All cell membranes, including those of the pulmonary endothelium, are composed in large part of phospholipids structurally derived from glycerol. In addition to serving a barrier function, these compounds are metabolized by a family of phospholipases, which liberates precursors of biologically active compounds that act in both an endocrine and paracrine fashion to alter physiologic responses. These phospholipids are enriched in the sn-2 position with arachi-donic acid, a 20-carbon alkane with four double bonds. Metabolism of arachidonic acid proceeds by oxygen insertion catalyzed by several different enzyme families, including cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2), yielding an unstable intermediate prostaglandin (PG) H2. Further metabolism of this compound depends on the complement of available enzymes in a given cell type (e.g., prostacyclin synthetase in endothelial cells and thromboxane synthase in platelets), but can result in formation of a variety of prostaglandins, including PGD2, PGE2, PGF2a prostacyclin (PGI2), and TxA2. In the vascular bed of the lung, the predominant prostaglandins made by vascular endothelium are prostacyclin and PGE2. Smooth muscle cells in lung vessels are net producers of both thromboxane A2 and prostacyclin.

It is also possible for arachidonic acid to be metabolized by lipoxygenases (yielding leukotrienes and lipoxins) and specific cytochrome P450 enzymes [yielding epoxye-icosatrienoic acids (EETs), and stereospecific hydroxye-icosatetraenoic acids, (HETEs)]. Alternately, during situations of enhanced oxidant stress, reactive oxygen species can directly interact with arachidonic acid esterified to membrane phospholipids to form isoprostanes (IsoPs), compounds analogous to prostaglandins but with different stereochemistry (Figure 1). All of these molecules derived from arachidonic acid are known as eicosanoids.

In vitro studies document that specific enzymes critical for synthesis of unique prostaglandins and leukotrienes are restricted in their cell distribution. As such, the appearance of end products in biological samples allows inference about which cells have been activated in vivo. In the case of intact tissues and organs perfused with blood containing cells with different metabolic capacities, additional opportunities for mediator synthesis are created. For example, early in the pulmonary inflammatory process, upregulation of adhesion molecules facilitates neutrophil binding to pulmonary vascular endothelial cells. Neither cell in isolation is capable of metabolizing arachidonic acid into cysteinyl leukotri-enes (LTC4, LTD4, LTE4). However, an unstable epoxide intermediate, LTA4, can be formed by the actions of 5-lipoxygenase in the neutrophil and transferred to the endothelial cell because of intimate cellular apposition. The endothelial cell lacks 5-lipoxygenase but does contain abundant LTC4 synthase and is able to complete formation of a cysteinyl leukotriene [1]. Such transcellular biosynthesis with transfer of arachidonic can also occur between platelets and endothelium to facilitate formation of prostacyclin. This is part of the biochemical basis for the beneficial effects of antiplatelet agents, such as aspirin, in prevention of myo-cardial infarction and stroke. Such processes emphasize the dynamic and complex nature of pulmonary vascular eicosanoids biosynthesis and, with waxing and waning of cell apposition, allows some plasticity in the eicosanoid synthetic capability of the lung.

Figure 1 Enzymatic metabolism of membrane phospholipid, via arachidonic acid, to form eicosanoids. Specific enzymes are shown in italics (LOX = lipoxygenase; COX = cyclo-oxygenase; P450 = cytochrome P450; "Synthases" includes PGD synthase, PGE synthase, PGF2 isomerase, prostacyclin synthase, and thromboxane synthase. (see color insert)

Figure 1 Enzymatic metabolism of membrane phospholipid, via arachidonic acid, to form eicosanoids. Specific enzymes are shown in italics (LOX = lipoxygenase; COX = cyclo-oxygenase; P450 = cytochrome P450; "Synthases" includes PGD synthase, PGE synthase, PGF2 isomerase, prostacyclin synthase, and thromboxane synthase. (see color insert)

The pulmonary microvasculature serves not only as an important source of prostaglandins but also as a major site of metabolism of these molecules. The standard bisenoic prostaglandins (PGE2, PGD2, PGF2a) are rapidly metabolized (60% to 90%) on first passage through the lung. This proceeds via oxidation of the 15-hydoxyl function followed by reduction of the 13,14-double bond, resulting in the respective 15-keto-13,14-dihydro metabolites [2]. Presumably such rapid metabolism by the lung limits systemic effects of prostaglandins generated in the splanchnic circulation. However, during periods of oxidant stress, such as in prolonged hyperoxia, or when lung endothelial function has been compromised (e.g., sepsis with ARDS), transpul-monary clearance of PGE2 has been shown to be markedly reduced. Such loss of the normal, homeostatic clearance function of the pulmonary vascular bed may result in significant alteration in function of systemic organs during times of prostaglandin overproduction. Interestingly, metabolism of both prostacyclin and thromboxane A2 tends to occur via both spontaneous hydrolysis, which curtails the circulating half-life of both autocoids to a few minutes, and hepatic metabolism by beta oxidation pathways in the liver.

The biological effects of prostaglandins depends on binding to specific cell surface receptors. There are subtypes of many of the prostaglandin receptors, which may signal by entirely different mechanisms. For example, of the four different E-prostaglandin (EP) receptors, one signals by altering calcium flux, two increase cellular cyclic adenosine monophosphate (AMP) levels, and one decreases c-AMP [3]. Relative regional differences in density of such receptors on target tissues and effector cells modulates the effects of eicosanoids on vascular tone and microvascular fluid and solute exchange. In addition, the structural similarity that exists between the different eicosanoids can result in "receptor promiscuity"—high levels of a structurally similar eicosanoid ligand can activate a related receptor. In vivo and in vitro studies have documented such cross-talk between PGD2 and the thromboxane (TP) receptor and between the prostacyclin and the EP receptor.

As described previously, a wide array of eicosanoid metabolites, often with opposing actions and synthesized by multiple cell types, are produced in, and act on, the pulmonary microvasculature. The predominant endothelial cell product is prostacyclin, a key endogenous vasodilator that results in rapid elevation of intracellular c-AMP after binding to its receptor. This in turn activates protein kinase A and decreases intracellular calcium concentration, leading to relaxation of smooth muscle. Both vasoconstrictors (angiotensin II) and vasodilators (nitric oxide) have been shown to increase the synthesis of prostacyclin. In addition, increased shear force is a potent stimulus for prostacyclin synthesis by endothelium. Of note, in precapillary PAH, the ordinary pulsatile capillary flow in the lung is lost. This may contribute to the decrease in synthesis observed in many forms of pulmonary arterial hypertension (PAH).

Prostacyclin has additional properties that are important. These include inhibition of platelet aggregation and effects on muscle growth and cardiac contractility [4]. PGI2 is a well documented inhibitor of platelet aggregation, which is mediated, similar to its vasodilatory actions, through an increase in intracellular c-AMP. Importantly, prostacyclin does not inhibit initial platelet adhesion at the site of vessel wall injury and thus allows hemostasis without vessel occlusion. When used in pharmacological doses for the treatment of PAH (see following discussion), although bleeding time increases, clinical evidence of excessive bleeding in patients does not occur. PGI2 also modulates leukocyte adhesion to abnormal vascular surfaces, preventing local leukostasis and limiting inflammation. The intracellular increase in c-AMP resulting from engagement of PGI2 to its receptor has also been shown to inhibit DNA synthesis in vascular smooth muscle cells. Direct inotropic effects of prostacyclin have been demonstrated in animal studies evaluating isolated ventricular muscle preparations, and clinically important improvement in cardiac output following administration of epoprostenol, the synthetic salt of prostacyclin, has been reported in patients with pulmonary hypertension receiving chronic therapy.

Thromboxane A2 (TxA2) is the principle eicosanoid produced by platelets, but significant production in alveolar macrophages and monocytes occurs as well, especially when they are activated [5]. TxA2 has properties opposite to those of PGI2 in that it is a potent vasoconstrictor and promotes platelet aggregation and smooth muscle growth, the latter possibly through a protein kinase C-linked pathway. Numerous triggers of platelet activation, including acute lung injury, endotoxemia, shear force, and procoagulant alterations of the endothelial surface, lead to increased TxA2 synthesis.

It is well known that PGD2 causes bronchoconstriction. Its effects on the pulmonary vasculature are less well documented, but in sheep PGD2 causes vasoconstriction, likely through binding to the TxA2 receptor. Conversely, and similar to prostacyclin, PGD2 also inhibits DNA synthesis in vascular smooth muscle cells, but this is independent of an increase in c-AMP. In humans, whether PGD2 has a beneficial or deleterious effect on the development, maintenance, or regression of pulmonary arteriopathy is unknown. PGD2 also may tend to oppose platelet aggregation.

Leukotrienes, eicosanoid products resulting from the action of lipoxygenases on arachidonic acid, are potent mediators of inflammation with well-documented involvement in the pathogenesis of asthma. Recently, increased levels of 5-lipoxygenase and 5-lipoxygenase activating protein have been found in small and medium-sized pulmonary arteries by immunohistochemical staining of lung tissue from patients with pulmonary hypertension [6]. These results, in combination with the finding of increased leukotriene E4 in lavage fluid from newborn infants with persistent pulmonary hypertension, suggests a possible role for leukotrienes in pulmonary vasculopathies.

Isoprostanes, the result of nonenzymatic, free-radical catalyzed peroxidation of arachidonic acid, are excellent markers of in vivo oxidant stress. Evidence of lipid peroxidation has been found in numerous vascular disorders, including pulmonary vascular disease. Isoprostanes may also cause vasoconstriction and modulate platelet function, possibly by binding to receptors for TxA2. Furthermore, they can stimulate proliferation of endothelial cells and synthesis of endothelin-1, an exceptionally potent endogenous vasoconstrictor and smooth muscle mitogen. Previously believed to be exclusively formed by cyclo-oxygenases, a recent study indicates that as much as 30 percent of PGD2 is synthesized via the isoprostane pathway. This suggests that oxidant stress can increase prostaglandin production by two independent mechanisms: (1) facilitation of enzymatic catalysis by COX, and (2) direct peroxidation of arachidonate-containing phospholipids with phospholipase (or PAF acetylhydrolase) induced release of prostanoids.

A large body of information has examined the role of eicosanoids in endotemia and acute lung injury [7]. Increased levels of the major metabolite of thromboxane, a powerful vasoconstrictor and platelet aggregating cyclo-oxygenase product produced exuberantly by platelets, have been demonstrated in experimental models of endotoxemia. However, neither inhibition of thromboxane A2 synthesis nor blockade of the TP receptor abrogates the decrease in vascular barrier function (10#26,69,74). Furthermore, platelet depletion does not significantly modify the pulmonary vascular permeability response to endotoxin in chronically instrumented, nonanesthetized sheep. Both humans and large animals develop pulmonary hypertension following endotoxemia. Brigham and coworkers administered E. coli endotoxin to awake, chronically instrumented sheep and noted a two-phased response consisting of an early phase of acute pulmonary hypertension, characterized by increased flow of lung lymph that was relatively low in protein. This was followed by a longer phase of increased vascular permeability (i.e., high lymph/plasma protein ratio), during which mean pulmonary pressure remained only slightly elevated. Both analytical and pharmacologic studies using cyclo-oxygenase inhibitors suggest that thromboxane A2 mediates the early pulmonary vasopressor response but does not play a role in the later alteration in microvascular permeability.

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