Primary Brain Microvascular Endothelial Cells Research Articles

PM2.5 Temporally Decreases Human Brain Endothelial Barrier Proteins and Concomitantly Increases Inflammation and Autophagy in a Dose Dependent Manner

Exposure to airborne particulate matter 2.5 (particle size <PM2.5 um) pollution is a significant co-morbidity for stroke and other cardio- and cerebrovascular diseases, causing premature mortality. However, mechanisms underlying PM2.5-mediated endothelial dysfunction and integrity loss are not known. Our goal was to elucidate how PM2.5 alters brain endothelial phenotype causing inflammation and loss of barrier integrity to provide better insight to the potential pathogenesis of PM2.5. We hypothesized that PM2.5 would alter integral endothelial barrier proteins as well as inflammatory mediators in a dose and temporal dependent manner, subsequently exacerbating the impact of acute ischemic-like injury. Primary adult male human brain microvascular endothelial cells (HBMEC) were preconditioned with PM2.5 (300, 75, or 15 μg/m3; 12, 24, or 36h) or vehicle, then exposed to normoxia (21% O2) or ischemic-like injury, hypoxia plus glucose deprivation (HGD; 1% O2), for 3h. HBMEC barrier function was assessed via trans-endothelial electrical resistance (TEER). HBMEC levels or expression of barrier markers (claudin-5, occludin, ZO-1), adhesion molecules (ICAM-1 and PECAM-1), inflammatory proteins (iNOS, COX-2, LOX-1, TNF-α, IL-1β), and autophagic protein (Beclin-1) were examined using qRT-PCR, in cell western, and standard immunoblotting. Ischemia-like injury as well as PM2.5 decreased HBMEC barrier function. PM2.5 also induced a concomitant temporal and dose dependent decrease in claudin-5 and increase in iNOS as well as TNF-α levels. Additionally, PM2.5 increased protein levels of beclin-1 and LOX-1 like that of HGD alone; however, combination of PM2.5 and HGD decreased levels of beclin-1. HGD alone or in combination with PM2.5 decreased levels of PECAM-1, but PM2.5 alone did not alter PECAM-1. PM2.5 in the presence or absence of HGD induced differential alteration in claudin-5 isoform protein levels. Intriguingly, PM2.5 decreased claudin-5 mRNA levels like that observed in HBMECs exposed to HGD alone. PM2.5 plus HGD significantly increased mRNA levels of both ICAM-1 and IL-1β. In conclusion, PM2.5 mediated inflammatory and vascular pathogenesis involves a potential complex intracellular molecular framework associated with the brain endothelium which is compounded by ischemic like injury. These findings have revealed detrimental effects of PM2.5 on the brain vascular endothelium which will help to identify targets in the endothelium to restore or preserve cerebrovascular homeostasis. University of Arizona Valley Research Partnership Grant VRP37 P2 (RJG), American Heart Association 19AIREA34480018 (RJG), UA VRP Grant VRP55 P1a (TSW). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Junctional adhesion molecule-A deficient mice are protected from severe experimental autoimmune encephalomyelitis.

In multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), early pathological features include immune cell infiltration into the central nervous system (CNS) and blood-brain barrier (BBB) disruption. We investigated the role of junctional adhesion molecule-A (JAM-A), a tight junction protein, in active EAE (aEAE) pathogenesis. Our study confirms JAM-A expression at the blood-brain barrier and its luminal redistribution during aEAE. JAM-A deficient (JAM-A-/-) C57BL/6J mice exhibited milder aEAE, unrelated to myelin oligodendrocyte glycoprotein-specific CD4+ T-cell priming. While JAM-A absence influenced macrophage behavior on primary mouse brain microvascular endothelial cells (pMBMECs) under flow in vitro, it did not impact T-cell extravasation across primary mouse brain microvascular endothelial cells. At aEAE onset, we observed reduced lymphocyte and CCR2+ macrophage infiltration into the spinal cord of JAM-A-/- mice compared to control littermates. This correlated with increased CD3+ T-cell accumulation in spinal cord perivascular spaces and brain leptomeninges, suggesting JAM-A absence leads to T-cell trapping in central nervous system border compartments. In summary, JAM-A plays a role in immune cell infiltration and clinical disease progression in aEAE.

A 3D Bioprinted Human Neurovascular Unit Model of Glioblastoma Tumor Growth.

A 3D bioprinted neurovascular unit (NVU) model was developed to study glioblastoma (GBM) tumor growth in a brain-like microenvironment. The NVU model included human primary astrocytes, pericytes and brain microvascular endothelial cells, and patient-derived glioblastoma cells (JHH-520) were used for this study. We used fluorescence reporters with confocal high content imaging to quantitate real-time microvascular network formation and tumor growth. Extensive validation of the NVU-GBM model included immunostaining for brain relevant cellular markers and extracellular matrix components; single cell RNA sequencing to establish physiologically relevant transcriptomics changes; and secretion of NVU and GBM-relevant cytokines. The scRNAseq revealed changes in gene expression and cytokines secretion associated with wound healing/angiogenesis, including the appearance of an endothelial mesenchymal transition (EndMT) cell population. The NVU-GBM model was used to test 18 chemotherapeutics and anti-cancer drugs to assess the pharmacological relevance of the model and robustness for high throughput screening. This article is protected by copyright. All rights reserved.

Abstract TP307: oxLDL Preconditioning Intensifies Ischemic-Like Injury Mediated Alterations in Human Male Brain Endothelial Cell Tight Junction Protein Levels and Proinflammatory Mediators

Introduction: Acute ischemic stroke (AIS) triggers endothelial activation and induces cerebrovascular inflammation which can result in vascular function and integrity loss leading to worsened stroke outcome. A clinically correlated risk factor for stroke shown to mediate vascular dysfunction and injury is elevated levels of oxidized low density lipoprotein (oxLDL). We hypothesized that oxLDL would exacerbate acute ischemic injury mediated alteration of endothelial proinflammatory mediator and integral barrier protein levels. Methods: Primary adult male human brain microvascular endothelial cells (HBMEC) were preconditioned with human oxLDL (50 or 100ug/dL; 6, 12, 24, or 36h) or vehicle, using a serum dose reported in AIS patients. HBMECs were then exposed to normoxia (21% O 2 ) or ischemic-like injury, hypoxia plus glucose deprivation (HGD; 1% O 2 ), for 3 or 6h. HBMEC levels or expression of barrier markers, adhesion molecules, and inflammatory mediators were examined using qRT-PCR, in cell western, and FIJI mediated immunocytochemical analysis. Secondary analysis was performed on RNA sequencing of adult male aortic endothelial cells (HAoECs) exposed to oxLDL (50ug/mL; 3, 6, 12, 24h) obtained from GEO dataset GSE13139. Results: HGD injury concomitantly decreased HBMEC claudin-5, occludin, and ZO-1 as well as increased IL-1b, ICAM-1, VCAM-1, iNOS, and TNF-a. OxLDL exacerbated an HGD mediated decrease in HBMEC barrier levels and increase in proinflammatory levels in a dose and time dependent manner. HAoEC barrier, adhesion, and proinflammatory levels were not altered by oxLDL. Conclusion: OxLDL may preferentially alter brain endothelial barrier and proinflammatory levels, as opposed to aortic endothelial cells in dose and time dependent manner. Mitigating the effect of oxLDL on brain endothelial permeability and inflammation, possibly via receptor inhibition may be a novel, viable therapeutic target in the treatment of AIS.

The Ohm-azing custom-made Transendothelial Electrical Resistance measuring device (and why is it a current sensation?)

The endothelial and epithelial cells form biological barriers, the unique anatomical structures that control substance movement between circulation and i.e., organs. Measuring trans-endothelial electrical resistance (TEER) is a standard method that enables the evaluation of barrier integrity. The capability of a novel measuring system (MS) that combines innovative components, customized electrodes, and user-friendly software tailored to provide accurate and adjustable measurements of endothelial barrier integrity. We demonstrated MS usage in TEER measurement of rat brain microvascular endothelial cell line (RBE-4), primary rat brain microvascular endothelial cells (PBMEC), rat brain microvascular endothelial cells (BMEC), and human intestinal epithelial cell line (HIEC-6). The MS was successfully applied to measure the TEER of cultured cell monolayers, finding that i) the device records stable values; ii) cells treated with ammonium chloride had lower TEER values compared to untreated cells, which was verified with permeability measurements with fluorescein dye and confocal microscopy. In conclusion, we invented a low-cost MS capable of accurate TEER measures in relevant biological ranges. The MS instrument facilitates long-time, real-time monitoring of the cellular barriers, giving vital insights into cell barrier stability, permeability, and the effects of pharmacological substances. The customized measurement duration and electrode configuration meet a broad range of experimental designs.

A20 regulates lymphocyte adhesion in murine neuroinflammation by restricting endothelial ICOSL expression in the CNS.

A20 is a ubiquitin-modifying protein that negatively regulates NF-κB signaling. Mutations in A20/TNFAIP3 are associated with a variety of autoimmune diseases, including multiple sclerosis (MS). We found that deletion of A20 in central nervous system (CNS) endothelial cells (ECs) enhances experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. A20ΔCNS-EC mice showed increased numbers of CNS-infiltrating immune cells during neuroinflammation and in the steady state. While the integrity of the blood-brain barrier (BBB) was not impaired, we observed a strong activation of CNS-ECs in these mice, with dramatically increased levels of the adhesion molecules ICAM-1 and VCAM-1. We discovered ICOSL to be expressed by A20-deficient CNS-ECs, which we found to function as adhesion molecules. Silencing of ICOSL in CNS microvascular ECs partly reversed the phenotype of A20ΔCNS-EC mice without reaching statistical significance and delayed the onset of EAE symptoms in WT mice. In addition, blocking of ICOSL on primary mouse brain microvascular ECs impaired the adhesion of T cells in vitro. Taken together, we propose that CNS EC-ICOSL contributes to the firm adhesion of T cells to the BBB, promoting their entry into the CNS and eventually driving neuroinflammation.

TMIC-08. CLAUDIN-1 UPREGULATION IN GLIOBLASTOMA INDUCED BLOOD-BRAIN-BARRIER DYSFUNCTION VIA A PARACRINE MECHANISM

Abstract BACKGROUND Blood-brain-barrier (BBB) disruption is a well-known phenomenon in glioblastoma. However, the mechanism driving BBB dysfunction in GBM is still unknown, especially in the previously established vasculature near the invasive edge. In this study, we aimed to determine if GBM paracrine signaling is sufficient to induce BBB dysfunction and identify changes in the tight junctions of the BBB. METHODS An in vivo xenograft model was established through an intracranial injection of U-87 MG cells in nude mice. Weekly MRIs were performed to monitor tumor growth. Two weeks after tumor appearance, brains were collected following a 4% PFA perfusion for immunostaining and confocal imaging. An in vitro model of the BBB was created by seeding primary mouse brain microvascular endothelial cells (BMECs) on transwell inserts. Once the BMECs reached confluency with a TEER value of 170-175Ω*cm2, the media was replaced with filtered conditioned media from U-87 MG cell line, an IDH mutant cell line, or BMECs for three days with TEER values monitored each day. RESULTS Immunofluorescent staining revealed a significant increase in claudin-1 expression and decrease in claudin-5 expression in the tumor vs normal brain tissue of our in vivo model (p < 0.01). After 24 hours of exposure to conditioned media from both U-87 MG and IDH mutant cells, BMECs had a significant decrease in TEER compared to control and BMEC conditioned media (p < .0001). After 72 hours, TEER values continued to decrease, reaching around 155Ω*cm2, with control BMECs remaining around 187Ω*cm2. CONCLUSIONS These results suggest GBM cells secrete factors capable of inducing changes in the tight junction proteins of the BBB and increasing its permeability. Future studies will aim to identify the mechanism in which these changes occur.

Abstract 12294: Poldip2 Mediates Brain Vascular Permeability by Regulating ZO-1 Phosphorylation and Localization at the Interendothelial Border

Introduction: Poldip2 is a novel regulator of cerebral vascular permeability involved in aggravating blood-brain barrier disruption following ischemic stroke; however, the underlying mechanisms are incompletely understood. Tight junctions are crucial for the formation of endothelial barriers and are composed of transmembrane proteins and adaptor proteins such as ZO-1. Cerebral ischemia and inflammation lead to ZO-1 redistribution resulting in vascular permeability. Hypothesis: We hypothesized that Poldip2 may mediate ZO-1 phosphorylation and promote brain endothelial monolayer permeability following cerebral ischemia. Methods: Cerebral ischemia was induced in endothelial-specific Poldip2 knockout and control mice. Vascular permeability was assessed by Evans blue dye extravasation. For in vitro analysis, primary rat brain microvascular endothelial cells were used to investigate the effect of Poldip2 depletion by siRNA on TNF-α-induced permeability and trans-endothelial electrical resistance (TEER). Immunocytochemistry was carried out to investigate the effects of Poldip2 depletion or overexpression on ZO-1 localization. Results: Endothelial-specific Poldip2 deletion abolished Evans blue dye extravasation after cerebral ischemia induction (p<0.05). Poldip2 knockdown in vitro suppressed TNF-α-induced endothelial cell (EC) monolayer permeability (FITC-dextran and biotin-avidin assays, p<0.05 and p<0.001) and prevented decreases in TEER (p<0.01). Poldip2 depletion prevented TNF-α-induced ZO-1 disruption at interendothelial junctions (p<0.05). Poldip2 overexpression increased endothelial permeability (p<0.05), decreased TEER (p<0.05), reduced ZO-1 localization at cell-cell junctions (p<0.0001) and enhanced reactive oxygen species (ROS) production (p<0.05). Treatment with the antioxidant N-acetyl cysteine (NAC) abrogated Poldip2-induced ZO-1 disruption (p<0.0001). Immunoprecipitation studies demonstrated that Poldip2 overexpression induced tyrosine phosphorylation of ZO-1 (p<0.0001), an effect prevented by NAC treatment (p<0.01). Conclusions: Poldip2 induces ROS-mediated tyrosine phosphorylation of the TJ protein ZO-1 and promotes EC permeability following cerebral ischemia

Fibrinogen in mice cerebral microvessels induces blood–brain barrier dysregulation with aging via a dynamin-related protein 1–dependent pathway

We previously reported evidence that oxidative stress during aging leads to adverse protein profile changes of brain cortical microvessels (MVs: end arterioles, capillaries, and venules) that affect mRNA/protein stability, basement membrane integrity, and ATP synthesis capacity in mice. As an extension of our previous study, we also found that proteins which comprise the blood–brain barrier (BBB) and regulate mitochondrial quality control were also significantly decreased in the mice’s cortical MVs with aging. Interestingly, the neuroinflammatory protein fibrinogen (Fgn) was increased in mice brain MVs, which corresponds with clinical reports indicating that the plasma Fgn concentration increased progressively with aging. In this study, protein–protein interaction network analysis indicated that high expression of Fgn is linked with downregulated expression of both BBB- and mitochondrial fission/fusion–related proteins in mice cortical MVs with aging. To investigate the mechanism of Fgn action, we observed that 2 mg/mL or higher concentration of human plasma Fgn changed cell morphology, induced cytotoxicity, and increased BBB permeability in primary human brain microvascular endothelial cells (HBMECs). The BBB tight junction proteins were significantly decreased with increasing concentration of human plasma Fgn in primary HBMECs. Similarly, the expression of phosphorylated dynamin-related protein 1 (pDRP1) and other mitochondrial fission/fusion–related proteins were also significantly reduced in Fgn-treated HBMECs. Interestingly, DRP1 knockdown by shRNA(h) resulted in the reduction of both BBB- and mitochondrial fission/fusion–related proteins in HBMECs. Our results suggest that elevated Fgn downregulates DRP1, leading to mitochondrial-dependent endothelial and BBB dysfunction in the brain microvasculature.

Compromised Blood-Brain Barrier Junctions Enhance Melanoma Cell Intercalation and Extravasation.

Melanoma frequently metastasises to the brain, and a detailed understanding of the molecular and cellular mechanisms underlying melanoma cell extravasation across the blood-brain barrier (BBB) is important for preventing brain metastasis formation. Making use of primary mouse brain microvascular endothelial cells (pMBMECs) as an in vitro BBB model, we imaged the interaction of melanoma cells into pMBMEC monolayers. We observed exclusive junctional intercalation of melanoma cells and confirmed that melanoma-induced pMBMEC barrier disruption can be rescued by protease inhibition. Interleukin (IL)-1β stimulated pMBMECs or PECAM-1-knockout (-ko) pMBMECs were employed to model compromised BBB barrier properties in vitro and to determine increased melanoma cell intercalation compared to pMBMECs with intact junctions. The newly generated brain-homing melanoma cell line YUMM1.1-BrM4 was used to reveal increased in vivo extravasation of melanoma cells across the BBB of barrier-compromised PECAM-1-deficient mice compared to controls. Taken together, our data indicate that preserving BBB integrity is an important measure to limit the formation of melanoma-brain metastasis.

A Next-Generation 3D Tissue-Engineered Model of the Human Brain Microvasculature to Study the Blood-Brain Barrier.

More than a billion people are affected by neurological disorders, and few have effective therapeutic options. A key challenge that has prevented promising preclinically proven strategies is the translation gap to the clinic. Humanized tissue engineering models that recreate the brain environment may aid in bridging this translational gap. Here, we showcase the methodology that allows for the practical fabrication of a comprehensive microphysicological system (MPS) of the blood-brain barrier (BBB). Compared to other existing 2D and 3D models of the BBB, this model features relevant cytoarchitecture and multicellular arrangement, with branching and network topologies of the vascular bed. This process utilizes 3D bioprinting with digital light processing to generate a vasculature lumen network surrounded by embedded human astrocytes. The lumens are then cellularized with primary human brain microvascular endothelial cells and pericytes. To initiate mechanotransduction pathways and complete maturation, vascular structures are continuously perfused for 7 days. Constructs are validated for complete endothelialization with viability dyes prior to functional assessments that include barrier integrity (permeability) and immune-endothelial interactions. This MPS has applications for the study of novel therapeutics, toxins, and elucidating mechanisms of pathophysiology.

Cerebral Microvascular Injury Induced by Lag3-Dependent α-Synuclein Fibril Endocytosis Exacerbates Cognitive Impairment in a Mouse Model of α-Synucleinopathies.

The pathological accumulation of α-synuclein (α-Syn) and the transmission of misfolded α-Syn underlie α-synucleinopathies. Increased plasma α-Syn levels are associated with cognitive impairment in Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies, but it is still unknown whether the cognitive deficits in α-synucleinopathies have a common vascular pathological origin. Here, it is reported that combined injection of α-Syn preformed fibrils (PFFs) in the unilateral substantia nigra pars compacta, hippocampus, and cerebral cortex results in impaired spatial learning and memory abilities at 6 months post-injection and that this cognitive decline is related to cerebral microvascular injury. Moreover, insoluble α-Syn inclusions are found to form in primary mouse brain microvascular endothelial cells (BMVECs) through lymphocyte-activation gene 3 (Lag3)-dependent α-Syn PFFs endocytosis, causing poly(ADP-ribose)-driven cell death and reducing the expression of tight junction proteins in BMVECs. Knockout of Lag3 in vitro prevents α-Syn PFFs from entering BMVECs, thereby reducing the abovementioned response induced by α-Syn PFFs. Deletion of endothelial cell-specific Lag3 in vivo reverses the negative effects of α-Syn PFFs on cerebral microvessels and cognitive function. In short, this study reveals the effectiveness of targeting Lag3 to block the spread of α-Syn fibrils to endothelial cells in order to improve cognition.

Differentiation of Human Induced Pluripotent Stem Cells to Brain Microvascular Endothelial Cell-Like Cells with a Mature Immune Phenotype

Blood-brain barrier (BBB) dysfunction is a pathological hallmark of many neurodegenerative and neuroinflammatory diseases affecting the central nervous system (CNS). Due to the limited access to disease-related BBB samples, it is still not well understood whether BBB malfunction is causative for disease development or rather a consequence of the neuroinflammatory or neurodegenerative process. Human induced pluripotent stem cells (hiPSCs) therefore provide a novel opportunity to establish in vitro BBB models from healthy donors and patients, and thus to study disease-specific BBB characteristics from individual patients. Several differentiation protocols have been established for deriving brain microvascular endothelial cell (BMEC)-like cells from hiPSCs. Consideration of the specific research question is mandatory for the correct choice of the respective BMEC-differentiation protocol. Here, we describe the extended endothelial cell culture method (EECM), which is optimized to differentiate hiPSCs into BMEC-like cells with a mature immune phenotype, allowing the study of immune cell-BBB interactions. In this protocol, hiPSCs are first differentiated into endothelial progenitor cells (EPCs) by activating Wnt/β-catenin signaling. The resulting culture, which contains smooth muscle-like cells (SMLCs), is then sequentially passaged to increase the purity of endothelial cells (ECs) and to induce BBB-specific properties. Co-culture of EECM-BMECs with these SMLCs or conditioned medium from SMLCs allows for the reproducible, constitutive, and cytokine-regulated expression of EC adhesion molecules. Importantly, EECM-BMEC-like cells establish barrier properties comparable to primary human BMECs, and due to their expression of all EC adhesion molecules, EECM-BMEC-like cells are different from other hiPSC-derived in vitro BBB models. EECM-BMEC-like cells are thus the model of choice for investigating the potential impact of disease processes at the level of the BBB, with an impact on immune cell interaction in a personalized fashion.

FGL2 deficiency alleviates maternal inflammation-induced blood-brain barrier damage by blocking PI3K/NF-κB mediated endothelial oxidative stress.

The impairment of blood-brain barrier (BBB) is one of the key contributors to maternal inflammation induced brain damage in offspring. Our previous studies showed Fibrinogen-like protein 2 (FGL2) deficiency alleviated maternal inflammation induced perinatal brain damage. However, its role in BBB remains undefined. Lipopolysaccharide (LPS) was intraperitoneally injected to dams at Embryonic day 17 to establish maternal inflammation model. FGL2 knockout mice and primary brain microvascular endothelial cells (BMECs) were used for the in-vivo and in-vitro experiments. BBB integrity was assessed by sodium fluorescein extravasation and tight junction (TJ) protein expression. Oxidative stress and the activation of PI3K/NF-κB pathway were evaluated to explore the mechanisms underlying. Upon maternal inflammation, BBB integrity was remarkedly reduced in neonatal mice. Meanwhile, FGL2 expression was consistently increased in BBB-impaired brain as well as in LPS-treated BMECs. Moreover, FGL2 deficiency attenuated the hyperpermeability of BBB, prevented the decline of TJ proteins, and reduced the cytokine expressions in LPS-exposed pups. Mechanistically, the indicators of oxidative stress, as well as the activation of PI3K/NF-κB pathway, were upregulated after LPS exposure in vivo and in vitro. FGL2 deletion decreased the generation of ROS and NO, reduced the endothelial iNOS and NOX2 expressions, and suppressed the PI3K/NF-κB pathway activation. Besides, inhibition of PI3K by LY294002 decreased the oxidative stress in LPS-treated wild-type BMECs. While, overexpression of PI3K by lentivirus reemerged the induction of NOX2 and iNOS as well as NF-κB activation in FGL2-deleted BMECs. Our findings indicate that FGL2 deficiency alleviates the maternal inflammation-induced BBB disruption by inhibiting PI3K/NF-κB mediated oxidative stress in BMECs. Targeting FGL2 may provide a new therapy for prenatal brain damage of offspring.

Circulating Plasma Exosomal Proteins of Either SHIV-Infected Rhesus Macaque or HIV-Infected Patient Indicates a Link to Neuropathogenesis.

Despite the suppression of human immunodeficiency virus (HIV) replication by combined antiretroviral therapy (cART), 50-60% of HIV-infected patients suffer from HIV-associated neurocognitive disorders (HAND). Studies are uncovering the role of extracellular vesicles (EVs), especially exosomes, in the central nervous system (CNS) due to HIV infection. We investigated links among circulating plasma exosomal (crExo) proteins and neuropathogenesis in simian/human immunodeficiency virus (SHIV)-infected rhesus macaques (RM) and HIV-infected and cART treated patients (Patient-Exo). Isolated EVs from SHIV-infected (SHIV-Exo) and uninfected (CTL-Exo) RM were predominantly exosomes (particle size < 150 nm). Proteomic analysis quantified 5654 proteins, of which 236 proteins (~4%) were significantly, differentially expressed (DE) between SHIV-/CTL-Exo. Interestingly, different CNS cell specific markers were abundantly expressed in crExo. Proteins involved in latent viral reactivation, neuroinflammation, neuropathology-associated interactive as well as signaling molecules were expressed at significantly higher levels in SHIV-Exo than CTL-Exo. However, proteins involved in mitochondrial biogenesis, ATP production, autophagy, endocytosis, exocytosis, and cytoskeleton organization were significantly less expressed in SHIV-Exo than CTL-Exo. Interestingly, proteins involved in oxidative stress, mitochondrial biogenesis, ATP production, and autophagy were significantly downregulated in primary human brain microvascular endothelial cells exposed with HIV+/cART+ Patient-Exo. We showed that Patient-Exo significantly increased blood-brain barrier permeability, possibly due to loss of platelet endothelial cell adhesion molecule-1 protein and actin cytoskeleton structure. Our novel findings suggest that circulating exosomal proteins expressed CNS cell markers-possibly associated with viral reactivation and neuropathogenesis-that may elucidate the etiology of HAND.

PRIMARY CULTURE AND IDENTIFICATION OF MOUSE BRAIN MICROVASCULAR ENDOTHELIAL CELLS

Brain microvascular endothelial cells are the basic components of the blood-brain barrier. Many neurological diseases are related to the loss of blood brain barrier (BBB) function. Isolating and culturing primary BMVEC is an important means to study the function and regulation of BBB in vitro. To establish a method for isolation and culture of primary mouse brain microvascular endothelial cells (BMECs). The 7-10-days mice were sacrificed by neck removal, the cranial cavity was opened, the brain was aseptically removed and the cerebral cortex was retained. To extract brain microvascular segments, the brain underwent three D-Hank solution rinses. It was then homogenized, twice digested by enzyme, and centrifuged using a Bovine Serum Albumin (BSA) gradient. For primary culture, the brain microvascular segments were injected into gelatin-coated culture plates using DMED complete media. When the cell density reaches 90 %, the media is removed, and the cells are then given two PBS washes. A new medium was introduced after adding 1ml of the trypsin-EDTA solution to digest for 2–5 minutes for passage. Cell culture plates were rinsed and pre-cooled 95 % ethanol was added for 20 minutes after passaged cells had grown to 80–90 % of their original size. After a third wash, 1 ml of mouse factor VIII antibody was added to the culture wells, where it was left for 4 hours at 37 °C. FITC-labeled rabbit anti-mouse antibody in a volume of 1 mL was added. Under a confocal laser microscope, the plates were examined and taken pictures of. The outcome demonstrates positive expression of the marker factor VIII associated antigen. To create a cell suspension, monolayer-growing microvascular endothelial cells were chosen. 100 L of cell suspension was used to inoculate each well in 96-well plates, and the cells were then grown. On days 1, 2, 3, 4, 5, 6, and 7, the original medium was removed, and then 180 μL of DMEM and 20 μL of MTT were simultaneously added to each well for a 4-hour culture. After adding dimethyl sulfoxide (DMSO), each well's absorbance value (A value) was measured at 492 nm. The outcome demonstrates that the growth peak is attained between 6 and 8 days. This method can successfully isolate and culture primary BMECs, which lay a foundation for the study of BMEC in vitro.

Functional and targeted proteomics characterization of a human primary endothelial cell model of the blood-brain barrier (BBB) for drug permeability studies

The blood-brain barrier (BBB) protects the brain from toxins but hinders the penetration of neurotherapeutic drugs. Therefore, the blood-to-brain permeability of chemotherapeutics must be carefully evaluated. Here, we aimed to establish a workflow to generate primary cultures of human brain microvascular endothelial cells (BMVECs) to study drug brain permeability and bioavailability. Furthermore, we characterized and validated this BBB model in terms of quantitative expression of junction and drug-transport proteins, and drug permeability. We isolated brain microvessels (MVs) and cultured BMVECs from glioma patient biopsies. Then, we employed targeted LC-MS proteomics for absolute protein quantification and immunostaining to characterize protein localization and radiolabeled drugs to predict drug behavior at the Human BBB. The abundance levels of ABC transporters, junction proteins, and cell markers in the cultured BMVECs were similar to the MVs and correctly localized to the cell membrane. Permeability values (entrance and exit) and efflux ratios tested in vitro using the primary BMVECs were within the expected in vivo values. They correctly reflected the transport mechanism for 20 drugs (carbamazepine, diazepam, imipramine, ketoprofen, paracetamol, propranolol, sulfasalazine, terbutaline, warfarin, cimetidine, ciprofloxacin, digoxin, indinavir, methotrexate, ofloxacin, azidothymidine (AZT), indomethacin, verapamil, quinidine, and prazosin). We established a human primary in vitro model suitable for studying blood-to-brain drug permeability with a characterized quantitative abundance of transport and junction proteins, and drug permeability profiles, mimicking the human BBB. Our results indicate that this approach could be employed to generate patient-specific BMVEC cultures to evaluate BBB drug permeability and develop personalized therapeutic strategies.

Abstract WMP115: Stroke Patient Cerebral Endothelial Cells And Their Exosomes Impair Cerebral Endothelial Cell Function

Background: Cerebral endothelial cells (CECs) play a key role in mediating cerebral vascular hemostasis. The biological function of CECs in stroke patients remains unknown due to technical challenges to acquire patient CECs. We isolated and analyzed the function of CECs from living stroke patients. Methods: Cells were isolated from stent retrievers used for mechanical thrombectomy (MT) for patients with large vessel occlusion (LVO) after completion of the intervention as part of an IRB approved protocol. Patient endothelial cells (pCECs) were analyzed. Results: We isolated 300-400 primary brain endothelial cells per retriever from 5 patients with LVO with ages ranging from 69 to 86 years. The pCECs exhibited a typical monolayer cobblestone appearance and expressed VE-cadherin, but not α-smooth muscle actin, indicating that these cells are CECs. Western blot analysis showed that compared to human primary brain microvascular endothelial cells (hBMECs, Cell System), pCECs expressed significantly increased iNOS, VEGF, and VEGFR2, but reduced eNOS and Ang1. In vitro endothelial permeability and angiogenesis assays showed that pCECs robustly increased cell leakage and angiogenesis, respectively. Moreover, treatment of healthy hBMECs with exosomes derived from patient CECs (pCEC-Exos) significantly increased cell leakage and reduced cell integrity, which were associated with the reduction of tight junction proteins ZO1 and Occludin. Blockage of the caveolin endocytotic pathway by nystatin abolished the effect of pCEC-Exos on hBMEC function. Conclusions: Our data demonstrate that: 1) pCECs from stroke patients can be harvested; 2) pCECs are dysfunctional, and 3) pCEC-Exos induce dysfunction of healthy CECs. These data provide novel insights into the mediation of stroke-induced neurovascular dysfunction by pCECs.

Abstract TMP33: Obesity And Hyperinsulinemia Impair Insulin Receptor Transport In The Brain Microvascular Endothelium

Introduction: Obesity afflicts over 650 million individuals worldwide and is a strong risk factor for both stroke and dementia. Hyperinsulinemia-induced insulin resistance, a finding commonly seen in obesity, is linked to brain insulin deficiency. As insulin is neuroprotective, impaired transport from the peripheral circulation into the brain may underlie the enhanced risk of vascular cognitive impairment in obese individuals. Thus, the focus of this study is to understand the role of aberrant insulin receptor endocytosis in impaired brain insulin transport associated with diet-induced obesity. Methods: C57Bl/6J mice were placed on a control or high fat diet beginning at 6 weeks of age for 24 weeks (n=3-5) and hippocampi collected to image microvascular receptor transport. To isolate the effects of insulin resistance, primary murine brain microvascular endothelial cells (MBEC) were exposed to hyperinsulinemia (HI; 20nM insulin for 12 hours). Naïve and HI cells were stimulated for 15 minutes with 20nM insulin and processed for immunoblotting, microscopy, and flow cytometry (n=3-12). Results: High fat diet fed mice exhibited reduced internalization of insulin receptor-positive vesicles in the vascular endothelium as compared to controls (0.27+/-0.04 v. 0.37+/-0.02). Correspondingly, HI MBECs displayed reduced insulin receptor internalization, disrupted actin architecture, and a decreased abundance of early endosomes as compared to naïve cells (0.90+/-0.12 v. 2.07+/-0.29). HI cells further demonstrated a shift towards increased endocytic vesicle formation and swelling relative to naïve cells (1.90+/-0.21 v. 1.04+/-0.08), the result of which may be due to an observed increase in endoplasmic reticulum stress and impaired nitric oxide synthase capacity. Conclusion: Diet-induced obesity impairs insulin receptor endocytosis into the brain vascular endothelium. These data suggest that hyperinsulinemia induces a compensatory shift in endocytic machinery, with receptors potentially becoming trapped within endocytic buds as opposed to trafficking into the cell. By uncovering the mechanisms linking obesity to cognitive decline, this study will help identify novel therapeutic targets for vascular cognitive impairment and dementia.

Detecting physiological concentrations of Alzheimer's associated amyloid-β oligomers with a cell-based response.

Alzheimer's disease (AD) is characterized by blood-brain barrier (BBB) breakdown and neuronal cell death, associated with aggregated amyloid-β (Aβ) plaques deposited around brain vasculature and within the brain. Changes in the brain occur up to 20 years before symptoms arise; however, there is currently no pre-symptomatic detection method available for AD. An intact BBB requires tight junctions (TJ) that render relatively high transendothelial electrical resistance (TEER). AD-associated breakdown of the BBB correlates with vascular Aβ deposition, suggesting that aggregated, pathological Aβ can modulate TEER. We explore leveraging this cellular response for early disease diagnosis via detection of early Aβ aggregates. Primary human brain microvascular endothelial cells (HBMVECs) were cultured on a suspended membrane and supplemented to mimic the BBB. Once TEER values reflected TJ formation, HBMVEC monolayers were treated with oligomeric Ab at picomolar to nanomolar concentrations, and TEER was monitored. Oligomeric Aβ at picomolar and nanomolar concentrations, but not monomer, induced a reduction of TEER and disruption of TJs. These results demonstrate that pathogenic Aβ aggregates are uniquely responsible for inducing endothelial monolayer permeability and reducing monolayer electrical resistance, and this response can be detected at physiological concentrations. This work also validates that introduction of physiologically active Aβ oligomers can increase permeability of an endothelial monolayer, while non-pathogenetic monomer is inert. These results provide evidence that oligomers, the pathogenic form of Aβ, selectively break down the BBB. Future work will include establishing a threshold of Aβ oligomer concentrations that reduce TEER to establish cellular sensitivity, and sensitizing cells to obtain a more pronounced response. Translation of this experimental system to a cell-based biosensor platform will lay the groundwork for the development of cost-effective, early AD detection.