OBJECTIVETo compare the angiogenesis behaviors of vascular endothelial growth factor (VEGF) and Chinese medicine Xuefu Zhuyu Decoction (, XZD) treatments.

METHODSHuman microvascular endothelial cells (HMEC-1) were treated with various concentrations of either XZD-containing serum (XZD-CS) or VEGF for 24, 48, and 72 h, respectively. Cell viability, proliferation, migration, adhesion, and in vitro tube formation assays were used to assess their angiogenic effects.

RESULTSVEGF promoted all cellular phases involved in angiogenesis including cell viability, proliferation, migration, adhesion, and tube formation (<0.05 or <0.01). Unlike the continuous promotion effects of VEGF at the above stages, XZD inhibited cell viability and proliferation (<0.05 or <0.01) and only promoted tube formation in the early phase of angiogenesis (<0.01).

CONCLUSIONSThese two medications promote different angiogenesis behaviors, which might be an important reason for their distinct therapeutic profile in clinical usage.

Cell Adhesion ; drug effects ; Cell Cycle ; drug effects ; Cell Line ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; drug effects ; metabolism ; Humans ; Microvessels ; cytology ; Neovascularization, Physiologic ; drug effects ; Vascular Endothelial Growth Factor A ; pharmacology

OBJECTIVETo investigate lipopolysaccharide (LPS)-induced changes of cytoskeletal filamentous actin in primary isolated pulmonary microvascular endothelial cells (PMVECs) from wild-type and RAGE knock-out mouse.

METHODSThe lungs of wild-type and RAGE knock-out mice were digested with collagenase type I to obtain endothelial cells purified by anti-CD31-coupled magnetic beads. The PMVEC identified by factor VIII labeling were stimulated with LPS at different concentrations and the changes of filamentous actin were observed by confocal microscopy.

RESULTSThe cultured primary cells showed typical endothelial cell phenotype as examined with factor VIII labeling. LPS stimulation caused rearrangement of the cytoskeletal filament F-actin in wild-type mouse PMVECs with stress fiber formation, but such changes were not obvious in RAGE knock-out mouse PMVECs.

CONCLUSIONMouse PMVECs of a high purity can be obtained by immune magnetic beads. RAGE is involved in LPS-induced destruction of mouse PMVEC cytoskeletons.

Actins ; metabolism ; Animals ; Cells, Cultured ; Cytoskeleton ; metabolism ; Endothelial Cells ; cytology ; Lipopolysaccharides ; Lung ; cytology ; Mice ; Mice, Knockout ; Microvessels ; cytology ; Phenotype ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; genetics ; metabolism

BACKGROUND: Angiogenesis is important for the proliferation and survival of multiple myeloma (MM) cells. Bone marrow (BM) microvessel density (MVD) is a useful marker of angiogenesis and is determined by immunohistochemical staining with anti-CD34 antibody. This study investigated the prognostic impact of MVD and demonstrated the relationship between MVD and previously mentioned prognostic factors in patients with MM. METHODS: The study included 107 patients with MM. MVD was assessed at initial diagnosis in a blinded manner by two hematopathologists who examined three CD34-positive hot spots per patient and counted the number of vessels in BM samples. Patients were divided into three groups according to MVD tertiles. Cumulative progression-free survival (PFS) and overall survival (OS) curves, calculated by using Kaplan-Meier method, were compared among the three groups. Prognostic impact of MVD was assessed by calculating Cox proportional hazard ratio (HR). RESULTS: Median MVDs in the three groups were 16.8, 33.9, and 54.7. MVDs were correlated with other prognostic factors, including beta2-microglobulin concentration, plasma cell percentage in the BM, and cancer stage according to the International Staging System. Multivariate Cox regression analysis showed that high MVD was an independent predictor of PFS (HR=2.57; 95% confidence interval, 1.22-5.42; P=0.013). PFS was significantly lower in the high MVD group than in the low MVD group (P=0.025). However, no difference was observed in the OS (P=0.428). CONCLUSIONS: Increased BM MVD is a marker of poor prognosis in patients newly diagnosed with MM. BM MVD should be assessed at the initial diagnosis of MM.
Aged ; Antigens, CD34/metabolism ; Bone Marrow/metabolism/*pathology ; Disease-Free Survival ; Female ; Humans ; Immunohistochemistry ; Kaplan-Meier Estimate ; Male ; Microvessels/*physiopathology ; Middle Aged ; Multiple Myeloma/*diagnosis/mortality ; Neoplasm Staging ; Neovascularization, Pathologic ; Plasma Cells/cytology ; Prognosis ; Proportional Hazards Models ; Regression Analysis ; Risk Factors

BACKGROUNDPericytes, located on microvessels, help to maintain vascular stability and blood-brain barrier integrity. The influence of pericytes on microvessels after spinal cord injury (SCI) is less clear. Therefore, the aim of this study was to investigate whether pericytes took a protective effect on microvessels in melatonin-treated SCI.

METHODSC57BL/6 mice were randomly divided into three groups: sham group, SCI group, and melatonin group (n = 27 per group). Functional recovery was evaluated using the Basso Mouse Scale. Motor neurons were observed using hematoxylin and eosin staining. Pericyte coverage was analyzed using immunofluorescence. Permeability of blood-spinal cord barrier (BSCB) was assessed by administration of Evan's Blue. Protein levels of occludin, aquaporin-4 (AQP4), angiopoietin-1 (Ang1), intercellular cell adhesion molecule-1 (ICAM-1), Bcl-2, and Bax were determined using Western blotting. Mimicking the pathological conditions of SCI, melatonin-treated primary pericytes were subjected to oxygen-glucose deprivation/reperfusion (OGD/R). Secretion of Ang1 was analyzed using an enzyme-linked immunosorbent assay, and the expression of ICAM-1 was detected by immunofluorescence.

RESULTSMelatonin treatment improved locomotor functional outcome and rescued motor neurons. Pericyte coverage was significantly reduced after SCI; melatonin treatment alleviated the loss of pericyte coverage and rescued perfused microvessels 7 days after injury. The permeability of BSCB and loss of occludin were attenuated, and edema formation and upregulation of AQP4 were inhibited, after melatonin treatment. The expression of Ang1 and Bcl-2 was improved, while the expression of ICAM-1 and Bax was inhibited, in melatonin-treated SCI mice. Furthermore, the secretion of Ang1 was increased and the expression of ICAM-1 was inhibited in melatonin-treated pericytes after OGD/R.

CONCLUSIONSMelatonin ameliorated the loss of blood vessels and disruption of BSCB to exert a protective effect on SCI, which might be mediated by increased pericyte coverage. The upregulation of Ang1 in pericytes could inhibit inflammation and apoptosis to protect the microvessels.

Angiopoietin-1 ; metabolism ; Animals ; Enzyme-Linked Immunosorbent Assay ; Intercellular Adhesion Molecule-1 ; metabolism ; Male ; Melatonin ; pharmacology ; therapeutic use ; Mice ; Mice, Inbred C57BL ; Microvessels ; cytology ; Occludin ; metabolism ; Pericytes ; drug effects ; metabolism ; Random Allocation ; Spinal Cord Injuries ; drug therapy ; metabolism

OBJCETIVETo investigate the method of separation of culture of bone microvascular endothelial cells (BMECs) of human femoral head in vitro.

METHODSFrom October 2013 to January 2014,15 femoral heads without pathologic change from patients resected during hip replacement were selected involving 2 males and 13 females with a mean age of 71.2 years old ranging from 38 to 92. Cancellous bone in femoral head was bited into broken bone grain and transfered into medium in aseptic contidion. Cells were isolated by the methods of enzymic digestion and density gradient centrifugation,purified by differiential attachment. The characteristics of cells was observed by inverted microscope. vWF and CD31 immunofluorescence analysis was applied for identification of cells.

RESULTSThe number of cells was positively correlated with patients' age after 24 hours in primary culture. The older patients had the less cells numbered. After 4 to 5 days' culture, primary cells appeared short spindle,polygon shaped and cobblestone-like morphology. After 7 to 10 days' culture, primary cells proliferated densely, became fusion, arranged in swirl, and contact inhibition appeared significantly. Immunofluorescence staining revealed the cells were 100% positive for vWF and CD31, and it showed that the cultured cells were BMECs.

CONCLUSIONIt was a simple, steady, effective method with good reproducibility, by which highly purified human BMECs can be obtained.

Adult ; Aged ; Aged, 80 and over ; Cell Culture Techniques ; Cell Proliferation ; Cell Separation ; methods ; Cells, Cultured ; Endothelial Cells ; cytology ; Female ; Femur Head ; blood supply ; Humans ; Male ; Microvessels ; cytology ; Middle Aged

OBJECTIVE: To study the expression of CD68 antibody marked tumor associated macrophage TAMs and matrix solution element MMP-7 in laryngeal squamous carcinoma tissue and the relationship with clinicopathological parameters, so that to explore the relationship between the expression of the two molecular markers and laryngeal cancer tissue microvascular density (MVD). METHOD: Immunohistochemical method was employed to detect the expression of CD68 and MMP-7 in 65 cases (laryngeal squamous carcinoma tissue in 45 cases; peritumoral nontumor tissue in 20 cases) and CD 34 antibody marked MVD expression. RESULT: CD68 positive rate in squamous carcinoma tissue (82.2%, 37/45) is obviously higher than that in the peritumoral tissue (15%, 3/20) (P < 0.05), and MMP-7 positive rate in squamous carcinoma tissue is significantly different from that in peritumoral tissue (71.1%; 25%) (P < 0.05). The expression rate of CD34-MVD in laryngeal squamous carcinoma tissue( 26.52 +/- 6.36 )is higher than that in peritumoral tissue (12.23 +/- 4.01) (P < 0.05). In lymph node metastasis group, the positive expression rates of CD68 and MMP-7 are higher than those in the group without lymph node metastasis. MMP-7 showed no correlation with cancer stage, and CD68 was related with cancer stage; CD68, MMP-7 and CD34- MVD have positive correlation. CONCLUSION The high level of expression of TAMs and MMP-7 in laryngeal cancer tissue and the positive correlation with MVD illustrate that both of the markers play important roles in promoting laryngeal squamous carcinoma tissue metastasis and angiogenesis, which can be used as important markers to evaluate the invasion and metastasis of laryngeal cancer.
Adult ; Aged ; Antigens, CD ; metabolism ; Antigens, CD34 ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Carcinoma, Squamous Cell ; blood supply ; metabolism ; pathology ; Humans ; Laryngeal Neoplasms ; blood supply ; metabolism ; pathology ; Lymphatic Metastasis ; Macrophages ; cytology ; metabolism ; Male ; Matrix Metalloproteinase 7 ; metabolism ; Microvessels ; Middle Aged ; Neoplasm Staging ; Neovascularization, Pathologic

Various stem cells, including neural stem cells (NSCs), have been extensively studied in stroke models, but how to increase neuronal differentiation rate of NSCs remains unresolved, particularly in a damaged environment. The purpose of this study was to investigate the effects of cerebral microvascular endothelial cells (CMECs) on the neurogenesis of NSCs with or without oxygen-glucose deprivation (OGD). The NSCs acquired from primary culture were immunostained to prove cell purity. Survival and proliferation of NSCs were determined after the co-culture with CMECs for 7 days. After removing the CMECs, NSCs were randomly divided into two groups as follows: OGD and non-OGD groups. Both groups were maintained in differentiation culture for 4 days to evaluate the differentiation rate. Mouse embryo fibroblast (MEF) cells co-cultured with NSCs served as control group. NSCs co-cultured with CMECs had an increase in size (on the 7th day: 89.80±26.12 μm vs. 73.08±15.01 μm, P<0.001) (n=12) and number [on the 7th day: 6.33±5.61/high power objective (HP) vs. 2.23±1.61/HP, P<0.001] (n=12) as compared with those co-cultured with MEF cells. After further differentiation culture for 4 days, NSCs co-cultured with CMECs had an increase in neuronal differentiation rate in OGD and non-OGD groups, but not in the control group (15.16% and 16.07% vs. 8.81%; both P<0.001) (n=6). This study provided evidence that OGD could not alter the effects of CMECs in promoting the neuronal differentiation potential of NSCs. These findings may have important implications for the development of new cell therapies for cerebral vascular diseases.
Animals ; Animals, Newborn ; Brain ; blood supply ; Cell Differentiation ; physiology ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; methods ; Endothelial Cells ; cytology ; metabolism ; Glucose ; metabolism ; Mice ; Mice, Inbred C57BL ; Microvessels ; cytology ; metabolism ; Neural Stem Cells ; cytology ; metabolism ; Oxygen ; metabolism

OBJECTIVETo investigate the effects of a recombinant adenovirus-mediated triple mutant hypoxia-inducible factor-1α (HIF-1α) on the proliferation and vascular endothelial growth factor (VEGF) expression in human microvascular endothelial cells (hMVECs).

METHODSThe adenovirus vector of the triple mutant HIF-1α (Ad-HIF-1α(564/402/803)), adenovirus vector of wild-type HIF-1α (Ad-HIF-1α(nature)), Ad-lacZ and Ad-Null were amplified in HEK293A cells, and the adenoviruses were purified and titrated. Dual luciferase reporter assay system was employed to detect the transcriptional activities of wild-type and triple mutant HIF-1α. After infection of the hMVECs with the adenoviruses, the cellular protein expressions of HIF-1α and VEGF were detected using Western blotting, and the cell proliferation was assessed by MTS assay.

RESULTSThe transcriptional activity of the triple mutant HIF-1α was significantly higher than that of wildtype HIF-1α in the infected hMVECs (P<0.001). The protein levels of HIF-1α and VEGF in cells infected with Ad-HIF-1α(564/402/803) were significantly higher than those in cells infected with other adenoviruses, and HIF-1α dose-dependently up-regulated VEGF protein expression. The absorbance was significantly higher in Ad-HIF-1α(564/402/803) group than in the other groups (P<0.01) on the third and fifth days after infection.

CONCLUSIONThe recombinant adenovirus-mediated triple mutant HIF-1α expression is stable under normoxic condition. The triple mutant HIF-1α can up-regulate the expression of VEGF protein in hMVECs to promote the cell proliferation.

Adenoviridae ; genetics ; Cell Proliferation ; Endothelial Cells ; cytology ; metabolism ; Endothelium, Vascular ; cytology ; Genetic Vectors ; HEK293 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; pharmacology ; Microvessels ; cytology ; Recombinant Proteins ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism

PURPOSE: Endothelial cells maintain the homeostasis of blood, which consists of plasma and cellular components, and regulate the interaction between blood and the surrounding tissues. They also have essential roles in vascular permeability, the circulation, coagulation, inflammation, wound healing, and tissue growth. The senescence of endothelial cells is closely related to the aging of the adjacent tissues and to age-related vascular disease. Recently, the expression of moesin was found to be decreased in elderly human dermal microvascular endothelial cells (HDMECs), and an association between moesin and senescence has been suggested. This study examined the functional role of moesin in cellular senescence. MATERIALS AND METHODS: To study the effects of decreased moesin expression on cellular senescence and metabolism, HDMECs were transfected with short hairpin-RNA (shRNA) lentivirus to silence moesin gene expression. In addition, specimens from young and old human skin were stained with anti-moesin and anti-p16 antibodies as an in vivo study. RESULTS: Using shRNAl-entivirus, moesin knock-down HDMECs developed characteristics associated with aging and expressed senescence associated-beta-galactosidase during early passages. They also showed increased p16 expression, decreased metabolic activity, and cell growth retardation. Human skin tissue from elderly persons showed decreased moesin expression and increased p16 expression. CONCLUSION: These findings suggest that there is a functional association between moesin expression and cellular senescence. Further study of the functional mechanism of moesin in the cytoskeleton and cellular senescence is needed. In addition, this study provides a useful model for developing anti-aging treatments.
Aged, 80 and over ; Antigens, CD31/metabolism ; Blotting, Western ; Cell Aging/genetics/*physiology ; Cell Line ; Child ; Endothelial Cells/*cytology/*metabolism ; Humans ; Immunohistochemistry ; Microfilament Proteins/genetics/*physiology ; Microscopy, Phase-Contrast ; Microvessels/*cytology ; RNA, Small Interfering/genetics/physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Skin/*blood supply

OBJECTIVETo observe and analyze the antitumor effect of endostar combined with docetaxel under different administration sequences.

METHODSNude mice with xenograft tumor (A549 cell line) were randomized into 3 groups, 8 mice/group: (1) Concurrent administration group (each mouse: endostar 400 µg/d, d1-d35, docetaxel 10 mg/kg, every 3 days, d1-d19); (2) Endo-first group (each mouse: endostar 400 µg/d, d1-d35, docetaxel 10 mg/kg, every 3 days, d16-d34); (3) Model group (positive control, tumor-bearing mice without treatment, each mouse: physiological saline, 100 µl/d, d1-d35, water for injection, 200 µl/d, d1-d35, every 3 days), and blank control group (negative control, normal mice without treatment, 8 mice), the administration method was the same to the model group. The volume of tumor and the weight of mouse were measured during treatment. Circulating endothelial cells (CECs) were detected by flowcytometry, and the expression of matrix metalloproteinase (MMP-2, MMP-9), the tissue inhibitor of MMP (TIMP-1, TIMP-2), the extracellular MMP inducer (EMMPRIN), CD34, α-smooth muscle actin (α-SMA) were determined by immunohistochemistry.

RESULTSThe tumor growth of concurrent administration group (39.94 mm(3)) was lower than that of the endo-first group [(99.57 ± 74.48) mm(3)] during treatment, both of them were smaller than that of the model group [(217.67 ± 95.44) mm(3), P < 0.05]. The amount of CECs in the endo-first group [(77.25 ± 24.02) cells/10(4) cells] was more than that of the concurrent administration group [(25.86 ± 11.77) cells/10(4) cells], the model group [(14.71 ± 11.07) cells/10(4) cells], and the blank control group [(12.90 ± 11.20) cells/10(4) cells, P < 0.01]. The expression of MMPs in the treatment groups was obviously downregulated. The expressions of TIMP-1 in the endo-first group and TIMP-2 in the concurrent administration group were upregulated (P < 0.05). The expression of EMMPRIN was significantly down-regulated in the concurrent administration group (P < 0.05). The MVD and α-SMA expressions of the treatment groups were less than that of the model group (P < 0.05).

CONCLUSIONIn comparison with the endo-first group, the anti-tumor effect and survival quality of the concurrent administration group are better. Both of the administration groups may have "vascular normalization effect" by down-regulating MMPs expression through different points, and inhibit the cancer-induced stromal reaction, restraining the cancer progress to a certain extent. The changes of CECs should be a dynamic process with an initial rise in the early-stage suggesting the decrease of vascular bed and subsequent decline ascribed to apoptosis of CECs and the tumor-regression after combined therapy. Investigation of its dynamic changes may be helpful to know the change of tumor burden and vascular bed and predict the antitumor effect.

Actins ; metabolism ; Angiogenesis Inhibitors ; administration & dosage ; pharmacology ; Animals ; Antineoplastic Agents ; administration & dosage ; pharmacology ; Antineoplastic Agents, Phytogenic ; administration & dosage ; pharmacology ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Basigin ; metabolism ; Cell Line, Tumor ; Drug Administration Schedule ; Endostatins ; administration & dosage ; pharmacology ; Endothelial Cells ; cytology ; Female ; Lung Neoplasms ; metabolism ; pathology ; Matrix Metalloproteinase 2 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microvessels ; drug effects ; Neoplasm Transplantation ; Taxoids ; administration & dosage ; pharmacology ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; metabolism ; Tumor Burden ; drug effects