Poster X8 - miR29 Loaded Extracellular Vesicles Reduce Inflammatory Signaling and Collagen Gel Contraction in Human Dermal Fibroblasts

Introduction: Fibrotic processes contribute to ~45% of all deaths in the developed world [1], due to aberrant deposition of extracellular matrix (ECM) molecules that cause tissue dysfunction. The mechanosensitive YAP/TAZ pathway and the inflammatory NFkB pathway are both directly involved in this process. Fibrotic remodeling results in stiffening of the local tissue environment, driving a self-amplifying positive feedback loop of disease progression [2]. Recent work has identified several miRNAs that target fibrosis related genes in response to fibrotic stimuli, indicating their potential to slow or reverse fibrotic processes. In particular, miR29 has been identified as a potential anti-fibrotic miRNA [4]. Previously, we showed that mesenchymal stem cell derived EVs loaded with miR29 can deliver cargo to fibroblasts [5]. The goal of the current work was to test these EVs using in vitro models of fibrotic ECM remodeling and inflammation.

Materials and

Methods: Human mesenchymal stem cells (hMSCs) were cultured in αMEM supplemented with 10% FBS and 1% pen-strep. NIH/3T3 fibroblasts and human dermal fibroblasts (HDFs) were cultured in high-glucose DMEM supplemented with 10% FBS and 1% pen-strep. MSCs were transfected with a scrambled miRNA sequence (Scr) or miR29a mimic using INTERFERin transfection reagent with a non-transfected control (Neg.). Transfected cells were switched to αMEM made with EV-free FBS, media was harvested after 48 hrs, and cells were re-seeded on fibronectin-coated glass coverslips. Cells were fixed (4% PFA) for immunostaining to quantify YAP activation based on nuclear/cytoplasmic ratio (MATLAB). MSC culture media was enriched with 24% PEG and spun following a differential centrifugation protocol [6]. EV size distribution and count was determined using nanoparticle tracking analysis (NTA). 3T3 cells expressing LSB-hsa-miR-29a-3p miRNA sensor [7, Addgene 103391] were treated with EVs for 24 hrs to test delivery by quantification of the mKate/BFP ratio (MATLAB) which indicates the relative activity of miRNA (low ratio=high targeting and activity). HDFs were exposed to IL-1β at various doses, and 1 ng/ml was used for testing isolated EVs. Cells were fixed and immunostained for assessing NFκΒ activation by quantification of the nuclear/cytoplasmic ratio of p65. HDFs were seeded within a 1% collagen gel to create a fibrotic model for cell contractility. Collagen gels were treated with EVs every 2 days, and the gel area was quantified after 7 days. Fixed gels were stained with Alexa-488 Phalloidin, YAP antibody, and Hoechst for imaging on an Andor spinning disc confocal microscope.

Results, Conclusions, and Discussions: Isolated EVs from transfected MSC culture followed an expected size distribution and showed no significant changes in the amount of EVs produced after transfection (Fig. 1A&B). Sensor experiments in the 3T3 reporter cell line show that with the addition of miR29a EVs, there is a decrease in the mKate/BFP ratio, indicating delivery of miRNA29a and targeting of the mKate reporter (Fig 1C&D). The addition of IL-1β was able to activate NFκB in HDFs and treatment with miR29a EVs significantly decreased NFκB nuclear localization when added at the same time as IL-1β (Fig 1E,F&G). Addition of both SCR and miR29a MSC EVs reduced HDF collagen gel contraction and decreased YAP nuclear localization (Fig. 1H,I,&J)

The current results indicate the potential for both native and miR29a loaded EVs to regulate fibrotic and inflammatory signaling. This is shown through reduced mKate/BFP ratio in reporter cells, reduced inflammatory signaling in HDFs, and decreased contractility and YAP activation in collagen gels. These engineered EVs have potential for therapeutic applications in a variety of fibrotic diseases.

Acknowledgements (Optional): [1] Wynn+ Journal of Pathology (2008), [2] Distler+ Nat Rev Rheum (2019), [3] O’Reily+ Arthritis Res and Ther (2016), [4] Rooij+ PNAS (2008), [5] McDaniel+ BMES (2023), [6] Rider+ Sci Rep (2016), [7] Gam+ (2018).