OBJECTIVETo evaluate the effects of aminoguanidine on methylglyoxal-mediated oxygen-glucose deprivation (OGD) injury in the cultured human brain microvascular endothelial cells (HBMEC).

METHODSCultured HBMEC cells were pretreated with methylglyoxal before oxygen-glucose deprivation injury. Cell vitality was determined by MTT method, cell mortality was assessed by LDH release method, cell apoptosis was examined by Annexin V/PI formation method, and the advanced glycation end products (AGEs) were detected by Western-blot.

RESULTSMethylglyoxal induced HBMEC injury in a dose-dependent manner. At 2 mmol/L of methylglyoxal, the cell viability was 56.1% when methylglyoxal-pretreated cells exposed to oxygen-glucose deprivation, the cell inhibition rate was 90.0%. Aminoguanidine (1 mmol/L) inhibited methylglyoxal and OGD induced LDH release and Annexin V/PI formation. Furthermore, aminoguanidine (1 mmol/L) also decreased advanced glycation end products (AGEs) formation induced by methylglyoxal and oxygen-glucose deprivation.

CONCLUSIONAminoguanidine protected methylglyoxal mediated-oxygen-glucose deprivation injury in the cultured HBMEC, which may be associated with anti-glycation activity.

Apoptosis ; drug effects ; Cell Hypoxia ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Drug Antagonism ; Endothelial Cells ; drug effects ; metabolism ; pathology ; Endothelium, Vascular ; cytology ; Glycation End Products, Advanced ; metabolism ; Guanidines ; pharmacology ; Humans ; Pyruvaldehyde ; pharmacology

OBJECTIVETo study the effect of diamide on the expression of macrophage inflammatory protein-1 alpha (MIP-1 alpha) in cultured human umbilical vein endothelial cells.

METHODSAfter exposure of the endothelial cells (ECs) to different concentrations of diamide for 4 hours, the MIP-1 alpha mRNA in the cells was detected by nuclease S1 protection assay and the MIP-1 alpha protein in those cells was determined by cell enzyme-linked immunosorbent assay. The chemotactic activity of MIP-1 alpha in the conditioned medium of ECs treated with diamide for peripheral blood monocytes was tested by microfilter method using modified Boyden chambers.

RESULTSIncubation of ECs with 5 micro mol/L diamide resulted in a 2.4-fold increase in the level of MIP-1 alpha mRNA expression as compared with the control group (t = 8.70, P < 0.05). Exposure of ECs to 1 micro mol/L, 5 micro mol/L and 10 micro mol/L diamide resulted in a 0.9-fold, 1.2-fold, and 0.7-fold increase in the level of MIP-1 alpha protein expression respectively, as compared with the control group (F = 35.65, P < 0.05). Chemotactic assay showed that the migration distance of monocytes towards the conditioned medium (CM) of ECs treated with 5 micromol/L diamide was 99.50 microm +/- 4.31 microm, which was significantly more than the 66.47 microm +/- 3.25 microm towards the conditioned medium of ECs in the non-diamide group, the chemokinetic group (67.03 microm +/- 6.83 microm) and the random migration group (65.40 microm +/- 3.36 microm) (F = 404.31, P < 0.05). The results revealed that there might be chemotactic substances in the conditioned medium of 5 micro mol/L diamide treated ECs. The migration distance of monocytes towards the conditioned medium of the ECs exposed to 5 micromol/L diamide was significantly reduced to 82.80 microm +/- 6.88 microm after the addition of goat anti-human MIP-1 alpha antibody (F = 192.25, P < 0.05), which indicates the chemotactic activity of MIP-1 alpha in the conditioned medium of the ECs in the diamide group.

CONCLUSIONSDiamide, a lipid peroxidation inducer, could stimulate ECs to produce high levels of MIP-1 alpha with chemotactic activity, and may play an important role in atherogenesis through attraction of peripheral blood monocytes into arterial intima.

Arteriosclerosis ; pathology ; Cells, Cultured ; Chemokine CCL4 ; Diamide ; pharmacology ; Endothelium, Vascular ; drug effects ; metabolism ; Gene Expression ; drug effects ; Humans ; Macrophage Inflammatory Proteins ; metabolism ; RNA, Messenger ; drug effects ; metabolism ; Radiation-Sensitizing Agents ; pharmacology

OBJECTIVETo explore the renal protective effect of Tongxinluo (TXL) and its mechanism of action.

METHODSEight-week old SD rats were divided into the sham-operated group (A), the model group (B) and the TXL group, 6 rats in each group. Angiotensin II (Ang II) was administered slowly (200 ng/kg per min) to rats in group B and C via subcutaneously embedded osmotic pump to make them stimulative model of renal injury, while to rats in group A, pump embedding with saline infusion. After modeling, TXL was given to group C by gastric perfusion in dosage of 0.8 g/kg per day. And the following indexes were observed 14 days later: configuration of renal arterial endothelium by transmission electron microscope; pathologic figure of kidney with HE stain; renal apoptosis by TUNEL; expression of p53 and p22phox by RT-PCR;and level of reactive oxygen species (ROS) in kidney.

RESULTSDifferent degree of congestion, swelling, denudation of endothelial cell in renal arterial endothelial cell; glomerular matrix proliferation and partial glomerular atrophy with tendency of fibrosis; increased renal parenchymal apoptosis; enhanced expression of p53 and p22phox; and elevated ROS were found in model animals. All the above-mentioned abnormalities, including glomerular injury, renal cell apoptosis, as well as the increased p53, p22phox expressions and ROS production were all alleviated in group C after TXL treatment.

CONCLUSIONTXL could protect renal against Ang II injury, and it may be realized by inhibiting NADPH-ROS/p53 pathways and suppressing cell apoptosis in renal parenchyma.

Angiotensin II ; metabolism ; Animals ; Apoptosis ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Endothelium, Vascular ; drug effects ; metabolism ; Kidney ; blood supply ; pathology ; Male ; NADPH Oxidases ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo study the effect of HIV-1 gp41 ectodomain (gp41-I90) on the cytoskeletal changes in human brain microvascular endothelial cells (HBMECs) induced by Cryptococcus neoformans.

METHODSHBMECs were cultured on collagen-coated chamber slide or transwell to allow the formation of cell monolayers. After pre-treatment with gp41-I90 and infection with Cryptococcus neoformans, the HBMECs were examined for the expression of actin or filamin by immunofluorescence assay. HRP permeability of the HBMECs treated with gp41-I90 was detected by ELISA. Transcytosis of Cryptococcus neoformans through the gp41-I90-treated HBMECs was detected by direct counting from a hemocytometer.

RESULTSgp41-I90 obviously enhanced the cytoskeletal changes of the HBMECs infected by Cryptococcus neoformans, causing curved and sparse filamentous arrangement of actin and filamin. gp41-I90 treatment also resulted in obviously increased HRP permeability of the cells and transcytosis of Cryptococcus neoformans.

CONCLUSIONgp41- I90 enhances Cryptococcus neoformans binding to HBMECs, which is related to its effect in enhancing Cryptococcus neoformans-induced cytoskeletal changes of the cells.

Brain ; blood supply ; Cells, Cultured ; Cryptococcosis ; pathology ; Cryptococcus neoformans ; pathogenicity ; Cytoskeleton ; drug effects ; metabolism ; Endothelial Cells ; cytology ; drug effects ; microbiology ; Endothelium, Vascular ; cytology ; drug effects ; metabolism ; HIV Envelope Protein gp41 ; pharmacology ; Humans ; Microcirculation

OBJECTIVETo explore the protective mechanism of officeihale on the vascular pathological process in diabetes mellitus (DM) rats.

METHODAfter the DM rat model was established, 24 DM rats were randomly divided into model group (12 DM rats) and Rheum officeinale group (12 DM rats). Rheum officeinale was orally given in 10 g kg(-1) per day, and the other two groups were given equal pure water. 8 weeks later, blood samples were collected to determine the level of nitric oxide (NO) and endothelin-1 (ET-1). Thoracic aortic rings was prepared to observe the inhibiting effect of Ach with different concentration on contraction caused by NE. Another part of aorta was made to observe the expression of ICAM-1 and VCAM-1 by method of SP immunohistochemistry staining,

RESULTRheum officeinale group obviously decreased the level of ET-1 and increased the NO compared with model group (P <0.05). The expression of ICAM-1 and VCAM-1 could be obviously inhibited in Rheum officeinale group compared with model group. (P <0.05).

CONCLUSIONRheum officeinale could decrease the level of ET-1 with increased the NO in diabetes rats, and inhibit the expression of ICAM-1 and VCAM-1, which may be mechanisms of protecting the endothelium of vessel in diabetes rats.

Animals ; Aorta ; drug effects ; metabolism ; pathology ; Blood Glucose ; metabolism ; Blood Vessels ; drug effects ; metabolism ; pathology ; Diabetes Mellitus ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Endothelin-1 ; metabolism ; Endothelium, Vascular ; drug effects ; metabolism ; pathology ; Gene Expression Regulation ; drug effects ; Intercellular Adhesion Molecule-1 ; metabolism ; Male ; Nitric Oxide ; metabolism ; Protective Agents ; pharmacology ; Rats ; Rheum ; chemistry

To observe the effect of oxidized low density lipoprotein (OxLDL) on arterial endothelial cells apoptosis in vivo, we established a model in which Sprague-Dawley rats were given intraperitoneal and intravenous injection of unmodified LDL (8 mg/kg every day) via the tail vein. Seven days after the injection, the aortic endothelial cells specimens were prepared by an en face preparation of rat aorta. The apoptotic cells were identified and counted by in situ nick and labelling (TUNEL) method and light microscopy. The numbers of the apoptotic cells were 12.52 +/- 4.71/field in the intraperitoneal injection control group, 11.41 +/- 2.94/field in the intravenous injection control group, 22.98 +/- 8.01/field in the intraperitoneal injection LDL group and 103.8 +/- 11.5/field in the intravenous injection LDL group, respectively. The difference was significant between injection LDL group and control (P < 0.01), and the difference was also significant between two LDL injection groups (P < 0.01). These findings suggest that injection of LDL can induce apoptosis in arterial endothelial cells and the effect is especially significant with intravenous injection LDL. After injection, oxidative modification of LDL may occur in local arteries and causes injury to the endothelial cells.
Aorta ; Apoptosis/*drug effects ; Endothelium, Vascular/*pathology ; In Situ Nick-End Labeling ; Injections, Intraperitoneal ; Injections, Intravenous ; Lipoproteins, LDL/*metabolism ; Lipoproteins, LDL/*pharmacology ; Oxidation-Reduction ; Random Allocation ; Rats, Sprague-Dawley

OBJECTIVEThis study was aimed to investigate the effects of carbon monoxide releasing molecule (CORM-2), a novel carbon monoxide carrier, on the reendothelialization of carotid artery in rat endothelial denudation model.

METHODSMale rats subjected to carotid artery balloon injury were treated with CORM-2, inactive CORM-2 (iCORM-2) or dimethyl sulfoxide (DMSO). The reendothelialization capacity was evaluated by Evans Blue dye and the immunostaining with anti-CD31 antibody. The number of circulating endothelial progenitor cells (EPCs) was detected by flow cytometry. The proliferation, migration, and adhesion of human umbilical vein endothelial cells (HUVECs) were assessed by using [3H]thymidine, Boyden chamber and human fibronectin respectively. The expressions of protein were detected by using western blot analysis.

RESULTSCORM-2 remarkably accelerated the re-endothelialization 5 d later and inhibited neointima formation 28 d later. In addition, the number of peripheral EPCs significantly increased in CORM-2-treated rats than that in iCORM-2 or DMSO-treated rats after 5 d later. In vitro experiments, CORM-2 significantly enhanced the proliferation, migration and adhesion of HUVECs. The levels of Akt, eNOS phosphorylation, and NO generation in HUVECs were also much higher in CORM-2 treated group. Blocking of PI3K/Akt/eNOS signaling pathway markedly suppressed the enhanced migration and adhesion of HUVECs induced by CORM-2.

CONCLUSIONCORM-2 could promote endothelial repair, and inhibit neointima formation after carotid artery balloon injury, which might be associated with the function changes of HUVECs regulated by PI3K/Akt/eNOS pathway.

Animals ; Carbon Monoxide ; metabolism ; pharmacology ; Carotid Artery Injuries ; drug therapy ; immunology ; metabolism ; pathology ; Carotid Artery, Common ; drug effects ; immunology ; metabolism ; pathology ; Cell Adhesion ; drug effects ; Disease Models, Animal ; Endothelial Cells ; drug effects ; immunology ; metabolism ; pathology ; Endothelium, Vascular ; drug effects ; metabolism ; pathology ; Humans ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo observe the expression of CD(14) protein and CD(14) gene in liver sinusoidal endothelial cells (LSECs) of rats during endotoxemia and the role of CD(14) protein in the activation of lipopolysaccharide (LPS)-induced LSECs.

METHODSWistar rat endotoxemia model was established by injection of a dose of LPS (5 mg/kg, Escherichia coli O111:B4) via the tail vein of the rats, then sacrificed immediately, at 3, 6, 12, and 24 h, respectively. LSECs were isolated from normal and LPS-injected rats by the in situ collagenase perfusion technique. The isolated LSECs were incubated with anti CD(14) polyclonal antibody, then followed by staining with goat anti-rabbit IgG conjugated fluorescein isothiocyanate (FITC). The percentage and mean fluorescence intensity (MFI) of CD(14)-positive cells were detected by the flow cytometric analysis (FCM). LSECs were collected to measure the expression of CD(14) mRNA by the in situ hybridization analysis. The isolated LSECs from normal rats were divided into two groups. Group of LPS: LSECs were induced with different concentration of LPS (0, 0.01 microg/ml, 1 microg/ml, 10 microg/ml, and 100 microg/ml). Group of anti-CD(14) blockade: LSECs were pre-incubated for 30 min with CD(14) antibody before different concentrations of LPS were added. The supernatants of these cells were then collected for measuring the levels of tumor necrosis factor (TNF)- alpha and interleukin (IL)-6.

RESULTSIn rats with endotoxemia, LSECs displayed a strong MFI distinct from that of control rats. The number of FITC-CD(14) positive LSECs was 54.32%, 65.83%, 85.61%, and 45.65% at 3, 6, 12, and 24 h, respectively, which increased markedly when compared to control rats (4.45%, P<0.01). The expression of CD(14) mRNA in LSECs was stronger than that in control rats. The levels of TNF-alpha were significantly increased in group of LPS (54.49 +/- 6.02 pg/ml, 84.65 +/- 10.16 pg/ml, 206.54 +/- 23.55 pg/ml, 349.87 +/- 39.47 pg/ml, and 365.76 +/- 40.31 pg/ml) than those in group of anti-CD(14) blockade (55.93 +/- 6.95 pg/ml, 63.32 +/- 7.81 pg/ml, 85.34 +/- 9.72 pg/ml, 112.75 +/- 13.54 pg/ml, and 198.66 +/- 21.54 pg/ml) (P<0.01). The levels of IL-6 also increased significantly in group of LPS (103.34 +/- 12.52 pg/ml, 187.39 +/- 20.31 pg/ml, 243.87 +/- 27.83 pg/ml, 289.51 +/- 30.15 pg/ml, and 298.53 +/- 31.94 pg/ml) than those in group of anti-CD(14) blockade (104.37 +/- 11.49 pg/ml, 125.02 +/- 13.58 pg/ml, 164.59 +/- 19.47 pg/ml, 183.47 +/- 20.17 pg/ml, and 221.76 +/- 26.43pg/ml) (P<0.01).

CONCLUSIONSLSEC can synthesize CD(14) protein and express CD(14) gene during endotoxemia. Anti CD(14) antibody can inhibit the production of TNF-alpha and IL-6 in LSECs induced by LPS. The expression of CD(14) protein may take an important part in the activation of LSECs induced by LPS.

Animals ; Dose-Response Relationship, Drug ; Endothelium, Vascular ; drug effects ; metabolism ; pathology ; Endotoxemia ; metabolism ; pathology ; Escherichia coli Infections ; metabolism ; pathology ; Gene Expression Regulation ; drug effects ; Interleukin-6 ; metabolism ; Lipopolysaccharide Receptors ; biosynthesis ; genetics ; Lipopolysaccharides ; administration & dosage ; Liver ; drug effects ; metabolism ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo explore the new mechanism of propranolol for treatment of hemangioma and the effects of propranolol on proliferation of hemangioma-derived mesenchymal stem cells ( Hem- MSCs).

METHODSWe isolated Hem-MSCs from hemangioma in the proliferating phase by their selective adhesion to plastic culture dishes. Immunofluorescence staining was used to examine the expression of marker antigens in Hem-MSCs. Human umbilical vein endothelial cells(HUVECs) were used as control. Indiuction of multi-lineage differentiation including osteogenesis and adipogeneis was performed with appropriate medium to identify the multi-lineage differentiation potential. MTT cell counting was used to observe the effects of different concentrations of propranolol on proliferation of Hem-MSCs.

RESULTSHem- MSCs were fibroblast-like morphology. All of them expressed vimentin, most expressed α-SMA,CD133, some expressed Glutl, and none of them expressed VEGF. Osteogenic, adipogenic differentiations of Hem- MSCs were induced successfully. Effects of low concentration of propranolol on proliferation of Hem-MSCs were not obvious, while high concentration of propranolol can inhibit the proliferation of Hem-MSCs.

CONCLUSIONSThe cells we isolated from hemangioma are Hem-MSCs. High concentration of propranolol can inhibit the proliferation of Hem-MSCs.

Adipogenesis ; Antigens ; metabolism ; Cell Differentiation ; Cell Proliferation ; drug effects ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Fibroblasts ; cytology ; Hemangioma ; pathology ; Humans ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Osteogenesis ; Propranolol ; pharmacology ; Umbilical Veins ; Vimentin ; metabolism

Vibrio vulnificus cytolysin forms transmembrane pores that are permeable to calcium ions in pulmonary endothelial cells, and has been suggested as an important virulence factor that sequestrate neutrophils primarily in the lung. To elucidate the mechanism we investigated whether the cytolysin affect the expression of endothelial P-selectin and adhesiveness of pulmonary endothelial cells for neutrophils. The cytolysin increased the adhesiveness of CPAE cell, a pulmonary endothelial cell line, for neutrophils in a concentrationand time-dependent manner. The increase of adhesiveness occurred within several minutes after the cytolysin exposure, persisted up to 90 min, and was not affected by cycloheximide. Furthermore, flow cytometric analyses showed that cytolysin enhanced the level of P-selectin on CPAE cell surface. Therefore, these results suggest that the cytolysin-induced hyperadhesiveness of pulmonary endothelial cells for neutrophils is mediated by the mobilization of endothelial P-selectin to the cell surface.
Animals ; Cattle ; Cell Adhesion/*drug effects ; Cell Line ; Cycloheximide/pharmacology ; Cytotoxins/*toxicity ; Dose-Response Relationship, Drug ; Endothelium, Vascular/cytology/*drug effects/metabolism ; Kinetics ; Neutrophils/*drug effects/pathology ; P-Selectin/*metabolism ; Protein Synthesis Inhibitors/pharmacology ; Pulmonary Artery/cytology/*drug effects/metabolism ; Rats ; Vibrio Infections/etiology/pathology ; Vibrio vulnificus/*pathogenicity