In Vivo Assessment of Protease Dynamics in Cutaneous Wound Healing by Degradomics Analysis of Porcine Wound Exudates

Abstract

Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ϳ450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease-substrate relations in the healing skin wound in vivo.

The kindlin family: functions, signaling properties and implications for human disease

“…In addition to calpain, also caspase-3 has recently been reported to cleave kindlin-3. The cleavage site has been mapped to the N-terminus at aspartic acid 344 and was detected in wound exudates where it, potentially, can promote apoptosis of hematopoietic cells leading to suppression of the inflammatory response (Sabino et al, 2015).…”

Autocrine and Paracrine Regulation of Keratinocyte Proliferation through a Novel Nrf2–IL-36γ Pathway

“…Soluble proteins were separated from insoluble material by two rounds of high-speed centrifugation. Selected reaction monitoring (SRM) measurements were essentially performed as described (13). For this purpose, 20 mg epidermal protein extract was reduced with 5 mM TCEP for 30 min and alkylated with 40 mM IAA at room temperature.…”

Growth factor pathways in hypertrophic scars: Molecular pathogenesis and therapeutic implications

“…Typically, the growth factors PDGF and EGF play essential roles in the recruitment, proliferation, and functional activities of fibroblasts and pericytes during the early phases of wound healing, but exert pro-fibrogenic effects promoting the formation of hypertrophic scars. Thirdly, topical treatment with a single growth factor has not been as effective as predicted for the treatment of chronic wounds, because the wounds usually have high activity of proteases that can cleave and inactivate proteins [104]. Development of proteaseresistant forms of growth factors or delivery of growth factors embedded in ECM components may overcome these obstacles [105,106].…”