内皮间质转化的信号机制

内皮间质转化是上皮间质转化的一种,是新近发现的一种细胞转化类型,和上皮间质转化一样也参与体内各种病理生理过程.已经证实,在转化过程中内皮细胞首先发生细胞与细胞脱联系过程,失去内皮细胞特异性标志物CD31和VE-钙粘蛋白,获得间质细胞特异性标志物α-SMA和FSP1,由原来的鹅卵石样结构转变为细长梭形结构,同时其迁移力和侵袭力明显增强.早期研究显示内皮间质转化在胚胎期心内膜的发育中发挥了十分关键的作用.近些年来,人们逐渐认识到内皮间质转化也参与各种疾病的发生发展,包括心、肺、肾等重要器官纤维化、特发性门脉高压、动脉粥样硬化、肺动脉高压以及肿瘤等.转化生长因子-β、Notch信号、Wnt信号以及MicroRNAs等参与对内皮间质转化的调节,然而,其具体机制尚未完全明确.通过探索内皮间质转化过程中重要信号通路及各因子的作用机制,可能为多种疾病的预防和治疗提供新的指导意见.     

Relocalization of Junctional Adhesion Molecule A during Inflammatory Stimulation of Brain Endothelial Cells

Junctional adhesion molecule A (JAM-A) is a unique tight junction (TJ) transmembrane protein that under basal conditions maintains endothelial cell-cell interactions but under inflammatory conditions acts as a leukocyte adhesion molecule. This study investigates the fate of JAM-A during inflammatory TJ complex remodeling and paracellular route formation in brain endothelial cells. The chemokine (C-C motif) ligand 2 (CCL2) induced JAM-A redistribution from the interendothelial cell area to the apical surface, where JAM-A played a role as a leukocyte adhesion molecule participating in transendothelial cell migration of neutrophils and monocytes. JAM-A redistribution was associated with internalization via macropinocytosis during paracellular route opening. A tracer study with dextran-Texas Red indicated that internalization occurred within a short time period (～10 min) by dextran-positive vesicles and then became sorted to dextran-positive/Rab34-positive/Rab5-positive vesicles and then Rab4-positive endosomes. By ～20 min, most internalized JAM-A moved to the brain endothelial cell apical membrane. Treatment with a macropinocytosis inhibitor, 5-(N-ethyl-N-isopropyl)amiloride, or Rab5/Rab4 depletion with small interfering RNA oligonucleotides prevented JAM-A relocalization, suggesting that macropinocytosis and recycling to the membrane surface occur during JAM-A redistribution. Analysis of the signaling pathways indicated involvement of RhoA and Rho kinase in JAM-A relocalization. These data provide new insights into the molecular and cellular mechanisms involved in blood-brain barrier remodeling during inflammation.     

Activated Brain Endothelial Cells Cross-Present Malaria Antigen

In the murine model of cerebral malaria caused by P. berghei ANKA (PbA), parasite-specific CD8⁺ T cells directly induce pathology and have long been hypothesized to kill brain endothelial cells that have internalized PbA antigen. We previously reported that brain microvessel fragments from infected mice cross-present PbA epitopes, using reporter cells transduced with epitope-specific T cell receptors. Here, we confirm that endothelial cells are the population responsible for cross-presentation in vivo, not pericytes or microglia. PbA antigen cross-presentation by primary brain endothelial cells in vitro confers susceptibility to killing by CD8⁺ T cells from infected mice. IFNγ stimulation is required for brain endothelial cross-presentation in vivo and in vitro, which occurs by a proteasome- and TAP-dependent mechanism. Parasite strains that do not induce cerebral malaria were phagocytosed and cross-presented less efficiently than PbA in vitro. The main source of antigen appears to be free merozoites, which were avidly phagocytosed. A human brain endothelial cell line also phagocytosed P. falciparum merozoites. Besides being the first demonstration of cross-presentation by brain endothelial cells, our results suggest that interfering with merozoite phagocytosis or antigen processing may be effective strategies for cerebral malaria intervention.     

大鼠脑微血管内皮细胞培养及其药物转运体Oatp2和P-gp的表达

贴块法培养脑微血管内皮细胞(BMECs),倒置显微镜动态观察细胞生长及形态,Ⅷ因子相关抗原、CD34免疫细胞化学联合鉴定细胞并确定纯度。免疫细胞化学和Western印迹法检测药物转运体有机阴离子转运多肽亚型2(Oatp2)及P-糖蛋白(P-gp)在培养内皮细胞上的表达。结果显示,获得的BMECs呈多角形或铺路石形,单层贴壁生长;培养细胞Ⅷ因子相关抗原免疫细胞化学、CD34免疫荧光染色均为阳性,细胞纯度90%;培养细胞有Oatp2及P-gp表达,且二者均主要表达于BMECs细胞膜。提示贴块法可获得原代培养BMECs,方法简便易行,细胞纯度较高。原代培养的BMECs上有药物转运体Oatp2及P-gp的表达,为血脑屏障上药物转运体的体外研究提供了可能途径。     

Reoxygenation Injury of Human Brain Capillary Endothelial Cells

1. Many studies have demonstrated that endothelial cells from several species can generate oxygen free radicals when subjected to anoxia and reoxygenation. However, due to the heterogeneity of the endothelium within different organs and species, the effects of superoxide dismutase (SOD), catalase, and allopurinol on reoxygenated cultured cells remain quite controversial.2. This review outlines the possible sources of oxygen free radicals within brain endothelial cells.3. We examine the aspects of the effects of SOD catalase and allopurinol on cultured human brain capillary endothelial cells upon reoxygenation.4. Also, we introduce briefly a method of culturing human brain capillary endothelial cells and present our experimental results on the effects of SOD, catalase, and allopurinol in these cultured cells following anoxia and reoxygenation.     

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.     

Involvement of Glial Cells in Cyclosporine-Increased Permeability of Brain Endothelial Cells

SUMMARY 1. To test whether astrocytes participate in cyclosporine-induced dysfunction of the blood-brain barrier, we examined the effects of cyclosporine on the permeability of the mouse brain endothelial (MBEC4) cells cocultured with C6 glioma cells, each cell layer placed on the top and bottom of the insert membrane, respectively.2. The presence of C6 cells remarkably aggravated cyclosporine-increased permeability of MBEC4 cells to sodium fluorescein.3. In light of these findings, the possibility that astroglial cells could contribute to the occurrence of cyclosporine-induced dysfunction of the blood-brain barrier triggering neurotoxicity should be considered.     

Possible Role of Nitric Oxide in Catecholamine Secretion by Chromaffin Cells in the Presence and Absence of Cultured Endothelial Cells

Abstract: We studied the effect of cultured endothelial cells on the secretion of catecholamines by cultured bovine chromaffin cells. Chromaffin cell catecholamine secretion was stimulated by either boluses of potassium (K⁺) or the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP). Endothelial cells inhibited the catecholamine release and stimulatory effects of K⁺ and DMPP. This inhibition increased with time, and in 25 min the initial stimulated secretory response (100%) to 30 m M K⁺ or 25 μ M DMPP dropped to 45 ± 3% and 53.5 ± 2.3%, respectively. This endothelial cell-induced inhibition was blocked by the nitric oxide synthase inhibitors N -nitro- l -arginine methyl ester ( l -NAME) and N -monoethyl- l -arginine ( l -NMMA), and by the guanylate cyclase inhibitor methylene blue, indicating that the l -arginine/nitric oxide/ cyclic GMP pathway is involved in this endothelial cell-chromaffin cell interaction. In the absence of endothelial cells, incubation of chromaffin cells with l -NAME, l -NMMA, or methylene blue also augmented the secretagogue-induced catecholamine secretion, indicating that nitric oxide from chromaffin cells could be implicated in an autoinhibitory process of catecholamine release. These results provide indirect evidence for the presence of nitric oxide synthase in bovine adrenomedullary chromaffin cells. Our results show that there is an autoinhibitory mechanism of catecholamine release in chromaffin cells and that an additional level of inhibition is observed when cultured vascular endothelial cells are present. These two inhibitory processes may have different origins, but they appear to converge into a common pathway, the l -arginine/nitric oxide synthase/guanylate cyclase pathway.     

Cilostazol Strengthens Barrier Integrity in Brain Endothelial Cells

We studied the effect of cilostazol, a selective inhibitor of phosphodiesterase 3, on barrier functions of blood–brain barrier (BBB)-related endothelial cells, primary rat brain capillary endothelial cells (RBEC), and the immortalized human brain endothelial cell line hCMEC/D3. The pharmacological potency of cilostazol was also evaluated on ischemia-related BBB dysfunction using a triple co-culture BBB model (BBB Kit?) subjected to 6-h oxygen glucose deprivation (OGD) and 3-h reoxygenation. There was expression of phosphodiesterase 3B mRNA in RBEC, and a significant increase in intracellular cyclic AMP (cAMP) content was detected in RBEC treated with both 1 and 10 μM cilostazol. Cilostazol increased the transendothelial electrical resistance (TEER), an index of barrier tightness of interendothelial tight junctions (TJs), and decreased the endothelial permeability of sodium fluorescein through the RBEC monolayer. The effects on these barrier functions were significantly reduced in the presence of protein kinase A (PKA) inhibitor H-89. Microscopic observation revealed smooth and even localization of occludin immunostaining at TJs and F-actin fibers at the cell borders in cilostazol-treated RBEC. In hCMEC/D3 cells treated with 1 and 10 μM cilostazol for 24 and 96 h, P-glycoprotein transporter activity was increased, as assessed by rhodamine 123 accumulation. Cilostazol improved the TEER in our triple co-culture BBB model with 6-h OGD and 3-h reoxygenation. As cilostazol stabilized barrier integrity in BBB-related endothelial cells, probably via cAMP/PKA signaling, the possibility that cilostazol acts as a BBB-protective drug against cerebral ischemic insults to neurons has to be considered.     

GSH Transport in Immortalized Mouse Brain Endothelial Cells

We have previously shown GSH transport across the blood-brain barrier in vivo and expression of transport in Xenopus laevis oocytes injected with bovine brain capillary mRNA. In the present study, we have used MBEC-4, an immortalized mouse brain endothelial cell line, to establish the presence of Na+-dependent and Na+-independent GSH transport and have localized the Na+-dependent transporter using domain-enriched plasma membrane vesicles. In cells depleted of GSH with buthionine sulfoximine, a significant increase of intracellular GSH could be demonstrated only in the presence of Na+. Partial but significant Na+ dependency of [35S]GSH uptake was observed for two GSH concentrations in MBEC-4 cells in which gamma-glutamyltranspeptidase and gamma-glutamylcysteine synthetase were inhibited to ensure absence of breakdown and resynthesis of GSH. Uniqueness of Na+-dependent uptake in MBEC-4 cells was confirmed with parallel uptake studies with Cos-7 cells that did not show this activity. Molecular form of uptake was verified as predominantly GSH, and very little conversion of [35S]cysteine to GSH occurred under the same incubation conditions. Poly(A)+ RNA from MBEC expressed GSH uptake with significant (approximately 40-70%) Na+ dependency, whereas uptake expressed by poly(A)+ RNA from HepG2 and Cos-1 cells was Na+ independent. Plasma membrane vesicles from MBEC were separated into three fractions (30, 34, and 38% sucrose, by wt) by density gradient centrifugation. Na+-dependent glucose transport, reported to be localized to the abluminal membrane, was found to be associated with the 38% fraction (abluminal). Na+-dependent GSH transport was present in the 30% fraction, which was identified as the apical (luminal) membrane by localization of P-glycoprotein 170 by western blot analysis. Localization of Na+-dependent GSH transport to the luminal membrane and its ability to drive up intracellular GSH may find application in the delivery of supplemented GSH to the brain in vivo.     

Delayed Contrast Extravasation MRI for Depicting Tumor and Non-Tumoral Tissues in Primary and Metastatic Brain Tumors

The current standard of care for newly diagnosed glioblastoma multiforme (GBM) is resection followed by radiotherapy with concomitant and adjuvant temozolomide. Recent studies suggest that nearly half of the patients with early radiological deterioration post treatment do not suffer from tumor recurrence but from pseudoprogression. Similarly, a significant number of patients with brain metastases suffer from radiation necrosis following radiation treatments. Conventional MRI is currently unable to differentiate tumor progression from treatment-induced effects. The ability to clearly differentiate tumor from non-tumoral tissues is crucial for appropriate patient management. Ten patients with primary brain tumors and 10 patients with brain metastases were scanned by delayed contrast extravasation MRI prior to surgery. Enhancement subtraction maps calculated from high resolution MR images acquired up to 75 min after contrast administration were used for obtaining stereotactic biopsies. Histological assessment was then compared with the pre-surgical calculated maps. In addition, the application of our maps for prediction of progression was studied in a small cohort of 13 newly diagnosed GBM patients undergoing standard chemoradiation and followed up to 19.7 months post therapy. The maps showed two primary enhancement populations: the slow population where contrast clearance from the tissue was slower than contrast accumulation and the fast population where clearance was faster than accumulation. Comparison with histology confirmed the fast population to consist of morphologically active tumor and the slow population to consist of non-tumoral tissues. Our maps demonstrated significant correlation with perfusion-weighted MR data acquired simultaneously, although contradicting examples were shown. Preliminary results suggest that early changes in the fast volumes may serve as a predictor for time to progression. These preliminary results suggest that our high resolution MRI-based delayed enhancement subtraction maps may be applied for clear depiction of tumor and non-tumoral tissues in patients with primary brain tumors and patients with brain metastases.     

Lysosomal Degradation of CD44 Mediates Ceramide Nanoliposome-induced Anoikis and Diminished Extravasation in Metastatic Carcinoma Cells

The ceramide nanoliposome (CNL) has shown promise in being able to treat a variety of primary tumors. However, its potential for treating metastatic cancer remains unknown. In this study, we demonstrate that CNL increases anoikis while preventing cancer cell extravasation under both static and physiological fluid flow conditions. Mechanistically, CNL limits metastases by decreasing CD44 protein levels in human breast and pancreatic cancer cells via lysosomal degradation of CD44, independent of palmitoylation or proteasome targeting. siRNA down-regulation of CD44 mimics CNL-induced anoikis and diminished extravasation of cancer cells. Taken together, our data indicate that ceramide limits CD44-dependent cancer cell migration, suggesting that CNL could be used to prevent and treat solid tumor metastasis.     

Impact of Calcium Signaling during Infection of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells

The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells.     

Isolation and Characterization of Brain Endothelial Cells: Morphology and Enzyme Activity

Microvessels were isolated from rat brain using a double collagenase treatment which removed the endothelial basement membranes. The isolate was characterized by intact luminal and abluminal membranes and an absence of pericytes and astrocyte membranes. Minimal contamination by 5′-nucleotidase, an enzyme believed exclusively localized within the plasma membranes of neuroglia, established the purity of the isolated microvessels. Enrichment of alkaline phosphatase and -γ-glutamyl transpeptidase activity in microvessel preparations supports the endothelial localization of these enzymes.     

运用植块法培养脑微血管内皮细胞

探讨简易可行的脑微血管内皮细胞(brain microvascular endothelial cells,BMECs培)养方法,为研究BMECs细胞在脑血管疾病中的重要作用提供技术支持。分离出生后1~7天内的SD乳鼠大脑皮质区,植块法培养BMECs细胞。用倒置显微镜观察BMECs细胞的形态以及从皮质块迁出的过程;MTT比色法检测BMECs细胞的生长曲线;采用免疫组化染色检测VIII因子相关抗原和CD34抗原,以鉴定内皮细胞。结果发现,大脑皮质块植块法培养的大鼠BMECs细胞呈单层贴壁生长,细胞形态以长梭形、多角形三角形、四边形为主,呈典型的“铺路石”样征象,经鉴定为内皮细胞,第三代纯度达95%以上。提示该方法具有经济、简便、要求条件不高,易于纯化的优点,可作为大鼠BMECs细胞体外培养的良好模型。     

Brain Endothelial Cells Are Exquisite Sensors of Age-Related Circulatory Cues

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