Proteomics Identification of Targets for Intervention in Pressure Ulcers

Abstract

Pressure ulcers (PUs) are chronic wounds that lead to amputations and death. Little is known about why PUs are recalcitrant to healing. Wound healing is mediated by matrix metalloproteinases (MMPs). The 24 MMPs in humans each exist in three forms, of which only one is catalytically competent. We analyzed human PU samples using an affinity resin that exclusively binds to the catalytically competent MMPs. We identified by mass spectrometry the active forms of MMP-1, MMP-8, MMP-9, and MMP-14. Concentrations of MMP-8, MMP-9, and MMP-14 were higher in human PUs compared to the healthy tissue, whereas those for MMP-1 did not change. Decreasing levels of active MMP-9 as the PU improved argued for a detrimental role for this enzyme. In a mouse model of PUs, a highly selective inhibitor for MMP-9 and MMP-14, (R)-ND-336, accelerated wound closure in parallel with significant amelioration of ulcer stage. (R)-ND-336 holds promise as a first-in-class treatment for PUs.

Matrix metalloproteinase profiling and their roles in disease

“…Analyses of wound tissue from patients with PUs ulcer stage 3–4 (where stage 3 is full-thickness deep open ulcer and stage 4 is full-thickness with muscle or bone exposure) using the batimastat affinity resin and proteomics, revealed the presence of active MMP-8 and MMP-9. 54 MMP-8 was present at 16-fold higher levels in PUs relative to controls, while MMP-9 was 14-fold higher ( Fig. 8 ).…”

“…8 ). 54 The amounts of MMP-9 decreased with improvement of PUs, indicating that MMP-9 played a detrimental role in PUs. The higher levels of MMP-8 and MMP-9 in PUs were not surprising, given that PUs share similar pathology with DFUs; in both diseases wound repair is stalled with persistent inflammation, and development of both wounds are due to neuropathy and trauma.…”

“…62,63 Bioanalytical methods have been developed to quantify the levels of active MMPs and ADAMs identified by the batimastat affinity resin. 14,54,62,63…”

“…For the batimastat resin, cysteines in MMPs are reduced and alkylated, followed by trypsin digestion, and analysis of the resulting peptides by tandem mass spectrometry. 14,54 Batimastat is a potent broad-spectrum MMP/ADAM inhibitor with IC 50 values of 3 nM for MMP-1, 55 4 nM for MMP-2, 55 20 nM for MMP-3, 55 6 nM for MMP-7, 56 10 nM for MMP-8, 55 10 nM for MMP-9, 55 , 1 nM for MMP-13, 56 2.8 nM for MMP-14, 56 29 nM for MMP-16, 57 50 nM for ADAM8, 58 50 nM for ADAM9, 59 and 230 nM for ADAM17. 60 The batimastat affinity resin has a limit of quanti-cation of 6 femtomoles and recoveries for MMPs and ADAMs are similar, ranging from 75% to 100%.…”

“…62,63 Bioanalytical methods have been developed to quantify the levels of active MMPs and ADAMs identied by the batimastat affinity resin. 14,54,62,63 Once the active MMPs and ADAMs are identied in human diseased tissues and quantied relative to control tissues, the roles of the identied MMPs/ADAMs can be ascertained by use of selective inhibitors for that particular MMP/ADAM in in vitro or in vivo models of the disease. A challenge is that the 24 human MMPs and 25 human ADAMs are closely related, so selective inhibitors may not be available.…”