Plasmin Modulates Vascular Endothelial Growth Factor-A-Mediated Angiogenesis during Wound Repair
2006; Elsevier BV; Volume: 168; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2006.050372
ISSN1525-2191
AutoresDetlev Roth, Michael Piekarek, Mats Paulsson, Hildegard Christ, Wilhelm Bloch, Thomas Krieg, Jeffrey M. Davidson, Sabine A. Eming,
Tópico(s)Lymphatic System and Diseases
ResumoPlasmin-catalyzed cleavage of the vascular endothelial growth factor (VEGF)-A isoform VEGF165 results in loss of its carboxyl-terminal heparin-binding domain and significant loss in its bioactivity. Little is known about the in vivo significance of this process. To investigate the biological relevance of the protease sensitivity of VEGF165 in wound healing we assessed the activity of a VEGF165 mutant resistant to plasmin proteolysis (VEGF165A111P) in a genetic mouse model of impaired wound healing (db/db mouse). In the present study we demonstrate that in this mouse model plasmin activity is increased at the wound site. The stability of the mutant VEGF165 was substantially increased in wound tissue lysates in comparison to wild-type VEGF165, thus indicating a prolonged activity of the plasmin-resistant VEGF165 mutant. The db/db delayed healing phenotype could be reversed by topical application of wild-type VEGF165 or VEGF165A111P. However, resistance of VEGF165 to plasmin cleavage resulted in the increased stability of vascular structures during the late phase of healing due to increased recruitment of perivascular cells and delayed and reduced endothelial cell apoptosis. Our data provide the first indication that plasmin-catalyzed cleavage regulates VEGF165-mediated angiogenesis in vivo. Inactivation of the plasmin cleavage site Arg110/Ala111 may preserve the biological function of VEGF165 in therapeutic angiogenesis under conditions in which proteases are highly active, such as wound repair and inflammation. Plasmin-catalyzed cleavage of the vascular endothelial growth factor (VEGF)-A isoform VEGF165 results in loss of its carboxyl-terminal heparin-binding domain and significant loss in its bioactivity. Little is known about the in vivo significance of this process. To investigate the biological relevance of the protease sensitivity of VEGF165 in wound healing we assessed the activity of a VEGF165 mutant resistant to plasmin proteolysis (VEGF165A111P) in a genetic mouse model of impaired wound healing (db/db mouse). In the present study we demonstrate that in this mouse model plasmin activity is increased at the wound site. The stability of the mutant VEGF165 was substantially increased in wound tissue lysates in comparison to wild-type VEGF165, thus indicating a prolonged activity of the plasmin-resistant VEGF165 mutant. The db/db delayed healing phenotype could be reversed by topical application of wild-type VEGF165 or VEGF165A111P. However, resistance of VEGF165 to plasmin cleavage resulted in the increased stability of vascular structures during the late phase of healing due to increased recruitment of perivascular cells and delayed and reduced endothelial cell apoptosis. Our data provide the first indication that plasmin-catalyzed cleavage regulates VEGF165-mediated angiogenesis in vivo. Inactivation of the plasmin cleavage site Arg110/Ala111 may preserve the biological function of VEGF165 in therapeutic angiogenesis under conditions in which proteases are highly active, such as wound repair and inflammation. Vascular endothelial growth factor-A (VEGF-A) is the most potent and specific vascular growth factor and a key regulator in physiological and pathological processes of angiogenic remodeling.1Ferrara N Gerber HP LeCouter J The biology of VEGF and its receptors.Nat Med. 2003; 9: 669-676Crossref PubMed Scopus (8025) Google Scholar VEGF-A levels are regulated through transcriptional control and mRNA stability. Moreover, using differential mRNA splicing, the single human VEGF-A gene can give rise to at least eight isoforms (VEGF121, 145, 148, 162, 165, 183, 189, and 206) whose relative levels vary among different tissues.1Ferrara N Gerber HP LeCouter J The biology of VEGF and its receptors.Nat Med. 2003; 9: 669-676Crossref PubMed Scopus (8025) Google Scholar, 2Tischer E Mitchell R Hartmann T Silvia M Gospodarowicz D Fiddes JC Abraham JA The human gene for VEGF.J Biol Chem. 1991; 266: 11947-11954Abstract Full Text PDF PubMed Google Scholar The most abundant isoform found in human tissue is VEGF165. All isoforms contain the binding site for VEGF tyrosine kinase receptors VEGF receptors 1 (VEGFR-1/Flt-1) and 2 (VEGFR-2/KDR/Flk-1), which is encoded by exons 1 to 5.3Keyt BA Nguyen HV Berleau LT Duarte CM Park J Chen H Ferrara N Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors.J Biol Chem. 1996; 271: 5638-5646Crossref PubMed Scopus (424) Google Scholar The isoforms are distinguished by the presence or absence of peptides encoded by exons 6 and 7 of the VEGF-A gene encoding two independent heparin-binding domains. Substantial evidence indicates that differences in the expression of the heparin-binding domains are crucially involved in the diverse biochemical and functional properties of the VEGF-A splice forms such as binding to cell surfaces and extracellular matrix,4Park J Keller GA Ferrara N The VEGF isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix bound VEGF.Mol Biol Cell. 1993; 4: 1317-1326Crossref PubMed Scopus (975) Google Scholar, 5Ortega N L'Faqihi FE Plouet J Control of VEGF activity by the extracellular matrix.Biol Cell. 1998; 90: 381-390Crossref PubMed Google Scholar, 6Carmeliet P Ng YS Nuyens D Theilmeier G Brusselmans K Cornelissen I Ehler E Kakkar VV Stalmans I Mattot V Perriard JC Dewerchin M Flameng W Nagy A Lupu F Moons L Collen D D'Amore PA Shima DT Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the VEGF isoform VEGF164 and VEGF188.Nat Med. 1999; 5: 495-502Crossref PubMed Scopus (566) Google Scholar, 7Hutchings H Ortega N Plouet J Extracellular matrix-bound VEGF promotes endothelial cell adhesion, migration, and survival through integrin ligation.FASEB J. 2003; 17: 1520-1522Crossref PubMed Scopus (186) Google Scholar receptor binding characteristics,8Soker S Takashima S Miao HQ Neufeld G Klagsbrun M Neuropilin-1 is expressed by endothelial and tumor cells as isoform-specific receptor for VEGF.Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2106) Google Scholar endothelial cell adhesion and survival,7Hutchings H Ortega N Plouet J Extracellular matrix-bound VEGF promotes endothelial cell adhesion, migration, and survival through integrin ligation.FASEB J. 2003; 17: 1520-1522Crossref PubMed Scopus (186) Google Scholar, 9Dor Y Djonov V Abramovitch R Itin A Fishman GI Carmeliet P Goelman G Keshet E Conditional switching of VEGF provides new insights into adult neovascularization and pro-angiogenic therapy.EMBO J. 2002; 21: 1939-1947Crossref PubMed Scopus (348) Google Scholar and vascular branch formation.10Ruhrberg C Gerhardt H Golding M Watson R Ioannidou S Fujisawa H Betsholtz C Shima DT Spatially restricted patterning cues provided by heparin-binding VEGF-A morphogenesis.Gene Dev. 2002; 16: 2684-2698Crossref PubMed Scopus (725) Google Scholar In addition to mRNA-splicing, proteolytic mechanisms also generate VEGF-A variants lacking peptides encoded by exon 6 and/or 7, thereby generating VEGF-A cleavage products with different biological activities.11Houck KA Leung DW Rowland AM Winer J Ferrara N Dual regulation of VEGF bioavailability by genetic and proteolytic mechanisms.J Biol Chem. 1992; 267: 26031-26037Abstract Full Text PDF PubMed Google Scholar, 12Keyt BA Berleau LT Nguyen HV Chen H Heinsoh H Vandlen R Ferrara N The carboxyl-terminal domain (111-165) of VEGF is critical for its mitogenic potency.J Biol Chem. 1996; 271: 7788-7795Crossref PubMed Scopus (541) Google Scholar, 13Plouet J Moro F Bertagnolli S Coldeboeuf N Mazarguil H Clamens S Bayard F Extracellular cleavage of the VEGF189-amino acid form by urokinase is required for its mitogenic effect.J Biol Chem. 1997; 272: 13390-13396Crossref PubMed Scopus (213) Google Scholar This suggests that the proteolytic microenvironment could function as a critical determinant controlling VEGF-A-mediated activities. We and other investigators have demonstrated the sensitivity of VEGF165 to serine proteases, in particular plasmin.11Houck KA Leung DW Rowland AM Winer J Ferrara N Dual regulation of VEGF bioavailability by genetic and proteolytic mechanisms.J Biol Chem. 1992; 267: 26031-26037Abstract Full Text PDF PubMed Google Scholar, 12Keyt BA Berleau LT Nguyen HV Chen H Heinsoh H Vandlen R Ferrara N The carboxyl-terminal domain (111-165) of VEGF is critical for its mitogenic potency.J Biol Chem. 1996; 271: 7788-7795Crossref PubMed Scopus (541) Google Scholar, 14Lauer G Sollberg S Cole M Flamme I Stürzebecher J Mann K Krieg T Eming SA Expression and proteolysis of VEGF is increased in chronic wounds.J Invest Dermatol. 2000; 115: 12-18Crossref PubMed Scopus (284) Google Scholar, 15Lauer G Sollberg S Cole M Krieg T Eming SA Generation of a novel proteolysis resistant VEGF 165 variant by a site-directed mutation at the plasmin sensitive cleavage site.FEBS Lett. 2002; 531: 309-313Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar Plasmin digestion of VEGF165 yields two fragments: an amino-terminal homodimer (VEGF1-110) containing the VEGF receptor binding site and a carboxyl-terminal polypeptide comprising the heparin-binding domains (VEGF111-165).12Keyt BA Berleau LT Nguyen HV Chen H Heinsoh H Vandlen R Ferrara N The carboxyl-terminal domain (111-165) of VEGF is critical for its mitogenic potency.J Biol Chem. 1996; 271: 7788-7795Crossref PubMed Scopus (541) Google Scholar, 14Lauer G Sollberg S Cole M Flamme I Stürzebecher J Mann K Krieg T Eming SA Expression and proteolysis of VEGF is increased in chronic wounds.J Invest Dermatol. 2000; 115: 12-18Crossref PubMed Scopus (284) Google Scholar, 15Lauer G Sollberg S Cole M Krieg T Eming SA Generation of a novel proteolysis resistant VEGF 165 variant by a site-directed mutation at the plasmin sensitive cleavage site.FEBS Lett. 2002; 531: 309-313Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar Loss of the carboxyl-terminal heparin binding domain through plasmin digestion significantly reduces VEGF165 mitogenic activity on human umbilical vein endothelial cells, supporting the crucial significance of the heparin-binding domains for VEGF-A function.12Keyt BA Berleau LT Nguyen HV Chen H Heinsoh H Vandlen R Ferrara N The carboxyl-terminal domain (111-165) of VEGF is critical for its mitogenic potency.J Biol Chem. 1996; 271: 7788-7795Crossref PubMed Scopus (541) Google Scholar, 15Lauer G Sollberg S Cole M Krieg T Eming SA Generation of a novel proteolysis resistant VEGF 165 variant by a site-directed mutation at the plasmin sensitive cleavage site.FEBS Lett. 2002; 531: 309-313Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar So far, little is known about the prevalence and biological significance of proteolytic digestion of VEGF-A in vivo. VEGF-A plays a pivotal role during the angiogenic response in tissue repair, by regulating vascular permeability, the influx of inflammatory cells into the site of injury, migration and proliferation of pre-existing endothelial cells and, as suggested recently, the recruitment of marrow-derived endothelial progenitor cells to the local wound site where they are able to accelerate repair.16Brown LF Yeo KT Berse B Senger DR Dvorak HF Van De Water L Expression of VEGF by epidermal keratinocytes during wound healing.J Exp Med. 1992; 176: 1375-1379Crossref PubMed Scopus (789) Google Scholar, 17Nissen NN Polverini PJ Koch AE Volin MV Gamelli RL DiPietro LA VEGF mediates angiogenic activity during the proliferative phase of wound healing.Am J Pathol. 1998; 152: 1445-1452PubMed Google Scholar, 18Kishimoto J Ehama R Ge Y Kobayashi T Nishiyama T Detmar M Burgeson RE In vivo detection of human VEGF promoter activity in transgenic mouse skin.Am J Pathol. 2000; 157: 103-110Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 19Galiano RD Tepper OM Pelo CR Bhatt KA Calaghan M Bastidas N Bunting S Steinmetz HG Gurtner GC Topical VEGF 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 (649) Google Scholar, 20Rossiter H Barresi C Pammer J Rendl M Haigh J Wagner EF Tschachler E Loss of VEGF A activity in murine epidermal keratinocytes delays wound healing and inhibits tumor formation.Cancer Res. 2004; 64: 3508-3516Crossref PubMed Scopus (93) Google Scholar We recently provided evidence that in the highly proteolytic environment of human nonhealing skin ulcers VEGF165 protein degradation is increased as compared to normal-healing wounds.14Lauer G Sollberg S Cole M Flamme I Stürzebecher J Mann K Krieg T Eming SA Expression and proteolysis of VEGF is increased in chronic wounds.J Invest Dermatol. 2000; 115: 12-18Crossref PubMed Scopus (284) Google Scholar Protease inhibitor studies and increased stability of a plasmin-resistant VEGF165 mutant after exposure to wound fluid of nonhealing human chronic wounds indicated that plasmin is one of the serine proteases critically involved in this process.15Lauer G Sollberg S Cole M Krieg T Eming SA Generation of a novel proteolysis resistant VEGF 165 variant by a site-directed mutation at the plasmin sensitive cleavage site.FEBS Lett. 2002; 531: 309-313Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar Hence, plasmin-catalyzed VEGF165 proteolysis and inactivation may lead to reduced VEGF165 availability at the wound site and contribute to an impaired healing response. To further characterize the biological relevance of the protease sensitivity of VEGF165 during cutaneous repair, in the present study we tested the stability and activity of a locally applied VEGF165 mutant resistant to plasmin proteolysis (VEGF165A111P) in a genetic mouse model of impaired healing (db/db mouse). Our experiments provide the first in vivo data indicating that plasmin-catalyzed cleavage is critical to regulate VEGF165-mediated angiogenesis. Our data indicate that inactivation of the plasmin cleavage site Arg110/Ala111 increases the overall integrity of the VEGF165 molecule with significant consequences to blood vessel persistence. Plasmid DNA encoding human wild-type VEGF165 (CMV-VEGF165-Wt) or a plasmin-resistant VEGF165 mutant VEGF165A111P (CMV-VEGF165A111P) was generated as previously described.15Lauer G Sollberg S Cole M Krieg T Eming SA Generation of a novel proteolysis resistant VEGF 165 variant by a site-directed mutation at the plasmin sensitive cleavage site.FEBS Lett. 2002; 531: 309-313Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar Briefly, mutagenesis was performed using a base mismatched oligonucleotide as indicated: 5′-GACCAAAGAAAGATAGACCAAGACAAG-3′ (nucleotide difference between wild-type VEGF165 and mutant is underlined) (MWG-Biotech, Ebersberg, Germany). This mutation results in the substitution of alanine at position 111 by proline and yields a plasmin-resistant and biologically active VEGF165 molecule as demonstrated recently.15Lauer G Sollberg S Cole M Krieg T Eming SA Generation of a novel proteolysis resistant VEGF 165 variant by a site-directed mutation at the plasmin sensitive cleavage site.FEBS Lett. 2002; 531: 309-313Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar VEGF165A111P was generated using the Gene-Editor in vitro site-directed mutagenesis system (Promega, Mannheim, Germany). A cDNA encoding human VEGF165-Wt or VEGF165A111P was ligated into the BamH1 and EcoR1 sites of the expression plasmid pcDNA 3.1 (Invitrogen, De Schelp, The Netherlands) containing a cytomegalovirus (CMV) promoter. To follow VEGF165 transgene expression in vivo a VEGF165-Wt-myc-his fusion protein was generated. A cDNA coding for VEGF165-Wt was inserted into a pcDNA 3.1 myc-his expression plasmid containing a CMV promoter (Invitrogen); the myc-tag was added to the carboxyl-terminus of the VEGF165 molecule. The β-galactosidase expression plasmid containing a cytomegalovirus promoter (CMV-LacZ) was a gift from Dr. M. Hafner (National Research Center for Biotechnology, Braunschweig, Germany). The sequences of expression constructs were verified by DNA sequencing. Plasmids were propagated in Escherichia coli DH1α, and plasmid DNA was prepared on Qiagen chromatographic columns as recommended by the manufacturer (Qiagen Plasmid midi kit; Qiagen, Hilden, Germany). VEGF165-proteins were generated and fractionated as recently described.15Lauer G Sollberg S Cole M Krieg T Eming SA Generation of a novel proteolysis resistant VEGF 165 variant by a site-directed mutation at the plasmin sensitive cleavage site.FEBS Lett. 2002; 531: 309-313Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar Briefly, COS-1 cells were transfected using the Superfect transfection reagent (Qiagen) following the manufacturer's instructions. Conditioned medium was incubated with heparin-Sepharose (Amersham Bioscience, Braunschweig, Germany) and was packed in a column. Elution of bound proteins was performed by 10 mmol/L Tris-HCl, pH 7.2, 0.9 mol/L NaCl. Fractions were pooled, desalted (D-Salt Excellulose plastic desalting columns; Pierce, St. Augustin, Germany), lyophilized, and the concentration of VEGF165 was determined using a commercially available hVEGF165-specific enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN). The assay was performed following the manufacturer's instructions. The mitogenic activity of both VEGF165 variants was similar when tested on human umbilical vein endothelial cells.15Lauer G Sollberg S Cole M Krieg T Eming SA Generation of a novel proteolysis resistant VEGF 165 variant by a site-directed mutation at the plasmin sensitive cleavage site.FEBS Lett. 2002; 531: 309-313Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar Be-cause VEGF165 proteins were purified by heparin-Sepharose they are termed partially purified VEGF. Cutaneous transfections were performed as described previously using the Bio-Rad Helios delivery device (Bio-Rad, München, Germany).21Benn SI Whitsitt JS Broadly KN Nanney LB Perkins D He L Patel M Morgan JR Swain WF Davidson JM Enhancement of wound healing in rat skin following particle bombardment with cDNAs encoding TGF-b1.J Clin Invest. 1996; 98: 2894-2902Crossref PubMed Scopus (97) Google Scholar, 22Eming SA Whitsitt JS He L Krieg T Morgan JR Davidson JM Particle-mediated gene transfer of PDGF isoforms promotes wound repair.J Invest Dermatol. 1999; 112: 297-302Crossref PubMed Scopus (106) Google Scholar, 23Davidson JM Krieg T Eming SA Particle-mediated gene therapy of wounds.Wound Rep Reg. 2000; 8: 452-459Crossref PubMed Google Scholar Briefly, supercoiled plasmid DNA was precipitated onto gold particles (0.95 mm in diameter) in the presence of 50 mmol/L spermidine and 0.5 mol/L CaCl2 at a concentration of 5 μg of DNA per mg of gold. The DNA/gold complexes were washed with absolute ethanol and resuspended in absolute ethanol. The suspension was used to coat the interior of 1/8-inch Tefzel tubing (Bio-Rad) in a specially designed apparatus (Bio-Rad). After the particles adhered to the tubing, it was cut into 1/2-inch lengths such that 0.25 mg of DNA/gold complexes would be delivered with each shot. DNA/gold particles were accelerated into the tissue surface by the rapid release of a pulse of helium at 300 psi. Total RNA was isolated from transfected skin by the guanidine isothiocyanate-acid phenol method. Residual plasmid DNA was destroyed by digestion of the RNA preparation with 1 U per RNase-free deoxyribonuclease (30 minutes, 37°C). To generate cDNA, RNA was reverse-transcribed using Superscript reverse transcriptase and oligo (dT)12-18 primers (Life Technologies, Gibco BRL, Eggenstein, Germany). The reaction mixture was incubated for 1 hour at 42°C and chilled on ice. The RNA template was hydrolyzed by digesting the reaction product with RNaseH before amplification. Portions of 1/10 vol of the first-strand synthesis reaction were then amplified by 30 cycles of PCR for VEGF164/165 or GAPDH adding primers specific for either VEGF164/165 (EMBL, accession no. M32977) (5′-GAGTCCAACATCACCATGCAG-3′, 5′-TCACCGCCTTGGCTTCTCACA-3′), or GAPDH (EMBL, accession no. M33197) (5′-TCATGACCACAGTCCATGCCATCA-3′, 5′-GCCAAATTCGTTGTCATACCAGGAAATGA-3′) (MWG Biotech) using a Thermocycler (Trio-Thermocycler; Biometra, Göttingen, Germany). The primers were chosen such that only the resulting amplification product of the transfected human VEGF165 would contain the DdeI endonuclease site. The amplified products were digested with DdeI, size fractionated by electrophoresis in a 2% TBE-agarose gel, transferred to a nitrocellulose membrane (Hybond-N, Amersham), and subjected to Southern blot hybridization with a random-primed 32P-dCTP-labeled VEGF probe (Ladderman labeling kit; Takara Bio Inc., Shiga, Japan). C57BLKS/J-m+/+Leprdb (db/db) and C57BL/6 mice were maintained and bred in the animal care facility of the Center for Molecular Medicine, University of Cologne, (Cologne, Germany). Genotyping of db/db mice was achieved by PCR analysis; primers were chosen so that the PCR product contains the db/db mutation that leads to loss of the RsaI restriction site in exon 12 (EMBL, accession no. MMU58861) (5′-AGAACGGACACTCTTTGAAGTCTC-3′, 5′-CATTCAAACCATAGTTTAGGTTTGTGT-3′). Male mice were 10 to 12 weeks of age at the start of the experiments. Four independent wound-healing experiments were performed; mice were anesthetized under Ketanest/Rompun (Ketanest S from Park Davies GmbH, Karlsruhe, Germany, and Rompun 2% from Bayer, Leverkusen, Germany). The animals' backs were shaved and four full-thickness punch-biopsy wounds (6 mm in diameter and 5 mm apart) were created. Wounds were either created immediately after transfection within the transfected target side or created in nontransfected skin and treated daily from day 1 through day 7 after injury with recombinant human VEGF165 protein (rhVEGF165) (1 μg in 100 μg of methylcellulose) or methylcellulose (control) (IntraSite Gel; Smith and Nephew, Lohfelden, Germany) and covered with a semiocclusive dressing. Treatment of wounds was rotated among sites to avoid site-specific bias. Analysis of wound closure was achieved through digital processing of photographs taken daily during the time of healing using the Imaging Software Lucia G 4.80 (Dental Eye, Olympus, Japan and Imaging Software Lucia G 4.80 from Laboratory Imaging Ltd., Prague, Czechchoslovakia); wound closure was calculated as the percentage of the wound area at day 1 after wounding. For histological analysis animals were sacrificed and wounds were harvested at 1, 3, 5, 8, 11, 12, 15, and 18 days after wounding. An area of 8 μm in diameter, which includes the complete epithelial margins, was excised at each time point; wounds were cut exactly in half and embedded in OCT compound (Tissue Tek; Miles, Elkhart, IN), immediately frozen in liquid nitrogen, and stored at −80°C. Complete histological analysis was performed on serial sections (5-μm cryosections) from the central portion of the wound. The extent of re-epithelialization and granulation tissue formation was measured by histomorphometric analysis of tissue sections (hematoxylin and eosin stain) using the Imaging Software Lucia G 4.80. For analysis of re-epithelialization, the distance that the epithelium had traveled across the wound was measured; the muscle edges of the panniculus carnosus were used as indicator for the wound edges; re-epithelialization was calculated as the percentage of the distance of muscle edges of the panniculus carnosus. For the quantification of granulation tissue, the wound site was identified, outlined, and the area covered by a highly cellular and vascularized tissue was determined. All histomorphometric analyses were performed blinded by two independent investigators. To process tissue sections for the immunodetection of CD31 (PECAM-1) (eight wounds per time point), VEGFR-2 (eight wounds per time point), F4/80 (five wounds per time point), or the fusion protein VEGF165-Wt-myc (three wounds per time point), 5-μm cryosections were fixed in acetone, endogenous peroxidase was blocked (0.03% H2O2, 0.15 mol/L NaN3), and samples were then blocked with 3% bovine serum albumin in phosphate-buffered saline (PBS). Sections were incubated with polyclonal rat antisera against murine CD31 (1 hour at room temperature, 1:500) (Pharmingen, Heidelberg, Germany), murine VEGFR-2 (1 hour at room temperature, 1:200) (Pharmingen), murine F4/80 (24 hours at 4°C, 1:10) (Serotec, Eching, Germany), or myc (1 hour at room temperature, 1:500) (Invitrogen). Bound primary antibodies were detected using a peroxidase-conjugated goat anti-rat antibody (Southern Biotechnology, Birmingham, AL). 3-Amino-9-ethyl-carbazol was used as a substrate, and sections were counterstained with hemalaun. As a control for specificity, primary antibodies were omitted and replaced by an irrelevant isotype-matched rat antibody. For semiquantitative analysis of immunohistochemical staining, video images of at least three serial wound sections per wound were captured and analyzed using the Imaging Software Lucia G 4.8; data are expressed as percentage of granulation tissue area. To determine average vessel size in CD31-stained sections, the capillary lumen was examined in three different fields on each wound section at a 400-fold magnification. All histomorphometric analysis was performed blinded by two independent investigators. To detect LacZ gene expression, cryosections were processed as described recently.24Eming SA Medalie DA Tompkins RG Yarmush ML Morgan JR Genetically modified human keratinocytes overexpressing PDGF-A enhance the performance of a composite skin graft.Hum Gene Ther. 1998; 9: 529-539Crossref PubMed Scopus (109) Google Scholar The presence of plasmin activity during wound repair was analyzed in lysates prepared from wound tissue. Wounds were harvested at 1, 3, 5, 8, and 14 days after wounding; an area of 8 mm in diameter, which includes the complete epithelial margins, was excised at each time point. Wound tissue was immediately frozen in liquid nitrogen and stored at −80°C until use. The frozen tissue was powdered and resuspended in 200 μl of buffer (50 mmol/L Tris, pH 7.8). The tissue was disrupted by sonication for 10 seconds at 50 W and the suspension was centrifuged (5 minutes at 15,000 × g). Plasmin activity was quantified in the supernatant using a fluorescent assay based on a plasmin-specific substrate; the difference in fluorescence between the substance formed by plasmin cleavage and the original nonfluorescent substrate is determined (H-D-Val-Leu-Lys-AMC) (Bachem, Heidelberg, Germany). Lysates generated from individual wounds were analyzed. To determine substratedegrading enzymes, α2-antiplasmin (0.7 IU/ml) (Sigma, Deisenhof, Germany) was added. Plasmin activity was normalized to total protein concentration. SDS-PAGE was performed following the protocol of Laemmli. VEGF165 variants (5 ng) expressed in COS-1 cells were incubated at 37°C with wound tissue lysates obtained from db/db mice 3, 5, 8, and 14 days after injury. Reactions were terminated by the addition of Pefabloc (1 mmol/L) and frozen at −20°C. To identify VEGF165-degrading enzymes, α2-antiplasmin (0.7 IU/ml) was added to wound lysates 30 minutes before VEGF165 protein incubation. For Western blotting fragments were resolved on 12% nonreducing SDS-PAGE gels and transferred to nitrocellulose (Hybond C-Super, Amersham Biosciences). VEGF165 amino terminal integrity was determined by detecting immunoreactive products with a polyclonal rabbit antibody that recognizes the amino-terminal VEGF epitopes (raised against a 20-amino acid amino-terminal peptide; Santa Cruz Biotechnology, Santa Cruz, CA). Detection of intact and VEGF165-derived degradation products was accomplished using the LumiLight plus chemiluminescence detection system (Roche Diagnostics, Mannheim, Germany). Densitometric analysis of Western blots was performed using a FluroS-Multiimager and Quantity One analysis software (Bio-Rad). All immunofluorescent staining was performed on 5 μm cryosections; particular caution was taken to store and treat all tissues and tissue sections equally. Terminal dUTP nick-end labeling (TUNEL) staining was performed with a commercial kit (DeadEnd Fluorometric TUNEL System; Promega, Madison, WI) following the instructions of the manufacturer. After TUNEL staining sections were fixed in paraformaldehyde (1% in PBS) and rinsed, and immunodetection of CD31 was performed as described above. Bound primary antibody was detected using an Alexa Fluor 594-conjugated polyclonal goat anti-rat antibody (1 hour, 1:500) (Molecular Probes, Leiden, The Netherlands). As a control for positive TUNEL staining the TDT enzyme was omitted. Immunofluorescent microscopy was conducted at ×600 magnification (Eclipse 800E; Nikon, Düsseldorf, Germany); images were captured with a digital camera (DXM1200 F; Nikon) and Imaging Software Lucia G4.8. Endothelial cells were identified by red fluorescence, and DNA fragmentation was detected by localized green fluorescence within the nucleus of apoptotic cells. Apoptotic endothelial cells were counted in three individual fields per wound, and serial sections were analyzed (CMV-VEGF165-Wt wounds, n = 4; CMV-VEGF165A111P wounds, n = 4). To detect the presence of endothelial cells and pericytes, immunodetection of CD31 was performed as described above; detection of α-smooth muscle actin (α-SMA) or desmin for pericytes was performed using an antiserum Cy3-conjugated murine anti-α-SMA (1 hour, 1:200) (Sigma) or a monoclonal IgG1 mouse anti-desmin (24 hours at 4°C, 1:200) (Dako Cytomation GmbH, Hamburg, Germany). Bound primary antibody was detected using an Alexa Fluor 488-conjugated polyclonal goat anti-mouse IgG1 antibody (1 hour, 1:500) (Molecular Probes); double fluorescence was anal
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