Cerebral ischemia induces microvascular pro-inflammatory cytokine expression via the MEK/ERK pathway

<p>Abstract</p> <p>Background</p> <p>Cerebral ischemia from middle cerebral artery wall (MCA) occlusion results in increased expression of cerebrovascular endothelin and angiotensin receptors and activation of the mitogen-activated protein kinase (MAPK) pathway, as well as reduced local cerebral blood flow and increased levels of pro-inflammatory mediators in the infarct region. In this study, we hypothesised that inhibition of the cerebrovascular inflammatory reaction with a specific MEK1/2 inhibitor (U0126) to block transcription or a combined receptor blockade would reduce infarct size and improve neurological score.</p> <p>Methods</p> <p>Rats were subjected to a 2-hours middle cerebral artery occlusion (MCAO) followed by reperfusion for 48 hours. Two groups of treated animals were studied; (i) one group received intraperitoneal administration of a specific MEK1/2 inhibitor (U0126) starting at 0, 6, or 12 hours after the occlusion, and (ii) a second group received two specific receptor antagonists (a combination of the angiotensin AT₁receptor inhibitor Candesartan and the endothelin ET_Areceptor antagonist ZD1611), given immediately after occlusion. The middle cerebral arteries, microvessels and brain tissue were harvested; and the expressions of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS) and phosphorylated ERK1/2, p38 and JNK were analysed using immunohistochemistry.</p> <p>Results</p> <p>We observed an infarct volume of 25 ± 2% of total brain volume, and reduced neurological function 2 days after MCAO followed by 48 hours of recirculation. Immunohistochemistry revealed enhanced expression of TNF-α, IL-1ß, IL-6 and iNOS, as well as elevated levels of phosphorylated ERK1/2 in smooth muscle cells of ischemic MCA and in associated intracerebral microvessels. U0126, given intraperitoneal at zero or 6 hours after the ischemic event, but not at 12 hours, reduced the infarct volume (11.7 ± 2% and 15 ± 3%, respectively), normalized pERK1/2, and prevented elevation of the expressions of TNF-α IL-1ß, IL-6 and iNOS. Combined inhibition of angiotensin AT₁and endothelin ET_Areceptors decreased the volume of brain damaged (12.3 ± 3; <it>P </it>< 0.05) but only slightly reduced MCAO-induced enhanced expression of iNOS and cytokines</p> <p>Conclusion</p> <p>The present study shows elevated microvascular expression of TNF-α, IL-1ß, IL-6 and iNOS following focal ischemia, and shows that this expression is transcriptionally regulated via the MEK/ERK pathway.</p

Enhanced cerebrovascular expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 via the MEK/ERK pathway during cerebral ischemia in the rat.

BACKGROUND: Cerebral ischemia is usually characterized by a reduction in local blood flow and metabolism and by disruption of the blood-brain barrier in the infarct region. The formation of oedema and opening of the blood-brain barrier in stroke is associated with enhanced expression of metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). RESULTS: Here, we found an infarct volume of 24.8 +/- 2% and a reduced neurological function after two hours of middle cerebral artery occlusion (MCAO), followed by 48 hours of recirculation in rat. Immunocytochemistry and confocal microscopy revealed enhanced expression of MMP-9, TIMP-1, and phosphorylated ERK1/2 in the smooth muscle cells of the ischemic MCA and associated intracerebral microvessels. The specific MEK1/2 inhibitor U0126, given intraperitoneal zero or 6 hours after the ischemic event, reduced the infarct volume significantly (11.8 +/- 2% and 14.6 +/- 3%, respectively; P < 0.05), improved neurological function, normalized expression of phosphorylated ERK1/2, and reduced expression of MMP-9 and TIMP-1 in the vessel walls. Administration of U0126 12 hours after MCAO did not alter the expression of MMP-9. Immunocytochemistry showed no overlap in expression between MMP-9/TIMP-1 and the astrocyte/glial cell marker GFAP in the vessel walls. CONCLUSION: These data are the first to show that the elevated vascular expression of MMP-9 and TIMP-1, associated with breakdown of the blood-brain barrier following focal ischemia, are transcriptionally regulated via the MEK/ERK pathway

Organ culture: a new model for vascular endothelium dysfunction.

BACKGROUND: Endothelium dysfunction is believed to play a role in the development of cardiovascular disease. The aim of the present study was to evaluate the suitability of organ culture as a model for endothelium dysfunction. METHODS: The isometric tension was recorded in isolated segments of the rat mesenteric artery branch, before and after organ culture for 20 h. Vasodilatation was expressed as % of preconstriction with U46619. The acetylcholine (ACh) induced nitric oxide (NO) mediated dilatation was studied in the presence of 10 &mgr;M indomethacin, 50 nM charybdotoxin and 1 &mgr;M apamin. Endothelium-derived hyperpolarising factor (EDHF) was studied in the presence of 0.1 mM L-NOARG and indomethacin. Prostaglandins were studied in the presence of L-NOARG, charybdotoxin and apamin. RESULTS: The ACh-induced NO and prostaglandin-mediated dilatations decreased significantly during organ culture (NO: 84% in control and 36% in cultured; prostaglandins: 48% in control and 16% in cultured). Notably, the total ACh-dilatation was not changed. This might be explained by the finding that EDHF alone stimulated a full dilatation even after organ culture (83% in control and 80% in cultured). EDHF may thereby compensate for the loss in NO and prostaglandin-mediated dilatation. Dilatations induced by forskolin or sodium nitroprusside did not change after organ culture, indicating intact smooth muscle cell function. CONCLUSIONS: Organ culture induces a loss in NO and prostaglandin-mediated dilatation, which is compensated for by EDHF. This shift in mediator profile resembles that in endothelium dysfunction. Organ culture provides an easily accessible model where the molecular changes that take place, when endothelium dysfunction is developed, can be examined over time

The role of tumor necrosis factor-α and TNF-α receptors in cerebral arteries following cerebral ischemia in rat

<p>Abstract</p> <p>Background</p> <p>Tumour 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.</p> <p>Methods</p> <p>The hypothesis was tested <it>in vivo </it>after subarachnoid hemorrhage (SAH) and middle cerebral artery occlusion (MCAO), and <it>in vitro </it>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.</p> <p>Results</p> <p>Immunohistochemistry 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.</p> <p>Conclusion</p> <p>The 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.</p

Lipid-soluble cigarette smoking particles induce expression of inflammatory and extracellular-matrix-related genes in rat cerebral arteries

Petter Vikman, Cang-Bao Xu, Lars EdvinssonDepartment of Clinical Sciences, Experimental Vascular Research, Lund, SwedenAims: Cigarette smoking is one of the strongest risk factors for stroke. However, the underlying molecular mechanisms that smoke leads to the pathogenesis of stroke are incompletely understood.Methods: Dimethyl sulfoxide (DMSO)-soluble (lipid-soluble) cigarette smoking particles (DSP) were extracted from cigarette smoke (0.8 mg nicotine per cigarette; Marlboro&amp;reg;). Rat cerebral arteries were isolated and organ cultured in the presence of DSP (0.2 &amp;mu;l/ml, equivalent to the plasma level in smokers) for 24 h. The expression of matrix metalloproteinase 9 and 13 (MMP9 and MMP13), angiotensin receptor 1 and 2 (AT1 and AT2), interleukin 6 and inducible nitric oxide synthase (iNOS) were investigated at mRNA level by real-time PCR and/or at protein level by immunohistochemistry. In addition, the activity of three mitogen-activated protein kinases (p38, ERK 1/2 and SAPK/JNK) and their downstream transcription factors (ATF-2, Elk-1 and c-Jun) were examined.Results: We observed that compared with control (DMSO-treated cerebral arteries), the cerebral arteries treated by DSP exhibited enhanced expression of MMP13 and AT1 receptors, but not of AT2 receptors, at both mRNA and protein levels, suggesting that a transcriptional mechanism is most likely involved in the DSP effects. This is further supported by the findings that DSP induced phosphorylation of p38 mitogen-activated protein kinases inflammatory signal protein in parallel with activation of its downstream transcription factor ATF-2 and Elk-1. However, ERK 1/2 and SAPK/JNK activities were markedly expressed in the control (organ culture per se with DMSO), and DSP failed to further enhance the activation of ERK 1/2 and SAPK/JNK in the cerebral arteries.Conclusions: DSP induces cerebral vessel inflammation with activation of p38 MAPK inflammatory signal and the downstream transcriptional factors (ATF-2 and Elk-1) in parallel with enhanced extracellular-matrix-related gene transcription and increased AT1 receptor expression in the cerebral arteries, which are key events in stroke pathogenesis.Keywords: cigarette smoking, vascular inflammation, signal transduction, extracellular matrix, MMP, strok

Role of ERK/MAPK in endothelin receptor signaling in human aortic smooth muscle cells

<p>Abstract</p> <p>Background</p> <p>Endothelin-1 (ET-1) is a potent vasoactive peptide, which induces vasoconstriction and proliferation in vascular smooth muscle cells (VSMCs) through activation of endothelin type A (ET_A) and type B (ET_B) receptors. The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein kinases (MAPK) are involved in ET-1-induced VSMC contraction and proliferation. This study was designed to investigate the ET_Aand ET_Breceptor intracellular signaling in human VSMCs and used phosphorylation (activation) of ERK1/2 as a functional signal molecule for endothelin receptor activity.</p> <p>Results</p> <p>Subconfluent human VSMCs were stimulated by ET-1 at different concentrations (1 nM-1 μM). The activation of ERK1/2 was examined by immunofluorescence, Western blot and phosphoELISA using specific antibody against phosphorylated ERK1/2 protein. ET-1 induced a concentration- and time- dependent activation of ERK1/2 with a maximal effect at 10 min. It declined to baseline level at 30 min. The ET-1-induced activation of ERK1/2 was completely abolished by MEK1/2 inhibitors U0126 and SL327, and partially inhibited by the MEK1 inhibitor PD98059. A dual endothelin receptor antagonist bosentan or the ET_Aantagonist BQ123 blocked the ET-1 effect, while the ET_Bantagonist BQ788 had no significant effect. However, a selective ET_Breceptor agonist, Sarafotoxin 6c (S6c) caused a time-dependent ERK1/2 activation with a maximal effect by less than 20% of the ET-1-induced activation of ERK1/2. Increase in bosentan concentration up to 10 μM further inhibited ET-1-induced activation of ERK1/2 and had a stronger inhibitory effect than BQ123 or the combined use of BQ123 and BQ788. To further explore ET-1 intracellular signaling, PKC inhibitors (staurosporin and GF109203X), PKC-delta inhibitor (rottlerin), PKA inhibitor (H-89), and phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin) were applied. The inhibitors showed significant inhibitory effects on ET-1-induced activation of ERK1/2. However, blockage of L-type Ca²⁺channels or calcium/calmodulin-dependent protein kinase II, chelating extracellular Ca²⁺or emptying internal Ca²⁺stores, did not affect ET-1-induced activation of ERK1/2.</p> <p>Conclusion</p> <p>The ET_Areceptors predominate in the ET-1-induced activation of ERK1/2 in human VSMCs, which associates with increments in intracellular PKC, PKA and PI3K activities, but not Ca²⁺signalling.</p

Neuropeptide expression in the human trigeminal nucleus caudalis and in the cervical spinal cord C1 and C2.

In migraine and other primary headaches there is a strong vascular component. Besides the trigeminovascular components some of the associated symptoms point to the involvement of brain stem regions. The central limb of the trigeminal vascular pathway is its projection to the trigeminal nucleus caudalis (TNC) and to the C1-C2 levels of the spinal cord. The aim of the present study was to demonstrate the occurrence of some neurotransmitters in these regions in man. In both the TNC and in the Rexed's laminae I and II of the dorsal horns at the C1 and C2 levels there were numerous substance P immunoreactive fibres. Fibres containing calcitonin gene-related peptide (CGRP) and pituitary adenylate cyclase-activating peptide (PACAP) were moderately dense in number. Fibres containing vasoactive intestinal peptide (VIP) or nitric oxide synthase (NOS) were not seen in the TNC or at the C1 and C2 levels of the spinal cord

Potent P2Y(6 )receptor mediated contractions in human cerebral arteries

BACKGROUND: Extracellular nucleotides play an important role in the regulation of vascular tone and may be involved in cerebral vasospasm after subarachnoidal haemorrhage. This study was designed to characterise the contractile P2 receptors in endothelium-denuded human cerebral and omental arteries. The isometric tension of isolated vessel segments was recorded in vitro. P2 receptor mRNA expression was examined by RT-PCR. RESULTS: In human cerebral arteries, the selective P2Y(6 )receptor agonist, UDPβS was the most potent of all the agonists tested (pEC(50 )= 6.8 ± 0.7). The agonist potency; UDPβS > αβ-MeATP > UTPγS > ATPγS > ADPβS = 0, indicated the presence of contractile P2X(1 )P2Y(2), P2Y(4 )and P2Y(6), but not P2Y(1 )receptors, in human cerebral arteries. In human omental arteries, UDPβS was inactive. The agonist potency; αβ-MeATP > ATPγS = UTPγS > ADPβS = UDPβS = 0, indicated the presence of contractile P2X(1), and P2Y(2 )receptors, but not P2Y(1 )or P2Y(6 )receptors, in human omental arteries. RT-PCR analysis of endothelium-denuded human cerebral and omental arteries demonstrated P2X(1), P2Y(1), P2Y(2 )and P2Y(6 )receptor mRNA expression. There were no bands for the P2Y(4 )receptor mRNA in the omental arteries, while barely detectable in the cerebral arteries. CONCLUSIONS: P2Y(6 )receptors play a prominent role in mediating contraction of human cerebral arteries. Conversely, no such effect can be observed in human omental arteries and previous results confirm the absence of P2Y(6 )receptors in human coronary arteries. The P2Y(6 )receptor might be a suitable target for the treatment of cerebral vasospasm