Renal Function Underpins the Cyclooxygenase-2: Asymmetric Dimethylarginine Axis in Mouse and Man.

Introduction: Through the production of prostacyclin, cyclooxygenase (COX)-2 protects the cardiorenal system. Asymmetric dimethylarginine (ADMA), is a biomarker of cardiovascular and renal disease. Here we determined the relationship between COX-2/prostacyclin, ADMA, and renal function in mouse and human models. Methods: We used plasma from COX-2 or prostacyclin synthase knockout mice and from a unique individual lacking COX-derived prostaglandins (PGs) because of a loss of function mutation in cytosolic phospholipase A2 (cPLA2), before and after receiving a cPLA2-replete transplanted donor kidney. ADMA, arginine, and citrulline were measured using ultra-high performance liquid-chromatography tandem mass spectrometry. ADMA and arginine were also measured by enzyme-linked immunosorbent assay (ELISA). Renal function was assessed by measuring cystatin C by ELISA. ADMA and prostacyclin release from organotypic kidney slices were also measured by ELISA. Results: Loss of COX-2 or prostacyclin synthase in mice increased plasma levels of ADMA, citrulline, arginine, and cystatin C. ADMA, citrulline, and arginine positively correlated with cystatin C. Plasma ADMA, citrulline, and cystatin C, but not arginine, were elevated in samples from the patient lacking COX/prostacyclin capacity compared to levels in healthy volunteers. Renal function, ADMA, and citrulline were returned toward normal range when the patient received a genetically normal kidney, capable of COX/prostacyclin activity; and cystatin C positively correlated with ADMA and citrulline. Levels of ADMA and prostacyclin in conditioned media of kidney slices were not altered in tissue from COX-2 knockout mice compared to wildtype controls. Conclusion: In human and mouse models, where renal function is compromised because of loss of COX-2/PGI2 signaling, ADMA levels are increased.

Activation and Contraction of Human "Vascular" Smooth Muscle Cells Grown From Circulating Blood Progenitors.

Blood outgrowth smooth muscle cells (BO-SMCs) offer the means to study vascular cells without the requirement for surgery providing opportunities for drug discovery, tissue engineering, and personalized medicine. However, little is known about these cells which meant that their therapeutic potential remains unexplored. Our objective was to investigate for the first time the ability of BO-SMCs and vessel-derived smooth muscle cells to sense the thromboxane mimetic U46619 by measuring intracellular calcium elevation and contraction. U46619 (10-6 M) increased cytosolic calcium in BO-SMCs and vascular smooth muscle cells (VSMCs) but not in fibroblasts. Increased calcium signal peaked between 10 and 20 s after U46619 in both smooth muscle cell types. Importantly, U46619 (10-9 to 10-6 M) induced concentration-dependent contractions of both BO-SMCs and VSMCs but not in fibroblasts. In summary, we show that functional responses of BO-SMCs are in line with VSMCs providing critical evidence of their application in biomedical research.

Platelet inhibition by P2Y12 antagonists is potentiated by adenosine signalling activators.

BACKGROUND AND PURPOSE: P2Y12 receptor antagonists reduce platelet aggregation and the incidence of arterial thrombosis. Adenosine signalling in platelets directly affects cyclic nucleotide tone, which we have shown to have a synergistic relationship with P2Y12 inhibition. Several studies suggest that ticagrelor inhibits erythrocyte uptake of adenosine and that this could also contribute to its antiplatelet effects. We therefore examined the effects on platelet activation of adenosine signalling activators in combination with the P2Y12 receptor antagonists ticagrelor and prasugrel. EXPERIMENTAL APPROACH: Human washed platelets, platelet-rich plasma and whole blood were used to test the interactions between ticagrelor or prasugrel and adenosine or 5'-N-ethylcarboxamidoadenosine (NECA). Platelet reactivity to thrombin, protease-activated receptor 1 (PAR-1) activation or collagen was assessed by a combination of 96-well plate aggregometry, light transmission aggregometry, whole blood aggregometry, ATP release assay and levels of cAMP. KEY RESULTS: The inhibitory effects of ticagrelor and prasugrel on platelet aggregation and ATP release were enhanced in the presence of adenosine or NECA. Isobolographic analysis indicated a powerful synergy between P2Y12 receptor inhibition and adenosine signalling activators. Increased levels of cAMP in platelets were also observed. In all cases, ticagrelor showed similar synergistic effects on platelet inhibition as prasugrel in the presence of adenosine or NECA. CONCLUSION AND IMPLICATIONS: These results indicate that P2Y12 antagonists have a synergistic relationship with adenosine signalling and that their efficacy may depend partly upon the presence of endogenous adenosine. This effect was common for prasugrel and ticagrelor despite reports of differences in their effects upon adenosine reuptake.

Combination of cyclic nucleotide modulators with P2Y12 receptor antagonists as anti-platelet therapy.

BACKGROUND: Endothelium-derived prostacyclin and nitric oxide elevate platelet cyclic nucleotide levels and maintain quiescence. We previously demonstrated that a synergistic relationship exists between cyclic nucleotides and P2Y12 receptor inhibition. A number of clinically approved drug classes can modulate cyclic nucleotide tone in platelets including activators of NO-sensitive guanylyl cyclase (GC) and phosphodiesterase (PDE) inhibitors. However, the doses required to inhibit platelets produce numerous side effects including headache. OBJECTIVE: We investigated using GC-activators in combination with P2Y12 receptor antagonists as a way to selectively amplify the anti-thrombotic effect of both drugs. METHODS: In vitro light transmission aggregation and platelet adhesion under flow were performed on washed platelets and platelet rich plasma. Aggregation in whole blood and a ferric chloride-induced arterial thrombosis model were also performed. RESULTS: The GC-activator BAY-70 potentiated the action of the P2Y12 receptor inhibitor prasugrel active metabolite in aggregation and adhesion studies and was associated with raised intra-platelet cyclic nucleotide levels. Furthermore, mice administered sub-maximal doses of the GC activator cinaciguat together with the PDE inhibitor dipyridamole and prasugrel, showed significant inhibition of ex vivo platelet aggregation and significantly reduced in vivo arterial thrombosis in response to injury without alteration in basal carotid artery blood flow. CONCLUSIONS: Using in vitro, ex vivo, and in vivo functional studies, we show that low dose GC activators synergize with P2Y12 inhibition to produce powerful anti-platelet effects without altering blood flow. Therefore, modulation of intra-platelet cyclic nucleotide levels alongside P2Y12 inhibition can provide a strong, focused anti-thrombotic regimen while minimizing vasodilator side effects.

A bioassay system of autologous human endothelial, smooth muscle cells, and leukocytes for use in drug discovery, phenotyping, and tissue engineering.

Blood vessels are comprised of endothelial and smooth muscle cells. Obtaining both types of cells from vessels of living donors is not possible without invasive surgery. To address this, we have devised a strategy whereby human endothelial and smooth muscle cells derived from blood progenitors from the same donor could be cultured with autologous leukocytes to generate a same donor "vessel in a dish" bioassay. Autologous sets of blood outgrowth endothelial cells (BOECs), smooth muscle cells (BO-SMCs), and leukocytes were obtained from four donors. Cells were treated in monoculture and cumulative coculture conditions. The endothelial specific mediator endothelin-1 along with interleukin (IL)-6, IL-8, tumor necrosis factor α, and interferon gamma-induced protein 10 were measured under control culture conditions and after stimulation with cytokines. Cocultures remained viable throughout. The profile of individual mediators released from cells was consistent with what we know of endothelial and smooth muscle cells cultured from blood vessels. For the first time, we report a proof of concept study where autologous blood outgrowth "vascular" cells and leukocytes were studied alone and in coculture. This novel bioassay has usefulness in vascular biology research, patient phenotyping, drug testing, and tissue engineering.

Mechanistic definition of the cardiovascular mPGES-1/COX-2/ADMA axis.

AIMS: Cardiovascular side effects caused by non-steroidal anti-inflammatory drugs (NSAIDs), which all inhibit cyclooxygenase (COX)-2, have prevented development of new drugs that target prostaglandins to treat inflammation and cancer. Microsomal prostaglandin E synthase-1 (mPGES-1) inhibitors have efficacy in the NSAID arena but their cardiovascular safety is not known. Our previous work identified asymmetric dimethylarginine (ADMA), an inhibitor of endothelial nitric oxide synthase, as a potential biomarker of cardiovascular toxicity associated with blockade of COX-2. Here, we have used pharmacological tools and genetically modified mice to delineate mPGES-1 and COX-2 in the regulation of ADMA. METHODS AND RESULTS: Inhibition of COX-2 but not mPGES-1 deletion resulted in increased plasma ADMA levels. mPGES-1 deletion but not COX-2 inhibition resulted in increased plasma prostacyclin levels. These differences were explained by distinct compartmentalization of COX-2 and mPGES-1 in the kidney. Data from prostanoid synthase/receptor knockout mice showed that the COX-2/ADMA axis is controlled by prostacyclin receptors (IP and PPARß/δ) and the inhibitory PGE2 receptor EP4, but not other PGE2 receptors. CONCLUSION: These data demonstrate that inhibition of mPGES-1 spares the renal COX-2/ADMA pathway and define mechanistically how COX-2 regulates ADMA.

Imaging of Tissue-Specific and Temporal Activation of GPCR Signaling Using DREADD Knock-In Mice.

Engineered G protein-coupled receptors (DREADDs, designer receptors exclusively activated by designer drugs) are convenient tools for specific activation of GPCR signaling in many cell types. DREADDs have been utilized as research tools to study numerous cellular and physiologic processes, including regulation of neuronal activity, behavior, and metabolism. Mice with random insertion transgenes and adeno-associated viruses have been widely used to express DREADDs in individual cell types. We recently created and characterized ROSA26-GsDREADD knock-in mice to allow Cre recombinase-dependent expression of a Gαs-coupled DREADD (GsD) fused to GFP in distinct cell populations in vivo. These animals also harbor a CREB-activated luciferase reporter gene for analysis of CREB activity by in vivo imaging, ex vivo imaging, or biochemical reporter assays. In this chapter, we provide detailed methods for breeding GsD animals, inducing GsD expression, stimulating GsD activity, and measuring basal and stimulated CREB reporter bioluminescence in tissues in vivo, ex vivo, and in vitro. These animals are available from our laboratory for non-profit research.

Development of a novel UHPLC-MS/MS-based platform to quantify amines, amino acids and methylarginines for applications in human disease phenotyping.

Amine quantification is an important strategy in patient stratification and personalised medicine. This is because amines, including amino acids and methylarginines impact on many homeostatic processes. One important pathway regulated by amine levels is nitric oxide synthase (NOS). NOS is regulated by levels of (i) the substrate, arginine, (ii) amino acids which cycle with arginine and (iii) methylarginine inhibitors of NOS. However, biomarker research in this area is hindered by the lack of a unified analytical platform. Thus, the development of a common metabolomics platform, where a wide range of amino acids and methylarginines can be measured constitutes an important unmet need. Here we report a novel high-throughput ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) platform where ≈40 amine analytes, including arginine and methylarginines can be detected and quantified on a molar basis, in a single sample of human plasma. To validate the platform and to generate biomarkers, human plasma from a well-defined cohort of patients before and after coronary artery bypass surgery, who developed systemic inflammatory response syndrome (SIRS), were analysed. Bypass surgery with SIRS significantly altered 26 amine analytes, including arginine and ADMA. Consequently, pathway analysis revealed significant changes in a range of pathways including those associated with NOS.

Astragaloside VI and cycloastragenol-6-O-beta-D-glucoside promote wound healing in vitro and in vivo.

BACKGROUND: Astragalus genus includes most of the common, historical herbal medicines that have various applications in Asian countries. However, clinical data and mechanistic insights into their actions are still lacking. PURPOSE: In this study, we aimed to examine the effects of astragalosides on wound healing in vitro and in vivo, as well as the underlying mechanisms of these actions. METHODS: The wound healing activity of astragalosides was investigated in human HaCaT keratinocytes, human dermal fibroblast (HDF) cells, and murine models of wound healing. RESULTS: All eight astragalosides studied enhanced epidermal growth factor receptor (EGFR) activity in HaCaT cells. Among them, astragaloside VI (AS-VI) showed the strongest EGFR activation. Consistently, AS-VI and cycloastragenol-6-O-beta-D-glucoside (CMG), which is the major metabolite of astragalosides, enhanced extracellular signal-regulated kinase (ERK) activity in a concentration-dependent manner. In agreement, both compounds induced EGFR-dependent cell proliferation and migration in HaCaT and HDF cells. In addition, we showed that AS-VI and CMG accelerated the healing of both sterile and infected wounds in vivo. These effects were associated with increased angiogenesis in the scar tissue. CONCLUSION: AS-VI and CMG increased the proliferation and migration of skin cells via activation of the EGFR/ERK signalling pathway, resulting in the improvement of wound healing in vitro and in vivo. These findings indicate the therapeutic potential of AS-VI and CMG to accelerate wound healing; additionally, they suggest the mechanistic basis of this activity.

Cyclooxygenase-2 Selectively Controls Renal Blood Flow Through a Novel PPARß/δ-Dependent Vasodilator Pathway.

Cyclooxygenase-2 (COX-2) is an inducible enzyme expressed in inflammation and cancer targeted by nonsteroidal anti-inflammatory drugs. COX-2 is also expressed constitutively in discreet locations where its inhibition drives gastrointestinal and cardiovascular/renal side effects. Constitutive COX-2 expression in the kidney regulates renal function and blood flow; however, the global relevance of the kidney versus other tissues to COX-2-dependent blood flow regulation is not known. Here, we used a microsphere deposition technique and pharmacological COX-2 inhibition to map the contribution of COX-2 to regional blood flow in mice and compared this to COX-2 expression patterns using luciferase reporter mice. Across all tissues studied, COX-2 inhibition altered blood flow predominantly in the kidney, with some effects also seen in the spleen, adipose, and testes. Of these sites, only the kidney displayed appreciable local COX-2 expression. As the main site where COX-2 regulates blood flow, we next analyzed the pathways involved in kidney vascular responses using a novel technique of video imaging small arteries in living tissue slices. We found that the protective effect of COX-2 on renal vascular function was associated with prostacyclin signaling through PPARß/δ (peroxisome proliferator-activated receptor-ß/δ). These data demonstrate the kidney as the principle site in the body where local COX-2 controls blood flow and identifies a previously unreported PPARß/δ-mediated renal vasodilator pathway as the mechanism. These findings have direct relevance to the renal and cardiovascular side effects of drugs that inhibit COX-2, as well as the potential of the COX-2/prostacyclin/PPARß/δ axis as a therapeutic target in renal disease.

Differential role of pannexin-1/ATP/P2X7 axis in IL-1ß release by human monocytes.

IL-1ß release is integral to the innate immune system. The release of mature IL-1ß depends on 2 regulated events: the de novo induction of pro-IL-1ß, generally via NF-κB-dependent transduction pathways; and the assembly and activation of the NLRP3 inflammasome. This latter step is reliant on active caspase-1, pannexin-1, and P2X7 receptor activation. Pathogen-associated molecular patterns in gram-positive and gram-negative bacteria activate IL-1ß release from immune cells via TLR2 and TLR4 receptors, respectively. We found that pro-IL-1ß and mature IL-1ß release from human monocytes is stimulated by the TLR2 agonists Pam3CSK4 or FSL-1, as well as the TLR4 agonist LPS in the absence of additional ATP. TLR2 agonists required pannexin-1 and P2X7 receptor activation to stimulate IL-1ß release. In contrast, IL-1ß release stimulated by the TLR4 agonist LPS is independent of both pannexin-1 and P2X7 activation. In the absence of exogenous ATP, P2X7 activation requires endogenous ATP release, which occurs in some cells via pannexin-1. In line with this, we found that LPS-stimulated human monocytes released relatively low levels of ATP, whereas cells stimulated with TLR2 agonists released high levels of ATP. These findings suggest that in human monocytes, both TLR2 and TLR4 signaling induce pro-IL-1ß expression, but the mechanism by which they activate caspase-1 diverges at the level of the pannexin-1/ATP/P2X7 axis.-Parzych, K., Zetterqvist, A. V., Wright, W. R., Kirkby, N. S., Mitchell, J. A., Paul-Clark, M. J. Differential role of pannexin-1/ATP/P2X7 axis in IL-1ß release by human monocytes.

Aspirin inhibits the production of proangiogenic 15(S)-HETE by platelet cyclooxygenase-1.

Regular consumption of low-dose aspirin reduces the occurrence of colorectal, esophageal, stomach, and gastrointestinal cancers. The underlying mechanism is unknown but may be linked to inhibition of angiogenesis. Because the effective doses of aspirin are consistent with the inhibition of cyclooxygenase-1 in platelets, we used liquid chromatography with tandem mass spectrometry analyses and immunoassays of human platelet releasates coupled with angiogenesis assays to search for the mediators of these effects. Blood or platelet-rich plasma from healthy volunteers stimulated with platelet activators produced a broad range of eicosanoids. Notably, preincubation of platelets with aspirin, but not with a P2Y12 receptor antagonist, caused a marked reduction in the production of 11-hydroxyeicosatetraenoic acid (HETE) and 15(S)-HETE, in addition to prostanoids such as thromboxane A2 Releasates from activated platelets caused cell migration and tube formation in cultured human endothelial cells and stimulated the sprouting of rat aortic rings in culture. These proangiogenic effects were absent when platelets were treated with aspirin but returned by coincubation with exogenous 15(S)-HETE. These results reveal 15(S)-HETE as a major platelet cyclooxygenase-1 product with strong proangiogenic effects. Thus, 15(S)-HETE represents a potential target for the development of novel antiangiogenic therapeutics, and blockade of its production may provide a mechanism for the anticancer effects of aspirin.-Rauzi, F., Kirkby, N. S., Edin, M. L., Whiteford, J. Zeldin, D. C., Mitchell, J. A., Warner, T. D. Aspirin inhibits the production of proangiogenic 15(S)-HETE by platelet cyclooxygenase-1.

P2Y12 receptor blockade synergizes strongly with nitric oxide and prostacyclin to inhibit platelet activation.

AIMS: In vivo platelet function is a product of intrinsic platelet reactivity, modifiable by dual antiplatelet therapy (DAPT), and the extrinsic inhibitory endothelial mediators, nitric oxide (NO) and prostacyclin (PGI2 ), that are powerfully potentiated by P2Y12 receptor blockade. This implies that for individual patients endothelial mediator production is an important determinant of DAPT effectiveness. Here, we have investigated this idea using platelets taken from healthy volunteers treated with anti-platelet drugs. METHODS: Three groups of male volunteers (n = 8) received either prasugrel (10 mg), aspirin (75 mg) or DAPT (prasugrel + aspirin) once daily for 7 days. Platelet reactivity in the presence of diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NONOate) and PGI2 was studied before and following treatment. RESULTS: Ex vivo, PGI2 and/or DEA/NONOate had little inhibitory effect on TRAP-6-induced platelet reactivity in control conditions. However, in the presence of DAPT, combination of DEA/NONOate + PGI2 reduced platelet aggregation (74 ± 3% to 19 ± 6%, P < 0.05). In vitro studies showed even partial (25%) P2Y12 receptor blockade produced a significant (67 ± 2% to 39 ± 10%, P < 0.05) inhibition when DEA/NONOate + PGI2 was present. CONCLUSIONS: We have demonstrated that PGI2 and NO synergize with P2Y12 receptor antagonists to produce powerful platelet inhibition. Furthermore, even with submaximal P2Y12 blockade the presence of PGI2 and NO greatly enhances platelet inhibition. Our findings highlight the importance of endothelial mediator in vivo modulation of P2Y12 inhibition and introduces the concept of refining ex vivo platelet function testing by incorporating an assessment of endothelial function to predict thrombotic outcomes better and adjust therapy to prevent adverse outcomes in individual patients.

Selective endothelin A receptor antagonism with sitaxentan reduces neointimal lesion size in a mouse model of intraluminal injury.

BACKGROUND AND PURPOSE: Endothelin (ET) receptor antagonism reduces neointimal lesion formation in animal models. This investigation addressed the hypothesis that the selective ETA receptor antagonist sitaxentan would be more effective than mixed ETA / B receptor antagonism at inhibiting neointimal proliferation in a mouse model of intraluminal injury. EXPERIMENTAL APPROACH: Antagonism of ETA receptors by sitaxentan (1-100 nM) was assessed in femoral arteries isolated from adult, male C57Bl6 mice using isometric wire myography. Neointimal lesion development was induced by intraluminal injury in mice receiving sitaxentan (ETA antagonist; 15 mg·kg(-1) ·day(-1) ), A192621 (ETB antagonist; 30 mg·kg(-1) ·day(-1) ), the combination of both antagonists or vehicle. Treatment began 1 week before, and continued for 28 days after, surgery. Femoral arteries were then harvested for analysis of lesion size and composition. KEY RESULTS: Sitaxentan produced a selective, concentration-dependent parallel rightward shift of ET-1-mediated contraction in isolated femoral arteries. Sitaxentan reduced neointimal lesion size, whereas ETB and combined ETA / B receptor antagonism did not. Macrophage and α-smooth muscle actin content were unaltered by ET receptor antagonism but sitaxentan reduced the amount of collagen in lesions. CONCLUSIONS AND IMPLICATIONS: These results suggest that ETA receptor antagonism would be more effective than combined ETA /ETB receptor antagonism at reducing neointimal lesion formation.

Blockade of the purinergic P2Y12 receptor greatly increases the platelet inhibitory actions of nitric oxide.

Circulating platelets are constantly exposed to nitric oxide (NO) released from the vascular endothelium. This NO acts to reduce platelet reactivity, and in so doing blunts platelet aggregation and thrombus formation. For successful hemostasis, platelet activation and aggregation must occur at sites of vascular injury despite the constant presence of NO. As platelets aggregate, they release secondary mediators that drive further aggregation. Particularly significant among these secondary mediators is ADP, which, acting through platelet P2Y12 receptors, strongly amplifies aggregation. Platelet P2Y12 receptors are the targets of very widely used antithrombotic drugs such as clopidogrel, prasugrel, and ticagrelor. Here we show that blockade of platelet P2Y12 receptors dramatically enhances the antiplatelet potency of NO, causing a 1,000- to 100,000-fold increase in inhibitory activity against platelet aggregation and release reactions in response to activation of receptors for either thrombin or collagen. This powerful synergism is explained by blockade of a P2Y12 receptor-dependent, NO/cGMP-insensitive phosphatidylinositol 3-kinase pathway of platelet activation. These studies demonstrate that activation of the platelet ADP receptor, P2Y12, severely blunts the inhibitory effects of NO. The powerful antithrombotic effects of P2Y12 receptor blockers may, in part, be mediated by profound potentiation of the effects of endogenous NO.

Hydrogen sulphide pathway contributes to the enhanced human platelet aggregation in hyperhomocysteinemia.

Homocysteine is metabolized to methionine by the action of 5,10 methylenetetrahydrofolate reductase (MTHFR). Alternatively, by the transulfuration pathway, homocysteine is transformed to hydrogen sulphide (H2S), through multiple steps involving cystathionine ß-synthase and cystathionine γ-lyase. Here we have evaluated the involvement of H2S in the thrombotic events associated with hyperhomocysteinemia. To this purpose we have used platelets harvested from healthy volunteers or patients newly diagnosed with hyperhomocysteinemia with a C677T polymorphism of the MTHFR gene (MTHFR++). NaHS (0.1-100 µM) or l-cysteine (0.1-100 µM) significantly increased platelet aggregation harvested from healthy volunteers induced by thrombin receptor activator peptide-6 amide (2 µM) in a concentration-dependent manner. This increase was significantly potentiated in platelets harvested from MTHFR++ carriers, and it was reversed by the inhibition of either cystathionine ß-synthase or cystathionine γ-lyase. Similarly, in MTHFR++ carriers, the content of H2S was significantly higher in either platelets or plasma compared with healthy volunteers. Interestingly, thromboxane A2 production was markedly increased in response to both NaHS or l-cysteine in platelets of healthy volunteers. The inhibition of phospholipase A2, cyclooxygenase, or blockade of the thromboxane receptor markedly reduced the effects of H2S. Finally, phosphorylated-phospholipase A2 expression was significantly higher in MTHFR++ carriers compared with healthy volunteers. In conclusion, the H2S pathway is involved in the prothrombotic events occurring in hyperhomocysteinemic patients.

Cyclooxygenase and cytokine regulation in lung fibroblasts activated with viral versus bacterial pathogen associated molecular patterns.

Cyclooxygenase (COX) is required for prostanoid (e.g. prostaglandin PGE2) production. Constitutive COX-1 and inducible COX-2 are implicated in lung diseases, such as idiopathic pulmonary fibrosis (IPF). Using lung fibroblasts from humans and wild type, COX-1(-/-) and COX-2(-/-) mice, we investigated how COX activity modulates cell growth and inflammatory responses induced by activators of Toll-like receptors (TLRs) 1-8. In mouse tissue, PGE2 release from fresh lung was COX-1 driven, in lung in culture (24h) COX-1 and COX-2 driven, and from proliferating lung fibroblasts exclusively COX-2 driven. COX-2 limited proliferation in lung fibroblasts and both isoforms limited KC release induced by a range of TLR agonists. Less effect of COX was seen on TLR-induced IP-10 release. In human lung fibroblasts inhibition of COX with diclofenac was associated with increased release of IL-8 and IP-10. Our results may have implications for the treatment of IPF.

Optical multichannel (optimul) platelet aggregometry in 96-well plates as an additional method of platelet reactivity testing.

Platelet reactivity testing is important for the diagnosis of bleeding disorders, and increasingly to optimise anti-platelet therapy. Traditional light transmission aggregometry is considered the gold standard, whilst 96-well plate aggregometry, founded on similar principles, provides a higher throughput screening method. Despite the widespread use of both, methodologies and outputs vary widely between laboratories. We report a methodological approach towards providing a standardised optical detection of platelet aggregation (optimul method) based upon 96-well plate aggregometry. Individual wells of half-area 96-well plates were coated with gelatine and one of seven concentrations of arachidonic acid (AA), adenosine diphosphate (ADP), collagen, epinephrine (EPI), ristocetin, TRAP-6 amide or U46619, before being lyophilised, vacuum-sealed, foil-packed and stored at room temperature for up to 24 weeks. For platelet testing, 40 µl of platelet-rich plasma was added to each well. Platelet aggregation was determined by changes in light absorbance, release of ATP/ADP by luminescence and release of thromboxane (TX) A(2) by ELISA. Some experiments were conducted in the presence of aspirin (30 µM) or prasugrel active metabolite (PAM; 3 µM). Optimul plates stored for up to 12 weeks permitted reliable detection of concentration-dependent platelet aggregation, ATP/ADP release and TXA2 production. PAM caused reductions in platelet responses to AA, ADP, collagen, EPI, TRAP-6 and U46619, whilst aspirin inhibited responses to AA, collagen and EPI. We conclude that the optimul method offers a viable, standardised approach, allowing platelet reactivity testing and could provide a broad platelet function analysis without the need for dedicated equipment.