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(The FASEB Journal. 2009;23:1447-1458.)
© 2009 FASEB

An acellular matrix-bound ligand enhances the mobilization, recruitment and therapeutic effects of circulating progenitor cells in a hindlimb ischemia model

Erik J. Suuronen*,{dagger},1, Pingchuan Zhang*, Drew Kuraitis*,{dagger}, Xudong Cao{ddagger}, Angela Melhuish{ddagger}, Daniel McKee*,{dagger}, Fengfu Li, Thierry G. Mesana*, John P. Veinot§ and Marc Ruel*,{dagger}

* Division of Cardiac Surgery, University of Ottawa Heart Institute, Ottawa, Ontario, Canada;

{dagger} Department of Cellular and Molecular Medicine,

{ddagger} Department of Chemical Engineering, and

§ Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario, Canada; and

Department of Vision, Ottawa Health Research Institute, Ottawa, Ontario, Canada

1 Correspondence: Erik J. Suuronen, Division of Cardiac Surgery, University of Ottawa Heart Institute, 40 Ruskin St., Ottawa, ON, K1Y 4W7, Canada. E-mail: esuuronen{at}ottawaheart.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Circulating progenitor cells home to and engraft to sites of ischemia, mediated in part by the adhesion molecule L-selectin; however, accumulation in tissues such as the heart is low. In this study, an acellular collagen-based matrix containing sialyl LewisX (sLeX), which binds L-selectin, was developed in order to enhance the endogenous progenitor cell therapeutic response. Its effect on progenitor cells and angiogenesis were assessed in vitro and using a hindlimb ischemia model with rats. In culture, the sLeX-collagen matrix recruited more CD133+CD34+L-selectin+ cells than collagen-only matrix, with adhesion mediated by L-selectin binding. Increased angiogenic/chemotactic cytokine production and improved resistance to apoptosis appeared in cells cultured on sLeX-collagen matrix. In vivo, mobilization of endogenous circulating progenitor cells was increased, and greater recruitment of these and systemically injected human peripheral blood CXCR4+L-selectin+ cells to sLeX-collagen treated limbs was observed compared to collagen-only. This condition was associated with differences in angiogenic/chemotactic cytokine levels, with greater arteriole density and increased perfusion in sLeX-collagen treated hindlimbs. With these factors taken together, we demonstrated that an acellular matrix-bound ligand approach can enhance the mobilization, recruitment, and therapeutic effects of endogenous and/or transplanted progenitor cells, possibly through paracrine and antiapoptotic mechanisms, and could be used to improve cell-based regenerative therapies.—Suuronen, E. J., Zhang, P., Kuraitis, D., Cao, X., Melhuish, A., McKee, D., Li, F., Mesana, T. G., Veinot, J. P., Ruel, M. An acellular matrix-bound ligand enhances the mobilization, recruitment and therapeutic effects of circulating progenitor cells in a hindlimb ischemia model.


Key Words: tissue engineering • stem cells • angiogenesis • cell homing • paracrine effects • apoptosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE HUMAN HEART HAS A LIMITED capacity for self-repair or regeneration. The use of stem or progenitor cells to augment regeneration of the myocardium could reverse or prevent heart failure in affected patients, making cell-based therapy a promising approach to rebuild the damaged heart. Cardiac cell-based therapies, therefore, aim at restoring perfusion and function to chronically ischemic, stunned, hibernating, or scarred myocardial areas. A number of cell therapy clinical trials have been performed to date, most of them using unselected bone marrow mononuclear cells; however, their results have been mixed (1 , 2) .

Preclinical studies have so far indicated that transplanted progenitor cells form few or no neosyncytial contractile units, in contrast to what had initially been hoped for (3 4 5) . Rather, it is believed that neovascularization of the dysfunctional myocardium from paracrine/humoral factors and secondary recruitment of host stem/progenitor cells may be the mechanisms leading to functional improvement (3 4 5 6) . Considering this, cell-based therapeutic "angiogenesis" [or "vasculogenesis"—a distinction in nomenclature (7) not continued further in this article] represents a goal within scientific reach, arguably more feasible than cardiomyocyte regeneration from any known nonembryonic stem cell source (8 , 9) . Therefore, the optimization of cell-based angiogenesis constitutes one plausible approach to improve the results of cardiac cell therapy in ischemic as well as in infarcted myocardium.

The modest objective benefits observed in human cell therapy trials to date may also be explained by other biological issues. Low cell engraftment, survival, phenotype, and function of the transplanted cells within the target tissue, as well as nonspecific cell delivery, remain significant problems (6 , 10 11 12) . It has been shown that marrow-derived circulating progenitor cells (CPCs) are augmented in response to cardiac events and that they home to sites of injured heart tissue (13 , 14) ; however, the increase in cell numbers is short-lived, and cell accumulation is low (3 , 15) . Notably, increased numbers of CPCs are associated with improved vascular function and recovery following a cardiac event (16 , 17) , and a reduced number predicts future cardiovascular events (18) . Therefore, strategies to augment the endogenous progenitor cell response and its effects may help improve tissue recovery and function.

We present a strategy to overcome some of the obstacles that minimize the success of cell therapy, here by using an acellular biomaterial designed to enhance the mobilization, recruitment, and effects of endogenous CPCs. To this end, we used the oligosaccharide sialyl LewisX (sLeX), a high-affinity ligand for L-selectin (19) . L-selectin is an adhesion molecule expressed on CPCs and implicated in their homing and adhesion (20) . For instance, inhibition of the interaction between sLeX and selectins has been investigated as a treatment to prevent tumor angiogenesis and growth, which can be stimulated by CPCs (20 , 21) . We hypothesized that the immobilization of sLeX into a collagen matrix previously shown to support vascularization (22) would promote mobilization, recruitment, and adhesion of progenitor cells, and angiogenesis and perfusion in ischemic tissue, thereby achieving some of the elusive goals of cell-based therapy, without the actual transplantation of cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Circulating progenitor cells
Procurement of human blood was approved by the Human Research Ethics Board of the University of Ottawa Heart Institute. Informed consent was obtained from all participants. Total peripheral blood mononuclear cells (PBMCs) were isolated from healthy young adults and were used fresh, or were cultured to yield CPCs, as described previously (ref. 23 ; Supplemental Materials and Methods).

Sialyl LewisX-collagen matrix
Solutions of sLeX (Cedarlane Laboratories, Hornby, ON, Canada; 0.125, 0.25, 0.5, 1.0, and 5.0 mM concentrations) were prepared in 0.1 M 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH ~5.0) containing a 1:1 (molar equivalent) cross-linking mixture of N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS; 13 mM; Sigma, Oakville, ON, Canada). EDC/NHS was used to activate and conjugate sLeX to collagen and subsequently to cross-link the collagen. On ice, activated sLeX/crosslinker mixture (200 µl) was added to 1 ml of 1% porcine type I atelocollagen (w/v; Nippon Ham, Tskuba, Japan) with 200 µl of 20% chondroitin 6-sulfate (w/v; Sigma). The mixture was diluted with 400 µl of PBS, the pH was adjusted to 7.4, and gels were formed at 37°C. Final concentrations of sLeX in the collagen mix were 0.0125, 0.025, 0.05, 0.1, and 0.5 mM. Collagen-only matrices were prepared identically, but without sLeX in the cross-linker mixture.

Cell adhesion to different concentrations of sLeX in collagen matrices
To evaluate cell adhesion at different concentrations of sLeX in collagen matrices, 2 x 106 CPCs labeled with 4',6-diamidino-2'-phenylindole (DAPI; Sigma) were cultured on matrices for 30 min at 37°C and then fixed with 4% paraformaldehyde (PFA). The number of adherent DAPI+ cells was determined per field of view (FOV), and adhesion was calculated as the number of adherent cells relative to the average number counted in lowest concentration of sLeX tested. It was determined that the 0.1 mM concentration of sLeX was optimal, and this concentration was used in all subsequent experiments.

Evaluation of sLeX-collagen matrix
Each gel was placed in its own perforated centrifuge tube (~2 mm holes) in a covered ddH2O bath and stirred for 2 days. The water, containing unbound sLeX, was then freeze-dried for 36 h. To assess grafting efficiency, a glycoprotein detection kit (Pierce, Rockford, IL, USA) was used according to the manufacturer’s protocol. The kit works on the principle that sodium metaperiodate oxidizes the glycoprotein’s carbohydrate moieties, and the resultant aldehyde reacts with the detection reagent. Briefly, samples (in triplicate) were incubated with 10 mM sodium metaperiodate for 10 min, and then with 0.5% glycoprotein detection reagent for 1 h. Concentrations of sLeX (n=5) were determined against a standard curve and normalized to readings for collagen-only matrix (Kodak ID imager; Kodak, Rochester, NY, USA). Grafting efficiency was calculated as the ratio of actual over maximum theoretical sLeX content in the matrix.

Adhesion and blocking assays
For static adhesion, 2 x 106 CPCs labeled with DAPI were cultured on matrices for 30 or 90 min at 37°C. For adhesion under flow conditions, CPCs were cultured on matrices for 0 or 60 min and then subjected to shear stress, applied for 30 min at a fluid rate of 300 s–1, using a cone-and-plate apparatus (24) . For blocking, CPCs were treated with either sLeX (0.1 mM) or monoclonal anti-human L-selectin antibody (20 µl/5 x 105 cells; Beckman Coulter, Mississauga, ON, Canada) for 30 min, then centrifuged and rinsed prior to culture on the matrix. The adherent cells were fixed with 4% PFA, and the percentage of adherent cells was calculated as the number of adherent DAPI+ cells divided by the initial number of plated cells. In a subset of experiments, adherent cells were lifted for analysis by flow cytometry.

Flow cytometry
As described previously (23) , the percentage of cells expressing a particular surface marker was determined by labeling cells with antibodies against one or more of the following antigens: human cells with CD34, L-selectin (both Beckman Coulter), and CD133 (Miltenyi Biotec, Bergisch-Gladbach, Germany); and rat cells with c-kit (Santa Cruz Biotechnology, Santa Cruz, CA, USA), CXCR4, VEGFR2, and CD45 (all Abcam, Cambridge, UK). Cells were analyzed with a FACSAriaTM (BD Biosciences, Mississauga, ON, Canada) or Cytomics FC500 (Beckman Coulter) flow cytometer. In controls, isotype-matched Pacific blue-, Alexa Fluor 430-, FITC-, PE-, and APC-conjugated antibodies were used.

Animal model
All experimental procedures were performed with the approval of the University of Ottawa Animal Care Committee, in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. To compare sLeX-collagen and collagen-only matrices in vivo, a double hindlimb ischemia rat model was used to eliminate variability between animals. Proximal femoral arteries in both hindlimbs of 8–9 wk old Sprague-Dawley rats (Charles River, Wilmington, MA, USA) were ligated to induce ischemia (n=8). One limb subsequently received 200 µl of sLeX-collagen matrix, and the other limb received 200 µl of collagen-only matrix, by multiple injections into the ischemic main adductor muscle using a 27-gauge needle. Similarly, a unilateral hindlimb ischemia procedure was performed on additional animals to evaluate whether the results observed in the double hindlimb ischemia model were improvements or merely differences in the level of negative effects between treatments. Animals each received one of the following treatments administered as described above: 200 µl of sLeX-collagen matrix (n=6); 200 µl of collagen-only matrix (n=12); or 200 µl PBS (n=10). These rats also received a tail-vein injection, via 26-gauge catheter, of 3 x 106 human CXCR4+L-selectin+ peripheral blood cells 1 day after the ligation and treatment.

Blood samples, ~0.1–0.2 ml/time point, were obtained from a subset of animals (n=5–7) via saphenous bleeds on days 0 (preoperative), 4, 7, and 14 after ligation surgery. PBMCs were immediately isolated and characterized by flow cytometry, as described above. Reported values have been normalized to the baseline average for all treatment groups. For all studies, rats were sacrificed after 14 days, and sections of hindlimb muscle were prepared as described below.

Laser doppler perfusion analysis
The sLeX-collagen/collagen matrix hindlimb blood perfusion ratio was measured before and after ligation and 7 and 14 days postoperatively by using a multifiber needle probe (8 separate collecting fibers) and a laser Doppler blood flow monitor (Moor Instruments, Axminster, UK). Similarly, for unilateral hindlimb ischemia animals, the ischemic/nonischemic hindlimb blood flow perfusion ratio was performed before and after femoral artery ligation as well as on days 7 and 14 postoperatively. See Supplemental Materials and Methods.

Histology and immunohistochemistry
For sLeX staining in matrices, 1–2 mm gel sections were prepared and incubated with monoclonal antisialyl LewisX antibody (1:50; Calbiochem, La Jolla, CA, USA), followed by a Cy2 secondary antibody (1:200; Amersham, Baie D'Urfé, QC, Canada). For controls, the same protocol was performed using an IgM antibody or without the primary antibody. For hindlimbs, arteriole density and the inflammatory cell reaction were calculated from hematoxylin phloxine saffron (HPS)-stained sections. Arterioles were identified further by direct staining using an {alpha}-smooth muscle actin antibody (1:400; Chemicon, Temecula, CA, USA). Additional sections were incubated with monoclonal anti-CD133 (1:50; Miltenyi Biotec), polyclonal anti-c-kit (1:100; Santa Cruz Biotechnology), monoclonal anti-CD68 (1:100; Abcam), polyclonal anti-CXCR4 (1:100; Abcam); monoclonal anti-human mitochondria (1:40; Chemicon), or polyclonal anti-human CXCR4 (1:200; Affinity Bioreagents, Golden, CO, USA) primary antibodies followed by secondary antibody or immunoperoxidase staining (ABC System, Santa Cruz Biotechnology). Imaging was performed using fluorescence microscopy. All density measures and cell counts were determined from 6 random microscopic fields and averaged from 2 masked observers. See Supplemental Materials and Methods.

Cytokine antibody arrays
Following the manufacturer’s recommended protocol, relative cytokine and growth factor levels (measured in arbitrary chemiluminescence units) in lysates from equal weight samples of hindlimb muscle tissue collected from a subset of animals (n=4–5 per group) at 2 wk were detected using the Raybio® Rat Cytokine Antibody Array kit (RayBiotech, Norcross, GA, USA). Fold differences in cytokine levels in the supernatant of human CPCs after 24 h of culture on sLeX-collagen or collagen-only matrix for individual blood donors (n=5) were detected using the Raybio human cytokine antibody array V (RayBiotech), according to the manufacturer’s recommended protocol. See Supplemental Materials and Methods.

Apoptosis assay
Human PBMCs were plated on either sLeX-collagen or collagen matrix coated 6-well plates (3x106 cells/well; n=5) and supplemented with EBM-2 media that was depleted of serum and growth factors. After 48 h, cultured cells were labeled with anti-CD133 and anti-CD34 antibodies, as described above. This was followed by incubation with 4 nM SYTO 16 (Molecular Probes, Invitrogen, Burlington, ON, Canada) and 30 µM verapamil, according to manufacturer’s protocol. Flow cytometry analysis was then performed for the detection of viable and apoptotic cells.

Statistical analysis
Values are expressed as means ± SE. Statistical analyses were performed in Intercooled Stata 9.2 (Stata, College Station, TX, USA). Comparisons of continuous data between groups were performed with a one-way analysis of variance adjusted for repeat measures, and individual two-group comparisons were examined with a two-tailed Student’s t test, using a Bonferroni correction as per the number of tests. For comparisons of parametric data between treatments for individual rats or blood donors, paired t tests were used. To determine the association between recruitment and arteriole density, linear regression was performed. Probability values of P < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of sLeX-collagen matrix
The collagen-based matrix was thermoresponsive and gelled at body temperature, allowing for direct injection as a liquid followed by gel formation within the target site. Immunohistochemical staining for sLeX in gels formed in vitro at 37°C showed uniform incorporation of sLeX throughout the matrix (Supplemental Fig. 1A). Controls (IgM or no primary antibody) showed little to no staining (Supplemental Fig. 1B). As determined by a glycoprotein detection assay, the average grafting efficiency of sLeX to the collagen was 80.2 ± 4.3%, further demonstrating sLeX incorporation into the matrix.

Adhesion of CPCs on sLeX-collagen matrix
After a 30-min static incubation on the different sLeX-collagen matrices, the number of adhered CPCs was significantly greater on the 0.1 and 0.5 mM concentration gels compared to the lower tested concentrations (Fig. 1A ). Overall, increasing concentrations of sLeX corresponded with an increase in adhered CPCs (P<0.001). Compared to the lowest concentration of sLeX (0.0125 mM), the 0.1 and 0.5 mM sLeX concentrations resulted in a significant increase in the relative number of adherent cells adhered (by 3.1- and 3.7-fold, respectively; P≤0.04). However, no significant difference was observed between these concentrations (0.1 mM vs. 0.5 mM; P=0.2). Therefore, for cost effectiveness, the 0.1 mM concentration sLeX-collagen matrix was used in all subsequent experiments.


Figure 1
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  		Figure 1. A) Number of adherent cells on sLeX-collagen matrices containing different concentrations of sLeX, relative to the lowest sLeX concentration (n=6–8). B) Number of adherent cells on collagen-only and sLeX-collagen matrix was evaluated after 30 and 90 min under static or flow conditions (n=6–8). C) Adhesion on collagen-only matrix (collagen) and sLeX-collagen matrix (sLeX), and of cells preincubated with sLeX (sLeX-primed) or anti-L-selectin on sLeX-collagen matrix after 30 min under flow conditions (n=6–8). *P < 0.05; **P ≤ 0.04; #P < 0.001.

Under static conditions, attachment of CPCs to the sLeX-collagen matrix (0.1 mM) was 2.4- and 2.1-fold greater than to collagen-only matrix after 30 and 90 min of incubation, respectively (P<0.05; Fig. 1B ). Under flow conditions, the number of adherent cells was 2.2- and 2.0-fold greater on the sLeX-collagen matrix compared to the collagen-only matrix for the 30 and 90 min groups, respectively (P<0.05; Fig. 1B ). Pretreatment of the CPC binding sites with sLeX or anti-L-selectin reduced the adhesion of cells on the sLeX-collagen matrix to levels observed on the collagen-only matrix (Fig. 1C ). Cell viability did not differ between groups (Supplemental Table 1).

Analysis of adherent cell phenotype
Prior to plating on matrix, 92.0 ± 1.3% of all CPCs expressed L-selectin, and 1.7 ± 0.6 and 7.0 ± 1.1% expressed the progenitor cell markers CD133 and CD34, respectively. CD133 and CD34 coexpression has been proposed as a phenotype defining one of the most potentially vasculogenic endothelial progenitor cells (25 , 26) . Two populations of cells were observed based on the level of L-selectin expression: L-selectinlow+ and L-selectinhigh+ (Fig. 2A ). The L-selectinlow+ population contained 94.7 ± 2.8% of the total number of CD133+CD34+ progenitor cells present in the CPC population prior to plating (Fig. 2B-D ). After plating and shear flow, the cells adhering to the sLeX-collagen matrix contained an increased proportion of L-selectinlow+ cells (Fig. 2E ) compared to the initial plated population, suggesting greater recruitment of CD133+CD34+ vasculogenic progenitors. Indeed, after culture on the sLeX-collagen matrix, the number of CD133+ and CD34+ cells was greater in the adherent population, compared to the initial cell population (Fig. 2F, G ). Also, 8.1 ± 1.4% of the total adherent cells was CD133+CD34+L-selectin+, compared to 1.9 ± 0.6% in the nonadherent population (a 4-fold increase; P<0.001; Fig. 2H ). Compared to the collagen-only matrix, the sLeX-collagen matrix recruited a 3.1- to 3.3-fold greater number of L-selectin+, CD133+, CD34+, and CD133+CD34+L-selectin+ cells (P<0.05; Fig. 2I ).


Figure 2
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  		Figure 2. Phenotype analysis of cultured cells. A) Flow cytometry of 4-day adherent CPCs prior to seeding revealed two populations based on L-selectin+ expression. B–D) Expression of progenitor cell markers CD133 and CD34 gated on the negative (B), L-selectinlow+ (C), and L-selectinhigh+ (D) populations in A revealed that ~95% of the CD133+CD34+L-selectin+ cells are in the L-selectinlow+ population. E) Proportion of adherent L-selectinlow+ cells increased when cultured on sLeX-collagen matrix. F, G) Number of CD133+ (F) and CD34+ (G) cells adherent to the sLeX-collagen matrix is increased compared to the initial cell population. H) Percentage of CD133+CD34+L-selectin+ cells was increased ~4-fold on the sLeX-collagen vs. the initial control and nonadherent cell populations (n=6). I) sLeX-collagen matrix recruited greater numbers of L-selectin+, CD133+, CD34+, and CD133+CD34+L-selectin+ cells compared to collagen-only matrix (n=7). #P < 0.001 vs. all other groups; *P < 0.05, sLeX-collagen vs. collagen-only matrix for each phenotype.

In vivo vascularization and perfusion
In bilateral hindlimb ischemia animals, intramuscular arteriole density was greater in hindlimbs treated with the sLeX-collagen matrix (12.2±1.2 /mm2) than in collagen-only treated limbs (3.8±0.4 /mm2), representing a 3.2-fold increase (P<0.001; Fig. 3A-G ). Both the sLeX-collagen and collagen-only treated hindlimbs had greater arteriole density than the PBS-treated group (2.2±0.4 /mm2; P<0.001). Corroborating the observation of improved vascularization in the sLeX-collagen treatment group, laser Doppler analysis revealed a significant increase in perfusion, measured as the ratio of sLeX-collagen/collagen limb blood flow, from 0.9 ± 0.1 (baseline postoperative) to 1.4 ± 0.2 at 2 wk after treatment (Fig. 3H ). This finding represents an increase in perfusion of 58% in hindlimbs treated with sLeX-collagen matrix vs. collagen-only matrix (P=0.04).


Figure 3
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  		Figure 3. Arteriolar density and limb perfusion. A–C) Representative images of HPS-stained sections of ischemic hindlimb muscle 2 wk after treatment with PBS (A), collagen-only (B), or sLeX-collagen matrix (C). Scale bars = 75 µm. D–F) Representative images of arteriole density in sections stained with {alpha}-smooth muscle actin antibody after treatment with PBS (D), collagen-only (E), or sLeX-collagen matrix (F). G) Number of arterioles/mm2 in ischemic hindlimb muscle of rats 2 wk after treatment (n=6–8). H) Laser Doppler analysis of perfusion preoperatively and 0, 7, and 14 days postoperatively after bilateral hindlimb ischemia. Data are presented as ratio of sLeX-collagen/collagen matrix limb blood flow (n=8). I) Laser Doppler analysis of perfusion preoperatively and at 0, 7, and 14 days after unilateral hindlimb ischemia (ischemic/nonischemic ratio; n=6–12). J) Change in ischemic/nonischemic perfusion ratio (from day 0 postop) at days 7 and 14 (n=6–12). *P < 0.001, {dagger}P ≤ 0.01, **P < 0.04 vs. all other treatments; #P = 0.04 vs. postoperative baseline.

In unilateral hindlimb ischemia animals, significantly greater perfusion (ischemic to nonischemic hindlimb ratio) was observed at 14 days in the sLeX-collagen matrix-treated hindlimbs (1.4±0.1), compared to hindlimbs receiving collagen-only matrix (0.9±0.1; P=0.01) and PBS (0.8±0.1; P=0.002; Fig. 3I ). In terms of the perfusion ratio difference between day 0 (postoperative) and day 14, the greatest increase was observed in the sLeX-collagen matrix-treated group (by 0.9±0.2), compared to the collagen-only and PBS groups (by 0.5±0.1 and by 0.3±0.1; P<0.04 and 0.005, respectively; Fig. 3J ).

Endogenous circulating progenitor cell mobilization in vivo
To examine the effect of the sLeX-collagen matrix on cell mobilization, flow cytometry analysis of blood samples was performed to determine the percentage of circulating cells expressing c-kit, CXCR4, VEGFR2, and CD45 at baseline (prior to surgery), and at 4, 7, and 14 days (Fig. 4 ). At baseline, no difference was observed in the absolute numbers of c-kit+, CXCR4+, VEGFR2+, or CD45+ cells between treatment groups (P=0.8, 0.9, 0.5, and 0.6, respectively). Generally, in all groups, the number of c-kit+, CXCR4+, VEGFR2+, and CD45+ cells in the circulation increased over the first 7 days and then decreased by day 14. Notably, the number of c-kit+, VEGFR2+, and CD45+ cells was greater at 7 days in the sLeX-collagen treatment group compared to the collagen-only and/or PBS groups. Furthermore, treatment with sLeX-collagen matrix significantly minimized the loss in circulating c-kit+, CXCR4+, VEGFR2+, and CD45+ cell numbers at day 14, compared to the collagen-only and PBS groups.


Figure 4
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  		Figure 4. Cell mobilization to the peripheral circulation. Fold change (relative to preoperative baseline) in number of circulating cells expressing c-kit (A), CXCR4 (B), VEGFR2 (C), and CD45 (D) at 4, 7, and 14 days after ligation surgery and treatment (n=5–8). Baseline values are normalized to a value of 1.0 for each group. To avoid superimposition, depicted SEM values at baseline correspond to the largest baseline SEM of the 3 groups; therefore, the baseline SEM for each group is equal to or less than the depicted SEM. A) *P = 0.04, sLeX vs. PBS at day 7; #P < 0.03, sLeX vs. all other groups at day 14. B) *P < 0.05, PBS vs. all other groups at day 4; #P < 0.05, sLeX vs. PBS at day 14. C) *P = 0.03, sLeX vs. PBS at day 14. D) *P < 0.03, PBS vs. all other groups at day 7; #P = 0.02, sLeX vs. PBS at day 14.

Endogenous circulating progenitor cell recruitment in vivo
In terms of cell recruitment, the inflammatory cell reaction at 2 wk was estimated by grading the number of leukocytes per HPS-stained tissue section (0=none, 1=low, 3=medium, and 5=severe), and no significant difference was observed between treatments (1.5±0.7, 1.4±0.6, and 1.8±0.5 for PBS, collagen-only, and sLeX-collagen groups, respectively; P=0.9), as determined by pathological assessment. In addition, specific immunohistochemical analysis of CD68 macrophage staining revealed no difference in the number of CD68+ cells/mm2 between sLeX-collagen (15.6±2.1), collagen-only (13.5±2.0), and PBS-treated hindlimbs (12.3±0.6; P=0.6) at 2 wk.

Immunostaining revealed significantly more CD133+ and c-kit+ cells in the sLeX-collagen-treated hindlimb compared to collagen-only and PBS treatments (Fig. 5A-F ; Supplemental Fig. 2). The number of CD133+ and c-kit+ cells recruited to sLeX-collagen treated hindlimbs was 3.0- and 2.1-fold greater compared to collagen-only treatment and 6.2- and 4.6-fold greater compared to hindlimbs receiving PBS injection, respectively (P<0.05; Fig. 5G ). CXCR4 was expressed mainly in proximity to vasculature between myofibers (Fig. 6A-D ), and the number of CXCR4+ cells in hindlimbs receiving sLeX-collagen was 2.0- and 3.8-fold greater than collagen and PBS-treated hindlimbs, respectively (P<0.05; Fig. 6E ). Moreover, the number of recruited CXCR4+ cells correlated with arteriole density of the hindlimb (P<0.001; Fig. 6F ).


Figure 5
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  		Figure 5. CD133+ and c-kit+ cell numbers in ischemic hindlimbs. A–F) Representative images of CD133+ (A–C; red; arrowheads) and c-kit+ (D–F; dark brown, arrowheads) cells in hindlimbs treated with PBS (A, D), sLeX-collagen (B, E), or collagen-only matrix (C, F). Cell nuclei are stained with DAPI (blue) in A–C; double-positive cells appear purple. Scale bars = 50 µm. G) Fold increase (relative to PBS treatment) was determined for numbers of CD133+ and c-kit+ cells in ischemic hindlimbs (n=6–8). *P < 0.05 vs. all other treatments within cell phenotype.


Figure 6
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  		Figure 6. Recruitment of CXCR4+ cells in the ischemic hindlimb. A–D) Representative images at low (A, C) and high magnification (B, D) of CXCR4+ cells in hindlimbs treated with collagen-only (A, B) or sLeX-collagen matrix (C, D). CXCR4+ cells (arrowheads) were often found to colocalize with the vasculature (B, D). Scale bars = 150 µm (A, C); 25 µm (B, D). E) Fold increase (relative to PBS treatment) for number of CXCR4+ cells in ischemic hindlimbs (n=6–8). *P < 0.05 vs. all other treatments. F) Arteriole density (no./mm2) observed within ischemic muscle correlated with CXCR4+ cell numbers; use of collagen-only and sLeX-collagen matrix increased cell recruitment (n=6–8).

Exogenous circulating progenitor cell recruitment in vivo
To further confirm the recruitment of cells from the circulation to the ischemic tissue, human peripheral blood CXCR4+L-selectin+ cells were injected systemically into rats from all 3 groups, 1 day after matrix implantation, and the number of cells staining positive for human mitochondria was determined in tissue sections at 14 days (Fig. 7A-C ). Recruited human cells were observed to either maintain or lose CXCR4 expression within the muscle tissue. Recruitment of human CXCR4+L-selectin+ cells to hindlimbs receiving sLeX-collagen matrix was 1.9- and 1.6-fold greater than to collagen-only and PBS-treated hindlimbs, respectively (P<0.05; Fig. 7D ). No difference in human cell recruitment was observed between the PBS group and the collagen-only group (P=0.6).


Figure 7
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  		Figure 7. Recruitment of systemically administered human CXCR4+L-selectin+ blood cells in the rat ischemic hindlimb. A–C) Sample images of human mitochondria+ (hMito+) cells (A), CXCR4+ cells (B), and the merged image with DAPI nuclear stain (C). Recruited hMito+ cells were found to either maintain (arrowheads) or lose (arrow) CXCR4 expression. Scale bars = 25 µm. D) Fold difference (relative to PBS treatment; n=5–7) in number of recruited human hMito+ cells from the circulation. *P < 0.05 vs. all other treatments.

Cytokines and growth factors
To examine the possibility that a paracrine mechanism may be involved in the observed differences between treatments, a rat-specific antibody array was used to examine protein expression of cytokines in hindlimb tissues 2 wk after matrix implantation. Compared to the PBS and/or collagen-only groups was a trend for the sLeX-collagen treated hindlimbs to contain elevated levels of the mobilizing agent GM-CSF, the chemoattractants MCP-1 and MIP-3{alpha}, and the antiapoptotic factor TIMP-1 (Fig. 8A ). In vitro studies using a human antibody cytokine array were conducted to explore further the specific effect of sLeX-collagen and collagen-only matrices on cytokine and growth factor production. The supernatant of CPCs cultured for 24 h on the sLeX-collagen matrix contained elevated levels of several cytokines and growth factors compared to the collagen-only cultures (Fig. 8B ). Among these were included (percentage increase and P value in parentheses): the mobilizing agent stromal cell-derived factor-1 (70.3%; P=0.04); the arteriogenic platelet-derived growth factor-(PDGF)-BB (22.2%; P=0.08); and the angiogenic cytokines angiogenin (107.6%; P=0.15), fibroblast growth factor (FGF)-4 (25.0%; P=0.15), FGF-6 (31.0%; P=0.04), and FGF-7 (50.7%; P=0.07).


Figure 8
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  		Figure 8. Cytokines and growth factors. A) Level of expression of selected cytokines in rat hindlimb tissue 2 wk after ligation surgery and treatment (n=4–5). P values indicated are for sLeX-collagen group vs. PBS group, except for TIMP-1, which is calculated for sLeX-collagen group vs. other groups combined. B) Fold difference in expression of selected cytokines and growth factors by human CPCs cultured on sLeX-collagen relative to culture on collagen-only matrix, after 24 h (n=5).

In vitro apoptosis
To investigate the possibility that the sLeX-collagen matrix improved the ability of recruited progenitor cells to resist apoptosis, an in vitro serum deprivation assay was performed. After 48 h of serum deprivation, the viability of total human peripheral blood mononuclear cells was equivalent between those cultured on sLeX-collagen vs. collagen-only matrix (n=5; P=0.8). However, the number of viable CD133+CD34+ progenitor cells was 1.6-fold greater on the sLeX-collagen matrix compared to collagen-only (n=5; P=0.02).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we developed and tested a novel material that could augment the endogenous progenitor cell response to ischemia. The addition of sLeX to a collagen matrix enhanced the adhesion of progenitor cells via an interaction with L-selectin on their cell surface. On injection into rat ischemic hindlimb muscle, the sLeX-collagen matrix increased the number and duration of mobilized circulating progenitors; promoted the accumulation of endogenous, as well as exogenous, progenitors within the target tissue; and may have enhanced the resistance of recruited cells to apoptosis. This was associated with increased neovascularization and improved tissue perfusion in hindlimbs receiving sLeX-collagen treatment. Taken together, the present studies mechanistically supported that this experimental approach may address at least three obstacles (mobilization, recruitment and survival) to the efficacy of progenitor cells in restoring tissue vascularity and function. In addition, a paracrine mechanism acting on other cell populations may have been responsible for some of the effects observed with the sLeX-collagen matrix.

A cell recruitment system is a promising alternative that could help to circumvent some of the current limitations associated with cell transplantation. For example, no consensus exists on the optimal cell choice, dose, and timing of injection for cell therapy for cardiovascular regeneration (4) . It is likely that our sLeX-collagen matrix augmented physiological regenerative processes through increased mobilization (level and duration) of the host’s own progenitor cells, and through the cells’ improved recruitment, and likely survival, in the target tissue. This could include many types of circulating progenitors that have demonstrated a capacity for regeneration (4) , thus eliminating the need to isolate and characterize a particular cell or mixed cell population. Analysis of hindlimb tissue demonstrated increased numbers of c-kit+, CD133+, and CXCR4+ cells in those treated with the sLeX-collagen matrix. In addition, greater numbers of human CXCR4+L-selectin+ cells, which were delivered systemically one day after ligation, were observed in the hindlimbs receiving sLeX-collagen matrix. These observations indicate an ability of the material to promote the recruitment of greater numbers of several progenitor cell types, of endogenous as well as exogenous origin, each with a documented contribution to enhancing angiogenesis (3 , 26 , 27) .

Of particular interest is the greater presence of CXCR4+ cells. Previous studies have found that recruited CXCR4+ cells colocalize with angiogenic vessels and that CXCR4 expression correlates with vascular density (28 , 29) , and both of these observations were made in the current study. In addition, it has been reported that a cytokine-mediated release of stromal cell-derived factor-1 (SDF-1) induces revascularization through enhanced mobilization and recruitment of CXCR4+ cells (30) . SDF-1 expression is increased in ischemic tissue and is further up-regulated after progenitor cell transplantation (6) . This finding suggests the possibility that greater accumulation of progenitor cells with sLeX-collagen matrix treatment may in turn increase paracrine recruitment of CXCR4+ cells via SDF-1 signaling. For example, c-kit+ and CD133+ progenitor cells, which were more abundant in the sLeX-collagen treated hindlimbs, may have enhanced vascular potential and/or production of SDF-1, via a hypoxia-inducible factor-1{alpha} (HIF-1{alpha}) mechanism in ischemic tissue (31 32 33) . In the sLeX-collagen treatment group, elevated levels of GM-CSF were observed, which has been shown to increase SDF-1 release (30) . SDF-1 was not included in the rat cytokine array, and hence its expression was not examined in the hindlimb tissue. However, the supernatant of human CPCs cultured on sLeX-collagen matrix in vitro contained elevated levels of SDF-1 compared to collagen-only cultures. This finding suggests that enhanced paracrine mechanisms may play a role in the ability of the sLeX-collagen matrix to increase mobilization and recruitment of circulating angiogenic cells and constitutes an area for more targeted investigation in future evaluation of this therapy.

Cell transplantation strategies also suffer from low implant capability and survival of the transplanted cells (11 , 14) . With no actual cell transplantation, our recruitment system alleviates the immediate implant and survival concerns associated with the lack of matrix attachments, or anoikis (34) . In addition, rat hindlimbs receiving sLeX-collagen demonstrated a trend for elevated expression of TIMP-1 and MCP-1, which have been shown to be involved in preventing cell apoptosis (35 , 36) . Furthermore, in vitro serum deprivation assays revealed increased survival of CD133+CD34+ progenitor cells when PBMCs were cultured on the sLeX-collagen matrix. This finding suggests that the sLeX-collagen matrix may potentially improve the recruited cell’s ability to resist apoptosis. It was also observed that human CXCR4+L-selectin+ cells transplanted at only 1 day after the ischemic event were found in greater numbers in the hindlimbs treated with sLeX-collagen matrix. Therefore, the sLeX-collagen matrix may act to improve the recruitment and/or persistence of the early recruited cells. This possibly represents a significant function of the biomaterial, since the early depletion of angiogenic cells from the target tissue has been shown to suppress neovascularization and tissue function, whereas their sustained persistence within the target site contributes to the maintenance of cell-mediated improvements (37) .

L-selectin has also been found on muscle-derived stem cells and has been shown to play a role in their homing and adhesion into dystrophic muscles (38) . Therefore, the application of the sLeX-collagen matrix may not be limited to circulatory populations. L-selectin also has a role in leukocyte biology, as it is involved in the recruitment of leukocytes to the endothelial cell surface, including sites of inflammation (39) . Circulating CD45+ leukocyte numbers were greater in the matrix-treated rats at 7 and 14 days compared to controls, which may suggest greater accumulation of leukocytes in the ischemic hindlimb also. However, other than L-selectin ligands, leukocytes require additional downstream events to participate effectively in inflammation (40) . Several of the proinflammatory stimuli involved in these downstream events, including IL-1{alpha}, IL-1β, IL-6, and TNF- {alpha}, were not expressed differentially between the treatment groups after 2 wk. Also, no adverse inflammatory response to the matrix was demonstrated as determined by pathological assessment and macrophage-specific CD68 staining. These observations suggest that, despite the possibility of increased CD45+ cell recruitment, inflammation was not induced by our material.

Although other circulating angiogenic populations of monocytic origin, such as those characterized by CD14 expression, were not evaluated in the present study, it is plausible that these cells were also recruited to the matrix-treated hindlimbs, as these and the cell types investigated in our study may respond to similar homing signals (41 , 42) . It has been shown that the presence of progenitor cells can augment the differentiation of monocytes into endothelial cells, leading to enhanced angiogenesis, and that paracrine regulation is involved in this phenomenon (41) . It could therefore be hypothesized that the increased number of progenitor cells observed in the sLeX-collagen treated hindlimbs may have a comparable effect on additionally recruited monocytic cells. Furthermore, the observation of increased expression of angiogenic cytokines, such as PDGF-BB, angiogenin, GM-CSF, FGFs, and MCP-1, suggests that this could similarly occur through paracrine mechanisms.

A therapy such as the one demonstrated in this study could also be used to augment stem/progenitor cell mobilization strategies, where long-term safety and efficacy of treatment currently remain uncertain. The effect of therapies that raise the number of circulating progenitors such as granulocyte colony stimulating factor, SDF-1, or erythropoietin (43 , 44) could potentially be magnified by the enhanced recruitment and function conferred by the sLeX-collagen matrix. In addition, the sLeX-collagen matrix may serve to improve the accumulation of exogenously administered cell populations, which have the ability to home to injured tissues, as was demonstrated in the current study. Therefore, a strategy for enhanced cell recruitment, such as the sLeX-collagen matrix, may boost responses to current cell-based therapies for tissue repair.

Overall, we developed a matrix that enhanced the adhesion of progenitor cells, mediated through a specific interaction with L-selectin. On injection into rat ischemic hindlimbs, the sLeX-collagen matrix increased progenitor cell mobilization and recruitment, enhanced neovascularization, and improved tissue perfusion, and these effects may be mediated at least in part through paracrine and antiapoptotic mechanisms. We conclude that the sLeX-collagen matrix serves as an example of how tissue-engineered materials can be developed in order to enhance the response and effects of endogenous progenitor cells and improve cell-based regenerative therapies.


   ACKNOWLEDGMENTS
 
We thank Suzanne Crowe, Samir Hazra, Dan deVette, and Drs. Fraser Rubens and Naoshi Shinozaki for their assistance and Drs. Ross Milne and Robert Roberts for their helpful insights. This work was supported by grant NA6121 from the Heart and Stroke Foundation of Ontario (to E.S.), by grant MOP-77536 from the Canadian Institutes of Health Research (to M.R. and E.S.), by a University of Ottawa Interfaculty research grant (E.S. and X.C.) and by a Natural Sciences and Engineering Research Council of Canada Grant (to X.C.). P.Z. is recipient of the Lawrence Soloway Research Fellowship, and D.K. is supported by a master’s studentship award from the Heart and Stroke Foundation of Ontario.

Received for publication June 25, 2008. Accepted for publication December 11, 2008.


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 DISCUSSION
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