A standardized method for plasma extracellular vesicle isolation and size distribution analysis.

The following protocol describes our workflow for isolation and quantification of plasma extracellular vesicles (EVs). It requires limited sample volume so that the scientific value of specimens is maximized. These steps include isolation of vesicles by automated size exclusion chromatography and quantification by tunable resistive pulse sensing. This workflow optimizes reproducibility by minimizing variations in processing, handling, and storage of EVs. EVs have significant diagnostic and therapeutic potential, but clinical application is limited by disparate methods of data collection. This standardized protocol is scalable and ensures efficient recovery of physiologically intact EVs that may be used in a variety of downstream biochemical and functional analyses. Simultaneous measurement quantifies EV concentration and size distribution absolutely. Absolute quantification corrects for variations in EV number and size, offering a novel method of standardization in downstream applications.

miR-133a Replacement Attenuates Thoracic Aortic Aneurysm in Mice.

Background Thoracic aortic aneurysms (TAAs) occur because of abnormal remodeling of aortic extracellular matrix and are accompanied by the emergence of proteolytically active myofibroblasts. The microRNA miR-133a regulates cellular phenotypes and is reduced in clinical TAA specimens. This study tested the hypothesis that miR-133a modulates aortic fibroblast phenotype, and overexpression by lentivirus attenuates the development of TAA in a murine model. Methods and Results TAA was induced in mice. Copy number of miR-133a was reduced in TAA tissue and linear regression analysis confirmed an inverse correlation between aortic diameter and miR-133a. Analyses of phenotypic markers revealed an mRNA expression profile consistent with myofibroblasts in TAA tissue. Fibroblasts were isolated from the thoracic aortae of mice with/without TAA. When compared with controls, miR-133a was reduced, migration was increased, adhesion was reduced, and the ability to contract a collagen disk was increased. Overexpression/knockdown of miR-133a controlled these phenotypes. After TAA induction in mice, a single tail-vein injection of either miR-133a overexpression or scrambled sequence (control) lentivirus was performed. Overexpression of miR-133a attenuated TAA development. The pro-protein convertase furin was confirmed to be a target of miR-133a by luciferase reporter assay. Furin was elevated in this murine model of TAA and repressed by miR-133a replacement in vivo resulting in reduced proteolytic activation. Conclusions miR-133a regulates aortic fibroblast phenotype and over-expression prevented the development of TAA in a murine model. These findings suggest that stable alterations in aortic fibroblasts are associated with development of TAA and regulation by miR-133a may lead to a novel therapeutic strategy.