Rapamycin Treatment Reduces Brain Pericyte Constriction in Ischemic Stroke.

The contraction and subsequent death of brain pericytes may play a role in microvascular no-reflow following the reopening of an occluded artery during ischemic stroke. Mammalian target of rapamycin (mTOR) inhibition has been shown to reduce motility/contractility of various cancer cell lines and reduce neuronal cell death in stroke. However, the effects of mTOR inhibition on brain pericyte contraction and death during ischemia have not yet been investigated. Cultured pericytes exposed to simulated ischemia for 12 h in vitro contracted after less than 1 h, which was about 7 h prior to cell death. Rapamycin significantly reduced the rate of pericyte contraction during ischemia; however, it did not have a significant effect on pericyte viability at any time point. Rapamycin appeared to reduce pericyte contraction through a mechanism that is independent of changes in intracellular calcium. Using a mouse model of middle cerebral artery occlusion, we showed that rapamycin significantly increased the diameter of capillaries underneath pericytes and increased the number of open capillaries 30 min following recanalisation. Our findings suggest that rapamycin may be a useful adjuvant therapeutic to reduce pericyte contraction and improve cerebral reperfusion post-stroke.

25-Hydroxycholesterol attenuates tumor necrosis factor alpha-induced blood-brain barrier breakdown in vitro.

Intracellular cholesterol metabolism is regulated by the SREBP-2 and LXR signaling pathways. The effects of inflammation on these molecular mechanisms remain poorly studied, especially at the blood-brain barrier (BBB) level. Tumor necrosis factor α (TNFα) is a proinflammatory cytokine associated with BBB dysfunction. Therefore, the aim of our study was to investigate the effects of TNFα on BBB cholesterol metabolism, focusing on its underlying signaling pathways. Using a human in vitro BBB model composed of human brain-like endothelial cells (hBLECs) and brain pericytes (HBPs), we observed that TNFα increases BBB permeability by degrading the tight junction protein CLAUDIN-5 and activating stress signaling pathways in both cell types. TNFα also promotes cholesterol release and decreases cholesterol accumulation and APOE secretion. In hBLECs, the expression of SREBP-2 targets (LDLR and HMGCR) is increased, while ABCA1 expression is decreased. In HBPs, only LDLR and ABCA1 expression is increased. TNFα treatment also induces 25-hydroxycholesterol (25-HC) production, a cholesterol metabolite involved in the immune response and intracellular cholesterol metabolism. 25-HC pretreatment attenuates TNFα-induced BBB leakage and partially alleviates the effects of TNFα on ABCA1, LDLR, and HMGCR expression. Overall, our results suggest that TNFα favors cholesterol efflux via an LXR/ABCA1-independent mechanism at the BBB, while it activates the SREBP-2 pathway. Treatment with 25-HC partially reversed the effect of TNFα on the LXR/SREBP-2 pathways. Our study provides novel perspectives for better understanding cerebrovascular signaling events linked to BBB dysfunction and cholesterol metabolism in neuroinflammatory diseases.

Hydrogen alleviates hypoxic-ischaemic brain damage in neonatal rats by inhibiting injury of brain pericytes.

Hypoxia-ischaemia (HI) can induce the death of cerebrovascular constituent cells through oxidative stress. Hydrogen is a powerful antioxidant which can activate the antioxidant system. A hypoxia-ischaemia brain damage (HIBD) model was established in 7-day-old SD rats. Rats were treated with different doses of hydrogen-rich water (HRW), and brain pericyte oxidative stress damage, cerebrovascular function and brain tissue damage were assessed. Meanwhile, in vitro-cultured pericytes were subjected to oxygen-glucose deprivation and treated with different concentrations of HRW. Oxidative injury was measured and the molecular mechanism of how HRW alleviated oxidative injury of pericytes was also examined. The results showed that HRW significantly attenuated HI-induced oxidative stress in the brain pericytes of neonatal rats, partly through the Nrf2-HO-1 pathway, further improving cerebrovascular function and reducing brain injury and dysfunction. Furthermore, HRW is superior to a single-cell death inhibitor for apoptosis, ferroptosis, parthanatos, necroptosis and autophagy and can better inhibit HI-induced pericyte death. The liver and kidney functions of rats were not affected by present used HRW dose. This study elucidates the role and mechanism of hydrogen in treating HIBD from the perspective of pericytes, providing new theoretical evidence and mechanistic references for the clinical application of hydrogen in neonatal HIE.

A switch in the pathway of TRPC3-mediated calcium influx into brain pericytes contributes to capillary spasms after subarachnoid hemorrhage.

Calcium influx and subsequent elevation of the intracellular calcium concentration ([Ca2+]i) induce contractions of brain pericytes and capillary spasms following subarachnoid hemorrhage. This calcium influx is exerted through cation channels. However, the specific calcium influx pathways in brain pericytes after subarachnoid hemorrhage remain unknown. Transient receptor potential canonical 3 (TRPC3) is the most abundant cation channel potentially involved in calcium influx into brain pericytes and is involved in calcium influx into other cell types either via store-operated calcium entry (SOCE) or receptor-operated calcium entry (ROCE). Therefore, we hypothesized that TRPC3 is associated with [Ca2+]i elevation in brain pericytes, potentially mediating brain pericyte contraction and capillary spasms after subarachnoid hemorrhage. In this study, we isolated rat brain pericytes and demonstrated increased TRPC3 expression and its currents in brain pericytes after subarachnoid hemorrhage. Calcium imaging of brain pericytes revealed that changes in TRPC3 expression mediated a switch from SOCE-dominant to ROCE-dominant calcium influx after subarachnoid hemorrhage, resulting in significantly higher [Ca2+]i levels after SAH. TRPC3 activity in brain pericytes also contributed to capillary spasms and reduction in cerebral blood flow in an in vivo rat model of subarachnoid hemorrhage. Therefore, we suggest that the switch in TRPC3-mediated calcium influx pathways plays a crucial role in the [Ca2+]i elevation in brain pericytes after subarachnoid hemorrhage, ultimately leading to capillary spasms and a reduction in cerebral blood flow.

Blood-brain barrier biomarkers.

The blood-brain barrier (BBB) is a dynamic interface that regulates the exchange of molecules and cells between the brain parenchyma and the peripheral blood. The BBB is mainly composed of endothelial cells, astrocytes and pericytes. The integrity of this structure is essential for maintaining brain and spinal cord homeostasis and protection from injury or disease. However, in various neurological disorders, such as traumatic brain injury, Alzheimer's disease, and multiple sclerosis, the BBB can become compromised thus allowing passage of molecules and cells in and out of the central nervous system parenchyma. These agents, however, can serve as biomarkers of BBB permeability and neuronal damage, and provide valuable information for diagnosis, prognosis and treatment. Herein, we provide an overview of the BBB and changes due to aging, and summarize current knowledge on biomarkers of BBB disruption and neurodegeneration, including permeability, cellular, molecular and imaging biomarkers. We also discuss the challenges and opportunities for developing a biomarker toolkit that can reliably assess the BBB in physiologic and pathophysiologic states.

Breast adenocarcinoma cells adhere stronger to brain pericytes than to endothelial cells.

Most of the malignancies detected within the brain parenchyma are of metastatic origin. As the brain lacks classical lymphatic circulation, the primary way for metastasis relies on hematogenous routes. Dissemination of metastatic cells to the brain implies attachment to the luminal surface of brain endothelial cells, transmigration through the vessel wall, and adhesion to the brain surface of the vasculature. During this process, tumor cells must interact with brain endothelial cells and later on with pericytes. Physical interaction between tumor cells and brain vascular cells might be crucial in the successful extravasation of metastatic cells through blood vessels and later in their survival within the brain environment. Therefore, we applied single-cell force spectroscopy to investigate the nanoscale adhesive properties of living breast adenocarcinoma cells to brain endothelial cells and pericytes. We found target cell type-dependent adhesion characteristics, i.e. increased adhesion of the tumor cells to pericytes in comparison to endothelial cells, which underlines the existence of metastatic potential-related nanomechanical differences relying partly on membrane tether dynamics. Varying adhesion strength of the tumor cells to different cell types of brain vessels presumably reflects the transitory adhesion to endothelial cells before extravasation and the long-lasting strong interaction with pericytes during survival and proliferation in the brain. Our results highlight the importance of specific mechanical interactions between tumor cells and host cells during metastasis formation.

Brain pericyte biology: from physiopathological mechanisms to potential therapeutic applications in ischemic stroke.

Pericytes play an indispensable role in various organs and biological processes, such as promoting angiogenesis, regulating microvascular blood flow, and participating in immune responses. Therefore, in this review, we will first introduce the discovery and development of pericytes, identification methods and functional characteristics, then focus on brain pericytes, on the one hand, to summarize the functions of brain pericytes under physiological conditions, mainly discussing from the aspects of stem cell characteristics, contractile characteristics and paracrine characteristics; on the other hand, to summarize the role of brain pericytes under pathological conditions, mainly taking ischemic stroke as an example. Finally, we will discuss and analyze the application and development of pericytes as therapeutic targets, providing the research basis and direction for future microvascular diseases, especially ischemic stroke treatment.

Highlighting In Vitro the Role of Brain-like Endothelial Cells on the Maturation and Metabolism of Brain Pericytes by SWATH Proteomics.

Within the neurovascular unit, brain pericytes (BPs) are of major importance for the induction and maintenance of the properties of the blood-brain barrier (BBB) carried by the brain microvessel endothelial cells (ECs). Throughout barriergenesis, ECs take advantage of soluble elements or contact with BPs to maintain BBB integrity and the regulation of their cellular homeostasis. However, very few studies have focused on the role of ECs in the maturation of BPs. The aim of this study is to shed light on the proteome of BPs solocultured (hBP-solo) or cocultured with ECs (hBP-coc) to model the human BBB in a non-contact manner. We first generated protein libraries for each condition and identified 2233 proteins in hBP-solo versus 2492 in hBP-coc and 2035 common proteins. We performed a quantification of the enriched proteins in each condition by sequential window acquisition of all theoretical mass spectra (SWATH) analysis. We found 51 proteins enriched in hBP-solo related to cell proliferation, contractility, adhesion and extracellular matrix element production, a protein pattern related to an immature cell. In contrast, 90 proteins are enriched in hBP-coc associated with a reduction in contractile activities as observed in vivo in 'mature' BPs, and a significant gain in different metabolic functions, particularly related to mitochondrial activities and sterol metabolism. This study highlights that BPs take advantage of ECs during barriergenesis to make a metabolic switch in favor of BBB homeostasis in vitro.

TNFα Activates the Liver X Receptor Signaling Pathway and Promotes Cholesterol Efflux from Human Brain Pericytes Independently of ABCA1.

Neuroinflammation and brain lipid imbalances are observed in Alzheimer's disease (AD). Tumor necrosis factor-α (TNFα) and the liver X receptor (LXR) signaling pathways are involved in both processes. However, limited information is currently available regarding their relationships in human brain pericytes (HBP) of the neurovascular unit. In cultivated HBP, TNFα activates the LXR pathway and increases the expression of one of its target genes, the transporter ATP-binding cassette family A member 1 (ABCA1), while ABCG1 is not expressed. Apolipoprotein E (APOE) synthesis and release are diminished. The cholesterol efflux is promoted, but is not inhibited, when ABCA1 or LXR are blocked. Moreover, as for TNFα, direct LXR activation by the agonist (T0901317) increases ABCA1 expression and the associated cholesterol efflux. However, this process is abolished when LXR/ABCA1 are both inhibited. Neither the other ABC transporters nor the SR-BI are involved in this TNFα-mediated lipid efflux regulation. We also report that inflammation increases ABCB1 expression and function. In conclusion, our data suggest that inflammation increases HBP protection against xenobiotics and triggers an LXR/ABCA1 independent cholesterol release. Understanding the molecular mechanisms regulating this efflux at the level of the neurovascular unit remains fundamental to the characterization of links between neuroinflammation, cholesterol and HBP function in neurodegenerative disorders.

Uptake of severe acute respiratory syndrome coronavirus 2 spike protein mediated by angiotensin converting enzyme 2 and ganglioside in human cerebrovascular cells.

Introduction: There is clinical evidence of neurological manifestations in coronavirus disease-19 (COVID-19). However, it is unclear whether differences in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/spike protein (SP) uptake by cells of the cerebrovasculature contribute to significant viral uptake to cause these symptoms. Methods: Since the initial step in viral invasion is binding/uptake, we used fluorescently labeled wild type and mutant SARS-CoV-2/SP to study this process. Three cerebrovascular cell types were used (endothelial cells, pericytes, and vascular smooth muscle cells), in vitro. Results: There was differential SARS-CoV-2/SP uptake by these cell types. Endothelial cells had the least uptake, which may limit SARS-CoV-2 uptake into brain from blood. Uptake was time and concentration dependent, and mediated by angiotensin converting enzyme 2 receptor (ACE2), and ganglioside (mono-sialotetrahexasylganglioside, GM1) that is predominantly expressed in the central nervous system and the cerebrovasculature. SARS-CoV-2/SPs with mutation sites, N501Y, E484K, and D614G, as seen in variants of interest, were also differentially taken up by these cell types. There was greater uptake compared to that of the wild type SARS-CoV-2/SP, but neutralization with anti-ACE2 or anti-GM1 antibodies was less effective. Conclusion: The data suggested that in addition to ACE2, gangliosides are also an important entry point of SARS-CoV-2/SP into these cells. Since SARS-CoV-2/SP binding/uptake is the initial step in the viral penetration into cells, a longer exposure and higher titer are required for significant uptake into the normal brain. Gangliosides, including GM1, could be an additional potential SARS-CoV-2 and therapeutic target at the cerebrovasculature.

Studying the Inflammatory Responses to Amyloid Beta Oligomers in Brain-Specific Pericyte and Endothelial Co-culture from Human Stem Cells.

Background: Recently, the in vitro blood brain barrier (BBB) models derived from human pluripotent stem cells have been given extensive attention in therapeutics due to the implications it has with the health of the central nervous system. It is essential to create an accurate BBB model in vitro in order to better understand the properties of the BBB and how it can respond to inflammatory stimulation and be passed by targeted or non-targeted cell therapeutics, more specifically extracellular vesicles. Methods: Brain-specific pericytes (iPCs) were differentiated from iPSK3 cells using dual SMAD signaling inhibitors and Wnt activation plus fibroblast growth factor 2 (FGF-2). The derived cells were characterized by immunostaining, flow cytometry and RT-PCR. In parallel, blood vessels organoids were derived using Wnt activation, BMP4, FGF2, VEGF and SB431542. The organoids were replated and treated with retinoic acid to enhance the blood brain barrier (BBB) features in the differentiated brain endothelial cells (iECs). Co-culture was performed for the iPCs and iECs in transwell system and 3-D microfluidics channels. Results: The derived iPCs expressed common markers PDGFRb and NG2, as well as brain-specific genes FOXF2, ABCC9, KCNJ8, and ZIC1. The derived iECs expressed common endothelial cell markers CD31, VE-cadherin, as well as BBB-associated genes BRCP, GLUT-1, PGP, ABCC1, OCLN, SLC2A1. The co-culture of the two cell types responded to the stimulation of amyloid ß42 oligomers by the upregulation of expression of TNFa, IL6, NFKB, Casp3, SOD2 and TP53. The co-culture also showed the property of trans-endothelial electrical resistance. The proof-of-concept vascularization strategy was demonstrated in a 3-D microfluidics-based device. Conclusion: The derived iPCs and iECs have brain-specific properties and the co-culture of iPCs and iECs provides an in vitro BBB model that show inflammatory response. This study has significance in establishing micro-physiological systems for neurological disease modeling and drug screening.

Differentiation of Brain Pericyte-Like Cells from Human Pluripotent Stem Cell-Derived Neural Crest.

Brain pericytes regulate diverse aspects of neurovascular development and function, including blood-brain barrier (BBB) induction and maintenance. Primary brain pericytes have been widely employed in coculture-based in vitro models of the BBB, and a method to generate brain pericytes from human pluripotent stem cells (hPSCs) could provide a renewable, genetically tractable source of cells for BBB modeling and studying pericyte roles in development and disease. Here, we describe a protocol to differentiate hPSCs to NG2+ PDGFRß+ αSMAlow brain pericyte-like cells in 22-25 days through a p75-NGFR+ HNK-1+ neural crest intermediate, which mimics the developmental origin of forebrain pericytes. The resulting brain pericyte-like cells have molecular and functional attributes of brain pericytes. We also provide protocols for maintenance, cryopreservation, and recovery of the neural crest intermediate, and for molecular and functional characterization of the resulting cells. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Differentiation of hPSCs to neural crest Basic Protocol 2: Differentiation of neural crest to brain pericyte-like cells Support Protocol 1: Flow cytometry analysis of neural crest cells Support Protocol 2: Maintenance, cryopreservation, and recovery of neural crest cells Support Protocol 3: Molecular characterization of brain pericyte-like cells Support Protocol 4: Cord formation assay with endothelial cells and brain pericyte-like cells.

Contribution of brain pericytes in blood-brain barrier formation and maintenance: a transcriptomic study of cocultured human endothelial cells derived from hematopoietic stem cells.

Formation, maintenance, and repair of the blood-brain barrier (BBB) are critical for central nervous system homeostasis. The interaction of endothelial cells (ECs) with brain pericytes is known to induce BBB characteristics in brain ECs during embryogenesis and can be used to differentiate human ECs from stem cell source in in vitro BBB models. However, the molecular events involved in BBB maturation are not fully understood. To this end, human ECs derived from hematopoietic stem cells were cultivated with either primary bovine or cell line-derived human brain pericytes to induce BBB formation. Subsequently, the transcriptomic profiles of solocultured vs. cocultured ECs were analysed over time by Massive Analysis of cDNA Ends (MACE) technology. This RNA sequencing method is a 3'-end targeted, tag-based, reduced representation transcriptome profiling technique, that can reliably quantify all polyadenylated transcripts including those with low expression. By analysing the generated transcriptomic profiles, we can explore the molecular processes responsible for the functional changes observed in ECs in coculture with brain pericytes (e.g. barrier tightening, changes in the expression of transporters and receptors). Our results identified several up- and downregulated genes and signaling pathways that provide a valuable data source to further delineate complex molecular processes that are involved in BBB formation and BBB maintenance. In addition, this data provides a source to identify novel targets for central nervous system drug delivery strategies.

An In Vitro Partial Lesion Model of Differentiated Human Mesencephalic Neurons: Effect of Pericyte Secretome on Phenotypic Markers.

Parkinson's disease (PD) is characterised by the progressive degeneration of dopaminergic (DA) neurons in the substantia nigra pars compacta. Post-mortem data suggests that the loss of DA markers may long precede the cell death, leaving a window to rescue the DA phenotype. Screening for potential neuroprotective or restorative therapies, however, requires that partial lesions of DA neurons can be modelled in vitro. In order to establish a partial lesion model of DA neurons in vitro, we evaluated the effects of different exposure times to 1-methyl-4-phenylpyridinium (MPP+) and 6-hydroxydopamine (6-OHDA) on the cell survival and DA marker expression using DA neurons derived from the Lund human mesencephalic (LUHMES) cell line. We show that 24-h incubation with 50 µM of MPP+ or 6-h incubation with 100 µM of 6-OHDA leads to a significant decrease in the protein expression of DA markers without affecting overall cell death, consistent with a mild DA lesion. Using conditioned medium of human brain-derived pericytes stimulated with platelet-derived growth factor BB (PDGF-BB), we demonstrate a significant upregulation of DA markers. In conclusion, we provide an experimental model of an in vitro DA neuron partial lesion suitable to study different molecules and their potential neuroprotective or neurorestorative effects on the DA phenotype. We provide evidence that the secretome of brain pericytes stimulated via PDGF-BB/PDGFRß affects DA marker expression and may represent one possible mechanism contributing to the neurorestoration previously observed in PD by this growth factor.

Pericytes secrete pro-regenerative molecules in response to platelet-derived growth factor-BB.

Brain pericytes not only maintain the anatomical, biochemical and immune blood-brain barrier, but display features of mesenchymal stem cells (MSCs) in vitro. MSCs have pro-regenerative properties attributed to their secretome. However, whether also brain pericytes possess such pro-regenerative capacities is largely unknown. Here we characterize the secretome and microvesicle (MV) release of human brain pericytes mediated by platelet-derived growth factor-BB (PDGF-BB)/PDGF receptor beta (PDGFRß) signalling. Upon PDGF-BB, pericytes release not only a plethora of growth factors and a panel of cytokines, but also MVs containing BDNF, FGFb, ßNGF, VEGF and PLGF, a response that is specific for PDGFRß signalling and activation of the ERK 1/2 pathway. In contrast, lipopolysaccharide (LPS), an activator of the innate immune system, stimulates the secretion of much higher amounts of mainly inflammatory cytokines and activates the NFκB pathway. Pericytes change their morphology and undergo opposite changes in surface marker expression, respectively. Our findings provide evidence that the secretome of human brain pericytes varies greatly depending on the exogenous stimulus. The differential secretory functions of pericytes may play an important role in either regulating neuroinflammation or contributing to neurorestoration and identify a possible new target cell for neuroregeneration.

Identification of neuronal and angiogenic growth factors in an in vitro blood-brain barrier model system: Relevance in barrier integrity and tight junction formation and complexity.

We previously demonstrated that the co-cultivation of endothelial cells with neural cells resulted in an improved integrity of the in vitro blood-brain barrier (BBB), and that this model could be useful to evaluate the transport properties of potential central nervous system disease drugs through the microvascular brain endothelial. In this study we have used real-time PCR, fluorescent microscopy, protein arrays and enzyme-linked immunosorbent assays to determine which neural- and endothelial cell-derived factors are produced in the co-culture and improve the integrity of the BBB. In addition, a further improvement of the BBB integrity was achieved by adjusting serum concentrations and growth factors or by the addition of brain pericytes. Under specific conditions expression of angiogenic, angiostatic and neurotrophic factors such as endostatin, pigment epithelium derived factor (PEDF/serpins-F1), tissue inhibitor of metalloproteinases (TIMP-1), and vascular endothelial cell growth factor (VEGF) closely mimicked the in vivo situation. Freeze-fracture analysis of these cultures demonstrated the quality and organization of the endothelial tight junction structures and their association to the two different lipidic leaflets of the membrane. Finally, a multi-cell culture model of the BBB with a transendothelial electrical resistance up to 371 (±15) Ω×cm2 was developed, which may be useful for preliminary screening of drug transport across the BBB and to evaluate cellular crosstalk of cells involved in the neurovascular unit.

Perivascular Mesenchymal Stem Cells From the Adult Human Brain Harbor No Instrinsic Neuroectodermal but High Mesodermal Differentiation Potential.

UNLABELLED: Brain perivascular cells have recently been identified as a novel mesodermal cell type in the human brain. These cells reside in the perivascular niche and were shown to have mesodermal and, to a lesser extent, tissue-specific differentiation potential. Mesenchymal stem cells (MSCs) are widely proposed for use in cell therapy in many neurological disorders; therefore, it is of importance to better understand the "intrinsic" MSC population of the human brain. We systematically characterized adult human brain-derived pericytes during in vitro expansion and differentiation and compared these cells with fetal and adult human brain-derived neural stem cells (NSCs) and adult human bone marrow-derived MSCs. We found that adult human brain pericytes, which can be isolated from the hippocampus and from subcortical white matter, are-in contrast to adult human NSCs-easily expandable in monolayer cultures and show many similarities to human bone marrow-derived MSCs both regarding both surface marker expression and after whole transcriptome profile. Human brain pericytes showed a negligible propensity for neuroectodermal differentiation under various differentiation conditions but efficiently generated mesodermal progeny. Consequently, human brain pericytes resemble bone marrow-derived MSCs and might be very interesting for possible autologous and endogenous stem cell-based treatment strategies and cell therapeutic approaches for treating neurological diseases. SIGNIFICANCE: Perivascular mesenchymal stem cells (MSCs) recently gained significant interest because of their appearance in many tissues including the human brain. MSCs were often reported as being beneficial after transplantation in the central nervous system in different neurological diseases; therefore, adult brain perivascular cells derived from human neural tissue were systematically characterized concerning neural stem cell and MSC marker expression, transcriptomics, and mesodermal and inherent neuroectodermal differentiation potential in vitro and in vivo after in utero transplantation. This study showed the lack of an innate neuronal but high mesodermal differentiation potential. Because of their relationship to mesenchymal stem cells, these adult brain perivascular mesodermal cells are of great interest for possible autologous therapeutic use.

Topiramate Protects Pericytes from Glucotoxicity: Role for Mitochondrial CA VA in Cerebromicrovascular Disease in Diabetes.

Hyperglycemia in diabetes mellitus causes oxidative stress and pericyte depletion from the microvasculature of the brain thus leading to the Blood-Brain Barrier (BBB) disruption. The compromised BBB exposes the brain to circulating substances, resulting in neurotoxicity and neuronal cell death. The decline in pericyte numbers in diabetic mouse brain and pericyte apoptosis in high glucose cultures are caused by excess superoxide produced during enhanced respiration (mitochondrial oxidative metabolism of glucose). Superoxide is precursor to all Reactive Oxygen Species (ROS) which, in turn, cause oxidative stress. The rate of respiration and thus the ROS production is regulated by mitochondrial carbonic anhydrases (mCA) VA and VB, the two isoforms expressed in the mitochondria. Inhibition of both mCA: decreases the oxidative stress and restores the pericyte numbers in diabetic brain; and reduces high glucose-induced respiration, ROS, oxidative stress, and apoptosis in cultured brain pericytes. However, the individual role of the two isoforms has not been established. To investigate the contribution of mCA VA in ROS production and apoptosis, a mCA VA overexpressing brain pericyte cell line was engineered. These cells were exposed to high glucose and analyzed for the changes in ROS and apoptosis. Overexpression of mCA VA significantly increased pericyte ROS and apoptosis. Inhibition of mCA VA with topiramate prevented increases both in glucose-induced ROS and pericyte death. These results demonstrate, for the first time, that mCA VA regulates the rate of pericyte respiration. These findings identify mCA VA as a novel and specific therapeutic target to protect the cerebromicrovascular bed in diabetes.

Brain pericytes are the most thrombin-sensitive matrix metalloproteinase-9-releasing cell type constituting the blood-brain barrier in vitro.

In the acute phase of intracerebral hemorrhage (ICH), hemorrhagic transformation and brain edema are associated with blood-brain barrier (BBB) disruption. Elevated levels of thrombin, a coagulation factor, contribute to the development of brain edema during ICH through matrix metalloproteinase (MMP)-9 production. Thrombin directly induces a variety of cellular responses through its specific receptors known as protease-activated receptors (PARs). However, it remains unclear which cell types constituting the BBB mainly produce MMP-9 in response to thrombin. Here, we compared the MMP-9 release induced by thrombin using primary cultures of rat brain microvascular endothelial cells, astrocytes, and pericytes. Brain pericytes exhibited the highest levels of MMP-9 release due to thrombin stimulation among the BBB cells. The pattern of PAR mRNA expression in pericytes was characterized by high expression of PAR1 and moderate expression of PAR4. Heat-inactivated thrombin failed to stimulate pericytes to release MMP-9. A selective PAR1 inhibitor SCH79797 blocked the thrombin-induced MMP-9 release from pericytes. These findings suggest that both PAR1 and PAR4 mediate thrombin-induced MMP-9 release from pericytes. The present study raises the possibility that brain pericytes could play a pivotal role as a highly thrombin-sensitive and MMP-9-producing cell type at the BBB in brain damage including ICH.

In vitro discrimination of the role of LRP1 at the BBB cellular level: focus on brain capillary endothelial cells and brain pericytes.

Several studies have demonstrated that the blood-brain barrier (BBB) (dynamic cellular complex composed by brain capillary endothelial cells (BCECs) and surrounded by astrocytic end feet and pericytes) regulates the exchanges of amyloid ß (Aß) peptide between the blood and the brain. Deregulation of these exchanges seems to be a key trigger for the brain accumulation of Aß peptide observed in Alzheimer's disease (AD). Whereas the involvement of receptor for advanced glycation end-products in Aß peptide transcytosis has been demonstrated in our laboratory, low-density lipoprotein receptor's role at the cellular level needs to be clarified. For this, we used an in vitro BBB model that consists of a co-culture of bovine BCECs and rat glial cells. This model has already been used to characterize low-density lipoprotein receptor-related peptide (LRP)'s involvement in the transcytosis of molecules such as tPA and angiopep-2. Our results suggest that Aß peptide efflux across the BCEC monolayer involves a transcellular transport. However, the experiments with RAP discard an involvement of LRP family members at BCECs level. In contrast, our results show a strong transcriptional expression of LRP1 in pericytes and suggest its implication in Aß endocytosis. Moreover, the observations of pericytes contraction and local downregulation of LRP1 in response to Aß treatment opens up perspectives for studying this cell type with respect to Aß peptide metabolism and AD.