Saturated, but not unsaturated, fatty acids induce apoptosis of human coronary artery endothelial cells via nuclear factor-kappaB activation

High nonesterified fatty acid (NEFA) concentrations, as observed in the metabolic syndrome, trigger apoptosis of human umbilical vein endothelial cells. Since endothelial apoptosis may contribute to atherothrombosis, we studied the apoptotic susceptibility of human coronary artery endothelial cells (HCAECs) toward selected NEFAs and the underlying mechanisms. HCAECs were treated with single or combined NEFAs. Apoptosis was quantified by flow cytometry, nuclear factor kappaB (NFkappaB) activation by electrophoretic mobility shift assay, and secreted cytokines by enzyme-linked immunosorbent assay. Treatment of HCAECs with saturated NEFAs (palmitate and stearate) increased apoptosis up to fivefold (P < 0.05; n = 4). Unsaturated NEFAs (palmitoleate, oleate, and linoleate) did not promote apoptosis but prevented stearate-induced apoptosis (P < 0.05; n = 4). Saturated NEFA-induced apoptosis neither depended on ceramide formation nor on oxidative NEFA catabolism. However, NEFA activation via acyl-CoA formation was essential. Stearate activated NFkappaB and linoleate impaired stearate-induced NFkappaB activation. Pharmacological inhibition of NFkappaB and inhibitor of kappaB kinase (IKK) also blocked stearate-induced apoptosis. Finally, the saturated NEFA effect on NFkappaB was not attributable to NEFA-induced cytokine production. In conclusion, NEFAs display differential effects on HCAEC survival; saturated NEFAs (palmitate and stearate) are proapoptotic, and unsaturated NEFAs (palmitoleate, oleate, and linoleate) are antilipoapoptotic. Mechanistically, promotion of HCAEC apoptosis by saturated NEFA requires acyl-CoA formation, IKK, and NFkappaB activation.     

Epstein-Barr virus latent membrane protein 1 activates nuclear factor-kappa B in human endothelial cells and inhibits apoptosis

BACKGROUND: Although vascular changes and transplant vasculopathy have been described with cytomegalovirus, the impact of Epstein-Barr virus (EBV) on the vascular endothelium of the transplanted allograft is largely unknown. We recently reported that EBV (+) patients taken off immunosuppressive medications for periods of time had a low incidence of chronic rejection. In another report, we noted that there was expression of the "protective" antiapoptotic factor Bcl-2 in the vascular endothelium of transplant allografts from EBV (+) patients. In this report, we determined the effect of latent EBV infection on endothelial cell activation and apoptosis. METHODS: Cultured human umbilical vein endothelial cells (HUVEC) were either infected with EBV or transduced with EBV latent membrane protein 1 and examined for apoptosis, nuclear factor (NF)-kappaB activation, and expression of chemokines, cytokines, and adhesion molecules. RESULTS: EBV infection and latent membrane protein 1 expression in HUVEC resulted in NF-kappaB activation and increased expression of the cytokines interleukin (IL)-1alpha, IL-1beta, and IL-6; the chemokines IL-8, monocyte chemotactic protein-1, and RANTES; and the adhesion molecules intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin. There was increased expression of the antiapoptotic genes A1, c-IAP2, and TRAF1; inhibition of caspase 3; and protection from apoptosis. CONCLUSIONS: Latent EBV in HUVEC results in constitutive NF-kappaB activation, protection from apoptosis, and increased basal expression of inflammatory factors. The in vivo effect of latent EBV in the vascular endothelium of the transplanted allograft and its resultant impact on transplant vasculopathy are the subject of further investigations in our laboratory.     

Prostate-specific antigen stimulates osteoprotegerin production and inhibits receptor activator of nuclear factor-kappaB ligand expression by human osteoblasts

BACKGROUND: Prostate cancer cells produce a large amount of prostate-specific antigen (PSA), which is widely used as a marker for this cancer. Even though it is widely used in the diagnosis of prostate cancer, many aspects of the pathophysiologic role of PSA in bone metastasis remain obscure. The receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for the activation of osteoclasts, while osteoprotegerin (OPG) neutralizes the action of RANKL. Various substances that act on bone have been shown to modulate the production of RANKL and OPG by osteoblasts. METHODS: In this study, we investigated the effect of PSA on the expression of OPG and RANKL mRNA and on protein production in human osteoblast-like cells. RESULTS: After addition of PSA and culture for 72 h, OPG mRNA expression and protein secretion by MG-63 and SaOS-2 cells showed a concentration-dependent increase. When osteoblasts were incubated with PSA (100 ng/ml), OPG mRNA expression and protein secretion increased with the passage of time. alpha1 -antichymotrypsin (ACT), which inactivates the serine protease activity of PSA, inhibited the increase of OPG mRNA expression and protein production in response to PSA, and this effect of PSA was also inhibited by anti-transforming growth factor-beta antibody. CONCLUSIONS: Based on our findings, PSA acts on human osteoblast-like cells via its own serine protease activity and promotes osteoblast differentiation. In addition, PSA stimulates OPG production and inhibits RANKL expression of osteoblasts, and inhibits bone resorption by osteoclasts, suggesting that it contributes to the characteristic osteoblastic features of bone metastases of prostate cancer.     

Cell-mediated cytotoxicity patterns of cloned cytotoxic T lymphocytes. Cytotoxicity directed against canine lymphoblasts, monocytes, and endothelial cells

Knowledge of the antigens recognized during allograft rejection is still incomplete. Cloned cytotoxic T lymphocytes were used to study the distribution of target determinants in the dog. CTL clones were obtained with a limiting dilution technique from effector cells generated in mixed lymphocyte culture. The clones have been tested for cell-mediated cytotoxicity against PHA-stimulated lymphoblasts, monocytes, and arterial and venous endothelial cells. A limited number of patterns of lysis of one, or more than one, of the four different target cells was observed. The nature of the possible target determinants recognized by these CTL-clones is discussed.     

Hypoxia regulates PDGF-B interactions between glomerular capillary endothelial and mesangial cells

BACKGROUND: Platelet-derived growth factor (PDGF)-B regulates mesangial cell and vessel development during embryogenesis, and contributes to the pathogenesis of adult renal and vascular diseases. Endothelial cell PDGF-B exerts paracrine effects on mesangial cells, but its regulation is not well defined. We examined the impact of hypoxia on PDGF-B-mediated interactions between glomerular endothelial and mesangial cells, a condition of potential relevance in developing, and diseased adult, kidneys. METHODS: Glomerular endothelial or mesangial cells were subjected to hypoxia and responses compared to normoxic cells. Endothelial PDGF-B was studied by Northern and Western analysis. Mesangial proliferative responses to PDGF-B were assessed by (3)H-thymidine incorporation, and migration by a modified Boyden chamber assay. Hypoxia-induced changes in receptor specific binding capacity were studied by saturation binding assays. RESULTS: Hypoxia stimulated increases in endothelial PDGF-B mRNA and protein. In normoxic mesangial cells, PDGF-B stimulated dose-dependent proliferation, but the proliferative response of hypoxic cells was two to three times greater. Exogenous PDGF-B also caused prompter migration in hypoxic mesangial cells. Mesangial cells were treated with endothelial cell-conditioned medium. More cells migrated when hypoxic cells were stimulated with hypoxic conditioned medium, than when normoxic cells were stimulated with normoxic conditioned medium. Preincubating conditioned medium with PDGF-B neutralizing antibody greatly decreased the chemoattractant activity. Binding studies demonstrated increased specific binding capacity in hypoxic cells. CONCLUSION: Hypoxia enhances PDGF-B paracrine interactions between glomerular endothelial and mesangial cells. These hypoxia-regulated interactions may be important during glomerulogenesis in fetal life and during the pathogenesis of adult glomerular disease.     

T lymphocyte activation by cytomegalovirus-infected, allogeneic cultured human endothelial cells.

Cytomegalovirus, a source of serious complications among immunosuppressed individuals, infects endothelial cells in vivo, and has been epidemiologically associated with allograft rejection and transplantation-associated accelerated arteriosclerosis (TxAA). As the interface between the host immune system and allograft tissues, the endothelium may be of primary importance in mediating pathogenic immune responses to CMV. We have therefore investigated T lymphocyte responses to CMV-infected allogeneic human umbilical vein endothelial cells (HUVE) in vitro. Proliferation assays demonstrated dramatically enhanced responses by CMV-seropositive donor-derived PBMC or purified T cells cocultured with CMV-infected HUVE, as compared with those elicited by noninfected stimulator cells. As determined by limiting dilution analysis of IL-2-producing cells, the frequency of T cells responding to infected HUVE was generally found to exceed by approximately one order of magnitude those responding to uninfected cells. Similar analyses of isolated T cell subsets revealed that these responses (proliferation and IL-2 production) were nearly entirely accountable to the CD4+ fraction. Responses of CD8+ populations, however, varied among donors. The marked activation of CD4+ cells is particularly intriguing since we have shown that CMV-infected HUVE do not express HLA class II antigens. Responses of lymphocytes derived from CMV-seronegative donors were minimal in all assays. These studies show that purified T cells can respond to CMV in the exclusive context of allogeneic endothelial cells. Furthermore, we demonstrate the heretofore undescribed phenomenon of CD4+ T cell activation in the absence of serologically detectable HLA class II antigen. As vascular rejection and TxAA are characterized by subendothelial T cell infiltration, these findings support a role for CMV/endothelial interaction in their pathogenesis.     

低温缺氧条件下血管内皮细胞的基因表达及与单核细胞间的相互作用

目的探讨在低温缺氧条件下血管内皮细胞RNA转录变化以及血管内皮细胞和单核细胞间的相互作用在再灌注损伤早期过程中的意义。方法将人主动脉内皮细胞经过90min低温缺氧处理后培养4h和8h，通过逆转录聚合酶链反应（RT—PCR）评估相关基因的RNA转录变化；通过细胞粘附实验来检查纯化的单核细胞、CD4^＋ T淋巴细胞与内皮细胞早期粘附水平；通过流式细胞术检测单核细胞吞噬内皮细胞膜的情况。结果经低温缺氧处理的血管内皮细胞上调一系列的炎性细胞介素、共刺激分子以及内皮细胞素和内皮细胞素转换酶-1（ECE-I）、粘附因子等RNA转录水平，而对CD54的表达上调有限。CD4^＋T淋巴细胞与正常内皮细胞和经低温缺氧处理的内皮细胞呈低水平的早期粘附，而单核细胞则表现出与血管内皮细胞呈高水平的早期粘附，且对低温缺氧处理的内皮细胞的早期粘附水平明显增加。单核细胞在和血管内皮细胞的早期相互作用过程中可吞噬内皮细胞膜，但对经低温缺氧处理的内皮细胞膜的吞噬作用并没有增加；CD4^＋T淋巴细胞不具备乔噬内皮细胞膜的功能。结论血管内皮细胞经低温缺氧处理后上调多种炎性介素、共刺激分子、粘附因子等相关的RNA转录；单核细胞与血管内皮细胞间的相互作用在缺血再灌注损伤早期起始过程中扮演着重要的角色。     

Vitamin E inhibits proliferation of primary cultured human mesangial and endothelial cells

BACKGROUND: Vitamin E (VE) has been used as an antioxidant and has been suggested to inhibit the proliferation of mesangial cells in rat and vascular endothelial cells. The direct effect of VE on primary cultures of mesangial cells (MC) and endothelial cells (EC) from the human glomerulus was studied. METHODS: (1) MC (in 17 or 2.5% FCS DMEM) or EC (in 10 or 5% FCS CSC) at 5,000 cells/well was incubated with serial concentrations of VE from 0.05 to 50 microg/ml (0.06 to 60 IU/l). (2) MC was cocultured with 160, 80, 40 or 20 microg/ml of low-density lipoprotein (LDL) or oxidized LDL (ox-LDL) in 17 or 2.5% FCS DMEM with or without VE. After 3 days of incubation at 37 degrees C in 5% CO(2), cell proliferation was measured by the Premix WST-1 Assay System. RESULTS: The concentration of VE that significantly inhibited the proliferation of MC cultured in 17 or 2.5% FCS DMEM was 50 or 2.5 microg/ml (60 or 3.0 IU/l), respectively, and that of EC in 10 or 5% FCS medium was 50 or 25 microg/ml (60 or 30 IU/l). VE at 25 microg/ml (30 IU/l) inhibited the LDL proliferative effect on MC cultured in 2.5 FCS DMEM by 21.79-93.21% in a LDL concentration-dependent manner. There was little difference between the effects of LDL and ox-LDL on the VE inhibitory effect on MC under our experimental conditions. CONCLUSION: VE at low concentrations had no effect on the proliferation of both MC and EC, but at high concentrations, it showed an inhibitory effect on both cells.     

C-reactive protein activates the nuclear factor-kappaB signal transduction pathway in saphenous vein endothelial cells: implications for atherosclerosis and restenosis

OBJECTIVES: Elevated levels of C-reactive protein are one of the strongest prognostic factors in atherosclerosis. In addition to predicting vascular disease, C-reactive protein may directly facilitate the development of a proinflammatory and proatherosclerotic phenotype. Recent studies have demonstrated marked up-regulation of various adhesion molecules and inflammatory responses in endothelial cells subjected to C-reactive protein. The nuclear factor-kappaB signal transduction is known to play a key role in the expression of these proatherogenic entities. This study examines the direct effects of C-reactive protein on nuclear factor-kappaB activation and related mechanisms in saphenous vein endothelial cells. METHODS: The activation of nuclear factor-kappaB was determined by confocal microscopy assessing the nuclear localization of nuclear factor-kappaB in endothelial cells incubated with C-reactive protein (50 microg/mL) for 30 minutes and 3 hours. Cells not incubated with C-reactive protein were used as negative controls, and cells incubated with tumor necrosis factor-alpha (10 ng/mL) for 15 minutes were used as positive controls in all studies. The degradation of IkappaB-alpha and IkappaB-beta was assessed by Western blotting of the cell lysates obtained from cells incubated with human recombinant C-reactive protein (50 microg/mL) for 15 minutes, 30 minutes, and 1 hour. RESULTS: Nuclear factor-kappaB nuclear translocation in endothelial cells increased significantly after 30 minutes of incubation with C-reactive protein (P <.01). Nuclear localization of nuclear factor-kappaB returned to baseline levels after 3 hours of incubation with C-reactive protein. Incubation with C-reactive protein resulted in degradation of IkappaB-alpha that was maximal at 30 minutes (P <.05). C-reactive protein showed no significant effect on IkappaB-beta degradation. CONCLUSIONS: These data demonstrate, for the first time, that C-reactive protein activates the nuclear factor-kappaB signal transduction pathway in endothelial cells. Degradation of IkappaB-alpha, but not IkappaB-beta, seems to be the major pathway leading to nuclear factor-kappaB nuclear translocation and activation induced by C-reactive protein. These data support the concept that C-reactive protein, at concentrations known to predict diverse vascular insults, directly facilitates a proinflammatory and proatherosclerotic phenotype through activation of nuclear factor-kappaB. These data have important implications for saphenous vein atherosclerosis in patients with elevated C-reactive protein levels.     

Evidence that genistein, a protein-tyrosine kinase inhibitor, inhibits CD28 monoclonal-antibody-stimulated human T cell proliferation.

In the present investigation, we compared the immunosuppressive effects of genistein and CsA on anti-CD28 stimulated human T cell proliferation, IL-2 production, and IL-2R expression. Genistein, an isoflavanoid compound, is a specific protein tyrosine kinase inhibitor and inhibited the PMA plus anti-CD28 stimulated T cell proliferation. In contrast, proliferation of T cells stimulated with PMA plus anti-CD28 is resistant to the inhibitory effects of CsA. Similar results were obtained with IL-2 synthesis and IL-2R expression. PHA plus anti-CD28 or PMA plus anti-CD28-induced IL-2 synthesis was inhibited by genistein, and CsA, though it inhibited the PHA plus PMA-stimulated IL-2 synthesis, failed to have any effect on PMA plus anti-CD28-induced IL-2 synthesis. Genistein at the concentration that inhibited T cell proliferation and IL-2 synthesis also showed significant inhibitory effects on PMA plus anti-CD28 stimulated IL-2R expression while CsA had no effect on IL-2R from these cultures. Our data suggest that genistein is a powerful immunosuppressive agent, with no toxic effects on T cells, and has the potential for use in the prophylaxis and treatment of allograft rejection. Since genistein blocks the CsA-resistant pathway of T cell proliferation, the combined usage of these two agents may provide better immunosuppressive effect and a lesser degree of CsA-induced nephrotoxicity.     

Glomerular endothelial cells and podocytes jointly synthesize laminin-1 and -11 chains

BACKGROUND: The glomerular basement membrane (GBM) originates in development from fusion of subendothelial and subepithelial matrices. Subsequently, newly synthesized subepithelial matrix is added as glomerular capillary loops expand. During GBM assembly, the laminin-1 heterotrimer (alpha 1, beta 1, and gamma 1 chains), initially expressed in vascular clefts of comma- and S-shaped bodies, is eventually replaced by laminin-11 (alpha 5, beta 2, and gamma 1 chains), which persists into maturation. The cellular source(s) of these laminins is not known and prompted this study. METHODS: To determine which cells synthesize the various laminin chains, postfixation immunoelectron microscopy of developing mouse kidney was performed using monoclonal and polyclonal antibodies that specifically recognized laminin alpha 1, beta 1, alpha 5, or beta 2 chains. RESULTS: Intracellular labeling for laminin alpha 1, beta 1 (laminin-1), and alpha 5 and beta 2 (laminin-11) chains was observed in developing glomerular endothelial cells and podocytes of comma- and S-shaped nephric figures. Laminin-1 was also seen in unfused GBMs at this stage, whereas laminin-11 was only found intracellularly. In capillary loop stage GBMs, laminin alpha 1 chain was completely absent, whereas labeling for laminin alpha 5 was intense, indicating rapid substitution between alpha chains. In contrast, laminin beta 1 chain labeling remained strong both intracellularly and in GBMs of capillary loop stage glomeruli, and beta 2 was up-regulated as well. In maturing stage glomeruli, beta 1 labeling declined, and alpha 5 and beta 2 remained at high levels intracellularly in both endothelial cells and podocytes and in GBMs. CONCLUSIONS: Our results show that both endothelial cells and podocytes synthesize laminin-1 and -11 chains throughout glomerular development. The sustained and comparatively high level of laminin synthesis by endothelial cells was unexpected, suggesting that the endothelium may be an important source of GBM proteins in glomerular disease.     

Interaction between proximal tubular epithelial cells and infiltrating monocytes/T cells in the proteinuric state

We hypothesize an interaction between T cells/monocytes and the tubules in the development of tubulointerstitial injury in chronic proteinuric nephropathy. We established in vitro co-culture systems of proximal tubular epithelial cells (PTEC) and T cells/monocytes to study the contribution of soluble factors and cell-to-cell contact in the development of tubulointerstitial injury. The release of monocyte chemoattractant protein-1 (MCP1 or CCL2), Regulated upon Activation, normal T cell Expressed and Secreted (RANTES or CCL5), soluble intracellular adhesion molecules-1 (sICAM-1), or interleukin-6 (IL-6) was increased in PTEC following apical exposure to human serum albumin (HSA). The release of CCL2, CCL5, or sICAM-1 from PTEC was enhanced by contact of monocytes/T cells on the basolateral surface. Tumor necrosis factor-alpha (TNF-alpha) and IL-1beta are important soluble factors as suggested by the blocking effect of antibodies (Abs) against TNF-alpha or IL-1beta but not against other cytokines. The percentage of CD4+ T cells expressing both chemokine receptors, CCR2 and CCR5, was increased after culturing with supernatant from the apical or basolateral surface of PTEC following apical exposure to HSA. However, only CCR2 was upregulated in CD8+ T cells, whereas CCR5 expression was increased in monocytes. The chemotaxis of CD4+ or CD8+ T cells to supernatant from PTEC upon apical exposure to HSA was reduced with neutralizing Abs against CCL5 and/or CCL2, whereas the chemotaxis of monocytes was only reduced by anti-CCL5 but not by anti-CCL2. In summary, chemokines released by HSA-activated PTEC are amplified by monocytes/T cells. Mediators released by HSA-activated PTEC can differentially modulate the expression of chemokine receptors in monocytes/T cells and hence, alter their chemotaxis towards activated PTEC. These interactions are pivotal in the development of tubulointerstitial injury.