✍🏼 Dipan Kumar Kundu, Matthew Kiedrowski, James Gadd, Min Gao, Madeline Evan, Yang Wang, Liya Yin, Vahagn Ohanyan, William M. Chilian and Feng Dong
🏠 Department of Biomedical Sciences, Northeast Ohio Medical University, Rootstown, OH, USA
📑 International Journal of Molecular Sciences (2025)
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Abstract
The aim of this study was to determine whether exosomes from Nicotinamide phosphoribosyltransferase (NAMPT)-overexpressing mesenchymal stem cells (MSC NAMPT-Exo) can attenuate aortic stenosis (AS) and explored the underlying mechanism. NAMPT expression was examined in EC CXCR4 KO (AS) mouse hearts. Six-week-old AS mice received weekly injections of NAMPT-Exo, MSC-Exo, or PBS for three weeks, followed by echocardiography and histological examination of the valves (H&E, Alizarin Red, immunofluorescence). Cardiac ECs from control, AS, and NAMPT-Exo-treated mice were analyzed for miRNA expression (miR-146a-3p/5p, miR-125b-5p, miR-142a-5p). NAMPT expression was decreased in AS hearts. Treatment with NAMPT-Exo reduced aortic valve peak velocity, valvular thickening, and microcalcifications, while improving ejection fraction, fractional shortening, and ventricular dimensions. AS endothelial cells showed elevated levels of miR-146a-3p, miR-146a-5p, and miR-142a-5p, NAMPT-Exo specifically normalized miR-146a-3p. Histology revealed EndMT in AS valves, which was diminished by NAMPT-Exo. In vitro, inhibiting miR-146a-3p suppressed TGF-β-induced EndMT. Our results demonstrate that NAMPT-enriched MSC-derived exosomes effectively slow the progression of AS. Additionally, our findings highlight miR-146a-3p as a key regulator of EndMT, suggesting it as a potential molecular target for future therapies.
How the WOLF was used in this study
The researchers used the WOLF Cell Sorter to purify a population of stably transduced mesenchymal stem cells (MSCs) that overexpress NAMPT. After introducing lentiviral vectors encoding a tdTomato fluorescent reporter along with the NAMPT construct into MSCs, they screened the cultures for tdTomato fluorescence and then employed the WOLF instrument’s fluorescence‑activated cell sorting to isolate and enrich tdTomato‑positive cells, effectively removing untransduced cells from the sample. This step ensured that downstream analyses (such as confirming NAMPT protein expression and evaluating functional effects of the engineered MSCs) were conducted on a highly pure, genetically modified cell population, improving the rigor and interpretability of the study’s findings.
