Screening for Interacting Proteins with Peptide Biomarker of Blood-Brain Barrier Alteration under Inflammatory Conditions.
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ABSTRACT:
SUBMITTER: Vargas-Sanchez K
PROVIDER: S-EPMC8124558 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Breakdown of the blood-brain barrier (BBB) is an early hallmark of multiple sclerosis (MS), a progressive inflammatory disease of the central nervous system. Cell adhesion in the BBB is modulated by sphingosine-1-phosphate (S1P), a signaling protein, via S1P receptors (S1P?). Fingolimod phosphate (FTY720-P) a functional S1P? antagonist has been shown to improve the relapse rate in relapsing-remitting MS by preventing the egress of lymphocytes from lymph nodes. However, its role in modulating BBB permeability-in particular, on the tight junction proteins occludin, claudin 5 and ZO-1-has not been well elucidated to date. In the present study, FTY720-P did not change the transendothelial electrical resistance in a rat brain microvascular endothelial cell (RBMEC) culture exposed to inflammatory conditions and thus did not decrease endothelial barrier permeability. In contrast, occludin was reduced in RBMEC culture after adding FTY720-P. Additionally, FTY720-P did not alter the amount of endothelial matrix metalloproteinase (MMP)-9 and MMP-2 in RBMEC cultures. Taken together, our observations support the assumption that S1P? plays a dual role in vascular permeability, depending on its ligand. Thus, S1P? provides a mechanistic basis for FTY720-P-associated disruption of endothelial barriers-such as the blood-retinal barrier-which might result in macular edema.
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Project description:Pathologic conditions in the central nervous system, regardless of the underlying injury mechanism, show a certain level of blood-brain barrier (BBB) impairment. Endothelial dysfunction is the earliest event in the initiation of vascular damage caused by inflammation due to stroke, atherosclerosis, trauma, or brain infections. Recently, microRNAs (miRNAs) have emerged as a class of gene expression regulators. The relationship between neuroinflammation and miRNA expression in brain endothelium remains unexplored. Previously, we showed the BBB-protective and anti-inflammatory effects of glycogen synthase kinase (GSK) 3β inhibition in brain endothelium in in vitro and in vivo models of neuroinflammation. Using microarray screening, we identified miRNAs induced in primary human brain microvascular endothelial cells after exposure to the pro-inflammatory cytokine, tumor necrosis factor-α, with/out GSK3β inhibition. Among the highly modified miRNAs, let-7 and miR-98 were predicted to target the inflammatory molecules, CCL2 and CCL5. Overexpression of let-7 and miR-98 in vitro and in vivo resulted in reduced leukocyte adhesion to and migration across endothelium, diminished expression of pro-inflammatory cytokines, and increased BBB tightness, attenuating barrier 'leakiness' in neuroinflammation conditions. For the first time, we showed that miRNAs could be used as a therapeutic tool to prevent the BBB dysfunction in neuroinflammation.
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Project description:Many neuroinflammatory diseases are characterized by massive immune cell infiltration into the central nervous system. Identifying the underlying mechanisms could aid in the development of therapeutic strategies specifically interfering with inflammatory cell trafficking. To achieve this, we implemented and validated a blood-brain barrier (BBB) model to study chemokine secretion, chemokine transport, and leukocyte trafficking in vitro. In a coculture model consisting of a human cerebral microvascular endothelial cell line and human astrocytes, proinflammatory stimulation downregulated the expression of tight junction proteins, while the expression of adhesion molecules and chemokines was upregulated. Moreover, chemokine transport across BBB cocultures was upregulated, as evidenced by a significantly increased concentration of the inflammatory chemokine CCL3 at the luminal side following proinflammatory stimulation. CCL3 transport occurred independently of the chemokine receptors CCR1 and CCR5, albeit that migrated cells displayed increased expression of CCR1 and CCR5. However, overall leukocyte transmigration was reduced in inflammatory conditions, although higher numbers of leukocytes adhered to activated endothelial cells. Altogether, our findings demonstrate that prominent barrier activation following proinflammatory stimulation is insufficient to drive immune cell recruitment, suggesting that additional traffic cues are crucial to mediate the increased immune cell infiltration seen in vivo during neuroinflammation.
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Project description:Background and Objectives: Preclinical studies have shown cannabidiol is protective in models of ischemic stroke. Based on results from our recent systematic review, we investigated the effects of two promising neuroprotective phytocannabinoids, cannabigerol (CBG) and cannabidivarin (CBDV), on cells of the blood-brain barrier (BBB), namely human brain microvascular endothelial cells (HBMECs), pericytes, and astrocytes. Experimental Approach: Cultures were subjected to oxygen-glucose deprivation (OGD) protocol to model ischemic stroke and cell culture medium was assessed for cytokines and adhesion molecules post-OGD. Astrocyte cell lysates were also analyzed for DNA damage markers. Antagonist studies were conducted where appropriate to study receptor mechanisms. Results: In astrocytes CBG and CBDV attenuated levels of interleukin-6 (IL-6) and lactate dehydrogenase (LDH), whereas CBDV (10 nM-10 μM) also decreased vascular endothelial growth factor (VEGF) secretion. CBDV (300 nM-10 μM) attenuated levels of monocyte chemoattractant protein (MCP)-1 in HBMECs. In astrocytes, CBG decreased levels of DNA damage proteins, including p53, whereas CBDV increased levels of DNA damage markers. Antagonists for CB1, CB2, PPAR-γ, PPAR-α, 5-HT1A, and TRPV1 had no effect on CBG (3 μM) or CBDV (1 μM)-mediated decreases in LDH in astrocytes. GPR55 and GPR18 were partially implicated in the effects of CBDV, but no molecular target was identified for CBG. Conclusions: We show that CBG and CBDV were protective against OG mediated injury in three different cells that constitute the BBB, modulating different hallmarks of ischemic stroke pathophysiology. These data enhance our understanding of the protective effects of CBG and CBDV and warrant further investigation into these compounds in ischemic stroke. Future studies should identify other possible neuroprotective effects of CBG and CBDV and their corresponding mechanisms of action.
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Project description:ObjectiveTo determine the structure of pulmonary tissue under conditions of high oxygen concentration.MethodsTen-week-old C57BL male mice and control mice were exposed to 100% oxygen and to room air for 72 hours, respectively. To follow the progression of lesions, the mice were sacrificed at 6, 12, 24, 48, and 72 hours after 100% oxygen administration. Lung specimens obtained from these mice underwent morphologic analysis and immunofluorescence studies. We used scanning and transmission electron microscopy to determine the ultrastructure of the pulmonary capillaries, including the endothelial glycocalyx. To visualize the endothelial glycocalyx, we performed lanthanum nitrate staining.ResultsThe survival rate of the 100% oxygen administration group was 5% (2/40) and that of the control group was 100%. Perivascular cavity enlargement was detected 12 hours after 100% oxygen administration and expanded over time. Ultrastructural analysis using electron microscopy revealed collapsed alveoli and pulmonary capillary wall and alveolar wall thickening in the 100% oxygen group. The pulmonary capillary endothelial glycocalyx was injured in the 100% oxygen group. The perivascular cavity decreased in mice that were returned to room air after 48 hours of 100% oxygen administration.ConclusionHigh-concentration oxygen causes perivascular cavity enlargement; this is thought to be a special characteristic of high oxygen damage. In addition, high-concentration oxygen may be involved in pulmonary endothelial glycocalyx injury.
| S-EPMC7791376 | biostudies-literature
Project description:The TLR4-interacting SPA4 peptide suppresses inflammation. We assessed the structural and physicochemical properties and binding of SPA4 peptide to TLR4-MD2. We also studied the changes at the whole transcriptome level, cell morphology, viability, secreted cytokines and chemokines, and cell influx in cell systems and mouse models challenged with LPS and treated with SPA4 peptide. Our results demonstrated that the SPA4 peptide did not alter the cell viability and size and only moderately affected the transcriptome of the cells. Computational docking and rendering suggested that the SPA4 peptide intercalates with LPS-induced TLR4-MD2 complex. Results with alanine mutations of D-2 amino acid and NYTXXXRG-12-19 motif of SPA4 peptide suggested their role in binding to TLR4 and in reducing the cytokine response against LPS stimulus. Furthermore, therapeutically administered SPA4 peptide significantly suppressed the secreted levels of cytokines and chemokines in cells and bronchoalveolar lavage fluids of LPS-challenged mice. The results suggest that the SPA4 peptide intercalates with LPS-induced TLR4 complex and signaling for the suppression of inflammation.
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Project description:Blood-brain barrier (BBB) dysfunction seen in neuroinflammation contributes to mortality and morbidity in multiple sclerosis, encephalitis, traumatic brain injury, and stroke. Identification of molecular targets maintaining barrier function is of clinical relevance. We used a novel in vivo model of localized aseptic meningitis where tumor necrosis factor alpha (TNFα) was introduced intracerebrally and surveyed cerebral vascular changes and leukocyte-endothelium interactions by intravital videomicroscopy. Poly(ADP-ribose) polymerase-1 (PARP) inhibition significantly reduced leukocyte adhesion to and migration across brain endothelium in cortical microvessels. PARP inactivation diminished BBB permeability in an in vivo model of systemic inflammation. PARP suppression in primary human brain microvascular endothelial cells (BMVEC), an in vitro model of BBB, enhanced barrier integrity and augmented expression of tight junction proteins. PARP inhibition in BMVEC diminished human monocyte adhesion to TNFα-activated BMVEC (up to 65%) and migration (80-100%) across BBB models. PARP suppression decreased expression of adhesion molecules and decreased activity of GTPases (controlling BBB integrity and monocyte migration across the BBB). PARP inhibitors down-regulated expression of inflammatory genes and dampened secretion of pro-inflammatory factors increased by TNFα in BMVEC. These results point to PARP suppression as a novel approach to BBB protection in the setting of endothelial dysfunction caused by inflammation.
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Project description:ObjectiveThe primary goals of this study were to investigate the potential roles of ZNF22 and HDAC3 as a histone deacetylase in regulating an increases in blood-tumor barrier (BTB) permeability and some of the possible molecular mechanisms associated with this effect.MethodsThe expression of ZNF22 and HDAC3 in glioma-exposed endothelial cells (GECs) of BTB were detected transcription real-time PCR or western blot. The interaction of ZNF22 and HDAC3 in GECs associated with transcript effect was analyzed by means of Co-Immunoprecipitation and luciferase reporter assay.ResultsIn the present investigation, GECs expressed higher levels of ZNF22 as a zinc finger transcription factor and HDAC3 than endothelial cells. We then affirmed that silencing HDAC3 or ZNF22 led to a reduction in BTB permeability. By bioinformatics analysis, chromatin immunoprecipitation (ChIP) assays and luciferase assay, we found that ZNF22 had a target binding relationship with the promoter regions of ZO-1, Occludin, and Claudin-5 and negatively regulated the expression of ZO-1, Occludin, and Claudin-5. Furthermore, we revealed that HDAC3, as a co-transcript repressor with histone deacetylase activity, could interact with ZNF22 to hinder the expression of TJ-associated proteins, thereby further facilitating the permeability of BTB.ConclusionZNF22 acted as a transcription factor in conjunction with HDAC3 to modulate the expression of TJ-associated proteins, which was correlated with an increase in BTB permeability. These results may provide new strategies and targets for the chemotherapy of gliomas as well as intracranial infections.
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Project description:Ischemic stroke (IS) is a major neurological disease with high fatality and residual disability burdens. Long noncoding RNAs (lncRNAs) have been found to play an important role in IS. However, the roles and significance of most lncRNAs in IS are still unknown. This study was performed to identify differentially expressed (DE) lncRNAs using a lncRNA microarray in whole blood samples of patients suffering from acute cerebral ischemia. Bioinformatics analyses, including GO, KEGG pathway enrichment analysis, and proximity to putative stroke risk location analysis were performed. The novel lncRNA, ENST00000530525, significantly decreased after IS. Furthermore, we evaluated lncRNA ENST00000530525 expression in cultured hCMEC/D3 cells under oxygen-glucose deprivation/reoxygenation (OGD/R) conditions using fluorescent in situ hybridization (FISH) and quantitative real-time polymerase chain reaction (RT-qPCR) analysis. To investigate the function of lncRNA ENST00000530525, its over-expression (OE) and negative control (NC) plasmids were transfected into hCMEC/D3 cells, and cell viability was detected by a cell counting kit-8 (CCK-8) assay after OGD/R. LncRNA ENST00000530525 and ANO1 expression were investigated using RT-qPCR and immunofluorescence. For blood-brain barrier (BBB) permeability, FITC-dextran transendothelial permeability assay and tight junction (TJ) protein immunofluorescence assays were performed. There were 3352 DE lncRNAs in the blood samples of acute IS patients. The validation results were consistent with the gene chip data. The GO and KEGG results showed that these lncRNAs were mainly related to oxygen and glucose metabolism, leukocyte transendothelial migration, mitophagy and cellular senescence. Among these, lncRNA ENST00000530525 was the most highly downregulated lncRNA and it was mapped within the IS-associated gene anoctamin-1 (ANO1). We further found that lncRNA ENST00000530525 was downregulated in hCMEC/D3 cells under 4 h OGD and 20 h reoxygenation (OGD4/R20) conditions. Upregulating lncRNA ENST00000530525 by plasmid transfection decreased cell viability while increasing ANO1 expression and it contributed to BBB injury in hCMEC/D3 cells after OGD4/R20. The lncRNA ENST00000530525 might play deleterious roles in post-stroke pathogenesis. These results show that some DE lncRNAs in humans participate through characteristic roles in post-stroke pathogenesis; thus, the roles and significance of some novel lncRNAs in IS warrant further study.
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Project description:Cartilage tissue engineering aims to repair damaged cartilage tissue in arthritic joints. As arthritic joints have significantly higher levels of pro-inflammatory cytokines (such as IL-1? and TNF? that cause cartilage destruction, it is critical to engineer stable cartilage in an inflammatory environment. Biomaterial scaffolds constitute an important component of the microenvironment for chondrocytes in engineered cartilage. However, it remains unclear how the scaffold material influences the response of chondrocytes seeded in these scaffolds under inflammatory stimuli. Here we have compared the responses of articular chondrocytes seeded within three different polymeric scaffolding materials (silk, collagen and polylactic acid (PLA)) to IL-1? and TNF?. These scaffolds have different physical characteristics and yielded significant differences in the expression of genes associated with cartilage matrix production and degradation, cell adhesion and cell death. The silk and collagen scaffolds released pro-inflammatory cytokines faster and had higher uptake water abilities than PLA scaffolds. Correspondingly, chondrocytes cultured in silk and collagen scaffolds maintained higher levels of cartilage matrix than those in PLA, suggesting that these biophysical properties of scaffolds may regulate gene expression and the response to inflammatory stimuli in chondrocytes. Based on this study we conclude that selecting the proper scaffold material will aid in the engineering of more stable cartilage tissues for cartilage repair, and that silk and collagen are better scaffolds in terms of supporting the stability of three-dimensional cartilage under inflammatory conditions.
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