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In brain, excess cholesterol is metabolized into 24S-hydroxycholesterol (24S-OH-chol) and eliminated into the circulation across the blood-brain barrier. 24S-OH-chol is a natural agonist of the nuclear liver X receptors (LXRs) involved in peripheral cholesterol homeostasis. The effects of this oxysterol on the pericytes embedded in the basal lamina of this barrier (close to the brain compartment) have not been previously studied. We used primary cultures of brain pericytes to demonstrate that the latter express LXR nuclear receptors and their target gene ATP-binding cassette, sub-family A, member 1 (ABCA1), known to be one of the major transporters involved in peripheral lipid homeostasis. Treatment with 24S-OH-chol caused an increase in ABCA1 expression that correlated with a reverse cholesterol transfer to apolipoprotein E, apolipoprotein A-I, and high density lipoprotein particles. Inhibition of ABCA1 decreased this efflux. As pericytes are able to internalize the amyloid-β peptides which accumulate in brain of Alzheimer's disease patients, we then investigated the effects of 24S-OH-chol on this process. We found that the cellular accumulation process was not modified by 24S-OH-chol treatment. Overall, our results highlight the importance of the LXR/ABCA1 system in brain pericytes and suggest a new role for these cells in brain cholesterol homeostasis.

P-glycoprotein (P-gp) and the multidrug resistance-associated proteins (MRP), whose expression is associated with multidrug resistance, have been recently located in the brain capillary endothelial cells (BCEC) forming the blood-brain barrier (BBB), without taking into account a possible influence or contribution of glial cells and pericytes. Using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), the present study analysed the transcriptional expression of P-gp and the seven homologues of MRP transporters in BCECs in solo culture or in an in vitro model of the BBB consisting of a co-culture of BCECs and glial cells. Pericytes, glial cells, isolated brain capillaries and bovine grey matter extracts were also tested. P-gp mRNA, absent in glial cells, was found in brain capillaries and in co-cultured BCECs with an increased signal compared to the in solo culture. No amplification was observed in pericytes or grey matter. While MRP2, MRP3 and MRP7 remained undetected, MRP1, absent in capillaries or grey matter, was amplified in BCECs, glial cells and pericytes. MRP4 gave a low signal in most cultures. MRP5 was ubiquitously expressed, displaying a potent signal in all conditions. In spite of its presence in cultured glial cells, MRP6 mRNA expression appeared to be restricted to BCECs, with the same upregulation in the co-cultured condition as observed with P-gp. Moreover, MRP6 was the only transporter whose endothelial mRNA expression was influenced by the presence of pericytes. The tissue distribution of the expression of these transporters and the contribution of the different cell populations are discussed.

Abstract Background The function of pericytes remains questionable but with improved cultured technique and the use of genetically modified animals, it has become increasingly clear that pericytes are an integral part of blood–brain barrier (BBB) function, and the involvement of pericyte dysfunction in certain cerebrovascular diseases is now emerging. The porcine stress syndrome (PSS) is the only confirmed, homologous model of malignant hyperthermia (MH) in veterinary medicine. Affected animals can experience upon slaughter a range of symptoms, including skeletal muscle rigidity, metabolic acidosis, tachycardia and fever, similar to the human syndrome. Symptoms are due to an enhanced calcium release from intracellular stores. These conditions are associated with a point mutation in ryr1/hal gene, encoding the ryanodine receptor, a calcium channel. Important blood vessel wall muscle modifications have been described in PSS, but potential brain vessel changes have never been documented in this syndrome. Methods In the present work, histological and ultrastructural analyses of brain capillaries from wild type and ryr1 mutated pigs were conducted to investigate the potential impairment of pericytes, in this pathology. In addition, brain pericytes were isolated from the three porcine genotypes (wild-type NN pigs; Nn and nn pigs, bearing one or two (n) mutant ryr1/hal alleles, respectively), and tested in vitro for their influence on the permeability of BBB endothelial monolayers. Results Enlarged perivascular spaces were observed in ryr1-mutant samples, corresponding to a partial or total detachment of the astrocytic endfeet. These spaces were electron lucent and sometimes filled with lipid deposits and swollen astrocytic feet. At the ultrastructural level, brain pericytes did not seem to be affected because they showed regular morphology and characteristics, so we aimed to check their ability to maintain BBB properties in vitro. Our results indicated that pericytes from the three genotypes of pigs had differing influences on the BBB. Unlike pericytes from NN pigs, pericytes from Nn and nn pigs were not able to maintain low BBB permeability. Conclusions Electron microscopy observations demonstrated brain capillary modifications in PSS condition, but no change in pericyte morphology. Results from in vitro experiments suggest that brain pericytes from ryr1 mutated pigs, even if they are not affected by this condition at the ultrastructural level, are not able to maintain BBB integrity in comparison with pericytes from wild-type animals.

Around 7–17% of metastatic breast cancer patients will develop brain metastases, associated with a poor prognosis. To reach the brain parenchyma, cancer cells need to cross the highly restrictive endothelium of the Blood-Brain Barrier (BBB). As treatments for brain metastases are mostly inefficient, preventing cancer cells to reach the brain could provide a relevant and important strategy. For that purpose an in vitro approach is required to identify cellular and molecular interaction mechanisms between breast cancer cells and BBB endothelium, notably at the early steps of the interaction. However, while numerous studies are performed with in vitro models, the heterogeneity and the quality of BBB models used is a limitation to the extrapolation of the obtained results to in vivo context, showing that the choice of a model that fulfills the biological BBB characteristics is essential. Therefore, we compared pre-established and currently used in vitro models from different origins (bovine, mice, human) in order to define the most appropriate tool to study interactions between breast cancer cells and the BBB. On each model, the BBB properties and the adhesion capacities of breast cancer cell lines were evaluated. As endothelial cells represent the physical restriction site of the BBB, all the models consisted of endothelial cells from animal or human origins. Among these models, only the in vitro BBB model derived from human stem cells both displayed BBB properties and allowed measurement of meaningful different interaction capacities of the cancer cell lines. Importantly, the measured adhesion and transmigration were found to be in accordance with the cancer cell lines molecular subtypes. In addition, at a molecular level, the inhibition of ganglioside biosynthesis highlights the potential role of glycosylation in breast cancer cells adhesion capacities. [ABSTRACT FROM AUTHOR]