Search

Aims Damage of the blood–brain barrier (BBB) is a hallmark of brain injury during the early stages of ischemic stroke. The subsequent endothelial hyperpermeability drives the initial pathological changes and aggravates neuronal death. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable nonselective cation channel activated by oxidative stress. However, whether TRPM2 is involved in BBB degradation during ischemic stroke remains unknown. We aimed to investigate the role of TRPM2 in BBB degradation during ischemic stroke and the underlying molecular mechanisms. Methods and results Specific deletion of Trpm2 in endothelial cells using Cdh5 Cre produces a potent protective effect against brain injury in mice subjected to middle cerebral artery occlusion (MCAO), which is characterized by reduced infarction size, mitigated plasma extravasation, suppressed immune cell invasion, and inhibited oxidative stress. In vitro experiments using cultured cerebral endothelial cells (CECs) demonstrated that either Trpm2 deletion or inhibition of TRPM2 activation attenuates oxidative stress, Ca2+ overload, and endothelial hyperpermeability induced by oxygen–glucose deprivation (OGD) and CD36 ligand thrombospondin-1 (TSP1). In transfected HEK293T cells, OGD and TSP1 activate TRPM2 in a CD36-dependent manner. Noticeably, in cultured CECs, deleting Trpm2 or inhibiting TRPM2 activation also suppresses the activation of CD36 and cellular dysfunction induced by OGD or TSP1. Conclusions In conclusion, our data reveal a novel molecular mechanism in which TRPM2 and CD36 promote the activation of each other, which exacerbates endothelial dysfunction during ischemic stroke. Our study suggests that TRPM2 in endothelial cells is a promising target for developing more effective and safer therapies for ischemic stroke. [ABSTRACT FROM AUTHOR]

Background: The mechanism of leukocyte transendothelial migration (TEM) across the highly restrictive blood-brain barrier (BBB) remains enigmatic, with paracellular TEM thought to require leukocytes to somehow navigate the obstructive endothelial tight junctions (TJs). Transient interactions between TJ proteins on the respective leukocyte and endothelial surfaces have been proposed as one mechanism for TEM. Given the expanding role of extracellular vesicles (EVs) in intercellular communication, we investigated whether EVs derived from brain microvascular endothelial cells (BMEC) of the BBB may play a role in transferring a major TJ protein, claudin-5 (CLN-5), to leukocytes as a possible basis for such a mechanism during neuroinflammation. Methods: High-resolution 3D confocal imaging was used to highlight CLN-5 immunoreactivity in the central nervous system (CNS) and on leukocytes of mice with the neuroinflammatory condition experimental autoimmune encephalomyelitis (EAE). Both Western blotting of circulating leukocytes from wild-type mice and fluorescence imaging of leukocyte-associated eGFP-CLN-5 in the blood and CNS of endothelial-targeted, Tie-2-eGFP-CLN-5 transgenic mice were used to confirm the presence of CLN-5 protein on these cells. EVs were isolated from TNF-α-stimulated BMEC cultures and blood plasma of Tie-2-eGFP-CLN-5 mice with EAE and evaluated for CLN-5 protein by Western blotting and fluorescence-activated cell sorting (FACS), respectively. Confocal imaging and FACS were used to detect binding of endothelial-derived EVs from these two sources to leukocytes in vitro. Serial electron microscopy (serial EM) and 3D contour-based surface reconstruction were employed to view EV-like structures at the leukocyte:BBB interface in situ in inflamed CNS microvessels. Results: A subpopulation of leukocytes immunoreactive for CLN-5 on their surface was seen to infiltrate the CNS of mice with EAE and reside in close apposition to inflamed vessels. Confocal imaging of immunostained samples and Western blotting established the presence of CLN-5+ leukocytes in blood as well, implying these cells are present prior to TEM. Moreover, imaging of inflamed CNS vessels and the associated perivascular cell infiltrates from Tie-2-eGFP-CLN-5 mice with EAE revealed leukocytes bearing the eGFP label, further supporting the hypothesis CLN-5 is transferred from endothelial cells to circulating leukocytes in vivo. Western blotting of BMEC-derived EVs, corresponding in size to both exosomes and microvesicles, and FACS analysis of plasma-derived EVs from Tie-2-eGFPCLN- 5 mice with EAE validated expression of CLN-5 by EVs of endothelial origin. Confocal imaging and FACS further revealed both PKH-67-labeled EVs from cultured BMECs and eGFP-CLN-5+ EVs from plasma of Tie-2-eGFP-CLN-5 mice with EAE can bind to leukocytes. Lastly, serial EM and 3D contour-based surface reconstruction revealed a close association of EV-like structures between the marginating leukocytes and BMECs in situ during EAE. Conclusions: During neuroinflammation, CLN-5+ leukocytes appear in the CNS, and both CLN-5+ leukocytes and CLN-5+ EVs are detected in the blood. As endothelial cells transfer CLN-5+ to leukocytes in vivo, and EVs released from BMEC bind to leukocytes in vitro, EVs may serve as the vehicles to transfer CLN-5 protein at sites of leukocyte:endothelial contact along the BBB. This action may be a prelude to facilitate TEM through the formation of temporary TJ protein bridges between these two cell types. [ABSTRACT FROM AUTHOR]

Background: The brain's initial innate response to stroke is primarily mediated by microglia, the resident macrophage of the CNS. However, as early as 4 h after stroke, the blood-brain barrier is compromised and monocyte infiltration occurs. The lack of discriminating markers between these two myeloid populations has led many studies to generate conclusions based on the grouping of these two populations. A growing body of evidence now supports the distinct roles played by microglia and monocytes in many disease models. Methods: Using a flow cytometry approach, combined with ex-vivo functional assays, we were able to distinguish microglia from monocytes using the relative expression of CD45 and assess the function of each cell type following stroke over the course of 7 days. Results: We found that at 72 h after a 90-min middle cerebral artery occlusion (MCAO), microglia populations decrease whereas monocytes significantly increase in the stroke brain compared to sham. After stroke, BRDU incorporation into monocytes in the bone marrow increased. After recruitment to the ischemic brain, these monocytes accounted for nearly all BRDU-positive macrophages. Inflammatory activity peaked at 72 h. Microglia produced relatively higher reactive oxygen species and TNF, whereas monocytes were the predominant IL-1β producer. Although microglia showed enhanced phagocytic activity after stroke, monocytes had significantly higher phagocytic capacity at 72 h. Interestingly, we found a positive correlation between TNF expression levels and phagocytic activity of microglia after stroke. Conclusions: In summary, the resident microglia population is vulnerable to the effects of severe ischemia, show compromised cell cycle progression, and adopt a largely pro-inflammatory phenotype after stroke. Infiltrating monocytes are primarily involved with early debris clearance of dying cells. These findings suggest that the early wave of infiltrating monocytes may be beneficial to stroke repair and future therapies aimed at mitigating microglia cell death may prove more effective than attempting to elicit targeted anti-inflammatory responses from damaged cells. [ABSTRACT FROM AUTHOR]

Background Expression of chemokine CCL2 in the normal central nervous system (CNS) is nearly undetectable, but is significantly upregulated and drives neuroinflammation during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis which is considered a contributing factor in the human disease. As astrocytes and brain microvascular endothelial cells (BMEC) forming the blood-brain barrier (BBB) are sources of CCL2 in EAE and other neuroinflammatory conditions, it is unclear if one or both CCL2 pools are critical to disease and by what mechanism(s). Methods Mice with selective CCL2 gene knockout (KO) in astrocytes (Astro KO) or endothelial cells (Endo KO) were used to evaluate the respective contributions of these sources to neuroinflammation, i.e., clinical disease progression, BBB damage, and parenchymal leukocyte invasion in a myelin oligodendrocyte glycoprotein peptide (MOG35-55)-induced EAE model. High-resolution 3-dimensional (3D) immunofluorescence confocal microscopy and colloidal gold immuno-electron microscopy were employed to confirm sites of CCL2 expression, and 3D immunofluorescence confocal microscopy utilized to assess inflammatory responses along the CNS microvasculature. Results Cell-selective loss of CCL2 immunoreactivity was demonstrated in the respective KO mice. Compared to wild-type (WT) mice, Astro KO mice showed reduced EAE severity but similar onset, while Endo KO mice displayed near normal severity but significantly delayed onset. Neither of the KO mice showed deficits in T cell proliferation, or IL-17 and IFN-γ production, following MOG35-55 exposure in vitro, or altered MOG-major histocompatibility complexclass II tetramer binding. 3D confocal imaging further revealed distinct actions of the two CCL2 pools in the CNS. Astro KOs lacked the CNS leukocyte penetration and disrupted immunostaining of CLN-5 at the BBB seen during early EAE in WT mice, while Endo KOs uniquely displayed leukocytes stalled in the microvascular lumen. Conclusions These results point to astrocyte and endothelial pools of CCL2 each regulating different stages of neuroinflammation in EAE, and carry implications for drug delivery in neuroinflammatory disease. [ABSTRACT FROM AUTHOR]