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TISSUE-SPECIFIC STEM CELLS |
aCenter for Transgene Technology and Gene Therapy, Flanders Institute for Biotechnology, Katholieke Universiteit Leuven, Leuven, Belgium;
bCardiology Division, University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Leuven, Belgium
Key Words. Endothelial progenitor cells • Cell transplantation • Bone marrow transplantation • Pulmonary hypertension • Microvasculature • Chronic hypoxia
Correspondence: Correspondence: Stefan Janssens, M.D., Ph.D., Center for Transgene Technology and Gene Therapy, Flanders Institute for Biotechnology, Katholieke Universiteit Leuven, P.O. Box 912, Herestraat 49, B-3000 Leuven, Belgium. Telephone: 32-16-344235; Fax: 32-16-344240; e-mail: stefan.janssens{at}med.kuleuven.be
Received on July 13, 2007;
accepted for publication on January 25, 2008.
First published online in STEM CELLS EXPRESS February 7, 2008.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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, adhesion to fibronectin, incorporation into a vascular network, and nitric oxide production (–41%, –29%, –30%, and –32%, respectively, vs. normoxic EPCs; p < .05 for all). The dysfunctional phenotype of hypoxic EPCs significantly impaired their neovascularization capacity in chronic hind limb ischemia, contrary to normoxic EPCs cultured in identical conditions. Mechanisms contributing to EPC dysfunction include reduced integrin v and β1 expression, decreased mitochondrial membrane potential, and enhanced senescence. Novel insights from chronic hypoxia-induced EPC dysfunction may provide important cues for improved future cell repair strategies. Disclosure of potential conflicts of interest is found at the end of this article.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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Pulmonary hypertension (PH) is a devastating disease with significant morbidity and mortality, characterized by increased pulmonary artery pressure, right ventricular hypertrophy, and pulmonary vascular remodeling [6, 7]. In rat and canine models of monocrotaline-induced pulmonary endothelial injury, early-outgrowth EPCs have been shown to mitigate toxin-induced PH [8–10], spurring increased interest in culture-expanded EPCs as a promising tissue-engineering tool in PH. Recently, circulating progenitor cells were shown to participate in hypoxic pulmonary vascular remodeling with recruitment of c-Kit+ cells to the adventitia of hypoxic bovine lung vessels [11] and of green fluorescent protein (GFP)-labeled bone marrow-derived EPCs to the endothelium of hypoxic murine lung vessels [12]. The biological significance of endogenous bone marrow cell recruitment to hypoxic lungs remains unknown. Also, whether or not bone marrow-derived progenitor cells can be considered in cell transfer strategies for hypoxia-induced PH is unknown and depends in part on adhesive, migratory, and vasculogenic properties of EPCs under these pathophysiologic conditions.
Therefore, we proposed to study the endogenous bone marrow-derived cellular response to pulmonary vascular remodeling in bone marrow chimeric mice, as well as the potential of exogenous EPC transfer to reduce PH. We observed that chronic hypoxia significantly impairs EPC phenotype compared with normoxic (Nx) mice, limiting therapeutic efficacy of EPCs, and we investigated several mechanisms that could contribute to EPC dysfunction. We identified reduced integrin v and β1 expression, impaired mitochondrial transmembrane potential, and enhanced cellular senescence in EPCs from mice exposed to chronic hypoxia. These findings could provide novel cues to reverse EPC dysfunction, not only in chronic hypoxia but also, by analogy, in patients with cardiovascular risk factors.
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MATERIALS AND METHODS |
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Generation of Chimeric Mice
Wild-type C57Bl6/N mice, 8 weeks of age, were lethally irradiated (9.5 Gy of total-body irradiation) using a linear accelerator (GE Healthcare, Little Chalfont, U.K., http://www.gehealthcare.com) at 3.9 Gy/minute. Irradiation was followed within 24 hours by injection of 5 x 106 total bone marrow cells harvested from femurs of C57Bl6/N mice ubiquitously expressing GFP. Bone marrow reconstitution was allowed for 8 weeks.
EPC Isolation
After homogenization of the spleen, mononuclear cells (MNCs) were isolated from CHx and age-matched Nx mice by density centrifugation (Histopaque 1083; Sigma-Aldrich). MNCs (40 x 106) were plated on fibronectin-coated (Tebu-Bio, Boechout, Belgium, http://www.tebu-bio.com/) six-well plates in endothelial basal medium 2 (EBM-2; Cambrex, Walkersville, MD, http://www.cambrex.com) supplemented with 5% fetal bovine serum, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), recombinant insulin-like growth factor, epidermal growth factor, ascorbic acid, and gentamicin/amphotericin-B. After 4 days of culture, nonadherent cells were removed, and cells were cultured for 3 additional days. These early-outgrowth EPCs were used for in vitro analysis of EPC function.
EPC Transfer During PH
After 1 or 2 weeks of exposure to hypoxia (FIO2 0.10), C57BL6/N mice were randomized to receive either saline or 5 x 105 EPCs via jugular vein injection (n = 6–8 in each group). After a total of 3 weeks of hypoxia, RVSP and RV hypertrophy were measured. In cell tracking experiments, EPCs derived from luciferase-expressing mice (described in the supplemental online Methods) or EPCs labeled with CM-1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) according to the manufacturer's instructions (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) were transplanted at the indicated time points. Homing of EPCs was determined at different time points using bioluminescence imaging [13, 14] (described in the supplemental online Methods). For detection of CM-DiI-labeled EPCs, lungs and spleens were paraffin-embedded, and sectioned. Sections were stained with 4',6-diamidino-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) to detect nuclei or for vWF using an Alexa647-conjugated secondary antibody to detect blood vessels. Images were obtained with a LSM510 confocal microscope (Carl Zeiss). All animal experiments were approved by the ethical committee of the University of Leuven.
EPC Characterization and Enumeration
Characterization of In Vitro-Cultured EPCs. After 7 days of culture, adherent spleen-derived cells were incubated with 12 µg/ml DiI-labeled acetylated low-density lipoprotein (acLDL) (Molecular Probes) for 4 hours. After fixation with 1% paraformaldehyde, cells were incubated with fluorescein-isothiocyanate-conjugated lectin from Bandeiraea simplicifolia (BS1 lectin; 1 hour; 10 µg/ml; Sigma-Aldrich), and fluorescent images were obtained (LSM510 confocal microscope; Carl Zeiss). Expression analysis was done by flow cytometry using a FACSCalibur (BD Biosciences, San Diego, http://www.bdbiosciences.com). Antibodies for vascular endothelial growth factor receptor-2 (VEGFR-2), Sca-1, c-Kit (CD117), and CXCR-4 were from BD Pharmingen, and isotype controls were run in parallel.
Analysis of Circulating EPC Number. Blood was collected from the vena cava, and Fc block (BD Pharmingen) was added to the samples prior to antibody incubation. Fluorescently labeled antibodies against CD45/VEGFR-2 and CD45/VEGFR-2/c-Kit (all from BD Pharmingen) were used to enumerate circulating endothelial cells and circulating endothelial progenitors, respectively. After 30 minutes of incubation, red blood cells were lysed (PharmLyse; BD Pharmingen), and samples were evaluated by flow cytometry. Isotype controls were run in parallel. Approximately 50,000 events were recorded. Following culture of spleen-derived MNCs, adhering acetylated LDL- and BS1 lectin-positive EPCs from Nx and CHx animals were counted in 10 high-power fields (HPFs), and the number of EPCs per mouse was determined from the surface area of the culture dish and total number of MNCs isolated.
EPC Function In Vitro
Migration.
Migration toward stromal cell-derived factor-1 (SDF-1) was determined by resuspending 1 x 105 EPCs derived from Nx and CHx mice in 100 µl of EBM-2 plus 0.5% bovine serum albumin (BSA) in the upper chamber of a modified Boyden chamber (8 µm pore size; Corning Life Sciences, Schiphol-Rijk, The Netherlands, http://www.corning.com/lifesciences). The upper chamber was transferred to a 24-well containing EBM-2/BSA and 150 ng/ml SDF-1. After 5 hours at 37°C, membrane inserts were fixed with 4% formaldehyde. SDF-1 was kindly provided by M. Tjwa (Center for Transgene Technology and Gene Therapy, Leuven, Belgium). Cell nuclei of migrated EPCs were stained using DAPI, and the difference in migrating EPCs toward EBM-2/BSA with or without SDF-1 was counted in four HPFs.
Adhesion. To evaluate adhesion, 50,000 EPCs were resuspended in EBM-2; plated onto fibronectin-coated (5 µg/cm2; Tebu-Bio), 1% BSA-blocked 24-wells; and incubated for 30 minutes. The number of adherent cells per 10 HPFs was counted and compared between EPCs derived from Nx and CHx mice.
Incorporation into a Vascular Network. EPCs from Nx and CHx mice were incubated with DiI-labeled acetylated LDL for 4 hours and coplated with human umbilical vein endothelial cells (HUVECs; Cambrex) in a 1:2 proportion on presolidified Matrigel (BD Biosciences). After 16 hours, the number of EPCs incorporating into the vascular network per HPF were determined. The average of six HPFs per experiment was calculated.
Nitric Oxide Production. Nitrite, nitrate, and S-nitrosothiol (NOx) levels were determined as stable oxidative products of nitric oxide (NO) in the supernatants of Nx and CHx EPCs using a Sievers 280 chemiluminescent NO analyzer (GE Analytical Instruments, Boulder, CO, http://www.geinstruments.com) [15]. Negative control values (unconditioned medium) were subtracted from the sample values (24 hours of conditioning), and NOx concentrations, normalized to cell number, were calculated. HUVECs and the murine endothelial cell line MS1 (CRL-2279; American Type Culture Collection, Manassas, VA, http://www.atcc.org) were used in control experiments. To demonstrate nitric oxide synthase (NOS)-dependent NO production, experiments were performed in the presence and absence of 2 mM NG-mono-methyl-L-arginine (L-NMA; Sigma-Aldrich).
Integrin and Nitric Oxide Synthase 3 (NOS3) Expression Analysis.
Transcript levels of integrins 5, v, β1, β2, β3, and NOS3 were determined by reverse transcriptase-polymerase chain reaction using specific primers and fam-labeled probes (supplemental online Table 1). Results were expressed as relative copy numbers standardized to levels of the housekeeping 18S rRNA gene (Applied Biosystems), and the average values for EPCs derived from CHx mice were normalized to the average values in EPCs derived from Nx mice.
Senescence-Associated β-Galactosidase Staining. Gently fixed EPCs were incubated for 6 hours at 37°C (without CO2) with fresh senescence-associated 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) staining solution (1 mg/ml X-gal [Immunosource, Halle-Zoersel, Belgium, http://www.immunosource.com], 40 mM citric acid/sodium phosphate, pH 6, 5 mM K3Fe[CN]6, 5 mM K4Fe[CN]6, 150 mM NaCl, 2 mM MgCl2) [16]. After incubation, nuclei were counterstained with nuclear fast red, and the percentage of cells showing senescence was determined on 10 HPFs per condition.
Mitochondrial Membrane Potential Analysis, Lactate Production, and Cellular ATP Content
EPCs cultured from Nx and CHx mice were loaded with the potentiometric mitochondrial dye JC-1 (Molecular Probes) at 4 µM for 20 minutes at 37°C. After two washing steps, green and red mean fluorescence intensities were evaluated using flow cytometry or confocal microscopy. To exclude any influence of different mitochondrial size, shape, or density, we calculated the ratio of red to green fluorescence for each experiment. In control experiments, the mitochondrial membrane potential was dissipated with carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP; 10 µM; Sigma-Aldrich) [17]. Lactate concentration in conditioned supernatants was measured using an enzymatic method on an automatic analyzer (Dimension analyzer; Siemens Healthcare Diagnostics, Brussels, Belgium, http://diagnostics.siemens.com/). In the presence of excess NAD+ and lactate dehydrogenase, lactate was converted to pyruvate and NADH. The formation of NADH results in increased absorbance at 340 nm, and this value was corrected for nonspecific signal by subtracting absorbance at 383 nm. Cellular ATP concentrations in EPCs from CHx and Nx mice were determined by luciferine/luciferase luminometry according to the manufacturer's instructions (ATPLite; PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com).
EPC Function In Vivo
To induce limb ischemia, the left femoral artery and vein were excised in 8–10-week-old athymic BALB/cJ nude mice as described previously [18]. Perfusion was measured using a Lisca PIM II camera (Perimed, Stockholm, Sweden, http://www.perimed.se/) and expressed as the ratio of ischemic to normal limb perfusion. After 24 hours, animals with significant impairment of perfusion (Doppler signal ratio <25%) and without signs of severe necrosis were randomized to i.v. injection of 5 x 105 EPCs from Nx or CHx mice or saline vehicle (n = 10 per group). After 14 days, animals were euthanized, and adductor muscles were perfused with phosphate-buffered saline, fixed in Z-fix, and embedded in paraffin. Capillary density was determined in four HPFs after visualization of BS1 lectin (Sigma-Aldrich) with diaminobenzidine.
Statistical Analysis
All data are expressed as mean ± SEM. Normality of data was confirmed using the Kolmogorov-Smirnov test. Since circulating endothelial and circulating EPC numbers were not normally distributed, data from Nx and CHx mice were compared using a Wilcoxon rank-sum test. All other differences in EPC function between Nx and CHx mice were compared using a paired t test, and a p value <.05 was considered statistically significant. For evaluation of EPC transfer in vivo, a one-way analysis of variance was used to compare perfusion ratios or vascular densities among the three treatment groups.
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RESULTS |
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The percentage of CD45–/VEGFR-2+/c-Kit+ cells in the peripheral blood was significantly higher in CHx mice, as determined by three-color flow cytometry (0.33% ± 0.10% of all nucleated cells vs. 0.07% ± 0.01% in Nx mice; n = 14; p < .001; supplemental online Fig. 1). Also, the percentage of CD45–/VEGFR-2+ circulating endothelial cells, of which endothelial progenitor cells are a subset, was higher in CHx mice (0.53% ± 0.12% vs. 0.21% ± 0.05% in Nx mice; n = 14; p < .001). The total number of circulating white blood cells did not differ between CHx and Nx mice (data not shown).
In addition, spleen-derived MNCs from both Nx and CHx mice, cultured for 7 days, express VEGFR-2, Sca-1, CXCR-4, and moderate levels of c-Kit (Fig. 2A), with more than 95% of these early-outgrowth EPCs being positive for acLDL and BS1 lectin, regardless of whether EPCs were derived from Nx or CHx mice. The number of acLDL/BS1 lectin double-positive EPCs per mm2 was significantly increased in CHx mice (220 ± 49 vs. 106 ± 25 EPCs per mm2 in Nx mice; n = 7; p < .01; Fig. 2B). The total number of EPCs (taking into account the number of MNCs in the spleen) was also higher in CHx mice (352 ± 87 vs. 240 ± 67 x 103 EPCs per mouse in Nx mice; n = 7; p < .05). Taken together, flow cytometry and cell culture experiments indicate that failure of EPCs to integrate in hypertensive lung vessels is not caused by impaired EPC mobilization during chronic hypoxia.
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expression levels by enzyme-linked immunosorbent assay. Although SDF-1 was significantly higher in lungs from CHx mice (131 ± 13 vs. 72 ± 11 pg of SDF-1 per mg of protein in Nx lungs; p < .01; n = 9 in each group), we did not observe upregulation of ICAM-1 expression in CHx lungs (n = 6 in each group; supplemental online Fig. 2).
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Migration.
In ischemic tissues, SDF-1 is induced and promotes the recruitment of CXCR-4-positive progenitor cells [22]. The ability of EPCs to migrate toward SDF-1 was therefore investigated using a modified Boyden chamber (Fig. 4A). EPCs from CHx mice were less responsive to SDF-1 compared with EPCs from Nx mice (99 ± 28 vs. 169 ± 27 migrating EPCs per HPF, respectively; n = 7; p < .05). In contrast, flow cytometry revealed no significant difference in CXCR-4 expression (mean fluorescence intensity of CHx EPCs was 110 ± 26 vs. 91 ± 20 arbitrary units in Nx EPCs; n = 7; p = not significant).
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Adhesion to Fibronectin and Incorporation into a Vascular Network. Chronic hypoxia caused a 29% decrease in adhesive capacity of EPCs to fibronectin, an important extracellular matrix component involved in the initial steps during vasculogenesis (52 ± 6 vs. 73 ± 10 adhering EPCs per HPF from Nx mice; n = 7; p < .05). Similarly, incorporation into a developing vascular network in vitro mimics in vivo incorporation of EPCs during vasculogenesis. To study the effect of chronic hypoxia on vasculogenic potential of EPCs, we plated DiI-labeled EPCs on Matrigel in the presence of HUVECs and determined incorporation after overnight incubation (Fig. 4B). The total number of incorporating EPCs per HPF was lower for EPCs derived from CHx mice (46 ± 3 vs. 66 ± 6 incorporating EPCs per HPF from Nx mice; n = 7; p < .05).
NO Production. To investigate whether chronic hypoxia influences the ability of EPCs to release NO, a downstream effector in VEGF-induced angiogenesis, we measured NO concentrations in 24-hour-conditioned medium. Although NOS3 mRNA expression did not differ between CHx and Nx EPCs (supplemental online Table 2), NOS activity was significantly impaired, as indicated by the approximately 30% lower NOx concentrations in the supernatants of EPCs derived from CHx mice compared with Nx EPCs (2.3 ± 0.4 vs. 3.4 ± 0.4 nmol per 106 EPCs, respectively; n = 9; p < .05). These concentrations are similar to concentrations released from HUVECs (4.6 ± 0.7 nmol per 106 HUVECs) and the murine endothelial MS1 cell line (3.9 ± 1.0 nmol per 106 cells) under identical conditions. NOx concentrations are reduced approximately 80% by the NOS inhibitor L-NMA.
Hind Limb Ischemia. To investigate whether or not the dysfunctional phenotype of CHx EPCs limits vascular repair capacity in vivo, we performed EPC transfer in mice with hind limb ischemia. Whereas EPCs derived from Nx mice significantly improved blood flow recovery in the ischemic hind limb compared with saline (recovery to 69% ± 2% of flow in the nonoperated limb; n = 10; p < .01), an equal number of EPCs from CHx mice failed to do so (Fig. 5A, 5B). Correspondingly, capillary density in the adductor muscle was significantly higher in mice receiving Nx EPCs than in mice receiving CHx EPCs or saline (Fig. 5C, 5D).
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Integrin Expression Levels.
To investigate potential molecular mechanisms for reduced adhesive capacity, we measured integrin expression levels of CHx and Nx EPCs. As shown in supplemental online Table 2, integrin v and β1 transcript levels were significantly lower in EPCs derived from CHx compared with Nx mice (–61% and –44% respectively; p < .05 for both). The expression of integrins 5, β2, and β3 was not significantly affected by chronic hypoxia.
Senescence-Associated β-Galactosidase Staining. To explore whether chronic hypoxia induces senescence of EPCs, we compared senescence-associated β-galactosidase staining of EPCs cultured from Nx and CHx mice. The percentage of dark blue-stained EPCs, indicative of a higher content of the lysosomal hydrolase β-galactosidase, was significantly higher in EPCs from CHx mice (9.6% ± 1.4%) compared with EPCs from Nx mice (5.2% ± 1.4%; n = 7; p < .01; Fig. 6A).
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Mitochondrial Membrane Potential Analysis and Cellular ATP Content. To evaluate mitochondrial membrane potential of EPCs, an important rheostat in determining EPC survival and function, we compared JC-1 aggregate fluorescence in EPCs cultured from Nx or CHx mice. Ratiometric analysis of JC-1 aggregated forms (red fluorescence) versus the monomeric form (green fluorescence) indicated a 30% lower ratio in CHx EPCs (0.61 ± 0.08 vs. 0.86 ± 0.12 in Nx EPCs; n = 10; p < .05; Fig. 6B). In EPCs treated with the uncoupler FCCP, this ratio decreased to 0.30 for both Nx and CHx EPCs. Since anaerobic ATP synthesis is coupled to the formation of lactate, we measured lactate concentrations in conditioned medium from CHx and Nx EPCs. CHx EPCs did not upregulate glycolysis for enhanced ATP generation, as lactate production was not increased in CHx EPCs (3.0 ± 0.8 vs. 5.2 ± 1.1 µmol per 106 Nx EPCs; n = 6; p = .11). Total cellular ATP concentrations were 10% lower in EPCs from CHx mice (1.99 ± 0.21 vs. 2.17 ± 0.24 µmol per 106 EPCs; n = 9; p < .05).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosure of Potential...AcknowledgmentsReferences |
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During prolonged hypoxia/hypoxemia, greater numbers of EPCs are mobilized into peripheral blood, as determined by flow cytometry (CD45–/VEGFR-2+/c-Kit+ MNCs) and cell culture (acetylated LDL/BS1 double-positive cells). However, EPCs from these hypoxic mice demonstrated reduced migration toward SDF-1, a critical ligand for EPC homing; reduced adhesive capacity to the subendothelial extracellular matrix component fibronectin (a first necessary step in vascular integration); and reduced angiogenic potential and NO release. These data suggest a sustained effect of chronic hypoxia on the functionality of early-outgrowth EPCs and have important therapeutic consequences. Indeed, functional impairment of hypoxic EPCs was also obvious in a well-standardized in vivo neovascularization assay, where CHx EPCs failed to improve perfusion in the ischemic hind limb model, contrary to identically cultured Nx EPCs. Taken together, these data provide evidence that the hypoxic milieu significantly impairs the tissue repair capacity of EPCs.
Our data of EPC function in chronic hypoxia are in marked contrast with the traditional view of the vasculoprotective or reparative role of EPCs at sites of vascular injury. EPC transfer accelerates re-endothelialization in the denuded vessel wall, improves endothelium-dependent vasorelaxation, and reduces neointima formation in rabbits [23] and mice [3]. EPCs also appear to have a promising tissue repair capacity in monocrotaline-induced PH in rats [8, 9]. Contrary to the transient engraftment in precapillary vessels in this inflammatory model of PH, we failed to observe a similar endogenous homing response of EPCs to the remodeling hypoxic pulmonary circulation.
The lack of an endogenous tissue repair response was not attributable to impaired mobilization of EPCs from the bone marrow compartment to the pulmonary circulation, as evidenced by the significant increase in circulating EPC numbers during chronic hypoxia. Moreover, i.v. transfer of fully functional Nx EPCs also failed to incorporate in remodeling vessels and to reduce PH. These observations suggest that either the extent of pulmonary endothelial damage or activation is insufficient to elicit effective engraftment of EPCs or, alternatively, that EPCs become dysfunctional during sustained exposure to chronic hypoxia and lose their ability to adhere and transmigrate.
To investigate the former possibility, we compared ICAM-1 and SDF-1 expression in CHx and Nx lungs. The interactions between integrin β2 on EPCs and ICAM-1 in vascular tissue and between CXCR-4 on EPCs and SDF-1 in tissue are important determinants for EPC homing [22, 24–26]. We observed increased SDF-1 generation but no upregulation of ICAM-1 in CHx lungs compared with Nx lungs. This contrasts with the significant upregulation of ICAM-1 in ischemic skeletal muscles [26]. These findings suggest that failure to upregulate ICAM-1 expression might contribute to the impaired homing and incorporation of circulating EPCs in chronic hypoxia.
To test the latter hypothesis (that EPCs become dysfunctional during sustained exposure to CHx), we performed several in vitro functionality assays, corresponding to essential steps during vasculogenesis: migration of chronic hypoxia EPCs toward the chemokine SDF-1, a critical ligand for EPC homing, was impaired; adhesion to the important extracellular matrix component fibronectin was decreased; and the cells were less likely to incorporate into a developing vascular network. In addition, EPCs from CHx mice showed reduced NO release. Importantly, the dysfunctional phenotype did not result from specific in vitro conditions or tissue culture artifacts during cell expansion, as simultaneously expanded Nx EPCs significantly improved neovascularization after hind limb ischemia.
The exact mechanisms of EPC dysfunction remain unknown, despite extensive studies in atherosclerosis and ischemic heart disease. Persistent dysfunction after 7 days of in vitro culture has also been described for EPCs derived from type I diabetics (reduced secretion of angiogenic growth factors) [27], type II diabetics (reduced incorporation into a developing vascular network) [5], and patients with chronic renal failure (reduced migration and incorporation into a developing vascular network) [28]. Moreover, a higher percentage of EPCs from patients with cardiovascular risk factors are senescent compared with EPCs from healthy persons after 7 days of culture [29]. In contrast to the extensive description of EPC dysfunction in cardiovascular diseases, animal models of EPC dysfunction are limited. EPCs from cathepsin L knockout mice have impaired in vivo neovascularization capacity, yet they preserved migration and growth factor secretion [30]. In contrast, EPCs from glutathione peroxidase 1-deficient mice incorporate less in a vascular network and have an impaired migratory response [31]. In our study, we have characterized EPC dysfunction associated with chronic hypoxia, which is relatively easy to use and well standardized in a genetically homogeneous population of inbred mice.
Our data indicate that chronic hypoxia is a useful experimental condition to investigate basic mechanisms of EPC dysfunction, including altered integrin expression, enhanced senescence, and perturbed energy metabolism. First, integrins regulate important cellular interactions of progenitor cells, including transendothelial migration toward SDF-1 (integrin β1) [32], bFGF- or VEGF-induced angiogenesis (integrin v) [33], and retention of progenitor cells in the bone marrow compartment (integrin β1 via very-late antigen 4 or integrin 4β1) [34]. We observed significantly reduced expression levels of v and β1 integrins in CHx EPCs, which contribute to reduced adhesion to fibronectin and migration toward SDF-1 and to increased circulating EPC numbers. In contrast, NOS3 expression was not different between Nx and CHx EPCs, whereas NOS activity was impaired, which is in accordance with previously published results of unaltered expression but impaired post-transcriptional activation during chronic hypoxia [35]. Second, the impaired function of CHx EPCs may be attributable in part to enhanced senescence, as evidenced by a twofold increase in the percentage of β-galactosidase-positive EPCs. Other environmental risk factors, including smoking, have been shown to promote a similar degree of EPC senescence [29]. Finally, the ratiometric analysis of the mitochondrial membrane potential in Nx and CHx EPCs demonstrated a 30% reduction in transmembrane potential. A similar depolarization of the mitochondrial membrane has recently been documented in isolated cells exposed to ischemic preconditioning [36, 37] and is thought to represent a cell defense mechanism against a lethal insult. Indeed, cellular ATP levels in EPCs from CHx mice were only moderately reduced despite the significant loss of mitochondrial membrane potential, which is normally required for maintaining respiratory ATP generation. Preservation of cellular ATP concentrations could be accounted for by previously described adaptive survival mechanisms of cells in response to chronic hypoxia in vitro and moderate ischemia in vivo (i.e., downregulation of ATP requiring processes, including ion pump activity and protein synthesis, to induce a hypometabolic state [38–41]). Our results are therefore consistent with the view that EPCs from chronically hypoxic mice are less metabolically active and consume less ATP, which in turn could account for their dysfunctional phenotype.
Several limitations of our study should be mentioned. First, we used a standardized study protocol using spleen-derived EPCs and cannot rule out differential in vitro behavior of spleen-derived EPCs versus circulating EPCs. The latter are routinely studied in patients, but limited numbers preclude a similar approach in mice. Second, although our molecular and cellular studies clearly establish altered integrin expression, senescence, and mitochondrial function in EPCs from CHx mice, the intracellular downstream signaling pathways remain to be determined. Third, although hypoxia-induced EPC dysfunction represents a useful paradigm to develop improved cell-based therapeutic strategies, we need to recognize that multiple, complex disease pathways likely cooperate in patients.
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CONCLUSION |
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DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST |
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ACKNOWLEDGMENTS |
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REFERENCES |
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