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• W1520623323 abstract "Reduced microcirculation and diminished expression of growth factors contribute to wound healing impairment in diabetes. Placenta growth factor (PlGF), an angiogenic mediator promoting pathophysiological neovascularization, is expressed during cutaneous wound healing and improves wound closure by enhancing angiogenesis. By using streptozotocin-induced diabetic mice, we here demonstrate that PlGF induction is strongly reduced in diabetic wounds. Diabetic transgenic mice overexpressing PlGF in the skin displayed accelerated wound closure compared with diabetic wild-type littermates. Moreover, diabetic wound treatment with an adenovirus vector expressing the human PlGF gene (AdCMV.PlGF) significantly accelerated the healing process compared with wounds treated with a control vector. The analysis of treated wounds showed that PlGF gene transfer improved granulation tissue formation, maturation, and vascularization, as well as monocytes/macrophages local recruitment. Platelet-derived growth factor, fibroblast growth factor-2, and vascular endothelial growth factor mRNA levels were increased in AdCMV.PlGF-treated wounds, possibly enhancing PlGF-mediated effects. Finally, PlGF treatment stimulated cultured dermal fibroblast migration, pointing to a direct role of PlGF in accelerating granulation tissue maturation. In conclusion, our data indicate that reduced PlGF expression contributes to impaired wound healing in diabetes and that PlGF gene transfer to diabetic wounds exerts therapeutic activity by promoting different aspects of the repair process. Reduced microcirculation and diminished expression of growth factors contribute to wound healing impairment in diabetes. Placenta growth factor (PlGF), an angiogenic mediator promoting pathophysiological neovascularization, is expressed during cutaneous wound healing and improves wound closure by enhancing angiogenesis. By using streptozotocin-induced diabetic mice, we here demonstrate that PlGF induction is strongly reduced in diabetic wounds. Diabetic transgenic mice overexpressing PlGF in the skin displayed accelerated wound closure compared with diabetic wild-type littermates. Moreover, diabetic wound treatment with an adenovirus vector expressing the human PlGF gene (AdCMV.PlGF) significantly accelerated the healing process compared with wounds treated with a control vector. The analysis of treated wounds showed that PlGF gene transfer improved granulation tissue formation, maturation, and vascularization, as well as monocytes/macrophages local recruitment. Platelet-derived growth factor, fibroblast growth factor-2, and vascular endothelial growth factor mRNA levels were increased in AdCMV.PlGF-treated wounds, possibly enhancing PlGF-mediated effects. Finally, PlGF treatment stimulated cultured dermal fibroblast migration, pointing to a direct role of PlGF in accelerating granulation tissue maturation. In conclusion, our data indicate that reduced PlGF expression contributes to impaired wound healing in diabetes and that PlGF gene transfer to diabetic wounds exerts therapeutic activity by promoting different aspects of the repair process. Wound healing is a highly dynamic and complex process that can be divided into three main and partially overlapping phases: the early inflammatory, the intermediate proliferative, and the late tissue remodeling phases.1Martin P Wound healing-aiming for perfect skin regeneration.Science. 1997; 276: 75-81Crossref PubMed Scopus (3626) Google Scholar In diabetes, wound healing is defective, often resulting in ulcer formation. Numerous aspects of the wound repair process are altered in diabetes, including dysfunction in the inflammatory response, reduced granulation tissue formation, and impaired angiogenesis. The mechanisms underlying such defects are only partially understood. Among them, decreased growth factor production has been shown to play an important role.2Werner S Grose R Regulation of wound healing by growth factors and cytokines.Physiol Rev. 2003; 83: 835-870Crossref PubMed Scopus (2508) Google Scholar Neoangiogenesis of the granulation tissue, which transiently fills the wound bed reconstituting skin continuity, is a step occurring during the proliferative phase, necessary to sustain the metabolic demand and to allow the recruitment of inflammatory cells. Reduced vascularization strongly contributes to ulcer formation in diabetes,3Abaci A Oguzhan A Kahraman S Eryol N Unal S Arinc H Ergin A Effect of diabetes mellitus on formation of coronary collateral vessels.Circulation. 1999; 99: 2239-2242Crossref PubMed Scopus (564) Google Scholar and therapies aimed at enhancing angiogenesis have proved beneficial in accelerating diabetic wound closure. Different proangiogenic growth factors directly or indirectly mediate new vessel formation during skin repair. Among them, the placenta growth factor (PlGF), a member of the vascular endothelial growth factor (VEGF) family, plays an important role in promoting adult pathophysiological neovascularization and has been shown to contribute to wound healing by enhancing angiogenesis.4Carmeliet P Moons L Luttun A Vincenti V Compernolle V De Mol M Wu Y Bono F Devy L Beck H Scholz D Acker T DiPalma T Dewerchin M Noel A Stalmans I Barra A Blacher S Vandendriessche T Ponten A Eriksson U Plate KH Foidart JM Schaper W Charnock-Jones DS Hicklin DJ Herbert JM Collen D Persico MG Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.Nat Med. 2001; 7: 575-583Crossref PubMed Scopus (1364) Google Scholar PlGF acts by binding and activating the vascular endothelial growth factor receptor-1 (VEGFR-1)5Park JE Chen HH Winer J Houck KA Ferrara N Placenta growth factor. Potentiation of vascular growth factor bioactivity, in vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR.J Biol Chem. 1994; 269: 25646-25654Abstract Full Text PDF PubMed Google Scholar and, in human, exists as four isoforms (PlGF-1, −2, −3, and −4) generated by alternative splicing that results in their different binding capability to heparin and the neuropilins.6Maglione D Guerriero V Viglietto G Ferraro M Aprelikova O Alitalo K Del Vecchio S Lei K Chou J Persico M Two alternative mRNAs coding for the angiogenic factor, placenta growth factor (PlGF), are transcribed from a single gene of chromosome 14.Oncogene. 1993; 8: 925-931PubMed Google Scholar, 7Cao Y Ji W-R Qi P Rosin A Cao Y Placenta growth factor: identification and characterization of a novel isoform generated by RNA alternative splicing.Biochem Biophys Res Commun. 1997; 235: 493-498Crossref PubMed Scopus (153) Google Scholar, 8Migdal M Huppertz B Tessler S Comforti A Shibuya M Reich R Baumann H Neufeld G Neuropilin-1 is a placenta growth factor-2 receptor.J Biol Chem. 1998; 273: 22272-22278Crossref PubMed Scopus (278) Google Scholar, 9Gluzman-Poltorak Z Cohen T Herzog Y Neufeld G Neuropilin-2 and neuropilin-1 are receptors for the 165-amino acid form of vascular endothelial growth factor (VEGF) and of placenta growth factor-2, but only neuropilin-2 functions as a receptor for the 145-amino acid form of VEGF.J Biol Chem. 2000; 275: 18040-18045Crossref PubMed Scopus (305) Google Scholar PlGF promotes monocyte chemiotaxis, collateral vessel growth, and bone marrow-derived precursor cell mobilization.10Clauss M Weich H Breier G Knies U Rockl W Waltenberger J Risau W The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implication for a functional role of placenta growth factor in monocyte activation and chemotaxis.J Biol Chem. 1996; 271: 17629-17634Crossref PubMed Scopus (751) Google Scholar, 11Pipp F Heil M Issbrucker K Ziegelhoeffer T Martin S van de Heuvel J Weich H Fernandez B Golomb G Carmeliet P Schaper W Clauss M VEGFR-1-selective VEGF homologue PlGF is arteriogenic. Evidence for a monocyte-mediated mechanism.Circ Res. 2003; 92: 378-385Crossref PubMed Scopus (263) Google Scholar, 12Hattori K Heissig B Wu Y Dias S Tejada R Ferris B Hicklin DJ Zhu Z Bohlen P Witte L Hendrikx J Hackett NR Crystal RG Moore MA Werb Z Lyden D Rafii S Placental growth factor reconstitutes hematopoiesis by recruiting VEGFR1+ stem cells from bonemarrow microenvironment.Nat Med. 2002; 8: 841-849Crossref PubMed Scopus (554) Google Scholar Some data indicate that PlGF may have a direct action on endothelial cells mediated by VEGFR-1 receptor binding.13Autiero M Waltenberger J Communi D Kranz A Moons L Lambrechts D Kroll J Plaisance S De Mol M Bono F Kliche S Fellbrich G Ballmer-Hofer K Maglione D Mayr-Beyrle U Dewerchin M Dombrowski S Stanimirovic D Van Hummelen P Dehio C Hicklin DJ Persico G Herbert JM Communi D Shibuya M Collen D Conway EM Carmeliet P Role of PlGF in the intra- and intermolecular crosstalk between the VEGF receptors Flt-1 and Flk-1.Nat Med. 2003; 9: 936-943Crossref PubMed Scopus (640) Google Scholar However, in vivo experimental findings show that PlGF angiogenic potential also relies on the potentiation of VEGF activity.4Carmeliet P Moons L Luttun A Vincenti V Compernolle V De Mol M Wu Y Bono F Devy L Beck H Scholz D Acker T DiPalma T Dewerchin M Noel A Stalmans I Barra A Blacher S Vandendriessche T Ponten A Eriksson U Plate KH Foidart JM Schaper W Charnock-Jones DS Hicklin DJ Herbert JM Collen D Persico MG Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.Nat Med. 2001; 7: 575-583Crossref PubMed Scopus (1364) Google Scholar Based on its role in adult angiogenesis, PlGF has been regarded as an attractive candidate for the therapeutic modulation of adult angiogenesis, and studies on heart and hindlimb ischemia animal models have confirmed the therapeutic potential of PlGF administration.11Pipp F Heil M Issbrucker K Ziegelhoeffer T Martin S van de Heuvel J Weich H Fernandez B Golomb G Carmeliet P Schaper W Clauss M VEGFR-1-selective VEGF homologue PlGF is arteriogenic. Evidence for a monocyte-mediated mechanism.Circ Res. 2003; 92: 378-385Crossref PubMed Scopus (263) Google Scholar, 14Luttun A Tiwa M Moons L Wu Y Angelillo-Scherrer A Liao F Nagy JA Hooper A Priller J De Clerck B Compernolle V Daci E Bohlen P Dewerchin M Herbert J-M Fava R Matthys P Carmeliet G Collen D Dvorak HF Hicklin DJ Carmeliet P Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1.Nat Med. 2002; 8: 831-840Crossref PubMed Scopus (931) Google Scholar, 15Tamarat R Silvestre J-S Le Ricousse-Roussanne S Barateau V Lecomte-Raclet L Clergue M Duriez M Tobelem G Lévy BI Impairment in ischemia-induced neovascularization in diabetes. Bone marrow mononuclear cell dysfunction and therapeutic potential of placenta growth factor treatment.Am J Pathol. 2004; 164: 457-466Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar In vivo, PlGF exerts a strong angiogenic activity. Transgenic mice overexpressing PlGF under the keratin 14 promoter (K14-PlGF mice) exhibit a robust increase in dermal vascularization that is formed of markedly enlarged and also more numerous and permeable vessels.16Odorisio T Schietroma C Zaccaria ML Cianfarani F Tiveron C Tatangelo L Failla CM Zambruno G Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability.J Cell Sci. 2002; 115: 2559-2567PubMed Google Scholar In keeping with this finding, adeno-mediated PlGF gene transfer in the skin is able to elicit a strong angiogenic response, giving rise to more numerous and strikingly larger vessels.14Luttun A Tiwa M Moons L Wu Y Angelillo-Scherrer A Liao F Nagy JA Hooper A Priller J De Clerck B Compernolle V Daci E Bohlen P Dewerchin M Herbert J-M Fava R Matthys P Carmeliet G Collen D Dvorak HF Hicklin DJ Carmeliet P Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1.Nat Med. 2002; 8: 831-840Crossref PubMed Scopus (931) Google Scholar PlGF gene transfer does not affect lymphatic vasculature and induces blood vessel permeability only when high adenovirus concentrations are used.17Nagy JA Vasile E Feng D Sundberg C Brown LF Detmar MJ Lawitts JA Benjamin L Tan X Manseau EJ Dvorak AM Dvorak HF Vascular permeability factor/vascular endothelial growth factor induces lymphangiogenesis as well as angiogenesis.J Exp Med. 2002; 196: 1497-1506Crossref PubMed Scopus (449) Google Scholar, 18Nagy JA Dvorak AM Dvorak HF VEGF-A164/165 and PlGF. Roles in angiogenesis and arteriogenesis.Trends Cardiovasc Med. 2003; 13: 169-175Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar In adult skin, PlGF expression is barely detectable, and it is up-regulated in association with both physiological and pathological neoangiogenesis, such as in the hair follicle cycle,19Cianfarani F, Zaccaria ML, Odorisio T, Zambruno G: Expression of placenta growth factor in mouse hair follicle cycle. Gior Ital Dermatol Venereol 140:497–503Google Scholar during wound healing,20Failla CM Odorisio T Cianfarani F Schietroma C Puddu P Zambruno G Placenta growth factor is induced in human keratinocytes during wound healing.J Invest Dermatol. 2000; 115: 388-395Crossref PubMed Scopus (91) Google Scholar and in melanoma cells.21Lacal PM Failla CM Pagani E Odorisio T Schietroma C Falcinelli S Zambruno G D'Atri S Human melanoma cells secrete and respond to placenta growth factor and vascular endothelial growth factor.J Invest Dermatol. 2000; 115: 1000-1007Crossref PubMed Scopus (155) Google Scholar, 22Graeven U Rodeck U Karpinski S Jost M Andre N Schmiegel W Expression patterns of placenta growth factor in human melanocytic cell lines.J Invest Dermatol. 2000; 115: 118-123Crossref PubMed Scopus (24) Google Scholar During the angiogenic phase of wound healing, PlGF is expressed by migrating keratinocytes and endothelial cells of small blood vessels, acting in a paracrine and autocrine way on VEGFR-1-expressing endothelium.20Failla CM Odorisio T Cianfarani F Schietroma C Puddu P Zambruno G Placenta growth factor is induced in human keratinocytes during wound healing.J Invest Dermatol. 2000; 115: 388-395Crossref PubMed Scopus (91) Google Scholar Moreover, lack of PlGF results in delayed wound closure, indicating that this factor is required for optimal skin repair.4Carmeliet P Moons L Luttun A Vincenti V Compernolle V De Mol M Wu Y Bono F Devy L Beck H Scholz D Acker T DiPalma T Dewerchin M Noel A Stalmans I Barra A Blacher S Vandendriessche T Ponten A Eriksson U Plate KH Foidart JM Schaper W Charnock-Jones DS Hicklin DJ Herbert JM Collen D Persico MG Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions.Nat Med. 2001; 7: 575-583Crossref PubMed Scopus (1364) Google Scholar By using streptozotocin-induced diabetic mice, a mouse model of type-1 diabetes, we investigated whether PlGF may be involved in the healing impairment of diabetic cutaneous wounds. We also evaluated the activity of adenovirus-mediated PlGF gene transfer in promoting diabetic skin repair. C57Bl/6 mice were purchased from Charles River (Calco, Italy). Transgenic mice overexpressing PlGF under the control of the human keratin 14 promoter (K14-PlGF mice) have a BDF1 background and have been produced and phenotypically characterized in our laboratory.16Odorisio T Schietroma C Zaccaria ML Cianfarani F Tiveron C Tatangelo L Failla CM Zambruno G Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability.J Cell Sci. 2002; 115: 2559-2567PubMed Google Scholar The full cDNA for the human PlGF-1 isoform6Maglione D Guerriero V Viglietto G Ferraro M Aprelikova O Alitalo K Del Vecchio S Lei K Chou J Persico M Two alternative mRNAs coding for the angiogenic factor, placenta growth factor (PlGF), are transcribed from a single gene of chromosome 14.Oncogene. 1993; 8: 925-931PubMed Google Scholar was inserted into the pAdTrack-constitutive cytomegalovirus (CMV) expression vector (Stratagene, La Jolla, CA) under the control of the CMV immediate-early promoter/enhancer. The pAdTrack-CMV plasmid contains the gene for the green fluorescent protein (GFP) under the control of a second CMV promoter and sequences for homologous recombination with the replication-deficient serotype 5 adenovirus (Ad5). The plasmid containing the human PlGF-1 was linearized and transformed with the pADEasy-1 plasmid (Stratagene) in BJ5138 Escherichia coli cells. The pAdCMV.PlGF, obtained following homologous recombination between pAdTrack-CMV.PlGF and pAdEasy-1, was selected for its resistance to kanamycin and sequenced to check the recombination event. The control vector, carrying the cDNA for the E. coli LacZ gene and coding for the enzyme β-galactosidase (AdCMV.LacZ), was obtained following the same procedure. AdCMV.PlGF and AdCMV.LacZ were propagated in 293 cells (American Type Culture Collection, Manassas, VA), modified to constitutively express viral genes for the formation of viral particles deleted in the pAdEasy-1 vector, and were purified by a CsCl density gradient. The preparation was dialyzed and stored in dialysis buffer (10 mmol/L Tris-HCl and 1 mmol/L MgCl2, pH 7.4) with 10% glycerol at −70°C. The titer of viral stocks was determined by plaque assay in 293 cells. Five-week-old C57Bl/6 male mice were rendered diabetic by a single intraperitoneal injection of 180 mg/kg streptozotocin (Sigma-Aldrich, St. Louis, MO). After 7 days, glucose level was measured in the peripheral blood, and mice with glycemia over 250 mg/dl were selected for the study. Two weeks later, the dorsum of anesthetized diabetic mice and age-matched healthy controls was shaved, and a 6 mm-diameter full-thickness wound was performed on the dorsal midline using a biopsy punch. One day after injury, the wounds of diabetic mice were treated with 50 μl of saline solution alone or containing 5 × 108 pfu of AdCMV.PlGF or 5 × 108 pfu of AdCMV.LacZ, delivered with a Hamilton's syringe under the clot covering the wound. The wound area of healthy controls was treated with an equivalent volume of saline solution. The wound margin was traced on a transparency film put on the back of treated mice immediately after wounding (day 0) and every 2nd day thereafter (days 2, 4, 6, etc. after wounding, corresponding to day 1, 3, 5, etc. after treatment). A blinded operator measured traced areas of the wounds using a computer-assisted image analyzer (KS300; Zeiss, Jena, Germany). The rate of healing was expressed as the percentage of the area at day 0. Sixteen to 22 animals/group were analyzed as a total of three independent experiments. Eight- to 10-week-old transgenic mice and wild-type littermates were used for the analysis of K14-PlGF mice. Diabetes induction, wounds, and closure analysis were performed on groups of nine animals as described above, apart from the timing of wound closure analysis that was performed every 2nd or 3rd day. Cultured human dermal fibroblasts, human umbilical vein endothelial cells, HaCaT cells, and wounds with surrounding normal skin were homogenized in TRIzol (Life Technologies, Invitrogen, Gröningen, The Netherlands) and total RNA extracted and purified following the manufacturer's instructions. RNA was converted into cDNA using the first Strand cDNA Synthesis Kit (Roche Bioproducts, Basel, Switzerland). The cDNAs were used as templates in RT-PCR or real-time RT-PCR. For RT-PCR of VEGFR-1 and VEGFR-2, the following primers were used: VEGFR-1 forward (5′-CTCCTGAGTACTCTACTCCT-3′) and reverse (5′-GAGTACAGGACCACCGAGTT-3′), amplifying a 632-bp fragment, and VEGFR-2 forward (5′-GTCTATGCCATTCCTCCCCC-3′) and reverse (5′-GAGACAGCTTGGCTGGGCT-3′), amplifying a cDNA fragment of 85 bp. For real-time RT-PCR, the SYBR Green PCR Master Mix (Applied Biosystem, Foster City, CA) was used following the manufacturer's instructions, and the following gene-specific primers were designed using the Primer Express software (Applied Biosystems): PlGF forward (5′-GTGTGCCGATAAAGACAGCCA-3′) and reverse (5′-GAAATGTGGATCCCGATTGG-3′); VEGF forward (5′-TGTGCAGGCTGCTGTAACGAT-3′) and reverse (5′-GCATGATCTGCATGGTGATGTT-3′); VEGFR-1, transmembrane form, forward (5′-AAACTAGGCAAATCGCTCGG-3′) and reverse (5′-GGCTTGAACGACTTTCCCAA-3′); and VEGFR-1, soluble form, forward (5′-TCAGGCCCAGAGGAAAGC-3′) and reverse (5′-GAGGGCACTGGGCTTTCTTA-3′). For the transforming growth factor β-1 (TGFβ-1), fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor-B (PDGF-B), the forward and reverse primers reported by Galiano et al23Galiano RD Tepper OM Pelo CR Bhatt KA Callaghan M Bastidas N Bunting S Steinmetz HG Gurtner GC Topical vascular endothelial growth factor accelerates diabetic wound healing through increased angiogenesis and by mobilizing and recruiting bone marrow-derived cells.Am J Pathol. 2004; 164: 1935-1947Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar were used. Gene-specific PCR products were measured by means of the ABI PRISM 5700 detection system (Perkin-Elmer, Norwalk, CT) and normalized to the glycer-aldehyde-3-phosphate dehydrogenase (GAPDH) amplification obtained with specific primers (forward-5′-GTATGACTCCACTCACGGCAAA-3′ and reverse-5′-TTC-CCATTCTCGGCCTTG-3′). Quantification was performed using the comparative CT method.24Pfaffl M A new mathematical model for relative quantification in real time RT-PCR.Nucl Ac Res. 2001; 29: 2002-2007Crossref Scopus (24586) Google Scholar A non-template control was run with every assay by adding water in place of cDNA, and all determinations were performed in triplicate. Protein extracts from skin wound biopsies were obtained as described.16Odorisio T Schietroma C Zaccaria ML Cianfarani F Tiveron C Tatangelo L Failla CM Zambruno G Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability.J Cell Sci. 2002; 115: 2559-2567PubMed Google Scholar Plasma samples used for the detection of human PlGF were obtained from blood collected from the jugular vein using a syringe containing an anticoagulant (Liquemin, 100,000 u.i.; Roche Bioproducts). Blood was immediately transferred in a vial containing EDTA at a concentration of 1.6 mg/ml and mixed with an equal volume of PBS without calcium and magnesium. After adding 3 vol of Lymphoprep (Axis-Shield PoC As, Oslo, Norway) to the mixture, a discontinuous density gradient was obtained by centrifugation, and the upper phase containing the plasma was collected. Mouse PlGF and VEGF were quantified in wound extracts of diabetic and healthy mice using specific murine Quantikine ELISA Kit (R&D Systems, Abingdon, UK), following the manufacturer's instructions. Human PlGF was detected in wound extracts after AdCMV.PlGF, AdCMV.LacZ, or saline local application or in plasma samples by using Quantikine human PlGF ELISA kit (R&D Systems). VEGFR-1 was quantified in 96-well plates coated with 50 μl of an anti-mouse VEGFR-1 antibody (AF471; R&D Systems) at a concentration of 10 μg/ml overnight at room temperature. The plates were then washed and incubated for 2 hours with 3% bovine serum albumin in PBS. After washing, 80 μg of protein extracts from diabetic and healthy mouse wounds were added to the plates for 2 hours at room temperature, followed by a 2-hour incubation with biotinylated anti-mouse VEGFR-1 antibody (BAF471; R&D Systems). The plates were then incubated overnight with horseradish peroxidase-conjugated streptavidin, diluted 1:1000. Colorimetric reactions were read at 490 nm in a Microplate Reader 3550-UV (Bio-Rad, Life Science Group, Hercules, CA). Wounds surrounded by normal skin were dissected 1 day after adenovirus application and frozen in Tissue-Tek OCT Compound (Sakura Finetek, Torrance, CA). Ten-μm cryostat sections were obtained and immediately analyzed at the fluorescence microscope. Paraformaldehyde-fixed, paraffin-embedded wound specimens were processed as previously described.20Failla CM Odorisio T Cianfarani F Schietroma C Puddu P Zambruno G Placenta growth factor is induced in human keratinocytes during wound healing.J Invest Dermatol. 2000; 115: 388-395Crossref PubMed Scopus (91) Google Scholar The mouse PlGF riboprobe containing the entire coding sequence and the human PlGF riboprobe spanning the cDNA region common to all of the human PlGF isoforms were previously described.16Odorisio T Schietroma C Zaccaria ML Cianfarani F Tiveron C Tatangelo L Failla CM Zambruno G Mice overexpressing placenta growth factor exhibit increased vascularization and vessel permeability.J Cell Sci. 2002; 115: 2559-2567PubMed Google Scholar, 20Failla CM Odorisio T Cianfarani F Schietroma C Puddu P Zambruno G Placenta growth factor is induced in human keratinocytes during wound healing.J Invest Dermatol. 2000; 115: 388-395Crossref PubMed Scopus (91) Google Scholar Four-μm-thick paraformaldehyde-fixed, paraffin-embedded sections from skin biopsies containing the wounds were processed as described.20Failla CM Odorisio T Cianfarani F Schietroma C Puddu P Zambruno G Placenta growth factor is induced in human keratinocytes during wound healing.J Invest Dermatol. 2000; 115: 388-395Crossref PubMed Scopus (91) Google Scholar To detect endothelial cells, an anti-mouse platelet endothelial cell adhesion molecule-1 (PECAM/CD31) polyclonal antibody (M 20; Santa Cruz Biotechnology, Santa Cruz, CA) was used at a concentration of 1 μg/ml for 14 hours at 4°C following antigen retrieval with a microwave treatment at 650 W for 2 plus 5 minutes. Smooth muscle cells/pericytes surrounding blood vessels were stained by using a mouse monoclonal anti-α-smooth muscle actin (clone 1A4; DakoCytomation, Milan, Italy) diluted 1:50, for 1 hour at room temperature after antigen retrieval in microwave at 650 W for 3 minutes using the Dakoark kit (DakoCytomation). Monocytes/macrophages were identified with an anti-mouse monoclonal antibody (Mac3, clone M3/84; PharMingen, BD Biosciences, Franklin Lakes, NJ) at a concentration of 5 μg/ml for 2 hours at room temperature after microwave treatment at 600 W for 3 minutes. Human PlGF was detected using an anti-goat polyclonal antibody (C-20; Santa Cruz Biotechnology) as described.20Failla CM Odorisio T Cianfarani F Schietroma C Puddu P Zambruno G Placenta growth factor is induced in human keratinocytes during wound healing.J Invest Dermatol. 2000; 115: 388-395Crossref PubMed Scopus (91) Google Scholar Negative controls were obtained by omitting the primary antibody. Two blinded observers independently analyzed granulation tissue area, CD31-positive vessel density and area, α-smooth muscle actin-positive vessel density, and Mac3-positive cell density by computer-assisted image analysis using a Zeiss KS300 Version 3.0 program. CD31-positive vessel density and area, α-smooth muscle actin-positive vessel density, and Mac3-positive cell density were calculated on 8 to 12 noncontiguous, randomly selected fields within the granulation tissue at ×100 magnification. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood after centrifugation over a discontinuous gradient using Lymphoprep (Axis-Shield PoC As). After centrifugation, PBMC present in the intermediate phase were resuspended in Iscove modified Dulbecco's medium (Gibco, Invitrogen). PBMC were seeded at a density of 1.2 × 105 cells/well in six multi-well plates containing methyl-cellulose culture medium (Methocult; StemCell Technologies, Grenoble, France) and the number of colonies/well was counted after 2 weeks of culture. Primary human fibroblast cultures previously established from skin biopsies from healthy volunteers were grown in F10 medium supplemented with 10% fetal calf serum. The human keratinocyte cell line HaCaT was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 4 mmol/L glutamine. Human umbilical vein endothelial cells were isolated from freshly delivered umbilical cords as previously described25Gimbrone MA Culture of vascular endothelium.Prog Hemost Thromb. 1976; 3: 1-28PubMed Google Scholar and cultured in Endothelial Cell Growth Medium-2 kit from Clonetics (BioWhittaker Inc., Walkersville, MD). Semiconfluent normal human fibroblasts were lysed and boiled in SDS sample buffer (50 mmol/L Tris-HCl, pH 6.8, 100 mmol/L dithiothreitol, 2% SDS, 0.1% bromphenol blue, and 10% glycerol). Equal amounts of protein per sample were analyzed in 7 or 10% SDS-polyacrylamide gels. Proteins were then transferred to nitrocellulose membranes (Hybond-C; Amersham Life Science, Buckinghamshire, UK), using a Transblot apparatus (Bio-Rad). Membranes were blocked in blocking solution (2% nonfat dry milk, 1% Triton X-100, 10 mmol/L EDTA, and 50 mmol/L Tris-HCl, pH 7.5) and incubated overnight at 4°C with the primary antibody, diluted in blocking solution. The anti-VEGFR-1 rabbit polyclonal antibody (C-17; Santa Cruz Biotechnology) was used at 2 μg/ml, the anti-VEGFR-2 goat polyclonal antibody (AF357; R&D Systems) was used at 0.2 μg/ml, and the anti-β-actin monoclonal antibody (AC-40; Sigma-Aldrich) was diluted 1:1000. After washing twice with blocking solution, membranes were incubated for 1 hour at room temperature with the secondary antibody (anti-rabbit or anti-mouse Ig/horseradish peroxidase, Amersham Life Science; or anti-goat Ig/horseradish peroxidase, Sigma-Aldrich; all diluted 1:5000 in blocking solution). Membranes were then washed with 0.1% Tween 20/TBS, and detection was performed using the ECL Western blotting detection reagents (Amersham Life Science). Fibroblast migration was analyzed using Boyden chambers (Neuroprobe Inc., Gaithersburg, MD) equipped with 8-μm pore diameter polycarbonate filters (Nuclepore; Whatman Incorporated, Clifton, NJ) coated with 5 μg/ml of a gelatin solution. In brief, cells were suspended in migra" @default.
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• W1520623323 title "Placenta Growth Factor in Diabetic Wound Healing" @default.
• W1520623323 cites W10457224 @default.
• W1520623323 cites W1531839415 @default.
• W1520623323 cites W1550364580 @default.
• W1520623323 cites W1587020653 @default.
• W1520623323 cites W1776420614 @default.
• W1520623323 cites W1875554126 @default.
• W1520623323 cites W1911505241 @default.
• W1520623323 cites W1931315964 @default.
• W1520623323 cites W1963876488 @default.
• W1520623323 cites W1964361550 @default.
• W1520623323 cites W1971248512 @default.
• W1520623323 cites W1978891988 @default.
• W1520623323 cites W1986469266 @default.
• W1520623323 cites W1989021197 @default.
• W1520623323 cites W1989483643 @default.
• W1520623323 cites W2009081283 @default.
• W1520623323 cites W2014148719 @default.
• W1520623323 cites W2018768026 @default.
• W1520623323 cites W2034646933 @default.
• W1520623323 cites W2039284766 @default.
• W1520623323 cites W2042373581 @default.
• W1520623323 cites W2043002662 @default.
• W1520623323 cites W2044144820 @default.
• W1520623323 cites W2050358495 @default.
• W1520623323 cites W2051268134 @default.
• W1520623323 cites W2070995184 @default.
• W1520623323 cites W2077733587 @default.
• W1520623323 cites W2102774859 @default.
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