The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after stroke.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-012-1076-3) contains supplementary material, which is available to authorized users.

Recent studies have shown that free fatty acids are associated with chronic inflammation, which may be involved in vascular injury. The intake of eicosapentaenoic acid (EPA) can decrease cardiovascular disease risks, but the protective mechanisms of EPA on endothelial cells remain unclear. In this study, primary human umbilical vein endothelial cells (HUVECs) treated with palmitic acid (PA) were used to explore the protective effects of EPA. The results revealed that EPA attenuated PA-induced cell death and activation of apoptosis-related proteins, such as caspase-3, p53 and Bax. Additionally, EPA reduced the PA-induced increase in the generation of reactive oxygen species, the activation of NADPH oxidase, and the upregulation of inducible nitric oxide synthase (iNOS). EPA also restored the PA-mediated reduction of endothelial nitric oxide synthase (eNOS) and AMP-activated protein kinase (AMPK) phosphorylation. Using AMPK siRNA and the specific inhibitor compound C, we found that EPA restored the PA-mediated inhibitions of eNOS and AKT activities via activation of AMPK. Furthermore, the NF-κB signals that are mediated by p38 mitogen-activated protein kinase (MAPK) were involved in protective effects of EPA. In summary, these results provide new insight into the possible molecular mechanisms by which EPA protects against atherogenesis via the AMPK/eNOS-related pathway.

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Although lead and lipopolysaccharide (LPS), both important environmental pollutants, activate cells through different receptors and participate in distinct upstream signaling pathways, Pb increases the amount of LPS-induced tumor necrosis factor-α (TNF-α). We examined the cells responsible for the excess production of Pb-increased LPS-induced TNF-α and liver injury, and the roles of protein kinase C (PKC) and p42/44 mitogen-activated protein kinase (MAPK) in the induction of TNF-α. Peritoneal injection of Pb alone (100 μmol/kg) or a low dose of LPS (5 mg/kg) did not affect serum TNF-α or liver functions in A/J mice. In contrast, coexposure to these noneffective doses of Pb plus LPS (Pb+LPS) strongly induced TNF-α expression and resulted in profound liver injury. Direct inhibition of TNF-α or functional inactivation of monocytes/macrophages significantly decreased the level of Pb+LPS-induced serum TNF-α and concurrently ameliorated liver injury. Pb+LPS coexposure stimulated the phosphorylation of p42/44 MAPK and the expression of TNF-α in CD14+ cells of cultured mouse whole blood, peritoneal macrophages, and RAW264.7 cells. Moreover, blocking PKC or MAPK effectively reduced Pb+LPS-induced TNF-α expression and liver injury. In summary, monocytes/macrophages were the cells primarily responsible for producing, through the PKC/MAPK pathway, the excess Pb-increased/LPS-induced TNF-α that caused liver injury.

Although current studies indicate that resveratrol exhibits potential antitumor activities, the precise mechanisms of its beneficial effects combined with chemotherapy are not fully understood. This work is warranted to elucidate the underlying mechanism of antitumor effects by the combination therapy of resveratrol and cisplatin. The presence of functional gap junctions is highly relevant for the success of chemotherapy. Gap junctions mediate cell communication by allowing the passage of molecules from one cell to another. Connexin (Cx) 43 is ubiquitous and reduced in a variety of tumor cells. Cx43 may influence the response of tumor cells to treatments by facilitating the passage of antitumor drugs or death signals between neighboring tumor cells. Following resveratrol treatment, dose-dependent upregulation of Cx43 expressions was observed. In addition, gap junction intercellular communication was increased. To study the mechanism underlying these resveratrol-induced Cx43 expressions, we found that resveratrol induced a significant increase in mitogen-activated protein kinases (MAPK) signaling pathways. The MAPK inhibitors significantly reduced the expression of Cx43 protein after resveratrol treatment. Specific knockdown of Cx43 resulted in a reduction of cell death after resveratrol and cisplatin treatment. Our results suggest that treatment of resveratrol in tumor leads to increase Cx43 gap junction communication and enhances the combination of resveratrol and cisplatin therapeutic effects. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 877-886, 2015.