ABSTRACT Pharmacological tools to selectively modulate extracellular vesicle (EV) secretion are scarce. Here, we identify the ALK5 (TGF‐β receptor I) inhibitor SD‐208 as a potent suppressor of small EV (sEV) secretion that acts independently of its canonical anti‐fibrotic activity. SD‐208 not only reversed myofibroblast activation but also markedly inhibited sEV secretion. Strikingly, this inhibitory effect persisted in non‐activated cardiac fibroblasts and non‐fibrotic HEK293 cells, demonstrating that SD‐208 regulates EV secretion through mechanisms uncoupled from TGF‐β/Smad signalling. Mechanistic analyses revealed that SD‐208 disrupts vesicle trafficking rather than EV biogenesis. Reduced secretion of CD63+ EVs was accompanied by intracellular accumulation of CD63+ structures and their selective diversion into LAMP1+ lysosomes. Proteomic profiling of SD‐208‐treated and control HEK293 cells and cardiac fibroblasts revealed dysregulation of vesicle trafficking pathways, enrichment of ubiquitin ligase complexes, and enhanced endosome‐to‐lysosome transport. Together, these findings demonstrate that SD‐208 diverts CD63+ multivesicular bodies (MVBs) from a secretory fate toward lysosomal degradation. This work identifies SD‐208 as a small‐molecule tool to interrogate the secretory‐versus‐degradative fate of MVBs and uncovers a new regulatory link between lysosomal pathways and EV trafficking. Beyond its established role as an anti‐fibrotic agent, SD‐208 provides mechanistic and therapeutic opportunities for the control of EV secretion in diseases such as fibrosis, cardiac remodelling, hypertrophic cardiomyopathy, and cancer.
Rahul Sanwlani, Georgina H. Thompson, Kyle Bramich et al.· Journal of Extracellular Ves...· 0 citations
Nanofibers-reinforced hydrogels offer improved mechanical properties as compared to classic hydrogels and can potentially better support cell viability and cell therapy outcomes. Here, we optimized polycaprolactone (PCL) short nanofibers production and combined them with a soft hydrogel based on kappa-carrageenan and gelatin type B (kCGb) to perform umbilical cord pericyte (UCP) delivery and promote re-endothelialization of injured blood vessels. Electrospinning of PCL identified 12.5% w/v concentration, 0.5 mL/h flow rate, 21 kV voltage, and 6.8 m/s drum collector speed as optimal parameters for aligned beadless nanofibers. Coaxial PCL/gelatin type A (Ga) fibers were successfully produced with diameters of 180-190 nm (core) and 225-245 nm (shell). The Ga acts as a temporary shell, which was removed during postfabrication processing by washing the nanofibers with deionized water (DIW) at 37°C for 30 min. Mechanical cryostat cutting generated short nanofibers (~75 μm ± 28 μm) with higher yields from coaxial mats (2 × 106 fibers/mL ± 1.6 × 105 fibers/mL) compared to single PCL mats (5 × 104 fibers/mL ± 1.3 × 104 fibers/mL). kCGb hydrogel showed a significant improvement in mechanical properties when reinforced with short PCL nanofibers. The recovery percentage, measured using rheology, increased from 12.1% ± 0.3% to 90.9% ± 0.4% when a small amount (0.05 wt%) of short PCL nanofibers was added. The injection force using clinically relevant needles of 0.5kC2Gb0.05PCL was about ~37 N, and there was no significant reduction in UCPs viability after injection up to 1 week of culture. Injection delivery of kCGbPCL mixed with UCPs (1 × 106 cells/mL) in an ex vivo model of porcine artery injury led to a marked improvement in endothelial coverage from 47.2% ± 19.1% in the injured artery to 76.5% ± 13.2% after cell therapy, with a restoration index of about 89.8% after 1 week of culture. These results represent a further step toward developing physically cross-linked and mechanically stable injectable scaffolds for different applications, such as drug delivery, 3D printing, in vitro modeling, and tissue engineering.
A. Jawad, C. Heiss, V. Stolojan et al.· Journal of Biomedical Materi...· 0 citations
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