Abstract
BackgroundTumour necrosis factor-α (TNF-α) is a pleiotropic pro-inflammatory cytokine, which is rapidly upregulated in the brain after injury. TNF-α acts by binding to its receptors, TNF-R1 (p55) and TNF-R2 (p75), on the cell surface. The aim of this study was first to investigate if there is altered expression of TNF-α and TNF-α receptors in cerebral artery walls following global or focal ischemia, and after organ culture. Secondly, we asked if the expression was regulated via activation of the MEK-ERK1/2 pathway.MethodsThe hypothesis was tested in vivo after subarachnoid hemorrhage (SAH) and middle cerebral artery occlusion (MCAO), and in vitro by organ culture of isolated cerebral arteries. The localization and amount of TNF-α, TNF-α receptor 1 and 2 proteins were analysed by immunohistochemistry and western blot after 24 and 48 h of organ culture and at 48 h following SAH or MCAO. In addition, cerebral arteries were incubated for 24 or 48 h in the absence or presence of a B-Raf inhibitor (SB386023-b), a MEK- inhibitor (U0126) or an NF-κB inhibitor (IMD-0354), and protein expression evaluated.ResultsImmunohistochemistry revealed enhanced expression of TNF-α, TNF-R1 and TNF-R2 in the walls of cerebral arteries at 48 h after MCAO and SAH compared with control. Co-localization studies showed that TNF-α, TNF-R1 and TNF-R2 were primarily localized to the cell membrane and the cytoplasm of the smooth muscle cells (SMC). There was, in addition, some expression of TNF-R2 in the endothelial cells. Immunohistochemistry and western blot analysis showed that these proteins were upregulated after 24 and 48 h in culture, and this upregulation reached an apparent maximum at 48 h of organ culture. Treatment with U0126 significantly reduced the enhanced SMC expression of TNF-α, TNF-R1 and TNF-R2 immunoreactivities after 24 and 48 h of organ culture. The Raf and NF-κB inhibitors significantly reduced organ culture induced TNF-α expression while they had minor effects on the TNF-α receptors.ConclusionThe present study shows that cerebral ischemia and organ culture induce expression of TNF-α and its receptors in the walls of cerebral arteries and that upregulation is transcriptionally regulated via the MEK/ERK pathway.
Highlights
Tumour necrosis factor-a (TNF-a) is a pleiotropic pro-inflammatory cytokine, which is rapidly upregulated in the brain after injury
The present study aimed to address two questions: First, is the expression of TNF-a, TNF-R1 and TNF-R2 altered in cerebrovascular smooth muscle cells (SMCs) following middle cerebral artery occlusion (MCAO), subarachnoid hemorrhage (SAH) and organ culture? Second, what intracellular signaling events are involved in regulating the expression of these molecules? This was examined by in vitro application of signal transduction blockers, such as the MEK/ERK1/2 inhibitor U0126, the B-Raf inhibitor SB3860-b, and the nuclear factor-B (NF-B) inhibitor IMD-0354 [18,19]
Middle cerebral artery occlusion The levels of TNF-a, TNF-R1 and TNF-R2 proteins expressed in the cerebral arteries after 48 h post MCAO for 2 h and reperfusion were investigated by immunofluorescence
Summary
Tumour necrosis factor-a (TNF-a) is a pleiotropic pro-inflammatory cytokine, which is rapidly upregulated in the brain after injury. There are two major kinds of stroke: ischemic stroke and hemorrhagic stroke Both are associated with disruption of the blood flow to the brain with rapid depletion of cellular energy and glucose, resulting in ionic cytokines such as tumor necrosis factor-a (TNF-a), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS), by a variety of activated cell types; endothelial cells, microglia, neurons, leukocytes platelets, monocytes, macrophages and fibroblasts [3,4]. TNF-a has been suggested to stimulate angiogenesis following ischemia through induced expression of angiogenesis-related genes [8,9] It is known as a strong immunomediator and pro-inflammatory cytokine, which is rapidly upregulated in the brain after injury and is associated with necrosis or apoptosis [10]. Increased TNF-a level has been observed in brain tissue, plasma and cerebrospinal fluid in Alzheimer’s disease, multiple sclerosis and Parkinson’s disease [15,16,17]
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