High glucose increases B1-kinin receptor expression and signaling in endothelial cells

The loss of endothelial function is the initiating factor in the development of diabetic vascular disease. Kinins control endothelial function by the activation of two receptors: the B2 which is constitutively expressed, and the B1 which is highly induced in pathological conditions. In the present study, we observed that the levels of B1-receptor mRNA and protein are induced in endothelial cells incubated in high glucose. An increase in B1-receptor was also observed in the endothelial layer of aortas, from 4-week diabetic rats. When cells were grown in high glucose, the B1 agonist des-Arg9-BK increased nitrite levels, whereas in normal glucose nitrite levels were unchanged. Nitrite increase was blocked by L-NAME and 1400W indicating the participation of the inducible Nitric Oxide Synthase (iNOS). iNOS protein levels were also increased in high glucose. These results demonstrate the participation of the B1 receptor in the signaling pathways mediated by kinins in high glucose.     

Characterization of somatostatin receptor subtype 2 expression in stably transfected A-427 human cancer cells

Although radiolabeled somatostatin analogs have become highly prevalent in the diagnosis and treatment of somatostatin receptor subtype (sst)-positive tumors, there are relatively few options with respect to sst-positive tumor cell lines and animal models. It would be highly beneficial, particularly for therapeutic purposes, to have several clones of one human sst2-positive cell line that express a range of sst2 concentrations for evaluating the dose response and intracellular processing of radiolabeled somatostatin analogs. The human non-small cell lung cancer line A-427 was stably transfected with a hemagglutinin-tagged human sst2. Expression of the receptor was evaluated in vitro using flow cytometry, saturation binding analysis, internalization assays, and quantitative polymerase chain reaction. The receptor expression was also validated in an in vivo mouse model in biodistribution and micro-positron emission tomography (microPET) studies using the somatostatin analog octreotide (OC), which was linked to the (64)Cu chelator 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), or (64)Cu-TETA-OC. Stable clones were isolated, and four clones (2, 4, 5, and 7) were chosen for further examination. In vitro assays showed that clone 4 had no expression of sst2, whereas the others had various levels in the order of 7 > 2 > 5. Biodistribution studies with (64)Cu-TETA-OC showed the same rank order, with tumor uptake of the clones ranging from 0.8 to 6.5% injected dose/g. These studies showed that there was a strong correlation among the in vitro assays and between the in vitro assays and the biodistribution. MicroPET confirmed significant uptake of (64)Cu-TETA-OC in clone 7 and background uptake in clone 4. These studies show that clones of a human cell line can be produced expressing various levels of sst2 that should be useful for the future evaluation of radiolabeled somatostatin analogs.     

Modulation of pulmonary endothelial endothelin B receptor expression and signaling: implications for experimental hepatopulmonary syndrome

The hepatopulmonary syndrome (HPS) results from intrapulmonary vasodilation in the setting of cirrhosis and portal hypertension. In experimental HPS, pulmonary endothelial endothelin B (ET(B)) receptor overexpression and increased circulating endothelin-1 (ET-1) contribute to vasodilation through enhanced endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) production. In both experimental cirrhosis and prehepatic portal hypertension, ET(B) receptor overexpression correlates with increased vascular shear stress, a known modulator of ET(B) receptor expression. We investigated the mechanisms of pulmonary endothelial ET(B) receptor-mediated eNOS activation by ET-1 in vitro and in vivo. The effect of shear stress on ET(B) receptor expression was assessed in rat pulmonary microvascular endothelial cells (RPMVECs). The consequences of ET(B) receptor overexpression on ET-1-dependent ET(B) receptor-mediated eNOS activation were evaluated in RPMVECs and in prehepatic portal hypertensive animals exposed to exogenous ET-1. Laminar shear stress increased ET(B) receptor expression in RPMVECs without altering mRNA stability. Both shear-mediated and targeted overexpression of the ET(B) receptor enhanced ET-1-mediated ET(B) receptor-dependent eNOS activation in RPMVECs through Ca(2+)-mediated signaling pathways and independent of Akt activation. In prehepatic portal hypertensive animals relative to control, ET-1 administration also activated eNOS independent of Akt activation and triggered HPS. These findings support that increased pulmonary microvascular endothelial ET(B) receptor expression modulates ET-1-mediated eNOS activation, independent of Akt, and contributes to the development of HPS.     

Vascular endothelial growth factor KDR receptor signaling potentiates tumor necrosis factor-induced tissue factor expression in endothelial cells

Vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF-alpha) have been shown to synergistically increase tissue factor (TF) expression in endothelial cells; however, the role of the VEGF receptors (KDR, Flt-1, and neuropilin) in this process is unclear. Here we report that VEGF binding to the KDR receptor is necessary and sufficient for the potentiation of TNF-induced TF expression in human umbilical vein endothelial cells. TF expression was evaluated by Western blot analysis and fluorescence-activated cell sorting. In the absence of TNF-alpha, wild-type VEGF- or KDR receptor-selective variants induced an approximate 7-fold increase in total TF expression. Treatment with TNF alone produced an approximate 110-fold increase in total TF expression, whereas coincubation of TNF-alpha with wild-type VEGF- or KDR-selective variants resulted in an approximate 250-fold increase in TF expression. VEGF lacking the heparin binding domain was also able to potentiate TF expression, indicating that heparin-sulfate proteoglycan or neuropilin binding is not required for TF up-regulation. Neither placental growth factor nor an Flt-1-selective variant was capable of inducing TF expression in the presence or absence of TNF. Inhibition of protein-tyrosine kinase or protein kinase C activity significantly blocked the TNF/VEGF potentiation of TF up-regulation, whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, increased TF expression. These data demonstrate that KDR receptor signaling governs both VEGF-induced TF expression and the potentiation of TNF-induced up-regulation of TF.     

HIV-1 Tat protein-induced VCAM-1 expression in human pulmonary artery endothelial cells and its signaling

Expression of cell adhesion molecule in endothelial cells upon activation by human immunodeficiency virus (HIV) infection is associated with the development of atherosclerotic vasculopathy. We postulated that induction of vascular cell adhesion molecule-1 (VCAM-1) by HIV-1 Tat protein in endothelial cells might represent an early event that could culminate in inflammatory cell recruitment and vascular injury. We determined the role of HIV-1 Tat protein in VCAM-1 expression in human pulmonary artery endothelial cells (HPAEC). HIV-1 Tat protein treatment significantly increased cell-surface expression of VCAM-1 in HPAEC. Consistently, mRNA expression of VCAM-1 was also increased by HIV-1 Tat protein as measured by RT-PCR. HIV-1 Tat protein-induced VCAM-1 expression was abolished by the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) and the p38 MAPK inhibitor SB-203580. Furthermore, HIV-1 Tat protein enhanced DNA binding activity of NF-kappaB, facilitated nuclear translocation of NF-kappaB subunit p65, and increased production of reactive oxygen species (ROS). Similarly to VCAM-1 expression, HIV-1 Tat protein-induced NF-kappaB activation and ROS generation were abrogated by PDTC and SB-203580. These data indicate that HIV-1 Tat protein is able to induce VCAM-1 expression in HPAEC, which may represent a pivotal early molecular event in HIV-induced vascular/pulmonary injury. These data also suggest that the molecular mechanism underlying the HIV-1 Tat protein-induced VCAM-1 expression may involve ROS generation, p38 MAPK activation, and NF-kappaB translocation, which are the characteristics of pulmonary endothelial cell activation.     

Progesterone receptor activation of extranuclear signaling pathways in regulating p53 expression in vascular endothelial cells

We previously showed that progesterone (P4) inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) through a p53-dependent pathway. Now we investigated further the molecular mechanism underlying the hormone activity. In cultured HUVECs, P4 increased the protein levels of phosphorylated Src (p-Src), Raf-1, and ERK. The levels of p-Src and p-Src-progesterone receptor complex in HUVECs were increased by P4 treatment. These effects were blocked by pretreatment with a progesterone receptor antagonist, RU486. The P4-induced increase in p53 transactivity was abolished by pretreatment with Src kinase inhibitors. Moreover, administration with cSrc antisense oligonucleotide prevented the P4-induced increases of the levels of p53 mRNA and protein. These data suggest that P4-induced up-regulation of p53 might be mediated through activation of cSrc. Pretreatment with Src kinase inhibitors also prevented P4-induced membrane translocation of Kras and increases of the protein levels of phosphorylated Raf and phosphorylated ERK. Transfection with dominant-negative ERK2 prevented the P4-induced increases of protein level and promoter activity of p53 and a decrease of thymidine incorporation. P4 also increased nuclear factor-κB (NF-κB) nuclear translocation and NF-κB binding onto the p53 promoter. These effects were abolished by pretreatment with ERK inhibitors. The P4-induced up-regulation of the p53 promoter activity was prevented by preadministration with dominant-negative ERK2 or NF-κB inhibitors. Taken together, our data suggest that the cSrc/Kras/Raf-1/ERK2/NF-κB signaling pathway contributes to the P4-induced up-regulation of p53 in HUVECs. These findings highlight progesterone receptor activation of extranuclear signaling pathways in regulating p53 and cell cycle progression in HUVECs.     

Upregulation of urokinase receptor expression on migrating endothelial cells

One of the phenotypic hallmarks of migrating endothelial cells, both in vivo and in vitro, is expression of the urokinase-type plasminogen activator (u-PA), a key mediator of extracellular proteolysis. In the study reported here, we have used an in vitro model of endothelial cell migration to explore the mechanism of this phenomenon. We have found that wounding of an endothelial cell monolayer triggers a marked, rapid and sustained increase in expression of a specific high-affinity receptor for u-PA (u-PAr) on the surface of migrating cells. Migrating cells displayed an increase in the levels of u-PA and u-PAr mRNAs, and this increase was mediated by endogenous basic fibroblast growth factor (bFGF). We also show that the increase in u-PA activity on migrating cells can be accounted for by an increase in receptor-bound u-PA, and that the increase in activity is also dependent on endogenous bFGF. These results demonstrate that the expression of plasmin-mediated proteolytic activity by migrating endothelial cells is a consequence of increased production of both u-PA and its receptor, and that this in turn is mediated by endogenous bFGF. This suggests that u-PA, produced at increased levels by migrating cells, binds to u-PAr whose expression is upregulated on the same cells. These observations are in accord with the postulated role of u-PAr in mediating efficient and spatially restricted extracellular proteolysis, particularly in the context of cell migration.     

Albumin endocytosis in endothelial cells induces TGF-beta receptor II signaling

Vascular endothelial cells undergo albumin endocytosis using a set of albumin binding proteins. This process is important for maintaining cellular homeostasis. We showed by several criteria that the previously described 73-kDa endothelial cell surface albumin binding protein is the 75-kDa transforming growth factor (TGF)-beta receptor type II (TbetaRII). Albumin coimmunoprecipitated with TbetaRII from a membrane fraction from rat lung microvascular endothelial cells. Albumin endocytosis-negative COS-7 cells became albumin endocytosis competent when transfected with wild-type TbetaRII but not when transfected with a domain-negative kinase mutant of TbetaRII. An antibody specific for TbetaRII inhibited albumin endocytosis. A mink lung epithelial cell line, which expresses both the TGF-beta receptor type I (TbetaRI) and the TbetaRII receptor, exhibited albumin binding to the cell surface and endocytosis. In contrast, mutant L-17 and DR-26 cells lacking TbetaRI or TbetaRII, respectively, each showed a dramatic reduction in binding and endocytosis. Albumin endocytosis induced Smad2 phosphorylation and Smad4 translocation as well as increased protein expression of the inhibitory Smad, Smad7. We identified regions of significant homology between amino acid sequences of albumin and TGF-beta, suggesting a structural basis for the interaction of albumin with the TGF-beta receptors and subsequent activation of TbetaRII signaling. The observed albumin-induced internalization of TbetaRII signaling may be an important mechanism in the vessel wall for controlling TGF-beta responses in endothelial cells.     

组胺对肺动脉内皮细胞一氧化氮合酶基因表达的影响

本实验研究了组胺对原代培养的肺动脉内皮细胞一氧化氮合酶(nitric oxidCsynthase,NOS)基因表达的影响及分子机制。采用RT-PCR和免疫印迹技术分别检测mRNA和蛋白质的表达水平,用荧光素酶报告基因实验检测eNOS基因转录起始点上游长1.6-kb的启动子活性,用硝酸还原酶法检测NO的产量。结果发现,组胺增强eNOS表达,呈浓度和时间依赖性,10μmol/L组胺处理肺动脉内皮细胞24h可使eNOS mRNA和蛋白质的表达达到高峰,eNOS mRNA水平为正常对照组的160.8±12.2%(P<0.05),蛋白质水平为正常对照组的136.2±11.2%(P<0.05)。特异性CaMK Ⅱ抑制剂KN-93可抑制组胺的这一效应,表明组胺可通过激活CaMK Ⅱ增强肺动脉内皮细胞eNOS基因的表达。报告基因实验表明,10μmol/L组胺处理24h后肺动脉内皮细胞eNOS基因启动子的活性增强,为正常对照组的148.2±33.7%(P<0.05)。组胺可使肺动脉内皮细胞产生NO增加。这些结果表明组胺在转录水平增强肺动脉内皮细胞eNOS基因的表达,并使细胞产生NO增加,这可能是组胺调节肺血管张力的机制之一。CaMK Ⅱ可能是组胺增强肺动脉内皮细胞eNOS基因表达的途径之一。     

Crosstalk between TNF and glucocorticoid receptor signaling pathways

TNF is a Janus-faced protein. It possesses impressive anti-tumor activities, but it is also one of the strongest known pro-inflammatory cytokines, which hampers its use as a systemic anti-cancer agent. TNF has been shown to play a detrimental role in inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Glucocorticoids are strongly anti-inflammatory and exert their therapeutic effects through binding to their receptor, the glucocorticoid receptor. Therefore, glucocorticoids have been used for over half a century for the treatment of inflammatory diseases. However, many patients are or become resistant to the therapeutic effects of glucocorticoids. Inflammatory cytokines have been suggested to play an important role in this steroid insensitivity or glucocorticoid resistance. This review aims to highlight the mechanisms of mutual inhibition between TNF and GR signaling pathways.     

Characterization of the tachykinin NK2 receptor subtype in the rabbit pulmonary artery.

Contractile responses to neurokinin A (NKA), neuropeptide gamma(NP gamma), and the NK2 receptor-selective analogs [Lys5,MeLeu9,Nle10]NKA(4-10) and MDL 28,564 were determined in the endothelium-denuded rabbit pulmonary artery. Responses to NKA, NP gamma, and [Lys5,MeLeu9,Nle10]NKA(4-10) were antagonized by the NK2 receptor antagonist MDL 29,913, with pA2 values of 6.67, 6.46, and 7.32, respectively. Autoradiographic studies failed to demonstrate any specific binding sites for [125I]-iodohistidyl NKA (INKA) over the pulmonary artery. These data suggest the presence in rabbit pulmonary artery of an unusual "nonclassical" NK2 receptor subtype, which appears to lack affinity for INKA.     

Chronic inflammation and protection from acute hepatitis in transgenic mice expressing TNF in endothelial cells

Endothelial activation is an important feature of many inflammatory diseases and has been implicated as the cause of vascular complications in disorders such as diabetes, atherosclerosis, and transplant rejection. One of the most potent activators of the endothelium is TNF, which can also be expressed by endothelial cells, causing a permanent, autocrine stimulatory signal. To establish a model of continuous endothelial activation and to elucidate the role of endothelial derived TNF in vivo, we generated transgenic mice expressing a noncleavable transmembrane form of TNF under the control of the endothelial-specific tie2 promoter. Adult tie2-transmembrane TNF-transgenic mice developed chronic inflammatory pathology in kidney and liver, characterized by perivascular infiltration of mononuclear cells into these organs. Along with the infiltrate, an up-regulation of the adhesion molecules ICAM-1 and VCAM-1, but not E-selectin, in the endothelium was observed. Despite predisposition to chronic inflammation these mice were protected from immune-mediated liver injury in a model of Con A-induced acute hepatitis. Although the blood levels of soluble TNF and IFN-gamma were increased in transgenic animals after challenge with Con A, no damage of hepatocytes could be detected, as assessed by the lack of increase in plasma transaminase activities and the absence of TUNEL staining in the liver. We conclude that expression of transmembrane TNF in the endothelium causes continuous endothelial activation, leading to both proinflammatory and protective events.     

Characterization of heparin affin regulatory peptide signaling in human endothelial cells

Heparin affin regulatory peptide (HARP) is an 18-kDa secreted growth factor that has a high affinity for heparin and a potent role on tumor growth and angiogenesis. We have previously reported that HARP is mitogenic for different types of endothelial cells and also affects cell migration and differentiation (12). In this study we examined the signaling pathways involved in the migration and tube formation on matrigel of human umbilical vein endothelial cells (HUVEC) induced by HARP. We report for the first time that receptor-type protein-tyrosine phosphatase beta/zeta (RPTPbeta/zeta), which is a receptor for HARP in neuronal cell types, is also expressed in HUVEC. We also document that HARP signaling through RPTPbeta/zeta leads to activation of Src kinase, focal adhesion kinase, phosphatidylinositol 3-kinase, and Erk1/2. Sodium orthovanadate, chondroitin sulfate-C, PP1, wortmannin, LY294002, and U0126 inhibit HARP-mediated signaling and HUVEC migration and tube formation. In addition, RPTPbeta/zeta suppression using small interfering RNA technology interrupts intracellular signals and HUVEC migration and tube formation induced by HARP. These results establish the role of RPTPbeta/zeta as a receptor of HARP in HUVEC and elucidate the HARP signaling pathway in endothelial cells.     

Cxc chemokine receptor expression on human endothelial cells.

CXC chemokines play a important role in the process of leukocyte recruitment and activation at sites of inflammation. However, recent evidence suggests that these molecules can also regulate endothelial cell functions such as migration, angiogenesis and proliferation. In this study we have investigated CXC chemokine receptor expression in both primary cultures of human umbilical vein endothelial cells (HUVEC) and the spontaneously transformed HUVEC cell line, ECV304. We found that both cell types express mRNA for chemokine receptors CXCR1, CXCR2 and CXCR4, but not CXCR3. Flow cytometric analysis revealed low levels of CXCR1 but higher levels of CXCR4 cell surface expression. HUVECs responded to SDF-1alpha with a rapid and robust calcium flux, however no calcium flux was seen with either IL-8 or Gro-alpha. HUVECs and ECV304 cells did not proliferate in response to CXC chemokines, although ECV304 cells did migrate towards SDF-1alpha and IL-8. These data demonstrate that HUVECs and the endothelial cell line, ECV304 express functional CXC chemokine receptors.     

Effect of steroid on hyperoxia-induced ICAM-1 expression in pulmonary endothelial cells

Intercellular adhesion molecule-1 (ICAM-1) of the vascular endothelium plays a key role in the development of pulmonary oxygen toxicity. We studied the effect of steroid on hyperoxia-induced ICAM-1 expression using cultured endothelial cells in vitro. Human pulmonary artery endothelial cells (HPAECs) were cultured to confluence, and then the monolayers were exposed to either control (21% O(2)-5% CO(2)) or hyperoxic (90% O(2)-5% CO(2)) conditions with and without a synthetic glucocorticoid, methylprednisolone (MP). MP reduced hyperoxia-induced ICAM-1 and ICAM-1 mRNA expression in a dose-dependent manner. Neutrophil adhesion to hyperoxia-exposed endothelial cells was also inhibited by MP treatment. In addition, MP attenuated hyperoxia-induced H(2)O(2) production in HPAECs as assessed by flow cytometry. An electrophoretic mobility shift assay demonstrated that hyperoxia activated nuclear factor-kappaB (NF-kappaB) but not activator protein-1 (AP-1) and that MP attenuated hyperoxia-induced NF-kappaB activation dose dependently. With Western immunoblot analysis, IkappaB-alpha expression was decreased by hyperoxia and increased by MP treatment. These results suggest that MP downregulates hyperoxia-induced ICAM-1 expression by inhibiting NF-kappaB activation via increased IkappaB-alpha expression.     

Internalization-defective mutants of somatostatin receptor subtype 2 exert normal signaling functions in hematopoietic cells

The regulatory peptide somatostatin (SST) acts via a family of G-protein-coupled receptors comprising five subtypes (SSTR1-5). G-protein-coupled receptors activate multiple signaling mechanisms, which variably depend on internalization and intracellular routing of activated receptors. We have recently demonstrated that hematopoietic precursors express SSTR2 and that SST is a chemoattractant for these cells. Herein, we characterize critical regions in SSTR2 involved in endocytosis and describe how ligand-induced internalization impacts on two major signaling functions of SSTR2 in hematopoietic cells, the activation of the Erk pathway and the induction of promigratory responses.     

Acute ethanol exposure disrupts VEGF receptor cell signaling in endothelial cells

Physiological angiogenesis is regulated by various factors, including signaling through vascular endothelial growth factor (VEGF) receptors. We previously reported that a single dose of ethanol (1.4 g/kg), yielding a blood alcohol concentration of 100 mg/dl, significantly impairs angiogenesis in murine wounds, despite adequate levels of VEGF, suggesting direct effects of ethanol on endothelial cell signaling (40). To examine the mechanism by which ethanol influences angiogenesis in wounds, we employed two different in vitro angiogenesis assays to determine whether acute ethanol exposure (100 mg/dl) would have long-lasting effects on VEGF-induced capillary network formation. Ethanol exposure resulted in reduced VEGF-induced cord formation on collagen and reduced capillary network structure on Matrigel in vitro. In addition, ethanol exposure decreased expression of endothelial VEGF receptor-2, as well as VEGF receptor-2 phosphorylation in vitro. Inhibition of ethanol metabolism by 4-methylpyrazole partially abrogated the effect of ethanol on endothelial cell cord formation. However, mice treated with t-butanol, an alcohol not metabolized by alcohol dehydrogenase, exhibited no change in wound vascularity. These results suggest that products of ethanol metabolism are important factors in the development of ethanol-induced changes in endothelial cell responsiveness to VEGF. In vivo, ethanol exposure caused both decreased angiogenesis and increased hypoxia in wounds. Moreover, in vitro experiments demonstrated a direct effect of ethanol on the response to hypoxia in endothelial cells, as ethanol diminished nuclear hypoxia-inducible factor-1alpha protein levels. Together, the data establish that acute ethanol exposure significantly impairs angiogenesis and suggest that this effect is mediated by changes in endothelial cell responsiveness to both VEGF and hypoxia.     

Synergy and antagonism between Notch and BMP receptor signaling pathways in endothelial cells

Notch and bone morphogenetic protein signaling pathways are important for cellular differentiation, and both have been implicated in vascular development. In many cases the two pathways act similarly, but antagonistic effects have also been reported. The underlying mechanisms and whether this is caused by an interplay between Notch and BMP signaling is unknown. Here we report that expression of the Notch target gene, Herp2, is synergistically induced upon activation of Notch and BMP receptor signaling pathways in endothelial cells. The synergy is mediated via RBP-Jkappa/CBF-1 and GC-rich palindromic sites in the Herp2 promoter, as well as via interactions between the Notch intracellular domain and Smad that are stabilized by p/CAF. Activated Notch and its downstream effector Herp2 were found to inhibit endothelial cell (EC) migration. In contrast, BMP via upregulation of Id1 expression has been reported to promote EC migration. Interestingly, Herp2 was found to antagonize BMP receptor/Id1-induced migration by inhibiting Id1 expression. Our results support the notion that Herp2 functions as a critical switch downstream of Notch and BMP receptor signaling pathways in ECs.