α6-Integrin Subunit Plays a Major Role in the Proangiogenic Properties of Endothelial Progenitor Cells
Objective— Alpha6 integrin subunit (α6) expression is increased by proangiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor. This increase correlates with enhanced in vitro tube formation by endothelial cells and their progenitors called Endothelial Colony-Forming Cells (ECFCs). We thus studied the role of α6 in vasculogenesis induced by human ECFCs, in a mouse model of hindlimb ischemia.
Methods and Results— We used small interfering RNA (siRNA) to inhibit α6 expression on the surface of ECFCs. For in vivo studies, human ECFCs were injected intravenously into a nude mouse model of unilateral hind limb ischemia. Transfection with siRNA α6 abrogated neovessel formation and reperfusion of the ischemic hind limb induced by ECFCs (P<0.01 and P<0.001, respectively). It also inhibited ECFC incorporation into the vasculature of the ischemic muscle (P<0.001). In vitro, siRNA α6 inhibited ECFC adhesion (P<0.01), pseudotube formation on Matrigel, migration, and AKT phosphorylation (P<0.0001), with no effect on cell proliferation or apoptosis.
Conclusion— α6 Expression is required for ECFC migration, adhesion, recruitment at the site of ischemia, and the promotion of the postischemic vascular repair. Thus, we have demonstrated a major role of α6 in the proangiogenic properties of ECFCs.
The α6-integrin subunit (α6) is a 140-kDa protein that can associate with β1- or β4-integrin subunits. Integrin α6β1 is a receptor for laminin, the main component of the basement membrane, but can also bind other extracellular matrix (ECM) proteins, such as the angiogenic inducer CYR61.1 It is expressed on platelets, monocytes/macrophages, neutrophils, and endothelial cells. Integrin α6β4 primarily binds laminin; its expression is restricted to epithelial tissues, endothelia, and peripheral nerves; and it is responsible for adhesion junctions called hemidesmosomes.
In mice, deletion of the gene coding for α6 leads to an absence of hemidesmosomes and, consequently, to severe skin blistering and neonatal death.2 In humans, defects in α6 result in hemidesmosome deficiency, causing epidermolysis bullosa or pyloric atresia and, in most cases, early postnatal death.3,4 No obvious vascular abnormalities have been reported in such patients or in α6 knockout mice, suggesting that α6 does not have a major role in vasculogenesis during embryogenesis. In contrast, α6 is involved in the angiogenic properties of mature cells. For example, α6 is required for the formation of vascular networks in vitro by human brain microvascular endothelial cells5 and human umbilical vein endothelial cells.6 Regarding immature cells, activation of CD117-positive cells localized in the subepicardium of adult human heart is associated with α6 expression.7 Cell transplantation experiments on irradiated mice have shown that α6 plays a role in hematopoietic stem cell homing to bone marrow.8 Because hematopoietic cells and endothelial cells might arise from a common precursor (the hemangioblast), we investigated the possible role of α6 in the homing of endothelial progenitor cells (EPCs).
EPCs are marrow-derived circulating cells involved in postnatal vasculogenesis.9 Unlike mature endothelial cells, EPCs are a candidate cell therapy product for postischemic vascular regeneration,9 but EPC injection has shown limited efficacy in the clinical setting, probably because of insufficient homing and engraftment into newly formed vessels.10
However, different subpopulations of EPCs have been characterized: “early” EPCs called colony-forming unit endothelial cells and “late” EPCs called endothelial colony-forming cells (ECFCs). Only ECFCs can differentiate into functionally active mature endothelial cells and form functional blood vessels.11 ECFC surface expression of α6 is upregulated by growth factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 212; this correlates with enhanced formation of vascular tubes in vitro.13 To our knowledge, the role of α6 expression by ECFCs in adult vasculogenesis has not been investigated. Therefore, we examined the influence of α6 expression on the proangiogenic properties of ECFCs and especially on its possible involvement in ECFC recruitment to sites of ischemia. We used small interfering RNA (siRNA) duplexes to inhibit α6 expression at the surface of human ECFCs and then examined the effect of this inhibition on adhesion, migration and pseudotube network formation in vitro, and revascularization in a nude mouse model of hind limb ischemia.
ECFC Isolation and Culture and RNA Interference
ECFCs were isolated, cultured, and characterized as previously described13 and as detailed in supplemental Figure I (available online at http://atvb.ahajournals.org).
The ECFCs were transfected as described in the Supplemental Data.
Mouse Model of Unilateral Hind Limb Ischemia
All the protocols were approved by the Regional Ethics Committee on Animal Experimentation (ref P2.CBV.031.07), and all experiments conformed to European Community guidelines for the care and use of laboratory animals.
Male athymic nude Foxn-1 mice (Harlan, Gannat, France), aged 7 weeks and weighing 20 to 30 g, were anesthetized by isoflurane inhalation; the left femoral artery was ligatured. Five hours after the onset of ischemia, the mice received an intravenous injection of 100 μL of vehicle (PBS), 105 transfected ECFCs, or 105 untransfected ECFCs. Cells were transfected 96 hours before being injected into the animals.
After 2 weeks, mice were anesthetized with pentobarbital and placed on a heating pad at 37°C; laser Doppler perfusion imaging was used to assess leg tissue perfusion, as previously described.14 The results are expressed as the ratio of perfusion in the ischemic (left) leg to perfusion in the nonischemic (right) leg.
Ischemic and nonischemic gastrocnemius muscles were then collected and slowly frozen in isopentane solution cooled in liquid nitrogen, before being stored at −80°C until histological analysis.
Capillary Density Determination
Frozen 10-μm-thick sections of the distal part of the gastrocnemius muscle were fixed in ice-cold acetone for 10 minutes and incubated for 1 hour with a rat anti–mouse CD31 monoclonal antibody (clone MEC 13.3; BD Biosciences, Franklin Lakes, NJ) and with a goat anti–rat secondary antibody coupled to fluorescein isothiocyanate (Abcam, Cambridge, Mass). Sections were observed by a blinded observer (C.B.) with a confocal microscope (TCS SP2; Leica, Wetzlar, Germany). Ten fields were analyzed per section, and vessels number was quantified with computer software (Histolab; Microvision Instruments, Evry, France). The results are expressed as the ratio of the ischemic (left) leg to the nonischemic (right) leg.
Frozen 10-μm-thick sections of ischemic gastrocnemius were fixed in acetone and stained with hematoxylin-eosin; a blinded observer (C.B.) analyzed the sections under a microscope linked to a computer. Necrotic areas were circled, and the areas of the selected surfaces were calculated with Histolab software. The results are expressed as the ratio of the necrotic surface area to the total surface area of the section.
Human ECFC Detection in Mouse Muscles
To evaluate ECFC incorporation into the vasculature of ischemic muscles, 1 million human ECFCs in 100 μL of PBS were injected intravenously 5 hours after the onset of ischemia. Four days later, the gastrocnemius muscles were collected as previously described. To detect ECFCs, frozen tissue sections (10 μm) were fixed in ice-cold acetone, immunostained with a biotinylated anti–human CD31 antibody (clone JC70A; DAKO, S-32355, Glostrup, Denmark), and incubated with streptavidin-Alexa 555 (Invitrogen, Carlsbad, Calif). The mouse vasculature was stained with a rat anti–mouse CD31 monoclonal antibody. Nuclei were stained with TOPRO3 iodide. Incorporated ECFCs were detected with a confocal microscope, and the results were expressed as the number of incorporated ECFCs per section.
In Vitro Assay 72 Hours After Transfection of ECFCs
Plates (with 96 wells) were coated with Matrigel, and nonspecific binding sites were saturated with BSA in PBS for 1 hour at room temperature. The wells were then washed 3 times with adhesion buffer (10-mmol/L Hepes; 140-mmol/L NaCl; 5.56 mmol/L glucose; 1% BSA; 5.4 mmol/L potassium chloride; 2 mmol/L calcium chloride; and 1 mmol/L magnesium chloride, pH 7.4) and then ECFCs suspended in adhesion buffer were distributed (30 000 cells per well). After 20 minutes at 37°C with 5% CO2, nonadherent cells were removed and the wells were washed 3 times. The number of adherent cells was determined by a p-nitrophenyl phosphate colorimetric assay. The results were normalized to the untransfected ECFC group.
For the Boyden chamber migration assay, cell culture inserts (8.0 μm pore size) were placed in 24-well plates and coated with laminin. Endothelial basal medium (EBM)-2–containing 5% FCS, with or without 40-ng/mL VEGF, was placed in the lower compartment of the Boyden chamber. ECFCs suspended in EBM-2 containing 5% FCS were placed in the upper compartment (15 000 cells per well). After 6 hours at 37°C with 5% CO2, cells adhering to the lower surface of the inserts were counted in 10 different fields per well using a microscope with a grid eyepiece. Results are expressed as the difference between experiments with and without VEGF and are normalized to the untransfected ECFC group.
Matrigel Tube Formation Assay
ECFCs suspended in EBM-2 containing 5% FCS were distributed (30 000 per well) on 48-well plates coated with growth factor–reduced Matrigel. They were allowed to form pseudotubes for 18 hours at 37°C with 5% CO2. The total length of the pseudotubes was quantified with computer software (Videomet; Microvision Instruments) and normalized to the untransfected ECFC control group.
ECFCs were detached and seeded (10 000 per well) on 48-well laminin-coated plates (1 μg/cm2). After 48 hours of proliferation, the number of cells was determined by a p-nitrophenyl phosphate colorimetric assay. The results were normalized to the untransfected ECFC group.
Evaluation of AKT Phosphorylation
After 96 hours of transfection with siRNA, ECFCs were detached and seeded on laminin-coated plates. One hour later, wells were washed with PBS and cells were lysed in lysis buffer (NuPAGE). Equal amounts of protein from each group were resolved by SDS-PAGE on an 8% acrylamide gel and probed by immunoblotting using anti–phosphorylated AKT and anti-AKT antibodies (Cell Signaling, Danvers, Mass). Densitometric readings were obtained using computer software (ImageJ). Results are expressed as the ratio of phosphorylated AKT to total AKT, normalized to the untransfected ECFC group.
Results are expressed as mean±SEM of at least 3 experiments. Data were analyzed by ANOVA, followed by the Fisher protected least significant difference post hoc test, and implemented with computer software (StatView). Differences were assumed to be significant at P<0.05.
Evaluation of siRNA Efficiency and Specificity
As described in the supplemental Data, siRNA α6 efficiently inhibited α6 expression (supplemental Figure II) and did not affect the expression of other integrins (supplemental Figure III).
Loss of α6 Expression on ECFCs Reduces Postischemic Hind Limb Vascular Repair
Laser Doppler Perfusion Imaging
A single intravenous injection of control ECFCs (n=8) increased the ischemic to nonischemic leg blood flow ratio relative to PBS-treated mice by 60% (P<0.001). ECFCs transfected with scramble siRNA (n=10) also enhanced the perfusion in the ischemic hind limb, with no significant difference from untransfected ECFCs. In contrast, ECFCs transfected with siRNA α6 (n=11) did not improve reperfusion, with an ischemic to nonischemic leg blood flow ratio as low as PBS-injected mice (n=8). Thus, the inhibition of α6 expression significantly inhibited the ability of ECFCs to promote the reperfusion of the ischemic hind limb (P<0.001 versus untransfected ECFCs and P<0.01 versus ECFCs transfected with scrambled siRNA) (Figure 1).
A single injection of untransfected ECFCs (n=6) or ECFCs transfected with scramble siRNA (n=10) increased capillary density in the ischemic gastrocnemius muscle by a factor of 2 relative to PBS-injected mice (P<0.01). Once again, for the group injected with ECFCs transfected with siRNA α6 (n=12), the ratio of ischemic to nonischemic leg capillary density was as low as PBS-injected mice (n=8). The inhibition of α6 expression by the siRNA cancelled the beneficial effects of ECFCs on capillary density (P<0.01 versus untransfected ECFCs, and P<0.001 versus ECFCs transfected with scramble siRNA) (Figure 2).
The left gastrocnemius was more severely necrotic in animals injected with PBS (n=4) or with ECFCs transfected with siRNA α6 (n=6) than in animals injected with untransfected ECFCs (n=4) or ECFCs transfected with scramble siRNA (n=5). Quantitative analysis showed that the injection of ECFCs transfected with scramble siRNA and untransfected ECFCs reduced the percentage of necrotic tissue by a factor of 2 relative to the PBS-treated animals (P<0.05 and P<0.01, respectively). In contrast, ECFCs transfected with siRNA α6 had no beneficial effect relative to PBS-treated controls (P<0.001 versus untransfected ECFCs, and P<0.01 versus ECFCs transfected with scramble siRNA) (Figure 3). These results are in accordance with TUNEL analysis (supplemental Figure IV).
Loss of α6 Reduces ECFC Incorporation Into the Microvasculature of Ischemic Skeletal Muscle
We evaluated the number of human ECFCs (labeled red with an anti–human CD31 antibody) incorporated into the mouse microvasculature (labeled green). ECFCs were found in the ischemic leg but not in the healthy leg. siRNA α6–transfected ECFCs were incorporated 5 times less efficiently than untransfected ECFCs and ECFCs transfected with scramble siRNA (n=5 per group, P<0.001) (Figure 4).
Loss of α6 Expression Reduces ECFC Adhesion to ECM
The basement membrane of blood vessels damaged during ischemia may be exposed, and integrin α6β1 mediates cell attachment to laminin, the main component of the basement membrane. Therefore, we used an adhesion assay on Matrigel (61% laminin, 30% collagen IV, and 7% entactin) to study the role of α6 in ECFC adhesion to this substitute of basement membrane. Transfection with siRNA α6 reduced ECFC adhesion to Matrigel by 50% (P<0.01) compared with all the control groups. As expected, there was no difference between untransfected ECFCs, ECFCs treated with the transfection reagent DharmaFECT (Df) alone, and ECFCs transfected with scramble siRNA (Figure 5A).
Loss of α6 Expression Reduces VEGF-Induced ECFC Migration In Vitro
After the onset of ischemia, VEGF is released and functions as a chemoattractant to recruit cells involved in vascular repair. Therefore, we used Boyden chambers to study the role of α6 in ECFC migration toward VEGF. ECFCs transfected with siRNA α6 migrated 40 times less efficiently than untransfected ECFCs, ECFCs transfected with Df alone, and ECFCs transfected with scramble siRNA (P<0.0001 for all) (Figure 5B).
Loss of α6 Expression Reduces Vascular Tube Formation by ECFCs in Matrigel and AKT Phosphorylation After ECFC Adhesion on Laminin
When ECFCs were transfected with siRNA α6, pseudotube length was reduced 15-fold compared with all the other groups: untransfected ECFCs, ECFCs transfected with Df alone, and ECFCs transfected with scramble siRNA (P<0.0001 for all) (Figure 5C).
siRNA α6 Has No Effect on ECFC Proliferation, Apopotosis or Viability
Transfection with scramble siRNA or siRNA α6 has no significant effect on ECFC proliferation (Figure 5D), apoptosis, or viability (supplemental Figure V).
When ECFCs were transfected with siRNA α6, AKT phosphorylation after adhesion on laminin was reduced by 50% compared with untransfected ECFCs or ECFCs transfected with scramble siRNA (P<0.0001 for all) (Figure 6).
This study demonstrates the importance of α6 in the proangiogenic properties of ECFCs. As previously reported, ECFCs injected intravenously into a nude mouse model of hind limb ischemia improved neovessel formation and reperfusion15–17 and provided protection toward necrosis. However, inhibition of ECFC cell surface α6 expression by using specific siRNA abrogated all these beneficial effects.
To understand why the cells lacking α6 were unable to improve neovessel formation and reperfusion, we investigated the role of α6 in the homing of ECFCs, which is a key step in cell therapy. Indeed, some studies showed that soon after the injection, the major part of the cells is removed from the blood circulation and found mainly in the spleen, liver, and kidneys. Despite this loss, the remaining cells are located in the ischemic area.18 ECFCs that have been attracted there by VEGF or stromal cell derived factor (SDF)-1 can incorporate the damaged vasculature and form new blood vessels, unlike colony-forming unit endothelial cells, which promote angiogenesis only through the release of proangiogenic factors and cytokines.11 Even if it is still unclear, ECFCs may also secrete proangiogenic factors, such as placental growth factor-1 (PlGF1)17 and prostaglandin,16 which could explain why ECFC injection can increase neovessel formation even with few cells found in the ischemic area.
Qian et al8 showed that α6 is involved in the homing of hematopoietic stem cells to bone marrow in a model of cell transplantation in irradiated mice. These researchers suggested that α6 contributes to hematopoietic stem cell transmigration to bone marrow because it serves as a receptor for ECM laminins, which are involved in regulating tissue organization, cell adhesion, differentiation, and migration.19 Other researchers reported that a subpopulation of mesenchymal stem cells, expressing high levels of α6, showed increased migration to infarcted heart in mice.20 To determine whether α6 is involved in the homing of ECFCs, we quantified the number of ECFCs incorporated into the vasculature of skeletal muscles 4 days after their injection, as described by Foubert et al.15 When cell surface α6 expression was inhibited by siRNA, the number of ECFCs integrated into the mouse microvasculature of the ischemic muscle was reduced 5-fold. To understand why the cells lacking α6 were not recruited and integrated to the ischemic mouse vasculature, we performed in vitro assays.
After the obstruction of an artery, the oxygen supply is reduced and, therefore, the endothelial cells lining the walls of the downstream vessels undergo hypoxia. The death of these endothelial cells leaves the basement membrane partially uncovered. The integrin α6β1 is a receptor for laminin, the main component of the basement membrane. Consequently, α6 could be implicated in ECFC adhesion to the basement membrane of the injured blood vessels located in the ischemic area (supplemental Figure VI and supplemental Figure VII). Although other integrins or adhesion proteins are also involved, when cell surface α6 expression was inhibited by siRNA, ECFC adhesion to Matrigel, a substitute of the basement membrane, was reduced by a factor of 2. Moreover, we verified that siRNA α6 had no effect on ECFC proliferation, apoptosis, or viability. These results suggest that the low number of ECFCs transfected with siRNA α6 found in the ischemic muscles was the result of poor attachment and was not a bias due to decreased cell proliferation or viability.
Once endothelial progenitors have adhered, they must migrate to participate to the remodeling and to form new blood vessels. At sites of ischemia, VEGF and other angiogenic factors act as chemoattractants for cells involved in neovascularization.21 By using a Boyden chamber migration assay, we found that a lack of α6 expression inhibited ECFC migration induced by VEGF. These observations are in keeping with results previously obtained with other cell types. On human brain microvascular endothelial cells, α6β1 is involved in VEGF-induced adhesion, migration, and in vitro angiogenesis.5 α6 Overexpression on hepatocarcinoma cells leads them to acquire an invasive phenotype.22 Integrin α6β1 is necessary for matrix-dependent focal adhesion kinase (FAK) activation and, therefore, for the migration of hepatocarcinoma cells.23 It is also involved in the attachment of these cells to laminin.24 The same phenomenon has been observed in breast cancer, where α6 promotes carcinoma survival and progression.25,26 By using a different approach, our findings support the hypothesis that α6 could be involved in the mobilization and migration of the progenitors or stem cells from their niches.
When ECFCs are recruited to sites of ischemia, they can participate in vascular repair by either exerting paracrine effects or directly forming new blood vessels. Previous experiments with anti–α6 antibodies have shown that α6 is involved in endothelial cell cord formation in vitro.6,13,27 Thus, we used the Matrigel model to examine the role of α6 in vessel formation. We found that ECFC transfection with siRNA α6 strongly inhibited vascular network formation, suggesting a crucial role of α6 in ECM-mediated migration and differentiation and, consequently, in new blood vessel sprouting, orientation, and stabilization during angiogenesis. Also, siRNA α6 inhibits cordlike network formation by human breast cancer cells.28
α6 Is involved in ECFC adhesion, migration, and pseudotube formation. To understand why, we investigated different signaling pathways (AKT, extracellular signal regulated kinase, and p38), and we observed that AKT phosphorylation was reduced by a factor of 2, 1 hour after adhesion on laminin, when α6 was knocked down. This result could explain the observed cellular effects because the phosphatidylinositol 3-kinase/AKT pathway has been shown to be implicated in endothelial progenitor migration and adhesion.29,30
However, ECFCs may also promote angiogenesis via indirect effects, such as interactions with other cell types. Interestingly, α6 can mediate cell-cell interactions independently of laminin. For example, α6β1 has a key role in gamete fusion,31 resulting from an interaction with membrane-anchored cell surface ligands from the A Disintegrin and Metalloproteinase (ADAM) family. Interaction with ADAM-9 is also responsible for the induction of fibroblast motility.32 The role of α6 in ECFC interaction with other cell types should be further investigated.
In conclusion, α6 plays a major role in the proangiogenic properties of ECFCs. α6 Is involved in ECFC adhesion to the basement membrane and in migration toward VEGF, explaining why this integrin subunit is required for ECFC recruitment to the site of ischemia and for the formation of vascular tube networks. A better understanding of the phenomenon involved in cell recruitment at the site of injury could allow us to find new strategies to enhance cell therapy efficiency. Regarding human cell therapy, our results suggest that enhancing α6 expression on ECFCs might improve their recruitment to sites of ischemia and promote vascular repair.33 On the other hand, ECFCs are involved in tumor angiogenesis,34 and α6 plays a role in both tumor angiogenesis and growth.5 Therefore, our findings support the possibility that α6, like other integrins, might be an interesting therapeutic target for strategies designed to disrupt tumor angiogenesis.35
We thank the staff of Hôpital des Diaconesses for providing cord blood samples, Bruno Saubaméa and the imaging platform for their advice on microscopy, Françoise Grelac and Véronique Remones for their excellent technical assistance, and the staff of the Institut Médicament, Toxicologie, Chimie, Environnement animal facility.
Sources of Funding
C. Bouvard was supported (or paid) by a research grant from Ministère de l'Enseignement Supérieur et de la Recherche. Dr Boisson-Vidal was paid by Centre National de la Recherche Scientifique.
Received on: December 14, 2009; final version accepted on: May 12, 2010.
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