2-Desaza-annomontine (C81) impedes angiogenesis through reduced VEGFR2 expression derived from inhibition of CDC2-like kinases.

Angiogenesis is a crucial process in the progression of various pathologies, like solid tumors, wet age-related macular degeneration, and chronic inflammation. Current anti-angiogenic treatments still have major drawbacks like limited efficacy in diseases that also rely on inflammation. Therefore, new anti-angiogenic approaches are sorely needed, and simultaneous inhibition of angiogenesis and inflammation is desirable. Here, we show that 2-desaza-annomontine (C81), a derivative of the plant alkaloid annomontine previously shown to inhibit endothelial inflammation, impedes angiogenesis by inhibiting CDC2-like kinases (CLKs) and WNT/ß-catenin signaling. C81 reduced choroidal neovascularization in a laser-induced murine in vivo model, inhibited sprouting from vascular endothelial growth factor A (VEGF-A)-activated murine aortic rings ex vivo, and reduced angiogenesis-related activities of endothelial cells in multiple functional assays. This was largely phenocopied by CLK inhibitors and knockdowns, but not by inhibitors of the other known targets of C81. Mechanistically, CLK inhibition reduced VEGF receptor 2 (VEGFR2) mRNA and protein expression as well as downstream signaling. This was partly caused by a reduction of WNT/ß-catenin pathway activity, as activating the pathway induced, while ß-catenin knockdown impeded VEGFR2 expression. Surprisingly, alternative splicing of VEGFR2 was not detected. In summary, C81 and other CLK inhibitors could be promising compounds in the treatment of diseases that depend on angiogenesis and inflammation due to their impairment of both processes.

Transport of lipids from golgi to plasma membrane is defective in tangier disease patients and Abc1-deficient mice.

Mutations in the gene encoding ATP-binding cassette transporter 1 ( ABC1) have been reported in Tangier disease (TD), an autosomal recessive disorder that is characterized by almost complete absence of plasma high-density lipoprotein (HDL), deposition of cholesteryl esters in the reticulo-endothelial system (RES) and aberrant cellular lipid trafficking. We demonstrate here that mice with a targeted inactivation of Abc1 display morphologic abnormalities and perturbations in their lipoprotein metabolism concordant with TD. ABC1 is expressed on the plasma membrane and the Golgi complex, mediates apo-AI associated export of cholesterol and phospholipids from the cell, and is regulated by cholesterol flux. Structural and functional abnormalities in caveolar processing and the trans-Golgi secretory pathway of cells lacking functional ABC1 indicate that lipid export processes involving vesicular budding between the Golgi and the plasma membrane are severely disturbed.

The gene encoding ATP-binding cassette transporter 1 is mutated in Tangier disease.

Tangier disease (TD) is an autosomal recessive disorder of lipid metabolism. It is characterized by absence of plasma high-density lipoprotein (HDL) and deposition of cholesteryl esters in the reticulo-endothelial system with splenomegaly and enlargement of tonsils and lymph nodes. Although low HDL cholesterol is associated with an increased risk for coronary artery disease, this condition is not consistently found in TD pedigrees. Metabolic studies in TD patients have revealed a rapid catabolism of HDL and its precursors. In contrast to normal mononuclear phagocytes (MNP), MNP from TD individuals degrade internalized HDL in unusual lysosomes, indicating a defect in cellular lipid metabolism. HDL-mediated cholesterol efflux and intracellular lipid trafficking and turnover are abnormal in TD fibroblasts, which have a reduced in vitro growth rate. The TD locus has been mapped to chromosome 9q31. Here we present evidence that TD is caused by mutations in ABC1, encoding a member of the ATP-binding cassette (ABC) transporter family, located on chromosome 9q22-31. We have analysed five kindreds with TD and identified seven different mutations, including three that are expected to impair the function of the gene product. The identification of ABC1 as the TD locus has implications for the understanding of cellular HDL metabolism and reverse cholesterol transport, and its association with premature cardiovascular disease.

[Immune mechanisms in retinal degeneration]. / Immunmechanismen bei Netzhautdegeneration.

Hereditary retinal dystrophies are characterised by a common apoptotic pathway leading to progressive retinal degeneration. Similar degenerative processes are evident in multifactorial and complex retinal disorders including age-related macular degeneration. To understand early triggers of these mechanisms, genetic and experimental mouse models have been developed that mimic various forms of human retinal degeneration. In most of these models, early chronic activation of the innate immune system has been documented. This process mainly involves the complement cascade as humoral component and microglial cells as sensors and effectors of the retinal immune response. Current evidence suggests that the genetic predisposition and individual factors like age or diet critically influence the immune homeostasis in the retina. Based on their effectiveness, innate immune mechanisms are thought to support or even provoke retinal degeneration. This review article summarises recent progress in understanding the role of innate immunity in retinal degenerative diseases. We especially focus on human studies and attempt to provide a link between activation of the complement system and microglial function. Moreover, concepts are presented that highlight the retinal immunopathology as potential therapeutic target to prevent vision loss.

[Neuroinflammation in neuronal ceroid lipofuscinosis]. / Neuroinflammation bei neuronalen Ceroid-Lipofuszinosen.

BACKGROUND: Retinal degeneration and neuroinflammation are often early hallmarks of different subtypes of neuronal ceroid lipofuscinosis (NCL) in patients and genetic animal models. OBJECTIVE: This article gives a summary of recently published research articles and novel concepts in the field of NCL-related neuroinflammation. MATERIAL AND METHODS: A search was carried out in PubMed for relevant publications and the results as well as own NCL-related research are discussed. RESULTS: Microglia and other glial cells are chronically activated and show various dysfunctions in the central nervous system (CNS) and retina of NCL patients and animal models. This is accompanied by significant changes in the transcriptome and proteome. In NCL there is also involvement of the adaptive immune response, as demonstrated by the influx of autoantibodies and activated T cells. CONCLUSION: A deeper understanding of the molecular processes that contribute to neuroinflammation and ultimately lead to neuronal cell death is an important basis for the discovery of possible biomarkers and the development of immunotherapies in NCL.

Blockade of microglial adenosine A2A receptor impacts inflammatory mechanisms, reduces ARPE-19 cell dysfunction and prevents photoreceptor loss in vitro.

Age-related macular degeneration (AMD) is characterized by pathological changes in the retinal pigment epithelium (RPE) and loss of photoreceptors. Growing evidence has demonstrated that reactive microglial cells trigger RPE dysfunction and loss of photoreceptors, and inflammasome pathways and complement activation contribute to AMD pathogenesis. We and others have previously shown that adenosine A2A receptor (A2AR) blockade prevents microglia-mediated neuroinflammatory processes and mediates protection to the retina. However, it is still unknown whether blocking A2AR in microglia protects against the pathological features of AMD. Herein, we show that an A2AR antagonist, SCH58261, prevents the upregulation of the expression of pro-inflammatory mediators and the alterations in the complement system triggered by an inflammatory challenge in human microglial cells. Furthermore, blockade of A2AR in microglia decreases the inflammatory response, as well as complement and inflammasome activation, in ARPE-19 cells exposed to conditioned medium of activated microglia. Finally, we also show that blocking A2AR in human microglia increases the clearance of apoptotic photoreceptors. This study opens the possibility of using selective A2AR antagonists in therapy for AMD, by modulating the interplay between microglia, RPE and photoreceptors.

Local complement activation in aqueous humor in patients with age-related macular degeneration.

PurposeTo investigate complement activation in aqueous humor and in plasma of patients with neovascular age-related macular degeneration (nAMD).Patients and methodsAqueous humor and EDTA-plasma of 31 nAMD patients and 30 age-matched controls was collected. The levels of the complement factor 3 (C3), the regulators factor H (FH), and factor I (FI), and of the complement activation products Ba, C3a, and the terminal complement complex (sC5b-9) were measured. Associations between complement levels and phenotype were determined using Mann-Whitney U-test.ResultsIn plasma, no significant differences were found between the nAMD group and the control group. In aqueous humor, significantly increased levels of Ba (P=0.002), and C3a (P=0.002) indicate local complement activation in nAMD patients and a trend for a concomitant upregulation of the complement regulators FH (P=0.02) and FI (P=0.04).ConclusionsOur findings provide strong evidence for a local complement dysregulation in nAMD patients.

Genomic organization and characterization of the promoter of the human ATP-binding cassette transporter-G1 (ABCG1) gene.

The ATP-binding cassette transporter G1 (ABCG1) was recently identified as a regulator of macrophage cholesterol and phospholipid transport. This transporter together with ABCA1 belongs to a group of sterol-sensitive ABC proteins which are induced by lipid loading or specific oxysterols. We report here the genomic structure of ABCG1 along with the 5' flanking sequence using library screening and BLAST search analysis. The ABCG1 gene spans more than 70 kb and contains 15 exons. The exon size is between 30 and 1081 bp and the introns range in size from 137 bp to more than 45 kb. All exon-intron boundaries display the canonical GT/AG sequences. Using promoter-luciferase reporter assays in the myeloid cell lines THP-1 and RAW246.7 and the hepatoma cell line HepG2 we could demonstrate the functionality of the ABCG1 promoter and the minimal sequence requirements for gene expression. The TATA-less proximal promoter contains multiple Sp1 binding sites and a consensus sequence for sterol regulatory element binding protein.

Identification and characterization of the human adipocyte apM-1 promoter.

The human adipocyte-specific apM-1 gene encodes a secretory protein of the adipose tissue and seems to play a role in the pathogenesis of obesity. A 1.3 kb amount of the proximal promoter region has been cloned and analyzed for the presence of putative transcription factor binding sites. Several binding sites known to be involved in adipogenesis and regulation of adipocyte-specific genes (C/EBP, SREBP) are present. No TATA box, but a classical CCAAT box could be identified. To confirm functionality and cell specificity of the 1.3 kb promoter, a series of 5'-deleted fragments were ligated in front of the luciferase gene and the constructs were transfected into 3T3-L1 adipocytes. The reporter gene was effectively transcribed, as demonstrated by the expression of enzyme activity. The 5'-end of the human cDNA was completed by 5'-RACE-PCR. Several alternative transcription start sites were detected by RNase protection assay and primer extension analysis. In addition, an exon/intron boundary was mapped at the extreme 5'-end of the cDNA sequence. Genomic Southern blotting suggests that the human apM-1 gene is a single copy gene.

Structural organization and characterization of the promoter region of a human carboxylesterase gene.

A gene encoding a human liver carboxylesterase has been isolated and characterized. Analysis of three overlapping genomic lambda clones revealed that the gene spans about 30 kb and is made of 14 exons being 39 to 379 bp in length. The encoded protein is 550 amino acids long and is highly homologous to carboxylesterases of various mammalian species. The transcription start site was determined by 5'-RACE PCR. An additional 900 bp of DNA from the 5' flanking region of the gene was cloned and sequenced in order to elucidate the structure of the promoter. In this sequence several possible binding sites for transcription factors have been identified, but no TATA-box was present. When different parts of the putative promoter region were ligated in front of the luciferase gene and the constructs were transfected into CHO cells, the reporter gene was effectively transcribed, as demonstrated by the expression of enzyme activity.

Regulation of scavenger receptor CD163 expression in human monocytes and macrophages by pro- and antiinflammatory stimuli.

CD163, also referred to as M130, a member of the scavenger receptor cysteine-rich family (SRCR) is exclusively expressed on cells of the monocyte lineage. In freshly isolated monocytes the CD14bright CD16+ monocyte subset revealed the highest expression of CD163 among all monocyte subsets. CD163 mRNA and protein expression is up-regulated during macrophage colony-stimulating factor (M-CSF)-dependent phagocytic differentiation of human blood monocytes. In contrast, monocytic cells treated with GM-CSF and interleukin-4 (IL-4) for dendritic differentiation down-regulate this antigen. CD163 expression is also suppressed by proinflammatory mediators like lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), and tumor necrosis factor alpha, whereas IL-6 and the antiinflammatory cytokine interleukin-10 (IL-10) strongly up-regulate CD163 mRNA in monocytes and macrophages. The effects of the immunosuppressants dexamethasone, cyclosporin A (CA), and cortisol differ in their capacity to influence CD163 mRNA levels. Our results demonstrate that CD163 expression in monocytes/macrophages is regulated by proinflammatory and antiinflammatory mediators. This expression pattern implies a functional role of CD 163 in the antiinflammatory response of monocytes.

Characterization of the vaccinia MVA hemagglutinin gene locus and its evaluation as an insertion site for foreign genes.

The 'Modified Vaccinia Ankara' (MVA) strain is a potential live vaccine vector. The use of the hemagglutinin (ha) gene of the MVA strain as an insertion site for foreign genes was evaluated. To identify the molecular basis of the hemagglutinin-negative (HA-) phenotype of MVA, the ha gene and the region around this gene were sequenced. Amino acid (aa) sequence comparisons with functional hemagglutinins of other vaccinia strains predicted a functional polypeptide. The late part of the promoter region of the ha gene, however, was deleted, causing the apparent loss of the ha gene function. Nevertheless, insertion of foreign DNA into the ha gene allowed generation of functional recombinant viruses, indicating that the ha-gene region is a suitable insertion site.

The satiety factor apolipoprotein A-IV modulates intestinal epithelial permeability through its interaction with alpha-catenin: implications for inflammatory bowel diseases.

INTRODUCTION: Apolipoprotein A-IV (apoA-IV), an intestinally and cerebrally synthesized satiety factor and anti-atherogenic plasma apolipoprotein, was recently identified as an anti-inflammatory protein. In order to elucidate whether intestinal apoA-IV exerts similar repair function as its hepatic homologue apolipoprotein A-V (apoA-V), apoA-IV-interactive proteins were searched and in vitro functional studies were performed with apoA-IV overexpressing cells. ApoA-IV was also analyzed in the intestinal mucosa of patients with inflammatory bowel diseases (IBD), together with other genes involved in epithelial junctional integrity. METHODS: A yeast-two-hybrid screening was used to identify apoA-IV-interactors. ApoA-IV was overexpressed in Caco-2 and HT-29 mucosal cells for colocalization and in vitro epithelial permeability studies. Mucosal biopsies from quiescent regions of colon transversum and terminal ileum were subjected to DNA-microarray analysis and pathway-related data mining. RESULTS: Four proteins interacting with apoA-IV were identified, including apolipoprotein B-100, alpha1-antichymotrypsin, cyclin C, and the cytosolic adaptor alpha-catenin, thus linking apoA-IV to adherens junctions. Overexpression of apoA-IV was paralleled with a differentiated phenotype of intestinal epithelial cells, upregulation of junctional proteins, and decreased paracellular permeability. Colocalization between alpha-catenin and apoA-IV occurred exclusively in junctional complexes. ApoA-IV was downregulated in quiescent mucosal tissues from patients suffering from IBD. In parallel, only a distinct set of junctional genes was dysregulated in non-inflamed regions of IBD gut. CONCLUSIONS: ApoA-IV may act as a stabilizer of adherens junctions interacting with alpha-catenin, and is likely involved in the maintenance of junctional integrity. ApoA-IV expression is significantly impaired in IBD mucosa, even in non-inflamed regions.

Metabolic learning in the intestine: adaptation to nutrition and luminal factors.

There is increasing evidence that the magnitude and potential of intestinal nutrient absorption (sugars, fatty acids, cholesterol and triglycerides) and intestinal defense function are regulated by metabolic learning phenomena, and are influenced by dietary energy content and exercise. Metabolic overload syndromes, mainly obesity, and chronic malabsorption disorders such as inflammatory bowel disease and celiac disease have been defined as extreme phenotypes. Metabolic learning processes depend on developmental and transcriptional control systems of intestinal epithelial cell differentiation. The physiological differentiation zone of enterocytes is linked to the beta-catenin system, apolipoprotein apoA-IV and the master transcription factors Cdx2, HNF1alpha, and GATA4. In addition to these developmental regulatory transcription factors, nuclear receptors including RXR, LXR, PPAR, PXR, and CAR have been implicated in the generation of more absorptive enterocytes with a more differentiated phenotype on the one hand, and dedifferentiated cells with reduced capacity of detoxification and defense causing loss of junction control and barrier defects on the other. Large-scale analysis of gene expression profiles and identification of key pathways and master regulatory transcription factors will help dissect the role of nutritional and environmental factors as well as pharmacological intervention on mucosal homeostasis and disease, with potential applications for diagnosis and therapy.

Structure, function and regulation of the ABC1 gene product.

The role of the ATP-binding cassette transporter 1 (ABCA1) in cellular lipid efflux and high density lipoprotein metabolism has been recently documented by mutations in genetic HDL deficiency syndromes such as classical Tangier disease. Analysis of ABCA1 knockout mice and overexpression studies have established the importance of ABCA1 as a major determinant of HDL cholesterol in plasma. These studies also indicate that ABCA1 is critically involved in cellular trafficking of cholesterol and choline-phospholipids and in total body lipid homeostasis, such as intestinal cholesterol and fat-soluble vitamin absorption and in the modulation of steroidogenesis. First insights into the upregulation of ABCA1 gene expression by cellular cholesterol and cAMP have identified critical ABCA1 promoter elements, which bind the transcription factors liver X receptor, retinoid X receptor, Sp1 and E-box proteins. The finding that a lipid sensitive subgroup of ABC transporters is able to translocate cholesterol and phospholipids supports the concept that in ABCA1 deficiency, compensatory mechanisms possibly involving MDR1, MDR3 and MRP-family members could be active. This suggests that a network of ABC transporters involved in cellular lipid transport exists, which is under the tight control of energy pathways directly linked to high density lipoprotein metabolism and atherogenesis.

Role of ABCG1 and other ABCG family members in lipid metabolism.

The molecular cloning and identification of mutations in ATP-binding cassette transporters in hereditary diseases have greatly expanded our knowledge of the normal physiology of intracellular lipid transport processes. In addition to the well-known ATP-binding cassette transporter A1 (ABCA1) molecule, ABC transporters belonging to the ABCG (White) subfamily (ABCG1, ABCG5, and ABCG8) have been shown to be critically involved in the regulation of lipid-trafficking mechanisms in macrophages, hepatocytes, and intestinal mucosa cells. ABCG1, the product of a sterol-induced gene, participates in cholesterol and phospholipid efflux. The ABCG5 and ABCG8 transporters, defective in beta-sitosterolemia, are also now considered interesting targets in the control and influence of total body sterol homeostasis. In this review, advances referring to the regulation and function of ABCG half-size transporters are summarized and discussed. In addition, new implications for the transcriptional control, as well as the intracellular routing and localization, of these proteins are presented.

Differentiation-dependent expression of a human carboxylesterase in monocytic cells and transcription factor binding to the promoter.

Carboxylesterases play an important role in defense and clearance mechanisms of the monocyte/macrophage system. During the differentiation process of cells from the monocytic cell line THP-1 we observed a transient transcriptional upregulation of a human carboxylesterase analyzed by means of Northern blots. In PMA-treated THP-1 cells we could detect three major transcription initiation sites as revealed by Nuclease Protection Assay carried out with two overlapping antisense RNA probes. We have recently cloned the carboxylesterase upstream sequence and showed its basal promoter activity in CHO cells. Using electrophoretic mobility shift analysis we demonstrated that the promoter region spanning base pairs -1 to -275, which contains several putative binding sites for transcription factors, is bound by nuclear factors Sp1 and IRBP but not by C/EBPs. Taken together these data indicate that carboxylesterase gene transcription in THP-1 cells starts at multiple initiation sites and that Sp1 and IRBP may be critical factors for modulating the differentiation-dependent transcription of this human carboxylesterase gene.

Genomic organization and chromosomal localization of the human CD163 (M130) gene: a member of the scavenger receptor cysteine-rich superfamily.

The human protein CD163 (M130) is a member of the scavenger receptor cysteine-rich (SRCR) superfamily, which is exclusively expressed by monocytes and macrophages. Here, we investigated the genomic organization and the chromosomal localization of the human CD163 gene. The CD163 gene is composed of 17 exons and 16 introns and spans over 35 kb. Each of its nine SRCR domains is encoded by a separate exon, which is similar to other members of the group B SRCR subfamily. Two cytoplasmic variants of CD163 arise from alternative splicing of intron 15, while a truncated and an extracellular variant results from alternative splicing of intron 5 or intron 7, respectively. Using fluorescence in situ hybridization we mapped this gene to the human chromosome 12p13. The transcription initiation sites of the CD163 gene were determined and the 5'-flanking region was sequenced. The nucleotide analysis revealed several putative binding sites for transcription factors, which have been shown to play an important role in myeloid specific gene expression. In addition, we identified a L1 element located 1.4 kb upstream of the major transcription initiation site.

Identification of regulatory elements in the human adipose most abundant gene transcript-1 ( apM-1) promoter: role of SP1/SP3 and TNF-alpha as regulatory pathways.

AIMS/HYPOTHESIS: The human adipocyte-specific apM-1 (adipose most abundant gene transcript-1) gene encodes for a secretory protein of the adipose tissue that seems to play a role in the pathogenesis of obesity-related insulin resistance and its expression is inhibited by TNF-alpha. Our aim was to characterize the tissue-specific regulation of the recently cloned apM-1 promoter and the mechanisms of TNF-alpha-induced downregulation of the apM-1 gene. METHODS: We characterised the apM-1 gene by electrophoretic mobility shift assays (EMSA) and luciferase reporter gene assays (LRA). RESULTS: Although several putative binding sites for transcription factors known to be involved in adipogenesis such as C/EBP and PPARgamma are present in the promoter, we could not detect any binding of these nuclear proteins from differentiated adipocytes. However, a proximal SP1 binding site specifically binds both, recombinant SP1 protein and SP1 derived from adipocyte nuclear extracts. Since the expression of SP1 during adipocyte differentiation has not yet been analysed, we could show by using EMSA, that binding activity of SP1 is increased during adipocyte differentiation. The stimulatory activity of SP1 was confirmed in LRA by cotransfection experiments in S2 Schneider cells lacking endogenous SP factors. An inhibitory activity of SP3 on the stimulatory effect of SP1 could be confirmed in LRA by contransfection experiments in adipocytes. Nuclear extracts from adipocytes incubated with TNF-alpha showed a reduced binding activity of SP1. CONCLUSION/INTERPRETATION: SP1 is expressed and its binding activity is enhanced during adipocyte differentiation. SP1 has stimulatory effects, SP3 has inhibitory effects on apM-1 promoter activity, mediated by a proximal SP1 binding site. The mechanism of TNF-alpha-induced inhibition of apM-1 gene expression is, at least in part, due to a decrease of transcriptional SP1 binding activity caused by TNF-alpha and thus provides a new mechanism of TNF-alpha-dependent signalling.