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

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

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

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

The present study aimed to investigate the effects of basic fibroblast growth factor (bFGF) on the expressions of angioge-nesis-related genes in a mouse brain microvascular endothelial cell line, namely bEnd.3, using cDNA microarray. The effects of bFGF (10 ng/ml) on mRNA and protein expressions of cyclooxygenase-2 (COX-2), an angiogenesis bystander molecule, were further investigated. cDNA microarray was employed to study the effects of bFGF on the expressions of angiogenic genes in a high throughput pattern. RT-PCR was used to study the effect of bFGF on COX-2 mRNA expression. Western blot and immunocytochemistry were utilized to study the effect of bFGF on COX-2 protein expression. The results showed that, 2 h after bFGF treatment, pro-angiogenic genes (Adamts1, MMP-9, Ang-1, PDGF B, G-CSF, FGF16, IGF-1, etc.) were significantly upregulated, whereas anti-angiogenic genes (TIMP-2, TSP-3, etc.) were significantly downregulated. The bystander molecule in angiogenic pathway COX-2 mRNA and protein expressions were significantly upregulated after bFGF treatment. It is suggested that triggering angiogensis switch through upregulating pro-angiogenic gene and downregulating anti-angiogenic gene expression is one of the major mechanisms of bFGF-induced angiogenesis. The expression change of COX-2, as a bystander molecule, was observed after bFGF treatment in bEnd.3 cells and the significance was discussed.
Animals ; Cell Line ; Cerebrovascular Circulation ; drug effects ; genetics ; Cyclooxygenase 2 ; genetics ; metabolism ; Endothelial Cells ; drug effects ; metabolism ; Fibroblast Growth Factor 2 ; pharmacology ; Mice ; Microvessels ; cytology ; metabolism ; Neovascularization, Physiologic ; drug effects ; Transcriptome

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

OBJECTIVEThe effect of human bone marrow mesenchymal stem cells (hMSCs) on tumor neovascularization were studied.

METHODShMSCs were isolated from human bone marrow by density gradient fractionation and adherence to plastic flasks. hMSCs-EGFP were obtained by pLEGFP-N1 retroviral vector. Flow cytometry was used to detect the cell surface antigen and the differentiation potential of hMSCs-EGFP was investigated under conditioned media. The effect of hMSCs on tumor neovascularization were observed by establishing solid tumor models in BALB/C nude mice. In addition, effect of the conditioned medium used for tumor cells and endothelial cells (EC) cultivation was collected, to detect its effect on the growth and migration rates of hMSC. hMSCs were induced to differentiate into EC in vitro and the migratory effect on HUVEC was also evaluated.

RESULTShMSCs-EGFP, like hMSC, exhibited a fibroblast-like morphological feature, and both had the similar cell surface antigens. They could be induced into osteocytes or adipocytes under the conditioned media. The results not only suggested that hMSCs contributed to tumor neovascularization, but also indicated that most of vessels were host-derived angiogenesis mediated by hMSCs. The mean vascular density (MVD) in suspension group (13.67 ± 1.53) was strikely higher than that in MCF-7 group (5.33 ± 1.42), which showed statistical significance (P < 0.05). Only very few vessels were attributed to hMSCs transdifferentiation into ECs. Tumor cells and ECs can promote hMSCs proliferation and migration through paracrine action. Furthermore, hMSCs were positive for CD31 after 2 weeks induction and HUVEC migration can be facilitated by hMSCs.

CONCLUSIONMSCs have the effect of promoting tumor neovascularization.

Animals ; Bone Marrow Cells ; cytology ; Breast Neoplasms ; pathology ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Epithelial Cells ; cytology ; Humans ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; cytology ; physiology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microvessels ; pathology ; Neoplasm Transplantation ; Neovascularization, Pathologic ; pathology

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

To observe the effects of phenylallyl compounds on prostaglandin E2 (PGE2) release in mouse cerebral microvascular endothelial cells (bEnd. 3) stimulated by IL-1beta, and to analyze their structure-activity relationship. Different concentrations of phenylallyl compounds were added separately, and the content of PGE2 induced by IL-1beta in the culture media was measured by ELISA assay. The 50% inhibitory concentration (IC50) of PGE2 was calculated. Studies showed that phenylallyl compounds could affect the PGE2 release differently in bEnd. 3 cells induced by IL-1beta. Close relationships were shown between the inhibitory activities and the location and number of the substituent groups. In conclusion, phenylallyl compounds exhibited inhibitory activities at different extent on PGE2 release in bEnd. 3 cells stimulated by IL-1beta and presented certain structure-activity relationship.
Acrolein ; analogs & derivatives ; isolation & purification ; pharmacology ; Animals ; Brain ; blood supply ; Cells, Cultured ; Cinnamates ; isolation & purification ; pharmacology ; Dinoprostone ; antagonists & inhibitors ; secretion ; Drugs, Chinese Herbal ; chemistry ; Endothelial Cells ; cytology ; metabolism ; Inhibitory Concentration 50 ; Interleukin-1beta ; pharmacology ; Mice ; Microvessels ; cytology ; Propanols ; isolation & purification ; pharmacology ; Structure-Activity Relationship

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