Ischemic stroke is a major public health problem worldwide. Although the circadian clock is involved in the process of ischemic stroke, the exact mechanism of the circadian clock in regulating angiogenesis after cerebral infarction remains unclear. In the present study, we determined that environmental circadian disruption (ECD) increased the stroke severity and impaired angiogenesis in the rat middle cerebral artery occlusion model, by measuring the infarct volume, neurological tests, and angiogenesis-related protein. We further report that Bmal1 plays an irreplaceable role in angiogenesis. Overexpression of Bmal1 promoted tube-forming, migration, and wound healing, and upregulated the vascular endothelial growth factor (VEGF) and Notch pathway protein levels. This promoting effect was reversed by the Notch pathway inhibitor DAPT, according to the results of angiogenesis capacity and VEGF pathway protein level. In conclusion, our study reveals the intervention of ECD in angiogenesis in ischemic stroke and further identifies the exact mechanism by which Bmal1 regulates angiogenesis through the VEGF-Notch1 pathway.
Rats ; Animals ; Vascular Endothelial Growth Factor A/pharmacology* ; Brain Ischemia/metabolism* ; Ischemic Stroke ; Signal Transduction ; ARNTL Transcription Factors/pharmacology* ; Neovascularization, Physiologic/physiology*

PURPOSE: Wound represents a major health challenge as they consume a large amount of healthcare resources to improve patient's quality of life. Many scientific studies have been conducted in search of ideal biomaterials with wound-healing activity for clinical use and collagen has been proven to be a suitable candidate biomaterial. This study intended to investigate the wound healing activity of collagen peptides derived from jellyfish following oral administration. METHODS: In this study, collagen was extracted from the jellyfish--Rhopilema esculentum using 1% pepsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fourier transform infrared (FTIR) were used to identify and determine the molecular weight of the jellyfish collagen. Collagenase II, papain and alkaline proteinase were used to breakdown jellyfish collagen into collagen peptides. Wound scratch assay (in vitro) was done to determine migration potential of human umbilical vein endothelial cells (HUVEC) covering the artificial wound created on the cell monolayer following treatment with collagen peptides. In vivo studies were conducted to determine the effects of collagen peptides on wound healing by examining wound contraction, re-epithelialization, tissue regeneration and collagen deposition on the wounded skin of mice. Confidence level (p < 0.05) was considered significant using GraphPad Prism software. RESULTS: The yield of collagen was 4.31%. The SDS-PAGE and FTIR showed that extracted collagen from jellyfish was type I. Enzymatic hydrolysis of this collagen using collagenase II produced collagen peptides (CP) and hydrolysis with alkaline proteinase/papain resulted into collagen peptides (CP). Tricine SDS-PAGE revealed that collagen peptides consisted of protein fragments with molecular weight <25 kDa. Wound scratch assay showed that there were significant effects on the scratch closure on cells treated with collagen peptides at a concentration of 6.25 μg/mL for 48 h as compared to the vehicle treated cells. Overall treatment with collagen peptide on mice with full thickness excised wounds had a positive result in wound contraction as compared with the control. Histological assessment of peptides treated mice models showed remarkable sign of re-epithelialization, tissue regeneration and increased collagen deposition. Immunohistochemistry of the skin sections showed a significant increase in β-fibroblast growth factor (β-FGF) and the transforming growth factor-β (TGF-β) expression on collagen peptides treated group. CONCLUSION Collagen peptides derived from the jellyfish-Rhopilema esculentum can accelerate the wound healing process thus could be a therapeutic potential product that may be beneficial in wound clinics in the future.
Administration, Oral ; Animals ; Collagen ; administration & dosage ; isolation & purification ; metabolism ; pharmacology ; Fibroblast Growth Factors ; metabolism ; Human Umbilical Vein Endothelial Cells ; Humans ; Male ; Regeneration ; Scyphozoa ; chemistry ; Skin ; metabolism ; Skin Physiological Phenomena ; Stimulation, Chemical ; Transforming Growth Factor beta1 ; metabolism ; Wound Healing ; drug effects

Angiogenesis is a crucial process in the development of inflammatory diseases, including cancer, psoriasis and rheumatoid arthritis. Recently, several alkaloids from Picrasma quassioides had been screened for angiogenic activity in the zebrafish model, and the results indicated that 1-methoxycarbony-β-carboline (MCC) could effectively inhibit blood vessel formation. In this study, we further confirmed that MCC can inhibit, in a concentration-dependent manner, the viability, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro, as well as the regenerative vascular outgrowth of zebrafish caudal fin in vivo. In the zebrafish xenograft assay, MCC inhibited the growth of tumor masses and the metastatic transplanted DU145 tumor cells. The proteome profile array of the MCC-treated HUVECs showed that MCC could down-regulate several angiogenesis-related self-secreted proteins, including ANG, EGF, bFGF, GRO, IGF-1, PLG and MMP-1. In addition, the expression of two key membrane receptor proteins in angiogenesis, TIE-2 and uPAR, were also down-regulated after MCC treatment. Taken together, these results shed light on the potential therapeutic application of MCC as a potent natural angiogenesis inhibitor via multiple molecular targets.
Angiogenesis Inhibitors ; chemistry ; pharmacology ; Animals ; Carbolines ; chemistry ; pharmacology ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Epidermal Growth Factor ; genetics ; metabolism ; Fibroblast Growth Factors ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; metabolism ; Humans ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Neovascularization, Physiologic ; drug effects ; Picrasma ; chemistry ; Plant Extracts ; chemistry ; pharmacology ; Receptor, TIE-2 ; genetics ; metabolism ; Zebrafish ; embryology

OBJECTIVETo investigate the effect of extracellular signal-regulated kinase (ERK) signaling pathway on the endothelial differentiation of periodontal ligament stem cells (PDLSC).

METHODSHuman PDLSC was cultured in the medium with vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b-FGF) to induce endothelial differentiation. Endothelial inducing cells was incubated with U0126, a specific p-ERK1/2 inhibitor. PDLSC from one person were randomly divided into four groups: control group, endothelial induced group, endothelial induced+DMSO group and endothelial induced+U0126 group. The protein expression of the p-EKR1/2 was analyzed by Western blotting at 0, 1, 3, 6 and 12 hours during endonthelial induction. The mRNA expressions of CD31, VE-cadherin, and VEGF were detected by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) after a 7-day induction. The proportion of CD31(+) to VE-cadherin(+) cells was identified by flow cytometry, and the ability of capillary-like tubes formation was detected by Matrigel assay after a 14-day induction. The measurement data were statistically analyzed.

RESULTSPhosphorylated ERK1/2 protein level in PDLSC was increased to 1.24±0.12 and 1.03±0.24 at 1 h and 3 h respectively, during the endothelial induction (P<0.01). The mRNA expressions of CD31 and VEGF in induced+U0126 group were decreased to 0.09±0.18 and 0.49±0.17, which were both significantly different with those in induced group (P<0.05). The proportion of CD31(+) to VE-cadherin(+) cells of induced+U0126 group were decreased to 5.22±0.85 and 3.56±0.87, which were both significantly different with those in induced group (P<0.05). In Matrigel assay, the branching points, tube number and tube length were decreased to 7.0±2.7, 33.5±6.4, and (15 951.0±758.1) pixels, which were all significantly different with those in induced group (P<0.05).

CONCLUSIONSThe endothelial differentiation of PDLSC is positively regulated by ERK signaling pathway. Inhibition of ERK1/2 phosphorylation could suppress endothelial differentiation of PDLSC.

Antigens, CD ; genetics ; metabolism ; Butadienes ; pharmacology ; Cadherins ; genetics ; metabolism ; Cell Differentiation ; Endothelial Cells ; cytology ; physiology ; Enzyme Inhibitors ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; physiology ; Fibroblast Growth Factor 2 ; pharmacology ; Humans ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Periodontal Ligament ; cytology ; metabolism ; Phosphorylation ; Platelet Endothelial Cell Adhesion Molecule-1 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Random Allocation ; Signal Transduction ; Stem Cells ; cytology ; physiology ; Time Factors ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; pharmacology

Angiogenesis, the formation of new blood vessels, is critical for tumor growth and metastasis. Notably, tumors themselves can lead to angiogenesis by inducing vascular endothelial growth factor (VEGF), which is one of the most potent angiogenic factors. Inhibition of angiogenesis is currently perceived as one of the most promising strategies for the blockage of tumor growth. In this study, we investigated the effects of Acer tegmentosum maxim water extract (ATME) on angiogenesis and its underlying signal mechanism. We studied the antiangiogenic activity of ATME by using human umbilical vein endothelial cells (HUVECs). ATME strongly inhibited VEGF-induced endothelial cell proliferation, migration, invasion, and tube formation, as well as vessel sprouting in a rat aortic ring sprouting assay. Moreover, we found that the p44/42 mitogen activated protein (MAP) kinase signaling pathway is involved in the inhibition of angiogenesis by ATME. Moreover, when we performed the in vivo matrigel plug assay, VEGF-induced angiogenesis was potently reduced when compared to that for the control group. Taken together, these results suggest that ATME exhibits potent antiangiogenic activity in vivo and in vitro and that these effects are regulated by the extracellular regulated kinase (ERK) pathway.
Acer/*metabolism ; Angiogenesis Inhibitors/*pharmacology ; Animals ; Cell Line, Tumor ; Cell Movement/drug effects ; Cell Proliferation/drug effects ; Cell Survival ; Extracellular Signal-Regulated MAP Kinases/*metabolism ; Hep G2 Cells ; Human Umbilical Vein Endothelial Cells/*drug effects ; Humans ; MAP Kinase Signaling System/drug effects ; Mice ; Mice, Inbred C57BL ; Mitogen-Activated Protein Kinase 1/metabolism ; Neoplasm Invasiveness/pathology ; Neovascularization, Pathologic/*drug therapy/prevention & control ; Nitric Oxide Synthase Type III/metabolism ; Phosphorylation/drug effects ; Plant Extracts/pharmacology ; Rats ; Rats, Sprague-Dawley ; Transcription Factors/metabolism ; Vascular Endothelial Growth Factor A/antagonists & inhibitors/metabolism

OBJECTIVE: To evaluate the regulatory effect of recombinant peroxisome-proliferatoractivatedreceptor- γ coactivator-1α (PGC-1α) on retinal neovascularization in mice. METHODS: Forty 7-day-old C57BL/6J mice were randomly divided into 2 groups including a normal injection group and a normal control group. Additional 40 7-day-old C57BL/6J mice were randomly divided into 2 groups including a model injection group and a model control group, in which the mice were induced retinal neovascularization by hypoxia. Liposome with recombinant PGC-1α protein was injected into the vitreous of mice in the normal injection group and the model injection group at postnatal day 12 (P12). No injection was performed in the control group. Fluorescein angiography was used to assess the vascular pattern. The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in cross-sections. PGC-1α levels in retina were measured by Western blot, and the vascular endothelial growth factor (VEGF) level in retina was measured by quantitative Real-time polymerase chain reaction and Western blot. RESULTS: Neovascular tuft was found in the normal injection group, but there was almost no neovascular tuft in the normal control group. Neovascular tuft and fluorescein leakage were increased in the model injection group compared with the model control group. The neovascular nuclei were increased both in the normal injection group and the model injection group compared with the control group (P<0.01). The expression of PGC-1α protein in retina was increased significantly both in the normal injection group and the model injection group as compared with the normal control group and the model control group, respectively (P<0.01). The expression of VEGF mRNA and protein in retina was increased significantly both in the normal injection group and the model injection group as compared with the normal control group and the model control group, respectively (P<0.01). CONCLUSION PGC-1α can induce the formation of retinal neovascularization in the mice.
Animals ; Blotting, Western ; Hypoxia ; Mice ; Mice, Inbred C57BL ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Recombinant Proteins ; pharmacology ; Retina ; drug effects ; metabolism ; Retinal Neovascularization ; Transcription Factors ; pharmacology ; Vascular Endothelial Growth Factor A

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with anti-inflammatory, antioxidant and antithrombotic properties and plays a protective role against atherosclerosis (AS). The purpose of the present study is to explore the effects of oxidized low density lipoprotein (ox-LDL) on the expression of PEDF in cultured human umbilical vein endothelial cells (HUVECs). HUVECs were cultured and incubated with ox-LDL at different concentrations (6.25, 12.5, 25, 50, 100 and 150 mg/L) for 24 h. Apoptosis of endothelial cells were assayed by morphological staining and flow cytometry. The intracellular reactive oxygen species (ROS) levels were measured by flow cytometry. Cell viability was assayed by MTT assay. PEDF protein and mRNA expressions in HUVECs were analyzed by Western blot and quantitative real-time PCR, respectively. The results showed that ox-LDL significantly induced apoptosis, reduced cell viability, increased intracellular ROS levels and decreased the PEDF expression in HUVECs in a concentration-dependent manner. Ox-LDL at 50 mg/L obviously decreased the PEDF protein expression compared with control group (P < 0.05), whereas 25 mg/L ox-LDL already markedly reduced the PEDF mRNA expression (P < 0.05). In conclusion, the results suggest that ox-LDL down-regulates the PEDF expression through an increased ox-LDL-induced intracellular production of ROS.
Apoptosis ; Cells, Cultured ; Down-Regulation ; Eye Proteins ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; Lipoproteins, LDL ; pharmacology ; Nerve Growth Factors ; metabolism ; Reactive Oxygen Species ; metabolism ; Serpins ; metabolism

Abnormal proliferation of vascular smooth muscle cells (VSMCs) plays an important role in several pathological processes of cardiovascular diseases. In this study, the effects of XCT790, a potent and selective inverse agonist of estrogen-related receptor alpha (ERRalpha), on rat VSMCs proliferation and related signal pathways were investigated. The proliferative activity of VSMCs was determined by CCK-8 assay. The mRNA levels of ERRalpha, PGC-1alpha, OPN and MCAD were assayed by RT-PCR. The protein levels of ERRalpha, ERK2 and p-ERK1/2 were evaluated by Western blotting. ELISA was used to assess the protein expression of VEGF. The results showed that XCT790 (5-20 micromol x L(-1)) inhibited rat VSMCs proliferation, and the expression of ERRalpha and its target genes, as well as p-ERK1/2, were also inhibited. XCT790 inhibited VSMCs proliferation in a dose-dependent manner at the dose range from 5 to 20 micromol x L(-1) and in a time-dependent manner at the dose range from 10 to 20 micromol x L(-1). These findings demonstrate that XCT790 inhibits rat VSMCs proliferation by down-regulating the gene level of ERRalpha and thus inhibiting the ERK signal pathway, suggesting that ERRalpha may be a novel potential target for therapeutic approaches to inhibit VSMCs proliferation, which plays an important role in several cardiovascular diseases.
Animals ; Cadherins ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cytoskeletal Proteins ; genetics ; metabolism ; Dose-Response Relationship, Drug ; GTPase-Activating Proteins ; genetics ; metabolism ; MAP Kinase Signaling System ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; metabolism ; Nitriles ; administration & dosage ; pharmacology ; Nuclear Proteins ; genetics ; metabolism ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; Phosphorylation ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Estrogen ; genetics ; metabolism ; Thiazoles ; administration & dosage ; pharmacology ; Transcription Factors ; genetics ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

To explore the feasibility of mesenchymal stem cells (MSCs) acting as seed cells in tissue engineering, we isolated human bone marrow MSCs and differentiated them into vascular endothelial-like cells (ELCs) in vitro. Bone marrow mononuclear cells (BMSCs) were isolated by the method of percoll density centrifugation, and seeded in Dulbecco Modified Eagle Medium supplemented with 10% fetal bovine serum. MSCs were purified through multiple adherent cultures, and differentiated into ELCs induced by endothelial cell growth medium-2 (EBM-2) medium containing vascular endothelial growth factor (VEGF), human fibroblast growth factor (hFGF), insulin like growth factors 1 (IGF-1), and human epidermal growth factor (hEGF). The relative biologic characteristics of ELCs including cell morphology and phenotype were studied by inverted microscope and flow cytometry. The induced cells were identified by immunofluorescence with CD31 and Von Willebrand factor (vWF). The results showed that the morphology of MSCs was long-spindle and vortex-like growth. After induction of differentiation, the cells were round, and similar to vascular endothelial cells (ECs). Flow cytometric analysis revealed that ELCs expressed ECs specific surface markers of CD31 and vascular endothelial cadherin (VE-cadherin), but not CD133. Immunofluorescence results also confirmed that ELCs expressed CD31 and vWF. The results suggested that ELCs possed similar cell biological characteristics with ECs. In one word, human MSCs derived from bone marrow have the potential to differentiate into ECs in vitro, and show clinical feasibility acting as ideal donor cells of vascular tissue engineering.
Antigens, CD ; metabolism ; Bone Marrow Cells ; Cadherins ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; Cells, Cultured ; Culture Media ; chemistry ; Endothelial Cells ; cytology ; Epidermal Growth Factor ; pharmacology ; Fibroblast Growth Factors ; pharmacology ; Flow Cytometry ; Humans ; Insulin-Like Growth Factor I ; pharmacology ; Mesenchymal Stromal Cells ; cytology ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Tissue Engineering ; Vascular Endothelial Growth Factor A ; pharmacology ; von Willebrand Factor ; metabolism

BACKGROUNDThe common pathological characteristics of corneal injury include inflammatory factors activation, vascular endothelial cells or inflammatory cells infiltration into lesions, corneal edema, corneal neovascularization (CNV), and scar formation. PEDF-34 is the functional fragment of pigment epithelium-derived factor (PEDF) that has anti-angiogenic and anti-inflammatory properties and contains an N-terminal 34-amino acid peptide. This study was to investigate the anti-inflammatory effects of PEDF-34 on H2O2-induced corneal injury in vitro.

METHODSAfter cultured in H2O2 (0.1 mmol/L) for 2 hours, human corneal fibroblasts (HCFs) and human umbilical vein endothelial cells (HUVECs) were treated with PEDF-34-nanoparticles (NPs) at different concentrations (0.1, 0.5, 1.0, 2.0 µg/ml) or 2.0 µg/ml control-NPs for 24 hours. The viable cells were quantified using the MTT assay. Western blotting or ELISA analysis was performed for measuring the human vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1) expression of both HCFs and HUVECs. VEGF and nuclear factor κB (NF-κB) mRNA levels of HCFs were semi-quantified by RT-PCR.

RESULTSThe survival rates of HCFs or HUVECs stimulated by H2O2 did not decrease significantly (P > 0.05) compared to those in the normal conditions. As compared to control-NP group, PEDF-34-NPs had dose-dependent inhibitive effect on HUVECs with the MTT assay, but not HCFs. Western blotting analysis showed that the VEGF and ICAM-1 levels in the HCFs and HUVECs stimulated by H2O2 were significantly higher than those in the normal conditions, which were decreased dramatically in those treated with PEDF-34-NPs. RT-PCR analysis revealed that the VEGF mRNA and NF-κB mRNA levels increased in H2O2-stimulated HCFs, while both of them decreased in PEDF-34-NP groups dose dependently.

CONCLUSIONSPEDF-34-NPs may play an important role in regulating the NF-κB pathway, inhibiting inflammatory activity. PEDF-34-NPs may be a potential new drug for treating corneal injury in the future.

Anti-Inflammatory Agents ; chemistry ; pharmacology ; Cells, Cultured ; Corneal Injuries ; chemically induced ; metabolism ; Eye Proteins ; chemistry ; Human Umbilical Vein Endothelial Cells ; drug effects ; metabolism ; Humans ; Hydrogen Peroxide ; pharmacology ; Nerve Growth Factors ; chemistry ; Peptides ; chemistry ; pharmacology ; Serpins ; chemistry