Exogenous arachidonic acid mediates permeability of human brain microvessel endothelial cells through prostaglandin E2 activation of EP3 and EP4 receptors

The blood–brain barrier, formed by microvessel endothelial cells, is the restrictive barrier between the brain parenchyma and the circulating blood. Arachidonic acid (ARA; 5,8,11,14‐cis‐eicosatetraenoic acid) is a conditionally essential polyunsaturated fatty acid [20:4(n ? 6)] and is a major constituent of brain lipids. The current study examined the transport processes for ARA in confluent monolayers of human brain microvascular endothelial cells (HBMEC). Addition of radioactive ARA to the apical compartment of HBMEC cultured on Transwell^® inserts resulted in rapid incorporation of radioactivity into the basolateral medium. Knock down of fatty acid transport proteins did not alter ARA passage into the basolateral medium as a result of the rapid generation of prostaglandin E₂ (PGE₂), an eicosanoid known to facilitate opening of the blood–brain barrier. Permeability following ARA or PGE₂ exposure was confirmed by an increased movement of fluorescein‐labeled dextran from apical to basolateral medium. ARA‐mediated permeability was attenuated by specific cyclooxygenase‐2 inhibitors. EP₃ and EP₄ receptor antagonists attenuated the ARA‐mediated permeability of HBMEC. The results indicate that ARA increases permeability of HBMEC monolayers likely via increased production of PGE₂ which acts upon EP₃ and EP₄ receptors to mediate permeability. These observations may explain the rapid influx of ARA into the brain previously observed upon plasma infusion with ARA.

     

Expression of vascular endothelial growth factor receptors in smooth muscle cells

Vascular Endothelial Growth Factor (VEGF) has been typically considered to be an endothelial-specific growth factor. However, it was recently demonstrated that VEGF can interact with non endothelial cells. In this study, we tested whether vascular smooth muscles cells (VSMCs) can express VEGF receptors, such as flk-1, flt-1, and neuropilin (NP)-1, and respond to VEGF in vitro. In cultured VSMCs, flk-1 and flt-1 expression was inversely related to cell density. The expression of flk-1 was down-regulated with increasing passage numbers. However, NP-1 levels were not affected by cell density or passage numbers. Flk-1, Flt-1, and NP-1 protein levels were confirmed by Western Blotting. Although the functional mature form of Flk-1 protein is expressed at low levels in VSMCs, phosphorylation of Flk-1 following VEGF(165) stimulation was still observed. SMCs migrated significantly in response to VEGF(165) and VEGF-E, whereas Placenta Growth Factor (PlGF) induced migration only at higher concentrations. Since VEGF-E is a specific activator of flk-1 while PlGF specifically activates only flt-1, SMC migration induced by VEGF(165) is likely to be mediated primarily through the flk-1 receptor. VSMCs did not significantly proliferate in response to VEGF(165), PlGF, and VEGF-E. In conclusion, our studies demonstrate the presence of VEGF receptors on VSMCs that are functional. These studies also indicate that in vivo, VEGF may play a role in modulating the response of VSMCs.     

Vascular endothelial growth factor inhibits the function of human mature dendritic cells mediated by VEGF receptor-2

Dendritic cells (DCs) are the most potent antigen-presenting cells and play a central role in the host-antitumor immunity. Since it has been reported that vascular endothelial growth factor (VEGF) inhibits the functional maturation of immature-DCs and impairs DC differentiation, it is important to elucidate the mechanisms of VEGF-induced DC-dysfunction. To investigate the effects of VEGF against human mature DCs, we investigated how VEGF affects mature DCs with regards to phenotype, induction of apoptosis, IL-12(p70) production and the antigen-presenting function evaluated by allogeneic mixed leukocyte reaction (allo-MLR). We generated monocyte-derived DCs matured with lipopolysaccharide, OK-432 or pro-inflammatory cytokine cocktails. As a result, VEGF treatment did not alter the mature DCs with regard to phenotype, IL-12(p70) production and induction of apoptosis. As a novel and important finding, VEGF inhibited the ability of mature DCs to stimulate allogeneic T cells. Furthermore, this VEGF-induced DC dysfunction was mainly mediated by VEGF receptor-2 (VEGF R2). These observations were confirmed by the findings that the VEGF-induced DC dysfunction was recovered by anti-human VEGF neutralizing mAb or anti-human VEGF R2 blocking mAb, and that placenta growth factor (PlGF), VEGF R1-specific ligand, did not have any effect against mature DCs. Some modalities aiming at reversing mature-DC dysfunction induced by VEGF will be needed in order to induce the effective antitumor immunity. This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology in Japan.     

Endothelin-1-induced prostaglandin E2-EP2, EP4 signaling regulates vascular endothelial growth factor production and ovarian carcinoma cell invasion

Cyclooxygenase (COX)-1- and COX-2-derived prostaglandins are implicated in the development and progression of several malignancies. We have recently demonstrated that treatment of ovarian carcinoma cells with endothelin-1 (ET-1) induces expression of both COX-1 and COX-2, which contributes to vascular endothelial growth factor (VEGF) production. In this study, we show that in HEY and OVCA 433 ovarian carcinoma cells, ET-1, through the binding with ETA receptor (ETAR), induces prostaglandin E2 (PGE2) production, as the more represented PG types, and increases the expression of PGE2 receptor type 2 (EP2) and type 4 (EP4). The use of pharmacological EP agonists and antagonists indicates that ET-1 and PGE2 stimulate VEGF production principally through EP2 and EP4 receptors. At the mechanistic level, we prove that the induction of PGE2 and VEGF by ET-1 involves Src-mediated epidermal growth factor receptor transactivation. Finally, we demonstrate that ETAR-mediated activation of PGE2-dependent signaling participates in the regulation of the invasive behavior of ovarian carcinoma cells by activating tumor-associated matrix metalloproteinase. These results implicate EP2 and EP4 receptors in the induction of VEGF expression and cell invasiveness by ET-1 and provide a mechanism by which ETAR/ET-1 can promote and interact with PGE2-dependent machinery to amplify its proangiogenic and invasive phenotype in ovarian carcinoma cells. Pharmacological blockade of ETAR can therefore represent an additional strategy to control PGE2 signaling, which has been associated with ovarian carcinoma progression.     

LXR-induced reverse cholesterol transport in human airway smooth muscle is mediated exclusively by ABCA1

The association of hypercholesterolemia and obesity with airway hyperresponsiveness has drawn increasing attention to the potential role of cholesterol and lipid homeostasis in lung physiology and in chronic pulmonary diseases such as asthma. We have recently shown that activation of the nuclear hormone receptor liver X receptor (LXR) stimulates cholesterol efflux in human airway smooth muscle (hASM) cells and induces expression of the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, members of a family of proteins that mediate reverse cholesterol and phospholipid transport. We show here that ABCA1 is responsible for all LXR-mediated cholesterol and phospholipid efflux to both apolipoprotein AI and high-density lipoprotein acceptors. In contrast, ABCG1 does not appear to be required for this process. Moreover, we show that hASM cells respond to increased levels of cholesterol by inducing expression of ABCA1 and ABCG1 transporters, a process that is dependent on LXR expression. These findings establish a critical role for ABCA1 in reverse cholesterol and phospholipid transport in airway smooth muscle cells and suggest that dysregulation of cholesterol homeostasis in these cells may be important in the pathogenesis of diseases such as asthma.     

Vascular endothelial growth factor stimulates osteoblastic differentiation of cultured human periosteal-derived cells expressing vascular endothelial growth factor receptors

Angiogenesis plays an important role in bone development and postnatal bone fracture repair. Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFRs) are primarily involved in angiogenesis. This study investigated the expression of VEGF isoforms, VEGFR-1, and VEGFR-2 during the osteoblastic differentiation of cultured human periosteal-derived cells. In addition, the effect of exogenous VEGF on the osteoblastic differentiation of cultured human periosteal-derived cells was also examined. The expression of the VEGF isoforms (VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆), VEGFR-1, and VEGFR-2 was observed in the periosteal-derived cells. Administration of KRN633, a VEGFR-1 and VEGFR-2 inhibitor, decreased the alkaline phosphatase (ALP) activity during the osteoblastic differentiation of cultured human periosteal-derived cells. However, the administration of VEGFR2 Kinase Inhibitor IV, a VEGFR-2 inhibitor, did not affect the ALP activity. The addition of recombinant human VEGF₁₆₅ elevated the ALP activity and increased the calcium content in the periosteal-derived cells. Treating the periosteal-derived cells with recombinant human VEGF₁₆₅ resulted in an increase in Runx2 transactivation in the periosteal-derived cells. These results suggest that exogenous VEGF stimulates the osteoblastic differentiation of cultured human periosteal-derived cells and VEGF might act as an autocrine growth factor for the osteoblastic differentiation of cultured human periosteal-derived cells.     

Regulatory features of interleukin-1beta-mediated prostaglandin E2 synthesis in airway smooth muscle

Exposure of airway smooth muscle (ASM) cells to the cytokine IL-1beta results in an induction of PGE2 synthesis that affects numerous cell functions. Current dogma posits induction of COX-2 protein as the critical, obligatory event in cytokine-induced PGE2 production, although PGE2 induction can be inhibited without a concomitant inhibition of COX-2. To explore other putative regulatory features we examined the role of phospholipase A2 (PLA2) and PGE synthase (PGES) enzymes in IL-1beta-induced PGE2 production. Treatment of human ASM cultures with IL-1beta caused a time-dependent induction of both cytosolic PLA2 (cPLA2) and microsomal PGES (mPGES) similar to that observed for COX-2. Regulation of COX-2 and mPGES induction was similar, being significantly reduced by inhibition of p42/p44 or p38, whereas cPLA2 induction was only minimally reduced by inhibition of p38 or PKC. COX-2 and mPGES induction was subject to feed-forward regulation by PKA, whereas cPLA2 induction was not. SB-202474, an SB-203580 analog lacking the ability to inhibit p38 but capable of inhibiting IL-1beta-induced PGE2 production, was effective in inhibiting mPGES but not COX-2 or cPLA2 induction. These data suggest that although COX-2, cPLA2, and mPGES are all induced by IL-beta in human ASM cells, regulatory features of cPLA2 are dissociated, whereas those of COX-2 and mPGES are primarily associated, with regulation of PGE2 production. mPGES induction and, possibly, cPLA2 induction appear to cooperate with COX-2 to determine IL-1beta-mediated PGE2 production in human ASM cells.     

The induction of cyclooxygenase-2 in IL-1beta-treated endothelial cells is inhibited by prostaglandin E2 through cAMP

Prostaglandins (PGs) have numerous cardiovascular and inflammatory effects. Cyclooxygenase (COX), which exists as COX-1 and COX-2 isoforms, is the first enzyme in the pathway in which arachidonic acid is converted to PGs. Prostaglandin E2 (PGE2) exerts a variety of biological activities for the maintenance of local homeostasis in the body. Elucidation of PGE2 involvement in the signalling molecules such as COX could lead to potential therapeutic interventions. Here, we have investigated the effects of PGE2 on the induction of COX-2 in human umbilical vein endothelial cells (HUVEC) treated with interleukin-1beta (IL-1beta 1 ng/ml). COX activity was measured by the production of 6-keto-PGF1alpha, PGE2, PGF2alpha and thromboxane B2 (TXB2) in the presence of exogenous arachidonic acids (10 microM for 10 min) using enzyme immunoassay (EIA). COX-1 and COX-2 protein was measured by immunoblotting using specific antibody. Untreated HUVEC contained only COX-1 protein while IL-1beta treated HUVEC contained COX-1 and COX-2 protein. PGE2 (3 microM for 24h) did not affect on COX activity and protein in untreated HUVEC. Interestingly, PGE2 (3 microM for 24h) can inhibit COX-2 protein, but not COX-1 protein, expressed in HUVEC treated with IL-1beta. This inhibition was reversed by coincubation with forskolin (100 microM). The increased COX activity in HUVEC treated with IL-1beta was also inhibited by PGE2 (0.03, 0.3 and 3 microM for 24h) in a dose-dependent manner. Similarly, forskolin (10, 50 or 100 microM) can also reverse the inhibition of PGE2 on increased COX activity in IL-1beta treated HUVEC. The results suggested that (i) PGE2 can initiate negative feedback regulation in the induction of COX-2 elicited by IL-1beta in endothelial cells, (ii) the inhibition of PGE2 on COX-2 protein and activity in IL-1beta treated HUVEC is mediated by cAMP and (iii) the therapeutic use of PGE2 in the condition which COX-2 has been involved may have different roles.     

Vasoactive peptides upregulate mRNA expression and secretion of vascular endothelial growth factor in human airway smooth muscle cells

Airway remodeling and associated angiogenesis are documented features of asthma, of which the molecular mechanisms are not fully understood. Angiotensin (ANG)II and endothelin (ET)-1 are potent vasoconstricting circulatory hormones implicated in asthma. We investigated the effects of ANG II and ET-1 on human airway smooth muscle (ASM) cells proliferation and growth and examined the mRNA expression and release of the angiogenic peptide, vascular endothelial growth factor (VEGF). Serum deprived (48 h) human ASM cells were incubated with ANG II (100 nM) or ET-1 (10nM) for 30 min, 1, 2, 4, 8, 16, and 24 h and the endogenous synthesis of VEGF was examined in relation to control cells receiving serum free culture medium. ET-1 induced time dependent DNA biosynthesis as determined by [³H]-thymidine incorporation assay. Using northern blot hybridization, we detected two mRNA species of 3.9 and 1.7 kb encoding VEGF in the cultured smooth muscle cells. Both ANG II and ET-1 induced the mRNA expression (two-to threefold) and secretion (1.8-to 2.8-fold) of VEGF reaching maximal levels between 4–8 h of incubation. Induced expression and release of VEGF declined after 8 h of ANG II incubation while levels remained elevated in the case of ET-1. The conditioned medium derived from ET-1-treated ASM cells induced [³H]-thymidine incorporation and cell number in porcine pulmonary artery endothelial as well as human umbilical vein endothelial cells. Moreover, the VEGF tyrosine kinase receptor inhibitor blocked the conditioned medium induced mitogenesis in endothelial cells. Our results suggest a potential role for ANG II and ET-1 in ASM cell growth and upregulation of VEGF that may participate in endothelial cell proliferation via paracrine mechanisms and thus causing pathological angiogenesis and vascular remodelling seen during asthma.     

Gene expression changes of prostanoid synthases in endothelial cells and prostanoid receptors in vascular smooth muscle cells caused by aging and hypertension.

The present study was designed to assess whether or not changes in genomic expression of cyclooxygenases (COX-1, COX-2), endothelial nitric oxide synthase (eNOS), and prostanoid synthases in the endothelium and of prostanoid receptors in vascular smooth muscle contribute to the occurrence of endothelium-dependent contractions during aging and hypertension. Gene expression was quantified by real-time PCR using isolated endothelial cells and smooth muscle cells (SMC) from the aorta of Wistar-Kyoto and spontaneously hypertensive rats. Genes for all known prostanoid synthases and receptors were present in endothelial cells and SMC, respectively. Aging caused overexpression of eNOS, COX-1, COX-2, thromboxane synthase, hematopoietic-type prostaglandin D synthase, membrane prostaglandin E synthase-2, and prostaglandin F synthase in endothelial cells and COX-1 and prostaglandin E(2) (EP)(4) receptors in SMC. Hypertension augmented the expression of COX-1, prostacyclin synthase, thromboxane synthase, and hematopoietic-type prostaglandin D synthase in endothelial cells and prostaglandin D(2) (DP), EP(3), and EP(4) receptors in SMC. The increase in genomic expression of endothelial COX-1 explains why in aging and hypertension the endothelium has greater propensity to release cyclooxygenase-derived vasoconstrictive prostanoids. The expression of prostacyclin synthase was by far the most abundant, explaining why the majority of the COX-1-derived endoperoxides are transformed into prostacyclin, substantiating the role of prostacyclin as an endothelium-derived contracting factor. The expression of thromboxane synthase was increased in the cells of aging or hypertensive rats, explaining why the prostanoid can contribute to endothelium-dependent contractions. It is uncertain whether the gene modifications caused by aging and hypertension directly contribute to endothelium-dependent contractions or rather to vascular aging and the vascular complications of the hypertensive process.     

Regulation of vascular endothelial cell growth factor expression in mouse mammary tumor cells by the EP2 subtype of the prostaglandin E2 receptor

Prostaglandin E(2) (PGE(2)), a major metabolite of the cyclooxygenase pathway in the mammary gland, induces angiogenesis during mammary tumor progression. To better define the molecular mechanisms involved, we examined the role of the G protein-coupled receptors (GPCR) for PGE(2) in mammary tumor cell lines isolated from MMTV-cyclooxygenase-2 (COX-2) transgenic mice. Expression of the EP2 subtype of the PGE(2) receptor was correlated with the tumorigenic phenotype and the ability to induce vascular endothelial growth factor (VEGF). Overexpression of EP2 by adenoviral transduction into EP2-null cells resulted in the induction of VEGF expression in response to PGE(2) and CAY10399, an EP2 receptor agonist. The induction of VEGF by the EP2 receptor did not require the hypoxia inducible factor (HIF)-1alpha pathway, MAP kinase pathway, or phosphoinositide-3-kinase/Akt pathway, but required the cAMP/protein kinase A pathway. These results suggest that EP2 receptor is a critical element for PGE(2) mediated VEGF induction in mouse mammary tumor cells.     

Proinflammatory cytokines upregulate mRNA expression and secretion of vascular endothelial growth factor in cultured human airway smooth muscle cells

Airflow obstruction in chronic airway disease is associated with airway and pulmonary vascular remodeling, of which the molecular mechanisms are poorly understood. Paracrine actions of angiogenic factors released by resident or infiltrating inflammatory cells following activation by proinflammatory cytokines in diseased airways could play a major role in the airway vascular remodeling process. Here, the proinflammatory cytokines interleukin (IL)-1β, and tumor necrosis factor (TNF)-α were investigated on cell cultures of human airway smooth muscle (ASM) for their effects on mRNA induction and protein release of the angiogenic peptide, vascular endothelial growth factor (VEGF). IL-1β (0.5 ng/mL) and TNF-α (10ng/mL) each increased VEGF mRNA (3.9 and 1.7 kb) expression in human ASM cells, reaching maximal levels between 16 and 24 and 4 and 8h, respectively. Both cytokines also induced a time-dependent release of VEGF, which was not associated with increased ASM growth. Preincubation of cells with 1μM dexamethasone abolished enhanced release of VEGF by TNF-α. The data suggest that human ASM cells express and secrete VEGF in response to proinflammatory cytokines and may participate in paracrine inflammatory mechanisms of vascular remodeling in chronic airway disease.