scholarly journals C.02 Whole genome expression profiling of blood-brain barrier endothelial cells after experimental subarachnoid hemorrhage

2016 ◽  
Vol 43 (S2) ◽  
pp. S11-S11
Author(s):  
MK Tso ◽  
P Turgeon ◽  
B Bosche ◽  
J Ai ◽  
P Marsden ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Subarachnoid Hemorrhage ◽  
Blood Brain Barrier ◽  
Expression Profiling ◽  
Brain Barrier ◽  
Whole Genome ◽  
Genome Expression ◽  
Blood Brain ◽  
Whole Genome Expression

Background: The pathophysiology of subarachnoid hemorrhage (SAH) is complex and includes disruption of the blood-brain barrier (BBB). We freshly isolated BBB endothelial cells (BECs) by 2 distinct methods after experimental SAH and then interrogated their gene expression profiles with the goal of uncovering new therapeutic targets. Methods: SAH was induced using the prechiasmatic blood injection mouse model. BBB permeability studies were performed by administering intraperitoneal cadaverine dye injections at 24h and 48h. BECs were isolated either by sequential magnetic-based sorting for CD45-CD31+ cells or by fluorescence-activated cell sorting (FACS) for Tie2+Pdgfrb- cells. Total RNA was extracted and analyzed using Affymetrix Mouse Gene 2.0 ST Arrays. Results: BBB impairment occurred at 24h and resolved by 48h after SAH. Analysis of gene expression patterns in BECs at 24h reveal clustering of SAH and sham samples. We identified 707 (2.8%) significant differentially-expressed genes (403 upregulated, 304 downregulated) out of 24,865 interrogated probe sets. Many significantly upregulated genes were involved in inflammatory pathways. These microarray results were validated with real-time polymerase chain reaction (RT-PCR). Conclusions: This study is the first to investigate in an unbiased manner, whole genome expression profiling of freshly-isolated BECs in an SAH animal model, yielding targets for novel therapeutic intervention.

Short regulatory DNA sequences to target brain endothelial cells for gene therapy

2021 ◽  
pp. 0271678X2110396 ◽  
Author(s):  
Hanna Graßhoff ◽  
Helge Müller-Fielitz ◽  
Godwin K Dogbevia ◽  
Jakob Körbelin ◽  
Jacqueline Bannach ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Regulatory Elements ◽  
Brain Barrier ◽  
Regulatory Sequences ◽  
Brain Endothelial Cells ◽  
Enhancer Element ◽  
Blood Brain

Gene vectors targeting CNS endothelial cells allow to manipulate the blood-brain barrier and to correct genetic defects in the CNS. Because vectors based on the adeno-associated virus (AAV) have a limited capacity, it is essential that the DNA sequence controlling gene expression is short. In addition, it must be specific for endothelial cells to avoid off-target effects. To develop improved regulatory sequences with selectivity for brain endothelial cells, we tested the transcriptional activity of truncated promoters of eleven (brain) endothelial-specific genes in combination with short regulatory elements, i.e., the woodchuck post-transcriptional regulatory element (W), the CMV enhancer element (C), and a fragment of the first intron of the Tie2 gene (S), by transfecting brain endothelial cells of three species. Four combinations of regulatory elements and short promoters ( Cdh5, Ocln, Slc2a1, and Slco1c1) progressed through this in-vitro pipeline displaying suitable activity. When tested in mice, the regulatory sequences C- Ocln-W and C- Slc2a1-S-W enabled a stronger and more specific gene expression in brain endothelial cells than the frequently used CAG promoter. In summary, the new regulatory elements efficiently control gene expression in brain endothelial cells and may help to specifically target the blood-brain barrier with gene therapy vectors.

scholarly journals Temporal genomewide expression profiling of DSS colitis reveals novel inflammatory and angiogenesis genes similar to ulcerative colitis

2011 ◽  
Vol 43 (1) ◽  
pp. 43-56 ◽  
Author(s):  
Kai Fang ◽  
Megan Bruce ◽  
Christopher B. Pattillo ◽  
Songlin Zhang ◽  
Randolph Stone ◽  
...  
Keyword(s):  
Gene Expression ◽  
Ulcerative Colitis ◽  
Expression Profiling ◽  
Profile Analysis ◽  
Whole Genome ◽  
Qrt Pcr ◽  
Dss Colitis ◽  
Genome Expression ◽  
Expression Profile Analysis ◽  
Whole Genome Expression

Dextran sodium sulfate (DSS)-induced colitis is widely used to study pathological mechanisms and potential treatments of inflammatory bowel disease. Because temporal changes in genome expression profiles remain unknown in this model, we performed whole genome expression profile analysis during the development of DSS colitis in comparison with ulcerative colitis (UC) specimens to identify novel and common responses during disease. Colon tissue from DSS-treated mice was collected at days 0, 2, 4, and 6. Half of each specimen was used for histopathological analysis and half for Affymetrix whole genome expression profiling and qRT-PCR validation. Genesifter and Ingenuity software analysis was used to identify differentially expressed genes and perform interactive network analysis. Identified DSS-associated genes in mice were also compared with UC patient data. We identified 1,609 genes that were significantly altered during DSS colitis; the majority were functionally related to inflammation, angiogenesis, metabolism, biological adhesion, cellular growth and proliferation, and cell-to-cell signaling responses. Five hundred and one genes were progressively upregulated, while one hundred seventy-three genes were progressively downregulated. Changes in gene expression were validated in a subset of 33 genes by qRT-PCR, with r2 = 0.925. Ingenuity gene interaction network analysis revealed novel relationships among antigen presentation, cell morphology, and other biological functions in the DSS mouse. Finally, DSS colitis gene array data were compared with UC patient array data: 152 genes were similarly upregulated, and 22 genes were downregulated. Temporal genomewide expression profile analysis of DSS-induced colitis revealed novel associations with various immune responses and tissue remodeling events such as angiogenesis similar to those in UC patients. This study provides a comprehensive view of DSS colitis changes in colon gene expression and identifies common molecules with clinical specimens that are interesting targets for further investigation.

scholarly journals Intranasal administration of vitamin D attenuates blood–brain barrier disruption through endogenous upregulation of osteopontin and activation of CD44/P-gp glycosylation signaling after subarachnoid hemorrhage in rats

2016 ◽  
Vol 37 (7) ◽  
pp. 2555-2566 ◽  
Author(s):  
Budbazar Enkhjargal ◽  
Devin W McBride ◽  
Anatol Manaenko ◽  
Cesar Reis ◽  
Yasushi Sakai ◽  
...  
Keyword(s):  
Vitamin D ◽  
Endothelial Cells ◽  
Subarachnoid Hemorrhage ◽  
Blood Brain Barrier ◽  
Vitamin D3 ◽  
Brain Barrier ◽  
Protective Effects ◽  
Brain Endothelial Cells ◽  
Sprague Dawley ◽  
Blood Brain

In this study, we investigated the role of vitamin D3 (VitD3) on endogenous osteopontin (OPN), a neuroprotective glycoprotein, after subarachnoid hemorrhage (SAH). The endovascular perforation SAH model in Sprague-Dawley rats was used to study the effect of intranasal VitD3 (30 ng/kg) before (Pre-SAH + VitD3) and after (Post-SAH + VitD3) subarachnoid hemorrhage. Vitamin D3 (30, 60, 120 ng/kg/day) increased more than one fold endogenous OPN expression in astrocytes and endothelial cells of rat brain. Vitamin D3 significantly decreased brain edema and Evans blue extravasation. In addition, neurobehavioral scores were significantly higher in Pre-SAH + VitD3, but partly higher in Post-SAH + VitD3, group compared with SAH group. These protective effects of vitamin D3 were completely attenuated by intracerebroventricular injection of transcription inhibitor Actinomycin D and significantly inhibited by small interfering ribonucleic acid (siRNA) for vitamin D receptor and OPN in Pre-SAH + VitD3 rats. OPN expression was significantly higher in Pre-SAH + VitD3 rats, specifically A and C, but not B, isomers were upregulated in the astrocytes, leading to CD44 splicing, and P-gp glycosylation in brain endothelial cells. The results show that intranasal vitamin D3 attenuates blood–brain barrier (BBB) disruption through endogenous upregulation of OPN and subsequent CD44 and P-gp glycosylation signals in brain endothelial cells. Furthermore, this study identifies a novel strategy for the cost-effective management of subarachnoid hemorrhage.

Gene expression profiling of prostate tissue based on variable expression of 5-alpha reductase 2.

2016 ◽  
Vol 34 (2_suppl) ◽  
pp. 143-143
Author(s):  
Keyan Salari ◽  
Seth Bechis ◽  
Rongbin Ge ◽  
Jian Hong ◽  
Aleksander Otsetov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hierarchical Clustering ◽  
Promoter Methylation ◽  
Expression Profiling ◽  
Molecular Subtypes ◽  
Prostatic Tissue ◽  
Whole Genome ◽  
Variable Expression ◽  
Genome Expression ◽  
Whole Genome Expression

143 Background: Androgen signaling plays a central role in the pathophysiology of both benign and malignant prostatic disease. We previously showed that some men carry epigenetic modifications of the 5-alpha reductase (AR) promoter that modulate gene expression, raising the hypothesis that there may be molecular heterogeneity underlying the variability in clinical response to 5-AR inhibitors for management of benign and malignant prostatic diseases. In this study, we performed whole-genome expression profiling of prostatic tissue based on variable expression of 5-AR2 to determine whether molecular subtypes of clinical relevance could be identified. Methods: Prostatic tissue specimens were obtained from 22 patients with symptomatic BPH undergoing prostate debulking surgery. 5-AR2 protein expression was measured by immunohistochemistry, and 5-AR2 promoter methylation determined by PCR. RNA was extracted from each specimen and whole-genome expression profiling was performed using Illumina Human BeadChip Arrays. Gene expression data was analyzed by unsupervised hierarchical clustering and Gene Set Enrichment Analysis. Results: Unsupervised hierarchical clustering identified two subtypes of patients with distinct gene expression signatures. Among the most differentially expressed genes were several notable androgen-regulated genes, including TMPRSS2, NKX3-1, AZGP1, KLK3 (encoding the PSA protein) and multiple other members of the kallikrein family of serine proteases (all P< 0.001, FDR < 0.02). Multiple gene sets composed of androgen-regulated genes and androgen receptor target genes were found to be significantly enriched in one of the two subtypes. Interestingly, patients from this Androgen-Up subtype were also noted to have a higher frequency of 5-AR2 promoter methylation (though not statistically significant). Conclusions: Our findings suggest that there are distinct molecular subtypes among prostatic tissues with variable expression of 5-AR2, driven by differences in androgen gene regulation. These differences may partially explain the variable response to 5-AR inhibitors and thereby inform clinical strategies for devising therapies for benign and malignant prostatic disease.

scholarly journals Screening for Interacting Proteins with Peptide Biomarker of Blood–Brain Barrier Alteration under Inflammatory Conditions

2021 ◽  
Vol 22 (9) ◽  
pp. 4725
Author(s):  
Karina Vargas-Sanchez ◽  
Monica Losada-Barragán ◽  
Maria Mogilevskaya ◽  
Susana Novoa-Herrán ◽  
Yehidi Medina ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Neurovascular Unit ◽  
Specific Interaction ◽  
Brain Barrier ◽  
Mass Spectrometry Analysis ◽  
Peptide Ligand ◽  
Inflammatory Conditions ◽  
Blood Brain ◽  
Potential Biomarker

Neurodegenerative diseases are characterized by increased permeability of the blood–brain barrier (BBB) due to alterations in cellular and structural components of the neurovascular unit, particularly in association with neuroinflammation. A previous screening study of peptide ligands to identify molecular alterations of the BBB in neuroinflammation by phage-display, revealed that phage clone 88 presented specific binding affinity to endothelial cells under inflammatory conditions in vivo and in vitro. Here, we aimed to identify the possible target receptor of the peptide ligand 88 expressed under inflammatory conditions. A cross-link test between phage-peptide-88 with IL-1β-stimulated human hCMEC cells, followed by mass spectrometry analysis, was used to identify the target of peptide-88. We modeled the epitope–receptor molecular interaction between peptide-88 and its target by using docking simulations. Three proteins were selected as potential target candidates and tested in enzyme-linked immunosorbent assays with peptide-88: fibronectin, laminin subunit α5 and laminin subunit β-1. Among them, only laminin subunit β-1 presented measurable interaction with peptide-88. Peptide-88 showed specific interaction with laminin subunit β-1, highlighting its importance as a potential biomarker of the laminin changes that may occur at the BBB endothelial cells under pathological inflammation conditions.

scholarly journals CdSe/ZnS Core-Shell-Type Quantum Dot Nanoparticles Disrupt the Cellular Homeostasis in Cellular Blood–Brain Barrier Models

2021 ◽  
Vol 22 (3) ◽  
pp. 1068
Author(s):  
Katarzyna Dominika Kania ◽  
Waldemar Wagner ◽  
Łukasz Pułaski
Keyword(s):  
Gene Expression ◽  
Quantum Dot ◽  
Blood Brain Barrier ◽  
Protein Gene ◽  
Brain Barrier ◽  
Core Shell ◽  
Cellular Homeostasis ◽  
Blood Brain ◽  
Shell Type ◽  
The Impact

Two immortalized brain microvascular endothelial cell lines (hCMEC/D3 and RBE4, of human and rat origin, respectively) were applied as an in vitro model of cellular elements of the blood–brain barrier in a nanotoxicological study. We evaluated the impact of CdSe/ZnS core-shell-type quantum dot nanoparticles on cellular homeostasis, using gold nanoparticles as a largely bioorthogonal control. While the investigated nanoparticles had surprisingly negligible acute cytotoxicity in the evaluated models, a multi-faceted study of barrier-related phenotypes and cell condition revealed a complex pattern of homeostasis disruption. Interestingly, some features of the paracellular barrier phenotype (transendothelial electrical resistance, tight junction protein gene expression) were improved by exposure to nanoparticles in a potential hormetic mechanism. However, mitochondrial potential and antioxidant defences largely collapsed under these conditions, paralleled by a strong pro-apoptotic shift in a significant proportion of cells (evidenced by apoptotic protein gene expression, chromosomal DNA fragmentation, and membrane phosphatidylserine exposure). Taken together, our results suggest a reactive oxygen species-mediated cellular mechanism of blood–brain barrier damage by quantum dots, which may be toxicologically significant in the face of increasing human exposure to this type of nanoparticles, both intended (in medical applications) and more often unintended (from consumer goods-derived environmental pollution).

scholarly journals Blood-Brain Barrier Deterioration and Hippocampal Gene Expression in Polymicrobial Sepsis: An Evaluation of Endothelial MyD88 and the Vagus Nerve

PLoS ONE ◽  
2016 ◽  
Vol 11 (1) ◽  
pp. e0144215 ◽  
Author(s):  
Gerard Honig ◽  
Simone Mader ◽  
Huiyi Chen ◽  
Amit Porat ◽  
Mahendar Ochani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Vagus Nerve ◽  
Blood Brain Barrier ◽  
Polymicrobial Sepsis ◽  
Brain Barrier ◽  
Blood Brain
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