Tenascin C upregulates interleukin-6 expression in human cardiac myofibroblasts via toll-like receptor 4.

AIM: To investigate the effect of Tenascin C (TNC) on the expression of pro-inflammatory cytokines and matrix metalloproteinases in human cardiac myofibroblasts (CMF). METHODS: CMF were isolated and cultured from patients undergoing coronary artery bypass grafting. Cultured cells were treated with either TNC (0.1 μmol/L, 24 h) or a recombinant protein corresponding to different domains of the TNC protein; fibrinogen-like globe (FBG) and fibronectin type III-like repeats (TNIII 5-7) (both 1 μmol/L, 24 h). The expression of the pro-inflammatory cytokines; interleukin (IL)-6, IL-1β, TNFα and the matrix metalloproteinases; MMPs (MMP1, 2, 3, 9, 10, MT1-MMP) was assessed using real time RT-PCR and western blot analysis. RESULTS: TNC increased both IL-6 and MMP3 (P < 0.01) mRNA levels in cultured human CMF but had no significant effect on the other markers studied. The increase in IL-6 mRNA expression was mirrored by an increase in protein secretion as assessed by enzyme-linked immunosorbant assay (P < 0.01). Treating CMF with the recombinant protein FBG increased IL-6 mRNA and protein (P < 0.01) whereas the recombinant protein TNIII 5-7 had no effect. Neither FBG nor TNIII 5-7 had any significant effect on MMP3 expression. The expression of toll-like receptor 4 (TLR4) in human CMF was confirmed by real time RT-PCR, western blot and immunohistochemistry. Pre-incubation of cells with TLR4 neutralising antisera attenuated the effect of both TNC and FBG on IL-6 mRNA and protein expression. CONCLUSION: TNC up-regulates IL-6 expression in human CMF, an effect mediated through the FBG domain of TNC and via the TLR4 receptor

Changes in glutamate transporter expression in mouse forebrain areas following focal ischemia

Dysfunction of glutamate transporters has been proposed to promote neuronal death in modelled cerebral ischemia. However, these studies have produced conflicting results and the changes in glutamate transporter expression have not yet been examined in a mouse focal ischemic stroke model. This study used quantitative real-time reverse-transcription polymerase chain reaction to examine glutamate transporter mRNA expression in the hippocampus, cortex and striatum in a mouse model of focal ischemic stroke induced by middle cerebral artery occlusion (MCAO). Effects on mRNA expression of glial (GLT-1, GLAST) and neuronal (EAAC1) glutamate transporters in these brain areas were assessed by comparing MCAO brains with sham-operated control brains. Changes in transporter proteins were also assessed by immunohistochemistry using specific antibodies to GLT-1 and GLAST. Following focal ischemia, GLT-1 mRNA expression was decreased significantly in the ipsilateral hippocampus and cortex compared to the sham-operated brains (p<0.05). There were no significant differences in GLAST or EAAC1 mRNA expression between MCAO and sham-operated brains. Immunohistochemistry also confirmed a marked reduction in GLT-1 immunoreactivity in the cortex and hippocampus. Down regulation of GLT-1 in these brain areas may impair normal clearance of synaptically-released glutamate and contribute to neural damage following focal ischemic insult

Demonstration of estrogen receptor alpha protein in glutamatergic (vesicular glutamate transporter 2 immunoreactive) neurons of the female rat hypothalamus and amygdala using double-label immunocytochemistry.

By means of double-label immunocytochemistry, authors studied the presence of estrogen receptor alpha (ER-alpha) protein in vesicular glutamate transporter 2 (VGluT2) protein-immunoreactive neurons in the female rat hypothalamus and amygdala. They examined colocalization of the 2 immunoreactive proteins in structures in which they found a significant overlap in the localization of the distribution of ER-alpha- and VGluT2-immunopositive nerve cells, namely in the medial preoptic area, the ventral subdivision of the ventromedial hypothalamic nucleus, and the medial amygdaloid nucleus. In the medial preoptic area, only 2.74 % of ER-alpha-immunoreactive neurons were VGluT2 positive, and conversely, 5 % of VGluT2-immunoreactive neurons contained ER-alpha immunofluorescent labeling. Highest degree of colocalization was detected in the ventral subdivision of the ventromedial hypothalamic nucleus, where 22.81 % of the ER-alpha-immunopositive neurons were VGluT2 immunoreactive and 37.14 % of the VGluT2-immunolabeled neurons contained ER-alpha-positive nucleus. In the medial amygdaloid nucleus, 15.38 % of the ER-alpha and 18.1 % of the VGluT2-immunoreactive neurons were double labeled. The colocalizations suggest that glutamatergic (VGluT2 protein immunoreactive) neurons are involved in the mediation of the action of estrogen on the rat brain