RESUMEN
Background: Thoracic aortic aneurysms (TAAs) associated with Marfan syndrome (MFS) are unique in that extracellular matrix metalloproteinase inducer (EMMPRIN) levels do not behave the way they do in other cardiovascular pathologies. EMMPRIN is shed into the circulation through the secretion of extracellular vesicles. This has been demonstrated to be dependent upon the Membrane Type-1 MMP (MT1-MMP). We investigated this relationship in MFS TAA tissue and plasma to discern why unique profiles may exist. Methods: Protein targets were measured in aortic tissue and plasma from MFS patients with TAAs and were compared to healthy controls. The abundance and location of MT1-MMP was modified in aortic fibroblasts and secreted EMMPRIN was measured in conditioned culture media. Results: EMMPRIN levels were elevated in MFS TAA tissue but reduced in plasma, compared to the controls. Tissue EMMPRIN elevation did not induce MMP-3, MMP-8, or TIMP-1 expression, while MT1-MMP and TIMP-2 were elevated. MMP-2 and MMP-9 were reduced in TAA tissue but increased in plasma. In aortic fibroblasts, EMMPRIN secretion required the internalization of MT1-MMP. Conclusions: In MFS, impaired EMMPRIN secretion likely contributes to higher tissue levels, influenced by MT1-MMP cellular localization. Low EMMPRIN levels, in conjunction with other MMP analytes, distinguished MFS TAAs from controls, suggesting diagnostic potential.
RESUMEN
MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate translation and are involved in many pathological processes. They have emerged as promising biomarkers for diagnosis of conditions such as aortic aneurysm disease. Quantifying miRNAs in plasma is uniquely challenging because of the lack of standardized reproducible protocols. To facilitate the independent verification of conclusions, it is necessary to provide a thorough disclosure of all pertinent experimental details. In this technical note, we present a comprehensive protocol for quantifying plasma miRNAs using droplet digital PCR. We detail the entire workflow, including blood collection, plasma processing, cryo-storage, miRNA isolation, reverse transcription, droplet generation, PCR amplification, fluorescence reading, and data analysis. We offer comprehensive guidance regarding optimization, assay conditions, expected results, and insight into the troubleshooting of common issues. The stepwise normalization and detailed methodological guide enhance reproducibility. Moreover, multiple portions of this protocol may be automated. The data provided in this technical note is demonstrative of the values typically obtained when following its steps. To facilitate standardization in data reporting, we include a table of expected aortic aneurysm-related miRNA levels in healthy human plasma. This versatile protocol can be easily adapted to quantify most circulating miRNAs in plasma, making it a valuable resource for diagnostic development.
RESUMEN
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.
