Suppression of cytokine-induced expression of adrenomedullin and endothelin-1 by dexamethasone in T98G human glioblastoma cells

There is accumulating evidence showing that glial cells and gliomas secrete some neuropeptides and vasoactive peptides, such as adrenomedullin and endothelin-1. We have previously shown that expression of these two peptides is induced by inflammatory cytokines in T98G human glioblastoma cells. Glucocorticoids are frequently used for the treatment of inflammatory diseases and glioblastomas. We therefore studied effects of dexamethasone on expression of adrenomedullin and endothelin-1 in T98G human glioblastoma cells. Dexamethasone dose-dependently increased adrenomedullin mRNA levels and immunoreactive-adrenomedullin levels in the medium in T98G cells, whereas it decreased immunoreactive-endothelin levels in the medium. A combination of three cytokines, interferon-gamma (100 U/ml), tumor necrosis factor-alpha (20 ng/ml) and interleukin-1beta (10 ng/ml) induced expression of adrenomedullin and endothelin-1 in T98G cells. Dexamethasone (10(-8) mol/l) suppressed increases in expression of both adrenomedullin and endothelin-1 induced by these three cytokines. Thus, dexamethasone alone increased adrenomedullin expression whereas it suppressed the cytokine-induced expression of adrenomedullin in T98G cells. These findings raised the possibility that effects of dexamethasone on brain inflammation and glioblastomas may be partly mediated or modulated by its effects on expression of adrenomedullin and endothelin-1.     

Leptin-induced transphosphorylation of vascular endothelial growth factor receptor increases Notch and stimulates endothelial cell angiogenic transformation

Leptin increases vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), and Notch expression in cancer cells, and transphosphorylates VEGFR-2 in endothelial cells. However, the mechanisms involved in leptin’s actions in endothelial cells are not completely known. Here we investigated whether a leptin-VEGFR-Notch axis is involved in these leptin’s actions. To this end, human umbilical vein and porcine aortic endothelial cells (wild type and genetically modified to overexpress VEGFR-1 or -2) were cultured in the absence of VEGF and treated with leptin and inhibitors of Notch (gamma-secretase inhibitors: DAPT and S2188, and silencing RNA), VEGFR (kinase inhibitor: SU5416, and silencing RNA) and leptin receptor, OB-R (pegylated leptin peptide receptor antagonist 2: PEG-LPrA2). Interestingly, in the absence of VEGF, leptin induced the expression of several components of Notch signaling pathway in endothelial cells. Inhibition of VEGFR and Notch signaling significantly decreased leptin-induced S-phase progression, proliferation, and tube formation in endothelial cells. Moreover, leptin/OB-R induced transphosphorylation of VEGFR-1 and VEGFR-2 was essential for leptin’s effects. These results unveil for the first time a novel mechanism by which leptin could induce angiogenic features via upregulation/trans-activation of VEGFR and downstream expression/activation of Notch in endothelial cells. Thus, high levels of leptin found in overweight and obese patients might lead to increased angiogenesis by activating VEGFR-Notch signaling crosstalk in endothelial cells. These observations might be highly relevant for obese patients with cancer, where leptin/VEGFR/Notch crosstalk could play an important role in cancer growth, and could be a new target for the control of tumor angiogenesis.     

Proteasome from cytokine-treated human cells shows stimulated BrAAP activity and depressed PGPH activity.

The branched chain amino acid-preferring (BrAAP) activity of multicatalytic proteinase complex isolated from human umbilical vein endothelial cells and treated with interferon-gamma was increased more than 2-fold, which was associated with a marked increase in LMP7 expression and decreased peptidylglutamyl peptide-hydrolyzing activity. Increases in BrAAP activity in supernatants from cells treated with interferon-gamma, tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6, or lipopolysaccharide paralleled the increases in LMP7 expression. These findings are consistent with the conclusion that the increased BrAAP activity of LMP-containing multicatalytic proteinase complex results from incorporation of LMP7 or other LMP subunits.     

Cryptotanshinone inhibits endothelin-1 expression and stimulates nitric oxide production in human vascular endothelial cells

The Chinese herb Salvia miltiorrhiza (SM) has been found to have beneficial effects on the circulatory system. In the present study, we investigated the effects of cryptotanshinone (derived from SM) on endothelin-1 (ET-1) expression in human umbilical vein endothelial cells (HUVECs). The effect of cryptotanshinone on nitric oxide (NO) in HUVECs was also examined. We found that cryptotanshinone inhibited basal and tumor necrosis factor-alpha (TNF-alpha) stimulated ET-1 secretion in a concentration-dependent manner. Cryptotanshinone also induced a concentration-dependent decrease in ET-1 mRNA expression. Cryptotanshinone increased basal and TNF-alpha-attenuated NO production in a dose-dependent fashion. Cryptotanshinone induced a concentration-dependent increase in endothelial nitric oxide synthase (eNOS) expression without significantly changing neuronal nitric oxide synthase (nNOS) expression in HUVECs in the presence or absence of TNF-alpha. NOS activities in the HUVECs were also induced by cryptotanshinone. Furthermore, decreased ET-1 expression in response to cryptotanshinone was not antagonized by the NOS inhibitor l-NAME. A gel shift assay further showed that TNF-alpha-induced Nuclear Factor-kappaB (NF-kappaB) activity was significantly reduced by cryptotanshinone. These data suggest that cryptotanshinone inhibits ET-1 production, at least in part, through a mechanism that involves NF-kappaB but not NO production.     

CNS melanocortin and leptin effects on stearoyl-CoA desaturase-1 and resistin expression

The objective of this study was to determine whether centrally administered leptin decreased liver and adipose SCD1 expression or adipose resistin expression, and whether these effects were mediated by central melanocortin receptors. Male Sprague-Dawley rats were injected into the lateral cerebral ventricle (i.c.v.) once daily for 4 days with artificial cerebrospinal fluid (aCSF, 5 microl), leptin (10 microg) or MTII (0.1 nmol); two other groups were pretreated icv with the melanocortin antagonist, SHU9119 (1.0 nmol), followed by leptin or MTII. Epididymal and inguinal adipose tissue and liver were collected after rats were killed and mRNA expression of SCD1 and resistin was measured. Both leptin and MTII reduced SCD1 expression and pretreatment with SHU9119 reversed their effects. Neither leptin nor MTII affected resistin expression, but it was increased by SHU9119. These results show that CNS melanocortin receptors are likely mediators of leptin's effects on SCD1 expression in liver and adipose tissue, The findings were inconclusive concerning the effects of leptin and melanocortins on adipose resistin expression.     

Pulmonary vasoreactivity in spontaneously hypertensive rats - Effects of endothelin-1 and leptin

Background

Systemic hypertension may be associated with an increased pulmonary vascular resistance, which we hypothesized could be, at least in part, mediated by increased leptin.

Methods

Vascular reactivity to phenylephrine (1 μmol/L), endothelin-1 (10 nmol/L) and leptin (0.001–100 nmol/L) was evaluated in endothelium-intact and -denuded isolated thoracic aorta and pulmonary arteries from spontaneously hypertensive versus control Wistar rats. Arteries were sampled for pathobiological evaluation and lung tissue for morphometric evaluation.

Results

In control rats, endothelin-1 induced a higher level of contraction in the pulmonary artery than in the aorta. After phenylephrine or endothelin-1 precontraction, leptin relaxed intact pulmonary artery and aortic rings, while no response was observed in denuded arteries. Spontaneously hypertensive rats presented with increased reactivity to phenylephrine and endothelin-1 in endothelium-intact pulmonary arteries. After endothelin-1 precontraction, endothelium-dependent relaxation to leptin was impaired in pulmonary arteries from hypertensive rats. In both strains of rats, aortic segments were more responsive to leptin than pulmonary artery. In hypertensive rats, pulmonary arteries exhibited increased pulmonary artery medial thickness, associated with increased expressions of preproendothelin-1, endothelin-1 receptors type A and B, inducible nitric oxide synthase and decreased endothelial nitric oxide synthase, together with decreased leptin receptor and increased suppressor of cytokine signaling 3 expressions.

Conclusions

Altered pulmonary vascular reactivity in hypertension may be related to a loss of endothelial buffering of vasoconstriction and decreased leptin-induced vasodilation in conditions of increased endothelin-1.     

High-density lipoprotein subfraction 3 decreases ADAMTS-1 expression induced by lipopolysaccharide and tumor necrosis factor-alpha in human endothelial cells.

Endothelial expression of matrix metalloproteinases has been implicated in angiogenesis and endothelial cell proliferation. Recently, it has been shown that high-density lipoproteins (HDLs) promote angiogenesis. In the present study, we investigated the effects of native HDLs on the expression of several proteases and their inhibitors in human umbilical vein endothelial cells. We show that ADAMTS-1 (a disintegrin and metalloproteinase with thrombospondin motif) was potently induced by incubation with lipopolysaccharide or tumor necrosis factor-alpha and that the expression was significantly reduced in the presence of HDL subfraction 3. Since ADAMTS-1 has recently been shown to inhibit endothelial cell proliferation, the result of the present work may represent a new mechanism by which HDL could have a positive effect on endothelial cell and vascular wall function.     

Mammalian target of rapamycin mediates the angiogenic effects of leptin in human hepatic stellate cells

Leptin modulates the angiogenic properties of hepatic stellate cells (HSC), but the molecular mechanisms involved are poorly understood. We investigated the pathways regulating hypoxia-inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) in leptin-stimulated myofibroblastic HSC. Exposure to leptin enhanced the phosphorylation of TSC2 on T1462 residues and of p70 S6 kinase and the translational inhibitor 4E-binding protein-1, indicating the ability of leptin to activate the mammalian target of rapamycin (mTOR) pathway. Similar findings were observed when HSC were exposed to PDGF. Both leptin and PDGF increased the expression of HIF-1α and VEGF in HSC. In the presence of rapamycin, a specific mTOR inhibitor, leptin and PDGF were no longer able to activate mTOR, and expression of VEGF was reduced, whereas HIF-1α abundance was not affected. Moreover, knockdown of Raptor, a component of the mTORC1 complex, reduced the ability of leptin to increase VEGF. mTOR was also necessary for leptin- and PDGF-dependent increase in HSC migration. Leptin increased the generation of reactive oxygen species in HSC, which was reduced by NADP(H) oxidase inhibitors. Both N-acetyl cysteine and diphenylene iodonium, a NADP(H) inhibitor, inhibited the expression of HIF-1α and VEGF stimulated by leptin or PDGF. Finally, conditioned media from HSC treated with leptin or PDGF induced tube formation in cultured human umbilical vein endothelial cells. In conclusion, in HSC exposed to leptin or PDGF, increased expression of VEGF requires both activation of mTOR and generation of reactive oxygen species via NADPH-oxidase. Induction of HIF-1α requires NADP(H) oxidase but not mTOR activation.     

Cytokine-Induced Expression of Parathyroid Hormone-Related Peptide in Cultured Human Vascular Endothelial Cells

Parathyroid hormone-related peptide (PTHrP) is a potent vasodilatory peptide whose expression has been demonstrated in various tissues. The present study was undertaken to examine the regulation of PTHrP expression in cultured endothelial cells derived from human umbilical vein. Immunoradiometric assay revealed that the amount of PTHrP in the conditioned medium was increased by both tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in both a time- and a dose-dependent manner. The induction of PTHrP mRNA was also observed, with a peak after 2 hours of incubation with both TNF-α and IL-1β. Angiotensin II, endothelin-1, and arginine vasopressin had no affect on PTHrP production. Our results suggest that PTHrP produced in vascular endothelial cells in response to cytokines may modulate vascular function as a local factor.     

Expression of adrenomedullin mRNA in adrenocortical tumors and secretion of adrenomedullin by cultured adrenocortical carcinoma cells

Immunoreactive-adrenomedullin concentrations and the expression of adrenomedullin mRNA were studied in the tumor tissues of adrenocortical tumors. Northern blot analysis showed the expression of adrenomedullin mRNA in tumor tissues of adrenocortical tumors, including aldosterone-producing adenomas, cortisol-producing adenomas, a non-functioning adenoma and adrenocortical carcinomas, as well as normal parts of adrenal glands and pheochromocytomas. On the other hand, immunoreactive-adrenomedullin was not detected in about 90% cases of adrenocortical tumors (<0.12 pmol/g wet weight (ww)). Immunoreactive-adrenomedullin concentrations ranged from 0.44 to 198.2 pmol/g ww in tumor tissues of pheochromocytomas and were 9.2 ± 1.2 pmol/g ww (mean ± SD, n = 4) in normal parts of adrenal glands. Adrenomedullin mRNA was expressed in an adrenocortical adenocarcinoma cell line, SW-13 and immunoreactive-adrenomedullin was detected in the culture medium of SW-13 (48.9 ± 1.8 fmol/10⁵ cells/24h, mean ± SEM, n = 4). On the other hand, immunoreactive-adrenomedullin was not detectable in the extract of SW-13 cells (<0.09 fmol/10⁵ cells), suggesting that adrenomedullin was actively secreted from SW-13 cells without long-term storage. These findings indicate that adrenomedullin is produced and secreted, not only by pheochromocytomas, but also by adrenocortical tumors. Undetectable or low levels of immunoreactive-adrenomedullin in the tumor tissues of adrenocortical tumors may be due to very rapid secretion of this peptide soon after the translation from these tumors.     

缺氧条件下血管活性因子对血管内皮细胞中VEGF表达的影响

 探讨在缺氧条件下人脐静脉血管内皮细胞对血管内皮生长因子 (vascular endothelialgrowth factor,VEGF)表达及缩血管活性物质内皮素 (ET)、舒血管活性物质一氧化氮 (NO)和 NO抑制剂 LNNA对 VEGF基因表达的影响 .体外培养人脐静脉血管内皮细胞 ,经缺氧及血管活性物质处理 .Northern杂交、酶联免疫检测和计算机图象分析等观察 VEGF m RNA和蛋白表达水平 .发现缺氧 6h内皮细胞可见 VEGF表达 .ET可促进 VEGF m RNA的表达 ,NO可明显抑制 VEGFm RNA的表达 ,NO抑制剂 LNNA也影响 VEGF m RNA的表达 .ELISA检测 VEGF蛋白水平分别为 6h组 8.2± 1 .1 ng/ L,ET+6h组 9.37± 1 .0 2 ng/ L,NO+6h组 2 .86± 0 .91 ng/ L,L - NNA+6h组 1 4.75± 1 .87ng/ L.缺氧可诱导人脐静脉血管内皮细胞分泌 VEGF并受血管活性物质ET和 NO的调控 ,ET促进其表达 ,NO抑制其表达 .     

EndoPDI,a novel protein-disulfide isomerase-like protein that is preferentially expressed in endothelial cells acts as a stress survival factor

We have identified a novel protein-disulfide isomerase and named it endothelial protein-disulfide isomerase (EndoPDI) because of its high expression in endothelial cells. Isolation of the full-length cDNA showed EndoPDI to be a 48 kDa protein that has three APWCGHC thioredoxin motifs in contrast to the two present in archetypal PDI. Ribonuclease protection and Western analysis has shown that hypoxia induces EndoPDI mRNA and protein expression. In situ hybridization analysis showed that EndoPDI expression is rare in normal tissues, except for keratinocytes of the hair bulb and syncytiotrophoblasts of the placenta, but was present in the endothelium of tumors and in other hypoxic lesions such as atherosclerotic plaques. We have compared the function of EndoPDI to that of PDI in endothelial cells using specific siRNA. PDI was shown to have a protective effect on endothelial cells under both normoxia and hypoxia. In contrast, EndoPDI has a protective effect only in endothelial cells exposed to hypoxia. The loss of EndoPDI expression under hypoxia caused a significant decrease in the secretion of adrenomedullin, endothelin-1, and CD105; molecules that protect endothelial cells from hypoxia-initiated apoptosis. The identification of an endothelial PDI further extends this increasing multigene family and EndoPDI, unlike archetypal PDI, may be a molecule with which to target tumor endothelium.     

Hypoxia/Aglycemia-Induced Endothelial Barrier Dysfunction and Tight Junction Protein Downregulation Can Be Ameliorated by Citicoline

This study explores the effect of citicoline on the permeability and expression of tight junction proteins (TJPs) in endothelial cells under hypoxia/aglycemia conditions. Hypoxia or oxygen and glucose deprivation (OGD) was utilized to induce endothelial barrier breakdown model on human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (bEnd.3s). The effect of citicoline on endothelial barrier breakdown models was determined at either low or high concentrations. FITC-Dextran flux was used to examine the endothelial permeability. The expression of TJPs was measured by immunofluorescence, Real-time PCR and Western Blot methods. Results showed that hypoxia or OGD increased the permeability of HUVECs accompanied with down-regulation of occludens-1 (ZO-1) and occludin at both mRNA and protein levels. Similarly in bEnd.3s, hypoxia increased the permeability and decreased the expression of ZO-1 and claudin-5. Citicoline treatment dose-dependently decreased the permeability in these two models, which paralleled with elevated expression of TJPs. The data demonstrate that citicoline restores the barrier function of endothelial cells compromised by hypoxia/aglycemia probably via up-regulating the expression of TJPs.     

Differences in mRNA expression of adipocyte-derived factors in response to fasting, refeeding and leptin

The present study examines whether and to what extent the profiles of adipose-derived factors are altered in epididymal and subcutaneous adipose tissues of long-term fasted/refed and of fasted rats treated by recombinant leptin. Fasting was characterized by three successive metabolic phases. Minor differences in the time-course and magnitude of response were detected between the two adipose sites. Leptin, adiponectin, resistin, adiponutrin, and insulin-like growth factor-1 (IGF-1) gene expressions differentially decreased according to the fasting duration. mRNA levels reached a minimum in late fasting for these secreted factors, being decreased by 60-90% for adiponectin, resistin, and IGF-1, 95-98% for leptin and by 100% for adiponutrin. Refeeding partially or totally restored their mRNA expression in epididymal adipose. Expression levels of apolipoprotein E (ApoE), angiotensinogen (AGT), adipsin and macrophage migration inhibitory factor (MIF) were either unchanged or slightly affected. In leptin-treated rats, leptin mRNA concentrations were significantly decreased in phase 2 of fasting (by 85%) from levels in control phosphate-buffered saline (PBS)-treated rats in both tissues. Leptin treatment also decreased resistin mRNA levels (by 78% in P2L and 63% in P3L relative to control groups) in subcutaneous adipose. These data suggest that adiponectin, resistin, adiponutrin, and IGF-1 could be involved in overall energy homeostasis during prolonged fasting, as leptin is. The mechanisms that underlie the expressions of these adipose-secreted factors remain to be determined.     

Differential distribution and modulation of expression of alpha 1/beta 1 integrin on human endothelial cells

In this paper we report that the integrin complex alpha 1/beta 1, a laminin/collagen receptor, is expressed on cultured foreskin microvascular endothelium, but is absent on endothelial cells from large vessels such as the aorta and umbilical and femoral veins. The restricted expression of integrin alpha 1/beta 1 to microvascular endothelium was also demonstrated in vivo, by immunohistochemical staining of human tissue sections. Alpha 1 specific antibodies reacted strongly with endothelial cells of small blood vessels and capillaries in several tissues, but not with endothelium of vein and arteries of umbilical cord. Expression of integrin alpha 1 can be induced in cultured umbilical vein endothelial cells by treatment with 5 ng/ml tumor necrosis factor alpha (TNF alpha). Induction of alpha 1 subunit expression also occurred after treatment of umbilical vein endothelium with 10(-5) M retinoic acid or with 10 nM PMA; Maximal induction of alpha 1 integrin was reached after 48 h of treatment and costimulation with TNF alpha and PMA resulted in a synergistic effect. The induction of alpha 1 integrin changed the adhesive properties of umbilical vein endothelial cells, by increasing the adhesiveness to collagen, laminin, and laminin fragment P1, while adhesion to fibronectin and laminin fragment E8 remained constant. The alpha 1 integrin is thus a marker of a specific population of endothelial cells and its expression confers distinctive properties of interaction with the underlying basal membrane.     

LPS and cytokine-induced endothelial cell IL-6 release and ELAM-1 expression; involvement of serum.

In this in vitro study, the influence of serum-concentration, heat inactivation of the serum and the origin of the serum on the responsiveness of cultured human umbilical vein endothelial cells (HUVEC) to immunological challenges was investigated. Addition of human serum during stimulation with 1 microgram/ml bacterial lipopolysaccharide (LPS) increased endothelial cell ELAM-1 expression and interleukin (IL)-6 release five to ten-fold. Full endothelial cell responsiveness to LPS required 10 to 50% human serum and was largely abrogated after heating the serum for 30 minutes at 56 degrees C. Addition of newborn or fetal bovine serum instead of human serum, induced even higher IL-6 release and ELAM-1 expression in response to LPS, whilst heat-inactivation of these serum-batches only moderately decreased endothelial cell responses. Endothelial cell IL-6 release and ELAM-1 expression after stimulation with IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) were less influenced by heat inactivation of the serum and by omission of serum, whilst responses to PMA remained completely unaffected by such modifications in assay media. Finally, we demonstrated that endothelial cell IL-8 release also and ICAM-1 expression in response to LPS and cytokines were increased by addition of human serum, indicating that the use of serum-free assay media, or the use of media enriched with heat-inactivated (HI) human serum interferes with physiological endothelial cell responsiveness.(ABSTRACT TRUNCATED AT 250 WORDS)     

RORalpha1 and RORalpha4 suppress TNF-alpha-induced VCAM-1 and ICAM-1 expression in human endothelial cells

Retinoic acid receptor-related orphan receptor-alpha (RORalpha) is a nuclear orphan receptor. Adenovirus-mediated overexpression of RORalpha1 and RORalpha4 suppressed tumor necrosis factor-alpha (TNF-alpha)-induced expression of vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells. Overexpression of RORalpha1 and RORalpha4 also suppressed TNF-alpha-stimulated translocation of p50 and p65 to the nucleus. In contrast, dominant-negative deletion mutants of RORalpha1 and RORalpha4 failed to suppress the induction of VCAM-1 and ICAM-1 and translocations of p50 and p65. These results suggest that RORalpha1 and RORalpha4 regulate the inflammatory responses via inhibition of the nuclear factor-kappaB signaling pathway in endothelial cells.