ABSTRACT
OBJECTIVE: To unravel the potential function of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) as the matrix metalloproteinase (MMP) inhibitor in the development of chronic obstructive pulmonary disease (COPD). MATERIALS AND METHODS: Twelve specific pathogen-free Sprague Dawley rats were randomly assigned to the control cohort (n = 6) or the COPD cohort (n = 6). COPD model was developed by tobacco smoke exposure. Functional residual capacity (FRC), static lung compliance (Cchord), ratio of forced expiratory volume in 0.1 s to forced vital capacity (FEV0.1/FVC), and peak expiratory flow (PEF) were detected by respiratory function tests. Immunohistochemistry was performed to determine the pathological changes as well as the expression and localization of RECK in pulmonary tissue. RECK expression was further quantified by real-time polymerase chain reaction (PCR) and Western blot assays. RESULTS: COPD rats had significantly reduced FEV0.1/FVC% and PEF values but increased FRC and Cchord levels, as compared to the control cohort (p < 0.05). Hematoxylin and eosin (HE) staining indicated typical COPD pathological changes, including leukocyte infiltration, airway thickening, alveoli fusion, etc., in the COPD rats. IHC indicated reduced expression of RECK in the COPD cohort, which was mainly expressed on the epithelium and partly expressed on subepithelial cells and inflammatory cells. Real-time PCR and Western blot assays further revealed the significantly lower expression of RECK in lung tissue from the COPD cohort. CONCLUSIONS: RECK is mainly expressed on airway epithelial cells. COPD rats expressed significantly lower RECK levels, indicating that RECK exhibits a protective function in the development of COPD.
Subject(s)
GPI-Linked Proteins/metabolism , Matrix Metalloproteinase Inhibitors/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Animals , GPI-Linked Proteins/analysis , GPI-Linked Proteins/genetics , Male , Matrix Metalloproteinase Inhibitors/analysis , Pulmonary Disease, Chronic Obstructive/genetics , Rats , Rats, Sprague-DawleyABSTRACT
OBJECTIVE: To investigate the heterogeneity of decidual endothelial cells and their changes during delivery. PATIENTS AND METHODS: Single-cell RNA sequencing was used to characterize the transcriptomes of decidual endothelial cells before and after the onset of labor. RESULTS: Decidual endothelial cells (9748 cells) were divided into five subgroups with different functions according to differences in the transcriptome. The functions of cluster 5 were enriched in vascular development and response to growth factors. After the onset of labor, the activities of each cluster were different, including the interleukin 17 pathway and regulation of ERK1 and ERK2 cascade. The downregulated genes were related to scavenger receptor (cluster 5), which may reflect the process of endothelial activation. In terms of genetic changes, cluster 5 may be more actively involved in labor than the other clusters. CONCLUSIONS: Peripartum decidual endothelial cells are heterogeneous and participate in labor to varying degrees. One of the five subtypes of endothelial cells may be more actively involved in labor onset. Our findings may enable the assessment of decidual endothelial cells and labor onset.
Subject(s)
Decidua/cytology , Endothelial Cells/cytology , Labor Onset , Single-Cell Analysis , Female , Humans , Pregnancy , Sequence Analysis, RNA , TranscriptomeABSTRACT
Pluripotency makes human pluripotent stem cells (hPSCs) promising for regenerative medicine, but the teratoma formation has been considered to be a major obstacle for their clinical applications. Here, we determined that the downregulation of miR-302 suppresses the teratoma formation, hampers the self-renewal and pluripotency, and promotes hPSC differentiation. The underlying mechanism is that the high endogenous expression of miR-302 suppresses the AKT1 expression by directly targeting its 3'UTR and subsequently maintains the pluripotent factor OCT4 at high level. Our findings reveal that miR-302 regulates OCT4 by suppressing AKT1, which provides hPSCs two characteristics related to their potential for clinical applications: the benefit of pluripotency and the hindrance of teratoma formation. More importantly, we demonstrate that miR-302 upregulation cannot lead OCT4 negative human adult mesenchymal stem cells (hMSCs) to acquire the teratoma formation in vivo. Whether miR-302 upregulation can drive hMSCs to acquire a higher differentiation potential is worthy of deep investigation.
Subject(s)
MicroRNAs/physiology , Octamer Transcription Factor-3/metabolism , Pluripotent Stem Cells/physiology , Proto-Oncogene Proteins c-akt/metabolism , Adult Stem Cells/cytology , Adult Stem Cells/metabolism , Adult Stem Cells/physiology , Animals , Cell Cycle/genetics , Cell Differentiation/physiology , Heterografts , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred NOD , Mice, Nude , Mice, SCID , MicroRNAs/genetics , MicroRNAs/metabolism , Octamer Transcription Factor-3/genetics , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Proto-Oncogene Proteins c-akt/genetics , Teratoma/genetics , Teratoma/pathology , TransfectionABSTRACT
Endostatin, a natural angiogenesis inhibitor, had been identified for years. It opened a new approach for cancer therapy. Sequence analysis revealed that endostatin is the NC1 domain (non-triple-helical domain) of collagen XVIII. In this report, the cDNA of NC1 domain of type VIII collagen (alpha 1) was cloned and expressed as soluble form in Escherichia coli. The recombinant protein was purified with Ni-NTA agarose column and named as vastatin. It inhibited the proliferation of bovine aortic endothelial (BAE) cell stimulated by basic fibroblast growth factor (bFGF) in a dose-dependent manner. The ED(50) of vastatin was 0.6 microg/ml, while the ED(50) of endostatin was 0.5 microg/ml. Treatment of BAE cell with vastatin caused G(0)-G(1) arrest and cell apoptosis. It is interesting that sequence analysis showed that there was only about 12% amino acid sequence homology between vastatin and endostatin. The structure-function relationship of these angiogenesis molecules remains to be elucidated.
Subject(s)
Collagen Type VIII/chemistry , Collagen Type VIII/pharmacology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Amino Acid Sequence , Angiogenesis Inhibitors/chemistry , Angiogenesis Inhibitors/pharmacology , Animals , Apoptosis/drug effects , Base Sequence , Cattle , Cell Cycle/drug effects , Cell Division/drug effects , Cloning, Molecular , Collagen/chemistry , Collagen/genetics , Collagen/pharmacology , Collagen Type VIII/genetics , Collagen Type XVIII , DNA Primers/genetics , DNA, Complementary/genetics , Endostatins , Escherichia coli/genetics , Humans , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/pharmacology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Sequence Homology, Amino AcidABSTRACT
Human primary hepatocellular carcinoma (HCC)is one of the highly prevalent malignant diseases worldwide, the identification of HCC-associated genes has been a major approach in elucidating the molecular mechanism of tumorigenesis of HCC. In our previous studies, a function-unknown gene, which displayed marked expression difference between the HCC sample and normal liver control has been detected by cDNA microarray. This gene was named after fup1 (function-unknown protein 1), and was cloned according to the data of GenBank. The cDNA of fup1 has an open-reading frame 1233 base pairs in size. Here, the function analysis of FUP1 related to HCC is being reported. The NIH3T3 cells transiently transfected with FLAG-conjugated FUP1 revealed strong nuclear staining in immunofluorescent assay. Furthermore, cell proliferation enhancing activity of fup1 was shown by MTT assay in stable transfectant NIH3T3 cell line with pcDNA3-derived plasmid having fup1 under the regulation of pCMV, while cell proliferation repressing activity of antisense fup1 was observed in BEL7404 stable transfectant cells. Tumorigenicity of the above stable transfectant cells was analyzed in nude mice compared with appropriate controls. The result was in good agreement with MTT assay. Elevated tumorigenicity of fup1 transfected NIH3T3 cell and repressed tumorigenicity of antisense fup1 transfected BEL7404 cell were clearly demonstrated. The results above suggested that fup1 might be a critical gene related to carcinogenesis of HCC. Detailed molecular function of fup1 remains to be elucidated.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , 3T3 Cells , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Base Sequence , Cell Division , Cell Line , Cell Nucleus/metabolism , Cloning, Molecular , DNA/metabolism , DNA, Complementary/metabolism , Humans , Mice , Mice, Nude , Microscopy, Fluorescence , Molecular Sequence Data , Neoplasm Transplantation , Oligonucleotide Array Sequence Analysis , Oligonucleotides, Antisense/metabolism , Open Reading Frames , Polymerase Chain Reaction , Promoter Regions, Genetic , Time Factors , TransfectionABSTRACT
Hepatocyte growth factor (HGF), also known as scatter factor, is a mesenchymal or stromal-derived mediator with angiogenic activity. There are four kringle domains in its amino terminus. They display considerable sequence similarity with those of angiostatin, an angiogenesis inhibitor. We now describe that the recombinant kringle1 of HGF (HGFK1) inhibits bovine aortic endothelial (BAE) cell proliferation stimulated by basic fibroblast growth factor in a dose-dependent manner, with an ED(50) of approximately 0.7 microg/ml, while ED(50) of angiostatin is 3 microg/ml. Treatment of BAE cell with HGFK1 caused cell apoptosis. This report thus constitutes the first demonstration that kringle1 of HGF is a selective inhibitor for BAE cell proliferation stimulated by bFGF.