Blood–Brain Barrier Pericytes Are the Main Source of γ-Glutamyltranspeptidase Activity in Brain Capillaries

Cerebral endothelial cells form the selective permeability barrier between brain and blood by virtue of their impermeable tight junctions and the presence of specific carrier systems. These specialized properties of brain capillaries are reflected in the presence of proteins that are not found in other capillaries of the body. gamma-Glutamyltranspeptidase (GGT) has been widely used as a marker for brain capillaries and differentiated properties of brain endothelial cells. By using histochemical and biochemical methods we have investigated the expression of GGT in isolated capillaries, cultured brain endothelial cells and pericytes, and cocultures of astrocytes and brain endothelial cells. It was surprising that the majority of GGT activity was associated with pericytes, but not endothelial cells, suggesting that GGT is a specific marker for brain pericytes. The remaining GGT activity that was associated with endothelial cells rapidly disappeared from cultured cells but was reinduced in cocultures with astrocytes. Our results emphasize the need for pure endothelial cells for the investigation of blood-brain barrier characteristics.     

The Possible Roles of Brain Pericytes in Brain Ischemia and Stroke

Brain pericytes regulate a variety of functions, such as microcirculation, angiogenesis, and the blood brain barrier in the brain. Recent studies have also shown that they are pluripotent in a manner similar to mesenchymal stem cells. Since, brain pericytes actively control these functions, these cells probably play an important role not only during brain ischemia, but also in the post-stroke period.     

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.     

Brain Pericytes: Emerging Concepts and Functional Roles in Brain Homeostasis

Brain pericytes are an important constituent of neurovascular unit. They encircle endothelial cells and contribute to the maturation and stabilization of the capillaries in the brain. Recent studies have revealed that brain pericytes play pivotal roles in a variety of brain functions, such as regulation of capillary flow, angiogenesis, blood brain barrier, immune responses, and hemostasis. In addition, brain pericytes are pluripotent and can differentiate into different lineages similar to mesenchymal stem cells. The brain pericytes are revisited as a key player to maintain brain function and repair brain damage.     

Eukaryotic DING Proteins Are Endogenous: An Immunohistological Study in Mouse Tissues

Background

DING proteins encompass an intriguing protein family first characterized by their conserved N-terminal sequences. Some of these proteins seem to have key roles in various human diseases, e.g., rheumatoid arthritis, atherosclerosis, HIV suppression. Although this protein family seems to be ubiquitous in eukaryotes, their genes are consistently lacking from genomic databases. Such a lack has considerably hampered functional studies and has fostered therefore the hypothesis that DING proteins isolated from eukaryotes were in fact prokaryotic contaminants.

Principal Findings

In the framework of our study, we have performed a comprehensive immunological detection of DING proteins in mice. We demonstrate that DING proteins are present in all tissues tested as isoforms of various molecular weights (MWs). Their intracellular localization is tissue-dependant, being exclusively nuclear in neurons, but cytoplasmic and nuclear in other tissues. We also provide evidence that germ-free mouse plasma contains as much DING protein as wild-type.

Significance

Hence, data herein provide a valuable basis for future investigations aimed at eukaryotic DING proteins, revealing that these proteins seem ubiquitous in mouse tissue. Our results strongly suggest that mouse DING proteins are endogenous. Moreover, the determination in this study of the precise cellular localization of DING proteins constitute a precious evidence to understand their molecular involvements in their related human diseases.     

Oncogenicity of an Endogenous C-Type Virus Chemically Activated from Mouse Cells in Culture

RNA C-type viruses are known to exist in an unexpressed form in all mouse cells. These endogenous viruses can be activated from cells in tissue culture under certain experimental conditions. The biologic activity in vivo of one class of chemically-activated C-type virus has been studied. This virus is shown to induce a specific tumor, lymphatic leukemia, in mice of a low leukemic incidence strain.     

小窝蛋白-1与脑胶质瘤

小窝蛋白-1（caveolin-1,Cav-1）是胞膜窖（caveolae）的标志性蛋白质。Cav-1在多种细胞的生命活动中起重要作用。大量证据表明,Cav-1参与乳腺癌、肝细胞癌、胰腺癌、前列腺癌、肾透明细胞癌等多种肿瘤的发生发展过程。胶质瘤是中枢神经系统恶性肿瘤之一,由于脑血屏障的存在,很多药物很难到达病灶,因而死亡率极高。近年来发现,Cav-1是胶质瘤细胞增殖的负调控因子,能够降低胶质瘤的迁移和侵袭能力。此外,Cav-1能够增加胶质瘤血瘤屏障的通透性。本文简要综述了近年来Cav-1在脑胶质瘤发生发展及其对血瘤屏障的调节作用的新进展,旨在为胶质瘤的临床治疗提供新的思路。     

Mitochondria Contribute to NADPH Generation in Mouse Rod Photoreceptors

NADPH is the primary source of reducing equivalents in the cytosol. Its major source is considered to be the pentose phosphate pathway, but cytosolic NADP⁺-dependent dehydrogenases using intermediates of mitochondrial pathways for substrates have been known to contribute. Photoreceptors, a nonproliferating cell type, provide a unique model for measuring the functional utilization of NADPH at the single cell level. In these cells, NADPH availability can be monitored from the reduction of the all-trans-retinal generated by light to all-trans-retinol using single cell fluorescence imaging. We have used mouse rod photoreceptors to investigate the generation of NADPH by different metabolic pathways. In the absence of extracellular metabolic substrates, NADPH generation was severely compromised. Extracellular glutamine supported NADPH generation to levels comparable to those of glucose, but pyruvate and lactate were relatively ineffective. At low extracellular substrate concentrations, partial inhibition of ATP synthesis lowered, whereas suppression of ATP consumption augmented NADPH availability. Blocking pyruvate transport into mitochondria decreased NADPH availability, and addition of glutamine restored it. Our findings demonstrate that in a nonproliferating cell type, mitochondria-linked pathways can generate substantial amounts of NADPH and do so even when the pentose phosphate pathway is operational. Competing demands for ATP and NADPH at low metabolic substrate concentrations indicate a vulnerability to nutrient shortages. By supporting substantial NADPH generation, mitochondria provide alternative metabolic pathways that may support cell function and maintain viability under transient nutrient shortages. Such pathways may play an important role in protecting against retinal degeneration.     

Pain Facilitation Brain Regions Activated by Nalbuphine Are Revealed by Pharmacological fMRI

Nalbuphine, an agonist-antagonist kappa-opioid, produces brief analgesia followed by enhanced pain/hyperalgesia in male postsurgical patients. However, it produces profound analgesia without pain enhancement when co-administration with low dose naloxone. To examine the effect of nalbuphine or nalbuphine plus naloxone on activity in brain regions that may explain these differences, we employed pharmacological magnetic resonance imaging (phMRI) in a double blind cross-over study with 13 healthy male volunteers. In separate imaging sessions subjects were administered nalbuphine (5 mg/70 kg) preceded by either saline (Sal-Nalb) or naloxone 0.4 mg (Nalox-Nalb). Blood oxygen level-dependent (BOLD) activation maps followed by contrast and connectivity analyses revealed marked differences. Sal-Nalb produced significantly increased activity in 60 brain regions and decreased activity in 9; in contrast, Nalox-Nalb activated only 14 regions and deactivated only 3. Nalbuphine, like morphine in a previous study, attenuated activity in the inferior orbital cortex, and, like noxious stimulation, increased activity in temporal cortex, insula, pulvinar, caudate, and pons. Co-administration/pretreatment of naloxone selectively blocked activity in pulvinar, pons and posterior insula. Nalbuphine induced functional connectivity between caudate and regions in the frontal, occipital, temporal, insular, middle cingulate cortices, and putamen; naloxone co-admistration reduced all connectivity to non-significant levels, and, like phMRI measures of morphine, increased activation in other areas (e.g., putamen). Naloxone pretreatment to nalbuphine produced changes in brain activity possess characteristics of both analgesia and algesia; naloxone selectively blocks activity in areas associated with algesia. Given these findings, we suggest that nalbuphine interacts with a pain salience system, which can modulate perceived pain intensity.     

Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation

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ProSAAS-Derived Peptides Are Differentially Processed and Sorted in Mouse Brain and AtT-20 Cells

ProSAAS is the precursor for some of the most abundant peptides found in mouse brain and other tissues, including peptides named SAAS, PEN, and LEN. Both SAAS and LEN are found in big and little forms due to differential processing. Initial processing of proSAAS is mediated by furin (and/or furin-like enzymes) and carboxypeptidase D, while the smaller forms are generated by secretory granule prohormone convertases and carboxypeptidase E. In mouse hypothalamus, PEN and big LEN colocalize with neuropeptide Y. In the present study, little LEN and SAAS were detected in mouse hypothalamus but not in cell bodies of neuropeptide Y-expressing neurons. PEN and big LEN show substantial colocalization in hypothalamus, but big LEN and little LEN do not. An antiserum to SAAS that detects both big and little forms of this peptide did not show substantial colocalization with PEN or big LEN. To further study this, the AtT-20 cells mouse pituitary corticotrophic cell line was transfected with rat proSAAS and the distribution of peptides examined. As found in mouse hypothalamus, only some of the proSAAS-derived peptides colocalized with each other in AtT-20 cells. The two sites within proSAAS that are known to be efficiently cleaved by furin were altered by site-directed mutagenesis to convert the P4 Arg into Lys; this change converts the sequences from furin consensus sites into prohormone convertase consensus sites. Upon expression of the mutated form of proSAAS in AtT-20 cells, there was significantly more colocalization of proSAAS-derived peptides PEN and SAAS. Taken together, these results indicate that proSAAS is initially cleaved in the Golgi or trans-Golgi network by furin and/or furin-like enzymes and the resulting fragments are sorted into distinct vesicles and further processed by additional enzymes into the mature peptides.     

Acetylcholinesterases from Musca domestica and Drosophila melanogaster Brain Are Linked to Membranes by a Glycophospholipid Anchor Sensitive to an Endogenous Phospholipase

The sensitivity of acetylcholinesterases (AChEs) from Musca domestica and from Drosophila melanogaster to the phosphatidylinositol-specific phospholipase C from Bacillus cereus and to the glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei was investigated. B. cereus phospholipase C solubilizes membrane-bound AChE, and both phospholipases convert amphiphilic AChEs into hydrophilic forms of the enzyme. The lipases uncover an immunological determinant that is found on other glycosylphosphatidylinositol-anchored membrane proteins after the same treatment. This immunological determinant is also present on the native hydrophilic form of AChE. The polypeptide bearing the active site of the membrane-bound enzyme migrates faster during sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the same polypeptide from the soluble enzyme. We conclude that AChE from insect brain is attached to membranes via a glycophospholipid anchor. This anchor is covalently linked to the polypeptide bearing the active esterase site of the enzyme and can be cleaved by an endogenous lipase.     

Pericytes Contribute to the Disruption of the Cerebral Endothelial Barrier via Increasing VEGF Expression: Implications for Stroke

Disruption of the blood-brain barrier (BBB) integrity occurring during the early onset of stroke is not only a consequence of, but also contributes to the further progression of stroke. Although it has been well documented that brain microvascular endothelial cells and astrocytes play a critical role in the maintenance of BBB integrity, pericytes, sandwiched between endothelial cells and astrocytes, remain poorly studied in the pathogenesis of stroke. Our findings demonstrated that treatment of human brain microvascular pericytes with sodium cyanide (NaCN) and glucose deprivation resulted in increased expression of vascular endothelial growth factor (VEGF) via the activation of tyrosine kinase Src, with downstream activation of mitogen activated protein kinase and PI3K/Akt pathways and subsequent translocation of NF-κB into the nucleus. Conditioned medium from NaCN-treated pericytes led to increased permeability of endothelial cells, and this effect was significantly inhibited by VEGF-neutralizing antibody. The in vivo relevance of these findings was further corroborated in the stroke model of mice wherein the mice, demonstrated disruption of the BBB integrity and concomitant increase in the expression of VEGF in the brain tissue as well as in the isolated microvessel. These findings thus suggest the role of pericyte-derived VEGF in modulating increased permeability of BBB during stroke. Understanding the regulation of VEGF expression could open new avenues for the development of potential therapeutic targets for stroke and other neurological disease.     

Brain and Kidney d-Amino Acid Oxidases Are Coded by a Single Gene in the Mouse

Mutant mice (ddY/DAO-) lacking D-amino acid oxidase in the kidney also lacked this enzyme in the brain. Genetic cross experiments showed that the inheritance of the enzyme in the brain was the same as that in the kidney. The deficiency in the enzyme in the brain could not be separated from that in the kidney. The brain and kidney enzymes showed similar substrate specificities. These results suggest that brain and kidney D-amino acid oxidases are coded by the same gene in the mouse.     

Platelet Specific Promoters Are Insufficient to Express Protease Activated Receptor 1 (PAR1) Transgene in Mouse Platelets

The in vivo study of protease activated receptors (PARs) in platelets is complicated due to species specific expression profiles. Human platelets express PAR1 and PAR4 whereas mouse platelets express PAR3 and PAR4. Further, PAR subtypes interact with one another to influence activation and signaling. The goal of the current study was to generate mice expressing PAR1 on their platelets using transgenic approaches to mimic PAR expression found in human platelets. This system would allow us to examine specific signaling from PAR1 and the PAR1-PAR4 heterodimer in vivo. Our first approach used the mouse GPIbα promoter to drive expression of mouse PAR1 in platelets (GPIbα-Tg-mPAR1). We obtained the expected frequency of founders carrying the transgene and had the expected Mendelian distribution of the transgene in multiple founders. However, we did not observe expression or a functional response of PAR1. As a second approach, we targeted human PAR1 with the same promoter (GPIbα-Tg-hPAR1). Once again we observed the expected frequency and distributing of the transgene. Human PAR1 expression was detected in platelets from the GPIbα-Tg-hPAR1 mice by flow cytometry, however, at a lower level than for human platelets. Despite a low level of PAR1 expression, platelets from the GPIbα-Tg-hPAR1 mice did not respond to the PAR1 agonist peptide (SFLLRN). In addition, they did not respond to thrombin when crossed to the PAR4^−/− mice. Finally, we used an alternative platelet specific promoter, human α_IIb, to express human PAR1 (α_IIb-Tg-hPAR1). Similar to our previous attempts, we obtained the expected number of founders but did not detect PAR1 expression or response in platelets from α_IIb-Tg-hPAR1 mice. Although unsuccessful, the experiments described in this report provide a resource for future efforts in generating mice expressing PAR1 on their platelets. We provide an experimental framework and offer considerations that will save time and research funds.     

Osteoprotegerin,Pericytes and Bone-Like Vascular Calcification Are Associated with Carotid Plaque Stability

Background and Purpose

Vascular calcification, recapitulating bone formation, has a profound impact on plaque stability. The aim of the present study was to determine the influence of bone-like vascular calcification (named osteoid metaplasia = OM) and of osteoprotegerin on plaque stability.

Methods

Tissue from carotid endarterectomies were analysed for the presence of calcification and signs of vulnerability according to AHA grading system. Osteoprotegerin (OPG), pericytes and endothelial cells were sought using immuno-histochemistry. Symptoms and preoperative imaging findings (CT-scan, MRI and Doppler-scan) were analyzed. Human pericytes were cultured to evaluate their ability to secrete OPG and to influence mineralization in the plaque.

Results

Seventy-three carotid plaques (49 asymptomatic and 24 symptomatic) were harvested. A significantly higher presence of OM (18.4% vs 0%, p<0.01), OPG (10.2% of ROI vs 3.4% of ROI, p<0.05) and pericytes (19% of ROI vs 3.8% of ROI, p<0.05) were noted in asymptomatic compared to symptomatic plaques. Consistently, circulating OPG levels were higher in the plasma of asymptomatic patients (3.2 ng/mL vs 2.5 ng/mL, p = 0.05). In vitro, human vascular pericytes secreted considerable amounts of OPG and underwent osteoblastic differentiation. Pericytes also inhibited the osteoclastic differentiation of CD14+ cells through their secretion of OPG.

Conclusions

OPG (intraplaque an plasmatic) and OM are associated with carotid plaque stability. Pericytes may be involved in the secretion of intraplaque OPG and in the formation of OM.     

Stellate Cells from Rat Pancreas Are Stem Cells and Can Contribute to Liver Regeneration

The identity of pancreatic stem/progenitor cells is still under discussion. They were suggested to derive from the pancreatic ductal epithelium and/or islets. Here we report that rat pancreatic stellate cells (PSC), which are thought to contribute to pancreatic fibrosis, have stem cell characteristics. PSC reside in islets and between acini and display a gene expression pattern similar to umbilical cord blood stem cells and mesenchymal stem cells. Cytokine treatment of isolated PSC induced the expression of typical hepatocyte markers. The PSC-derived hepatocyte-like cells expressed endodermal proteins such as bile salt export pump along with the mesodermal protein vimentin. The transplantation of culture-activated PSC from enhanced green fluorescent protein-expressing rats into wild type rats after partial hepatectomy in the presence of 2-acetylaminofluorene revealed that PSC were able to reconstitute large areas of the host liver through differentiation into hepatocytes and cholangiocytes. This developmental fate of transplanted PSC was confirmed by fluorescence in situ hybridization of chromosome Y after gender-mismatched transplantation of male PSC into female rats. Transplanted PSC displayed long-lasting survival, whereas muscle fibroblasts were unable to integrate into the host liver. The differentiation potential of PSC was further verified by the transplantation of clonally expanded PSC. PSC clones maintained the expression of stellate cell and stem cell markers and preserved their differentiation potential, which indicated self-renewal potential of PSC. These findings demonstrate that PSC have stem cell characteristics and can contribute to the regeneration of injured organs through differentiation across tissue boundaries.     

Elevated Endogenous Erythropoietin Concentrations Are Associated with Increased Risk of Brain Damage in Extremely Preterm Neonates

Background

We sought to determine, in very preterm infants, whether elevated perinatal erythropoietin (EPO) concentrations are associated with increased risks of indicators of brain damage, and whether this risk differs by the co-occurrence or absence of intermittent or sustained systemic inflammation (ISSI).

Methods

Protein concentrations were measured in blood collected from 786 infants born before the 28th week of gestation. EPO was measured on postnatal day 14, and 25 inflammation-related proteins were measured weekly during the first 2 postnatal weeks. We defined ISSI as a concentration in the top quartile of each of 25 inflammation-related proteins on two separate days a week apart. Hypererythropoietinemia (hyperEPO) was defined as the highest quartile for gestational age on postnatal day 14. Using logistic regression and multinomial logistic regression models, we compared risks of brain damage among neonates with hyperEPO only, ISSI only, and hyperEPO+ISSI, to those who had neither hyperEPO nor ISSI, adjusting for gestational age.

Results

Newborns with hyperEPO, regardless of ISSI, were more than twice as likely as those without to have very low (< 55) Mental (OR 2.3; 95% CI 1.5-3.5) and/or Psychomotor (OR 2.4; 95% CI 1.6-3.7) Development Indices (MDI, PDI), and microcephaly at age two years (OR 2.4; 95%CI 1.5-3.8). Newborns with both hyperEPO and ISSI had significantly increased risks of ventriculomegaly, hemiparetic cerebral palsy, microcephaly, and MDI and PDI < 55 (ORs ranged from 2.2-6.3), but not hypoechoic lesions or other forms of cerebral palsy, relative to newborns with neither hyperEPO nor ISSI.

Conclusion

hyperEPO, regardless of ISSI, is associated with elevated risks of very low MDI and PDI, and microcephaly, but not with any form of cerebral palsy. Children with both hyperEPO and ISSI are at higher risk than others of very low MDI and PDI, ventriculomegaly, hemiparetic cerebral palsy, and microcephaly.     

Multifactorial Effects on Different Types of Brain Cells Contribute to Ammonia Toxicity

Effects of ammonia on astrocytes play a major role in hepatic encephalopathy, acute liver failure and other diseases caused by increased arterial ammonia concentrations (e.g., inborn errors of metabolism, drug or mushroom poisoning). There is a direct correlation between arterial ammonia concentration, brain ammonia level and disease severity. However, the pathophysiology of hyperammonemic diseases is disputed. One long recognized factor is that increased brain ammonia triggers its own detoxification by glutamine formation from glutamate. This is an astrocytic process due to the selective expression of the glutamine synthetase in astrocytes. A possible deleterious effect of the resulting increase in glutamine concentration has repeatedly been discussed and is supported by improvement of some pathologic effects by GS inhibition. However, this procedure also inhibits a large part of astrocytic energy metabolism and may prevent astrocytes from responding to pathogenic factors. A decrease of the already low glutamate concentration in astrocytes due to increased synthesis of glutamine inhibits the malate–aspartate shuttle and energy metabolism. A more recently described pathogenic factor is the resemblance between NH₄ ⁺ and K⁺ in their effects on the Na⁺,K⁺-ATPase and the Na⁺,K⁺, 2 Cl^? and water transporter NKCC1. Stimulation of the Na⁺,K⁺-ATPase driven NKCC1 in both astrocytes and endothelial cells is essential for the development of brain edema. Na⁺,K⁺-ATPase stimulation also activates production of endogenous ouabains. This leads to oxidative and nitrosative damage and sensitizes NKCC1. Administration of ouabain antagonists may accordingly have therapeutic potential in hyperammonemic diseases.     

Jasmonate-Inducible Genes Are Activated in Rice by Pathogen Attack without a Concomitant Increase in Endogenous Jasmonic Acid Levels

The possible role of the octadecanoid signaling pathway with jasmonic acid (JA) as the central component in defense-gene regulation of pathogen-attacked rice was studied. Rice (Oryza sativa L.) seedlings were treated with JA or inoculated with the rice blast fungus Magnaporthe grisea (Hebert) Barr., and gene-expression patterns were compared between the two treatments. JA application induced the accumulation of a number of pathogenesis-related (PR) gene products at the mRNA and protein levels, but pathogen attack did not enhance the levels of (-)-JA during the time required for PR gene expression. Pathogen-induced accumulation of PR1-like proteins was reduced in plants treated with tetcyclacis, a novel inhibitor of jasmonate biosynthesis. There was an additive and negative interaction between JA and an elicitor from M. grisea with respect to induction of PR1-like proteins and of an abundant JA-and wound-induced protein of 26 kD, respectively. Finally, activation of the octadecanoid signaling pathway and induction of a number of PR genes by exogenous application of JA did not confer local acquired resistance to rice. The data suggest that accumulation of nonconjugated (-)-JA is not necessary for induction of PR genes and that JA does not orchestrate localized defense responses in pathogen-attacked rice. Instead, JA appears to be embedded in a signaling network with another pathogen-induced pathway(s) and may be required at a certain minimal level for induction of some PR genes.