RESUMEN
Diabetic retinopathy (DR) is a leading cause of blindness worldwide with limited treatment options. Mesenchymal stem cell-derived small extracellular vesicles (MSC-sEVs) hold promise as a cell-free therapy for retinal diseases. In this study, we present evidence that the intravitreal injection of MSC-sEVs improved retinal function and alleviated retinal apoptosis, inflammation, and angiogenesis in both db/db mice and streptozotocin-induced diabetic rats. Mechanistically, hyperglycemia-induced activation of hypoxia-inducible factor-1α (HIF-1α) inhibited the tripartite motif 21 (TRIM21)-mediated ubiquitination and degradation of enhancer of zeste homologue 2 (EZH2), ultimately resulting in the downregulation of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) through EZH2-induced methylation modification. The presence of miR-5068 and miR-10228 in MSC-sEVs targeted the HIF-1α/EZH2/PGC-1α pathway. The blockade of miR-5068 and miR-10228 abolished the retinal therapeutic effects of MSC-sEVs. Additionally, we engineered MSC-sEVs with elevated levels of miR-5068 and miR-10228 to enhance retinal repair efficiency. Together, our findings provide novel insights into the mechanism underlying DR progress and highlight the potential of MSC-sEVs, especially engineered MSC-sEVs, as a therapeutic option for DR.
RESUMEN
Photoreceptor apoptosis is an important pathogenesis of retinal degeneration and a primary cause of vision loss with limited treatment methods. Mesenchymal stem/stromal cells-derived small extracellular vesicles (MSC-sEVs) have shown therapeutic value in various ocular disorders. Recent studies have revealed that hypoxic preconditioning can improve the effectiveness of MSC-sEVs in tissue regeneration. However, whether hypoxic preconditioned MSC-sEVs (Hyp-sEVs) exert superior effects on photoreceptor protection relative to normoxic conditioned MSC-sEVs (Nor-sEVs) remains unclear. Here, we reported that Hyp-sEVs further improved retinal structure, recovered retinal function, and suppressed photoreceptor apoptosis in N-methyl-N-nitrosourea (MNU)-induced mouse model compared with Nor-sEVs. Hyp-sEVs also exhibited enhanced anti-apoptotic roles in MNU-provoked 661 W cell injury in vitro. We then analyzed the protein profiles of Nor-sEVs and Hyp-sEVs by LC-MS/MS and found that growth-associated protein 43 (GAP43) was enriched in Hyp-sEVs. The knockdown of GAP43 abolished the retinal therapeutic effects of Hyp-sEVs. Mechanistically, hypoxic stimulation-induced hypoxia-inducible factor-1α (HIF-1α) activation was responsible for preventing tripartite motif-containing protein 25 (TRIM25)-mediated GAP43 ubiquitination and degradation, leading to the upregulation of GAP43 in Hyp-sEVs. Together, our findings uncover the efficacy and mechanism of Hyp-sEVs-based photoreceptor protection and highlight the potential of Hyp-sEVs as optimized therapeutics for retinal degeneration.
Asunto(s)
Vesículas Extracelulares , Degeneración Retiniana , Ratones , Animales , Degeneración Retiniana/prevención & control , Degeneración Retiniana/metabolismo , Cromatografía Liquida , Espectrometría de Masas en Tándem , Retina/metabolismo , Vesículas Extracelulares/metabolismo , Hipoxia/metabolismoRESUMEN
Spinal cord injury (SCI) is a traumatic condition of the central nervous system that causes paralysis of the limbs. Micro electric fields (EF) have been implicated in a novel therapeutic approach for nerve injury repair and regeneration, but the effects of human umbilical cord mesenchymal stem cell-derived small extracellular vesicles that are induced by micro electric fields (EF-sEVs) stimulation on SCI remain unknown. The aim of the present study was to investigate whether EF-sEVs have therapeutic effects a rat model of SCI. EF-sEVs and normally conditioned human umbilical cord mesenchymal stem cells-derived small extracellular vesicles (CON-sEVs) were collected and injected intralesionally into SCI model rats to evaluate the therapeutic effects. We detect the expression of candidate long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (lncRNA-MALAT1) in EF-sEVs and CON-sEVs. The targets and downstream effectors of lncRNA-MALAT1 were investigated using luciferase reporter assays. Using both in vivo and in vitro experiments, we demonstrated that EF-sEVs increased autophagy and decreased apoptosis after SCI, which promoted the recovery of motor function. We further confirmed that the neuroprotective effects of EF-sEVs in vitro and in vivo correlated with the presence of encapsulated lncRNA-MALAT1 in sEVs. lncRNA-MALAT1 targeted miR-22-3p via sponging, reducing miR-22-3p's suppressive effects on its target, SIRT1, and this translated into AMPK phosphorylation and increased levels of the antiapoptotic protein Bcl-2. Collectively, the present study identified that the lncRNA-MALAT1 in EF-sEVs plays a neuroprotective role via the miRNA-22-3p/SIRT1/AMPK axis and offers a fresh perspective and a potential therapeutic approach using sEVs to improve SCI.
Asunto(s)
Neoplasias Pulmonares , MicroARNs , ARN Largo no Codificante , Traumatismos de la Médula Espinal , Ratas , Humanos , Animales , ARN Largo no Codificante/metabolismo , Proteínas Quinasas Activadas por AMP , Sirtuina 1/genética , Sirtuina 1/metabolismo , Apoptosis , Traumatismos de la Médula Espinal/metabolismo , MicroARNs/metabolismo , AutofagiaRESUMEN
BACKGROUND: As a leading cause of vision decline and severe blindness in adults, diabetic retinopathy (DR) is characterized by the aggravation of retinal oxidative stress and apoptosis in the early stage. Emerging studies reveal that mesenchymal stem cells-derived small extracellular vesicles (MSC-sEV) treatment represents a promising cell-free approach to alleviate ocular disorders. However, the repairing effects of MSC-sEV in DR remain largely unclear. This study aimed at exploring the role and the underlying mechanism of MSC-sEV in hyperglycemia-induced retinal degeneration. METHODS: In vivo, we used streptozotocin (STZ) to establish diabetic rat model, followed by the intravitreal injection of MSC-sEV to determine the curative effect. The cell viability and antioxidant capacity of retinal pigment epithelium (RPE) cells stimulated with high-glucose (HG) medium after MSC-sEV treatment were analyzed in vitro. By detecting the response of cell signaling pathways in MSC-sEV-treated RPE cells, we explored the functional mechanism of MSC-sEV. Mass spectrometry was performed to reveal the bioactive protein which mediated the role of MSC-sEV. RESULTS: The intravitreal injection of MSC-sEV elicited antioxidant effects and counteracted retinal apoptosis in STZ-induced DR rat model. MSC-sEV treatment also reduced the oxidative level and enhanced the proliferation ability of RPE cells cultured in HG conditions in vitro. Further studies showed that the increased level of phosphatase and tensin homolog (PTEN) inhibited AKT phosphorylation and nuclear factor erythroid 2-related factor 2 (NRF2) expression in RPE cells stimulated with HG medium, which could be reversed by MSC-sEV intervention. Through mass spectrometry, we illustrated that MSC-sEV-delivered neuronal precursor cell-expressed developmentally downregulated 4 (NEDD4) could cause PTEN ubiquitination and degradation, activate AKT signaling and upregulate NRF2 level to prevent DR progress. Moreover, NEDD4 knockdown impaired MSC-sEV-mediated retinal therapeutic effects. CONCLUSIONS: Our findings indicated that MSC-sEV ameliorated DR through NEDD4-induced regulation on PTEN/AKT/NRF2 signaling pathway, thus revealing the efficiency and mechanism of MSC-sEV-based retinal protection and providing new insights into the treatment of DR.
Asunto(s)
Diabetes Mellitus Experimental , Retinopatía Diabética , Vesículas Extracelulares , Células Madre Mesenquimatosas , Ubiquitina-Proteína Ligasas Nedd4 , Animales , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/terapia , Retinopatía Diabética/terapia , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/administración & dosificación , Proteínas Proto-Oncogénicas c-akt/metabolismo , RatasRESUMEN
Mesenchymal stem cells (MSCs) have been widely applied to regenerative medicine owing to their multiple differentiation, self-renewal, and immunomodulatory abilities. Exosomes are cell-secreted natural nanovesicles and thought to be mediators of intercellular communication and material transport. The therapeutic potential of MSCs can be largely attributed to MSC-derived exosomes (MSC-exosomes). Emerging evidence suggests that the therapeutic efficacy of MSC-exosomes is highly dependent on the status of MSCs, and optimization of the extracellular environment affects the exosomal content. Pretreatment methods including three-dimensional cultures, hypoxia, and other biochemical cues have been shown to potentially enhance the biological activity of MSC-exosomes while maintaining or enhancing their production. On the other hand, engineering means to enhance the desired function of MSC-exosomes has been rapidly gaining attention. In particular, biologically active molecule encapsulation and membrane modification can alter or enhance biological functions and targeting of MSC-exosomes. In this review, we summarize two possible strategies to improve the therapeutic activity of MSC-exosomes: preconditioning approaches and engineering exosomes. We also explore the underlying mechanisms of different strategies and discuss their advantages and limitations of the upcoming clinical applications.
RESUMEN
Prostate cancer (CaP) is a common male malignancy. Using prostate specific antigen (PSA) and prostate cancer antigen 3 (PCA3) in the diagnosis of prostate cancer, sensitivity and specificity still require improvement. Additional targets are urgently needed for the diagnosis, prognosis, and prediction of therapeutic response, leading to better treatments in order to reduce the mortality of CaP. Here, we utilized a solid-phase antibody array, which can simultaneously detect 200 proteins, for the screening of novel blood-based biomarkers. The proteins differentially expressed in the pathogenesis of CaP were further analyzed using bioinformatics methods. The identified targets were further validated by the enzyme-linked immunosorbent assay (ELISA). A total of 38 proteins were identified with significantly differential levels in CaP serum compared to healthy control serum, including 21 up-regulated and 17 down-regulated cytokines. ELISA result showed that validated six ones of these differential cytokines were significantly differential between CaP and control, consistent with the antibody array result. The protein-protein interaction (PPI) analysis for these differentially expressed cytokines showed the top five cytokines interacting with most other cytokines were insulin, SDF-1a, CD40L, IL-18 and NCAM-1, suggesting these five targets are important in the pathogenesis of CaP, and more sensitive for the early diagnosis and prognosis of CaP. Targeting these cytokines may be more effective therapies against CaP. Among these differentially expressed cytokines, it was found that AR, BTC, IL-1 F8, IL-31, Marapsin, b-NGF, EDA-A2, MCP-3, MCP-4, MIP-3a, PIGF, and TECK decreased, while Fas, Flt-3L, and NCAM-1 increased in CaP when compared to the controls. Taken together, those 38 differentially expressed cytokines may service as novel serum biomarkers for CaP, which will be further validated with more clinical samples.
Asunto(s)
Biomarcadores/sangre , Citocinas/sangre , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/metabolismo , Ligando de CD40/metabolismo , Antígeno CD56/metabolismo , Quimiocina CXCL12/metabolismo , Biología Computacional , Regulación hacia Abajo , Ontología de Genes , Humanos , Insulina/metabolismo , Interleucina-18/metabolismo , Masculino , Persona de Mediana Edad , Neoplasias de la Próstata/patología , Análisis por Matrices de Proteínas , Unión Proteica , Mapas de Interacción de Proteínas , Regulación hacia ArribaRESUMEN
Upon receptor activator of NF-κB ligand (RANKL) binding, RANK promotes osteoclast formation through the recruitment of tumor necrosis factor (TNF) receptor-associated factors (TRAFs). In vitro assays identified two RANK intracellular motifs that bind TRAFs: PVQEET560-565 (Motif 2) and PVQEQG604-609 (Motif 3), which potently mediate osteoclast formation in vitro. To validate the in vitro findings, we have generated knock-in (KI) mice harboring inactivating mutations in RANK Motifs 2 and 3. Homozygous KI (RANKKI/KI ) mice are born at the predicted Mendelian frequency and normal in tooth eruption. However, RANKKI/KI mice exhibit significantly more trabecular bone mass than age- and sex-matched heterozygous KI (RANK+/KI ) and wild-type (RANK+/+ ) counterparts. Bone marrow macrophages (BMMs) from RANKKI/KI mice do not form osteoclasts when they are stimulated with macrophage colony-stimulating factor (M-CSF) and RANKL in vitro. RANKL is able to activate the NF-κB, ERK, p38, and JNK pathways in RANKKI/KI BMMs, but it cannot stimulate c-Fos or NFATc1 in the RANKKI/KI cells. Previously, we showed that RANK signaling plays an important role in Porphyromonas gingivalis (Pg)-mediated osteoclast formation by committing BMMs into the osteoclast lineage. Here, we show that RANKL-primed RANKKI/KI BMMs are unable to differentiate into osteoclasts in response to Pg stimulation, indicating that the two RANK motifs are required for Pg-induced osteoclastogenesis. Mechanistically, RANK Motifs 2 and 3 facilitate Pg-induced osteoclastogenesis by stimulating c-Fos and NFATc1 expression during the RANKL pretreatment phase as well as rendering c-Fos and NFATc1 genes responsive to subsequent Pg stimulation. Cell-penetrating peptides (CPPs) conjugated with RANK segments containing Motif 2 or 3 block RANKL- and Pg-mediated osteoclastogenesis. The CPP conjugates abrogate RANKL-stimulated c-Fos and NFATc1 expression but do not affect RANKL-induced activation of NF-κB, ERK, p38, JNK, or Akt signaling pathway. Taken together, our current findings demonstrate that RANK Motifs 2 and 3 play pivotal roles in osteoclast formation in vivo and mediate Pg-induced osteoclastogenesis in vitro.
Asunto(s)
Diferenciación Celular , Sistema de Señalización de MAP Quinasas , Osteoclastos/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Secuencias de Aminoácidos , Animales , Infecciones por Bacteroidaceae/genética , Infecciones por Bacteroidaceae/metabolismo , Infecciones por Bacteroidaceae/patología , Ratones , Ratones Mutantes , Osteoclastos/patología , Porphyromonas gingivalis/metabolismo , Receptor Activador del Factor Nuclear kappa-B/genéticaRESUMEN
OBJECTIVE: 5-Hydroxytryptophan (5-HTP) is the precursor of serotonin and 5-HTP has been widely used as a dietary supplement to raise serotonin level. Serotonin has recently been discovered to be a novel and important player in bone metabolism. As peripheral serotonin negatively regulates bone, the regular take of 5-HTP may affect the alveolar bone metabolism and therefore influence the alveolar bone loss induced by periodontitis. The aim of this study was to investigate the effect of 5-HTP on alveolar bone destruction in periodontitis. DESIGN: Male Sprague-Dawley rats were randomly divided into the following four groups: (1) the control group (without ligature); (2) the 5-HTP group (5-HTP at 25 mg/kg/day without ligature); (3) the L group (ligature+saline placebo); and (4) the L+5-HTP group (ligature+5-HTP at 25 mg/kg/day). Serum serotonin levels were determined by ELISA. The alveolar bones were evaluated with micro-computed tomography and histology. Tartrate-resistant acid phosphatase staining was used to assess osteoclastogenesis. The receptor activator of NF-kB ligand (RANKL) and osteoprotegerin (OPG) expression in the periodontium as well as the interleukin-6 positive osteocytes were analysed immunohistochemically. RESULTS: 5-HTP significantly increased serum serotonin levels. In rats with experimental periodontitis, 5-HTP increased alveolar bone resorption and worsened the micro-structural destruction of the alveolar bone. 5-HTP also stimulated osteoclastogenesis and increased RANKL/OPG ratio and the number of IL-6 positive osteocytes. However, 5-HTP treatment alone did not cause alveolar bone loss in healthy rats. CONCLUSION: The present study showed that 5-HTP aggravated alveolar bone loss, deteriorated alveolar bone micro-structure in the presence of periodontitis, which suggests 5-HTP administration may increase the severity of periodontitis.
Asunto(s)
5-Hidroxitriptófano/administración & dosificación , 5-Hidroxitriptófano/farmacología , Pérdida de Hueso Alveolar/inducido químicamente , Periodontitis/complicaciones , Administración Oral , Pérdida de Hueso Alveolar/diagnóstico por imagen , Animales , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Técnicas para Inmunoenzimas , Interleucina-6/metabolismo , Masculino , Osteoclastos/efectos de los fármacos , Osteocitos/efectos de los fármacos , Ligando RANK/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Serotonina/sangre , Microtomografía por Rayos XRESUMEN
OBJECTIVE: The local delivery of growth factors such as bone morphogenetic protein-7 (BMP-7) into the tissues around dental implants may improve their osseointegration. We have designed a new method of attaching BMP-7 to a titanium surface and assessed both the retention of the BMP-7 and its effect on osteoblast differentiation. DESIGN: Adenoviral vector expressing BMP-7 was attached to dental titanium discs by hexon-specific antibodies in a type I collagen-avidin gel. FITC-labelled secondary antibody was used to measure the continuing adherence of the coating after repeated rinsing. Osteoblasts were harvested and seeded on the titanium discs. Gene transduction efficiency and targeting ability were assessed after 24h. Surface morphology was observed by SEM. Cell proliferation and alkaline phosphatase (ALP) activities were measured. RESULTS: The anti-adenohexon antibody adhered strongly to the collagen-avidin gels. BMP-7 gene expression was localized precisely to cells growing on the gels bound by the hexon-specific antibody. Osteoblasts on the titanium containing Ad-BMP-7 had a higher ALP activity than those without Ad-BMP-7. CONCLUSIONS: This study describes a novel technique for the precise attachment of BMP-7 to titanium surfaces. The process may enhance the osseointegration of dental implants.