ABSTRACT
The limited cardiomyocyte proliferation is insufficient for repair of the myocardium. Therefore, activating cardiomyocyte proliferation might be a reasonable option for myocardial regeneration. Here, we investigated effect of retinoic acid (RA) on inducing adult cardiomyocyte proliferation and assessed efficacy of self-assembling peptide (SAP)-released RA in activating regeneration of the infarcted myocardium. Effect of RA on inducing cardiomyocyte proliferation was examined with the isolated cardiomyocytes. Expression of the cell cycle-associated genes and paracrine factors in the infarcted myocardium was examined at one week after treatment with SAP-carried RA. Cardiomyocyte proliferation, myocardial regeneration and improvement of cardiac function were assessed at four weeks after treatment. In the adult rat myocardium, expression of RA synthetase gene Raldh2 and RA concentration were decreased significantly. After treatment with RA, the proliferated cardiomyocytes were increased. The formulated SAP could sustainedly release RA. After treatment with SAP-carried RA, expression of the pro-proliferative genes in cell cycle and paracrine factors in the infarcted myocardium were up-regulated. Myocardial regeneration was enhanced, and cardiac function was improved significantly. These results demonstrate that RA can induce adult cardiomyocytes to proliferate effectively. The sustained release of RA with SAP is a promise strategy to enhance repair of the infarcted myocardium.
Subject(s)
Myocardial Infarction , Myocytes, Cardiac , Rats , Animals , Myocytes, Cardiac/metabolism , Myocardial Infarction/metabolism , Tretinoin/pharmacology , Tretinoin/metabolism , Myocardium/metabolism , Peptides/pharmacology , Peptides/metabolism , Cell ProliferationABSTRACT
Cardiomyocytes are particularly prone to lipofuscin accumulation. In the aging heart, lipofuscin accumulation is augmented. This study examined distribution of lipofuscin and senescent cardiomyocytes and evaluated improvement of lipofuscin accumulation and cardiomyocytic senescence of the aging heart after treatment with rapamycin. The results of Schmorl staining, Sudan black staining and autofluorescence detection showed that there was more lipofuscin in the myocardium of the ventricles especially in the left ventricle. The conductive tissue contained less lipofuscin than the myocardium. In the aged hearts, lipofuscin accumulation and senescent cardiomyocytes were increased, and the level of autophagy was reduced. In double staining of Sudan black B and senescence-associated ß-galactosidase, 10%-20% lipofuscin-loaded cardiomyocytes became senescent. All senescent cardiomyocytes contained lipofuscin deposits. After enhancing autophagy with feed of rapamycin for six months, lipofuscin accumulation and senescence of cardiomyocytes were improved in old rats. Colocalization of autophagic structure and lipofuscin as well as electron micrographs showed that some lipofuscin-loaded lysosomes were sequestrated by autophagic structures. This study suggests that rapamycin-enhanced autopahgy is effective for reducing lipofuscinogenesis and promoting degradation of lipofuscin. Therefore, enhancing autophagy is a novel therapy for alleviating lipofuscin accumulation and myocardial senescence.
Subject(s)
Aging/metabolism , Autophagy/physiology , Lysosomes/metabolism , Myocytes, Cardiac/metabolism , Animals , Cells, Cultured , Cellular Senescence/physiology , Male , Myocardium/metabolism , Rats, Sprague-Dawley , Staining and Labeling/methodsABSTRACT
Impairment of cardiac lymphatic vessels leads to cardiac lymphedema. Recent studies have suggested that stimulation of lymphangiogenesis may reduce cardiac lymphedema. However, effects of lymphatic endothelial progenitor cells (LEPCs) on cardiac lymphangiogenesis are poorly understood. Therefore, this study investigated effectiveness of LEPC transplantation and VEGF-C release with self-assembling peptide (SAP) on cardiac lymphangiogenesis after myocardial infarction (MI). CD34+VEGFR-3+ EPCs isolated from rat bone marrow differentiated into lymphatic endothelial cells after VEGF-C induction. VEGF-C also stimulated the cells to incorporate into the lymphatic capillary-like structures. The functionalized SAP could adhere with the cells and released VEGF-C sustainedly. In the condition of hypoxia and serum deprivation or abdominal pouch assay, the SAP hydrogel protected the cells from apoptosis and necrosis. At 4 weeks after intramyocardial transplantation of the cells and VEGF-C loaded with SAP hydrogel in rat MI models, cardiac lymphangiogenesis was increased, cardiac edema and reverse remodeling were reduced, and cardiac function was improved significantly. Delivery with SAP hydrogel favored survival of the engrafted cells. VEGF-C released from the hydrogel promoted differentiation and incorporation of the cells as well as growth of pre-existed lymphatic vessels. Cardiac lymphangiogenesis was beneficial for elimination of the inflammatory cells in the infarcted myocardium. Moreover, angiogenesis and myocardial regeneration were enhanced after reduction of lymphedema. These results demonstrate that the combined delivery of LEPCs and VEGF-C with the functionalized SAP promotes cardiac lymphangiogenesis and repair of the infarcted myocardium effectively. This study represents a novel therapy for relieving myocardial edema in cardiovascular diseases.
Subject(s)
Edema, Cardiac/therapy , Endothelial Progenitor Cells/transplantation , Lymphangiogenesis , Vascular Endothelial Growth Factor C/therapeutic use , Animals , Antigens, CD34/metabolism , Endothelial Progenitor Cells/metabolism , Male , Myocardium/metabolism , Neovascularization, Physiologic , Rats, Sprague-Dawley , Vascular Endothelial Growth Factor A/blood , Vascular Endothelial Growth Factor C/blood , Vascular Endothelial Growth Factor Receptor-3/metabolismABSTRACT
Serelaxin, a recombinant form of human relaxin-2, is currently regarded as a novel drug for treatment of acute heart failure. However, whether therapeutic effects of serelaxin are achieved by inhibiting cardiac fibrosis remains unclear. In this study, we investigate effects of serelaxin on inhibiting cardiac fibrosis. Cardiac fibroblasts (CFs) were isolated from the hearts of adult rats. Effects of serelaxin on differentiation of CFs towards myofibroblasts (MFs) and their fibrotic behaviors after induction with TGF-ß1 were examined. Synthesis and degradation of collagens, secretion of IL-10, and expression of ALK-5 and p-Smad2/3 of TGF-ß1-induced cells were assessed after treatment with serelaxin. Serelaxin inhibited differentiation of TGF-ß1-induced CFs towards MFs, and reduced proliferation and migration of the induced cells. Moreover, serelaxin down-regulated expression of collagen I/III and TIMP-2, and up-regulated expression of MMP-2 and MMP-9 in the cells. After treatment with serelaxin, activity of MMP-2 and MMP-9 and secretion of IL-10 increased, expression of ALK-5 and the level of Smad2/3 phosphorylation was reduced significantly. These results suggest that serelaxin can inhibit differentiation of TGF-ß1-induced CFs towards MFs, reduce production of collagens by suppressing ALK-5/Smad2/3 signaling pathway, and enhance extracellular matrix degradation by increasing MMP-2/TIMP-2 ratio and IL-10 secretion. Serelaxin may be a potential therapeutic drug for inhibiting cardiac fibrosis.
Subject(s)
Cell Differentiation/drug effects , Myocardium/pathology , Myocytes, Cardiac/drug effects , Myofibroblasts/drug effects , Relaxin/pharmacology , Animals , Cells, Cultured , Down-Regulation/drug effects , Female , Fibrosis/metabolism , Fibrosis/prevention & control , Humans , Myocardium/metabolism , Myocytes, Cardiac/pathology , Myocytes, Cardiac/physiology , Protein Serine-Threonine Kinases/metabolism , Rats , Rats, Sprague-Dawley , Receptor, Transforming Growth Factor-beta Type I , Receptors, Transforming Growth Factor beta/metabolism , Recombinant Proteins/pharmacology , Signal Transduction/drug effects , Smad2 Protein/metabolism , Smad3 Protein/metabolismABSTRACT
Recent clinical studies have suggested that endothelial progenitor cells (EPCs) transplantation provides a modest benefit for treatment of the ischaemic diseases such as limb ischaemia. However, cell-based therapies have been limited by poor survival of the engrafted cells. This investigation was designed to establish optimal hypoxia preconditioning and evaluate effects of hypoxic preconditioning-induced autophagy on survival of the engrafted EPCs. Autophagy of CD34+ VEGFR-2+ EPCs isolated from rat bone marrow increased after treatment with 1% O2 . The number of the apoptotic cells in the hypoxic cells increased significantly after autophagy was inhibited with 3-methyladenine. According to balance of autophagy and apoptosis, treatment with 1% O2 for 2 hrs was determined as optimal preconditioning for EPC transplantation. To examine survival of the hypoxic cells, the cells were implanted into the ischaemic pouch of the abdominal wall in rats. The number of the survived cells was greater in the hypoxic group. After the cells loaded with fibrin were transplanted with intramuscular injection, blood perfusion, arteriogenesis and angiogenesis in the ischaemic hindlimb were analysed with laser Doppler-based perfusion measurement, angiogram and the density of the microvessels in histological sections, respectively. Repair of the ischaemic tissue was improved significantly in the hypoxic preconditioning group. Loading the cells with fibrin has cytoprotective effect on survival of the engrafted cells. These results suggest that activation of autophagy with hypoxic preconditioning is an optimizing strategy for EPC therapy of limb ischaemia.
Subject(s)
Autophagy/physiology , Endothelial Progenitor Cells/transplantation , Ischemic Preconditioning/methods , Stem Cell Transplantation/methods , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Apoptosis/drug effects , Apoptosis/physiology , Autophagy/drug effects , Cell Hypoxia , Cell Survival/physiology , Cells, Cultured , Endothelial Progenitor Cells/metabolism , Endothelial Progenitor Cells/physiology , Hindlimb/blood supply , Hindlimb/physiopathology , Hypoxia , Ischemia/physiopathology , Rats, Sprague-Dawley , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Cardiac patch is considered a promising strategy for enhancing stem cell therapy of myocardial infarction (MI). However, the underlying mechanisms for cardiac patch repairing infarcted myocardium remain unclear. In this study, we investigated the mechanisms of PCL/gelatin patch loaded with MSCs on activating endogenous cardiac repair. PCL/gelatin patch was fabricated by electrospun. The patch enhanced the survival of the seeded MSCs and their HIF-1α, Tß4, VEGF and SDF-1 expression and decreased CXCL14 expression in hypoxic and serum-deprived conditions. In murine MI models, the survival and distribution of the engrafted MSCs and the activation of the epicardium were examined, respectively. At 4 weeks after transplantation of the cell patch, the cardiac functions were significantly improved. The engrafted MSCs migrated across the epicardium and into the myocardium. Tendency of HIF-1α, Tß4, VEGF, SDF-1 and CXCL14 expression in the infarcted myocardium was similar with expression in vitro. The epicardium was activated and epicardial-derived cells (EPDCs) migrated into deep tissue. The EPDCs differentiated into endothelial cells and smooth muscle cells, and some of EPDCs showed to have differentiated into cardiomyocytes. Density of blood and lymphatic capillaries increased significantly. More c-kit+ cells were recruited into the infarcted myocardium after transplantation of the cell patch. The results suggest that epicardial transplantation of the cell patch promotes repair of the infarcted myocardium and improves cardiac functions by enhancing the survival of the transplanted cells, accelerating locality paracrine, and then activating the epicardium and recruiting endogenous c-kit+ cells. Epicardial transplantation of the cell patch may be applied as a novel effective MI therapy.
Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Myocardial Infarction/pathology , Myocardial Infarction/therapy , Myocardium/pathology , Regeneration , Animals , Biocompatible Materials/chemistry , Capillaries/pathology , Cell Differentiation/genetics , Cell Survival/genetics , Chemokines/metabolism , Cytoprotection , Gelatin/chemistry , Gene Expression Regulation , Heart Function Tests , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lymphangiogenesis/genetics , Male , Mesenchymal Stem Cells/ultrastructure , Mice , Myocardial Infarction/physiopathology , Neovascularization, Physiologic , Polyesters/chemistry , Proto-Oncogene Proteins c-kit/metabolism , Rats, Sprague-Dawley , Thymosin/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Clearance of the apoptotic cells by phagocytes plays pivotal roles in maintenance of tissue homeostasis, promotion of immunological tolerance and anti-inflammatory response. Recent studies show that autophagy is involved in phagocytosis of the apoptotic cells. However, contribution of autophagy to phagocytosis of the apoptotic cells by macrophages is not clearly defined. Here, we assessed cytoprotective effect of autophagy on clearance of the apoptotic cells. Apoptosis of murine splenic lymphocytes and human T-cell leukemia cells was induced with cyclophosphamide. After engulfment of the apoptotic cells, expression of Belin-1 and LC3 in macrophages was upregulated, the number of MDC-positive vesicles, LC3-positive autophagosomes and autophagic ultrastructures increased significantly. Autophagosome was fused with phagosome containing fragments of the nuclei or other debris of the apoptotic cells to form amphisome. Some cells in macrophages phagocytosing the apoptotic cells became apoptotic. After autophagy of macrophages was inhibited with 3-MA, viability and survival of macrophages reduced, phagocytosis of the apoptotic cells by macrophages deceased significantly. These results demonstrate that autophagy plays an important role in promoting clearance of the apoptotic cells by protecting macrophages from apoptosis during phagocytosis as well as degrading the contents of phagosomes via amphisome formation.
Subject(s)
Apoptosis , Autophagy , Cytoprotection , Macrophages/cytology , Phagocytosis , Animals , Beclin-1/genetics , Beclin-1/metabolism , Cadaverine/analogs & derivatives , Cadaverine/metabolism , Cell Survival , Cytoplasmic Vesicles/metabolism , Humans , Jurkat Cells , Lymphocytes/cytology , Macrophages/ultrastructure , Male , Microtubule-Associated Proteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats, Sprague-DawleyABSTRACT
Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization. However, it is poorly understood whether EPCs contribute to lymphangiogenesis. Here, we assessed differentiation of a novel population of EPCs towards lymphatic endothelial cells and their lymphatic formation. CD34(+) VEGFR-3(+) EPCs were isolated from mononuclear cells of human cord blood by fluorescence-activated cell sorting. These cells expressed CD133 and displayed the phenotype of the endothelial cells. Cell colonies appeared at 7-10 days after incubation. The cells of the colonies grew rapidly and could be repeatedly subcultured. After induction with VEGF-C for 2 weeks, CD34(+) VEGFR-3(+) EPCs could differentiate into lymphatic endothelial cells expressing specific markers 5'-nucleotidase, LYVE-1 and Prox-1. The cells also expressed hyaluronan receptor CD44. The differentiated cells had properties of proliferation, migration and formation of lymphatic capillary-like structures in three-dimensional collagen gel and Matrigel. VEGF-C enhanced VEGFR-3 mRNA expression. After interfering with VEGFR-3 siRNA, the effects of VEGF-C were diminished. These results demonstrate that there is a population of CD34(+) VEGFR-3(+) EPCs with lymphatic potential in human cord blood. VEGF-C/VEGFR-3 signalling pathway mediates differentiation of CD34(+) VEGFR-3(+) EPCs towards lymphatic endothelial cells and lymphangiogenesis. Cord blood-derived CD34(+) VEGFR-3(+) EPCs may be a reliable source in transplantation therapy for lymphatic regenerative diseases.
Subject(s)
Antigens, CD34/metabolism , Cell Differentiation , Endothelial Cells/cytology , Stem Cells/cytology , Vascular Endothelial Growth Factor Receptor-3/metabolism , Animals , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Separation , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/ultrastructure , Fibroblast Growth Factor 2/pharmacology , Flow Cytometry , Gels , Gene Expression Regulation/drug effects , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Rats , Stem Cells/drug effects , Stem Cells/metabolism , Stem Cells/ultrastructure , Vascular Endothelial Growth Factor A/pharmacology , Vascular Endothelial Growth Factor C/pharmacology , Vascular Endothelial Growth Factor Receptor-3/geneticsABSTRACT
Stem cell transplantation is a promising therapeutic strategy for myocardial infarction (MI). However, engraftment, survival and differentiation of the transplanted stem cells in ischemic and inflammatory microenvironment are poor. We designed a novel self-assembly peptide (SAP) by modifying the peptide RADA16 with cell-adhesive motif and BMP-2 (bone morphogenetic protein-2)-binding motif. Effects of the functionalized SAP on adhesion, survival and differentiation of c-kit+ MSCs (mesenchymal stem cells) were examined. Myocardial regeneration, neovascularization and cardiac function were assessed after transplantation of the SAP loading c-kit+ MSCs and BMP-2 in rat MI models. The SAP could spontaneously assemble into well-ordered nanofibrous scaffolds. The cells adhered to the SAP scaffolds and spread well. The SAP protected the cells in the condition of hypoxia and serum deprivation. Following degradation of the SAP, BMP-2 was released sustainedly and induced c-kit+ MSCs to differentiate into cardiomyocytes. At four weeks after transplantation of the SAP loading c-kit+ MSCs and BMP-2, myocardial regeneration and angiogenesis were enhanced, and cardiac function was improved significantly. The cardiomyocytes differentiated from the engrafted c-kit+ MSCs were increased markedly. The differentiated cells connected with recipient cardiomyocytes to form gap junctions. Collagen volume was decreased dramatically. These results suggest that the functionalized SAP promotes engraftment, survival and differentiation of stem cells effectively. Local sustained release of BMP-2 with SAP is a viable strategy to enhance differentiation of the engrafted stem cells and repair of the infarcted myocardium.
Subject(s)
Bone Morphogenetic Protein 2 , Cell Differentiation , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Myocardial Infarction , Peptides , Regeneration , Animals , Myocardial Infarction/therapy , Myocardial Infarction/pathology , Bone Morphogenetic Protein 2/pharmacology , Bone Morphogenetic Protein 2/metabolism , Cell Differentiation/drug effects , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/drug effects , Rats , Regeneration/drug effects , Peptides/pharmacology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/cytology , Male , Rats, Sprague-Dawley , Neovascularization, Physiologic/drug effects , Myocardium/pathology , Myocardium/metabolism , Tissue Scaffolds/chemistry , Nanofibers/chemistry , Proto-Oncogene Proteins c-kit/metabolism , Cell Adhesion/drug effectsABSTRACT
Endothelial progenitor cells (EPCs) may be incorporated into local vessels to enhance angiogenesis within ischemic tissue. Recently, EPC transplantation has become a potential therapy for improving tissue function in cardiovascular disease. However, the mechanisms of proliferation, differentiation, and survival of EPCs in a hypoxic microenvironment remain unclear. In this study, CD34(+)VEGFR-2(+) EPCs were isolated from mononuclear cells of human umbilical cord blood, and differentiation to endothelial cells was induced with VEGF. When EPC autophagy was inhibited with 3-methyladenine (3-MA) under normoxic conditions, proliferation and viability of the cells were decreased, and the cells failed to differentiate into endothelial cells. Under hypoxic conditions (1% O(2)), Beclin-1 expression of the cells was upregulated and both MDC-labeled and LC3-positive puncta and autophagic ultrastructures in the cells increased significantly. The number of lysosomes also increased in hypoxia-exposed cells. When autophagy was inhibited with 3-MA under hypoxic conditions, the number of apoptotic cells increased, and the number and size of lysosomes decreased. Conversely, apoptosis of the hypoxic EPCs was reduced when autophagy was induced by pretreatment with rapamycin. These results demonstrate that autophagy is involved in proliferation and differentiation of EPCs. Furthermore, hypoxia activates autophagy, promoting EPC survival by inhibiting apoptosis. Enhancing autophagy with hypoxic preconditioning may be beneficial for survival of the transplanted EPCs in a local hypoxic environment.
Subject(s)
Apoptosis/physiology , Autophagy/physiology , Endothelial Cells/physiology , Oxygen/physiology , Stem Cells/physiology , Apoptosis Regulatory Proteins/metabolism , Beclin-1 , Cell Hypoxia/physiology , Cell Survival/physiology , Endothelial Cells/ultrastructure , Fetal Blood/cytology , Flow Cytometry , Humans , Lysosomes/physiology , Membrane Proteins/metabolism , Stem Cells/ultrastructureABSTRACT
Resent study suggests that c-kit+ cells in bone marrow-derived MSCs may differentiate toward cardiamyocytes. However, the properties of c-kit+ MSCs remain unclear. This study isolated c-kit+VEGFR-2+ cells from rat bone marrow-derived MSCs, and assessed potential of c-kit+VEGFR-2+ MSCs to differentiate towards cardiovascular cells and their efficiency of repairing the infarcted myocardium after transplantation. Gene expression profile of the cells was analyzed with RNA-sequencing. Potential of differentiation of the cells was determined after induction. Rat models of myocardial infarction were established by ligation of the left anterior descending coronary artery. The cells were treated with hypoxia and serum deprivation for four hours before transplantation. Improvement of cardiac function and repair of the infarcted myocardium were assessed at four weeks after transplantation. Gene expression profile revealed that c-kit+VEGFR-2+ MSCs expressed most smooth muscle-specific and myocardium-specific genes, while expression of endothelium-specific genes was upregulated significantly. After induction with VEGF or TGF-ß for two weeks, the cells expressed CD31 and α-SMA respectively. At three weeks, BMP-2-induced cells expressed cTnT. After transplantation of the cells, cardiac function was improved, scar size of the infarcted myocardium was decreased, and angiogenesis and myocardial regeneration were enhanced significantly. Moreover, paracrine in the myocardium was increased after transplantation. These results suggest that c-kit+VEGFR-2+ MSCs have a potential of differentiation towards cardiovascular cells. Transplantation of c-kit+VEGFR-2+ MSCs is effective for repair of the infarcted myocardium. c-kit+VEGFR-2+ MSCs may be a reliable source for cell therapy of ischaemic diseases.
Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Myocardial Infarction , Rats , Animals , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism , Mesenchymal Stem Cell Transplantation/methods , Myocardium/metabolism , Proto-Oncogene Proteins c-kit/genetics , Proto-Oncogene Proteins c-kit/metabolismABSTRACT
Objective: Polypyrimidine tract-binding protein 1 (PTBP1) is an RNA-binding protein, which plays a role in pre-mRNA splicing and in the regulation of alternative splicing events. However, little was known about the correlation between PTBP1 and glioma and its prognostic significance in glioma patients. Our aim was to investigate the expression, functional role, and prognostic value of PTBP1 in glioma. Methods: We explored the expression of PTBP1 protein using immunohistochemistry in 150 adult malignant glioma tissues and 20 normal brain tissues and evaluated its association with clinicopathological parameters by chi-square test. Kaplan-Meier method was used to evaluate the prognostic effect of PTBP1 in glioma. Univariate/multivariate Cox analyses were used to identify independent prognostic factors. Transcriptional regulation network was constructed based on differentially expressed genes (DEGs) of PTBP1 from TCGA/CGGA database. GO and KEGG enrichment analyses were used to explore the function and pathways of DEGs. Results: Out of the 150 malignant glioma tissues (60 LGG and 90 GBMs) and 20 normal brain tissues in our cohort, PTBP1 protein was high expressed in glioma tissues (79/150, 52.7%), but no expression was detected in normal brain tissues (0/20, 0%). The expression of PTBP1 was significantly higher in GBMs (P < 0.001). More than half of GBMs (62/90, 68.9%) were PTBP1 high expression. Chi-square test showed that the expression of PTBP1 was correlated with patient age, WHO grade, Ki-67 index, and IDH status. High expression of PTBP1 was significantly associated with poor prognosis in glioma, and it was an independent risk factor in glioma patients. Furthermore, we shed light on the underlying mechanism of PTBP1 by constructing a miR-218-TCF3-PTBP1 transcriptional network in glioma. Conclusion: PTBP1 was high expressed in glioma, and it significantly correlated with poor prognosis, suggesting a potential therapeutic target for glioma, particularly for GBM.
Subject(s)
Biomarkers, Tumor/genetics , Brain Neoplasms/genetics , Glioma/genetics , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Polypyrimidine Tract-Binding Protein/genetics , Adult , Brain Neoplasms/pathology , Female , Glioma/pathology , Humans , Male , PrognosisABSTRACT
BACKGROUND: The high death rate of the transplanted stem cells in the infarcted heart and low efficiency of differentiation toward cardiomyocytes show that mesenchymal stem cell (MSC) transplantation after myocardial infarction (MI) is not effective. Csx/Nkx2.5 and GATA-4 are considered to be key regulators of cardiogenesis. The aim of the present study was to investigate the effect of transplanting MSC overexpressing Csx/Nkx2.5 and GATA-4 (MSCs-CG) after MI. METHODS AND RESULTS: According to acridine orange/ethidium bromide staining, MSCs-CG were more resistant to anoxia as compared with MSCs in vitro. In a mouse MI model, ejection fraction and fractional shortening were higher in the MSC-CG group than in the MSC or phosphate-buffered saline group. Wall thickness of the infarct area was increased and collagen deposition was clearly reduced in the MSC-CG group as compared with the other groups. There were more surviving MSCs in the MSC-CG group than in the MSC group. Most of the Y chromosome-positive cells expressed cardiac troponin T and connexin43 (Cx-43). Cx-43 was localized between Y chromosome-positive cells and recipient cardiomyocytes. Microvessel density in the peri-infarct regions and infarct regions increased significantly in the MSC-CG group. CONCLUSIONS: Transplantation of MSCs overexpressing Csx/Nkx2.5 and GATA-4 represents a new treatment strategy with the potential to improve cardiac function after MI.
Subject(s)
GATA4 Transcription Factor/biosynthesis , Gene Expression , Homeodomain Proteins/biosynthesis , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/metabolism , Myocardial Infarction/therapy , Transcription Factors/biosynthesis , Animals , Connexin 43/biosynthesis , Connexin 43/genetics , Female , GATA4 Transcription Factor/genetics , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/genetics , Male , Mice , Myocardial Infarction/genetics , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocardium/metabolism , Myocardium/pathology , Stroke Volume , Transcription Factors/genetics , Transplantation, Homologous , Troponin T/biosynthesis , Troponin T/geneticsABSTRACT
ABSTRACT: Enhancer RNAs (eRNAs), a subclass of lncRNAs, are derived from enhancer regions. The function of eRNAs has been reported by many previous studies. However, the role of eRNAs in gastric cancer, especially the prognosis-associated eRNAs, has not been studied yet.In this study, we have used a novel approach to screened key eRNAs in gastric cancer. Kaplan-Meier correlation analysis and Co-expression analysis were used to find the most significant survival-associated eRNAs. Enrichment analysis is applied to explore the key functions and pathways of screened eRNAs. The correlation and survival analysis are used to evaluate targeted genes in the pan-cancer analysisA total of 63 prognostic-associated eRNAs in gastric cancer were identified, the top 6 eRNAs were LINC01714, ZNF192P1, AC079760.2, LINC01645, EMX2OS, and AC114489.2. The correlation analysis demonstrated the top 10 screened eRNAs and their targeted genes. The results demonstrated that EMX2OS was ranked as the top eRNA according to the results of the Kaplan-Meier analysis. The correlation analysis demonstrated that eRNA EMX2OS is correlated with age, grade, stage, and cancer status. The pan-cancer analysis demonstrated that EMX2OS was associated with poor survival outcomes in adrenocortical carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, kidney renal clear cell carcinoma, stomach adenocarcinoma, and uveal melanoma.In this study, survival-related eRNAs were screened and the correlation between survival-related eRNAs and their targeted genes was demonstrated. EMX2OS plays a prognosis-associated eRNA role in gastric cancer, which might be a novel therapeutic target in clinical practice.
Subject(s)
Adenocarcinoma/genetics , Homeodomain Proteins/genetics , RNA, Long Noncoding/genetics , Stomach Neoplasms/genetics , Transcription Factors/genetics , Adenocarcinoma/diagnosis , Aged , Biomarkers, Tumor/genetics , Enhancer Elements, Genetic/genetics , Female , Gene Expression Regulation, Neoplastic , Humans , Kaplan-Meier Estimate , Male , Middle Aged , Neoplasm Staging/methods , Prognosis , RNA, Antisense/genetics , Stomach Neoplasms/mortality , Stomach Neoplasms/pathology , Survival AnalysisABSTRACT
The epicardium plays an important role in cardiomyogenesis during development, while it becomes quiescent in adult heart during homeostasis. This study investigates the efficiency of thymosin ß4 (Tß4) release with RPRHQGVM conjugated to the C-terminus of RADA16-I (RADA-RPR), the functionalized self-assembling peptide (SAP), to activate the epicardium and repairing the infarcted myocardium. Methods: The functionalized SAP was constituted with self-assembling motif, Tß4-binding site, and cell adhesive ligand. Myocardial infarction (MI) models of the transgenic mice were established by ligation of the left anterior descending coronary artery. At one week after intramyocardial injection of Tß4-conjugated SAP, the activation of the epicardium was assessed. At four weeks after implantation, the migration and differentiation of epicardium-derived cells (EPDCs) as well as angiogenesis, lymphangiogenesis and myocardial regeneration were examined. Results: We found that the designer RADA-RPR bound Tß4 and adhered to EPDCs and that Tß4 released from the functionalized SAP could effectively activate the epicardium and induce EPDCs to differentiate towards cardiovascular cells as well as lymphatic endothelial cells. Moreover, SAP-released Tß4 (SAP-Tß4) promoted proliferation of cardiomyocytes. Furthermore, angiogenesis, lymphangiogenesis and myocardial regeneration were enhanced in the MI models at 4 weeks after delivery of SAP-Tß4 along with attenuation of adverse myocardial remodeling and significantly improved cardiac function. Conclusions: These results demonstrate that sustained release of Tß4 from the functionalized SAP can activate the epicardium and effectively enhance the repair of infarcted myocardium. We believe the delivery of SAP-Tß4 may be a promising strategy for MI therapy.
Subject(s)
Myocardial Infarction/drug therapy , Myocardium/pathology , Peptides/pharmacology , Pericardium/drug effects , Thymosin/pharmacology , Animals , Cell Differentiation/drug effects , Cell Movement/drug effects , Cell Proliferation/drug effects , Endothelial Cells/drug effects , Lymphangiogenesis/drug effects , Mice , Mice, Transgenic , Myocardium/metabolism , Myocytes, Cardiac/drug effects , Neovascularization, Physiologic/drug effectsABSTRACT
Progress in stem cell transplantation for the treatment of myocardial infarction is hampered by the poor retention and survival of the implanted cells. To enhance cell survival and differentiation and thereby improve the efficiency of stem cell therapy, we constructed a novel self-assembling peptide by attaching an RGDSP cell-adhesion motif to the self-assembling peptide RADA16. c-kit(pos)/Nkx2.5(low)/GATA4(low) marrow-derived cardiac stem cells (MCSCs), which have a specific potential to differentiate into cardiomyocytes, were isolated from rat bone marrow. The cytoprotective effects of RGDSP scaffolds were assessed by exposure of MCSCs to anoxia in vitro. The efficacy of transplanting MCSCs in RGDSP scaffolds was evaluated in a female rat MI model. The designer self-assembling peptide self-assembled into RGDSP nanofiber scaffolds under physiological conditions. RGDSP scaffolds were beneficial for the growth of MCSCs and protected them from apoptosis and necrosis caused by anoxia. In a rat MI model, cardiac function was improved and collagen deposition was markedly reduced in the group receiving MCSCs in RGDSP scaffolds compared with groups receiving MCSCs alone, RGDSP scaffolds alone or MCSCs in RADA16 scaffolds. There were more surviving MCSCs in the group receiving MCSCs in RGDSP scaffolds than in the groups receiving MCSCs alone or MCSCs in RADA16 scaffolds. Most of the Y chromosome-positive cells expressed cardiac troponin T and connexin43 (Cx-43). These results suggest that RGDSP scaffolds provide a suitable microenvironment for the survival and differentiation of MCSCs. RGDSP scaffolds enhanced the efficacy of MCSC transplantation to repair myocardium and improve cardiac function.
Subject(s)
Bone Marrow Cells/physiology , Bone Marrow Transplantation/methods , Myocardial Infarction/surgery , Myocytes, Cardiac/transplantation , Nanofibers , Stem Cell Transplantation/methods , Animals , Bone Marrow Cells/metabolism , Cell Culture Techniques , Cell Differentiation , Cell Survival , Fibrosis , Male , Myocardial Contraction , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/physiology , Oligopeptides/metabolism , Peptides/metabolism , Rats , Rats, Sprague-Dawley , Ventricular Function, LeftABSTRACT
The poor survival and differentiation of the donor cells in the infarcted myocardium has hampered the therapeutic efficacy of cell transplantation. A self-assembling polypeptide RAD16-II (Ac-RARADADARARADADA-CONH2) spontaneously assembles into stable nanofiber scaffolds, which mimic natural extracellular matrix at 0.1-1% peptide concentrations in the myocardium. We isolated mesenchymal stem cells from the bone marrow of adult male rats that express both c-kit and Nkx2.5, a cardiac transcription factor, yielding selected mesenchymal stem cells (SMSCs). We initially confirmed that the self-assembling polypeptide scaffolds are conducive to growth, survival and differentiation of SMSCs in vitro. Subsequently, SMSCs mixed with the self-assembling polypeptide were injected into the infarcted area at 30 min after the establishment of myocardial infarction in female rats. The donor cells were tracked with Y chromosome in the myocardium. The changes of cardiac function, myocardial structure and capillary density were detected at 4 weeks after cell transplantation. The hearts transplanted with SMSCs incorporated into the nanofiber scaffolds showed smaller infarction size (19.55 ± 2.1%) than the hearts injected with SMSCs (27.37 ± 4.8%). Importantly, the systolic function indices, left ventricle ejection fraction and left ventricle fractional shortening, were significantly improved in the animals transplanted with SMSCs mixed with the nanofiber scaffolds (59.31 ± 4.9% and 31.91 ± 8.1%), compared to those with SMSCs alone (48.31 ± 9.2% and 23.58 ± 8.5%). In conclusion, transplantation of SMSCs mixed with the self-assembling polypeptide RAD16-II is more effective to promote myocardial regeneration and attenuate cardiac injury in a rat model of myocardial infarction.
Subject(s)
Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells/cytology , Myocardial Infarction/therapy , Peptides/therapeutic use , Amino Acid Sequence , Animals , Apoptosis/drug effects , Capillaries/drug effects , Capillaries/pathology , Cell Differentiation/drug effects , Cell Survival/drug effects , Female , Heart Function Tests/drug effects , Male , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/ultrastructure , Molecular Sequence Data , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Nanofibers/ultrastructure , Peptides/chemistry , Peptides/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
Myocardial infarction (MI) causes myocardium injury and scar formation, and the transmural infarction is associated with ventricular hypofunction. Stem cell transplantation therapy has improved cardiac function in animal models of MI. However, the poor survival of the donor cells in the host myocardium hampers the therapeutic efficacy of stem cell transplantation. Diazoxide, a mitochondrial ATP-sensitive potassium channel opener, has been applied to suppress cell apoptosis and promote cell survival. We therefore assessed the effects of diazoxide on the selected mesenchymal stem cells (SMSCs). Pretreatment of SMSCs with diazoxide (200 micromol/L) for 30 min protected cells from oxidative stress injury by upregulating the expression of basic fibroblast growth factor and hepatocyte growth factor mRNAs and phospho-Akt and by preventing mitochondral cytochrome c translocation into the cytoplasm. Expression of mRNAs and proteins was detected by RT-PCR and western blot analyses. Thirty min after establishment of MI (the ligation of the left anterior descending of coronary artery) in female rats, the male rat SMSCs preconditioned with diazoxide were injected at four sites on the edge of the infarcted area. At 4 weeks after cell tranplantation, the donor cells in the recipient myocardium were tracked with Y chromosome. Preconditioning with diazoxide improved the survival rate of the transplanted SMSCs, compared to the untreated SMSCs. Moreover, transplantation of the diazoxide-pretreated SMSCs reduced the infarct size and increased left ventricular function, as judged by transthoracic echocardiography. In conclusion, diazoxide preconditioning is effective to promote SMSCs survival under oxidative stress and attenuates cardiac injury in MI.
Subject(s)
Cytoprotection/drug effects , Diazoxide/pharmacology , Ion Channel Gating/drug effects , Mesenchymal Stem Cell Transplantation/methods , Mesenchymal Stem Cells/drug effects , Myocardial Infarction/therapy , Potassium Channels/metabolism , Animals , Apoptosis/drug effects , Cell Count , Cell Survival/drug effects , Collagen/metabolism , Cytochromes c/metabolism , Decanoic Acids/pharmacology , Female , Fibroblast Growth Factor 2/genetics , Gene Expression/drug effects , Gene Expression/genetics , Heart Ventricles/metabolism , Heart Ventricles/pathology , Hepatocyte Growth Factor/genetics , Hydrogen Peroxide/pharmacology , Hydroxy Acids/pharmacology , Male , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Mitochondrial Membranes/drug effects , Mitochondrial Membranes/metabolism , Muscle Development/physiology , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Myocardial Infarction/physiopathology , Myocardium/metabolism , Myocardium/pathology , Neovascularization, Physiologic/physiology , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Rats , Rats, Sprague-Dawley , Troponin T/metabolism , Vascular Endothelial Growth Factor A/genetics , Ventricular Function, Left/physiologyABSTRACT
OBJECTIVE: To investigate the change of autophagy and morphological characteristics of the autophagic structures in free-living amoeba under nutritional stress. METHODS: Free-living amoebae were incubated on the agaric solid medium which had been covered with Escherichia cdi in control group. In the experiment group, amoebae incubated on the agaric solid medium with E. coli were collected and moved to another solid medium without E. coli and incubated for 12 h. The morphological changes of free-living amoeba in the medium without E. coli were viewed with scanning electron microscope. The changes of autophagy and the structural features of the autophagosome precursors, autophagosomes and autophagolysosomes in amoeba were examined with transmission electron microscope, and the cross-section areas of the autophagic structures and cytoplasm were measured with an image analyzer. The autophagosomes in the organism were labeled with monodansylcadaverine (MDC) staining and quantitated using laser scanning confocal microscope. RESULTS: In the control group, free-living amoebae were all in the form of trophozoite. In the experiment group, trophozoites were induced to transform to cysts gradually. In control group, amoeba was full of fragment of E. coli. There was merely little autophagy with fewer autophagic structures in amoeba. When compared with the control group, the autophagic abilities of amoeba were enhanced significantly, number of autophagic structures increased in the experiment group. In addition, the ratio of the cross-sectional areas of the autophagic structures to that of the cytoplasm of amoeba was greater (P < 0.05 or 0.01). There was fragment of E. coli that was not digested in some of the amoebae. CONCLUSION: In the circumstance of nutritional stress, amoebic trophozoites were induced to transform to cysts gradually. The autophagic ability of free-living amoeba significantly enhanced.
Subject(s)
Amoeba/physiology , Autophagy , Trophozoites/physiology , Amoeba/ultrastructure , Culture Media , Escherichia coliABSTRACT
Stem cell transplantation has been limited by poor survival of the engrafted cells in hostile microenvironment of the infarcted myocardium. This study investigated cytoprotective effect of rapamycin-preactivated autophagy on survival of the transplanted mesemchymal stem cells (MSCs). MSCs isolated from rat bone marrow were treated with 50 nmol/L rapamycin for 2 h, and then the cytoprotective effect of rapamycin was examined. After intramyocardial transplantation in rat ischemia/reperfusion models, the survival and differentiation of the rapamycin-pretreated calls were accessed. After treatment with rapamycin, autophagic activities and lysososme production of the cells were increased significantly. In the condition of short-term or long-term hypoxia and serum deprivation, the apoptotic cells in rapamycin-pretreated cells were less, and secretion of HGF, IGF-1, SCF, SDF-1 and VEGF was increased. After transplantation of rapamycin-pretreated cells, repair of the infarcted myocardium and restoration of cardial function were enhanced dramatically. Expression of HGF, IGF-1, SCF, SDF-1, VEGF, HIF-1α and IL-10 in the myocardium was upregulated, while expression of IL-1ß and TNF-α was downregulated. Tracing of GFP and Sry gene showed that the survival of rapamycin-pretreated cells was increased. Cardiomyogenesis and angiogenesis in the infarcted myocardium were strengthened. Some rapamycin-pretreated cells differentiated into cardiomyocytes or endothelial cells. These results demonstrate that moderate preactivation of autophagy with rapamycin enhances the survival and differentiation of the transplanted MSCs. Rapamycin-primed MSCs can promote repair of the infarcted myocardium and improvement of cardiac function effectively.