artery wire injury as previously described,23 with the following modifications. Briefly, the saphenous artery was ligated distally, an arteriotomy was created on the saphenous artery, and a 0.014-inch wire (Cook Medical, Bloomington, Ind; catalog #G02426) was passed through an arteriotomy. The saphenous artery was then ligated above the arteriotomy, leaving the profunda and epigastric vessels intact. A separate group of ligation-only controls underwent ligation
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of the saphenous artery at similar locations but without wire injury. A sham surgery group consisted of femoral artery exposure without vessel injury. After wound closure, the animal was recovered and received postoperative analgesia. Antibodies. Rat antibody reactive to murine sca-1, immunoglobulin G2a (IgG2a) k isotype, was prepared from hybridoma E13 161-7 (ATCC, Manassas, Va; catalog #HB-215). Rat IgG2a k isotype, clone RTK2758, was purchased from BioLegend (San Diego, Calif; catalog #400502). Both antibodies were conjugated to biotin with EZ-Link Sulfo-NHS-Biotin per the manufacturer’s instructions (Pierce, Rockford, Ill; catalog #21326) at 20-fold molar excess and then dialyzed against phosphatebuffered saline and frozen until use. Mice received intraperitoneal doses of rat anti-sca-1 IgG2a antibody E13 161-7 at 0.5 mg/d for 1 day before surgery and 2 days after surgery. Maintenance doses of 0.25 mg/d were given on days 3, 5, 7, and 9 after injury. Isotype control mice received isotype IgG (cIg) on the same schedule. Mice in the naive group received saline injection without antibody. Inclusion of doses given preoperatively, on the operative day, and postoperatively represents a total duration of treatment of 11 days. Confocal microscopy of endothelium. Naive and wire-injured femoral arteries (n ¼ 4 per each group) were explanted after paraformaldehyde (Affymetrix, Santa Clara, Calif; catalog #19943) perfusion fixation, cryoprotected with 30% sucrose (Sigma-Aldrich, St. Louis, Mo; catalog #S0389-1 KG), opened lengthwise, and pinned back to expose the vessel intima. The arteries were incubated in a 1:100 dilution of isolectin GS-IB4, Alexa Fluor 568 conjugate (Molecular Probes, Grand Island, NY; catalog #I21412) overnight at 4 C. The artery was then stained with Hoechst dye (Sigma-Aldrich, catalog #B-2883) and mounted with gelvatol. Images were captured on the Nikon 90i confocal microscope, using NIS-Elements software (Nikon Instruments, Melville, NY) at the Center for Biologic Imaging, University of Pittsburgh. Images were further analyzed in Imaris image processing and analysis software (Bitplane, South Windsor, Conn). Specimen collection. At the time of collection, all mice received a small dose of subcutaneous heparin (APP Pharmaceuticals, Schaumburg, Ill; catalog #401796I). This was selected to avoid adverse effects of ethylenediaminetetraacetic acid on enzyme-linked immunosorbent assay (ELISA) analyses. Terminal blood samples were drawn within 5 minutes, and blood was centrifuged at 1800 rpm to yield plasma and a cellular pellet. Plasma was centrifuged a second time, aliquoted, and frozen for ELISA analysis. The cellular pellet underwent lysis of red cells and was stained as described later. Bone marrow was collected by flushing marrow from tibia and femurs of euthanized animals. Cellular suspensions were filtered with a 100-mm mesh filter (Fisher, Pittsburgh, Pa; catalog #22-363-549) before staining. ELISA. Plasma specimens stored at 80 C were analyzed by ELISA for sdf1a (R&D Systems, Minneapolis,
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Minn; catalog #MCX120) per the manufacturer’s instructions. After substrate incubation, plates were read with a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, Calif) at 450 nm. Flow cytometry. After removal of plasma from blood, cellular pellets were mixed with ammonium chloride solution (pH 7.4) for lysis of red blood cells and filtered with a 100-mm mesh filter. The specimens were centrifuged at 1800 rpm and resuspended in staining buffer (0.5% bovine serum albumin, 0.1% azide in phosphate-buffered saline, pH 7.4) twice, followed by aspiration of supernatant and incubation with FcR Blocking reagent (Miltenyi, Auburn, Calif; catalog #130-092-575). Cells were stained with antibodies APC/ Cy7 CD117 (c-kit; BioLegend, catalog #105826), Alexa700 anti-CD34 (Becton Dickinson, Franklin Lakes, NJ; catalog #BDB560518), PE-Texas Red CD45 (Invitrogen, Grand Island, NY; catalog #MCD4517), and phycoerythrin-labeled lineage cocktail (CD3E; Ly-6 G/Ly-6C [Gr-1]; CD45 R/ B220, CD11 b; and TER-119/erythroid cells; BioLegend, catalog #133303). APC streptavidin (BioLegend, catalog #405207) was used for detection of biotinylated primary sca1 and isotype control antibodies. After incubation, cells were washed and fixed with paraformaldehyde. Washed cells were permeabilized, followed by incubation with DAPI (Molecular Probes, catalog #D1306). Cells were examined by flow cytometry for the markers CD117 (c-kit), CD34, sca-1, CD45, and lineage markers using a Gallios flow cytometer (Beckman Coulter, Brea, Calif). Data were processed using VenturiOne software (Applied Cytometry, Dinnington, Sheffield, UK). Flow compensation was calculated by using single antibody-stained mouse IgG capture beads (BD CompBeads; BD Biosciences, San Jose, Calif; catalog #BDB552845). Debris, doublets, lineage-positive cells, and residual red blood cells were exclusion gated, and all downstream gating was performed on the DAPI-positive population. Circulating progenitors were defined as cells expressing CD34 and c-kit in the absence of the maturity marker CD45. Quantification of restenosis. For determination of restenosis, explantation of wire-injured vessels was performed on postoperative day 28 consistent with previous reports. Paraformaldehyde perfusion-fixed, sucrose-cryopreserved sections were sectioned and stained with elastin and hematoxylin and eosin. Area within the external elastic lamina, within the internal elastic lamina, and within the lumen was calculated with ImageJ software (National Institutes of Health, Bethesda, Md). Intimal area was calculated as internal elastic lamina area minus lumen area. Medial area was calculated as external elastic lamina area minus internal elastic lamina area. The intima/media ratio was then determined as intimal area divided by medial area. Statistical analysis. Comparison of ELISA results, flow cytometric analysis, and histologic data was performed by the Student t-test. Statistical significance was defined as P < .05. RESULTS Whereas we have previously reported a surge among circulating progenitors after vascular injury in an ovine
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model,21 it was necessary to determine if similar changes also occur in a murine model, specifically among STHSCs. Wire injury of the femoral artery is a welldescribed vascular injury model for the investigation of restenosis. To confirm the degree of endothelial injury in this laboratory, C57BL6 mice underwent unilateral wire injury of the femoral artery as previously described23 (Fig 1, A). Vessels were examined on postoperative day 1 using en face fluorescent confocal techniques with lectin staining. As shown, wire injury expectedly created a significant injury to the endothelial monolayer compared with uninjured artery (Fig 1, B and C). Given the expected role of vascular injury signaling molecules in the pathologic process of restenosis, the early injury marker sdf1a was assayed by ELISA (Fig 2). Blood was collected from animals on postoperative day 1 after femoral wire injury and examined by ELISA and flow cytometry. As our wire injury model inherently includes a ligation of the distal femoral artery and potentially a degree of ischemic tissue injury, we included an experimental group consisting of ligation alone of the distal femoral artery without wire injury. Importantly, there were no obvious ischemic complications among our study animals from this limited arterial ligation. In addition, a sham surgery group underwent vascular exposure without vascular injury or ligation. Our results demonstrated a 2.2-fold increase in sdf1a after femoral wire injury (P ¼ .0003) compared with nonsurgical animals. Ligation-alone also increased sdf1a by twofold (P ¼ .001) compared with the nonsurgical group. There was no significant difference between sham surgery and nonsurgical animals or between ligation and wire injury groups. Next, flow cytometric gating was used to examine cells for the progenitor markers CD34 and c-kit (Fig 3), known as ST-HSCs.14 On postoperative day 1, animals with wire injury revealed a significant 2.3-fold increase among CD34þ/c-kitþ circulating progenitor populations relative to nonsurgical controls (P ¼ .005), sham surgery (P ¼ .016), or distal ligation alone (P ¼ .018) as shown in Fig 3. Sca-1 is a glycosylphosphatidylinositol-anchored cell surface protein that is a marker of primitive murine hematopoietic cells,24 which are precursors to CD34þ/ckitþ ST-HSCs. Toward the goal of mitigating surge of these cells after injury, we next examined an IgG2a antibody directed toward sca-1 using methodology similar to classic T-cell depletion.25,26 Mice were given empirical antibody dosing with an initial 3 days of induction followed by maintenance dosing every other day until postoperative day 9. This time frame was based on our initial findings of sdf1a surge, which appeared to taper during this period. An isotype antibody was administered to a separate control group (cIg) using the same administration schedule. In addition, distinct nonsurgical groups of mice were treated with either isotype and sca-1 antibodies, as additional controls. We observed no evidence of physiologic impact of this antibody among treated animals. The vascular injury signal sdf1a was assayed after treatment with isotype and sca antibody. Compared with nonsurgical naive animals,
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Fig 1. Wire injury of the murine femoral artery injures endothelium of the femoral segment. A, Representative image of murine femoral wire injury with ligation of the saphenous artery (arrow) and wire dilation of the common femoral artery (arrowhead). B and C, Confocal fluorescent microscopy of lectin (red) and Hoechst (blue) staining revealed an en face intact endothelial monolayer of a control vessel (B) that was absent on a wire-injured vessel (C). Scale bar is shown.
wire injury with control antibody (Wi cIg) and wire injury with sca (Wi sca) increased sdf1a 1.7- and 2.2-fold, respectively, on postoperative day 1 (Fig 4). As might be expected from a therapy directed at downstream events, treatment with sca-1 antibody did not affect levels of sdf1a compared with isotype control-treated animals. The sdf1a levels for isotype wire-injured animals remained elevated at 3 and 9 days postoperatively (Fig 5). Flow cytometric gating after treatment of nonsurgical and wire-injured mice treated with isotype or sca-1 antibody is shown in Fig 6. Similar to the trend of untreated animals after wire injury, we observed that wire-injured mice treated with isotype antibody (Wi cIg) revealed a surge among CD34þ/c-kitþ circulating progenitors after wire injury relative to naive animals on postoperative day 1 (P ¼ .004). In contrast, these progenitors were significantly reduced among wire-injured mice treated with sca1 depletion (Wi sca) relative to isotype-treated animals (P ¼ .02) and not significantly increased relative to naive nonsurgical animals (P ¼ .38; Fig 7). As part of an extended time course to identify rebound phenomenon, sca-1 therapy was noted to mitigate the surge of circulating progenitors at additional time points of 3 and 9 days after injury (P ¼ NS relative to naive nonsurgical animals at all time points; Fig 7). Pluripotent progenitors have important physiologic roles in hematopoiesis27 and endothelial repair. For this reason, preservation of the bone marrow reservoir is essential for survival. Given the theoretical concerns about bone marrow depletion from this approach, bone marrow was compared between nonsurgical sca-1- and isotype-treated
Fig 2. Stromal cell-derived factor 1a (sdf1a) increases after vascular injury. Compared with nonsurgical controls or mice with a sham surgery, mice with distal femoral artery ligation revealed a significant increase of sdf1a expression by enzyme-linked immunosorbent assay (ELISA; P ¼ .001), whereas mice with femoral wire injury revealed a similar 2.2-fold increase in sdf1a (P ¼ .003; n ¼ 6 per condition).
animals with flow cytometry. As shown in Fig 8, whereas sca-1þ progenitor cells were significantly reduced by sca1 depletion at an early time point of day 3, they quickly rebounded by day 11 of therapy to levels equivalent to those of isotype-treated animals. Based on previous reports of histologic and clinical associations of progenitor cells with restenosis, we hypothesized that mitigation of the ST-HSC surge would reduce restenotic disease. Wire-injured mice were divided into three
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Fig 3. Wire injury of the femoral artery increased circulating CD34þ/c-kitþ cells (short-term hematopoietic stem cells [ST-HSCs]). Flow cytometry included (A) exclusion of debris followed by (B) gating of DAPI-positive events. Lineage-positive and CD45-positive events were then excluded. Shown are representative gating of CD34þ/c-kitþ events among nonsurgical (C) and wire-injured (D) animals. Compared with mice with no surgery, animals after wire injury of the femoral artery revealed a statistically significant 2.3-fold increase among CD34þ/c-kitþ cells (P ¼ .005; E). Sham surgery and vessel ligation did not result in a significant rise in progenitors. Shown are percentage of cells with standard error bars (n ¼ 6 per condition).
groups: saline treated (Wi saline), treatment with an isotype control (Wi cIg), or treatment with sca-1-depleting antibody (Wi sca) using the same 11-day dosing protocol of the cellular analysis. At 28 days after injury, iliac and femoral vessels were explanted and examined histologically. As demonstrated in Fig 9, intima to media ratios were calculated and revealed a statistically significant reduction in the intima to media ratio compared with both saline-treated mice (P ¼ .04) and isotype controls (P ¼ .036). DISCUSSION Restenosis remains a major threat to the longevity of interventions after cardiovascular and hemodialysis access procedures. With failure of up to half of interventions in 5 years, this pathologic process has increased the number
of procedures, escalated costs to the health care system, and compromised long-term patient outcome. Although trends among EPCs have been well documented after vascular injury,28 these cells represent only a narrow subset of progenitors. In this study, we instead examined the trends of more primitive ST-HSC14 progenitors that have not been previously characterized after vascular injury. The role of these primitive progenitors as precursors to myeloid cells is especially fascinating, given the important association of myeloid cells with vascular disease such as atherosclerosis15,16 and restenosis.17,29 Our findings in the murine model suggest a surge among STHSC (CD34þ/c-kitþ) cells after wire injury of the femoral artery. We did not observe a significant change in ST-HSC after a sham surgery of the same compartment, suggesting that microvascular injury inherent in surgical exposure
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Fig 4. Antibody treatment does not prevent increases in plasma stromal cell-derived factor 1a (sdf1a) 1 day after vascular injury. Vascular wire injury among isotype (Wi cIg) or sca-treated (Wi sca) mice increased plasma sdf1a relative to nonsurgical, non-antibodytreated controls (naive) and to nonsurgical isotype (cIg, treated with control antibody without injury) on day 1. The sdf1a levels in Wi sca mice were not significantly different from those of Wi cIg animals. Means with standard error are shown (n ¼ 8 per condition).
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Fig 6. Sca-1 antibody treatment reduced a surge among shortterm hematopoietic stem cells (ST-HSCs) 1 day after wire injury. Compared with naive nonsurgical mice (n ¼ 6) or nonsurgical isotype controls (cIg, n ¼ 8), wire injury after isotype treatment (Wi cIg, n ¼ 8) increased progenitor cells by more than threefold (P ¼ .004 and .0001, respectively). Animals treated with sca-1 antibody (Wi sca, n ¼ 8) prevented the surge among progenitors relative to naive animals (P ¼ NS). Shown are mean percentage of CD34þ/c-kitþ/Lin cells with standard error.
Fig 5. Stromal cell-derived factor 1a (sdf1a) levels remain elevated over 9 days after wire injury and are not affected by sca-1 treatment (Wi sca). The sdf1a levels at extended time points of 3 and 9 days after wire injury remained significantly elevated compared with either naive nonsurgical or nonsurgical isotype (cIg) controls. Treatment with sca-1 antibody did not affect sdf1a levels (P ¼ NS relative to wire injury with control antibody [Wi cIg]; n ¼ 8 per condition for naive and postoperative day 1; n ¼ 6 for all other time points).
Fig 7. Sca-1 antibody mitigates surge of CD34þ/c-kitþ short-term hematopoietic stem cells (ST-HSCs) out to 9 days. Whereas the surge of CD34þ/c-kitþ cells remained elevated through postoperative day (POD) 9, CD34þ/c-kitþ cells among sca-1-treated wire-injured animals (Wi sca) were nonsignificant relative to naive nonsurgical animals at all time points. Shown are mean percentage CD34þ/c-kitþ/Lin with standard error. cIg, Isotype control immunoglobulin; Wi, wire injured.
alone is not sufficient to elicit a progenitor response. Although increases among EPCs have been observed by others after ischemic injury,30 we did not observe a similar trend with ST-HSCs after ligation of the saphenous artery. Importantly, the ST-HSCs of this study reflect a distinct progenitor from the EPCs quantified in other studies.
In addition, our model of wire injury involves ligation of only the distal femoral artery with preservation of collaterals, which the detailed report by Hellingman et al31 would suggest does not create a significant ischemic picture. As a result, the absence of significant changes in progenitors in our study would not necessarily imply
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Fig 8. Murine bone marrow is preserved after sca antibody at late time points. By flow cytometric analysis, sca-1 cells in sca-treated mice were significantly reduced at day 3 of therapy relative to isotype (cIg) animals. By day 11, despite ongoing antibody treatment, there was no difference in scaþ cells between cIg- and scatreated mice (n ¼ 6 per condition on day 3, n ¼ 9 on day 11). Shown are mean percentage with standard error.
absence of progenitor surge in a more authentic ischemic model. Looking upstream of progenitor cells, vascular injury results in the release of multiple signals related to progenitor mobilization.32,33 The particular importance of sdf1a (also known as CXCL12) in the context of progenitors has previously been described in the setting of ischemic injury34 with specific contributions in both mobilization35,36 and homing of EPCs.18,37 Whereas our findings reveal an increase in sdf1a that parallels the surge of CD34þ/c-kitþ progenitors, our data fall short of concluding that sdf1a is a stimulus for these CD34þ/ckitþ cells. In fact, our findings of sdf1a could instead be explained as a result of the limited ischemia caused by the distal ligation inherent in this model. Notably, our results suggest sdf1a surge after a distal ligation but without accompanying cellular surge. Whereas this may seem to contrast with previous reports of progenitors after ischemic injury,34,38 it is essential to note the significant differences in the ischemic model as well as differences in the phenotype of cells being examined (EPCs vs STHSCs). Our study has several limitations. First, our dosing strategy was empirically derived from classic T-cell depletion protocols, and further investigation may be needed to optimize the dosing for this application. Although our protocol captured changes among progenitor cells out to 9 days, we did not examine cells outside of this time frame. Finally, it is possible that our antibody-based depletion of progenitors merely postponed restenosis as we did not examine specimens beyond a single histologic time point of 28 days. To date, it has been unclear whether circulating progenitor cells actually play a meaningful and active role in
restenotic disease. The absence of an adult model in which to deplete progenitor cells has been an obstacle to answering this question. We used a depleting IgG2a antibody toward a marker of primitive murine progenitors, sca-1. Our results confirmed that this approach can indeed mitigate CD34þ/c-kitþ progenitor surge after vascular injury. Based on concerns that a rebound surge might overwhelm this approach, we examined animals out to 9 days and yet treatment with a depleting sca-1 antibody consistently prevented an increase in progenitors. There are theoretical concerns that an approach to deplete a key marker of pluripotent progenitors could precipitate bone marrow failure. In fact, despite a minor reduction in sca-1þ cells at an early time point, we confirmed preservation of the sca-1þ marrow reservoir at a later time point, still amid sca-1 depletion therapy. The high proliferative capacity of bone marrow may have been protective in this setting. This model of sca-1 depletion may have utility in other studies for reduction of circulating progenitors. Our study further concludes that mitigation of ST-HSC progenitor surge is effective toward reducing restenosis in the murine model. Whereas this study does not address the specific mechanism of how ST-HSCs contribute to restenosis, several previous studies have demonstrated that circulating progenitors can differentiate into smooth muscle cells.12,39,40 In addition, ST-HSCs are known precursors to monocytes/macrophages,14 cell types with roles in both atherosclerotic and restenotic vascular disease.15-17,29 As a result, further investigations will be needed to clarify whether progenitor cells contribute either by direct cellular contribution or by modifying the inflammatory environment. In particular, we are already examining cell types known to be derived from ST-HSCs, including monocytic lineages. CONCLUSIONS This study demonstrates important changes among circulating ST-HSCs that appear to be limited to macrovascular injury. We have developed a successful adult murine model to attenuate the response of CD34þ/c-kitþ progenitor cells and demonstrated that mitigation of the progenitor response leads to a significant reduction in vascular restenosis. Further studies on the identity of ST-HSCderived effector cells that contribute to restenosis may offer new approaches to this life- and limb-threatening complication. The authors gratefully acknowledge The Center for Biologic Imaging (University of Pittsburgh) for imaging support. AUTHOR CONTRIBUTIONS Conception and design: BT, JK, TR, AC, AD, VD, ET Analysis and interpretation: BT, JK, TR, AC, AD, VD, ET Data collection: BT, JK, TR, AD, VD Writing the article: BT, AC, ET Critical revision of the article: BT, ET Final approval of the article: BT
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Fig 9. Depletion of short-term hematopoietic stem cells (ST-HSCs) reduces restenosis after wire injury. Compared with mice treated with control immunoglobulin G (Wi cIg) at the time of wire injury (A), mice treated with sca1-depleting antibody (Wi sca) revealed reduced restenosis at 28 days (B; hematoxylin and eosin, 400). Compared with either control mice with saline (n ¼ 8) or mice with treatment of isotype control Ig (cIg, n ¼ 5), mice treated with sca-1-depleting antibody (n ¼ 8) revealed a significant decrease in the intima/media ratio. C, Means with standard error bars are shown. IEL, Internal elastic lamina; L, lumen; I, intima; M, media; Wi, wire injury.
Statistical analysis: BT Obtained funding: BT Overall responsibility: BT
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deleterious effects of granulocyte colony-stimulating factor. J Thorac Cardiovasc Surg 2006;131:843-52. Rotmans JI, Heyligers JM, Verhagen HJ, Velema E, Nagtegaal MM, de Kleijn DP, et al. In vivo cell seeding with anti-CD34 antibodies successfully accelerates endothelialization but stimulates intimal hyperplasia in porcine arteriovenous expanded polytetrafluoroethylene grafts. Circulation 2005;112:12-8. Schober A, Hoffmann R, Opree N, Knarren S, Iofina E, Hutschenreuter G, et al. Peripheral CD34þ cells and the risk of in-stent restenosis in patients with coronary heart disease. Am J Cardiol 2005;96:1116-22. Pelliccia F, Cianfrocca C, Rosano G, Mercuro G, Speciale G, Pasceri V. Role of endothelial progenitor cells in restenosis and progression of coronary atherosclerosis after percutaneous coronary intervention: a prospective study. JACC Cardiovasc Interv 2010;3:78-86. Inoue T, Sata M, Hikichi Y, Sohma R, Fukuda D, Uchida T, et al. Mobilization of CD34-positive bone marrow-derived cells after coronary stent implantation: impact on restenosis. Circulation 2007;115:553-61. Yang L, Bryder D, Adolfsson J, Nygren J, Mansson R, Sigvardsson M, et al. Identification of LinSca1þkitþCD34þFlt3 short-term hematopoietic stem cells capable of rapidly reconstituting and rescuing myeloablated transplant recipients. Blood 2005;105:2717-23. Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001;414:105-11. Hanna RN, Carlin LM, Hubbeling HG, Nackiewicz D, Green AM, Punt JA, et al. The transcription factor NR4A1 (Nur77) controls bone marrow differentiation and the survival of Ly6C-monocytes. Nat Immunol 2011;12:778-85. Stoneman V, Braganza D, Figg N, Mercer J, Lang R, Goddard M, et al. Monocyte/macrophage suppression in CD11b diphtheria toxin receptor transgenic mice differentially affects atherogenesis and established plaques. Circ Res 2007;100:884-93. Danenberg HD, Fishbein I, Gao J, Monkkonen J, Reich R, Gati I, et al. Macrophage depletion by clodronate-containing liposomes reduces neointimal formation after balloon injury in rats and rabbits. Circulation 2002;106:599-605. Gallagher KA, Liu ZJ, Xiao M, Chen H, Goldstein LJ, Buerk DG, et al. Diabetic impairments in NO-mediated endothelial progenitor cell
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22. 23.
24. 25.
26.
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28. 29.
mobilization and homing are reversed by hyperoxia and SDF-1a. J Clin Invest 2007;117:1249-59. Ceradini DJ, Kulkarni AR, Callaghan MJ, Tepper OM, Bastidas N, Kleinman ME, et al. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1. Nat Med 2004;10: 858-64. De Falco E, Porcelli D, Torella AR, Straino S, Iachininoto MG, Orlandi A, et al. SDF-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells. Blood 2004;104:3472-82. Harbuzariu A, Kim J, Meyer EM, Donnenberg AD, Tillman BW. CD34 affinity pheresis attenuates a surge among circulating progenitor cells following vascular injury. J Vasc Surg 2014;59:1686-94. Benjamin RJ, Waldmann H. Induction of tolerance by monoclonal antibody therapy. Nature 1986;320:449-51. Sata M, Maejima Y, Adachi F, Fukino K, Saiura A, Sugiura S, et al. A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia. J Mol Cell Cardiol 2000;32:2097-104. Holmes C, Stanford WL. Concise review: stem cell antigen-1: expression, function, and enigma. Stem Cells 2007;25:1339-47. Benjamin RJ, Cobbold SP, Clark MR, Waldmann H. Tolerance to rat monoclonal antibodies. Implications for serotherapy. J Exp Med 1986;163:1539-52. Cobbold S, Martin G, Waldmann H. Monoclonal antibodies for the prevention of graft-versus-host disease and marrow graft rejection. The depletion of T cell subsets in vitro and in vivo. Transplantation 1986;42:239-47. Domen J, Wagers A, Weissman IL. Bone marrow (hematopoietic) stem cells. 2. Bone marrow (hematopoietic) stem cells. Stem Cell Information. Bethesda, Md: National Institutes of Health, U.S. Department of Health and Human Services; 2011. Available at: http://stemcells.nih.gov/info/Regenerative_Medicine/pages/2006 chapter2.aspx. Accessed January 4, 2015. Lamping K. Endothelial progenitor cells: sowing the seeds for vascular repair. Circ Res 2007;100:1243-5. Cohen-Sela E, Rosenzweig O, Gao J, Epstein H, Gati I, Reich R, et al. Alendronate-loaded nanoparticles deplete monocytes and attenuate restenosis. J Control Release 2006;113:23-30.
30. Liu ZJ, Velazquez OC. Hyperoxia, endothelial progenitor cell mobilization, and diabetic wound healing. Antioxid Redox Signal 2008;10:1869-82. 31. Hellingman AA, Bastiaansen AJ, de Vries MR, Seghers L, Lijkwan MA, Lowik CW, et al. Variations in surgical procedures for hind limb ischaemia mouse models result in differences in collateral formation. Eur J Vasc Endovasc Surg 2010;40:796-803. 32. Tsai S, Butler J, Rafii S, Liu B, Kent KC. The role of progenitor cells in the development of intimal hyperplasia. J Vasc Surg 2009;49:502-10. 33. Zernecke A, Schober A, Bot I, von Hundelshausen P, Liehn EA, Mopps B, et al. SDF-1a/CXCR4 axis is instrumental in neointimal hyperplasia and recruitment of smooth muscle progenitor cells. Circ Res 2005;96:784-91. 34. Penn MS. Importance of the SDF-1:CXCR4 axis in myocardial repair. Circ Res 2009;104:1133-5. 35. Hattori K, Heissig B, Rafii S. The regulation of hematopoietic stem cell and progenitor mobilization by chemokine SDF-1. Leuk Lymphoma 2003;44:575-82. 36. Moore MA, Hattori K, Heissig B, Shieh JH, Dias S, Crystal RG, et al. Mobilization of endothelial and hematopoietic stem and progenitor cells by adenovector-mediated elevation of serum levels of SDF-1, VEGF, and angiopoietin-1. Ann N Y Acad Sci 2001;938:36-45; discussion: 45-7. 37. Liu ZJ, Tian R, An W, Zhuge Y, Li Y, Shao H, et al. Identification of Eselectin as a novel target for the regulation of postnatal neovascularization: implications for diabetic wound healing. Ann Surg 2010;252:625-34. 38. Bauer SM, Goldstein LJ, Bauer RJ, Chen H, Putt M, Velazquez OC. The bone marrow-derived endothelial progenitor cell response is impaired in delayed wound healing from ischemia. J Vasc Surg 2006;43:134-41. 39. Simper D, Stalboerger PG, Panetta CJ, Wang S, Caplice NM. Smooth muscle progenitor cells in human blood. Circulation 2002;106:1199-204. 40. Wang CH, Anderson N, Li SH, Szmitko PE, Cherng WJ, Fedak PW, et al. Stem cell factor deficiency is vasculoprotective: unraveling a new therapeutic potential of imatinib mesylate. Circ Res 2006;99:617-25.
Submitted Feb 23, 2015; accepted May 3, 2015.
INVITED COMMENTARY
Gale L. Tang, MD, Seattle, Wash Restenosis from intimal hyperplasia remains the Achilles heel of lower extremity endovascular and open revascularization procedures despite recent advances in drug-coated balloons and stents. The source of the cells leading to intimal hyperplasia and the most effective strategy to inhibit intimal hyperplasia while also allowing normal arterial wall healing remain in doubt. The first problem is a significant limiting factor for future molecularly targeted therapies because the correct cell type to target is unknown. The second problem can likely be addressed only by correctly answering the first. In an attempt to address the source of cells, Tillman and colleagues demonstrated that there is a surge in circulating vascular progenitor cells at the time of arterial injury in a femoral wire injury mouse model and that this surge is ablated by periprocedural treatment using an antibody to sca-1. They further demonstrated that mice treated with the antibody to sca-1 have significantly less intimal hyperplasia than control mice. This is supported by previous studies showing that the neointimal cells after a more severe femoral wire injury were primarily bone marrow derived.1 However, the source of neointimal cells in mice is heavily dependent on the model being used,1 and it remains an open question which
model best reflects what happens in humans after angioplasty or open bypass. What is also left to be determined is whether the surge in vascular progenitor cells actually causes the intimal hyperplasia. Other groups have shown a significant proportion of sca-1þ cells within the arterial adventitia,2 suggesting the alternative hypothesis that the antibody to sca-1 may instead lead to decreased intimal hyperplasia because of suppression of adventitial sca-1þ progenitors. Unfortunately, there is no recognized sca-1 homologue in humans, so translating this work from mice to humans may be problematic.
REFERENCES 1. Tanaka K, Sata M, Hirata Y, Nagai R. Diverse contribution of bone marrow cells to neointimal hyperplasia after mechanical vascular injuries. Circ Res 2003;93:783-90. 2. Hu Y, Zhang Z, Torsney E, Afzal AR, Davison F, Metzler B, et al. Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice. J Clin Invest 2004;113:1258-64.
Restenosis is a pathologic process of subintimal cellular proliferation and matrix deposition that threatens up to half of all vascular interventions in under 5 years. It affects a From the Division of Vascular Surgerya and Department of Surgery,b University of Pittsburgh Medical Center; the McGowan Institute for Regenerative Medicine, University of Pittsburghc; the Department of Veterans Affairs Medical Center, University of Pittsburghd; the University of Pittsburgh Cancer Institute, University of Pittsburghe; and the Division of Hematology/Oncologyf and Department of Cardiothoracic Surgery,g University of Pittsburgh School of Medicine. Support for this project includes the Vascular Cures Wylie Scholar Award; the American Surgical Association Fellowship Research Award; the Vascular Medicine Institute, Pilot Project Program in Hemostasis and Vascular Biology (University of Pittsburgh); and the Division of Vascular Surgery, Department of Surgery (University of Pittsburgh Medical Center). Author conflict of interest: none. Correspondence: Bryan W. Tillman, MD, PhD, Division of Vascular Surgery, University of Pittsburgh Medical Center Shadyside, 5200 Centre Ave, Ste 307, Pittsburgh, PA 15232 (e-mail: [email protected]). 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 Copyright Ó 2016 by the Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jvs.2015.05.002
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wide spectrum of interventions spanning the coronary circulation, peripheral vasculature, and even hemodialysis access.1-4 Whereas drug-eluting stents have been an important development, they are contraindicated in a variety of contexts, including anatomic points of flexion, bifurcations, and open surgical bypass. Evidence of involvement of progenitor cells in restenotic disease has been reported by several groups, including histologic studies,5,6 murine cellular transplant models,7 animal models,8,9 and even human clinical studies.10-12 The last, specifically, have revealed associations between increased circulating progenitor cells and the occurrence of restenosis after human coronary interventions. As further evidence, the study of Inoue et al12 described an increase among progenitor cells after a bare-metal coronary stent placement in humans and yet observed a reduction in progenitors after placement of a drug-eluting stent. Unfortunately, most of these studies have been observational, and skepticism has remained as to whether progenitors are active participants or merely bystanders in the pathologic process of restenosis. To examine the role of these cells, we have developed an approach in which progenitor cells can be downmodulated in an adult murine model. In addition, multiple previous
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reports have characterized the subset of progenitors known as endothelial progenitor cells (EPCs). An important distinction of the present study is that we sought to examine a more primitive progenitor cell population known as short-term hematopoietic stem cells (STHSCs).13,14 These cells expressing CD34 and c-kit are derived from sca-1þ cells and have not been previously examined after vascular injury. In targeting this more primitive progenitor, the impact of nonendothelial progenitors could be examined; this includes myeloid lineages that have become a cornerstone of other vascular disease.15-17 As one of the potential linkages between vascular injury and progenitors, stromal cell-derived growth factor a/ CXCL12 (sdf1a) has an essential role in progenitor cell release from the bone marrow to the circulation as well as in progenitor homing,18,19 with importance for disease contexts such as diabetic wounds and cancer metastasis. Ischemia is one of the traditional vascular triggers for sdf1a, which culminates in recruitment of EPCs for angiogenesis.20 We postulated that similar increases would be observed after endoluminal injury of the artery and may represent an early event related to more primitive progenitor recruitment after vascular injury. Our group has previously reported a surge of CD34 progenitors limited to macrovascular injury and also detailed an effective affinity pheresis approach for mitigating progenitor surge in an ovine model.21 We hypothesized that similar approaches to attenuate progenitor surge would reduce restenotic disease. In the present murine model, a pheresis approach was not practical, and therefore an antibody-based depletion modeled on classic T-cell depletion approaches was instead pursued.22 We selected an antibody to sca-1, a marker of murine ST-HSCs, toward the goal of depleting these cells. In summary, the goals for this study were to characterize the pattern of both the vascular injury signal sdf1a and circulating progenitor cells after vascular injury as well as to determine the impact of sca-1 depletion therapy on both ST-HSC progenitor surge and restenotic outcome. METHODS Animals. C57BL6 mice between the ages of 8 and 12 weeks were purchased from Jackson Laboratory (Bar Harbor, Me; catalog #000664). All procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Femoral wire injury. Consistent with an Institutional Animal Care and Use Committee-approved protocol, mice anesthetized with inhaled isoflurane underwent femoral artery exposure with or without femoral artery wire injury as previously described,23 with the following modifications. Briefly, the saphenous artery was ligated distally, an arteriotomy was created on the saphenous artery, and a 0.014-inch wire (Cook Medical, Bloomington, Ind; catalog #G02426) was passed through an arteriotomy. The saphenous artery was then ligated above the arteriotomy, leaving the profunda and epigastric vessels intact. A separate group of ligation-only controls underwent ligation
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of the saphenous artery at similar locations but without wire injury. A sham surgery group consisted of femoral artery exposure without vessel injury. After wound closure, the animal was recovered and received postoperative analgesia. Antibodies. Rat antibody reactive to murine sca-1, immunoglobulin G2a (IgG2a) k isotype, was prepared from hybridoma E13 161-7 (ATCC, Manassas, Va; catalog #HB-215). Rat IgG2a k isotype, clone RTK2758, was purchased from BioLegend (San Diego, Calif; catalog #400502). Both antibodies were conjugated to biotin with EZ-Link Sulfo-NHS-Biotin per the manufacturer’s instructions (Pierce, Rockford, Ill; catalog #21326) at 20-fold molar excess and then dialyzed against phosphatebuffered saline and frozen until use. Mice received intraperitoneal doses of rat anti-sca-1 IgG2a antibody E13 161-7 at 0.5 mg/d for 1 day before surgery and 2 days after surgery. Maintenance doses of 0.25 mg/d were given on days 3, 5, 7, and 9 after injury. Isotype control mice received isotype IgG (cIg) on the same schedule. Mice in the naive group received saline injection without antibody. Inclusion of doses given preoperatively, on the operative day, and postoperatively represents a total duration of treatment of 11 days. Confocal microscopy of endothelium. Naive and wire-injured femoral arteries (n ¼ 4 per each group) were explanted after paraformaldehyde (Affymetrix, Santa Clara, Calif; catalog #19943) perfusion fixation, cryoprotected with 30% sucrose (Sigma-Aldrich, St. Louis, Mo; catalog #S0389-1 KG), opened lengthwise, and pinned back to expose the vessel intima. The arteries were incubated in a 1:100 dilution of isolectin GS-IB4, Alexa Fluor 568 conjugate (Molecular Probes, Grand Island, NY; catalog #I21412) overnight at 4 C. The artery was then stained with Hoechst dye (Sigma-Aldrich, catalog #B-2883) and mounted with gelvatol. Images were captured on the Nikon 90i confocal microscope, using NIS-Elements software (Nikon Instruments, Melville, NY) at the Center for Biologic Imaging, University of Pittsburgh. Images were further analyzed in Imaris image processing and analysis software (Bitplane, South Windsor, Conn). Specimen collection. At the time of collection, all mice received a small dose of subcutaneous heparin (APP Pharmaceuticals, Schaumburg, Ill; catalog #401796I). This was selected to avoid adverse effects of ethylenediaminetetraacetic acid on enzyme-linked immunosorbent assay (ELISA) analyses. Terminal blood samples were drawn within 5 minutes, and blood was centrifuged at 1800 rpm to yield plasma and a cellular pellet. Plasma was centrifuged a second time, aliquoted, and frozen for ELISA analysis. The cellular pellet underwent lysis of red cells and was stained as described later. Bone marrow was collected by flushing marrow from tibia and femurs of euthanized animals. Cellular suspensions were filtered with a 100-mm mesh filter (Fisher, Pittsburgh, Pa; catalog #22-363-549) before staining. ELISA. Plasma specimens stored at 80 C were analyzed by ELISA for sdf1a (R&D Systems, Minneapolis,
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Minn; catalog #MCX120) per the manufacturer’s instructions. After substrate incubation, plates were read with a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, Calif) at 450 nm. Flow cytometry. After removal of plasma from blood, cellular pellets were mixed with ammonium chloride solution (pH 7.4) for lysis of red blood cells and filtered with a 100-mm mesh filter. The specimens were centrifuged at 1800 rpm and resuspended in staining buffer (0.5% bovine serum albumin, 0.1% azide in phosphate-buffered saline, pH 7.4) twice, followed by aspiration of supernatant and incubation with FcR Blocking reagent (Miltenyi, Auburn, Calif; catalog #130-092-575). Cells were stained with antibodies APC/ Cy7 CD117 (c-kit; BioLegend, catalog #105826), Alexa700 anti-CD34 (Becton Dickinson, Franklin Lakes, NJ; catalog #BDB560518), PE-Texas Red CD45 (Invitrogen, Grand Island, NY; catalog #MCD4517), and phycoerythrin-labeled lineage cocktail (CD3E; Ly-6 G/Ly-6C [Gr-1]; CD45 R/ B220, CD11 b; and TER-119/erythroid cells; BioLegend, catalog #133303). APC streptavidin (BioLegend, catalog #405207) was used for detection of biotinylated primary sca1 and isotype control antibodies. After incubation, cells were washed and fixed with paraformaldehyde. Washed cells were permeabilized, followed by incubation with DAPI (Molecular Probes, catalog #D1306). Cells were examined by flow cytometry for the markers CD117 (c-kit), CD34, sca-1, CD45, and lineage markers using a Gallios flow cytometer (Beckman Coulter, Brea, Calif). Data were processed using VenturiOne software (Applied Cytometry, Dinnington, Sheffield, UK). Flow compensation was calculated by using single antibody-stained mouse IgG capture beads (BD CompBeads; BD Biosciences, San Jose, Calif; catalog #BDB552845). Debris, doublets, lineage-positive cells, and residual red blood cells were exclusion gated, and all downstream gating was performed on the DAPI-positive population. Circulating progenitors were defined as cells expressing CD34 and c-kit in the absence of the maturity marker CD45. Quantification of restenosis. For determination of restenosis, explantation of wire-injured vessels was performed on postoperative day 28 consistent with previous reports. Paraformaldehyde perfusion-fixed, sucrose-cryopreserved sections were sectioned and stained with elastin and hematoxylin and eosin. Area within the external elastic lamina, within the internal elastic lamina, and within the lumen was calculated with ImageJ software (National Institutes of Health, Bethesda, Md). Intimal area was calculated as internal elastic lamina area minus lumen area. Medial area was calculated as external elastic lamina area minus internal elastic lamina area. The intima/media ratio was then determined as intimal area divided by medial area. Statistical analysis. Comparison of ELISA results, flow cytometric analysis, and histologic data was performed by the Student t-test. Statistical significance was defined as P < .05. RESULTS Whereas we have previously reported a surge among circulating progenitors after vascular injury in an ovine
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model,21 it was necessary to determine if similar changes also occur in a murine model, specifically among STHSCs. Wire injury of the femoral artery is a welldescribed vascular injury model for the investigation of restenosis. To confirm the degree of endothelial injury in this laboratory, C57BL6 mice underwent unilateral wire injury of the femoral artery as previously described23 (Fig 1, A). Vessels were examined on postoperative day 1 using en face fluorescent confocal techniques with lectin staining. As shown, wire injury expectedly created a significant injury to the endothelial monolayer compared with uninjured artery (Fig 1, B and C). Given the expected role of vascular injury signaling molecules in the pathologic process of restenosis, the early injury marker sdf1a was assayed by ELISA (Fig 2). Blood was collected from animals on postoperative day 1 after femoral wire injury and examined by ELISA and flow cytometry. As our wire injury model inherently includes a ligation of the distal femoral artery and potentially a degree of ischemic tissue injury, we included an experimental group consisting of ligation alone of the distal femoral artery without wire injury. Importantly, there were no obvious ischemic complications among our study animals from this limited arterial ligation. In addition, a sham surgery group underwent vascular exposure without vascular injury or ligation. Our results demonstrated a 2.2-fold increase in sdf1a after femoral wire injury (P ¼ .0003) compared with nonsurgical animals. Ligation-alone also increased sdf1a by twofold (P ¼ .001) compared with the nonsurgical group. There was no significant difference between sham surgery and nonsurgical animals or between ligation and wire injury groups. Next, flow cytometric gating was used to examine cells for the progenitor markers CD34 and c-kit (Fig 3), known as ST-HSCs.14 On postoperative day 1, animals with wire injury revealed a significant 2.3-fold increase among CD34þ/c-kitþ circulating progenitor populations relative to nonsurgical controls (P ¼ .005), sham surgery (P ¼ .016), or distal ligation alone (P ¼ .018) as shown in Fig 3. Sca-1 is a glycosylphosphatidylinositol-anchored cell surface protein that is a marker of primitive murine hematopoietic cells,24 which are precursors to CD34þ/ckitþ ST-HSCs. Toward the goal of mitigating surge of these cells after injury, we next examined an IgG2a antibody directed toward sca-1 using methodology similar to classic T-cell depletion.25,26 Mice were given empirical antibody dosing with an initial 3 days of induction followed by maintenance dosing every other day until postoperative day 9. This time frame was based on our initial findings of sdf1a surge, which appeared to taper during this period. An isotype antibody was administered to a separate control group (cIg) using the same administration schedule. In addition, distinct nonsurgical groups of mice were treated with either isotype and sca-1 antibodies, as additional controls. We observed no evidence of physiologic impact of this antibody among treated animals. The vascular injury signal sdf1a was assayed after treatment with isotype and sca antibody. Compared with nonsurgical naive animals,
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Fig 1. Wire injury of the murine femoral artery injures endothelium of the femoral segment. A, Representative image of murine femoral wire injury with ligation of the saphenous artery (arrow) and wire dilation of the common femoral artery (arrowhead). B and C, Confocal fluorescent microscopy of lectin (red) and Hoechst (blue) staining revealed an en face intact endothelial monolayer of a control vessel (B) that was absent on a wire-injured vessel (C). Scale bar is shown.
wire injury with control antibody (Wi cIg) and wire injury with sca (Wi sca) increased sdf1a 1.7- and 2.2-fold, respectively, on postoperative day 1 (Fig 4). As might be expected from a therapy directed at downstream events, treatment with sca-1 antibody did not affect levels of sdf1a compared with isotype control-treated animals. The sdf1a levels for isotype wire-injured animals remained elevated at 3 and 9 days postoperatively (Fig 5). Flow cytometric gating after treatment of nonsurgical and wire-injured mice treated with isotype or sca-1 antibody is shown in Fig 6. Similar to the trend of untreated animals after wire injury, we observed that wire-injured mice treated with isotype antibody (Wi cIg) revealed a surge among CD34þ/c-kitþ circulating progenitors after wire injury relative to naive animals on postoperative day 1 (P ¼ .004). In contrast, these progenitors were significantly reduced among wire-injured mice treated with sca1 depletion (Wi sca) relative to isotype-treated animals (P ¼ .02) and not significantly increased relative to naive nonsurgical animals (P ¼ .38; Fig 7). As part of an extended time course to identify rebound phenomenon, sca-1 therapy was noted to mitigate the surge of circulating progenitors at additional time points of 3 and 9 days after injury (P ¼ NS relative to naive nonsurgical animals at all time points; Fig 7). Pluripotent progenitors have important physiologic roles in hematopoiesis27 and endothelial repair. For this reason, preservation of the bone marrow reservoir is essential for survival. Given the theoretical concerns about bone marrow depletion from this approach, bone marrow was compared between nonsurgical sca-1- and isotype-treated
Fig 2. Stromal cell-derived factor 1a (sdf1a) increases after vascular injury. Compared with nonsurgical controls or mice with a sham surgery, mice with distal femoral artery ligation revealed a significant increase of sdf1a expression by enzyme-linked immunosorbent assay (ELISA; P ¼ .001), whereas mice with femoral wire injury revealed a similar 2.2-fold increase in sdf1a (P ¼ .003; n ¼ 6 per condition).
animals with flow cytometry. As shown in Fig 8, whereas sca-1þ progenitor cells were significantly reduced by sca1 depletion at an early time point of day 3, they quickly rebounded by day 11 of therapy to levels equivalent to those of isotype-treated animals. Based on previous reports of histologic and clinical associations of progenitor cells with restenosis, we hypothesized that mitigation of the ST-HSC surge would reduce restenotic disease. Wire-injured mice were divided into three
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Fig 3. Wire injury of the femoral artery increased circulating CD34þ/c-kitþ cells (short-term hematopoietic stem cells [ST-HSCs]). Flow cytometry included (A) exclusion of debris followed by (B) gating of DAPI-positive events. Lineage-positive and CD45-positive events were then excluded. Shown are representative gating of CD34þ/c-kitþ events among nonsurgical (C) and wire-injured (D) animals. Compared with mice with no surgery, animals after wire injury of the femoral artery revealed a statistically significant 2.3-fold increase among CD34þ/c-kitþ cells (P ¼ .005; E). Sham surgery and vessel ligation did not result in a significant rise in progenitors. Shown are percentage of cells with standard error bars (n ¼ 6 per condition).
groups: saline treated (Wi saline), treatment with an isotype control (Wi cIg), or treatment with sca-1-depleting antibody (Wi sca) using the same 11-day dosing protocol of the cellular analysis. At 28 days after injury, iliac and femoral vessels were explanted and examined histologically. As demonstrated in Fig 9, intima to media ratios were calculated and revealed a statistically significant reduction in the intima to media ratio compared with both saline-treated mice (P ¼ .04) and isotype controls (P ¼ .036). DISCUSSION Restenosis remains a major threat to the longevity of interventions after cardiovascular and hemodialysis access procedures. With failure of up to half of interventions in 5 years, this pathologic process has increased the number
of procedures, escalated costs to the health care system, and compromised long-term patient outcome. Although trends among EPCs have been well documented after vascular injury,28 these cells represent only a narrow subset of progenitors. In this study, we instead examined the trends of more primitive ST-HSC14 progenitors that have not been previously characterized after vascular injury. The role of these primitive progenitors as precursors to myeloid cells is especially fascinating, given the important association of myeloid cells with vascular disease such as atherosclerosis15,16 and restenosis.17,29 Our findings in the murine model suggest a surge among STHSC (CD34þ/c-kitþ) cells after wire injury of the femoral artery. We did not observe a significant change in ST-HSC after a sham surgery of the same compartment, suggesting that microvascular injury inherent in surgical exposure
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Fig 4. Antibody treatment does not prevent increases in plasma stromal cell-derived factor 1a (sdf1a) 1 day after vascular injury. Vascular wire injury among isotype (Wi cIg) or sca-treated (Wi sca) mice increased plasma sdf1a relative to nonsurgical, non-antibodytreated controls (naive) and to nonsurgical isotype (cIg, treated with control antibody without injury) on day 1. The sdf1a levels in Wi sca mice were not significantly different from those of Wi cIg animals. Means with standard error are shown (n ¼ 8 per condition).
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Fig 6. Sca-1 antibody treatment reduced a surge among shortterm hematopoietic stem cells (ST-HSCs) 1 day after wire injury. Compared with naive nonsurgical mice (n ¼ 6) or nonsurgical isotype controls (cIg, n ¼ 8), wire injury after isotype treatment (Wi cIg, n ¼ 8) increased progenitor cells by more than threefold (P ¼ .004 and .0001, respectively). Animals treated with sca-1 antibody (Wi sca, n ¼ 8) prevented the surge among progenitors relative to naive animals (P ¼ NS). Shown are mean percentage of CD34þ/c-kitþ/Lin cells with standard error.
Fig 5. Stromal cell-derived factor 1a (sdf1a) levels remain elevated over 9 days after wire injury and are not affected by sca-1 treatment (Wi sca). The sdf1a levels at extended time points of 3 and 9 days after wire injury remained significantly elevated compared with either naive nonsurgical or nonsurgical isotype (cIg) controls. Treatment with sca-1 antibody did not affect sdf1a levels (P ¼ NS relative to wire injury with control antibody [Wi cIg]; n ¼ 8 per condition for naive and postoperative day 1; n ¼ 6 for all other time points).
Fig 7. Sca-1 antibody mitigates surge of CD34þ/c-kitþ short-term hematopoietic stem cells (ST-HSCs) out to 9 days. Whereas the surge of CD34þ/c-kitþ cells remained elevated through postoperative day (POD) 9, CD34þ/c-kitþ cells among sca-1-treated wire-injured animals (Wi sca) were nonsignificant relative to naive nonsurgical animals at all time points. Shown are mean percentage CD34þ/c-kitþ/Lin with standard error. cIg, Isotype control immunoglobulin; Wi, wire injured.
alone is not sufficient to elicit a progenitor response. Although increases among EPCs have been observed by others after ischemic injury,30 we did not observe a similar trend with ST-HSCs after ligation of the saphenous artery. Importantly, the ST-HSCs of this study reflect a distinct progenitor from the EPCs quantified in other studies.
In addition, our model of wire injury involves ligation of only the distal femoral artery with preservation of collaterals, which the detailed report by Hellingman et al31 would suggest does not create a significant ischemic picture. As a result, the absence of significant changes in progenitors in our study would not necessarily imply
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Fig 8. Murine bone marrow is preserved after sca antibody at late time points. By flow cytometric analysis, sca-1 cells in sca-treated mice were significantly reduced at day 3 of therapy relative to isotype (cIg) animals. By day 11, despite ongoing antibody treatment, there was no difference in scaþ cells between cIg- and scatreated mice (n ¼ 6 per condition on day 3, n ¼ 9 on day 11). Shown are mean percentage with standard error.
absence of progenitor surge in a more authentic ischemic model. Looking upstream of progenitor cells, vascular injury results in the release of multiple signals related to progenitor mobilization.32,33 The particular importance of sdf1a (also known as CXCL12) in the context of progenitors has previously been described in the setting of ischemic injury34 with specific contributions in both mobilization35,36 and homing of EPCs.18,37 Whereas our findings reveal an increase in sdf1a that parallels the surge of CD34þ/c-kitþ progenitors, our data fall short of concluding that sdf1a is a stimulus for these CD34þ/ckitþ cells. In fact, our findings of sdf1a could instead be explained as a result of the limited ischemia caused by the distal ligation inherent in this model. Notably, our results suggest sdf1a surge after a distal ligation but without accompanying cellular surge. Whereas this may seem to contrast with previous reports of progenitors after ischemic injury,34,38 it is essential to note the significant differences in the ischemic model as well as differences in the phenotype of cells being examined (EPCs vs STHSCs). Our study has several limitations. First, our dosing strategy was empirically derived from classic T-cell depletion protocols, and further investigation may be needed to optimize the dosing for this application. Although our protocol captured changes among progenitor cells out to 9 days, we did not examine cells outside of this time frame. Finally, it is possible that our antibody-based depletion of progenitors merely postponed restenosis as we did not examine specimens beyond a single histologic time point of 28 days. To date, it has been unclear whether circulating progenitor cells actually play a meaningful and active role in
restenotic disease. The absence of an adult model in which to deplete progenitor cells has been an obstacle to answering this question. We used a depleting IgG2a antibody toward a marker of primitive murine progenitors, sca-1. Our results confirmed that this approach can indeed mitigate CD34þ/c-kitþ progenitor surge after vascular injury. Based on concerns that a rebound surge might overwhelm this approach, we examined animals out to 9 days and yet treatment with a depleting sca-1 antibody consistently prevented an increase in progenitors. There are theoretical concerns that an approach to deplete a key marker of pluripotent progenitors could precipitate bone marrow failure. In fact, despite a minor reduction in sca-1þ cells at an early time point, we confirmed preservation of the sca-1þ marrow reservoir at a later time point, still amid sca-1 depletion therapy. The high proliferative capacity of bone marrow may have been protective in this setting. This model of sca-1 depletion may have utility in other studies for reduction of circulating progenitors. Our study further concludes that mitigation of ST-HSC progenitor surge is effective toward reducing restenosis in the murine model. Whereas this study does not address the specific mechanism of how ST-HSCs contribute to restenosis, several previous studies have demonstrated that circulating progenitors can differentiate into smooth muscle cells.12,39,40 In addition, ST-HSCs are known precursors to monocytes/macrophages,14 cell types with roles in both atherosclerotic and restenotic vascular disease.15-17,29 As a result, further investigations will be needed to clarify whether progenitor cells contribute either by direct cellular contribution or by modifying the inflammatory environment. In particular, we are already examining cell types known to be derived from ST-HSCs, including monocytic lineages. CONCLUSIONS This study demonstrates important changes among circulating ST-HSCs that appear to be limited to macrovascular injury. We have developed a successful adult murine model to attenuate the response of CD34þ/c-kitþ progenitor cells and demonstrated that mitigation of the progenitor response leads to a significant reduction in vascular restenosis. Further studies on the identity of ST-HSCderived effector cells that contribute to restenosis may offer new approaches to this life- and limb-threatening complication. The authors gratefully acknowledge The Center for Biologic Imaging (University of Pittsburgh) for imaging support. AUTHOR CONTRIBUTIONS Conception and design: BT, JK, TR, AC, AD, VD, ET Analysis and interpretation: BT, JK, TR, AC, AD, VD, ET Data collection: BT, JK, TR, AD, VD Writing the article: BT, AC, ET Critical revision of the article: BT, ET Final approval of the article: BT
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Fig 9. Depletion of short-term hematopoietic stem cells (ST-HSCs) reduces restenosis after wire injury. Compared with mice treated with control immunoglobulin G (Wi cIg) at the time of wire injury (A), mice treated with sca1-depleting antibody (Wi sca) revealed reduced restenosis at 28 days (B; hematoxylin and eosin, 400). Compared with either control mice with saline (n ¼ 8) or mice with treatment of isotype control Ig (cIg, n ¼ 5), mice treated with sca-1-depleting antibody (n ¼ 8) revealed a significant decrease in the intima/media ratio. C, Means with standard error bars are shown. IEL, Internal elastic lamina; L, lumen; I, intima; M, media; Wi, wire injury.
Statistical analysis: BT Obtained funding: BT Overall responsibility: BT
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30. Liu ZJ, Velazquez OC. Hyperoxia, endothelial progenitor cell mobilization, and diabetic wound healing. Antioxid Redox Signal 2008;10:1869-82. 31. Hellingman AA, Bastiaansen AJ, de Vries MR, Seghers L, Lijkwan MA, Lowik CW, et al. Variations in surgical procedures for hind limb ischaemia mouse models result in differences in collateral formation. Eur J Vasc Endovasc Surg 2010;40:796-803. 32. Tsai S, Butler J, Rafii S, Liu B, Kent KC. The role of progenitor cells in the development of intimal hyperplasia. J Vasc Surg 2009;49:502-10. 33. Zernecke A, Schober A, Bot I, von Hundelshausen P, Liehn EA, Mopps B, et al. SDF-1a/CXCR4 axis is instrumental in neointimal hyperplasia and recruitment of smooth muscle progenitor cells. Circ Res 2005;96:784-91. 34. Penn MS. Importance of the SDF-1:CXCR4 axis in myocardial repair. Circ Res 2009;104:1133-5. 35. Hattori K, Heissig B, Rafii S. The regulation of hematopoietic stem cell and progenitor mobilization by chemokine SDF-1. Leuk Lymphoma 2003;44:575-82. 36. Moore MA, Hattori K, Heissig B, Shieh JH, Dias S, Crystal RG, et al. Mobilization of endothelial and hematopoietic stem and progenitor cells by adenovector-mediated elevation of serum levels of SDF-1, VEGF, and angiopoietin-1. Ann N Y Acad Sci 2001;938:36-45; discussion: 45-7. 37. Liu ZJ, Tian R, An W, Zhuge Y, Li Y, Shao H, et al. Identification of Eselectin as a novel target for the regulation of postnatal neovascularization: implications for diabetic wound healing. Ann Surg 2010;252:625-34. 38. Bauer SM, Goldstein LJ, Bauer RJ, Chen H, Putt M, Velazquez OC. The bone marrow-derived endothelial progenitor cell response is impaired in delayed wound healing from ischemia. J Vasc Surg 2006;43:134-41. 39. Simper D, Stalboerger PG, Panetta CJ, Wang S, Caplice NM. Smooth muscle progenitor cells in human blood. Circulation 2002;106:1199-204. 40. Wang CH, Anderson N, Li SH, Szmitko PE, Cherng WJ, Fedak PW, et al. Stem cell factor deficiency is vasculoprotective: unraveling a new therapeutic potential of imatinib mesylate. Circ Res 2006;99:617-25.
Submitted Feb 23, 2015; accepted May 3, 2015.
INVITED COMMENTARY
Gale L. Tang, MD, Seattle, Wash Restenosis from intimal hyperplasia remains the Achilles heel of lower extremity endovascular and open revascularization procedures despite recent advances in drug-coated balloons and stents. The source of the cells leading to intimal hyperplasia and the most effective strategy to inhibit intimal hyperplasia while also allowing normal arterial wall healing remain in doubt. The first problem is a significant limiting factor for future molecularly targeted therapies because the correct cell type to target is unknown. The second problem can likely be addressed only by correctly answering the first. In an attempt to address the source of cells, Tillman and colleagues demonstrated that there is a surge in circulating vascular progenitor cells at the time of arterial injury in a femoral wire injury mouse model and that this surge is ablated by periprocedural treatment using an antibody to sca-1. They further demonstrated that mice treated with the antibody to sca-1 have significantly less intimal hyperplasia than control mice. This is supported by previous studies showing that the neointimal cells after a more severe femoral wire injury were primarily bone marrow derived.1 However, the source of neointimal cells in mice is heavily dependent on the model being used,1 and it remains an open question which
model best reflects what happens in humans after angioplasty or open bypass. What is also left to be determined is whether the surge in vascular progenitor cells actually causes the intimal hyperplasia. Other groups have shown a significant proportion of sca-1þ cells within the arterial adventitia,2 suggesting the alternative hypothesis that the antibody to sca-1 may instead lead to decreased intimal hyperplasia because of suppression of adventitial sca-1þ progenitors. Unfortunately, there is no recognized sca-1 homologue in humans, so translating this work from mice to humans may be problematic.
REFERENCES 1. Tanaka K, Sata M, Hirata Y, Nagai R. Diverse contribution of bone marrow cells to neointimal hyperplasia after mechanical vascular injuries. Circ Res 2003;93:783-90. 2. Hu Y, Zhang Z, Torsney E, Afzal AR, Davison F, Metzler B, et al. Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice. J Clin Invest 2004;113:1258-64.