Ischemic stroke is currently treated with
thrombolytic therapy with a drawback to induce hemorrhagic transformation (HT) if applied beyond its relatively narrow treatment time window. The present study was designed to examine the role of
IMM-H004, a derivative of
coumarin, in recombinant
tissue plasminogen activator (tPA)-induced HT. Rats subjected to 6 h of thromboembolic occlusion or
middle cerebral artery occlusion received tPA with or without
IMM-H004. Delayed tPA intervention drastically increased the risk of HT and exaggerated the ischemic injury. To assess the effect of
IMM-H004 on
delayed treatment of tPA-induced toxicity after
ischemia and reperfusion, various approaches were used, including a behavior test, TTC-staining, determination of
cerebral hemorrhage,
laser speckle imaging, Western blot,
gelatin zymogram, immunohistochemistry and immunofluorescence staining. Experiments were also conducted in vitro in human brain microvascular endothelial cells (HBMECs) and PC12 cells to explore the mechanism for the role of
IMM-H004. Combination
therapy of tPA and
IMM-H004 prevented the development of HT, and reduced the mortality rate,
infarct volume and
brain edema.
IMM-H004 also exerted a protective role by decreasing
matrix metalloproteinases, the co-localization of
matrix metalloproteinase-2 with astrocytes and increasing
occludin. Experiments in HBMECs and PC12 revealed an elevation in
ATP level and a
protein kinase A- and PI3K-dependent activation of Akt by
IMM-H004 after tPA administration. These results suggest
IMM-H004 as a promising adjuvant to alleviate the detrimental side effects of tPA in clinical
therapy of
ischemic stroke, and contribute to better understand the mechanism for the beneficial role of this novel remedy.