Biomaterials composed of extracellular matrix (ECM) provide both mechanical support and a reservoir of constructive signaling molecules that promote functional tissue repair. Recently, matrix-bound nanovesicles (MBVs) have been reported as an integral component of ECM bioscaffolds. Although liquid-phase extracellular vesicles (EVs) have been the subject of intense investigation, their similarity to MBV is limited to size and shape. Liquid chromatography–mass spectrometry (LC-MS)–based lipidomics and redox lipidomics were used to conduct a detailed comparison of liquid-phase EV and MBV phospholipids. Combined with comprehensive RNA sequencing and bioinformatic analysis of the intravesicular cargo, we show that MBVs are a distinct and unique subpopulation of EV and a distinguishing feature of ECM-based biomaterials. The results begin to identify the differential biologic activities mediated by EV that are secreted by tissue-resident cells and deposited within the ECM.

temic expansion of ST2 + Tregs (29,30). IL-33 expressed by fibrogenic/adipogenic progenitors in skeletal muscle has also been shown to regulate skeletal muscle Treg homeostasis and support muscle regeneration (31). Related studies have suggested a direct, cardioprotective role for rIL-33 against hypertrophy resulting from cardiac overload (32) and fibrosis after myocardial infarction (33). However, delivery of rIL-33 also aggravates autoimmune eosinophilic pericarditis during coxsackievirus B3 infection (34), suggesting that IL-33 can contribute to cardiac inflammation. IL-33 expression has been reported in cardiac fibroblasts (32) and the vasculature ( 35), yet how the expression of this alarmin is modulated in cardiac allografts or impacts outcomes was unknown.Using IL-33-deficient heart grafts in a mouse chronic rejection model we have established that IL-33 stands out among identified alarmins and limits differentiation of proinflammatory macrophages to prevent chronic rejection. Specifically, transplants lacking IL-33 displayed dramatically accelerated chronic rejectionassociated vasculopathy and subsequent fibrosis orchestrated by graft-infiltrating recipient proinflammatory macrophages. IL-33expressing heart grafts in recipients with ST2-deficient macrophages also displayed increased graft infiltration by proinflammatory macrophages and accelerated graft loss. Mechanistic studies demonstrated that IL-33 promoted a reparative macrophage phenotype through a metabolic reprograming involving augmented oxidative phosphorylation (OXPHOS) and fatty acid (FA) uptake. We also revealed that IL-33 prevents proinflammatory stimuli-induced disruption of the tricarboxylic acid (TCA) cycle that shifts macrophage metabolism to anaerobic glycolysis and generates proinflammatory metabolites (36,37). Restoration of IL-33 to IL-33-deficient heart transplants using vesicles in ECM-derived hydrogel immediately after transplantation profoundly reduced the frequency of proinflammatory myeloid cells in the graft and prevented graft loss to chronic rejection. Thus, the local delivery of IL-33 in ECM-based materials after transplantation may be a practical and promising biologic for chronic rejection prophylaxis.

The regenerative healing response of injured skeletal muscle is dependent upon an appropriately timed switch from a local type-I to a type-II immune response. Biologic scaffolds derived from extracellular matrix (ECM) have been shown to facilitate a macrophage phenotype transition that leads to downstream site-appropriate functional tissue deposition and myogenesis. However, the mechanisms by which ECM directs the switching of immune cell phenotype are only partially understood. Herein, we provide the first evidence that matrix bound nanovesicles (MBV) embedded within ECM-scaffolds are a rich and stable source of interleukin-33 (IL-33), an alarmin/ cytokine with emerging reparative properties. We show that IL-33 encapsulated within MBV bypass the classical IL33/ST2 receptor signaling pathway to direct macrophage differentiation into the reparative, pro-remodeling M2 phenotype, which in turn facilitates myogenesis of skeletal muscle progenitor cells. Our results suggest the potential of IL-33 + MBV as a clinical therapy to augment the restorative efficacy of existing ECM-based and non-ECM based approaches.