Combined radiation-wound injury (CRWI) is characterized by blood vessel damage and pro-inflammatory cytokine deficiency. Studies have identified that the direct application of leptin plays a significant role in angiogenesis and inflammation. We established a sustained and stable leptin expression system to study the mechanism. A lentivirus method was employed to explore the angiogenic potential and peripheral inflammation of irradiated human umbilical vein endothelial cells (HUVECs). Leptin was transfected into human placenta-derived mesenchymal stem cells (HPMSCs) with lentiviral vectors. HUVECs were irradiated by X-ray at a single dose of 20 Gy. Transwell migration assay was performed to assess the migration of irradiated HUVECs. Based on the Transwell systems, co-culture systems of HPMSCs and irradiated HUVECs were established. Cell proliferation was measured by cell counting kit-8 (CCK-8) assay. The secretion of pro-inflammatory cytokines (human granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-1α, IL-6, and IL-8) was detected by enzyme-linked immunosorbent assay (ELISA). The expression of pro-angiogenic factors (vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF)) mRNA was detected by real-time quantitative polymerase chain reaction (RT-qPCR) assay. Relevant molecules of the nuclear factor-κB (NF-κB) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathways were detected by western blot assay. Results showed that leptin-modified HPMSCs (HPMSCs/ leptin) exhibited better cell proliferation, migration, and angiogenic potential (expressed more VEGF and bFGF). In both the single HPMSCs/leptin and the co-culture systems of HPMSCs/leptin and irradiated HUVECs, the increased secretion of pro-inflammatory cytokines (human GM-CSF, IL-1α, and IL-6) was associated with the interaction of the NF-κB and JAK/STAT signaling pathways. We conclude that HPMSCs/leptin could promote angiogenic potential and peripheral inflammation of HUVECs after X-ray radiation.
Cell Proliferation ; Cells, Cultured ; Cytokines/biosynthesis* ; Female ; Human Umbilical Vein Endothelial Cells/radiation effects* ; Humans ; Inflammation/etiology* ; Leptin/pharmacology* ; Mesenchymal Stem Cells/physiology* ; Neovascularization, Physiologic/physiology* ; Placenta/cytology* ; Pregnancy ; STAT3 Transcription Factor/genetics* ; Transcription Factor RelA/genetics* ; X-Rays

OBJECTIVE: To assess the effect of miR-137 on the proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) induced by high glucose and its mechanism. METHODS: HUVECs cells were divided into low-glucose group (5.5 mmol/L glucose-treated cells), high-glucose group (33.36 mmol/L glucose-treated cells), anti-NC group (cells treated with 33.36 mmol/L glucose after anti-NC transfection) and anti-miR-137 group (cells treated with 33.36 mmol/L glucose after anti-miR-137 transfection). After 48 hours, qRT-PCR was used to determine the expression of miR-137. CCK-8 assay and flow cytometry were used to detect cell proliferation and apoptosis rate, respectively. The targeting relationship between miR-137 and AKT2 was validated by dual fluorescence reporter gene detection system and AKT2 protein expression after overexpression or inhibition of miR-137. RESULTS: High glucose could significantly up-regulate the expression of miR-137 in HUVECs cells, and the expression of miR-137 in HUVECs cells transfected with miR-137 inhibitor was significantly decreased (P<0.05). High glucose can significantly inhibit HUVECs cell proliferation and induce apoptosis, while inhibition of miR-137 expression can weaken the effect of high glucose on HUVECs cell proliferation inhibition and apoptosis promotion (P<0.05). Inhibiting AKT2 expression could weaken the inhibitory effect of miR-137 inhibitor on HUVECs cell proliferation and apoptosis (P<0.05). CONCLUSION Inhibiting the expression of miR-137 gene can attenuate the proliferation inhibition and apoptosis promotion of HUVECs induced by high glucose, and the mechanism is related to activating the expression of AKT2.
Apoptosis ; Cell Proliferation ; Cells, Cultured ; Glucose ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; MicroRNAs ; genetics ; Proto-Oncogene Proteins c-akt ; genetics

This study aimed to investigate the effect and mechanism of ophiopogonin D (OP-D) on Ang Ⅱ-induced HUVECs apoptosis, in order to provide a reliable basis for the safety and efficacy of traditional Chinese medicines. The effect of Ang Ⅱ on survival and total proteins content of HUVECs were measured by MTT and Western blotting. The effect of OP-D on Ang Ⅱ-induced lactate dehydrogenase (LDH) release rate in HUVECs was measured by enzyme standard instrument. The effects of OP-D and 11,12-EET on phosphorylation of JNK/c-Jun induced by Ang Ⅱ were measured by Western blot and RT-PCR with the help of JNK specific inhibitor SP600125 and CYP450 isozymes selective inhibitor 6-(2-propargyloxyphenyl) hexanoic acid (PPOH). The cell apoptosis was assayed by flow cytometry. According to the results, different doses of Ang Ⅱ had no significant effect on cell survival; treatment with Ang Ⅱ at 1×10⁻⁶ mol·L⁻¹ could increase the release of LDH (<0.001), improve the JNK and c-Jun phosphorylation levels(<0.01, <0.001), increase the expression of caspase-3(<0.01), and promote the apoptosis of HUVECs(<0.001). The phosphorylation of JNK and c-Jun could be inhibited by the pre-treatment with SP600125, 11,12-EET and OP-D. Pre-treatment with OP-D could significantly reduce the release of LDH induced by Ang Ⅱ stimulation, decrease the expression of caspase-3, and diminish the apoptosis of cells. The protective effect of OP-D was suppressed, when being pretreated with PPOH. The experimental results showed that the apoptosis of HUVECs induced by Ang Ⅱ may be associated with JNK/c-Jun signaling pathway. OP-D-mediated CYP2J2 expression increased 11,12-EET levels, and could remarkably resist Ang Ⅱ-induced injury and apoptosis of cells, which is associated with the maintenance of endothelium homeostasis.
Angiotensin II ; Apoptosis ; Arachidonic Acids ; metabolism ; Cells, Cultured ; Cytochrome P-450 Enzyme System ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; Phosphorylation ; Saponins ; pharmacology ; Signal Transduction ; Spirostans ; pharmacology

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

PURPOSE: Apoptosis of vascular endothelial cells is a type of endothelial damage that is associated with the pathogenesis of cardiovascular diseases such as atherosclerosis. Heterotrimeric GTP-binding proteins (G proteins), including the alpha 12 subunit of G protein (Galpha12), have been found to modulate cellular proliferation, differentiation, and apoptosis of numerous cell types. However, the role of Galpha12 in the regulation of apoptosis of vascular cells has not been elucidated. We investigated the role of Galpha12 in serum withdrawal-induced apoptosis of human umbilical vein endothelial cells (HUVECs) and its underlying mechanisms. MATERIALS AND METHODS: HUVECs were transfected with Galpha12 small-interfering RNA (siRNA) to knockdown the endogenous Galpha12 expression and were serum-deprived for 6 h to induce apoptosis. The apoptosis of HUVECs were assessed by Western blotting and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expressions of microRNAs were analyzed by quantitative real-time PCR. RESULTS: Knockdown of Galpha12 with siRNA augmented the serum withdrawal-induced apoptosis of HUVECs and markedly repressed the expression of microRNA-155 (miR-155). Serum withdrawal-induced apoptosis of HUVECs was inhibited by the overexpression of miR-155 and increased significantly due to the inhibition of miR-155. Notably, the elevation of miR-155 expression prevented increased apoptosis of Galpha12-deficient HUVECs. CONCLUSION: From these results, we conclude that Galpha12 protects HUVECs from serum withdrawal-induced apoptosis by retaining miR-155 expression. This suggests that Galpha12 might play a protective role in vascular endothelial cells by regulating the expression of microRNAs.
*Apoptosis ; Atherosclerosis/*blood/genetics/immunology ; Cell Proliferation ; Endothelial Cells/*metabolism ; GTP-Binding Protein alpha Subunits, G12-G13/*genetics ; Gene Expression Profiling ; Gene Expression Regulation ; Human Umbilical Vein Endothelial Cells/cytology ; Humans ; MicroRNAs/*metabolism ; Protective Agents ; *RNA, Small Interfering ; Real-Time Polymerase Chain Reaction ; *Transfection

OBJECTIVETo investigate the role of p38MAPK signaling pathway in the mechanism by which glucagon-like peptide-1 (GLP-1) inhibits endothelial cell damage induced by AGEs.

METHODSHuman umbilical vein endothelial cells were divided into control group, AGEs group, GLP-1 group, AGEs+GLP-1 group, AGEs+inhibitor group, and AGEs+GLP-1+inhibitor group. The expressions of p-p38MAPK/p38MAPK and p-eNOS/eNOS protein were examined by Western blotting, and the cell apoptosis rates were tested by flow cytometry.

RESULTSCompared with the control group, AGEs significantly enhanced the expression of p-p38 MAPK protein (P=0.001) while GLP-1 significantly inhibited its expression (P<0.001). AGEs significantly inhibited the expression of p-eNOS protein (P=0.007), which was enhanced by GLP-1 and p38 MAPK inhibitor (SB203580) (P=0.004). Both SB203580 and GLP-1 treatment decreased the apoptosis rate of AGEs-treated cells (P<0.001).

CONCLUSIONGLP-1 can protect human umbilical vein endothelial cells against AGEs-induced apoptosis partially by inhibiting the phosphorylation of p38MAPK protein and promoting the expression of p-eNOS protein.

Apoptosis ; Cells, Cultured ; Glucagon-Like Peptide 1 ; pharmacology ; Glycation End Products, Advanced ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; Humans ; Nitric Oxide Synthase Type III ; metabolism ; Phosphorylation ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases ; metabolism

Axl encodes the tyrosine-protein kinase receptor, participating in the proliferation and migration of many cells. This study examined the role of Axl in functions of endothelial progenitor cells (EPCs). Axl was detected by RT-PCR and Western blotting in both placentas and EPCs from normal pregnancy and preeclampsia patients. The Axl inhibitor, BMS777-607, was used to inhibit the Axl signalling pathway in EPCs. Cell proliferation, differentiation, migration and adhesion were measured by CCK-8 assay, cell differentiation assay, Transwell assay, and cell adhesion assay, respectively. Results showed the expression levels of Axl mRNA and protein were significantly higher in both placentas and EPCs from preeclampsia patients than from normal pregnancy (P<0.05). After treatment with BMS777-607, proliferation, differentiation, migration and adhesion capability of EPCs were all significantly decreased. Our study suggests Axl may play a role in the function of EPCs, thereby involving in the pathogenesis of preeclampsia.
Adult ; Aminopyridines ; pharmacology ; Blood Pressure ; Case-Control Studies ; Cell Adhesion ; drug effects ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Female ; Fetal Blood ; cytology ; enzymology ; Gene Expression Regulation ; Gestational Age ; Human Umbilical Vein Endothelial Cells ; drug effects ; enzymology ; pathology ; Humans ; Placenta ; metabolism ; physiopathology ; Pre-Eclampsia ; blood ; genetics ; physiopathology ; Pregnancy ; Primary Cell Culture ; Protein Kinase Inhibitors ; pharmacology ; Proto-Oncogene Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Pyridones ; pharmacology ; RNA, Messenger ; antagonists & inhibitors ; genetics ; metabolism ; Receptor Protein-Tyrosine Kinases ; antagonists & inhibitors ; genetics ; metabolism ; Stem Cells ; drug effects ; enzymology ; pathology

Progressive tumor growth is dependent on angiogenesis. The mechanisms by which endothelial cells (ECs) are incorporated to develop new blood vessels are not well understood. Recent studies reveal that the ezrin radixin moesin (ERM) family members are key regulators of cellular activities such as adhesion, morphogenetic change, and migration. We hypothesized that ezrin, one of the ERM family members, may play important roles in ECs organization during angiogenesis, and new vessels formation in preexisting tissues. To test this hypothesis, in this study, we investigated the effects of ezrin gene silencing on the migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. HUVECs were transfected with plasmids with ezrin-targeting short hairpin RNA by using the lipofectamine-2000 system. Wound assay in vitro and three-dimensional culture were used to detect the migration and angiogenesis capacity of HUVECs. The morphological changes of transfected cells were observed by confocal and phase contrast microscopy. Our results demonstrated that the decreased expression of ezrin in HUVECs significantly induced the morphogenetic changes and cytoskeletal reorganization of the transfected cells, and also reduced cell migration and angiogenesis capacity in vitro, suggesting that ezrin play an important role in the process of HUVECs migration and angiogenesis.
Cell Movement ; genetics ; Cytoskeletal Proteins ; genetics ; metabolism ; Cytoskeleton ; metabolism ; Human Umbilical Vein Endothelial Cells ; cytology ; metabolism ; physiology ; Humans ; Neovascularization, Physiologic ; genetics

OBJECTIVETo investigate the optimal starvation conditions of human umbilical vein endothelial cells (HUVECs) and establish a highly efficient and stable method for separating HUVECs.

METHODSHUVECs harvested from human umbilical cords by digestion with 0.1% collagenase II for 15 min were cultured in endothelial culture medium (ECM) containing 5% fetal bovine serum (FBS), 1% endothelial cell growth factor (ECGS) and 1% penicillin/streptomycin solution(P/S) at 37 degrees celsius; in 5% CO2. The cells were observed for cell morphology under an inverted microscope and identified with immunofluorescence assay. The purity of HUVECs was detected using flow cytometry (FCM). The cell cycles of HUVECs cultured in the presence of 0, 0.1%, 0.5%, and 1% FBS for 0, 6, 12, 18, and 24 h were analyzed with flow cytometry.

RESULTSs The purity of HUVECs harvested by digestion with 0.1% collagenase II reached 99.67%. The primary HUVECs showed a cobblestone or volute appearance in vitro. Immunocytochemistry showed that HUVECs highly expressed VIII-related antigen. Cell culture in the presence of different concentrations of FBS for 6 h resulted in 70% G0/G1 phase cells, which increased to 80%-90% at 12 h of cell culture, and further to around 95% at 18 and 24 h.

CONCLUSIONDigestion with 0.1% collagenase II can obtain high-purity primary HUVECs. Culturing HUVECs in serum-free medium for 12 h can result in a high purity (over 80%) of G0/G1 phase cells.

Cell Culture Techniques ; Cell Cycle ; Cells, Cultured ; Culture Media ; chemistry ; Flow Cytometry ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Matrix Metalloproteinase 8 ; chemistry ; Serum

OBJECTIVETo investigate the effect of prostaglandins E2 (PGE2) in enhancing vascular endothelial growth factor (VEGF) expression in a rat macrophage cell line and the effect of the media from PGE2-inuced rat macrophages on angiogenetic ability of human umbilical vein endothelial cells (HUVECs) in vitro.

METHODSWestern blotting and qPCR were employed to investigate the expressions of VEGF protein and mRNAs in rat macrophage cell line NR8383 stimulated by PGE2 in the presence or absence of EP2 receptor inhibitor (AH6809) and EP4 receptor inhibitor (AH23848). Conditioned supernatants were obtained from different NR8383 subsets to stimulate HUVECs, and the tube formation ability and migration of the HUVECs were assessed with Transwell assay.

RESULTSPGE2 stimulation significantly enhanced the expression of VEGF protein and mRNAs in NR8383 cells in a dose-dependent manner. The supernatants from NR8383 cells stimulated by PGE2 significantly enhanced tube formation ability of HUVECs (P<0.05) and promoted the cell migration. Such effects of PGE2 were blocked by the application of AH6809 and AH23848.

CONCLUSIONPGE2 can dose-dependently increase VEGF expression in NR8383 cells, and the supernatants derived from PGE2-stimulated NR8383 cells can induce HUVEC migration and accelerate the growth of tube like structures. PGE2 are essential to corpus luteum formation by stimulating macrophages to induce angiogenesis through EP2/EP4.

Animals ; Cell Line ; Cell Movement ; Cells, Cultured ; Culture Media, Conditioned ; pharmacology ; Dinoprostone ; pharmacology ; Human Umbilical Vein Endothelial Cells ; cytology ; drug effects ; Humans ; Macrophages ; chemistry ; Neovascularization, Pathologic ; RNA, Messenger ; Rats ; Receptors, Prostaglandin E, EP2 Subtype ; metabolism ; Receptors, Prostaglandin E, EP4 Subtype ; metabolism ; Vascular Endothelial Growth Factor A ; Xanthones ; pharmacology