Collagenase derived from the gram-positive bacterium Clostridium histolyticum
has been used for wound debridement since the 1960s (28
). More recently, it has been suggested that bacterial collagenase promotes wound healing by stimulating post-injury cellular responses within the epidermal and dermal compartments, which enhances re-epithelialization, wound healing angiogenesis and wound closure (13
We have identified several fragments that are uniquely present in extracellular matrix digested with bacterial collagenase (). The differences in matrix degradation patterns reflect differential cleavage sites between the host (mammalian) and bacterial enzymes (29
). The mammalian collagenase cleaves specific Gly-Ile or Gly-Leu bonds of α1 and α2 chains releasing two fragments that represent 1/3 and 2/3 the intact parent collagenous substrate. Bacterial collagenase on the other hand, attacks the parent collagenous backbone much more frequently, especially at Y-Gly bonds, therein releasing multiple fragments (29
). Our data validate these earlier reports and lend credence to the notion that the resultant products of matrix digestion may be linked to key Clostridial collagenase wound healing properties.
Isolation of the specific peptides released by bacterial collagenase revealed the presence of fragments of several ECM molecules that have been reported to be produced by endothelial cells (26
). Furthermore, matrix macromolecules (tenascin X, collagen IV and fibrillin 1) are glycoproteins that possess multiple domains, including regions that respectively interact with collagen I, III, V, decorin, and fibronectin, elastin and latent transforming growth factor β-binding protein (33
). That tenascin X is present within the anti-collagen I IPs is of particular interest since this matrix component has been previously implicated in tissue injury and repair in humans and mice (35
Using both 2- and 3D models we found that the peptides released from the ECM by Clostridial collagenase induce key wound healing responses embodied by purified Clostridial collagenase (). Several peptides significantly increase the rate of sprout formation in Matrigel-based 2D model. Interestingly, the effects of the peptides on endothelial morphogenesis in this model are more pronounced at the earlier time point 7h as compared to 24h post-plating. This suggests that in addition to enhancement of the rate of cell-cell interactions, which leads to cellular alignment and tube formation, the peptides also enhance endothelial motility - an early step of angiogenic process.
Our results agree with several earlier reports showing biological activity of matrices degraded by bacterial collagenase. It has been demonstrated that fragments of human eschar degraded by bacterial collagenase stimulate fibroblast proliferation both in vitro
and in vivo
). Moreover, it has been reported that bacterial collagenase degraded collagens, are chemotactic for human fibroblasts (38
) and neutrophils (39
). The effects of these fragments on endothelial cells have not been reported. To our knowledge no ECM fragments released by Clostridial collagenase have anti-angiogenic effects. This is in striking contrast to mammalian matrix degrading enzymes that liberate both pro- and anti-angiogenic moieties from naturally occurring matrices (10
It has been reported that several elastin-derived peptides containing GXXPG sequence enhance endothelial migration, tube formation on collagen type I in vitro and in chick chorio-allantoic membrane model ex vivo via induction of MT1-MMP production by endothelial cells (11
). Two of our peptides comb1 and ten2 also contain this stretch of amino acids, and both induce morphogenesis in a Matrigel-based assay. However, only a larger combinatorial peptide (comb1), with presumably more complex tertiary structure and two GXXPG domains, also stimulated cellular proliferation. While both ten2 and comb1 can probably stimulate MMP production by cells necessary for endothelial morphogenesis, comb1 is likely to activate other yet undetermined pathways mediating cell proliferation.
Importantly, the chemical structure of other bioactive ECM fragments released by bacterial collagenase and described here have not been reported by others. The receptors and signaling pathways activated by the peptides remain to be discovered. The receptors could include pro-angiogenic integrins αvβ3, αvβ5, mediating both migration and proliferation of endothelial cells necessary for angiogenesis. Other potential receptors may include well-established growth factor/tyrosine kinase-dependent receptors (e.g. epidermal factor receptor), that can be activated by proteolytically exposed cryptic sites within matrix molecules (40
The experiments performed using a novel 3D model of injury repair () we demonstrate that the peptides can also endothelial responses to injury in complex environment (–). To our knowledge, this is the first report where this unique model (25
) is used to compare the compounds with different angiogenic potential. Unlike standard Matrigel-based assays (27
) here the cells remain viable for a prolonged time (up to 1 week). Thus, in addition to the identification of promising pro-angiogenic or anti-angiogenic therapeutics, such 3D constructs can be employed to study the stability of wound healing compounds and to evaluate drug delivery systems.
In conclusion, we have shown that degradation of endothelial extracellular matrices with bacterial collagenase releases pro-angiogenic peptide fragments. Based on our previous observations ((13
); Demidova-Rice, Herman unpublished data) we suggest that the peptides described here can also stimulate epithelial responses to injury and wound healing in vivo
. Therefore we propose () that bacterial collagenase has multiple direct and indirect positive effects on cellular responses to injury and wound healing: it degrades wound matrix easing dermal and epidermal cell motility, releases ECM and cell surface-bound growth factors. Moreover, it liberates biologically active ECM fragments, which in turn can stimulate angiogenesis and epithelialization during wound healing. Future work will determine whether the peptides can stimulate cellular responses to injury in vivo
and establish the mechanisms of their activity.
Proposed mechanism of wound healing activity of bacterial collagenase: the role of ECM fragments