Altered long non-coding RNA transcriptomic profiles in brain microvascular endothelium after cerebral ischemia.
ABSTRACT: The brain endothelium is an important therapeutic target for the inhibition of cerebrovascular dysfunction in ischemic stroke. Previously, we documented the important regulatory roles of microRNAs in the cerebral vasculature, in particular the cerebral vascular endothelium. However, the functional significance and molecular mechanisms of other classes of non-coding RNAs in the regulation of cerebrovascular endothelial pathophysiology after stroke are completely unknown. Using RNA sequencing (RNA-seq) technology, we profiled long non-coding RNA (lncRNA) expressional signatures in primary brain microvascular endothelial cells (BMECs) after oxygen-glucose deprivation (OGD), an in vitro mimic of ischemic stroke conditions. After 16h of OGD exposure, the expression levels for 362 of the 10,677 lncRNAs analyzed changed significantly, including a total of 147 lncRNAs increased and 70 lncRNAs decreased by more than 2-fold. Among them, the most highly upregulated lncRNAs include Snhg12, Malat1, and lnc-OGD 1006, whereas the most highly downregulated lncRNAs include 281008D09Rik, Peg13, and lnc-OGD 3916. Alteration of the most highly upregulated/downregulated ODG-responsive lncRNAs was further confirmed in cultured BMECs after OGD as well as isolated cerebral microvessels in mice following transient middle cerebral artery occlusion (MCAO) and 24h reperfusion by the quantitative real-time PCR approach. Moreover, promoter analysis of altered ODG-responsive endothelial lncRNA genes by bioinformatics showed substantial transcription factor binding sites on lncRNAs, implying potential transcriptional regulation of those lncRNAs. These findings are the first to identify OGD-responsive brain endothelial lncRNAs, which suggest potential pathological roles for these lncRNAs in mediating endothelial responses to ischemic stimuli. Endothelial-selective lncRNAs may function as a class of novel master regulators in cerebrovascular endothelial pathologies after ischemic stroke.
Project description:The study was designed to determine the role of long noncoding RNA (lncRNA), metastasis-associated lung adenocarcinoma transcript 1 (Malat1), in ischemic stroke outcome. Primary mouse brain microvascular endothelial cells (BMECs) were cultured and treated with Malat1 GapmeR before 16 h oxygen and glucose depravation (OGD). Cell death was assayed by LDH and MTT methods. Malat1 knock-out and wild-type mice were subjected to 1 h of middle cerebral artery occlusion (MCAO) and 24-72 h of reperfusion. To explore the underlying mechanism, apoptotic and inflammatory factors were measured by qPCR, ELISA, and Western blotting. The physical interaction between Malat1 and apoptotic or inflammatory factors was measured by RNA immunoprecipitation. Increased Malat1 levels were found in cultured mouse BMECs after OGD as well as in isolated cerebral microvessels in mice after MCAO. Silencing of Malat1 by Malat1 GapmeR significantly increased OGD-induced cell death and Caspase 3 activity in BMECs. Silencing of Malat1 also significantly aggravated OGD-induced expression of the proapoptotic factor Bim and proinflammatory cytokines MCP-1, IL-6, and E-selectin. Moreover, Malat1 KO mice presented larger brain infarct size, worsened neurological scores, and reduced sensorimotor functions. Consistent with in vitro findings, significantly increased expression of proapoptotic and proinflammatory factors was also found in the cerebral cortex of Malat1 KO mice after ischemic stroke compared with WT controls. Finally, we demonstrated that Malat1 binds to Bim and E-selectin both in vitro and in vivo Our study suggests that Malat1 plays critical protective roles in ischemic stroke.SIGNIFICANCE STATEMENT Accumulative studies have demonstrated the important regulatory roles of microRNAs in vascular and neural damage after ischemic stroke. However, the functional significance and mechanisms of other classes of noncoding RNAs in cerebrovascular pathophysiology after stroke are less studied. Here we demonstrate a novel role of Malat1, a long noncoding RNA that has been originally identified as a prognostic marker for non-small cell lung cancer, in cerebrovascular pathogenesis of ischemic stroke. Our experiments have provided the first evidence that Malat1 plays anti-apoptotic and anti-inflammatory roles in brain microvasculature to reduce ischemic cerebral vascular and parenchymal damages. Our studies also suggest that lncRNAs can be therapeutically targeted to minimize poststroke brain damage.
Project description:Stroke is one of the leading causes of mortality and disability worldwide. Uncovering the cellular and molecular pathophysiological processes in stroke have been a top priority. Long non-coding (lnc) RNAs play critical roles in different kinds of diseases. In recent years, a bulk of aberrantly expressed lncRNAs have been screened out in ischemic stroke patients or ischemia insulted animals using new technologies such as RNA-seq, deep sequencing, and microarrays. Nine specific lncRNAs, antisense non-coding RNA in the INK4 locus (ANRIL), metastasis-associate lung adenocarcinoma transcript 1 (MALAT1), N1LR, maternally expressed gene 3 (MEG3), H19, CaMK2D-associated transcript 1 (C2dat1), Fos downstream transcript (FosDT), small nucleolar RNA host gene 14 (SNHG14), and taurine-upregulated gene 1 (TUG1), were found increased in cerebral ischemic animals and/or oxygen-glucose deprived (OGD) cells. These lncRNAs were suggested to promote cell apoptosis, angiogenesis, inflammation, and cell death. Our Gene Ontology (GO) enrichment analysis predicted that MEG3, H19, and MALAT1 might also be related to functions such as neurogenesis, angiogenesis, and inflammation through mechanisms of gene regulation (DNA transcription, RNA folding, methylation, and gene imprinting). This knowledge may provide a better understanding of the functions and mechanisms of lncRNAs in ischemic stroke. Further elucidating the functions and mechanisms of these lncRNAs in biological systems under normal and pathological conditions may lead to opportunities for identifying biomarkers and novel therapeutic targets of ischemic stroke.
Project description:Ischemic stroke induces neuronal death in the core of the infarct within a few hours and the secondary damage in the surrounding regions over a long period of time. Reduction of inflammation using pharmacological reagents has become a target of research for the treatment of stroke. Cyclooxygenase 2 (COX-2), a marker of inflammation, is induced during stroke and enhances inflammatory reactions through the release of enzymatic products, such as prostaglandin (PG) E2.Wild-type (WT) and COX-2 knockout (COX-2KO) mice were subjected to middle cerebral artery occlusion (MCAO). Additionally, brain slices derived from these mice or brain microvascular endothelial cells (BMECs) were exposed to oxygen-glucose deprivation (OGD) conditions. The expression levels of extracellular matrix (ECM) proteins were assessed and correlated with the state of inflammation.We found that components of the ECM, and specifically laminin, are transiently highly upregulated on endothelial cells after MCAO or OGD. This upregulation is not observed in COX-2KO mice or WT mice treated with COX-2 inhibitor, celecoxib, suggesting that COX-2 is associated with changes in the levels of laminins.Taken together, we report that transient ECM remodeling takes place early after stroke and suggest that this increase in ECM protein expression may constitute an effort to revascularize and oxygenate the tissue.
Project description:Stroke is a neurological disease with high disability and fatality rates, and ischemic stroke accounts for 75% of all stroke cases. The underlying pathophysiologic processes of ischemic stroke include oxidative stress, toxicity of excitatory amino acids, excess calcium ions, increased apoptosis and inflammation. Long non-coding RNAs (lncRNAs) may participate in the regulation of the pathophysiologic processes of ischemic stroke as indicated by altered expression of lncRNAs in blood samples of acute ischemic stroke patients, animal models of focal cerebral ischemia and oxygen-glucose deprivation (OGD) cell models. Because of the potentially important role, lncRNAs might be useful as biomarkers for the diagnosis, treatment and prognosis of ischemic stroke. This article reviews the functions of lncRNAs in different pathophysiology events of ischemic stroke with a focus on specific lncRNAs that may underlie ischemic stroke pathophysiology and that could therefore serve as potential diagnostic biomarkers and therapeutic targets.
Project description:Cerebral vascular endothelial cell (CEC) degeneration significantly contributes to blood-brain barrier (BBB) breakdown and neuronal loss after cerebral ischemia. Recently, emerging data suggest that peroxisome proliferator-activated receptor delta (PPARdelta) activation has a potential neuroprotective role in ischemic stroke. Here we report for the first time that PPARdelta is significantly reduced in oxygen-glucose deprivation (OGD)-induced mouse CEC death. Interestingly, PPARdelta overexpression can suppress OGD-induced caspase-3 activity, Golgi fragmentation, and CEC death through an increase of bcl-2 protein levels without change of bcl-2 mRNA levels. To explore the molecular mechanisms, we have identified that upregulation of PPARdelta can alleviate ODG-activated microRNA-15a (miR-15a) expression in CECs. Moreover, we have demonstrated that bcl-2 is a translationally repressed target of miR-15a. Intriguingly, gain- or loss-of-miR-15a function can significantly reduce or increase OGD-induced CEC death, respectively. Furthermore, we have identified that miR-15a is a transcriptional target of PPARdelta. Consistent with the in vitro findings, we found that intracerebroventricular infusion of a specific PPARdelta agonist, GW 501516 (2-[2-methyl-4-[[4-methyl-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl]methylsulfanyl]phenoxy]acetic acid), significantly reduced ischemia-induced miR-15a expression, increased bcl-2 protein levels, and attenuated caspase-3 activity and subsequent DNA fragmentation in isolated cerebral microvessels, leading to decreased BBB disruption and reduced cerebral infarction in mice after transient focal cerebral ischemia. Together, these results suggest that PPARdelta plays a vascular-protective role in ischemia-like insults via transcriptional repression of miR-15a, resulting in subsequent release of its posttranscriptional inhibition of bcl-2. Thus, regulation of PPARdelta-mediated miR-15a inhibition of bcl-2 could provide a novel therapeutic strategy for the treatment of stroke-related vascular dysfunction.
Project description:Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin ?-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin ?-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin ?-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke.
Project description:Cell therapies that invoke pleiotropic mechanisms may facilitate functional recovery in patients with stroke. Based on previous experiments using microglia preconditioned by oxygen-glucose deprivation, we hypothesized that the administration of peripheral blood mononuclear cells (PBMCs) preconditioned by oxygen-glucose deprivation (OGD-PBMCs) to be a therapeutic strategy for ischemic stroke. Here, OGD-PBMCs were identified to secrete remodelling factors, including the vascular endothelial growth factor and transforming growth factor-? in vitro, while intra-arterial administration of OGD-PBMCs at 7 days after focal cerebral ischemia prompted expression of such factors in the brain parenchyma at 28 days following focal cerebral ischemia in vivo. Furthermore, administration of OGD-PBMCs induced an increasing number of stage-specific embryonic antigen-3-positive cells both in vitro and in vivo. Finally, it was found to prompt angiogenesis and axonal outgrowth, and functional recovery after cerebral ischemia. In conclusion, the administration of OGD-PBMCs might be a novel therapeutic strategy against ischemic stroke.
Project description:This study examines the regulating effect of Sonic Hedgehog (Shh) on the permeability of the blood-brain barrier (BBB) in cerebral ischemia. By employing permanent middle cerebral artery occlusion (pMCAO) model, we find that Shh significantly decreases brain edema and preserves BBB permeability. Moreover, Shh increases zonula occludens-1 (ZO-1), occludin and angiopiotetin-1 (Ang-1) expression in the ischemic penumbra. Blockage of Shh with cyclopamine abolishes the effects of Shh on brain edema, BBB permeability and ZO-1, occludin, Ang-1 expression. Primary brain microvessel endothelial cells (BMECs) and astrocytes were pre-treated with Shh, cyclopamine, Ang-1-neutralizing antibody, and subjected to oxygen-glucose deprivation (OGD). Results show that the Ang-1 protein level in the culture medium of Shh-treated astrocytes is significantly higher. Shh also increased ZO-1, occludin and Ang-1 expression in BMECs, while cyclopamine and Ang-1-neutralizing antibody inhibited the effects of Shh on the ZO-1 and occludin expression, respectively. This study suggests that, under ischemic insults, Shh triggers Ang-1 production predominantly in astrocytes, and the secreted Ang-1 acts on BMECs, thereby upregulating ZO-1 and occludin to repair the tight junction and ameliorate the brain edema and BBB leakage.
Project description:The blood-brain barrier (BBB) is a selectively permeable cerebrovascular endothelial barrier that maintains homeostasis between the periphery and the central nervous system. BBB disruption is a consequence of ischemic stroke and BBB permeability can be altered by infection/inflammation, but the complex cellular and molecular changes that result in this BBB alteration need to be elucidated to determine mechanisms.Infection mimic (lipopolysaccharide) challenge on infarct volume, BBB permeability, infiltrated neutrophils, and functional outcomes after murine transient middle cerebral artery occlusion in vivo; mitochondrial evaluation of cerebrovascular endothelial cells challenged by lipopolysaccharide in vitro; pharmacological inhibition of mitochondria on BBB permeability in vitro and in vivo; the effects of mitochondrial inhibitor on BBB permeability, infarct volume, and functional outcomes after transient middle cerebral artery occlusion.We report here that lipopolysaccharide worsens ischemic stroke outcome and increases BBB permeability after transient middle cerebral artery occlusion in mice. Furthermore, we elucidate a novel mechanism that compromised mitochondrial function accounts for increased BBB permeability as evidenced by: lipopolysaccharide-induced reductions in oxidative phosphorylation and subunit expression of respiratory chain complexes in cerebrovascular endothelial cells, a compromised BBB permeability induced by pharmacological inhibition of mitochondrial function in cerebrovascular endothelial cells in vitro and in an in vivo animal model, and worsened stroke outcomes in transient middle cerebral artery occlusion mice after inhibition of mitochondrial function.We concluded that mitochondria are key players in BBB permeability. These novel findings suggest a potential new therapeutic strategy for ischemic stroke by endothelial cell mitochondrial regulation.
Project description:Cell-therapies that invoke pleiotropic mechanisms may facilitate functional recovery in stroke patients. We hypothesized that a cell therapy using microglia preconditioned by optimal oxygen-glucose deprivation (OGD) is a therapeutic strategy for ischemic stroke because optimal ischemia induces anti-inflammatory M2 microglia. We first delineated changes in angiogenesis and axonal outgrowth in the ischemic cortex using rats. We found that slight angiogenesis without axonal outgrowth were activated at the border area within the ischemic core from 7 to 14 days after ischemia. Next, we demonstrated that administration of primary microglia preconditioned by 18?hours of OGD at 7 days prompted functional recovery at 28 days after focal cerebral ischemia compared to control therapies by marked secretion of remodelling factors such as vascular endothelial growth factor, matrix metalloproteinase-9, and transforming growth factor-? polarized to M2 microglia in vitro/vivo. In conclusion, intravascular administration of M2 microglia preconditioned by optimal OGD may be a novel therapeutic strategy against ischemic stroke.