Decreasing Intracellular Superoxide Corrects Defective Ischemia-induced New Vessel Formation in Diabetic Mice
2008; Elsevier BV; Volume: 283; Issue: 16 Linguagem: Inglês
10.1074/jbc.m707451200
ISSN1083-351X
AutoresDaniel J. Ceradini, Dachun Yao, Raymon H. Grogan, Matthew J. Callaghan, Diane Edelstein, Michael Brownlee, Geoffrey C. Gurtner,
Tópico(s)Adipose Tissue and Metabolism
ResumoTissue ischemia promotes vasculogenesis through chemokine-induced recruitment of bone marrow-derived endothelial progenitor cells (EPCs). Diabetes significantly impairs this process. Because hyperglycemia increases reactive oxygen species in a number of cell types, and because many of the defects responsible for impaired vasculogenesis involve HIF1-regulated genes, we hypothesized that HIF1 function is impaired in diabetes because of reactive oxygen species-induced modification of HIF1α by the glyoxalase 1 (GLO1) substrate methylglyoxal. Decreasing superoxide in diabetic mice by either transgenic expression of manganese superoxide dismutase or by administration of an superoxide dismutase mimetic corrected post-ischemic defects in neovascularization, oxygen delivery, and chemokine expression, and normalized tissue survival. In hypoxic fibroblasts cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the EPC mobilizing chemokine stromal cell-derived factor-1 (SDF-1) and of vascular epidermal growth factor, which modulates growth and differentiation of recruited EPCs. In hypoxic EPCs cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the SDF-1 receptor CXCR4, and endothelial nitric-oxide synthase, an enzyme essential for EPC mobilization. HIF1α modification by methylglyoxal reduced heterodimer formation and HIF1α binding to all relevant promoters. These results provide a basis for the rational design of new therapeutics to normalize impaired ischemia-induced vasculogenesis in patients with diabetes. Tissue ischemia promotes vasculogenesis through chemokine-induced recruitment of bone marrow-derived endothelial progenitor cells (EPCs). Diabetes significantly impairs this process. Because hyperglycemia increases reactive oxygen species in a number of cell types, and because many of the defects responsible for impaired vasculogenesis involve HIF1-regulated genes, we hypothesized that HIF1 function is impaired in diabetes because of reactive oxygen species-induced modification of HIF1α by the glyoxalase 1 (GLO1) substrate methylglyoxal. Decreasing superoxide in diabetic mice by either transgenic expression of manganese superoxide dismutase or by administration of an superoxide dismutase mimetic corrected post-ischemic defects in neovascularization, oxygen delivery, and chemokine expression, and normalized tissue survival. In hypoxic fibroblasts cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the EPC mobilizing chemokine stromal cell-derived factor-1 (SDF-1) and of vascular epidermal growth factor, which modulates growth and differentiation of recruited EPCs. In hypoxic EPCs cultured in high glucose, overexpression of GLO1 prevented reduced expression of both the SDF-1 receptor CXCR4, and endothelial nitric-oxide synthase, an enzyme essential for EPC mobilization. HIF1α modification by methylglyoxal reduced heterodimer formation and HIF1α binding to all relevant promoters. These results provide a basis for the rational design of new therapeutics to normalize impaired ischemia-induced vasculogenesis in patients with diabetes. Studies in both experimental animals and humans have shown that diabetes impairs ischemia-driven neovascularization (1Abaci A. Oguzhan A. Kahraman S. Eryol N.K. Unal S. Arinc H. Ergin A. Circulation. 1999; 99: 2239-2242Crossref PubMed Scopus (573) Google Scholar, 2Yarom R. Zirkin H. Stammler G. Rose A.G. J. Pathol. 1992; 166: 265-270Crossref PubMed Scopus (136) Google Scholar). Diabetic animals have a decreased vascular density following hind limb ischemia (3Rivard A. Silver M. Chen D. Kearney M. Magner M. Annex B. Peters K. Isner J.M. Am. J. Pathol. 1999; 154: 355-363Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 4Schatteman G.C. Hanlon H.D. Jiao C. Dodds S.G. Christy B.A. J. Clin. Investig. 2000; 106: 571-578Crossref PubMed Scopus (431) Google Scholar) and impaired wound healing (5Gallagher K.A. Liu Z.J. Xiao M. Chen H. Goldstein L.J. Buerk D.G. Nedeau A. Thom S.R. Velazquez O.C. J. Clin. Investig. 2007; 117: 1249-1259Crossref PubMed Scopus (555) Google Scholar). Human angiograms demonstrate fewer collateral vessels in diabetic patients compared with non-diabetic controls (1Abaci A. Oguzhan A. Kahraman S. Eryol N.K. Unal S. Arinc H. Ergin A. Circulation. 1999; 99: 2239-2242Crossref PubMed Scopus (573) Google Scholar). Clinically, this contributes to increased rates of lower limb amputation, heart failure, and increased mortality after ischemic events. Ischemic tissue selectively recruits endothelial progenitor cells (EPCs) 3The abbreviations used are: EPCs, endothelial progenitor cells; ARNT, aryl hydrocarbon receptor nuclear translocator; ChIP, chromatin immunoprecipitation; eNOS, endothelial nitric-oxide synthase; GLO1, glyoxalase 1; HIF1α, hypoxia-inducible factor-1α; MG, methylglyoxal; Mn-SOD, manganese superoxide dismutase; ROS, reactive oxygen species; SDF1, stromal cell-derived factor-1; VEGF, vascular endothelial growth factor; qPCR, quantitative PCR; HRE, hypoxia response element; CREB, cAMP-response element-binding protein; Mn-TBAP, manganese(III) meso-tetrakis(4-carboxyphenyl)porphyrin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild type; bHLH, basic helix loop helix. from the bone marrow compartment by up-regulating the chemokine stromal cell-derived factor-1 (SDF-1). SDF-1 expression acts as a signal indicating the presence of tissue ischemia, and its expression is directly regulated by hypoxia-inducible factor-1 (5Gallagher K.A. Liu Z.J. Xiao M. Chen H. Goldstein L.J. Buerk D.G. Nedeau A. Thom S.R. Velazquez O.C. J. Clin. Investig. 2007; 117: 1249-1259Crossref PubMed Scopus (555) Google Scholar, 6Tepper O. Galiano R. Capla J. Kalka C. Gagne P. Jacobowitz G. Levine J. Gurtner G. Circulation. 2002; 106: 2781-2786Crossref PubMed Scopus (1292) Google Scholar). Ischemic tissue also up-regulates expression of VEGF, which modulates growth and differentiation of recruited EPCs (7Asahara T. Takahashi T. Masuda H. Kalka C. Chen D. Iwaguro H. Inai Y. Silver M. Isner J.M. EMBO J. 1999; 18: 3964-3972Crossref PubMed Scopus (1666) Google Scholar). Endothelial cell mobilization from the bone marrow compartment is mediated by the SDF-1 receptor CXCR4, and endothelial nitric-oxide synthase plays an essential role in this mobilization (8Aicher A. Heeschen C. Mildner-Rihm C. Urbich C. Ihling C. Technau-Ihling K. Zeiher A.M. Dimmeler S. Nat. Med. 2003; 9: 1370-1376Crossref PubMed Scopus (1208) Google Scholar). In diabetic mice, expression of SDF-1 in peripheral wound tissue is decreased, and in diabetic mice and humans, there is a significant decrease in circulating EPCs (5Gallagher K.A. Liu Z.J. Xiao M. Chen H. Goldstein L.J. Buerk D.G. Nedeau A. Thom S.R. Velazquez O.C. J. Clin. Investig. 2007; 117: 1249-1259Crossref PubMed Scopus (555) Google Scholar, 9Ceradini D.J. Kulkarni A.R. Callaghan M.J. Tepper O.M. Bastidas N. Kleinman M.E. Capla J.M. Galiano R.D. Levine J.P. Gurtner G.C. Nat. Med. 2004; 10: 858-864Crossref PubMed Scopus (2213) Google Scholar), which exhibit impaired proliferation, adhesion, and incorporation into vascular structures (9Ceradini D.J. Kulkarni A.R. Callaghan M.J. Tepper O.M. Bastidas N. Kleinman M.E. Capla J.M. Galiano R.D. Levine J.P. Gurtner G.C. Nat. Med. 2004; 10: 858-864Crossref PubMed Scopus (2213) Google Scholar). The mechanisms underlying defective ischemia-induced vasculogenesis in diabetes remain unclear. One common element in the different environments where vasculogenesis is believed to occur in the presence of a hypoxic stimulus (10Ceradini D.J. Gurtner G.C. Trends Cardiovasc. Med. 2005; 15: 57-63Crossref PubMed Scopus (295) Google Scholar). Hypoxia-inducible gene transcription is normally regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric master regulator of hypoxia-inducible gene transcription (11Kaelin W.G. Annu. Rev. Biochem. 2005; 74: 115-128Crossref PubMed Scopus (368) Google Scholar) composed of an α subunit, HIF-1α, which is labile in the presence of oxygen, and a constitutively expressed stable β subunit, aryl hydrocarbon receptor nuclear translocator (ARNT). In the presence of normal oxygen concentrations, HIF-1α is hydroxylated at two conserved proline residues, ubiquinated, and then degraded by the proteosome. This process is mediated by the von Hippel-Lindau tumor suppressor protein, which binds to hydroxylated HIF-1α. When oxygen concentration is low, hydroxylation does not occur, allowing HIF-1α protein levels to rise. The HIF-1α/ARNT heterodimer then binds to a consensus hypoxia response element (HRE), where it recruits the coactivator p300/CREB-binding protein and activates transcription. Because HIF1α regulates transcription of SDF-1, VEGF, CXCR4, and endothelial nitric-oxide synthase (eNOS) in response to hypoxia, and because the bone marrow niche is normally hypoxic, we hypothesized that a diabetes-induced defect in HIF1α could cause both defective cell signaling in response to ischemia, and defective EPC response to those signals. Hyperglycemia increases reactive oxygen species (ROS) in cell types affected by diabetic complications, and these ROS initiate several complex series of molecular events that result in diabetic tissue damage (12Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (7064) Google Scholar). In non-diabetic glutathione peroxidase 1-deficient mice, increased ROS causes impaired revascularization in the ischemic hindlimb model, accompanied by impaired EPC mobilization and function (13Galasso G. Schiekofer S. Sato K. Shibata R. Handy D.E. Ouchi N. Leopold J.A. Loscalzo J. Walsh K. Circ. Res. 2006; 98: 254-261Crossref PubMed Scopus (136) Google Scholar). One consequence of increased intracellular ROS is accumulation of the highly reactive dicarbonyl methylglyoxal, a degradation product formed from triose phosphates during glycolysis, which forms stable adducts primarily with arginine residues of intracellular proteins. Because high glucose and diabetes increase angiopoietin-2 transcription in endothelial cells through methylglyoxal modification of the corepressor mSin3A (14Yao D. Taguchi T. Matsumura T. Pestell R. Edelstein D. Giardino I. Suske G. Rabbani N. Thornalley P.J. Sarthy V.P. Hammes H.P. Brownlee M. J. Biol. Chem. 2007; 282: 31038-31045Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar), we further postulated that this consequence of increased ROS affects the HIF1-dependent expression of genes involved in ischemia-induced vasculogenesis. Murine Diabetes and Ischemia Model—Diabetes was induced in 2-4-month old C57Bl/6 mice with streptozotocin, and stable hyperglycemia was confirmed 14 days after treatment with monitoring at regular intervals thereafter. Age-matched non-diabetic littermates served as controls. Three weeks after induction of diabetes, animals began daily treatment with manganese(III) meso-tetrakis(4-carboxyphenyl)porphyrin (Mn-TBAP, 10 mg/kg per day, dissolved in 0.005 m NaOH solution) or carrier (control), which was continued for the duration of the experiments. In parallel, transgenic mice that overexpress Mn-SOD (Mn-SOD+/-) underwent identical diabetes induction with similar age-matched controls. This formed six experimental groups: non-diabetic wild type, non-diabetic Mn-TBAP-treated, non-diabetic Mn-SOD transgenic, diabetic wild type, diabetic Mn-TBAP-treated, and diabetic Mn-SOD transgenics. After 4 weeks of diabetes, all animals were used in the mouse ischemia model as previously described in full accordance with the New York University Institutional Animal Care and Use Committee. Briefly, a peninsular shaped incision was made on the dorsum of mice, generating a reproducible gradient of soft tissue ischemia (6Tepper O. Galiano R. Capla J. Kalka C. Gagne P. Jacobowitz G. Levine J. Gurtner G. Circulation. 2002; 106: 2781-2786Crossref PubMed Scopus (1292) Google Scholar, 9Ceradini D.J. Kulkarni A.R. Callaghan M.J. Tepper O.M. Bastidas N. Kleinman M.E. Capla J.M. Galiano R.D. Levine J.P. Gurtner G.C. Nat. Med. 2004; 10: 858-864Crossref PubMed Scopus (2213) Google Scholar). Over the course of 14 days, gene expression, blood flow, tissue oxygenation, mobilization, and recruitment of endothelial progenitors, tissue viability, and neovascularization were assessed. Mouse blood glucose was determined by testing 5 μl of tail vein blood using a One Touch Blood Glucose monitoring system (Life Scan, Johnson and Johnson). Tissue Perfusion, Oxygen Tension Measurements, and Viability—Blood flow and tissue oxygenation was determined as described previously on postoperative days 7 and 14 (9Ceradini D.J. Kulkarni A.R. Callaghan M.J. Tepper O.M. Bastidas N. Kleinman M.E. Capla J.M. Galiano R.D. Levine J.P. Gurtner G.C. Nat. Med. 2004; 10: 858-864Crossref PubMed Scopus (2213) Google Scholar). An optical fiber probe (100-μm radius, Oxford Optronix) matched with a thermocoupler was directly inserted into tissue, allowing for continuous temperature-compensated oxygen tension measurements (10 values/s). The probe was positioned at each of four reference points (points 1-4) for 60 s, generating an average of 600 values per trial. Perfusion was measured with color laser Doppler (Moor Instruments). Relative blood flow was calculated as previously described (9Ceradini D.J. Kulkarni A.R. Callaghan M.J. Tepper O.M. Bastidas N. Kleinman M.E. Capla J.M. Galiano R.D. Levine J.P. Gurtner G.C. Nat. Med. 2004; 10: 858-864Crossref PubMed Scopus (2213) Google Scholar). Gross tissue viability was determined by digital imaging techniques, and the percentage of clinically viable tissue was calculated using the Sigma-Scan software package. Immunohistochemistry—Samples from each ischemic tissue area (A-C) as well as a non-ischemic control (D) were harvested at regular intervals as indicated, and either snap-frozen in liquid nitrogen or embedded in paraffin. Capillary density in each area was determined from five nonconsecutive 10-μm sections using phosphatidylethanolamine-conjugated CD31 immunostaining (Pharmingen) as previously described (9Ceradini D.J. Kulkarni A.R. Callaghan M.J. Tepper O.M. Bastidas N. Kleinman M.E. Capla J.M. Galiano R.D. Levine J.P. Gurtner G.C. Nat. Med. 2004; 10: 858-864Crossref PubMed Scopus (2213) Google Scholar). Mobilization of Endothelial Progenitor Cells—On postoperative day 7, the number of circulating endothelial progenitor cells was determined by flow cytometry. Following red blood cell lysis with ammonium chloride, whole blood was stained with antibodies for flk-1-PE and CD11b-fluorescein isothiocyanate to identify endothelial progenitor cells as flk-1+/CD11b-, as described previously (15Tepper O.M. Capla J.M. Galiano R.D. Ceradini D.J. Callaghan M.J. Kleinman M.E. Gurtner G.C. Blood. 2005; 105: 1068-1077Crossref PubMed Scopus (393) Google Scholar). The mean percentage of circulating EPCs in each experimental group was calculated using four independent experiments. Bone Marrow Transplant Model—Following sublethal radiation (gray), a group of wild type FVB mice were transplanted with bone marrow harvested from FVB Tie2/LacZ transgenic mice, which feature the LacZ gene driven by the endothelial-specific Tie2 promoter. In this transplanted group, all newly formed endothelial cells derived from the bone marrow specifically express β-galactosidase, and can be used to readily identify neovascularization that proceeds via endothelial progenitor cells during postnatal vasculogenesis (15Tepper O.M. Capla J.M. Galiano R.D. Ceradini D.J. Callaghan M.J. Kleinman M.E. Gurtner G.C. Blood. 2005; 105: 1068-1077Crossref PubMed Scopus (393) Google Scholar). Following reconstitution over 4 weeks, diabetes induction, treatment with Mn-TBAP, and ischemic surgery proceeded as above. Intact tissue was harvested and stained with the β-galactosidase staining kit (Roche), followed by sectioning for histologic analysis. The number of β-galactosidase positive endothelial cells in each tissue area was enumerated in five nonconsecutive sections at ×200. Serum Enzyme-linked Immunosorbent Assay—Enzyme-linked immunosorbent assay was performed using the human/mouse SDF-1 and VEGF Quantikine kit according to the manufacturer's protocol (R&D). Cell culture supernatants were used following standardization of each sample by total protein content using the BCA Protein Assay Kit (Pierce). Results are representative of four independent experiments. Migration Assays—Migration was studied using a modified transwell assay. Lin-/Sca-1+/c-kit+ (5 × 104) were seeded onto Chemotx filters (5.7-mm, 8-μm pore, Neuro Probe) in endothelial basal medium, 0.5% fetal bovine serum. Recombinant human SDF-1β/pre-β cell growth-stimulating factor (Sigma) was then added to the lower chamber. Following the 6-h migration period, nonmigrating cells were completely wiped from the top surface of the membrane. Migrating cells were quantified using Kodak One-dimensional software. Results are indicative of four independent experiments. In Vitro Materials—Antibodies for HIF-1α (sc-10790), VEGF (sc-507), SDF-1 (sc-28876), Arnt1 (sc-17811), Gal4-DBD (sc-577), CXCR4 (sc-9046), β-actin (sc-10731, sc-8432), Flt-1 (sc-316), eNOS (sc-653), and Flk-1 (sc-504) were purchased from Santa Cruz Biotechnology. A monoclonal antibody to the major intracellular methylglyoxal-derived epitope, N-acetyl-N-(5-hydro-5-methyl)-4-imidazolone (MG), was previously generated and characterized by our laboratory (by M. B.). Primary fibroblasts were collected from dorsal skins of either diabetic or wild type mice and maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and antibiotics. Cells were treated with LG (5 mm glucose), HG (30 mm glucose), or HG for 3 days after a 24-h infection with adGLO1. 30 mml-glucose was used as a negative control. Hypoxia was induced for 18 h prior to analysis in a hypoxic chamber in which O2 was removed by flushing with 95% CO2 plus 5% N2 gas for 15 min. Primary bone marrow cells were collected from the tibia from either diabetic or wild type mice, and maintained in endothelial basal medium with supplements of hydrocortisone, endothelial growth factor, and 10% fetal calf serum on fibronectin/gelatin-coated dishes. The media was refreshed every day for 3 days, and then the cells were stimulated with human recombinant VEGF for 2 days as described previously (16Dimmeler S. Aicher A. Vasa M. Mildner-Rihm C. Adler K. Tiemann M. Rütten H. Fichtlscherer S. Martin H. Zeiher A. J. Clin. Investig. 2001; 108: 391-397Crossref PubMed Scopus (1141) Google Scholar). The adherent cells were characterized by washing with medium and incubating with 2.4 μg/ml 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbo-cyanine-labeled acetylated low deinsity lipoprotein (Dil-Ac-LDL; Biomedical Technologies Inc., MA) for 1 h. Cells were fixed in 2% paraformaldehyde and counterstained with fluorescein isothiocyanate-labeled lectin from Ulex europaeus (Sigma). Double positive staining cells were considered to be EPCs (16Dimmeler S. Aicher A. Vasa M. Mildner-Rihm C. Adler K. Tiemann M. Rütten H. Fichtlscherer S. Martin H. Zeiher A. J. Clin. Investig. 2001; 108: 391-397Crossref PubMed Scopus (1141) Google Scholar). Animal protocols used for these experiments were approved by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee. Plasmids—For construction of luciferase reporters, murine genomic DNA was purified from primary fibroblasts by DNeasy Tissue Kit (Qiagen), and the Sdf-1, Vegf, and Cxcr4 promoters were amplified by PCR. Restriction sites for XhoI and HindIII were introduced, and the fragments were then subcloned into pGL3 basic vector (Promega). For mapping of the HIF-1α-MG modification sites, the murine HIF-1α full-length cDNA or indicated deletions were generated by PCR methods, and the fragments were subcloned into pM vector (Clontech) by introduction of EcoRI and BamHI restriction sites. The indicated single point mutations were generated using the Site-directed Mutagenesis Kit from Promega (Madison, WI). All constructs were verified by sequencing. Detailed information regarding each construct is available upon request. Plasmid DNA was transfected with Lipofectamine™ Reagent (Invitrogen). Luciferase activity assays were carried out using the Dual-Luciferase™ Assay System (Promega) and transfection efficiencies were normalized using a cotransfected Renilla plasmid. Nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce). Protein concentration was measured by the Coomassie Protein Assay Kit (Pierce) using bovine serum albumin as a standard. Immunoprecipitation (IP) and Western Blotting—Cell lysates or nuclear extracts were cleared by preimmune IgG plus Protein A-agarose beads for 2 h, and the supernatants were immunoprecipitated by the indicated antibodies and a 50% slurry of Protein A-agarose beads overnight at 4 °C. After washing with buffer containing 50 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, and 0.5% deoxycholate with protease inhibitors, proteins were released and separated on 10% SDS-PAGE gels. After transfer to membranes, the blots were simultaneously incubated with the differentially labeled species-specific secondary antibodies, anti-rabbit IRDye™ 800CW (green) and anti-mouse Alexa 680 (red). Membranes were scanned and quantitated by the ODYSSEY Infrared Imaging System (LI-COR, NE) and normalized to β-actin. Chromatin Immunoprecipitation (ChIP)—Treated cells were cross-linked by 1% formaldehyde for 20 min, and terminated by addition of 0.1 m glycine. Cell lysates were sonicated and centrifuged. 500 μg of protein were pre-cleared by bovine serum albumin/salmon sperm DNA plus preimmune IgG and a slurry of Protein A-agarose beads as previously described (17Metivier R. Penot G. Hubner M.R. Reid G. Brand H. Kos M. Gannon F. Cell. 2003; 115: 751-763Abstract Full Text Full Text PDF PubMed Scopus (1248) Google Scholar). Immunoprecipitations were performed with the indicated antibodies, bovine serum albumin/salmon sperm DNA, and a 50% slurry of Protein A-agarose beads. Input and immunoprecipitated DNA were washed and eluted, then incubated 2 h at 42 °C in the presence of Proteinase K followed by 6 h at 65 °C to reverse the formaldehyde cross-linking. DNA fragments were recovered by phenol/chloroform extraction and ethanol precipitation. A 150-bp fragment from the murine Sdf-1, Vegf, Cxcr4, and eNos promoters were amplified by real-time PCR (qPCR). Reverse Transcriptase Reaction and Real-time Quantitative PCR—Total RNA from treated cells was extracted using the RNeasy Mini Kit (Qiagen), and the mRNA from ischemic flaps and normal skin was isolated using an RNeasy® Fibrous Tissue Midi Kit (Qiagen, CA). Either the entire peninsular skin flap (ischemic tissue) or a corresponding 1.25 × 2.5-cm segment of skin and soft tissue was harvested and immersed in 2 ml of RLT buffer with β-mercaptoethanol and immediately homogenized and disrupted with a Polytron PT 10-35 (Brinkmann). Total RNA was then isolated per the manufacturer's instructions. The mRNA was reverse transcribed by the SuperScript™ III First Strand Synthesis System (Invitrogen). Real-time quantitative PCR (qPCR) was run on a LightCycler (Roche Molecular Systems) with the LightCycler FastStart DNA Master SYBR Green I kit (Roche). PCR was performed by denaturing at 95 °C for 7 min, followed by 45 cycles of denaturation at 95 °C, annealing at 60 °C, and extension at 72 °C for 10 s, respectively. 1 μl of each cDNA was used to measure target genes, and the results were normalized to β-actin or GAPDH. Measurement of Reactive Oxygen Species—The levels of superoxide production in primitive bone marrow progenitor cells was determined preoperatively and 14 days following surgery. Bone marrow cells were flushed from the lower extremity long bones with phosphate-buffered saline, 10% fetal calf serum, and lineage depleted using MACS magnetic beads according to the manufacturer's protocol. Lineage-depleted bone marrow was then stained with dihydroethidium (Molecular Probes), c-kit, and Sca-1 antibodies and mean dihydroethidium fluorescence in the Lin-ckit+Sca-1+ population was determined by flow cytometry. Statistical Analysis—Results are given as mean ± S.D. All experiments were performed at least in triplicate. Data distribution was analyzed, and statistical differences for different treatments were evaluated by analysis of variance and the Tukey-Kramer test using SPSS 15 software. Decreasing Intracellular Superoxide Corrects Defective Ischemia-induced New Vessel Formation in Diabetic Mice—We first determined that the most general mechanism underlying our more specific hypothesis, diabetes-induced superoxide production, played a central role in the diabetes defect in ischemia-induced neovascularization, using a previously characterized murine model of soft tissue ischemia. Diabetic mice exhibited a significant impairment in ischemia-induced neovascularization compared with non-diabetic controls, resulting in necrosis of the ischemic soft tissue (Fig. 1a, upper panels). We evaluated the possible role of diabetes-induced intracellular superoxide formation in defective ischemia-induced neovascularization by using two complementary murine models: diabetic transgenic mice overexpressing manganese superoxide dismutase, the mitochondrial isoform of this enzyme, and diabetic WT mice treated with a cell-permeable superoxide dismutase/catalase mimetic, Mn-TBAP. There was no effect of either the Mn-SOD transgene or Mn-TBAP treatment on blood glucose levels in diabetic mice (WT diabetic = 474 + 23 mg/dl, Mn-SOD diabetic = 439 + 15 mg/dl, Mn-TBAP diabetic = 508 + 15 mg/dl). In both of these diabetic models, the ischemic soft tissue healed normally (Fig. 1a, lower panels), and the 70% reduction in ischemic tissue capillary density induced by diabetes was prevented (Fig. 1b). Similarly, the 50% reduction in perfusion of the ischemic tissue (Fig. 1c) and the resultant decrease in oxygen delivery to the ischemic tissue were also normalized (Fig. 1d). Thus, diabetes-induced overproduction of superoxide plays a central role in the pathogenesis of impaired ischemia-induced neovascularization in this disease. Because humans with diabetes have dramatically reduced levels of circulating EPCs (6Tepper O. Galiano R. Capla J. Kalka C. Gagne P. Jacobowitz G. Levine J. Gurtner G. Circulation. 2002; 106: 2781-2786Crossref PubMed Scopus (1292) Google Scholar), we next determined the level of circulating EPCs mobilized from the bone marrow following an ischemic injury in diabetic mice, diabetic Mn-SOD transgenic mice, and diabetic mice treated with Mn-TBAP (Fig. 1e). Diabetes dramatically reduced the level of mobilized EPCs, to only 12% of levels observed in non-diabetic mice. Notably, this decrease was not attributable to fewer progenitor cells in the bone marrow compartment of diabetic mice, as the number of Lin-/Sca-1+/c-Kit+ progenitor cells (18Asahara T. Murohara T. Sullivan A. Silver M. van der Zee R. Li T. Witzenbichler B. Schatteman G. Isner J. Science. 1997; 275: 964-967Crossref PubMed Scopus (7721) Google Scholar, 19Awad O. Jiao C. Ma N. Dunnwald M. Schatteman G.C. Stem Cells. 2005; 23: 575-583Crossref PubMed Scopus (82) Google Scholar) from both non-diabetic and diabetic mice was not statistically different. Transgenic expression of Mn-SOD in diabetic mice increased the level of mobilized EPCs to 63% of non-diabetic levels, whereas treatment of diabetics with a SOD mimetic increased the number to 58% of non-diabetic levels. To confirm that the diabetes-induced reduction in mobilized EPCs resulted from a failure to recruit bone marrow-derived EPCs to the site of ischemia, we utilized a transplant model in which bone marrow harvested from Tie-2/LacZ mice was transplanted into wild type mice (15Tepper O.M. Capla J.M. Galiano R.D. Ceradini D.J. Callaghan M.J. Kleinman M.E. Gurtner G.C. Blood. 2005; 105: 1068-1077Crossref PubMed Scopus (393) Google Scholar, 20Suri C. Jones P.F. Patan S. Bartunkova S. Maisonpierre P.C. Davis S. Sato T.N. Yancopoulos G.D. Cell. 1996; 87: 1171-1180Abstract Full Text Full Text PDF PubMed Scopus (2395) Google Scholar). In this model, endothelial cells derived from bone marrow progenitor cells during neovascularization stain β-galactosidase positive. Because the effect of Mn-SOD overexpression and Mn-TBAP treatment were identical (Fig. 1, a-e), in this experiment and mouse experiments that followed, only Mn-TBAP-treated diabetic mice were used. Following reconstitution and induction of diabetes, animals underwent ischemic surgery. Prior to surgery, there was no difference in capillary density of diabetics compared with non-diabetics, nor in transplanted diabetics compared with transplanted non-diabetics. However, after surgery, diabetic animals had a greater than 50% reduction in the number of lacZ positive cells recruited to the ischemic tissue (Fig. 1f). In contrast, the number of lacZ positive cells recruited in diabetic animals treated with Mn-TBAP was increased 2.2-fold over untreated diabetics, to 77% that observed in non-diabetic mice. The observed failure of diabetic animals to mobilize and recruit reparative progenitor cells in a peripheral wound model reflects both a failure of the ischemic tissue to generate appropriate levels of at least one chemokine signal, SDF-1α, and a failure of progenitor cells to respond to ischemia-specific signals due in part to a decrease in eNOS activation (5Gallagher K.A. Liu Z.J. Xiao M. Chen H. Goldstein L.J. Buerk D.G. Nedeau A. Thom S.R. Velazquez O.C. J. Clin. Investig. 2007; 117: 1249-1259Crossref PubMed Scopus (555) Google Scholar). In our ischemic flap model, diabetic mice showed a significantly blunted up-regulation of SDF-1 mRNA in the ischemic skin flap compared with skin from non-diabetic mice (Fig. 2a). Treatment of d
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