Birgit Ebner

Synthetic LXR agonist attenuates plaque formation in apoE-/- mice without inducing liver steatosis and hypertriglyceridemia

Liver X receptors (LXRs) are important regulators of cholesterol and lipid metabolism. LXR agonists have been shown to limit the cellular cholesterol content by inducing reverse cholesterol transport, increasing bile acid production, and inhibiting intestinal cholesterol absorption. Most of them, however, also increase lipogenesis via sterol regulatory element-binding protein-1c (SREBP1c) and carbohydrate response element-binding protein activation resulting in hypertriglyceridemia and liver steatosis. We report on the antiatherogenic properties of the steroidal liver X receptor agonist N,N-dimethyl-3β-hydroxy-cholenamide (DMHCA) in apolipoprotein E (apoE)-deficient mice. Long-term administration of DMHCA (11 weeks) significantly reduced lesion formation in male and female apoE-null mice. Notably, DMHCA neither increased hepatic triglyceride (TG) levels in male nor female apoE-deficient mice. ATP binding cassette transporter A1 and G1 and cholesterol 7α-hydroxylase mRNA abundances were increased, whereas SREBP1c mRNA expression was unchanged in liver, and even decreased in macrophages and intestine. Short-term treatment revealed even higher changes on mRNA regulation. Our data provide evidence that DMHCA is a strong candidate as therapeutic agent for the treatment or prevention of atherosclerosis, circumventing the negative side effects of other LXR agonists.

Action of polystyrene nanoparticles of different sizes on lysosomal function and integrity.

BackgroundData from environmental exposure to nanoparticles (NPs) suggest that chronic exposure may increase the incidence of lung, cardiovascular and neurodegenerative diseases. Impairment of cell function by intracellular accumulation of NPs is also suspected. Many types of NPs have been detected in the endosomal-lysosomal system and, upon repeated exposure, alterations of the endosomal-lysosomal system may occur. To identify such effects we compared the effect of carboxyl polystyrene particles (CPS) of different sizes (20-500 nm) on lysosomes of the endothelial cell line EAhy926 after short (24h) and long (72h-96h) exposure times. Lysosomal localization of CPS, as well as lysosomal pH, lysosomal membrane integrity, morphology of the endosomal-lysosomal system and activities of the lysosomal enzymes,cathepsin B and sulfatases, upon exposure to CPS were recorded.ResultsCPS in sizes ≤100 nm showed high co-localization with lysosomes already after 4h, larger CPS after 24h. None of the particles at non-cytotoxic concentrations caused marked changes in lysosomal pH or destroyed lysosomal membrane integrity. At 24h of exposure, 20 nm CPS induced significant dilatation of the endosomal-lysosomal system and reduced activity of lysosomal sulfatases. After 72h, these alterations were less pronounced.ConclusionsDespite accumulation in lysosomes CPS induced only small changes in lysosomes. Upon longer contact, these changes are even less pronounced. The presented panel of assays may serve to identify effects on lysosomes also for other NPs.

More than cell dust: microparticles isolated from cerebrospinal fluid of brain injured patients are messengers carrying mRNAs, miRNAs, and proteins.

Microparticles are cell-derived, membrane-sheathed structures that are believed to shuttle proteins, mRNA, and miRNA to specific local or remote target cells. To date best described in blood, we now show that cerebrospinal fluid (CSF) contains similar structures that can deliver RNAs and proteins to target cells. These are, in particular, molecules associated with neuronal RNA granules and miRNAs known to regulate neuronal processes. Small RNA molecules constituted 50% of the shuttled ribonucleic acid. Using microarray analysis, we identified 81 mature miRNA molecules in CSF microparticles. Microparticles from brain injured patients were more abundant than in non-injured subjects and contained distinct genetic information suggesting that they play a role in the adaptive response to injury. Notably, miR-9 and miR-451 were differentially packed into CSF microparticles derived from patients versus non-injured subjects. We confirmed the transfer of genetic material from CSF microparticles to adult neuronal stem cells in vitro and a subsequent microRNA-specific repression of distinct genes. This first indication of a regulated transport of functional genetic material in human CSF may facilitate the diagnosis and analysis of cerebral modulation in an otherwise inaccessible organ.

LXR-Agonists Regulate ApoM Expression Differentially in Liver and Intestine

Apolipoprotein M (apoM) has been suggested to play a role in reverse cholesterol transport. Here we studied the influence of liver X-receptor (LXR) agonist on the transcriptional regulation of apoM. Studies were performed in murine liver and intestinal mucosal cells in vivo and in human intestinal Caco-2 cells in vitro. The expression of apoM was analyzed by quantitative real time PCR, and compared to well-established LXR target genes. Mice fed with TO901317 for six days showed a downregulation of apoM and apoAI in the liver to 40 % and 60 % respectively and an upregulation of Cyp7A1 to 280 %. In the small intestine, however, apoM and apoAI were upregulated by 30-60 % and ABCA1 by 250-430 %. In Caco-2 cells TO901317 caused a 60 % upregulation and the natural LXR agonist 22-hydroxycholesterol a 40 % upregulation of apoM. Possible causes for the differential effects in liver and intestine are discussed.

Fetal HDL/apoE: a novel regulator of gene expression in human placental endothelial cells

Maternal lipoproteins have been studied extensively in human pregnancies, but little is known about the role of fetal lipoproteins. The vascularized human placenta interfaces between the mother and fetus to transfer nutrients for sustaining pregnancy. Unlike that of adults, fetal high-density lipoprotein (HDL), which is in contact with placental vessels, is characterized by a high proportion of apolipoprotein E (apoE). We hypothesize this unique composition of fetal HDL affects key functions of the growing fetal tissues. The aim was to identify genes regulated by apoE-HDL by incubating human placental endothelial cells (HPEC) with either fetal HDL or apoE-rich reconstituted HDL particles (apoE-rHDL). HPEC were exposed to 15 μg/ml fetal HDL, 15 μg/ml apoE-rHDL, or medium for 16 h, respectively. Microarray analysis determined genes regulated by fetal HDL and apoE. Characterization of HDL particles revealed a different hydrodynamic radius for apoE-rHDL (13.70 nm) compared with fetal HDL (18.11 nm). Stepwise gene clustering after microarray experiments identified 79 differentially expressed genes (P < 0.05) when cells were exposed to HDL compared with controls. Among them 16 genes were downregulated, whereas five genes were upregulated by twofold, respectively. When HPEC were incubated with apoE-rHDL 18-fold more genes (1,417, 12% of transcripts) were regulated (P < 0.05) in contrast to HDL. Thereof, 172 genes were downregulated and 376 genes upregulated (twofold). In the common subset of 38 genes regulated by both HDL particles, genes involved in cholesterol biosynthesis and cell protection prevailed. Strikingly, results suggest that HDL has the capability of regulating metallothioneins, which may have an effect on oxidative stress in HPEC.

Differential response of arterial and venous endothelial cells to extracellular matrix is modulated by oxygen

Binding of endothelial cell (EC) integrins to extracellular-matrix (ECM) components is one of the key events to trigger intracellular signaling that will ultimately result in proper vascular development. Even within one tissue, the endothelial phenotype differs between arteries and veins. Here, we tested the hypothesis that anchorage-dependent processes, such as proliferation, viability, survival and actin organization of venous (VEC) and arterial EC (AEC) differently depend on ECM proteins. Moreover, because of different oxygen tension in AEC and VEC, we tested oxygen as a co-modulator of ECM effects. Primary human placental VEC and AEC were grown in collagens I and IV, fibronectin, laminin, gelatin and uncoated plates and exposed to 12 and 21% oxygen. Our main findings revealed that VEC are more sensitive than AEC to changes in the ECM composition. Proliferation and survival of VEC, in contrast to AEC, were profoundly increased by the presence of collagen I and fibronectin when compared with gelatin or uncoated plates. These effects were reversed by inhibition of focal adhesion kinase (Fak) and modulated by oxygen. VEC were more susceptible to the oxygen-dependent ECM effects than AEC. However, no differential ECM effect on actin organization was observed between the two cell types. These data provide first evidence that AEC and VEC from the same vascular loop respond differently to ECM and oxygen in a Fak-dependent manner.

Lim1, an Embryonal Transcription Factor, Is Absent in Multicystic Renal Dysplasia, but Reactivated in Nephroblastomas

Objective:Lim1 (Lim homeobox 1) plays an important role during rodent renal development; however, its rolein human kidney development and disease is still unclear. Methods: We investigated LIM1 expression during human renal development, in dysplastic kidneys and in renal neoplasms using immunohistochemistry. RNA levels in renal carcinomas were determined by quantitative RT-PCR, and the potential roles of LIM1 in mesenchymal-epithelial transition and cell cycle were investigated in a cell culture model. Results: LIM1 was detected in pretubular aggregates, S-shaped and comma-shaped bodies as well as immature glomeruli between 10 and 30 weeks of gestation. Eleven dysplastic kidneys showed no expression of LIM1. In contrast, 12 of 32 nephroblastomas showed nuclear positivity. One regressive nephroblastoma had diffuse expression of LIM1 in tubular structures, all others showed focal positivity in mesenchymal, blastemal and epithelial structures. Renal cell carcinomas revealed no expression of LIM1. Overexpression of LIM1 in a cell culture model led to an increase in KERATIN7 expression but no change in the cell cycle. Conclusion: Our study supports the concept of a causative role of LIM1 deficiency in the development of multicystic kidney. In a small subset of nephroblastomas with a more diffuse expression pattern LIM1 might also contribute to the pathogenesis of these lesions.

BMP‐6 and BMPR‐1a are up‐regulated in the growth plate of the fractured tibia

Bone overgrowth is a known phenomenon occurring after fracture of growing long bones with possible long‐term physical consequences for affected children. Here, the physeal expression of bone morphogenetic proteins (BMPs) was investigated in a fracture‐animal model to test the hypothesis that a diaphyseal fracture stimulates the physeal expression of these known key regulators of bone formation, thus stimulating bone overgrowth. Sprague–Dawley rats (male, 4 weeks old), were subjected to a unilateral mid‐diaphyseal tibial fracture. Kinetic expression of physeal BMP‐2, ‐4, ‐6, ‐7, and BMP receptor‐1a (BMPR‐1a) was analyzed in a monthly period by quantitative real time‐polymerase chain reaction and immunohistochemistry. On Days 1, 3, 10, and 14 post‐fracture, no changes in physeal BMPs gene‐expression were detected. Twenty‐nine days post‐fracture, when the fracture was consolidated, physeal expression of BMP‐6 and BMPR‐1a was significantly upregulated in the growth plate of the fractured and contra‐lateral intact bone compared to control (p < 0.005). This study demonstrates a late role of BMP‐6 and BMPR‐1a in fracture‐induced physeal growth alterations and furthermore, may have discovered the existence of a regulatory “cross‐talk” mechanism between the lower limbs whose function could be to limit leg‐length‐discrepancies following the breakage of growing bones.

Is Predisposition for Nephroblastoma Linked to Polymorphisms of the WTX Gene

Inactivation of Wilms´ tumor X (WTX) gene has been linked to the pathogenesis of a varying percentage of nephroblastomas. In contrast, germline mutations of WTX were identified to cause bone dysplasia, but not to induce the development of nephroblastomas. In our study we investigated whether tumor promotion of nephroblastoma by inactivation of WTX gene is linked to certain single nucleotide polymorphisms (SNPs). Therefore 8 SNPs—distributed over the whole length of the WTX gene—were investigated by high resolution melting curve analysis (HRMA) and sequencing of genomic DNA from nephroblastoma patients (NB) and controls. No difference was detected in the 8 SNPs investigated, which were distributed over the whole length of the gene. Additionally, sequence analysis of the coding part of the WTX gene of the tumor samples revealed no chromosomal aberration. Our study indicates, that inactivation of WTX appears to be a late event in tumorigenesis of nephroblastoma in a subgroup of nephroblastomas.

Loss of PTEN/MMAC1 activity is a rare and late event in the pathogenesis of nephroblastomas

Recent genetic investigations of nephroblastomas point to an activation of the Wnt pathway. Data indicate however that activation might be partly due to cross talk of different signaling pathways including the tumor suppressor gene PTEN (phosphatase and tensin homolog on chromosome 10). Therefore, we examined expression and chromosomal aberrations of PTEN in nephroblastomas of different subtypes and the corresponding nephrogenic rests. Loss of heterozygosity was analyzed by high-resolution melting analysis of 4 different single nucleotide polymorphisms. Results were confirmed by sequence analysis of the polymerase chain reaction products. In addition, an intragenic insertion-deletion polymorphism of the PTEN gene was investigated. Protein expression was assessed by immunohistochemistry. Twenty-two nephroblastomas and their corresponding nephrogenic rests were included in the study. In the high-resolution melting analysis, 15 samples were homozygous, 6 were heterozygous, and for 1 sample results could not be obtained for technical reasons. None of the samples showed loss of heterozygosity. Nineteen of the tumors and corresponding nephrogenic rests were also examined immunohistochemically. All tumors showed cytoplasmic positivity, with the exception of 1 tumor that showed complete loss of staining. In 1 tumor, the epithelial component showed distinct cytoplasmic staining, whereas the immature muscle and hyaline cartilage were negative. All nephrogenic rests exhibited positive cytoplasmic staining of all components. Our results establish that inactivation of PTEN is a rare and late event in the pathogenesis of nephroblastomas.