S100A8/A9 deficiency in nonhealing venous leg ulcers uncovered by multiplexed antibody microarray profiling

Abstract

Knowledge on the underlying mechanisms for nonhealing chronic wounds is fragmentary. To increase our understanding of the pathogenesis, the relationship between healing ability and a large panel of proteins was studied using a specially designed wound-healing antibody-based microarray. Wound fluid from nondiabetic patients with nonhealing venous leg ulcers was compared with that from patients with healing open granulating acute wounds. The high-throughput method enabled simultaneous measurement of the relative levels of 48 different proteins representing major categories of wound-healing modulators. Unexpectedly, several of the examined proteins, including various proinflammatory cytokines, proteinases and antiproteinases, were not significantly (P>0·001) changed in chronic wound fluid. For example, levels of matrix metalloproteinase-9 and one of its substrates type IV collagen were similar in the two groups. The wound fluid samples displayed similar degrees of fragmentation of fibronectin by Western blot analysis and the total fibronectin levels were doubled (P<0·001) in chronic compared with acute wounds. The increased fibronectin originated from α-smooth muscle actin-positive myofibroblasts and not from the circulation. S100A8/A9 was the sole protein that was reduced (P<0·001) in wound fluid from venous ulcers [median 226 μg mL(-1) (interquartile range 213-278)] compared with healing wounds [455 μg mL(-1) (382-504)], probably reflecting a difference in inflammatory cell composition. The molecular anomalies in chronic wounds are more subtle than the current paradigm and neither excessive proteinase activity nor deficiencies of examined extracellular matrix proteins, growth factors or angiogenic/angiostatic factors appear to contribute significantly to the nonhealing state of venous leg ulcers.