Endothelial Prostacyclin Production Research Articles

A randomized trial to assess the pharmacodynamics and pharmacokinetics of a single dose of an extended-release aspirin formulation

Aims. Low-dose acetylsalicylic acid (ASA; aspirin) for secondary prevention reduces cardiovascular disease mortality risk. ASA acetylates cyclooxygenase in the portal circulation and is rapidly (half-life, 20 min) hydrolyzed. Certain patients with cardiovascular disease may exhibit high on-therapy platelet reactivity as a result of high platelet turnover, a process whereby platelets are produced and are active beyond the duration of antiplatelet coverage provided by once-daily immediate-release (IR) ASA. A once-daily, extended-release (ER) ASA formulation using ER microcapsule technology was developed to release ASA over the 24-h dosing interval and reduce maximal plasma concentrations to spare peripheral endogenous endothelial prostacyclin production. Methods. Healthy adults (n = 50) were randomized in a crossover study to receive two different ER-ASA single doses (up to 325 mg) and two different IR-ASA single doses (up to 81 mg) in four periods, each separated by ≥14 days. Pharmacodynamics was assessed by measuring serum thromboxane B2 (TXB2), urine 11-dehydro-TXB2, and arachidonic acid-induced platelet aggregation. Pharmacokinetics was determined for ASA and salicylic acid (SA). Results: Both formulations produced dose-dependent inhibition on all pharmacodynamic parameters. Marked inhibition of TXB2 and 11-dehydro-TXB2 was maintained over the 24-h dosing interval after a dose of ≥81 mg ER-ASA or ≥40 mg IR-ASA. The dose required to achieve 50% of maximum TXB2 inhibition with ER-ASA was 49.9 mg versus 29.6 mg for IR-ASA, for a similar maximum pharmacodynamic effect (98.9% TXB2 inhibition). This suggests that an approximately twofold greater ER-ASA dose (162.5 mg) is necessary to obtain the same response as that of IR-ASA 81 mg. Peak ASA concentrations were lower and Tmax was longer with ER-ASA versus IR-ASA. Administration of IR-ASA resulted in a dose-normalized mean Cmax of ASA that was approximately sixfold higher than that for ER-ASA and a Cmax of SA approximately two- to threefold higher than that for ER-ASA. Conclusion. Both ASA formulations showed dose-dependent antiplatelet activity. Compared with the IR-ASA, ER-ASA released active drug more slowly, resulting in prolonged absorption and lower systemic drug concentrations, which is expected for an ER (24-h) formulation.

Oleoyl-lysophosphatidylcholine limits endothelial nitric oxide bioavailability by induction of reactive oxygen species.

Previously we reported modulation of endothelial prostacyclin and interleukin-8 production, cyclooxygenase-2 expression and vasorelaxation by oleoyl- lysophosphatidylcholine (LPC 18:1). In the present study, we examined the impact of this LPC on nitric oxide (NO) bioavailability in vascular endothelial EA.hy926 cells. Basal NO formation in these cells was decreased by LPC 18:1. This was accompanied with a partial disruption of the active endothelial nitric oxide synthase (eNOS)- dimer, leading to eNOS uncoupling and increased formation of reactive oxygen species (ROS). The LPC 18:1-induced ROS formation was attenuated by the superoxide scavenger Tiron, as well as by the pharmacological inhibitors of eNOS, NADPH oxidases, flavin-containing enzymes and superoxide dismutase (SOD). Intracellular ROS-formation was most prominent in mitochondria, less pronounced in cytosol and undetectable in endoplasmic reticulum. Importantly, Tiron completely prevented the LPC 18:1-induced decrease in NO bioavailability in EA.hy926 cells. The importance of the discovered findings for more in vivo like situations was analyzed by organ bath experiments in mouse aortic rings. LPC 18:1 attenuated the acetylcholine-induced, endothelium dependent vasorelaxation and massively decreased NO bioavailability. We conclude that LPC 18:1 induces eNOS uncoupling and unspecific superoxide production. This results in NO scavenging by ROS, a limited endothelial NO bioavailability and impaired vascular function.

High-density lipoprotein as a modulator of platelet and coagulation responses.

Platelets and coagulation factors are involved in the process of haemostasis, which ensures undisturbed blood flow upon vessel wall damage. However, excessive platelet aggregation and/or coagulation may lead to arterial or venous thrombosis. Pro-atherogenic lipoproteins, including native and oxidized low-density lipoprotein (LDL), are associated with an increased susceptibility to thrombosis. In contrast, numerous epidemiological studies have established an inverse correlation between high-density lipoprotein (HDL) levels and the risk for thrombosis. In addition to its role in reverse cholesterol transport, HDL also interacts with platelets, the coagulation cascade, and the vascular endothelium. Native HDL prevents platelet hyperreactivity by limiting intraplatelet cholesterol overload, as well as by modulating platelet signalling pathways after binding platelet HDL receptors such as scavenger receptor class B type I (SR-BI) and apoER2'. The antithrombotic properties of native HDL are also related to the suppression of the coagulation cascade and stimulation of clot fibrinolysis. Furthermore, HDL stimulates the endothelial production of nitric oxide and prostacyclin, which are potent inhibitors of platelet activation. Thus, HDL's antithrombotic actions are multiple and therefore, raising HDL may be an important therapeutic strategy to reduce the risk of arterial and venous thrombosis.

Mitigating the Cardiovascular and Renal Effects of NSAIDs: Table 1

Nonsteroidal anti-inflammatory drugs (NSAIDs) are principal pharmacologic agents for symptom relief in patients with arthritis and other inflammatory conditions. Cardiovascular risk is associated with all NSAIDs, excluding aspirin. Selective inhibition of cyclo-oxygenase-2 (COX)-2 could produce a relative reduction in endothelial production of prostacyclin, while leaving the platelet production of thromboxane A2 (TXA2 ) intact. It has been speculated that this imbalance of homeostatic prostanoids might increase the risk for thrombotic events. The goal of this review is to provide physicians guidelines to mitigate cardiovascular and nephrotoxicity of NSAIDs. We conducted a systematic literature review to determine what information is available to guide treatment decisions in this patient population. Selective inhibition of COX-2 could produce a relative reduction in endothelial production of prostacyclin, while leaving the platelet production of TXA2 intact. Increasing degrees of selectivity for COX-2 are associated with augmented cardiovascular risk, whereas increasing degrees of selectivity for cyclo-oxygenase-2 (COX-1) are associated with augmented gastrointestinal risk. Some NSAIDs (such as ibuprofen) can interfere with the cardioprotective effects of aspirin by competitively binding to COX-1 enzyme, resulting in increased TXA2 production Naproxen may differ from other NSAIDs in sustaining functionally important degrees of inhibition of platelet cyclooxygenase-1 activity throughout the dosing interval. It is of paramount importance to consider individual health factors when choosing therapy with NSAIDs.

Estradiol-17β and its Cytochrome P450- and Catechol-O-Methyltransferase–Derived Metabolites Selectively Stimulate Production of Prostacyclin in Uterine Artery Endothelial Cells

Metabolism of estradiol-17β to 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol, and 4-methoxyestradiol contributes importantly to the vascular effects of estradiol-17β in several vascular beds. However, little is known about the role of estradiol-17β metabolites via the different estrogen receptors (ER-α/ER-β) on de novo endothelial prostacyclin and thromboxane production. We hypothesized that estradiol-17β and its metabolites, via ER-α or ER-β, can enhance the prostacyclin/thromboxane ratio through the classic phospholipase A 2 , cyclooxygenase-1, and prostacyclin synthase pathway in ovine uterine artery endothelial cells (UAECs) derived from pregnant (P-UAECs) versus nonpregnant (NP-UAECs) ewes. Western analyses showed higher expression of phospholipase A 2 , cyclooxygenase-1, and prostacyclin synthase in UAECs from the pregnant state, whereas thromboxane synthase was lowered in UAECs from the pregnant state. In UAECs from the pregnant state, estradiol-17β, 2-hydroxyestradiol, 4-hydroxyestradiol, 2-methoxyestradiol and 4-methoxyestradiol concentration and time-dependently increased prostacyclin compared with controls. Prostacyclin increases in UAECs from the nonpregnant state were of a lower magnitude. Estradiol-17β and its metabolites stimulated higher prostacyclin/thromboxane ratios in UAECs from the pregnant state compared with UAECs from the nonpregnant state. Estradiol-17β–induced prostacyclin increases were abrogated by the antagonists SC-560 (cyclooxygenase-1), U-51605 (Prostacyclin synthase), ICI 182 780 (ICI; both ER-α/β), and 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinyleth oxy)phenol]-1H-pyrazole dihydrochloride (MPP; ER-α), but not by 4-[2-phenyl-5,7-bis (trifluoromethyl) pyrazolo[1,5-a]pyrim idin-3-yl]phenol (PHTPP; ER-β). Prostacyclin increases induced by its metabolites were abolished by SC-560 and U-51605, but unaltered by ICI, MPP, or PHTPP. Our findings demonstrate that estrogen via primarily ER-α and its metabolites via ER-independent mechanisms influence the de novo endothelial biosynthesis of prostacyclin, which may be important in the regulation of vascular tone. These findings also shed light on the complexities of estrogen signaling via its metabolism and the functional heterogeneity of the ERs.

Estrogen Receptors and Endothelium

Estrogens, and in particular 17beta-estradiol (E2), play a pivotal role in sexual development and reproduction and are also implicated in a large number of physiological processes, including the cardiovascular system. Both acetylcholine-induced and flow-dependent vasodilation are preserved or potentiated by estrogen treatment in both animal models and humans. Indeed, E2 increases the endothelial production of nitric oxide and prostacyclin and prevents early atheroma through endothelial-mediated mechanisms. Furthermore, whereas it prevents endothelial activation, E2 potentiates the ability of several subpopulations of the circulating or resident immune cells to produce proinflammatory cytokines. The balance between these 2 actions could determine the final effect in a given pathophysiological process. E2 also promotes endothelial healing, as well as angiogenesis. Estrogen actions are essentially mediated by 2 molecular targets: estrogen receptor-alpha (ERalpha) and ERbeta. The analysis of mouse models targeted for ERalpha or ERbeta demonstrated a prominent role of ERalpha in vascular biology. ERalpha directly modulates transcription of target genes through 2 activation functions (AFs), AF-1 and AF-2. Interestingly, an AF-1-deficient ERalpha isoform can be physiologically expressed in the endothelium and appears sufficient to mediate most of the vasculoprotective actions of E2. In contrast, AF-1 is necessary for the E2 actions in reproductive targets. Thus, it appears conceivable to uncouple the vasculoprotective and sexual actions with appropriate selective ER modulators.

Neutrophil elastase contributes to the development of ischemia/reperfusion-induced liver injury by decreasing the production of insulin-like growth factor-I in rats

Neutrophil elastase (NE) decreases the endothelial production of prostacyclin (PGI(2)) through the inhibition of endothelial nitric oxide synthase (NOS) activation and thereby contributes to the development of ischemia/reperfusion (I/R)-induced liver injury. We previously demonstrated that calcitonin gene-related peptide (CGRP) released from sensory neurons increases the insulin-like growth factor- I (IGF-I) production and thereby reduces I/R-induced liver injury. Because PGI(2) is capable of stimulating sensory neurons, we hypothesized that NE contributes to the development of I/R-induced liver injury by decreasing IGF-I production. In the present study, we examined this hypothesis in rats subjected to hepatic I/R. Ischemia/reperfusion-induced decreases of hepatic tissue levels of CGRP and IGF-I were prevented significantly by NE inhibitors, sivelestat, and L-658, 758, and these effects of NE inhibitors were reversed completely by the nonselective cyclooxygenase inhibitor indomethacin (IM) and the nonselective NOS inhibitor L-NAME but not by the selective inducible NOS inhibitor 1400W. I/R-induced increases of hepatic tissue levels of caspase-3, myeloperoxidase and the number of apoptotic cells were inhibited by NE inhibitors, and these effects of NE inhibitors were reversed by IM and L-NAME but not by 1400W. Administration of iloprost, a stable PGI(2) analog, produced effects similar to those induced by NE inhibitors. Taken together, these observations strongly suggest that NE may play a critical role in the development of I/R-induced liver injury by decreasing the IGF-I production through the inhibition of sensory neuron stimulation, which may lead to an increase of neutrophil accumulation and hepatic apoptosis through activation of caspase-3 in rats.

Microsomal prostaglandin E synthase‐1 inhibition blocks proliferation and enhances apoptosis in oesophageal adenocarcinoma cells without affecting endothelial prostacyclin production

Prostaglandins are important in the progression of various gastrointestinal cancers including oesophageal adenocarcinoma (OAC). Cyclo-oxygenase (COX)-2 inhibitors reduce OAC prostaglandin production but also have potentially detrimental effects on vascular endothelial function by reducing prostacyclin production and increasing the risk of cardiovascular events. We have examined the effects of inhibiting microsomal prostaglandin synthase-1 (mPGES-1), the enzyme downstream of COX-2 in the prostaglandin synthetic cascade. In OAC cells, reduction of mPGES-1 with RNA interference blocked PGE2 production, inhibited serum-induced proliferation and enhanced apoptosis in the COX-2 expressing cell lines (OE33 and FLO) but had no effect in COX-2 deficient BIC-1 cells. Three different methods of inhibiting mPGES-1 (RNA interference, a novel small molecule inhibitor and the endogenous inhibitor 15-deoxy-Delta(12,14)-PGJ(2)) also blocked leptin induced mPGES-1 expression and PGE2 production and abolished the leptin-induced proliferative and anti-apoptotic effects in OE33 cells, without affecting COX-2 expression. The anti-proliferative effects were equivalent to those produced by COX-2 inhibitory concentrations of celecoxib and NS-398. However, unlike the two COX-2 inhibitors, mPGES-1 inhibition did not reduce endothelial prostacyclin production. In contrast to the effects of the COX-2 inhibitor celecoxib, mPGES-1 inhibition had no effects on Akt kinase activity in OAC cells. We conclude that inhibition of mPGES-1 has potentially beneficial effects in OAC without the potentially detrimental effects on vascular endothelial prostacyclin synthesis. We have also confirmed that celecoxib has anticancer actions separate from the inhibition of COX-2. Inhibition of mPGES-1 may be therapeutically useful in the treatment and prevention of OAC.

Putting the vasoactive effects of COX-2-derived prostanoids into clinical perspective

Ever since the recognition that the selective inhibitor of cyclooxygenase (COX)-2, rofecoxib, increases the risk for adverse cardiovascular events, enormous attention has been given to the mechanisms underlying these adverse vascular effects of the coxibs.1,2 Many recent studies that have investigated the biological consequences of COX-2 inhibition in the vasculature have focussed either on the prothrombotic side effects of decreased endothelial prostacyclin production during selective COX-2 blockade3,4 or on detrimental effects of such an intervention on atherosclerosis.5,6 Although such studies have greatly broadened our understanding of the pathophysiological roles of COX-2-mediated prostanoid production in vascular biology, the important vasodilatory functions of COX-2-derived prostanoids have been somewhat eclipsed by them. Foudi et al. presents data that address the topic of detrimental vascular effects of COX-2 inhibition from this different, yet important, point of view.7 They show in isolated human internal mammary arteries (IMA) that under conditions of acute septic inflammation, which is induced by simultaneous administration of interleukin-1β (IL-1β) and lipopolysaccharide (LPS), COX-2 is upregulated and produces prostacyclin and prostaglandin E2 (PGE2) rather than thromboxane A2 (TxA2). COX-2 expression is shown to be enhanced not only in the intima, but also in the smooth muscle cell-containing media of the vasculature. When vasoconstriction is induced by norepinephrine in the IMA preparations, COX-2 inhibition leads … Corresponding author. Tel: +49 89 5160 2111; fax: +49 89 5160 2410. E-mail address : florian.kroetz{at}med.uni-muenchen.de

ROLE OF NEUTROPHIL ELASTASE IN DEVELOPMENT OF PULMONARY VASCULAR INJURY AND SEPTIC SHOCK IN RATS

Prostacyclin prevents pulmonary vascular injury and shock by inhibiting increases in lung tissue levels of TNF in rats administered endotoxin. We previously reported that NO derived from eNOS increases endothelial production of prostacyclin. Because neutrophil elastase has been shown to decrease endothelial production of prostacyclin by inhibiting NOS activity, we examined whether neutrophil elastase inhibitors reduce pulmonary vascular injury and hypotension by inhibiting the decrease in pulmonary endothelial production of prostacyclin in rats administered endotoxin. Animals were pretreated with sivelestat or L-658,758, neutrophil elastase inhibitors, before endotoxin administration. Lung tissue levels of 6-keto-prostaglandin F1alpha were markedly increased after endotoxin administration, followed by a rapid decrease to baseline levels. Sivelestat and L-658,758 inhibited these decreases as well as inhibiting increases in lung tissue levels of TNF and lung wet-to-dry weight ratios in animals administered endotoxin. These inhibitors also reduced hypotension and inhibited increases in lung tissue levels of mRNA of the inducible form of NOS in animals administered endotoxin. The effects of neutrophil elastase inhibitors were completely reversed by pretreatment with nitro-L-arginine methyl ester, an inhibitor of NOS, or indomethacin, a nonspecific cyclooxygenase inhibitor. These observations suggested that neutrophil elastase might decrease the pulmonary endothelial production of prostacyclin by inhibiting endothelial NO production, thereby contributing to the development of pulmonary vascular injury and shock through increases in lung tissue levels of TNF in rats administered endotoxin.

Atrial Natriuretic Peptide Reduces Ischemia/Reperfusion-Induced Spinal Cord Injury in Rats by Enhancing Sensory Neuron Activation

We recently demonstrated that calcitonin gene-related peptide (CGRP) released from sensory neurons reduces spinal cord injury (SCI) by inhibiting neutrophil activation through an increase in the endothelial production of prostacyclin (PGI(2)). Carperitide, a synthetic alpha-human atrial natriuretic peptide (ANP), reduces ischemia/reperfusion (I/R)-induced tissue injury. However, its precise therapeutic mechanism(s) remains to be elucidated. In the present study, we examined whether ANP reduces I/R-induced spinal cord injury by enhancing sensory neuron activation using rats. ANP increased CGRP release and cellular cAMP levels in dorsal root ganglion neurons isolated from rats in vitro. The increase in CGRP release induced by ANP was reversed by pretreatment with capsazepine, an inhibitor of vanilloid receptor-1 activation, or with (9S, 10S, 12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]-benzodiazocine-10-carboxylic acid hexyl ester (KT5720), an inhibitor of protein kinase A (PKA), suggesting that ANP might increase CGRP release from sensory neurons by activating PKA through an increase in the cellular cAMP level. Spinal cord ischemia was induced in rats using a balloon catheter placed in the aorta. ANP reduced mortality and motor disturbances by inhibiting reduction of the number of motor neurons in animals subjected to SCI. ANP significantly enhanced I/R-induced increases in spinal cord tissue levels of CGRP and 6-keto-prostaglandin F(1alpha). a stable metabolite of PGI(2). ANP inhibited I/R-induced increases in spinal cord tissue levels of tumor necrosis factor and myeloperoxidase. Pretreatment with 4'-chloro-3-methoxycinnamanilide (SB366791), a specific vanilloid receptor-1 antagonist, and indomethacin reversed the effects of ANP. These results strongly suggest that ANP might reduce I/R-induced SCI in rats by inhibiting neutrophil activation through enhancement of sensory neuron activation.

Use of Nonsteroidal Antiinflammatory Drugs

Clinical trial data have prompted questions about the degree to which patients and their physicians should consider an increased risk of cardiovascular or cerebrovascular events when selecting medications for pain relief. Since the 2005 publication of a Science Advisory on the use of nonsteroidal antiinflammatory drugs (NSAIDs) by the American Heart Association,1 several important events have occurred that have served as the catalyst for this update for clinicians. (1) Additional data from randomized controlled trials of cyclooxygenase (COX)-2–selective agents have been reported and summarized in meta-analyses, which has reinforced the concern about cardiovascular events with COX-2 inhibitors (coxibs; Figure 1). (2) Several reports have appeared that have identified an increased risk of cardiovascular events even with the nonselective NSAIDs, which has raised concern about the use of those agents as well (Table). (3) Regulatory authorities in several regions of the world have introduced warning statements and advisories to both healthcare professionals and the lay public about the use of various NSAIDs (Figures 2 and 3⇓). Figure 1. Comparison of effects of different selective COX-2 inhibitors vs placebo on myocardial infarction. Event numbers and person-years of exposure, with corresponding mean annual event rates in parentheses, are presented for patients allocated to selective COX-2 inhibitor or placebo. Event rate ratios for pooled data with 95% CIs are indicated by a diamond; rate ratios for individual selective COX-2 inhibitors, with 99% CIs, are indicated by a square and horizontal line. Diamonds to the right of the solid line indicate hazard with a selective COX-2 inhibitor compared with placebo. As noted, there was a significant increase in the rate ratio for myocardial infarction with COX-2 inhibitors compared with placebo. Similar analyses (data not shown) include rate ratios of 1.42 (1.13 to 1.78; P =0.003) for vascular events, 1.02 (0.71 to 1.47; P …

Inhibition of Neutrophil Activation by Lafutidine, an H2-receptor Antagonist, Through Enhancement of Sensory Neuron Activation Contributes to the Reduction of Stress-Induced Gastric Mucosal Injury in Rats

Sensory neuron activation reduces water-immersion restraint stress (WIR)-induced gastric mucosal injury by inhibiting neutrophil activation through increase in endothelial production of prostacyclin. This study was designed to examine whether lafutidine, which is an H(2)-receptor antagonist and activates sensory neurons, inhibits neutrophil activation, thereby reducing WIR-induced gastric mucosal injury. Lafutidine enhanced WIR-induced increases in gastric tissue levels of calcitonin gene-related peptide (CGRP) and 6-keto-PGF(1alpha), a stable metabolite of prostacyclin, whereas famotidine, another H(2)-receptor antagonist, did not. Such lafutidine-induced increases in gastric tissue levels of 6-keto-PGF(1alpha) were reversed by pretreatment with capsazepine, an inhibitor of sensory neuron activation, CGRP(8-37), a CGRP antagonist, and indomethacin. Lafutidine inhibited acid-induced exacerbation of gastric mucosal injury in animals subjected to WIR by inhibiting neutrophil activation, whereas famotidine did not. Lafutidine synergistically increased CGRP release from isolated rat dorsal root ganglion neurons in the presence of anandamide, but famotidine did not. These observations suggest that lafutidine might reduce WIR-induced gastric mucosal injury not only by inhibiting acid secretion but also by inhibiting neutrophil activation through enhancement of sensory neuron activation.

Atrial natriuretic peptide reduces the ischemia/reperfusion-induced renal injury in rats by enhancing sensory neuron activation

We recently demonstrated that calcitonin gene-related peptide (CGRP) released from sensory neurons reduces spinal cord injury (SCI) by inhibiting neutrophil activation through an increase in the endothelial production of prostacyclin (PGI2). Carperitide, a synthetic -human atrial natriuretic peptide (ANP), reduces ischemia/reperfusion (I/R)-induced tissue injury. However, its precise therapeutic mechanism(s) remains to be elucidated. In the present study, we examined whether ANP reduces I/R-induced spinal cord injury by enhancing sensory neuron activation using rats. ANP increased CGRP release and cellular cAMP levels in dorsal root ganglion neurons isolated from rats in vitro. The increase in CGRP release induced by ANP was reversed by pretreatment with capsazepine, an inhibitor of vanilloid receptor-1 activation, or with (9S,10S,12R)2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12epoxy-1H-diindolo[1,2,3-fg:3,2,1-kl]pyrrolo[3,4-i][1,6]-benzodiazocine-10-carboxylic acid hexyl ester (KT5720), an inhibitor of protein kinase A (PKA), suggesting that ANP might increase CGRP release from sensory neurons by activating PKA through an increase in the cellular cAMP level. Spinal cord ischemia was induced in rats using a balloon catheter placed in the aorta. ANP reduced mortality and motor disturbances by inhibiting reduction of the number of motor neurons in animals subjected to SCI. ANP significantly enhanced I/R-induced increases in spinal cord tissue levels of CGRP and 6-keto-prostaglandin F1. a stable metabolite of PGI2. ANP inhibited I/R-induced increases in spinal cord tissue levels of tumor necrosis factor and myeloperoxidase. Pretreatment with 4-chloro-3-methoxycinnamanilide (SB366791), a specific vanilloid receptor-1 antagonist, and indomethacin reversed the effects of ANP. These results strongly suggest that ANP might reduce I/R-induced SCI in rats by inhibiting neutrophil activation through enhancement of sensory neuron activation.

Regulation of Inflammatory Responses by Sensory Neurons: Molecular Mechanism(s) and Possible Therapeutic Applications

Capsaicin-sensitive sensory neurons are nociceptive neurons that release calcitonin gene-related peptide (CGRP) on activation. Since CGRP has potent vasodilatory activity, it has long been considered to be involved in aggravation of inflammation such as tissue hyperemia and edema. However, since ablation of the sensory fibers can result in a marked increase in the severity of inflammation and reperfusion-induced tissue inflammatory responses are enhanced in congenital CGRP-knockout mice, the sensory neurons have been shown to play a role in the maintenance of tissue integrity by regulating local inflammatory responses. We demonstrated in rodents that stimulation of sensory neurons reduces hypertension, stress-induced gastric mucosal injury, reperfusion-induced liver injury, and endotoxin-induced shock responses by attenuating inflammatory responses such as increases in both tissue levels of tumor necrosis factor (TNF) and tissue accumulation of neutrophils. Attenuation of inflammatory responses by sensory neuron activation can be explained mainly by CGRP-induced increase in the endothelial production of prostacyclin (PGI(2)). Since inflammatory responses are critically involved in the development of a wide variety of diseases, pharmacological stimulation of sensory neurons might contribute to treatment of various pathologic conditions. In this review, the authors describe molecular mechanism(s) by which sensory neuron activation inhibits TNF production, thereby attenuating inflammatory responses. Furthermore, the authors discuss some clinically useful therapeutic agents that are capable of activating sensory neurons and raise the possibility that pharmacological stimulation of sensory neurons is the new paradigm for future therapeutic strategies.

Dalteparin, a low molecular weight heparin, attenuates inflammatory responses and reduces ischemia-reperfusion–induced liver injury in rats*

To examine whether dalteparin, a low molecular weight heparin, prevents hepatic damage by inhibiting leukocyte activation, we analyzed its effect on ischemia/reperfusion (I/R) injury of rat liver in which activated leukocytes play a critical role. Prospective, randomized, controlled study. Research laboratory at a university medical center. Male Wistar rats weighing 220-280 g. Hepatic damage was evaluated by changes in serum transaminase concentrations after I/R. Coagulation abnormalities were evaluated by changes in serum concentrations of fragment E of fibrin and fibrinogen degradation products after I/R. Hepatic tissue blood flow was measured by laser-Doppler flow meter. Hepatic edema was evaluated by determination of the change in the wet/dry tissue weight ratio. Rats were intravenously injected with dalteparin or unfractionated heparin (300 units/kg) and subcutaneously injected with DX9056a, a selective inhibitor of activated factor X (3 mg/kg). To determine whether dalteparin inhibits leukocyte activation, we examined the effect of dalteparin on hepatic concentrations of interleukin-12, tumor necrosis factor-alpha, and hepatic myeloperoxidase activity after I/R in vivo. In addition, we examined increases in tumor necrosis factor-alpha production in rat monocytes and in intracellular calcium concentrations in neutrophils in vitro. We also examined the effect of dalteparin on endothelial production of prostacyclin using isolated rat hepatic sinusoidal cells in vitro. Intravenous administration of dalteparin inhibited increases in serum levels of both transaminases and serum concentrations of fragment E of fibrin and fibrinogen degradation products in animals subjected to hepatic I/R. Hepatic tissue blood flow after reperfusion was increased by dalteparin. Dalteparin inhibited hepatic edema, increases in hepatic tissue levels of interleukin-12 and tumor necrosis factor-alpha, and accumulation of neutrophils in animals subjected to hepatic I/R. Neither DX9065a nor unfractionated heparin showed any therapeutic effects, despite potent inhibition of increases in serum levels of fragment E of fibrin and fibrinogen degradation products. Neither monocytic tumor necrosis factor-alpha production nor neutrophil activation was inhibited by dalteparin in vitro. Dalteparin enhanced the hepatic I/R-induced increases in hepatic tissue levels of 6-keto-prostaglandin (PG) F1alpha, a stable metabolite of prostacyclin, which is capable of inhibiting monocytic tumor necrosis factor-alpha production. Pretreatment with indomethacin completely reversed both of the therapeutic effects of dalteparin, whereas pretreatment with NS-398, a selective inhibitor of cyclooxygenase-2, did not. Dalteparin did not directly increase the endothelial production of prostacyclin in vitro. Dalteparin might reduce I/R-induced liver injury in rats by attenuating inflammatory responses. These therapeutic effects might be independent of its anticoagulant activity but dependent on its capacity to enhance endothelial production of prostacyclin via cyclooxygenase-1 activation. Furthermore, the mechanism or mechanisms by which dalteparin promotes the endothelial production of prostacyclin in vivo might involve unknown factors other than endothelial cells.

Coxibs and Cancer Prevention

Preventive medicine is becoming a cornerstone in our concept of health. This is especially significant in regard to cancer, as cancer is predicted to become the leading cause of death, surpassing heart disease, by the end of this decade. The prevention of colorectal cancer (CRC) has become an important public health goal because of the high incidence of CRC, with more than 945,000 new cases expected worldwide in 2006, and the considerable mortality and morbidity associated, with more than 492,000 deaths expected worldwide in the same year. The past 2 decades have seen the emergence of chemopreventive agents that have 1 of 3 effects: inhibiting, delaying, or reversing carcinogenesis. Notwithstanding a substantial body of evidence suggesting an inverse relationship between aspirin or nonsteroidal anti-inflammatory drug use and CRC incidence and mortality, the use of traditional nonsteroidal anti-inflammatory drugs in the chemoprevention of CRC is limited by their gastrointestinal toxicity. The favorable gastrointestinal safety profile of selective cyclooxygenase-2 inhibitors has therefore made them particularly attractive for this purpose. There has been concern, however, that selective cyclooxygenase-2 inhibitors may increase the risk of cardiovascular events, possibly by reducing endothelial prostacyclin production while leaving platelet thromboxane A2 generation unopposed. In the intriguing jigsaw puzzle of cancer prevention, we now have a definite positive answer for the basic question "if," but several other parts of the equation (proper patient selection, ultimate drug, optimal dosage, and duration) are missing. The most challenging task is to find the proper place for these interventions in the entire effort of cancer prevention, in subjects at risk for colorectal neoplasia, and in those at risk for other tumors. The achievement of this important goal may contribute to the conversion of CRC into a truly preventable disease.

Cyclooxygenase-2 Induction and Prostacyclin Release by Protease-activated Receptors in Endothelial Cells Require Cooperation between Mitogen-activated Protein Kinase and NF-κB Pathways

The functional significance of protease-activated receptors (PARs) in endothelial cells is largely undefined, and the intracellular consequences of their activation are poorly understood. Here, we show that the serine protease thrombin, a PAR-1-selective peptide (TFLLRN), and SLIGKV (PAR-2-selective peptide) induce cyclooxygenase-2 (COX-2) protein and mRNA expression in human endothelial cells without modifying COX-1 expression. COX-2 induction was accompanied by sustained production of 6-keto-PGF1alpha, the stable hydrolysis product of prostacyclin, and this was inhibited by indomethacin and the COX-2-selective inhibitor NS398. PAR-1 and PAR-2 stimulation rapidly activated both ERK1/2 and p38MAPK, and pharmacological blockade of MEK with either PD98059 or U0126 or of p38MAPK by SB203580 or SB202190 strongly inhibited thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha formation. Thrombin and peptide agonists of PAR-1 and PAR-2 increased luciferase activity in human umbilical vein endothelial cells infected with an NF-kappaB-dependent luciferase reporter adenovirus, and this, as well as PAR-induced 6-keto-PGF1alpha synthesis, was inhibited by co-infection with adenovirus encoding wild-type or mutated (Y42F) IkappaBalpha. Thrombin- and SLIGKV-induced COX-2 expression and 6-keto-PGF1alpha generation were markedly attenuated by the NF-kappaB inhibitor PG490 and partially inhibited by the proteasome pathway inhibitor MG-132. Activation of PAR-1 or PAR-2 promoted nuclear translocation and phosphorylation of p65-NF-kappaB, and thrombin-induced but not PAR-2-induced p65-NF-kappaB phosphorylation was reduced by inhibition of MEK or p38MAPK. Activation of PAR-4 by AYPGKF increased phosphorylation of ERK1/2 and p38MAPK without modifying NF-kappaB activation or COX-2 induction. Our data show that PAR-1 and PAR-2, but not PAR-4, are coupled with COX-2 expression and sustained endothelial production of vasculoprotective prostacyclin by mechanisms that depend on ERK1/2, p38MAPK, and IkappaBalpha-dependent NF-kappaB activation.

Antithrombin reduces endotoxin-induced hypotension by enhancing pulmonary sensory neuron activation in rats

We recently demonstrated that activation of the pulmonary sensory neurons plays a critical role in prevention of endotoxin-induced shock by releasing calcitonin gene-related peptide (CGRP) in rats. CGRP increased the endothelial production of prostacyclin (PGI(2)) in the lungs, thereby preventing endotoxin-induced shock response by inhibiting tumor necrosis factor-alpha (TNF-alpha) production. Since antithrombin (AT) enhances sensory neuron activation, we hypothesized that AT might reduce endotoxin-induced hypotension by enhancing the activation of pulmonary sensory neurons in rats. We examined this possibility using a rat model of endotoxin shock. AT-induced effects including reduction of hypotension (n = 5) and inhibition of induction of iNOS (n = 4 or 5) and TNF- alpha (n = 5) in the lungs of endotoxin-treated animals were completely reversed by pretreatment with capsazepine (CPZ) (n = 4 or 5), a vanilloid receptor antagonist, or CGRP(8-37), a CGRP receptor antagonist (n = 4 or 5). AT enhanced endotoxin-induced increases in lung tissue levels of CGRP (n = 4), but this effect of AT was not seen in animals pretreated with CPZ (n = 4). CGRP produced therapeutic effects (n = 5) similar to those induced by AT, and such therapeutic effects were completely abrogated by pretreatment with indomethacin (n = 4). AT increased CGRP release from cultured dorsal root ganglion neurons only in the presence of anandamide (n = 5), and AT-induced increase in CGRP release was not observed in the presence KT5720, an inhibitor of protein kinase A (n = 5). AT markedly increased intracellular levels of cAMP in the presence of anandamide (n = 5). These results strongly suggested that AT might reduce endotoxin-induced hypotension in rats by enhancing activation of sensory neurons via activation of protein kinase A.

EDHF Redux: EETs, TRPV4, and Ca 2+ Sparks

See related article, pages 1270–1279 Over the past two decades the complexity of the molecular processes underlying endothelial-dependent vascular relaxation has gradually become apparent. After the ground breaking discovery of the role of NO as a major endothelium-derived relaxant factor (EDRF), evidence emerged indicating the existence of a hyperpolarizing factor (endothelium-derived hyperpolarizing factor [EDHF]) with actions distinct from the cyclase-dependent relaxations associated with endothelial production of NO and prostacyclin.1–3 The specific molecules and vascular relaxation mechanisms underlying this intensely studied phenomenon have been difficult to establish, however. H2O2, K ions, and epoxygenase products have all been implicated as the EDHF, and evidence has suggested in turn that the mechanism by which EDHF relaxes arteries involves diffusion of the substance to smooth muscle and direct gating effects on calcium-activated K+ (maxiK) channels,4,5 actions mediated through endothelial small and intermediate conductance Ca2+-activated K+ (SK and IK) channels,6–8 and passive ionic effects associated with an extracellular accumulation of K+ ions.9 To further muddy these dark waters, recent evidence has implicated TRPV4 channels in EDHF signaling in several vascular preparations. In this issue of Circulation Research , Earley and colleagues10 identify a new putative mechanism and one that links EDHF to the regulation of Ca2+ release by ryanodine receptors (RYR), thereby forging a mechanistic link between EDHF and the well described local gating of large conductance Ca2+-activated K+ …