Endothelium : journal of endothelial cell research最新文献

The study has been designed to investigate the effect of 8-Br-cAMP, an activator of protein kinase A (PKA), in diabetes mellitus- and hyperhomocysteinemia-induced vascular endothelial dysfunction. Streptozotocin (55 mg kg-1, i.v.) and methionine (1.7% w/w, p.o., 4 weeks) were administered to rats to produce diabetes mellitus (serum glucose >200 mg dL-1) and hyperhomocysteinemia (serum homocysteine >10 microM), respectively. Vascular endothelial dysfunction was assessed using isolated aortic ring preparation, electron microscopy of thoracic aorta, and serum concentration of nitrite/nitrate. The expression of mRNA for p22phox and endothelial nitric oxide synthase (eNOS) was assessed by using reverse transcriptase-polymerase chain reaction (TBARS) (RT-PCR). Serum thiobarbituric acid-reactive substances (TBARS) concentration and aortic superoxide anion concentration were estimated to assess oxidative stress. 8-Br-cAMP (5 mg kg-1, i.p.) or atorvastatin (30 mg kg-1, p.o.) prevented diabetes mellitus- and hyperhomocysteinemia-induced attenuation of acetylcholine-induced endothelium-dependent relaxation, impairment of vascular endothelial lining, decrease in expression of mRNA for eNOS, serum nitrite/nitrate concentration, and increase in expression of mRNA for p22phox, superoxide anion, and serum TBARS. The ameliorative effect of 8-Br-cAMP was prevented by N omega-nitro-L-arginine methyl ester (L-NAME) (25 mg kg-1, i.p.) and glibenclamide (5 mg kg-1, i.p.). Therefore, it may be concluded that 8-Br-cAMP-induced activation of PKA may improve vascular endothelial dysfunction.

Vascular hypotheses provide compelling pathogenic mechanisms for the etiology of avascular necrosis of the femoral head (ANFH). A decrease in local blood flow of the femoral head has been postulated to be the cause of the disease. Several studies in human and animal models of ANFH have shown microvascular thrombosis. Endothelial cell damage could be followed by abnormal blood coagulation and thrombus formation with any resulting degeneration distal to the site of vascular occlusion. Other studies suggest that thrombophilia, particularly impaired fibrinolysis, plays a potential role in thrombus formation in ANFH. Reduction in shear stress due to decreased blood flow could lead to apoptosis of endothelial cells, which can ultimately contribute to plaque erosion and thrombus formation. Dysregulation of endothelial cell activating factors and stimulators of angiogenesis or repair processes could also affect the progression and outcome of ANFH. Likewise, regional endothelium dysfunction (RED), referred to as a potential defect in endothelial cells located in the feeding vessels of the femoral head itself, may also have a crucial role in the pathogenesis of ANFH. Molecular gene analysis of regional endothelial cells could also help to determine potential pathways important in the pathogenesis of ANFH.

Diabetes is associated with augmentation of prothrombogenic von Willebrand factor (vWF) content in plasma. Earlier, the author and colleagues have shown that high glucose and insulin do not appreciably influence deposition of vWF into the subendothelial extracellular matrix (SECM) produced by cultured human umbilical vein endothelial cells (HUVECs). In the present work, the author used this model to test the effects of nonenzymatically glycated albumin (Glyc-HSA) and two lectins, concanavalin A (ConA) and wheat germ agglutinin (WGA), on vWF deposition into the SECM. First-passage HUVECs were seeded into gelatin-coated 96-well plates and cultured for 6 to 7 days. HSA or Glyc-HSA (at concentrations 25, 50, and 100 microg/mL), and WGA or ConA (4, 8, and 16 microg/mL) were added 3 h after seeding. Cell viability was tested by the MTT method. To determine vWF contents in the SECM, HUVECs were detached by treatment with NH4OH and the residual material was used as a solid phase in an enzyme-linked immunosorbent assay (ELISA)-like assay with primary (anti-vWF) and secondary (peroxidase-conjugated) antibodies. Addition of Glyc-HSA did not essentially influence VWF contents in the SECM (A490 was 0.226 versus 0.268 at 0 and 100 microg/mL, respectively; p > .05, n = 16). Cultivation in the presence of WGA led to the deterioration of cell viability, which was accompanied by a significant decrease of vWF in the SECM (0.248 versus 0.128 at 0 and 16 microg/mL, respectively; p < .001, n = 16). ConA did not influence viability of HUVECs, but this lectin at all concentrations consistently increased the deposition of vWF (up to 164% relative to control, p <.001; n = 16). These data indicate that endothelial carbohydrate determinants and corresponding ligands (namely, mannose-specific lectins) may be involved in the regulation of production and deposition of vWF.

Vascular endothelial cells (ECs) are constantly exposed to blood flow-induced shear stress; these forces strongly influence the behaviors of neighboring vascular smooth muscle cells (VSMCs). VSMC migration is a key event in vascular wall remodeling. In this study, the authors assessed the difference between VSMC migration in VSMC/EC coculture under static and shear stress conditions. Utilizing a parallel-plate coculture flow chamber system and Transwell migration assays, they demonstrated that human ECs cocultured with VSMCs under static conditions induced VSMC migration, whereas laminar shear stress (1.5 Pa, 15 dynes/cm2) applied to the EC side for 12 h significantly inhibited this process. The changes in VSMC migration is mainly dependent on the close interactions between ECs and VSMCs. Western blotting showed that there was a consistent correlation between the level of Akt phosphorylation and the efficacy of shear stress-mediated EC regulation of VSMC migration. Wortmannin and Akti significantly inhibited the EC-induced effect on VSMC Akt phosphorylation and migration. These results indicate that shear stress protects against endothelial regulation of VSMC migration, which may be an atheroprotective function on the vessel wall.

Nuclear factor of activated T cells, Cytoplasmic 1 (NFATc1) is required for heart valve formation. Vascular endothelial growth factor (VEGF) signaling, mediated by NFATc1 activation, positively regulates growth of valvular endothelial cells. However, regulators of VEGF/NFATc1 signaling in valve endothelium are poorly understood. Peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits NFATc1 activity in T cells and cardiomyocytes, but it is not known if PPARgamma controls NFATc1 function in endothelial cells. The authors hypothesize PPARgamma antagonizes VEGF signaling in valve endothelium by inhibiting NFATc1. Endothelial cells isolated from human valve leaflet tissue were shown by immunocytochemistry to express the endothelial-specific markers von Willebrand factor (vWF) and platelet endothelial cell adhesion molecule (PECAM)-1. VEGF-induced proliferation and migration of human pulmonary valve endothelial cells (HPVECs) were inhibited by rosiglitazone (ROSI), a specific ligand of PPARgamma activation, suggesting that PPARgamma disrupts VEGF signaling in the valve endothelium. ROSI also antagonized VEGF-mediated NFATc1 nuclear translocation in HPVECs, suggesting that PPARgamma inhibits VEGF signaling of NFATc1 activation in the valve. The effect of ROSI on nonvalve human umbilical vein endothelial cells (HUVECs) was tested in parallel and a similar inhibition of NFATc1 activation was observed. These data provide the first demonstration that ROSI negatively regulates VEGF signaling in the valve endothelium by a mechanism involving NFATc1 activation and nuclear translocation.

Lysophosphatidylcholine (LPC) and 7-ketocholesterol (7-KC) are two key components of oxidized low-density lipoprotein (oxLDL) and have been shown to injure endothelial cells derived from various species. This report examines LPC- and 7-KC-induced cell death in mouse aorta endothelial cells (MAECs). The presence and the mechanism of cell death were assessed with morphological criteria, Hoechst 33342 and propidium iodide fluorescence staining, and caspase-3 activity. The authors observed that 7-KC induced cell shrinkage, nuclear condensation, and caspase-3 activity. In contrast, LPC induced membrane rupture, nuclear expansion, and cell lysis. In addition, 7-KC induced a transient increase, whereas LPC induced a sustained increase in intracellular Ca2+ levels and production of reactive oxygen species (ROS). Antioxidants and calcium antagonists attenuated both 7-KC- and LPC-induced cell death. These findings suggest that 7-KC and LPC injure MAECs through differential mechanisms; LPC induces necrosis, 7-KC induces apoptosis, and the increase in intracellular Ca2+ levels and production of ROS are common mechanisms for these cytotoxic injuries.

It has become increasingly clear that stress-activated protein kinases have cytoplasmic substrates in addition to well-established transcription factor substrates in cell nuclei. The present study documented specific cytoplasmic locations of these enzymes in proliferating vascular cells. Immunofluorescent staining for active c-jun NH2-terminal kinase (JNK), the precipitation of JNK with microfilaments, and the loss of fiber-associated active JNK after cytochalasin treatment, but not nocodazole treatment, together indicate that active JNK is associated with stress fibers. The lack of complete scaffold colocalization and the total lack of immediate upsteam kinase colocalization along with the inability of JNK inhibitors to alter JNK-microfilament associations suggest that the microfilament association is not simply involved in enzyme activation. In addition, active p38 was found along with vinculin in focal adhesions. Although the p38 in focal adhesions could also be disrupted by cytochalasin treatment, it remained stable after nocodazole treatment. These results support the hypothesis that vascular cell stress kinase enzymes are important for signal transduction in the cytoplasm. The localization of active stress-activated protein kinases to specific cytoskeletal structures in proliferating cells suggests that subsets of these enzymes are involved in signal transduction to and/or from the cytoskeleton under conditions that include vascular cell proliferation.

In this study, the authors isolated morphologically different capillary endothelial cells, designated as BCE-1 and BCE-2 cells, from bovine adrenal cortex. By a series of experiments involving proliferation, migration, and tubular-like structure formation assays, the authors found that the two BCE clones showed a clearly different response to basic fibroblast growth factor (bFGF). Similar to these results, the ERK-1/2 in the BCE-1 cells was phosphorylated by bFGF or vascular endothelial growth factor (VEGF), whereas that of the BCE-2 cells was phosphorylated only by VEGF. However, when the BCE-2 cells were transfected with FGF receptor 1 cDNA, the ERK-1/2 of these cells was phosphorylated by exogenous bFGF. Receptor binding experiments revealed that BCE-2 cells expressed high-affinity tyrosine-kinase FGF receptors approximately twofold less than BCE-1 cells. Transfection and receptor binding studies suggest a possibility that the poor response of the BCE-2 cells to exogenous bFGF is derived from the limitation of functional availability of high affinity FGF receptors. On the other hand, when both BCE clones were treated with anti-bFGF antibodies, basal formation of tubular-like structure in both clones were strongly inhibited, indicating that endogenous bFGF plays a role in in vitro angiogenesis of both BCE clones. Taken together, these data show that the isolated capillary endothelial cells are heterogeneous for paracrine but not autocrine bFGF signaling, and suggest that the diversity of capillary endothelial cells can occur by angiogenic factors, such as bFGF.

In the authors' previous studies, they found that phosphatidylcholine-specific phospholipase C (PC-PLC) and phosphatidylinositol-specific phospholipase C (PI-PLC) played contrary roles in the apoptosis of vascular endothelial cells (VECs), but the mechanism underlying the phenomenon remains unclear. To address this question, in this study, the authors investigated the changes of cell cycle distribution, the expression of P53, and the phosphorylation of Akt when PI-PLC was inhibited by its specific inhibitor compound 48/80, and they also examined the phosphorylation of Akt when VEC apoptosis was inhibited by D609, a specific inhibitor of PC-PLC. The results showed that suppression of PI-PLC promoted VEC apoptosis by inhibiting Akt phosphorylation, elevating P53 expression, and affecting the cell cycle distribution. Contrarily, suppression of PC-PLC promoted the phosphorylation of Akt. The data suggested that PI-PLC and PC-PLC might control the apoptosis by jointly regulating Akt phosphorylation, P53 expression, and affecting cell cycle in VECs.