The suppressive effect of anti-KDR antibody against VEGF on proliferation of hemangioma-derived vascular endothelial cells (HVECs) was investigated. HVECs from one case of hemangioma in proliferative phase were cultured. Both primary culture and sub-culture were conducted in M199 medium. The HVECs of passage 3 were divided into 4 groups based on the concentrations of anti-KDR antibody. Cell count was performed and inhibitory rate of HVECs was measured before and 9 days after interference. The results showed that the number of HVECs in the anti-KDR antibody-treated groups was significantly decreased and the inhibitory rate of HVECs by anti-KDR antibody (50, 10 and 2 microg/mL) was 84%, 63% and 39% respectively at 9th day after interference, with the difference being significant. In the control group, the number of HVECs was increased significantly. In was concluded that the anti-KDR antibody could suppress the activity of VEGF through blocking the KDR, indicating the potential clinical applications of anti-KDR antibody in the treatment of hemangioma.
Antibodies/*pharmacology ; Cell Proliferation/drug effects ; Endothelium, Vascular/*pathology ; Hemangioma/*pathology ; Tumor Cells, Cultured ; Vascular Endothelial Growth Factor Receptor-2/*immunology

OBJECTIVETo investigate the autoimmune injuries of diabetic macrovascular disease (aorta) and the protective effects of immunosuppressive agent (cyclosporine A, CsA) on aortic injuries in streptozotocin (STZ)-induced diabetic rats.

METHODSSTZ-induced diabetic rats were assigned randomly to 6 groups which received low (BML or AML, 1 mgxkg(-1)xd(-1)), middle (BMM or AMM, 4 mgxkg(-1)xd(-1)) or high (BMH or AMH, 8 mgxkg(-1)xd(-1)) dose of CsA from 1 week before or after STZ for 8 weeks. Diabetic rats without any treatment, insulin-treated diabetic rats and normal rats were also monitored simultaneously and served as control groups. The pathologic abnormalities of the aorta were verified by HE, Masson staining and electronmicroscopy. The depositions of immunoglobulins (IgG, IgM and IgA) were determined by immunohistochemistry and immunofluorescence methods.

RESULTSAt the end of study, lymphocytes infiltration and collagen content (26 582 +/- 6901) were significantly higher in diabetic aorta than those in non-diabetic aorta (Collagen: 7482 +/- 3491, P < 0.01). The deposited IgG and IgA were also significantly increased in diabetic aorta compared with non-diabetic aorta (IgG: 11 789 +/- 2491 vs. 2518 +/- 1066, P < 0.01; IgA: 17 430 +/- 3159 vs. 1135 +/- 758, P < 0.01). These changes were not affected by insulin while CsA intervention significantly reduced aortic collagen content (BMH: 13 518 +/- 5440, P < 0.01 vs. STZ) and immunoglobulin deposition (BMH: IgG: 7584 +/- 4462; IgA: 6176 +/- 1900, all P < 0.01 vs. STZ). These immunoglobulin deposition changes were confirmed by results of immunofluorescence. Aortic collagen accumulation was positively correlated to aortic immunoglobulin deposition (IgG, r = 0.556, P < 0.01; IgA, r = 0.661, P < 0.01).

CONCLUSIONSOur data suggest that the autoimmune injuries might be a promoting factor in the pathogenesis of the diabetic macrovascular disease which could lead to the development of macrovascular disease. Immunosuppressive agent, such as CsA, could inhibit the abnormal deposition of immunoglobulins and therefore, delay the development of diabetic macrovascular disease in this model.

Animals ; Aorta ; immunology ; pathology ; Aortic Diseases ; etiology ; Cyclosporine ; pharmacology ; Diabetes Mellitus, Experimental ; immunology ; pathology ; Endothelium, Vascular ; drug effects ; pathology ; Immunosuppressive Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley

FTY720 is a novel immunomodulator that has proven effective in animal models of transplantation and autoimmunity, has achieved promising results in Phase I and Phase II studies of renal transplantation in humans, and is currently undergoing phase III studies. FTY720 acts as a high-affinity agonist at the sphingosine 1-phosphate receptor-1 (S1P1), where it internalises the receptor and causes alterations to the normal circulation of lymphocytes between the blood and lymphoid tissue. Unlike conventional immunosuppressants, FTY720 does not impair the activation, proliferation or effector functions of T- and B-cells. Further development of FTY720 is in progress, including trials in autoimmune disorders as well as transplantation. This review summarises the mechanism of action of FTY720, its effects in models of transplantation and autoimmunity, and results from clinical trials in humans.
Animals ; Autoimmune Diseases/drug therapy ; Clinical Trials ; Endothelium, Vascular/drug effects ; Humans ; Immune System/drug effects ; Immunosuppressive Agents/*therapeutic use ; *Organ Transplantation ; Propylene Glycols/*therapeutic use ; Receptors, Lysosphingolipid/agonists ; Transplantation Immunology

We investigated the effects of mixed leukocyte reaction (MLR), hydrocortisone (HC) and cyclosporine A (CsA) on the expression of leukocyte adhesion molecules on the mesangial (MC) and endothelial cells (EnC). Cell surface enzyme immunoassay showed that INFnu, IL-1beta, or TNF alpha stimulated expression of ICAM-1, or VCAM-1 on MC after 24 hours. Flow cytometric analysis demonstrated that MLR supernatant induced a marked increase in mean fluorescence of or % of cells highly expressing intercellular adhesion molecule(ICAM)-1 or vascular cell adhesion molecule (VCAM)-1 on both cells after 24 hours (p<0.001). HC treatment(300 ng/ml) during MLR effectively inhibited MLR-induced upregulation of ICAM-1 and VCAM-1 on both cells (p<0.005). When MLR supernatant with HC was added to adhesion molecule assay, there was no inhibitory effect of HC on VCAM-1. CsA treatment (500 ng/ml) during MLR caused a modest decrease in upregulation of VCAM-1 on EnC (p<0.05), but had no effects on ICAM-1 on both cells. CsA directly decreased expression of VCAM-1 on MC. In conclusion, alloreactive lymphocytes and monocytes upregulate the expression of VCAM-1 and ICAM-1 on target cells probably by the mediation of cytokines. HC effectively prevents MLR-induced upregulation of VCAM-1 and ICAM-1. CsA does not increase the expression of VCAM-1 and ICAM-1.
Cells, Cultured ; Cyclosporine/*pharmacology ; Endothelium, Vascular/drug effects/*immunology ; Glomerular Mesangium/drug effects/*immunology ; Human ; Hydrocortisone/*pharmacology ; Intercellular Adhesion Molecule-1/*biosynthesis ; Interleukin-1/pharmacology ; Leukocytes/drug effects/*metabolism ; Lymphocyte Culture Test, Mixed ; Monocytes/drug effects/metabolism ; Tumor Necrosis Factor/pharmacology ; Vascular Cell Adhesion Molecule-1/*biosynthesis

We investigated the effects of mixed leukocyte reaction (MLR), hydrocortisone (HC) and cyclosporine A (CsA) on the expression of leukocyte adhesion molecules on the mesangial (MC) and endothelial cells (EnC). Cell surface enzyme immunoassay showed that INFnu, IL-1beta, or TNF alpha stimulated expression of ICAM-1, or VCAM-1 on MC after 24 hours. Flow cytometric analysis demonstrated that MLR supernatant induced a marked increase in mean fluorescence of or % of cells highly expressing intercellular adhesion molecule(ICAM)-1 or vascular cell adhesion molecule (VCAM)-1 on both cells after 24 hours (p<0.001). HC treatment(300 ng/ml) during MLR effectively inhibited MLR-induced upregulation of ICAM-1 and VCAM-1 on both cells (p<0.005). When MLR supernatant with HC was added to adhesion molecule assay, there was no inhibitory effect of HC on VCAM-1. CsA treatment (500 ng/ml) during MLR caused a modest decrease in upregulation of VCAM-1 on EnC (p<0.05), but had no effects on ICAM-1 on both cells. CsA directly decreased expression of VCAM-1 on MC. In conclusion, alloreactive lymphocytes and monocytes upregulate the expression of VCAM-1 and ICAM-1 on target cells probably by the mediation of cytokines. HC effectively prevents MLR-induced upregulation of VCAM-1 and ICAM-1. CsA does not increase the expression of VCAM-1 and ICAM-1.
Cells, Cultured ; Cyclosporine/*pharmacology ; Endothelium, Vascular/drug effects/*immunology ; Glomerular Mesangium/drug effects/*immunology ; Human ; Hydrocortisone/*pharmacology ; Intercellular Adhesion Molecule-1/*biosynthesis ; Interleukin-1/pharmacology ; Leukocytes/drug effects/*metabolism ; Lymphocyte Culture Test, Mixed ; Monocytes/drug effects/metabolism ; Tumor Necrosis Factor/pharmacology ; Vascular Cell Adhesion Molecule-1/*biosynthesis

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 investigate the influence of endothelial dysfunction induced by inoculated dendritic cells (DCs) loaded heat shock protein 60 (HSP60) in apolipoprotein (Apo) E-null mice, and the effect of Puerarin on it.

METHODSHSP60 DC (DChsp) acquired after prepared bone marrow-derived DCs of ApoE-null mice and treated with HSP60. In vitro, the function of DCs and the effect of Puerarin were detected. While in vivo, ApoE-null mice fed with high-cholesterol forage were divided into two groups and intravenous inoculated with DCh-sp or normal saline via vein twice respectively. The mice in the two groups were subdivided into the Puerarin group and non-treated group, and they were injected intraperitoneally with Puerarin and normal saline at the beginning of inoculation and the following 3 weeks, respectively. In addition, C57BL/6 mice without inoculation were taken as the normal control group. Two weeks after the last time inoculated, the response of T lymphocytes to HSP60 and endothelial-dependent diastolic function of aortic ring were detected.

RESULTSHSP60 could promote DCs expressed CD86 and stimulate T lymphocytes proliferation in vitro, while Puerarin had significantly inhibitory effect. After inoculated, DChsp activated inflammatory response in vivo and aggravated endothelium-dependent dilation in mice. Puerarin could significantly inhibit inflammatory reaction caused by DChsp and improve endothelium dilation.

CONCLUSIONHsp60 could activate DCs in vitro and in vivo, Puerarin could significantly inhibit specific immunity induced by HSP60 and improve vascular endothelium-dependent dilation.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Apolipoproteins E ; genetics ; B7-2 Antigen ; immunology ; Cell Proliferation ; drug effects ; Chaperonin 60 ; metabolism ; Dendritic Cells ; drug effects ; enzymology ; immunology ; Endothelium, Vascular ; drug effects ; physiology ; Immunity ; drug effects ; Inflammation ; chemically induced ; Isoflavones ; pharmacology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Protective Agents ; pharmacology ; T-Lymphocytes ; drug effects ; Vasodilator Agents ; pharmacology

OBJECTIVETo investigate the direct effects of the Flt3 ligand (FL) on hematopoiesis, such as the stimulation of the formation of hematopoietic colonies and the proliferation of dendritic cells, as well as the indirect stimulation of hematopoiesis, especially via the proliferation of endothelial cells.

METHODSMononuclear cells from human cord blood were plated in methylcellulose medium containing different cytokines to induce hematopoietic colony formation. Dendritic cells (DCs) were induced from the mononuclear cells with a cytokine cocktail with or without recombinant human soluble FL (rhFL; 100 ng/ml). The Flt3 receptors on the surface of a human microvascular endothelial cell line (ECV) were analyzed by flow cytometry. The proliferation of ECV stimulated by rhFL was measured with the microculture tetrazolium assay. The levels of FL, IL-6, IL-8, G-CSF and GM-CSF in the supernatant of ECV cultures were measured by enzyme linked immunoabsorbent assay (ELISA).

RESULTSrhFL stimulates colony formation from cord blood when used as a sole stimulant. FL in combination with other cytokines increased colony formation significantly. The number of DCs was approximately 2.5 times higher when rhFL was used. rhFL stimulates the proliferation of ECV on which Flt3 receptors are expressed. Furthermore, ECV secretes FL, IL-6, IL-8, G-CSF and GM-CSF, which were augmented by tumor necrosis factor-alpha and rhFL.

CONCLUSIONSrhFL enhances hematopoietic colony formation and DC proliferation from human cord blood cells. FL not only stimulates the proliferation of ECV, but is also secreted by ECV. FL may exert direct and indirect effects on hematopoiesis.

Cell Division ; drug effects ; Cell Line ; Dendritic Cells ; cytology ; drug effects ; immunology ; Dexamethasone ; pharmacology ; Dose-Response Relationship, Drug ; Endothelium, Vascular ; cytology ; drug effects ; metabolism ; Fetal Blood ; cytology ; drug effects ; Hematopoiesis ; drug effects ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Immunophenotyping ; Membrane Proteins ; pharmacology ; Recombinant Proteins ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo investigate the effect of anti-VEGF antibody on angiogenesis induced by osteosarcoma OS-732 cell line and tumor growth.

METHODSWith a tumor model on the chick embryo chorioallantoic membrane (CAM), the inhibition of angiogenesis and tumor growth by anti-VEGF antibody were observed under a stero-microscope and a light microscope. Furthermore, the proliferation and apoptosis in tumor cells and endothelial cells (EC) were studied by TdT-mediated duTP nick and labeling (TUNEL) and immunohistochemical staining using proliferating cell nuclear antigen (PCNA) monoclonal antibody.

RESULTSVEGF polyclonal antibody administration in tumor-bearing chick embryo resulted in growth arrest of xenografts and a markedly reduction in the new capillaries which converged upon the tumor. The tumor cell apoptotic index was higher in the anti-VEGF antibody treated group than the negative control group, but the proliferation index was not significantly different between them. At the same time, increased apoptosis and decreased proliferation in EC were also noted.

CONCLUSIONVEGF polyclonal antibody is able to inhibit the angiogenesis induced by OS-732 obviously, probably by the mechanism of inhibition of EC proliferation and promotion of their apoptosis, furtherly, which may contribute to the apoptosis of tumor cells and result in suppression of tumor growth.

Animals ; Antibodies ; therapeutic use ; Apoptosis ; Bone Neoplasms ; therapy ; Cell Division ; drug effects ; Chick Embryo ; Disease Models, Animal ; Endothelial Growth Factors ; immunology ; Endothelium, Vascular ; cytology ; drug effects ; Immunotherapy ; Lymphokines ; immunology ; Microscopy ; Neovascularization, Pathologic ; prevention & control ; Osteosarcoma ; therapy ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo explore the postburn adhesion properties of polymorphonuclear leukocyte (PMN) onto pulmonary vascular endothelial cells (PVEC) in burn patients with acute lung injury (ALI), so as to determine the role of C5a on PVEC-PMN adhesion.

METHODSMicrotubule sucking technique was employed to determine the PVEC-PMN adhesion. The myeloperoxidase (MPO) was also assayed to reflect the magnitude of PVEC-PMN adhesion.

RESULTSThe magnitude of PVEC-PMN adhesion increased and the adhesion force increased along with an increase in rh-C5a concentration. Simultaneously, the MPO activity was increased, which could be inhibited by anti-C5aR McAb in a concentration 1:104.

CONCLUSIONBoth C5a and C5aR participated in PVEC-PMN adhesion, which might be important in the pathogenesis of ALI.

Acute Disease ; Antibodies, Monoclonal ; pharmacology ; Antigens, CD ; immunology ; Burns ; blood ; complications ; Cell Adhesion ; drug effects ; Cells, Cultured ; Complement C5a ; pharmacology ; Dose-Response Relationship, Drug ; Endothelium, Vascular ; cytology ; drug effects ; Fetus ; Humans ; Lung ; Lung Diseases ; complications ; Neutrophils ; cytology ; drug effects ; enzymology ; Peroxidase ; antagonists & inhibitors ; drug effects ; metabolism ; Receptor, Anaphylatoxin C5a ; Receptors, Complement ; immunology