An important stage in cutaneous wound healing is the formation of a granulation tissue. Firstly associated with the latter is the migration in of newly formed vessels (neoangiogenesis). Numerous experimental and clinical studies show that chronic wounds are characterized by impaired angiogenesis and thus diminished formation of granulation tissue.
A large number of mediators which stimulate angiogenesis during wound healing are known. They include firstly the factors which, besides stimulating endothelial cells, also activate mesenchymal and/or epidermal cells (bFGF, aFGF, TGF-a, PDGF), and secondly so-called endothelial cell-specific factors whose receptors are substantially confined to endothelial cells (VEGF, angiopoietin). A large number of physiological and pathological reactions involving the blood vessels correlates with an increased expression of VEGF and its receptors, so that VEGF assumes a central role in angiogenesis of the skin. The first indications of the possible importance of VEGF in wound healing impairments were provided on the basis of experiments on VEGF expression in diabetic mice (db/db mice) (Frank et al. 1995). It was possible to show in this model that the wound healing impairment correlates with a diminished VEGF expression. It has recently been possible to provide support for the role of VEGF in wound healing by a further transgenic animal model (Fukumura et al., 1998) and detection of VEGF in the wound discharge from acute human wounds (Nissen et al., 1998).
It has further been shown that there is increased expression of the mRNA of VEGF and its receptors in the tissue of chronic wounds (Lauer et al., 2000). Investigations by SDS-PAGE show, however, breakdown of the VEGF protein in the chronic wound environment, in contrast to the acute wound. This breakdown leads to a significant loss of the biological activity and may thus, despite the increased expression of the VEGF receptors, underlie a deficient stimulation of neoangiogenesis in the chronic wound environment. As explained above, it was possible to show that plasmin is involved in the cleavage of VEGF in the chronic wound environment (Lauer et al., 2000).
Cleavage of VEGF165 via plasmin leads to detachment of the carboxyl-terminal domain which is encoded by Exon 7. Whereas Exons 3 and 4 determine the binding properties of VEGF to the VEGF receptors Flt-1 and Flk-1/KDR, Exon 7 has a critical importance in the interaction of VEGF with neuropilin-1 (Keyt et al. 1996). Neuropilin-1 is a 130 kDa cell surface glycoprotein. Its role in the potentiation of the mitogenic effect of VEGF on endothelial cells was described only recently (Soker et al. 1998). In this connection, the interaction of neuropilin-1 with Flk-1/KDR appears to be important because binding solely of VEGF to neuropilin-1 has no signal effect.
Plasmin belongs to the class of serine proteases. These enzymes are able to cleave peptide linkages. The cleavage takes place by a so-called catalytic triad. In the catalytic centre thereof an essential part is played in particular by the eponymous serine, but also by the amino acids histidine and aspartate, because the process of peptide cleavage takes place by means of them (Stryer 1987, pp. 231 et seq.). Although the mechanism of the linkage cleavage is identical in all serine proteases, they differ markedly in their substrate specificity. Thus, plasmin, just like trypsin, cleaves peptide linkages after the basic amino acids lysine and arginine. However, the substrate specificity of plasmin, which is determined by the structure of the catalytic centre, leads to plasmin being unable to cleave all these linkages. Catalysis of peptide-linkage cleavage is possible only if the corresponding protein segments are able to interact with the catalytic centre of the enzyme (Powers et al. 1993; Stryer 1987). To date, no unambiguous consensus sequence of a plasmin cleavage site is known.
The present invention is based on the object of providing improved means for healing chronic wounds. Surprisingly, this object is achieved by the provision according to the invention of a vascular endothelial growth factor (VEGF) variant which is characterized in that at least one amino acid in the sequence of the native vascular endothelial growth factor at positions 109 to 112 of the native vascular endothelial growth factor is replaced by another amino acid or a deletion.
USPTO Backgrounds
USPTO Backgrounds