Platelet-activating factor and metastasis: calcium-independent phospholipase A2β deficiency protects against breast cancer metastasis to the lung

We determined the contribution of calcium-independent phospholipase A(2)β (iPLA(2)β) to lung metastasis development following breast cancer injection into wild-type (WT) and iPLA(2)β-knockout (iPLA(2)β-KO) mice. WT and iPLA(2)β-KO mice were injected in the mammary pad with 200,000 E0771 breast cancer cells. There was no difference in primary tumor size between WT and iPLA(2)β-KO mice at 27 days postinjection. However, we observed an 11-fold greater number of breast cancer cells in the lungs of WT mice compared with iPLA(2)β-KO animals (P < 0.05). Isolated WT lung endothelial cells demonstrated a significant increase in platelet-activating factor (PAF) production when stimulated with thrombin [1 IU/ml, 10 min, 4,330 ± 555 vs. 15,227 ± 1,043 disintegrations per minute (dpm), P < 0.01] or TNF-α (10 ng/ml, 2 h, 16,532 ± 538 dpm, P < 0.01). Adherence of E0771 cells to WT endothelial cells was increased by thrombin (4.8 ± 0.3% vs. 70.9 ± 6.3, P < 0.01) or TNF-α (60.5 ± 4.3, P < 0.01). These responses were blocked by pretreatment with the iPLA(2)β-selective inhibitor (S)-bromoenol lactone and absent in lung endothelial cells from iPLA(2)β-KO mice. These data indicate that endothelial cell iPLA(2)β is responsible for PAF production and adherence of E0771 cells and may play a role in cancer cell migration to distal locations.     

Lung endothelial cell platelet-activating factor production and inflammatory cell adherence are increased in response to cigarette smoke component exposure

An early event in the pathogenesis of emphysema is the development of inflammation associated with accumulation of polymorphonuclear leukocytes (PMN) in small airways, and inflammatory cell recruitment from the circulation involves migration across endothelial and epithelial cell barriers. Platelet-activating factor (PAF) promotes transendothelial migration in several vascular beds, and we postulated that increased PAF production in the airways of smokers might enhance inflammatory cell recruitment and exacerbate inflammation. To examine this possibility, we incubated human lung microvascular endothelial cells (HMVEC-L) with cigarette smoke extract (CSE) and found that CSE inhibits PAF-acetylhydrolase (PAF-AH) activity. This enhances HMVEC-L PAF production and PMN adherence, and adherence is blocked by PAF receptor antagonists (CV3988 or ginkgolide B). CSE also inhibited PAF-AH activity of lung endothelial cells isolated from wild-type (WT) and iPLA(2)β knockout mice, and with WT cells, CSE enhanced PAF production and RAW 264.7 cell adherence. In contrast, CSE did not affect PAF production or RAW 264.7 cell adherence to iPLA(2)β-null cells, suggesting that iPLA(2)β plays an important role in PAF production by lung endothelial cells. These findings suggest that inhibition of PAF-AH by components of cigarette smoke may initiate or exacerbate inflammatory lung disease by enhancing PAF production and promoting accumulation of inflammatory cells in small airways. In addition, iPLA(2)β is identified as a potential target for therapeutic interventions to reduce airway inflammation and the progression of chronic lung disease.     

Potential role for mast cell tryptase in recruitment of inflammatory cells to endothelium

Recent research suggests that activation of protease-activated receptors (PARs) on the surface of endothelial and epithelial cells may play a role in general mechanisms of inflammation. We hypothesized that mast cell tryptase activation of endothelial cell PAR-2 is coupled to increased calcium-independent PLA₂ (iPLA₂) activity and increased platelet-activating factor (PAF) production that may play a role in inflammatory cell recruitment at sites of vascular injury. Stimulation of human coronary artery endothelial cells (HCAEC) with 20 ng/ml tryptase increased iPLA₂ activity, arachidonic acid release, and PAF production. These tryptase-stimulated responses were inhibited by pretreatment with the iPLA₂-selective inhibitor bromoenol lactone (BEL; 5 µM, 10 min). Similar patterns of increased iPLA₂ activity and PAF production were also seen when HCAEC were treated with SLIGKV, which represents the tethered ligand sequence for the human PAR-2 once the receptor is cleaved by tryptase. Tryptase stimulation also increased cell surface expression of P-selectin, decreased electrical resistance, and increased neutrophil adherence to the endothelial cell monolayer. The tryptase-stimulated increases in both cell surface P-selectin expression and neutrophil adhesion were also inhibited with BEL pretreatment. We conclude that tryptase stimulation of HCAEC contributes importantly to early inflammatory events after vascular injury by activation of iPLA₂, leading to arachidonic acid release, PAF production, cell surface P-selectin expression, and increased neutrophil adherence. atherosclerosis; endothelial cells     

Endothelial cell PAF synthesis following thrombin stimulation utilizes Ca(2+)-independent phospholipase A(2).

Platelet activating factor (PAF) is a potent lipid autocoid that is rapidly synthesized and presented on the surface of endothelial cells following thrombin stimulation. PAF production may occur via de novo synthesis or by the combined direct action of phospholipase A(2) (PLA(2)) and acetyl-CoA:lyso-PAF acetyltransferase or via the remodeling pathway. This study was undertaken to define the role of PLA(2) and plasmalogen phospholipid hydrolysis in PAF synthesis in thrombin-treated human umbilical artery endothelial cells (HUAEC). Basal PLA(2) activity in HUAEC was primarily found to be Ca(2+)-independent (iPLA(2)), membrane-associated, and selective for arachidonylated plasmenylcholine substrate. Thrombin stimulation of HUAEC resulted in a preferential 3-fold increase in membrane-associated iPLA(2) activity utilizing plasmenylcholine substrates with a minimal increase in activity with alkylacyl glycerophospholipids. No change in cystolic iPLA(2) activity in thrombin-stimulated HUAEC was observed. The thrombin-stimulated activation of iPLA(2) and associated hydrolysis of plasmalogen phospholipids was accompanied by increased levels of arachidonic acid (from 1.1 +/- 0.1 to 2.8 +/- 0.1%) and prostacyclin release (from 38 +/- 12 to 512 +/- 24%) as well as an increased level of production of lysoplasmenylcholine (from 0.6 +/- 0.1 to 2.1 +/- 0.3 nmol/mg of protein), lysophosphatidylcholine (from 0.3 +/- 0.1 to 0.6 +/- 0.1 nmol/mg of protein), and PAF (from 790 +/- 108 to 3380 +/- 306 dpm). Inhibition of iPLA(2) with bromoenol lactone resulted in inhibition of iPLA(2) activity, plasmalogen phospholipid hydrolysis, production of choline lysophospholipids, and PAF synthesis. These data indicate that PAF production requires iPLA(2) activation in thrombin-stimulated HUAEC and may occur through the CoA-independent transacylase remodeling pathway rather than as a direct result of the PLA(2)-catalyzed hydrolysis of membrane alkylacyl glycerophosphocholine.     

Enhancement of thrombin- and ionomycin-stimulated prostacyclin and platelet-activating factor production in cultured endothelial cells by a tumor-promoting phorbol ester

Tumor-promoting phorbol esters such as 4 beta-phorbol 12-myristate 13-acetate (PMA) have been shown to act synergistically with Ca2+ ionophores in cell activation, including stimulation of arachidonic acid metabolism. The effects of PMA on unstimulated and Ca2+ ionophore- or thrombin-stimulated PGI2 and platelet-activating factor (PAF) production in cultured bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were investigated. Incubation of BAEC or HUVEC for 5-10 min with 100 nM PMA alone slightly increased basal PGI2 production. PGI2 production was rapidly stimulated in BAEC and HUVEC treated with the Ca2+ ionophore ionomycin. Preincubation of BAEC or HUVEC with 100 nM PMA for 5-10 min followed by ionomycin for up to 60 min enhanced PGI2 production up to 2.5-fold. Pretreatment with 100 nM PMA for 5 min also caused a 2-fold enhancement of thrombin-stimulated (1 U/ml) PGI2 production in HUVEC. The production of other prostaglandins, PGF2 alpha, PGE2, and PGD2, was also enhanced. In contrast, PMA had no effect on PGI2 synthesized directly from exogenous arachidonic acid or PGH2. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate was without effect. Since the biosyntheses of both PGI2 and PAF share a common first step, the hydrolysis of their respective phospholipid precursors by phospholipase A2, we investigated whether PMA preincubation could also enhance PAF biosynthesis. Incubation of HUVEC with 100 nM PMA alone had a negligible effect on PAF production. However, thrombin-stimulated (1 U/ml) PAF production was enhanced 2.6-fold by preincubation with 100 nM PMA. The protein kinase C inhibitors H-7 and staurosporine ablated the enhancing effect of PMA on thrombin-stimulated PGI2 and PAF biosynthesis. These results demonstrate that PMA can significantly alter the production of PGI2 and PAF in vascular endothelial cells, and suggest that protein kinase C activation modulates phospholipase A2 activity in this cell type.     

Roles of cytosolic phospholipase A(2) and platelet-activating factor receptor in the Ca-induced biosynthesis of PAF

Casein-elicited peritoneal exudate cells (PEC), mainly consisted of neutrophils, were collected from platelet-activating factor receptor-knock-out (PAFR-KO), cytosolic phospholipase A(2) knock-out (cPLA(2)-KO), and wild-type (WT) mice. After stimulation of PEC with calcium ionophore A 23187, PAF levels were measured by radio-ligand binding assay using receptor-rich membrane fraction prepared from the PAF receptor transgenic mice. We found that the level of PAF production by PEC was not different between WT and PAFR-KO mice. On the other hand, cPLA(2)-KO mice were deficient in the PAF production. These results provide the direct evidence while cPLA(2) is essential in the production of PAF, PAF receptor deficiency has little effect on the PAF production.     

Endothelin-1 attenuates increases in hydraulic conductivity due to platelet-activating factor via prostacyclin release

We previously showed that endothelin-1 (ET-1) and prostacyclin (PGI(2)) similarly attenuate increases in microvascular permeability induced by platelet-activating factor (PAF). This led us to hypothesize that ET-1 attenuates trans-endothelial fluid flux during PAF through PGI(2) release. We tested this hypothesis in three phases. First, bovine pulmonary artery endothelial cells were exposed to 0.008-8 μM ET-1 and assayed for PGI(2) release. Second, to determine whether increased transmonolayer flux after PAF could be attenuated by ET-1 or PGI(2) and reversed by PGI(2) synthesis inhibition or PGI(2) receptor blockade, we measured endothelial cell transmonolayer flux after cells were exposed to 10 nM PAF plus 10 μM PGI(2) or 80 pM ET-1, with or without 500 μM tranylcypromine (PGI(2) synthase inhibitor) or 20 μM CAY-10441 (PGI(2) receptor blocker). Finally, hydraulic conductivity (L(p)) was measured in rat mesenteric venules in vivo after exposure to 10 nM PAF and 80 pM ET-1 with or without tranylcypromine (100 and 500 μM) or CAY-10441 (2 and 20 μM). We found that in vitro, ET-1 stimulated a dose-dependent increase in PGI(2) production (from 126 to 217 pg/ml, P < 0.01). Compared with PAF alone, PGI(2) plus PAF and ET-1 plus PAF decreased transmonolayer flux similarly by 52 and 46%, respectively (P < 0.01), while tranylcypromine and CAY-10441 reversed these effects by 92 and 47%, respectively (P < 0.05). In vivo, PAF increased L(p) fourfold (P < 0.01) and ET-1 attenuated this effect by 83% (P < 0.01). Tranylcypromine and CAY-10441 reversed the ET-1 attenuation in L(p) during PAF by 55 and 45%, respectively (P < 0.01). We conclude that ET-1 may stimulate endothelial cell PGI(2) release to attenuate the increases in transmonolayer flux and hydraulic conductivity secondary to PAF.     

Prostaglandin production in human coronary artery endothelial cells is modulated differentially by selective phospholipase A(2) inhibitors

Atherosclerotic plaque formation is a dynamic process involving repeated injury and inflammation of the endothelium. We have demonstrated previously that thrombin and tryptase stimulation of human coronary artery endothelial cells (HCAEC) leads to increased phospholipase A(2) (PLA(2)) activity and generation of membrane phospholipid derived inflammatory metabolites, including eicosanoids and platelet activating factor. Thus, our hypothesis is that selective PLA(2) inhibitors have therapeutic potential as anti-inflammatory agents. Stimulation of confluent HCAEC monolayers with thrombin or tryptase resulted in a concentration and time-dependent increase in both prostaglandin E(2) (PGE(2)) and prostacyclin (PGI(2)) production. Pretreatment with PX-18 to inhibit secretory PLA(2) or BEL to inhibit calcium-independent PLA(2) prior to thrombin or tryptase stimulation resulted in a significant inhibition of both PGI(2) and PGE(2) release. However, pretreatment with methyl arachidonyl fluorophosphonate (MAFP), a widely used inhibitor of cytosolic PLA(2) isoforms, resulted in a significant potentiation of both thrombin and tryptase stimulated PGI(2) and PGE(2) release as a consequence of increased free arachidonic acid production. We conclude that the use of selective PLA(2) inhibitors may be of therapeutic benefit in the development and progression of atherosclerosis, however, the development of such an agent requires rigorous screening.     

Priming effect of lipopolysaccharide on acetyl-coenzyme A:lyso-platelet-activating factor acetyltransferase is MyD88 and TRIF independent

LPS has a priming effect on various stimuli. For instance, LPS priming enhances the production of platelet-activating factor (PAF), a proinflammatory lipid mediator that is induced by PAF itself. Among various enzymes responsible for PAF biosynthesis, acetyl-coenzyme A:1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyltransferase is one of the enzymes activated by PAF receptor stimulation. In this study we investigated the priming effect of LPS on the acetyltransferase activation by PAF in TLR4-knockout (KO) mice, MyD88-KO mice, and Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF)-KO mice. This enzyme was biphasically activated by LPS. Although the first peak occurred within 30 min in wild-type (WT), but not TLR4-KO or MyD88-KO, macrophages, the second phase reached a maximum within hours in WT, MyD88-KO, and TRIF-KO, but not in TLR4-KO, macrophages. Only in the second phase was the increase in acetyltransferase activity upon PAF receptor activation remarkably enhanced in WT, MyD88-KO, and TRIF-KO cells, but not in TLR4-KO cells. These data demonstrated that LPS exerted a priming effect on PAF receptor-mediated acetyltransferase activation through the TLR4-dependent, but MyD88- and TRIF-independent, pathway.     

Tumor necrosis factor-alpha priming of phospholipase A2 activation in human neutrophils. An alternative mechanism of priming.

The cytokine, TNF-alpha, interacts with human neutrophils (PMN) via specific membrane receptors and primes leukotriene B4 (LTB4) production in PMN for subsequent stimulation by calcium ionophores. We have further examined the effects of TNF-alpha on arachidonic acid (AA) release, LTB4 production, and platelet-activating factor (PAF) formation in PMN by prelabeling cells with either [3H]AA or [3H]lyso-PAF, priming with human rTNF-alpha, and then stimulating with the chemotactic peptide, FMLP. TNF-alpha, alone, had little effect; minimal AA release, LTB4 or PAF production occurred after PMN were incubated with 0 to 1000 U/ml TNF-alpha. However, when PMN were first preincubated with 100 U/ml TNF-alpha for 30 min and subsequently challenged with 1 microM FMLP, both [3H] AA release and LTB4 production were elevated two- to threefold over control values. Measurement of AA mass by gas chromatography and LTB4 production by RIA confirmed the radiolabeled results. TNF-alpha priming also increased PAF formation after FMLP stimulation. These results demonstrate that TNF-alpha priming before stimulation with a physiologic agonist can enhance activation of phospholipase A2 (PLA2) resulting in increased AA release and can facilitate the activities of 5-lipoxygenase (LTB4 production) and acetyltransferase (PAF formation). Reports in the literature have hypothesized that the priming mechanism involves either production of PLA2 metabolites, increased diglyceride (DG) levels, or enhanced cytosolic calcium levels induced by the priming agent. We investigated these possibilities in TNF-alpha priming of PMN and report that TNF-alpha had no direct effect on PLA2 activation or metabolite formation. Treatment of PMN with TNF-alpha did not induce DG formation and, in the absence of cytochalasin B, no increased DG production (measured by both radiolabel techniques and mass determinations) occurred after TNF-alpha priming followed by FMLP stimulation. TNF-alpha also had no effect on basal cytosolic calcium and did not enhance intracellular calcium levels after FMLP stimulation. These results suggest that an alternative, as yet undefined, mechanism is active in TNF-alpha priming of human PMN.     

Thromboxane and prostacyclin generation by human umbilical veins after thrombin.

Prostacyclin (PGI2) and Thromboxane B2 (TxB2) production induced by thrombin in human umbilical veins (HUV) was studied. Successive stimulations of HUV segments were performed with and without restoration of arachidonic acid (AA). Thrombin consistently stimulated the production of both substances. The magnitude of the increment declined with progressive stimuli. The addition of exogenous AA could restore the production of TXB2 but not that of PGI2. These results suggest that sustained stimulation of AA release may lead to an imbalance in the TXA2/PGI2 ratio perhaps through an effect of unknown products of AA oxidation on PGI2 synthase.     

Arachidonic acid strongly stimulates prostaglandin I3 (PGI3) production from eicosapentaenoic acid in human endothelial cells

Eicosapentaenoic acid (EPA) is a prominent polyunsaturated fatty acid in fish oil which inhibits blood platelet aggregation and thromboxane A2 formation but not prostacyclin-like material generation from vascular endothelium. In this study we investigated interaction between EPA and arachidonic acid (AA) during their oxygenation by cultured endothelial cells. As measured by gas chromatography-mass spectrometry (GC-MS), AA increased markedly prostaglandin I3 (PGI3) production from EPA while that of PGI2 from AA was decreased by EPA. However, increasing the ratio AA/EPA over one almost suppressed the inhibition of PGI2 formation by EPA, and the stimulation of PGI3 production by AA was even higher. The effect of AA on EPA conversion to minor prostaglandins like PGE3 and PGF3 alpha was similar then confirming the stimulating effect and suggesting it is occurring at the cyclooxygenase instead of the prostacyclin synthase level. Altogether these data indicate that, in certain nutritional states where the liberation of EPA from endothelial cells will be accompanied with that of endogenous AA, substantial amounts of PGI3 could contribute to the prostacyclin-like activity of the vessel wall in addition to PGI2.     

Analysis of two major intracellular phospholipases A(2) (PLA(2)) in mast cells reveals crucial contribution of cytosolic PLA(2)α, not Ca(2+)-independent PLA(2)β, to lipid mobilization in proximal mast cells and distal fibroblasts

Mast cells release a variety of mediators, including arachidonic acid (AA) metabolites, to regulate allergy, inflammation, and host defense, and their differentiation and maturation within extravascular microenvironments depend on the stromal cytokine stem cell factor. Mouse mast cells express two major intracellular phospholipases A(2) (PLA(2)s), namely group IVA cytosolic PLA(2) (cPLA(2)α) and group VIA Ca(2+)-independent PLA(2) (iPLA(2)β), and the role of cPLA(2)α in eicosanoid synthesis by mast cells has been well documented. Lipidomic analyses of mouse bone marrow-derived mast cells (BMMCs) lacking cPLA(2)α (Pla2g4a(-/-)) or iPLA(2)β (Pla2g6(-/-)) revealed that phospholipids with AA were selectively hydrolyzed by cPLA(2)α, not by iPLA(2)β, during FcεRI-mediated activation and even during fibroblast-dependent maturation. Neither FcεRI-dependent effector functions nor maturation-driven phospholipid remodeling was impaired in Pla2g6(-/-) BMMCs. Although BMMCs did not produce prostaglandin E(2) (PGE(2)), the AA released by cPLA(2)α from BMMCs during maturation was converted to PGE(2) by microsomal PGE synthase-1 (mPGES-1) in cocultured fibroblasts, and accordingly, Pla2g4a(-/-) BMMCs promoted microenvironmental PGE(2) synthesis less efficiently than wild-type BMMCs both in vitro and in vivo. Mice deficient in mPGES-1 (Ptges(-/-)) had an augmented local anaphylactic response. These results suggest that cPLA(2)α in mast cells is functionally coupled, through the AA transfer mechanism, with stromal mPGES-1 to provide anti-anaphylactic PGE(2). Although iPLA(2)β is partially responsible for PGE(2) production by macrophages and dendritic cells, it is dispensable for mast cell maturation and function.     

Inhibitory effects of interleukin 6 on prostaglandin I2 production in cultured bovine vascular endothelial cells.

The effect of interleukin 6 (IL-6) upon arachidonic acid (AA) metabolism was studied in cultured bovine vascular endothelial cells (BVECs). Although, in those BVECs pretreated with IL-6 for 48 h, there was no effect upon cell proliferation, it was discovered that IL-6 suppressed the conversion of AA to prostaglandin I2 (PGI2) in cellular homogenates. First signs of the suppression occurred 3 h after incubation, and thereafter the suppression increased with time until it leveled off at 12 h. This effect of IL-6 was concentration-dependent, ranging from 20 ng/ml to a maximum of 100 ng/ml with IL-6 at 47% of control. IL-6 had no effect upon AA conversion when exogenously added to the BVEC homogenates. To investigate the mechanism of the suppression induced by IL-6, we studied its effect upon PGI2 synthesizing enzymes. We found that IL-6 had no effect upon the release of AA from phospholipids, but inhibited cyclooxygenase, the key enzyme for PGI2 production from AA. To amplify PGI2 production, AA was added exogenously to both control and IL-6-treated BVECs, and in this case also, the conversion activity in IL-6-treated BVEC's was suppressed. Through immunoblot analysis with an antibody to cyclooxygenase, it was determined that the level of cyclooxygenase protein was lowered in IL-6-treated cells. This is the first report to confirm down expression of cyclooxygenase in addition to the first observation as to the effect of IL-6 on endothelial cells.     

Tryptase activates calcium-independent phospholipase A2 and releases PGE2 in airway epithelial cells

Human small airway epithelial cells (HSAEC) form the boundary between the external environmental allergens and the internal lung milieu. Mast cells are present in human lung tissue interspersed within the pulmonary epithelium and can secrete a host of pre- and newly formed mediators from their granules, which may propagate small airway inflammation. In this study, tryptase stimulation of HSAEC increased membrane-associated, calcium-independent phospholipase A(2)gamma (iPLA(2)gamma) activity, resulting in increased arachidonic acid and PGE(2) release. These responses were inhibited by pretreating HSAEC with the iPLA(2)-selective inhibitor bromoenol lactone. The tryptase-stimulated PGE(2) production was inhibited by treating HSAEC with the cyclooxygenase (COX)-1-selective inhibitor SC-560 and the nonselective COX inhibitor aspirin but not by the COX-2-selective inhibitor CAY10404, indicating that the early release of arachidonic acid is metabolized by constitutive COX-1 to form PGE(2) in tryptase-stimulated HSAEC. Additionally, platelet-activating factor production and neutrophil adherence to tryptase-stimulated HSAEC was also increased. This complex response can set up a cascade of inflammatory mediator production in small airways. We speculate that selective inhibition of iPLA(2)gamma-mediated phospholipid hydrolysis may prove beneficial in inflammatory airway diseases.     

Macrophage migration inhibitory factor is induced by thrombin and factor Xa in endothelial cells

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, has been shown to play a role in wound-healing processes. In this study, we investigated whether protease-activated receptor (PAR)-1 and PAR-2 mediated MIF expression in human endothelial cells. Thrombin, factor Xa (FXa), and trypsin induced MIF expression in human dermal microvascular endothelial cells and human umbilical vein endothelial cells, but other proteases, including kallikrein and urokinase, failed to do so. Thrombin-induced MIF mRNA expression was significantly reduced by the thrombin-specific inhibitor hirudin. Thrombin receptor activation peptide-6, a synthetic PAR-1 peptide, induced MIF mRNA expression, suggesting that PAR-1 mediates MIF expression in response to thrombin. The effects of FXa were blocked by antithrombin III, but not by hirudin, indicating that FXa might enhance MIF production directly rather than via thrombin stimulation. The synthetic PAR-2 peptide SLIGRL-NH(2) induced MIF mRNA expression, showing that PAR-2 mediated MIF expression in response to FXa. Concerning the signal transduction, a mitogen-activated protein kinase kinase inhibitor (PD98089) and a nuclear factor (NF)-kappaB inhibitor (SN50) suppressed the up-regulation of MIF mRNA in response to thrombin, FXa, and PAR-2 agonist stimulation, whereas a p38 inhibitor (SB203580) had little effect. These facts indicate that up-regulation of MIF by thrombin or FXa is regulated by p44/p42 mitogen-activated protein kinase-dependent pathways and NF-kappaB-dependent pathways. Moreover, we found that PAR-1 and PAR-2 mRNA expression in endothelial cells was enhanced by MIF. Furthermore, we examined the inflammatory response induced by PAR-1 and PAR-2 agonists injected into the mouse footpad. As shown by footpad thickness, an indicator of inflammation, MIF-deficient mice (C57BL/6) were much less sensitive to either PAR-1 or PAR-2 agonists than wild-type mice. Taken together, these results suggest that MIF contributes to the inflammatory phase of the wound healing process in concert with thrombin and FXa via PAR-1 and PAR-2.     

Involvement of proteinase-activated receptor-2 in mast cell tryptase-induced barrier dysfunction in bovine aortic endothelial cells

We report here a direct modulation by mast cell tryptase of endothelial barrier function through activation of proteinase-activated receptor-2 (PAR-2). In cultured bovine aortic endothelial cells (BAECs), tryptase, trypsin and PAR-2 activating peptide impaired the barrier function as determined by the permeability of protein-conjugated Evans blue. The tryptase-induced barrier dysfunction was completely blocked by U73122, and partially reversed by xestospongin C, calphostin C or Y27632. The intracellular Ca(2+) was elevated by tryptase. It was notable that ioxaglate, a contrast material that degranulates mast cells, markedly increased the permeability when applied to BAECs in combination with mast cells, an action that was blocked by nafamostat, a potent tryptase inhibitor. Immunofluorescence analysis showed that actin stress fibre formation and disruption of VE-cadherin were observed after exposure to tryptase or ioxaglate in combination with mast cells. Therefore, it is suggested that mast cell tryptase impairs endothelial barrier function through activation of endothelial PAR-2 in a manner dependent on the phospholipase C activity.     

sPLA(2) cooperates with cPLA(2)alpha to regulate prostacyclin synthesis in human endothelial cells

The first step in prostacyclin (PGI(2)) synthesis involves the generation of arachidonic acid (AA) from membrane phospholipids mediated by the 85 kDa cytosolic phospholipase A(2) (cPLA(2)alpha). The current study examined the effects of secretory PLA(2)s (sPLA(2)s) on PGI(2) production by human umbilical vein endothelial cells (HUVEC). We demonstrate that exposure of HUVEC to sPLA(2) dose- and time-dependently enhances AA release and PGI(2) generation. sPLA(2)-stimulated AA mobilisation was blocked by AACOCF(3), an inhibitor of cPLA(2)alpha, suggesting cross-talk between the two classes of PLA(2). sPLA(2) induced the phosphorylation of cPLA(2)alpha and enhanced the phosphorylation states of p42/44(mapk), p38(mapk), and JNK, concomitant with elevated AA and PGI(2) release. The MEK inhibitor PD98059 attenuated sPLA(2)-stimulated cPLA(2)alpha phosphorylation and PGI(2) release. These data show that sPLA(2) cooperates with cPLA(2)alpha in a MAPK-dependent manner to regulate PGI(2) generation and suggests that cross-talk between sPLA(2) and cPLA(2)alpha is a physiologically important mechanism for enhancing prostanoid production in endothelial cells.     

The fibrinogen anion-binding exosite of thrombin is necessary for induction of rises in intracellular calcium and prostacyclin production in endothelial cells.

Thrombin stimulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelial cells (HUVEC) requires the active site of thrombin and involves rapid and transient rises in cytoplasmic free calcium [Ca2+]i. In this study, we investigated whether or not the anion-binding exosite for fibrinogen recognition of thrombin (which confers certain substrate specificities) is also necessary for the induction of rises in [Ca2+]i and PGI2 production. Thrombin variants which lack either the catalytic site (DIP-alpha-thrombin) or anion-binding exosite (gamma-thrombin) either alone or in combination failed to induce rises in [Ca2+]i or PGI2 production in HUVEC. To further study the role of the anion-binding exosite of thrombin in the activation of HUVEC, COOH-terminal fragments of hirudin were used. This portion of hirudin interacts with the anion-binding exosite of thrombin and inhibits thrombin-induced fibrinogen coagulation while leaving the catalytic activity of thrombin intact. A 21-amino acid COOH-terminal peptide of hirudin (N alpha-acetyldesulfato-hirudin45-65 or Hir45-65) inhibited thrombin-induced (0.5 U/ml) rises in [Ca2+]i and PGI2 production with IC50 of 0.13 and 0.71 microM, respectively. Similar results were obtained using shorter hirudin-derived peptides. Thus, the fibrinogen anion-binding exosite of thrombin is required for alpha-thrombin-induced rises in [Ca2+]i and PGI2 production in HUVEC.