Inflammatory extracellular vesicles prompt heart dysfunction via TRL4-dependent NF-κB activation.

Background: After myocardial infarction, necrotic cardiomyocytes release damage-associated proteins that stimulate innate immune pathways and macrophage tissue infiltration, which drives inflammation and myocardial remodeling. Circulating inflammatory extracellular vesicles play a crucial role in the acute and chronic phases of ischemia, in terms of inflammatory progression. In this study, we hypothesize that the paracrine effect mediated by these vesicles induces direct cytotoxicity in cardiomyocytes. Thus, we examined whether reducing the generation of inflammatory vesicles within the first few hours after the ischemic event ameliorates cardiac outcome at short and long time points. Methods: Myocardial infarction was induced in rats that were previously injected intraperitoneally with a chemical inhibitor of extracellular-vesicle biogenesis. Heart global function was assessed by echocardiography performed at 7, 14 and 28 days after MI. Cardiac outcome was also evaluated by hemodynamic analysis at sacrifice. Cytotoxic effects of circulating EV were evaluated ex-vivo in a Langendorff, system by measuring the level of cardiac troponin I (cTnI) in the perfusate. Mechanisms undergoing cytotoxic effects of EV derived from pro-inflammatory macrophages (M1) were studied in-vitro in primary rat neonatal cardiomyocytes. Results: Inflammatory response following myocardial infarction dramatically increased the number of circulating extracellular vesicles carrying alarmins such as IL-1α, IL-1β and Rantes. Reducing the boost in inflammatory vesicles during the acute phase of ischemia resulted in preserved left ventricular ejection fraction in vivo. Hemodynamic analysis confirmed functional recovery by displaying higher velocity of left ventricular relaxation and improved contractility. When added to the perfusate of isolated hearts, post-infarction circulating vesicles induced significantly more cell death in adult cardiomyocytes, as assessed by cTnI release, comparing to circulating vesicles isolated from healthy (non-infarcted) rats. In vitro inflammatory extracellular vesicles induce cell death by driving nuclear translocation of NF-κB into nuclei of cardiomyocytes. Conclusion: Our data suggest that targeting circulating extracellular vesicles during the acute phase of myocardial infarction may offer an effective therapeutic approach to preserve function of ischemic heart

Effect of synthesis conditions on formation pathways of metal organic framework (MOF-5) Crystals

Metal Organic Frameworks (MOFs) represent a class of nanoporous crystalline materials with far reaching potential in gas storage, catalysis, and medical devices. We investigated the effects of synthesis process parameters on production of MOF-5 from terephthalic acid and zinc nitrate in diethylformamide. Under favorable synthesis conditions, we systematically mapped a solid formation diagram in terms of time and temperature for both stirred and unstirred conditions. The synthesis of MOF-5 has been previously reported as a straightforward reaction progressing from precursor compounds in solution directly to the final MOF-5 solid phase product. However, we show that the solid phase formation process is far more complex, invariably transferring through metastable intermediate crystalline phases before the final MOF-5 phase is reached, providing new insights into the formation pathways of MOFs. We also identify process parameters suitable for scale-up and continuous manufacturing of high purity MOF-5

Exosomal Expression of CXCR4 Targets Cardioprotective Vesicles to Myocardial Infarction and Improves Outcome after Systemic Administration.

Cell therapy has been evaluated to enhance heart function after injury. Delivered cells mostly act via paracrine mechanisms, including secreted growth factors, cytokines, and vesicles, such as exosomes (Exo). Intramyocardial injection of cardiac-resident progenitor cells (CPC)-derived Exo reduced scarring and improved cardiac function after myocardial infarction in rats. Here, we explore a clinically relevant approach to enhance the homing process to cardiomyocytes (CM), which is crucial for therapeutic efficacy upon systemic delivery of Exo. By overexpressing exosomal CXCR4, we increased the efficacy of plasmatic injection of cardioprotective Exo-CPC by increasing their bioavailability to ischemic hearts. Intravenous injection of Exo <sup>CXCR4</sup> significantly reduced infarct size and improved left ventricle ejection fraction at 4 weeks compared to Exo <sup>CTRL</sup> (p < 0.01). Hemodynamic measurements showed that Exo <sup>CXCR4</sup> improved dp/dt min, as compared to Exo <sup>CTRL</sup> and PBS group. In vitro, Exo <sup>CXCR4</sup> was more bioactive than Exo <sup>CTRL</sup> in preventing CM death. This in vitro effect was independent from SDF-1α, as shown by using AMD3100 as specific CXCR4 antagonist. We showed, for the first time, that systemic administration of Exo derived from CXCR4-overexpressing CPC improves heart function in a rat model of ischemia reperfusion injury These data represent a substantial step toward clinical application of Exo-based therapeutics in cardiovascular disease

Exosomes From Human Cardiac Progenitor Cells for Therapeutic Applications: Development of a GMP-Grade Manufacturing Method.

Exosomes, nanosized membrane vesicles secreted by cardiac progenitor cells (Exo-CPC), inhibit cardiomyocyte apoptosis under stress conditions, promote angiogenesis <i>in vitro</i> , and prevent the early decline in cardiac function after myocardial infarction <i>in vivo</i> in preclinical rat models. The recognition of exosome-mediated effects has moved attempts at developing cell-free approaches for cardiac repair. Such approaches offer major advantages including the fact that exosomes can be stored as ready-to-use agents and delivered to patients with acute coronary syndromes. The aim of the present work was the development of a good manufacturing practice (GMP)-grade method for the large-scale preparation of Exo-CPC as a medicinal product, for a future clinical translation. A GMP-compliant manufacturing method was set up, based on large-scale cell culture in xeno-free conditions, collection of up to 8 l of exosome-containing conditioned medium and isolation of Exo-CPC through tangential flow filtration. Quality control tests were developed and carried out to evaluate safety, identity, and potency of both cardiac progenitor cells (CPC) as cell source and Exo-CPC as final product (GMP-Exo-CPC). CPC, cultured in xeno-free conditions, showed a lower doubling-time than observed in research-grade condition, while producing exosomes with similar features. Cells showed the typical phenotype of mesenchymal progenitor cells (CD73/CD90/CD105 positive, CD14/CD20/CD34/CD45/HLA-DR negative), and expressed mesodermal (TBX5/TBX18) and cardiac-specific (GATA4/MESP1) transcription factors. Purified GMP-Exo-CPC showed the typical nanoparticle tracking analysis profile and expressed main exosome markers (CD9/CD63/CD81/TSG101). The GMP manufacturing method guaranteed high exosome yield (>10 <sup>13</sup> particles) and consistent removal (≥97%) of contaminating proteins. The resulting GMP-Exo-CPC were tested for safety, purity, identity, and potency <i>in vitro</i> , showing functional anti-apoptotic and pro-angiogenic activity. The therapeutic efficacy was validated <i>in vivo</i> in rats, where GMP-Exo-CPC ameliorated heart function after myocardial infarction. Our standardized production method and testing strategy for large-scale manufacturing of GMP-Exo-CPC open new perspectives for reliable human therapeutic applications for acute myocardial infarction syndrome and can be easily applied to other cell sources for different therapeutic areas

Superior exosome-mediated paracrine effects of cardiac progenitor cells compared to bone marrow mesenchymal stem cells derived from the same patient for cardiac repair

Background: Both bone marrow-derived mesenchymal stem cells (BM-MSCs) and cardiac progenitor cells (CPCs) have shown promising results in clinical trials in patients after acute myocardial infarction (MI). Available evidence supports paracrine effects as the mechanism of benefit of both cell types. Recently, we have shown that exosomes (Exo), secreted extracellular nanovesicles, is the critical component of the paracrine activity of CPCs. However, exosomes derived from BM-MSC (Exo-MSC) and those derived from CPCs (Exo-CPC) have not been compared thus far. Methods and results: Both a sternal BM aspirate and a right atrial appendage explant were obtained from patients who underwent heart surgery for valve disease to derive BM-MSCs and CPCs, respectively. Exo was purified by ultracentrifugation from the respective conditioned media (CM). Exo-CPC inhibited staurosporin-induced in vitro apoptosis in mouse HL-1 cardiomyocytes more effectively than Exo-MSC. Exo-depleted CM from either cell type was inactive. Both Exo-CPC and Exo-MSC promoted angiogenesis in vitro. Infarcted rat hearts injected with Exo-CPC showed significantly less cardiomyocyte apoptosis and scar, more angiogenesis, higher end-diastolic thickness, and a significant improvement in LV ejection fraction (LVEF) at 4 weeks post-MI compared with those injected with Exo-MSC, exosomes derived from human dermal fibroblasts (Exo-F) as an inert cell control, or saline. Exo-MSC showed intermediate efficacy between Exo-CPC and Exo-F. Both Exo-CPC and Exo-MSC were enriched for cardioprotective and/or proangiogenic miRNAs such as miR-146a-3p, miR-210 and miR-132 compared to Exo-F. Proteomics analyses identified a number of proteins overexpressed in Exo-CPC relative to Exo-MSC, including potentially beneficial proteins such as pregnancy-associated plasma protein-A (PAPP-A, or pappalysin), which releases active interleukin-like growth factor-1 (IGF-1) from IGF binding proteins, and superoxide dismutase-2 (SOD2). Using PAPP-A-specific silencing RNAs, we showed that Exo from PAPP-A knockdown CPCs were significantly less efficient than Exo from naïve CPCs using the in vivo infarct model. Conclusion: Exo fully accounts for paracrine cardioprotective effects by CPCs and BM-MSCs. On a same patient-basis, Exo-CPC was more cardioprotective than Exo-MSC both in vitro and in vivo. Enrichment of Exo-CPCs with PAPP-A significantly contributes to its beneficial effect