Gereon Lauer, Stephan Sollberg, Melanie Cole, Thomas Krieg, Sabine A

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Expression and Proteolysis of Vascular Endothelial Growth Factor is Increased in Chronic Wounds Gereon Lauer, Stephan Sollberg, Melanie Cole, Thomas Krieg, Sabine A. Eming Journal of Investigative Dermatology Volume 115, Issue 1, Pages 12-18 (July 2000) DOI: 10.1046/j.1523-1747.2000.00036.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

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 specific 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 μm. Journal of Investigative Dermatology 2000 115, 12-18DOI: (10.1046/j.1523-1747.2000.00036.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Flt-1 and KDR mRNA is expressed in chronic leg ulcers. Analysis of VEGF receptor 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 control 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. Ethidiumbromide agarose gel electrophoresis demonstrates the amplification 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 expression in the papillary dermis distant to the ulcer edge (C, F). Occasionally, dermal infiltrating mononuclear cells, most probably macrophages, stain positive for Flt-1 (D, arrows). E, epidermis; D, dermis. Scale bar: 50 μm. Journal of Investigative Dermatology 2000 115, 12-18DOI: (10.1046/j.1523-1747.2000.00036.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 rVEGF165 is degraded in chronic wound fluid. rVEGF165 was incubated in wound fluid harvested from chronic (cWF) and acute (aWF) wounds for time periods as indicated; rVEGF165 degradation was monitored by SDS-PAGE under nonreducing conditions. Journal of Investigative Dermatology 2000 115, 12-18DOI: (10.1046/j.1523-1747.2000.00036.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Protease inhibitors enhance rVEGF165 stability in chronic wound fluid. rVEGF165 was incubated (8 h) in chronic wound fluid with increasing concentrations of EDTA or Pefabloc as indicated, in mM; rVEGF165 degradation was monitored by SDS-PAGE under nonreducing conditions. Journal of Investigative Dermatology 2000 115, 12-18DOI: (10.1046/j.1523-1747.2000.00036.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Limited proteolysis of rVEGF165 by plasmin. (A) rVEGF165 was incubated with plasmin or chronic wound fluid (cWF) for increasing time periods as indicated. rVEGF165 degradation was monitored by SDS-PAGE under nonreducing conditions. Incubation of rVEGF165 in chronic wound fluid in the presence of increasing concentrations of (B) α2-antiplasmin (AP) or (C) PL-I for increasing time periods as indicated. Journal of Investigative Dermatology 2000 115, 12-18DOI: (10.1046/j.1523-1747.2000.00036.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions