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 (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

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: Microvascular endothelial integrity is important for maintaining the blood-brain barrier (BBB). However, subarachnoid hemorrhage (SAH) disrupts this integrity, making the BBB dysfunctional—an important pathophysiological change after SAH. Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) regulate microvascular permeability by balancing each other’s expression. METHODS: This study investigated the dynamics of Ang-1 and Ang-2 expression after SAH and the protective effect of Ang-1 on BBB functioning using an endovascular puncture model of rat SAH. The Ang-1 and Ang-2 expression in brain tissue was determined by immunohistochemistry. In addition, Western blotting was used to estimate Ang-1 and Ang-2 concentration and to compare them at 6–72 hours post-SAH cortex and hippocampus. Evans blue viability assay was used to evaluate BBB permeability, and neurological testing was implemented to evaluate neurological impairment during SAH. RESULTS: It was found that following SAH, Ang-1 expression decreases and Ang-2 expression increases in the cortex, hippocampus, and microvessels. The Ang-1/Ang-2 ratio decreased as quickly as 6 hours after SAH and reached its lowest 1 day after SAH. Finally, it was found that exogenous Ang-1 reduces SAH-associated BBB leakage and improves neurological function in post-SAH rats. CONCLUSIONS: Our findings suggest that the equilibrium between Ang-1 and Ang-2 is broken in a period shortly after SAH, and the treatment of exogenous Ang-1 injection alleviates neurological dysfunctions through decreasing BBB destruction.
Angiopoietin-1* ; Angiopoietin-2* ; Animals ; Blood-Brain Barrier ; Blotting, Western ; Brain ; Brain Injuries ; Capillary Permeability ; Evans Blue ; Hippocampus ; Immunohistochemistry ; Microvessels ; Permeability ; Punctures ; Rats ; Subarachnoid Hemorrhage*

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

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

PURPOSE: The aim of this study was to investigate whether plasma angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) concentrations in pregnant women with chronic hypertension are different from those of normotensive pregnant women. METHODS: This hospital-based case-control study consisted of 35 pregnant women with chronic hypertension who delivered at Seoul National University Hospital. Normotensive pregnant women (n=70) were selected as controls, matched with maternal age, gestational age at delivery and birthweight. Maternal blood was drawn at the time of admission for delivery and plasma was separated and stored. The plasma Ang-1 and Ang-2 levels were measured by ELISA. Statistical analysis was done with Mann-Whitney U test, Fisher's exact test and Spearman rank correlation test using SPSS. RESULTS: Median (range) maternal age, gestational age and birthweight were 33 years (24-42), 38 weeks (32-41), and 3.08 kg (1.13-4.01). Pregnant women with chronic hypertension had significantly higher median Ang-1 and Ang-2 levels than normotensive pregnant women (for Ang-1 : median 4,111 pg/mL, range 1,415-30,172 vs. median 2,824 pg/mL range 662-14,512, P=0.015, for Ang-2 : median 5,637 pg/mL, range 1,131-29,327 vs. median 3,345 pg/mL, range 609-24,467, P=0.039). CONCLUSION: Maternal plasma Ang-1 and Ang-2 levels were elevated in pregnant women with chronic hypertension compared with normotensive pregnant women. Further study is needed to determine if this change is a cause or a compensatory mechanism to chronic hypertension.
Angiopoietin-1* ; Angiopoietin-2* ; Case-Control Studies ; Enzyme-Linked Immunosorbent Assay ; Female ; Gestational Age ; Humans ; Hypertension* ; Maternal Age ; Plasma* ; Pregnant Women* ; Seoul

Acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the mechanisms of aGVHD are not well understood. We aim to investigate the roles of the three angiogenic factors: angiopoietin-1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) in the development of aGVHD. Twenty-one patients who underwent allo-HSCT were included in our study. The dynamic changes of Ang-1, Ang-2 and VEGF were monitored in patients before and after allo-HSCT. In vitro, endothelial cells (ECs) were treated with TNF-β in the presence or absence of Ang-1, and then the Ang-2 level in the cell culture medium and the tubule formation by ECs were evaluated. After allo-HSCT, Ang-1, Ang-2 and VEGF all exhibited significant variation, suggesting these factors might be involved in the endothelial damage in transplantation. Patients with aGVHD had lower Ang-1 level at day 7 but higher Ang-2 level at day 21 than those without aGVHD, implying that Ang-1 may play a protective role in early phase yet Ang-2 is a promotion factor to aGVHD. In vitro, TNF-β promoted the release of Ang-2 by ECs and impaired tubule formation of ECs, which were both weakened by Ang-1, suggesting that Ang-1 may play a protective role in aGVHD by influencing the secretion of Ang-2, consistent with our in vivo tests. It is concluded that monitoring changes of these factors following allo-HSCT might help to identify patients at a high risk for aGVHD.
Acute Disease ; Adolescent ; Adult ; Angiogenesis Inducing Agents ; immunology ; metabolism ; pharmacology ; Angiopoietin-1 ; genetics ; immunology ; pharmacology ; Angiopoietin-2 ; genetics ; immunology ; pharmacology ; Antineoplastic Agents ; therapeutic use ; Female ; Gene Expression Regulation, Neoplastic ; Graft vs Host Disease ; genetics ; immunology ; pathology ; Hematopoietic Stem Cell Transplantation ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; immunology ; Humans ; Leukemia, Myeloid ; genetics ; immunology ; pathology ; therapy ; Lymphoma, Non-Hodgkin ; genetics ; immunology ; pathology ; therapy ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; immunology ; pathology ; therapy ; Retrospective Studies ; Signal Transduction ; Transplantation, Homologous ; Tumor Necrosis Factor-alpha ; pharmacology ; Vascular Endothelial Growth Factor A ; genetics ; immunology