Inhibition of ABCC1 Decreases cAMP Egress and Promotes Human Airway Smooth Muscle Cell Relaxation.

In most living cells, the second-messenger roles for adenosine 3',5'-cyclic monophosphate (cAMP) are short-lived, confined to the intracellular space, and tightly controlled by the binary switch-like actions of Gαs (stimulatory G protein)-activated adenylyl cyclase (cAMP production) and cAMP-specific PDE (cAMP breakdown). Here, by using human airway smooth muscle (HASM) cells in culture as a model, we report that activation of the cell-surface ß2AR (ß2-adrenoceptor), a Gs-coupled GPCR (G protein-coupled receptor), evokes cAMP egress to the extracellular space. Increased extracellular cAMP levels ([cAMP]e) are long-lived in culture and are induced by receptor-dependent and receptor-independent mechanisms in such a way as to define a universal response class of increased intracellular cAMP levels ([cAMP]i). We find that HASM cells express multiple ATP-binding cassette (ABC) membrane transporters, with ABCC1 (ABC subfamily member C 1) being the most highly enriched transcript mapped to MRPs (multidrug resistance-associated proteins). We show that pharmacological inhibition or downregulation of ABCC1 with siRNA markedly reduces ß2AR-evoked cAMP release from HASM cells. Furthermore, inhibition of ABCC1 activity or expression decreases basal tone and increases ß-agonist-induced HASM cellular relaxation. These findings identify a previously unrecognized role for ABCC1 in the homeostatic regulation of [cAMP]i in HASM that may be conserved traits of the Gs-GPCRs (Gs-coupled family of GPCRs). Hence, the general features of this activation mechanism may uncover new disease-modifying targets in the treatment of airflow obstruction in asthma. Surprisingly, we find that serum cAMP levels are elevated in a small cohort of patients with asthma as compared with control subjects, which warrants further investigation.

Cilostazol increases adenosine plasma concentration in patients with acute coronary syndrome.

WHAT IS KNOWN AND OBJECTIVE: Cilostazol is a specific and strong inhibitor of phosphodiesterase (PDE) type III which can suppress the platelet aggregation by increasing cyclic adenosine monophosphate (cAMP) levels. The clinical benefit of cilostazol in ACS patients suggested that the drug may have non-platelet-directed properties. Some in vitro and animal studies also indicated that the 'pleiotropic' properties of cilostazol might be related to the interaction with adenosine metabolism. Adenosine is an important regulatory metabolite and an inhibitor of platelet activation. However, no human study has been conducted to determine whether cilostazol could increase the adenosine plasma concentration in vivo. As a result, this study aimed to investigate the impact of cilostazol on adenosine plasma concentration (APC) in acute coronary syndrome (ACS) patients. METHODS: We prospectively analysed 149 ACS patients undergoing percutaneous coronary intervention (PCI) with drug-eluting stents. The included patients were divided into two groups according to the presence (cilostazol group, n = 64) or absence (aspirin group, n = 85) of aspirin intolerance. The inhibition of platelet aggregation (IPA), APC and cAMP concentration was measured. Patient characteristics, medications and 30-day clinical outcomes were examined. RESULTS: Patients receiving cilostazol had a significantly higher adenosine and cAMP plasma concentration than patients receiving aspirin (3.00 ± 0.67 vs 2.56 ± 0.74 mol/L, P < .001; 28.10 ± 14.74 vs 20.48 ± 11.35 pmol/mL, P = .0014). Cilostazol was associated with a higher inhibition rate of ADP induced platelet aggregation than aspirin (63.35 ± 26.71 vs 52.2 ± 28.35, P = .036). The plasma levels of adenosine and cAMP showed a positive correlation with ADP induced platelet aggregation. WHAT IS NEW AND CONCLUSION: Cilostazol increases adenosine concentration compared with aspirin. Its potent antiplatelet effect in ACS patients may be partly mediated by adenosine.

Influence of rs1042713 and rs1042714 polymorphisms of ß2-adrenergic receptor gene with erythrocyte cAMP in sickle cell disease patients from Odisha State, India.

The vaso-occlusive crisis (VOCs) in sickle cell disease (SCD) is often associated with stress. Epinephrine released during stress acts via beta 2-adrenergic receptors (ß2-AR or ADRB2) to stimulate the synthesis of cyclic adenosine monophosphate (cAMP) in the red blood cells (RBCs). Higher cAMP levels promote adhesion of sickled RBCs to vascular endothelium, a major contributor for VOCs. Several single-nucleotide polymorphisms (SNPs) of the ß2-AR gene have been reported; two of them at codon 16 (rs1042713) and codon 27 (rs1042714) have been extensively studied for their clinical relevance. Therefore, we assessed the influence of polymorphism at these two sites of the ß2-AR gene on the RBC cAMP concentrations with and without epinephrine stimulation in SCD subjects. We determined the frequency distribution of different genotypes of codon 16 and codon 27 of the ß2-AR gene using the Sanger sequencing method in the SCD subjects. We measured the RBC-cAMP levels at baseline and after stimulation with epinephrine, to ascertain the influence of different genotypes in determining cAMP levels. There was no difference in the socio-demographic and hematological indicators in different genotypes of both codon 16 and 27. In the sham-treated erythrocytes, the cAMP levels were significantly different with three genotypes of codon 16 (F = 3.39, P = 0.036; one way ANOVA) but not with different genotypes of codon 27. A significant increase in cAMP levels was noticed with epinephrine treatment in all genotypes of codons 16 and 27 (P = 0.001; Wilcoxon signed-rank test). However, the extent of increase in the epinephrine-treated cAMP values from the sham-treated (baseline) cAMP values was significantly different between the three genotypes of codon 16 (H = 8.74; P = 0.012; Kruskal-Wallis test) but not in codon 27 genotypes. Polymorphism in codon 16 (rs1042713) of the ß2-AR gene influences cAMP concentrations in the RBC both before and after epinephrine treatment. Higher cAMP levels may lead to increased adhesion of sickle cell RBCs to vascular endothelium and may increase the frequency of VOCs.

Cyclic AMP in human preterm infant blood is associated with increased TLR-mediated production of acute-phase and anti-inflammatory cytokines in vitro.

BACKGROUND: Preterm infants are at high risk of infection and have distinct pathogen recognition responses. Suggested mechanisms include soluble mediators that enhance cellular levels of cAMP. The aim of this study was to assess the relationship between blood cAMP concentrations and TLR-mediated cytokine production in infants during the first month of life. METHODS: Cord and serial peripheral blood samples (days of life 1-28) were obtained from a cohort of very preterm (<30 weeks' gestational age) and term human infants. Whole-blood concentrations of cAMP and FSL-1 and LPS in vitro stimulated cytokine concentrations were measured by ELISA and multiplex bead assay. RESULTS: cAMP concentrations were higher in cord than in peripheral blood, higher in cord blood of female preterm infants, and lower at Days 1 and 7 in infants exposed to chorioamnionitis, even after adjusting for leukocyte counts. TLR2 and TLR4-mediated TNF-α, IL-1ß, IL-6, IL-12p70, and IL-10 production in vitro increased over the first month of life in preterm infants and were positively correlated with leukocyte-adjusted cAMP levels and reduced by exposure to chorioamnionitis. CONCLUSIONS: The ontogeny of blood cAMP concentrations and associations with chorioamnionitis and TLR-mediated production of cytokines suggest that this secondary messenger helps shape distinct neonatal pathogen responses in early life.

Biomarker-specific differences between transpulmonary and peripheral arterial-venous blood sampling in patients with pulmonary hypertension.

Purpose: Transpulmonary biomarkers may provide insight into pulmonary hypertension (PH) pathophysiology, but require cardiac catheterization. We investigated whether the peripheral arterial-venous ratio (PR) could substitute for the transpulmonary ratio (TPR).Materials and methods: Blood from the pulmonary artery (PA), pulmonary arterial wedge (PAW), peripheral venous, and peripheral arterial positions was analysed for ET-1, NT-pro-BNP and cAMP levels in subjects with no PH (n = 18) and PH due to left heart disease (PH-LHD), which included combined pre- and post-capillary PH (Cpc-PH; n = 7) and isolated post-capillary PH (Ipc-PH; n = 9). Bland-Altman comparisons were made between peripheral venous and PA samples and between peripheral arterial and PAW samples. TPR was defined as [PAW]/[PA].Results: For ET-1, Bland-Altman analysis indicated negative bias (-24%) in peripheral arterial compared to PAW concentration and positive bias (23%) in peripheral venous compared to PA concentration. There was <10% absolute bias for NT-pro-BNP and cAMP. For ET-1, there was no difference in PR between Cpc-PH and Ipc-PH (0.87 ± 0.4 vs. 0.94 ± 0.6, p = 0.8), whereas there was a difference in TPR (2.2 ± 1.1 vs. 1.1 ± 0.2, p < 0.05).Conclusions: In PH-LHD, peripheral samples may be inadequate surrogates for transpulmonary samples, particularly when measuring mediators with prominent pulmonary secretion or clearance, such as ET-1.

A Pharmacodynamic Study of CN-218, a Novel Antiplatelet and Antithrombotic Agent Primarily Targeting the P2Y12 Receptor.

PURPOSE: Drugs inhibiting the platelet P2Y12 receptor, such as clopidogrel and prasugrel, are potent antithrombotic agents and are widely used in cardiovascular disease. However, the adverse effects of these drugs have limited their clinical use. For example, clopidogrel resistance occurs in approximately one third of patients, while prasugrel increases the risk of major bleeding. Therefore, new generations of such drugs are of clinical interest. METHODS: In this study, the pharmacodynamics of a new P2Y12 antagonist, CN-218, was compared with that of clopidogrel and prasugrel in rats and mice. The differences between CN-218 and clopidogrel include deuteration of the 7-position methyl carboxylate and the introduction of cinnamate in the 2-position of thiophene. RESULTS: CN-218 had an antiaggregatory efficacy that was at least five times more potent than that of clopidogrel but not as potent as that of prasugrel. It had a significant impact on activated partial thromboplastin time (APTT), whereby the APTT of CN-218-treated rats was approximately 9 s longer than that of the vehicle- or clopidogrel-treated group, while it had no impact on prothrombin time (PT) in rats. CN-218 had a similar potent antithrombotic effect to that of prasugrel and clopidogrel and also reduced the risk of bleeding compared to prasugrel. CONCLUSION: CN-218 may be a promising antithrombotic agent, with potent antiplatelet and significant anticoagulant activity, as well as a lower risk of bleeding compared to clopidogrel and prasugrel.

Analgesic action of Rubimaillin in vitro and in vivo.

Pain, a common symptom in clinics, is a serious impediment to quality of life. The analgesic drugs presently in use have poor efficacy, and are associated with undesirable side effects. Rubimaillin (Rub) is a naphthoquinone compound extracted from Chinese herbal medicine, and it has various biological activities. In this study, the analgesic effect of Rub, and its mechanism of action were investigated using glacial acetic acid-induced mice writhing model and a mice model of neurogenic and inflammatory bipolar pain. Analgesic effects were measured in different experimental groups. In vitro, RAW 264.7 cells were used to investigate the release of nitric oxide (NO), iNOS and COX-2 protein in RAW 264.7 cells stimulated with lipopolysaccharide (LPS). The results revealed that Rub reduced the number of acetic acid-induced writhing in mice, inhibited formalin-induced biphasic pain response, and suppressed the production of NO in RAW 264.7 cells. The mechanisms involved in the analgesic and anti-inflammatory effects of rub may be related to the inhibition of cyclooxygenase-2 (COX-2), endogenous inflammatory mediators, and reduction in the content of pain-induced mediators.

Acute Enhancement of Cardiac Function by Phosphodiesterase Type 1 Inhibition.

BACKGROUND: Phosphodiesterase type-1 (PDE1) hydrolyzes cAMP and cGMP and is constitutively expressed in the heart, although cardiac effects from its acute inhibition in vivo are largely unknown. Existing data are limited to rodents expressing mostly the cGMP-favoring PDE1A isoform. Human heart predominantly expresses PDE1C with balanced selectivity for cAMP and cGMP. Here, we determined the acute effects of PDE1 inhibition in PDE1C-expressing mammals, dogs, and rabbits, in normal and failing hearts, and explored its regulatory pathways. METHODS: Conscious dogs chronically instrumented for pressure-volume relations were studied before and after tachypacing-induced heart failure (HF). A selective PDE1 inhibitor (ITI-214) was administered orally or intravenously±dobutamine. Pressure-volume analysis in anesthetized rabbits tested the role of ß-adrenergic and adenosine receptor signaling on ITI-214 effects. Sarcomere and calcium dynamics were studied in rabbit left ventricular myocytes. RESULTS: In normal and HF dogs, ITI-214 increased load-independent contractility, improved relaxation, and reduced systemic arterial resistance, raising cardiac output without altering systolic blood pressure. Heart rate increased, but less so in HF dogs. ITI-214 effects were additive to ß-adrenergic receptor agonism (dobutamine). Dobutamine but not ITI-214 increased plasma cAMP. ITI-214 induced similar cardiovascular effects in rabbits, whereas mice displayed only mild vasodilation and no contractility effects. In rabbits, ß-adrenergic receptor blockade (esmolol) prevented ITI-214-mediated chronotropy, but inotropy and vasodilation remained unchanged. By contrast, adenosine A2B-receptor blockade (MRS-1754) suppressed ITI-214 cardiovascular effects. Adding fixed-rate atrial pacing did not alter the findings. ITI-214 alone did not affect sarcomere or whole-cell calcium dynamics, whereas ß-adrenergic receptor agonism (isoproterenol) or PDE3 inhibition (cilostamide) increased both. Unlike cilostamide, which further enhanced shortening and peak calcium when combined with isoproterenol, ITI-214 had no impact on these responses. Both PDE1 and PDE3 inhibitors increased shortening and accelerated calcium decay when combined with forskolin, yet only cilostamide increased calcium transients. CONCLUSIONS: PDE1 inhibition by ITI-214 in vivo confers acute inotropic, lusitropic, and arterial vasodilatory effects in PDE1C-expressing mammals with and without HF. The effects appear related to cAMP signaling that is different from that provided via ß-adrenergic receptors or PDE3 modulation. ITI-214, which has completed phase I trials, may provide a novel therapy for HF.

Global expression profiling and pathway analysis in two different population groups in relation to high altitude.

High altitude (HA) is associated with number of stresses. Response of these stresses may vary in different populations depending upon altitude, duration of residency, ancestry, geographical variation, lifestyle, and ethnicities. For understanding population variability in transcriptome, array-based global gene expression profiling was performed on extracted RNA of male volunteers of two different lowland population groups, i.e., Indians and Kyrgyz, at baseline and day 7 of HA exposure (3200 m). A total of 97 genes were differentially expressed at basal in Kyrgyz as compared to Indians (82 downregulated and 15 upregulated), and 196 were differentially expressed on day 7 of HA (118 downregulated and 78 upregulated). Ingenuity Pathway Analysis and gene ontology highlighted eIF2 signaling with most significant negative activation z score at basal in Kyrgyz compared to Indians with downregulation of various L- and S-ribosomal proteins indicating marked translational repression. On day 7, cAMP-mediated signaling is most enriched with positive activation z score in Kyrgyz compared to Indians. Plasma cAMP levels were higher in Kyrgyz on day 7 compared to Indians. Extracellular adenosine levels were elevated in both the groups upon HA, but higher in Kyrgyz compared to Indians. Valedictory qRT-PCR showed upregulation of ADORA2B and CD73 along with downregulation of ENTs in Kyrgyz compared to Indians indicating elevated levels of extracellular nucleotides mainly adenosine and activation of extracellular cAMP-adenosine pathway which as per literature triggers endogenous protective mechanisms under stress conditions like hypoxia. Thus, transcriptome changes at HA are population-specific, and it may be necessary to take care while interposing similar results in different populations.

Effect of Different Doses of Acetylsalicylic Acid on the Antithrombotic Activity of Clopidogrel in a Mouse Arterial Thrombosis Model.

Objective- Dual-antiplatelet therapy with acetylsalicylic acid and a P2Y12 antagonist, such as clopidogrel, is the standard of care for acute coronary syndromes. However, the drugs have divergent effects on the formation of cAMP, an inhibitory second messenger. Thus, by inhibiting the synthesis of prostacyclin, acetylsalicylic acid reduces cAMP formation, whereas clopidogrel potentiates it. Therefore, with higher doses of acetylsalicylic acid, the potentiation of cAMP production by clopidogrel may be attenuated, which could limit the antithrombotic potential of the drug combination. The purpose of this study was to examine this possibility in vivo. Approach and Results- Mice were given oral acetylsalicylic acid at varying doses, oral clopidogrel (5 mg/kg body weight), or both. At doses of 0.15 and 0.6 mg/kg, acetylsalicylic acid inhibited arachidonic acid-induced platelet aggregation, but only 0.6 mg/kg acetylsalicylic acid, or higher, decreased the plasma levels of 6-keto-prostaglandin-F1α, the stable metabolite of prostacyclin. When given with clopidogrel, laser injury-induced arterial thrombi were significantly larger with the 0.6 mg/kg dose of acetylsalicylic acid than with the 0.15 mg/kg dose. Thrombi in mice treated with clopidogrel and the 0.15 mg/kg dose of acetylsalicylic acid were smaller than in mice treated with clopidogrel alone, suggesting that acetylsalicylic acid can add to the antithrombotic effect of clopidogrel but that higher doses of acetylsalicylic acid blunt the antithrombotic effect of clopidogrel. Conclusions- These findings support the use of lower, prostacyclin-preserving, doses of acetylsalicylic acid in conjunction with clopidogrel.

The impact of the rs8005161 polymorphism on G protein-coupled receptor GPR65 (TDAG8) pH-associated activation in intestinal inflammation.

BACKGROUND: Tissue inflammation in inflammatory bowel diseases (IBD) is associated with a decrease in local pH. The gene encoding G-protein-coupled receptor 65 (GPR65) has recently been reported to be a genetic risk factor for IBD. In response to extracellular acidification, proton activation of GPR65 stimulates cAMP and Rho signalling pathways. We aimed to analyse the clinical and functional relevance of the GPR65 associated single nucleotide polymorphism (SNP) rs8005161. METHODS: 1138 individuals from a mixed cohort of IBD patients and healthy volunteers were genotyped for SNPs associated with GPR65 (rs8005161, rs3742704) and galactosylceramidase (rs1805078) by Taqman SNP assays. 2300 patients from the Swiss IBD Cohort Study (SIBDC) were genotyped for rs8005161 by mass spectrometry based SNP genotyping. IBD patients from the SIBDC carrying rs8005161 TT, CT, CC and non-IBD controls (CC) were recruited for functional studies. Human CD14+ cells were isolated from blood samples and subjected to an extracellular acidic pH shift, cAMP accumulation and RhoA activation were measured. RESULTS: In our mixed cohort, but not in SIBDC patients, the minor variant rs8005161 was significantly associated with UC. In SIBDC patients, we observed a consistent trend in increased disease severity in patients carrying the rs8005161-TT and rs8005161-CT alleles. No significant differences were observed in the pH associated activation of cAMP production between IBD (TT, CT, WT/CC) and non-IBD (WT/CC) genotype carriers upon an acidic extracellular pH shift. However, we observed significantly impaired RhoA activation after an extracellular acidic pH shift in IBD patients, irrespective of the rs8005161 allele. CONCLUSIONS: The T allele of rs8005161 might confer a more severe disease course in IBD patients. Human monocytes from IBD patients showed impaired pH associated RhoA activation upon an acidic pH shift.

Possible roles for ATP release from RBCs exclude the cAMP-mediated Panx1 pathway.

Red blood cell (RBC)-derived adenosine triphosphate (ATP) has been proposed as an integral component in the regulation of oxygen supply to skeletal muscle. In ex vivo settings RBCs have been shown to release ATP in response to a number of stimuli, including stimulation of adrenergic receptors. Further evidence suggested that ATP release from RBCs was dependent on activation of adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)-dependent pathways and involved the pannexin 1 (Panx1) channel. Here we show that RBCs express Panx1 and confirm its absence in Panx1 knockout (-/-) RBCs. However, Panx1-/- mice lack any decrease in exercise performance, challenging the assumptions that Panx1 plays an essential role in increased blood perfusion to exercising skeletal muscle and therefore in ATP release from RBCs. We therefore tested the role of Panx1 in ATP release from RBCs ex vivo in RBC suspensions. We found that stimulation with hypotonic potassium gluconate buffer resulted in a significant increase in ATP in the supernatant, but this was highly correlated with RBC lysis. Next, we treated RBCs with a stable cAMP analog, which did not induce ATP release from wild-type or Panx1-/- mice. Similarly, multiple pharmacological treatments activating AC in RBCs increased intracellular cAMP levels (as measured via mass spectrometry) but did not induce ATP release. The data presented here question the importance of Panx1 for exercise performance and dispute the general assumption that ATP release from RBCs via Panx1 is regulated via cAMP.

Diminished responsiveness to dobutamine as an inotrope in mice with cecal ligation and puncture-induced sepsis: attribution to phosphodiesterase 4 upregulation.

Dobutamine has been used in septic shock for many years as an only inotrope, but its benefit has been questioned. We weighed the effects of dobutamine and milrinone as inotropes in mice with cecal ligation and puncture (CLP)-induced polymicrobial sepsis. CLP-induced septic mice exhibited significant cardiac inflammation, as indicated by greatly increased mRNAs of proinflammatory cytokines and robust infiltration of inflammatory cells in the ventricular myocardium. Elevations of plasma cardiac troponin-I showed cardiac injury in CLP mice. Noninvasive echocardiographic assessment of cardiac function revealed that despite preserved left ventricular function in the presence of fluid replacement, the dobutamine inotropic response was significantly impaired in CLP mice compared with sham-operated controls. By contrast, milrinone exerted inotropic effects in sham-operated and CLP mice in an equally effective manner. Surface expression levels of ß1-adrenoceptors and α-subunits of three main G protein families in the myocardium were unaffected by CLP-induced sepsis. Plasma cAMP levels were significantly elevated in both sham-operated and CLP mice in response to milrinone but only in sham-operated controls in response to dobutamine. Of phosphodiesterase (PDE) isoforms, PDE4D, but not PDE3A, both of which are responsible for cardiac cAMP hydrolysis, was significantly upregulated in CLP mouse myocardium. We define a novel mechanism for the impaired responsiveness to dobutamine as an inotrope in sepsis, and understanding the role of PDE4D in modulating cardiac functional responsiveness in sepsis may open the potential of a PDE4D-targeted therapeutic option in septic patients with low cardiac output who have a need for inotropic support.NEW & NOTEWORTHY Advisability of the usefulness of dobutamine in septic shock management is limited. Here, we reveal that the effect of dobutamine as a positive inotrope is impaired in mice with cecal ligation and puncture-induced sepsis without changes in cardiac ß1-adrenoceptor signaling as a result of cAMP breakdown achieved by upregulated phosphodiesterase 4D.

Exenatide induces an increase in vasodilatory and a decrease in vasoconstrictive mediators.

In view of the known vasodilatory effects of glucagon-like peptide-1 and exenatide, we investigated the effects of exenatide on vasoactive factors. We analysed blood samples and mononuclear cells (MNCs) from a previous study, collected after a single dose and 12 weeks of exenatide or placebo treatment in a series of 24 patients with type 2 diabetes mellitus. After exenatide treatment, plasma concentrations of atrial natriuretic peptide, cyclic guanyl monophosphate (cGMP) and cyclic adenyl monophosphate increased significantly at 12 weeks. Plasma cGMP and adenylate cyclase expression in MNCs increased significantly after a single dose. Angiotensinogen concentration fell significantly 2 hours after a single dose and at 12 weeks, while renin and angiotensin II levels fell significantly only after a single dose and not after 12 weeks of treatment. Exenatide also suppressed the plasma concentration of transforming growth factor-ß and the expression of P311 in MNCs at 12 weeks. Thus, exenatide induces an increase in a series of vasodilators, while suppressing the renin-angiotensin system. These changes may contribute to the overall vasodilatory effect of exenatide.

12(S)-HETrE, a 12-Lipoxygenase Oxylipin of Dihomo-γ-Linolenic Acid, Inhibits Thrombosis via Gαs Signaling in Platelets.

OBJECTIVE: Dietary supplementation with polyunsaturated fatty acids has been widely used for primary and secondary prevention of cardiovascular disease in individuals at risk; however, the cardioprotective benefits of polyunsaturated fatty acids remain controversial because of lack of mechanistic and in vivo evidence. We present direct evidence that an omega-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), exhibits in vivo cardioprotection through 12-lipoxygenase (12-LOX) oxidation of DGLA to its reduced oxidized lipid form, 12(S)-hydroxy-8Z,10E,14Z-eicosatrienoic acid (12(S)-HETrE), inhibiting platelet activation and thrombosis. APPROACH AND RESULTS: DGLA inhibited ex vivo platelet aggregation and Rap1 activation in wild-type mice, but not in mice lacking 12-LOX expression (12-LOX(-/-)). Similarly, wild-type mice treated with DGLA were able to reduce thrombus growth (platelet and fibrin accumulation) after laser-induced injury of the arteriole of the cremaster muscle, but not 12-LOX(-/-) mice, supporting a 12-LOX requirement for mediating the inhibitory effects of DGLA on platelet-mediated thrombus formation. Platelet activation and thrombus formation were also suppressed when directly treated with 12(S)-HETrE. Importantly, 2 hemostatic models, tail bleeding and arteriole rupture of the cremaster muscle, showed no alteration in hemostasis after 12(S)-HETrE treatment. Finally, the mechanism for 12(S)-HETrE protection was shown to be mediated via a Gαs-linked G-protein-coupled receptor pathway in human platelets. CONCLUSIONS: This study provides the direct evidence that an omega-6 polyunsaturated fatty acid, DGLA, inhibits injury-induced thrombosis through its 12-LOX oxylipin, 12(S)-HETrE, which strongly supports the potential cardioprotective benefits of DGLA supplementation through its regulation of platelet function. Furthermore, this is the first evidence of a 12-LOX oxylipin regulating platelet function in a Gs α subunit-linked G-protein-coupled receptor-dependent manner.

Role of P2Y12 Receptor in Thrombosis.

P2Y12 receptor is a 342 amino acid Gi-coupled receptor predominantly expressed on platelets. P2Y12 receptor is physiologically activated by ADP and inhibits adenyl cyclase (AC) to decrease cyclic AMP (cAMP) level, resulting in platelet aggregation. It also activates PI3 kinase (PI3K) pathway leading to fibrinogen receptor activation, and may protect platelets from apoptosis. Abnormalities of P2Y12 receptor include congenital deficiencies or high activity in diseases like diabetes mellitus (DM) and chronic kidney disease (CKD), exposing such patients to a prothrombotic condition. A series of clinical antiplatelet drugs, such as clopidogrel and ticagrelor, are designed as indirect or direct antagonists of P2Y12 receptor to reduce incidence of thrombosis mainly for patients of acute coronary syndrome (ACS) who are at high risk of thrombotic events. Studies on novel dual-/multi-target antiplatelet agents consider P2Y12 receptor as a promising part in combined targets. However, the clinical practical phenomena, such as "clopidogrel resistance" due to gene variations of cytochrome P450 or P2Y12 receptor constitutive activation, call for better antiplatelet agents. Researches also showed inverse agonist of P2Y12 receptor could play a better role over neutral antagonists. Personalized antiplatelet therapy is the most ideal destination for antiplatelet therapy in ACS patients with or without other underlying diseases like DM or CKD, however, there is still a long way to go.

Inhibition of histone deacetylase 6 activity reduces cyst growth in polycystic kidney disease.

Abnormal proliferation of cyst-lining epithelium and increased intracystic fluid secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) are thought to contribute to cyst growth in autosomal dominant polycystic kidney disease (ADPKD). Histone deacetylase 6 (HDAC6) expression and activity are increased in certain cancers, neurodegenerative diseases, and in Pkd1-mutant renal epithelial cells. Inhibition of HDAC6 activity with specific inhibitors slows cancer growth. Here we studied the effect of tubacin, a specific HDAC6 inhibitor, on cyst growth in polycystic kidney disease. Treatment with tubacin prevented cyst formation in MDCK cells, an in vitro model of cystogenesis. Cyclic AMP stimulates cell proliferation and activates intracystic CFTR-mediated chloride secretion in ADPKD. Treatment with tubacin downregulated cyclic AMP levels, inhibited cell proliferation, and inhibited cyclic AMP-activated CFTR chloride currents in MDCK cells. We also found that tubacin reduced cyst growth by inhibiting proliferation of cyst-lining epithelial cells, downregulated cyclic AMP levels, and improved renal function in a Pkd1-conditional mouse model of ADPKD. Thus, HDAC6 could play a role in cyst formation and could serve as a potential therapeutic target in ADPKD.

Critical role of parathyroid hormone (PTH) receptor-1 phosphorylation in regulating acute responses to PTH.

Agonist-induced phosphorylation of the parathyroid hormone (PTH) receptor 1 (PTHR1) regulates receptor signaling in vitro, but the role of this phosphorylation in vivo is uncertain. We investigated this role by injecting "knock-in" mice expressing a phosphorylation-deficient (PD) PTHR1 with PTH ligands and assessing acute biologic responses. Following injection with PTH (1-34), or with a unique, long-acting PTH analog, PD mice, compared with WT mice, exhibited enhanced increases in cAMP levels in the blood, as well as enhanced cAMP production and gene expression responses in bone and kidney tissue. Surprisingly, however, the hallmark hypercalcemic and hypophosphatemic responses were markedly absent in the PD mice, such that paradoxical hypocalcemic and hyperphosphatemic responses were observed, quite strikingly with the long-acting PTH analog. Spot urine analyses revealed a marked defect in the capacity of the PD mice to excrete phosphate, as well as cAMP, into the urine in response to PTH injection. This defect in renal excretion was associated with a severe, PTH-induced impairment in glomerular filtration, as assessed by the rate of FITC-inulin clearance from the blood, which, in turn, was explainable by an overly exuberant systemic hypotensive response. The overall findings demonstrate the importance in vivo of PTH-induced phosphorylation of the PTHR1 in regulating acute ligand responses, and they serve to focus attention on mechanisms that underlie the acute calcemic response to PTH and factors, such as blood phosphate levels, that influence it.

Treatment-related biomarkers in pulmonary hypertension.

Significant advances in the treatment of pulmonary arterial hypertension (PAH) over the last two decades have led to the introduction of multiple classes of oral therapy, but the disease remains devastating for many patients. Disease progression, in spite of oral monotherapy, is a major problem, and alternative therapy, such as infusion of prostacyclins, is cumbersome and carries considerable potential morbidity. Use of combination oral therapy, including drugs from both the endothelin receptor antagonist and phosphodiesterase-5 inhibitor classes, has increased, and there is some evidence to support this approach. Given the multiple options now available in pulmonary hypertension (PH) therapy, biomarkers to guide treatment decisions could be helpful. Here, we review the evidence for and against the clinical use of molecular biomarkers relevant to PH pathogenesis, emphasizing assayable markers that may also inform more rational selection of agents that influence pathways targeted by treatment. We emphasize the interactive nature of changes in mediators and messengers, such as endothelin-1, prostacyclin, brain natriuretic peptide (which has demonstrated biomarker utility), nitric oxide derivatives, and cyclic guanosine monophosphate, which play important roles in processes central to progression of PAH, such as vascular remodeling, vasoconstriction, and maladaptive right ventricular changes, and are relevant to its therapy. Accordingly, we propose that the identification and use of a molecular biomarker panel that assays these molecules in parallel and serially might, if validated, better inform unique patient phenotypes, prognosis, and the rational selection and titration of combination oral and other therapy in individual patients with PH/PAH.

Highly electronegative LDL from patients with ST-elevation myocardial infarction triggers platelet activation and aggregation.

Platelet activation and aggregation underlie acute thrombosis that leads to ST-elevation myocardial infarction (STEMI). L5-highly electronegative low-density lipoprotein (LDL)-is significantly elevated in patients with STEMI. Thus, we examined the role of L5 in thrombogenesis. Plasma LDL from patients with STEMI (n = 30) was chromatographically resolved into 5 subfractions (L1-L5) with increasing electronegativity. In vitro, L5 enhanced adenosine diphosphate-stimulated platelet aggregation twofold more than did L1 and induced platelet-endothelial cell (EC) adhesion. L5 also increased P-selectin expression and glycoprotein (GP)IIb/IIIa activation and decreased cyclic adenosine monophosphate levels (n = 6, P < .01) in platelets. In vivo, injection of L5 (5 mg/kg) into C57BL/6 mice twice weekly for 6 weeks shortened tail bleeding time by 43% (n = 3; P < .01 vs L1-injected mice) and increased P-selectin expression and GPIIb/IIIa activation in platelets. Pharmacologic blockade experiments revealed that L5 signals through platelet-activating factor receptor and lectin-like oxidized LDL receptor-1 to attenuate Akt activation and trigger granule release and GPIIb/IIIa activation via protein kinase C-α. L5 but not L1 induced tissue factor and P-selectin expression in human aortic ECs (P < .01), thereby triggering platelet activation and aggregation with activated ECs. These findings indicate that elevated plasma levels of L5 may promote thrombosis that leads to STEMI.