110 results found
Shih C-C, Chan MV, Kirkby NS, et al., 2021, Platelet inhibition by P2Y12 antagonists is potentiated by adenosine signalling activators., Br J Pharmacol
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 previously 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, 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 to both prasugrel and ticagrelor despite reports of differences in their effects upon adenosine reuptake.
Mitchell J, Shala F, Lopes Pires M, et al., 2021, Endothelial cyclooxygenase-1 paradoxically drives local vasoconstriction and atherogenesis despite underpinning prostacyclin generation, Science Advances, Vol: 7, ISSN: 2375-2548
Endothelial cyclooxygenase-1–derived prostanoids, including prostacyclin, have clear cardioprotective roles associated with their anti-thrombotic potential but have also been suggested to have paradoxical pathological activities within arteries. To date it has not been possible to test the importance of this because no models have been available that separate vascular cyclooxygenase-1 products from those generated elsewhere. Here, we have used unique endothelial-specific cyclooxygenase-1 knockout mice to show that endothelial cyclooxygenase-1 produces both protective and pathological products. Functionally, however, the overall effect of these was to drive pathological responses in the context of both vasoconstriction in vitro and the development of atherosclerosis and vascular inflammation in vivo. These data provide the first demonstration of a pathological role for the vascular cyclooxygenase-1 pathway, highlighting its potential as a therapeutic target. They also emphasize that, across biology, the role of prostanoids is not always predictable due to unique balances of context, products, and receptors.
Mohamed NA, Abou-Saleh H, Kameno Y, et al., 2021, Studies on metal-organic framework (MOF) nanomedicine preparations of sildenafil for the future treatment of pulmonary arterial hypertension, Scientific Reports, Vol: 11, Pages: 1-8, ISSN: 2045-2322
Pulmonary arterial hypertension (PAH) is an incurable disease, although symptoms are treated with a range of dilator drugs. Despite their clinical benefits, these drugs are limited by systemic side-effects. It is, therefore, increasingly recognised that using controlled drug-release nanoformulation, with future modifications for targeted drug delivery, may overcome these limitations. This study presents the first evaluation of a promising nanoformulation (highly porous iron-based metal–organic framework (MOF); nanoMIL-89) as a carrier for the PAH-drug sildenafil, which we have previously shown to be relatively non-toxic in vitro and well-tolerated in vivo. In this study, nanoMIL-89 was prepared and charged with a payload of sildenafil (generating Sil@nanoMIL-89). Sildenafil release was measured by Enzyme-Linked Immunosorbent Assay (ELISA), and its effect on cell viability and dilator function in mouse aorta were assessed. Results showed that Sil@nanoMIL-89 released sildenafil over 6 h, followed by a more sustained release over 72 h. Sil@nanoMIL-89 showed no significant toxicity in human blood outgrowth endothelial cells for concentrations up to100µg/ml; however, it reduced the viability of the human pulmonary artery smooth muscle cells (HPASMCs) at concentrations > 3 µg/ml without inducing cellular cytotoxicity. Finally, Sil@nanoMIL-89 induced vasodilation of mouse aorta after a lag phase of 2–4 h. To our knowledge, this study represents the first demonstration of a novel nanoformulation displaying delayed drug release corresponding to vasodilator activity. Further pharmacological assessment of our nanoformulation, including in PAH models, is required and constitutes the subject of ongoing investigations.
Cyclooxygenase (COX)-1 and COX-2 are centrally important enzymes within the cardiovascular system with a range of diverse, sometimes opposing, functions. Through the production of thromboxane, COX in platelets is a pro-thrombotic enzyme. By contrast, through the production of prostacyclin, COX in endothelial cells is antithrombotic and in the kidney regulates renal function and blood pressure. Drug inhibition of COX within the cardiovascular system is important for both therapeutic intervention with low dose aspirin and for the manifestation of side effects caused by nonsteroidal anti-inflammatory drugs. This review focuses on the role that COX enzymes and drugs that act on COX pathways have within the cardiovascular system and provides an in-depth resource covering COX biology and pharmacology. The review goes on to consider the role of COX in both discrete cardiovascular locations and in associated organs that contribute to cardiovascular health. We discuss the importance of, and strategies to manipulate, the thromboxane: prostacyclin balance. Finally within this review the authors discuss testable COX-2-hypotheses intended to stimulate debate and facilitate future research and therapeutic opportunities within the field.
Ahmetaj-Shala B, Ricky V, Santosh A, et al., 2020, Cardiorenal tissues express SARS-CoV-2 entry genes and basigin (BSG/CD147) increases with age in endothelial cells, JACC: Basic to Translational Science, Vol: 5, Pages: 1111-1123, ISSN: 2452-302X
Objectives: To obtain mechanistic insight into COVID-19 within a cardiovascular setting.Background: Thrombosis and vascular dysfunction are part of the complex pathology seen in severe COVID-19 and advancing age is the most significant risk factor. Little is known about age and expression of pathways utilised by the COVID-19 virus, SARS-CoV-2, in cardiovascular tissues.Methods: We used publicly available databases (GTEx, GEO and Array Express) to investigate gene expression levels, in adult tissues, of the two putative SARS-CoV-2 receptors, ACE2 and BSG along with a selected range of genes thought to be involved in virus binding/processing. Our analysis included; vessels (aorta and coronary artery), heart (atrial appendage and left ventricle), kidney (cortex), whole blood, lung, colon and spleen along with endothelial cells, nasal and bronchial epithelium and peripheral blood mononuclear cells. Gene expression levels were then analysed for age associations.Results: We found: (i) cardiovascular tissues/endothelial cells express the required genes for SARS-CoV-2 infection, (ii) SARS-CoV-2 receptor pathways, ACE2/TMPRSS2 and BSG/PPIB(A) polarise to lung/epithelium and vessel/endothelium respectively, (iii) expression of host genes are relatively stable with age and (iv) notable exceptions are ACE2 which decreases with age in some tissues and BSG which increases with age in endothelial cells.Conclusion: Our data identifies a positive correlation of BSG with age in endothelial cells. Since BSG is utilised by other pathogens and is implicated in a range of cardiovascular disease, our observations may have relevance to our understanding of mechanisms associated with other pathogens and in the diseases associated with aging respectively.
Kirkby N, Raouf J, Ahmetaj-Shala B, et al., 2020, Mechanistic definition of the cardiovascular mPGES-1/COX-2/ADMA axis, Cardiovascular Research, Vol: 116, Pages: 1972-1980, ISSN: 0008-6363
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 eNOS, 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 compartmentalisation 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 PPARβ/δ) and the inhibitory PGE2 receptor EP4, but not other PGE2 receptors.Conclusions:These data demonstrate that inhibition of mPGES-1 spares the renal COX-2/ADMA pathway and define mechanistically how COX-2 regulates ADMA.
Vaja R, Chan J, Ferreira P, et al., 2020, The COVID-19 ibuprofen controversy: A systematic review of NSAIDs in adult acute lower respiratory tract infections, BRITISH JOURNAL OF CLINICAL PHARMACOLOGY, Vol: 87, Pages: 776-784, ISSN: 0306-5251
Armstrong PC, Ferreira PM, Chan MV, et al., 2020, Combination of cyclic nucleotide modulators with P2Y(12) receptor antagonists as anti-platelet therapy, Journal of Thrombosis and Haemostasis, Vol: 18, Pages: 1705-1713, ISSN: 1538-7836
BackgroundEndothelium‐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.ObjectiveWe investigated using GC‐activators in combination with P2Y12 receptor antagonists as a way to selectively amplify the anti‐thrombotic effect of both drugs.MethodsIn 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.ResultsThe 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.ConclusionsUsing 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.
Crescente M, Armstrong P, Kirkby N, et al., 2020, Profiling the eicosanoid networks that underlie the anti- and pro-thrombotic effects of aspirin, The FASEB Journal, ISSN: 0892-6638
Aspirin preventsthrombosisby inhibiting platelet cyclooxygenase (COX)-1 activity and the production of thromboxane (Tx)A2, a pro-thrombotic eicosanoid. However, thenon-platelet actions ofaspirin limit its anti-thrombotic effects. Here we used platelet-COX-1-komice to define the platelet andnon-platelet eicosanoids affected by aspirin.Mass-spectrometry analysis demonstrated blood fromplatelet-COX-1-ko and global-COX-1-ko mice produced similar eicosanoid profiles in vitro: e.g. formation of TxA2,prostaglandin (PG) F2a, 11-HETE and 15-HETE was absent in both platelet-and global-COX-1-komice. Conversely, in vivo,platelet-COX-1-ko mice had a distinctly different profile from global-COX-1-koor aspirin-treated controlmice, notably significantly higher levels of PGI2metabolite. Ingenuity Pathway Analysis predicted that platelet-COX-1-ko mice would be protected from thrombosis,formingless prothrombotic TxA2 and PGE2. Conversely, aspirinor lack of systemic COX-1 activity decreased the synthesis of anti-aggregatory PGI2and PGD2 at non-platelet sites leading to predictedthrombosis increase. In vitroand in vivothrombosis studies proved these predictions. Overall, we have established the eicosanoid profileslinked to inhibition of COX-1 in platelets and in the remainder of the cardiovascular systemand linked them to anti-and pro-thrombotic effects of aspirin. These results explainwhy increasing aspirin dosage or aspirin addition to other drugs may lessenanti-thrombotic protection.
Ahmetaj-Shala B, Kawai R, Marei I, et al., 2020, A bioassay system of autologous human endothelial, smooth muscle cells and leucocytes for use in drug discovery, phenotyping and tissue engineering, The FASEB Journal, Vol: 34, Pages: 1745-1754, ISSN: 0892-6638
Purpose: 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 leucocytes to generate a same donor ‘vessel in a dish’ bioassay. Basic procedures: Autologous sets of blood outgrowth endothelial cells (BOECs), smooth muscle cells (BO-SMCs) and leucocytes were obtained from 4 donors. Cells were treated in mono and cumulative co-culture conditions. The endothelial specific mediator endothelin-1 along with interleukin (IL)-6, IL-8, tumour necrosis factor α, and interferon gamma-induced protein 10 were measured under control culture conditions and after stimulation with cytokines.Main findings: Co-cultures 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.Principle conclusions: For the first time, we report a proof of concept study where autologous blood outgrowth ‘vascular’ cells and leucocytes were studied alone and in co-culture. This novel bioassay has utility in vascular biology research, patient phenotyping, drug testing and tissue engineering.
Mitchell J, Shala F, Elghazouli Y, et al., 2019, Cell-Specific Gene Deletion Reveals the Antithrombotic Function of COX1 and Explains the Vascular COX1/Prostacyclin Paradox, Circulation Research, Vol: 125, Pages: 847-854, ISSN: 0009-7330
Rationale: Endothelial cells and platelets, which respectively produce anti-thrombotic prostacyclin and pro-thrombotic thromboxane A2, both express COX1. Consequently, there has been no way to delineate any anti-thrombotic role for COX1-derived prostacyclin from the pro-thrombotic effects of platelet COX1. By contrast an anti-thrombotic role for COX2, which is absent in platelets, is straightforward to demonstrate. This has resulted in an incomplete understanding of the relative importance of COX1 versus COX2 in prostacyclin production and anti-thrombotic protection in vivo.Objective: We sought to identify the role, if any, of COX1-derived prostacyclin in anti-thrombotic protection in vivo and compare this to the established protective role of COX2.Methods and Results: We developed vascular-specific COX1 knockout mice and studied them alongside endothelial-specific COX2 knockout mice. COX1 immunoreactivity and prostacyclin production were primarily associated with the endothelial layer of aortae; freshly isolated aortic endothelial cells released >10-fold more prostacyclin than smooth muscle cells. Moreover, aortic prostacyclin production, the ability of aortic rings to inhibit platelet aggregation and plasma prostacyclin levels were reduced when COX1 was knocked out in endothelial cells but not in smooth muscle cells. When thrombosis was measured in vivo after FeCl3 carotid artery injury, endothelial COX1 deletion accelerated thrombosis to a similar extent as prostacyclin receptor blockade. However, but this effect was lost when COX1 was deleted from both endothelial cells and platelets. Deletion of COX2 from endothelial cells also resulted in a pro-thrombotic phenotype that was independent of local vascular prostacyclin production.Conclusions:These data demonstrate for the first time that, in healthy animals, endothelial COX1 provides an essential anti-thrombotic tone, which is masked when COX1 activity is lost in both endothelial cells and platelets. These res
Mohamed NA, Davies RP, Lickiss PD, et al., 2019, Mil-89 nanoformulation as a platform to improve pulmonary arterial hypertension treatment, British-Pharmacology-Society Meeting (Pharmacology), Publisher: WILEY, Pages: 3075-3075, ISSN: 0007-1188
Ahmetaj-Shala B, Kawai R, Marei I, et al., 2019, A bioassay system of autologous human endothelial and smooth muscle cells for use in cardiovascular drug discovery and patient phenotyping, British-Pharmacology-Society Meeting (Pharmacology), Publisher: WILEY, Pages: 3040-3041, ISSN: 0007-1188
Mazi SI, Ahmetaj-Shala B, Warner TD, et al., 2019, Omic profiling in healthy volunteers taking celecoxib reveals novel biomarkers regulated by cyclooxygenase-2, British-Pharmacology-Society Meeting (Pharmacology), Publisher: WILEY, Pages: 1628-1628, ISSN: 0306-5251
Akhmedov D, Kirkby NS, Mitchell JA, et al., 2019, Imaging of Tissue-Specific and Temporal Activation of GPCR Signaling Using DREADD Knock-In Mice., Pages: 361-376
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.
Mitchell J, Kirkby NS, 2019, Eicosanoids, prostacyclin and cyclooxygenase in the cardiovascular system, British Journal of Pharmacology, Vol: 176, Pages: 1038-1050, ISSN: 1476-5381
Eicosanoids represent a diverse family of lipid mediators with fundamental roles in controlling physiology and disease. Within the eicosanoid super family are prostanoids, which are specifically derived from arachidonic acid by the enzyme cyclooxygenase(COX). COX has two isoforms; COX-1 and COX-2. COX-2 is the therapeutic target for the nonsteroidal anti-inflammatory drug (NSAID) class of pain medications. Of the prostanoids,prostacyclin, first discovered by Sir Professor John Vane in 1976,remains amongst the best studied and retains an impressive pedigree as onethe bodies fundamental cardiovascular protective pathways. Since this time, we have learnt much about how eicosanoids, COXenzymes and prostacyclin function in the cardiovascular system which has allowed us to, for example, harness the power of prostacyclin as therapy to treat pulmonary arterial hypertension and peripheral vascular disease. However, there remain many unanswered questions in our basic understanding of the pathways and how they can be usedto improve human health. Perhaps the most importantand controversial outstanding question in the field remains;‘how do NSAIDsproduce their much publicized cardiovascular side effects?’This review summarises the history, biology and cardiovascular function of key eicosanoids with particular focus on prostacyclin and other COXproducts and discusses how our knowledge of these pathways can applied in future drug discovery and be used to explain the cardiovascular side effects of NSAIDs.
Kawai R, Ahmetaj-Shala B, Shih CC, et al., 2018, Development of a human autologous 3-cell cytokine release assay that models the vascular wall in vitro, 54th Congress of the European-Societies-of-Toxicology (EUROTOX) - Toxicology Out of the Box, Publisher: ELSEVIER IRELAND LTD, Pages: S114-S114, ISSN: 0378-4274
Ahmetaj-Shala B, Olanipekun M, Tesfai A, et al., 2018, Development of a novel UPLC-MS/MS-based platform to quantify amines, amino acids and methylarginines for applications in human disease phenotyping, Scientific Reports, Vol: 8, ISSN: 2045-2322
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.
Mitchell JA, Knowles RB, Kirkby NS, et al., 2018, Letter by Mitchell et al Regarding Article, "Urinary Prostaglandin Metabolites: An Incomplete Reckoning and a Flush to Judgment"., Circulation Research, Vol: 122, Pages: e84-e85, ISSN: 0009-7330
Mitchell JA, Knowles RB, Kirkby NS, et al., 2018, Kidney transplantation in a patient lacking cytosolic phospholipase A2Proves renal origins of urinary PGI-M and TX-M., Circulation Research, Vol: 122, Pages: 555-559, ISSN: 0009-7330
RATIONALE: The balance between vascular prostacyclin, which is antithrombotic, and platelet thromboxane A2, which is prothrombotic, is fundamental to cardiovascular health. Prostacyclin and thromboxane A2are formed after the concerted actions of cPLA2α (cytosolic phospholipase A2) and COX (cyclooxygenase). Urinary 2,3-dinor-6-keto-PGF1α(PGI-M) and 11-dehydro-TXB2(TX-M) have been taken as biomarkers of prostacyclin and thromboxane A2formation within the circulation and used to explain COX biology and patient phenotypes, despite concerns that urinary PGI-M and TX-M originate in the kidney. OBJECTIVE: We report data from a remarkable patient carrying an extremely rare genetic mutation in cPLA2α, causing almost complete loss of prostacyclin and thromboxane A2, who was transplanted with a normal kidney resulting in an experimental scenario of whole-body cPLA2α knockout, kidney-specific knockin. By studying this patient, we can determine definitively the contribution of the kidney to the productions of PGI-M and TX-M and test their validity as markers of prostacyclin and thromboxane A2in the circulation. METHODS AND RESULTS: Metabolites were measured using liquid chromatography-tandem mass spectrometry. Endothelial cells were grown from blood progenitors. Before kidney transplantation, the patient's endothelial cells and platelets released negligible levels of prostacyclin (measured as 6-keto-prostaglandin F1α) and thromboxane A2(measured as TXB2), respectively. Likewise, the urinary levels of PGI-M and TX-M were very low. After transplantation and the establishment of normal renal function, the levels of PGI-M and TX-M in the patient's urine rose to within normal ranges, whereas endothelial production of prostacyclin and platelet production of thromboxane A2remained negligible. CONCLUSIONS: These data show that PGI-M and TX-M can be derived exclusively from the kidney without contribution from prostacyclin made by endothelial cells or thromb
Kirkby NS, Sampaio W, Etelvino G, et al., 2018, Cyclooxygenase-2 selectively controls renal blood flow through a novel PPARβ/δ-dependent renal vasodilator pathway, Hypertension, Vol: 71, Pages: 297-305, ISSN: 0194-911X
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 PPARβ/δ (peroxisome proliferator-activated receptor-β/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 PPARβ/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/PPARβ/δ axis as a therapeutic target in renal disease.
Lee S-Y, Chang W-L, Li Z-X, et al., 2018, Astragaloside VI and cycloastragenol-6-O-beta-D-glucoside promote wound healing in vitro and in vivo, PHYTOMEDICINE, Vol: 38, Pages: 183-191, ISSN: 0944-7113
BackgroundAstragalus 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.PurposeIn 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.MethodsThe wound healing activity of astragalosides was investigated in human HaCaT keratinocytes, human dermal fibroblast (HDF) cells, and murine models of wound healing.ResultsAll 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.ConclusionAS-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.
Mitchell JA, Shala F, Ahmetaj-Shala B, et al., 2017, Novel Tissue-specific Cyclooxygenase-1 Knockout Mice Demonstrate a Dominant Role for Endothelial Cyclooxygenase-1 in Prostacyclin Production, Scientific Sessions of the American-Heart-Association / Resuscitation Science Symposium, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Kirkby NS, Morris AP, Lytton J, et al., 2017, Genome-Wide Association Study Links Variants With Occurrence of Cardiovascular Events in People Taking the COX-2 Inhibitor Celecoxib: Identification of NCKX2 as a Novel Protective Pathway in Renal Vessels, Scientific Sessions of the American-Heart-Association / Resuscitation Science Symposium, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Kirkby NS, Jiao J, Herschman HR, et al., 2017, Production of High Levels of PGI-M in the Kidney and Bladder Explains the Renal Origin of Urinary Markers of Prostacyclin, Scientific Sessions of the American-Heart-Association / Resuscitation Science Symposium, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Mitchell JA, Benson J, Shala F, et al., 2017, Vascular Prostanoids Paradoxically Amplify Vasoconstriction During Platelet Activation, Scientific Sessions of the American-Heart-Association / Resuscitation Science Symposium, Publisher: LIPPINCOTT WILLIAMS & WILKINS, ISSN: 0009-7322
Lee S-Y, Chen P-Y, Lin J-C, et al., 2017, Melaleuca alternifolia Induces Heme Oxygenase-1 Expression in Murine RAW264.7 Cells through Activation of the Nrf2-ARE Pathway, AMERICAN JOURNAL OF CHINESE MEDICINE, Vol: 45, Pages: 1631-1648, ISSN: 0192-415X
Melaleuca alternifolia concentrate (MAC) is the refined essential oil of the Australian native plant Melaleuca alternifolia. MAC has been reported to suppress the production of pro-inflammatory cytokines in both murine RAW264.7 macrophages and human monocytes stimulated with lipopolysaccharide (LPS). However, the mechanisms involved in this effect remain unclear. This study aims to delineate the molecular mechanisms that drive the anti-inflammatory activity of MAC and its active component, terpinen-4-ol, in macrophages. The effects of MAC on RAW264.7 cells were studied using western blotting, real-time PCR, an electrophoretic mobility shift assay (EMSA), and NF-κB luciferase reporter assays. Our results showed that MAC significantly increased both the mRNA and protein levels of heme oxygenase-1 (HO-1) via p38 and JNK MAPK activation. In addition, we showed that MAC significantly increased the activation and nuclear translocation of NF-E2-related factor 2 (Nrf2), a key transcription factor regulating HO-1 induction. MAC was also associated with significant inhibition of iNOS expression, NO production, and NF-κB activation. HO-1 was required for these anti-inflammatory effects as tin protoporphyrin IX (SnPPIX), an HO-1 inhibitor, abolished the effects of MAC on LPS-induced iNOS, NO, and NF-κB activation. Our results indicate that MAC induces HO-1 expression in murine macrophages via the p38 MAPK and JNK pathways and that this induction is required for its anti-inflammatory activity.
Slingsby MHL, Nyberg M, Egelund J, et al., 2017, Aerobic exercise training lowers platelet reactivity and improves platelet sensitivity to prostacyclin in pre- and postmenopausal women, Journal of Thrombosis and Haemostasis, Vol: 15, Pages: 2419-2431, ISSN: 1538-7836
BackgroundThe risk of atherothrombotic events increases after the menopause. Regular physical activity has been shown to reduce platelet reactivity in younger women, but it is unknown how regular exercise affects platelet function after the menopause.ObjectivesTo examine the effects of regular aerobic exercise in late premenopausal and recent postmenopausal women by testing basal platelet reactivity and platelet sensitivity to prostacyclin and nitric oxide.MethodsTwenty-five sedentary, but healthy, late premenopausal and 24 matched recently postmenopausal women, mean (95% confidence interval) 49.1 (48.2–49.9) and 53.7 (52.5–55.0) years old, participated in an intervention study: 3-month high-intensity supervised aerobic spinning-cycle training (1 h, × 3/week). Basal platelet reactivity was analyzed in platelet-rich plasma from venous blood as agonist-induced % aggregation. In a subgroup of 13 premenopausal and 14 postmenopausal women, platelet reactivity was tested ex vivo after femoral arterial infusion of prostacyclin, acetylcholine, a cyclooxygenase inhibitor, and after acute one-leg knee extensor exercise.ResultsBasal platelet reactivity (%aggregation) to TRAP-6 (1 μm) was higher in the postmenopausal, 59% (50–68), than the premenopausal women, 45% (35–55). Exercise training reduced basal platelet reactivity to collagen (1 μg mL−1) in the premenopausal women only: from 63% (55–71%) to 51% (41–62%). After the training intervention, platelet aggregation was more inhibited by the arterial prostacyclin infusion and the acute exercise in both premenopausal and postmenopausal women.ConclusionsThese results highlight previously unknown cardioprotective aspects of regular aerobic exercise in premenopausal and postmenopausal women, improving their regulation of platelet reactivity through an increased platelet sensitivity to prostacyclin, which may counterbalance the increased atherothrombotic risk associated with the
Tesfai A, MacCallum N, Kirkby NS, et al., 2017, Metabolomic profiling of amines in sepsis predicts changes in NOS canonical pathways, PLoS ONE, Vol: 12, ISSN: 1932-6203
RationaleNitric oxide synthase (NOS) is a biomarker/target in sepsis. NOS activity is driven by amino acids, which cycle to regulate the substrate L-arginine in parallel with cycles which regulate the endogenous inhibitors ADMA and L-NMMA. The relationship between amines and the consequence of plasma changes on iNOS activity in early sepsis is not known.ObjectiveOur objective was to apply a metabolomics approach to determine the influence of sepsis on a full array of amines and what consequence these changes may have on predicted iNOS activity.Methods and measurements34 amino acids were measured using ultra purification mass spectrometry in the plasma of septic patients (n = 38) taken at the time of diagnosis and 24–72 hours post diagnosis and of healthy volunteers (n = 21). L-arginine and methylarginines were measured using liquid-chromatography mass spectrometry and ELISA. A top down approach was also taken to examine the most changed metabolic pathways by Ingenuity Pathway Analysis. The iNOS supporting capacity of plasma was determined using a mouse macrophage cell-based bioassay.Main resultsOf all the amines measured 22, including L-arginine and ADMA, displayed significant differences in samples from patients with sepsis. The functional consequence of increased ADMA and decreased L-arginine in context of all cumulative metabolic changes in plasma resulted in reduced iNOS supporting activity associated with sepsis.ConclusionsIn early sepsis profound changes in amine levels were defined by dominant changes in the iNOS canonical pathway resulting in functionally meaningful changes in the ability of plasma to regulate iNOS activity ex vivo.
Mohamed NA, Davies RP, Lickiss PD, et al., 2017, Chemical and biological assessment of metal organic frameworks (MOFs) in pulmonary cells and in an acute in vivo model: relevance to pulmonary arterial hypertension therapy, Pulmonary Circulation, Vol: 7, Pages: 1-11, ISSN: 2045-8940
Pulmonary arterial hypertension (PAH) is a progressive and debilitating condition. Despite promoting vasodilation, current drugs have a therapeutic window within which they are limited by systemic side effects. Nanomedicine uses nanoparticles to improve drug delivery and/or reduce side effects. We hypothesize that this approach could be used to deliver PAH drugs avoiding the systemic circulation. Here we report the use of iron metal organic framework (MOF) MIL-89 and PEGylated MIL-89 (MIL-89 PEG) as suitable carriers for PAH drugs. We assessed their effects on viability and inflammatory responses in a wide range of lung cells including endothelial cells grown from blood of donors with/without PAH. Both MOFs conformed to the predicted structures with MIL-89 PEG being more stable at room temperature. At concentrations up to 10 or 30 µg/mL, toxicity was only seen in pulmonary artery smooth muscle cells where both MOFs reduced cell viability and CXCL8 release. In endothelial cells from both control donors and PAH patients, both preparations inhibited the release of CXCL8 and endothelin-1 and in macrophages inhibited inducible nitric oxide synthase activity. Finally, MIL-89 was well-tolerated and accumulated in the rat lungs when given in vivo. Thus, the prototypes MIL-89 and MIL-89 PEG with core capacity suitable to accommodate PAH drugs are relatively non-toxic and may have the added advantage of being anti-inflammatory and reducing the release of endothelin-1. These data are consistent with the idea that these materials may not only be useful as drug carriers in PAH but also offer some therapeutic benefit in their own right.
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