Published online before print July 24, 2008, doi: 10.1161/CIRCRESAHA.107.169375
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© 2008 American Heart Association, Inc.
Integrative Physiology |
Leptin Enhances the Recruitment of Endothelial Progenitor Cells Into Neointimal Lesions After Vascular Injury by Promoting Integrin-Mediated Adhesion
From the Departments of Cardiology and Pulmonary Medicine (M.R.S., M.L., P.S., N.-M.H., C.D., I.R., S.R., M.P., G.H., S.K., K.S.) and Hematology and Oncology (F.A., M.Z.), Georg August University of Goettingen, Germany. Present address for S.R.: First Division of Internal Medicine, Democritus University of Thrace, Alexandroupolis, Greece.
Correspondence to Katrin Schaefer, MD, Department of Cardiology and Pulmonary Medicine, University of Goettingen, Robert Koch Strasse 40, D-37099 Goettingen, Germany. E-mail katrin.schaefer{at}med.uni-goettingen.de
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Abstract |
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The adipocytokine leptin modulates vascular remodeling and neointima formation. Because endothelial progenitor cells (EPCs) participate in vascular repair, we analyzed the effects of leptin on human EPC function in vitro and in vivo. After 7 days in culture, EPCs expressed the leptin receptor and responded to leptin stimulation with increased STAT3 phosphorylation. Incubation of EPCs with leptin (at concentrations between 1 and 100 ng/mL) increased the number of EPCs adhering to vitronectin and fibronectin in a receptor-specific manner. It also enhanced the capacity of EPCs to incorporate into a monolayer of human endothelial cells and the adherence of these cells to activated platelets. Leptin upregulated
vβ5 and 4 integrin expression in EPCs, and the effects of leptin on EPC function could be prevented, at least in part, by RGD peptides and function-blocking antibodies. Intravenous injection of fluorescently labeled human EPCs into athymic nude mice shortly after vascular injury revealed that preincubation of EPCs with leptin augmented their accumulation within intimal lesions, accelerating reendothelialization and decreasing neointima formation in an vβ5 and 4 integrin-dependent manner. Our findings suggest that leptin specifically modulates the adhesive properties and the homing potential of EPCs and may thus enhance their capacity to promote vascular regeneration in vivo.
Key Words: leptin • endothelial progenitor cells • neointima formation • integrins
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Introduction |
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Leptin is an adipose tissue–secreted hormone acting primarily on hypothalamic neurons to regulate body weight and energy expenditure. However, isoforms of the leptin receptor are also expressed on a number of cell types outside of the central nervous system. For example, leptin potentiates agonist-induced platelet aggregation,1 and it promotes arterial1,2 and venous3 thrombosis in vivo. Moreover, the hormone has been shown to promote the proliferation, differentiation, and functional activation of hematopoietic cells,4 and it may function as an angiogenic factor for mature endothelial cells.5,6
Accumulating evidence suggests that endothelial progenitor cells (EPCs) play an important role in maintaining vascular integrity and regulating neovascularization. For example, EPCs, which either were exogenously administered or mobilized from the bone marrow in response to stimulation with various cytokines and angiogenic growth factors, contributed to postnatal vasculogenesis associated with myocardial and skeletal muscle ischemia, wound healing, and tumor growth.7–9 In the vasculature, circulating EPCs migrate to sites of endothelial injury, and it has been suggested that they are capable of promoting reendothelialization, improving endothelium-dependent vasoreactivity, and reducing intimal hyperplasia during vascular remodeling.10,11 Importantly, the ability of EPCs to confer vascular protection appears to be related not only to their number in the circulation but also to their functional properties and particularly their ability to adhere (home) to areas of tissue damage.12,13 Based on these previous reports, and on the recent detection of the leptin receptor on human EPCs,14 we sought to determine whether leptin may affect the ability of EPCs to interact with endothelial cells and the extracellular matrix (ECM) of the vessel wall and to participate in vascular remodeling. Our results indicate that ex vivo stimulation of culture-expanded human EPCs with leptin at concentrations ranging between 10 and 100 ng/mL results in upregulation of 4 and vβ5 integrins. These effects promoted EPCs adhesion to ECM proteins, activated platelets, and mature endothelial cells in vitro, and they increased EPC recruitment to sites of vascular injury in vivo. Interestingly, although EPCs did not appear to incorporate into the neoendothelium itself, leptin treatment enhanced reendothelialization and decreased neointima formation. Our findings provide novel insights into the mechanisms underlying the pleiotropic effects of leptin, and perhaps other adipocytokines, on vascular homeostasis. They may help modify existing pathophysiological concepts and improve our understanding of the links between obesity and cardiovascular disease.
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Materials and Methods |
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An expanded Materials and Methods is available in the online data supplement at http://circres.ahajournals.org.
Mononuclear cells were isolated from the peripheral blood of human volunteers by density-gradient centrifugation. After 4 days in culture, nonadherent cells were removed and adherent cells were maintained in endothelial cell medium until analysis on day 7. EPCs were characterized and expression of the leptin receptor was determined using flow cytometry, immunofluorescence, RT-PCR, and Western blot analysis. The effects of leptin (1, 10, 100, or 1000 ng/mL for 24 hour) or neutralizing antibodies against leptin, leptin receptor, and 4 or vβ5 integrins on EPC function in vitro was assessed by analyzing EPC adhesion to ECM proteins, tumor necrosis factor (TNF)-–stimulated human umbilical vein endothelial cells (HUVECs), or collagen-activated platelets. The in vivo effects of leptin on the recruitment of EPCs to the site of vascular injury and the degree of neointima formation or reendothelialization was examined using laser fluorescence microscopy, immunofluorescence, and morphometric quantification.
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Results |
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Characterization of Endothelial Progenitor Cells
Human mononuclear cells were isolated and cultivated under endothelial cell conditions. On day 7, numerous spindle-shaped cells were observed, and the majority of adherent cells (>95%) were found to be double-positive for uptake of DiI-labeled acetylated LDL (acLDL) and binding of Ulex europaeus lectin (Figure IA in the online data supplement). Flow cytometric analysis of endothelial cell surface antigens revealed that 85.9±3.5% of the adherent cells were positive for vascular endothelial growth factor receptor (VEGFR)2, 62.1±4.2% for VE-cadherin (CD144), 56.7±5.0% for CD31, and 9.5±1.4% for von Willebrand factor (vWF) (supplemental Figure IB). In addition, the adherent cells expressed surface markers consistent with leukocytic (CD45; 97.5±0.7%) and monocytic (CD14; 20.3±11.2%) cell lineage, whereas only very few cells (<2.0%) expressed the hematopoietic markers CD34 and CD133. RT-PCR analysis confirmed the expression of endothelial cell lineage markers (supplemental Figure IC), including VEGFR1 and endothelial NO synthase. Moreover, cells were capable of incorporating into a network of tubular structures provided by cocultivated mature endothelial cells (supplemental Figure ID). On the basis of these morphological and functional characteristics, and in line with published data,8,15–19 cells were characterized as peripheral blood–derived early outgrowth or culture-expanded EPCs.
EPCs Express the Leptin Receptor
Expression of the leptin receptor (ObR) was analyzed on EPCs cultured for 7 days. Flow cytometric analysis revealed that 24.9±3.0% of the EPCs expressed the leptin receptor (supplemental Figure II), and the majority (>90%) of the ObR-expressing cells were also found to be positive for VEGFR2 or M integrin (Figure 1A). ObR expression was confirmed using immunofluorescence (Figure 1B) and RT-PCR (Figure 1C). Leptin resulted in upregulation of both ObR mRNA (Figure 1C) and surface (Figure 1D) expression. Moreover, Western blot analysis revealed increased STAT3 phosphorylation in response to leptin (Figure 1E), indicating that EPCs possess the long isoform of the leptin receptor.20
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Leptin Potentiates the Adherence of EPCs to ECM Proteins
EPCs homing to the vessel wall involves the adhesion of circulating cells to ECM proteins following vascular injury. To assess whether leptin alters the adhesive properties of EPCs, cells were incubated with leptin and then allowed to attach to different ECM proteins. Preincubation of EPCs for 24 hours with leptin (1, 10, and 100 ng/mL) significantly increased the number of cells firmly adherent to vitronectin (VN) compared to PBS-treated cells (supplemental Figure IIIA), whereas stimulation with 1000 ng/mL leptin appeared to have the opposite effect. Time curve experiments (ranging from 1 to 72 hours) revealed that the effect of leptin on EPC adhesion was most pronounced after stimulation for 24 hours (data not shown). Of note, coincubation with leptin- or leptin receptor–neutralizing antibodies both prevented the potentiating effect of leptin on EPC adhesion to VN (supplemental Figure IIIB). Stimulation with leptin also increased the number of EPCs binding to fibronectin (FN)-coated culture dishes (supplemental Figure IIIC), whereas it did not alter their ability to adhere to collagen, gelatin, or plastic (data not shown).
Leptin Increases the Ability of EPCs to Adhere to Endothelial Cell Monolayers and Enhances Transendothelial Migration
Apart from the interaction of EPCs with ECM proteins, their homing to the vessel wall requires adhesion and transmigration through endothelial cells. Pretreatment of EPCs with leptin (10 and 100 ng/mL) significantly enhanced their capacity to incorporate into a monolayer of TNF-–activated HUVECs (supplemental Figure IIID). Also, preincubation with leptin, at concentrations of 10 and 100 ng/mL, increased the migration of EPCs through a HUVEC monolayer toward a chemotactic gradient induced by stromal cell–derived factor-1 (199±33% and 152±40% versus control, respectively; P=0.01 for the difference between 10 ng/mL leptin and PBS-treated controls).
Stimulation of EPCs With Leptin Upregulates the Expression of vβ5 and 4 Integrins
EPCs adhere to ECM proteins or cells via specific integrin receptors. For example, binding of cells to VN is mediated primarily by vβ3 and vβ5 integrins, whereas at least 10 different integrins are involved in mediating adhesion to FN, among which 5β1, 4β1, and IIbβ3 have been studied in more detail.21 Integrins capable of mediating cell–cell contacts include 4β1 and members of the β2 family; they enable binding to vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule-1, and endothelial surface–associated fibrinogen.22 In addition, vβ3 and vβ5 integrins may participate in cell–cell interactions using platelet endothelial cell adhesion molecule23 and endothelial surface–bound VN24 as ligands. We analyzed the surface expression of integrin adhesion receptors on EPCs after 7 days in culture using flow cytometry. As summarized in Figure 2A, EPCs mostly expressed β2 (94±4.4% of gated mononuclear cells), M (89±3.7%), and L (89±3.0%), and, to a lesser extent, β1 (66±11%) integrins. EPCs also expressed vβ5 (26±2.6%) and 4 integrins (12±3.7%), whereas the expression of vβ3 on EPCs was low (7.0±1.9%).
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Figure 2B shows that stimulation of EPCs with 10 or 100 ng/mL leptin for 24 hours significantly increased surface expression of 4 and vβ5 integrins, as assessed by flow cytometry, compared to unstimulated controls (representative histograms shown in Figure 2C), whereas the expression of vβ3, β1, β2, and the β2-associated subunits L and M remained unchanged. Fluorescence microscopy confirmed that leptin increased 4 and vβ5 integrin expression (Figure 2D), and the majority of the vβ5- and 4-expressing cells were found to also express VEGFR2, M integrin, and ObR (Figure 2E). Upregulation of 4 and vβ5 integrin expression appeared to be related, at least in part, to an increase in their mRNA levels (supplemental Figure IVA); however, this effect was not consistently observed in EPCs of all individuals studied (supplemental Figure IVB).
The Effects of Leptin on Integrin-Mediated EPC Adhesion Involve Both RGD-Dependent and RGD-Independent Mechanisms
Given the fact that the Arg-Gly-Asp (RGD) sequence is a major recognition motif for integrin-mediated cell adhesion to ECM proteins, experiments were performed to test the effect of RGD peptides on EPC adhesion with and without leptin stimulation. Our findings, which are summarized in Figure 3, show that the addition of RGD peptides (0.25 mg/mL) reduced the ability of EPCs to adhere to VN (P=0.004; A) and mature endothelial cells (P=0.006; Figure 3E), whereas they did not detectably affect their adhesion to FN (P=0.944; Figure 3C). Moreover, addition of RGD peptides significantly reduced the leptin-mediated increase of EPC adhesion to VN (Figure 3A and representative findings in Figure 3B) and to HUVECs (Figure 3E and representative findings in Figure 3F), although adhesion to HUVECs was still elevated compared to unstimulated, RGD-treated controls (P<0.006). Further experiments revealed that the effect of leptin on EPC adhesion to FN continued to be significant in the presence of RGD peptides (P<0.002 versus unstimulated controls; Figure 3C and representative findings in Figure 3D). These results suggest that both RGD-dependent (adhesion to VN and HUVECs) and -independent mechanisms (adhesion to FN and, partly, to HUVECs) are involved in mediating the effects of leptin on EPC adhesion.
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P<0.05 vs RGD alone. Representative photomicrographs show the adhesion of fluorescently labeled PBS- or leptin-treated EPCs to VN-coated (B) or FN-coated (D) culture plates or HUVECs (F). Magnification, x50 (B and D) or x200 (F).
Further experiments revealed that monoclonal vβ5-inhibiting antibodies (MAB1961; 10 µg/mL) significantly reduced the adhesion of both untreated and leptin-stimulated EPCs to VN (Figure 3A and representative findings in Figure 3B), whereas monoclonal 4-inhibiting antibodies (MAB16983; 10 µg/mL) significantly reduced EPC adhesion to FN (Figure 3C and representative findings in Figure 3D). Adhesion of leptin-stimulated EPCs to a monolayer of TNF-–stimulated HUVECs could completely be abolished by 4- and VCAM-function-blocking antibodies (Figure 3E and representative findings in Figure 3F), suggesting that the leptin-mediated increase of EPC adhesion involves binding via 4 integrins to VCAM, expressed on activated HUVECs.
Leptin Enhances the Incorporation of EPCs After Vascular Injury and Reduces the Size of Neointimal Lesions In Vivo
To determine the effects of leptin on EPCs homing to the vessel wall in vivo, vascular injury was induced at the carotid artery using the ferric chloride model.1 Fluorescent DiD-labeled EPCs, pretreated with 10 ng/mL leptin or PBS for 24 hours, were intravenously injected into athymic nude mice within 10 minutes after induction of vascular injury. Whole body fluorescence laser imaging at different time points (up to 3 weeks) revealed that DiD-labeled EPCs already began to accumulate at the injured carotid artery within the first hour. A significantly enhanced specific fluorescence intensity projected on the injured carotid artery could be observed between days 1 and 3 after injury in mice that had received leptin-treated EPCs compared to those injected with control-treated cells (representative findings shown in Figure 4A; results summarized in B). At later time points, the differences in the specific fluorescence intensity were no longer significant. Additional experiments using CM-DiI-labeled cells and fluorescence microscopy confirmed the presence of EPCs at the injured carotid artery. Fluorescent cells were detected accumulating within the thrombotic material occluding the vascular lumen, and a higher cell number was detected 3 days after injection when the EPCs had been pretreated with leptin (Figure 4C). In further experiments, leptin (0.1 µg/g body weight1) was injected into mice 30 minutes before induction of arterial injury followed by injection of untreated EPCs as described above. Morphometric quantification revealed that not only ex vivo leptin prestimulation but also leptin IP injection before injury was capable of significantly enhancing the accumulation of EPCs to the site of vascular injury (supplemental Figure V).
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Because ferric chloride–induced injury results in the formation of platelet-rich thrombi, and platelet -granules contain ligands for both 4 (fibronectin or thrombospondin) and vβ5 (VN) integrins, we also assessed the adhesion of leptin- or control-stimulated EPCs to a monolayer of collagen-activated human platelets or releasates from maximally stimulated platelets in vitro. These analyses revealed that preincubation with leptin significantly enhanced the adhesion of EPCs to collagen-activated platelets (236±46.3% versus PBS-treated cells; n=5; P=0.019) or platelet releasates (182±15.6%; n=5; P<0.001). Importantly, the leptin effects were receptor-specific, because they could be inhibited by preincubation of EPCs with ObR-neutralizing antibodies (86.5±16.8%; P=0.39; and 84.3±33.2%; P=0.54, respectively).
Histological analysis of neointimal lesions 3 weeks after injury revealed that CM-DiD-labeled EPCs were located within the neointima, predominantly in the subendothelial layer (Figure 5A). Immunofluorescence labeling confirmed that these were injected human EPCs, because they colocalized with human leukocyte antigen (data not shown). Double staining with antibodies against cell type–specific antigens further showed that 46.4±5.8% of CM-DiI-labeled EPCs colocalized with CD31 and 53.2±7.7% with M, whereas costaining with -actin was rarely observed (Figure 5A). Importantly, mice treated with leptin-stimulated EPCs exhibited increased reendothelialization of the neointimal lesions, as suggested by a higher degree of luminal vWF expression (Figure 5B). In this regard, binding studies using Ulex europaeus lectin failed to detect the presence of human (ie, EPC-derived) endothelial cells, whereas strong staining of murine endothelial cells could be observed (Bandeiraea simplicifolia lectin; supplemental Figure VI). Finally, morphometric analysis of arterial cross sections through the neointima 3 weeks after injury revealed a significant reduction of both the neointima area and the degree of luminal stenosis in mice injected with leptin-treated EPCs (10 and 100 ng/mL) compared to those injected with PBS-treated EPCs (Figure 5C). Of note, lesions from mice injected with EPCs that had been treated with supraphysiological leptin concentrations (ie, 1000 ng/mL) did not differ from those receiving control-treated cells (data not shown). Finally, preincubation of leptin-stimulated EPCs with specific integrin function-blocking antibodies or control IgG, 30 minutes before their intravenous injection after injury, confirmed that the observed effects of leptin on the neointima formation and reendothelialization are mediated by vβ5 or 4 integrins (Figure 5D and 5E).
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Discussion |
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The results of the present study indicate that the adipokine leptin may enhance the ability of endothelial progenitor cells to participate in vascular remodeling. Pretreatment of culture-expanded EPCs with leptin at concentrations ranging between 10 and 100 ng/mL promoted the adhesion of these cells to ECM proteins, activated platelets, and mature endothelial cells. In vivo, leptin stimulation increased the recruitment of EPCs to sites of vascular injury and platelet-rich thrombi, and this effect was associated with enhanced reendothelialization and decreased neointima formation. These findings unveil novel mechanisms that may contribute to the pleiotropic effects of the adipokine on vascular homeostasis.
Experimental studies have suggested beneficial effects of EPCs on intimal hyperplasia. For example, implantation of autologous circulating EPCs into balloon-injured carotid arteries of rabbits accelerated endothelialization, reduced neointima formation, and improved endothelial-dependent vasoreactivity.10,11 Similarly, spleen-derived EPCs enhanced reendothelialization and reduced neointima formation after carotid artery injury in mice.15 Importantly, homing and incorporation of EPCs into sites of vascular injury and ischemia appears to depend not only on their circulating numbers but also on their functional properties. Thus, EPCs that were culture-expanded from diabetic humans and transplanted into mice after wire-induced endothelial denudation exhibited impaired recruitment,25 whereas transgenic overexpression of vasculoprotective genes enhanced the ability of rabbit EPCs to reduce neointima hyperplasia.26
The attachment of circulating EPCs to matrix proteins and (activated) endothelial cells is a critical step in the arrest and extravasation of these cells at the site of injury and appears to be mediated, at least in part, by integrin adhesion receptors.22,27 The VN receptor, which has been shown to be upregulated in response to hypoxia28 or vascular injury,29 promotes the adhesion of cells to the plasma protein VN, released from activated platelets30 and accumulating in the vessel wall after injury.31 VN may, thus, function as an adhesive substrate guiding circulating progenitor cells to the site of tissue injury, and its role appears to be similar to that recently ascribed to fibrin or activated platelets.32,33 Notably, it also has been suggested that VN may function as a bridging molecule between circulating cells and the endothelium itself.24 Two functionally homologous integrins (vβ3 and vβ5) have been shown to bind VN in an RGD-dependent manner. Blocking antibodies to vβ3, or to VN itself, suppressed neointima formation after balloon injury in rats,34,35 and a preclinical study reported that cyclic RGD-coated stents reduced intimal hyperplasia, possibly by enhancing the recruitment of endothelial progenitor cells.36 Although vβ5 expression also is increased in response to balloon injury,29 its role during vascular remodeling is less well examined. In the present study, we demonstrate that EPCs express vβ5 integrins and that stimulation of EPCs with leptin significantly upregulated vβ5 surface expression resulting in enhanced, RGD-dependent EPC adhesion to VN. Importantly, in vivo studies using function-blocking antibodies suggest that vβ5 integrins are involved in the beneficial effect of leptin prestimulation of EPCs on neointima formation.
In addition to the vβ5-VN interaction, the results of our study also suggest a critical role for 4 integrins, most likely as part of the 4β1 or very late antigen-4 complex, in mediating the effects of leptin on EPC homing. It has been reported that EPCs, but not smooth muscle cell progenitors, express vβ5 and 4β1.37 In the present study, treatment of EPCs with leptin upregulated the expression of 4 integrins, and 4-neutralizing antibodies not only prevented the binding of leptin-stimulated EPCs to FN or HUVECs but also the leptin-mediated reduction of neointima formation. Binding of leptin-stimulated EPCs to HUVECs could also be inhibited by VCAM-neutralizing antibodies, suggesting that leptin promotes adhesion of 4-expressing EPCs to VCAM present on activated endothelial cells. In this regard, ex vivo preincubation of EPCs with 4-neutralizing antibodies was shown to reduce the recovery of hindlimb blood flow and capillary density and incorporation of EPCs into ischemic tissues.38
In accordance with the effects of leptin on integrin-mediated adhesion of EPCs to mature endothelial cells, activated platelets, and ECM proteins, our in vivo studies showed that pretreatment of human EPCs with leptin before their injection into athymic nude mice resulted in more pronounced accumulation of these cells at the site of vascular injury. This effect was associated with accelerated reendothelialization and, importantly, reduced neointima formation. Previous reports suggested that EPCs may facilitate endothelial cell repair by transdifferentiation into endothelial cells.15,39 However, in our experiments using 2 different fluorescent membrane labels, as well as indirect detection methods such as the visualization of human leukocyte antigen, we could not detect human (injected) EPCs lining the luminal surface of injured mouse arteries, although CD31- or M-positive EPCs were found to be present within the neointimal lesions. In this regard, it has been suggested that EPCs may stimulate the recruitment and proliferation of local mature cells through paracrine mechanisms, including the secretion of angiogenic factors.40,41 Our findings confirm and extend these reports by showing that pretreatment of human EPCs with leptin enhanced reendothelialization of injured arteries with murine, ie, endogenous cells. It remains to be determined whether these endogenous cells originate from the proliferation of local endothelial cells or circulating progenitors.
We and others have previously shown that once-daily intraperitoneal administration of recombinant leptin into wild-type and leptin-deficient mice enhanced (rather than reduced) intimal hyperplasia after experimental injury.42,43 The explanation for the apparent disagreement between these earlier observations and the results of the present study is partly related to the mode of leptin application, ie, once-daily injection of the hormone into mice over several weeks, as opposed to a single ex vivo stimulation of culture-expanded, human EPCs. Also, the leptin dosages that have been used to rescue the phenotype of leptin-deficient mice42 result in transient but pronounced (and partly supraphysiological) elevations of plasma leptin concentrations.1 Notably, the EPCs used in our experiments were isolated from lean individuals, and it is possible that the effects of continuously elevated circulating leptin levels, such as those encountered in human obesity, on EPCs or other cells of the vessel wall may differ from those described in the present study.
In conclusion, we could show that the adipokine leptin enhanced the functional capacity of peripheral blood-derived human EPCs in vitro and improved their homing capacity in vivo. These findings, which were partly unexpected, point to novel mechanisms possibly contributing to the effects of leptin on vascular remodeling, and they also raise the possibility that pretreatment of progenitor cells could therapeutically modify (enhance) their regenerative properties following vascular injury. Future studies will have to examine in more detail how interaction of leptin with its receptor on endothelial (progenitor) cells upregulates specific integrins. Moreover, it needs to be clarified how obesity-associated hyperleptinemia affects the functional activity of EPCs and other vascular cells and, particularly, whether the increased cardiovascular risk associated with excess body weight is related to the hyperleptinemia itself or resistance to the possible vasoprotective and vasoregenerative effects of the hormone.
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Acknowledgments |
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We thank Sarah Henkel and Stephanie Minne for expert technical assistance. We are also grateful to Drs Zaverio Ruggeri and Yuichi Kamikubo (The Scripps Research Institute, La Jolla, Calif) for providing the human leptin receptor cDNA plasmid vector and to Mario Baumgard for assistance with near-infrared fluorescence microscopy.
Sources of Funding
This work was supported by a grant from the Novartis Stiftung für therapeutische Forschung (to K.S.) and a MSD Sharp & Dohme Fellowship (to M.R.S.).
Disclosures
None.
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Footnotes |
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Original received April 16, 2007; resubmission received December 4, 2007; revised resubmission received June 2, 2008; accepted July 10, 2008.
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References |
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