Superoxide anions and hydrogen peroxide inhibit proliferation of activated rat stellate cells and induce different modes of cell death

Background: In chronic liver injury, hepatic stellate cells (HSCs) proliferate and produce excessive amounts of connective tissue causing liver fibrosis and cirrhosis. Oxidative stress has been implicated as a driving force of HSC activation and proliferation, although contradictory results have been described. Aim: To determine the effects of oxidative stress on activated HSC proliferation, survival and signalling pathways. Methods: Serum‐starved culture‐activated rat HSCs were exposed to the superoxide anion donor menadione (5–25 μmol/L) or hydrogen peroxide (0.2–5 mmol/L). Haem oxygenase‐1 mRNA expression, glutathione status, cell death, phosphorylation of mitogen‐activated protein (MAP) kinases and proliferation were investigated. Results: Menadione induced apoptosis in a dose‐ and time‐dependent, but caspase‐independent manner. Hydrogen peroxide induced necrosis only at extremely high concentrations. Both menadione and hydrogen peroxide activated Jun N‐terminal kinase (JNK) and p38. Hydrogen peroxide also activated extracellular signal‐regulated protein. Menadione, but not hydrogen peroxide, reduced cellular glutathione levels. Inhibition of JNK or supplementation of glutathione reduced menadione‐induced apoptosis. Non‐toxic concentrations of menadione or hydrogen peroxide inhibited platelet‐derived growth factor‐ or/and serum‐induced proliferation. Conclusion: Reactive oxygen species (ROS) inhibit HSC proliferation and promote HSC cell death in vitro. Different ROS induce different modes of cell death. Superoxide anion‐induced HSC apoptosis is dependent on JNK activation and glutathione status.     

Antioxidants stimulate endothelial cell proliferation in culture

The effect of antioxidants on the proliferation of cultured endothelial cells (ECs) were studied. Probucol, a lipid-lowering drug with antioxidant properties, added to the growth medium stimulated the proliferation of ECs. D1-alpha-tocopherol (tocopherol), a natural antioxidant, and butylated hydroxytoluene, a synthetic antioxidant also had the same effect on the proliferation of ECs. There was no significant difference in the content of thiobarbituric acid-reacting substances between the probucol-containing growth medium and control medium during the growth assay. Pretreatment of ECs with probucol or tocopherol also enhanced ECs proliferation in medium without any added antioxidants. The addition of free radical scavengers superoxide dismutase, catalase and mannitol to the growth medium did not enhance the proliferation of ECs. These findings indicate that the stimulatory effect on ECs proliferation of antioxidants in the growth medium appears to be unrelated to preventing free radical reactions in the medium itself, but rather may be related to the uptake of antioxidants by ECs.     

α硫辛酸抑制血管内皮细胞增殖的实验研究

目的 探讨α硫辛酸(LA)对血管内皮细胞增殖的影响.方法 采用人脐静脉内皮细胞(HU-VECs)培养模型,分析LA对HUVECs细胞活力(MTT测定)、细胞增殖(EdU掺入实验)、增殖相关基因表达(定量PCR分析)、细胞死亡[乳酸脱氢酶(LDH)渗漏实验、Hoechst33342细胞核染色]的影响.结果 LA呈剂量依赖...     

Superoxide anions induce the maturation of human dendritic cells

Dendritic cells play a key role in immune responses. There is growing evidence that reactive oxygen species participate in signaling pathways involving nuclear factor (NF)-kappaB, leading to expression of important immune system genes. We found that, unlike H2O2, reactive oxygen species generated by the reaction of oxidase on xanthine induced early phenotypic maturation of dendritic cells by upregulating specific markers CD80, CD83, and CD86 and downregulating mannose receptor-mediated endocytosis. Maturation induced by xanthine oxidase was prevented by allopurinol, an inhibitor of xanthine oxidase activity, and by N-acetylcysteine. The proteasome inhibitor MG-132, which blocks NF-kappaB activation, also inhibited CD86 upregulation, but not endocytosis downregulation by reactive oxygen species. Finally, xanthine-xanthine oxidase enhanced or blocked antigen presentation by dendritic cells depending on whether they had been prepulsed or not with the antigen. Taken together, these results demonstrate that oxidative stress induces phenotypic and functional maturation of dendritic cells, partly through an NF-kappaB-dependent mechanism.     

Role of alphav integrin in osteoprotegerin-induced endothelial cell migration and proliferation

Osteoprotegerin (OPG) is a decoy receptor for the receptor activator of nuclear factor kappaB ligand (RANKL). However, the role of OPG in the endothelium remains unknown. In this study, we demonstrate that OPG stimulates the proliferation and migration of human microvascular endothelial cells (HMVECs). In addition, we show that treatment with integrin alpha(v)beta(3) or integrin alpha(v)beta(5) blocking antibody inhibits endothelial cell migration. In contrast, treatment with anti-alpha(v)beta(3) antibody or anti-alpha(v)beta(5) antibody alone did not inhibit OPG-induced proliferation. However, OPG-induced proliferation was inhibited when these antibodies were applied simultaneously. Furthermore, OPG evoked activation of extracellular signal-regulated kinase (ERK) 1/2, which has been linked to integrin alpha(v) activity. Taken together, these results suggest that integrins alpha(v)beta(3) and/or alpha(v)beta(5) contribute to endothelial cell proliferation and migration induced by OPG.     

Effects of aspirin and indomethacin on endothelial cell proliferation in vitro

BACKGROUND AND AIM: Non-steroidal anti-inflammatory drugs (NSAID) are associated with delayed peptic ulcer healing. Ulcer healing is dependent on angiogenesis, which requires endothelial cell (EC) proliferation. The present study aimed to determine whether NSAID and prostaglandin E2 (PGE2) inhibited EC proliferation in vitro. METHODS: Effects of 50 micro L aspirin (10 micro M-1 mM), indomethacin (10 micro M-1 mM) and PGE2 (1 micro M-0.1 mM) on the proliferation, viability and cell cycle of human dermal microvascular (HuDMEC) and human umbilical vein (HUVEC) EC were assessed using dual staining cell viability, 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide and flow cytometry assays. RESULTS: Proliferation of HuDMEC and HUVEC was significantly inhibited by 0.1 mM/1 mM indomethacin, 1 mM aspirin and 100 micro M PGE2, with a significant (P < 0.05) increase in EC necrosis with 1 mM indomethacin and 100 micro M PGE2. No effects on cell cycle were demonstrated. CONCLUSIONS: High concentrations of NSAID inhibit both HuDMEC and HUVEC proliferation in vitro by cytotoxic (indomethacin) or cytostatic (aspirin and indomethacin) mechanisms. Interestingly, PGE2 was also antiproliferative. Inhibition of EC proliferation may prevent angiogenesis at the ulcer site, which may in part explain the delayed ulcer healing associated with NSAID.     

Vascular endothelial growth factor binds to fibrinogen and fibrin and stimulates endothelial cell proliferation

Vascular development and response to injury are regulated by several cytokines and growth factors including the members of the fibroblast growth factor and vascular endothelial cell growth factor (VEGF) families. Fibrinogen and fibrin are also important in these processes and affect many endothelial cell properties. Possible specific interactions between VEGF and fibrinogen that could play a role in coordinating vascular responses to injury are investigated. Binding studies using the 165 amino acid form of VEGF immobilized on Sepharose beads and soluble iodine 125 ((125)I)-labeled fibrinogen demonstrated saturable and specific binding. Scatchard analysis indicated 2 classes of binding sites with dissociation constants (K(d)s) of 5.9 and 462 nmol/L. The maximum molar binding ratio of VEGF:fibrinogen was 3.8:1. Further studies characterized binding to fibrin using (125)I-labeled VEGF- and Sepharose-immobilized fibrin monomer. These also demonstrated specific and saturable binding with 2 classes of sites having K(d)s of 0.13 and 97 nmol/L and a molar binding ratio of 3.6:1. Binding to polymerized fibrin demonstrated one binding site with a K(d) of 9.3 nmol/L. Binding of VEGF to fibrin(ogen) was independent of FGF-2, indicating that there are distinct binding sites for each angiogenic peptide. VEGF bound to soluble fibrinogen in medium and to surface immobilized fibrinogen or fibrin retained its capacity to support endothelial cell proliferation. VEGF binds specifically and saturably to fibrinogen and fibrin with high affinity, and this may affect the localization and activity of VEGF at sites of tissue injury. (Blood. 2000;96:3772-3778)     

Eggs of Schistosoma mansoni stimulate endothelial cell proliferation in vitro

In humans chronically infected with Schistosoma mansoni, total hepatic blood flow and normal hepatic function appear to be maintained by neovascularization of the periportal fibrotic tissue. To identify possible stimuli for this neovascularization, we evaluated parasite-derived products for their ability to activate endothelial cells in vitro. Soluble egg antigen (SEA) of S. mansoni at concentrations as low as 30 ng/mL induced a marked proliferation of human umbilical vein endothelial cells (HUVE) that increased in a dose-dependent fashion; equivalent effects were seen with bovine adrenal and murine lung endothelial cells. Live, intact S. mansoni eggs secreted a soluble factor that stimulated HUVE in a manner similar to crude SEA. In contrast, adult worm extracts of S. mansoni had no significant effect on endothelial cell proliferation in any of the three cell lines tested. Preliminary biochemical characterization showed the activity to be protease sensitive and heat stable. S. mansoni SEA, in the absence of inflammatory cells, can stimulate endothelial cell proliferation in vitro.     

Inhibition of endothelial cell proliferation and tumor-induced angiogenesis by pentoxifylline

PURPOSE: In this study we investigated the effect of pentoxifylline (PTX) on tumor-induced neovascularization as well as on different steps involved in the angiogenic process. METHODS: To assess angiogenesis inhibition. we injected intradermally (i.d.) 10 B16-F10 melanoma cells into C57BL/6J mice which were subsequently intraperitoneally (i.p.) inoculated with PTX or saline. On day 7 the number of blood vessels converging to the remnant of injected material was counted and the volumes of incipient tumors were calculated in each case. In vitro growth inhibition by PTX was evaluated in two different cell lines of endothelial origin and in human umbilical vein endothelial cells. Motility assays, as well as zymographic assays carried out to analyze gelatinolytic metalloproteinases and urokinase-type plasminogen activator, were performed in one of the endothelial cell lines. RESULTS: A significant inhibition of tumor-induced angiogenesis was observed in C57B1/6 mice i.p. inoculated with PTX, that paralleled reduced incipient tumor volumes. The endothelial cells derived from different sources were inhibited in a dose-response manner by PTX in vitro. Non-cytotoxic PTX concentrations assayed in one of the endothelial cell lines did not inhibit its in vitro cell motility nor its gelatinase secretion, but its low molecular weight urokinase-type plasminogen activator expression. CONCLUSIONS: Our findings suggest that the inhibitory effect of PTX on tumor angiogenesis is related to antiproliferative action on endothelial cells, as well as to down regulation of u-PA secreted by them.     

Effects of Helicobacter pylori on endothelial cell proliferation and chemotaxis

BACKGROUND/AIM: Helicobacter pylori is associated with an increased risk of peptic ulcer disease development and recurrence. Ulcer healing is dependent upon angiogenesis, requiring endothelial cell (EC) proliferation and chemotaxis. This study determined whether extracts of H. pylori affected EC proliferation and/or chemotaxis in vitro. METHODS: The effects of water and broth extracts of three genotypically different strains of H. pylori and of single strains of Campylobacter jejuni and Escherichia coli on human dermal microvascular EC (HuDMEC) and human umbilical vein EC (HUVEC) were assessed. Tetrazolium dye (MTT) proliferation, dual staining cell viability, flow cytometry, and microchemotaxis assays were performed. RESULTS: H. pylori (all strains) and C. jejuni inhibited HuDMEC (p < 0.01) and HUVEC (p < 0.01) proliferation and decreased the proportion of HUVECs in the S phase of the cell cycle. E. coli had no effect on EC proliferation. The levels of vascular endothelial growth factor stimulated chemotaxis were significantly greater (p < 0.01) than the levels of basal migration for both control and extract-treated ECs. However, none of the bacterial extracts affected EC basal migration or chemotaxis. CONCLUSION: H. pylori extracts inhibit HuDMEC and HUVEC proliferation in vitro by a cytostatic, strain-independent mechanism. A similar antiproliferative effect of C. jejuni was observed. Our findings suggest that both H. pylori and C. jejuni have the ability to inhibit one of the key stages of angiogenesis which may have implications in peptic ulcer disease.     

The effects of hyperglycemia on the directed migration of wounded endothelial cell monolayers

To determine if cellular motility is affected by abnormal concentrations of ambient glucose, we utilized an endothelial cell monolayer model of wounding to observe the effect of hyperglycemia on the serum-mediated polarization and migration responses of cells at the edge of the wound. We found that hyperglycemia inhibited the rapid polarization response and the consequent cellular migration induced by 20% fetal bovine serum. The inhibitory effect of glucose was significant at a concentration of 11 mmol/L and became more pronounced at higher glucose concentrations. The effect of hyperglycemia on the two components of cell locomotion differed from the inhibitory action of mannitol, another hyperosmolar agent, in two ways (1) the glucose-induced inhibition persisted in cells reincubated in medium containing physiologic glucose (5.5 mmol/L); and (2) the inhibitory effect of glucose was blocked in cells preincubated in medium containing either the sulfonylurea glyburide, or D-myo-inositol. These observations may be relevant to the pathogenesis and management of accelerated atherosclerosis associated with the diabetic state.     

Role of αv integrin in osteoprotegerin-induced endothelial cell migration and proliferation

Osteoprotegerin (OPG) is a decoy receptor for the receptor activator of nuclear factor κB ligand (RANKL). However, the role of OPG in the endothelium remains unknown. In this study, we demonstrate that OPG stimulates the proliferation and migration of human microvascular endothelial cells (HMVECs). In addition, we show that treatment with integrin α_vβ₃ or integrin α_vβ₅ blocking antibody inhibits endothelial cell migration. In contrast, treatment with anti-α_vβ₃ antibody or anti-α_vβ₅ antibody alone did not inhibit OPG-induced proliferation. However, OPG-induced proliferation was inhibited when these antibodies were applied simultaneously. Furthermore, OPG evoked activation of extracellular signal-regulated kinase (ERK) 1/2, which has been linked to integrin α_v activity. Taken together, these results suggest that integrins α_vβ₃ and/or α_vβ₅ contribute to endothelial cell proliferation and migration induced by OPG.     

Cleaved bovine prolactin-related protein-I stimulates vascular endothelial cell proliferation

Prolactin-related protein-I (PRP1) is a member of a non-classical prolactin (PRL)/growth hormone family in cattle. However, its function is still unknown. PRL, when cleaved by cathepsin D and matrix metalloproteinases (MMPs), resulted in cleaved N-terminal 16 kDa fragments (16K-PRL) that have antiangiogenetic properties in human and rodents. We examined the possibility of similar activity of bovine PRP1. PRP1 (normally 33 kDa) was cleaved by cathepsins (CTSs), MMPs, and bovine cotyledonary-conditioned medium (BCCM), and generated mainly 26 kDa N-terminal fragments. Two specific enzyme families, CTSs and MMPs cleaved intact PRP1, and BCCM also contained PRP1 cleavage activity. Bioactivity for pro- or anti-angiogenesis of the cleaved PRP1 was examined in a cell proliferation assay using bovine brain vascular endothelial cells. The cleaved PRP1 proliferated the endothelial cells in vitro. The endothelial cell proliferation activity of cleaved PRP1 may be shared in specific bovine placentomal angiogenesis.     

家兔颈总动脉内皮剥脱术后细胞凋亡与增殖的动态研究

目的　探讨细胞凋亡在血管损伤后内膜增厚过程中的作用。方法　采用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法和免疫组化技术对内皮剥脱术后 3、9、1 4、2 8天家兔颈总动脉壁的凋亡及增生细胞分别进行检测 ,并采用计算机图像分析系统进行定量分析。结果　术后各损伤组凋亡及增生指数示 9天时均显著高于 3天 ,1 4天达峰值 ,2 8天呈下降趋势 ,其中凋亡指数仍较高 ;阳性表达面积率示高峰及之前的凋亡 /增殖比 <1 ,而之后 >1。对照组染色呈阴性。结论　细胞凋亡调节着损伤动脉壁的细胞数目 ;它与细胞增生伴存 ,在增生后期占主导地位 ,调控细胞不再增多。     

环氧合酶2对血管内皮细胞增殖和凋亡的影响

环氧合酶(cyclooxygenase,COX)是以花生四烯酸为底物合成前列腺素的首要限速酶,COX-2作为其亚型之一,主要受细胞内外的各种刺激因素诱导表达.大量的研究表明,COX-2与血管的发生密切相关,它能够通过其衍生的各种前列腺素来影响血管内皮细胞的增殖与凋亡,并由此来参与各种慢性炎症性疾病和肿瘤的发生.弄清COX-2对血管内皮细胞增殖与凋亡的影响,及其相关的作用机制,有助于我们更好的认识其在疾病发生中的作用,从而避弊取利,为临床治疗提供更多更好的方案.     

Stimulation of endothelial cell proliferation by rat granulosa cell-conditioned medium

The ability of granulosa cells to produce mitogenic factors for vascular endothelial cells, factors which could potentially mediate angiogenesis in the ovary, was examined. Granulosa cells were obtained from preovulatory follicles of immature rats 48 h after priming with PMSG (20 IU). The cells (1 X 10(6)/well) were cultured in 3 ml serum-free medium 199 (M199) at 37 C without further treatment or in the presence of LH (100 ng/ml) or FSH (20 ng/ml). Since oxygen tension has been shown to regulate the production of angiogenic factors by other cell types, the cultures were carried out with either a high (20%) or a low (2%) oxygen concentration in the culture chamber. After 48 h, the medium was collected, filtered (0.2 micron), and frozen until tested for mitogenic effects on sparsely plated fetal bovine aortic endothelial cells. A 1:1 mixture of granulosa cell-conditioned M199 with fresh M199 plus 1% dialyzed fetal bovine serum resulted in 7- to 8-fold increases in endothelial cell numbers over the 4-day test period compared to controls (fresh M199 + 1% dialyzed fetal bovine serum only). Neither gonadotropin treatment nor the oxygen concentration during the conditioning period influenced the proliferation-stimulating activity of the medium. Medium conditioned by granulosa cells in 2% oxygen, however, did have an additional effect on endothelial cell morphology; the cells were more elongated and aligned than those treated with medium conditioned by granulosa cells in 20% oxygen, which showed a typical cobblestone morphology. Preliminary characterization studies indicate that both high (greater than 30,000) and low (less than 10,000) mol wt mitogenic factors are present. The mitogenic activity is heat resistant but partially destroyed by trypsin. The morphology-altering activity is confined to high mol wt fractions (greater than 30,000). These studies demonstrate that granulosa cells from preovulatory follicles release one or more factors in vitro which are mitogenic for endothelial cells. Furthermore, conditioned medium from granulosa cells cultured in low oxygen induces morphological changes in endothelial cells which suggest an increased propensity for migration.     

Prolyl hydroxylase domain 2 protein suppresses hypoxia-induced endothelial cell proliferation

Prolyl hydroxylase domain 2 protein (PHD2) signals the degradation of hypoxia-inducible factor (HIF)-1alpha by hydroxylating specific prolyl residues located within oxygen-dependent degradation domains. As expected, endothelial cells (ECs) overexpressing PHD2 had reduced HIF-1alpha and vascular endothelial growth factor-A expression and failed to accelerate their proliferation in response to hypoxia. Surprisingly, although these cells displayed further reductions in HIF-1alpha and vascular endothelial growth factor-A expression when cultured under normoxia, there was no further reduction in EC proliferation. Thus, there seemed to be no consistent correlation between PHD2 hydroxylase-mediated suppression of HIF-1alpha expression and inhibition of EC growth. Indeed, overexpression of a mutant PHD2 lacking hydroxylase activity also greatly diminished EC response to hypoxia-induced increase in proliferation, in spite of the fact that hypoxia-induced HIF-1alpha accumulation was not affected by mutant PHD2. These data strongly suggest the existence of a hydroxylase-independent mechanism for PHD2-mediated inhibition of EC proliferation under hypoxia. In support of a physiological relevance of PHD2 overexpression, we found that endogenous PHD2 expression was significantly upregulated by hypoxia and that silencing of the Phd2 gene by RNA interference significantly enhanced hypoxia-induced EC proliferation. In conclusion, this study demonstrates that PHD2 may act as a negative feedback regulator to antagonize hypoxia-induced EC proliferation.