Topical Vascular Endothelial Growth Factor Accelerates Diabetic Wound Healing through Increased Angiogenesis and by Mobilizing and Recruiting Bone Marrow-Derived Cells
2004; Elsevier BV; Volume: 164; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)63754-6
ISSN1525-2191
AutoresRobert D. Galiano, Oren M. Tepper, Catherine R. Pelo, Kirit A. Bhatt, Matthew J. Callaghan, Nicholas Bastidas, Stuart Bunting, Hope Steinmetz, Geoffrey C. Gurtner,
Tópico(s)Tendon Structure and Treatment
ResumoDiminished production of vascular endothelial growth factor (VEGF) and decreased angiogenesis are thought to contribute to impaired tissue repair in diabetic patients. We examined whether recombinant human VEGF165 protein would reverse the impaired wound healing phenotype in genetically diabetic mice. Paired full-thickness skin wounds on the dorsum of db/db mice received 20 μg of VEGF every other day for five doses to one wound and vehicle (phosphate-buffered saline) to the other. We demonstrate significantly accelerated repair in VEGF-treated wounds with an average time to resurfacing of 12 days versus 25 days in untreated mice. VEGF-treated wounds were characterized by an early leaky, malformed vasculature followed by abundant granulation tissue deposition. The VEGF-treated wounds demonstrated increased epithelialization, increased matrix deposition, and enhanced cellular proliferation, as assessed by uptake of 5-bromodeoxyuridine. Analysis of gene expression by real-time reverse transcriptase-polymerase chain reaction demonstrates a significant up-regulation of platelet-derived growth factor-B and fibroblast growth factor-2 in VEGF-treated wounds, which corresponds with the increased granulation tissue in these wounds. These experiments also demonstrated an increase in the rate of repair of the contralateral phosphate-buffered saline-treated wound when compared to wounds in diabetic mice never exposed to VEGF (18 days versus 25 days), suggesting that topical VEGF had a systemic effect. We observed increased numbers of circulating VEGFR2+/CD11b− cells in the VEGF-treated mice by fluorescence-activated cell sorting analysis, which likely represent an endothelial precursor population. In diabetic mice with bone marrow replaced by that of tie2/lacZ mice we demonstrate that the local recruitment of bone marrow-derived endothelial lineage lacZ+ cells was augmented by topical VEGF. We conclude that topical VEGF is able to improve wound healing by locally up-regulating growth factors important for tissue repair and by systemically mobilizing bone marrow-derived cells, including a population that contributes to blood vessel formation, and recruiting these cells to the local wound environment where they are able to accelerate repair. Thus, VEGF therapy may be useful in the treatment of diabetic complications characterized by impaired neovascularization. Diminished production of vascular endothelial growth factor (VEGF) and decreased angiogenesis are thought to contribute to impaired tissue repair in diabetic patients. We examined whether recombinant human VEGF165 protein would reverse the impaired wound healing phenotype in genetically diabetic mice. Paired full-thickness skin wounds on the dorsum of db/db mice received 20 μg of VEGF every other day for five doses to one wound and vehicle (phosphate-buffered saline) to the other. We demonstrate significantly accelerated repair in VEGF-treated wounds with an average time to resurfacing of 12 days versus 25 days in untreated mice. VEGF-treated wounds were characterized by an early leaky, malformed vasculature followed by abundant granulation tissue deposition. The VEGF-treated wounds demonstrated increased epithelialization, increased matrix deposition, and enhanced cellular proliferation, as assessed by uptake of 5-bromodeoxyuridine. Analysis of gene expression by real-time reverse transcriptase-polymerase chain reaction demonstrates a significant up-regulation of platelet-derived growth factor-B and fibroblast growth factor-2 in VEGF-treated wounds, which corresponds with the increased granulation tissue in these wounds. These experiments also demonstrated an increase in the rate of repair of the contralateral phosphate-buffered saline-treated wound when compared to wounds in diabetic mice never exposed to VEGF (18 days versus 25 days), suggesting that topical VEGF had a systemic effect. We observed increased numbers of circulating VEGFR2+/CD11b− cells in the VEGF-treated mice by fluorescence-activated cell sorting analysis, which likely represent an endothelial precursor population. In diabetic mice with bone marrow replaced by that of tie2/lacZ mice we demonstrate that the local recruitment of bone marrow-derived endothelial lineage lacZ+ cells was augmented by topical VEGF. We conclude that topical VEGF is able to improve wound healing by locally up-regulating growth factors important for tissue repair and by systemically mobilizing bone marrow-derived cells, including a population that contributes to blood vessel formation, and recruiting these cells to the local wound environment where they are able to accelerate repair. Thus, VEGF therapy may be useful in the treatment of diabetic complications characterized by impaired neovascularization. Diverse processes such as embryonic development, tumor growth, and tissue repair are linked by the absolute requirement for a vascular bed to deliver nutrients and oxygen to metabolically active cells. The clinical importance of controlled vascular growth is illustrated by disease states resulting from imbalances in blood vessel formation, such as diabetes mellitus. Vasculopathies associated with diabetes include excessive blood vessel formation (eg, retinopathy, glomerular nephropathy) and accelerated atherosclerosis leading to coronary artery disease, peripheral vascular disease, and cerebrovascular disease.1Martin A Komada MR Sane DC Abnormal angiogenesis in diabetes mellitus.Med Res Rev. 2003; 23: 117-145Crossref PubMed Scopus (395) Google Scholar Microvascular dysfunction is also believed to contribute to morbidity in diabetes by impairing collateral formation, resulting in poor outcomes after vascular occlusive events.2The Diabetes Control and Complications Trial Study Group The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus.N Engl J Med. 1993; 329: 977-986Crossref PubMed Scopus (23169) Google Scholar, 3Abaci A Oguzhan A Kahraman S Eryol NK Unal S Arinc H Ergin A Effect of diabetes mellitus on formation of coronary collateral vessels.Circulation. 1999; 99: 2239-2242Crossref PubMed Scopus (581) Google Scholar Diabetes impairs numerous components of wound healing, including hemostasis and inflammation, matrix deposition, and angiogenesis. These impairments are present in a wide variety of tissues including myocardium, skeletal muscle, nerve, and skin. Cutaneous wounds in diabetics have been shown to have altered blood flow, impaired neutrophil anti-microbial activity, and a dysfunctional inflammatory state associated with abnormal chemokine expression.4Wetzler C Kampfer H Stallmeyer B Pfeilschifter J Frank S Large and sustained induction of chemokines during impaired wound healing in the genetically diabetic mouse: prolonged persistence of neutrophils and macrophages during the late phase of repair.J Invest Dermatol. 2000; 115: 245-253Crossref PubMed Scopus (454) Google Scholar A number of growth factors essential for wound healing, including FGF-2 and platelet-derived growth factor (PDGF)-B, have also been found to be reduced in experimental diabetic wounds.5Beer HD Longaker MT Werner S Reduced expression of PDGF and PDGF receptors during impaired wound healing.J Invest Dermatol. 1997; 109: 132-138Crossref PubMed Scopus (202) Google Scholar, 6Werner S Breeden M Hubner G Greenhalgh DG Longaker MT Induction of keratinocyte growth factor expression is reduced and delayed during wound healing in the genetically diabetic mouse.J Invest Dermatol. 1994; 103: 469-473Abstract Full Text PDF PubMed Scopus (234) Google Scholar, 7Bitar MS Labbad ZN Transforming growth factor-beta and insulin-like growth factor-I in relation to diabetes-induced impairment of wound healing.J Surg Res. 1996; 61: 113-119Abstract Full Text PDF PubMed Scopus (110) Google Scholar, 8Brown DL Kane CD Chernausek SD Greenhalgh DG Differential expression and localization of insulin-like growth factors I and II in cutaneous wounds of diabetic and nondiabetic mice.Am J Pathol. 1997; 151: 715-724PubMed Google Scholar Vascular endothelial growth factor (VEGF)-A, a member of a family of growth factors with essential roles in vascular and lymphatic growth and patterning, has been shown to be deficient in experimental and clinical diabetic wounds.9Frank S Hubner G Breier G Longaker MT Greenhalgh DG Werner S Regulation of vascular endothelial growth factor expression in cultured keratinocytes. Implications for normal and impaired wound healing.J Biol Chem. 1995; 270: 12607-12613Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar VEGF acts through at least two receptors, expressed primarily on endothelial cells, along with other vascular cytokines [fibroblast growth factor (FGF), PDGF, and the angiopoietins] to induce and maintain the vasculature.10Carmeliet P Mechanisms of angiogenesis and arteriogenesis.Nat Med. 2000; 6: 389-395Crossref PubMed Scopus (3536) Google Scholar VEGF-A (referred subsequently as simply VEGF) is the prototype member of this family and has been shown to be absolutely essential for vascular development.11Carmeliet P Ferreira V Breier G Pollefeyt S Kieckens L Gertsenstein M Fahrig M Vandenhoeck A Harpal K Eberhardt C Declercq C Pawling J Moons L Collen D Risau W Nagy A Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.Nature. 1996; 380: 435-439Crossref PubMed Scopus (3498) Google Scholar, 12Gerber HP Hillan KJ Ryan AM Kowalski J Keller GA Rangell L Wright BD Radtke F Aguet M Ferrara N VEGF is required for growth and survival in neonatal mice.Development. 1999; 126: 1149-1159Crossref PubMed Google Scholar VEGF facilitates tissue repair by both increasing vascular permeability, allowing the efflux of inflammatory cells into the site of injury, and increasing the migration and proliferation of pre-existing endothelial cells. However, exogenous administration of VEGF results in leaky, malformed vessels which has raised concerns regarding its therapeutic usefulness.13Lee RJ Springer ML Blanco-Bose WE Shaw R Ursell PC Blau HM VEGF gene delivery to myocardium: deleterious effects of unregulated expression.Circulation. 2000; 102: 898-901Crossref PubMed Scopus (634) Google Scholar, 14Drake CJ Little CD Exogenous vascular endothelial growth factor induces malformed and hyperfused vessels during embryonic neovascularization.Proc Natl Acad Sci USA. 1995; 92: 7657-7661Crossref PubMed Scopus (297) Google Scholar Recent studies have established that angiogenesis is not the sole mechanism by which new vessels are formed. It is now apparent that bone marrow-derived cells, including endothelial progenitor cells (EPCs), are mobilized in response to trauma or ischemia and are able to contribute to tissue repair and new blood vessel formation.15Asahara T Murohara T Sullivan A Silver M van der Zee R Li T Witzenbichler B Schatteman G Isner JM Isolation of putative progenitor endothelial cells for angiogenesis.Science. 1997; 275: 964-967Crossref PubMed Scopus (7834) Google Scholar, 16Gill M Dias S Hattori K Rivera ML Hicklin D Witte L Girardi L Yurt R Himel H Rafii S Vascular trauma induces rapid but transient mobilization of VEGFR2(+)AC133(+) endothelial precursor cells.Circ Res. 2001; 88: 167-174Crossref PubMed Scopus (750) Google Scholar, 17Peichev M Naiyer AJ Pereira D Zhu Z Lane WJ Williams M Oz MC Hicklin DJ Witte L Moore MA Rafii S Expression of VEGFR-2 and AC133 by circulating human CD34(+) cells identifies a population of functional endothelial precursors.Blood. 2000; 95: 952-958Crossref PubMed Google Scholar, 18Takahashi T Kalka C Masuda H Chen D Silver M Kearney M Magner M Isner JM Asahara T Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization.Nat Med. 1999; 5: 434-438Crossref PubMed Scopus (47) Google Scholar The development of blood vessels from blood-borne endothelial precursors, termed vasculogenesis, was previously thought to be restricted to embryonic development, but is now accepted to play a role in postnatal processes including tissue repair.18Takahashi T Kalka C Masuda H Chen D Silver M Kearney M Magner M Isner JM Asahara T Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization.Nat Med. 1999; 5: 434-438Crossref PubMed Scopus (47) Google Scholar, 19Crosby JR Kaminski WE Schatteman G Martin PJ Raines EW Seifert RA Bowen-Pope DF Endothelial cells of hematopoietic origin make a significant contribution to adult blood vessel formation.Circ Res. 2000; 87: 728-730Crossref PubMed Scopus (460) Google Scholar, 20Asahara T Takahashi T Masuda H Kalka C Chen D Iwaguro H Inai Y Silver M Isner JM VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells.EMBO J. 1999; 18: 3964-3972Crossref PubMed Scopus (1686) Google Scholar We have recently demonstrated that EPCs isolated from diabetic patients are functionally impaired,21Tepper OM Galiano RD Capla JM Kalka C Gagne PJ Jacobowitz GR Levine JP Gurtner GC Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures.Circulation. 2002; 106: 2781-2786Crossref PubMed Scopus (1311) Google Scholar but the contribution of EPC-mediated vasculogenesis on impaired diabetic wound healing remains poorly understood. In this study, we demonstrate that topical VEGF accelerates wound healing in diabetic mice to nearly the rate observed in nondiabetic mice. Unexpectedly, we found that VEGF also accelerated healing in untreated (control) wounds. This correlated with the systemic mobilization and recruitment of putative endothelial precursors from the bone marrow. Despite the disordered vasculature induced by VEGF administration, repair was accelerated and persisted after cessation of VEGF treatment, suggesting that these vessels are functional during wound healing. This may be explained by an up-regulation of PDGF and basic FGF present in VEGF-treated diabetic wounds. This supports the rational clinical use of VEGF in the treatment of diabetic vascular complications. Because the mechanism partly involves the mobilization and recruitment of bone marrow-derived progenitors, VEGF may be useful for injuries not amenable to topical therapy. This study was approved by the New York University Medical Center Animal Care Committee. All mice were obtained from Jackson Laboratories (Bar Harbor, ME) and housed in an approved animal care center with 12-hour light cycles and provided standard rodent chow and water ad libitum. For most experiments, the db/db mouse (BKS.Cg-m +/+ Leprdb, Jackson Laboratories stock no. 000642) was used. It is a model of type II diabetes with documented impairments in wound healing.22Tsuboi R Shi CM Rifkin DB Ogawa H A wound healing model using healing-impaired diabetic mice.J Dermatol. 1992; 19: 673-675Crossref PubMed Scopus (64) Google Scholar Female mice 10 to 12 weeks of age were used. At this age the mice are all diabetic, with blood glucose levels >350 g/dL. For bone marrow transplantation experiments, the FVB/NJ strain of mice was used (Jackson Laboratories stock no. 01800) along with tie2/lacZ transgenic donor mice of the same genetic background [strain FVB/N-TgN(TIE2-lacZ)182Sato, stock no. 002856]. A novel model of wound analysis was used (Galiano et al, submitted for publication). Under sterile conditions, paired 6-mm circular, full-thickness wounds were made on the dorsal skin of the mice after depilation. A donut-shaped 12-mm splint made of 0.5-mm-thick silicone sheeting (Grace Bio-Labs, Bend, OR) was then placed around the wounds and adhered to the skin with cyanoacrylate glue and interrupted 6-0 nylon sutures. Recombinant human VEGF165 protein (supplied by Genentech, South San Francisco, CA) or phosphate-buffered saline (PBS) vehicle was placed into the wound bed at a dose of 20 μg per wound. A transparent sterile occlusive dressing was then placed over the wound and the splint. The dressing and the splint were maintained on the wound throughout the entire course of the experiments. VEGF protein was applied sterilely to the wounds on days 0, 2, 4, 6, and 8 after wounding. Wounds were covered with an occlusive dressing after VEGF or PBS administration. Digital photographs were taken every 2 days. Time to closure was defined as the time until the wound bed was completely resurfaced with new tissue, and was determined by a blinded observer. Wound area was calculated as a percent area of the original wound size; because the splint has a constant area, it was used to normalize the wound sizes, even at different focal distances. Three to five mice were analyzed at each time point. Throughout this article, wounds were classified into three groups. The first group was the VEGF-treated wound group, and consisted of wounds that received topical VEGF using the dosing regimen given above. The contralateral PBS-treated wound group consisted of the paired wounds in these VEGF-treated mice; these wounds received PBS instead of VEGF. The final group consisted of vehicle-treated wounds in a separate group of mice. These mice did not receive VEGF in either wound, and are referred to as control wounds in mice not treated with VEGF. At time intervals ranging from 5 days through 21 days after wounding, wounds were excised with a 2-mm rim of surrounding tissue and placed either in Bouin's fixative overnight or snap-frozen in liquid nitrogen. The wounds were then bisected down the center, and 8-μm sections were processed for routine hematoxylin and eosin (H&E) staining. Digital imaging software (SigmaScan; SPSS Science, Chicago, IL) was used to measure granulation tissue area and epithelial gap histomorphometrically. Granulation tissue area is measured in pixels, and epithelialization is presented as the gap between the leading epithelial edges, as measured from the wound edges. This distance (epithelial gap) is presented in pixels. For these measurements, H&E-stained wound sections were analyzed at ×40 magnification. Three mice were analyzed at each time point. A subset of animals was injected with 100 mg of bromodeoxyuridine (BrdU) (Sigma Chemicals, St. Louis, MO) intraperitoneally 3 hours before sacrifice and wound harvest. BrdU incorporation into proliferating cells was detected with a biotinylated anti-BrdU antibody (Zymed, South Francisco, CA) after brief trypsin digestion of the paraffin sections. The number of proliferating cells was determined by manually scoring the number of positive staining cells at ×200 magnification. A total of six random fields from either the leading wound margin or the wound center (for closed wounds) were counted in each wound by a blinded observer. Wounds were harvested on day 12 and from unwounded skin from control and experimental mice by excising the wound and ∼1 mm of surrounding skin. The samples were immediately homogenized and purified using the RNeasy kit (Qiagen, Valencia, CA). Purified RNA was quantified by absorbance spectroscopy at 260/280 nm. After extraction and purification, RNA was converted to cDNA using the RNA PCR Core kit (Applied Biosystems, Foster City, CA) containing MuLV reverse transcriptase and stored at −20°C until use. Complementary DNA of murine GAPDH, PDGF-B, and FGF-2 was amplified for use in real-time PCR standardization by PCR of mouse genomic DNA, and purified using PCR product purification columns (Qiagen, Valencia, CA). The specificity of the primers used and product size was confirmed by electrophoresis on a 2% agarose gel. The number of copies of cDNA/μl of purified template was determined by UV spectroscopy. Serial dilutions of each purified product were made and standard curves encompassing from 10−8 to 102 copies were obtained on a real-time PCR cycler (Cepheid Smartcycler, Sunnyvale, CA) using Platinum Sybr Green Supermix as per the manufacturer's instructions. The amplification protocol for each gene was as follows: an initial denaturation step at 95°C for 2 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C annealing for 30 seconds, and 72°C extension for 30 seconds. The data acquisition was performed at 80°C for 10 seconds (optics ON), and a melting curve was designed at 0.2-degree increments. This four-step protocol was chosen to minimize primer-dimer formation. For each experiment, a negative control was included by placing water in place of cDNA. The following primers were used: GAPDH forward, (5′-ACCACAGTCCATGCCATCAC-3′) and reverse, (5′-TCCACCACCCTGTTGCTGTA-3′); connective tissue growth factor forward, (5′-TATCCCACCAAAGTGAGAACG-3′) and reverse, (5′-TGGAATCAGAATGGTCAGAGC-3′); insulin-like growth factor-I forward, (5′-ATGTACTGTGCCCCACTGAAG-3′) and reverse, (5′-GTGTTTCGATGTTTTGCAGGT-3′); transforming growth factor-β1 forward, (5′-AACAATTCCTGGCGTTACCTT-3′) and reverse, (5′-TTTGCTGTCACAAGAGCAGTG-3′); FGF-2 forward, (5′-GCTGCTGGCTTCTAAGTGTGT-3′) and reverse, (5′-CCAACTGGAGTATTTCCGTGA-3′); PDGF-B forward, (5′-GGTCAAACCTCTGAGGAAAGG-3′) and reverse, (5′-AGTACCATGGGCTCATTTCTGA-3′). After acquisition of a standard curve for each gene, gene expression was then determined by real-time RT-PCR and the absolute number of gene copies was quantified using each representative standard curve. Frozen sections were placed on glass slides, dried at 50°C for 1 hour and then rinsed in PBS. Cytokeratin 6 was detected with an anti-mouse keratin-specific antibody (MK6; Covance, Berkeley, CA) at a 1:500 dilution. The secondary antibody was an Alexa-Fluor 488-linked anti-mouse IgG antibody (Molecular Probes, Eugene, OR) used at a 1:100 dilution. Murine CD31 was detected with a rat monoclonal antibody (clone MEC 13.3; BD Biosciences, San Diego, CA). The secondary antibody was an Alexa-Fluor 594-linked anti-rat IgG antibody (Molecular Probes) used at a 1:100 dilution. All blocking steps were performed with SuperBlock reagent (Biogenex, San Ramon, CA). Processed sections were mounted in mounting media (VectaShield; Vector Laboratories, Burlingame, CA) and viewed on an Olympus BX51 epifluorescent microscope. For quantification of CD31-positive cells, wound edges or wound centers were analyzed under ×200 magnification, and total positive cells per high-power field (hpf) were counted by a blinded observer. The keratin staining was used to delineate the wound edges under immunofluorescence. For the in vivo measurement of wound vascular density the rat monoclonal anti-CD31 antibody used above was radiolabeled with 125I (Dupont NEN, Boston, MA); a nonspecific isotype control antibody (rat anti-mouse CD 35, clone 8C12; Pharmingen, San Diego, CA) was radiolabeled with 131I/125I (Dupont NEN). The control antibody was used to account for any nonspecific antibody binding, vascular leakage, or any blood left in the tissue. All antibodies were iodinated using the iodogen method in a ratio of 1 μg of antibody to 1 μCi of either 125I or 131I as described.23Eppihimer MJ Russell J Langley R Vallien G Anderson DC Granger DN Differential expression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) in murine tissues.Microcirculation. 1998; 5: 179-188Crossref PubMed Google Scholar Mice were anesthetized and the left carotid artery and right jugular vein were isolated and cannulated. The animals were heparinized with 40 U of sodium heparin (Elkins-Sinn, Cherry Hill, NJ). To measure CD31 antibody binding, a mixture of 125I-CD31 monoclonal antibody (10 μg) and 131I-nonbinding monoclonal antibody (equivalent to 500,000 cpm) was diluted with PBS to a volume of 200 μl. Initial radioactivity was counted in a 2-μl sample using a Wallac Wizard 3” gamma counter (model 1480; Perkin Elmer, Gaithersburg, MD). Thirty μg of unlabeled CD31 monoclonal antibody was added to the solution. The mixture was injected through the jugular vein catheter and allowed to circulate for 5 minutes. At the end of 5 minutes a blood sample was obtained from the carotid catheter to measure the circulating radiolabeled antibody level. The animal was then exsanguinated by perfusion with bicarbonate-buffered saline through the jugular catheter with simultaneous blood withdrawal from the carotid catheter. This was followed by perfusion of bicarbonate-buffered saline through the carotid catheter (15 ml) after severing the inferior vena cava at the thoracic level. Wounds were collected, weighed, and radioactivity measured with a gamma counter. Results are presented as μg antibody per g of tissue. Mice were sedated and positioned such that the wound was 25 cm from the reflection mirror. A 1 cm by 1 cm square of tissue was imaged in triplicate, with the wound centered in the square, using a MoorLDI laser Doppler imager (Moor Instruments Limited, Devon, UK). Flow is reported in relative units. In a subset of mice, peripheral blood (500 μl per animal) was drawn before sacrifice by intracardiac puncture. Mononuclear cells were separated by density centrifugation with Histopaque 1083 (Sigma) and plated on fibronectin-coated four-well glass slides. This EPC culture assay used has been described elsewhere.20Asahara T Takahashi T Masuda H Kalka C Chen D Iwaguro H Inai Y Silver M Isner JM VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells.EMBO J. 1999; 18: 3964-3972Crossref PubMed Scopus (1686) Google Scholar In brief, mononuclear cells were cultured in media supplemented with endothelial cell growth medium microvascular SingleQuots (EGM-2-MV; Cambrex BioProducts, East Rutherford, NJ). After 4 days, fluorescence staining with fluorescein isothiocyanate-conjugated BS1-lectin (Vector Laboratories) and the uptake of DiI-labeled acetylated LDL (ac-LDL) (Biomedical Technologies, Inc., Stoughton, MA) were used to detect EPCs (dual-staining cells). This has previously been shown to be representative of circulating EPCs.15Asahara T Murohara T Sullivan A Silver M van der Zee R Li T Witzenbichler B Schatteman G Isner JM Isolation of putative progenitor endothelial cells for angiogenesis.Science. 1997; 275: 964-967Crossref PubMed Scopus (7834) Google Scholar, 24Asahara T Masuda H Takahashi T Kalka C Pastore C Silver M Kearne M Magner M Isner JM Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization.Circ Res. 1999; 85: 221-228Crossref PubMed Scopus (2958) Google Scholar The number of dual-staining cells (EPCs) per hpf was determined by an independent reviewer analyzing 10 random fields. EPCs from three mice were analyzed for each group. To further quantify the effects of VEGF on mobilizing EPCS to the circulation, we performed FACS analysis on freshly isolated peripheral blood mononuclear cells. Peripheral blood mononuclear cells from VEGF-treated and nontreated animals were stained with a phycoerythrin-labeled anti-flk-1/VEGFR-2 (clone Avas 12α1, BD Biosciences) antibody (2 μg/ml) to delineate those circulating cells expressing VEGF receptor-2. Two other cell surface markers were used to further define the circulating endothelial progenitor population. One group of samples was co-incubated with a fluorescein isothiocyanate-labeled CD31 antibody (clone MEC 13.3, BD Biosciences) at 5 μg/ml. Because some monocytes also express VEGFR-2, we excluded cells of the myeloid/monocyte lineage by also staining another group of samples with a fluorescein isothiocyanate-labeled CD11b (clone M1/70, BD Biosciences) antibody (0.5 μg/ml). Circulating VEGFR-2+/CD11b− cells have previously been shown to represent an EPC population.16Gill M Dias S Hattori K Rivera ML Hicklin D Witte L Girardi L Yurt R Himel H Rafii S Vascular trauma induces rapid but transient mobilization of VEGFR2(+)AC133(+) endothelial precursor cells.Circ Res. 2001; 88: 167-174Crossref PubMed Scopus (750) Google Scholar, 25Hattori K Dias S Heissig B Hackett NR Lyden D Tateno M Hicklin DJ Zhu Z Witte L Crystal RG Moore MA Rafii S Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells.J Exp Med. 2001; 193: 1005-1014Crossref PubMed Scopus (610) Google Scholar Quantitative analysis was performed on a FACStar flow cytometer (BD Biosciences). Each experiment was repeated with peripheral blood mononuclear cells from four different mice. Bone marrow cells were collected from the tibia and femurs of transgenic mice that express lacZ under the control of the endothelial-specific tie2 promoter (tie2/lacZ mice) (Jackson Laboratories). Bone marrow cells were purified by density centrifugation (Histopaque 1083) and 2 × 106 cells were systemically transplanted to FVB/NJ wild-type mice that had been lethally irradiated (12 Gy). Four weeks after bone marrow transplantation, diabetes was induced in the transplanted mice by administering intraperitoneal injections of streptozotocin (40 mg/kg) (Sigma) for 5 consecutive days. Two weeks after streptozotocin treatment, blood glucose levels were assessed with a glucometer (Roche Bioproducts, Indianapolis IN) to confirm successful induction of diabetes by streptozotocin. Only mice with blood glucose levels >300 mg/dL were used for these experiments. Six weeks later, diabetic animals were wounded and treated with VEGF in an identical manner as described above. After surgery, the wounds from four different mice were harvested at days 14 and 21. They were fixed in 1% paraformaldehyde/0.5% gluteraldehyde in PBS for 4 hours, then washed (in PBS with 2 mmol/L MgCl2, 5 mmol/L ethylenediaminetetraacetic acid, 0.001% sodium deoxycholate, and 0.02% Nonidet P-40) and stained for β-galactosidase activity overnight at 37°C (β-gal staining kit, Roche). Before further processing, each skin sample was placed under a dissecting microscope to visualize and document foci of lacZ-positive cells. A blinded observer quantified the nu
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