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.

Altered protein expression of membrane transporters in isolated cerebral microvessels and brain cortex of a rat Alzheimer's disease model.

There is growing evidence that membrane transporters expressed at the blood-brain barrier (BBB) and brain parenchymal cells play an important role in Alzheimer's disease (AD) development and progression. However, quantitative information about changes in transporter protein expression at neurovascular unit cells in AD is limited. Here, we studied the changes in the absolute protein expression of five ATP-binding cassette (ABC) and thirteen solute carrier (SLC) transporters in the isolated brain microvessels and brain cortical tissue of TgF344-AD rats compared to age-matched wild-type (WT) animals using liquid chromatography tandem mass spectrometry based quantitative targeted absolute proteomic analysis. Moreover, sex-specific alterations in transporter expression in the brain cortical tissue of this model were examined. Protein expressions of Abcg2, Abcc1 and FATP1 (encoded by Slc27a1) in the isolated brain microvessels of TgF344-AD rats were 3.1-, 2.0-, 4.3-fold higher compared to WT controls, respectively (p < 0.05). Abcc1 and 4F2hc (encoded by Slc3a2) protein expression was significantly up-regulated in the brain cortical tissue of male TgF344-AD rats compared to male WT rats (p < 0.05). The study provides novel information for the elucidation of molecular mechanisms underlying AD and valuable knowledge about the optimal use of the TgF344-AD rat model in AD drug development and drug delivery research.

Tau induces blood vessel abnormalities and angiogenesis-related gene expression in P301L transgenic mice and human Alzheimer's disease.

Mixed pathology, with both Alzheimer's disease and vascular abnormalities, is the most common cause of clinical dementia in the elderly. While usually thought to be concurrent diseases, the fact that changes in cerebral blood flow are a prominent early and persistent alteration in Alzheimer's disease raises the possibility that vascular alterations and Alzheimer pathology are more directly linked. Here, we report that aged tau-overexpressing mice develop changes to blood vessels including abnormal, spiraling morphologies; reduced blood vessel diameters; and increased overall blood vessel density in cortex. Blood flow in these vessels was altered, with periods of obstructed flow rarely observed in normal capillaries. These changes were accompanied by cortical atrophy as well as increased expression of angiogenesis-related genes such as Vegfa, Serpine1, and Plau in CD31-positive endothelial cells. Interestingly, mice overexpressing nonmutant forms of tau in the absence of frank neurodegeneration also demonstrated similar changes. Furthermore, many of the genes we observe in mice are also altered in human RNA datasets from Alzheimer patients, particularly in brain regions classically associated with tau pathology such as the temporal lobe and limbic system regions. Together these data indicate that tau pathological changes in neurons can impact brain endothelial cell biology, altering the integrity of the brain's microvasculature.

Angiotensin II modulates amphetamine-induced glial and brain vascular responses, and attention deficit via angiotensin type 1 receptor: Evidence from brain regional sensitivity to amphetamine.

Amphetamine-induced neuroadaptations involve vascular damage, neuroinflammation, a hypo-functioning prefrontal cortex (PFC), and cognitive alterations. Brain angiotensin II, through angiotensin type 1 receptor (AT1 -R), mediates oxidative/inflammatory responses, promoting endothelial dysfunction, neuronal oxidative damage and glial reactivity. The present work aims to unmask the role of AT1 -R in the development of amphetamine-induced changes over glial and vascular components within PFC and hippocampus. Attention deficit was evaluated as a behavioral neuroadaptation induced by amphetamine. Brain microvessels were isolated to further evaluate vascular alterations after amphetamine exposure. Male Wistar rats were administered with AT1 -R antagonist, candesartan, followed by repeated amphetamine. After one week drug-off period, animals received a saline or amphetamine challenge and were evaluated in behavioral tests. Afterward, their brains were processed for cresyl violet staining, CD11b (microglia marker), GFAP (astrocyte marker) or von Willebrand factor (vascular marker) immunohistochemistry, and oxidative/cellular stress determinations in brain microvessels. Statistical analysis was performed by using factorial ANOVA followed by Bonferroni or Tukey tests. Repeated amphetamine administration increased astroglial and microglial markers immunoreactivity, increased apoptotic cells, and promoted vascular network rearrangement at the PFC concomitantly with an attention deficit. Although the amphetamine challenge improved the attentional performance, it triggers detrimental effects probably because of the exacerbated malondialdehyde levels and increased heat shock protein 70 expression in microvessels. All observed amphetamine-induced alterations were prevented by the AT1 -R blockade. Our results support the AT1 -R involvement in the development of oxidative/inflammatory conditions triggered by amphetamine exposure, affecting cortical areas and increasing vascular susceptibility to future challenges.

Interstitial flow regulates in vitro three-dimensional self-organized brain micro-vessels.

Cell culture under medium flow has been shown to favor human brain microvascular endothelial cells function and maturation. Here a three-dimensional in vitro model of the human brain microvasculature, comprising brain microvascular endothelial cells but also astrocytes, pericytes and a collagen type I microfiber - fibrin based matrix, was cultured under continuous medium flow in a pressure driven microphysiological system (10 kPa, in 60-30 s cycles). The cells self-organized in micro-vessels perpendicular to the shear flow. Comparison with static culture showed that the resulting interstitial flow enhanced a more defined micro-vasculature network, with slightly more numerous lumens, and a higher expression of transporters, carriers and tight junction genes and proteins, essential to the blood-brain barrier functions.

Identification and quantification of blood-brain barrier transporters in isolated rat brain microvessels.

The blood-brain barrier (BBB) maintains brain homeostasis by tightly regulating the exchange of molecules with systemic circulation. It consists primarily of microvascular endothelial cells surrounded by astrocytic endfeet, pericytes, and microglia. Understanding the make-up of transporters in rat BBB is essential to the translation of pharmacological and toxicological observations into humans. In this study, experimental workflows are presented in which the optimization of (a) isolation of rat brain microvessels (b) enrichment of endothelial cells, and (c) extraction and digestion of proteins were evaluated, followed by identification and quantification of BBB proteins. Optimization of microvessel isolation was indicated by 15-fold enrichment of endothelial cell marker Glut1 mRNA, whereas markers for other cell types were not enriched. Filter-aided sample preparation was shown to be superior to in-solution sample preparation (10251 peptides vs. 7533 peptides). Label-free proteomics was used to identify nearly 2000 proteins and quantify 1276 proteins in isolated microvessels. A combination of targeted and global proteomics was adopted to measure protein abundance of 6 ATP-binding cassette and 27 solute carrier transporters. Data analysis using proprietary Progenesis and open access MaxQuant software showed overall agreement; however, Abcb9 and Slc22a8 were quantified only by MaxQuant, whereas Abcc9 and Abcd3 were quantified only by Progenesis. Agreement between targeted and untargeted quantification was demonstrated for Abcb1 (19.7 ± 1.4 vs. 17.8 ± 2.3) and Abcc4 (2.2 ± 0.7 vs. 2.1 ± 0.4), respectively. Rigorous quantification of BBB proteins, as reported in this study, should assist with translational modeling efforts involving brain disposition of xenobiotics.

Cilostazol inhibits platelet-endothelial cell interaction in murine microvessels after transient bilateral common carotid artery occlusion.

BACKGROUND: The purpose of this study was to investigate the effects of cilostazol on platelet behavior (rolling and adhesion) in murine cerebral microvessels after transient bilateral carotid artery occlusion using intravital fluorescence microscopy. METHODS: We used 41 C57BL/6J mice for the experiment. Fourteen mice were used as sham group (no ischemia and reperfusion, no medication); an ischemia (induced by 15-minute occlusion of bilateral common carotid arteries) and reperfusion (I/R) group (n = 17); and an I/R + cilostazol (I/R + CZ) group (receiving 30 mg/kg of cilostazol orally at 30 minutes before ischemia) (n = 10). A cranial window was prepared in the right parietal region. Platelets obtained from donor mice were labeled with a fluorescent dye (carboxyfluorescein iodoacetate succinimidyl ester) in vitro. Labeled platelets were intravenously administered at 3 or 6 hours after reperfusion, and then platelet behavior (rolling and adhesion) in the brain microvessels was observed. The numbers of rolling and adhering platelets in the arteriole and venule were calculated. RESULTS: Numbers of rolling and adherent platelets at 3 and 6 hours after reperfusion were significantly higher in the I/R group than in the sham or I/R + CZ groups in both venule (P < .05) and arteriole (P < .05). CONCLUSIONS: Cilostazol inhibits platelet-endothelial interactions following cerebral ischemia and reperfusion.

Acute severe hypoglycemia alters mouse brain microvascular proteome.

Hypoglycemia increases the risk related to stroke and neurodegenerative diseases, however, the underlying mechanisms are unclear. For the first time, we studied the effect of a single episode (acute) of severe (ASH) and mild (AMH) hypoglycemia on mouse brain microvascular proteome. After four-hour fasting, insulin was administered (i.p) to lower mean blood glucose in mice and induce ∼30 minutes of ASH (∼30 mg/dL) or AMH (∼75 mg/dL), whereas a similar volume of saline was given to control mice (∼130 mg/dL). Blood glucose was allowed to recover over 60 minutes either spontaneously or by 20% dextrose administration (i.p). Twenty-four hours later, the brain microvessels (BMVs) were isolated, and tandem mass tag (TMT)-based quantitative proteomics was performed using liquid chromatography-mass spectrometry (LC/MS). When compared to control, ASH significantly downregulated 13 proteins (p ≤ 0.05) whereas 23 proteins showed a strong trend toward decrease (p ≤ 0.10). When compared to AMH, ASH significantly induced the expression of 35 proteins with 13 proteins showing an increasing trend. AMH downregulated only 3 proteins. ASH-induced downregulated proteins are involved in actin cytoskeleton maintenance needed for cell shape and migration which are critical for blood-brain barrier maintenance and angiogenesis. In contrast, ASH-induced upregulated proteins are RNA-binding proteins involved in RNA splicing, transport, and stability. Thus, ASH alters BMV proteomics to impair cytoskeletal integrity and RNA processing which are critical for cerebrovascular function.

Increased Expression of Transferrin Receptor 1 in the Brain Cortex of 5xFAD Mouse Model of Alzheimer's Disease Is Associated with Activation of HIF-1 Signaling Pathway.

Alzheimer's disease (AD) is the most common cause of dementia. Despite intensive research efforts, there are currently no effective treatments to cure and prevent AD. There is growing evidence that dysregulation of iron homeostasis may contribute to the pathogenesis of AD. Given the important role of the transferrin receptor 1 (TfR1) in regulating iron distribution in the brain, as well as in the drug delivery, we investigated its expression in the brain cortex and isolated brain microvessels from female 8-month-old 5xFAD mice mimicking advanced stage of AD. Moreover, we explored the association between the TfR1 expression and the activation of the HIF-1 signaling pathway, as well as oxidative stress and inflammation in 5xFAD mice. Finally, we studied the impact of Aß1-40 and Aß1-42 on TfR1 expression in the brain endothelial cell line hCMEC/D3. In the present study, we revealed that an increase in TfR1 protein levels observed in the brain cortex of 5xFAD mice was associated with activation of the HIF-1 signaling pathway as well as accompanied by oxidative stress and inflammation. Interestingly, incubation of Aß peptides in hCMEC/D3 cells did not affect the expression of TfR1, which supported our findings of unaltered TfR1 expression in the isolated brain microvessels in 5xFAD mice. In conclusion, the study provides important information about the expression of TfR1 in the 5xFAD mouse model and the potential role of HIF-1 signaling pathway in the regulation of TfR1 in AD, which could represent a promising strategy for the development of therapies for AD.

Protein Expression of Amino Acid Transporters Is Altered in Isolated Cerebral Microvessels of 5xFAD Mouse Model of Alzheimer's Disease.

Membrane transporters such as ATP-binding cassette (ABC) and solute carrier (SLC) transporters expressed at the neurovascular unit (NVU) play an important role in drug delivery to the brain and have been demonstrated to be involved in Alzheimer's disease (AD) pathogenesis. However, our knowledge of quantitative changes in transporter absolute protein expression and functionality in vivo in NVU in AD patients and animal models is limited. The study aim was to investigate alterations in protein expression of ABC and SLC transporters in the isolated brain microvessels and brain prefrontal cortices of a widely used model of familial AD, 5xFAD mice (8 months old), using a sensitive liquid chromatography tandem mass spectrometry-based quantitative targeted absolute proteomic approach. Moreover, we examined alterations in brain prefrontal cortical and plasmatic levels of transporter substrates in 5xFAD mice compared to age-matched wild-type (WT) controls. ASCT1 (encoded by Slc1a4) protein expression in the isolated brain microvessels and brain prefrontal cortices of 5xFAD mice was twice higher compared to WT controls (p = 0.01). Brain cortical levels of ASCT1 substrate, serine, were increased in 5xFAD mice compared to WT animals. LAT1 (encoded by Slc7a5) and 4F2hc (encoded by Slc3a2) protein expressions were significantly altered in the isolated brain microvessels of 5xFAD mice compared to WT controls (p = 0.008 and p = 0.05, respectively). Overall, the study provides important information, which is crucial for the optimal use of the 5xFAD mouse model in AD drug development and for investigating novel drug delivery approaches. In addition, the findings of the study shed light on the novel potential mechanisms underlying AD pathogenesis.

In mice and humans, brain microvascular contractility matures postnatally.

Although great efforts to characterize the embryonic phase of brain microvascular system development have been made, its postnatal maturation has barely been described. Here, we compared the molecular and functional properties of brain vascular cells on postnatal day (P)5 vs. P15, via a transcriptomic analysis of purified mouse cortical microvessels (MVs) and the identification of vascular-cell-type-specific or -preferentially expressed transcripts. We found that endothelial cells (EC), vascular smooth muscle cells (VSMC) and fibroblasts (FB) follow specific molecular maturation programs over this time period. Focusing on VSMCs, we showed that the arteriolar VSMC network expands and becomes contractile resulting in a greater cerebral blood flow (CBF), with heterogenous developmental trajectories within cortical regions. Samples of the human brain cortex showed the same postnatal maturation process. Thus, the postnatal phase is a critical period during which arteriolar VSMC contractility required for vessel tone and brain perfusion is acquired and mature.

Probing cerebral malaria inflammation in 3D human brain microvessels.

Sequestration of Plasmodium falciparum-infected erythrocytes (IEs) in the brain microcirculation is a hallmark of cerebral malaria (CM), which leads to endothelial activation, brain swelling, and death. Here, we probed CM inflammation in a perfusable 3D human brain microvessel model. 3D brain microvessels supported in vivo-like capacities for parasite binding and maturation in situ, leading to a distinct inflammatory response from the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). By combining transcriptional analysis, imaging, and leukocyte perfusion, we showed that whereas TNF-α promotes a reversible inflammatory phenotype with widespread leukocyte recruitment, parasites induce unique stress response pathways and cause localized cell adhesivity changes, focal endothelial disruptions, and apoptosis. Furthermore, parasites modified the temporal kinetics of the TNF transcriptional response, suggesting augmented inflammatory damage with the two sequential stimuli. Our findings offer mechanistic insights into CM biology in a 3D brain microvessel mimetic platform and suggest that multiple events intersect to promote brain barrier inflammation in CM.

Differential Expression of ABC Transporter Genes in Brain Vessels vs. Peripheral Tissues and Vessels from Human, Mouse and Rat.

BACKGROUND: ATP-binding cassette (ABC) transporters comprise a superfamily of genes encoding membrane proteins with nucleotide-binding domains (NBD). These transporters, including drug efflux across the blood-brain barrier (BBB), carry a variety of substrates through plasma membranes against substrate gradients, fueled by hydrolyzing ATP. The expression patterns/enrichment of ABC transporter genes in brain microvessels compared to peripheral vessels and tissues are largely uncharacterized. METHODS: In this study, the expression patterns of ABC transporter genes in brain microvessels, peripheral tissues (lung, liver and spleen) and lung vessels were investigated using RNA-seq and WesTM analyses in three species: human, mouse and rat. RESULTS: The study demonstrated that ABC drug efflux transporter genes (including ABCB1, ABCG2, ABCC4 and ABCC5) were highly expressed in isolated brain microvessels in all three species studied; the expression of ABCB1, ABCG2, ABCC1, ABCC4 and ABCC5 was generally higher in rodent brain microvessels compared to those of humans. In contrast, ABCC2 and ABCC3 expression was low in brain microvessels, but high in rodent liver and lung vessels. Overall, most ABC transporters (with the exception of drug efflux transporters) were enriched in peripheral tissues compared to brain microvessels in humans, while in rodent species, additional ABC transporters were found to be enriched in brain microvessels. CONCLUSIONS: This study furthers the understanding of species similarities and differences in the expression patterns of ABC transporter genes; this is important for translational studies in drug development. In particular, CNS drug delivery and toxicity may vary among species depending on their unique profiles of ABC transporter expression in brain microvessels and BBB.

Exposure of human cerebral microvascular endothelial cells hCMEC/D3 to laminar shear stress induces vascular protective responses.

Endothelial cells (ECs) are constantly submitted in vivo to hemodynamical forces derived from the blood circulation, including shear stress (SS). ECs are able to detect SS and consequently adapt their phenotype, thus affecting many endothelial functions. If a plethora of shear stress-regulated molecular networks have been described in peripheral ECs, less is known about the molecular responses of microvascular brain ECs which constitute the blood-brain barrier (BBB). In this work, we investigated the response of human cerebral microvascular ECs to laminar physiological shear stress using the well characterized hCMEC/D3 cell line. Interestingly, we showed that hCMEC/D3 cells responded to shear stress by aligning perpendicularly to the flow direction, contrary to peripheral endothelial cells which aligned in the flow direction. Whole proteomic profiles were compared between hCMEC/D3 cells cultured either in static condition or under 5 or 10 dyn.cm-2 SS for 3 days. 3592 proteins were identified and expression levels were significantly affected for 3% of them upon both SS conditions. Pathway analyses were performed which revealed that most proteins overexpressed by SS refer to the antioxidant defense, probably mediated by activation of the NRF2 transcriptional factor. Regarding down-regulated proteins, most of them participate to the pro-inflammatory response, cell motility and proliferation. These findings confirm the induction of EC quiescence by laminar physiological SS and reveal a strong protective effect of SS on hCMEC/D3 cells, suggesting a similar effect on the BBB. Our results also showed that SS did not significantly increase expression levels nor did it affect the localization of junctional proteins and did not afect either the functional activity of several ABC transporters (P-glycoprotein and MRPs). This work provides new insights on the response of microvascular brain ECs to SS and on the importance of SS for optimizing in vitro BBB models.

Quantitative Targeted Absolute Proteomics for Better Characterization of an In Vitro Human Blood-Brain Barrier Model Derived from Hematopoietic Stem Cells.

We previously developed an in vitro model of the human blood-brain barrier (BBB) based on the use of endothelial cells derived from CD34+-hematopoietic stem cells and cultured with brain pericytes. The purpose of the present study was to provide information on the protein expression levels of the transporters, receptors, tight junction/adherence junction molecules, and transporter-associated molecules of human brain-like endothelial cells (hBLECs). The absolute protein expression levels were determined by liquid chromatography-mass spectrometry-based quantitative targeted absolute proteomics and compared with those from human brain microvessels (hBMVs). The protein levels of CD144, CD147, MRP4, Annexin A6 and caveolin-1 showed more than 3-fold abundance in hBLECs, those of MCT1, Connexin 43, TfR1, and claudin-5 showed less than 3-fold differences, and the protein levels of other drug efflux transporters and nutrient transporters were less represented in hBLECs than in hBMVs. It is noteworthy that BCRP was more expressed than MDR1 in hBLECs, as this was the case for hBMVs. These results suggest that transports mediated by MCT1, TfR1, and claudin-5-related tight junction function reflect the in vivo BBB situation. The present study provided a better characterization of hBLECs and clarified the equivalence of the transport characteristics between in vitro BBB models and in vivo BBB models using LC-MS/MS-based protein quantification.

Identification of Reference Genes for Circadian Studies on Brain Microvessels and Choroid Plexus Samples Isolated from Rats.

Delivery of putative compounds of therapeutic value to the brain is limited by brain barriers: the blood-brain barrier located in the endothelium of the brain microvessels (BrMVs) and the blood-cerebrospinal fluid barrier located in the epithelium of the choroid plexus (ChP). Understanding their function and modulation by the circadian clock may enhance the efficacy of brain-targeting therapies. The aim of the present study was to evaluate the stability of 10 reference genes in the BrMV and ChP, isolated from male and female rats at six time points (ZT1, 5, 9, 13, 17, and 21). Gene evaluations were performed by qPCR, analyzed by RefFinder tool, and verified by analyzing the expression of the brain and muscle ARNT-like 1 (Bmal1) using the qPCR and digital PCR methods. We identified as the most stable genes for circadian studies tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and apolipoprotein E (Apoe) for BrMV, and beta actin (Actb) and hypoxanthine-guanine phosphoribosyltransferase (Hprt1) for ChP. After verification, ribosomal protein (Rps18) was also included as a sufficient reference gene. Additionally, the observed gender difference in the Bmal1 oscillations in both BrMV and ChP suggests that separate studies for each gender are recommended.

Ginseng gintonin attenuates the disruptions of brain microvascular permeability and microvascular endothelium junctional proteins in an APPswe/PSEN-1 double-transgenic mouse model of Αlzheimer's disease.

It has been previously indicated that gintonin, which is a novel exogenous ginseng-derived lysophosphatidic acid (LPA) receptor ligand, restores memory dysfunctions in an APPswe/PSEN-1 double-transgenic mouse model of Alzheimer's disease (AD Tg mice) by attenuating ß-amyloid plaque deposition, recovering cholinergic dysfunctions and upregulating hippocampal neurogenesis in the cortex and hippocampus. Although ß-amyloid plaque depositions in AD is accompanied with disruptions of brain microvessels, including the brain-blood barrier (BBB), it is unknown whether gintonin exerts protective effects on brain microvascular dysfunctions in AD Tg mice. In the present study, the effects of gintonin-enriched fraction (GEF) on the changes in ß-amyloid plaque depositions, brain permeability of Evans blue, and microvascular junctional proteins were investigated in AD Tg mice. Long-term oral administration of GEF reduced ß-amyloid plaque depositions in the cortex and hippocampus of AD Tg mice. GEF treatment also reduced the permeability of Evans blue through BBB and decreased immunoreactivity of platelet endothelial cell adhesion molecule-1 (a marker of BBB disruption) in the cortex and hippocampus of AD Tg mice in a dose-dependent manner. However, GEF elevated the protein expression of occludin, claudin-5 and zonula occludens-1, which are tight-junction proteins. The present results demonstrated that long-term oral GEF treatment not only attenuates ß-amyloid plaque depositions in the brain but also exhibits protective effects against microvascular disruptions in AD Tg mice. Finally, GEF exhibits anti-AD effects through attenuation of ß-amyloid plaque depositions and protection against brain microvascular damage in an AD animal model.

HIV Tat-mediated induction of autophagy regulates the disruption of ZO-1 in brain endothelial cells.

The blood-brain barrier (BBB) is a tight barrier that is critical for preventing the entry of pathogens and small molecules into the brain. HIV protein Tat (Tat) is known to disrupt the tight junctions of the BBB. Autophagy is an intracellular process that involves degradation and recycling of damaged organelles to the lysosome and has recently been implicated in the BBB disruption. The role of autophagy in Tat-mediated BBB disruption, however, remains elusive. Herein we hypothesized that Tat induces endothelial autophagy resulting in decreased expression of the tight junction protein ZO-1 leading to breach of the BBB. In this study, we demonstrated that exposure of human brain microvessel endothelial cells (HBMECs) to Tat resulted in induction of autophagy in a dose- and time-dependent manner, with upregulation of BECN1/Beclin 1, ATG5 and MAP1LC3B proteins and a concomitant downregulation of the tight junction protein ZO-1 ultimately leading to increased endothelial cell monolayer paracellular permeability in an in vitro BBB model. Pharmacological and genetic inhibition of autophagy resulted in the abrogation of Tat-mediated induction of MAP1LC3B with a concomitant restoration of tight junction proteins, thereby underscoring the role of autophagy in Tat-mediated breach of the BBB. Additionally, our data also demonstrated that Tat-mediated induction of autophagy involved PELI1/K63-linked ubiquitination of BECN1. Increased autophagy and decreased ZO-1 was further recapitulated in microvessels isolated from the brains of HIV Tg26 mice as well as the frontal cortex of HIV-infected autopsied brains. Overall, our findings identify autophagy as an important mechanism underlying Tat-mediated disruption of the BBB.

Tau reduction in aged mice does not impact Microangiopathy.

Microangiopathy, including proliferation of small diameter capillaries, increasing vessel tortuosity, and increased capillary blockage by leukocytes, was previously observed in the aged rTg4510 mouse model. Similar gene expression changes related to angiogenesis were observed in both rTg4510 and Alzheimer's disease (AD). It is uncertain if tau is directly responsible for these vascular changes by interacting directly with microvessels, and/or if it contributes indirectly via neurodegeneration and concurrent neuronal loss and inflammation. To better understand the nature of tau-related microangiopathy in human AD and in tau mice, we isolated capillaries and observed that bioactive soluble tau protein could be readily detected in association with vasculature. To examine whether this soluble tau is directly responsible for the microangiopathic changes, we made use of the tetracycline-repressible gene expression cassette in the rTg4510 mouse model and measured vascular pathology following tau reduction. These data suggest that reduction of tau is insufficient to alter established microvascular complications including morphological alterations, enhanced expression of inflammatory genes involved in leukocyte adherence, and blood brain barrier compromise. These data imply that 1) soluble bioactive tau surprisingly accumulates at the blood brain barrier in human brain and in mouse models, and 2) the morphological and molecular phenotype of microvascular disturbance does not resolve with reduction of whole brain soluble tau. Additional consideration of vascular-directed therapies and strategies that target tau in the vascular space may be required to restore normal function in neurodegenerative disease.

Differential expression of receptors mediating receptor-mediated transcytosis (RMT) in brain microvessels, brain parenchyma and peripheral tissues of the mouse and the human.

Receptor-mediated transcytosis (RMT) is a principal pathway for transport of macromolecules essential for brain function across the blood-brain barrier (BBB). Antibodies or peptide ligands which bind RMT receptors are often co-opted for brain delivery of biotherapeutics. Constitutively recycling transferrin receptor (TfR) is a prototype receptor utilized to shuttle therapeutic cargos across the BBB. Several other BBB-expressed receptors have been shown to mediate transcytosis of antibodies or protein ligands including insulin receptor (INSR) and insulin-like growth factor-1 receptor (IGF1R), lipid transporters LRP1, LDLR, LRP8 and TMEM30A, solute carrier family transporter SLC3A2/CD98hc and leptin receptor (LEPR). In this study, we analyzed expression patterns of genes encoding RMT receptors in isolated brain microvessels, brain parenchyma and peripheral organs of the mouse and the human using RNA-seq approach. IGF1R, INSR and LRP8 were highly enriched in mouse brain microvessels compared to peripheral tissues. In human brain microvessels only INSR was enriched compared to either the brain or the lung. The expression levels of SLC2A1, LRP1, IGF1R, LRP8 and TFRC were significantly higher in the mouse compared to human brain microvessels. The protein expression of these receptors analyzed by Western blot and immunofluorescent staining of the brain microvessels correlated with their transcript abundance. This study provides a molecular transcriptomics map of key RMT receptors in mouse and human brain microvessels and peripheral tissues, important to translational studies of biodistribution, efficacy and safety of antibodies developed against these receptors.