Abstract
Chronic, non-healing wounds are a major complication of diabetes and are characterized by chronic inflammation and excessive protease activity. Although once thought to function primarily as a pro-apoptotic serine protease, granzyme B (GzmB) can also accumulate in the extracellular matrix (ECM) during chronic inflammation and cleave ECM proteins that are essential for proper wound healing, including fibronectin. We hypothesized that GzmB contributes to the pathogenesis of impaired diabetic wound healing through excessive ECM degradation. In the present study, the murine serine protease inhibitor, serpina3n (SA3N), was administered to excisional wounds created on the dorsum of genetically induced type-II diabetic mice. Wound closure was monitored and skin wound samples were collected for analyses. Wound closure, including both re-epithelialization and contraction, were significantly increased in SA3N-treated wounds. Histological and immunohistochemical analyses of SA3N-treated wounds revealed a more mature, proliferative granulation tissue phenotype as indicated by increased cell proliferation, vascularization, fibroblast maturation and differentiation, and collagen deposition. Skin homogenates from SA3N-treated wounds also exhibited greater levels of full-length intact fibronectin compared with that of vehicle wounds. In addition, GzmB-induced detachment of mouse embryonic fibroblasts correlated with a rounded and clustered phenotype that was prevented by SA3N. In summary, topical administration of SA3N accelerated wound healing. Our findings suggest that GzmB contributes to the pathogenesis of diabetic wound healing through the proteolytic cleavage of fibronectin that is essential for normal wound closure, and that SA3N promotes granulation tissue maturation and collagen deposition.
Highlights
Wound healing is a complex process that involves overlapping and sequential phases involving haemostasis, inflammation, granulation tissue formation and tissue remodelling
As Granzyme B (GzmB) may accumulate in the extracellular milieu during chronic inflammation, GzmB levels were assessed in wounded tissues
No known endogenous extracellular GzmB inhibitor exists in humans, so the elevated levels of GzmB observed in bodily fluids may contribute to dysregulated proteolysis.[28,29,30,31,32,33,34]
Summary
Wound healing is a complex process that involves overlapping and sequential phases involving haemostasis, inflammation, granulation tissue formation and tissue remodelling. In diabetic patients, the normal continuum of wound healing is disrupted, and wounds enter a chronic, non-healing state characterized by persistent inflammation, enhanced proteolytic activity and impaired ECM deposition.[7] The roles of various proteases, primarily matrix metalloproteinases (MMPs), have been extensively studied. Granzyme B (GzmB) is a cytotoxic serine protease that is often viewed exclusively as a pro-apoptotic serine protease that is released from cytotoxic lymphocytes, along with the pore-forming, molecule perforin, to induce cell death.[11] because GzmB can be induced in other types of immune and non-immune cells that often do not express perforin and/or do not form immunological synapses with target cells, there is emerging evidence supporting the paradigm that GzmB can accumulate and function in the extracellular milieu.[12] In support, many ECM proteins are GzmB substrates and the consequences of such cleavage may be implicated in many diseases associated with aging and/or chronic inflammation such as abdominal aortic aneurysm (AAA), skin aging, atherosclerosis and wound healing using GzmB knockout mice.[13,14,15,16] Fibronectin is one such ECM glycoprotein that has an important role in cell attachment, differentiation and migration during wound healing and is cleaved by GzmB.[17]. A previous study by our group had demonstrated efficacy for SA3N as an in vivo GzmB inhibitor in a murine model of AAA.[20]
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