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

Background: Cell-based therapeutic strategies have been proposed as an alternative for brain repair after stroke, but their clinical application has been hampered by potential adverse effects in the long term. The present study was designed to test the effect of the secretome of endothelial progenitor cells (EPCs) from stroke patients (scCM) on in vitro human models of angiogenesis and vascular barrier. Methods: Two different scCM batches were analysed by mass spectrometry and a proteome profiler. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used for designing angiogenesis studies (proliferation, migration, and tubulogenesis) or in vitro models of EC monolayer (confluent monolayer ECs—CMECs) and blood–brain barrier (BBB; brain-like ECs—BLECs). Cells were treated with scCM (5 μg/mL) or protein-free endothelial basal medium (scEBM—control). CMECs or BLECs were exposed (6 h) to oxygen–glucose deprivation (OGD) conditions (1% oxygen and glucose-free medium) or normoxia (control—5% oxygen, 1 g/L of glucose) and treated with scCM or scEBM during reoxygenation (24 h). Results: The analysis of different scCM batches showed a good reproducibility in terms of protein yield and composition. scCM increased CD34+-EC proliferation, tubulogenesis, and migration compared to the control (scEBM). The proteomic analysis of scCM revealed the presence of growth factors and molecules modulating cell metabolism and inflammatory pathways. Further, scCM decreased the permeability of CMECs and upregulated the expression of the junctional proteins such as occludin, VE-cadherin, and ZO-1. Such effects were possibly mediated through the activation of the interferon pathway and a moderate downregulation of Wnt signalling. Furthermore, OGD increased the permeability of both CMECs and BLECs, while scCM prevented the OGD-induced vascular leakage in both models. These effects were possibly mediated through the upregulation of junctional proteins and the regulation of MAPK/VEGFR2 activity. Conclusion: Our results suggest that scCM promotes angiogenesis and the maturation of newly formed vessels while restoring the BBB function in ischemic conditions. In conclusion, our results highlight the possibility of using EPC-secretome as a therapeutic alternative to promote brain angiogenesis and protect from ischemia-induced vascular leakage. [ABSTRACT FROM AUTHOR]

Background: Cell-based therapeutic strategies have been proposed as an alternative for brain repair after stroke, but their clinical application has been hampered by potential adverse effects in the long term. The present study was designed to test the effect of the secretome of endothelial progenitor cells (EPCs) from stroke patients (scCM) on in vitro human models of angiogenesis and vascular barrier. Methods: Two different scCM batches were analysed by mass spectrometry and a proteome profiler. Human primary CD34+-derived endothelial cells (CD34+-ECs) were used for designing angiogenesis studies (proliferation, migration, and tubulogenesis) or in vitro models of EC monolayer (confluent monolayer ECs—CMECs) and blood–brain barrier (BBB; brain-like ECs—BLECs). Cells were treated with scCM (5 μg/mL) or protein-free endothelial basal medium (scEBM—control). CMECs or BLECs were exposed (6 h) to oxygen–glucose deprivation (OGD) conditions (1% oxygen and glucose-free medium) or normoxia (control—5% oxygen, 1 g/L of glucose) and treated with scCM or scEBM during reoxygenation (24 h). Results: The analysis of different scCM batches showed a good reproducibility in terms of protein yield and composition. scCM increased CD34+-EC proliferation, tubulogenesis, and migration compared to the control (scEBM). The proteomic analysis of scCM revealed the presence of growth factors and molecules modulating cell metabolism and inflammatory pathways. Further, scCM decreased the permeability of CMECs and upregulated the expression of the junctional proteins such as occludin, VE-cadherin, and ZO-1. Such effects were possibly mediated through the activation of the interferon pathway and a moderate downregulation of Wnt signalling. Furthermore, OGD increased the permeability of both CMECs and BLECs, while scCM prevented the OGD-induced vascular leakage in both models. These effects were possibly mediated through the upregulation of junctional proteins and the regulation of MAPK/VEGFR2 activity. Conclusion: Our results suggest that scCM promotes angiogenesis and the maturation of newly formed vessels while restoring the BBB function in ischemic conditions. In conclusion, our results highlight the possibility of using EPC-secretome as a therapeutic alternative to promote brain angiogenesis and protect from ischemia-induced vascular leakage. [ABSTRACT FROM AUTHOR]

The article titled "Retraction Note: Secretome of endothelial progenitor cells from stroke patients promotes endothelial barrier tightness and protects against hypoxia-induced vascular leakage" has been retracted by the authors. The retraction was prompted by an investigation that revealed the cells used in the study were not endothelial progenitor cells as expected, but rather hCMEC/D3 cells. The authors apologize for this unexpected finding and all authors agree with the retraction. Springer Nature, the publisher, remains neutral in this matter. [Extracted from the article]

Background: Pediatric diffuse intrinsic pontine glioma (DIPG) represents one of the most devastating and lethal brain tumors in children with a median survival of 12 months. The high mortality rate can be explained by the ineligibility of patients to surgical resection due to the diffuse growth pattern and midline localization of the tumor. While the therapeutic strategies are unfortunately palliative, the blood–brain barrier (BBB) is suspected to be responsible for the treatment inefficiency. Located at the brain capillary endothelial cells (ECs), the BBB has specific properties to tightly control and restrict the access of molecules to the brain parenchyma including chemotherapeutic compounds. However, these BBB specific properties can be modified in a pathological environment, thus modulating brain exposure to therapeutic drugs. Hence, this study aimed at developing a syngeneic human blood–brain tumor barrier model to understand how the presence of DIPG impacts the structure and function of brain capillary ECs. Methods: A human syngeneic in vitro BBB model consisting of a triple culture of human (ECs) (differentiated from CD34+-stem cells), pericytes and astrocytes was developed. Once validated in terms of BBB phenotype, this model was adapted to develop a blood–brain tumor barrier (BBTB) model specific to pediatric DIPG by replacing the astrocytes by DIPG-007, -013 and -014 cells. The physical and metabolic properties of the BBTB ECs were analyzed and compared to the BBB ECs. The permeability of both models to chemotherapeutic compounds was evaluated. Results: In line with clinical observation, the integrity of the BBTB ECs remained intact until 7 days of incubation. Both transcriptional expression and activity of efflux transporters were not strongly modified by the presence of DIPG. The permeability of ECs to the chemotherapeutic drugs temozolomide and panobinostat was not affected by the DIPG environment. Conclusions: This original human BBTB model allows a better understanding of the influence of DIPG on the BBTB ECs phenotype. Our data reveal that the chemoresistance described for DIPG does not come from the development of a "super BBB". These results, validated by the absence of modification of drug transport through the BBTB ECs, point out the importance of understanding the implication of the different protagonists in the pathology to have a chance to significantly improve treatment efficiency. [ABSTRACT FROM AUTHOR]

Objective: Autoimmune polyendocrine syndrome type 1 (APS 1) is caused by mutations in theAIREgene that induce intrathymic T-cell tolerance breakdown, which results in tissue-specific autoimmune diseases.Design: To evaluate the effect of a well-defined T-cell repertoire impairment on humoral self-reactive fingerprints, comparative serum self-IgG and self-IgM reactivities were analyzed using both one- and two-dimensional western blotting approaches against a broad spectrum of peripheral tissue antigens.Methods: Autoantibody patterns of APS 1 patients were compared with those of subjects affected by other autoimmune endocrinopathies (OAE) and healthy controls.Results: Using a Chi-square test, significant changes in the Ab repertoire were found when intergroup patterns were compared. A singular distortion of both serum self-IgG and self-IgM repertoires was noted in APS 1 patients. The molecular characterization of these antigenic targets was conducted using a proteomic approach. In this context, autoantibodies recognized more significantly either tissue-specific antigens, such as pancreatic amylase, pancreatic triacylglycerol lipase and pancreatic regenerating protein 1α, or widely distributed antigens, such as peroxiredoxin-2, heat shock cognate 71-kDa protein and aldose reductase. As expected, a well-defined self-reactive T-cell repertoire impairment, as described in APS 1 patients, affected the tissue-specific self-IgG repertoire. Interestingly, discriminant IgM reactivities targeting both tissue-specific and more widely expressed antigens were also specifically observed in APS 1 patients. Using recombinant targets, we observed that post translational modifications of these specific antigens impacted upon their recognition.Conclusions: The data suggest that T-cell-dependent but also T-cell-independent mechanisms are involved in the dynamic evolution of autoimmunity in APS 1. [ABSTRACT FROM PUBLISHER]

Serological proteome analysis (SERPA) combines classical proteomic technology with effective separation of cellular protein extracts on two-dimensional gel electrophoresis, western blotting, and identification of the antigenic spot of interest by mass spectrometry. A critical point is related to the antigenic target characterization by mass spectrometry, which depends on the accuracy of the matching of antigenic reactivities on the protein spots during the 2D immunoproteomic procedures. The superimposition, based essentially on visual criteria of antigenic and protein spots, remains the major limitation of SERPA. The introduction of fluorescent dyes in proteomic strategies, commonly known as 2D-DIGE (differential in-gel electrophoresis), has boosted the qualitative capabilities of 2D electrophoresis. Based on this 2D-DIGE strategy, we have improved the conventional SERPA by developing a new and entirely fluorescence-based bi-dimensional immunoproteomic (FBIP) analysis, performed with three fluorescent dyes. To optimize the alignment of the different antigenic maps, we introduced a landmark map composed of a combination of specific antibodies. This methodological development allows simultaneous revelation of the antigenic, landmark and proteomic maps on each immunoblot. A computer-assisted process using commercially available software automatically leads to the superimposition of the different maps, ensuring accurate localization of antigenic spots of interest. [ABSTRACT FROM AUTHOR]

Low birth weight is associated with an increased risk for developing type 2 diabetes and metabolic diseases. The placental capacity to supply nutrients and oxygen to the fetus represents the main determiner of fetal growth. However, few studies have investigated the effects of maternal diet on the placenta. We explored placental adaptive proteomic processes implicated in response to maternal undernutrition. Rat term placentas from 70% food-restricted (FR30) mothers were used for a proteomic screen. Placental mitochondrial functions were evaluated using molecular and functional approaches, and ATP production was measured. FR30 drastically reduced placental and fetal weights. FR30 placentas displayed 14 proteins that were differentially expressed, including several mitochondrial proteins. FR30 induced a marked increase in placental mtDNA content and changes in mitochondrial functions, including modulation of the expression of genes implicated in biogenesis and bioenergetic pathways. FR30 mitochondria showed higher oxygen consumption but failed to maintain their ATP production. Maternal undernutrition induces placental mitochondrial abnormalities. Although an increase in biogenesis and bioenergetic efficiency was noted, placental ATP level was reduced. Our data suggest that placental mitochondrial defects may be implicated in fetoplacental pathologies. [ABSTRACT FROM AUTHOR]

Although the physiological properties of the blood-brain barrier (BBB) are relatively well known, the phenotype of the component brain capillary endothelial cells (BCECs) has yet to be described in detail. Likewise, the molecular mechanisms that govern the establishment and maintenance of the BBB are largely unknown. Proteomics can be used to assess quantitative changes in protein levels and identify proteins involved in the molecular pathways responsible for cellular differentiation. Using the well-established in vitro BBB model developed in our laboratory, we performed a differential nano- LC MALDI-TOF/TOF-MS study of Triton X-100-soluble protein species from bovine BCECs displaying either limited BBB functions or BBB functions re-induced by glial cells. Due to the heterogeneity of the crude extract, we increased identification yields by applying a repeatable, reproducible fractionation process based on the proteins' relative hydrophobicity. We present proteomic and biochemical evidence to show that tissue non-specific alkaline phosphatase (TNAP) and Eps15 homology domain-containing protein 1(EDH1) are over-expressed by bovine BCECs after the re-induction of BBB properties. We discuss the impact of these findings on current knowledge of endothelial and BBB permeability. [ABSTRACT FROM AUTHOR]

Background: Brain capillary endothelial cells (BCECs) form the physiological basis of the blood-brain barrier (BBB). The barrier function is (at least in part) due to well-known proteins such as transporters, tight junctions and metabolic barrier proteins (e.g. monoamine oxidase, gamma glutamyltranspeptidase and P-glycoprotein). Our previous 2-dimensional gel proteome analysis had identified a large number of proteins and revealed the major role of dynamic cytoskeletal remodelling in the differentiation of bovine BCECs. The aim of the present study was to elaborate a reference proteome of Triton X-100-soluble species from bovine BCECs cultured in the wellestablished in vitro BBB model developed in our laboratory. Results: A total of 215 protein spots (corresponding to 130 distinct proteins) were identified by 2-dimensional gel electrophoresis, whereas over 350 proteins were identified by a shotgun approach. We classified around 430 distinct proteins expressed by bovine BCECs. Our large-scale gene expression analysis enabled the correction of mistakes referenced into protein databases (e.g. bovine vinculin) and constitutes valuable evidence for predictions based on genome annotation. Conclusions: Elaboration of a reference proteome constitutes the first step in creating a gene expression database dedicated to capillary endothelial cells displaying BBB characteristics. It improves of our knowledge of the BBB and the key proteins in cell structures, cytoskeleton organization, metabolism, detoxification and drug resistance. Moreover, our results emphasize the need for both appropriate experimental design and correct interpretation of proteome datasets. [ABSTRACT FROM AUTHOR]

The blood–brain barrier (BBB) is one of the most complex and selective barriers in the human organism. Its role is to protect the brain and preserve the homeostasis of the central nervous system (CNS). The central elements of this physical and physiological barrier are the endothelial cells that form a monolayer of tightly joined cells covering the brain capillaries. However, as endothelial cells regulate nutrient delivery and waste product elimination, they are very sensitive to signals sent by surrounding cells and their environment. Indeed, the neuro-vascular unit (NVU) that corresponds to the assembly of extracellular matrix, pericytes, astrocytes, oligodendrocytes, microglia and neurons have the ability to influence BBB physiology. Extracellular vesicles (EVs) play a central role in terms of communication between cells. The NVU is no exception, as each cell can produce EVs that could help in the communication between cells in short or long distances. Studies have shown that EVs are able to cross the BBB from the brain to the bloodstream as well as from the blood to the CNS. Furthermore, peripheral EVs can interact with the BBB leading to changes in the barrier's properties. This review focuses on current knowledge and potential applications regarding EVs associated with the BBB. [ABSTRACT FROM AUTHOR]