Macrophages engage in extracellular matrix remodeling both degrading and synthesizing hyaluronan. Hyaluronan is a high molecular weight ( > 1000 kDa) extracellular matrix component that is known to be upregulated in various forms of arterial injury. Inflammation can cause fragmentation of hyaluronan and circulating hyaluronan fragments have been found to stimulate chemokine production by macrophages through a TLR4- and TLR2-dependent mechanism.

We investigated the effects of hyaluronan in male B6 cardiac allografts in female B6 recipients. These grafts elicit acute inflammation that peaks at 2 weeks and progresses to chronic arterial rejection by 6 weeks. Increased amounts of hyaluronan were detected by ELISA in homogenates of allografts at 2 and 6 weeks compared to isografts. Immunohistology demonstrated increased hyaluronan in the interstitium at 2 weeks and in the neointimal lesions and surrounding adventitial infiltrates of arteries as well as the interstitium of cardiac allografts at 6 weeks. CD44 is the primary receptor for hyaluronan and CD44 positive cells co-localized with the hyaluronan.

To test whether hyaluronan fragments increased chemokines in cardiac allografts, we administered 100 μg of hyaluronan fragments daily to female recipients of male allografts. Controls were administered an equal volume of the PBS diluent. Both 4.7 or 35 kD fragments of hyaluronan stimulated MIG and MCP-1 production in the cardiac allografts by 2 weeks. F4/80 expressing macrophages were isolated from cardiac allografts to mice treated with PBS or hyaluronan fragments, mRNA was isolated and markers of M1 and M2 macrophages were probed by PCR. In addition to MIG and MCP-1, the M1 markers IL-6, IL-15, IL-18, TNFα and Nos2 were increased.

In summary, we demonstrated that increased amounts of hyaluronan were localized in the neointima of arteries in cardiac allografts, which contained activated macrophages that could contribute to fragmentation of the hyaluronan. The functional implications of this pathological finding was demonstrated by the administration of low molecular weight hyaluronan fragments, which stimulated MIG and MCP-1 expression as well as M1 markers in macrophages isolated from allografted hearts. Our data identify hyaluronan as an important mediator of intragraft inflammation after cardiac transplantation.

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