Atherosclerosis, the major cause of cardiovascular disease, is a chronic inflammatory disease characterized by the accumulation of lipids and inflammatory cells in the artery wall. Aberrant expression of microRNAs has been implicated in the pathophysiological processes underlying the progression of atherosclerosis. Here, we define the contribution of miR‐21 in hematopoietic cells during atherogenesis. Interestingly, we found that miR‐21 is the most abundant miRNA in macrophages and its absence results in accelerated atherosclerosis, plaque necrosis, and vascular inflammation. miR‐21 expression influences foam cell formation, sensitivity to ER‐stress‐induced apoptosis, and phagocytic clearance capacity. Mechanistically, we discovered that the absence of miR‐21 in macrophages increases the expression of the miR‐21 target gene, MKK3, promoting the induction of p38‐CHOP and JNK signaling. Both pathways enhance macrophage apoptosis and promote the post‐translational degradation of ABCG1, a transporter that regulates cholesterol efflux in macrophages. Altogether, these findings reveal a major role for hematopoietic miR‐21 in atherogenesis.

MicroRNAs (miRNAs) are small non-coding RNAs with a known role as mediators of gene expression in crucial biological processes, which converts them into high potential contenders in the ongoing search for effective therapeutic strategies. However, extracellular RNAs are unstable and rapidly degraded, reducing the possibility of successfully exerting a biological function in distant target cells. Strategies aimed at enhancing the therapeutic potential of miRNAs include the development of efficient, tissue-specific and nonimmunogenic delivery methods. Since miRNAs were discovered to be naturally transported within exosomes, a type of extracellular vesicle that confers protection against RNase degradation and increases miRNA stability have been proposed as ideal delivery vehicles for miRNA-based therapy. Although research in this field has grown rapidly in the last few years, a standard, reproducible and cost-effective protocol for exosome isolation and extracellular RNA delivery is lacking. We aimed to evaluate the use of milk-derived extracellular vesicles as vehicles for extracellular RNA drug delivery. With this purpose, exosomes were isolated from raw bovine milk, combining ultracentrifugation and size exclusion chromatography (SEC) methodology. Isolated exosomes were then loaded with exogenous hsa-miR148a-3p, a highly expressed miRNA in milk exosomes. The suitability of exosomes as delivery vehicles for extracellular RNAs was tested by evaluating the absorption of miR-148a-3p in hepatic (HepG2) and intestinal (Caco-2) cell lines. The potential exertion of a biological effect by miR-148a-3p was assessed by gene expression analysis, using microarrays. Results support that bovine milk is a cost-effective source of exosomes which can be used as nanocarriers of functional miRNAs with a potential use in RNA-based therapy. In addition, we show here that a combination of ultracentrifugation and SEC technics improve exosome enrichment, purity, and integrity for subsequent use.

Haloperidol, a typical antipsychotic, has been shown to inhibit cholesterol biosynthesis by affecting Δ⁷-reductase, Δ^8,7-isomerase, and Δ¹⁴-reductase activities, which results in the accumulation of different sterol intermediates. In the present work, we investigated the effects of atypical or second-generation antipsychotics (SGA), such as clozapine, risperidone, and ziprasidone, on intracellular lipid metabolism in different cell lines. All the SGAs tested inhibited cholesterol biosynthesis. Ziprasidone and risperidone had the same targets as haloperidol at inhibiting cholesterol biosynthesis, although with different relative activities (ziprasidone > haloperidol > risperidone). In contrast, clozapine mainly affected Δ²⁴-reductase and Δ^8,7-isomerase activities. These amphiphilic drugs also interfered with the LDL-derived cholesterol egress from the endosome/lysosome compartment, thus further reducing the cholesterol content in the endoplasmic reticulum. This triggered a homeostatic response with the stimulation of sterol regulatory element-binding protein (SREBP)-regulated gene expression. Treatment with SGAs also increased the synthesis of complex lipids (phospholipids and triacylglycerides). Once the antipsychotics were removed from the medium, a rebound in the cholesterol biosynthesis rate was detected, and the complex-lipid synthesis further increased. In this condition, apolipoprotein B secretion was also stimulated as demonstrated in HepG2 cells. These effects of SGAs on lipid homeostasis may be relevant in the metabolic side effects of antipsychotics, especially hypertriglyceridemia.

Splice variants (SVs) of receptors for growth hormone-releasing hormone (GHRH) have been found in primary human prostate cancers and diverse human cancer cell lines. GHRH antagonists inhibit growth of various experimental human cancers, including pancreatic and colorectal, xenografted into nude mice or cultured in vitro , and their antiproliferative action could be mediated in part through SVs of GHRH receptors. In this study we examined the expression of mRNA for GHRH and for SVs of its receptors in tumors of human pancreatic, colorectal, and gastric cancer cell lines grown in nude mice. mRNA for both GHRH and SV ₁ isoform of GHRH receptors was expressed in tumors of pancreatic (SW1990, PANC-1, MIA PaCa-2, Capan-1, Capan-2, and CFPAC1), colonic (COLO 320DM and HT-29), and gastric (NCI-N87, HS746T, and AGS) cancer cell lines; mRNA for SV ₂ was also present in Capan-1, Capan-2, CFPAC1, HT-29, and NCI-N87 tumors. In proliferation studies in vitro , the growth of pancreatic, colonic, and gastric cancer cells was stimulated by GHRH(1–29)NH ₂ and inhibited by GHRH antagonist JV-1–38. The stimulation of some gastroenteropancreatic cancer cells by GHRH was followed by an increase in cAMP production, and GHRH antagonist JV-1–38 competitively inhibited this effect. Our study indicates the presence of an autocrine/paracrine stimulatory loop based on GHRH and SV ₁ of GHRH receptors in human pancreatic, colorectal, and gastric cancers. The finding of SV ₁ receptor in human cancers provides an approach to an antitumor therapy based on the blockade of this receptor by specific GHRH antagonists.