Published online before print March 6, 2008, doi: 10.1161/CIRCRESAHA.107.164376
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© 2008 American Heart Association, Inc.
Cellular Biology |
Phosphatidylinositol-3-Kinase-
Is Integral to Homing Functions of Progenitor Cells
From Molecular Cardiology, Department of Internal Medicine III (E.C., G.C., C.U., A.M.Z., S.D.), and the DRK Institute of Transfusion Medicine and Immune Hematology (S.G., R.H.), J. W. Goethe University Frankfurt, Germany; the Institute for Molecular Biotechnology (J.M.P.), Vienna, Austria; and the Experimental Immunology Branch (T.C.), National Cancer Institute, NIH, Bethesda, Md.
Correspondence to Emmanouil Chavakis, MD, Molecular Cardiology, Department of Internal Medicine III, J. W. Goethe University of Frankfurt, Theodor Stern-Kai 7, 60590 Frankfurt, Germany. E-mail Chavakis{at}em.uni-frankfurt.de
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Abstract |
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Endothelial progenitor cells (EPCs) and hematopoietic progenitor cells are recruited to ischemic regions, improving neovascularization. β1 and β2 integrins play a crucial role for progenitor cell homing to ischemic tissues. Integrin activity is regulated by chemokines and their respective G protein–coupled receptors. The phosphatidylinositol-3-kinase catalytic subunit
(PI3K) is the PI3K isoform that selectively transduces signals from G protein–coupled receptors. Here, we investigated the role of PI3K as a signaling intermediate in the chemokine-induced integrin-dependent homing functions of progenitor cells. A pharmacological PI3K inhibitor significantly reduced chemokine-induced chemotaxis and stromal cell–derived factor (SDF)1
-induced transmigration of human EPCs. Moreover, the PI3K inhibitor significantly reduced SDF1-induced adhesion of EPCs to intercellular adhesion molecule-1 and human umbilical vein endothelial cell monolayers. These findings were corroborated with Lin– bone marrow–derived progenitor cells from PI3K-deficient mice that displayed reduced SDF1-induced migration and intercellular adhesion molecule-1 adhesion as compared with wild-type cells. Pharmacological inhibition or genetic ablation of PI3K reduced SDF1-induced integrin activation in human EPCs and in murine Lin– BM-derived progenitor cells, respectively. In vivo, the homing of PI3K-deficient Lin– progenitor cells to ischemic muscles after intravenous infusion in the model of hindlimb ischemia and their neovascularization-promoting capacity was reduced as compared with wild-type cells. In conclusion, PI3K is integral to the integrin-dependent homing of progenitor cells.
Key Words: progenitor cells • homing • integrins • adhesion • migration • PI3K • neovascularization
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Introduction |
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In 1997, cells isolated from peripheral blood were found to display properties of endothelial cells and progenitor cells and termed "endothelial progenitor cells" (EPCs).1 These cells promote postnatal neovascularization.1–5 Meanwhile, there exist different protocols for the isolation and ex vivo cultivation of distinct cell subpopulations from human blood, which are all called EPCs, although the isolated cells vary with respect to marker expression, proliferation, and endothelial differentiation.6,7 Despite the different culture assays and characteristics in vitro, various assays have yielded cells that enhance neovascularization after ischemia in vivo.6,7 Moreover, single-cell transplantation of hematopoietic stem cells gives rise in vivo to vascular endothelial cells and contributes to postnatal neovascularization.8,9 Not only the hematopoietic stem cells, but also other cells such as infused bone marrow Lin– progenitor cells incorporate into vessels and acquire an endothelial cell phenotype in vivo.10 Therapeutic administration of BM-derived progenitor cells and EPCs increases the neovascularization of ischemic myocardium and limbs and improves left ventricular function after myocardial infarction in animal models.11–13 Moreover, the first clinical studies demonstrated a beneficial effect of intracoronary administration of EPCs or BM cells on the left ventricular function in patients after myocardial infarction.14–16 BM-derived progenitor cells and EPCs are preferentially recruited to sites of ischemia and improve neovascularization by eliciting paracrine effects and/or by being directly incorporated into vascular structures.1,3,5,6,11,17 However, independently of the way of action (paracrine effects or differentiation to endothelial cells), the neovascularization promoting capacity of progenitor cells is dependent on the homing of progenitor cells to sites of ischemia.
In an in vivo intravital microscopy study, embryonic EPCs arrested within tumor microvessels extravasated into the interstitium and incorporated into neovessels, suggesting that a multistep cascade of events including adhesion and transendothelial migration is involved in the recruitment of embryonic EPCs.18 Recent evidence supports the involvement of integrins for the homing of progenitor cells to sites of active neovascularization. Specifically, we and other groups have found that β2 integrins are mediating the homing of human EPCs with neovascularization promoting-capacity and murine BM progenitor cells to ischemic tissues.19,20 Moreover, 4β1 integrin is involved in the homing of BM progenitor cells to sites of active tumor angiogenesis.10 Chemokines, like stromal cell–derived factor (SDF1) and high-mobility group box 1 protein (HMGB1), can increase integrin activity in human EPCs.21 However, the molecular mechanisms of regulation of integrin activity and homing in neovascularization-promoting progenitor cells by chemokines are unclear.
Phosphoinositide 3-kinases (PI3Ks) are important cellular lipid kinases that convert phosphatidylinositol-4,5-bisphosphate to phosphatidylinositol-3,4,5-trisphosphate, a second messenger involved in a variety of cellular functions and responses.22–26 The PI3K (p110 isoform of PI3K catalytic subunits), a class IB PI3K, is predominantly expressed in leukocytes and endothelial cells and transduces signals specifically from the Gβ subunits of G protein–coupled receptors.22–26 Previous studies have demonstrated that PI3K is involved in the chemokine-induced migration of neutrophils, eosinophils, and macrophages.27–30 However, in other subsets of leukocytes, such as T and B lymphocytes, PI3K seems to play only a minor role for migration.25,31,32
Because chemokines such as SDF1 and interleukin (IL)-8, which play an essential role for the recruitment of progenitor cells to sites of ischemia,33–35 act through G protein–coupled receptors, we investigated the role of PI3K for integrin-dependent homing functions of progenitor cells. We found that progenitor cells express the p110 catalytic subunit. Pharmacological inhibition of PI3K blocked the chemokine-induced integrin activation and the integrin-dependent adhesion, migration, and diapedesis of human EPCs derived from peripheral blood. In addition, murine PI3K-deficient BM Lin– progenitor cells displayed reduced migratory and adhesive responses to SDF1 in comparison to wild-type cells. Furthermore, in a mouse model of hindlimb ischemia, using murine BM Lin– progenitor cells from PI3K-deficient mice, we demonstrate that PI3K is involved in the homing of progenitor cells to sites of ischemia and is essential for their neovascularization-promoting capacity.
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Materials and Methods |
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Cell Culture
Approval was obtained from the J.W. Goethe University Hospital institutional review board for these studies. Informed consent was provided according to the Declaration of Helsinki. Peripheral blood mononuclear cells from healthy human volunteers were isolated by density-gradient centrifugation with Ficoll as described.19 Immediately after isolation, total mononuclear cells were plated on culture dishes coated with human fibronectin (10 µg/mL; Sigma) and maintained in endothelial basal medium (Cambrex) supplemented with 1 µg/mL hydrocortisone, 12 µg/mL bovine brain extract, 50 µg/mL gentamycin, 50 ng/mL amphotericin B, 10 ng/mL epidermal growth factor, and 20% FCS. After 3 days, nonadherent cells were removed and adherent cells were incubated for another 24 hours before the experiments. EPCs were characterized by dual-staining for 1,1'-dioctadecyl-3,3',3'-tetramethylindo-carbocyanine-labeled acetyl-low-density lipoprotein and lectin and expression of endothelial markers KDR, VE-cadherin, and vWF.5 Besides expressing endothelial cell markers, EPCs, isolated by this method, express also the hematopoietic cell marker CD45 and the myeloid marker CD14, indicating their relationship to myeloid cells. Human umbilical vein endothelial cells (HUVECs) were purchased from Cambrex and cultured until the third passage as described.36 Lin– progenitor cells were purified from BM from wild-type and PI3K
-deficient mice by negative selection using a cocktail of biotinylated antibodies to lineage markers (anti-CD3, anti-B220, anti-CD11b, anti-Gr-1, anti-TER119; BD Pharmingen, San Diego, Calif) for 10 minutes at 4°C, followed by anti-biotin microbeads for 15 minutes (Miltenyi Biotec, Bergisch-Gladbach, Germany). Murine BM Lin– progenitor cells express Sca-1 (23.9%), c-Kit (44.4%), the hematopoietic cell marker CD45 (95.5%), and the β2 integrin subunit (93.7%). The methods for oligonucleotide microarray analysis, Western blot, cell matrix migration, transendothelial migration, cell–cell adhesion, adhesion to intercellular adhesion molecule (ICAM)-1, detection of activation epitopes of integrins, binding of soluble vascular cell adhesion molecule (VCAM)-1, the model of hindlimb ischemia, and statistical analysis are described in detail in the expanded Materials and Methods section in the online data supplement, available at http://circres.ahajournals.org.
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Results |
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Expression of PI3K
in Human EPCsTo characterize the expression of PI3K
in human EPCs, we used a microarray assay comparing EPCs and HUVECs. The endothelial phenotype of the ex vivo cultivated human EPCs was confirmed by immunostaining, fluorescence-activated cell sorting analysis and functional response to shear stress as previously described.11,37,38 EPCs, circulating human CD34+ cells, HUVECs, and CD14+ monocytes expressed mRNA for PI3K (Figure 1A). We confirmed the expression of PI3K in human EPCs by Western blot (Figure 1B) and in murine BM Lin– progenitor cells by immunofluorescence (data not shown). Moreover, human EPCs express the catalytic subunits PI3K and PI3Kβ but not the catalytic subunit PI3K
(data not shown). Interestingly, EPCs display a lower expression of the catalytic subunits PI3K and PI3Kβ but a higher expression of PI3K in comparison to HUVECs (data not shown).
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Role of PI3K for Matrix Migration of Human EPCs and Murine BM Progenitor Cells
Because PI3K is downstream of G protein–coupled receptors (GPCR) and has been implicated in the regulation of cell migration, we first performed a Transwell migration assay with human EPCs and investigated the expression of PI3K mRNA in migrated (EPCs in the lower well) versus nonmigrated EPCs (EPCs in the upper well). Interestingly, migrated human EPCs demonstrated a higher level of mRNA expression for PI3K than nonmigrated EPCs (Figure 1C). These data prompted us to study the role of PI3K for EPC migration. For this purpose, we first used the unselective inhibitor of PI3K wortmannin. Preincubation of EPCs with wortmannin significantly blocked the SDF1-induced EPC migration on the matrix protein fibronectin, whereas the mitogen-activated protein kinase inhibitor PD98059 had no significant effect (Figure 2A). To specifically assess the role of PI3K on the chemokine-induced migration of human EPCs, we used AS605240, a pharmacological compound that preferentially inhibits the PI3K isoform.39,40 Preincubation of human EPCs with AS605240 led to a significant inhibition of their migration on fibronectin induced by SDF1 and IL-8, which is also a relevant chemokine for EPC homing (Figure 2B and 2C).35 Because the PI3K/Akt pathway is implicated in the regulation of cell survival and proliferation in many cellular systems, we also explored the effects of the PI3K inhibition on the proliferation and apoptosis rates of human EPCs. We found that incubation with AS605240 (200 nmol/L) for 16 hours did not significantly affect apoptosis and proliferation rates of human EPCs (data not shown). Because unspecific effects cannot be excluded by the use of chemical inhibitors, we additionally investigated the role of PI3K in chemokine-induced migration of murine BM Lin– progenitor cells by engaging the PI3K-deficient mice. Strikingly, deletion of the PI3K gene significantly reduced the SDF1-induced migration of BM Lin– progenitor cells on fibronectin in comparison to wild-type cells (Figure 2D). In conclusion, these data demonstrate that PI3K is an essential mediator of chemokine-induced migration in human EPCs and murine BM progenitor cells.
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P<0.05 vs WT+control, #P<0.05 vs PI3K
Role of PI3K for the Transendothelial Migration of Human EPCs
We then investigated the involvement of PI3K in the transendothelial migration of human EPCs in a Transwell transmigration assay. Chemoattraction of EPCs by SDF1 significantly increased the transmigration rate of EPCs through HUVEC monolayers (Figure 3). Preincubation of EPCs with AS605240 or LY294002 (an unselective PI3K inhibitor) led to a significant reduction of SDF1-induced transendothelial migration.
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Role of PI3K for Adhesion of Human EPCs and Murine BM Progenitor Cells
Adhesion to endothelial cells is an essential step for the extravasation of inflammatory cells to sites of inflammation41 and of progenitor cells to ischemic tissues.19 Therefore, we investigated whether PI3K mediates the chemokine-induced EPC adhesion to mature endothelial cells. Preincubation of EPCs with the PI3K inhibitor, AS605240, or with the unselective PI3K inhibitor Ly294002 significantly blocked the SDF1-induced adhesion of EPC to HUVEC monolayers (Figure 4A). The interaction of β2 integrins with endothelial ICAM-142 is crucial in mediating EPC-endothelial cell interactions.19 We, therefore, studied EPC adhesion to immobilized recombinant human ICAM-1. Preincubation of EPCs with the PI3K inhibitor AS605240 or with the unselective PI3K inhibitor Ly294002 significantly blocked the SDF1-induced β2-integrin–dependent adhesion of EPCs to ICAM-1 (Figure 4B). In addition, inhibition of PI3K blocked the SDF1-induced β2-integrin–dependent adhesion of human EPCs on ICAM-1 even under flow conditions (Figure 4C). In line with these results, the SDF1-induced adhesion of murine PI3K-deficient BM Lin– progenitor cells to ICAM-1 was reduced in comparison with the wild-type cells (Figure 4D). In contrast, the surface expression of β1 and β2 integrins or CXCR4 in BM Lin– progenitor cells was not significantly affected by PI3K deficiency (data not shown). Taken together, these data demonstrate that PI3K mediates the chemokine-induced β2-integrin–dependent adhesion of progenitor cells.
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Role of PI3K for the Chemokine-Induced Affinity Regulation of β2 Integrins
The findings so far demonstrated that PI3K is essential for the chemokine-induced integrin-dependent adhesion of progenitor cells without affecting integrin expression on cell surface. This suggested that PI3K regulates integrin activity in progenitor cells. Integrin activity is regulated by affinity and valency changes.43,44 Therefore, we investigated the role of PI3K in the chemokine-induced integrin-affinity regulation in human EPCs. To do so, we used specific antibodies that recognize activation-dependent epitopes on integrins. Stimulation of human EPCs with SDF1 rapidly increased the expression of the activation-dependent epitopes mAb24 (epitope on the β2-integrin I–like domain) and CBRM1/5 (epitope on the CD11b-subunit of β2 integrins), suggesting that SDF1 rapidly increases the affinity stage of β2 integrins (Figure 5A and 5B). Strikingly, preincubation of EPCs with AS605240 blocked the SDF1-induced increase of the expression of the β2 integrin activation epitopes mAb24 and CBRM1/5 (Figure 5A and 5B). None of the treatments (SDF1 without or with AS605240) affected the total protein expression of β1 and β2 integrins on the surface of human EPCs (data not shown). Taken together, these data suggest that PI3K is involved in integrin activation most likely by regulating integrin affinity in human EPCs. Additionally, we also assessed the binding of soluble murine VCAM-1/Fc chimera on murine Lin– progenitor cells as an indicator of integrin affinity. Manganese chloride a direct extrinsic activator of integrin affinity increased the binding of soluble VCAM-1/Fc (4β1 integrin ligand) to the same extent in progenitor cells from wild-type and PI3K-deficient mice (Figure 5C). In contrast, PI3K-deficient BM Lin– progenitor cells displayed a significantly reduced binding of soluble VCAM-1/Fc on stimulation with SDF1 in comparison to wild-type cells (Figure 5C), indicating that PI3K-mediated signaling is essential for the regulation of integrin affinity in mouse progenitor cells. Taken together, these data suggest that PI3K is necessary for the regulation of the β2 and β1 integrin activity in progenitor cells.
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Role of PI3K for the In Vivo Homing and Neovascularization Capacity of Murine BM Progenitor Cells
Our data underline that PI3K mediates chemokine-induced migratory and adhesive functions of different progenitor cell populations in vitro. Therefore, we questioned whether PI3K is also involved in the progenitor cell homing at sites of ischemia in vivo. BM Lin– progenitor cells were isolated from PI3K-deficient and wild-type mice. PI3K deficiency did not affect the surface expression of β2 integrins, the 4 and 5 integrin subunits of β1 integrins, or CXCR4 (receptor of SDF1) in progenitor cells (data not shown). BM Lin– progenitor cells from PI3K-deficient and wild-type mice were intravenously injected in nude mice 1 day after the induction of hindlimb ischemia. After 24 hours, the ischemic muscles were harvested. Strikingly, PI3K-deficient BM Lin– progenitor cells had a reduced ability to home to ischemic tissues in comparison to wild-type cells (Figure 6A), suggesting that PI3K is essential for the in vivo recruitment of progenitor cells to ischemic tissues. In line with these results, PI3K-deficient BM Lin– progenitor cells were significantly less effective in improving neovascularization of ischemic muscles in comparison to wild-type progenitor cells, as assessed 15 days after the induction of hindlimb ischemia (Figure 6B).
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Discussion |
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The present study underscores the importance of PI3K
for the regulation of integrin-dependent homing functions of human EPCs and BM-derived progenitor cells. Specifically, our investigations revealed that: (1) human EPCs express PI3K; (2) PI3K mediates the chemokine-induced migration and diapedesis of progenitor cells; (3) PI3K is essential for integrin activation and the chemokine-induced integrin-dependent adhesion of progenitor cells; (4) PI3K is involved in the in vivo homing of progenitor cells to sites of ischemia in the mouse model of hindlimb ischemia; and (5) PI3K is essential for the in vivo neovascularization-promoting capacity of progenitor cells in sites of ischemia. Thus, the present study provides insights into the regulation of integrin activity in progenitor cells and unravels a new function of PI3K as a key mediator of homing of progenitor cells to sites of ischemia.
Previous studies have demonstrated that PI3K is involved in the chemokine-induced migration of neutrophils, eosinophils, and macrophages.27–30 However, in other subsets of leukocytes, such as T and B lymphocytes, PI3K seems to play only a minor role for migration.25,31,32 In our present work, we demonstrated for the first time that pharmacological inhibition or genetic deficiency of PI3K significantly reduces the chemokine-induced migration of human EPCs and murine BM Lin– progenitor cells, respectively. These data support the importance of PI3K for the matrix migration of progenitor cells. However, because the inhibition of migration was not complete, alternative parallel signaling pathways (eg, a Dock-dependent pathway)31 may be additionally involved in the regulation of progenitor cell migration.
Our studies also have revealed a critical role of PI3K for the chemokine-induced adhesion of progenitor cells to the major β2-integrin–ligand ICAM-1. There is controversial evidence regarding the role of PI3K in the regulation of integrin-dependent adhesion. Two recent studies have suggested a role of PI3K in the chemokine-induced adhesion of neutrophils,29,45 whereas another study has shown only a minor contribution of PI3K in neutrophil adhesion.46 Moreover, in lymphocytes, inhibition of PI3K activity with unselective inhibitors did not affect the adhesion in vitro47 and in vivo.48 Nevertheless, our data clearly demonstrate that PI3K is essential for the chemokine-induced increase of progenitor cell adhesiveness. It is likely that distinct molecular intermediates may be required for the regulation of chemokine-induced integrin-dependent adhesion in different subsets of BM cells (progenitor cells versus neutrophils, monocytes or lymphocytes).
In line with these results, using antibodies recognizing the active conformation of β2 integrins in human EPCs and performing soluble VCAM-1-Fc binding assays in murine Lin– BM progenitor cells, our data indicate that PI3K is involved in the regulation of integrin affinity at least in progenitor cells. Interestingly, overexpression of a constitutively active PI3K catalytic subunit induced increases in LFA-1 and VLA-5 affinity.49,50 However, PI3K activity was shown to be dispensable for the chemokine-induced integrin affinity activation in lymphocytes.48 The role of PI3K for the chemokine-induced affinity regulation of integrins in different subpopulations of BM-derived cells requires further investigation.
The signaling intermediates involved in the regulation of the chemokine-induced integrin-dependent adhesivity by PI3K in progenitor cells remain unclear. Overexpression of PI3K was shown to increase specifically the β2-integrin–dependent adhesion in Jurkat cells via cytohesin-1.51 However, it is unclear whether PI3K mediates the chemokine-induced integrin activation in a cytohesin-1–dependent manner in progenitor cells. Moreover, SDF1 and HMGB1 increased the activity of the small GTPase Rap1 in human EPCs,21 which is involved in the stimulation of integrin affinity and avidity in many cellular systems.52,53 It is conceivable that Rap1 could be a downstream effector of PI3K mediating the chemokine-induced integrin activation in progenitor cells. Indeed, it has been demonstrated that PI3Ks can facilitate agonist-induced Rap1 activation in platelets.54–56 An additional possibility is that PI3K could affect the function of Rap1 effectors. Further studies are required to clarify the mechanism by which PI3K transduces the chemokine-induced integrin activation in progenitor cells.
In accordance with our in vitro data demonstrating the role of PI3K in progenitor cell migration and adhesion, the in vivo homing of PI3K-deficient progenitor cells to sites of ischemia was significantly reduced in comparison to wild-type cells. Our finding that PI3K deficiency does not completely block homing of infused BM progenitor cells suggests that additional mechanisms may be involved in the complementation of homing. Further studies are needed to elucidate a potentially synergistic role of other signaling pathways for the multistep recruitment process of progenitor cells to ischemic tissues. In accordance with the reduced homing of PI3K-deficient progenitor cells to sites of ischemia, we found that PI3K-deficient progenitor cells were less effective in improving neovascularization.
In summary, the present study demonstrates for the first time a critical role of PI3K for in vitro and in vivo homing of progenitor cells to ischemic tissues and for their neovascularization-promoting capacity and provides insight into the chemokine-induced integrin-activity regulation in these cells. A better understanding of the homing mechanisms of progenitor cells may lead to the development of new therapeutic strategies for improvement of neovascularization in patients with ischemic diseases.
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Acknowledgments |
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We thank Peggy Schuster for excellent technical assistance and Ariane Fischer for the animal experiments. We thank Dr N. Hogg for providing us the antibody mAb24.
Sources of Funding
This work was supported by Deutsche Forschungsgemeinschaft Transregional Collaborative Research Center SFB/TR23, Project A2 (to E.C. and S.D.), by EuGeneHeart (to J.M.P.), and by the Intramural Research Program of the NIH, National Cancer Institute (to T.C.).
Disclosures
None.
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Footnotes |
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Original received September 17, 2007; revision received February 7, 2008; accepted February 27, 2008.
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