RESUMEN
BACKGROUND: Testicular torsion is an urological emergency that may lead to infertility due to ischemic injury. Promptly surgical correction by orchiopexy is the only way to avoid infertility and no effective treatment for restoration of spermatogenesis. We previously reported that mesenchymal stem cells (MSCs), through local injection upon testicular torsion-detorsion, restored the spermatogenesis without differentiation into sperm. In this study, molecular mechanisms of MSCs in regulating germ cell activity induced by testicular torsion-detorsion were investigated. METHODS: Sixteen male Sprague-Dawley rats 6-8 weeks old received left testis 720° torsion for 3 h followed by detorsion with or without MSCs. Right inguinal skin incision without testicular torsion served as control. MSCs with 3 × 104 cells were locally injected into left testis 30 min before detorsion. Three days after the surgery, orchiectomy was executed and the testis, epididymis, and sperm were separated to each other. Functional assessments on sperm included counting sperm amount and sperm motility, staining F-actin, and quantifying adenosine triphosphate (ATP) content. The hallmarks of glycogenesis and glycolysis in each tissue segment were measured by Western blot. RESULTS: Testicular torsion-detorsion significantly decreased the amount of sperm, inhibited the motility, declined the F-actin expression, and reduced the content of ATP in sperm. Local injection of MSCs improved sperm function, particularly in sperm motility. With MSCs, ATP content and F-actin were preserved after testicular torsion-detorsion. MSCs significantly reversed the imbalance of glycolysis in sperm and testis induced by testicular torsion-detorsion, as evidenced by increasing the expression of phosphoglycerate kinase 2 and glyceraldehyde-3-phosphate dehydrogenase-spermatogenic, activating Akt, and increasing glycogen synthase kinase 3 (GSK3), which led to the increase in glycolysis cascades and ATP production. Human stem cell factor contributed the activation of Akt/GSK3 axis when sperm suffered from testicular torsion-detorsion-induced germ cell injury. CONCLUSIONS: Local injection of MSCs into a testis damaged by testicular torsion-detorsion restores sperm function mainly through the improvement of sperm motility and energy. MSCs reversed the imbalance of glycogenesis and glycolysis in sperm by regulating Akt/GSK3 axis. Thus, MSCs may potentially rescue torsion-detorsion-induced infertility via local injection.
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Glucosa/metabolismo , Células Madre Mesenquimatosas/metabolismo , Motilidad Espermática/fisiología , Torsión del Cordón Espermático/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Actinas/metabolismo , Animales , Células Germinativas/metabolismo , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Isoenzimas/metabolismo , Masculino , Fosfoglicerato Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/metabolismo , Espermatogénesis/fisiologíaRESUMEN
The healing of a corneal epithelial defect is essential for preventing infectious corneal ulcers and subsequent blindness. We previously demonstrated that mesenchymal stem cells (MSCs) in the corneal stroma, through a paracrine mechanism, yield a more favorable therapeutic benefit for corneal wound re-epithelialization than do MSCs in the corneal epithelium. In this study, MSCs were grown on a matrix with the rigidity of the physiological human vitreous (1 kPa), corneal epithelium (8 kPa), or corneal stroma (25 kPa) for investigating the role of corneal tissue rigidity in MSC functions regarding re-epithelialization promotion. MSC growth on a 25-kPa dish significantly promoted the wound healing of human corneal epithelial (HCE-T) cells. Among growth factors contributing to corneal epithelial wound healing, corneal stromal rigidity selectively enhanced transforming growth factor-beta (TGF-ß) secretion from MSCs. Inhibitors of TGF-ß pan receptor, TGF-ß receptor 1, and Smad2 dose dependently abrogated MSC-mediated HCE-T wound healing. Furthermore, MSCs growth on a matrix with corneal stromal rigidity enhanced the ability of themselves to promote corneal re-epithelialization by activating matrix metalloproteinase (MMP) expression and integrin ß1 production in HCE-T cells through TGF-ß signaling pathway activation. Smad2 activation resulted in the upregulation of MMP-2 and -13 expression in HCE-T cells, whereas integrin ß1 production favored a Smad2-independent TGF-ß pathway. Altogether, we conclude that corneal stromal rigidity is a critical factor for MSC-induced promotion of corneal re-epithelialization. The activation of the TGF-ß signaling pathway, which maintains the balance between integrin and MMP expression, in HCE-T cells is the major pathway responsible for MSC-mediated wound healing. Stem Cells 2016;34:2525-2535.
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Lesiones de la Cornea/patología , Lesiones de la Cornea/terapia , Sustancia Propia/patología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Cicatrización de Heridas , Línea Celular , Proliferación Celular , Humanos , Integrina beta1/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Células Madre Mesenquimatosas/metabolismo , Repitelización , Proteína Smad2/metabolismo , Regulación hacia ArribaRESUMEN
Lung fibrosis is a poor prognostic factor for pulmonary adenocarcinoma, and the effect of a rigid microenvironment on cancer behavior is unclear. We cultured A549 cells on matrices of 0.2, 2, and 25 kPa to mimic the rigidities of normal lung parenchyma, progressive fibrotic change, and lung fibrosis, respectively. Lung tissue from patients with pulmonary adenocarcinoma was used to confirm the in vitro findings. Increased matrix rigidity promoted cell proliferation and upregulated the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-Met), and Snail expression in A549 cells. A549 cells became more resistant to the EGFR inhibitor (Erlotinib) and c-Met inhibitor (PHA-665752) when matrix rigidity increased; however, a high concentration of PHA-665752 reversed the rigidity-induced morphological pleomorphism. In human lung tissue, expression of type I collagen was more consistent with clinical fibrosis than the expression of alpha-smooth muscle antibody was. c-Met- and Snail-expressing tumor cells, rather than EGFR-experssing cells, were localized with lung parenchyma rich in type I collagen. Our findings suggest that c-Met causes the rigidity-induced biophysical reaction in pulmonary adenocarcinoma. Treatment targeting both EGFR and c-Met should be considered for patients with lung fibrosis and who are abundant type I collagen expression in the tumor mass.
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Adenocarcinoma/tratamiento farmacológico , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Pulmón/efectos de los fármacos , Pulmón/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-met/metabolismo , Adenocarcinoma/diagnóstico , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Fenómenos Biomecánicos , Línea Celular Tumoral , Colágeno Tipo I/análisis , Colágeno Tipo I/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Receptores ErbB/análisis , Receptores ErbB/antagonistas & inhibidores , Clorhidrato de Erlotinib/farmacología , Clorhidrato de Erlotinib/uso terapéutico , Fibrosis , Humanos , Indoles/farmacología , Indoles/uso terapéutico , Integrinas/análisis , Integrinas/metabolismo , Pulmón/metabolismo , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Mecanotransducción Celular , Pronóstico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-met/análisis , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Sulfonas/farmacología , Sulfonas/uso terapéuticoRESUMEN
INTRODUCTION: Testicular torsion is a urological emergency and infertility is a common complication due to ischemic injury. Surgical reduction and orchiopexy is indicated, but to date there is no effective method for restoration of spermatogenesis. The effects of mesenchymal stem cells (MSCs) on acute tissue injury have been demonstrated, and the abilities of paracrine support, differentiation and immune-modulation may benefit to testicular torsion-induced infertility. We investigate the therapeutic efficacy and the mechanisms of MSCs in testicular torsion-induced germ cell injury when injected locally. METHODS: Six to eight-week-old Sprague-Dawley rats received surgical 720 degree torsion for 3 hours, followed by detorsion on the left testis. 20 µl of phosphate-buffered saline (PBS) without or with 3 x 10(4) MSCs from human orbital fat tissues (OFSCs) were given for 10 rats, respectively, via local injection into the left testis 30 minutes before detorsion. 20 µl of PBS injection for 6 rats with surgical exposure without torsion served as sham control. Histopathology with Johnsen's score analysis, Western blot analysis for superoxide dismutase 2, Bax, Caspase-3, human insulin growth factor-1 and human stem cell factor, malondialdehyde (MDA) assay in testis and plasma, hormones level including testosterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by ELISA Kits, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and fluorescence staining for P450, Sox-9 and VASA were performed. RESULTS: Animals were sacrificed and bilateral orchiectomy was performed 7 days after torsion-detorsion. Local injections of OFSCs prevented torsion-induced infertility judging from Johnsen's score. TUNEL assay and Western blot analysis on caspase 3 and Bax demonstrated that OFSCs prevented ischemic/reperfusion induced intrinsic apoptosis. MDA assay revealed that OFSCs significantly reduced the oxidative stress in the damaged testicular tissues. After the OFSC injection, serum testosterone secretion was increased, while the elevation of FSH triggered by testicular injury was balanced. OFSCs also produced stem cell factor in the damaged testis. Immunofluorescence staining revealed that most transplanted cells surrounded the Leydig cells. Some of transplanted cells differentiated into p450 expressing cells within 7 days. CONCLUSIONS: Local injection of allogenic MSCs before surgical detorsion is a simple, clinical friendly procedure to rescue torsion-induced infertility.
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Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Torsión del Cordón Espermático/terapia , Testículo/patología , Tejido Adiposo/citología , Animales , Apoptosis , Caspasa 3/metabolismo , Hormona Folículo Estimulante/sangre , Células Germinativas/citología , Células Germinativas/metabolismo , Humanos , Factor I del Crecimiento Similar a la Insulina/metabolismo , Hormona Luteinizante/sangre , Masculino , Malondialdehído/metabolismo , Células Madre Mesenquimatosas/metabolismo , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Superóxido Dismutasa/metabolismo , Testosterona/sangre , Trasplante HeterólogoRESUMEN
Mesenchymal stromal cells (MSCs) have great potential to maintain glucose homeostasis and metabolic balance. Here, we demonstrate that in mice continuously fed with high-fat diet (HFD) that developed non-insulin-dependent diabetes, two episodes of systemic MSC transplantations effectively improve glucose tolerance and blood glucose homeostasis and reduce body weight through targeting pancreas and insulin-sensitive tissues and organs via site-specific mechanisms. MSCs support pancreatic islet growth by direct differentiation into insulin-producing cells and by mitigating the cytotoxicity of interleukin 1 (IL-1) and tumor necrosis factor-α (TNF-α) in the pancreas. Localization of MSCs in the liver and skeletal muscles in diabetic animals is also enhanced and therefore improves glucose tolerance, although long-term engraftment is not observed. MSCs prevent HFD-induced fatty liver development and restore glycogen storage in hepatocytes. Increased expression of IL-1 receptor antagonist and Glut4 in skeletal muscles after MSC transplantation results in better blood glucose homeostasis. Intriguingly, systemic MSC transplantation does not alter adipocyte number, but it decreases HFD-induced cell infiltration in adipose tissues and reduces serum levels of adipokines, including leptin and TNF-α. Taken together, systemic MSC transplantation ameliorates HFD-induced obesity and restores metabolic balance through multisystemic regulations that are niche dependent. Such findings have supported systemic transplantation of MSCs to correct metabolic imbalance.
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Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/metabolismo , Dieta Alta en Grasa/efectos adversos , Células Madre Mesenquimatosas/fisiología , Adipocitos/metabolismo , Animales , Células Cultivadas , Diabetes Mellitus Experimental/sangre , Humanos , Insulina/sangre , Interleucina-1/sangre , Leptina/sangre , Masculino , Ratones , Factor de Necrosis Tumoral alfa/sangreRESUMEN
INTRODUCTION: Our previous works demonstrated that systemic orbital fat-derived stem cell (OFSC) transplantation was effective in ameliorating lipopolysaccharide (LPS)-induced extensive acute lung injury (ALI) in vivo mainly through paracrine regulation of macrophage-mediated cytokine-storm. In this study, we explore the molecular mechanism(s) of OFSCs regulating macrophage activity in a cytokine-inducible fashion. METHODS: LPS (100 ng/ml)-activated macrophages were treated by conditioned medium from OFSCs (OFSCs-CM) or non-contact cultured with OFSCs for 6 hours. The potency of OFSCs on macrophage proliferation and pro-inflammation ability were determined. Expression levels of pro-inflammatory cytokines in macrophages, inducible immuno-modulatory factors in OFSCs, were investigated. Deep sequencing analysis as well as interaction between microRNA (miRNA) and genes of immuno-modulators in OFSCs induced by activated macrophages was predicted by miRTar. Transfection of miRNA inhibitor into OFSCs was performed. Real-time RT-PCR and transplantation of OFSCs into mice with LPS-induced ALI confirmed the in vitro and in vivo mechanism. RESULTS: The paracrine effect of OFSCs on inhibition of macrophage pro-inflammatory cytokine release was more potent than induction of macrophage G0/G1 cell cycle arrest. OFSCs-CM suppressed LPS-induced inducible nitric oxide synthetase and the pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1 alpha, and IL-1 beta expression in macrophages. Under non-contact culture, LPS-activated macrophages effectively triggered the expression of soluble immuno-modulating factors in OFSCs, i.e., IL-10, IL-1 receptor antagonist (IL-1 RA), indoleamine 2,3-dioxygenase, and soluble TNF receptor type II (sTNF RII). Under miRTar prediction, miR-671-5p was identified as a critical microRNA in regulation of multiple immune-modulating factors in OFSCs response to macrophages. The baseline level of miR-671-5p was high in OFSCs, and down-regulation of miR-671-5p upon co-culture with activated macrophages was observed. MiR-671-5p inhibitor transfection into OFSCs selectively enhanced the IL-1 RA and sTNF RII expressions. In addition, inhibition of miR-671-5p in OFSCs enhanced the anti-inflammatory ability against LPS-induced ALI. CONCLUSION: The paracrine effect of OFSCs inhibits the pro-inflammatory ability and proliferation of macrophages. The immune-modulation capacity of OFSCs can be triggered by activated macrophages, and down-regulation of miR-671-5p enhances OFSC immuno-modulation ability by up-regulating IL-1 RA and sTNF RII expression.
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Macrófagos/fisiología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , MicroARNs/fisiología , Tejido Adiposo/citología , Animales , Técnicas de Cocultivo , Regulación hacia Abajo , Secuenciación de Nucleótidos de Alto Rendimiento , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lipopolisacáridos , Macrófagos/metabolismo , Ratones Endogámicos BALB C , MicroARNs/genética , MicroARNs/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Órbita/citología , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
INTRODUCTION: Topical administration of eye drops is the major route for drug delivery to the cornea. Orbital fat-derived stem cells (OFSCs) possess an in vitro corneal epithelial differentiation capacity. Both the safety and immunomodulatory ability of systemic OFSC transplantation were demonstrated in our previous work. In this study, we investigated the safety, therapeutic effect, and mechanism(s) of topical OFSC administration in an extensive alkali-induced corneal wound. METHODS: Corneal injury was created by contact of a piece of 0.5 N NaOH-containing filter paper on the corneal surface of a male Balb/c mouse for 30 s. The area of the filter paper covered the central 70% or 100% of the corneal surface. OFSCs (2 × 10(5)) in 5 µl phosphate-buffered saline (PBS) were given by topical administration (T) twice a day or by two intralimbal (IL) injections in the right cornea, while 5 µl of PBS in the left cornea served as the control. RESULTS: Topical OFSCs promoted corneal re-epithelialization of both the limbal-sparing and limbal-involved corneal wounds. In the first three days, topical OFSCs significantly reduced alkali-induced corneal edema and stromal infiltration according to a histopathological examination. Immunohistochemistry and immunofluorescence staining revealed that transplanted cells were easily detectable in the corneal epithelium, limbal epithelium and stroma, but only some of transplanted cells at the limbal epithelium had differentiated into cytokeratin 3-expressing cells. OFSCs did not alter neutrophil (Ly6G) levels in the cornea, but significantly reduced macrophage (CD68) infiltration and inducible nitrous oxide synthetase (iNOS) production during acute corneal injury as quantified by a Western blot analysis. Continuous topical administration of OFSCs for seven days improved corneal transparency, and this was accompanied by diffuse stromal engraftment of transplanted cells and differentiation into p63-expressing cells at the limbal area. The therapeutic effect of the topical administration of OFSCs was superior to that of the IL injection. OFSCs from the IL injection clustered in the limbal area and central corneal epithelium, which was associated with a persistent corneal haze. CONCLUSIONS: Topical OFSC administration is a simple, non-surgical route for stem cell delivery to promote corneal tissue regeneration through ameliorating acute inflammation and corneal epithelial differentiation. The limbal area serves as a niche for OFSCs differentiating into corneal epithelial cells in the first week, while the stroma is a potential site for anti-inflammation of OFSCs. Inhibition of corneal inflammation is related to corneal transparency.