Role of nitric oxide in the gastrointestinal tract

Worldwide osteoarthritis (OA) affects more than 9.6% of men and 18% of women older that 60 years. Treatment for OA often requires chronic use of selective or nonselective nonsteroidal anti-inflammatory drugs (NSAIDs), which have been associated with gastrointestinal and cardiovascular complications. An increased risk for upper gastrointestinal bleeding with NSAIDs alone and when combined with low-dose aspirin has been described in numerous studies. Although cyclo-oxygenase-2 inhibitors have been shown to carry a lower risk for gastrointestinal injury than nonselective NSAIDs, research continues to identify new treatments that not only are effective but also provide an improved benefit/risk profile, including better gastrointestinal tolerability. Nitric oxide (NO) is known to have a protective effect on the gastrointestinal tract. In preclinical studies NO was shown to help maintain gastric mucosal integrity, to inhibit leukocyte adherence to the endothelium, and to repair NSAID-induced damage. In addition, epidemiologic studies have shown that the use of NO-donating agents with NSAIDs or aspirin resulted in reduced risk for gastrointestinal bleeding. Recent studies have shown that cyclo-oxygenase inhibiting NO-donating drugs (CINODs), in which a NO molecule is chemically linked to an NSAID, are effective anti-inflammatory agents and may result in less gastrointestinal damage than is associated with NSAID use. Therefore, these agents provide a potential therapeutic option for patients with arthritis who require long-term NSAID therapy.     

The role of nitric oxide in cardiovascular diseases

Nitric oxide (NO) is a gaseous lipophilic free radical cellular messenger generated by three distinct isoforms of nitric oxide synthases (NOS), neuronal (nNOS), inducible (iNOS) and endothelial NOS (eNOS). NO plays an important role in the protection against the onset and progression of cardiovascular disease. Cardiovascular disease is associated with a number of different disorders including hypercholesterolaemia, hypertension and diabetes. The underlying pathology for most cardiovascular diseases is atherosclerosis, which is in turn associated with endothelial dysfunctional. The cardioprotective roles of NO include regulation of blood pressure and vascular tone, inhibition of platelet aggregation and leukocyte adhesion, and prevention smooth muscle cell proliferation. Reduced bioavailability of NO is thought to be one of the central factors common to cardiovascular disease, although it is unclear whether this is a cause of, or result of, endothelial dysfunction. Disturbances in NO bioavailability leads to a loss of the cardio protective actions and in some case may even increase disease progression. In this chapter the cellular and biochemical mechanisms leading to reduced NO bioavailability are discussed and evidence for the prevalence of these mechanisms in cardiovascular disease evaluated.     

Nitric oxide and atherosclerosis: an update.

Nitric oxide (NO) is a molecule that has gained recognition as a crucial modulator of vascular disease. NO has a number of intracellular effects that lead to vasorelaxation, endothelial regeneration, inhibition of leukocyte chemotaxis, and platelet adhesion. Endothelium damage induced by atherosclerosis leads to the reduction in bioactivity of endothelial NO synthase (eNOS) with subsequent impaired release of NO together with a local enhanced degradation of NO by increased generation of reactive oxygen species with subsequent cascade of oxidation-sensitive mechanisms in the arterial wall. Many commonly used vasculoprotective agents have their therapeutic actions through the production of NO. L-Arginine, the precursor of NO, has demonstrated beneficial effects in atherosclerosis and disturbed shear stress. Finally, eNOS gene polymorphism might be an additional risk factor that may contribute to predict cardiovascular events. However, further studies are needed to understand the possible clinical implications of these correlations.     

Sildenafil potentiates nitric oxide mediated inhibition of human platelet aggregation

Nitric oxide (NO) inhibits platelet aggregation primarily via a cyclic 3'5'-guanosine monophosphate (cGMP)-dependent process. Sildenafil is a phosphodiesterase type 5 (PDE5) inhibitor that potentiates NO action by reducing cGMP breakdown. We hypothesised that sildenafil would augment the inhibitory effects of NO on in vitro platelet aggregation. After incubation with sildenafil or the soluble guanylate cyclase inhibitor H-(1,2,4)oxadiazolo(4,3-a)quinoxallin-1-one (ODQ), collagen-mediated human platelet aggregation was assessed in the presence of two NO donors, the cGMP-dependent sodium nitroprusside (SNP) and the cGMP-independent diethylamine diazeniumdiolate (DEA/NO). SNP and DEA/NO caused a concentration-dependent inhibition of platelet aggregation. ODQ inhibited and sildenafil augmented the effect of SNP, and to a lesser extent the effect of DEA/NO. We conclude that sildenafil potentiates NO-mediated inhibition of platelet aggregation through blockade of cGMP metabolism and that PDE5 inhibitors may have important antiplatelet actions relevant to the prevention of cardiovascular disease.     

Effects of novel plant antioxidants on platelet superoxide production and aggregation in atherosclerosis.

Superoxide anion is produced in human platelets predominantly by Nox2-dependent NADPH oxidases. In vitro experiments have shown that it might play a role in modulating platelet functions. The relationship between platelet superoxide production and aggregation remains poorly defined. Accordingly, we aimed to study superoxide production and aggregation in platelets from subjects with significant cardiovascular risk factors (hypertension, hypercholesterolemia, smoking and diabetes mellitus) and from control individuals. Moreover, we studied the effects of novel polyphenol-rich extracts of Aronia melanocarpa (chokeberry) berries on platelet function in vitro. Superoxide production was significantly increased in patients with cardiovascular risk profile when compared to controls, while platelet aggregation in response to either collagen or thrombin were borderline higher, and did not reach statistical significance. Interestingly, no relationship was observed between platelet aggregation ex vivo and platelet superoxide production in either of studied groups. No correlation was found between endothelial function (measured by FMD) and platelet aggregation ex vivo either. Polyphenol-rich extracts of A. melanocarpa berries caused a significant concentration dependent decrease in superoxide production only in patients with cardiovascular risk factors, while no effect was observed in the control group. A. melanocarpa extracts abolished the difference in superoxide production between risk factor patients and controls. A. melanocarpa extracts exerted significant concentration dependent anti-aggregatory effects in both studied groups, which indicated that these effects may be independent of it's ability to modulate superoxide production. The anti-aggregatory effects of chokeberry extracts were similar irrespective of aggregation inducing agent (collagen or thrombin). Moreover, they appear to be independent of platelet NO release as NOS inhibition by L-NAME did not lead to their abrogation.     

Use of NSAIDs in treating patients with arthritis

Patients with rheumatic diseases, including rheumatoid arthritis and osteoarthritis, almost universally describe pain and stiffness as important contributors to reduced health-related quality of life. Of the treatment options available, NSAIDs are the most widely used agents for symptomatic treatment. NSAIDs are effective anti-inflammatory and analgesic drugs by virtue of their ability to inhibit biosynthesis of prostaglandins at the level of the cyclooxygenase enzyme. However, many of the adverse effects of NSAIDs are also related to inhibition of prostaglandin production, making their use problematic in some patient populations. For the clinician, understanding the biology of prostaglandin as it relates to gastrointestinal, renal, and cardiovascular physiology and the pharmacologic properties of specific NSAIDs is key to using these drugs safely. Of particular importance is the recognition of co-morbid conditions and concomitant drugs that may increase the risk of NSAIDs in particular patients. In patients with risk factors for NSAID toxicity, using the lowest dose of a drug with a short half-life only when it is needed is likely to be the safest treatment option. For those patients whose symptoms cannot be managed with intermittent treatment, using protective strategies is essential.     

Nitric oxide donors

Nitric oxide (NO) donors are pharmacologically active substances that release NO in vivo or in vitro. NO has a variety of functions such as the release of prostanoids, inhibition of platelet aggregation, effect on angiogenesis, and production of oxygen free radicals. This report discusses the chemical and pharmacological characteristics of NO donors, their effect on platelet function and cyclooxygenase, their cardiac action including myocardial infarction, and release of superoxide anions. This review stresses NO tolerance and the effect of NO donors on angiogenesis in myocardial infarction and in solid tumors.     

Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis

Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis.In the present investigation,we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis.The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver.Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis.Platelet aggregation was also measured in the presence of the eNOS inhibitor,diphenylene iodinium chloride (DIC).The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects.During the course of platelet aggregation,concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples,whereas the elevation was not significant in platelets of patients with cirrhosis.A parallel increase was observed in the levels of NO and NOS activity.In the presence of the eNOS inhibitor,platelet aggregation was enhanced and accompanied by an elevated calcium level.The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS.Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis.The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance,and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.     

Prostanoids in tumor angiogenesis: therapeutic intervention beyond COX-2

Prostanoids regulate angiogenesis in carcinoma and chronic inflammatory disease progression. Although prostanoid biosynthetic enzymes and signaling have been extensively analyzed in inflammation, little is known about how prostanoids mediate tumor-induced angiogenesis. Targeted cyclooxygenase (COX)-2 inhibition in tumor, stromal and endothelial cells is an attractive antiangiogenic strategy; however, the associated cardiovascular side effects have led to the development of a new generation of nonsteroidal anti-inflammatory drugs (NSAIDs) acting downstream of COX. These agents target terminal prostanoid synthases and prostanoid receptors, which may also include several peroxisome proliferator-activated receptors (PPARs). Here, we discuss the role of prostanoids as modulators of tumor angiogenesis and how prostanoid metabolism reflects complex cell-cell crosstalk that determines tumor growth. Finally, we discuss the potential of new NSAIDs for the treatment of angiogenesis-dependent tumor development.     

Endothelium-derived nitric oxide: actions and properties

Vascular smooth muscle relaxation in response to chemically diverse naturally occurring neurotransmitters and autacoids has been attributed to the formation and/or release of one or more vascular endothelium-derived relaxing factors (EDRFs) distinct from prostacyclin. The chemical, biochemical, and pharmacological properties of one such EDRF resemble closely the properties of nitric oxide (NO). Thus, both arterial and venous EDRFs as well as authentic NO cause heme-dependent activation of soluble guanylate cyclase, endothelium-independent vascular and nonvascular smooth muscle relaxation accompanied by tissue cyclic GMP formation, and inhibition of platelet aggregation and adhesion to endothelial cell surfaces. EDRF from artery, vein, and freshly harvested and cultured aortic endothelial cells was recently identified as NO or a labile nitroso species as assessed by chemical assay and bioassay. Endothelium-derived NO (EDNO) has an ultrashort half-life of 3-5 s due to spontaneous oxidation to nitrite and nitrate, both of which have only weak biological activity. EDNO can be synthesized from L-arginine and possibly other basic amino acids and polypeptides, perhaps by oxidative metabolic pathways that could involve polyunsaturated fatty acid-derived oxygen radicals. Inorganic nitrite could serve as both a stored precursor and an inactivation product of EDNO. EDNO and related EDRFs may serve physiological and/or pathophysiological roles in the regulation of local blood flow and platelet function.     

The dichotomous role of nitric oxide in the pathogenesis of accelerated atherosclerosis associated with systemic lupus erythematosus

Atherosclerosis is an inflammatory disorder, and the inflammation associated with systemic lupus erythematosus (SLE) accelerates the development of atherosclerosis. Nitric oxide (NO) is an important mediator of inflammation including the inflammation associated with atherosclerosis and SLE. Endothelial nitric oxide synthase (NOS3)-mediated constitutive expression of NO promotes endothelial integrity and normal vascular function. In contrast, inducible nitric oxide synthase- (NOS2) mediated expression of NO promotes endothelial dysfunction and atherogenesis. Statins appear to have anti-inflammatory properties and reverse many of the deleterious effects associated with NO metabolism in atherosclerosis. Statins augment NOS3 expression and inhibit the induction of NOS2. Therefore, the balance between normal vascular function and atherogenesis may be mediated by differences in the quantity, location, and timing of NO production within vessel walls.     

Prevention of alphaII(b)beta3 activation by non-steroidal antiinflammatory drugs

We have studied the effect of non-steroidal antiinflammatory drugs (NSAIDs) on alphaII(b)beta3 integrin activation and platelet aggregation. NSAIDs such as meloxicam, piroxicam, indomethacin and aspirin, but not aceclofenac or diclofenac interfered with the activation state of alphaII(b)beta3. NSAIDs that inhibited alphaII(b)beta3 activation were also able both to partially inhibit platelet primary aggregation and to accelerate platelet deaggregation. These effects of NSAIDs were not dependent on cyclooxygenase inhibition. The results obtained indicate that some NSAIDs exert a specific action on alphaII(b)beta3 activation, and provide an additional mechanism that accounts for their beneficial effects in diseases in which platelet activation is involved.     

Effect of Blood Nitrite and Nitrate Levels on Murine Platelet Function

Nitric oxide (NO) appears to play an important role in the regulation of thrombosis and hemostasis by inhibiting platelet function. The discovery of NO generation by reduction of nitrite (NO₂ ⁻) and nitrate (NO₃ ⁻) in mammals has led to increased attention to these anions with respect to potential beneficial effects in cardiovascular diseases. We have previously shown that nitrite anions at 0.1 µM inhibit aggregation and activation of human platelet preparations in vitro in the presence of red blood cells and this effect was enhanced by deoxygenation, an effect likely due to NO generation. In the present study, we hypothesized that nitrite and nitrate derived from the diet could also alter platelet function upon their conversion to NO in vivo. To manipulate the levels of nitrite and nitrate in mouse blood, we used antibiotics, NOS inhibitors, low nitrite/nitrate (NOx) diets, endothelial NOS knock-out mice and also supplementation with high levels of nitrite or nitrate in the drinking water. We found that all of these perturbations affected nitrite and nitrate levels but that the lowest whole blood values were obtained by dietary restriction. Platelet aggregation and ATP release were measured in whole blood and the results show an inverse correlation between nitrite/nitrate levels and platelet activity in aggregation and ATP release. Furthermore, we demonstrated that nitrite-supplemented group has a prolonged bleeding time compared with control or low NOx diet group. These results show that diet restriction contributes greatly to blood nitrite and nitrate levels and that platelet reactivity can be significantly affected by these manipulations. Our study suggests that endogenous levels of nitrite and nitrate may be used as a biomarker for predicting platelet function and that dietary manipulation may affect thrombotic processes.     

Gastric-sparing nitric oxide-releasable ‘true’ prodrugs of aspirin and naproxen

Nitric oxide-releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs) are gaining attention as potentially gastric-sparing NSAIDs. Herein, we report a novel class of ‘1-(nitrooxy)ethyl ester’ group-containing NSAIDS as efficient NO releasing ‘true’ prodrugs of aspirin and naproxen. While an aspirin prodrug exhibited comparable oral bioavailability and antiplatelet activity (i.e., TXB₂ inhibition) to those of aspirin, a naproxen prodrug exhibited better bioavailability than naproxen. These promising NO-NSAIDs protected experimental rats from gastric damage. We therefore believe that these promising NO-NSAIDs could represent a new class of potentially ‘Safe NSAIDs’ for the treatment of arthritic pain, inflammation and cardiovascular disorders in the case of NO-aspirin.     

Dehydroepiandrosterone stimulates nitric oxide release in vascular endothelial cells: evidence for a cell surface receptor

Dehydroepiandrosterone (DHEA) improves vascular function, but the mechanism of this effect is unclear. Since nitric oxide (NO) regulates vascular function, we hypothesized that DHEA affects the vasculature by increasing endothelial NO production. Physiological concentrations of DHEA stimulated NO release from intact bovine aortic endothelial cells (BAEC) within 5min. This effect was mediated by activation of endothelial nitric oxide synthase (eNOS) in BAEC and human umbilical vein endothelial cells (HUVEC). Dehydroepiandrosterone increased cyclic GMP (cGMP) levels in BAEC, consistent with its effect on NO production. Albumin-conjugated DHEA also stimulated NO release, suggesting that DHEA stimulates eNOS by a plasma membrane-initiated signal. Tamoxifen blocked estrogen-stimulated NO release from BAEC, but did not inhibit the DHEA effect. Pertussis toxin abolished the acute effect of DHEA on NO release. Dehydroepiandrosterone had no effect on intracellular calcium fluxes. However, inhibition of tyrosine kinases or the mitogen-activated protein (MAP) kinase kinase (MEK) blocked NO release and cGMP production in response to DHEA. These findings demonstrate that physiological concentrations of DHEA acutely increase NO release from intact vascular endothelial cells, by a plasma membrane-initiated mechanism. This action of DHEA is mediated by a steroid-specific, G-protein coupled receptor, which activates eNOS in both bovine and human cells. The release of NO is independent of intracellular calcium mobilization, but depends on tyrosine- and MAP kinases. This cellular mechanism may underlie some of the cardiovascular protective effects proposed for DHEA.     

Cyclooxygenase isoforms and atherosclerosis

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of arthritis and pain. However, their long-term use is limited by gastrointestinal (GI) side effects such as gastric ulcers. NSAIDs act by inhibiting an enzyme called cyclooxygenase. Cyclooxygenase (COX) catalyses the generation of prostaglandins from arachidonic acid. Two isoforms of the enzyme exist--COX-1 and COX-2--both of which are targets for NSAIDs. Although they are associated with GI toxicity, NSAIDs have important antithrombotic and anti-inflammatory effects. The GI injury has been attributed to COX-1 inhibition and the anti-inflammatory effects to COX-2 inhibition. As COX-2 is traditionally viewed as an inducible enzyme, selective inhibition of COX-2 by 'coxibs' (selective COX-2 inhibitors) has been employed to achieve anti-inflammatory and analgesic effects without GI side effects. However, recently there have been suggestions that chronic administration of coxibs might increase the risk of cardiovascular events, such as atherosclerosis, compared with traditional NSAIDs. In vascular disease, there is increased expression of both COX-1 and COX-2, resulting in enhanced prostaglandin generation. The specific role of COX-1 and COX-2 in vascular regulation is still unknown but such knowledge is essential for the effective use of coxibs. Although more evidence is pointing to selective COX-1 inhibition as a therapeutic measure in inflammatory atherosclerosis, there are some studies that suggest that inhibition of COX-2 might have a potential benefit on atherosclerosis.     

Effect of inhibition of nitric oxide synthase on the vasopressor response to ephedrine

Ephedrine is a mixed adrenergic agonist, stimulating both alpha- and beta-adrenergic receptors. The effects of ephedrine use include increases in heart rate, cardiac output, peripheral resistance, and blood pressure, and its use is associated with serious cardiovascular events such as stroke, arrhythmias, and myocardial infarction. The vascular endothelium plays a fundamental role in the regulation of vascular tone by releasing vasoactive factors such as nitric oxide (NO). The loss of NO bioactivity, often referred to as endothelial dysfunction, is characterized by the loss of endothelium-dependent vasodilation and is thought to be a common pathway for cardiovascular events such as vasospasm, hypertension, and myocardial infarction. Since endothelial dysfunction is characterized by loss of NO activity, and since ephedrine and endothelial dysfunction may be associated with similar cardiovascular events, the current study was undertaken to determine the effect of inhibition of NO production on responses to ephedrine in the rat. A sodium nitroprusside (SNP) infusion procedure was used to restore baseline vascular parameters to pre-L-NAME levels, allowing for direct comparison of agonist responses before and after NOS inhibition. The results demonstrate that the vascular response to ephedrine in the rat is modulated by NO and that NO production in response to ephedrine may be secondary to beta 2-receptor stimulation.     

Efficacy of topical non-steroidal anti-inflammatory drugs in the treatment of osteoarthritis: meta-analysis of randomised controlled trials

Objective To assess the efficacy of topical non-steroidal anti-inflammatory drugs (NSAIDs) in the treatment of osteoarthritis.Data sources Medline, Embase, Scientific Citation Index, CINAHL, Cochrane Library, and abstracts from conferences.Review methods Inclusion criterion was randomised controlled trials comparing topical NSAIDs with placebo or oral NSAIDs in osteoarthritis. Effect size was calculated for pain, function, and stiffness. Rate ratio was calculated for dichotomous data such as clinical response rate and adverse event rate. Number needed to treat to obtain the clinical response was estimated. Quality of trial was assessed, and sensitivity analyses were undertaken.Results Topical NSAIDs were superior to placebo in relieving pain due to osteoarthritis only in the first two weeks of treatment. Effect sizes for weeks 1 and 2 were 0.41 (95% confidence interval, 0.16 to 0.66) and 0.40 (0.15 to 0.65), respectively. No benefit was observed over placebo in weeks 3 and 4. A similar pattern was observed for function, stiffness, and clinical response rate ratio and number needed to treat. Topical NSAIDs were inferior to oral NSAIDs in the first week of treatment and associated with more local side effects such as rash, itch, or burning (rate ratio 5.29, 1.14 to 24.51).Conclusion Randomised controlled trials of short duration only (less than four weeks) have assessed the efficacy of topical NSAIDs in osteoarthritis. After two weeks there was no evidence of efficacy superior to placebo. No trial data support the long term use of topical NSAIDs in osteoarthritis.     

Antiplatelet,antioxidative, and anti-inflammatory effects of hydroquinone

Platelets play crucial roles in thrombosis and hemostasis through platelet activation and aggregation that are crucial in cardiovascular diseases. Hydroquinone (HQ) and its derivatives are present in many dermatological creams, paints, motor fuels, air, microorganisms, and plant products like wheat bread, fruit, coffee, and red wine. The effect of HQ on humans is not clear. In this study, we found that HQ (>25 μM) inhibited arachidonic acid (AA)-induced platelet aggregation. HQ suppressed AA-induced thromboxane B2 production of platelets. HQ (>10 μM) also attenuated ex vivo platelet-rich plasma aggregation. HQ prevented the interleukin (IL)-1β-induced 8-isoprostane, and PGE2 production, but not IL-8 production of pulp cells. These results indicate that HQ may have an antiplatelet effect via inhibition of thromboxane production. HQ has antioxidative and anti-inflammatory effects, and possible inhibition of COX. Exposure and consumption of HQ-containing products, food or drugs may have antiplatelet, antioxidative, and anti-inflammatory effects.     

Modulation of endothelial nitric oxide by plant-derived products

Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is recognised as a central anti-inflammatory and anti-atherogenic principle in the vasculature. Decreased availability of NO in the vasculature promotes the progression of cardiovascular diseases. Epidemiological and clinical studies have demonstrated that a growing list of natural products, as components of the daily diet or phytomedical preparations, may improve vascular function by enhancing NO bioavailability. In this article we first outline common pathways modulating endothelial NO production or bioavailability to provide a basis for subsequent mechanistic discussions. Then we comprehensively review natural products and plant extracts known to positively influence eNOS activity and/or endothelial function in vitro or in vivo.We will discuss red wine, highlighting polyphenols, oligomeric procyanidins (OPC) and resveratrol as modulators of endothelial NO production. Other dietary products and their active components known to activate eNOS include cocoa (OPC and its monomer (?)-epicatechin), pomegranates (polyphenols), black and green tea (flavanoids, especially epigallocatechin gallate), olive oil (oleic acid and polyphenols), soy (genistein), and quercetin, one of the most abundant flavonoids in plants. In addition, phytomedical preparations made from ginkgo, hawthorn and ginseng, as well as formulations used in traditional Chinese Medicine, have been shown to affect endothelial NO production. Recurring phytochemical patterns among active fractions and purified compounds are discussed.In summary, there is increasing evidence that several single natural products and plant extracts influence endothelial NO production. Identification of such compounds and characterisation of their cellular actions may increase our knowledge of the regulation of endothelial NO production and could provide valuable clues for the prevention or treatment of cardiovascular diseases.