Synthesis of Novel MicroRNA-30c Analogs to Reduce Apolipoprotein B Secretion in Human Hepatoma Cells.

Atherosclerosis, a condition characterized by thickening of the arteries due to lipid deposition, is the major contributor to and hallmark of cardiovascular disease. Although great progress has been made in lowering the lipid plaques in patients, the conventional therapies fail to address the needs of those that are intolerant or non-responsive to the treatment. Therefore, additional novel therapeutic approaches are warranted. We have previously shown that increasing the cellular amounts of microRNA-30c (miR-30c) with the aid of viral vectors or liposomes can successfully reduce plasma cholesterol and atherosclerosis in mice. To avoid the use of viruses and liposomes, we have developed new methods to synthesize novel miR-30c analogs with increasing potency and efficacy, including 2'-O-methyl (2'OMe), 2'-fluoro (2'F), pseudouridine (á´ª), phosphorothioate (PS), and N-acetylgalactosamine (GalNAc). The discovery of these modifications has profoundly impacted the modern RNA therapeutics, as evidenced by their increased nuclease stability and reduction in immune responses. We show that modifications on the passenger strand of miR-30c not only stabilize the duplex but also aid in a more readily uptake by the cells without the aid of viral vectors or lipid emulsions. After uptake, the analogs with PS linkages and GalNAc-modified ribonucleotides significantly reduce the secretion of apolipoprotein B (ApoB) without affecting apolipoprotein A1 (ApoA1) in human hepatoma Huh-7 cells. We envision an enormous potential for these modified miR-30c analogs in therapeutic intervention for treating cardiovascular diseases. This protocol was validated in: J Biol Chem (2021), DOI: 10.1016/j.jbc.2022.101813.

Glucagon-Like Peptide 2 (GLP-2) Stimulates Postprandial Chylomicron Production and Postabsorptive Release of Intestinal Triglyceride Storage Pools via Induction of Nitric Oxide Signaling in Male Hamsters and Mice.

The intestinal overproduction of apolipoprotein B48 (apoB48)-containing chylomicron particles is a common feature of diabetic dyslipidemia and contributes to cardiovascular risk in insulin resistant states. We previously reported that glucagon-like peptide-2 (GLP-2) is a key endocrine stimulator of enterocyte fat absorption and chylomicron output in the postprandial state. GLP-2's stimulatory effect on chylomicron production in the postabsorptive state has been confirmed in human studies. The mechanism by which GLP-2 regulates chylomicron production is unclear, because its receptor is not expressed on enterocytes. We provide evidence for a key role of nitric oxide (NO) in mediating the stimulatory effects of GLP-2 during the postprandial and postabsorptive periods. Intestinal chylomicron production was assessed in GLP-2-treated hamsters administered the pan-specific NO synthase (NOS) inhibitor L-N(G)-nitroarginine methyl ester (L-NAME), and in GLP-2-treated endothelial NOS knockout mice. L-NAME blocked GLP-2-stimulated apoB48 secretion and reduced triglycerides (TGs) in the TG-rich lipoprotein (TRL) fraction of the plasma in the postprandial state. Endothelial NOS-deficient mice were resistant to GLP-2 stimulation and secreted fewer large apoB48-particles. When TG storage pools were allowed to accumulate, L-NAME mitigated the GLP-2-mediated increase in TRL-TG, suggesting that NO is required for early mobilization and secretion of stored TG and preformed chylomicrons. Importantly, the NO donor S-nitroso-L-glutathione was able to elicit an increase in TRL-TG in vivo and stimulate chylomicron release in vitro in primary enterocytes. We describe a novel role for GLP-2-mediated NO-signaling as a critical regulator of intestinal lipid handling and a potential contributor to postprandial dyslipidemia.

The impact of phospholipid transfer protein (PLTP) on lipoprotein metabolism.

It has been reported that phospholipid transfer protein (PLTP) is an independent risk factor for human coronary artery disease. In mouse models, it has been demonstrated that PLTP overexpression induces atherosclerosis, while its deficiency reduces it. PLTP is considered a promising target for pharmacological intervention to treat atherosclerosis. However, we must still answer a number of questions before its pharmaceutical potential can be fully explored. In this review, we summarized the recent progresses made in the PLTP research field and focused on its effect on apoB-containing- triglyceride-rich particle and HDL metabolism.

Hepatic steatosis in hepatitis C is a storage disease due to HCV interaction with microsomal triglyceride transfer protein (MTP).

Liver steatosis is a frequent histological feature in patients chronically infected with hepatitis C virus (HCV). The relationship between HCV and hepatic steatosis seems to be the result of both epigenetic and genetic factors. In vivo and in vitro studies have shown that HCV can alter intrahepatic lipid metabolism by affecting lipid synthesis, oxidative stress, lipid peroxidation, insulin resistance and the assembly and secretion of VLDL. Many studies suggest that HCV-related steatosis might be the result of a direct interaction between the virus and MTP. It has been demonstrated that MTP is critical for the secretion of HCV particles and that inhibition of its lipid transfer activity reduces HCV production. However, higher degrees of hepatic steatosis were found in chronic hepatitis C patients carrying the T allele of MTP -493G/T polymorphism that seems to be associated with increased MTP transcription. We propose here that liver steatosis in hepatitis C could be a storage disease induced by the effects of the virus and of its proteins on the intracellular lipid machinery and on MTP. Available data support the hypothesis that HCV may modulate MTP expression and activity through a number of mechanisms such as inhibition of its activity and transcriptional control. Initial up regulation could favour propagation of HCV while down regulation in chronic phase could cause impairment of triglyceride secretion and excessive lipid accumulation, with abnormal lipid droplets facilitating the "storage" of virus particles for persistent infection.