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During stroke the blood–brain barrier (BBB) is damaged which can result in vasogenic brain edema and inflammation. The reduced blood supply leads to decreased delivery of oxygen and glucose to affected areas of the brain. Oxygen and glucose deprivation (OGD) can cause upregulation of glucose uptake of brain endothelial cells. In this letter, we investigated the influence of MK801, a non-competitive inhibitor of the NMDA-receptor, on the regulation of the glucose uptake and of the main glucose transporters glut1 and sglt1 in murine BBB cell line cerebEND during OGD. mRNA expression of glut1 was upregulated 68.7- fold after 6 h OGD, which was significantly reduced by 10 μM MK801 to 28.9-fold. Sglt1 mRNA expression decreased during OGD which was further reduced by MK801. Glucose uptake was significantly increased up to 907% after 6 h OGD and was still higher (210%) after the 20 h reoxygenation phase compared to normoxia. Ten micromolar MK801 during OGD was able to reduce upregulated glucose uptake after OGD and reoxygenation significantly. Presence of several NMDAR subunits was proven on the mRNA level in cerebEND cells. Furthermore, it was shown that NMDAR subunit NR1 was upregulated during OGD and that this was inhibitable by MK801. In conclusion, the addition of MK801 during the OGD phase reduced significantly the glucose uptake after the subsequent reoxygenation phase in brain endothelial cells.
Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the Förster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.
The blood-brain barrier (BBB), made up of endothelial cells of capillaries in the brain, maintains the microenvironment of the central nervous system. During ischemia and traumatic brain injury (TBI), cellular disruption leading to mechanical insult results to the BBB being compromised. Oxygen glucose deprivation (OGD) is the most commonly used in vitro model for ischemia. On the other hand, stretch injury is currently being used to model TBI in vitro. In this paper, the two methods are used alone or in combination, to assess their effects on cerebrovascular endothelial cells cEND in the presence or absence of astrocytic factors. Applying severe stretch and/or OGD to cEND cells in our experiments resulted to cell swelling and distortion. Damage to the cells induced release of lactate dehydrogenase enzyme (LDH) and nitric oxide (NO) into the cell culture medium. In addition, mRNA expression of inflammatory markers interleukin (I L)-6, IL-1\(\alpha\) chemokine (C-C motif) ligand 2 (CCL2) and tumor necrosis factor (TNF)-\(\alpha\) also increased. These events could lead to the opening of calcium ion channels resulting to excitotoxicity. This could be demonstrated by increased calcium level in OGD-subjected cEND cells incubated with astrocyte-conditioned medium. Furthermore, reduction of cell membrane integrity decreased tight junction proteins claudin-5 and occludin expression. In addition, permeability of the endothelial cell monolayer increased. Also, since cell damage requires an increased uptake of glucose, expression of glucose transporter glut1 was found to increase at the mRNA level after OGD. Overall, the effects of OGD on cEND cells appear to be more prominent than that of stretch with regards to TJ proteins, NO, glutl expression, and calcium level. Astrocytes potentiate these effects on calcium level in cEND cells. Combining both methods to model TBI in vitro shows a promising improvement to currently available models.
Microvascular endothelial cells are an essential part of many biological barriers, such as the blood–brain barrier (BBB) and the endothelium of the arteries and veins. A reversible opening strategy to increase the permeability of drugs across the BBB could lead to improved therapies due to enhanced drug bioavailability. Vanilloids, such as capsaicin, are known to reversibly open tight junctions of epithelial and endothelial cells. In this study, we used several in vitro assays with the murine endothelial capillary brain cells (line cEND) as a BBB model to characterize the interaction between capsaicin and endothelial tight junctions.
Protocadherins (PCDHs) belong to the cadherin superfamily and represent the largest subgroup of calcium-dependent adhesion molecules. In the genome, most PCDHs are arranged in three clusters, α, β, and γ on chromosome 5q31. PCDHs are highly expressed in the central nervous system (CNS). Several PCDHs have tumor suppressor functions, but their individual role in primary brain tumors has not yet been elucidated. Here, we examined the mRNA expression of PCDHGC3, a member of the PCDHγ cluster, in non-cancerous brain tissue and in gliomas of different World Health Organization (WHO) grades and correlated it with the clinical data of the patients. We generated a PCDHGC3 knockout U343 cell line and examined its growth rate and migration in a wound healing assay. We showed that PCDHGC3 mRNA and protein were significantly overexpressed in glioma tissue compared to a non-cancerous brain specimen. This could be confirmed in glioma cell lines. High PCDHGC3 mRNA expression correlated with longer progression-free survival (PFS) in glioma patients. PCDHGC3 knockout in U343 resulted in a slower growth rate but a significantly faster migration rate in the wound healing assay and decreased the expression of several genes involved in WNT signaling. PCDHGC3 expression should therefore be further investigated as a PFS-marker in gliomas. However, more studies are needed to elucidate the molecular mechanisms underlying the PCDHGC3 effects.
Progressive deterioration of the central nervous system (CNS) is commonly associated with aging. An important component of the neurovasculature is the blood-brain barrier (BBB), majorly made up of endothelial cells joined together by intercellular junctions. The relationship between senescence and changes in the BBB has not yet been thoroughly explored. Moreover, the lack of in vitro models for the study of the mechanisms involved in those changes impede further and more in-depth investigations in the field. For this reason, we herein present an in vitro model of the senescent BBB and an initial attempt to identify senescence-associated alterations within.
Brain metastases are the most severe tumorous spread during breast cancer disease. They are associated with a limited quality of life and a very poor overall survival. A subtype of extracellular vesicles, exosomes, are sequestered by all kinds of cells, including tumor cells, and play a role in cell-cell communication. Exosomes contain, among others, microRNAs (miRs). Exosomes can be taken up by other cells in the body, and their active molecules can affect the cellular process in target cells. Tumor-secreted exosomes can affect the integrity of the blood-brain barrier (BBB) and have an impact on brain metastases forming. Serum samples from healthy donors, breast cancer patients with primary tumors, or with brain, bone, or visceral metastases were used to isolate exosomes and exosomal miRs. Exosomes expressed exosomal markers CD63 and CD9, and their amount did not vary significantly between groups, as shown by Western blot and ELISA. The selected 48 miRs were detected using real-time PCR. Area under the receiver-operating characteristic curve (AUC) was used to evaluate the diagnostic accuracy. We identified two miRs with the potential to serve as prognostic markers for brain metastases. Hsa-miR-576-3p was significantly upregulated, and hsa-miR-130a-3p was significantly downregulated in exosomes from breast cancer patients with cerebral metastases with AUC: 0.705 and 0.699, respectively. Furthermore, correlation of miR levels with tumor markers revealed that hsa-miR-340-5p levels were significantly correlated with the percentage of Ki67-positive tumor cells, while hsa-miR-342-3p levels were inversely correlated with tumor staging. Analysis of the expression levels of miRs in serum exosomes from breast cancer patients has the potential to identify new, non-invasive, blood-borne prognostic molecular markers to predict the potential for brain metastasis in breast cancer. Additional functional analyzes and careful validation of the identified markers are required before their potential future diagnostic use.
Ischemic stroke is one of the leading causes of death worldwide. It damages neurons and other supporting cellular elements in the brain. However, the impairment is not only confined to the region of assault but the surrounding area as well. Besides, it also brings about damage to the blood-brain barrier (BBB) which in turn leads to microvascular failure and edema. Hence, this necessitates an on-going, continuous search for intervention strategies and effective treatment. Of late, the natural sweetener stevioside proved to exhibit neuroprotective effects and therapeutic benefits against cerebral ischemia-induced injury. Its injectable formulation, isosteviol sodium (STVNA) also demonstrated favorable results. Nonetheless, its effects on the BBB have not yet been investigated to date. As such, this present study was designed to assess the effects of STVNA in our in vitro stroke model of the BBB.The integrity and permeability of the BBB are governed and maintained by tight junction proteins (TJPs) such as claudin-5 and occludin. Our data show increased claudin-5 and occludin expression in oxygen and glucose (OGD)-deprived murine brain capillary cerebellar endothelial cells (cerebEND) after STVNa treatment. Likewise, the upregulation of the transmembrane protein integrin-αv was also observed. Finally, cell volume was reduced with the simultaneous administration of STVNA and OGD in cerebEND cells. In neuropathologies such as stroke, the failure of cell volume control is a major feature leading to loss of cells in the penumbra as well as adverse outcomes. Our initial findings, therefore, point to the neuroprotective effects of STVNA at the BBB in vitro, which warrant further investigation for a possible future clinical intervention.
Advances in breast cancer management and extracellular vesicle research, a bibliometric analysis
(2021)
Extracellular vesicles transport variable content and have crucial functions in cell–cell communication. The role of extracellular vesicles in cancer is a current hot topic, and no bibliometric study has ever analyzed research production regarding their role in breast cancer and indicated the trends in the field. In this way, we aimed to investigate the trends in breast cancer management involved with extracellular vesicle research. Articles were retrieved from Scopus, including all the documents published concerning breast cancer and extracellular vesicles. We analyzed authors, journals, citations, affiliations, and keywords, besides other bibliometric analyses, using R Studio version 3.6.2. and VOSviewer version 1.6.0. A total of 1151 articles were retrieved, and as the main result, our analysis revealed trending topics on biomarkers of liquid biopsy, drug delivery, chemotherapy, autophagy, and microRNA. Additionally, research related to extracellular vesicles in breast cancer has been focused on diagnosis, treatment, and mechanisms of action of breast tumor-derived vesicles. Future studies are expected to explore the role of extracellular vesicles on autophagy and microRNA, besides investigating the application of extracellular vesicles from liquid biopsies for biomarkers and drug delivery, enabling the development and validation of therapeutic strategies for specific cancers.
The blood-brain barrier (BBB) is a highly specialized structure that separates the brain from the blood and allows the exchange of molecules between these two compartments through selective channels. The breakdown of the BBB is implicated in the development of severe neurological diseases, especially stroke and traumatic brain injury. Oxygen-glucose deprivation is used to mimic stroke and traumatic brain injury in vitro. Pathways that trigger BBB dysfunction include an imbalance of oxidative stress, excitotoxicity, iron metabolism, cytokine release, cell injury, and cell death. MicroRNAs are small non-coding RNA molecules that regulate gene expression and are emerging as biomarkers for the diagnosis of central nervous system (CNS) injuries. In this review, the regulatory role of potential microRNA biomarkers and related therapeutic targets on the BBB is discussed. A thorough understanding of the potential role of various cellular and linker proteins, among others, in the BBB will open further therapeutic options for the treatment of neurological diseases.