Brain Vascular Pericytes Research Articles

Myt1l induced direct reprogramming of pericytes into cholinergic neurons.

The cholinergic deficit is thought to underlie progressed cognitive decline in Alzheimer Disease. The lineage reprogramming of somatic cells into cholinergic neurons may provide strategies toward cell-based therapy of neurodegenerative diseases. Here, we found that a combination of neuronal transcription factors, including Ascl1, Myt1l, Brn2, Tlx3, and miR124 (5Fs) were capable of directly converting human brain vascular pericytes (HBVPs) into cholinergic neuronal cells. Intriguingly, the inducible effect screening of reprogramming factors showed that a single reprogramming factor, Myt1l, induced cells to exhibit similarly positive staining for Tuj1, MAP2, ChAT, and VAChT upon lentivirus infection with the 5Fs after 30days. HBVP-converted neurons were rarely labeled even after long-term incubation with BrdU staining, suggesting that induced neurons were directly converted from HBVPs rather than passing through a proliferative state. In addition, the overexpression of Myt1l induced the elevation of Ascl1, Brn2, and Ngn2 levels that contributed to reprogramming. Our findings provided proof of the principle that cholinergic neurons could be produced from HBVPs by reprogramming factor-mediated fate instruction. Myt1l was a critical mediator of induced neuron cell reprogramming. HBVPs represent another excellent alternative cell resource for cell-based therapy to treat neurodegenerative disease.

Endothelial Activation and Blood-Brain Barrier Disruption in Neurotoxicity after CD19 CAR-T Cell Immunotherapy

CD19 chimeric antigen receptor (CAR)-modified T cell therapy has produced impressive results in patients (pts) with CD19+B cell malignancies; however, treatment can be complicated by neurologic adverse events (AEs). The presentation and pathogenesis of neurotoxicity (NT) are incompletely understood.We report a clinical and pathologic study evaluating NT in 133 B-ALL, NHL and CLL pts treated with lymphodepletion chemotherapy and CD19 CAR-T cells (NCT01865617). Neurologic AEs were observed in 53 of 133 pts (40%; 19% grade [gr] 1-2; 16% gr 3; 2% gr 4; 3% gr 5). Most neurologic AEs were reversible. Delirium, headache, and language disturbances were the most frequently observed neurologic AEs, presenting in 66%, 55%, and 34% of pts with NT, respectively. Multivariable analysis showed that higher burden of malignant B cells in marrow, lymphodepletion with cyclophosphamide and fludarabine, and higher infused CAR-T cell dose were associated with increased risk of NT and cytokine release syndrome (CRS).The severity of NT correlated with higher peak concentrations of cytokines that may activate endothelial cells (EC), such as IL-6, IFN-γ, and TNF-α. Pts who developed gr ≥3 NT had more severe CRS with higher CAR-T cell counts in blood and evidence of vascular dysfunction. In severe NT, hypofibrinogenemia with elevated PT, aPTT, and d-dimer were consistent with EC activation. We confirmed in vivo EC activation during NT by demonstrating high concentrations of serum angiopoietin-2 (Ang-2) and von Willebrand Factor (VWF), which are released from Weibel-Palade bodies on EC activation. In vitro, serum from pts with NT induced activation and VWF release from human umbilical vein ECs (HUVECs), which suggests that the finding of a reduced fraction of high molecular weight VWF multimers in vivo during gr ≥4 NT results from consumption on platelets and sequestration on activated EC. Consistent with this explanation, we found reduced activity of the VWF-cleaving protease ADAMTS13 relative to VWF levels and more severe thrombocytopenia in pts with gr ≥4 NT.We considered that cytokine-induced EC activation might alter the integrity of the blood-brain barrier (BBB) during NT. During severe NT, CSF analyses demonstrated increased permeability of the BBB to protein and leukocytes, including CAR-T cells. The gradient between serum and CSF cytokines observed before lymphodepletion was lost during acute NT, consistent with inability of the BBB to shield the CNS from high concentrations of plasma cytokines. Brain vascular pericytes (BVP) play a critical role in vascular and BBB support, and when exposed to high TNF-α or IFN-γ concentrations BVP exhibited stress (increased cleaved caspase-3 and reduced PDGFRβ) and secreted IL-6 and VEGF, further promoting EC activation and BBB permeability.Neuropathologic examination of the brain of a patient with fatal NT demonstrated disrupted endothelium by CD31 immunohistochemistry and EC activation was confirmed by intravascular VWF binding and CD61+ platelet microthrombi. Further evidence of breach of the BBB included red blood cell extravasation from multiple vessels, vascular lesions with karyorrhexis, perivascular CD8+ T cell infiltration, and fibrinoid vessel wall necrosis. CAR-T cells were detected in the CNS.We investigated strategies to reduce the risks of severe NT. Logistic probability curves demonstrated that reduction in CAR-T cell dose to reduce the peak in vivo CAR-T cell blood count was associated with reduced risk of NT, but that the narrow therapeutic index of this approach would lead to loss of anti-tumor efficacy. To maintain CAR-T cell peak counts in blood, we developed a strategy to identify pts early after CAR-T cell infusion who might be at risk of subsequent severe NT and could be candidates for early intervention. Classification tree modeling demonstrated that in the first 36 hours after CAR-T cell infusion pts with fever ≥38.9°C, serum IL-6 ≥16 pg/mL, and MCP-1 ≥1343.5 pg/mL were at high risk of subsequent gr ≥4 NT (sensitivity 100%; specificity 94%). We also investigated whether pts with pre-existing endothelial activation were at higher risk of NT. Before lymphodepletion, pts who developed gr ≥4 NT had higher Ang-2:Ang-1 ratios than those with gr ≤3 NT, indicating that endothelial activation before lymphodepletion or CAR-T cell infusion may be a risk factor for NT that identifies pts who would benefit from a modified treatment regimen. DisclosuresTurtle:Adaptive Biotechnologies: Other: Advisory board; Bluebird Bio: Other: Advisory board; Gilead Sciences: Other: Advisory board; Precision Biosciences: Other: Advisory board; Celgene: Other: Advisory board; Juno Therapeutics: Other: Advisory board, Patents & Royalties, Research Funding. Liles:Juno Therapeutics: Consultancy, Other: Advisory board. Li:Juno Therapeutics: Employment, Equity Ownership. Yeung:Gilead: Research Funding. Riddell:Juno Therapeutics: Equity Ownership, Patents & Royalties, Research Funding. Maloney:Celgene: Other: Advisory board; Kite Pharmaceuticals: Other: Advisory board; Juno Theraapeutics: Other: Advisory board, Patents & Royalties, Research Funding; Roche/Genetech: Other: Advisory board.

Abstract 5808: The role of breast cancer-derived exosomes in brain metastasis

Abstract The development of brain metastasis is associated with a significant reduction in the survival rate of breast cancer patients. Improving the prognosis of women with brain metastasis from breast cancer relies on the elucidation of the mechanisms underlying this process. Our group and others have shown that the formation of brain metastases occurs along the abluminal side of brain vessels, a process called “vessel co-option”. These observations prompted our hypothesis that the cellular and extracellular matrix (ECM) components of the blood-brain-barrier (BBB) can serve as a pre-metastatic niche for breast cancer brain metastases. The role of tumor-derived exosomes (TEx) in the preparation of a pre-metastatic niche in distant organs has been shown in a number of cancers. To investigate the mechanisms driving the vessel co-option of breast cancer brain metastases, we studied the role of breast cancer-derived exosomes in preparation of the BBB for metastasis formation. Exosomes were isolated from the MDA-MB-231 breast cancer cell line, a brain-seeking variant of these cells, and a bone-seeking variant as a non-brain metastatic control. Brain endothelial cells, astrocytes, and brain vascular pericytes, the three components of the BBB, were treated with exosomes for 3 days to recapitulate the continuous exposure of cells to the circulating TEx in vivo. The treated cells were analyzed for cellular activities relevant to pre-metastatic niche preparation in the brain such as the integrity of the BBB, expression of cytokines, and modulation of the ECM. An initial screen was performed using cytokine antibody arrays and PCR arrays for ECM and adhesion molecules and results were validated in three separate experiments using western blots, ELISA, and Multiplex assays. Exosomes derived from brain-seeking cells significantly increased astrocyte migration and decreased the expression of Integrin β1 in brain endothelial cells, both of which can lead to the disruption of the BBB integrity. Moreover, the expression of Interleukin 8 was increased by TEx in astrocytes. TEx also increased the expression of MMP-3 and -9 (Matrix Metalloproteinases) from an undetectable baseline level and decreased the expression of TIMP-1 and -2 (Tissue Inhibitors of MMPs) in astrocytes. While the importance of these two MMPs for brain metastasis from breast cancer has been previously reported, our study is the first to demonstrate that secretion of these MMPs is triggered by TEx. Surprisingly, we did not observe any significant TEx-derived modulations in the expression of cytokines, ECM, and adhesion molecules in brain pericytes. These findings indicate that within the BBB, astrocytes and endothelial cells, but not pericytes, can be affected by breast cancer-derived exosomes in such a way that can potentially lead to preparation of a suitable niche for future metastasis formation along the brain vasculature. (This work is supported by the Breast Cancer Research Foundation.) Citation Format: Golnaz Morad, Jiang Yang, Marsha A. Moses. The role of breast cancer-derived exosomes in brain metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5808. doi:10.1158/1538-7445.AM2017-5808

Luteolin inhibits angiogenesis by blocking Gas6/Axl signaling pathway.

Growth arrest-specific protein6 (Gas6) induces the activation of Axl receptor tyrosine kinase (Axl), which plays an important role in angiogenic processes, including proliferation, migration, invasion, tube formation and pericyte recruitment of endothelial cells. The inhibition of Gas6/Axl pathway has been demonstrated to be an effective anti-angiogenic therapy. Luteolin, which is a natural active flavonoid, has been reported to possess anti-angiogenic effects. However, the underlying mechanism of luteolin in anti-angiogenesis is not fully understood. Herein, we report that luteolin significantly inhibited the Gas6-induced proliferation, migration, invasion and tube formation of human microvascular endothelial cells (HMEC‑1s) invitro, and suppressed the Gas6-induced recruitment of human brain vascular pericytes (HBVPs) to the endothelial tubes. Luteolin also suppressed Gas6-induced microvessel sprouting in aortic ring assay and neovascularization in chick chorioallantoic membrane assay. The anti-angiogenic effect of luteolin may be associated with the inhibition of the Gas6/Axl pathway and its downstream phosphatidylinositol 3-kinase (PI3K)/protein kinaseB (Akt)/mammalian target of rapamycin (mTOR) signaling pathways. Taken together, the present study provides new evidence regarding an anti-angiogenic mechanism of luteolin, and supports the notion that the dietary intake of luteolin contributes to the treatment of pathological angiogenesis.

Expression of pattern recognition receptors and activation of the non-canonical inflammasome pathway in brain pericytes

Cerebral pericytes are mural cells embedded in the basement membrane of capillaries. Increasing evidence suggests that they play important role in controlling neurovascular functions, i.e. cerebral blood flow, angiogenesis and permeability of the blood–brain barrier. These cells can also influence neuroinflammation which is highly regulated by the innate immune system. Therefore, we systematically tested the pattern recognition receptor expression of brain pericytes. We detected expression of NOD1, NOD2, NLRC5, NLRP1-3, NLRP5, NLRP9, NLRP10 and NLRX mRNA in non-treated cells. Among the ten known human TLRs, TLR2, TLR4, TLR5, TLR6 and TLR10 were found to be expressed. Inflammatory mediators induced the expression of NLRA, NLRC4 and TLR9 and increased the levels of NOD2, TLR2, inflammasome-forming caspases and inflammasome-cleaved interleukins. Oxidative stress, on the other hand, upregulated expression of TLR10 and NLRP9. Activation of selected pattern recognition receptors can lead to inflammasome assembly and caspase-dependent secretion of IL-1β. TNF-α and IFN-γ increased the levels of pro-IL-1β and pro-caspase-1 proteins; however, no canonical activation of NLRP1, NLRP2, NLRP3 or NLRC4 inflammasomes could be observed in human brain vascular pericytes. On the other hand, we could demonstrate secretion of active IL-1β in response to non-canonical inflammasome activation, i.e. intracellular LPS or infection with E. coli bacteria. Our in vitro results indicate that pericytes might have an important regulatory role in neuroinflammation.

Nitration of TRPM2 as a Molecular Switch Induces Autophagy During Brain Pericyte Injury.

Dysfunction of neurovascular pericytes underlies breakdown of the blood-brain barrier, but the molecular mechanisms are largely unknown. In this study, we evaluated the role of the transient receptor potential melastatin-related 2 (TRPM2) channel and autophagy during brain pericyte injury both in vitro and in vivo. A rapid induction in autophagy in human brain vascular pericytes, in the zinc oxide nanoparticles (ZnO-NP)-induced cell stress model, was paralleled with an increase in the expression of the TRPM2-S truncated isoform, which was abolished by treatment with a nitric oxide synthase inhibitor and a peroxynitrite scavenger. Furthermore, Y1485 in the C-terminus of the TRPM2 protein was identified as the tyrosine nitration substrate by mass spectrometry. Overexpression of the Y1485S TRPM2 mutant reduced LC3-II accumulation and pericyte injury induced by ZnO-NP. Consistently, LC3-II accumulation was reduced and pericytes were better preserved in intact brain microvessels of the TRPM2 knockout mice after ZnO-NP-induced vascular injury. Innovation and Conclusions: Our present study has revealed a novel mechanism of autophagy disturbance secondary to nitrosative stress-induced tyrosine nitration of TRPM2 during pericyte injury. Antioxid. Redox Signal. 27, 1297-1316.

Mechanism underlying docosahexaenoic acid-induced regulation of angiopoietin expression in human brain vascular pericytes subjected to oxygen-glucose deprivation and restoration: the relationship with SSeCKS

Objective To evaluate the relationship between the mechanism underlying docosahexaenoic acid (DHA)-induced regulation of angiopoietin expression and Src-suppressed C kinase substrate (SSeCKS) in human brain vascular pericytes (HBVPs) subjected to oxygen-glucose deprivation and restoration (OGD/R). Methods HBVPs were seeded in 96-well or in 6-well plates at a density of 2×105 cells/ml and divided into 5 groups (n=18 each) using a random number table: control group (group C), OGD/R group, DHA group (group D), SSeCKS gene silencing group (group S) and SSeCKS gene silencing plus DHA group (group SD). The model of OGD/R injury was established as follows: the cells were subjected to O2-glucose deprivation for 24 h in glucose- and serum-free culture medium aerated with 94%N2-5%CO2-1%O2 followed by restoration of O2-glucose supply for 6 h in high-glucose DMEM culture medium in normal atmosphere.DHA was added at 1 h before hypoxia with the final concentration of 40 μmol/L in group D. Small interfering RNA induced SSeCKS gene silencing in S and SD groups.Subsequently, DHA with the final concentration of 40 μmol/L was added at 1 h before hypoxia in group SD.At 6 h of reoxygenation, the cell survival rate was determined by CCK-8 assay, the amount of LDH released was detected using ELISA, and the expression of SSeCKS, angiopoietin-1 (Ang-1) and Ang-2 was detected by Western blot. Results Compared with group C, the cell survival rate was significantly decreased, the amount of LDH released was increased, the expression of SSeCKS and Ang-1 was down-regulated, the expression of Ang-2 was up-regulated, and Ang-1/Ang-2 ratio was decreased in group OGD/R, and the expression of SSeCKS was down-regulated in group S (P 0.05). Compared with group D, the cell survival rate was significantly decreased, the amount of LDH released was increased, the expression of SSeCKS and Ang-1 was down-regulated, the expression of Ang-2 was up-regulated, and Ang-1/Ang-2 ratio was decreased in group SD (P<0.05). Conclusion The mechanism by which DHA increases the ratio of Ang-1/Ang-2 may be totally related to up-regulation of SSeCKS expression in HBVPs subjected to OGD/R. Key words: Docosahexaenoic acids; A kinase anchor proteins; Angiopoietins; Pericytes; Brain; Reperfusion injury

Cells responding to surface structure of calcium phosphate ceramics for bone regeneration.

Surface structure largely affects the inductive bone-forming potential of calcium phosphate (CaP) ceramics in ectopic sites and bone regeneration in critical-sized bone defects. Surface-dependent osteogenic differentiation of bone marrow stromal cells (BMSCs) partially explained the improved bone-forming ability of submicron surface structured CaP ceramics. In this study, we investigated the possible influence of surface structure on different bone-related cells, which may potentially participate in the process of improved bone formation in CaP ceramics. Besides BMSCs, the response of human brain vascular pericytes (HBVP), C2C12 (osteogenic inducible cells), MC3T3-E1 (osteogenic precursors), SV-HFO (pre-osteoblasts), MG63 (osteoblasts) and SAOS-2 (mature osteoblasts) to the surface structure was evaluated in terms of cell proliferation, osteogenic differentiation and gene expression. The cells were cultured on tricalcium phosphate (TCP) ceramics with either micron-scaled surface structure (TCP-B) or submicron-scaled surface structure (TCP-S) for up to 14 days, followed by DNA, alkaline phosphatase (ALP) and quantitative polymerase chain reaction gene assays. HBVP were not sensitive to surface structure with respect to cell proliferation and osteogenic differentiation, but had downregulated angiogenesis-related gene expression (i.e. vascular endothelial growth factor) on TCP-S. Without additional osteogenic inducing factors, submicron-scaled surface structure enhanced ALP activity and osteocalcin gene expression of human (h)BMSCs and C2C12 cells, favoured the proliferation of MC3T3-E1, MG63 and SAOS-2, and increased ALP activity of MC3T3-E1 and SV-HFO. The results herein indicate that cells with osteogenic potency (either osteogenic inducible cells or osteogenic cells) could be sensitive to surface structure and responded to osteoinductive submicron-structured CaP ceramics in cell proliferation, ALP production or osteogenic gene expression, which favour bone regeneration. Copyright © 2017 John Wiley & Sons, Ltd.

Quantitative assessment of angiogenesis and pericyte coverage in human cell-derived vascular sprouts

BackgroundPericytes, surrounding the endothelium, fulfill diverse functions that are crucial for vascular homeostasis. The loss of pericytes is associated with pathologies, such as diabetic retinopathy and Alzheimer’s disease. Thus, there exists a need for an experimental system that combines pharmacologic manipulation and quantification of pericyte coverage during sprouting angiogenesis. Here, we describe an in vitro angiogenesis assay that develops lumenized vascular sprouts composed of endothelial cells enveloped by pericytes, with the additional ability to comparatively screen the effect of multiple small molecules simultaneously. For automated analysis, we also present an ImageJ plugin tool we developed to quantify sprout morphology and pericyte coverage.MethodsHuman umbilical vein endothelial cells and human brain vascular pericytes were coated on microcarrier beads and embedded in fibrin gels in a 96-well plate to form lumenized vascular sprouts. After treatment with pharmacologic compounds, sprouts were fixed, stained, and imaged via optical z-sections over the area of each well. The maximum intensity projections of these images were stitched together to form montages of the wells, and those montages were processed and analyzed.ResultsVascular sprouts formed within 4–12 days and contained a patent lumen surrounded by a layer of human endothelial cells and pericytes. Using our workflow and image analysis, pericyte coverage after treatment with various compounds was successfully quantified.ConclusionsHere we present a robust in vitro assay using primary human vascular cells that allows researchers to analyze the effects of multiple compounds on sprouting angiogenesis and pericyte coverage. Our ImageJ plugin offers automated evaluation across multiple different vascular parameters, such as sprout length, cell density, branch points, and pericyte coverage.

Novel method to study pericyte contractility and responses to ischaemia in vitro using electrical impedance

Pericytes are contractile vascular mural cells overlying capillary endothelium, and they have been implicated in a variety of functions including regulation of cerebral blood flow. Recent work has suggested that both in vivo and ex vivo, ischaemia causes pericytes to constrict and die, which has implications for microvascular reperfusion. Assessing pericyte contractility in tissue slices and in vivo is technically challenging, while in vitro techniques remain unreliable. Here, we used isolated cultures of human brain vascular pericytes to examine their contractile potential in vitro using the iCelligence electrical impedance system. Contraction was induced using the vasoactive peptide endothelin-1, and relaxation was demonstrated using adenosine and sodium nitroprusside. Endothelin-1 treatment also resulted in increased proliferation, which we were able to monitor in the same cell population from which we recorded contractile responses. Finally, the observation of pericyte contraction in stroke was reproduced using chemical ischaemia, which caused a profound and irreversible contraction clearly preceding cell death. These data demonstrate that isolated pericytes retain a contractile phenotype in vitro, and that it is possible to quantify this contraction using real-time electrical impedance recordings, providing a significant new platform for assessing the effects of vasoactive and vasculoprotective compounds on pericyte contractility.

Axitinib inhibits retinal and choroidal neovascularization in in vitro and in vivo models

Age-related Macular Degeneration (AMD) is the leading cause of visual impairment and blindness in the elderly in developed countries. Neovascular/exudative (wet) AMD is the aggressive form of AMD and can involve choroidal neovascularization and vascular leakage. Anti-vascular endothelial growth factor (anti-VEGF) medications have significantly improved treatment of wet-AMD. However, only approximately 40% of patients obtain full benefit from anti-VEGF therapy and the medications are given by intravitreal injection. Axitinib, a small molecule multi-receptor tyrosine kinase inhibitor used for the treatment of advanced renal cell carcinoma, is taken orally and inhibits VEGF activity by blocking VEGF receptors. Axitinib also has the advantage of blocking platelet derived growth factor (PDGF) receptors which play a role in neovascularization. Using in vitro human retinal microvascular endothelial cells (HRMVECs), human brain vascular pericytes (HBVRs), 3D co-culture vessel sprout assay, and in vivo laser induced rat choroidal neovascularization (CNV) models, the effect of axitinib on neovascularization was evaluated. Axitinib inhibited neovascularization better than anti-VEGF and/or anti-hPDGF-B mAb in the in vitro models demonstrating that combined inhibition of both VEGF and PDGF pathways may be synergistic in treating wet-AMD. Additionally, axitinib showed good efficacy at a low dose (0.875 mg/day) in laser-induced CNV model in rats. In conclusion our data shows that axitinib, an inhibitor of VEGF and PDGF-B pathways may be useful in ameliorating wet-AMD therapy.

CBIO-21INFLUENCES OF BLOOD BRAIN BARRIER (BBB) BASEMENT MEMBRANE ECM MOLECULES ON HUMAN BRAIN VASCULAR PERICYTES

BACKGROUND: Pericytes are specialised cells found within the blood brain barrier (BBB) ensheathed by a basement membrane (BM) containing numerous extracellular matrix (ECM) proteins which regulate vessel stabilisation and barrier formation. Knowledge on ECM influence on pericyte behaviour is limited. This study aimed to determine the effects of BM-ECM components on pericyte proliferation, adhesion, motility and cell-to-cell contact. METHODS: Human brain vascular pericytes (HBVP) were used. Cell seeding number was optimised and cells were plated on different human-derived, previously optimised, ECM molecules including Fibronectin (5 µg/ml), Laminin (75 µg/ml), Collagen IV (10 µg/ml), Agrin (1 µg/ml) and Perlecan (10 µg/ml) and assessed for proliferation and both quantitative and qualitative adhesion. Motility assays were performed using live cell (time-lapse) imaging. Resistance values between pericytes were collated using an Electric Cell-Substrate Impedance Sensing (ECIS) system. Trans-Endothelial Electrical Resistance (TEER) changes when pericytes were seeded onto human endothelial cells under the different ECM culture conditions were also analysed by voltometer and ECIS. RESULTS: Cells adhered best when seeded on Perlecan and least well on Agrin (p < 0.05). Motility assays showed that pericytes travelled the longest distance and fastest on Agrin and least on Perlecan (p < 0.05). Higher resistance values were reached when cells were plated on Perlecan (∼400 Ω/cm2) and least on Agrin (∼300 Ω/cm2). A tighter barrier was noted when pericytes were co-cultured with endothelial cells (1200 Ω/cm2) compared to when pericytes were added to endothelial cell monolayers (1000 Ω/cm2). CONCLUSIONS: Results demonstrate that different BBB basement molecules have varied effects on TEER values, one indicator of BBB function. Findings also suggest that Agrin may function to recruit pericytes by migration while Perlecan may function to serve as an anchor. This study highlights the importance of BM-ECM molecules and suggests a concentration dependant role of the perlecan/agrin axis in BBB establishment and function.

Pyruvate and cilostazol protect cultured rat cortical pericytes against tissue plasminogen activator (tPA)-induced cell death

Since even a brief ischemia can cause permanent brain damage, rapid restoration of blood flow is critical to limiting damage. Although intravenous tPA during the acute stage is the treatment of choice for achieving reperfusion, this treatment is sometimes associated with brain hemorrhage. Agents that reduce tPA-related bleeding risk may help expand its therapeutic window. This study assessed whether zinc dyshomeostasis underlies the toxic effect of tPA on brain vascular pericytes; whether pyruvate, an inhibitor of zinc toxicity, protects pericytes against tPA-induced cell death; and whether cilostazol, which protects pericytes against tPA-induced cell death, affects zinc dyshomeostasis associated with tPA toxicity. Cultured pericytes from newborn rat brains were treated with 10–200μg/ml tPA for 24h, inducing cell death in a concentration-dependent manner. tPA-induced cell death was preceded by increases in intracellular free zinc levels, and was substantially attenuated by plasminogen activator inhibitor-1 (PAI-1) or TPEN. Pyruvate completely blocked direct zinc toxicity and tPA-induced pericyte cell death. Both cAMP and cilostazol, a PDE3 inhibitor that attenuates tPA-induced pericyte cell death in vitro and tPA-induced brain hemorrhage in vivo, reduced zinc- and tPA-induced pericyte cell death, suggesting that zinc dyshomeostasis may be targeted by cilostazol in tPA toxicity. These findings show that tPA-induced pericyte cell death may involve zinc dyshomeostasis, and that pyruvate and cilostazol attenuate tPA-induced cell death by reducing the toxic cascade triggered by zinc dyshomeostasis. Since pyruvate is an endogenous metabolite and cilostazol is an FDA-approved drug, in vivo testing of both as protectors against tPA-induced brain hemorrhage may be warranted.This article is part of a Special Issue entitled SI: Neuroprotection.

Brain vascular pericytes following ischemia have multipotential stem cell activity to differentiate into neural and vascular lineage cells.

Brain vascular pericytes (PCs) are a key component of the blood-brain barrier (BBB)/neurovascular unit, along with neural and endothelial cells. Besides their crucial role in maintaining the BBB, increasing evidence shows that PCs have multipotential stem cell activity. However, their multipotency has not been considered in the pathological brain, such as after an ischemic stroke. Here, we examined whether brain vascular PCs following ischemia (iPCs) have multipotential stem cell activity and differentiate into neural and vascular lineage cells to reconstruct the BBB/neurovascular unit. Using PCs extracted from ischemic regions (iPCs) from mouse brains and human brain PCs cultured under oxygen/glucose deprivation, we show that PCs developed stemness presumably through reprogramming. The iPCs revealed a complex phenotype of angioblasts, in addition to their original mesenchymal properties, and multidifferentiated into cells from both a neural and vascular lineage. These data indicate that under ischemic/hypoxic conditions, PCs can acquire multipotential stem cell activity and can differentiate into major components of the BBB/neurovascular unit. Thus, these findings support the novel concept that iPCs can contribute to both neurogenesis and vasculogenesis at the site of brain injuries.

Erratum：脳ペリサイトをめぐる脳保護と再生

Erratum：脳ペリサイトをめぐる脳保護と再生

脳ペリサイトをめぐる脳保護と再生

脳ペリサイトをめぐる脳保護と再生

P38 MAP kinase mediates transforming-growth factor-β1-induced upregulation of matrix metalloproteinase-9 but not -2 in human brain pericytes

Pericytes are vascular mural cells embedded within the basal lamina of blood micro-vessels. Within the neurovascular unit, pericytes play important roles in regulating neurovascular homeostasis by secreting soluble factors, such as matrix metalloproteinases (MMPs). However, little is known about the regulatory signaling pathways in brain pericytes. Here we show that transforming growth factor-β1 (TGF-β1) induces MMP-9 upregulation in pericytes via p38 mitogen-activated protein (MAP) kinase signaling. Cultured human brain vascular pericytes were used in this study. When the brain pericytes were treated with purified human TGF-β1 (0.1–10ng/mL for 24h), the levels of MMP-2 and MMP-9 in culture media were significantly increased in a concentration dependent manner as measured by gelatin zymography. WST assay confirmed that TGF-β1 did not affect cell survival of the brain pericytes. A TGF-β-receptor inhibitor SB431542 (0.5–5μM) decreased the TGF-β1-induced upregulation of MMP-2 and MMP-9. To assess the underlying intracellular mechanisms, we focused on p38 MAP kinase signaling, which is one of the major downstream kinases for TGF-β1. A well-validated p38 MAP kinase inhibitor SB203580 (0.5–5μM) cancelled the effect of TGF-β1 in upregulation of MMP-9 but not MMP-2. Western blotting confirmed that TGF-β1 treatment increased the level of p38 MAP kinase phosphorylation in pericytes, and again, the TGF-β-receptor inhibitor SB431542 (0.5–5μM) blocked the TGF-β1-induced phosphorylation of p38 MAP kinase. Both TGF-β1 and MMP-9 are major neurovascular mediators, and therefore, our current finding may suggest a novel mechanism for how pericytes regulate neurovascular homeostasis.

P50 * DEVELOPING A PHYSIOLOGICALLY RELEVANT BLOOD BRAIN BARRIER MODEL FOR THE STUDY OF DRUG DISPOSITION IN GLIOMA

INTRODUCTION: Many in vitro blood brain barrier (BBB) models have been designed to study the underlying mechanism involved in drug transport across the BBB, but most have not included glioma or cells of human origin. Trans-endothelial electrical resistance (TEER) is a key measure of membrane potential and resistance and can be used to quantify the tightness of the BBB. The aim of this study was to evaluate the integrity of various models of the in vitro BBB as determined by TEER measurements. METHOD: Varying combinations of co-culture models were set up using primary cells of human origin which included human macrovascular endothelial cells (HBMEC), human brain vascular pericytes (HBVP), human astrocytes (HA) and human glioma. All cells were grown as monolayers to 80 % confluency in respective media supplemented with 5 % foetal bovine serum and incubated at 37°C, 5 % CO2 prior to establishing the co-culture. Culture dishes and inserts were coated with extracellular matrix (poly-L-lysine or fibronectin). The TEER was measured across the insert for 15 days. RESULTS: A steady increase in TEER was exhibited by all the models up to day 10. The highest TEER value of 250 Ω was recorded for the tri-culture comprising of HBMEC on the insert, HBVP cells in contact and HA cells out of contact of the well. The lowest TEER value of 100 Ω was recorded for the monolayer culture of HBMEC and the co-culture model (HBMEC and HBVP cells). CONCLUSION: The TEER values suggested that the tri culture model showed the highest membrane potential. This model would further be tested by various permeability assays to assess its functionality as an in vitro BBB model.

P01.15 * ASSESSMENT OF A NOVEL MODIFIED KYTOZYME NANOPARTICLE TO CROSS AN 'ALL HUMAN' BLOOD BRAIN BARRIER MODEL

The blood brain barrier (BBB) consists mainly of endothelial cells, astrocytes and pericytes embedded in a specialized basement membrane. To properly evaluate the ability of novel brain tumour therapeutics to cross the BBB, an ‘all human’ model that better reflects in vivo conditions, critical for the BBB function and integrity has been developed to assess the delivery of modified Kytozyme nanoparticles (NPs). Human brain endothelial cells (hCMEC/D3), human cortical astrocytes (SC-1800) and human brain vascular pericytes (HBVP) in mono-, co- and tri- cultures were plated and TEER values were measured using an Electric Cell-Substrate Impedance Sensing (ECIS) system. Tight junction protein levels (ZO-1, occludin and β-catenin) were examined by Western blotting and immunocytochemistry. Response of culture combinations post NPs addition was assessed using cytotoxicity assay. Barrier integrity post-injection of NPs loaded in Evans Blue was monitored using live cell imaging. Co-cultures and tri-cultures resulted in significantly higher TEER values compared to endothelial cells alone (p < 0.05). Maximal TEER values for each condition were observed at 120 hours post plating. TEER values for co-culture (E + A) were 1250 Ω/cm2, and for tri-culture (E + A + P), 1190 Ω/cm2 compared to monoculture (E) (950 Ω/cm2). These peak values lasted for an additional 125 hours and provided plenty of time to test NP delivery. Western blot analysis revealed that ZO-1, occludin and β-catenin protein expression was higher in co- and tri-cultures compared to mono culture. In addition, ICC studies revealed localised expression of these tight junction proteins at cell-cell contacts whereas in monoculture, diffuse staining was observed. Following addition of Kytozyme NPs to the co-culture (E + A), at 120 hours, there was a decrease in TEER values (1250 Ω/cm2 to 1120 Ω/cm2) with a full recovery to peak TEER values within 12 hours suggesting passage through this BBB model. The addition of Evans Blue had no effect on TEER values. This study not only confirms that astrocytes contribute to BBB formation in terms of higher TEER values and in higher expression of ZO-1, occludin, and β-catenin proteins which are involved in tight junction formation but also provides an effective model with which to study nanoparticle drug delivery. We are now loading NPs with drugs to provide meaningful data that should help bridge the gap between drug discovery and pre-clinical studies.

The effect of a rock inhibitor ama0428 in a model of wet age‐related macular degeneration

AbstractPurpose Rho kinase (ROCK) is associated with VEGF‐driven angiogenesis and is involved in inflammation and fibrosis. Therefore, the effect of a locally acting ROCK inhibitor, AMA0428, was studied in wet age‐related macular degeneration (AMD).Methods The effect of AMA0428 on human brain microvascular endothelial cells (HBMEC), human brain vascular pericytes (HBVP) and human tenon fibroblasts (HTF) was determined by measuring cell viability (WST‐1), apoptosis (caspase 3/7) and 2 migration assays (scratch and under‐agarose) The in vivo response was investigated using a laser‐induced choroidal neovascularization (CNV) mouse model. Intravitreal injections were given on day 0, 4, 10 and 20 with AMA0428, murine anti‐VEGFR Ab (DC101) or placebo. Outcome was assessed by analysis of inflammation (CD45), angiogenesis (FITC‐dextran), vessel leakage (Texas Red‐conjugated Dextran and FITC‐labeled lectin) and fibrosis (Collagen I).Results AMA0428 dose‐dependently reduced proliferation and VEGF‐induced migration of HBMEC and HTF. No significant effect was seen on HBVP proliferation; however, migration and pericyte recruitment were increased. There was no apoptosis induction. AMA0428 significantly reduced CNV and vessel leakage 2 weeks after lasering, comparable to DC101. In addition, AMA0428 inhibited inflammation on day 5 by 20% and collagen deposition on day 30 by 39% while DC101 had no effect on inflammation nor fibrosis.Conclusion Our data suggest that targeting ROCK with AMA0428 not only reduces neoangiogenesis, but also blocks inflammation and fibrosis (contrary to anti‐VEGF). These results point to a potential therapeutic benefit of ROCK inhibition in wet AMD.