Chemokine expression is associated with reperfusion of infarcted myocardium in the setting of tissue
necrosis, intense
inflammation, and inflammatory
cytokine release. The specific synthesis of
monocyte chemotactic protein (MCP)-1
mRNA by cardiac venules in reperfused
infarcts corresponded to the region where leukocytes normally localize. MCP-1 could be induced by exogenous
tumor necrosis factor (
TNF)-alpha or by postischemic cardiac lymph containing
TNF-alpha. However, the release of
TNF-alpha during early reperfusion did not explain the venular localization of MCP-1 induction. To better understand the factors mediating MCP-1 induction, we examined the role of
ischemia/reperfusion in a model of brief
coronary occlusion in which no
necrosis or inflammatory response is seen. Adult mongrel dogs were subjected to 15 minutes of
coronary occlusion and 5 hours of reperfusion.
Ribonuclease protection assay revealed up-regulation of MCP-1
mRNA only in ischemic segments of reperfused canine myocardium. Pretreatment with the reactive
oxygen scavenger N-(2-mercaptopropionyl)-glycine completely inhibited MCP-1 induction. In situ hybridization localized MCP-1 message to small venular endothelium in ischemic areas without myocyte
necrosis. Gel shift analysis of nuclear extracts from the ischemic area showed enhanced
DNA binding of the
transcription factors AP-1 and nuclear factor (
NF)-kappaB, crucial for MCP-1 expression, in ischemic myocardial regions. Immunohistochemical staining demonstrated reperfusion-dependent nuclear translocation of c-Jun and
NF-kappaB (p65) in small venular endothelium, only in the ischemic regions of the myocardium, that was inhibited by N-(2-mercaptopropionyl)-glycine. In vitro, treatment of cultured canine jugular vein endothelial cells with the reactive
oxygen intermediate H2O2 induced a concentration-dependent increase in MCP-1
mRNA levels, which was inhibited by the
antioxidant N-acetyl-L-cysteine, a precursor of
glutathione, but not
pyrrolidine dithiocarbamate, an inhibitor of
NF-kappaB and activator of
AP-1. In contrast to our studies with
infarction, incubation of canine jugular vein endothelial cells with postischemic cardiac lymph did not induce MCP-1
mRNA expression suggesting the absence of
cytokine-mediated MCP-1 induction after a sublethal ischemic period. These results suggest that reactive
oxygen intermediate generation, after a brief ischemic episode, is capable of inducing MCP-1 expression in venular endothelium through
AP-1 and
NF-kappaB. Short periods of
ischemia/reperfusion, insufficient to produce a
myocardial infarction, induce MCP-1 expression, potentially mediating angiogenesis in the ischemic noninfarcted heart.