[show abstract][hide abstract] ABSTRACT: 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 β-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 β-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.
Proceedings of the National Academy of Sciences 09/2013; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 09/2013; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cyclooxygenase 2 (COX)-2 is induced by bacterial and viral infections and has complex, poorly understood roles in anti-pathogen immunity. Here, we use a knock-in luciferase reporter model to image Cox2 expression across a range of tissues in mice following treatment with the either the prototypical bacterial pathogen-associated molecular pattern (PAMP), LPS, which activates Toll-like receptor (TLR)4, or with poly(I:C), a viral PAMP, which activates TLR3. LPS induced Cox2 expression in all tissues examined. In contrast, poly(I:C) elicited a milder response, limited to a subset of tissues. A panel of cytokines and interferons was measured in plasma of wild-type, Cox1(-/-) and Cox2(-/-) mice treated with LPS, poly(I:C), MALP2 (TLR2/6), Pam3CSK4 (TLR2/1), R-848 (TLR7/8) or CpG ODN (TLR9), to establish whether/how each COX isoform modulates specific PAMP/TLR responses. Only LPS induced notable loss of condition in mice (inactivity, hunching, piloerection). However, all TLR agonists produced cytokine responses, many of which were modulated in specific fashions by Cox1 or Cox2 gene deletion. Notably we observed opposing effects of Cox2 gene deletion on the responses to the bacterial PAMP, LPS, and the viral PAMP, poly(I:C), consistent with the differing abilities of the PAMPs to induce Cox2 expression. Cox2 gene deletion limited the plasma IL-1β and interferon-γ responses and hypothermia produced by LPS. In contrast, in response to poly(I:C), Cox2(-/-) mice exhibited enhanced plasma interferon (IFNα,β,γ,λ) and related cytokine responses (IP-10,IL-12). These observations suggest that a COX-2 selective inhibitor, given early in infection, may enhance and/or prolong endogenous interferon responses, and thereby increase anti-viral immunity.
Biochemical and Biophysical Research Communications 07/2013; · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Inhibition of cyclooxygenase (COX)-2 increases cardiovascular deaths. Identifying a biomarker of COX-2 is desirable but difficult, since COX-1 and COX-2 ordinarily catalyze formation of an identical product, prostaglandin H2. When acetylated by aspirin, however, COX-2 (but not COX-1) can form 15(R)-HETE, which is metabolized to aspirin-triggered lipoxin (ATL), 15-epi-lipoxin A4. Here we have used COX-1- and COX-2-knockout mice to establish whether plasma ATL could be used as a biomarker of vascular COX-2 in vivo. Vascular COX-2 was low but increased by LPS (10 mg/kg; i.p). Aspirin (10 mg/kg; i.v.) inhibited COX-1, measured as blood thromboxane and COX-2, measured as lung PGE2. Aspirin also increased the levels of ATL in the lungs of LPS-treated wild-type C57Bl6 mice (vehicle: 25.5±9.3 ng/ml; 100 mg/kg: 112.0±7.4 ng/ml; P<0.05). Despite this, ATL was unchanged in plasma after LPS and aspirin. This was true in wild-type as well as COX-1(-/-) and COX-2(-/-) mice. Thus, in mice in which COX-2 has been induced by LPS treatment, aspirin triggers detectable 15-epi-lipoxin A4 in lung tissue, but not in plasma. This important study is the first to demonstrate that while ATL can be measured in tissue, plasma ATL is not a biomarker of vascular COX-2 expression.-Kirkby, N. S., Chan, M. V., Lundberg, M. H., Massey, K. A., Edmands, W. M. B., MacKenzie, L. S., Holmes, E., Nicolaou, A., Warner, T. D., Mitchell, J. A. Aspirin-triggered 15-epi-lipoxin A4 predicts cyclooxygenase-2 in the lungs of LPS-treated mice but not in the circulation: implications for a clinical test.
[show abstract][hide abstract] ABSTRACT: Acute renal failure (ARF) has high mortality and no effective treatment. Nitric oxide (NO) delivery represents a credible means of preventing the damaging effects of vasoconstriction, central to ARF, but design of drugs with the necessary renoselectivity is challenging. Here, we developed N-hydroxyguanidine NO donor drugs that were protected against spontaneous NO release by linkage to glutamyl adducts that could be cleaved by γ-glutamyl transpeptidase (γ-GT), found predominantly in renal tissue. Parent NO donor drug activity was optimized in advance of glutamyl adduct prodrug design. A lead compound that was a suitable substrate for γ-GT-mediated deprotection was identified. Metabolism of this prodrug to the active parent compound was confirmed in rat kidney homogenates, and the prodrug was shown to be an active vasodilator in rat isolated perfused kidneys (EC50 ∼50 μM). The data confirm that glutamate protection of N-hydroxyguanidines is an approach that might hold promise in ARF.
Journal of Medicinal Chemistry 06/2013; · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Cyclooxygenase (COX) is required for prostanoid (eg prostaglandin (PG)E2) 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 (24hours) 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.
Prostaglandins & other lipid mediators 06/2013; · 2.42 Impact Factor
[show abstract][hide abstract] ABSTRACT: Rationale: MicroRNA biomarkers are attracting considerable interest. Effects of medication, however, have not been investigated thus far. Objective: To analyse changes in plasma microRNAs in response to anti-platelet therapy. Methods and Results: Profiling for 377 microRNAs was performed in platelets, platelet microparticles, platelet-rich plasma, platelet-poor plasma and serum. Platelet-rich plasma showed markedly higher levels of microRNAs than serum and platelet-poor plasma. Few abundant platelet microRNAs, such as miR-24, miR-197, miR-191, and miR-223, were also increased in serum compared to platelet-poor plasma. In contrast, anti-platelet therapy significantly reduced microRNA levels. Using custom-made qPCR plates, 92 microRNAs were assessed in a dose-escalation study in healthy volunteers at four different time points: at baseline without therapy, at 1 week with 10mg prasugrel, at 2 weeks with 10mg prasugrel+75mg aspirin and at 3 weeks with 10mg prasugrel+300mg aspirin. Findings in healthy volunteers were confirmed by individual TaqMan qPCR assays (n=9). Validation was performed in an independent cohort of patients with symptomatic atherosclerosis (n=33) who received low dose aspirin at baseline. Plasma levels of platelet microRNAs, such as miR-223, miR-191 and others, i.e. miR-126 and miR-150, decreased upon further platelet inhibition. Conclusions: Our study demonstrated a substantial platelet contribution to the circulating microRNA pool and identified microRNAs responsive to anti-platelet therapy. It also highlights that anti-platelet therapy and preparation of blood samples could be confounding factors in case-control studies relating plasma microRNAs to cardiovascular disease.
Circulation Research 01/2013; · 11.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: There are two schools of thought regarding the cyclooxygenase (COX) isoform active in the vasculature. Using urinary prostacyclin markers some groups have proposed that vascular COX-2 drives prostacyclin release. In contrast, we and others have found that COX-1, not COX-2, is responsible for vascular prostacyclin production. Our experiments have relied on immunoassays to detect the prostacyclin breakdown product, 6-keto-PGF1α and antibodies to detect COX-2 protein. Whilst these are standard approaches, used by many laboratories, antibody-based techniques are inherently indirect and have been criticized as limiting the conclusions that can be drawn. To address this question, we measured production of prostanoids, including 6-keto-PGF1α, by isolated vessels and in the circulation in vivo using liquid chromatography tandem mass spectrometry and found values essentially identical to those obtained by immunoassay. In addition, we determined expression from the Cox2 gene using a knockin reporter mouse in which luciferase activity reflects Cox2 gene expression. Using this we confirm the aorta to be essentially devoid of Cox2 driven expression. In contrast, thymus, renal medulla, and regions of the brain and gut expressed substantial levels of luciferase activity, which correlated well with COX-2-dependent prostanoid production. These data are consistent with the conclusion that COX-1 drives vascular prostacyclin release and puts the sparse expression of Cox2 in the vasculature in the context of the rest of the body. In doing so, we have identified the thymus, gut, brain and other tissues as target organs for consideration in developing a new understanding of how COX-2 protects the cardiovascular system.
PLoS ONE 01/2013; 8(7):e69524. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: OBJECTIVE: Cryptogenic multifocal ulcerating stenosing enteritis (CMUSE) is an extremely rare, but devastating, disease of unknown aetiology. We investigated the genetic basis of this autosomal recessive condition in a pair of affected siblings who have 40-year histories of catastrophic gastrointestinal and extraintestinal disease. DESIGN: Genome-wide single-nucleotide polymorphism homozygosity mapping in the two affected family members combined with whole-exome sequencing of one affected sibling. This was followed by confirmatory Sanger sequencing of the likely disease-causing sequence variant and functional studies in affected and unaffected family members. RESULTS: Insertion/deletion variation analysis revealed the presence of a homozygous 4 bp deletion (g.155574_77delGTAA) in the PLA2G4A gene, located in the splice donor site directly after exon 17 (the penultimate exon) of the gene in both affected siblings. This introduces a frameshift of 10 amino acids before a premature stop codon (p.V707fsX10), which is predicted to result in the loss of 43 amino acids (residues 707-749) at the C-terminus of cytosolic phospholipase A2-α (cPLA(2)α). cPLA(2)α protein expression was undetectable in the gut of both siblings, with platelet aggregation and thromboxane A(2) production, as functional assays for cPLA(2)α activity, grossly impaired. CONCLUSIONS: We have identified mutations in PLA2G4A as a cause of CMUSE in two affected siblings. Further studies are needed to determine if mutations in this gene are also responsible for disease of a similar phenotype in other cases.
[show abstract][hide abstract] ABSTRACT: Prostacyclin is an antithrombotic hormone produced by the endothelium, whose production is dependent on cyclooxygenase (COX) enzymes of which two isoforms exist. It is widely believed that COX-2 drives prostacyclin production and that this explains the cardiovascular toxicity associated with COX-2 inhibition, yet the evidence for this relies on indirect evidence from urinary metabolites. Here we have used a range of experimental approaches to explore which isoform drives the production of prostacyclin in vitro and in vivo. Our data show unequivocally that under physiological conditions it is COX-1 and not COX-2 that drives prostacyclin production in the cardiovascular system, and that urinary metabolites do not reflect prostacyclin production in the systemic circulation. With the idea that COX-2 in endothelium drives prostacyclin production in healthy individuals removed, we must seek new answers to why COX-2 inhibitors increase the risk of cardiovascular events to move forward with drug discovery and to enable more informed prescribing advice.
Proceedings of the National Academy of Sciences 10/2012; 109(43):17597-602. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The potent vasoconstrictor endothelin-1 (ET-1), acting on the endothelin-A (ETA) receptor, promotes intimal lesion formation following vascular injury. The endothelin-B (ETB) receptor, which mediates nitric oxide release and ET-1 clearance in endothelial cells, may moderate lesion formation, but this is less clear. We used selective ET receptor antagonists and cell-specific deletion to address the hypothesis that ETB receptors in the endothelium inhibit lesion formation following arterial injury.
Neointimal proliferation was induced by wire or ligation injury to the femoral artery in mice treated with selective ETA (ABT-627) and/or ETB antagonists (A192621). Measurement of lesion formation by optical projection tomography and histology indicated that ETA blockade reduced lesion burden in both models. Although ETB blockade had little effect on ligation injury-induced lesion formation, after wire injury, blockade of the ETB receptor increased lesion burden (184% of vehicle; P < 0.05) and reversed the protective effects of an ETA antagonist. Selective deletion of ETB receptors from the endothelium, however, had no effect on neointimal lesion size.
These results are consistent with ETB receptor activation playing an important role in limiting neointimal lesion formation following acute vascular injury, but indicate that this protective effect is not mediated by those ETB receptors expressed by endothelial cells. These data support the proposal that selective ETA antagonists may be preferable to mixed ETA/ETB antagonists for targeting the arterial response to injury.
Cardiovascular research 03/2012; 95(1):19-28. · 5.80 Impact Factor
[show abstract][hide abstract] ABSTRACT: Increasing evidence suggests that strong P2Y12 receptor blockade may attenuate both ADP- and thromboxane (TX) A2-dependent pathways of platelet aggregation. To explore this, we compared the potency of two structurally distinct P2Y12 receptor antagonists for (i) inhibition of ADP-dependent aggregation, and (ii) inhibition of TXA2-dependent aggregation and TXA2-synthesis. Platelet-rich plasma from healthy human volunteers was treated with (i) ticagrelor (0.1-10 μM), (ii) prasugrel-active-metabolite (PAM; 0.1-10 μM) or (iii) vehicle. Aggregations to ADP, the TXA2 mimetic U46619 and arachidonic acid (AA) were recorded using 96-well plate light transmission aggregometry. TXA2 synthesis was measured by ELISA for TXB2. Ticagrelor and PAM produced concentration-dependent inhibitions of aggregation to ADP. Ticagrelor was more potent than PAM (-logIC50 against 20 μM ADP: 6.5±0.1 vs 5.6±0.1; p<0.01). As has been reported previously, the potency of ticagrelor, but not PAM, was greater at lower agonist concentration (eg, -logIC50 against 2.5 μM ADP: 7.1±0.3). Both drugs inhibited U46619-induced aggregation (logIC50 against 3 μM U46619, 6.5±0.3 and 5.6±0.1, respectively for ticagrelor and PAM), AA-induced aggregation (-logIC50 against 1 mM AA, 6.8±0.1 and 5.9±0.1) and AA-induced TXA2 formation -logIC50 against 1 mM AA, 6.9±0.9 and 5.9±0.3) with similar potencies as against ADP. P2Y12 antagonists inhibit TXA2 receptor (TP)-dependent platelet aggregation and TXA2 synthesis with similar potencies to those inhibiting ADP-induced aggregation. This is consistent with the idea that the ADP-P2Y12 interaction is crucial in supporting the activation mechanisms downstream of the TXA2 receptor, TP, and those driving TXA2 synthesis. These results question the necessity of anti-thrombotic aspirin therapy when used in combination with strong P2Y12 inhibition.
[show abstract][hide abstract] ABSTRACT: Inhibition of platelet aggregation following aspirin ingestion is thought to be overcome by the presence of a small minority of uninhibited platelets. Indeed, it is often quoted that more than 95% of platelets must be inhibited by aspirin for full anti-thrombotic protection.1 2 Aspirin is an irreversible inhibitor of platelet cyclooxygenase, while the thienopyridines, notably clopidogrel and prasugrel, are irreversible blockers of platelet P2Y12 receptors. In analogy to the effects of aspirin, we have investigated the relationships between the proportion of P2Y12 inhibited platelets and aggregation responses, using the agonists ADP, U46619, TRAP-6 amide and collagen. Platelet rich plasma obtained from healthy volunteers was incubated with either prasugrel-active metabolite (PAM; 3 μM) or vehicle. After washing, different combinations of PAM- and vehicle-treated platelets were transferred to 96-well plates containing fibrinogen (10 μM), CaCl(2) (2 μM) and increasing concentrations of ADP, U46619, TRAP-6 amide or collagen. To follow platelet aggregation, absorbance at 595 nm was measured over 16 min of vigorous shaking. In contrast to studies with aspirin, in this in vitro study we have found a linear relationship between the proportion of P2Y12 receptor uninhibited platelets and aggregation in responses to ADP, U46619 and TRAP-6 amide. As P2Y12 receptor antagonists are almost always used in conjunction with aspirin for anti-thrombotic protection this suggests a complex relationship between proportions of uninhibited platelets and platelet responsiveness which demands further examination, particularly with regard to at risk patient groups.
[show abstract][hide abstract] ABSTRACT: This study evaluated the effect of prasugrel alone, and in combination with low and high dose aspirin, on urinary metabolites of thromboxane A2 (TXA2) and prostaglandin I2 (PGI2), TX-M and PGI-M, respectively. 9 healthy males, aged 18-40, were enrolled in the 21-day study. Prasugrel was loaded at 60 mg on day 1 and maintained at 10 mg once daily until day 21. At day 8 aspirin 75 mg o.d. was introduced and the dose increased to 300 mg o.d. on day 15. On days 0, 7, 14 and 21 urine samples were obtained and TX-M and PGI-M assayed by mass spectrometry. Platelet aggregation to a TXA2-mimetic (U46619) was also determined at each time point. Data are presented as ng of urinary metabolite per mg creatine (mean±SEM). Data were analysed by one-way ANOVA with Bonferroni's post-test. Prasugrel alone did not reduce either TX-M (day 7, 0.24±0.02 ng per mg creatine; day 0, 0.28±0.04) or PGI-M (day 7, 0.15±0.03; day 0, 0.15±0.04), although both metabolites were reduced by the addition of low dose aspirin (day 14: TX-M, 0.09±0.01; PGI-M, 0.07±0.01; p<0.01 vs day 0). There were no further effects with the higher aspirin dose (p>0.05). Platelet aggregation to U46619 was largely inhibited by prasugrel, with no further effect of aspirin. Prasugrel alone preserves urinary PGI2 metabolites while inhibiting TXA2-driven platelet aggregation. Addition of aspirin reduces the excreted levels of metabolites of both TXA2 and vasoprotective PGI2.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: Strong P2Y(12) blockade, as can be achieved with novel anti-platelet agents such as prasugrel, has been shown in vitro to inhibit both ADP and thromboxane A(2) -mediated pathways of platelet aggregation, calling into question the need for the concomitant use of aspirin.
The present study investigated the hypothesis that aspirin provides little additional anti-aggregatory effect in a group of healthy volunteers taking prasugrel. STUDY PARTICIPANTS/METHODS: In all, 9 males, aged 18 to 40 years, enrolled into the 21-day study. Prasugrel was loaded at 60 mg on day 1 and maintained at 10 mg until day 21. At day 8, aspirin 75 mg was introduced and the dose increased to 300 mg on day 15. On days 0, 7, 14 and 21, platelet function was assessed by aggregometry, response to treatments was determined by VerifyNow and urine samples were collected for quantification of prostanoid metabolites.
At day 7, aggregation responses to a range of platelet agonists were reduced and there was only a small further inhibition of aggregation to TRAP-6, collagen and epinephrine at days 14 and 21, when aspirin was included with prasugrel. Urinary prostanoid metabolites were unaffected by prasugrel, and were reduced by the addition of aspirin, independent of dose.
In healthy volunteers, prasugrel produces a strong anti-aggregatory effect, which is little enhanced by the addition of aspirin. The addition of aspirin as a dual-therapy with potent P2Y(12) receptor inhibitors warrants further investigation.
Journal of Thrombosis and Haemostasis 07/2011; 9(10):2050-6. · 6.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Traditional methods for the analysis of vascular lesion formation are labour intensive to perform - restricting study to 'snapshots' within each vessel. This study was undertaken to determine the suitability of optical projection tomographic (OPT) imaging for the 3-dimensional representation and quantification of intimal lesions in mouse arteries.
Vascular injury was induced by wire-insertion or ligation of the mouse femoral artery or administration of an atherogenic diet to apoE-deficient mice. Lesion formation was examined by OPT imaging of autofluorescent emission. Lesions could be clearly identified and distinguished from the underlying vascular wall. Planimetric measurements of lesion area correlated well with those made from histological sections subsequently produced from the same vessels (wire-injury: R² = 0.92; ligation-injury: R² = 0.89; atherosclerosis: R² = 0.85), confirming both the accuracy of this methodology and its non-destructive nature. It was also possible to record volumetric measurements of lesion and lumen and these were highly reproducible between scans (coefficient of variation = 5.36%, 11.39% and 4.79% for wire- and ligation-injury and atherosclerosis, respectively).
These data demonstrate the eminent suitability of OPT for imaging of atherosclerotic and neointimal lesion formation, providing a much needed means for the routine 3-dimensional analysis of vascular morphology in studies of this type.
PLoS ONE 01/2011; 6(2):e16906. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Clinical use of selective inhibitors of cyclooxygenase (COX)-2 appears associated with increased risk of thrombotic events. This is often hypothesised to reflect reduction in anti-thrombotic prostanoids, notably PGI(2), formed by COX-2 present within endothelial cells. However, whether COX-2 is actually expressed to any significant extent within endothelial cells is controversial. Here we have tested the effects of acute inhibition of COX on platelet reactivity using a functional in vivo approach in mice.
A non-lethal model of platelet-driven thromboembolism in the mouse was used to assess the effects of aspirin (7 days orally as control) diclofenac (1 mg.kg(-1), i.v.) and parecoxib (0.5 mg.kg(-1), i.v.) on thrombus formation induced by collagen or the thromboxane (TX) A(2)-mimetic, U46619. The COX inhibitory profiles of the drugs were confirmed in mouse tissues ex vivo. Collagen and U46619 caused in vivo thrombus formation with the former, but not latter, sensitive to oral dosing with aspirin. Diclofenac inhibited COX-1 and COX-2 ex vivo and reduced thrombus formation in response to collagen, but not U46619. Parecoxib inhibited only COX-2 and had no effect upon thrombus formation caused by either agonist.
Inhibition of COX-1 by diclofenac or aspirin reduced thrombus formation induced by collagen, which is partly dependent upon platelet-derived TXA(2), but not that induced by U46619, which is independent of platelet TXA(2). These results are consistent with the model demonstrating the effects of COX-1 inhibition in platelets, but provide no support for the hypothesis that acute inhibition of COX-2 in the circulation increases thrombosis.
PLoS ONE 01/2011; 6(5):e20062. · 3.73 Impact Factor