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CD44 is a transmembrane glycoprotein known to be involved in endothelial cell recognition, lymphocyte trafficking, and regulation of cytokine gene expression in inflammatory diseases. In the present study, we demonstrated that the expression of CD44 mRNA was induced in a mouse model of cerebral ischemia. A potential role of CD44 in ischemic brain injury was investigated using CD44-deficient (CD44-/-) mice. Over 50% (p < 0.05, n ¼ 14) and 78% (p < 0.05, n ¼ 10) reduction in ischemic infarct was observed in CD44-/-mice compared with that of wild-type mice following transient (30 min ischemia) and permanent (24 h) occlusion of the middle cerebral artery (MCAO), respectively. Similarly, significant improvement was observed in neurological function in CD44-/-mice as evidenced by spontaneous and forced motor task scores. The marked protection from ischemic brain injury in CD44-/-mice was associated with normal physiological parameters, cytokine gene expression, astrocyte and microglia activation as compared with wild-type mice. However, in CD44-/-mice, significantly lower expression of soluble interleukin-1b protein was noted after brain ischemia. Our data provide new evidence on the potential role of CD44 in brain tissue in response to ischemia and may suggest that this effect might be associated with selective reduction in inflammatory cytokines such as interleukin-1b.

Tumor necrosis factor ␣ (TNF␣) is an immunomodulatory and proinflammatory cytokine implicated in neuroinflammation and neuronal damage in response to cerebral ischemia. Tumor necrosis factor-␣ converting enzyme (TACE or ADAM17) is a key sheddase that releases TNF␣ from its inactive cell-bound precursor. Using a selective small molecule inhibitor of TACE, DPH-067517, we tested the hypothesis that inhibition of TNF␣ formation might have a salutary effect in ischemic stroke induced by embolic occlusion of the middle cerebral artery (MCAO). DPH-067517 selectively inhibited TACE enzyme activity in vitro (K i ϭ 2.8 nM), and effectively suppressed ischemia-induced increase in soluble TNF␣ in brain tissue after systemic administration. DPH-067517 (3 and 30 mg/kg, i.p. administered 15 min before MCAO) produced 43% (n ϭ 8, p ϭ 0.16) and 58% (n ϭ 8, p Ͻ 0.05) reduction in infarct size and 36% (p Ͻ 0.05) and 23% (p Ͻ 0.05) reduction in neurological deficits, respectively. The salutary effect of DPH-067517 in ischemic brain injury was also observed when the first dose was administrated 60 min after the onset of ischemia. Inhibition of TACE had no effect on apoptosis measured by levels of active caspase-3 expression and DNA fragmentation. Our data suggest that inhibition of TACE might be a potential therapeutic strategy for neuroprotection after focal ischemic stroke.

Background and Purpose-CD44 is a transmembrane glycoprotein involved in endothelial cell recognition, lymphocyte trafficking, and regulation of cytokine gene expression in inflammatory diseases. The present report describes the discovery of upregulated CD44 gene expression and its spatial and temporal distribution in the brain after focal stroke. Methods-Rats were subjected to permanent occlusion of the middle cerebral artery (MCAO). Suppression subtractive hybridization (SSH) strategy was used to identify differentially expressed genes. Northern blotting and real-time polymerase chain reaction were used to evaluate the expression of CD44 and hyaluronan synthase 2 (HAS-2) mRNA. Western blotting and immunohistochemistry were used to examine CD44 expression and cellular distribution. Results-CD44 upregulation after focal stroke was discovered by the SSH approach and confirmed by DNA sequencing.Northern blot using a pooled poly(A)ϩ RNA revealed 3 splice variants of CD44 mRNA, and their inducible expression started at 6 hours (5.3-fold increase over sham operation), peaked at 24 hours (28.6-fold increase), and persisted up to 72 hours (17.8-fold increase) after MCAO. A parallel induction profile of HAS-2 mRNA was observed in the ischemic brain tissue. The levels of CD44 were markedly elevated at 6 hours (1.8-fold increase over sham; nϭ3), 24 hours (2.9-fold, peak induction; PϽ0.01), and 72 hours (2.4-fold increase; PϽ0.05) after MCAO by means of Western analysis. Immunohistochemical and confocal microscopy confirmed that constitutive expression of CD44 is limited to microvessels in normal brain but is strongly induced after ischemia, where the immunoreactive signal mainly resided in endothelial cells and monocytes. Double-labeling immunohistochemistry demonstrated that a marked induction of CD44 in the ischemic lesion is dominantly located in microglia and a subset of macrophages. Conclusions-The discovery of concomitant induction of CD44 and HAS-2 mRNA expression and the localization of CD44 in the microglia, macrophages, and microvessels of the ischemic brain tissue suggest that an active interaction between CD44 and hyaluronan may occur and play a role in the known inflammatory response and tissue remodeling after stroke.