PDGF receptor β signaling in pericytes following ischemic brain injury

Platelet derived growth factor (PDGF)-B plays a neuroprotective role in brain damages, including ischemic stroke. It has been suggested recently that PDGF receptor β (PDGFRβ) expressed in brain pericytes as well as in neurons and astrocytes may mediate the neuroprotective role of PDGF-B. The aims of this study were to elucidate the roles of PDGFRβ signaling in brain pericytes after ischemic stroke. In a rat middle cerebral artery occlusion (MCAO) model, PDGFRβ expression was induced specifically in the pericytes in peri-infarct areas and its level was gradually increased. PDGF-B induced marked phosphorylation of Akt in cultured brain pericytes. Consistently, PDGF-B was upregulated in endothelial cells in per-infarct areas and Akt was strongly phosphorylated in the PDGFRβ-expressing pericytes in periinfarct areas after MCAO. In the cultured pericytes, PDGF-B induced cell growth and anti-apoptotic responses through Akt. Furthermore, PDGF-B significantly increased the expression of nerve growth factor (NGF) and neurotrophin-3 (NT-3) through Akt in the pericytes. Thus, the PDGFRβ-Akt signaling in brain pericytes may play various important roles leading to neuroprotection after ischemic stroke.     

TGF‐β pro‐oligodendrogenic effects on adult SVZ progenitor cultures and its interaction with the Notch signaling pathway

Adult neural progenitor cells (NPCs) are capable of differentiating into neurons, astrocytes, and oligodendrocytes throughout life. Notch and transforming growth factor β1 (TGF‐β) signaling pathways play critical roles in controlling these cell fate decisions. TGF‐β has been previously shown to exert pro‐neurogenic effects on hippocampal and subventricular zone (SVZ) NPCs in vitro and to interact with Notch in different cellular types. Therefore, the aim of our work was to study the effect of TGF‐β on adult rat brain SVZ NPC glial commitment and its interaction with Notch signaling. Initial cell characterization revealed a large proportion of Olig2+, Nestin+, and glial fibrillary acidic protein (GFAP+) cells, a low percentage of platelet‐derived growth factor receptor α (PDGFRα+) or NG2+ cells, and <1% Tuj1+ cells. Immunocytochemical analyses showed a significant increase in the percentage of PDGFRα+, NG2+, and GFAP+ cells upon four‐day TGF‐β treatment, which demonstrates the pro‐gliogenic effect of this growth factor on adult brain SVZ NPCs. Real‐time polymerase chain reaction analyses showed that TGF‐β induced the expression of Notch ligand Jagged1 and downstream gene Hes1. Notch signaling inhibition in cultures treated with TGF‐β produced a decrease in the proportion of PDGFRα+ cells, while TGF‐β receptor II (TβRII) inhibition also rendered a decrease in the proportion of PDGFRα+ cells, concomitantly with a decrease in Jagged1 levels. These findings demonstrate the participation of Notch signaling in TGF‐β effects and illustrate the impact of TGF‐β on glial cell fate decisions of adult brain SVZ NPCs, as well as on oligodendroglial progenitor cell proliferation and maturation.     

VEGF‐dependent continuous angiogenesis in the median eminence of adult mice

Brain vasculature forms the blood–brain barrier (BBB) that restricts the movement of molecules between the brain and blood, but the capillary of the median eminence (ME) lacks the BBB for secretion of adenohypophysial hormone‐releasing peptides. In the present study, we aimed to elucidate whether continuous angiogenesis occurs in the ME of adult mice. By using a mitotic marker, bromodeoxyuridine (BrdU), we demonstrated that new endothelial cells were born continuously in the ME of adults. Prominent expression of NG2, platelet‐derived growth factor receptor B (PDGFRB), and delta‐like ligand 4 was observed at pericytes of adults, although the expression of these angiogenesis‐associated proteins has been shown to be at low or trace levels in adult mature capillary. In addition, vascular endothelial growth factor (VEGF), a key regulator of angiogenesis, was expressed highly in the nervous parenchyma of the ME. Expression of VEGF receptor 2 (VEGFR2) was observed at endothelial cells in the external zone and at somatodendrites in the internal zone. Finally, a VEGFR‐ and PDGFR‐associated tyrosine kinase inhibitor, SU11248, significantly decreased the number of BrdU‐positive proliferating endothelial cells and parenchyma cells. In conclusion, the present study demonstrates VEGF‐dependent continuous angiogenesis in the ME of adult mouse brains under normal conditions, which provides new insight into our understanding of neurosecretion in the ME.     

Extracellular vesicles of the blood-brain barrier: Role in the HIV-1 associated amyloid beta pathology

HIV-infected brains are characterized by increased amyloid beta (Aβ) deposition. It is believed that the blood-brain barrier (BBB) is critical for Aβ homeostasis and contributes to Aβ accumulation in the brain. Extracellular vesicles (ECV), like exosomes, recently gained a lot of attention as potentially playing a significant role in Aβ pathology. In addition, HIV-1 hijacks the exosomal pathway for budding and release. Therefore, we investigated the involvement of BBB-derived ECV in the HIV-1-induced Aβ pathology in the brain. Our results indicate that HIV-1 increases ECV release from brain endothelial cells as well as elevates their Aβ cargo when compared to controls. Interestingly, brain endothelial cell-derived ECV transferred Aβ to astrocytes and pericytes. Infusion of brain endothelial ECV carrying fluorescent Aβ into the internal carotid artery of mice resulted in Aβ fluorescence associated with brain microvessels and in the brain parenchyma. These results suggest that ECV carrying Aβ can be successfully transferred across the BBB into the brain. Based on these observations, we conclude that HIV-1 facilitates the shedding of brain endothelial ECV carrying Aβ; a process that may increase Aβ exposure of cells of neurovascular unit, and contribute to amyloid deposition in HIV-infected brain.     

Dynamic cell type‐specific expression of Nrf2 after traumatic brain injury in mice

Nrf2 plays a pivotal role in antioxidant response and anti‐inflammation after traumatic brain injury (TBI), and its deletion aggravates TBI‐induced brain damage. Previous studies have demonstrated that Nrf2 is activated post TBI, but dynamic changes in expression and cell type‐specific characteristics remain unclear. In this study, the Feeney weight‐drop contusion model was conducted to mimic TBI, and the ipsilateral cerebral cortex was collected at 1, 3, 7 and 14 days post TBI (dpi). Nrf2 protein levels were observed by western blot. Cell type‐specific localization of Nrf2 after TBI was detected at different time intervals by double immunofluorescence staining. NeuN, GFAP, IBA1 and NG2 were used as cell type‐specific markers to neurons, astrocytes, microglia and NG2 glia, respectively. After TBI, Nrf2 protein levels peaked at 1 dpi. Robust transient Nrf2 accumulation was co‐localized with neurons, which was predominant at 1 dpi. Continuous weak Nrf2 expression was detected in activated astrocytes, and the number of double positive cells peaked at 7 dpi. Inducible widespread immunostaining of Nrf2 was observed in the nucleus of the microglia, and the number of Nrf2+ microglia peaked at 7 dpi. In addition, we also explored colocalization of Nrf2 in NG2 glia, in which the percentage of Nrf2+ in NG2 glia reached a climax at 3 dpi. This study reveals that the accumulation of endogenous Nrf2 might mediate different pathophysical roles in neurons and glias after TBI, the cell‐type specific and time‐dependent expression provide insights to explain the roles of Nrf2 in different neural cells.     

Astrocytic β1-integrin affects cellular composition of murine blood brain barrier in the cerebral cortex

The blood brain barrier (BBB) is composed of endothelial cells, astrocytes, and pericytes and maintains functional homeostasis by regulating transport of ions, fluid and cells between blood and neural tissue. The cellular and molecular pathways that contribute to the formation of the BBB in the developing brain have not been fully deciphered. β1-integrin (β1-itg) within endothelial cells is known to play a critical role in vasculogenesis. However, the role of astrocytic β1-itg in BBB development is not known. Our study used a mouse glial fibrillary acidic protein (GFAP)-cre transgenic line to selectively ablate β1-itg within astrocytes. We found that deletion of astrocytic β1-itg had a striking effect on the different cell types that form the BBB. Mutant mice had a decreased density of aquaporin-4 immunoreactivity within the perivascular astrocytic end-feet. We also found decreases in immunoreactivity for vimentin and CD-31 within endothelial cells. These changes were not accompanied by functional changes in BBB under physiological conditions as assessed by extravasation of large and small molecular weight molecules. However, mutant mice had an increased incidence of severe cystic injury in response to neonatal hypoxia. Our findings show that astrocytic β1-itg has an important role in defining cellular properties of the blood brain barrier in the cerebral cortex.     

Lack of NG2 exacerbates neurological outcome and modulates glial responses after traumatic brain injury

Traumatic brain injury (TBI) is a major cause of death and disability. The underlying pathophysiology is characterized by secondary processes including neuronal death and gliosis. To elucidate the role of the NG2 proteoglycan we investigated the response of NG2‐knockout mice (NG2‐KO) to TBI. Seven days after TBI behavioral analysis, brain damage volumetry and assessment of blood brain barrier integrity demonstrated an exacerbated response of NG2‐KO compared to wild‐type (WT) mice. Reactive astrocytes and expression of the reactive astrocyte and neurotoxicity marker Lcn2 (Lipocalin‐2) were increased in the perilesional brain tissue of NG2‐KO mice. In addition, microglia/macrophages with activated morphology were increased in number and mRNA expression of the M2 marker Arg1 (Arginase 1) was enhanced in NG2‐KO mice. While TBI‐induced expression of pro‐inflammatory cytokine genes was unchanged between genotypes, PCR array screening revealed a marked TBI‐induced up‐regulation of the C‐X‐C motif chemokine 13 gene Cxcl13 in NG2‐KO mice. CXCL13, known to attract immune cells to the inflamed brain, was expressed by activated perilesional microglia/macrophages seven days after TBI. Thirty days after TBI, NG2‐KO mice still exhibited more pronounced neurological deficits than WT mice, up‐regulation of Cxcl13, enhanced CD45+ leukocyte infiltration and a relative increase of activated Iba‐1+/CD45+ microglia/macrophages. Our study demonstrates that lack of NG2 exacerbates the neurological outcome after TBI and associates with abnormal activation of astrocytes, microglia/macrophages and increased leukocyte recruitment to the injured brain. These findings suggest that NG2 may counteract neurological deficits and adverse glial responses in TBI. GLIA 2016;64:507–523     

Imatinib preserves blood–brain barrier integrity following experimental subarachnoid hemorrhage in rats

Blood–brain barrier (BBB) disruption and consequent edema formation contribute to the development of early brain injury following subarachnoid hemorrhage (SAH). Various cerebrovascular insults result in increased platelet‐derived growth factor receptor (PDGFR)‐α stimulation, which has been linked to BBB breakdown and edema formation. This study examines whether imatinib, a PDGFR inhibitor, can preserve BBB integrity in a rat endovascular perforation SAH model. Imatinib (40 or 120 mg/kg) or a vehicle was administered intraperitoneally at 1 hr after SAH induction. BBB leakage, brain edema, and neurological deficits were evaluated. Total and phosphorylated protein expressions of PDGFR‐α, c‐Src, c‐Jun N‐terminal kinase (JNK), and c‐Jun were measured, and enzymatic activities of matrix metalloproteinase (MMP)?2 and MMP‐9 were determined in the injured brain. Imatinib treatment significantly ameliorated BBB leakage and edema formation 24 hr after SAH, which was paralleled by improved neurological functions. Decreased brain expressions of phosphorylated PDGFR‐α, c‐Src, JNK, and c‐Jun as well as reduced MMP‐9 activities were found in treated animals. PDGFR‐α inhibition preserved BBB integrity following experimental SAH; however, the protective mechanisms remain to be elucidated. Targeting PDGFR‐α signaling might be advantageous to ameliorate early brain injury following SAH. © 2014 Wiley Periodicals, Inc.     

Simvastatin therapy prevents brain trauma‐induced increases in β‐amyloid peptide levels

Elevations in β‐amyloid peptide (Aβ) levels after traumatic brain injury (TBI) may confer risk for developing Alzheimer's disease in head trauma patients. We investigated the effects of simvastatin, a 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibitor, on hippocampal Aβ burden in a clinically relevant head injury/intervention model using mice expressing human Aβ. Simvastatin therapy blunted TBI‐induced increases in Aβ, reduced hippocampal tissue damage and microglial activation, and improved behavioral outcome. The ability of statins to reduce post‐injury Aβ load and ameliorate pathological sequelae of brain injury makes them potentially effective in reducing the risk of developing Alzheimer's disease in TBI patients. Ann Neurol 2009;66:407–414     

Lesion-associated accumulation of uPAR/CD87- expressing infiltrating granulocytes, activated microglial cells/macrophages and upregulation by endothelial cells following TBI and FCI in humans

Urokinase-type plasminogen activator receptor (uPAR/CD87) together with its ligand, urokinase-type plasminogen activator (uPA), constitutes a proteolytic system associated with tissue remodelling and leucocyte infiltration. uPAR is a member of the glycosyl phosphatidyl inositol (GPI) anchored protein family. The functional role of uPAR comprises fibrinolysis by conversion of plasminogen to plasmin. In addition, uPAR promotes cell adhesion, migration, proliferation, re-organization of the actin cytoskeleton, and angiogenesis. Furthermore, uPAR is involved in prevention of scar formation and is chemoattractant to macrophages and leucocytes. In order to investigate the pathophysiological role of uPAR following human CNS injury we examined necrotic brain lesions resulting from traumatic brain injury (TBI; n = 28) and focal cerebral infarctions (FCI; n = 17) by immunohistochemistry. Numbers of uPAR+ cells and uPAR+ blood vessels were counted. Following brain damage, uPAR+ cells increased significantly within 12 h, reached a maximum after 3-4 days and remained elevated until later stages. uPAR was expressed by infiltrating granulocytes, activated microglia/macrophages and endothelial cells. Numbers of uPAR+ vessels increased in parallel subsiding earlier following FCI than post TBI. The restricted, lesion-associated accumulation of uPAR+ cells in the brain parenchyma and upregulated expression by endothelial cells suggests a crucial role for the influx of inflammatory cells and blood-brain barrier (BBB) disturbance. Through a failure in BBB function, uPAR participates in formation of brain oedema and thus contributes to secondary brain damage. In conclusion, the study defines the localization, kinetic course and cellular source of uPAR as a potential pharmacological target following human TBI and FCI.     

Multipotent PDGFRβ-expressing cells in the circulation of stroke patients

Tissue pericytes respond to injury, and support vascular and tissue regeneration. The presence of pericytes in the circulation may provide an attractive framework for tissue regeneration. Here, we detected multipotent pericyte-like cells in the circulating blood and determined its profiles during cerebral ischemia. Pericyte-like cells were isolated from the peripheral blood of acute stroke patients or asymptomatic individuals with vascular risk factors by fluorescence or magnetic activated cell sorting with anti-PDGF receptor-beta (PDGFRβ) antibody. The morphologic and molecular features of circulating PDGFRβ(+) cells were compared with tissue pericytes, and the associations with respect to quantity in the blood, culture outcome, and patient characteristics were analyzed. We found an increase in circulating PDGFRβ(+) cells in acute stroke patients compared to controls and a correlation with neurologic impairment. The isolated PDGFRβ(+) cells expressed mesenchymal stem cell markers, proliferated, and were multipotent under permissive culture conditions. The multipotent nature of these cells was comparable to fat-derived PDGFRβ(+) cells. These cells could be obtained by pharmacologic stimulation using bone marrow mobilizer. Circulating PDGFRβ(+) cells will be useful for future research involving endogenous recovery or autologous cell-based therapy.     

Diffuse traumatic axonal injury in mice induces complex behavioural alterations that are normalized by neutralization of interleukin‐1β

Widespread traumatic axonal injury (TAI) results in brain network dysfunction, which commonly leads to persisting cognitive and behavioural impairments following traumatic brain injury (TBI). TBI induces a complex neuroinflammatory response, frequently located at sites of axonal pathology. The role of the pro‐inflammatory cytokine interleukin (IL)‐1β has not been established in TAI. An IL‐1β‐neutralizing or a control antibody was administered intraperitoneally at 30 min following central fluid percussion injury (cFPI), a mouse model of widespread TAI. Mice subjected to moderate cFPI (n = 41) were compared with sham‐injured controls (n = 20) and untreated, naive mice (n = 9). The anti‐IL‐1β antibody reached the target brain regions in adequate therapeutic concentrations (up to ~30 μg/brain tissue) at 24 h post‐injury in both cFPI (n = 5) and sham‐injured (n = 3) mice, with lower concentrations at 72 h post‐injury (up to ~18 μg/g brain tissue in three cFPI mice). Functional outcome was analysed with the multivariate concentric square field (MCSF) test at 2 and 9 days post‐injury, and the Morris water maze (MWM) at 14–21 days post‐injury. Following TAI, the IL‐1β‐neutralizing antibody resulted in an improved behavioural outcome, including normalized behavioural profiles in the MCSF test. The performance in the MWM probe (memory) trial was improved, although not in the learning trials. The IL‐1β‐neutralizing treatment did not influence cerebral ventricle size or the number of microglia/macrophages. These findings support the hypothesis that IL‐1β is an important contributor to the processes causing complex cognitive and behavioural disturbances following TAI.     

Long‐lasting pro‐ictogenic effects induced in vivo by rat brain exposure to serum albumin in the absence of concomitant pathology

Purpose: Dysfunction of the blood–brain barrier (BBB) is a common finding during seizures or following epileptogenic brain injuries, and experimentally induced BBB opening promotes seizures both in naive and epileptic animals. Brain albumin extravasation was reported to promote hyperexcitability by inducing astrocytes dysfunction. To provide in vivo evidence for a direct role of extravasated serum albumin in seizures independently on the pathologic context, we did the following: (1) quantified the amount of serum albumin extravasated in the rat brain parenchyma during status epilepticus (SE); (2) reproduced a similar concentration in the hippocampus by intracerebroventricular (i.c.v.) albumin injection in naive rats; (3) measured electroencephalography (EEG) activity in these rats, their susceptibility to kainic acid (KA)–induced seizures, and their hippocampal afterdischarge threshold (ADT). Methods: Brain albumin concentration was measured in the rat hippocampus and other forebrain regions 2 and 24 h after SE by western blot analysis. Brain distribution of serum albumin or fluorescein isothiocyanate (FITC)‐albumin was studied by immunohistochemistry and immunofluorescence, respectively. Naive rats were injected with rat albumin or FITC‐albumin, i.c.v., to mimic the brain concentration attained after SE, or with dextran used as control. Inflammation was evaluated by immunohistochemistry by measuring glial induction of interleukin (IL)‐1β. Western blot analysis was used to measure inward rectifying potassium channel subunit Kir4.1 protein levels in the hippocampus. Seizures were induced in rats by intrahippocampal injection of 80 ng KA and quantified by EEG analysis, 2 or 24 h after rat albumin or dextran administration. ADT was measured by electrical stimulation of the hippocampus 3 months after albumin injection. In these rats, EEG was continuously monitored for 2 weeks to search for spontaneous seizures. Key Findings: The hippocampal serum albumin concentration 24 h post‐SE was 0.76 ± 0.21 μm . Similar concentrations were measured in other forebrain regions, whereas no changes were found in cerebellum. The hippocampal albumin concentration was similarly reproduced in naive rats by i.c.v. administration of 500 μg/4 μl rat albumin: albumin was predominantly detected extracellularly 2 h after injection, whereas at 24 h it was visible inside pyramidal neurons and in only a few scattered chondroitin sulphate proteoglycan (NG2)‐positive cells, but not in glial fibrillary acidic protein (GFAP)‐positive astrocytes or CR‐3 complement receptor (OX‐42)‐positive microglia. The presence of albumin in naive rat hippocampus was associated with induced IL‐1β in GFAP‐positive astrocytes and a concomitant tissue down‐regulation of Kir4.1. Spiking activity was evoked by albumin in the hippocampus lasting for 2 h. When KA was intrahippocampally applied either 2 or 24 h after albumin injection, the number of total interictal spikes in 3 h EEG recording was significantly increased by twofold on average. Three months after albumin injection, neither albumin nor inflammation was detected in brain tissue; at this time, the ADT was reduced by 50% but no spontaneous seizures were observed. Significance: Transient hippocampal exposure to albumin levels similar to those attained after prominent BBB breakdown resulted in increased seizure susceptibility and long‐term reduction in seizure threshold, but it did not evoke spontaneous seizures. These effects may be mediated by albumin‐induced astrocytes dysfunction and the associated induction of proinflammatory molecules.     

Chronic cerebrovascular dysfunction after traumatic brain injury

Traumatic brain injuries (TBI) often involve vascular dysfunction that leads to long‐term alterations in physiological and cognitive functions of the brain. Indeed, all the cells that form blood vessels and that are involved in maintaining their proper function can be altered by TBI. This Review focuses on the different types of cerebrovascular dysfunction that occur after TBI, including cerebral blood flow alterations, autoregulation impairments, subarachnoid hemorrhage, vasospasms, blood–brain barrier disruption, and edema formation. We also discuss the mechanisms that mediate these dysfunctions, focusing on the cellular components of cerebral blood vessels (endothelial cells, smooth muscle cells, astrocytes, pericytes, perivascular nerves) and their known and potential roles in the secondary injury cascade. © 2016 Wiley Periodicals, Inc.     

Dysfunction of brain pericytes in chronic neuroinflammation

Brain pericytes are uniquely positioned within the neurovascular unit to provide support to blood brain barrier (BBB) maintenance. Neurologic conditions, such as HIV-1-associated neurocognitive disorder, are associated with BBB compromise due to chronic inflammation. Little is known about pericyte dysfunction during HIV-1 infection. We found decreased expression of pericyte markers in human brains from HIV-1-infected patients (even those on antiretroviral therapy). Using primary human brain pericytes, we assessed expression of pericyte markers (α1-integrin, α-smooth muscle actin, platelet-derived growth factor-B receptor β, CX-43) and found their downregulation after treatment with tumor necrosis factor-α (TNFα) or interleukin-1 β (IL-1β). Pericyte exposure to virus or cytokines resulted in decreased secretion of factors promoting BBB formation (angiopoietin-1, transforming growth factor-β1) and mRNA for basement membrane components. TNFα and IL-1β enhanced expression of adhesion molecules in pericytes paralleling increased monocyte adhesion to pericytes. Monocyte migration across BBB models composed of human brain endothelial cells and pericytes demonstrated a diminished rate in baseline migration compared to constructs composed only of brain endothelial cells. However, exposure to the relevant chemokine, CCL2, enhanced the magnitude of monocyte migration when compared to BBB models composed of brain endothelial cells only. These data suggest an important role of pericytes in BBB regulation in neuroinflammation.     

Developmental and post‐injury cortical gliogenesis: A Genetic fate‐mapping study with Nestin‐CreER mice

The primary sources of cortical gliogenesis, either during development or after adult brain injury, remain uncertain. We previously generated Nestin‐CreER mice to fate‐map the progeny of radial glial cells (RG), a source of astrocytes and oligodendrocytes in the nervous system. Here, we show that Nestin‐CreER mice label another population of glial progenitors, namely the perinatal subventricular zone (SVZ) glioblasts, if they are crossed with stop‐floxed EGFP mice and receive tamoxifen in late embryogenesis (E16–E18). Quantification showed E18 tamoxifen‐induction labeled more perinatal SVZ glioblasts than RG and transitional RG combined in the newborn brain (54% vs. 22%). Time‐lapse microscopy showed SVZ‐glioblasts underwent complex metamorphosis and often‐reciprocal transformation into transitional RG. Surprisingly, the E10‐dosed RG progenitors produced astrocytes, but no oligodendrocytes, whereas E18‐induction fate‐mapped both astrocytes and NG2+ oligodendrocyte precursors in the postnatal brain. These results suggest that cortical oligodendrocytes mostly derive from perinatal SVZ glioblast progenitors. Further, by combining genetic fate‐mapping and BrdU‐labeling, we showed that cortical astrocytes cease proliferation soon after birth (<P10) and only undergo nonproliferative gliosis (i.e., increased GFAP expression without cell‐division) after stab‐wound injury in adult brains. By contrast, 9.7% of cortical NG2+ progenitors remained mitotic at P29, and the ratio rose to 13.8% after stab‐wound injury. Together, these results suggest NG2+ progenitors, rather than GFAP+ astrocytes, are the primary source of proliferative gliosis after adult brain injury. © 2008 Wiley‐Liss, Inc.     

Brain pericytes contribute to the induction and up-regulation of blood-brain barrier functions through transforming growth factor-beta production

The blood-brain barrier (BBB) is a highly organized multicellular complex consisting of an endothelium, brain pericytes and astrocytes. The present study was aimed at evaluating the role of brain pericytes in the induction and maintenance of BBB functions and involvement of transforming growth factor-beta (TGF-beta) in the functional properties of pericytes. We used an in vitro BBB model established by coculturing immortalized mouse brain capillary endothelial (MBEC4) cells with a primary culture of rat brain pericytes. The coculture with rat pericytes significantly decreased the permeability to sodium fluorescein and the accumulation of rhodamine 123 in MBEC4 cells, suggesting that brain pericytes induce and up-regulate the BBB functions. Rat brain pericytes expressed TGF-beta1 mRNA. The pericyte-induced enhancement of BBB functions was significantly inhibited when cells were treated with anti-TGF-beta1 antibody (10 microg/ml) or a TGF-beta type I receptor antagonist (SB431542) (10 microM) for 12 h. In MBEC4 monolayers, a 12 h exposure to TGF-beta1 (1 ng/ml) significantly facilitated the BBB functions, this facilitation being blocked by SB431542. These findings suggest that brain pericytes contribute to the up-regulation of BBB functions through continuous TGF-beta production.     

The critical component to establish in vitro BBB model: Pericyte

The blood–brain barrier (BBB), a highly regulated membranous barrier of brain capillaries, consists of an intricate network of tight junctions (TJs) that segregate the central nervous system (CNS) from systemic blood circulation and maintain a delicate homeostasis of the CNS environment. While endothelial cells (ECs) of brain capillaries are clearly the principal cellular element of BBB, the formation and regulation of intact BBB structure appear to require the interactions of endothelial cells with other cellular components. Astrocytes, one of the major non-neural cells in the brain, associate closely and interact with capillary endothelial cells during the angiogenesis and BBB development. Current in vitro cellular models for the study of BBB functions often incorporate astrocytes with endothelial cells. However, another foremost cell type, CNS pericyte, which intimately embraces brain capillary endothelium, attracts relatively little attention for its role in developing the in vitro BBB system. This review will analyze the critical functions of pericytes in angiogenesis in various systems and discuss the relevance of these functions in mediating the development, maintenance, and regulation of BBB. The author will also discuss the functional role of actin in both ECs and pericytes, and further elaborate the molecular mechanisms of BBB permeability regulation that involves the transduction pathway-mediated actin remodeling process. Finally, the rationale of incorporating pericytes for establishing a better in vitro BBB model will be emphasized.     

Cilostazol attenuates ischemia–reperfusion-induced blood–brain barrier dysfunction enhanced by advanced glycation endproducts via transforming growth factor-β1 signaling

We investigated the effects of cilostazol, a selective inhibitor of phosphodiesterase 3, on blood–brain barrier (BBB) integrity against ischemia–reperfusion injury enhanced by advanced glycation endproducts (AGEs). We used in vitro BBB models with primarily cultured BBB-related cells from rats (brain capillary endothelial cells, astrocytes and pericytes), and subjected cells to either normoxia or 3-h oxygen glucose deprivation (OGD)/24-h reoxygenation with or without AGEs. Treatment of AGEs did not affect the transendothelial electrical resistance (TEER) in the BBB model under normoxia, but there was a significant decrease in TEER under 3-h OGD/24-h reoxygenation conditions with AGEs. Cilostazol inhibited decreases in TEER induced by 3-h OGD/24-h reoxygenation with AGEs. Immunocytochemical and Western blot analyses showed that AGEs reduced the expression of claudin-5, the main functional protein of tight junctions (TJs). In contrast, cilostazol increased the expression of claudin-5 under 3-h OGD/24-h reoxygenation with AGEs. Furthermore, while AGEs increased the production of extracellular transforming growth factor (TGF)-β1, cilostazol inhibited the production of extracellular TGF-β1 and restored the integrity of TJs. Thus, we found that AGEs enhanced ischemia–reperfusion injury, which mainly included decreases in the expression of proteins comprising TJs through the production of TGF-β1. Cilostazol appeared to limit ischemia–reperfusion injury with AGEs by improving the TJ proteins and inhibiting TGF-β1 signaling.