MicroRNA-541-3p alters lipoproteins to reduce atherosclerosis by degrading Znf101 and Casz1 transcription factors.

High apoB-containing low-density lipoproteins (LDL) and low apoA1-containing high-density lipoproteins (HDL) are associated with atherosclerosis. In search of a molecular regulator that could simultaneously and reciprocally control both LDL and HDL levels, we screened a microRNA (miR) library using human hepatoma Huh-7 cells. We identified miR-541-3p that both decreases apoB and increases apoA1 expression by inducing mRNA degradation of two different transcription factors, Znf101 and Casz1. Znf101 enhances apoB expression while Casz1 represses apoA1 expression. The hepatic knockdown of orthologous Zfp961 and Casz1 genes in mice altered plasma lipoproteins and reduced atherosclerosis without causing hepatic lipid accumulation, most likely by lowering hepatic triglyceride production, increasing HDL cholesterol efflux capacity, and reducing lipogenesis. Notably, human genetic variants in the MIR541, ZNF101, and CASZ1 loci are significantly associated with plasma lipids and lipoprotein levels. This study identifies miR-541-3p and Znf101/Casz1 as potential therapeutic agent and targets, respectively, to reduce plasma lipoproteins and atherosclerosis without causing liver steatosis.

Characterization of lipoproteins in human placenta and fetal circulation as well as gestational changes in lipoprotein assembly and secretion in human and mouse placentas.

In the maternal circulation, apoB-containing low-density lipoproteins (LDL) and apoA1-containing high-density lipoproteins (HDL) transport lipids. The production of lipoproteins in the placenta has been suggested, but the directionality of release has not been resolved. We compared apolipoprotein concentrations and size-exclusion chromatography elution profiles of lipoproteins in maternal/fetal circulations, and in umbilical arteries/veins; identified placental lipoprotein-producing cells; and studied temporal induction of lipoprotein-synthesizing machinery during pregnancy. We observed that maternal and fetal lipoproteins are different with respect to concentrations and elution profiles. Surprisingly, concentrations and elution profiles of lipoproteins in umbilical arteries and veins were similar indicating their homeostatic control. Human placental cultures synthesized apoB100-containing LDL-sized and apoA1-containing HDL-sized particles. Immunolocalization techniques revealed that ApoA1 was present mainly in syncytiotrophoblasts. MTP, a critical protein for lipoprotein assembly, was in these trophoblasts. ApoB was in the placental stroma indicating that trophoblasts secrete apoB-containing lipoproteins into the stroma. ApoB and MTP expressions increased in placentas from the 2nd trimester to term, whereas apoA1 expression was unchanged. Thus, our studies provide new information regarding the timing of lipoprotein gene induction during gestation, the cells involved in lipoprotein assembly and the gel filtration profiles of human placental lipoproteins. Next, we observed that mouse placenta produces MTP, apoB100, apoB48 and apoA1. The expression of genes gradually increased and peaked in late gestation. This information may be useful in identifying transcription factors regulating the induction of these genes in gestation and the importance of placental lipoprotein assembly in fetal development.

Human Pluripotent Stem Cell Differentiation to Microglia.

Microglia, the immune cells of the central nervous system (CNS), play critical roles in CNS homeostasis and disease. Mounting evidence has linked aberrant microglial functions to neurodevelopment, neuroinflammatory and neurodegenerative diseases, underlining the need for novel models to investigate human microglia biology. Here we describe a protocol for generating in vitro patient-specific microglia progenitors and microglia-like cells from induced pluripotent stem cells (iPSCs). Our protocol generates microglia progenitor cells in approximately 35 days, which then can further mature into microglia-like cells within two additional weeks. Microglia differentiation is driven by specific growth factors and cytokines in serum-free conditions, resulting in mesodermal progenitors that grow in a monolayer which releases free-floating microglia progenitors. Isolated progenitors can be used in co-culture systems with other neuronal cells, xenotransplanted to generate chimeric mouse models, or further differentiated into adherent microglia-like cells for functional studies.

GLP-2 Dysregulates Hepatic Lipoprotein Metabolism, Inducing Fatty Liver and VLDL Overproduction in Male Hamsters and Mice.

Fundamental complications of insulin resistance and type 2 diabetes include the development of nonalcoholic fatty liver disease and an atherogenic fasting dyslipidemic profile, primarily due to increases in hepatic very-low-density lipoprotein (VLDL) production. Recently, central glucagon-like peptide-2 receptor (GLP2R) signaling has been implicated in regulating hepatic insulin sensitivity; however, its role in hepatic lipid and lipoprotein metabolism is unknown. We investigated the role of glucagon-like peptide-2 (GLP-2) in regulating hepatic lipid and lipoprotein metabolism in Syrian golden hamsters, C57BL/6J mice, and Glp2r-/- mice consuming either a normal chow or high-fat diet (HFD). In the chow-fed hamsters, IP GLP-2 administration significantly increased fasting dyslipidemia, hepatic VLDL production, and the expression of key genes involved in hepatic de novo lipogenesis. In HFD-fed hamsters and chow-fed mice, GLP-2 administration exacerbated or induced hepatic lipid accumulation. HFD-fed Glp2r-/- mice displayed reduced glucose tolerance, VLDL secretion, and microsomal transfer protein lipid transfer activity, as well as exacerbated fatty liver. Thus, we conclude that GLP-2 plays a lipogenic role in the liver by increasing lipogenic gene expression and inducing hepatic steatosis, fasting dyslipidemia, and VLDL overproduction. In contrast, the lack of Glp2r appears to interfere with VLDL secretion, resulting in enhanced hepatic lipid accumulation. These studies have uncovered a role for GLP-2 in maintaining hepatic lipid and lipoprotein homeostasis.

microRNA-30c reduces plasma cholesterol in homozygous familial hypercholesterolemic and type 2 diabetic mouse models.

High plasma cholesterol levels are found in several metabolic disorders and their reductions are advocated to reduce the risk of atherosclerosis. A way to lower plasma lipids is to curtail lipoprotein production; however, this is associated with steatosis. We previously showed that microRNA (miR)-30c lowers diet-induced hypercholesterolemia and atherosclerosis in C57BL/6J and Apoe-/- mice. Here, we tested the effect of miR-30c on plasma lipids, transaminases, and hepatic lipids in different mouse models. Hepatic delivery of miR-30c to chow-fed leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) hypercholesterolemic and hyperglycemic mice reduced cholesterol in total plasma and VLDL/LDL by ∼28% and ∼25%, respectively, without affecting triglyceride and glucose levels. And these mice had lower plasma transaminases and creatine kinase activities than controls. Moreover, miR-30c significantly lowered plasma cholesterol and atherosclerosis in Western diet-fed Ldlr-/- mice with no effect on plasma triglyceride, glucose, and transaminases. In these studies, hepatic lipids were similar in control and miR-30c-injected mice. Mechanistic studies showed that miR-30c reduced hepatic microsomal triglyceride transfer protein activity and lipid synthesis. Thus miR-30c reduced plasma cholesterol in several diet-induced and diabetic hypercholesterolemic mice. We speculate that miR-30c may be beneficial in lowering plasma cholesterol in different metabolic disorders independent of the origin of hypercholesterolemia.