Liver X receptor alpha ensures blood-brain barrier function by suppressing SNAI2.

In Alzheimer's disease (AD) more than 50% of the patients are affected by capillary cerebral amyloid-angiopathy (capCAA), which is characterized by localized hypoxia, neuro-inflammation and loss of blood-brain barrier (BBB) function. Moreover, AD patients with or without capCAA display increased vessel number, indicating a reactivation of the angiogenic program. The molecular mechanism(s) responsible for BBB dysfunction and angiogenesis in capCAA is still unclear, preventing a full understanding of disease pathophysiology. The Liver X receptor (LXR) family, consisting of LXRα and LXRß, was reported to inhibit angiogenesis and particularly LXRα was shown to secure BBB stability, suggesting a major role in vascular function. In this study, we unravel the regulatory mechanism exerted by LXRα to preserve BBB integrity in human brain endothelial cells (BECs) and investigate its role during pathological conditions. We report that LXRα ensures BECs identity via constitutive inhibition of the transcription factor SNAI2. Accordingly, deletion of brain endothelial LXRα is associated with impaired DLL4-NOTCH signalling, a critical signalling pathway involved in vessel sprouting. A similar response was observed when BECs were exposed to hypoxia, with concomitant LXRα decrease and SNAI2 increase. In support of our cell-based observations, we report a general increase in vascular SNAI2 in the occipital cortex of AD patients with and without capCAA. Importantly, SNAI2 strongly associated with vascular amyloid-beta deposition and angiopoietin-like 4, a marker for hypoxia. In hypoxic capCAA vessels, the expression of LXRα may decrease leading to an increased expression of SNAI2, and consequently BECs de-differentiation and sprouting. Our findings indicate that LXRα is essential for BECs identity, thereby securing BBB stability and preventing aberrant angiogenesis. These results uncover a novel molecular pathway essential for BBB identity and vascular homeostasis providing new insights on the vascular pathology affecting AD patients.

Increased matrix metalloproteinases expression in tuberous sclerosis complex: modulation by microRNA 146a and 147b in vitro.

AIM: Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) control proteolysis within the extracellular matrix (ECM) of the brain. Dysfunction of this enzymatic system due to brain inflammation can disrupt the blood-brain barrier (BBB) and has been implicated in the pathogenesis of epilepsy. However, this has not been extensively studied in the epileptogenic human brain. METHODS: We investigated the expression and cellular localization of major MMPs (MMP2, MMP3, MMP9 and MMP14) and TIMPs (TIMP1, TIMP2, TIMP3 and TIMP4) using quantitative real-time polymerase chain reaction (RT-PCR) and immunohistochemistry in resected epileptogenic brain tissue from patients with tuberous sclerosis complex (TSC), a severe neurodevelopmental disorder characterized by intractable epilepsy and prominent neuroinflammation. Furthermore, we determined whether anti-inflammatory microRNAs, miR146a and miR147b, which can regulate gene expression at the transcriptional level, could attenuate dysregulated MMP and TIMP expression in TSC tuber-derived astroglial cultures. RESULTS: We demonstrated higher mRNA and protein expression of MMPs and TIMPs in TSC tubers compared to control and perituberal brain tissue, particularly in dysmorphic neurons and giant cells, as well as in reactive astrocytes, which was associated with BBB dysfunction. More importantly, IL-1ß-induced dysregulation of MMP3, TIMP2, TIMP3 and TIMP4 could be rescued by miR146a and miR147b in tuber-derived TSC cultures. CONCLUSIONS: This study provides evidence of dysregulation of the MMP/TIMP proteolytic system in TSC, which is associated with BBB dysfunction. As dysregulated MMP and TIMP expression can be ameliorated in vitro by miR146a and miR147b, these miRNAs deserve further investigation as a novel therapeutic approach.

Purinergic receptors P2Y12R and P2X7R: potential targets for PET imaging of microglia phenotypes in multiple sclerosis.

BACKGROUND: Microglia are major players in the pathogenesis of multiple sclerosis (MS) and may play a dual role in disease progression. The activation status of microglia in vivo is highly dynamic and occurs as a continuum, with the pro-inflammatory and anti-inflammatory phenotypes on either end of this spectrum. Little is known about in vivo dynamics of microglia phenotypes in MS due to the lack of diagnostic tools. Positron emission tomography (PET) imaging is a powerful non-invasive technique that allows real-time imaging of microglia activation phenotypes in the central nervous system, depending on the availability of selective PET tracers. Our objective is to investigate and characterize the expression of the purinergic receptors P2Y12R and P2X7R as potential targets for PET tracer development and subsequent PET imaging in order to evaluate the dynamics of microglia status in vivo. METHODS: We used immunohistochemical analysis to explore the expression of P2Y12R and P2X7R in experimental autoimmune encephalomyelitis (EAE) post-mortem tissues and different stages of well-characterized MS lesions. We evaluated by quantitative real-time polymerase chain reaction the expression of P2Y12R and P2X7R in human polarized microglia, and we performed autoradiography binding assay with radiolabeled P2Y12R and P2X7R antagonists using MS and rat EAE tissues. RESULTS: Here, we demonstrate that P2X7R is associated with a pro-inflammatory phenotype of human microglia in vitro, and is highly expressed in microglia in MS lesions as well as during the peak of EAE. In contrast, P2Y12R was associated with an anti-inflammatory phenotype in human microglia in vitro and was expressed at lower levels in active inflammatory MS lesions compared to normal-appearing white matter (NAWM) and similarly in EAE, while its expression increased in the remission phase of EAE. Binding of radiolabeled tracers specific for P2Y12R and P2X7R on ex vivo tissues validated the value of these receptors as PET imaging targets for microglia phenotypes in vivo. CONCLUSION: Our results suggest that P2Y12R and P2X7R are excellent targets for PET imaging to discriminate distinct microglia phenotypes in MS. Ultimately, this may provide insight into the role of microglia in disease progression and monitor novel treatment strategies to alter microglia phenotype.

Approach for defining endogenous reference genes in gene expression experiments.

The quantification of gene expression by real-time polymerase chain reaction (PCR) has revolutionized the field of gene expression analysis. Due to its sensitivity and flexibility it is becoming the method of choice for many investigators. However, good normalization protocols still have to be implemented to facilitate data exchange and comparison. We have designed primers for 10 unrelated genes and developed a simple protocol to detect genes with stable expression that are suitable for use as endogenous reference genes for further use in the normalization of gene expression data obtained by real-time PCR. Using this protocol, we were able to identify human proteosome subunit Y as a reliable endogenous reference gene for human umbilical vein endothelial cells treated for up to 18 h with TNFalpha, IL-4, or IFNgamma and for B cells isolated from healthy controls and patients suffering from IgA nephropathy. Other optional endogenous reference genes that can be considered are phosphomannomutase (PPMM) and actin for endothelial cells and glyceraldehyde-3-phosphate dehydrogenase and PPMM for B cells.

Increased alpha3-fucosylation of alpha1-acid glycoprotein in Type I diabetic patients is related to vascular function.

Diabetic mellitus is attended by the development of endothelial dysfunction which is suggested to be accompanied with a chronic low-degree of inflammation. During a chronic hepatic inflammatory response, specific changes in glycosylation of the acute phase protein alpha1-acid glycoprotein (AGP) can be detected. In this report we studied the changes in glycosylation of AGP in more detail and evaluated the relation between a change in glycosylation of AGP and urinary albumin secretion in Type I diabetic patients. The glycosylation of AGP, studied by crossed affinity immunoelectrophoresis (CAIE) and high pH anion exchange chromatography with pulse amperometric detection (HPAEC-PAD), showed an increase in alpha3-fucosylation. Staining with an antibody against sialyl Lewis(x) (sLe(x)) implied that part of the alpha3-fucosylation was present in a sLe(x)-conformation. In the group of Type I diabetic patients with increased urinary albumin excretion, a significant increase in alpha3-fucosylation of AGP (p<0.0005) could be detected. Therefore, the increased alpha3-fucosylation of AGP can be used as an additional marker for the development of vascular complications in Type I diabetic patients.

Differences in sulfation patterns of heparan sulfate derived from human bone marrow and umbilical vein endothelial cells.

OBJECTIVE: Heparan sulfates (HS), the polysaccharide side chains of HS proteoglycans, differ in structure and composition of sulfated domains among various tissue types, resulting in selective protein binding. HS proteoglycans on bone marrow endothelial cells (BMEC) could contribute to tissue specificity of the bone marrow endothelium and play a role in the presentation of chemokines such as stromal cell-derived factor-1 (SDF-1) and adhesion of hematopoietic progenitor cells after stem cell transplantations. We characterized differences in HS structure and SDF-1 binding between BMEC and human umbilical vein endothelial cells (HUVEC). MATERIALS AND METHODS: Expression of HS proteoglycans on human bone marrow microvessels was investigated by immunohistochemical staining. Comparison of three human BMEC cell lines with HUVEC and an HUVEC cell line was studied by flow cytometry using antibodies against different epitopes of the HS polysaccharide chain. HS proteoglycans were biochemically characterized after isolation from metabolically labeled cultures of the BMEC cell line 4LHBMEC and HUVEC. Binding of radiolabeled SDF-1 to 4LHBMEC and HUVEC and competition with heparins were investigated. RESULTS: Bone marrow microvessels constitutively expressed HS proteoglycans. Flow cytometric experiments showed differences in HS chain composition between BMEC and HUVEC. Biochemical characterization revealed more O-sulfation of the N-sulfated domains present in cell-associated HS glycosaminoglycans in 4LHBMEC compared to HUVEC. Binding experiments showed that 4LHBMEC bound more 125[I]-SDF-1 per cell than HUVEC. This could be inhibited largely by heparin and O-sulfated heparin and to a lesser extent by N-sulfated heparin. CONCLUSIONS: Cellular HS from BMEC differs in composition from HUVEC. We postulate that the presence of highly sulfated domains in the HS chains from BMEC contributes to tissue specificity of bone marrow endothelium in which HS may be involved in SDF-1 presentation and adhesion of hematopoietic progenitor cells.

Specific glycosylation of alpha(1)-acid glycoprotein characterises patients with familial Mediterranean fever and obligatory carriers of MEFV.

BACKGROUND: Familial Mediterranean fever (FMF) is a periodic febrile disorder, characterised by fever and serositis. The acute phase response during attacks of FMF results from the release of cytokines, which in turn induce increased expression and changed glycosylation of acute phase proteins. A recent study indicated that attacks in FMF are accompanied by a rise of plasma concentrations of serum amyloid A (SAA) and C reactive protein (CRP), which remain significantly raised during remission relative to healthy controls. Another study suggested that obligatory heterozygotes also display an inflammatory acute phase response. OBJECTIVE: To determine the state of inflammation in homozygotic and heterozygotic MEFV genotypes. METHODS: CRP and SAA were studied by enzyme linked immunosorbent assay (ELISA). The glycosylation of the acute phase protein, alpha(1)-acid glycoprotein (AGP), was visualised with crossed affinoimmunoelectrophoresis with concanavalin A as diantennary glycan-specific component and Aleuria aurantia lectin as fucose-specific affinity component. RESULTS: FMF attacks were associated with an increase (p<0.05) in the serum inflammation parameters CRP, SAA, and AGP. The glycosylation of AGP showed an increase (p<0.05) in fucosylated AGP glycoforms, whereas the branching of the glycans remained unaffected. The glycosylation of AGP in the MEFV carrier group, compared with that in a healthy control group, was characterised by a significant increase (p<0.05) in branching of the glycans, whereas the fucosylation remained unaffected. CONCLUSION: The findings suggest an FMF-specific release of cytokines, resulting in a different glycosylation of AGP between a homozygotic and heterozygotic MEFV genotype.

Increased alpha3-fucosylation of alpha(1)-acid glycoprotein in patients with congenital disorder of glycosylation type IA (CDG-Ia).

Increased fucosylation of the type (sialyl) Lewis(x) was detected on the acute-phase plasma protein alpha(1)-acid glycoprotein (AGP) in patients with the congenital disorder of glycosylation type IA. This is remarkable, because in these patients the biosynthesis of guanosine 5'-diphosphate (GDP)-D-mannose is strongly decreased, and GDP-D-mannose is the direct precursor for GDP-L-fucose, the substrate for fucosyltransferases. The concomitantly occurring increased branching of the glycans of AGP and the increased fucosyltransferase activity in plasma suggest that a chronic hepatic inflammatory reaction has induced the increase in fucosylation.

Severe rheumatoid arthritis prohibits the pregnancy-induced decrease in alpha3-fucosylation of alpha1-acid glycoprotein.

Patients suffering from rheumatoid arthritis (RA) may experience a temporary reduction of disease symptoms during pregnancy. As indicated by the occurrence of RA-disease symptoms during pregnancy, three categories of patients were defined, namely, remission, relapse and unchanged. In all three categories changes in the plasma level and glycosylation of alpha1-acid glycoprotein (AGP) were determined longitudinally in comparison to those occurring in pregnancy of healthy women. In healthy pregnancy, we observed: (i) a peak in the plasma concentration at week 18 and a minimum at week 30; (ii) a continuous increase in the degree of branching of the glycans during the entire pregnancy period, and (iii) a decrease in the degree of alpha3-fucosylation of AGP-glycans with a minimum occurring at week 25. Comparable pregnancy-induced changes in glycosylation were found for two other acute-phase proteins alpha1-protease inhibitor (PI) and alpha1-antichymotrypsin (ACT). Increased oestrogen levels, known to occur during pregnancy, may be one of the factors that induce these changes, because the increased branching and decreased alpha3-fucosylation is in agreement with our earlier findings regarding an involvement of this hormone in the regulation of acute phase protein glycosylation in oestrogen-treated males as well as females. In all three clinical categories in RA, pregnancy also induced a continuous increase in the degree of branching of the glycans of AGP. However, similar changes in concentration and fucosylation were only found during remission of the disease symptoms. In the relapse and unchanged categories in RA, the degree of fucosylation and the plasma concentration of AGP remained constant throughout pregnancy. This indicates a relationship between changes in alpha3-fucosylation of AGP and RA disease activity.