Platelets. Platelet aggregation leads to activation of membrane phospholipases, with the release of AA and consequent eicosanoid biosynthesis. In human platelets, TxA2 and 12-HETE are the two major eicosanoids formed, although eicosanoids from other sources (e.g., PGI2 derived from vascular endothelium) also affect platelet function. A naturally occurring mutation in the first intracellular loop of the TP receptor is associated with a mild bleeding diathesis and resistance of platelet aggregability to TP agonists (Hirata et al., 1994). The importance of the TxA2 pathway is evident from the efficacy of low-dose aspirin in the secondary prevention of myocardial infarction and ischemic stroke. The total biosynthesis of TxA2, as determined by excretion of its urinary metabolites, is augmented in clinical syndromes of platelet activation, including unstable angina, myocardial infarction, and stroke (Smyth et al., 2009). Deletion of the TP receptor in the mouse prolongs bleeding time, renders platelets unresponsive to TP agonists, modifies their response to collagen but not to ADP, and blunts the response to vasopressors and the proliferative response to vascular injury.
PGI2 inhibits platelet aggregation and disaggregates preformed clumps. Deficiency of the IP receptor in disease-free mice does not alter platelet aggregation significantly ex vivo, although increased responsiveness to thrombin was evident in a mouse model of atherosclerosis (Smyth and FitzGerald, 2009). Augmented biosynthesis of PGI2 in syndromes of platelet activation serves to constrain the effects platelet agonists, vasoconstrictors, and stimuli to platelet activation. However, PGI2 does limit platelet activation by TxA2 in vivo, reducing the thrombotic response to vascular injury (Cheng et al., 2002). The increased incidence of myocardial infarction and stroke in patients receiving selective inhibitors of COX-2, most parsimoniously explained by inhibition of COX-2-dependent PGI2 formation, supports this concept (Grosser et al., 2006b).
Low concentrations of PGE2 activate the EP3 receptor, leading to platelet aggregation (Fabre, 2001). Deletion of the EP3 in mice leads to an increased bleeding tendency and decreased susceptibility to thromboembolism. Deletion of mPGES-1 did not affect thrombogenesis in vivo, probably due to substrate rediversion and augmented formation of PGI2 (Cheng et al., 2006b).
Vascular Tone. Because of their short t1/2, prostanoids do not circulate and generally are considered not to impact directly on systemic vascular tone. They may, however, modulate vascular tone locally at their sites of biosynthesis or through renal or other indirect effects. PGI2, the major arachidonate metabolite released from the vascular endothelium, is derived primarily from COX-2 in humans (Catella-Lawson et al., 1999; McAdam et al., 1999). PGI2 generation and release is regulated by shear stress and by both vasoconstrictor and vasodilator autacoids. Deletion of the IP in mice augments vascular proliferation, remodeling, atherogenesis, and hypertension, while PGI synthase polymorphisms have been associated with essential hypertension and myocardial infarction (Smyth and FitzGerald, 2009). PGI2 limits pulmonary hypertension induced by hypoxia and systemic hypertension induced by AngII and lowers pulmonary resistance in patients with pulmonary hypertension.
Deficiency of EP1 or EP4 receptors reduces resting blood pressure in male mice; EP1-receptor deficiency is associated with elevated renin–angiotensin activity. Both EP2 and EP4 receptor–deficient animals develop hypertension in response to a high-salt diet, reflecting the importance of PGE2 in maintenance of renal blood flow and salt excretion. PGI2 and PGE2 are implicated in the hypotension associated with septic shock. PGs also may play a role in the maintenance of placental blood flow. Although conflicting data exist, it appears that loss of mPGES-1 in mice is less likely than loss of COX-2 to alter blood pressure (Smyth et al., 2009).
COX-2-derived PGE2, via the EP4 receptor, maintains the ductus arteriosus patent until birth, when reduced PGE2 levels (a consequence of increased PGE2 metabolism) permit closure. (Coggins et al., 2002). The tNSAIDs induce closure of a patent ductus in neonates (see Chapter 34). Contrary to expectation, animals lacking the EP4 receptor die with a patent ductus during the perinatal period (Table 33–1) because the mechanism for control of the ductus in utero, and its remodeling at birth, is absent.
Endogenous biosynthesis of EETs is increased in human syndromes of hypertension. An analog of 11,12-EET abrogated the enhanced renal microvascular reactivity to AngII associated with hypertension (Imig et al., 2001), and blood pressure is lower in mice deficient in soluble EH (Sinal et al., 2000); these findings suggest that EH enzyme may be a potential pharmacological target for hypertension. Much indirect evidence suggests the existence of EET receptors, although none has been cloned.
Inflammatory Vascular Disease. Studies with knockout mice strongly implicate prostanoids in the development of atherogenesis and abdominal aortic aneurism; both inflammatory cardiovascular diseases (Smyth et al., 2009; Smyth and FitzGerald, 2009). Suppression of TxA2 biosynthesis, as well as antagonism or deletion of the TP, retards atherogenesis in mice. Deletion of the FP receptor reduces blood pressure and retards atherogenesis, coincident with reduced renin. Conversely, PGI2 appears atheroprotective and also limits vascular proliferative and remodeling responses. In humans, an arginine212 to cysteine substitution in the fifth intracellular loop of the IP, which disrupts IP signaling, co-segregated with increased cardiovascular risk in a recent study (Arehart et al., 2008), concordant with a role for this prostanoid in modifying human cardiovascular disease.
The role of PGE2 effects on inflammatory cardiovascular disease is less clear. Deletion of mPGES-1 does not accelerate the response to a thrombogenic stimulus in vivo in rodents (in contrast to either selective inhibition of COX-2 or deletion of the IP (Cheng et al., 2006b) but does retard atherogenesis in fat-fed hyperlipidemic mice (Wang et al., 2006). It is unclear, however, whether this results from loss of PGE2 or because of concomitant elevations in PGI2 biosynthesis. Deletion, or selective inhibition, of COX-2, but not inhibition of COX-1, decreases abdominal aortic aneurism formation in hyperlipidemic mice (King et al., 2006). Similar results were seen in mPGES-1-deficient mice (Wang et al., 2008), although, again, it is unclear to what extent rediversion to biosynthesis of other prostanoids (e.g., PGI2) contributes.
There is growing evidence for a role of the LTs in cardiovascular disease (Peters-Golden and Henderson, 2007). Although conflicting data have been reported in animal studies, human genetic studies have demonstrated a link between cardiovascular disease and polymorphisms in the LT biosynthetic enzymes and FLAP.
Lung. A complex mixture of autacoids is released when sensitized lung tissue is challenged by the appropriate antigen. COX-derived bronchodilator (PGE2) and bronchoconstrictor (e.g., PGF2α, TxA2, PGD2) substances are released. IP deletion in mice exaggerates features of acute and chronic experimental asthma, including increased bronchial hyperresponsiveness. Inhaled iloprost (a PGI2 analog) suppresses the cardinal features of asthma in mice via inhibition of airway dendritic cell function.
Polymorphisms in the genes for PGD2 synthase and the TP receptor have been associated with asthma in humans. Deletion of either DP1 or DP2 in mice suggests an important role of this prostanoid in asthma (and in other allergic responses), although contradictory findings in DP2-deficient mice suggest significant complexity in the function of PGD2 in airway inflammation (Pettipher et al., 2007).
The CysLTs probably dominate during allergic constriction of the airway (Drazen, 1999). Deficiency of 5-LOX leads to reduced influx of eosinophils in airways and attenuates bronchoconstriction. Furthermore, unlike COX inhibitors and histaminergic antagonists, CysLT-receptor antagonists and 5-LOX inhibitors are effective in the treatment of human asthma (see "Inhibitors of Eicosanoid Biosynthesis"). The relatively slow LT metabolism in lung contributes to the long-lasting bronchoconstriction that follows challenge with antigen and may be a factor in the high bronchial tone that is observed in asthmatic patients in periods between acute attacks (see Chapter 36).
Kidney. Long-term use of all COX inhibitors is limited by the development of hypertension, edema, and congestive heart failure in a significant number of patients. PGE2, along with PGI2, apparently derived from COX-2, plays a critical role in maintaining renal blood flow and salt excretion, whereas there is some evidence that the COX-1-derived vasoconstrictor TxA2 may play a counterbalancing role. Biosynthesis of PGE2 and PGI2 is increased by factors that reduce renal blood flow (e.g., stimulation of sympathetic nerves; AngII).
Bartter's syndrome is an autosomal recessive trait that is manifested as hypokalemic metabolic alkalosis. The syndrome results from inappropriate renal salt absorption caused primarily by dysfunctional mutations in the Na+–K+–2Cl– co-transporter NKCC2, a target of loop diuretics in the ascending thick limb of the loop of Henle (Simon et al., 1996) (see Chapter 25). The syndrome also can result from dysfunctional alterations in proteins whose activities can limit NKCC2 function: the K+ channel ROMK2 (Kir1.1) that recycles K+ into the tubular fluid; the basolateral membrane Cl– channel, ClC–Kb; and Barttin, the integral membrane protein that forms the α-subunit of the ClC–Kb heteromer (O'Shaughnessy and Karet, 2004). The antenatal variant of Bartter's syndrome, owing to dysfunctional ROMK2, also is known as hyperprostaglandin E syndrome. The elevated PGE2 may exacerbate the symptoms of salt and water loss. The relationship between dysfunctional ROMK2 and elevated PGE2 synthesis is not clear. However, in patients with antenatal Bartter's syndrome, inhibition of COX-2 ameliorates many of the clinical symptoms (Nüsing et al., 2001).
Inflammatory and Immune Responses. PGs and LTs are synthesized in response to a host of stimuli that elicit inflammatory and immune responses, and contribute significantly to inflammation and immunity (Tilley et al., 2001; Brink et al., 2003; Kim and Luster, 2007). Prostanoids generally promote acute inflammation, although there are some exceptions, such as the inhibitory actions of PGE2 on mast cell activation (see "Inflammation and Immunity"). Data from animals deficient in either COX-1 or COX-2 yield conflicting results depending on the inflammatory model used, perhaps reflecting the contribution of both isozymes to inflammation. Deletion of mPGES markedly reduced inflammation in several mouse models.
LTs are potent mediators of inflammation. Deletion of either 5-LOX or FLAP reduces inflammatory responses. Generation of BLT1-deficient mice confirms the role of LTB4 in chemotaxis, adhesion, and recruitment of leukocytes to inflamed tissues (Toda et al., 2002). Increased vascular permeability resulting from innate and adaptive immune challenges is offset in mice deficient in CysLT1 or LTC4 synthase (Kanaoka and Boyce, 2004) (Table 33–1). Deletion either of LTC4 synthase (and thus loss of CysLT biosynthesis) or CysLT2 reduced chronic pulmonary inflammation and fibrosis in response to bleomycin. In contrast, absence of CysLT1 led to an exaggerated response. These findings demonstrate a role for CysLT2 in promoting, and an unexpected role for CysLT1 in counteracting, chronic inflammation.
Heart. Studies suggest a role for COX-2 in cardiac function (Smyth et al., 2009). PGI2 and PGE2, acting on the IP or the EP3, respectively (Dowd et al., 2001; Shinmura et al., 2005), protect against oxidative injury in cardiac tissue. IP deletion augments myocardial ischemia/reperfusion injury and both mPGES-1 deletion (Degousee et al., 2008) and cardiomyocyte specific deletion of the EP4 (Qian et al., 2008) exacerbate the decline in cardiac function after experimental myocardial infarction. COX-2-derived TxA2 contributed to oxidant stress, isoprostane generation, and activation of the TP, and also possibly the FP, to increase cardiomyocyte apoptosis and fibrosis in a model of heart failure (Zhang et al., 2003). Selective deletion of COX-2 in cardiomyocytes results in mild heart failure and a predisposition to arrhythmogenesis.
Reproduction and Parturition. Studies with knockout mice confirm a role for PGs in reproduction and parturition (Smyth and FitzGerald, 2009). COX-1-derived PGF2α appears important for luteolysis, consistent with delayed parturition in mice deficient in COX-1. Subsequent upregulation of COX-2 generates prostanoids, including PGF2α and TxA2, that are important in the final stages of parturition. Mice lacking both COX-1 and oxytocin undergo normal parturition, demonstrating the critical interplay between PGF2α and oxytocin in onset of labor. EP2 receptor–deficient mice demonstrate a preimplantation defect (Table 33–1), which likely underlies some of the breeding difficulties seen in COX-2 knockouts.
Cancer. Pharmacological inhibition or genetic deletion of COX-2 restrains tumor formation in models of colon, breast, lung, and other cancers. Large human epidemiological studies report that the incidental use of NSAIDs is associated with significant reductions in relative risk for developing these and other cancers (Harris et al., 2005). PGE2 has been implicated as the primary pro-oncogenic prostanoid in multiple studies. The pro- and anti-oncogenic roles of other prostanoids remain under investigation, with TxA2 emerging as another likely COX-2-derived pro-carcinogenic mediator. Studies in mice lacking EP1, EP2, and EP4 reduce disease in multiple carcinogenesis models. EP3, in contrast, may even play a protective role in some cancers. Three randomized controlled trials of COX-2 inhibitors reported a significant reduction in the reoccurrence of adenomas in patients receiving either celecoxib or rofecoxib compared to placebo (Bertagnolli, 2007), while polymorphisms in COX-2 have been associated with an increased risk of colon and other cancers. In mouse mammary tissue, COX-2 is pro-oncogenic (Liu et al., 2001), whereas aspirin use is associated with a reduced risk of breast cancer in women, especially for hormone receptor–positive tumors (Terry et al., 2004). Despite the emphasis on COX-2, studies support a role for both COX enzymes in pro-oncogenic processes, and it remains untested whether selective COX-2 inhibitors will prove superior to nonselective NSAIDs for the prevention or treatment of human cancer. The CysLT and LTB4 receptors also are implicated in cancer, raising interest in the use of LT inhibitors/antagonists in chemoprevention/therapy.