Biomarkers for predicting response to long‐term high dose aspirin therapy in aspirin‐exacerbated respiratory disease

IntroductionAspirin‐exacerbated respiratory disease (AERD) is a phenotype of asthma characterized by eosinophilic inflammation in the airways, mast cell activation, cysteinyl leukotriene overproduction, and acute respiratory reactions on exposure to cyclooxygenase‐1 inhibitors. Aspirin desensitization followed by daily high‐dose aspirin therapy is a safe and effective treatment option for the majority of patients with AERD. However, there is still some percentage of the population who do not derive benefits from daily aspirin use.MethodsBased on the current literature, the biomarkers, which might predict aspirin treatment outcomes in AERD patients, were evaluated.Results and conclusionsPatients with severe symptoms of chronic rhinosinusitis, type 2 asthma based on blood eosinophilia, non‐neutrophilic inflammatory phenotype based on sputum cells, as well as high plasma level of 15‐hydroxyeicosatetraenoic acid (15‐HETE) are potentially good responders to long term high‐dose aspirin therapy. Additionally, high expression of the hydroxyprostaglandin dehydrogenase gene, HPGD encoding prostaglandin‐degrading enzyme 15‐hydroxyprostaglandin dehydrogenase (15‐PGDH) and low expression of the proteoglycan 2 gene, PRG2 encoding constituent of the eosinophil granule in sputum cells might serve as a predictor of good response to aspirin therapy. Variations in the expression of cysteinyl leukotriene receptor 1 in the airways could additionally influence the response to long‐term aspirin therapy. Arachidonic acid metabolites levels via the 5‐lipoxygenase as well as via the cyclooxygenase pathways in induced sputum supernatant do not change during high dose long‐term aspirin therapy and do not influence outcomes of aspirin treatment.     

Selective Cumulative Inhibition of Platelet Thromboxane Production by Low-dose Aspirin in Healthy Subjects

Acetylation of platelet cyclooxygenase by oral aspirin is dose dependent and cumulative with repeated administration. However, no single dose of aspirin has been found to be completely selective of platelet thromboxane (TX) synthesis inhibition in man. We determined the dose dependence, cumulative nature and selectivity of aspirin effects on platelet TXB₂ and renal prostaglandin (PG) and prostacyclin (PGI₂) production. We measured, by radioimmunoassay, serum TXB₂ levels after whole blood clotting and urinary excretion of PGE₂, PGF_2α, and 6-keto-PGF_1α, before and after single or repeated oral aspirin doses given to 46 healthy subjects. Single doses of 6-100 mg aspirin resulted in a linear (r = 0.92, P < 0.01) inhibition of platelet TXB₂ production, ranging from 12 to 95% after 24 h. A daily dose of 0.45 mg/kg given for 7 d produced a cumulative and virtually complete inhibition of platelet TXB₂ production, without significantly reducing the urinary excretion of PGE₂, PGF_2α, and 6-keto-PGF_1α in both healthy men and women. The platelet inhibitory effect of this regimen was maintained unaltered throughout 1 mo of therapy, with no evidence of cumulative inhibition of renal PG-synthesis. Moreover, furosemide-induced renal PGI₂ synthesis and renin release were unaffected by chronic low-dose aspirin. Following cessation of aspirin therapy, platelet TXB₂ production returned toward control values at a similar rate as after a single higher dose.     

Greater Collagen‐Induced Platelet Aggregation Following Cyclooxygenase 1 Inhibition Predicts Incident Acute Coronary Syndromes

Greater ex vivo platelet aggregation to agonists may identify individuals at risk of acute coronary syndromes (ACS). However, increased aggregation to a specific agonist may be masked by inherent variability in other activation pathways. In this study, we inhibited the cyclooxygenase‐1 (COX1) pathway with 2‐week aspirin therapy and measured residual aggregation to collagen and ADP to determine whether increased aggregation in a non‐COX1 pathway is associated with incident ACS. We assessed ex vivo whole blood platelet aggregation in 1,699 healthy individuals with a family history of early‐onset coronary artery disease followed for 6±1.2 years. Incident ACS events were observed in 22 subjects. Baseline aggregation was not associated with ACS. After COX1 pathway inhibition, collagen‐induced aggregation was significantly greater in participants with ACS compared with those without (29.0 vs. 23.6 ohms, p < 0.001). In Cox proportional hazards models, this association remained significant after adjusting for traditional cardiovascular risk factors (HR = 1.10, 95%CI = 1.06–1.15; p < 0.001). In contrast, ADP‐induced aggregation after COX1 inhibition was not associated with ACS. After COX1 pathway inhibition, subjects with greater collagen‐induced platelet aggregation demonstrated a significant excess risk of incident ACS. These data suggest that platelet activation related to collagen may play an important role in the risk of ACS.     

Effect of oral aspirin on "ecto-ATPase" activity of washed human platelets.

Aspirin is a potent inhibitor of the platelet release reaction and the accompanying second phase of platelet aggregation. The platelet release reaction is an active, energy-dependent process which appears to require ATP. Eight men ingested 0.32 gm of aspirin daily for 7 days. Although the second phase of 1.7 micron ADP-induced platelet aggregation was absent after aspirin ingestion, the "ecto-ATPase" activities of washed human platelet suspensions were not significantly different before and after ingestion of aspirin. This suggests that the effect of aspirin on the second phase of platelet aggregation is not mediated through inhibition of "ecto-ATPase".     

Modulation of ADP-induced platelet activation by aspirin and pravastatin: role of lectin-like oxidized low-density lipoprotein receptor-1, nitric oxide, oxidative stress, and inside-out integrin signaling

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1), a receptor for oxidized-LDL, is up-regulated in activated endothelial cells, and it plays a role in atherothrombosis. However, its role in platelet aggregation is unclear. Both aspirin and HMG CoA reductase inhibitors (statins) reduce LOX-1 expression in endothelial cells. In this study, we investigated the effect of aspirin and pravastatin on LOX-1 expression on plate-lets. After ADP stimulation, mean fluorescence intensity of LOX-1 expression on platelets increased 1.5- to 2.0-fold. Blocking LOX-1 inhibited ADP-induced platelet aggregation in a concentration- and time-dependent manner. We also established that LOX-1 is important for ADP-stimulated inside-out activation of platelet alpha(IIb)beta(3) and alpha(2)beta(1) integrins (fibrinogen receptors). The specificity of this interaction was determined by arginine-glycine-aspartate-peptide inhibition. Furthermore, we found that LOX-1 inhibition of integrin activation is mediated by inhibition of protein kinase C activity. In other experiments, treatment with aspirin (1-10 mM) and pravastatin (1-5 microM) reduced platelet LOX-1 expression, with a synergistic effect of the combination of aspirin and pravastatin. Aspirin and pravastatin both reduced reactive oxygen species (ROS) released by activated platelets measured as malonyldialdehyde (MDA) release and nitrate/nitrite ratio. Aspirin and pravastatin also enhanced nitric oxide (NO) release measured as nitrite/nitrite + nitrate (NOx) ratio in platelet supernates. Small concentrations of aspirin and pravastatin had a synergistic effect on the inhibition of MDA release and enhancement of nitrite/NOx. Thus, LOX-1 is important for ADP-mediated platelet integrin activation, possibly through protein kinase C activation. Furthermore, aspirin and pravastatin inhibit LOX-1 expression on platelets in part by favorably affecting ROS and NO release from activated platelets.     

In vivo platelet activation is responsible for enhanced vascular endothelial growth factor levels in hypertensive patients

BACKGROUND: Essential hypertension may be a consequence of an abnormal regulation of vascular endothelial growth factor (VEGF). In vivo activation of platelets does result in the release of VEGF. Thus, we investigated whether VEGF production in hypertensive patients is related to in vivo platelet activation, and whether it may be modified by aspirin treatment. METHODS: Plasma VEGF, soluble (s)P-selectin and thrombin-anti-thrombin complex (TATc) were analyzed in 80 patients with therapeutically controlled essential hypertension and 40 age and sex-matched healthy normotensive controls. The effects of a 6-month treatment with aspirin 100 mg/day on VEGF levels of 20 hypertensive patients were also studied. RESULTS: Plasma VEGF (p<0.0001), sP-selectin (p=0.01) and TATc (p=0.02) levels were higher in hypertensives compared to controls. Multivariate analysis including age, sex, risk factors, cardiovascular disease, anti-hypertensive treatment, sP-selectin and TATc showed that only sP-selectin was an independent predictor of VEGF (beta=0.40, p<0.03). Aspirin treated hypertensives showed a significant reduction of sP-selectin (-26%, p<0.01) and VEGF (-33%, p<0.01) levels. Moreover, the reduction of plasma VEGF levels directly correlated with that of sP-selectin (Rho=0.46, p=0.04). CONCLUSIONS: In vivo activation of platelets in hypertensive patients is responsible for enhanced circulating VEGF levels, which are significantly lowered by aspirin treatment.     

Aspirin and salicylate inhibit colon cancer medium- and VEGF-induced endothelial tube formation: correlation with suppression of cyclooxygenase-2 expression

Summary.  To determine whether aspirin and salicylate suppress colon cancer cell-mediated angiogenesis, we evaluated the effects of aspirin and sodium salicylate on endothelial tube formation on Matrigel. Aspirin and sodium salicylate concentration-dependently inhibited human endothelial cell (EC) tube formation induced by conditioned medium collected from DLD-1, HT-29 or HCT-116 colon cancer cells. Aspirin and sodium salicylate at pharmacological concentrations were equally effective in blocking tube formation. Neutralizing antivascular endothelial growth factor (VEGF) antibodies blocked colon cancer medium-induced tube formation. VEGF receptor 2 but not receptor 1 antibodies inhibited tube formation to a similar extent as anti-VEGF antibodies. These results indicate that VEGF interaction with VEGF receptor 2 is the primary mechanism underlying colon cancer-induced angiogenesis. Aspirin or sodium salicylate inhibited VEGF-induced tube formation in a concentration-dependent manner comparable to that of inhibition of colon cancer medium-induced endothelial tube formation. It has been shown that cyclooxygenase-2 (COX-2) is pivotal in cancer angiogenesis. We found that colon cancer medium-induced COX-2 protein expression in EC and aspirin or sodium salicylate suppressed the cancer-induced COX-2 protein levels at concentrations correlated with those that suppressed endothelial tube formation. Furthermore, aspirin and sodium salicylate inhibited COX-2 expression stimulated by VEGF. These findings indicate that aspirin and other salicylate drugs at pharmacological concentrations inhibit colon cancer-induced angiogenesis which is correlated with COX-2 suppression.     

Urinary Biomarkers for Sensitive and Specific Detection of Acute Kidney Injury in Humans

Acute kidney injury (AKI) is associated with high morbidity and mortality. The lack of sensitive and specific injury biomarkers has greatly impeded the development of therapeutic strategies to improve outcomes of AKI.The unique objective of this study was to evaluate the diagnostic performance of nine urinary biomarkers of AKI—kidney injury molecule‐1 (KIM‐1), neutrophil gelatinase associated lipocalin (NGAL), interleukin‐18 (IL‐18), hepatocyte growth factor (HGF), cystatin C (Cys), N‐acetyl‐β‐D‐glucosaminidase (NAG), vascular endothelial growth factor (VEGF), chemokine interferon‐inducible protein 10 (IP‐10; CXCL10), and total protein—in a cross‐sectional comparison of 204 patients with or without AKI.Median urinary concentrations of each biomarker were significantly higher in patients with AKI than in those without AKI (p < 0.001). The area under the receiver operating characteristics curve (AUC‐ROC) for the combination of biomarkers using a logic regression model [risk score of 2.93*(NGAL > 5.72 and HGF > 0.17) + 2.93*(PROTEIN > 0.22) −2*(KIM < 0.58)] was greater (0.94) than individual biomarker AUC‐ROCs. Age‐adjusted levels of urinary KIM‐1, NAG, HGF, VEGF, and total protein were significantly higher in patients who died or required renal replacement therapy (RRT) when compared to those who survived and did not require RRT.Our results demonstrate the comparative value of multiple biomarkers in the diagnosis and prognosis of AKI.     

Delivery of siRNA Using CXCR4-targeted Nanoparticles Modulates Tumor Microenvironment and Achieves a Potent Antitumor Response in Liver Cancer

Antiangiogenic therapy has recently emerged as a highly promising therapeutic strategy for treating hepatocellular carcinoma (HCC). However, the only clinically approved systemic antiangiogenic agent for advanced HCC is sorafenib, which exerts considerable toxicity. Moreover, acquired resistance to antiangiogenic therapy often develops and restricts the therapeutic efficacy of this treatment. Hence, in this study, we develop a CXCR4-targeted lipid-based nanoparticle (NP) formulation to specifically deliver vascular endothelial growth factor (VEGF) siRNA as an antiangiogenic substance into HCC. AMD3100, a CXCR4 antagonist, is added into NPs to serve as both a targeting moiety and a sensitizer to antiangiogenic therapy. We demonstrate that AMD-modified NPs (AMD-NPs) can efficiently deliver VEGF siRNAs into HCC and downregulate VEGF expression in vitro and in vivo. Despite the upregulation of the SDF1α/CXCR4 axis upon the induction of hypoxia after antiangiogenic therapy, CXCR4 inhibition by AMD-NPs in combination with either conventional sorafenib treatment or VEGF siRNA prevents the infiltration of tumor-associated macrophages. These dual treatments also induce synergistic antiangiogenic effects and suppress local and distant tumor growth in HCC. In conclusion, the tumor-targeted multifunctional AMD-NPs that co-deliver VEGF siRNA and AMD3100 provide an effective approach for overcoming tumor evasion of antiangiogenic therapy, leading to delayed tumor progression in HCC.     

Kinetics of platelet adhesion to a fibrinogen‐coated surface in whole blood under flow conditions

AimTo test a novel method of assessment of platelet adhesion to a fibrinogen‐coated surface in whole blood under flow conditions.MethodsWe developed a fluidic device that mimics blood flow in vessels. The method of detection of platelet adhesion is based on recording of a scattered laser light signal from a fibrinogen‐covered surface. Testing was performed in platelet‐rich plasma (PRP) and whole blood of healthy volunteers. Control measurements were performed, followed by tests with inhibition of platelet GPIIa/IIIb and GPIb receptors. Then, the same testing sequence was performed in whole blood of persons with autoimmune thrombocytopenia and type 3 von Willebrand disease.ResultsThe change in intensity of scattered light was 2.7 (2.4; 4.1) times higher in whole blood (0.2 ± 0.08V, n = 7) than in PRP (0.05 ± 0.02 V, n = 7), p < 0.01. The blocking of GP IIb/IIIa receptors decreased the intensity of scattered light to 8.5 (6.5;12)%; the blocking of GPIb receptors decreased it to 34 (23;58)%, p < 0.01. In the whole blood of a person with autoimmune thrombocytopenia, the inhibition of GPIb receptors decreased platelet adhesion, but no effect was observed in type 3 von Willebrand disease. Inhibition of platelet GPIIb/IIIa receptors alone or combined inhibition of GPIb and GPIIb/IIIa receptors resulted in almost total suppression of adhesion in both cases.ConclusionOur system effectively registers platelet adhesion to a fibrinogen‐coated surface under controlled‐flow conditions and may successfully be applied to the investigation of platelet adhesion kinetics.     

Endogenous biosynthesis of prostacyclin and thromboxane and platelet function during chronic administration of aspirin in man

To assess the pharmacologic effects of aspirin on endogenous prostacyclin and thromboxane biosynthesis, 2,3-dinor-6-keto PGF1 alpha (PGI-M) and 2,3-dinor-thromboxane B2 (Tx-M) were measured in urine by mass spectrometry during continuing administration of aspirin. To define the relationship of aspirin intake to endogenous prostacyclin biosynthesis, sequential urines were initially collected in individuals prior to, during, and subsequent to administration of aspirin. Despite inter- and intra-individual variations, PGI-M excretion was significantly reduced by aspirin. However, full mass spectral identification confirmed continuing prostacyclin biosynthesis during aspirin therapy. Recovery of prostacyclin biosynthesis was incomplete 5 d after drug administration was discontinued. To relate aspirin intake to indices of thromboxane biosynthesis and platelet function, volunteers received 20 mg aspirin daily followed by 2,600 mg aspirin daily, each dose for 7 d in sequential weeks. Increasing aspirin dosage inhibited Tx-M excretion from 70 to 98% of pretreatment control values; platelet TxB2 formation from 4.9 to 0.5% and further inhibited platelet function. An extended study was performed to relate aspirin intake to both thromboxane and prostacyclin generation over a wide range of doses. Aspirin, in the range of 20 to 325 mg/d, resulted in a dose-dependent decline in both Tx-M and PGI-M excretion. At doses of 325-2,600 mg/d Tx-M excretion ranged from 5 to 3% of control values while PGI-M remained at 37-23% of control. 3 d after the last dose of aspirin (2,600 mg/d) mean Tx-M excretion had returned to 85% of control, whereas mean PGI-M remained at 40% of predosing values. Although the platelet aggregation response (Tmax) to ADP ex vivo was inhibited during administration of the lower doses of aspirin the aggregation response returned to control values during the final two weeks of aspirin administration (1,300 and 2,600 mg aspirin/d) despite continued inhibition of thromboxane biosynthesis. These results suggest that although chronic administration of aspirin results in inhibition of endogenous thromboxane and prostacyclin biosynthesis over a wide dose range, inhibition of thromboxane biosynthesis is more selective at 20 than at 2,600 mg aspirin/d. However, despite this, inhibition of platelet function is not maximal at the lower aspirin dosage. Doses of aspirin in excess of 80 mg/d resulted in substantial inhibition of endogenous prostacyclin biosynthesis. Thus, it is unlikely that any dose of aspirin can maximally inhibit thromboxane generation without also reducing endogenous prostacyclin biosynthesis. These results also indicate that recovery of endogenous prostacyclin biosynthesis is delayed following aspirin administration and that the usual effects of aspirin on platelet function ex vivo may be obscured during chronic aspirin administration in man.     

Immunostimulatory Effects of Mesenchymal Stem Cell‐Derived Neurons: Implications for Stem Cell Therapy in Allogeneic Transplantations

Mesenchymal stem cells (MSCs) differentiate along various lineages to specialized mesodermal cells and also transdifferentiate into cells such as ectodermal neurons. MSCs are among the leading adult stem cells for application in regenerative medicine. Advantages include their immune‐suppressive properties and reduced ethical concerns. MSCs also show immune‐enhancing functions. Major histocompatibility complex II (MHC‐II) is expected to be downregulated in MSCs during neurogenesis. Ideally, “off the shelf” MSCs would be suited for rapid delivery into patients. The question is whether these MSC‐derived neurons can reexpress MHC‐II in a milieu of inflammation. Western analyses demonstrated gradual decrease in MHC‐II during neurogenesis, which correlated with the expression of nuclear CIITA, the master regulator of MHC‐II expression. MHC‐II expression was reversed by exogenous IFNY. One‐way mixed lymphocyte reaction with partly differentiated neurons showed a stimulatory effect, which was partly explained by the release of the proinflammatory neurotransmitter substance P (SP), cytokines, and decreases in miR‐130a and miR‐206. The anti‐inflammatory neurotransmitters VIP and CGRP were decreased at the peak time of immune stimulation. In summary, MSC‐derived neurons show decreased MHC‐II expression, which could be reexpressed by IFNY. The release of neurotransmitters could be involved in initiating inflammation, underscoring the relevance of immune responses as consideration for stem cell therapies.     

Combined procalcitonin and hemogram parameters contribute to early differential diagnosis of Gram‐negative/Gram‐positive bloodstream infections

BackgroundHemogram parameters and procalcitonin (PCT) play auxiliary roles in the diagnosis and outcome of sepsis. However, it is not clear whether these indicators can quickly distinguish bacterial classification or guide the choice of empirical antibiotics.MethodsWe retrospectively enrolled 381 patients with bloodstream infections (BSI), divided into Gram‐positive bloodstream infections (GP‐BSI) and Gram‐negative bloodstream infections (GN‐BSI). Demographic parameters, hemogram parameters, and PCT were recorded and compared between the two groups.ResultsThe mean platelet volume (MPV), platelet distribution width (PDW), and PCT in the GN‐BSI group were significantly higher than those in the GP‐BSI group, while the platelet count (PLT), plateletcrit, platelet count‐to‐white blood cell count ratio (PWR), platelet count‐to‐neutrophil count ratio (PNR), platelet count‐to‐PCT ratio (PLT/PCT), and mean platelet volume‐to‐PCT ratio (MPV/PCT) were significantly lower in the GN‐BSI group. Multivariate stepwise logistic regression analysis revealed that the independent predictors of GN‐BSI were MPV, PWR, and PCT. The areas under the curve (AUC) for this prediction model was 0.79, with sensitivity =0.75 and specificity =0.71.ConclusionsThere were significant differences in terms of PCT, platelet parameters, and platelet‐related index‐PCT ratio between GN‐BSI and GP‐BSI. Combined PCT and hemogram parameters are more conducive to the early differential diagnosis of bacterial classification of BSI.     

Effect of Propranolol as Antiadhesive Therapy in Sickle Cell Disease

Sickle red blood cells (SSRBCs) adhere to both endothelial cells (ECs) and the extracellular matrix. Epinephrine elevates cyclic adenosine monophosphate in SSRBCs and increases adhesion of SSRBCs to ECs in a β‐adrenergic receptor and protein kinase A‐dependent manner. Studies in vitro as well as in vivo have suggested that adrenergic stimuli like epinephrine may contribute to vaso‐occlusion associated with physiologic stress. We conducted both animal studies and a Phase I dose‐escalation study in sickle cell disease (SCD) patients to investigate whether systemically administered propranolol inhibits SSRBC adhesion and to document the safety of propranolol in SCD. Systemically administered propranolol prevented SSRBC adhesion and associated vaso‐occlusion in a mouse model. In patients receiving a single oral dose of 10, 20, or 40 mg propranolol, SSRBC adhesion to ECs was studied before and after propranolol, with and without stimulation with epinephrine. Propranolol administration significantly reduced epinephrine‐stimulated SSRBC adhesion in a dose dependent manner (p = 0.03), with maximum inhibition achieved at 40 mg. Adverse events were not severe, did not show dose dependence, and were likely unrelated to drug. No significant heart rate changes occurred. These results imply that β‐blockers may have a role as antiadhesive therapy for SCD. Clin Trans Sci 2012; Volume 5: 437–444     

Negative Regulation of VEGF Signaling in Human Coronary Artery Endothelial Cells by G Protein‐Coupled Receptor Kinase 5

G protein‐coupled receptor kinase 5 (GRK5) is present in endothelial cells (ECs) and has the potential to regulate EC function through seven transmembrane‐spanning receptor (7TMR) signaling. Recently, it has been appreciated that GRKs can affect receptor tyrosine kinases (RTKs). VEGF, an RTK, is one of the most potent mediators for EC function and angiogenesis; therefore, we determined the role GRK5 plays in VEGF signaling in human coronary artery ECs (HCAECs). GRK5 levels were increased by VEGF treatment in HCAECs. Adenoviral overexpression of GRK5 inhibited migration and proliferation of HCAECs in response to VEGF. GRK5 overexpression in HCAECs significantly suppressed both acute and late activation of Akt and extracellular signal‐related kinase (ERKs) as well as the phosphorylationof GSK‐3β, an endogenous substrate of Akt. Coimmunoprecipitations revealed that GRK5 is physically associated with Akt. This study shows for the first time that GRK5 negatively regulates VEGF signaling in HCAECs and suggests that targeted intervention of GRK5 in ECs might be a novel therapeutic strategy to prevent and treat disorders involving altered EC function.     

Selective and nonselective inhibition of thromboxane formation

Thromboxane A2, the predominant cyclooxygenase product of arachidonic acid in the platelet, is a potent vasoconstrictor and stimulus of platelet aggregation. Prostacyclin, the principal cyclooxygenase metabolite formed in the vascular endothelium, inhibits platelet aggregation and dilates blood vessels. A therapeutic objective in the treatment of human vascular occlusive disease has been the inhibition of thromboxane formation without coincident reduction in prostacyclin biosynthesis. We compared the biochemical selectivity and platelet inhibitory actions of single doses of aspirin, a cyclooxygenase inhibitor, with imidazo(1,5-2)pyridine-5-hexanoic acid (CGS 13080), an inhibitor of thromboxane synthase. Aspirin, 325 mg, prolonged the bleeding time markedly, inhibited aggregation and nucleotide release in whole blood and platelet-rich plasma, and maximally inhibited thromboxane generation in serum. The effects of aspirin, 20 mg, were considerably less marked but, as with the higher dose, persisted throughout the study period (24 hr after dosing). CGS 13080 also prolonged bleeding time and inhibited thromboxane formation. In contrast to aspirin, these effects were reversible and inhibition of aggregation was less marked. Endogenous prostacyclin biosynthesis was measured by excretion of the major urinary metabolite 2,3-dinor-6-keto-PGF1 alpha (PGI-M). Whereas aspirin, 325 mg, reduced PGI-M excretion a mean 29%, excretion increased 48% and 100% after CGS 13080, 100 mg and 200 mg. Aspirin, 20 mg, did not alter prostacyclin biosynthesis. Inhibition of thromboxane synthase permits selective inhibition of thromboxane formation in man. Although drugs of greater potency and longer duration of action are desirable, enhanced prostacyclin synthesis may be an important component of the platelet inhibitory actions of thromboxane synthase inhibitors in man.     

Effects of celecoxib on prostanoid biosynthesis and circulating angiogenesis proteins in familial adenomatous polyposis

Vascular cyclooxygenase (COX)-2-dependent prostacyclin (PGI(2)) may affect angiogenesis by preventing endothelial activation and platelet release of angiogenic factors present in platelet α-granules. Thus, a profound inhibition of COX-2-dependent PGI(2) might be associated with changes in circulating markers of angiogenesis. We aimed to address this issue by performing a clinical study with celecoxib in familial adenomatous polyposis (FAP). In nine patients with FAP and healthy controls, pair-matched for gender and age, we compared systemic biosynthesis of PGI(2), thromboxane (TX) A(2), and prostaglandin (PG) E(2), assessing their urinary enzymatic metabolites, 2,3-dinor-6-keto PGF(1α) (PGI-M), 11-dehydro-TXB(2) (TX-M), and 11-α-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), respectively. The impact of celecoxib (400 mg b.i.d. for 7 days) on prostanoid biosynthesis and 14 circulating biomarkers of angiogenesis was evaluated in FAP. Intestinal tumorigenesis was associated with enhanced urinary TX-M levels, but unaffected by celecoxib, suggesting the involvement of a COX-1-dependent pathway, presumably from platelets. This was supported by the finding that in cocultures of a human colon adenocarcinoma cell line (HT-29) and platelets enhanced TXA(2) generation was almost completely inhibited by pretreatment of platelets with aspirin, a preferential inhibitor of COX-1. In FAP, celecoxib profoundly suppressed PGE(2) and PGI(2) biosynthesis that was associated with a significant increase in circulating levels of most proangiogenesis proteins but also the antiangiogenic tissue inhibitor of metalloproteinase 2. Urinary PGI-M, but not PGE-M, was negatively correlated with circulating levels of fibroblast growth factor 2 and angiogenin. In conclusion, inhibition of tumor COX-2-dependent PGE(2) by celecoxib may reduce tumor progression. However, the coincident depression of vascular PGI(2), in a context of enhanced TXA(2) biosynthesis, may modulate the attendant angiogenesis, contributing to variability in the chemopreventive efficacy of COX-2 inhibitors such as celecoxib.     

Aspirin ‘resistance’: role of pre‐existent platelet reactivity and correlation between tests

Summary. Background: Aspirin ‘resistance’ is a widely used term for hyporesponsiveness to aspirin in a platelet function test. Serum thromboxane (TX) B₂ is the most specific test of aspirin’s effect on platelets. Objectives: (i) To examine the role of pre‐existent platelet hyperreactivity in aspirin ‘resistance’. (ii) To determine the correlation between aspirin resistance defined by serum TXB₂ and other assays of platelet function. Methods: To enable pre‐aspirin samples to be drawn, platelet function was measured in normal subjects (n = 165) before and after aspirin 81 mg daily for seven days. Results: The proportion of the post‐aspirin platelet function predicted by the pre‐aspirin platelet function was 28.3 ± 7.5% (mean ± asymptotic standard error) for serum TXB₂, 39.3 ± 6.8% for urinary 11‐dehydro TXB₂, 4.4 ± 7.7% for arachidonic acid‐induced platelet aggregation, 40.4 ± 7.1% for adenosine diphosphate‐induced platelet aggregation, 26.3 ± 9.2% for the VerifyNow Aspirin Assay^®, and 45.0 ± 10.9% for the TEG^® PlateletMapping? System with arachidonic acid. There was poor agreement between aspirin‐resistant subjects identified by serum TXB₂ vs. aspirin‐resistant subjects identified by the other five assays, irrespective of whether the analysis was based on categorical or continuous variables. Platelet count correlated with pre‐aspirin serum TXB₂ and VerifyNow Aspirin Assay, but not with any post‐aspirin platelet function test. Conclusions: (i) Aspirin ‘resistance’ (i.e. hyporesponsiveness to aspirin in a laboratory test) is in part unrelated to aspirin but is the result of underlying platelet hyperreactivity prior to the institution of aspirin therapy. (ii) Aspirin resistance defined by serum TXB₂ shows a poor correlation with aspirin resistance defined by other commonly used assays.     

Identifying determinants of variability to tailor aspirin therapy

Once-daily, low-dose aspirin is a cornerstone in the prophylaxis and treatment of cardiovascular diseases. Aspirin ‘resistance’ still lacks definition, a standardized reference assay, underlying mechanisms, clinical impact or efficacy of alternative antiplatelet drugs. Aspirin response in several studies has been measured by different platelet function tests, not always reflecting aspirin pharmacodynamics, thus generating significantly heterogeneous results. The EMA indicated serum thromboxane B₂ as the only valid surrogate assay to study different aspirin formulations. Rather than resistance, recent studies focused on sources of intra- and inter-individual variability in response to aspirin, based on pharmacokinetic and/or pharmacodynamic mechanisms. Drug interactions, diabetes, conditions of increased platelet output, obesity and aging can potentially increase the variability of aspirin response. Preliminary studies testing different aspirin regimens showed that twice-daily low doses were more effective than once-daily higher aspirin doses on surrogate end points of platelet inhibition. Large studies are needed to test new disease-tailored, low-dose aspirin regimens.     

Topical inflammasome inhibition with disulfiram prevents irritant contact dermatitis

BackgroundThe pathogenesis of contact dermatitis, a common inflammatory skin disease with limited treatment options, is held to be driven by inflammasome activation induced by allergens and irritants. We here aim to identify inflammasome‐targeting treatment strategies for irritant contact dermatitis.MethodsA high content screen with 41,184 small molecules was performed using fluorescent Apoptosis associated speck‐like protein containing a CARD (ASC) speck formation as a readout for inflammasome activation. Hit compounds were validated for inhibition of interleukin (IL)‐1β secretion. Of these, the approved thiuramdisulfide derivative disulfiram was selected and tested in a patch test model of irritant contact dermatitis in 25 healthy volunteers. Topical application of disulfiram, mometasone or vehicle was followed by application of sodiumdodecylsulfate (SDS) for 24 h each. Eczema induction was quantified by mexameter and laser speckle imaging. Corneocyte sampling of lesional skin was performed to assess inflammasome‐mediated cytokines IL‐1β and IL‐18.ResultsDisulfiram induced a dose‐dependent inhibition of ASC speck formation and IL‐1β release in cellular assays in vitro. In vivo, treatment with disulfiram, but not with vehicle and less mometasone, inhibited SDS‐induced eczema. This was demonstrated by significantly lower erythema and total perfusion values assessed by mexameter and laser speckle imaging for disulfiram compared to vehicle (p < 0.001) and/or mometasone (p < 0.001). Also, corneocyte IL‐18 levels were significantly reduced after application of disulfiram compared to vehicle (p < 0.001).ConclusionWe show that disulfiram is a dose‐dependent inhibitor of inflammasome pathway activation in vitro and inhibitor of SDS‐induced eczema in vivo. Topical application of disulfiram represents a potential treatment option for irritant contact dermatitis.