Objective: To explore the effects of P311 on the angiogenesis ability of human microvascular endothelial cell 1 (HMEC-1) in vitro and the potential molecular mechanism. Methods: The experimental research method was used. HMEC-1 was collected and divided into P311 adenovirus group and empty adenovirus group according to the random number table (the same grouping method below), which were transfected correspondingly for 48 h. The cell proliferation activity was detected using the cell counting kit 8 on 1, 3, and 5 days of culture. The residual scratch area of cells at post scratch hour 6 and 11 was detected by scratch test, and the percentage of the residual scratch area was calculated. The blood vessel formation of cells at 8 h of culture was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. The protein expressions of vascular endothelial growth factor receptor 2 (VEGFR2), phosphorylated VEGFR2 (p-VEGFR2), extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated ERK1/2 (p-ERK1/2) in cells were detected by Western blotting. HMEC-1 was collected and divided into P311 adenovirus+small interfering RNA (siRNA) negative control group, empty adenovirus+siRNA negative control group, P311 adenovirus+siRNA-VEGFR2 group, and empty adenovirus+siRNA-VEGFG2 group, which were treated correspondingly. The protein expressions of VEGFR2, p-VEGFR2, ERK1/2, and p-ERK1/2 in cells were detected by Western blotting at 24 h of transfection. The blood vessel formation of cells at 24 h of transfection was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. HMEC-1 was collected and divided into P311 adenovirus+dimethylsulfoxide (DMSO) group, empty adenovirus+DMSO group, P311 adenovirus+ERK1/2 inhibitor group, and empty adenovirus+ERK1/2 inhibitor group, which were treated correspondingly. The protein expressions of ERK1/2 and p-ERK1/2 in cells were detected by Western blotting at 2 h of treatment. The blood vessel formation of cells at 2 h of treatment was observed by angiogenesis experiment in vitro, and the number of nodes and total length of the tubular structure were measured. The sample number at each time point in each group was 6. Data were statistically analyzed with independent sample t test, analysis of variance for repeated measurement, one-way analysis of variance, and least significant difference test. Results: Compared with that of empty adenovirus group, the proliferation activity of cells in P311 adenovirus group did not show significant difference on 1, 3, and 5 days of culture (with t values of -0.23, -1.30, and -1.52, respectively, P>0.05). The residual scratch area percentages of cells in P311 adenovirus group were significantly reduced at post scratch hour 6 and 11 compared with those of empty adenovirus group (with t values of -2.47 and -2.62, respectively, P<0.05). At 8 h of culture, compared with those of empty adenovirus group, the number of nodes and total length of the tubular structure of cells in P311 adenovirus group were significantly increased (with t values of 4.49 and 4.78, respectively, P<0.01). At 48 h of transfection, compared with those of empty adenovirus group, the protein expressions of VEGFR2 and ERK1/2 of cells in P311 adenovirus group showed no obvious changes (P>0.05), and the protein expressions of p-VEGFR2 and p-ERK1/2 of cells in P311 adenovirus group were significantly increased (with t values of 17.27 and 16.08, P<0.01). At 24 h of transfection, the protein expressions of p-VEGFR2 and p-ERK1/2 of cells in P311 adenovirus+siRNA negative control group were significantly higher than those in empty adenovirus+siRNA negative control group (P<0.01). The protein expressions of VEGFR2, p-VEGFR2, and p-ERK1/2 of cells in P311 adenovirus+siRNA negative control group were significantly higher than those in P311 adenovirus+siRNA-VEGFR2 group (P<0.01). The protein expressions of VEGFR2 and p-ERK1/2 of cells in empty adenovirus+siRNA negative control group were significantly higher than those in empty adenovirus+siRNA-VEGFR2 group (P<0.05 or P<0.01). At 24 h of transfection, the number of nodes of the tubular structure in cells of P311 adenovirus+siRNA negative control group was 720±62, which was significantly more than 428±38 in empty adenovirus+siRNA negative control group and 364±57 in P311 adenovirus+siRNA-VEGFR2 group (with P values both <0.01). The total length of the tubular structure of cells in P311 adenovirus+siRNA negative control group was (21 241±1 139) μm, which was significantly longer than (17 005±1 156) μm in empty adenovirus+siRNA negative control group and (13 494±2 465) μm in P311 adenovirus+siRNA-VEGFR2 group (with P values both <0.01). The number of nodes of the tubular structure in cells of empty adenovirus+siRNA negative control group was significantly more than 310±75 in empty adenovirus+siRNA-VEGFR2 group (P<0.01), and the total length of the tubular structure of cells in empty adenovirus+siRNA negative control group was significantly longer than (11 600±2 776) μm in empty adenovirus+siRNA-VEGFR2 group (P<0.01). At 2 h of treatment, the protein expression of p-ERK1/2 of cells in P311 adenovirus+DMSO group was significantly higher than that in empty adenovirus+DMSO group and P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01), and the protein expression of p-ERK1/2 of cells in empty adenovirus+DMSO group was significantly higher than that in empty adenovirus+ERK1/2 inhibitor group (P<0.05). At 2 h of treatment, the number of nodes of the tubular structure in cells of P311 adenovirus+DMSO group was 726±72, which was significantly more than 421±39 in empty adenovirus+DMSO group and 365±41 in P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01). The total length of the tubular structure of cells in P311 adenovirus+DMSO group was (20 318±1 433) μm, which was significantly longer than (16 846±1 464) μm in empty adenovirus+DMSO group and (15 114±1 950) μm in P311 adenovirus+ERK1/2 inhibitor group (with P values both <0.01). The number of nodes of the tubular structure in cells of empty adenovirus+DMSO group was significantly more than 317±67 in empty adenovirus+ERK1/2 inhibitor group (P<0.01), and the total length of the tubular structure of cells in empty adenovirus+DMSO group was significantly longer than (13 188±2 306) μm in empty adenovirus+ERK1/2 inhibitor group (P<0.01). Conclusions: P311 can enhance the angiogenesis ability of HMEC-1 by activating the VEGFR2/ERK1/2 signaling pathway.
Adenoviridae/genetics* ; Cell Line ; Endothelial Cells ; Endothelium, Vascular ; Humans ; Neovascularization, Physiologic ; Nerve Tissue Proteins ; Oncogene Proteins ; Signal Transduction ; Transfection ; Vascular Endothelial Growth Factor A

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

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

Alkanesulfonic Acids ; toxicity ; Animals ; Female ; Fluorocarbons ; toxicity ; Humans ; Male ; Mice ; Neovascularization, Physiologic ; drug effects ; Pedigree ; Placenta ; blood supply ; drug effects ; metabolism ; Pregnancy ; Prenatal Exposure Delayed Effects ; genetics ; metabolism ; physiopathology ; RNA, Long Noncoding ; genetics ; metabolism

Background: Microparticles (MPs) are small extracellular plasma membrane particles shed by activated and apoptotic cells, which are involved in the development of atherosclerosis. Our previous study found that microRNA (miR)-19b encapsulated within endothelial MPs (EMPs) may contribute to the upregulation of circulating miR-19b in unstable angina patients. Hypoxia is involved in atherosclerosis as a critical pathological stimulus. However, it still remains unclear whether the increase of miR-19b levels in EMPs is related to hypoxia and if the effect of miR-19b - wrapped within EMPs - stimulates hypoxia on vascular endothelial cells. This study aimed to explore the changes of miR-19b in EMPs induced by hypoxia as well as their effects on endothelial cells. Methods: Human umbilical vein endothelial cells (HUVECs) were cultured in vitro and arranged to harvest EMPs in two parts: the first part consisted of EMP and EMP and the second part included EMP, EMP, and EMP. Cell migration was detected by scratch migration and transwell chamber migration. Angiogenesis was assessed by tube formation assays. Furthermore, we predicted the target gene of miR-19b by bioinformatics analysis, and luciferase assay was used to verify the targeted gene of miR-19b. Data were analyzed by one-way analysis of variance. Student's t-test was used when two groups were compared. Results: Compared with EMP- and EMP-inhibited migration of cells by scratch migration assay (80.77 ± 1.10 vs. 28.37 ± 1.40, P < 0. 001) and transwell chamber migration assay (83.00 ± 3.46 vs. 235.00 ± 16.52, P < 0.01), the number of tube formations was markedly reduced by 70% in the EMP group (P < 0.001) in vitro analysis of HUVECs. Meanwhile, a strong inhibition of migration and tube formation of HUVECs in the presence of miR-19b-enriched EMP was observed. This effect might be due to the delivery of miR-19b in EMPs. Transforming growth factor-β2 (TGFβ2) was predicted to be one of the target genes of miR-19b, and we further confirmed that TGFβ2 was a direct target gene of miR-19b using the luciferase assay. The expression of TGFβ2 in HUVECs was inhibited by treatment with EMP and EMP. Conclusions MiR-19b in EMPs induced by hypoxia could reduce endothelial cell migration and angiogenesis by downregulating TGFβ2 expression, which may have inhibited the progression of atherosclerosis.
Cell Hypoxia ; genetics ; physiology ; Cell Movement ; genetics ; physiology ; Endothelial Cells ; metabolism ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; MicroRNAs ; genetics ; metabolism ; Neovascularization, Physiologic ; genetics ; physiology ; Transforming Growth Factor beta2 ; genetics ; metabolism

The suitable experimental animal model is important in research of pathogenesis and therapeutic strategies of diabetic foot ulcer, and the murine model is the most commonly used one at present. It can be divided into two types: the animal model simulating pathological conditions and the model simulating clinical symptoms. This article reviews the current research progress on the mechanisms of diabetic ulcer pathogenesis, and relevant treatment strategies, including the inhibition of matrix metalloproteinases (MMPs) expression, promotion of angiogenesis and anti-inflammatory therapy.
Animals ; Anti-Inflammatory Agents ; therapeutic use ; Diabetic Foot ; etiology ; genetics ; therapy ; Disease Models, Animal ; Humans ; Matrix Metalloproteinase Inhibitors ; therapeutic use ; Matrix Metalloproteinases ; genetics ; metabolism ; Mice ; Neovascularization, Physiologic ; physiology

This study aimed to investigate the expression of the natriuretic peptide precursor C coding gene nppc and its role in angiogenesis during embryonic period of the zebrafish. Whole mount in situ hybridization was performed to detect the expression pattern of nppc. nppc specific morpholino and nppc mRNA were injected respectively into the one-cell stage embryo to specifically knock-down and rescue the expression of nppc in Tg (flk1:GFP) and Tg (fli1a:nGFP) transgenic lines. The morphology and endothelial cell number of intersegmental vessel (ISV) were analyzed after imaging using the laser scanning confocal microscope. The results revealed that nppc was expressed in the brain, heart and vasculature of zebrafish larvae at 24 and 48 hours post-fertilization (hpf). Knock-down of nppc affected the development of ISV. Endothelial cell number was reduced after the knock-down of nppc. These results suggest that nppc controls zebrafish angiogenesis by affecting the endothelial cell proliferation and migration.
Animals ; Animals, Genetically Modified ; Cell Movement ; Cell Proliferation ; Endothelial Cells ; physiology ; Gene Knockdown Techniques ; Heart ; physiology ; Larva ; Natriuretic Peptides ; genetics ; physiology ; Neovascularization, Physiologic ; RNA, Messenger ; Zebrafish ; genetics ; physiology ; Zebrafish Proteins ; genetics ; physiology

BACKGROUNDRecombinant human-erythropoietin (rh-EPO) has therapeutic efficacy for premature infants with brain damage during the active rehabilitation and anti-inflammation. In the present study, we found that the rh-EPO was related to the promotion of neovascularization. Our aim was to investigate whether rh-EPO augments neovascularization in the neonatal rat model of premature brain damage through the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway.

METHODSPostnatal day 5 (PD5), rats underwent permanent ligation of the right common carotid artery and were exposed to hypoxia for 2 h. All the rat pups were randomized into five groups as follows: (1) control group; (2) hypoxia-ischemic (HI) group; (3) HI + LY294002 group; (4) HI + rh-EPO group; and (5) HI + rh-EPO + LY294002 group. The phospho-Akt protein was tested 90 min after the whole operation, and CD34, vascular endothelial growth factor receptor 2 (VEGFR2), and vascular endothelial growth factor (VEGF) were also tested 2 days after the whole operation.

RESULTSIn the hypoxic and ischemic zone of the premature rat brain, the rh-EPO induced CD34+ cells to immigrate to the HI brain zone (P < 0.05) and also upregulated the VEGFR2 protein expression (P < 0.05) and VEGF mRNA level (P < 0.05) through the PI3K/Akt (P < 0.05) signaling pathway when compared with other groups.

CONCLUSIONSThe rh-EPO treatment augments neovascularization responses in the neonatal rat model of premature brain damage through the PI3K/Akt signaling pathway. Besides, the endogenous EPO may exist in the HI zone of rat brain and also has neovascularization function through the PI3K/Akt signaling pathway.

Animals ; Animals, Newborn ; Antigens, CD34 ; metabolism ; Brain ; drug effects ; metabolism ; pathology ; Disease Models, Animal ; Erythropoietin ; genetics ; metabolism ; therapeutic use ; Female ; Humans ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; Neovascularization, Physiologic ; drug effects ; Phosphatidylinositol 3-Kinase ; metabolism ; Pregnancy ; Proto-Oncogene Proteins c-akt ; metabolism ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; genetics ; metabolism ; therapeutic use ; Signal Transduction ; drug effects ; Vascular Endothelial Growth Factor A ; genetics ; Vascular Endothelial Growth Factor Receptor-2 ; metabolism

OBJECTIVETo investigate the microRNAs (miRNAs) expression profile of acute myocardial infarction (AMI) rats and the regulating effects of Huoxue Anxin Recipe (, HAR) on angiogenesis-related miRNAs and genes.

METHODSForty-five Wistar rats were randomly assigned to 3 groups according to a random number table: sham, AMI, and AMI+HAR groups (15 in each group). AMI rats were established by ligation of the left descending coronary artery. HAR was intragastrically administered to rats of the AMI+HAR group for successive 21 days since modeling, meanwhile the same volume of 0.9% normal saline was administered to rats of the sham and AMI groups. Doppler echocardiography was used for noninvasive cardiac function test. Hematoxylin and eosin staining was used to observe the histopathological change. miRNAs expression profile was detected by quantitative realtime polymerase chain reaction (qRT-PCR). The mRNA and protein expressions of vascular endothelial growth factor (VEGF), and a target gene of miR-210 was further detected by qRT-PCR and Western blot, respectively. The microvessels density of myocardium was evaluated by CD31 immunostaining.

RESULTSCompared with the sham group, ejection fraction (EF) and fractional shortening (FS) values were decreased significantly in the AMI group (P<0.01), while the infarction area and the interstitial collagen deposition were increased obviously. As for the AMI+HAR group, EF and FS values were increased significantly (P<0.05 vs. AMI group), and the infarction area was reduced and the interstitial collagen deposition were alleviated significantly. Total of 23 miRNAs in the AMI group expressed differently by at least 1.5 folds compared with those in the sham group; 5 miRNAs in the AMI+HAR group expressed differently by at least 1.5 folds compared with those in the AMI group. Among them, miR-210 was low in the AMI group and high in the AMI+HAR group. The relative mRNA and protein expressions of VEGF were decreased significantly in the AMI group (P<0.05 vs. sham group), and increased significantly in the AMI+HAR group (P<0.01 vs. AMI group). CD31 expression area and optical intensity were decreased significantly in the AMI group (P<0.05 vs. sham group), and increased significantly in the AMI+HAR group (P<0.01 vs. AMI group).

CONCLUSIONSHAR could reduce the infarction area, alleviate the interstitial fibrosis and improve the cardiac function of AMI rats. Those effects could be related to promoting myocardium angiogenesis of HAR by up-regulating miR-210 and VEGF.

Animals ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Heart Function Tests ; Male ; MicroRNAs ; genetics ; metabolism ; Microvessels ; pathology ; Myocardial Infarction ; drug therapy ; genetics ; physiopathology ; Myocardium ; pathology ; Neovascularization, Physiologic ; drug effects ; genetics ; RNA, Messenger ; genetics ; metabolism ; Rats, Wistar ; Up-Regulation ; drug effects ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVETo evaluate the effect of Xuefu Zhuyu Capsule ()-containing serum (XFZY-CS) on EphB4/ephrinB2 and its reverse signal in human microvascular endothelial cell-1 (HMEC-1).

METHODSXFZY-CS and the blank control serum were collected. HMEC-1 cells were randomly assigned to 6 groups including the concentration 1.25%, 2.5%, and 5% XFZY-CS groups and their blank serum control ones. The angiogenesis effect of XFZY-CS was tested with an in vitro tube formation assay and the best condition of pro-angiogenesis was determined. The effect of XFZY-CS on EphB4/ephrinB2 and the reverse signal were determined by Western blot and real-time quantitative polymerase chain reaction, respectively; we also confifirmed the results through activating and inhibiting the reverse signal by EphB4/fc and pyrophosphatase/ phosphodiesterase2 (PP2).

RESULTSXFZY-CS promoted angiogenesis at the concentration of 2.5% corresponding serum after being cultured for 48 h, while inhibited angiogenesis at the concentration of 5% after culturing for 48 and 72 h. Under the 2.5% serum concentration, XFZY up-regulated the expression of EphB4-mRNA at 12 h (P<0.05), and down-regulates its expression at 24 h (P<0.01). Protein expression of EphB4 was apparently up-regulated at 12 h and down-regulated at 24 h. The phosphorylation of ephrinB2 increased at 9 h (P<0.05). In addition, 2.5% XFZY-CS played a similar role as the reverse signaling activator EphB4/Fc ranging from 0.5 to 5 μg/mL (P>0.05). XFZY-CS also reduced the inhibitive effect of PP2 in limited periods.

CONCLUSIONSEphB4/ephrinB2 was the upstream signal in the process of angiogenesis and its reverse signaling was responsible for XFZY's effect on promoting angiogenesis.

Adult ; Capsules ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; drug effects ; metabolism ; Ephrin-B2 ; metabolism ; Gene Expression Regulation ; drug effects ; Humans ; Male ; Microvessels ; pathology ; Middle Aged ; Neovascularization, Physiologic ; drug effects ; genetics ; Phosphoric Diester Hydrolases ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Receptor, EphB4 ; genetics ; metabolism ; Serum ; metabolism ; Time Factors ; Young Adult