Deficiency of inactive rhomboid protein 2 (iRhom2) attenuates diet-induced hyperlipidaemia and early atherogenesis.

AIMS: Atherosclerosis is a chronic inflammatory disease of the arterial vessel wall and anti-inflammatory treatment strategies are currently pursued to lower cardiovascular disease burden. Modulation of recently discovered inactive rhomboid protein 2 (iRhom2) attenuates shedding of tumour necrosis factor-alpha (TNF-α) selectively from immune cells. The present study aims at investigating the impact of iRhom2 deficiency on the development of atherosclerosis. METHODS AND RESULTS: Low-density lipoprotein receptor (LDLR)-deficient mice with additional deficiency of iRhom2 (LDLR-/-iRhom2-/-) and control (LDLR-/-) mice were fed a Western-type diet (WD) for 8 or 20 weeks to induce early or advanced atherosclerosis. Deficiency of iRhom2 resulted in a significant decrease in the size of early atherosclerotic plaques as determined in aortic root cross-sections. LDLR-/-iRhom2-/- mice exhibited significantly lower serum levels of TNF-α and lower circulating and hepatic levels of cholesterol and triglycerides compared to LDLR-/- mice at 8 weeks of WD. Analyses of hepatic bile acid concentration and gene expression at 8 weeks of WD revealed that iRhom2 deficiency prevented WD-induced repression of hepatic bile acid synthesis in LDLR-/- mice. In contrast, at 20 weeks of WD, plaque size, plaque composition, and serum levels of TNF-α or cholesterol were not different between genotypes. CONCLUSION: Modulation of inflammation by iRhom2 deficiency attenuated diet-induced hyperlipidaemia and early atherogenesis in LDLR-/- mice. iRhom2 deficiency did not affect diet-induced plaque burden and composition in advanced atherosclerosis in LDLR-/- mice.

Lipid Peroxidation Plays an Important Role in Chemotherapeutic Effects of Temozolomide and the Development of Therapy Resistance in Human Glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most malignant primary brain tumor. Relapse occurs regularly, and the clinical behavior seems to be due to a therapy-resistant subpopulation of glioma-initiating cells that belong to the group of cancer stem cells. Aldehyde dehydrogenase (ALDH) has been identified as a marker for this cell population, and we have shown previously that ALDH1A3-positive GBM cells are more resistant against temozolomide (TMZ) treatment. However, it is still unclear how ALDH expression mediates chemoresistance. MATERIALS AND METHODS: ALDH1A3 expression was analyzed in 112 specimens from primary and secondary surgical resections of 56 patients with GBM (WHO grade IV). All patients received combined adjuvant radiochemotherapy. For experimental analysis, CRISPR-Cas9-induced knockout cells from three established GBM cell lines (LN229, U87MG, T98G) and two glioma stem-like cell lines were investigated after TMZ treatment. RESULTS: ALDH1A3 knockout cells were more sensitive to TMZ, and oxidative stress seemed to be the molecular process where ALDH1A3 exerts its role in resistance against TMZ. Oxidative stress led to lipid peroxidation, yielding active aldehydes that were detoxified by ALDH enzymatic activity. During the metabolic process, autophagy was induced leading to downregulation of the enzyme, but ALDH1A3 is upregulated to even higher expression levels after finishing the TMZ therapy in vitro. Recurrent GBMs show significantly higher ALDH1A3 expression than the respective samples from the primary tumor, and patients suffering from GBM with high ALDH1A3 expression showed a shorter median survival time (12 months vs 21 months, P < .05). CONCLUSION: Oxidative stress is an important and clinically relevant component of TMZ-induced therapeutic effects. Cytotoxicity seems to be mediated by aldehydes resulting from lipid peroxidation, and ALDH1A3 is able to reduce the number of toxic aldehydes. Therefore, we present a molecular explanation of the role of ALDH1A3 in therapeutic resistance of human GBM cells.

PRAS40 suppresses atherogenesis through inhibition of mTORC1-dependent pro-inflammatory signaling in endothelial cells.

Endothelial pro-inflammatory activation plays a pivotal role in atherosclerosis, and many pro-inflammatory and atherogenic signals converge upon mechanistic target of rapamycin (mTOR). Inhibitors of mTOR complex 1 (mTORC1) reduced atherosclerosis in preclinical studies, but side effects including insulin resistance and dyslipidemia limit their clinical use in this context. Therefore, we investigated PRAS40, a cell type-specific endogenous modulator of mTORC1, as alternative target. Indeed, we previously found PRAS40 gene therapy to improve metabolic profile; however, its function in endothelial cells and its role in atherosclerosis remain unknown. Here we show that PRAS40 negatively regulates endothelial mTORC1 and pro-inflammatory signaling. Knockdown of PRAS40 in endothelial cells promoted TNFα-induced mTORC1 signaling, proliferation, upregulation of inflammatory markers and monocyte recruitment. In contrast, PRAS40-overexpression blocked mTORC1 and all measures of pro-inflammatory signaling. These effects were mimicked by pharmacological mTORC1-inhibition with torin1. In an in vivo model of atherogenic remodeling, mice with induced endothelium-specific PRAS40 deficiency showed enhanced endothelial pro-inflammatory activation as well as increased neointimal hyperplasia and atherosclerotic lesion formation. These data indicate that PRAS40 suppresses atherosclerosis via inhibition of endothelial mTORC1-mediated pro-inflammatory signaling. In conjunction with its favourable effects on metabolic homeostasis, this renders PRAS40 a potential target for the treatment of atherosclerosis.

Aldehyde dehydrogenase 1A3 (ALDH1A3) is regulated by autophagy in human glioblastoma cells.

Aldehyde dehydrogenase is a polymorphic enzyme, which responsible for the oxidation of aldehydes. It has been shown that ALDH1A3 is expressed in human glioblastomas and that its expression correlates with a worse prognosis. In our present study ALDH1A3 expression was associated with resistance against Temozolomide (TMZ) treatment and sensitivity could be re-established in ALDH1A3 knockout cells. TMZ treatment at high concentrations diminished ALDH1A3 protein and this downregulation made the tumor cells more sensitive to chemotherapy. ALDH1A3 was post-transcriptionally regulated since mRNA levels were not affected by TMZ treatment. With increasing concentrations of TMZ, autophagy was up-regulated, and we found evidence for a physical interaction between ALDH1A3 and p62, an important adaptor protein in autophagosomes indicating that ALDH1A3 protein was downregulated by autophagy. So far, the results of the exact role of autophagy in tumor development and tumor growth are inconsistent. Our data indicate that ALDH1A3, that is directly involved in therapy resistance of glioblastoma, is regulated by autophagy during chemotherapy.

Formation of ß-glucogallin, the precursor of ellagic acid in strawberry and raspberry.

Ellagic acid/ellagitannins are plant polyphenolic antioxidants that are synthesized from gallic acid and have been associated with a reduced risk of cancer and cardiovascular diseases. Here, we report the identification and characterization of five glycosyltransferases (GTs) from two genera of the Rosaceae family (Fragaria and Rubus; F. × ananassa FaGT2*, FaGT2, FaGT5, F. vesca FvGT2, and R. idaeus RiGT2) that catalyze the formation of 1-O-galloyl-ß-D-glucopyranose (ß-glucogallin) the precursor of ellagitannin biosynthesis. The enzymes showed substrate promiscuity as they formed glucose esters of a variety of (hydroxyl)benzoic and (hydroxyl)cinnamic acids. Determination of kinetic values and site-directed mutagenesis revealed amino acids that affected substrate preference and catalytic activity. Green immature strawberry fruits were identified as the main source of gallic acid, ß-glucogallin, and ellagic acid in accordance with the highest GT2 gene expression levels. Injection of isotopically labeled gallic acid into green fruits of stable transgenic antisense FaGT2 strawberry plants clearly confirmed the in planta function. Our results indicate that GT2 enzymes might contribute to the production of ellagic acid/ellagitannins in strawberry and raspberry, and are useful to develop strawberry fruit with additional health benefits and for the biotechnological production of bioactive polyphenols.