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Expression and Proteolysis of Vascular Endothelial Growth Factor is Increased in Chronic Wounds

Gereon Lauer, Stephan Sollberg, Melanie Cole, Ingo Flamme,* JoÈrg StuÈrzebecher,² Karlheinz Mann,³ Thomas Krieg, and Sabine A.Eming Department of Dermatology, University of Cologne, Cologne, Germany; *Center for Molecular Medicine, Cologne, Germany; ²Center for Vascular Biology and Medicine, Friedrich-Schiller-University Jena, Erfurt, Germany; ³Max-Planck-Institute of , Martinsried, Germany

Degradation of angiogenic mediators might be an wounds and psoriasis. To analyze whether this underlying cause of chronic wounds. To test this apparent decrease in immunoreactivity could be the hypothesis, we evaluated the expression and integrity result of degradation of vascular endothelial growth of vascular endothelial growth factor, a potent angio- factor by proteolytic activities from the wound genic mediator, and its receptors, Flt-1 and KDR, in environment, we examined the stability of recombi- chronic venous leg ulcerations. Immunohisto- nant vascular endothelial growth factor in wound chemical, in situ hybridization, and semiquantitative ¯uid from chronic leg ulcers. As demonstrated by reverse transcriptase polymerase chain reaction ana- polyacrylamide gel electro- lyses all indicate that expression of vascular endothe- phoresis, incubation of rVEGF165 with chronic, but lial growth factor is elevated in ulcerative tissue, with not acute, wound ¯uid resulted in rapid proteolytic vascular endothelial growth factor mRNA being degradation of rVEGF165. inhibitor studies especially pronounced in the hyperplastic epithelium indicate that , such as , are of the wound margin. Flt-1 and KDR and involved in this degradation. Together, our data mRNA were detected in the papillary vessels in close show that, although vascular endothelial growth fac- vicinity to the lesional epithelium of chronic tor expression is elevated in chronic wounds, wounds. Although increased expression of vascular increased proteolytic activity in this environment endothelial growth factor protein was detected in the results in its degradation, which may contribute to epidermis, the intensity of this staining was weak an impaired wound healing response. Key words: plas- compared with the epidermal staining in psoriatic min/serine proteases/VEGF/wound healing. J Invest lesions and compared with the strong vascular Dermatol 115:12±18, 2000 endothelial growth factor mRNA signal in chronic

uring cutaneous wound healing, the development of edge of this patient population documents endothelial alterations, granulation tissue requires the formation of new indicating endothelial cell damage that might lead to slow and capillaries, and the absence of this tissue is a typical insuf®cient capillary growth (Leu, 1991; Herrick et al, 1992; Scelsi feature of chronic wounds such as decubitus, et al, 1994; Leu et al, 1995). Ddiabetic, and venous ulcers.The development of A number of different growth factors are thought to stimulate new blood vessels () is tightly regulated by factors that angiogenesis during wound healing, including those that in addition act by either stimulating or inhibiting vessel growth (reviewed in to acting on endothelial cells also stimulate mesenchymal/epithelial Risau, 1997).Disturbance of this dynamic process may in¯uence cells, such as members of the ®broblast growth factor family (aFGF, the development of chronic wounds. bFGF), transforming growth factor a, and platelet-derived growth Little is known about local mechanisms leading to epidermal/ factor.In addition, other growth factors are also involved that are dermal injury causing and/or perpetuating wound healing failure thought to be largely speci®c for the vascular endothelium, because (reviewed in Falanga, 1993).In chronic venous insuf®ciency, the expression of their receptors is mainly restricted to endothelial cells. major cause for chronic leg ulcers, functional and organic changes These include vascular endothelial growth factor (VEGF) and its involving the macrocirculatory and microcirculatory system have relatives and angiopoietins (reviewed in Risau, 1997).Little is known been de®ned, which ultimately might lead to tissue and about the role of these endothelium-speci®c growth factors during tissue damage (Franzeck et al, 1984; Luetolf et al, 1993).Functional wound healing.Recently it has been shown that VEGF is critically and ultrastructural analysis of the microcirculation in the wound involved in cutaneous angiogenesis in normal and pathologic conditions (reviewed in Dvorak et al, 1995).This endothelial-cell- speci®c mitogen is a homodimer of which six distinct splice variants Manuscript received August 26, 1999; revised April 13, 2000; accepted have been described (VEGF 121, 145, 165, 183, 189, 206) (Houck for publication April 25, 2000. Reprint requests to: Dr.Sabine A.Eming, Department of Dermatology, et al, 1991; Tischer et al, 1991; Lei et al, 1998), with the 165-amino- University of Cologne, Joseph-Stelzmann-Straûe 9, 50924 Cologne, acid-long VEGF165 representing the major isoform found in human Germany. Email: [email protected] tissues.During normal wound healing (Brown et al, 1992; Frank et al, Abbreviation: VEGF, vascular endothelial growth factor. 1995; Fukumura et al, 1998; Nissen et al, 1998) and during the

0022-202X/00/$15.00 ´ Copyright # 2000 by The Society for Investigative Dermatology, Inc. 12 VOL.115, NO.1 JULY 2000 VEGF IN CHRONIC WOUNDS 13 development of pathologic processes such as psoriasis, allergic contact and then blocked with 3% bovine serum in phosphate-buffered dermatitis, and various bullous diseases, VEGF expression correlated saline (PBS) to reduce nonspeci®c antibody binding.Next, sections were both temporally and spatially with the proliferation of new blood incubated (1 h, 37°C) with an anti-VEGF polyclonal rabbit antibody (raised vessels (Brown et al, 1994, 1995a, b).In these studies, VEGF mRNA against either a 20-amino-acid amino-terminal VEGF or a peptide corresponding to amino acids 1±192 with a deletion from amino acids 142± and protein were localized to epidermal keratinoctyes and in®ltrating 185) (1:250, Santa Cruz Biotechnology, Santa Cruz, CA) or with .In addition, the receptors for VEGF, Flt-1 and KDR, monoclonal antisera against Flt-1 and KDR (1:50, kindly provided by were detected in dermal capillaries and macrophages.Recent in vivo Dr.H.Weich).Bound primary antibodies were detected using the alkaline studies supported the hypothesis that epidermally derived VEGF can phosphatase-antialkaline phosphatase complex (APAAP, DAKO, stimulate angiogenesis in a paracrine fashion by demonstrating Hamburg, Germany).Neufuchsin red was used as a substrate and increased microvascular density in the skin of VEGF transgenic mice sections were counterstained with Hemalaun.Where indicated, primary (Detmar et al, 1998).Finally, in a diabetic mouse model characterized antibodies were omitted and replaced by an irrelevant isotype-matched by a severe delay in wound healing, VEGF mRNA was dramatically rabbit antibody as a control for speci®city. decreased during the repair process (Frank et al, 1995).Together, these data indicate that VEGF expression and activity are critical In situ hybridization In situ hybridization was performed on regulators of wound repair. paraformaldehyde-®xed cryosections as described (chronic wounds, n = 4; psoriasis, n = 2; normal skin, n = 2).Brie¯y, sections (5 mm) were Several studies have shown that wound ¯uid obtained from hybridized at 48°C for 18 h with 25 3 103 cpm per mlof35S-dUTP chronic leg ulcers contains increased , labeled antisense or sense RNA.The VEGF cDNA fragment (517 bp) used , and plasmin activity compared with ¯uid from acute to generate these probes contains sequences common to all VEGF isoforms wounds (Palolathi et al, 1993; Weckroth et al, 1996; Yager et al, (Flamme et al, 1998).After hybridization, slides were washed under 1996; Vaalamo et al, 1997).In addition, the activity of protease stringent conditions, including a 2 h wash in wash buffer (50% formamide, inhibitors such as a1-proteinase inhibitor, tissue inhibitor of 2 3 sodium citrate/chloride buffer, 10 mM 2-mercaptoethanol), a 15 min metalloproteinase-1, and a2-macroglobulin has been shown to wash in RNase buffer [0.5 M NaCl, 10 mM Tris pH 7.5, 1 mM decrease under these conditions (Bullen et al, 1995; Rao et al, 1995; ethylenediamine tetraacetic acid (EDTA)], a 15 min digest with RNase A Grinnell and Zhu, 1996).Therefore, the failure of certain wounds (Boehringer, Mannheim, Germany; 20 mg per ml in RNase buffer), followed by a 15 min wash in RNAse buffer and overnight wash in to resolve successfully may be due to the increased proteolytic 5 mM dithiothreitol in wash buffer.All washing steps were performed at degradation of regulatory factors (Grinnell et al, 1992; Wlaschek 37°C in a water bath with gentle agitation.Slides were then processed for et al, 1997; Hoffman et al, 1998). autoradiography.The photographic emulsion was developed after 14 d and In this study, we evaluated the expression of VEGF in chronic the tissue sections were counterstained with hematoxylin. wounds and assessed whether the loss of VEGF activity might play a role in wound healing failure.Our results clearly indicate that the Reverse transcriptase polymerase chain reaction (RT-PCR) Total expression of VEGF and VEGF receptors, Flt-1 and KDR, are RNA was isolated from skin biopsies using the RNeasy kit according to the upregulated in chronic wounds compared with uninjured control manufacturers' instructions (Qiagen, Hilden, Germany).To generate skin of healthy individuals.In addition, evidence is presented that cDNA, RNA was reverse-transcribed using Superscript reverse serine proteases, in particular plasmin, are involved in the transcriptase and oligo-dT primers ( Technologies, Gibco BRL, degradation of VEGF in the chronic wound environment.Our Eggenstein, Germany).Portions of 1/10 and 1/40 volume of the ®rst data establish an important link between protease activity and strand synthesis reaction were then ampli®ed by 30 cycles for VEGF and 1/ 10 volume of the ®rst strand synthesis reaction was ampli®ed by 32 cycles growth factor instability as a contributing factor in chronic wound for Flt-1 and KDR-1 by PCR adding 2.5 U Taq-polymerase (Qiagen) and healing failure. primers speci®c for either VEGF (EMBL, Accession: M32977) (5¢-GAG TCC AAC ATC ACC ATG CAG-3¢,5¢-TCA CCG CCT TGG CTT MATERIALS AND METHODS CTC ACA-3¢) (sequence exon 4±8), GAPDH (EMBL, Accession: Biopsies Biopsies were taken by consent from patients presenting M33197) (5¢-TCA TGA CCA CAG TCC ATG CCA TCA-3¢,5¢- chronic ulcera crura (n = 8) of more than 6 mo duration, due to primary GCC AAA TTC GTT GTC ATA CCA GGA AAT GA-3¢), Flt-1 or secondary venous insuf®ciency (size of ulcer > 5 3 5cm2; mean age of (EMBL, Accession: X51602) (5¢-CAC CTT GGT TGT GGC TGA CTC patients 67 y).The biopsies were obtained from the wound edge of chronic TAG-3¢,5¢-CAA GTG ATC TGA GGC TCG GGG AC-3¢) (sequence of wounds and showed no clinical sign of infection.Biopsies taken from Ig-like domain 7), or KDR (EMBL, Accession: X61656) (5¢-GCC ACC uninjured skin of healthy volunteers (n = 4) and skin of psoriatic lesions ATG TTC TCT AAT AGC AC-3¢,5¢-CAC TGC ATG CCT GGC (n = 6) served as negative and positive control, respectively.Biopsies were AGG TGT AG-3¢) (sequence of Ig-like domain 7) (MWG-Biotech, embedded in OCT compound (Tissue Tek, Miles, IN), immediately Ebersberg, Germany) using a Thermocycler (Trio-Thermocycler frozen in liquid nitrogen, and stored at ±80°C. Biometra, GoÈttingen, Germany).The speci®city of the RT-PCR products was veri®ed by DNA sequencing.RT-PCR products were Wound ¯uid Chronic wound ¯uid was obtained from the same patients resolved through 2% TBE-agarose gels and transferred to nitrocellulose from whom biopsies were taken, in addition to seven other patients (Hybond-N, Amersham, Braunschweig, Germany).Southern suffering from chronic venous leg ulcers.For this purpose ulcers were hybridization was done using a random-primed 32P-dCTP-labeled VEGF covered with a semipermeable polyurethane ®lm (Hyalo®lm, Hartmannn, probe (Boehringer).The intensity of the PCR product was quanti®ed by Heidenheim, Germany) for a maximum of 8 h.Acute wound ¯uid was scanning densitometry. obtained from mesh-graft donor sites of patients undergoing split-thickness skin grafting (1±4 d postoperatively the wound was covered with a Sodium dodecyl sulfate polyacrylamide (SDS- polyurethane ®lm) and from patients undergoing surgical mastectomy (1± PAGE) immunoblotting SDS-PAGE was performed following the 6 d postoperatively by drainage).After collection, ¯uids were centrifuged protocol of Laemmli.Recombinant human VEGF 165 (rVEGF165) (10 min, 13,000 3 g,4°C) to remove insoluble material, and supernatants produced by a baculovirus/insect cell system (R&D Systems, were frozen at ±80°C until use. Minneapolis, MN) was incubated at 37°C with either acute (n = 8) or chronic (n = 8) wound ¯uid or, as a negative control, with PBS.At the -linked immunosorbent assay (ELISA) The level of VEGF indicated times, reactions were terminated by the addition of Pefabloc in wound ¯uid was determined using a commercially available ELISA that (1 mM in sample buffer), and fragments were resolved on 12% nonreducing detects the soluble isoforms of human VEGF (R&D Systems, Minneapolis, SDS-PAGE gels and transferred to nitrocellulose (Hybond C-super, MN).VEGF levels were normalized to the total protein concentration, Amersham).rVEGF 165 integrity was determined by detecting which was determined by the Bradford procedure (Bio-Rad Protein Assay, immunoreactive products with a polyclonal rabbit antibody that Bio-Rad, MuÈnchen, Germany). recognizes amino-terminal VEGF (Santa Cruz Biotechnology). Detection of intact and VEGF-derived degradation products was Immunohistochemistry To process tissue sections for the accomplished using the enhanced chemiluminescence Western blot immunodetection of VEGF (chronic wound, n = 8; psoriasis, n = 6; detection system (ECL, Amersham).The detection limit of the Western normal skin, n = 4) and its receptors Flt-1 and KDR (chronic wound, blot was approximately 1 ng per lane.For proteolysis studies 200 ng n = 5; normal skin, n = 4), 5 mm cryosections were ®xed in acetone, rinsed, rVEGF165 were incubated with 40 ml of wound ¯uid or PBS.With a 14 LAUER ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

loading maximum of 2 ml per slot 10±15 ng rVEGF165 were loaded per we performed immunohistochemistry studies of cryosections of lane.To determine potentially important VEGF-degrading , normal skin and chronic ulcerated wounds caused by primary or protease inhibitor studies were performed using commercially available secondary venous insuf®ciency.Consistent with previous studies Antipain-dichloride (10±100 mg per ml), a2-antiplasmin (1±2.5 U per ml) the staining of VEGF was weak in normal human epidermis (kindly provided by Dr.Klaus Preissner), E-64 (0.1±20 mg per ml in (Fig 1A).In contrast, VEGF staining was increased in basal and ethanol), EDTA-Na2 (0.2±5 mM), elastatinal (5±500 mM in acetic acid), leupeptin (0.05±2 mg per ml), Pefabloc (0.2±10 mM), phenylmethylsulfonyl suprabasal layers of the hyperplastic epidermis of chronic ulcers (Fig ¯uoride (PMSF) (0.2±5 mM in isopropanol), Pepstain (0.1±5 mg per ml in 1B).Its overall expression was not as high as in lesional psoriatic methanol), phenanthroline (0.2±20 mM), and TLCK (1.5±100 mg per ml), epidermis, however, where the strongest VEGF signal was found, either individually or in combination (Boehringer).In addition we tested a as previously described (Fig 1D) (Detmar et al, 1994).Identical recently developed synthetic plasmin inhibitor PL-I (Na-2- ®ndings were obtained using two different rabbit antisera directed naphthylsulfonyl-3-amidino-(D,L)-phenylalanyl-nipecotic acid- against distinct VEGF epitopes.Occasionally, in chronic wounds thiophene-2-methylamide hydrochloride) (0.8 mg per ml in ethanol) (Fig 1C) and psoriatic lesions (Fig 1E), dermal mononuclear (StuÈrzebecher et al, 1995; 1996).As a positive control, proteolysis of in®ltrating cells, judged to be macrophages by light microscopic rVEGF165 was performed with human plasma plasmin (Sigma, Deisenhofen, Germany) at a concentration of 0.01 U per ml at 37°C for criteria, stained weakly for VEGF. 4h. To investigate the levels of mRNA expression of VEGF in human chronic wounds, in situ hybridization studies of chronic Protein sequencing For amino-terminal sequencing the PAGE- wounds, psoriatic lesions, and normal skin were performed using a separated fragments were blotted onto Immobilan-P (Millipore, Bedford, 35S-labeled riboprobe speci®c for VEGF.In line with previous MA) according to Matsudaira (1987).Sequencing was done using the observations normal skin epidermal keratinocytes were found to Applied Biosystems Procise 492 sequencer. express low levels of VEGF (Fig 1F).In contrast, levels of mRNA Detection of plasmin activity To verify the presence of plasminogen for VEGF in chronic wounds were relatively strong in the and plasmin activity in wound ¯uids, ¯uids were analyzed using a epidermis (Fig 1G) and were comparable to that of lesional chromogenic assay speci®c for plasmin activity (Chromogenix, MoÈhndal, epithelium in psoriasis (Fig 1I).Hybridization with the VEGF- Sweden).This technique is based on the difference in adsorbance between sense control showed no signal in chronic wounds (Fig 1H), the substance formed (pNA) by plasmin cleavage and the original psoriasis (Fig 1J), and normal control skin (data not shown). chromogenic substrate S-2251. To con®rm the increased levels of mRNA for VEGF in chronic wounds a semiquantitative RT-PCR analysis was performed using RESULTS total RNA isolated from skin samples of chronic wound margins as VEGF expression is upregulated in chronic leg ulcers To well as from normal and psoriatic skin.Southern blot analysis of the assess the distribution of VEGF protein in human chronic wounds, RT-PCR products was consistent with increased levels of three

Figure 1. VEGF is expressed in chronic wounds. Analysis of VEGF expression by immunohistochemistry (A-E), in situ hybridization (F, J), and RT- PCR analysis (K) in uninjured skin (A, F), chronic wounds (B, C, G, H), and psoriasis (D, E, I, J).Note the weak VEGF expression in suprabasal layers of the epidermis in chronic wounds (B) in comparision with epidermal staining in psoriasis (D); in chronic wounds (C) and psoriatic lesions (E), macrophages stained positive for VEGF (arrow).VEGF mRNA was strongly expressed in the epithelium of chronic wounds (G ) and psoriatic lesions (I).Hybridization of chronic wounds (H) or psoriatic lesions (J) with the VEGF-sense control showed no signal.(K ) RT-PCR speci®c for VEGF.Total RNA was isolated from uninjured skin (lanes 1, 2), psoriatic lesions (lanes 3, 4), and chronic wounds (lanes 5, 6).Southern blotting of the RT-PCR products revealed the increased expression of three major VEGF splice variants VEGF121, VEGF165, and VEGF189 in psoriasis and chronic wounds.The RT-PCR was performed with two different amounts of RT product: lines 1, 3, 5, 1/40; lines 2, 4, 6, 1/10.RT-PCR of GAPDH served as a positive control.E, epidermis; D, dermis. Scale bar: 100 mm. VOL.115, NO.1 JULY 2000 VEGF IN CHRONIC WOUNDS 15

major VEGF splice variants, VEGF121, VEGF165, and VEGF189,in incubated in chronic wound ¯uid, the protein, which was initially chronic wounds and psoriatic lesions compared with normal skin identi®ed as a 42 kDa band, underwent a degradation with (Fig 1K). increasing incubation time to a 38 kDa protein band (Fig 3).In addition, prolonged incubation of VEGF165 in chronic wound ¯uid Flt-1 and KDR are expressed in chronic leg ulcers To for 8 h resulted in the loss of both the 42 and 38 kDa protein bands, analyze the expression of VEGF receptors on mRNA level, total suggesting further proteolytic degradation of rVEGF . RNA was isolated from chronic ulcers, uninjured skin, and 165 psoriatic lesions and an RT-PCR analysis for Flt-1 and KDR was Protease inhibitors enhance rVEGF stability in chronic performed (Fig 2A).The primers were chosen such that KDR and wound ¯uid In order to characterize the proteases in chronic Flt-1 ampli®cation products were 385 and 522 bp, respectively. wound ¯uid involved in the degradation of rVEGF165, rVEGF165 Expression of KDR and Flt-1 was demonstrated in both chronic was incubated with chronic wound ¯uid in the presence of various wounds and psoriatic lesions.To further examine the distribution protease inhibitors.Degradation of VEGF 165 in chronic wound of the VEGF receptors in chronic wounds, immunohistochemistry ¯uid could be signi®cantly inhibited by the addition of either studies were performed.The expression of Flt-1 and KDR Pefabloc or PMSF, two inhibitors of serine proteases (Fig 4).In receptors was increased in papillary dermal microvessels near the contrast, inhibitors of (EDTA, phenanthroline), ulcer edge (Fig 2B, E).In contrast, virtually no expression of these of cystein proteinases (elastatinal, leupeptin, E-64, TLCK, VEGF receptors was detected in capillaries distant to the ulcer edge Antipain), and of acidic proteases (Pepstatin) had no effect on the (Fig 2C, F).In addition, rare mononuclear cells, presumably proteolytic degradation of rVEGF165.These results strongly in®ltrating macrophages, stained positive for Flt-1 (Fig 2D). indicate that serine proteases are involved in rVEGF165 degradation in the chronic wound environment. rVEGF165 is degraded in chronic wound ¯uid As wound ¯uid re¯ects the microenvironment of the wounds from which it is Plasmin is involved in the proteolysis of rVEGF165 in collected, its study may provide important clues to the events chronic wound ¯uid Plasmin is a major extracellular occurring during wound repair.The presence of endogenous proteinase that mediates controlled breakdown of ®brin and VEGF in wound ¯uid, however, was demonstrated by ELISA at various extracellular .In accordance with previous levels too low to be detected by Western blotting (acute wound studies, acute and chronic wound ¯uid contained plasminogen and ¯uid 1.8 6 1.2 ng per ml, n = 10; chronic wound ¯uid plasmin activity as assessed by a speci®c chromogenic assay (data not 10.3 6 2.3 ng per ml, n = 15). To evaluate the hypothesis that shown).Incubation of rVEGF 165 in the presence of plasmin VEGF protein is proteolytically degraded in the chronic wound resulted in the degradation of the 42 kDa rVEGF165 protein to a environment, rVEGF165 was incubated in wound ¯uid obtained fragment of approximately 38 kDa, as indicated by Western blotting from either chronic ulcers or acute wounds.SDS-PAGE analysis of (Fig 5A).The 38 kDa protein band had exactly the same rVEGF165 following an 8 h incubation in either acute wound ¯uid electrophoretic mobility as the proteolytic product obtained or PBS showed that the protein migrated with an approximate following incubation of rVEGF165 with chronic wound ¯uid molecular weight of 42 kDa, indicating that the recombinant (Fig 5A).To further support the idea that the degradation of protein is stable (Fig 3).In contrast, when rVEGF 165 was rVEGF165 was due to the action of plasmin, a2-antiplasmin and a

Figure 2. Flt-1 and KDR mRNA is expressed in chronic leg ulcers. Analysis of VEGF recep- tor expression by RT-PCR analysis (A) and by immunohistochemistry (B-F).Representative RT- PCR analysis of total RNA extracted from normal skin (lanes 2, 6, 10), psoriasis (lanes 3, 7, 11), chronic wounds (lanes 4, 8, 12), and negative con- trol without cDNA (lanes 1, 5, 9).Primers were chosen such that the PCR products of Flt-1 and KDR were 522 bp and 385 bp, respectively.Ethi- diumbromide agarose gel electrophoresis demon- strates the ampli®cation products of the expected size.RT-PCR of GAPDH served as standard. Small capillaries in the papillary dermis next to the ulcer edge show strong expression of Flt-1 (B) and KDR (E), in contrast to virtually no expres- sion in the papillary dermis distant to the ulcer edge (C, F).Occasionally, dermal in®ltrating mononuclear cells, most probably macrophages, stain positive for Flt-1 (D, arrows).E, epidermis; D, dermis. Scale bar:50mm. 16 LAUER ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Figure 3. rVEGF165 is degraded in chronic wound ¯uid. rVEGF165 Figure 4. Protease inhibitors enhance rVEGF stability in chronic was incubated in wound ¯uid harvested from chronic (cWF) and acute 165 wound ¯uid. rVEGF165 was incubated (8 h) in chronic wound ¯uid with (aWF) wounds for time periods as indicated; rVEGF165 degradation was increasing concentrations of EDTA or Pefabloc as indicated, in mM; monitored by SDS-PAGE under nonreducing conditions. rVEGF165 degradation was monitored by SDS-PAGE under nonreducing conditions. synthetic inhibitor of plasmin, PL-I, were added to the reaction mixture. a2-antiplasmin and PL-I were found to partially inhibit the degradation of rVEGF165 in chronic wound ¯uid, even after an 8 h incubation (Fig 5B, C).In addition we could demonstrate that plasmin digest yields four fragments of rVEGF165 as indicated by SDS-PAGE analysis and amino-terminal sequencing of the fragments obtained.Amino-terminal sequencing of the fragments showed the sequence -ARQENP(C)GP, identifying one of the plasmin cleavage sites as Arg110/Ala111, as previously reported (Keyt et al, 1996).Other cleavage sites were between Arg123/ Arg124, Lys125/His126, and Lys147/Ala148.These four carboxy- terminal cleavage products were connected by disul®de bonds and thus appeared in one band after PAGE under nonreducing conditions.The amino-terminal fragment VEGF1±110 could be revealed as a 38 kDa fragment by Western blotting.These observations strongly suggest that plasmin is potentially involved in the proteolytic degradation of VEGF in chronic wounds.

DISCUSSION In this study we have evaluated the role of VEGF in chronic wound healing failure.Our immunohistochemistry and in situ hybridization studies as well as semiquantitative RT-PCR analyses provide strong evidence that the expression of VEGF is upregulated in chronic venous leg ulcerations.The levels of mRNA for VEGF appeared to be increased in the hyperplastic epithelium of the wound margin of the ulcer.Expression of both VEGF receptors, Flt-1 and KDR, is also upregulated in the papillary vessels in close vicinity to the lesional epithelium of chronic wounds as demonstrated by immunohistochemistry and RT-PCR studies. Recent animal studies suggest a temporal and spatial correlation between the expression of VEGF and its receptors in cutaneous wound healing (Brown et al, 1992; Frank et al, 1995; Fukumura et al, 1998).Consistent with and extending these observations, our results indicate that upregulation of both VEGF and its receptors is a feature of chronic wounds caused by venous insuf®ciency in humans.Several epidermal and dermal factors have been shown to enhance VEGF expression in vitro, including transforming growth factor a (Detmar et al, 1995), epidermal growth factor (Detmar et al, 1995; Frank et al, 1995), transforming growth factor b (Frank et al, 1995), keratinocyte-derived growth factor (Frank et al, 1995), tumor factor a (Frank et al, 1995), and scatter factor (HGF/SF) (Gille et al, 1998).These factors, which are also expressed during cutaneous wound healing, might thus serve as Figure 5. Limited proteolysis of rVEGF165 by plasmin. (A) rVEGF165 autocrine and paracrine regulators of VEGF expression and might was incubated with plasmin or chronic wound ¯uid (cWF) for increasing contribute to the upregulation of VEGF at chronic wound sites.In time periods as indicated.rVEGF 165 degradation was monitored by SDS- addition, hypoxia, which is a characteristic feature of the skin close PAGE under nonreducing conditions.Incubation of rVEGF 165 in chronic to chronic venous leg ulcerations, might act as a potent inducer of wound ¯uid in the presence of increasing concentrations of (B) a2- VEGF expression at the wound edge (Detmar et al, 1997). antiplasmin (AP) or (C) PL-I for increasing time periods as indicated. VOL.115, NO.1 JULY 2000 VEGF IN CHRONIC WOUNDS 17

Most remarkable was the ®nding that, despite signi®cant bacteria may contribute to proteolysis in the leg ulcer by facilitating increased levels of mRNA for VEGF in the epithelial rim of generation of plasmin on their surface or by producing various chronic venous wounds, the immunoreactivity for VEGF protein proteinases (Kuusela and Saksela, 1990; Kuus et al, 1992). appeared to be relatively reduced compared to that observed in Little is known about the biologic signi®cance of the interaction lesional psoriatic skin in which levels of mRNA for VEGF were between VEGF and proteases such as plasmin in vivo.Limited also strongly increased.These ®ndings raise the intriguing proteolysis of the larger VEGF splice variants VEGF165 and possibility that in the chronic wound environment at the wound VEGF189 by plasmin has been shown to remove the carboxy- margin the regulation of VEGF expression is disturbed as a result of terminal domain (Houck et al, 1992; Keyt et al, 1996; Plouet et al, either defective VEGF or intracellular/extracellular 1997).As this region is involved in binding of VEGF to degradation of VEGF.The possibility that antigen epitopes are extracellular molecules and its binding to the recently described masked in chronic wounds is unlikely, as identical results were neuropilin-1 receptor, it is possible that limited proteolysis of obtained using two different antisera directed against distinct VEGF VEGF alters its ability to diffuse in the tissue or its biologic activity epitopes.As immunohistochemistry and in situ hybridization do not as angiogenic signal.Recent studies have indicated that proteolysis allow quantitative analysis of VEGF expression, the data were of rVEGF165 by plasmin results in a nonheparin-binding isoform further supported by semiquantitative RT-PCR analysis.The VEGF110 and a heparin-binding fragment VEGF111±165 (Keyt et al, results obtained with these techniques prompted us to focus on the 1996).Whereas the af®nity of VEGF 110 to the VEGF receptors Flt- degradation pathway of VEGF in chronic wounds. 1 and KDR is only modestly decreased compared to that of rVEGF , its endothelial cell mitogenic potency is substantially The results of our incubation time-course of rVEGF165 with 165 chronic wound ¯uid demonstrate that this protein was ®rst cleaved reduced.There is recent evidence that impaired binding of to a 38 kDa intermediate fragment, which is lost after prolonged VEGF110 to the VEGF receptor neuropilin-1 might be responsible incubation.To demonstrate that enzymatic proteolysis of VEGF is for this decrease in mitogenicity (Soker et al, 1997, 1998). involved in VEGF instability in the chronic wound ¯uid, we Neuropilin-1 appears to act as a coreceptor that enhances VEGF binding to KDR and VEGF mitogenic and chemo- performed protease inhibitor studies.The proteolysis of rVEGF 165 165 165 upon incubation with chronic wound ¯uid could be inhibited by tactic activity.These studies suggest that proteolysis of VEGF by inhibitors, such as PMSF and Pefabloc.These plasmin represents a means by which the spatial distribution of ®ndings strongly indicate that chronic wound ¯uid contains VEGF fragments with distinct mitogenic potency is regulated.The enzymatic activity, in particular serine proteases, which might knowledge of the VEGF±plasmin interaction in wound healing affect VEGF stability. failure might be relevant for other processes in which extracellular To further de®ne the proteolytic enzyme involved in VEGF matrix-degrading enzymes as well as VEGF have been implicated, degration in chronic wound ¯uid, we tested additional protease including tumorinvasion, metastasis, and in¯ammation. inhibitors more speci®c for single proteases.These experiments and In conclusion, our ®ndings provide support to the idea that previous studies (Palolathi et al, 1993) strongly suggest that plasmin protease±cytokine interactions are important in the regulation of activity is increased in chronic wound ¯uid.Next we investigated angiogenesis during wound repair.Strategies aimed at controlling the ability of plasmin, a serine protease, to digest rVEGF .Our and modulating the proteolytic activity in chronic wounds may 165 constitute a novel approach for the treatment of wound healing results demonstrate that rVEGF165 is susceptible to proteolysis by plasmin, and amino-terminal sequencing of the fragments identi®ed failure. several plasmin cleavage sites.Following the by plasmin or chronic wound ¯uid VEGF165 appeared as a single protein band of 38 kDa by SDS-PAGE analysis.In line with these observations, We gratefully acknowledge Herbert Weich for providing the monoclonal antisera for a2-antiplasmin and PL-I, speci®c inhibitors for plasmin, could VEGF receptors and Klaus Preissner for providing a2-antiplasmin. We thank Magnus Agren for providing samples of acute wound ¯uid of split-thickness donor partially protect rVEGF165 against degradation in the chronic wound ¯uid.These ®ndings indicate that plasmin is one of the sites, Eckhard Lechler for help with the quantitation of plasmin activity in wound ¯uids, and Angelika Arora for help with the histologic preparations. We thank Luca serine proteases involved in the degradation of rVEGF165 in the chronic wound environment. Borradori and Barry Sudbeck for critically reading the manuscript. This work was supported by the German Research Society (DFG) (S.S. and S.A.E.) and in part The proteolytic degradation of rVEGF165 upon incubation with chronic wound ¯uid seems to be speci®c, as incubation with either by the Research Program KoÈln Fortune (S.A.E.) and Nolting Foundation acute wound ¯uid or PBS did not result in VEGF degradation.Our (S.A.E.). ®ndings provide evidence, however, indicating that plasmin activity is signi®cantly increased in chronic wound ¯uid compared with that REFERENCES in acute wound ¯uid.Thus it is possible that the plasmin activity present in acute wound ¯uid is too low to affect rVEGF .These Brown LF, Yeo KT, Berse B, Yeo TK, Senger DR, Dvorak HF, Van De Water L: 165 Expression of VEGF by epidermal keratinocytes during wound healing. 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