Shlomo Sasson

Apolipoprotein A1 (APOA1) and paraoxonase 1 (PON1) are key proteins of high-density lipoproteins (HDL). The aim of the present study was to obtain and characterize an in vitro model for endogenous APOA1 and PON1 longstanding upregulation in hepatocytes that can be further used to decipher the mechanisms of their protective action. Cultured human hepatocytes (HuH-7 cell line) were transfected with CRISPR/dCas9 activation plasmids targeting APOA1/PON1 genes. Following selection with specific antibiotics, RNA sequencing was used for the transcriptomic characterization of the transfected hepatocytes. The functionality of the secreted APOA1/PON1 was evaluated as the capacity of the conditioned medium (CM) from transfected HuH-7 to modulate the oxidative and inflammatory stress in TNFα-activated primary human umbilical endothelial cells (HUVEC). The results showed that: (1) a robust, longstanding upregulation (46 days) of endogenous APOA1/PON1 was obtained after CRISPR/dCas9 transfection and antibiotics selection; (2) APOA1/PON1 upregulation led to a modified transcriptomic profile and increased the expression of several antioxidant genes in transfected hepatocytes as demonstrated by RNAseq analysis; (3) secreted APOA1/PON1 were functional as demonstrated by the CM ability to reduce the levels of reactive oxygen species and inflammatory markers (VCAM-1, MCP-1) in TNFα-activated HUVEC. In conclusion, we achieved an experimental model of successful longstanding upregulation of endogenous APOA1 and PON1 in human hepatocytes. The targeted proteins are secreted in a functional form and can be used for deciphering their complex mechanism of protective action in various pathological conditions.

Apolipoprotein A1 (APOA1) and paraoxonase 1 (PON1) are key proteins of high-density lipoprotein (HDL). The aim of the present study was to obtain and characterize an in vitro model for endogenous APOA1 and PON1 long-time up-regulation in hepatocytes that can be further used to decipher the mechanism of their protective action. Cultured human hepatocytes (HuH-7 cell line) were transfected with CRISPR/dCas9 activation plasmids targeting APOA1/PON1 genes. Following selection with specific antibiotics, RNA sequencing was used for the transcriptomic characterization of the transfected hepatocytes. The functionality of the secreted APOAI/PON1 was evaluated as the capacity of the conditioned medium (CM) from transfected HuH-7 to modulate the oxidative and inflammatory stress in TNFα-activated primary human umbilical endothelial cells (HUVEC). The results showed that: (1) a robust, long-time up-regulation (46 days) of endogenous APOA1/PON1 was obtained after CRISPR/dCas9 transfection and antibiotics selection; (2) APOA1/PON1 up-regulation led to a modified transcriptomic profile and increased the expression of several antioxidant genes in transfected hepatocytes as demonstrated by RNAseq analysis; (3) secreted APOA1/PON1 were functional as demonstrated by the CM ability to reduce the levels of reactive oxygen species and inflammatory markers (VCAM-1, MCP-1) in TNF-α-activated HUVEC. In conclusion, we achieved an experimental model of successful long-term up-regulation of endogenous APOA1 and PON1 in human hepatocytes. The targeted proteins are secreted in a functional form and can be used for deciphering their complex mechanism of protective action in various pathological conditions.

Diabetes-induced oxidative stress induces the development of vascular complications, which are significant causes of morbidity and mortality in diabetic patients. Among these, diabetic retinopathy (DR) is often caused by functional changes in the blood–retinal barrier (BRB) due to harmful oxidative stress events in lipids, proteins, and DNA. Docosahexaenoic acid (DHA) has a potential therapeutic effect against hyperglycemia-induced oxidative damage and apoptotic pathways in the main constituents of BRB, retinal pigment epithelium cells (ARPE-19). Effective antioxidant response elicited by DHA is driven by the activation of the Nrf2/Nqo1 signaling cascade, which leads to the formation of NADH, a reductive agent found in the cytoplasm. Nrf2 also induces the expression of genes encoding enzymes involved in lipid metabolism. This study, therefore, aims at investigating the modulation of lipid metabolism induced by high-glucose (HG) on ARPE-19 cells through the integration of metabolic imaging and molecular biology to provide a comprehensive functional and molecular characterization of the mechanisms activated in the disease, as well the therapeutic role of DHA. This study shows that HG augments RPE metabolic processes by enhancing lipid metabolism, from fatty acid uptake and turnover to lipid biosynthesis and β-oxidation. DHA exerts its beneficial effect by ameliorating lipid metabolism and reducing the increased ROS production under HG conditions. This investigation may provide novel insight for formulating novel treatments for DR by targeting lipid metabolism pathways.

Diabetes-induced oxidative stress leads to the onset of vascular complications, which are major causes of disability and death in diabetic patients. Among these, diabetic retinopathy (DR) often arises from functional alterations of the blood-retinal barrier (BRB) due to damaging oxidative stress reactions in lipids, proteins, and DNA. This study aimed to investigate the impact of the ω3-polyunsaturated docosahexaenoic acid (DHA) on the regulation of redox homeostasis in the human retinal pigment epithelial (RPE) cell line (ARPE-19) under hyperglycemic-like conditions. The present results show that the treatment with DHA under high-glucose conditions activated erythroid 2-related factor Nrf2, which orchestrates the activation of cellular antioxidant pathways and ultimately inhibits apoptosis. This process was accompanied by a marked increase in the expression of NADH (Nicotinamide Adenine Dinucleotide plus Hydrogen) Quinone Oxidoreductase 1 (Nqo1), which is correlated with a contextual modulation and intracellular re-organization of the NAD+/NADH redox balance. This investigation of the mechanisms underlying the impairment induced by high levels of glucose on redox homeostasis of the BRB and the subsequent recovery provided by DHA provides both a powerful indicator for the detection of RPE cell impairment as well as a potential metabolic therapeutic target for the early intervention in its treatment.

Free fatty acids are essential structural components of the cell, and their intracellular distribution and effects on membrane organelles have crucial roles in regulating the metabolism, development, and cell cycle of most cell types. Here we engineered novel fluorescent, polarity-sensitive fatty acid derivatives, with the fatty acid aliphatic chain of increasing length (from 12 to 18 carbons). As in the laurdan probe, the lipophilic acyl tail is connected to the environmentally sensitive dimethylaminonaphthalene moiety. The fluorescence lifetime imaging analysis allowed us to monitor the intracellular distribution of the free fatty acids within the cell, and to simultaneously examine how the fluidity and the microviscosity of the membrane environment influence their localization. Each of these probes can thus be used to investigate the membrane fluidity regulation of the correspondent fatty acid intracellular distribution. We observed that, in PC-12 cells, fluorescent sensitive fatty acid derivatives with increased chain length compartmentalize more preferentially in the fluid regions, characterized by a low microviscosity. Moreover, fatty acid derivatives with the longest chain compartmentalize in lipid droplets and lysosomes with characteristic lifetimes, thus making these probes a promising tool for monitoring lipophagy and related events.

Insulin secretion from pancreatic beta cells is tightly regulated by glucose and paracrine signals within the microenvironment of islets of Langerhans. Extracellular matrix from islet microcapillary endothelial cells (IMEC) affect beta-cell spreading and amplify insulin secretion. This study was aimed at investigating the hypothesis that contact-independent paracrine signals generated from IMEC may also modulate beta-cell insulin secretory functions. For this purpose, conditioned medium (CMp) preparations were prepared from primary cultures of rat IMEC and were used to simulate contact-independent beta cell–endothelial cell communication. Glucose-stimulated insulin secretion (GSIS) assays were then performed on freshly isolated rat islets and the INS-1E insulinoma cell line, followed by fractionation of the CMp, mass spectroscopic identification of the factor, and characterization of the mechanism of action. The IMEC-derived CMp markedly attenuated first- and second-phase GSIS in a time- and dose-dependent manner without altering cellular insulin content and cell viability. Size exclusion fractionation, chromatographic and mass-spectroscopic analyses of the CMp identified the attenuating factor as the enzyme triosephosphate isomerase (TPI). An antibody against TPI abrogated the attenuating activity of the CMp while recombinant human TPI (hTPI) attenuated GSIS from beta cells. This effect was reversed in the presence of tolbutamide in the GSIS assay. In silico docking simulation identified regions on the TPI dimer that were important for potential interactions with the extracellular epitopes of the sulfonylurea receptor in the complex. This study supports the hypothesis that an effective paracrine interaction exists between IMEC and beta cells and modulates glucose-induced insulin secretion via TPI–sulfonylurea receptor–K_ATP channel (SUR1-Kir6.2) complex attenuating interactions.

Modern omics techniques reveal molecular structures and cellular networks of tissues and cells in unprecedented detail. Recent advances in single cell analysis have further revolutionized all disciplines in cellular and molecular biology. These methods have also been employed in current investigations on the structure and function of insulin secreting beta cells under normal and pathological conditions that lead to an impaired glucose tolerance and type 2 diabetes. Proteomic and transcriptomic analyses have pointed to significant alterations in protein expression and function in beta cells exposed to diabetes like conditions (e.g., high glucose and/or saturated fatty acids levels). These nutritional overload stressful conditions are often defined as glucolipotoxic due to the progressive damage they cause to the cells. Our recent studies on the rat insulinoma-derived INS-1E beta cell line point to differential effects of such conditions in the phospholipid bilayers in beta cells. This review focuses on confocal microscopy-based detection of these profound alterations in the plasma membrane and membranes of insulin granules and lipid droplets in single beta cells under such nutritional load conditions.

Hyperglycemia and hyperlipidemia synergistically and adversely impair insulin secretion and ultimately lead to pancreatic beta cell decomposition. We found that both nutrient overload conditions displace arachidonic and linoleic acids from membrane phospholipids and subject them to free radical-mediated peroxidation and generation of advanced lipid peroxidation end products (ALEs), of which the aldehyde 4-hydroxy-2-nonenal (4-HNE) is prominent. When present at high levels this electrophilic molecule binds covalently to nucleophilic moieties in proteins, phospholipids and nucleic acid, modifies their structure and function and leads to severe cellular dysfunction and apoptosis. However, when present at low and unharmful levels this same molecule activates the nuclear receptor PPARδ and augments insulin secretion. The level of endogenous 4-HNE is determined by the extent of lipid peroxidation on one hand, and by enzymatic neutralization of the aldehyde, on the other. The latter step is mediated by enzymatic processes of which the transformation of the aldehyde to the corresponding inactive carboxylic derivative 4-hydroxy-2-nonenoic acid (4-HNA) is significant. The enzyme responsible for this transformation, which belongs to the large family of aldehyde dehydrogenases and selectively neutralizes fatty acid-derived aldehydes, is ALDH3A2, which is also known as fatty aldehyde dehydrogenase (FALDH). Consequently, we hypothesized that the expression level and function of ALDH3A2 may determine the fate of beta cells under nutrient overload conditions: insufficient neutralization of 4-HNE by the enzyme will lead to cell demise, whereas increased expression and function will extend the adaptive response of beta cells. This adaptive response that is characterized with increased insulin secretion enables effective storage of the nutrient surplus in peripheral tissues and organs while minimizing the dire consequences of the nutrient overload. We aimed at investigating the expression pattern of ALDH3A2 in pancreatic beta cells (the INS-1E cell line) under hyperglycemic condition without or with supplementation with saturated fatty acids (e.g., palmitic acid). Our results show significant glucose- and palmitic acid-dependent induction of ALDH3A2 expression in the cells. We also found that the transformation of palmitic acid (16:1) to mono-unsaturated palmitoleic acid (16:1, cis 9) by the enzyme Stearoyl-CoA desaturase-1 (SCD1) decreased the burden of the lipid stress on the cells and abrogated the stimulus for the induction of ALDH3A2. Preliminary experiments indicated that the upregulation of the induction of ALDH3A2 was partly induced by PPARδ. These findings correlate to our previous discovery that the hormetic effects of 4HNE were mediated via activation of this nuclear receptor. In summary, this study assigns a central role to the enzyme ALDH3A2 in the protective mechanism beta cells employ to mitigate detrimental effects of ALEs, and divert them into hormetic agents, that by feedback mechanism through PPARδ increase ALDH3A2 expression.

Cardiovascular disease (CVD) remains the leading cause of death worldwide and, despite continuous advances, better diagnostic and prognostic tools, as well as therapy, are needed. The human transcriptome, which is the set of all RNA produced in a cell, is much more complex than previously thought and the lack of dialogue between researchers and industrials and consensus on guidelines to generate data make it harder to compare and reproduce results. This European Cooperation in Science and Technology (COST) Action aims to accelerate the understanding of transcriptomics in CVD and further the translation of experimental data into usable applications to improve personalized medicine in this field by creating an interdisciplinary network. It aims to provide opportunities for collaboration between stakeholders from complementary backgrounds, allowing the functions of different RNAs and their interactions to be more rapidly deciphered in the cardiovascular context for translation into the clinic, thus fostering personalized medicine and meeting a current public health challenge. Thus, this Action will advance studies on cardiovascular transcriptomics, generate innovative projects, and consolidate the leadership of European research groups in the field. COST (European Cooperation in Science and Technology) is a funding organization for research and innovation networks (www.cost.eu).