Seminar: Extracellular Vesicles as Mediators of Environmental Stress in Human Disease.

BACKGROUND: Extracellular vesicles (EVs), membrane-bound particles containing a variety of RNA types, DNA, proteins, and other macromolecules, are now appreciated as an important means of communication between cells and tissues, both in normal cellular physiology and as a potential indicator of cellular stress, environmental exposures, and early disease pathogenesis. Extracellular signaling through EVs is a growing field of research for understanding fundamental mechanisms of health and disease and for the potential for biomarker discovery and therapy development. EVs are also known to play important roles in mediating the effects of exposure to environmental stress. OBJECTIVES: This seminar addresses the application of new tools and approaches for EV research, developed in part through the National Institutes of Health (NIH) Extracellular RNA Communication Program, and reflects presentations and discussions from a workshop held 27-28 September 2021 by the National Institute of Environmental Health Sciences (NIEHS) and the National Center for Advancing Translational Sciences (NCATS) on "Extracellular Vesicles, Exosomes, and Cell-Cell Signaling in Response to Environmental Stress." The panel of experts discussed current research on EVs and environmental exposures, highlighted recent advances in EV isolation and characterization, and considered research gaps and opportunities toward identifying and characterizing the roles for EVs in environmentally related diseases, as well as the current challenges and opportunities in this field. DISCUSSION: The authors discuss the application of new experimental models, particularly organ-on-chip (OOC) systems and in vitro approaches and how these have the potential to extend findings in population-based studies of EVs in exposure-related diseases. Given the complex challenges of identifying cell-specific EVs related to environmental exposures, as well as the general heterogeneity and variability in EVs in blood and other accessible biological samples, there is a critical need for rigorous reporting of experimental methods and validation studies. The authors note that these efforts, combined with cross-disciplinary approaches, would ensure that future research efforts in environmental health studies on EV biomarkers are rigorous and reproducible. https://doi.org/10.1289/EHP12980.

MicroRNA-143 regulates adipocyte differentiation.

MicroRNAs (miRNAs) are endogenously expressed 20-24 nucleotide RNAs thought to repress protein translation through binding to a target mRNA (1-3). Only a few of the more than 250 predicted human miRNAs have been assigned any biological function. In an effort to uncover miRNAs important during adipocyte differentiation, antisense oligonucleotides (ASOs) targeting 86 human miRNAs were transfected into cultured human pre-adipocytes, and their ability to modulate adipocyte differentiation was evaluated. Expression of 254 miRNAs in differentiating adipocytes was also examined on a miRNA microarray. Here we report that the combination of expression data and functional assay results identified a role for miR-143 in adipocyte differentiation. miR-143 levels increased in differentiating adipocytes, and inhibition of miR-143 effectively inhibited adipocyte differentiation. In addition, protein levels of the proposed miR-143 target ERK5 (4) were higher in ASO-treated adipocytes. These results demonstrate that miR-143 is involved in adipocyte differentiation and may act through target gene ERK5.

Use of antisense oligonucleotides in functional genomics and target validation.

With the completion of sequencing of the human genome, a great deal of interest has been shifted toward functional genomics-based research for identification of novel drug targets for treatment of various diseases. The major challenge facing the pharmaceutical industry is to identify disease-causing genes and elucidate additional roles for genes of known functions. Gene functionalization and target validation are probably the most important steps involved in identifying novel potential drug targets. This review focuses on recent advances in antisense technology and its use for rapid identification and validation of new drug targets. The significance and applicability of this technology as a beginning of the drug discovery process are underscored by relevant cell culture-based assays and positive correlation in specific animal disease models. Some of the antisense inhibitors used to validate gene targets are themselves being developed as drugs. The current clinical trials based on such leads that were identified in a very short time further substantiate the importance of antisense technology-based functional genomics as an integral part of target validation and drug target identification.

Insulin receptor substrate-1 and phosphoinositide-dependent kinase-1 are required for insulin-stimulated production of nitric oxide in endothelial cells.

Vasodilator actions of insulin are mediated by signaling pathways involving phosphatidylinositol 3-kinase (PI 3-kinase) and Akt that lead to activation of endothelial nitric oxide synthase (eNOS) in endothelium. Signaling molecules immediately upstream and downstream from PI 3-kinase involved with production of NO in response to insulin have not been previously identified. In this study, we evaluated roles of insulin receptor substrate 1 (IRS-1) and phosphoinositide-dependent kinase 1 (PDK-1) in production of NO. The fluorescent dye 4,5-diamine fluorescein diacetate was used to directly measure NO in NIH-3T3(IR) cells transiently cotransfected with eNOS and various IRS-1 or PDK-1 constructs. In control cells, transfected with only eNOS, insulin stimulated a rapid dose-dependent increase in NO. Overexpression of wild-type IRS-1 increased the maximal insulin response 3-fold. Overexpression of IRS1-F6 (mutant that does not bind PI 3-kinase) or an antisense ribozyme against IRS-1 substantially inhibited insulin-stimulated production of NO. Likewise, overexpression of wild-type PDK-1 enhanced insulin-stimulated production of NO, whereas a kinase-inactive mutant PDK-1 inhibited this action of insulin. Qualitatively similar results were observed in vascular endothelial cells. Production of NO by a calcium-dependent mechanism in response to lysophosphatidic acid was unaffected by either wild-type or mutant IRS-1 and PDK-1. We conclude that IRS-1 and PDK-1 play necessary roles in insulin-signaling pathways leading to activation of eNOS. Furthermore, classical Ca2+-mediated pathways for activation of eNOS are separable from IRS-1- and PDK-1-dependent insulin-signaling pathways.