OBJECTIVE: To prepare clinical-grade human umbilical cord-derived mesenchymal stem cells (hUC-MSC) with high expression of hematopoietic supporting factors and evaluate their stem cell characteristics. METHODS: Fetal umbilical cord tissues were collected from healthy postpartum women during full-term cesarean section. Wharton's jelly was mechanically separated and hUC-MSCs were obtained by explant culture method and enzyme digestion method in an animal serum-free culture system with addition of human platelet lysate. The phenotypic characteristics of hUC-MSCs obtained by two methods were detected by flow cytometry. The differences in proliferation ability between the two groups of hUC-MSCs were identified through CCK-8 assay and colony forming unit-fibroblast (CFU-F) assay. The differences in multilineage differentiation potential between the two groups of hUC-MSCs were identified through induction of adipogenic, osteogenic, and chondrogenic differentiation. The mRNA expression levels of hematopoietic supporting factors such as SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in the two groups of hUC-MSCs were identified by real-time fluorescence quantiative PCR(RT-qPCR). RESULTS: The results of flow cytometry showed that hUC-MSCs obtained by the two methods both expressed high levels of CD73, CD90 and CD105, while lowly expressed CD31, CD45 and HLA-DR. The results of CCK-8 and CFU-F assay showed that the proliferation ability of hUC-MSCs obtained by explant culture method was better than those obtained by enzyme digestion method. The results of the triple lineage differentiation experiment showed that there was no significant difference in multilineage differentiation potential between the two grous of hUC-MSCs. The results of RT-qPCR showed that the mRNA expression levels of hematopoietic supporting factors SCF, IL-3, CXCL12, VCAM1 and ANGPT1 in hUC-MSCs obtained by explant cultrue method were higher than those obtained by enzyme digestion method. CONCLUSION Clinical-grade hUC-MSCs with high expression levels of hematopoietic supporting factors were successfully cultured in an animal serum-free culture system.
Humans ; Mesenchymal Stem Cells/metabolism* ; Umbilical Cord/cytology* ; Cell Differentiation ; Female ; Cell Proliferation ; Cells, Cultured ; Chemokine CXCL12/metabolism* ; Angiopoietin-1/metabolism* ; Vascular Cell Adhesion Molecule-1/metabolism* ; Stem Cell Factor/metabolism* ; Flow Cytometry ; Pregnancy

OBJECTIVE: To investigate the expression and possible regulatory mechanism of miR-34a in the lung tissue of neonatal rat model of bronchopulmonary dysplasia (BPD) induced by hyperoxia. METHODS: In the study, 80 newborn SD rats were randomly divided into hyperoxia group (FiO₂=60%) and air group (FiO₂=21%) within 2 hours after birth, 40 rats per group. Lung tissue samples of the SD rats in each group were extracted on the 1st, 7th, 14th and 21st days after birth, and the pathological changes of lung tissue were observed under light microscope after HE staining. The number of radial alveolar counts (RAC) and the mean alveolar diameter (MAD) and the thickness of alveolar septal thickness (AST) were measured to evaluate the development of alveoli. Real-time fluorescence quantitative PCR was used to detect the expression of miR-34a, angiopoietin-1 (Ang-1) and tyrosine kinase receptor-2 (Tie-2) in lung tissue of rats in hyperoxia group and air group at different time points. Enzyme-linked immunosorbent assay (ELISA) was used to detect the proteins expression of Ang-1 and Tie-2 in the lung tissues of the two groups at different time points. RESULTS: The weight of rats in the hyperoxia group on the 7th, 14th and 21st days after birth was significantly lower than that in the air group (P all < 0.05). With the prolongation of oxygen exposure, the number of alveoli decreased, the volume increased, the structure simplified, the alveolar cavity enlarged obviously and the alveolar septum thickened in the hyperoxia group. On the 7th, 14th and 21st days after birth, the RAC in the hyperoxia group was significantly lower than that in the air group (P all < 0.05). Compared with the air group, MAD and AST increased significantly on the 7th, 14th and 21st days after birth in the hyperoxia group, and the difference was statistically significant (P all < 0.05). The expression level of miR-34a in lung tissue of hyperoxia group was significantly higher than that of air group on the 7th, 14th and 21st days after birth, and the difference was statistically significant (P all < 0.05). Compared with the air group at the same time point, the expression levels of Ang-1 and Tie-2 mRNA and protein in the hyperoxia group were lower than those in the air group on the 14th and 21st days after birth (P all < 0.05). CONCLUSION The new BPD model of newborn SD rats can be successfully established by continuous exposure to 60% hyperoxia. The expression of miR-34a was up-regulated in the lung tissue of the new BPD model of neonatal rats. MiR-34a may play an important role in the occurrence and development of BPD by regulating Ang-1/Tie-2 signal pathway.
Animals ; MicroRNAs/metabolism* ; Bronchopulmonary Dysplasia/genetics* ; Hyperoxia/metabolism* ; Rats, Sprague-Dawley ; Animals, Newborn ; Rats ; Angiopoietin-1/genetics* ; Disease Models, Animal ; Receptor, TIE-2/genetics* ; Lung/pathology* ; Male

Hereditary angioedema (HAE) is a rare,unpredictable,autosomal dominant disorder characterized by recurrent swelling in subcutaneous and submucosal tissue.In recent years,the pathophysiology and pathogenesis of HAE have been continuously studied and elucidated.In addition to the genes encoding complement 1 esterase inhibitors,new pathogenic variants have been identified in the genes encoding coagulation factor Ⅻ,plasminogen,angiopoietin-1,kininogen,heparan sulfate 3-O-sulfotransferase 6,and myoferlin in HAE.Moreover,different pathogenic variants have different mechanisms in causing HAE.In addition,the pathogenic genes of some patients remain unknown.This review summarizes the recent progress in the classification,epidemiology,pathophysiology,and pathogenesis of HAE,aiming to provide ideas for further fundamental research,clinical diagnosis,and drug development of HAE.
Humans ; Angioedemas, Hereditary/diagnosis* ; Angiopoietin-1/metabolism* ; Plasminogen/metabolism* ; Kininogens/metabolism* ; Complement C1 Inhibitor Protein/metabolism*

OBJECTIVE: To detect whether Danlou Tablet (DLT) regulates the hypoxia-induced factor (HIF)-1α-angiopoietin-like 4 (Angptl4) mRNA signaling pathway and explore the role of DLT in treating chronic intermittent hypoxia (CIH)-induced dyslipidemia and arteriosclerosis. METHODS: The mature adipocytes were obtained from 3T3-L1 cell culturation and allocated into 8 groups including control groups (Groups 1 and 5, 0.1 mL of cell culture grade water); DLT groups (Groups 2 and 6, 0.1 mL of 1,000 µg/mL DLT submicron powder solution); dimethyloxalylglycine (DMOG) groups (Groups 3 and 7, DMOG and 0.1 mL of cell culture grade water); DMOG plus DLT groups (Groups 4 and 8, DMOG and 0.1 mL of 1,000 µg/mL DLT submicron powder solution). Groups 1-4 used mature adipocytes and groups 5-8 used HIF-1 α-siRNA lentivirus-transfected mature adipocytes. After 24-h treatment, real-time polymerase chain reaction and Western blot were employed to determine the mRNA and protein expression levels of HIF-1 α and Angptl4. In animal experiments, the CIH model in ApoE^-/- mice was established. Sixteen mice were complete randomly divided into 4 groups including sham group, CIH model group [intermittent hypoxia and normal saline (2 mL/time) gavage once a day]; Angptl4 Ab group [intermittent hypoxia and Angptl4 antibody (30 mg/kg) intraperitoneally injected every week]; DLT group [intermittent hypoxia and DLT (250 mg/kg) once a day], 4 mice in each group. After 4-week treatment, enzyme linked immunosorbent assay was used to detect the expression levels of serum total cholesterol (TC) and triglyceride (TG). Hematoxylin-eosin and CD68 staining were used to observe the morphological properties of arterial plaques. RESULTS: Angptl4 expression was dependent on HIF-1 α, with a reduction in mRNA expression and no response in protein level to DMOG or DLT treatment in relation to siHIF-1 α -transfected cells. DLT inhibited HIF-1 α and Angptl4 mRNA expression (P<0.05 or P<0.01) and reduced HIF-1 α and Angptl4 protein expressions with DMOG in mature adipocytes (all P<0.01), as the effect on HIF-1 α protein also existed in the presence of siHIF-1 α (P<0.01). ApoE^-/- mice treated with CIH had increased TG and TC levels (all P<0.01) and atherosclerotic plaque. Angptl4 antibody and DLT both reduce TG and TC levels (all P<0.01), as well as reducing atherosclerotic plaque areas, narrowing arterial wall thickness and alleviating atherosclerotic lesion symptoms to some extent. CONCLUSION DLT had positive effects in improving dyslipidemia and arteriosclerosis by inhibiting Angptl4 protein level through HIF-1 α-Angptl4 mRNA signaling pathway.
Angiopoietin-Like Protein 4/genetics* ; Animals ; Apolipoproteins E ; Atherosclerosis/metabolism* ; Drugs, Chinese Herbal ; Dyslipidemias/genetics* ; Hypoxia/metabolism* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Mice ; Plaque, Atherosclerotic ; Powders ; RNA, Messenger/genetics* ; Signal Transduction ; Triglycerides ; Water

OBJECTIVE: The favored method of preserving fertility in young female cancer survivors is cryopreservation and autotransplantation of ovarian tissue. Reducing hypoxia until angiogenesis takes place is essential for the survival of transplanted ovarian tissue. The aim of this study was to investigate the role of angiopoietin-1 (Angpt-1), angiopoietin-2 (Angpt-2), and vascular endothelial growth factor (VEGF) in ovarian tissue grafts that were cryopreserved using two methods. METHODS: Ovarian tissues harvested from ICR mice were divided into three groups: group I (control), no cryopreservation; group II, vitrification in EFS (ethylene-glycol, ficoll, and sucrose solution)-40; and group III, slow freezing in dimethyl sulfoxide. We extracted mRNA for VEGF, Angpt-1, and Angpt-2 from ovarian tissue 1 week following cryopreservation and again 2 weeks after autotransplantation. We used reverse transcriptase-polymerase chain reaction to quantify the levels of VEGF, Angpt-1, and Angpt-2 in the tissue. RESULTS: Angpt-1 and Angpt-2 expression decreased after cryopreservation in groups II and III. After autotransplantation, Angpt-1 and Angpt-2 expression in ovarian tissue showed different trends. Angpt-1 expression in groups II and III was lower than in group I, but Angpt-2 in groups II and III showed no significant difference from group I. The vitrified ovarian tissues had higher expression of VEGF and Angpt-2 than the slowfrozen ovarian tissues, but the difference was not statistically significant. CONCLUSION: Our results indicate that Angpt-2 may play an important role in ovarian tissue transplantation after cryopreservation although further studies are needed to understand its exact function.
Angiopoietin-1* ; Angiopoietin-2 ; Animals ; Anoxia ; Autografts ; Cryopreservation* ; Dimethyl Sulfoxide ; Female ; Fertility ; Fertility Preservation ; Ficoll ; Freezing ; Humans ; Methods* ; Mice ; Mice, Inbred ICR ; Ovary ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Messenger ; Sucrose ; Survivors ; Tissue Transplantation ; Transplantation, Autologous ; Transplants* ; Vascular Endothelial Growth Factor A* ; Vascular Endothelial Growth Factors ; Vitrification

Men with diabetic erectile dysfunction (ED) respond poorly to the currently available oral phosphodiesterase-5 inhibitors. Therefore, functional therapies for diabetic ED are needed. Stromal vascular fraction (SVF) and the adenovirus-mediated cartilage oligomeric matrix angiopoietin-1 (Ad-COMP-Ang1) gene are known to play critical roles in penile erection. We previously reported that SVF and Ad-COMP-Ang1 have only a short-term effect in restoring erectile function. Further improvements to ED therapy are needed for long-lasting effects. In the present study, we aimed to test if the combination of SVF and Ad-COMP-Ang1 could extend the erection effect in diabetic ED. We found that the combination therapy showed a long-term effect in restoring erectile function through enhanced penile endothelial and neural cell regeneration. Combination therapy with SVF and Ad-COMP-Ang1 notably restored cavernous endothelial cell numbers, pericyte numbers, endothelial cell-cell junctions, decreased cavernous endothelial cell permeability, and promoted neural regeneration for at least 4 weeks in diabetic mice. In summary, this is an initial description of the long-term effect of combination therapy with SVF and Ad-COMP-Ang1 in restoring erectile function through a dual effect on endothelial and neural cell regeneration. Such combination therapy may have therapeutic potential for the treatment of diabetic ED.
Angiopoietin-1/genetics* ; Animals ; Diabetes Mellitus, Experimental/metabolism* ; Endothelium, Vascular/metabolism* ; Erectile Dysfunction/therapy* ; Genetic Therapy/methods* ; Intercellular Junctions/metabolism* ; Male ; Mesenchymal Stem Cell Transplantation ; Mice ; Penile Erection/physiology* ; Permeability

PURPOSE: Angiopoietin-1 (Ang-1) plays a crucial role in vascular and hematopoietic development, mainly through its cognate receptor, Tie-2. Increased levels of Ang-2 have been shown to be correlated with abnormal tumor angiogenesis in several malignancies. Hence, we estimated the increased expression of Ang-2 relative to Ang-1 in patients with colorectal cancer and correlated our finding with prognosis in order to investigate the relationships between the expressions of Ang-1/Ang-2/Tie-2 receptor and the clinical parameters or overall survival of such patients. METHODS: We retrospectively analyzed 114 tissue samples from patients with colorectal cancer by using immunohistochemistry (IHC) to examine Ang-1, Ang-2, and Tie-2 expressions and to investigate the relationship between those expressions and clinical parameters or overall survival of such patients. A Western blot analysis was used for Ang-2 expression. RESULTS: IHC staining showed a link between Ang-1 and Tie-2 (P = 0.018), as well as meaningful correlations between Ang-2 and Tie-2 receptor (P = 0.022) and between lymph-node metastasis and Ang-2 (P = 0.025). The stronger the IHC staining for Ang-2 expression was, the shorter the cumulative survival was (P = 0.016). CONCLUSION: A relationship was found to exist between Ang-2 and Tie-2 expressions. The Ang-2 was correlated with lymph-node metastasis, and high expression of Ang-2 was indicative of poor overall survival. These findings suggest that Ang-2 is a useful prognostic marker in the management of patients with colorectal cancer. In addition, we suggest that Ang/Tie-2 signaling plays an important role in the progression of colorectal cancer.
Angiopoietin-1* ; Angiopoietin-2* ; Angiopoietins ; Blotting, Western ; Colorectal Neoplasms* ; Humans ; Immunohistochemistry ; Neoplasm Metastasis ; Prognosis ; Receptor, TIE-2* ; Retrospective Studies

PURPOSE: Angiopoietin-1 (Ang1) is a critical factor for vascular stabilization and endothelial survival via inhibition of endothelial permeability and leukocyte- endothelium interactions. Hence, we hypothesized that treatment with umbilical cord mesenchymal stem cells (UCMSCs) carrying the Ang1 gene (UCMSCs-Ang1) might be a potential approach for acute lung injury (ALI) induced by lipopolysaccharide (LPS). MATERIALS AND METHODS: UCMSCs with or without transfection with the human Ang1 gene were delivered intravenously into rats one hour after intra-abdominal instillation of LPS to induce ALI. After the rats were sacrificed at 6 hours, 24 hours, 48 hours, 8 days, and 15 days post-injection of LPS, the serum, the lung tissues, and bronchoalveolar lavage fluid (BALF) were harvested for analysis, respectively. RESULTS: Administration of fluorescence microscope confirmed the increased presence of UCMSCs in the injured lungs. The evaluation of UCMSCs and UCMSCs-Ang1 actions revealed that Ang1 overexpression further decreased the levels of the pro-inflammatory cytokines TNF-α, TGF-β1, and IL-6 and increased the expression of the anti-inflammatory cytokine IL-10 in the injured lungs. This synergy caused a substantial decrease in lung airspace inflammation and vascular leakage, characterized by significant reductions in wet/dry ratio, differential neutrophil counts, myeloperoxidase activity, and BALF. The rats treated by UCMSCs-Ang1 showed improved survival and lower ALI scores. CONCLUSION: UCMSCs-Ang1 could improve both systemic inflammation and alveolar permeability in ALI. UC-derived MSCs-based Ang1 gene therapy may be developed as a potential novel strategy for the treatment of ALI.
Acute Lung Injury/chemically induced/*therapy ; Angiopoietin-1/*genetics ; Animals ; Bronchoalveolar Lavage Fluid ; Cytokines/metabolism ; Endotoxins ; Genetic Therapy ; Interleukin-10/metabolism ; Interleukin-6/metabolism ; Leukocyte Count ; Lipopolysaccharides ; Lung/metabolism ; Male ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/metabolism ; Neutrophils/metabolism ; Rats ; Transforming Growth Factor beta1/metabolism ; Tumor Necrosis Factor-alpha/metabolism ; Umbilical Cord/*cytology

Minimal change disease (MCD) is a common pathological type of nephrotic syndrome. Its main histology is the fusion of podocyte foot process. The pathogenesis of MCD is not clear, but previously it was thought to be related to immune mechanism. In recent years more studies show that podocyte injury is the key link in the pathogenesis of MCD. In MCD mouse model and human kidney tissues, the expressions of podocyte slit membrane protein-nephrin and podocin, skeleton protein-synaptopodin are decreased, and the expression of synaptopodin is correlated with the response to hormone therapy. In addition, newest studies focused on another two potocyte associated proteins, CD80 and Angiopoietin-like-4. CD80, a T cell stimulating molecule, is expressed in potocyte. Kappa B gene sequences can be activated by external microbes, antigens through acting potocytes, which can induce the upregulation of CD80 expression, cytoskeletal protein damage and the glomerular filtration rate changes, resulting in proteinuria. Angiopoietin-like-4 can be expressed in normal potocytes, but over-expression of angiopoietin-like-4 may injure the GBM charge barrier and induce the foot process fusion, leading to MCD. However, further studies on the factors inducing CD80 and Angiopoietin-like-4 expression, and the interaction between glomerular basement membrane and the two proteins are needed. Based on the mechanism of MCD, NF-kappa B inhibitors and sialylation therapy would be a novel non-immune therapy for MCD.
Angiopoietin-like 4 Protein ; Angiopoietins ; metabolism ; Animals ; B7-1 Antigen ; metabolism ; Disease Models, Animal ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Kidney ; pathology ; Membrane Proteins ; metabolism ; Mice ; Microfilament Proteins ; metabolism ; NF-kappa B ; metabolism ; Nephrosis, Lipoid ; pathology ; Podocytes ; pathology

CTHRC1 (collagen triple-helix repeat-containing 1), a protein secreted during the tissue-repair process, is highly expressed in several malignant tumors, including pancreatic cancer. We recently showed that CTHRC1 has an important role in the progression and metastasis of pancreatic cancer. Although CTHRC1 secretion affects tumor cells, how it promotes tumorigenesis in the context of the microenvironment is largely unknown. Here we identified a novel role of CTHRC1 as a potent endothelial activator that promotes angiogenesis by recruiting bone marrow-derived cells to the tumor microenvironment during tumorigenesis. Recombinant CTHRC1 (rCTHRC1) enhanced endothelial cell (EC) proliferation, migration and capillary-like tube formation, which was consistent with the observed increases in neovascularization in vivo. Moreover, rCTHRC1 upregulated angiopoietin-2 (Ang-2), a Tie2 receptor ligand, through ERK-dependent activation of AP-1 in ECs, resulting in recruitment of Tie2-expressing monocytes (TEMs) to CTHRC1-overexpressing tumor tissues. Treatment with a CTHRC1-neutralizing antibody-abrogated Ang-2 expression in the ECs in vitro. Moreover, administration of a CTHRC1-neutralizing antibody to a xenograft mouse model reduced the tumor burden and infiltration of TEMs in the tumor tissues, indicating that blocking the CTHRC1/Ang-2/TEM axis during angiogenesis inhibits tumorigenesis. Collectively, our findings support the hypothesis that CTHRC1 induction of the Ang-2/Tie2 axis mediates the recruitment of TEMs, which are important for tumorigenesis and can be targeted to achieve effective antitumor responses in pancreatic cancers.
Angiopoietin-2 ; Animals ; Carcinogenesis ; Endothelial Cells ; Heterografts ; In Vitro Techniques ; Mice ; Monocytes* ; Neoplasm Metastasis ; Pancreatic Neoplasms ; Receptor, TIE-2 ; Transcription Factor AP-1 ; Tumor Burden ; Tumor Microenvironment