RESUMEN
Extracellular vesicles (EVs) are known as molecular carriers involved in cell communication and the regulation of (patho)physiological processes. miRNAs and growth factors are the main contents of EVs which make them a good candidate for the treatment of diseases caused by ischemia, but the low production of EVs by a cell producer and a significant variation of the molecular contents in EVs according to the cell source are the main limitations of their widespread use. Here, we show how to improve the therapeutic properties of mesenchymal stromal cell (MSC)-derived EVs (MSC-EVs) by modifying MSCs to enrich these EVs with specific angiomiRs (miR-135b or miR-210) using lentiviral vectors carrying miR-135b or miR-210. MSCs were obtained from the mouse bone marrow and transduced with a corresponding lentivector to overexpress miR-135b or miR-210. The EVs were then isolated by ultracentrifugation and characterized using a flow cytometer and a nanoparticle tracking analyzer. The levels of 20 genes in the MSCs and 12 microRNAs in both MSCs and EVs were assessed by RTâqPCR. The proangiogenic activity of EVs was subsequently assessed in human umbilical vein endothelial cells (HUVECs). The results confirmed the overexpression of the respective microRNA in modified MSCs. Moreover, miR-135b overexpression upregulated miR-210-5p and follistatin, whereas the overexpression of miR-210 downregulated miR-221 and upregulated miR-296. The tube formation assay showed that EVs from MSCs overexpressing miR-210-5p (EVmiR210) significantly promoted tubular structure formation in HUVECs. A significant increase in angiogenic proteins (PGF, endothelin 1, and artemin) and genes (VEGF, activin A, and IGFBP1) in HUVECs treated with VEmiR210 justifies the better tubular structure formation of these cells compared with that of EVmiR135b-treated HUVECs, which showed upregulated expression of only artemin. Collectively, our results show that the EV cargo can be modified by lentiviral vectors to enrich specific miRNAs to achieve a specific angiogenic potential.
Asunto(s)
Vesículas Extracelulares , Células Madre Mesenquimatosas , MicroARNs , Inductores de la Angiogénesis/metabolismo , Animales , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
CD8+ T lymphocytes play an important role in controlling infections by intracellular pathogens. Chemokines and their receptors are crucial for the migration of CD8+ T-lymphocytes, which are the main IFNγ producers and cytotoxic effectors cells. Although the participation of chemokine ligands and receptors has been largely explored in viral infection, much less is known in infection by Trypanosoma cruzi, the causative agent of Chagas disease. After T. cruzi infection, CXCR3 chemokine receptor is highly expressed on the surface of CD8+ T-lymphocytes. Here, we hypothesized that CXCR3 is a key molecule for migration of parasite-specific CD8+ T-cells towards infected tissues, where they may play their effector activities. Using a model of induction of resistance to highly susceptible A/Sn mice using an ASP2-carrying DNA/adenovirus prime-boost strategy, we showed that CXCR3 expression was upregulated on CD8+ T-cells, which selectively migrated towards its ligands CXCL9 and CXCL10. Anti-CXCR3 administration reversed the vaccine-induced resistance to T. cruzi infection in a way associated with hampered cytotoxic activity and increased proapoptotic markers on the H2KK-restricted TEWETGQI-specific CD8+ T-cells. Furthermore, CXCR3 receptor critically guided TEWETGQI-specific effector CD8+ T-cells to the infected heart tissue that express CXCL9 and CXCL10. Overall, our study pointed CXCR3 and its ligands as key molecules to drive T. cruzi-specific effector CD8+ T-cells into the infected heart tissue. The unveiling of the process driving cell migration and colonization of infected tissues by pathogen-specific effector T-cells is a crucial requirement to the development of vaccine strategies.
Asunto(s)
Vacunas contra el Adenovirus/inmunología , Linfocitos T CD8-positivos/inmunología , Cardiomiopatía Chagásica/inmunología , Quimiotaxis de Leucocito , Miocardio/metabolismo , Receptores CXCR3/metabolismo , Trypanosoma cruzi/inmunología , Animales , Apoptosis , Cardiomiopatía Chagásica/parasitología , Cardiomiopatía Chagásica/prevención & control , Femenino , Corazón/parasitología , Ligandos , Ratones , Ratones Endogámicos C57BL , Miocardio/inmunología , Receptores CCR2/metabolismo , Bazo/inmunología , Regulación hacia Arriba , Vacunas de ADN/inmunologíaRESUMEN
Regeneration of injured skeletal muscles is affected by fibrosis, which can be improved by the administration of angiotensin II (AngII) receptor (ATR) blockers in normotensive animals. However, the role of ATR in skeletal muscle fibrosis in hypertensive organisms has not been investigated yet. The tibialis anterior (TA) muscle of spontaneously hypertensive (SHR) and Wistar rats (WR) were lacerated and a lentivector encoding a microRNA targeting AngII receptor type 1 (At1) (Lv-mirAT1a) or control (Lv-mirCTL) was injected. The TA muscles were collected after 30 days to evaluate fibrosis by histology and gene expression by real-time quantitative PCR (RT-qPCR) and Western blot. SHR's myoblasts were analyzed by RT-qPCR, 48 h after transduction. In the SHR's TA, AT1 protein expression was 23.5-fold higher than in WR without injury, but no difference was observed in the angiotensin II receptor type 2 (AT2) protein expression. TA laceration followed by suture (LS) produced fibrosis in the SHR (23.3±8.5%) and WR (7.9±1.5%). Lv-mirAT1 treatment decreased At1 gene expression in 50% and reduced fibrosis to 7% 30 days after. RT-qPCR showed that reduction in At1 expression is due to downregulation of the At1a but not of the At1b. RT-qPCR of myoblasts from SHR transduced with Lv-mirAT1a showed downregulation of the Tgf-b1, Tgf-b2, Smad3, Col1a1, and Col3a1 genes by mirAT1a. In vivo and in vitro studies indicate that hypertension overproduces skeletal muscle fibrosis, and AngII-AT1a signaling is the main pathway of fibrosis in SHR. Moreover, muscle fibrosis can be treated specifically by in loco injection of Lv-mirAT1a without affecting other organs.
Asunto(s)
MicroARNs/genética , Músculo Esquelético/patología , Receptores de Angiotensina/genética , Animales , Regulación hacia Abajo , Fibrosis , Perfilación de la Expresión Génica , Ratas , Ratas Endogámicas SHR , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
PURPOSE: In this prospective, randomized, double-blinded study we sought to evaluate the efficacy and safety of combined use of intravenous ketorolac and acetaminophen in small children undergoing outpatient inguinal hernia repair. MATERIALS AND METHODS: We studied 55 children 1 to 5 years old who were undergoing elective repair of unilateral inguinal hernia. After induction of general anesthesia children in the experimental group (28 patients) received 1 mg/kg ketorolac and 20 mg/kg acetaminophen intravenously. In the control group (27 patients) the same volume of saline was administered. All patients received 1 microg/kg fentanyl intravenously before incision. We also evaluated the number of patients requiring postoperative rescue fentanyl, total fentanyl consumption, pain scores and side effects. RESULTS: Significantly fewer patients receiving ketorolac-acetaminophen received postoperative rescue fentanyl compared to controls (28.6% vs 81.5%). A significantly lower total dose of fentanyl was administered to patients receiving ketorolac-acetaminophen compared to controls (0.54 vs 1.37 microg/kg). Pain scores were significantly higher in the control group immediately postoperatively but eventually decreased. The incidences of sedation use (55.6% vs 25.0%) and vomiting (33.3% vs 10.7%) were significantly higher in controls. CONCLUSIONS: Preoperative intravenous coadministration of ketorolac and acetaminophen is a simple, safe and effective method for relieving postoperative pain, and demonstrates highly significant fentanyl sparing effects in small children after outpatient inguinal hernia repair.