SDF-1α-induced dual pairs of E-selectin/ligand mediate endothelial progenitor cell homing to critical ischemia

Homing of endothelial progenitor cells (EPC) to the ischemic tissues is a key event in neovascularization and tissue regeneration. In response to ischemic insult, injured tissues secrete several chemo-cytokines, including stromal cell-derived factor-1α (SDF-1α), which triggers mobilization and homing of bone marrow-derived EPC (BMD-EPC). We previously reported that SDF-1α-induced EPC homing is mediated by a panel of adhesion molecules highly or selectively expressed on the activated endothelium in ischemic tissues, including E-selectin. Elevated E-selectin on wound vasculature serve as docking sites for circulating EPC, which express counterpart E-selectin ligands. Here, we show that SDF-1α presented in wound tissue and released into circulation can act both locally and remotely to induce ischemic tissue endothelium and BMD-EPC to express both E-selectin and its ligands. By performing BM transplantation using E-selectin−/− and E-selectin+/+ mice as the donors and recipients respectively, we demonstrate that upregulated dual E-selectin/ligand pairs reciprocally expressed on ischemic tissue endothelium and BMD-EPC act as double-locks to secure targeted EPC- endothelium interactions by which to facilitate EPC homing and promote neovascularization and tissue repair. These findings describe a novel mechanism for BMD-EPC homing and indicate that dual E-selectin/ligand pairs may be effective targets/tools for therapeutic neovascularization and targeted cell delivery.


SDF-1α supplied to ischemic wound tissue has a remote role in inducing E-selectin expression on BMD-EPC.
Wound tissue SDF-1α , either secreted by local tissue cells in response to ischemic insult or therapeutically administrated, can be released into circulation. To study the remote effect of local SDF-1α in ischemic diabetic wound tissue on EPC resided in their BM niche and in circulation, ischemic hindlimb wounds were made in NOD mouse. Because the tissue levels of SDF-1α in diabetic murine wounds are significantly lower than in healthy tissue, as indicated in a previous study 19 , exogenous recombinant mouse SDF-1α (γ mSDF-1α ) (25 μ g/kg, this dosage was determined in our previous study 10 ) was injected into the wound bed. Equal volumes of solvent (PBS) were injected into wound bed in the control group (n = 8/group). Concentrations of SDF-1α in sera collected from the tail vein 4 h later were measured by ELISA. Local wound injection of γ mSDF-1α resulted in elevated serum levels of SDF-1α compared with PBS injection in control mice (Fig. 1A), indicating the release of locally administrated γ mSDF-1α into circulation. Elevated SDF-1α in sera can either stimulate circulating EPC or reach BM to stimulate EPC present in BM. Both BM and peripheral blood were harvested 24 h after wound tissue SDF-1α injection. Cells were incubated with FITC-CD34, APC-KDR, and PE-E-selectin antibodies (Abs). E-selectin levels in the gated EPC (EPC are defined as CD34 + /KDR + herein) fraction were measured using flow cytometry analysis. In response to elevated systemic levels of SDF-1α , expression of E-selectin in EPC from BM (Fig. 1B) and peripheral blood (Fig. 1C) was significantly increased, showing that local supplementation of SDF-1α in ischemic wound tissue can have a remote effect on expression of E-selectin in BM and circulating EPC. SDF-1α also appeared to promote EPC mobilization in that there were approximately 1-fold more EPC in peripheral blood (Fig. 1C) in response to locally administrated SDF-1α . The comparable amounts of EPC in BM (Fig. 1B) suggested a quick self-renewal of EPC to maintain a stable BM EPC pool after EPC mobilization.
To confirm these observations, we conducted immunoblotting analyses and confirmed upregulation of E-selectin in recombinant human SDF-1α (γ hSDF-1α )-stimulated human EPC in vitro (Fig. 1D). A RT 2 Profiler ™ PCR array was made to examine gene expression profile of expanded panel of cell adhesion molecules and extracellular matrix in EPC in response to SDF-1α . Of the 84 genes, 3 were downregulated (− ) and 22 were upregulated (+ ) (cut-off value: < or > 2-fold) (Supplemental Table 1), and 59 were unaltered. Notably, expression of the E-selectin gene was increased about 3.48-fold upon SDF-1α stimulation (Fig. 1E). PCR array data offered additional candidates for future elucidation of the mechanisms underlying SDF-1α -triggered BMD-EPC homing and mobilization.
Collectively, these data demonstrated that local administration of SDF-1α to ischemic wounds could have a remote effect on expression of E-selectin in BM and circulating EPC, and mobilization of BMD-EPC.

SDF-1α induces cultured EC and mouse wound endothelium to express E-selectin ligands in vitro and in vivo.
To determine whether wound endothelium expresses counterpart ligands of E-selectin in response to locally administrated γ mSDF-1α in wound tissue, the expression of the E-selectin ligand CD44 was specifically examined in the microvasculature of ischemic hindlimb wounds of NOD mouse 24 h after wound bed injection of γ mSDF-1α , because the presence of PSGL-1/CD162 on injured or inflamed endothelium has already been established 17 . We observed that CD44 expression in luminal EC was markedly higher in ischemic diabetic wounds than endothelium in the control hindlimb tissue (non-ischemia, PBS injection) of the same mouse (n = 8/group) ( Fig. 2A), demonstrating that elevated SDF-1α in wound tissue caused the local endothelium to increase expression of the E-selectin ligand CD44.
E-selectin ligands can be expressed as unmodified inactive form or modified active form. The active form can associate with E-selectin. To determine whether SDF-1α -induced E-selectin ligands are in the inactive or active form, a cell surface E-selectin ligand binding assay was conducted to determine whether FITC-conjugated HECA452, which is mAb directed against a sialyl Lewis X (sLeX) epitope on active E-selectin ligand CD162 20 and CD44 21 , can associate with SDF-1α -induced E-selectin ligands on HMVEC and EPC. It had already been demonstrated that SDF-1α also induces E-selectin ligands expressed in EPC 10 . Approximately 1-fold and 1.4-fold more FITC-HECA452 was found to be bound to SDF-1α -stimulated HMVEC (Fig. 2C) and EPC (Fig. 2D) compared to non-stimulated HMVEC and EPC, respectively, indicating that SDF-1α -induced E-selectin ligands expressed on HMVEC and EPC are modified as active form, which can be associated with HECA452. These experiments demonstrated that SDF-1α could cause HMVEC and EPC to express three E-selectin ligands, CD44, CD162, and ESL-1, which are likely to be modified into an active form.
Therefore, SDF-1α -induced E-selectin ligands expressed on the ischemic diabetic wound endothelium may serve as docking adhesion molecules to mediate anchorage of E-selectin + circulating cells, for example, SDF-1α -triggered circulating EPC, on the activated endothelium.
Up-regulated E-selectin on EPC and E-selectin ligands on EC are responsible for mediating SDF-1α-induced EPC-EC interactions. We previously reported that SDF-1α induced EPC-EC adhesion in vitro, which is mediated by up-regulated E-selectin on EC and E-selectin ligands on EPC 10 . To determine whether SDF-1α -induced E-selectin on EPC and E-selectin ligands on EC, which have been identified in the current study, are also involved in mediating SDF-1α -enhanced EPC-EC adhesion, the effect of antagonists against E-selectin and its ligands on inhibition of SDF-1α -enhanced EPC adhesion to EC monolayer was tested in vitro. Human EPC were pre-labeled with Dil-Ac-LDL. Dil-Ac-LDL + EPC were detached from dishes and cultured in agarose gel-coated dishes to prevent adhesion. These EPC were then stimulated with γ hSDF-1α or Levels of E-selectin in EPC from mice injected with PBS were established as "1" and relative levels of E-selectin in EPC from mice injected with γ mSDF-1α were normalized accordingly (n = 8 mice/group). (C) Measurement of % of EPC in peripheral blood MNC and relative levels of E-selectin in circulating EPC as described in (B) (n = 8 mice/group). (D) Immunoblotting analysis of E-selectin expression upon γ hSDF-1α (100 ng/ml) stimulation in human EPC at various time points. β -actin served as a loading control. (E) Human EPC were stimulated with γ hSDF-1α or BSA for 4 h, and total RNA was extracted. Expression of extracellular matrix and adhesion molecules were analyzed using RT2-PCRArray. Expression of E-selectin was upregulated upon γ mSDF-1α stimulation. Levels of mRNA in BSA-treated EPC were established as "1" and compared to those in γ mSDF-1α -treated EPC. Experiments were repeated three times in (D) and (E). Data are analyzed by 2-tailed Student's t-test and presented as mean ± SEM.
BSA. After 4 h, Dil-Ac-LDL + EPC were replaced with EGM2 medium containing either E-selectin neutralizing Ab or isotype-matched control Ab (2 μ g/ml) and incubated for 15 min at 37 °C. HMVEC were grown as a monolayer and stimulated with γ hSDF-1α or BSA for 4 h. The stimulated HMVEC monolayer was re-cultured in EGM2 media containing either soluble E-selectin (sE-sel) or BSA (2 μ g/ml) and incubated for 15 min at 37 °C. After washing both EPC and HMVEC with PBS to remove un-associated E-selectin-neutralizing Ab or sE-sel, non-adherent Dil-Ac-LDL + EPC were collected from agarose-gel-coated dishes and added to the wells containing the HMVEC monolayer to allow EPC-EC interactions. After 30 min, unbound EPC were washed out by PBS (twice) and Dil-Ac-LDL + EPC associated with HMVEC-monolayers were measured using a fluorescence scanner. As in previous reports, SDF-1α -stimulated EPC and HMVEC monolayers showed significantly more EPC-EC binding capability than un-stimulated control cells (Fig. 3). Combined treatments of EC and EPC with SDF-1α increased EPC-EC interactions. There were more EPC associated with EC monolayers compared to single treatment (EC or EPC treated with SDF-1α ). It suggested that stimulation of both EC and EPC with SDF-1α induce both types of cells to express elevated levels of E-selectin/lignad pairs that mediate stronger EPC-EC interactions. Both treatment of SDF-1α -stimulated EPC with E-selectin-neutralizing Ab and treatment of SDF-1α -stimulated HMVEC monolayers with sE-sel alone significantly inhibited EPC-EC interactions, but control Ab and BSA (Supplemental Figure 1) had no significant effect. Combination of E-selectin-neutralizing Ab and sE-sel showed Data are presented as mean ± SD of ratio of CD44:CD31 signals from 5 random selected sections of high power field (LPF, X 20) of each wound sample. CD31 signal was established as "1" in each section and relative amount of CD44 signal was normalized accordingly. (B) Immunoblotting analysis of SDF-1α -induced expression of three E-selectin ligands in HMVEC at various time points (γ hSDF-1α : 100 ng/ml). β -actin is used as loading control. Experiments were repeated three times and similar results were obtained. (C) Binding of FITC-conjugated HECA452 to HMVEC stimulated with γ hSDF-1α or BSA. Fluorescent signals were quantified (top). Representative fluorescent images were exhibited (bottom). (D) Binding of FITC-conjugated HECA452 to human EPC stimulated with γ hSDF-1α or BSA (100 ng/ml). Fluorescent signals were quantified (top). Representative fluorescent images were exhibited (bottom). Data are analyzed by 2-tailed Student's t-test and presented as mean ± SEM of fluorescent signals based on triplicate wells in each condition and totally three independent experiments in (C,D). even stronger inhibitory effect on EPC-EC interaction (Supplemental Figure 1). Overall, these results demonstrated that upregulated E-selectin on EPC and E-selecin ligands on EC are also responsible for mediating SDF-1α -enhanced EPC-EC interactions.

E-selectin on both EPC and wound endothelium are required for mediation of SDF-1α-induced EPC homing.
To assess the involvement of SDF-1α -induced E-selectin on both EPC and wound endothelium in mediating EPC homing to wound tissue, syngeneic BM transplantation (BMT) was used to assess the homing capability of EPC derived from two types of donor mice, Rosa26 +/− vs. Rosa26 +/− ;E-sel −/− in which LacZ + -BM cells, including EPC, either expressed or did not express E-selectin in response to SDF-1α (E-sel +/+ -EPC vs. E-sel −/− -EPC), to wound tissues of two types of recipient mice, C57BL6 vs. E-sel −/− in which all tissue cells, including wound endothelium, expressed or did not express E-selectin (E-sel +/+ -EC vs. E-sel −/− -EC). BM of γ -irradiated recipient mice (C57BL6 vs. E-sel −/− ) were cross-reconstituted using LacZ + -BM cells from donor mice (infusion of LacZ + -BM cells from Rosa26 +/− vs. Rosa26 +/− ;E-sel −/− mice (n = 6/group) into recipient mice (C57BL6 vs. E-sel −/− , n = 12/group)). Recipient mice were subjected to femoral ligation to create unilateral hindlimb ischemia and subsequent bilateral 4 mm cutaneous excision. γ mSDF-1α was administered to wound bed. Recipient mice were killed after 7 days, and half of mice (n = 6/group) were used to harvest wound tissues to perform IHC and the remaining mice (n = 6/group) were used for Dil-perfusion to measure neovascularization (see below). LacZ + -EPC that were recruited to wound tissues and incorporated into blood vessels were detected using double staining with X-gal (blue) and anti-CD31 (brown). We observed significantly less LacZ + / CD31 + -EPC in blood vessels of ischemic wounds in the recipient C57BL6 mice which were transplanted with BMC from Rosa26 +/− ;E-sel −/− mice than that from Rosa26 +/− mice (Fig. 4), indicating that E-selectin expressed on EPC is required for mediating SDF-1α -induced EPC homing. Consistent with previous observations, there were fewer LacZ + /CD31 + -EPC in blood vessels of ischemic wounds in the recipient E-sel −/− mice than in those of C57BL6 mice (Fig. 4), confirming that E-selectin expressed in wound endothelium is also essential to mediation of SDF-1α -induced EPC homing.

E-selectin on both EPC and EC is essential to mediation of SDF-1α-induced neovascularization and re-perfusion in ischemic limb.
The role of SDF-1α -induced E-selectin on EPC and on wound endothelium in regulation of neovascularization in wound tissue and perfusion in ischemic limb was explored. Neovascularization was evaluated by whole-body blood vessel Dil perfusion and subsequent laser scanning confocal microscopy in wound tissues harvested from recipient mice (n = 6/group) on day 7. Laser Doppler perfusion imaging (LDI) was used to confirm post-operative limb ischemia and quantify the spontaneous restoration of hindlimb blood flow over time (n = 12/group).
There was significantly higher blood vessel density in ischemic wounds (Fig. 5A) and increased mean flux measurements in the ischemic limb (Fig. 5B) of recipient C57BL6 mice transplanted with BMC (containing LacZ + -EPC) from Rosa26 +/− mice compared to that from Rosa26 +/− ;E-sel −/− mice, indicating that E-selectin expressed on EPC is required for mediation of SDF-1α -induced neovascularization. Consistent with previous observations, there were fewer blood vessels within wounds (Fig. 5A) and decreased mean flux measurements in the ischemic limbs (Fig. 5B) in recipient E-sel −/− mice than in C57BL6 mice, confirming that E-selectin was expressed in wound endothelium and essential to mediation of SDF-1α -induced neovascularization and reperfusion of the ischemic limbs. SDF-1α-induced E-selectin in both EPC and EC are critical for wound healing. The role of SDF-1α -induced E-selectin on EPC and on wound endothelium,in the regulation of wound healing was investigated. Wound areas were measured using daily digital photography until day 7 and calculated using ImageJ software. A delayed healing rate was observed in the recipient C57BL6 mice, which were transplanted with BMC from Rosa26 +/− ;E-sel −/− mice compared to that from Rosa26 +/− mice (Fig. 6), indicating that E-selectin expressed on EPC is required for mediating SDF-1α -induced wound healing. Similarly, healing rate in recipient E-sel −/− mice was slower than in C57BL6 mice (Fig. 6), confirming that E-selectin expressed in wound endothelium is also relevant to mediation of SDF-1α -induced wound healing.

Discussion
Luminal EC form a natural barrier between the blood and surrounding tissue. Under steady-state physiological conditions, luminal EC are mostly quiescent and form a tight, impermeable barrier. Under pathological conditions, such as tissue ischemia, inflammation, and tumors, a variety of cytokines/chemokines, such as SDF-1α , TGF-ß, and IL-1, are produced and released into tissue, and the local tissue endothelium is stimulated by these soluble factors. It causes upregulation and activation of a unique panel of cell adhesion molecules (CAMs), including selectins and integrins, in the endothelium within the local tissue. This causes the local endothelium to switch from a tight impermeable to permeable and "sticky" status. These adhesion molecules act as docking sites and mediate tethering of circulating inflammatory cells and stem/ progenitor cells, including BMD-EPC. The anchored circulating cells undergo rolling, tight adhesion to the endothelium and subsequent transendothelial migration, extravasation from permeable capillaries and infiltration into diseased tissues. We previously demonstrated that SDF-1α upregulates E-selectin expression on the local wound endothelium and induces EPC to express E-selectin ligands 10 . The current study extend these findings and show that SDF-1α elevated or supplied in ischemic wound tissue has not only local but also remote effects, when it is released into circulation, on the expression of both E-selectin and its cognate ligands in wound endothelium and BMD-EPC. Upregulated dual E-selectin/ligand pairs reciprocally expressed in activated endothelium in wounds and mobilized BMD circulating EPC act as double locks, securing EPC-EC interactions by which to enhance selective homing of EPC to ischemic wound tissue, which is essential to neovascularization and tissue repair. In this way, the current findings provide profound and novel insight into the molecular mechanisms underlying the biological effects of SDF-1α on EPC homing and suggest that E-selectin and its ligands may be suitable targets and tools for therapeutic manipulation of targeted EPC homing. Full molecules, fragments, and binding epitopes of E-selectin and its   ligands may also be utilized to direct therapeutic stem cells, for instance, mesenchymal stem cells (MSC), homing to sites of ischemic lesions for cell-based therapy.
It is worth mentioning that there still remain debate with respect to the definition and function of EPC in the field. Murine EPC are defined as CD34 + /KDR + (in flow cytomery analysis) and LacZ + /CD31 + (in in vivo bone marrow transplantation experiments) in our study although these cells may also be generally referred as pro-angiogenic bone marrow-derived mononuclear cells. BM-derived circulating progenitor/stem cells, including EPC, can be incorporated into newly formed capillaries, enhance neovascularization after hind limb ischemia and improve tissue function after ischemic injury. Alternatively, recruited progenitor/stem cells, including EPC, may also promote neovascularization and tissue regeneration by releasing soluble factors, which act in a paracrine manner to support local angiogenesis and mobilize tissue residing progenitor cells.
The current study identified SDF-1α as a trigger to induce dual E-selectin/ligand pairs (all three types of ligands) to be reciprocally expressed on both activated endothelium in wound tissue and BMD-EPC. SDF-1α -induced ligands were found to be functional on both EPC and endothelium because they undergo post-translational modification (glycosylation), as indicated by increased association with HECA452, which specifically recognizes sLeX epitope on active PSGL-1/CD162 and CD44 20,21 . In addition, SDF-1α can mobilize BMD-EPC into circulation, although the precise role of induced E-selectin/ligand pairs in EPC mobilization is unclear. In this way, SDF-1α produced or presented in the local wound tissues play a pivotal role in the orchestration of a dynamic pro-angiogenic process in which both local and remote events are involved.
Although SDF-1α causes both EC and EPC to express dual E-selectin/ligand pairs, the quantity of E-selectin and ligands, especially the activity of ligands (extent of glycosylation post translation) in EC and EPC, do not appear to be equal. SDF-1α -induced E-selectin ligands expressed on EPC showed less modification than those expressed on EC (a weaker binding activity of EPC than EC to HECA452 as showed in Fig. 2C,D). This may be attributed to the differences in microenvironment between wound endothelium (for EC modification) and BM niche or circulation (for EPC modification). The quantities and activity of enzymes responsible for glycosylation vary in different microenvironments and may not depend upon SDF-1α . In other words, expression and modification of E-selectin ligands may be determined by different mechanisms. Less activated E-selectin ligands on EPC could be important because this may not result in aggregation of EPC in circulation, especially under disruption of blood flow. However, such lowly activated E-selectin ligands on EPC may enable association with highly expressed E-selectin on activated endothelium in the swampy, low-flow capillaries in the ischemic tissues, where highly activated E-selectin ligands on EC can bind with the E-selectin on EPC to enhance EPC-EC interactions, which is essential to EPC homing, using what is here described as a "double-lock" mechanism (Fig. 7). In addition, the number of circulating EPC in blood is very low (approximately 1 EPC/μ l in peripheral blood according to our previous studies 19 . It must be pointed out that the number of EPC depends upon the methods used to define EPC). Thus, the chance for EPC to meet/interact in the circulating blood is low. Therefore, all these factors, including less activated E-selectin ligands on EPC, low amount of EPC in circulating blood, and disruption of blood flow, prevent EPC from forming aggregates in the circulating blood. However, when circulating EPC arrive at the "swampy", low-flow capillaries in the ischemic tissues, the local microenvironment (the quantities Figure 7. Illustration of a "double-lock" mechanism mediating EPC homing to ischemic wounds tissues for therapeutic neovascularization and wound repair. SDF-1α acts both locally and remotely to induce ischemic tissue endothelium and BMD-EPC to express E-selectin/ligands. Dual E-selectin/ligand pairs reciprocally expressed on activated endothelium and BMD-EPC mediate enhanced EPC-EC interactions and selective EPC homing. Scientific RepoRts | 6:34416 | DOI: 10.1038/srep34416 and activity of enzymes responsible for glycosylation of E-selectin ligands) may increase the activity of E-selectin ligands, which grants "double-lock" mechanism to work.
Roles of SDF-1α in retention of hematopoietic stem cells (HSC) in BM and recruited circulating mesenchymal stromal cells (MSC) in ischemic tissues have been reported 6,22,23 . SDF-1α -induced cell retention is ascribed to SDF-1α and its receptor CXCR4. Although it requires future study, our findings may suggest a potential role of up-regulated E-selectin/ligand pair on EPC in SDF-1α -induced retention of EPC in ischemic wound tissue. Because many tissue cells in ischemic limb and skin, including fibroblasts and inflammatory cells, express CD44 and/or CD162, E-selectin expressed on EPC can mediate cell-cell interaction between EPC and various tissue cells after EPC extravasate from blood vessels. Retention of EPC in ischemic tissue may be critical for neovascularization and tissue repair.
In summary, we report that SDF-1α elevated or therapeutically administered in ischemic wounded tissue can stimulate both local EC and BMD circulating EPC to express reciprocally E-selectin/ligand pairs and thereby enhance EPC-EC interactions. Our findings not only expand repertoire of SDF-1α and scope of SDF-1α -induced vascular adhesion molecules, but also identify a novel "double-lock" mechanism mediated by interaction of E-selectin/ligand pairs reciprocally expressed on the surface of both activated EC and EPC. Such a "double-lock" enhances and secures EPC-EC interactions by which to facilitate targeted EPC homing to ischemic wound tissue and subsequent EPC-associated neovascularization and repair responses. Further studies are warranted to address several important yet unanswered questions, for example, the molecular mechanisms underlying the regulation of E-selectin/ligand expression in EC and EPC by SDF-1α ; the roles of each of these E-selectin ligands in mediating EPC-EC interaction. . Subconfluent HMVEC and human EPC were stimulated with recombinant human (γ h) SDF-1α protein (350-NS/CF, R&D Systems, Minneapolis, MN) vs. BSA at 100 ng/ml for 4 h (The dosage of γ hSDF-1α (100 ng/ml) was pre-determined in our previous study 10 based on the lowest concentration that could induce E-selectin expression in HMVEC). Total RNA was extracted using Trizol ® (Invitrogen, Grand Island, NY). cDNA was synthesized using RT 2 First Strand Kit (Qiagen). PCR array was carried out according to the manufacturer's protocol. The threshold cycle (Ct) values were used to plot a standard curve. All samples were normalized to the relative levels of β-actin, and results are expressed as fluorescence intensity in relative levels.

Methods
Flow cytometry. Murine BMC was harvested as previously described 10 . Murine blood samples were immediately placed on ice at 4 °C. After red cell lysis, BMC were incubated with FITC-CD34, APC-KDR, and PE-E-selectin (#560238, 561252, #553751, BD Biosciences, San Jose, CA) or isotype-matched control Abs at 4 °C for 30 min using predetermined optimal concentrations of the test Abs. Cells were washed and analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). EPC (CD34 + /KDR + ) were gated, counted, and analyzed for levels of E-selectin. Five thousand cells were analyzed per sample.
ELISA. The concentrations of SDF-1α in the sera of mice were measured using Quantikine ® SDF-1α ELISA kit (DY460, R&D Systems) based on the manufacturer's protocol.
Active form of E-selectin ligand binding assay. Subconfluent HMVEC and human EPC were stimulated with γ hSDF-1α and BSA (100 ng/ml) for 16 h in 96-well plates. After washing with PBS, 200 ng/well of FITC-HECA452 (#321306, BioLegend) vs. FITC-BSA (P8779, Sigma-Aldrich, St. Louis, MO) were added into well and incubated at 37 °C for 30 min. After being washed twice with PBS, the plates were scanned using a fluorescence scanner and fluorescent imaging was recorded using a fluorescence microscope. Samples were triplicated and experiments were repeated three times.
Laser Doppler perfusion imaging (LDI). Limb perfusion was assessed daily using LDI (Periscan PIM II, Perimed AB, Sweden) as described previously 10 , Relative perfusion data are expressed as the ratio of the ischemic (right) to normal (left) limb blood flow.
Induction of mouse hindlimb ischemic wounds. Creation of mouse hindlimb ischemia (right limb), cutaneous wounds and wound bed injection of γ mSDF-1α protein (460-SD/CF, R&D Systems) was conducted as described previously 10 .
Tissue-level detection of recruited BMD EPC. Frozen ischemic wound tissue sections were stained by X-gal (Fermentas, Canada) and HRP-anti-CD31 (ab28364, Abcam) and counterstained with nuclear fast red (Vector Labs) as described 10 . The number of EPC was quantified by counting LacZ + cells in CD31 + vessels from 5 random high power fields (HPF, 20X) per section in at least 3 serial sections per wound sample (n = 6/group).
Blood vessel perfusion and laser scanning confocal microscopy. Mouse blood vessels were labeled by live perfusion with DiI (D-282, Invitrogen/Molecular Probes) solution, and the vascular density was visualized by scanning the entire wound tissue to a depth of 200 μ m, using laser scanning confocal microscopy (Vibratome (VT1000S, Leica Microsystems, Buffalo Grove, IL) as described 10,15,24 . Vessel density was quantified by assessing total number of Dil + vessels normalized to the entire scanned wound area, using ImageJ software (Imaging Processing and Analysis in Java, National Institutes of Health, MD).