Cell survival regulation during receptor-mediated endocytosis of chemically-modified lipoproteins associated to the formation of an Amphiphysin 2 (Bin1)/c-Myc complex.

Amphiphysin 2 and members of the BAR-domain family of proteins participate in a wide array of cellular processes including cell cycle and endocytosis. Given that amphiphysin 2 is related to diverse cell responses as a result of metabolic stress, we investigated in macrophages whether oxidative stress originated by the internalization of oxidized low density lipoproteins (oxLDL) affect both, the expression of amphiphysin 2 and its binding partner c-Myc. Here we report that under oxidative stress, a complex formation between amphiphysin 2(Bin1) and c-Myc allows the cell to develop a novel survival equilibrium state established between cell proliferation and cell death. We propose that under conditions of oxidative stress given by the internalization of oxLDL, macrophages employ the formation of the amphiphysin 2(Bin1)/c-Myc complex as a control mechanism to initially avoid the process of cell death in an attempt to prolong cell survival.

Therapeutic Intranasal Vaccine HB-ATV-8 Prevents Atherogenesis and Non-alcoholic Fatty Liver Disease in a Pig Model of Atherosclerosis.

BACKGROUND AND AIMS: Atherosclerosis as an inflammatory disease involved in the etiology of cardiovascular disease worldwide, in our days demands an array of different therapeutic approaches in order to soon be able to visualize an effective prevention. Based on an immunotherapeutic approach, we designed a non-invasive vaccine (HB-ATV-8), contained in a micellar nanoparticle composed of lipids and a peptide segment derived from the C-terminus of the cholesterol-ester transfer protein (CETP). Now we extend our successful proof of concept from the rabbit to a porcine model and investigated its effect in an attempt to undoubtedly establish the efficacy of vaccination in a model closer to the human. METHODS: A preclinical trial was designed to study the efficacy of vaccine HB-ATV-8 in pigs (Large White × Landrace). Male experimental animals were fed with standard diet (control), high fat diet (HFD) or the same HFD but treated with HB-ATV-8 (HFD + Vaccine) applied nasally for up to 7 months. All biochemical and enzymatic analyses were performed in peripheral venous blood and thoracic aorta and liver samples examined using conventional, two-photon excitation and second harmonic generation microscopy to identify atherosclerotic and hepatic lesions. mRNA concentrations for KLF2, ACTA2, SOD1, COL1A1 genes and protein levels for PPARα and ABCA1 were quantified in aorta and liver respectively using qPCR and Western blot analysis. RESULTS: The administration of vaccine HB-ATV-8 induced anti-CETP IgG antibodies and reduced atherosclerotic and hepatic lesions promoted by the high fat diet. In addition, plasma triglyceride levels of vaccine treated pigs fed the HFD were similar to those of control group, in contrast to high concentrations reached with animals exclusively fed with HFD. Moreover, HFD promotes a tendency to decrease hepatic PPARα levels and increase in aorta gene expression of KLF2, ACTA2, SOD1 and COL1A1, while vaccine application promotes recovery close to control values. CONCLUSIONS: Vaccine HB-ATV-8 administration constitutes a promissory preventive approach useful in the control of atherogenesis and fatty liver disease. The positive results obtained, the non-invasive characteristics of the vaccine, the simple design employed in its conception and its low production cost, support the novelty of this therapeutic strategy designed to prevent the process of atherogenesis and control the development of fatty liver disease.

Early Transcriptomic Response to LDL and oxLDL in Human Vascular Smooth Muscle Cells.

BACKGROUND: Although nowadays it is well known that the human transcriptome can importantly vary according to external or environmental condition, the reflection of this concept when studying oxidative stress and its direct relationship with gene expression profiling during the process of atherogenesis has not been thoroughly achieved. OBJECTIVE: The ability to analyze genome-wide gene expression through transcriptomics has shown that the genome responds dynamically to diverse stimuli. Here, we describe the transcriptome of human vascular smooth muscle cells (hVSMC) stimulated by native and oxidized low-density lipoprotein (nLDL and oxLDL respectively), with the aim of assessing the early molecular changes that induce a response in this cell type resulting in a transcriptomic transformation. This expression has been demonstrated in atherosclerotic plaques in vivo and in vitro, particularly in the light of the oxidative modification hypothesis of atherosclerosis. APPROACH AND RESULTS: Total RNA was isolated with TRIzol reagent (Life Technologies) and quality estimated using an Agilent 2100 bioanalyzer. The transcriptome of hVSMC under different experimental conditions (1,5 and 24 hours for nLDL and oxLDL) was obtained using the GeneChip Human Gene 1.0 ST (Affymetrix) designed to measure gene expression of 28,869 well-annotated genes. A fixed fold-change cut-off corresponding to ± 2 was used to identify genes exhibiting the most significant variation and statistical significance (P< 0.05), and 8 genes validated by qPCR using Taqman probes. CONCLUSIONS: 10 molecular processes were significantly affected in hVSMC: Apoptosis and cell cycle, extracellular matrix remodeling, DNA repair, cholesterol efflux, cGMP biosynthesis, endocytic mechanisms, calcium homeostasis, redox balance, membrane trafficking and finally, the immune response to inflammation. The evidence we present supporting the hypothesis for the involvement of oxidative modification of several processes and metabolic pathways in atherosclerosis is strengthen by the fact that gene expression patterns obtained when hVSMC are incubated for a long period of time in the presence of nLDL, correspond very much the same as when cells are incubated for a short period of time in the presence of chemically modified oxLDL. Our results indicate that under physiological conditions and directly related to specific environmental conditions, LDL particles most probably suffer chemical modifications that initially serve as an alert signal to overcome a harmful stimulus that with time might get transformed to a pathological pattern and therefore consolidate a pathological condition.

Osteopontin upregulation in atherogenesis is associated with cellular oxidative stress triggered by the activation of scavenger receptors.

BACKGROUND: Osteopontin (OPN) is a highly phosphorylated sialoprotein and a prominent component of mineralized extracellular matrices of bones and teeth. Although the structure of OPN has begun to be elucidated, the role of OPN overexpression in tissues distant from the bones and teeth remains poorly understood. In the present study, a rabbit model of hypercholesterolemia was employed to analyze the relationship between the vascular calcification process and OPN overexpression in the neointima of atherosclerotic plaques. METHODS: OPN identification in the aorta of experimental animals fed with a high cholesterol diet was carried out by immunohistochemical procedures and Western blot analysis of tissue homogenates. Transmission electron microscopy was employed to localize target-like extracellular structures of atherosclerotic aortas. The human cell line T/G HA-VSMC was employed in the establishment of a ROS generation model employing the internalization of OxLDL particles. RESULTS: Using immunohistochemical and Western blot analysis, OPN overexpression was detected in the aortas of rabbits fed a high-cholesterol diet. Results from the ultrastructural analysis of the rabbit neointima through transmission electron microscopy and from the detection of calcium phosphate precipitates by specific histochemical techniques, suggested that OPN may be functionally important as a regulator of vascular calcification. OPN was dramatically overexpressed by vascular smooth muscle cells in the presence of oxidized and acetylated LDL particles bound to scavenger receptors, thereby promoting cytosolic oxidative stress. CONCLUSIONS: This study establishes the in vivo role of OPN in the intima of the aorta regulating calcium phosphate precipitate deposition in response to oxidative stress.