Properties Of Vascular Endothelial Cells Research Articles

The effect of lipid-lowering therapies on the pro-inflammatory and anti-inflammatory properties of vascular endothelial cells.

Atherosclerosis and cardiovascular events can be prevented, or treated, using statin therapy, either alone or in combination with ezetimibe. Chronic inflammation, vascular proliferation, and the development of atherosclerosis are also influenced by 25-hydroxycholesterol (25-OHC). The aim of the study was to compare the direct pleiotropic effects of two commonly-used statins (atorvastatin, rosuvastatin), ezetimibe, and their combinations, on the mRNA expression of pro-inflammatory IL1β, IL-18 and IL-23 and anti-inflammatory TGFβ, IL-35 (EBI3, IL-12 subunits), IL-10 and IL-37, in endothelial cells damaged by 25-OHC. It also analyzed IL-35 expression at the protein level. HUVECs were stimulated with atorvastatin (5 μM), rosuvastatin (10 μM), ezetimibe (1.22 μM), atorvastatin-ezetimibe (5 μM + 1.22 μM) or rosuvastatin-ezetimibe (10 μM + 1.22 μM), with or without pre-incubation with 10 μg/mL 25-OHC. mRNA expression was analyzed by real-time PCR. The protein level of IL-35 was analyzed by ELISA. In the pre-stimulated HUVECs, atorvastatin and rosuvastatin decreased mRNA expression of IL1β, IL-18, IL-23, TGFβ, IL35 and increased mRNA expression of IL-10 and IL-37 compared to 25-OHC. Furthermore, only incubation with rosuvastatin and rosuvastatin-ezetimibe decreased IL-35 mRNA and protein levels. Ezetimibe down-regulated only IL1β. Treatment with rosuvastatin-ezetimibe and atorvastatin-ezetimibe reversed the effect of 25-OHC in IL1β, IL-18 and IL-35 mRNA expression. In conclusion, rosuvastatin has the strongest anti-inflammatory effects and is the best at reducing the effect of oxysterols. Both statins exert a greater anti-inflammatory effect than ezetimibe. The anti-inflammatory effect of the combination therapies appears to be based on the effects of the statins alone and not their combination with ezetimibe.

Crosstalk between H1975 tumor cells and platelets to induce the proliferation, migration and tube formation of vascular endothelial cells.

Activated platelets (PLTs) participate in the regulation of tumor angiogenesis, and tumors can activate PLTs. Whether co-culture of lung carcinoma with PLTs improves the function of human umbilical vein endothelial cells (HUVECs) requires further investigation. The present study aimed to investigate the impact of H1975 cell crosstalk with PLTs on the proliferation, migration and tube formation of HUVECs. Following generation of cell-derived supernatants and construction of the co-culture system, Cell Counting Kit-8, flow cytometry, transmission electron microscopy and a meter for epithelial measurement were performed to detect the proliferative ability of HUVECs. Furthermore, the wound healing and Transwell migration assays were performed to detect the migratory ability of HUVECs. A tube formation assay was performed to assess angiogenesis, ELISA was applied to detect the content of vascular endothelial growth factor (VEGF) and western blotting was carried out to measure the expression levels of VEGF receptor 2 (VEGFR2) in HUVECs. Compared with single-cultured HUVECs (control), co-culture with H1975 cells and PLTs (Exp_HP) improved cell proliferation, increased the proportion of cells in the S-phase, destroyed the cell ultrastructure and decreased transepithelial electrical resistance in HUVECs. In addition, a higher relative migration rate, greater number of migrated cells, stronger tube-forming ability and increased expression of VEGF and VEGFR2 were detected in the Exp_HP group compared with the control group. The properties of HUVECs in Exp_H (co-cultured with H1975 cells) were similar to those in Exp_HP, but significantly weaker. Taken together, the results of the present study suggest that tumor cells interacting with PLTs may play an important role in tumor angiogenesis by affecting or mediating changes in the properties of vascular endothelial cells (VECs).

SU5416 attenuated lipopolysaccharide-induced acute lung injury in mice by modulating properties of vascular endothelial cells.

Background and aim: A potent and selective vascular endothelial growth factor receptor (VEGFR) inhibitor SU5416, has been developed for the treatment of solid human tumors. The binding of VEGF to VEGFR plays a crucial role in the pathophysiology of respiratory disorders. However, the impact of SU5416 on lipopolysaccharide (LPS)-induced acute lung injury (ALI) remains unclear. Thus, this study aimed to illuminate the biofunction of SU5416 in the mouse model of ALI.Methods: Wild-type (WT) and toll-like receptor 4 (TLR4)-deficient (TLR4−/-) C57BL/6 mice were used to establish LPS-induced ALI model. The primary pulmonary microvascular endothelial cell (PMVEC) was extracted for detection of endothelial barrier function.Results: LPS significantly increased the number of inflammatory cells and inflammatory cytokines in bronchoalveolar lavage fluid (BALF). In addition, LPS increased alveolar epithelial cells injury, inflammation infiltration and vascular permeability of PMVEC in WT and TLR4−/- mice. Western blotting experiment indicated VEGF/VEGFR and TLR4/NF-κB pathways were involved in the progression of LPS-stimulated ALI. Consistent with previous research, dexamethasone treatment appeared to be an effective therapeutic for mice with ALI. Moreover, treatment with SU5416 dramatically attenuated LPS-induced immune responses in mice lung tissues via inhibiting VEGF/VEGFR and TLR4/NF-κB pathways. Finally, SU5416 also decreased vascular permeability of PMVEC in vitro.Conclusion: SU5416 ameliorated alveolar epithelial cells injury and histopathological changes in mice lung via inhibiting VEGF/VEGFR and TLR4/NF-κB signaling pathways. We also confirmed that SU5416 could restrain vascular permeability in PMVEC through improving the integrity of endothelial cell. These findings suggested that SU5416 may serve as a potential agent for the treatment of patients with ALI.

Dynamic monitoring of cell mechanical properties using profile microindentation.

We have developed a simple and relatively inexpensive system to visualize adherent cells in profile while measuring their mechanical properties using microindentation. The setup allows simultaneous control of cell microenvironment by introducing a micropipette for the delivery of soluble factors or other cell types. We validate this technique against atomic force microscopy measurements and, as a proof of concept, measure the viscoelastic properties of vascular endothelial cells in terms of an apparent stiffness and a dimensionless parameter that describes stress relaxation. Furthermore, we use this technique to monitor the time evolution of these mechanical properties as the cells’ actin is depolymerized using cytochalasin-D.

Dynamic Monitoring of Cell Mechanical Properties using Profile Microindentation

We have developed a simple and relatively inexpensive system to visualize adherent cells in profile while measuring their mechanical properties using microindentation. The setup allows simultaneous control of cell microenvironment by introducing a micropipette for the delivery of soluble factors or other cell types. We validate this technique against atomic force microscopy measurements and, as a proof of concept, measure the viscoelastic properties of vascular endothelial cells in terms of an apparent stiffness and a dimensionless parameter that describes stress relaxation. Furthermore, we use this technique to monitor the time evolution of these mechanical properties as the cells’ actin is depolymerized using cytochalasin-D.

Pivotal Roles of Shear Stress Stimulation in the Regulation of Biological Properties of Vascular Endothelial Cells

Pivotal Roles of Shear Stress Stimulation in the Regulation of Biological Properties of Vascular Endothelial Cells

Viscoelastic Properties of Vascular Endothelial Cells Exposed to Uniaxial Stretch

Vascular endothelial cells (VECs) that line the interior of blood vessels are continually stretched as the vessel walls expand and contract. It is widely accepted that VECs will remodel themselves in response to this mechanical stimuli, leading to changes in cell function. Few studies, however, have analyzed the mechanical properties of these cells under stretch. We hypothesize that uniaxial stretch will cause an anisotropic realignment of actin filaments, and a change in the viscoelastic properties of the cell. To test this hypothesis, VECs were grown on a thin, transparent membrane mounted on a microscope. The membrane was stretched, consequently stretching the cells. Time-lapse sequences of the cells were taken every hour with a time resolution of 10 Hz. The random trajectories of intracellular endogenous particles were tracked using in-house algorithms. Using a novel particle tracking microrheology formulation, that takes into account the anisotropic nature of the cytoplasm, these trajectories were analyzed and the mechanical properties of VECs subjected to various stretching conditions were calculated and compared.

Vascular endothelial cells cultured from patients with cerebral or uncomplicated malaria exhibit differential reactivity to TNF.

Plasmodium falciparum malaria is a major cause of morbidity and mortality in African children, and factors that determine the development of uncomplicated (UM) versus cerebral malaria (CM) are not fully understood. We studied the ex vivo responsiveness of microvascular endothelial cells to pro-inflammatory stimulation and compared the findings between CM and UM patients. In patients with fatal disease we compared the properties of vascular endothelial cells cultured from brain tissue to those cultured from subcutaneous tissue, and found them to be very similar. We then isolated, purified and cultured primary endothelial cells from aspirated subcutaneous tissue of patients with CM (ECCM) or UM (ECUM) and confirmed the identity of the cells before analysis. Upon TNF stimulation in vitro, ECCM displayed a significantly higher capacity to upregulate ICAM-1, VCAM-1 and CD61 and to produce IL-6 and MCP-1 but not RANTES compared with ECUM. The shedding of endothelial microparticles, a recently described parameter of severity in CM, and the cellular level of activated caspase-3 were both significantly greater in ECCM than in ECUM. These data suggest that inter-individual differences in the endothelial inflammatory response to TNF may be an additional factor influencing the clinical course of malaria.

Lipid Rafts in Membrane–Cytoskeleton Interactions and Control of Cellular Biomechanics: Actions of oxLDL

Membrane-cytoskeleton coupling is known to play major roles in a plethora of cellular responses, such as cell growth, differentiation, polarization, motility, and others. In this review, the authors discuss the growing amount of evidence indicating that membrane-cytoskeleton interactions are regulated by the lipid composition of the plasma membrane, suggesting that cholesterol-rich membrane domains (lipid rafts), including caveolae, are essential for membrane-cytoskeleton coupling. Several models for raft-cytoskeleton interactions are discussed. Also described is the evidence suggesting that raft-cytoskeleton interactions play key roles in several cytoskeleton-dependent processes, particularly in the regulation of cellular biomechanical properties. To address further the physiological significance of raft-cytoskeleton coupling, the authors focus on the impact of oxidized low density lipoproteins, one of the major cholesterol carriers and proatherogenic factors, on the integrity of lipid rafts/caveolae, and on the organization of the cytoskeleton. Finally, the authors review the recent studies showing that oxLDL and cholesterol depletion have similar impacts on the biomechanical properties of vascular endothelial cells, which in turn affect endothelial angiogenic potential.

Proteoglycans Predominantly Synthesized by Human Brain Microvascular Endothelial Cells in Culture are Perlecan and Biglycan

It has been suggested that the properties of vascular endothelial cells differ depending on the type of blood vessel. Proteoglycans are macromolecules that contribute to vascular properties through regulation of endothelial cell functions. In this study, we characterized proteoglycans synthesized by cultured human brain microvascular endothelial cells by biochemical techniques. The experiments indicated that the cells synthesize and secrete two types of proteoglycans: large heparan sulfate proteoglycans bearing heparan sulfate chains of approximately Mr~68000 and chondroitin/dermatan sulfate proteoglycans bearing chondroitin/dermatan sulfate chains of approximately Mr~48000. The heparan and chondroitin/dermatan sulfate proteoglycans were identified as perlecan and biglycan, respectively, by Western blot analysis. A part of perlecan was associated with the cell layer, while most of biglycan was secreted into the medium. These results suggest that the characteristics of proteoglycans synthesized by brain microvascular endothelial cells are similar to the characteristics of those synthesized by previously reported arterial endothelial cells. Regulation of endothelial cell functions by proteoglycans may be independent of blood vessel types.

Coagulopathic complications in breast cancer.

Patients with cancer are highly susceptible to thromboembolic complications, which account for a significant percentage of the morbidity and mortality of the disease. Up to 15% of patients with clinically overt cancer present with venous thromboembolism during the course of their disease. Moreover, patients with cancer represent 20% of all patients in whom deep venous thrombosis and pulmonary embolism are diagnosed. This prothrombotic state in cancer can occur due to the ability of tumor cells to directly activate the blood-clotting cascade and cause thrombosis or induce procoagulant properties and inhibit anticoagulant properties of vascular endothelial cells, platelets, monocytes, and macrophages. It also is well established that this prothrombotic tendency in patients with cancer can be enhanced greatly by anticancer treatments, such as surgery and chemotherapy. This phenomenon can be seen in patients with breast cancer, particularly after surgery and chemotherapy. Increased clotting risk also is associated with the use of central venous access devices, commonly used to administer chemotherapeutic agents in patients with cancer. Thrombosis prophylaxis, therefore, should be considered for patients with breast cancer who are at risk before and during intervention. In the current review, the authors discuss the problem of thromboembolism in patients with breast cancer who are undergoing therapy, the mechanisms by which thromboembolisms occur, and the potential strategies by which these events may be prevented. Better understanding of these pathogenetic pathways may lead to the development of more targeted strategies to prevent thromboembolism in patients with cancer.

Goniodomin A, an antifungal polyether macrolide, exhibits antiangiogenic activities via inhibition of actin reorganization in endothelial cells.

Goniodomin A (GDA) is an antifungal polyether macrolide isolated from the dinoflagellate Goniodoma pseudogoniaulax. Previous studies revealed that GDA profoundly affected cytoskeletal reorganization. We examined the effect of GDA on the angiogenic properties of vascular endothelial cells. GDA itself did not affect proliferation of, migration of, and tube formation in type I collagen gels by, bovine aortic endothelial cells (BAECs). Proliferation of BAECs stimulated by bFGF was not affected by GDA at concentrations of up to 10 nM. However, at similar concentrations, GDA significantly inhibited bFGF-induced migration and tube formation in type I collagen gels by BAECs. Actin reorganization is required for cell migration. GDA caused the perinuclear aggregation of filamentous actin and inhibited stress fiber formation in bFGF- or VEGF-stimulated BAECs and lysophosphatidic acid-stimulated HeLa cells. However, GDA did not affect stress fiber structures already formed through Gbetagamma expression or in constitutively active RhoA mutant HeLa cells. Finally, GDA inhibited forming of vasucular system in a chorioallantoic membrane. Our results indicated that GDA suppressed angiogenic properties of ECs at least in part through the inhibition of actin reorganization and inhibited angiogenesis in vivo.

423 マイクロピペット吸引法の接着細胞への応用と血管内皮細胞の力学的特性の計測

423 マイクロピペット吸引法の接着細胞への応用と血管内皮細胞の力学的特性の計測

Pathophysiology of the thrombophilic state in the cancer patient.

The "hypercoagulable state" of malignancy is due to a complex interaction of tumor cells and their products with host cells, leading to various degrees of impairment of the normal defense mechanisms that ordinarily protect the host against thrombogenesis. Tumor cells can activate directly the blood clotting cascade and cause thrombosis or can induce procoagulant properties and inhibit anticoagulant properties of vascular endothelial cells, platelets, and monocytes and macrophages. In the setting of the local and systemic effects of cancer (e.g., stasis induced by prolonged bed rest and/or vascular invasion by tumor), together with iatrogenic complications of the treatment of cancer (e.g., the use of central vein catheters and angiopathic chemotherapy), this basic pathophysiology conspires to make cancer perhaps the best example of "acquired thrombophilia." In this brief review, we have attempted to describe what is currently known about the mechanisms for the hypercoagulable state of cancer and provide a summary of the evidence that indicates the many levels of defects in patients with malignancies that predispose them to thrombosis. A better understanding of the pathophysiology of thrombophilia in cancer should provide clinicians with an improved rationale for more aggressive and specific anticoagulant strategies in selected patients.

Endothelial degradation of extracellular lyso-phosphatidylcholine

Formation of lysophospholipids, including lyso-phosphatidylcholine (lysoPC), is enhanced during oxidation of low-density lipoprotein, in ischaemic tissue and under inflammatory conditions. Besides being potentially cytotoxic, extracellular lysoPC induces changes in several properties of vascular endothelial cells. These include expression of endothelial adhesion molecules and interference with the endothelial production of nitrogen monoxide, prostacyclin and growth factors. One way of controlling the concentration of extracellular lysoPC is by the action of lysophospholipases, which degrade lysoPC into a free fatty acid and glycerophosphocholine. We therefore tested whether vascular endothelial cells have the ability to degrade extracellular lysoPC. Monolayers of primary cultures of human umbilical vein endothelial cells degraded an average of 84+/-24 nmol lysoPC/10(6) cells/2 h. By comparison, monocytes degraded 9.7+/-3.7 nmol lysoPC/10(6) cells/2 h, and erythrocytes and platelets < 1 nmol lysoPC/10(6) cells/2 h. The ability of endothelial cells to degrade extracellular phospholipids (diacylphosphatidyl choline) was found to be relatively low (9.5+/-6.4 nmol/10(6) cells/2 h). Triacylglycerol hydrolase activity was just above detection level. In conclusion, endothelial cells seem to degrade extracellular lysoPC effectively. This endothelial property may be important in controlling plasma and tissue levels of extracellular lysoPC as well as in the interaction between lysoPC and the vascular endothelium.

12-O-Tetradecanoylphorbol-13-Acetate Inhibits Aminophospholipid Translocase Activity and Modifies the Lateral Motions of Fluorescent Phospholipid Analogs in the Plasma Membrane of Bovine Aortic Endothelial Cells

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is a potent mitogenic factor which can replace the growth promoting activity of basic fibroblast growth factor (bFGF) on bovine aortic endothelial cells. However, TPA-treated cells lose their strict contact inhibition at confluence, which is a characteristic of cells grown in the presence of bFGF. We have examined whether these changes could be related to modifications of the transbilayer and lateral motions of fluorescent lipids, namely 1-acyl-2-[6-[N-(7-nitrobenz2-oxa-1,3-diazol-4-yl)amino]caproyl]-phosphatidyl-choline (C6-NBD-PC), -phosphatidylserine (C6-NBD-PS), and -phosphatidylethanolamine (C6-NBD-PE) inserted in the outer leaflet of the cell plasma membrane. In TPA-treated cells, the three fluorescent phospholipids remained located in the outer leaflet for at least 1 h at 20°C after their insertion, indicating a blockade of the aminophospholipid translocase activity which is normally present in the plasma membrane of bFGF-treated cells [1, 2]. TPA also induced a large increase in the percentage of C6-NBD-PC and C6-NBD-PE probes which were free to diffuse laterally. The mobile fractionsMreached values of ∼100% for the two lipids, while for bFGF-treated cells they were found around 85 and 75%, respectively. For the C6-NBD-PS probe,Mremained unchanged in bFGF and TPA-treated cells, at around 85%. TPA treatment also induced a twofold increase in the lateral diffusion coefficients of C6-NBD-PC and C6-NBD-PE, while that of C6-NBD-PS remained nearly unchanged. These effects of TPA may be related to the observed loss of differentiated properties of vascular endothelial cells and not to its mitogenic properties.

Tumor necrosis factor infusions have a procoagulant effect on the hemostatic mechanism of humans

Several investigators have reported that tumor necrosis factor (TNF) can alter the hemostatic properties of vascular endothelial cells in vitro. We have examined the in vivo effects on the hemostatic mechanism of recombinant human TNF administered as a continuous intravenous infusion to 23 cancer patients with active disease. A battery of sensitive and specific immunochemical techniques were used to monitor changes in blood coagulability. Serial determinations of F1 + 2, the protein C activation peptide (PCP), and fibrinopeptide A (FPA) were obtained prior to the initiation of the TNF infusions and at three and 24 hours after the start of therapy in 12 individuals who received greater than 3 x 10(5) U/m2/24h. The mean levels of F1 + 2, PCP, and FPA were significantly elevated at both time points as compared to the baseline values. The metabolic behavior of 125I-F1 + 2 in an animal model was not affected by infusions of the cytokine. We therefore conclude that the observed elevations in the concentration of this marker in humans receiving TNF result from hemostatic system hyperactivity. In 11 subjects infused with 1 x 10(5) to 2.4 x 10(5) U/m2/24 h of the cytokine, the mean levels of F1 + 2, PCP, and FPA were not significantly greater at 24 hours as compared with the baseline values, indicating that there is a threshold dose at which the cytokine can exert a biochemical effect on the coagulation system. Our studies demonstrate that TNF is able to provide a substantial net procoagulant stimulus to the hemostatic mechanism, and suggest that this cytokine may be a mediator of certain hypercoagulable states in humans.

Tumor Necrosis Factor Infusions Have a Procoagulant Effect on the Hemostatic Mechanism of Humans

AbstractSeveral investigators have reported that tumor necrosis factor (TNF) can alter the hemostatic properties of vascular endothelial cells in vitro. We have examined the in vivo effects on the hemostatic mechanism of recombinant human TNF administered as a continuous intravenous infusion to 23 cancer patients with active disease. A battery of sensitive and specific immunochemical techniques were used to monitor changes in blood coagulability. Serial determinations of F1+2, the protein C activation peptide (PCP), and fibrinopeptide A (FPA) were obtained prior to the initiation of the TNF infusions and at three and 24 hours after the start of therapy in 12 individuals who received greater than 3 × 105 U/m2/24 h. The mean levels of F1+2, PCP, and FPA were significantly elevated at both time points as compared to the baseline values. The metabolic behavior of 125I-F1+2 in an animal model was not affected by infusions of the cytokine. We therefore conclude that the observed elevations in the concentration of this marker in humans receiving TNF result from hemostatic system hyperactivity. In 11 subjects infused with 1 × 105 to 2.4 × 105 U/m2/24 h of the cytokine, the mean levels of F1+2, PCP, and FPA were not significantly greater at 24 hours as compared with the baseline values, indicating that there is a threshold dose at which the cytokine can exert a biochemical effect on the coagulation system. Our studies demonstrate that TNF is able to provide a substantial net procoagulant stimulus to the hemostatic mechanism, and suggest that this cytokine may be a mediator of certain hypercoagulable states in humans.

Phenotypic heterogeneity of vascular endothelial cells in the human kidney.

To clarify the structural base of immune response occurring in the kidney, we investigated the antigenic and functional properties of vascular endothelial cells. Peritubular capillary endothelial cells exhibited the same immunohistochemical characteristics (OKM5-positive, HLA-DR-positive, Factor VIII/von Willebrand factor antigen-negative, Interleukin 1-positive) as a peripheral blood macrophage subset capable of presenting soluble antigens and triggering the autologous mixed lymphocyte reaction. On the other hand, endothelial cells of glomerular capillary loops, considered to be involved in blood coagulation, were OKM5-negative, HLA-DR-positive, Factor VIII/von Willebrand factor antigen-positive, Interleukin 1-positive. Thus the results of this study suggest that vascular endothelial cells in different anatomic compartments of the kidney express surface antigens heterogenously and may play different roles in the immune reaction.

Cachectin (tumor necrosis factor): a macrophage hormone governing cellular metabolism and inflammatory response.

Introduction DURING the past few years, a variety of cytokines have been isolated and synthesized in abundance by molecular cloning techniques. One of these, a molecule known as “cachectin” or “tumor necrosis factor,” has emerged as an important component of the inflammatory response. Cachectin is produced by extravascular effector cells (principally macrophages) in response to invasive stimuli, and circulates throughout the body. Cachectin activates neutrophils, alters the properties of vascular endothelial cells, and regulates the metabolic activities of a variety of other tissues as well. Through its multiple effects, cachectin mediates many of the effects of endotoxin, both untoward and beneficial. Moreover, it induces a state of anorexia and wasting similar to that observed in chronic neoplastic and infectious diseases. Thus, this single protein mediator may play a central role in the pathogenesis of diverse disease states. Cachectin, an important factor in numerous physiological processes, was indep...