Prostaglandin production selectively in brain endothelial cells is both necessary and sufficient for eliciting fever.

Fever is known to be elicited by prostaglandin E2 acting on the brain, but its origin has remained disputed. We show in mice that selective deletion of prostaglandin synthesis in brain endothelial cells, but not in neural cells or myeloid cells, abolished fever induced by intravenous administration of lipopolysaccharide and that selective rescue of prostaglandin synthesis in brain endothelial cells reinstated fever. These data demonstrate that prostaglandin production in brain endothelial cells is both necessary and sufficient for eliciting fever.

Group B Streptococcus transcriptome when interacting with brain endothelial cells.

Bacterial meningitis is a life-threatening infection of the central nervous system (CNS) that occurs when bacteria are able to cross the blood-brain barrier (BBB) or the meningeal-cerebrospinal fluid barrier (mBCSFB). The BBB and mBCSFB comprise highly specialized brain endothelial cells (BECs) that typically restrict pathogen entry. Group B Streptococcus (GBS or Streptococcus agalactiae) is the leading cause of neonatal meningitis. Until recently, identification of GBS virulence factors has relied on genetic screening approaches. Instead, we here conducted RNA-seq analysis on GBS when interacting with induced pluripotent stem cell-derived BECs (iBECs) to pinpoint virulence-associated genes. Of the 2,068 annotated protein-coding genes of GBS, 430 transcripts displayed significant changes in expression after interacting with BECs. Notably, we found that the majority of differentially expressed GBS transcripts were downregulated (360 genes) during infection of iBECs. Interestingly, codY, encoding a pleiotropic transcriptional repressor in low-G + C Gram-positive bacteria, was identified as being highly downregulated. We conducted qPCR to confirm the codY downregulation observed via RNA-seq during the GBS-iBEC interaction and obtained codY mutants in three different GBS background parental strains. As anticipated from the RNA-seq results, the [Formula: see text]codY strains were more adherent and invasive in two in vitro BEC models. Together, this demonstrates the utility of RNA-seq during the BEC interaction to identify GBS virulence modulators. IMPORTANCE: Group B Streptococcus (GBS) meningitis remains the leading cause of neonatal meningitis. Research work has identified surface factors and two-component systems that contribute to GBS disruption of the blood-brain barrier (BBB). These discoveries often relied on genetic screening approaches. Here, we provide transcriptomic data describing how GBS changes its transcriptome when interacting with brain endothelial cells. Additionally, we have phenotypically validated these data by obtaining mutants of a select regulator that is highly down-regulated during infection and testing on our BBB model. This work provides the research field with a validated data set that can provide an insight into potential pathways that GBS requires to interact with the BBB and open the door to new discoveries.

Kv7 channel activation reduces brain endothelial cell permeability and prevents kainic acid-induced blood-brain barrier damage.

Ion channels in the blood-brain barrier (BBB) play a main role in controlling the interstitial fluid composition and cerebral blood flow, and their dysfunction contributes to the disruption of the BBB occurring in many neurological diseases such as epilepsy. In this study, using morphological and functional approaches, we evaluated the expression and role in the BBB of Kv7 channels, a family of voltage-gated potassium channels including five members (Kv7.1-5) that play a major role in the regulation of cell excitability and transmembrane flux of potassium ions. Immunofluorescence experiments showed that Kv7.1, Kv7.4, and Kv7.5 were expressed in rat brain microvessels (BMVs), as well as brain primary- and clonal (BEND-3) endothelial cells (ECs). Kv7.5 localized at the cell-to-cell junction sites, whereas Kv7.4 was also found in pericytes. The Kv7 activator retigabine increased transendothelial electrical resistance (TEER) in both primary ECs and BEND-3 cells; moreover, retigabine reduced paracellular dextran flux in BEND-3 cells. These effects were prevented by the selective Kv7 blocker XE-991. Exposure to retigabine also hyperpolarized cell membrane and increased tight junctions (TJs) integrity in BEND-3 cells. BMVs from rats treated with kainic acid (KA) showed a disruption of TJs and a selective reduction of Kv7.5 expression. In BEND-3 cells, retigabine prevented the increase of cell permeability and the reduction of TJs integrity induced by KA. Overall, these findings demonstrate that Kv7 channels are expressed in the BBB, where they modulate barrier properties both in physiological and pathological conditions.NEW & NOTEWORTHY This study describes for the first time the expression and the functional role of Kv7 potassium channels in the blood-brain barrier. We show that the opening of Kv7 channels reduces endothelial cell permeability both in physiological and pathological conditions via the hyperpolarization of cell membrane and the sealing of tight junctions. Therefore, activation of endothelial Kv7 channels might be a useful strategy to treat epilepsy and other neurological disorders characterized by blood-brain barrier dysfunction.

Viral entry and translation in brain endothelia provoke influenza-associated encephalopathy.

Influenza-associated encephalopathy (IAE) is extremely acute in onset, with high lethality and morbidity within a few days, while the direct pathogenesis by influenza virus in this acute phase in the brain is largely unknown. Here we show that influenza virus enters into the cerebral endothelium and thereby induces IAE. Three-weeks-old young mice were inoculated with influenza A virus (IAV). Physical and neurological scores were recorded and temporal-spatial analyses of histopathology and viral studies were performed up to 72 h post inoculation. Histopathological examinations were also performed using IAE human autopsy brains. Viral infection, proliferation and pathogenesis were analyzed in cell lines of endothelium and astrocyte. The effects of anti-influenza viral drugs were tested in the cell lines and animal models. Upon intravenous inoculation of IAV in mice, the mice developed encephalopathy with brain edema and pathological lesions represented by micro bleeding and injured astrocytic process (clasmatodendrosis) within 72 h. Histologically, massive deposits of viral nucleoprotein were observed as early as 24 h post infection in the brain endothelial cells of mouse models and the IAE patients. IAV inoculated endothelial cell lines showed deposition of viral proteins and provoked cell death, while IAV scarcely amplified. Inhibition of viral transcription and translation suppressed the endothelial cell death and the lethality of mouse models. These data suggest that the onset of encephalopathy should be induced by cerebral endothelial infection with IAV. Thus, IAV entry into the endothelium, and transcription and/or translation of viral RNA, but not viral proliferation, should be the key pathogenesis of IAE.

Nanomedicine: Patuletin-conjugated with zinc oxide exhibit potent effects against Gram-negative and Gram-positive bacterial pathogens.

With the emergence of drug-resistance, there is a need for novel anti-bacterials or to enhance the efficacy of existing drugs. In this study, Patuletin (PA), a flavanoid was loaded onto Gallic acid modified Zinc oxide nanoparticles (PA-GA-ZnO), and evaluated for antibacterial properties against Gram-positive (Bacillus cereus and Streptococcus pneumoniae) and Gram-negative (Samonella enterica and Escherichia coli) bacteria. Characterization of PA, GA-ZnO and PA-GA-ZnO' nanoparticles was accomplished utilizing fourier-transform infrared spectroscopy, efficiency of drug entrapment, polydispersity index, zeta potential, size, and surface morphology analysis through atomic force microscopy. Using bactericidal assays, the results revealed that ZnO conjugation displayed remarkable effects and enhanced Patuletin's effects against both Gram-positive and Gram-negative bacteria, with the minimum inhibitory concentration observed at micromolar concentrations. Cytopathogenicity assays exhibited that the drug-nanoconjugates reduced bacterial-mediated human cell death with minimal side effects to human cells. When tested alone, drug-nanoconjugates tested in this study showed limited toxic effects against human cells in vitro. These are promising findings, but future work is needed to understand the molecular mechanisms of effects of drug-nanoconjugates against bacterial pathogens, in addition to in vivo testing to determine their translational value. This study suggests that Patuletin-loaded nano-formulation (PA-GA-ZnO) may be implicated in a multi-target mechanism that affects both Gram-positive and Gram-negative pathogen cell structures, however this needs to be ascertained in future work.

Endothelial Cell-Derived Soluble CD200 Determines the Ability of Immune Cells to Cross the Blood-Brain Barrier.

The infiltration of immune cells into the central nervous system mediates the development of autoimmune neuroinflammatory diseases. We previously showed that the loss of either Fabp5 or calnexin causes resistance to the induction of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model of multiple sclerosis (MS). Here we show that brain endothelial cells lacking either Fabp5 or calnexin have an increased abundance of cell surface CD200 and soluble CD200 (sCD200) as well as decreased T-cell adhesion. In a tissue culture model of the blood-brain barrier, antagonizing the interaction of CD200 and sCD200 with T-cell CD200 receptor (CD200R1) via anti-CD200 blocking antibodies or the RNAi-mediated inhibition of CD200 production by endothelial cells increased T-cell adhesion and transmigration across monolayers of endothelial cells. Our findings demonstrate that sCD200 produced by brain endothelial cells regulates immune cell trafficking through the blood-brain barrier and is primarily responsible for preventing activated T-cells from entering the brain.

BACE2 beyond ß-processing of APP, its neuroprotective role in cerebrovascular endothelium.

Several proteases are involved in the proteolytic processing of the amyloid precursor protein (APP) generating the amyloidogenic Aß peptide, which can act as the triggering pathological effector of Alzheimer's disease (AD). Among these proteases, the ß-site amyloid precursor protein cleaving enzyme 2 (BACE2) is of particular interest because it was first proposed as an alternative ß-secretase to its homolog BACE1; however, accumulating evidence suggests that BACE2 acts as a non-amyloidogenic α-secretase and exerts neuroprotective effects. In this issue of J Neurochem, Katusic et al. present an interesting article reporting that BACE2 plays a role in preservation of cerebral vascular endothelial nitric oxide synthase (eNOS) function, thus exerting protective functions. Their data support that the process is mediated by the large soluble non-amyloidogenic APP fragment sAPPα through the γ-aminobutyric acid type B receptor 1, which enhances the expression of a major transcription factor for eNOS gene expression in endothelial cells, the Krüppel-like factor 2. These protective functions of BACE2 contrast with the pathogenic role of BACE1 as a key player in the AD amyloidogenic pathway. Indeed, many efforts have been invested in BACE1 inhibitors as potential disease modifiers for AD. Unfortunately, the results in clinical trials have been disappointing. In this scenario, a better understanding of the functions of BACE2, as well as the selectivity of BACE1 inhibitors with respect to other ß-secretases (mainly BACE2), is crucial for the development of new therapeutic agents. Furthermore, specific cellular targeting should also be considered to improve such therapies due to the diverse balance of secretases targeting APP and the complex cross-talk between them and the generated APP fragments.

Chronic kidney disease promotes cerebral microhemorrhage formation.

BACKGROUND: Chronic kidney disease (CKD) is increasingly recognized as a stroke risk factor, but its exact relationship with cerebrovascular disease is not well-understood. We investigated the development of cerebral small vessel disease using in vivo and in vitro models of CKD. METHODS: CKD was produced in aged C57BL/6J mice using an adenine-induced tubulointerstitial nephritis model. We analyzed brain histology using Prussian blue staining to examine formation of cerebral microhemorrhage (CMH), the hemorrhagic component of small vessel disease and the neuropathological substrate of MRI-demonstrable cerebral microbleeds. In cell culture studies, we examined effects of serum from healthy or CKD patients and gut-derived uremic toxins on brain microvascular endothelial barrier. RESULTS: CKD was induced in aged C57BL/6J mice with significant increases in both serum creatinine and cystatin C levels (p < 0.0001) without elevation of systolic or diastolic blood pressure. CMH was significantly increased and positively correlated with serum creatinine level (Spearman r = 0.37, p < 0.01). Moreover, CKD significantly increased Iba-1-positive immunoreactivity by 51% (p < 0.001), induced a phenotypic switch from resting to activated microglia, and enhanced fibrinogen extravasation across the blood-brain barrier (BBB) by 34% (p < 0.05). On analysis stratified by sex, the increase in CMH number was more pronounced in male mice and this correlated with greater creatinine elevation in male compared with female mice. Microglial depletion with PLX3397 diet significantly decreased CMH formation in CKD mice without affecting serum creatinine levels. Incubation of CKD serum significantly reduced transendothelial electrical resistance (TEER) (p < 0.01) and increased sodium fluorescein permeability (p < 0.05) across the endothelial monolayer. Uremic toxins (i.e., indoxyl sulfate, p-cresyl sulfate, and trimethylamine-N-oxide) in combination with urea and lipopolysaccharide induced a marked drop in TEER compared with the control group (p < 0.0001). CONCLUSIONS: CKD promotes the development of CMH in aged mice independent of blood pressure but directly proportional to the degree of renal impairment. These effects of CKD are likely mediated in part by microglia and are associated with BBB impairment. The latter is likely related to gut-derived bacteria-dependent toxins classically associated with CKD. Overall, these findings demonstrate an important role of CKD in the development of cerebral small vessel disease.

Impaired endothelium-mediated cerebrovascular reactivity promotes anxiety and respiration disorders in mice.

Carbon dioxide (CO2), the major product of metabolism, has a strong impact on cerebral blood vessels, a phenomenon known as cerebrovascular reactivity. Several vascular risk factors such as hypertension or diabetes dampen this response, making cerebrovascular reactivity a useful diagnostic marker for incipient vascular pathology, but its functional relevance, if any, is still unclear. Here, we found that GPR4, an endothelial H+ receptor, and endothelial Gαq/11 proteins mediate the CO2/H+ effect on cerebrovascular reactivity in mice. CO2/H+ leads to constriction of vessels in the brainstem area that controls respiration. The consequential washout of CO2, if cerebrovascular reactivity is impaired, reduces respiration. In contrast, CO2 dilates vessels in other brain areas such as the amygdala. Hence, an impaired cerebrovascular reactivity amplifies the CO2 effect on anxiety. Even at atmospheric CO2 concentrations, impaired cerebrovascular reactivity caused longer apneic episodes and more anxiety, indicating that cerebrovascular reactivity is essential for normal brain function. The site-specific reactivity of vessels to CO2 is reflected by regional differences in their gene expression and the release of vasoactive factors from endothelial cells. Our data suggest the central nervous system (CNS) endothelium as a target to treat respiratory and affective disorders associated with vascular diseases.

Effects of Prokineticins on Cerebral Cell Function and Blood-Brain Barrier Permeability.

Prokineticins are a family of small proteins with diverse roles in various tissues, including the brain. However, their specific effects on different cerebral cell types and blood-brain barrier (BBB) function remain unclear. The aim of this study was to investigate the effects of PROK1 and PROK2 on murine cerebral cell lines, bEnd.3, C8.D30, and N2a, corresponding to microvascular endothelial cells, astrocytes and neurons, respectively, and on an established BBB co-culture model. Western blot analysis showed that prokineticin receptors (PROKR1 and PROKR2) were differentially expressed in the considered cell lines. The effect of PROK1 and PROK2 on cell proliferation and migration were assessed using time-lapse microscopy. PROK1 decreased neural cells' proliferation, while it had no effect on the proliferation of endothelial cells and astrocytes. In contrast, PROK2 reduced the proliferation of all cell lines tested. Both PROK1 and PROK2 increased the migration of all cell lines. Blocking PROKRs with the PROKR1 antagonist (PC7) and the PROKR2 antagonist (PKR-A) inhibited astrocyte PROK2-mediated migration. Using the insert co-culture model of BBB, we demonstrated that PROKs increased BBB permeability, which could be prevented by PROKRs' antagonists.

Melanoma Cells Produce Large Vesicular-Bodies That Cause Rapid Disruption of Brain Endothelial Barrier-Integrity and Disassembly of Junctional Proteins.

It is known that many cells produce extracellular vesicles, and this includes a range of different cancer cell types. Here we demonstrate the profound effects of large vesicular-like bodies produced by melanoma cells on the barrier integrity of human brain endothelial cells. These vesicular-bodies have not been fully characterised but range in size from ~500 nm to >10 µm, are surrounded by membrane and are enzymatically active based on cell-tracker incorporation. Their size is consistent with previously reported large oncosomes and apoptotic bodies. We demonstrate that these melanoma-derived vesicular-bodies rapidly affect brain endothelial barrier integrity, measured using ECIS biosensor technology, where the disruption is evident within ~60 min. This disruption involves acquisition of the vesicles through transcellular uptake into the endothelial cells. We also observed extensive actin-rearrangement, actin removal from the paracellular boundary of the endothelial cells and envelopment of the vesicular-bodies by actin. This was concordant with widespread changes in CD144 localisation, which was consistent with the loss of junctional strength. High-resolution confocal imaging revealed proximity of the melanoma vesicular-bodies juxtaposed to the endothelial nucleus, often containing fragmented DNA themselves, raising speculation over this association and potential delivery of nuclear material into the brain endothelial cells. The disruption of the endothelial cells occurs in a manner that is faster and completely distinct to that of invasion by intact melanoma cells. Given the clinical observation of large vesicles in the circulation of melanoma patients by others, we hypothesize their involvement in weakening or priming the brain vasculature for melanoma invasion.

Establishing Co-Culture Blood-Brain Barrier Models for Different Neurodegeneration Conditions to Understand Its Effect on BBB Integrity.

The blood-brain barrier (BBB) is a functional interface that provides selective permeability, protection from toxic substances, transport of nutrients, and clearance of brain metabolites. Additionally, BBB disruption has been shown to play a role in many neurodegenerative conditions and diseases. Therefore, the aim of this study was to establish a functional, convenient, and efficient in vitro co-cultured BBB model that can be used for several physiological conditions related to BBB disruption. Mouse brain-derived endothelial (bEnd.3) and astrocyte (C8-D1A) cells were co-cultured on transwell membranes to establish an intact and functional in vitro model. The co-cultured model and its effects on different neurological diseases and stress conditions, including Alzheimer's disease (AD), neuroinflammation, and obesity, have been examined by transendothelial electrical resistance (TEER), fluorescein isothiocyanate (FITC) dextran, and tight junction protein analyses. Scanning electron microscope images showed evidence of astrocyte end-feet processes passing through the membrane of the transwell. Moreover, the co-cultured model showed effective barrier properties in the TEER, FITC, and solvent persistence and leakage tests when compared to the mono-cultured model. Additionally, the immunoblot results showed that the expression of tight junction proteins such as zonula occludens-1 (ZO-1), claudin-5, and occludin-1 was enhanced in the co-culture. Lastly, under disease conditions, the BBB structural and functional integrity was decreased. The present study demonstrated that the co-cultured in vitro model mimicked the BBB's structural and functional integrity and, under disease conditions, the co-cultured model showed similar BBB damages. Therefore, the present in vitro BBB model can be used as a convenient and efficient experimental tool to investigate a wide range of BBB-related pathological and physiological studies.

Ultrastructural Remodeling of the Blood-Brain Barrier and Neurovascular Unit by Lipopolysaccharide-Induced Neuroinflammation.

The blood-brain barrier (BBB) is an interface primarily comprised of brain endothelial cells (BECs), separating the central nervous system (CNS) from the systemic circulation while carefully regulating the transport of molecules and inflammatory cells, and maintaining the required steady-state environment. Inflammation modulates many BBB functions, but the ultrastructural cytoarchitectural changes of the BBB with inflammation are understudied. Inflammation was induced in male 8-10-week-old CD-1 mice with intraperitoneal lipopolysaccharide (LPS), using a regimen (3 mg/kg at 0, 6, and 24 h) that caused robust BBB disruption but had minimal lethality at the study timepoint of 28 h. Perfusion-fixed brains were collected and the frontal cortical layer III regions were analyzed using a transmission electron microscopy (TEM). The LPS-treated mice had pronounced ultrastructural remodeling changes in BECs that included plasma membrane ruffling, increased numbers of extracellular microvesicles, small exosome formation, aberrant BEC mitochondria, increased BEC transcytosis, while tight junctions appeared to be unaltered. Aberrant pericytes were contracted with rounded nuclei and a loss of their elongated cytoplasmic processes. Surveilling microglial cells were attracted to the neurovascular unit (NVU) of BECs, and astrocyte detachment and separation were associated with the formation of a perivascular space and pericapillary edema. The LPS treatment resulted in numerous ultrastructural aberrant remodeling changes to the neurovascular unit's BECs, microglia, pericytes, and astrocytes. In summary, a disturbance of the NVU morphology is a consequence of LPS treatment.

Brain Injury: Response to Injury Wound-Healing Mechanisms and Enlarged Perivascular Spaces in Obesity, Metabolic Syndrome, and Type 2 Diabetes Mellitus.

Embryonic genetic mechanisms are present in the brain and ready to be placed into action upon cellular injury, termed the response to injury wound-healing (RTIWH) mechanism. When injured, regional brain endothelial cells initially undergo activation and dysfunction with initiation of hemostasis, inflammation (peripheral leukocytes, innate microglia, and perivascular macrophage cells), proliferation (astrogliosis), remodeling, repair, and resolution phases if the injurious stimuli are removed. In conditions wherein the injurious stimuli are chronic, as occurs in obesity, metabolic syndrome, and type 2 diabetes mellitus, this process does not undergo resolution and there is persistent RTIWH with remodeling. Indeed, the brain is unique, in that it utilizes its neuroglia: the microglia cell, along with peripheral inflammatory cells and its astroglia, instead of peripheral scar-forming fibrocytes/fibroblasts. The brain undergoes astrogliosis to form a gliosis scar instead of a fibrosis scar to protect the surrounding neuropil from regional parenchymal injury. One of the unique and evolving remodeling changes in the brain is the development of enlarged perivascular spaces (EPVSs), which is the focus of this brief review. EPVSs are important since they serve as a biomarker for cerebral small vessel disease and also represent an impairment of the effluxing glymphatic system that is important for the clearance of metabolic waste from the interstitial fluid to the cerebrospinal fluid, and disposal. Therefore, it is important to better understand how the RTIWH mechanism is involved in the development of EPVSs that are closely associated with and important to the development of premature and age-related cerebrovascular and neurodegenerative diseases with impaired cognition.

Simplified in vitro 3D co-culture-based blood-brain barrier model using transwell.

The blood-brain barrier (BBB) is a major hurdle for treatment of brain diseases. To overcome this, precise and reproducible BBB model is one of the key factors for successful evaluation of BBB-penetrating efficacy of developmental drugs. Thus, in vitro BBB model recapitulating the physiological structure of the BBB is a valuable tool for drug discovery and development for brain diseases. Here, we develop a simplified 3D co-culture-based BBB model using immortalized human brain endothelial cells and immortalized human astrocytes mixed with Matrigel allowing model preparation within 30 min. We directly compare our 3D BBB model to a 2D BBB model comprised solely of immortalized brain endothelial cells, to demonstrate that our 3D BBB model blocks penetration of Dextran molecules with various molecular weights, remain durable and impermeable even in a BBB-degrading condition, and rapidly form tight junctions while the 2D BBB model do not. In conclusion, this establishes our simplified 3D BBB model as a valuable tool for high throughput screening of drug candidates for brain diseases.

Argonaute-2 protects the neurovascular unit from damage caused by systemic inflammation.

BACKGROUND: The brain vasculature plays a pivotal role in the inflammatory process by modulating the interaction between blood cells and the neurovascular unit. Argonaute-2 (Ago2) has been suggested as essential for endothelial survival but its role in the brain vasculature or in the endothelial-glial crosstalk has not been addressed. Thus, our aim was to clarify the significance of Ago2 in the inflammatory responses elicited by these cell types. METHODS: Mouse primary cultures of brain endothelial cells, astrocytes and microglia were used to evaluate cellular responses to the modulation of Ago2. Exposure of microglia to endothelial cell-conditioned media was used to assess the potential for in vivo studies. Adult mice were injected intraperitoneally with lipopolysaccharide (LPS) (2 mg/kg) followed by three daily intraperitoneal injections of Ago2 (0.4 nM) to assess markers of endothelial disruption, glial reactivity and neuronal function. RESULTS: Herein, we demonstrated that LPS activation disturbed the integrity of adherens junctions and downregulated Ago2 in primary brain endothelial cells. Exogenous treatment recovered intracellular Ago2 above control levels and recuperated vascular endothelial-cadherin expression, while downregulating LPS-induced nitric oxide release. Primary astrocytes did not show a significant change in Ago2 levels or response to the modulation of the Ago2 system, although endogenous Ago2 was shown to be critical in the maintenance of tumor necrosis factor-α basal levels. LPS-activated primary microglia overexpressed Ago2, and Ago2 silencing contained the inflammatory response to some extent, preventing interleukin-6 and nitric oxide release. Moreover, the secretome of Ago2-modulated brain endothelial cells had a protective effect over microglia. The intraperitoneal injection of LPS impaired blood-brain barrier and neuronal function, while triggering inflammation, and the subsequent systemic administration of Ago2 reduced or normalized endothelial, glial and neuronal markers of LPS damage. This outcome likely resulted from the direct action of Ago2 over the brain endothelium, which reestablished glial and neuronal function. CONCLUSIONS: Ago2 could be regarded as a putative therapeutic agent, or target, in the recuperation of the neurovascular unit in inflammatory conditions.

Activated microglia promote invasion and barrier dysfunction of brain endothelial cells via regulating the CXCL13/CXCR5 axis.

The blood brain barrier (BBB) is a protective border that prevents noxious substances from gaining access to the central nervous system (CNS). CXCL13 is a chemokine from the CXC chemokine family, which has been shown to destroy the barrier function of umbilical vein endothelial cells with its receptor CXCR5. Here, we aimed to investigate the role of CXCL13/CXCR5 signaling axis in BBB. The invasive ability of bEnd.3 cells was determined by the Transwell invasion assay. The barrier integrity of bEnd.3 cells was assessed by detecting trans-endothelial electrical resistance, the permeability to fluorescein isothiocyanate-dextran, and the expression levels of the tight junction protein E-cadherin. Lipopolysaccharide (LPS)-activated microglia promoted invasion and barrier dysfunction, and upregulated CXCR5 and p-p38 expression levels in cocultured bEnd.3 cells. However, the effects of activated microglia were alleviated by knocking down CXCR5 in cocultured bEnd.3 cells. Furthermore, recombinant CXCL13 promoted invasion and barrier dysfunction, and upregulated the expression levels of p-p38 in bEnd.3 cells; however, its effects were abolished by treating bEnd.3 cells with the p38 inhibitor SB203580. Our data tentatively demonstrated that LPS-activated microglial cells may promote invasion and barrier dysfunction in bEnd.3 cells by regulating the CXCL13/CXCR5 axis and p38 signaling.

Pro-angiogenic activity of isofuran.

Isofurans (IsoFs) are a series of novel discovered lipid peroxidation products. This study focused on the investigation of the angiogenic property of IsoF. MTT stain assay indicated that 1 µm IsoF had the most bioactivity in rat brain endothelial cells (RBECs). IsoF significantly promoted cellular proliferation and migration and remarkably decreased staurosporine-induced apoptosis by TUNEL assay in the RBECs. It successfully up-regulated rat aortic vascularization and choroid explant sprouting, extracellular regulated protein kinases (ERK)1/2, and triggered calcium release. RT-PCR examination indicated that IsoF up-regulated tumor necrosis factor (TNF)α, angiopoietin-1 receptor (Tie2), and vascular endothelial growth factor (VEGF)-A, but did not interfere with caspase 2 and VEGF-C in the RBECs. IsoF has pro-angiogenic activity. Calcium release and ERK1/2 phosphorylation may be involved in the signaling of the IsoF-induced up-regulation of TNFα, Tie2, and VEGF-A, which could be the molecular mechanism of the pro-angiogenic activity of the IsoF.

A ligand motif enables differential vascular targeting of endothelial junctions between brain and retina.

Endothelial heterogeneity has important implications in health and disease. Molecular markers selectively expressed in the vasculature of different organs and tissues are currently being explored in targeted therapies with promising results in preclinical and clinical studies. Noteworthy is the role that combinatorial approaches such as phage display have had in identifying such markers by using phage as nanoparticles and surrogates for billions of different peptides, screening noninvasively the vascular lumen for binding sites. Here, we show that a new peptide motif that emerged from such combinatorial screening of the vasculature binds selectively to blood vessels in the brain in vivo but not to vessels in other organs. Peptides containing a conserved motif in which amino acids Phenylalanine-Arginine-Tryptophan (FRW) predominate could be visualized by transmission electron microscopy bound to the junctions between endothelial cells in all areas of the brain, including the optic nerve, but not in other barrier-containing tissues, such as intestines and testis. Remarkably, peptides containing the motif do not bind to vessels in the retina, implying an important molecular difference between these two vascular barriers. Furthermore, the peptide allows for in vivo imaging, demonstrating that new tools for studying and imaging the brain are likely to emerge from this motif.

Cytoskeleton Elements Contribute to Prion Peptide-Induced Endothelial Barrier Breakdown in a Blood-Brain Barrier In Vitro System.

The mechanisms involved in the interaction of PrP 106-126, a peptide corresponding to the prion protein amyloidogenic region, with the blood-brain barrier (BBB) were studied. PrP 106-126 treatment that was previously shown to impair BBB function, reduced cAMP levels in cultured brain endothelial cells, increased nitric oxide (NO) levels, and changed the activation mode of the small GTPases Rac1 (inactivation) and RhoA (activation). The latter are well established regulators of endothelial barrier properties that act via cytoskeletal elements. Indeed, liquid chromatography-mass spectrometry (LC-MS)-based proteomic profiling study revealed extensive changes in expression of cytoskeleton-related proteins. These results shed light on the nature of the interaction between the prion peptide PrP 106-126 and the BBB and emphasize the importance of the cytoskeleton in endothelium response to prion- induced stress.