Endothelial cell regulation of pulmonary vascular tone, inflammation, and coagulation.

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Endothelial Cell Regulation of Pulmonary Vascular Tone, Inflammation, and Coagulation Neil M. Goldenberg1,6 and Wolfgang M. Kuebler*1–5 ABSTRACT The pulmonary endothelium represents a heterogeneous cell monolayer covering the luminal surface of the entire lung vasculature. As such, this cell layer lies at a critical interface between the blood, airways, and lung parenchyma, and must act as a selective barrier between these diverse compartments. Lung endothelial cells are able to produce and secrete mediators, display surface receptor, and cellular adhesion molecules, and metabolize circulating hormones to influence vasomotor tone, both local and systemic inflammation, and coagulation functions. In this review, we will explore the role of the pulmonary endothelium in each of these systems, highlighting key regulatory functions of the pulmonary endothelial cell, as well as novel aspects of the pulmonary endothelium in contrast to the systemic cell type. The interactions between pulmonary endothelial cells and both leukocytes and platelets will be discussed in detail, and wherever possible, elements of endothelial control over physiological and pathophysiological processes will C 2015 American Physiological Society. Compr Physiol 5:531-559, 2015. be examined.

Introduction Vascular endothelial cells (ECs) form a continuous monolayer that covers the inner surface of the entire vascular system. The human lung contains an estimated number of 68 · 109 EC each covering an average surface area of 1300 μm2 , thus amounting to a total lung endothelial surface area of approximately 90 m2 (37). While this large surface was long regarded to primarily constitute a passive anatomical barrier that separates the circulating blood from the body’s interstitium and parenchyma, the recent decades have revealed the fundamental role of the endothelium in vessel homeostasis and the regulation of vascular responses to physiological and pathophysiological stimuli. By identification of an endothelial-derived relaxing factor, the pioneering work by Furchgott and Zawadzki (75) paved the way for a new understanding of how ECs may regulate vascular tone. Lung ECs are also critical determinants and active regulators of the exchange of fluid and solutes between the blood and the pulmonary interstitium. Likewise, ECs play key roles in preventing or permitting coagulation of the blood, adherence and aggregation of platelets, or adhesion and emigration of leukocytes or circulating progenitor cells. And last but not least, pulmonary ECs have metabolic functions, for example, in that they avidly and selectively clear the blood from circulating monoamines such as noradrenaline or serotonin (138). In this review, we aim to highlight the role of the pulmonary endothelium as a key regulator of vascular tone, inflammation, and coagulation in the pulmonary circulation. To this end, we will summarize the basic principles of these vascular responses, and discuss in detail their regulation by the endothelial generation, release, metabolism, or presentation

Volume 5, April 2015

of mediators, signaling or adhesion molecules which facilitate the communication of the endothelium with respective effector cells such as vascular smooth muscle cells, platelets, and leukocytes. Within this context, it is important to bear in mind that ECs do not constitute a uniform cell type, but are highly heterogeneous (4, 5, 174) and differ morphologically and functionally not only between different species and organs, but also between macro- and microvascular compartments and—in the case of the pulmonary microvasculature—even within individual vascular segments (123, 258). The molecular basis of this heterogeneity has been subject to various expert reviews in the past (5, 177, 215), and we will hence address this topic only in view of the regulation of lung vascular tone, inflammation, and coagulation. Likewise, we will address the role of interendothelial communication and signal propagation specifically in this context.

* Correspondence

to [email protected] Keenan Research Centre for Biomedical Science of St. Michael’s, Toronto, Ontario, Canada 2 German Heart Institute Berlin, Germany 3 Institute of Physiology, Charit´ e-Universit¨ atsmedizin Berlin, Germany 4 Department of Surgery, University of Toronto, Ontario, Canada 5 Department of Physiology, University of Toronto, Ontario, Canada 6 Department of Anesthesia, University of Toronto, Ontario, Canada Published online, April 2015 (comprehensivephysiology.com) DOI: 10.1002/cphy.c140024 C American Physiological Society. Copyright 1 The

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Endothelial Cell Regulation of Pulmonary Vascular Tone, Inflammation, and Coagulation

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Endothelial control of pulmonary vascular tone. An increase in intracellular Ca2+ , either from internal stores or the extracellular fluid, triggers a series of intracellular signaling events that culminate in the binding of Ca2+ to calmodulin, activation of myosin light chain kinase (MLCK), and the phosphorylation of myosin. This, in turn, causes cell contraction and vasoconstriction. The most important regulator of this process in nitric oxide (NO). NO activates soluble guanylate cyclase (sGC), which produces cGMP. cGMP, acting via protein kinase G (PKG), can then inhibit various membrane channels to block the vasoconstrictive response. Additionally, PKG can inhibit sarcoplasmic reticulum (SR) Ca2+ channels, and can release myosin light chain phosphatase (MLCP) from inhibition by ROK, leading to dephosphorylation of MLC and the cessation of SMC contraction.

Figure 1

Basic Principles of Lung Vascular Tone, Inflammation and Coagulation Vascular tone Vascular tone refers to the degree of constriction of an individual blood vessel relative to its maximally dilated state. In adults, muscularized blood vessels of the systemic circulation generate considerable tone under physiological conditions, whereas basal vascular tone in the pulmonary circulation is remarkably low and the pulmonary circulation is almost completely dilated. Accordingly, inhaled or intravenous vasodilators have very little or no effect on the pulmonary vascular tone in healthy individuals (26, 73, 190, 257). The low basal tone of pulmonary blood vessels appears to be independent of endogenously formed vasodilators such as nitric oxide (NO), since NO synthase (NOS) inhibitors do not increase lung vascular tone in most species (perhaps with the exception of sheep and pigs) as elegantly reviewed by Hampl and Herget (90). However, a study in healthy humans demonstrated that pharmacological inhibition of NOS resulted in decreased pulmonary vascular resistance (212). Notably, the situation is quite different in the fetus, in which lung vascular tone is as high as in the systemic circulation resulting in the

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physiological shunting of the pulmonary circulation via the ductus arteriosus (Fig. 1). Vascular tone is the result of smooth muscle cell contraction brought about by the phosphorylation of the regulatory myosin light chain, thus facilitating the power stroke cycle. In a simplistic, yet however, expedient manner for our purpose myosin light chain phosphorylation can be viewed as the result of two regulatory pathways, the activation of myosin light chain kinase (MLCK) by Ca2+ and calmodulin, or the inhibition of myosin light chain phosphatase (MLCP) by activation of the small GTPase RhoA and the Rho-associated kinase ROCK (108). Accordingly, endothelial-derived signals may regulate vascular tone by one of the following mechanisms (Fig. 1): First, by altering the intracellular Ca2+ concentration by modulating (i) extracellular Ca2+ influx via plasmalemmal Ca2+ channels, (ii) Ca2+ release from endosomal stores, or (iii) Ca2+ removal from the cytosol. Second, by Ca2+ desensitization of MLCK, for example, by MLCK phosphorylation (217), and third, by regulation of MLCP activity typically by ROCK or protein kinase C (PKC)-dependent phosphorylation of both PKC-potentiated inhibitory protein for PP1 of 17 kDa (CPI-17) (54, 201) and the regulatory subunit myosin phosphatase target (MYPT1) (59). The regulation of these


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Pulmonary arteriole Alveolar capillary

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Figure 2 The pulmonary endothelial cell in inflammation. As highlighted in the text, the interaction between the pulmonary endothelium and circulating leukocytes differs greatly from that in the systemic circulation. Important physical factors exist which lead to leukocyte retention in the pulmonary capillary network. To traverse the lung vasculature, neutrophils must undergo cytoskeletal rearrangement so that they can fit through the small pulmonary vessels, which also have far lower flow rates than the systemic circulation. Because of this, the role of leukocyte rolling is diminished in the lung, and both CD18-dependent and CD18-independnet pathways exist for leukocyte extravasation.

different pathways and thus, vascular tone, by the pulmonary endothelium will be discussed individually for the various endothelial-derived vasoactive mediators in Section 3 of this review.

Inflammation Local inflammation relies upon the targeted recruitment of effector cells to sites of tissue injury or infection. To do so, inflammatory cells, such as leukocytes, must traverse the endothelial barrier and enter inflamed tissues. This process relies on both the ECs’ ability to signal nearby tissue damage, and their capacity to allow for the regulated passage of cells across the endothelial monolayer. This highly orchestrated process is coordinated by signaling events inside the EC, and by intercellular interaction between the EC and the inflammatory cells. Critical to this process is the regulated expression of surface adhesion molecules by the EC. Such adhesion proteins will be examined in detail in Section 4 of this review, and the mediators produced by the EC that regulate this process are to be described in Section 3. We will also examine the key differences between these processes in the systemic and pulmonary circulations. The unique physiology and geometry of the lung vasculature means that processes that are seen in the systemic circulation, such as leukocyte rolling, are not observed in the lung due to the small size of the pulmonary microvessels (51). Similarly, neutrophils spend


a disproportionately large amount of time in the pulmonary circulation, since the small caliber of pulmonary capillaries means that neutrophils must undergo a series of cytoskeletal rearrangements to negotiate the lung vasculature. The coordinated regulation of EC interactions with leukocytes, platelets, and circulating inflammatory mediators will be addressed in detail in the body of this review (Fig. 2).

Coagulation The pulmonary endothelium is an important regulator of clotting function, and the role of the EC in coagulation is intimately related to both inflammation and endothelial barrier function. In the resting state, the endothelial monolayer must provide an antithrombotic lining to the pulmonary vasculature so that inappropriate clotting does not occur. However, activation of the endothelium or tissue damage must be capable of activating both the coagulation cascade and platelet aggregation in a timely and spatially limited manner. Of fundamental importance in this system is EC-derived NO, which is a potent inhibitor of both platelet aggregation and adhesion to the EC monolayer (181). NO causes a decrease in platelet cytosolic Ca2+ , which influences platelet glycoprotein activation, and therefore, platelet adhesion (63). NO can also inhibit fibrin deposition, further attenuating thrombus formation (137). In contrast, EC-generated ROS acts to promote platelet activation and aggregation (42). Additionally, prostanoids produced

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The role of the pulmonary endothelium in platelet function. At rest, the pulmonary EC represents an antithrombogenic surface, repelling interactions with both platelets and clotting factors. This antiplatelet phenotype is largely due to constitutive secretion of PGI2 and NO by the resting lung EC. After endothelial injury, or inflammatory states of platelet and EC activation, the EC will secrete proplatelet aggregation factors, such as thromboxane A2 . Activated platelets have increased availability of GPIIb/IIIa, which binds to elevated surface levels of ICAM-1 and integrins. Furthermore, endothelial-derived von Willebrand Factor (VWF) is released, and assists in cross-linking platelets to form a mature platelet plug.

by the pulmonary endothelium have profound effects on platelet function. The prototypical antiplatelet prostanoid, prostacyclin, as well as the proplatelet mediator, thromboxane A2 , are secreted by activated ECs at sites of inflammation and tissue damage (154, 222). Section 3 of this review will enumerate the large number of mediators produced by the pulmonary EC, and will detail their roles in the regulation of platelets and the coagulation cascade (Fig. 3).

Endothelial-Generated Mediators Nitric oxide NO represents the most thoroughly studied mediator produced by the pulmonary EC, and as such, has been assigned a wide array of regulatory roles in the homeostasis and pathophysiology of vasomotor function, coagulation, and inflammation. NO has been termed a gasotransmitter, along with hydrogen sulfide and carbon monoxide (159). Since NO freely diffuses across cellular membranes, and has a half life of 2 to 3 s, it is well-suited for finely tuned, temporally and spatially confined paracrine signaling between its source in the EC and its target in the SMC (85). While freely diffusible, NO can be scavenged by ROS and hemoglobin, both of which have a large effect on NO bioavailability (85). NO is constitutively produced by the pulmonary endothelium, and while NO exerts a wide range of effects on the pulmonary vasculature, one of its most important roles is the maintenance of the low resistance of the pulmonary vascular bed (212). This effect, however, is somewhat controversial, with conflicting data arising from different groups. Indeed, loss of NO production through either pharmacological inhibition of eNOS in healthy humans (212),

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or via targeted disruption of the eNOS3 gene in mice, resulted in elevated baseline PAP and enhanced hypoxic pulmonary vasoconstriction (HPV) (212, 213). However, as reviewed in (90), there are extensive data demonstrating that acute inhibition of NO production in several animal models has no effect on basal pulmonary vascular tone. It is, therefore, possible that NO exerts different effects in different model systems. The effects of NO on pulmonary artery smooth muscle cell (PASMC) contraction are myriad, but many stem from the activation of soluble guanylate cyclase (sGC) (157). Activation of sGC leads to the production cGMP, which then activates protein kinase G (PKG). PKG activity lowers PASMC cytosolic Ca2+ through a variety of mechanisms, including the inhibition of the IP3 receptor (196), and activation of SERCA pumps (33,157). Additionally, NO-dependent PKG activation leads to the activation of Ca2+ -dependent K+ channels (BKCa channels) (58) to hyperpolarize PASMC and inhibit their contraction via inhibition of voltage-operated Ca2+ channels (VOCC). VOCC are also inactivated by cGMP, which provides another potential mechanism for NO-mediated pulmonary vasorelaxation. Notably, work done by our group has demonstrated that cGMP can inhibit the calcium channels TRPC6 (194) and TRPV4 (256) (to be reviewed below); which again mediate vasoconstrictor responses to hypoxia (248) or humoral stimuli (252). The primary site of pulmonary NO production is in the macrovascular EC (214). NO is produced via the oxygenation of a guanido nitrogen from L-arginine, creating a molecule of L-citrulline as a byproduct (90,169). This reaction is catalyzed by NOS, which exists in three isoforms; neuronal (nNOS), inducible (iNOS), and endothelial (eNOS), all of which are present in pulmonary ECs (55). However, the contribution of


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Figure 4 Regulation of nitric oxide production in the pulmonary endothelium. eNOS forms NO in the lung EC from Larginine, in the presence of the cofactor BH4 , as a byproduct of L-citrulline production. eNOS activity is enhanced by shear stress, intracellular Ca2+ , and signaling by cytokines such as TGF-β. Calcium enhances eNOS activity via direct binding of Ca2+ /calmodulin (CaM) to eNOS, while phosphorylation of eNOS at Ser1177 by PI3-Kinase and Akt also increases NO production. NO produced in this manner can freely diffuse into the PASMC, where it activates soluble guanylate cyclase to produce cGMP. cGMP then, via PKG function, produces PASMC relaxation. In the EC itself, NO inhibits neutrophil and platelet binding. On the other hand, reactive oxygen species (ROS), and conditions favoring uncoupling (including loss of BH4 availability and inflammatory states) decrease eNOS activity and NO production. Binding of eNOS to caveolin-1 in caveolae downregulates is activity, as does tyrosine phosphorylation by PKC. Under such conditions, in addition to diminished NO production, uncoupled eNOS will produce superoxide, which then leads to further uncoupling in a positive feedback loop.

iNOS and nNOS to overall NO production by the pulmonary endothelium is negligible under basal conditions (55). eNOS requires multiple cofactors, including NADPH and BH4 for normal function (70). In various pathological states, including the presence of certain cardiovascular risk factors, BH4 is depleted, leading to eNOS-mediated production of O2 − , via a process termed eNOS uncoupling (70). This establishes a positive feedback loop, whereby eNOS produces O2 − , which then further inhibits NO production and/or scavenges residually formed NO yielding peroxynitrite. Recently, this feedback loop has emerged as a central pathogenetic mechanism in a variety of cardiovascular diseases that affect both the pulmonary and systemic circulation (126). eNOS activity is modulated by a wide variety of signals at the transcriptional, translational, and posttranslational levels (Fig. 4). For example, expression of the eNOS transcript is


enhanced by TGF-β (131), estrogen (139), and shear stress (40). Of the multitude of important protein-protein interactions that influence eNOS function, two important activators of eNOS are calcium-calmodulin (90) and protein phosphorylation at serine 1177 (85). Conversely, translocation of eNOS to membrane microdomains containing caveolin-1, as well as other phosphorylation events, downregulate enzyme activity (60). Regulation of eNOS localization has profound effects on NO generation; mice deficient in caveolin-1, and therefore, deficient in caveolae, have constitutively increased NO production (265). Surprisingly, these animals develop pulmonary hypertension in spite of elevated NO levels. The mechanism of this effect is thought to be direct nitration of PKG leading to its inactivity (265). These data serve to illustrate the multiplicity of functions of eNOS in the pulmonary circulation.

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NO activity also has profound effects upon inflammation, specifically influencing the interaction of leukocytes with the activated endothelium, as well as mediating changes in vascular tone and barrier function during the inflammatory response. In a complex fashion, NO can exert both proand anti-inflammatory effects. Acute inflammation leads to increased EC cytosolic Ca2+ , which activates eNOS. NO produced in venular EC promotes the generation of tissue edema (24) [for a review of the mechanism of edema formation as a result of increased NO see (119)]. Proteins present in the edema fluid subsequently provide a scaffold for incoming neutrophils (43). Furthermore, the inflammatory milieu results in an environment that favors uncoupling: This is commonly due to the production of peroxynitrite, which oxidizes BH4 , resulting in eNOS-uncoupling and further generation of ROS and peroxynitrite (111, 253), which in turn amplifies inflammatory stress. In addition, inflammation can result in eNOS downregulation, as well as its functional inhibition, as evidenced by the loss of eNOS function during stimulation with platelet activating factor (116, 255). Conversely, NO dampens the inflammatory response by decreasing the expression of the adhesion molecules Intercellular adhesion molecule (ICAM) and Vascular cell adhesion molecule (VCAM), and by blocking the expression of tissue factor (116). The ability of NO to regulate the expression of adhesion molecules also plays a key role in the NO-mediated inhibition of coagulation and thrombus formation. It has long been appreciated that NO is a potent inhibitor of platelet aggregation and adhesion to ECs (181) by increasing intracellular cGMP, resulting in a subsequent decrease in intracellular Ca2+ (63). This suppression of intracellular Ca2+ influx inhibits a conformational change in the platelet glycoprotein IIb/IIIa, essentially decreasing the number of fibrinogen binding sites on the platelet surface, thereby limiting platelet cross-linking and clot strength (41, 154). Additionally, NO inhibits fibrin deposition, which is another level at which it can block thrombosis and formation of an atheroma (137). It is noteworthy to consider controversies in the literature regarding NO production, signaling, and the contribution of various segments of the pulmonary vasculature to NO signaling, as such seeming discrepancies may highlight important differences between EC subsets, study designs, or analytical methods, and provide an important focus for future investigation. One such area of exploration relates to the overall notion of endothelial heterogeneity. It is clear that EC from different organs possess fundamental phenotypic differences; additionally, EC in different vessel types (i.e. veins, capillaries, and arteries) within a given organ also demonstrate unique features (53). Important differences exist between different EC populations (such as micro- vs. macrovascular EC) with respect to barrier function, expression of surface molecules, and response to agonists [reviewed in (214)]. It is thus little surprising that relevant differences are also found with respect to NO signaling. First, pulmonary arteries, arterioles, and veins respond differentially to NO, and produce various quantities of NO. The precise contribution of pulmonary

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veins to overall pulmonary resistance remains controversial, ranging in estimates from 7% to 49%, likely due to differences in measuring techniques and model species [reviewed in (78)]. Additionally, age of the animal studied is a critical variable, with veins seeming to contribute less and less from the neonatal period through to adulthood (78). However, NO does play a critical role in the pulmonary veins, and potentially more so than in pulmonary arteries. In porcine models, pulmonary veins have been shown to both produce more NO than arteries, and to respond more robustly to NO than arteries (13). Additionally, it has long been recognized that NO is a more potent relaxer of bovine intrapulmonary veins than arteries (53). In pigs and lambs, eNOS expression and activity are highest in venous segments, rather than in arteries, highlighting the importance of pulmonary veins for the role of NO in the regulation of pulmonary vascular tone (17, 229). Furthermore, phosphodiesterase levels are highest in the arteries, which serves to limit the downstream effects of bioactive NO in these vessels (17). Finally, NO production by pulmonary macrovascular EC is known to be greater than in microvascular (i.e., capillary) EC (79). This finding fits the anatomical structure of the lung, since the smooth muscle cells, which are the primary effectors of NO, are only present in the microvasculature. Clearly NO is a fundamental regulator of lung physiology, and future work will uncover further details regarding this important molecule. It is clear that NO is indeed a master regulator of the pulmonary circulation. Through complex mechanisms, NO is capable of influencing vascular tone, clotting function, endothelial activation, and inflammation. The central role of the pulmonary EC in vascular homeostasis is due largely to its key function as a source and regulator of NO production. How lung EC-derived NO interacts with downstream effectors in various contexts remains a fertile research field.

Eicosanoids Eicosanoids are the 20 carbon-containing fatty acid derivatives of arachidonic acid (AA) metabolism. AA is produced via the action of phospholipases on phospholipids or diacylglycerol. In the pulmonary endothelium, cPLA2 is the predominant isoform. AA can then be further metabolized by one of three main pathways: the action of cyclo-oxygenase (COX) enzymes produces prostaglandins and thromboxanes, the action of lipoxygenases produces leukotrienes, and metabolism by cytochrome P450 enzymes produces epoxyeicosatrienoic acids (EET). The end result of these pathways is the generation of a variety of bioactive lipid species, the most important of which from the standpoint of pulmonary endothelial function will be discussed below.

PGI2 /prostacyclin One of the most important products of COX activity is prostacyclin, also known as PGI2 (Fig. 5). Prostacyclin is a potent pulmonary vasodilator, acting via the IP1 receptor to activate adenylyl cyclase, producing cyclic AMP (cAMP) and


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Arachidonic acid metabolism. The enzyme cytosolic phospholipase A2 (cPLA2 ), which is the dominant isoform in the pulmonary EC, liberates the 20 carbon fatty acid, arachidonic acid (AA) from plasma membrane phospholipids. Among several possible fates for AA, it can be metabolized by lipooxygenases (LOX) to produce Hydroperoxyeicosatetraenoic acid (HPETE), which can be further metabolized into leukotrienes. The activity of cyclooxygenase enzymes (COX) on AA produces prostaglandin H2 (PGH2 ) which can then be further processed into one of PGI2 , PGE2 , or thromboxane A2 (TxA2 ).

mediating PASMC relaxation (30). Prostacyclin synthase is downregulated in patients with primary pulmonary hypertension, resulting in diminished plasma levels of PGI2 (228). As such, synthetic prostacyclin has become a mainstay of treatment for severe pulmonary hypertension (179). Interestingly, prostacyclin levels increase dramatically during hypoxia (27). Prostacyclin plays an important role in HPV, with exogenous prostacyclin causing a decrease in basal PAP as well as abolishing the increase in PAP seen during hypoxic ventilation in rats (171). Taken together, these data suggest that prostacyclin plays a critical regulatory role in HPV, being activated to counterbalance the vasoconstrictive response to hypoxia. As such, prostacyclin modulation may represent an important means by which HPV can be modulated clinically. Prostacyclin is a potent inhibitor of platelet aggregation, and has actually been shown to be able to revert activated platelets to their basal state (155). Binding of prostacyclin to its receptor on platelets inhibits Ca2+ entry and degranulation through an increase in intracellular cAMP (9). In animal models of thrombosis, the lack of the platelet receptor for


prostacyclin led to an increase in the rate of thrombosis after experimental vascular injury, suggesting an important regulatory role for prostacyclin in the coagulation cascade (94). Prostacyclin is also a mediator of the acute antiinflammatory response. Initially identified as the basis for the anti-inflammatory effects of aspirin (235), further studies revealed the importance of prostacyclin in inhibiting the development of vascular permeability and edema formation in response to an acute inflammatory insult (94, 269). Specific mechanisms have been determined for these antiinflammatory effects, including the ability of prostacyclin to inhibit dendritic cell function (267). The role of prostacyclin, however, is complicated, since it has also been shown to be beneficial in preventing allergic responses (221). In a similarly complex fashion, prostacyclin appears to have beneficial, anti-inflammatory functions in preventing atherosclerosis, yet has a deleterious, proinflammatory role in rheumatoid arthritis [reviewed in (216)]. In summary, the potent pulmonary vasodilatory effect of prostacyclin, coupled with its relative paucity in patients

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with pulmonary hypertension, has made prostacyclin analogues into one of the most important recent advancements in the treatment of patients with pulmonary vascular disease. Its additional benefits in maintaining lung endothelial barrier function and controlling pulmonary arterial hypertension in response to hypoxia suggest that more therapeutics will emerge from future manipulation of prostacyclin function.

Thromboxane A2 In opposition to prostacyclin, thromboxane A2 (TxA2 ), produced by the activity of COX, is a potent pulmonary and systemic vasoconstrictor (225, 260). Acting through a G protein-coupled receptor, TxA2 stimulation results in Ca2+ influx and phospholipase C activation, leading to downstream stimulation of PKC (199). The end result in the SMC is contraction, as well as mitogenesis in situations of prolonged stimulation. TxA2 release from the EC as well as from platelets is involved in the pathogenesis of myocardial infarction, cerebral vasospasm, and both systemic and pulmonary hypertension [reviewed in (199)]. TxA2 is a powerful mediator of platelet aggregation, and is involved in thrombus formation in a host of disease states (41,128,222). In addition to mediating aggregation, TxA2 also stimulates platelet activation. An addition to aspirin, which is a TxA2 inhibitor, several newer drugs blocking TxA2 synthesis or signaling have been tested in a variety of pulmonary diseases, with varying degrees of success (6, 41). It should be noted, however, that the primary source of TxA2 in many disease states is the platelet itself, not the EC. In addition to its roles as a vasoconstrictor and platelet activator, TxA2 is also a proinflammatory molecule (219). Interactions between platelets and neutrophils in the presence of TxA2 result in increased EC expression of cellular adhesion molecules, leading to increased platelet-EC interaction and vascular tissue damage (118). Specifically, during murine acid-induced acute lung injury, blockade of TxA2 receptors resulted in improved oxygenation, reduced interstitial and alveolar macrophage accumulation, and near complete attenuation of lung edema accumulation (261). Furthermore, TxA2 can then bind EC thromboxane receptors, leading to increased endothelial ICAM-1 expression and, therefore, increased neutrophil-EC binding (118). These data and others demonstrate that TxA2 is a key regulator of both immune function and EC barrier properties in the lung.

Prostaglandin E2 The role of prostaglandin E2 (PGE2 ) in the vasomotor control of the pulmonary circulation remains controversial. Several studies have shown that PGE2 is a vasodilatory molecule (82,147). Others, however, suggest that PGE2 has differential effects on different vessels, even within the same organ (234). In the lung, PGE2 constricts rat intrapulmonary arteries (114, 254). In contrast, other data have shown that PGE2 is a relaxant for pulmonary veins (69, 239). Clearly much remains to be

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studied before we fully understand the vasomotor response to PGE2 stimulation, but it would appear that the wide variety of responses of different vessels in different organ preparations to PGE2 relates to the multiplicity of PGE2 receptor subtypes (94). PGE2 binds to the EP receptor subtypes, named EP14, all four of which are G protein-coupled receptors (94). EP receptor distribution is variable between species, but both all are present in the lung. Functionally, EP3 is most highly expressed in smooth muscle, and mediates contraction, while EP2 mediates smooth muscle relaxation (34) via Gs -mediated increases in cAMP (94). Finally, EP4 activation has been shown to be barrier protective in the lung, and can diminish neutrophil transmigration (112). The influence of PGE2 on the immune system is similarly controversial. PGE2 is often termed immuno-inhibitory, in that it, for example, inhibits TNF-α and IL-10 production in macrophages and other antigen presenting cells, and promotes T cell differentiation along the TH 1 lineage (91, 270). Furthermore, PGE2 stimulation suppresses T cell division (160). However, PGE2 has also been associated with proinflammatory effects mediated by the EP4 receptor, notably the ability to induce IL-1β and IL-6 in conjunction with LPS in macrophages (167). Presumably, again due to the presence of multiple receptor isoforms for PGE2 , often even within the same cell type, PGE2 is able to exert both stimulatory and inhibitory effects on the immune system [reviewed in (94)].

EETs and HETEs Metabolism of AA in the EC can also occur via cytochrome P450 (CYP) enzymes. Of significance in the pulmonary vasculature are the CYP2C and CYP2J families, which have epoxygenase activity (187). They produce the four biologically active species of EETs termed 5,6-, 8,9-, 11,12-, and 14,15-EET (Fig. 6). Additionally, the CYP4A family of ωhydrolases produces hydroxyeicosatetraenoic acids (19- and 20-HETE) (187). All of the CYP enzymes required for EET and HETE biosynthesis are expressed in the pulmonary EC (101, 262). EETs in the systemic circulation have anti-inflammatory and vasodilatory properties, and have been considered as an endothelium-derived hyperpolarizing factor (146). The halflives of EETs are short, owing to their rapid metabolism by the soluble epoxide hydrolase (sEH) (187). Experiments in mice have suggested a critical role for EETs in HPV; sEH inhibition dramatically increased the HPV response, and overexpression of CYP2C9 increased PAP and right ventricular systolic pressure (RVSP) both at baseline and after hypoxia (176). Further, exogenous 11,12-EET increased both baseline PAP and HPV in isolated perfused mouse lungs (106). Hypoxia also induced the expression of endogenous CYP2C9 in mice, and isolated pulmonary microspheres stimulated production of all four bioactive EETs in response to hypoxia (176). Taken together, these data suggest that EETs promote vasoconstriction in the lung—and, therefore, potentially promote pulmonary hypertension—while promoting vasodilation in the systemic


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Pulmonary endothelial synthesis of vasoactive EET and HETE species. Further pathways of AA metabolism in the pulmonary endothelial cell can produce EET and HETE. The action of the cytochrome P450 epoxygenases 2C and 2J can produce a family of EET species, with various vasomotor and inflammatory functions, detailed in the text. The main catabolizer of EET species is the soluble epoxide hydrolase (sEH), which breaks EET species down into dihydroxyeicosatrienoic acids (DHET). Alternatively, ω or ω-1 hydrolases can metabolize AA into 20- or 19-HETE.

circulation. Mechanistically, much remains to be resolved regarding the effect of EETs on EC and PASMC in the lung, and the reasons underlying their differential effects in the pulmonary and systemic circulation. However, it is thought that in the systemic circulation, EETs activate Ca2+ -dependent K+ -channels, leading to EC hyperpolarization, which then spreads to the SMC (67, 136). In the pulmonary vasculature, however, EET stimulation results in activation of K+ channels in the mitochondrial membrane, depolarizing the mitochondria and subsequently, the SMC itself (136). The situation becomes even more complex, with the responses to specific EET species seemingly varying between species and blood vessel size [reviewed in (99)]. Specific EET species have been shown to activate the transient receptor potential, canonical 6 (TRPC6) channel, which is a multimodal cation channel expressed in the EC (106, 136). In PASMC, TRPC6 translocates to caveolin-rich membrane microdomains in response to both hypoxia and 11,12-EET, leading to Ca2+ influx and SMC contraction (106). Similarly, in isolated EC, overexpression of CYP2C9 or exogenous EET activates TRPC6, stimulates Ca2+ influx, and prolongs the activation of Ca2+ -dependent K+ channels in response to agonists such as bradykinin (68). This role of TRPC6, as well as other TRP family members, is currently an intensive field of study in pulmonary vascular biology. For instance, the TRP vanilloid channel, TRPV4, has been implicated in the regulation of pulmonary EC barrier integrity, and is also thought to be an important player in EC Ca2+ signaling (103). Importantly, TRPV4 is activated by 5,6-EET (243), making it another candidate regulator of lung vascular tone and permeability. Analogous to EETs, HETE exerts differential effects on the pulmonary and systemic vasculature. While HETE is a systemic vasoconstrictor, it acts as a pulmonary vasodilator (18). In the systemic circulation, HETE activates Rho kinase, which results in phosphorylation of MLCK and SMC contraction (184). In the pulmonary EC, 20-HETE increases NO


production, leading to vasorelaxation via a PI3-Kinase- and Akt-dependent mechanism (20). Interestingly, 20-HETE has also been shown to activate TRPC6 in several in vitro systems (12,100). This effect is the opposite of that which is observed when TRPC6 is activated by 11,12-EET, and one could speculate that TRPC6 can be differentially regulated by various EETs. EETs have anti-inflammatory properties at the interface of the EC and the immune system. The first studies to show the magnitude of this effect demonstrated that 11,12-EET was capable of inhibiting leukocyte rolling and adhesion to the EC (161). Treatment with 11,12-EET, or overexpression of CYP2J2 led to decreased VCAM-1 expression and decreased NF-κB activation (161). Recent work in mice overexpressing CYP2J2 and CYP2C8 revealed that EET production attenuated lung injury in LPS-treated mice (46). These anti-inflammatory effects of EETs at the level of the EC are thought to occur through PPARγ activity (135). A final important role for EETs in the EC relates to their effects on platelet function. EETs produced by the EC inhibit platelet aggregation, and have been shown to activate platelet NO synthesis (66, 263). EETs also inhibit Ca2+ influx into platelets, and hyperpolarize the platelet membrane [reviewed in (99)]. Additionally, EETs inhibit P-selectin expression by platelets in response to ADP stimulation in a membrane potential-dependent fashion (115). In sum, these data show that EETs are potent inhibitors of platelet activation and aggregation through several mechanisms. The overall picture of EETs as pulmonary regulators of vascular tone, endothelial barrier function, immune function, and coagulation, coupled with the fact that they are generated during inflammatory stimulation, means that EETs could be critically important players in disease processes such as acute lung injury and ARDS. Further research into the roles of EETs and their pathophysiological modification may yield important novel therapeutics.

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Endothelin-1 Endothelin-1 (ET-1) is a pulmonary vasoconstrictor produced by the pulmonary EC that has generated much attention in the fields of systemic and pulmonary arterial hypertension. ET-1 is a small peptide secreted by the EC, and to a lesser extent by vascular SMC and lung fibroblasts. ET-1 is transcribed in an immature form, called prepro-ET-1. This peptide is then cleaved by neutral endopeptidase to form big ET-1, which is also known as pro-ET-1. Finally, the membrane-bound enzyme endothelin converting enzyme cleaves big ET-1 into mature ET-1 [reviewed in (52)]. The expression of ET-1 is stimulated by hypoxia, shear stress, angiotensin, and various growth factors [reviewed in (52)]. On the other hand, ET1 expression is downregulated by NO (21), a fact that may have clinical importance in pulmonary hypertension, when decreased NO production may lead to an increase in ET-1 and further elevation of pulmonary artery pressure. Indeed, ET-1 expression and secretion is increased in virtually every animal model of pulmonary arterial hypertension, and ET-1 receptor antagonism is now a mainstay in the treatment of PAH (203, 244) (Fig. 7).


EC-secreted ET-1 is primarily released from the basolateral plasma membrane (238), and as such is thought to predominantly act in a paracrine fashion at the PASMC (206). At its site of action, ET-1 binds to one of two G-protein coupled receptors, ETA R or ETB R (142). These two receptors are differentially expressed; EC only express ETB R, while SMC express both receptor subtypes (206). Furthermore, the ratio of ETA R to ETB R expression varies between lung regions (52). Binding of ET-1 to its receptor results in K+ channel inhibition, activation of Rho kinase and the Na+ channel, NHE1, and influx of Ca2+ (233, 249). Notably, ETB R stimulation on ECs can also stimulate NO production, so there is an interesting interplay between the two mediators. As a result, ET-1 may only have moderate vasoconstrictive effects in normal vessels, but causes massive constriction in vessels with endothelial dysfunction which lack stimulated NO production.

Reactive oxygen species The number of cellular processes known to be regulated by reactive oxygen species (ROS) has exploded over the past

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Figure 7 Pulmonary endothelial synthesis and secretion of ET-1. ET-1 is produced in the pulmonary endothelial cell in a prepro form, which is cleaved into pro-ET-1 (also called big ET-1). The enzyme endothelin converting enzyme (ECE) then cleaves big ET-1 into mature ET-1, which then has both autocrine and paracrine functions within the lung vasculature. In the EC itself, ET-1 can bind to the ETB receptor (ETB R), which results in nitric oxide (NO) and prostacyclin (PGI2 ) production. These two mediators can both subsequently act upon the smooth muscle cell to cause vasorelaxation. In a paracrine fashion, ET-1 itself can bind to its receptors on smooth muscle cells, ETA R and ETB R, to cause G-protein-mediated activation of phospholipase C (PLC). The resultant production of inositol triphosphate (IP3 ) and diacylglycerol (DAG) results in SMC migration, and contraction, or proliferation, respectively.

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decades. A comprehensive description of the many effects exerted by ROS, its various pathways of biosynthesis, and regulation of those pathways is beyond the scope of this review. Readers are directed to one of several outstanding reviews on this topic for a general discussion of the cellular role of ROS. We will focus on the specific functions of ROS that are germane to the pulmonary endothelium and the regulation of lung vascular physiology. ROS refer to a variety of highly ROS, either containing unpaired electrons (as in superoxide, O2 − ), or possessing a strong ability to oxidize other molecules (as in hydrogen peroxide, H2 O2 ). Most forms of ROS evolve from superoxide in the cellular context. In a general sense, cellular homeostasis depends on a fine balance of ROS; excessive ROS leads to widespread oxidation of macromolecules and cellular dysfunction, termed “oxidative stress,” while at appropriate levels, ROS serves a signaling function for fundamental processes. Oxidative stress results in aberrant gene expression, protein oxidation at cysteine residues, DNA mutagenesis, and lipid peroxidation. Many enzymatic pathways produce ROS: important in the lung are the enzymes of the mitochondrial respiratory chain, CYP enzymes, COX, xanthine oxidases, eNOS under conditions favoring uncoupling, and the family of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX). Interestingly, with the exception of NOX, these enzymes do not specifically produce ROS; rather, ROS is produced either as a byproduct of an enzymatic reaction, or when the enzyme activity is shunted away from its normal role. Equally plentiful are enzymatic antioxidant mechanisms, including catalase, superoxide dismutase, hemeoxygenases, and others (Fig. 8). EC possess several NOX isoforms (264), and ROS modulate various endothelial functions, including proliferation, vascular tone, and interaction with leukocytes. Some of these effects occur through modulation of redox-sensitive gene expression, such as NF-κ B, AP-1, and HIF-1α (200). Through these pathways, ROS is involved in inflammatory responses as characterized by the induction of ICAM-1 and VCAM-1 to agonists such as TNF-α (227), angiotensin II (36), and shear stress (211). Importantly, NOX2 has been implicated in acute lung injury in response to LPS in mice (144). Furthermore, loss of NOX2 suppressed LPS-induced upregulation of EC ICAM-1 in the same model system (144). Clearly, NOX2 is a key source of ROS in states favoring lung injury and pulmonary EC dysfunction. The effects of ROS on vasomotor tone are tightly linked to eNOS uncoupling, as discussed above. Superoxide, often produced in the EC by NOX isoforms, reacts vigorously with NO to produce the highly reactive species, peroxynitrite (15). This affects vascular tone in at least two ways: First, superoxide acts as a sink for NO, decreasing NO bioavailability and therefore diminishing NO-mediated vasorelaxation. Second, ROS can modify enzymatic pathways in such a manner as to promote further production of ROS, for instance by


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Figure 8 Sources of reactive oxygen species in the pulmonary endothelial cell. A critically important source of ROS in the pulmonary EC is the family of NADPH oxidase (NOX) enzymes. Activated by angiotensin II, TGF-β, and shear stress, NOX isoforms present in the lung can generate large quantities of ROS. Similarly, the mitochondrial electron transport chain can produce ROS, although controversy exists regarding whether hyperoxia or hypoxia favors this condition, and this controversy is discussed in the main text. Importantly, uncoupled eNOS produces ROS, and this ROS then leads to further eNOS uncoupling, which is an important propagator of inflammatory stress under a variety of pathological states. Elevated ROS levels leads to endothelial cytoskeletal changes, increased surface adhesion molecule expression, platelet adhesion, and a state referred to as endothelial dysfunction. Important enzymes such as superoxide dismutase exist to metabolize ROS and limit its accumulation.

uncoupling eNOS, or oxidation of xanthine dehydrogenase into xanthine oxidase (39). The role of ROS in HPV remains a controversial subject. ROS as a pulmonary oxygen sensor is one of several dominant theories, and discussion of such is beyond the scope of this review, and is covered in depth by Sylvester (218). To date, results from animal models have been inconclusive with respect to the role of ROS in HPV (39). As discussed above, multiple sources of ROS exist in the pulmonary vasculature. The mitochondria are an important potential source, and some superoxide is constantly generated as a byproduct of the mitochondrial electron transport chain (245). Since hyperoxia can result in an increase in ROS generated by the mitochondria, it is reasonable to speculate that hypoxia, and therefore the loss of substrate for ROS generation, would result in a decrease in ROS evolution from the mitochondria. Some data support this notion—notably the work of Archer and Weir, who have demonstrated that hypoxia results in a more reduced intracellular state, with less ROS, and subsequent changes in channel activity and SMC contraction (8, 247). On the other hand, equally persuasive data exist to support the opposite notion, whereby hypoxia results in dysfunction of the mitochondrial

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electron transport chain and the subsequent generation of ROS via cytochrome P450 enzymes (48, 245). Clearly, these two hypotheses are at odds with each other, and further work remains to be done to resolve this debate. The pulmonary hypertension literature, however, identifies ROS as an important mediator of both idiopathic PAH and PAH in response to chronic hypoxia. Chronic hypoxia increases superoxide production (268), and ROS is required for an appropriate vasoconstrictive response to ET-1 (134), which is a key mediator of PAH. Finally, NOX4 is upregulated in idiopathic PAH, and is involved in PASMC proliferation (39, 150). In the inflammatory state, ROS—even ROS derived from the EC—has many effects on the EC itself. ROS can induce apoptosis in EC, which then leads to dysregulated interaction between the immune system and the vessel wall, as well as the exposure of the prothrombotic subendothelial layer to the vessel lumen (49). This proapoptotic effect can be inhibited in vitro by antioxidant treatments (49). As stated above, ROS also upregulates expression of surface adhesion molecules characteristic of the activated EC, including VCAM-1 and ICAM-1 (223). ROS production during inflammation has profound effects on platelets and the clotting system. In an animal model of carotid vascular injury, overexpression of the ROS-metabolizing protein, glutathione peroxidase 1, limited thrombosis in response to hydrogen peroxide (42). Another study in animals overexpressing the antioxidant enzyme glutathione peroxidase-3 demonstrated that antioxidant activity decreased thrombosis in vivo (104). Several mechanisms for this effect have been proposed. Direct oxidation of AA produces a family of molecules known as isoprostanes, which are potent inducers of platelet activation (237). Oxidation of intracellular signaling molecules, such as the receptor-binding protein SHP-2, plays a role in the activation of platelets by exposed collagen (102). Hyperoxia, perhaps through generation of ROS, leads to Ca2+ influx and increased EC surface expression of P-selectin, resulting in platelet retention in intact rodent lungs (23). Further work remains to be completed, but it is clear that ROS are capable of influencing the platelet-EC interface in a manner that increases platelet activation and aggregation. Such interactions between platelets and the pulmonary EC are critical factors in the development and progression of inflammatory lung disease (219), and the modulation of ROS generation as a means of controlling this interaction may be exploited for the treatment of such conditions.

Von Willebrand factor von Willebrand Factor (vWF) is a glycoprotein of critical importance in hemostasis. Any qualitative or quantitative deficiency in vWF results in a bleeding disorder termed von Willebrand’s Disease (VWD) (132). vWF is synthesized at two primary sites; the megakaryocyte, and the EC. EC vWF is stored in granules along with P-selectin, Factor VIIIa, and IL8, termed Weibel-Palade Bodies (WPB) (162). The primary

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role for vWF is, indeed, a hemostatic one, in that it allows for the initiation of hemostasis in the presence of high shear stress. vWF binds exposed collagen in the subendothelium, as well as to platelet gpIb and gpIIb/IIIa, forming a functional bridge between a damaged EC and a circulating platelet (191). The binding of vWF to gpIIb/IIIa represents a critical step in platelet activation. Additionally, vWF binds inactive factor VIII in the circulation to prevent its degradation. Finally, vWF binds procoagulant molecules like thrombin when in their active states (191). It has been said that the multiplicity of vWF binding partners allows vWF to act as a molecular “bus,” shuttling clotting factors and inflammatory proteins and cells to their active sites in the vasculature (129). Endothelial WPB are an important source of vWF in the pulmonary circulation. Recent work has indicated that not all pulmonary ECs are created equally in this regard; WPB are absent from the EC of pulmonary capillaries, but are present in the pulmonary artery EC (74) (Fig. 9), an observation originally made as early as 1966 (74). However, pulmonary capillary EC still express vWF and produce vWF protein, and it is presently unclear as to how these cells store vWF and the other typical WPB contents. Additionally, the activation of vWF differs spatially in the pulmonary circulation. As stated above, shear stress is an important activator of vWF, inducing a conformational change in the protein that improves platelet binding (207). Shear stress and flow conditions vary greatly over space and time in the lung—different lung areas are exposed to vastly different shear conditions, and flow also varies with the respiratory cycle and body position (supine vs. upright). Therefore, vWF activation is likely to vary similarly in different lung vascular beds (162). In addition to the critical role played by vWF in clotting, vWF also is an important mediator of inflammation. At an early stage, vWF is required for the correct targeting of Pselectin to the WPB (47), and loss of this vWF function leads to impaired surface expression of P-selectin, and therefore, impaired endothelial activation (129). Further downstream, it has been shown that vWF-platelet aggregates are necessary for leukocyte recruitment and binding to the EC (16), as well as for the extravasation of leukocytes at sites of inflammation (175). The importance of interactions between vWF and immune cells has been highlighted by the recent discovery of a leukocyte receptor for vWF, called Siglec-5 (174).

Clinical relevance of endothelium-derived mediators Our understanding of many of the factors secreted by the endothelium, as detailed above, has led to numerous efforts to exploit these physiological insights in the clinical context. While a detailed discussion of all such studies goes beyond the scope of this review, we will highlight a selection of those upon which the greatest clinical focus has been drawn, including NO, prostacyclin, and ET-1. Due to its efficacy as a pulmonary vasodilator, as well as its anti-inflammatory properties, NO was heralded as a


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Heterogeneous distribution of Weibel-Pallade Bodies (WPB) in the lung endothelium. Representative electron micrographs of pulmonary endothelial cells from human lungs. WPB are indicated by arrowheads. Upper panels: High and low magnification images of extra-alveolar endothelial cells, showing readily detectable WPB. Lower panels depict alveolar capillary endothelial cells, which are notably devoid of WPB. EC, endothelial cells, N, nucleus, BM, basement membrane, ∗, caveolae. Reproduced, with permission, from (266).

potentially transformative therapy for pulmonary hypertension and other disorders, such as ARDS. Unfortunately, the initial enthusiasm for NO in these and other groups of adult patients has now diminished. In fact, the only FDA approved clinical indication for inhaled NO currently is for the treatment of persistent pulmonary hypertension of the newborn, a condition for which NO reduces the need for extracorporeal membrane oxygenation without affecting overall survival (10). In all adult patients, the effect of NO on pulmonary hemodynamics is highly variable between patients, and in a single patient over a period of time (86). Furthermore, several large human trials have failed to demonstrate a significant benefit when comparing inhaled NO to regular care (2,3). The lack of substantial improvements in outcome, coupled with high cost and difficulty of administration in chronic diseases such as pulmonary hypertension, have severely limited the potential uses for inhaled NO in pulmonary disease. Downstream of NO, several therapeutic interventions have targeted stimulation of sGC, or inhibition of phosphodiesterase 5 (PDE5) to enhance intracellular levels of the second messenger cGMP. PDE5 inhibitors, such as sildenafil, have been used extensively for the treatment of pulmonary hypertension. When specifically used for Type 1 pulmonary arterial hypertension, sildenafil was well tolerated, and produced improvements in 6-min walk tests, pulmonary


hemodynamics, and quality of life (76, 189). Mortality was unchanged in these studies. In spite of these benefits from sildenafil, activation of sGC did not yield such results clinically. Results of two major trials, one in patients with chronic thromboembolic disease leading to pulmonary hypertension (80), and one in patients with pulmonary arterial hypertension (81) yielded modest functional improvements in patients treated with the sGC activator, riociguat. However, the overall effect size was low (7). While potentially promising, this treatment strategy requires further refinement before being broadly applicable. A further avenue that has been pursued clinically for the treatment of pulmonary hypertension involves antagonism of ET-1 receptors. Currently, three drugs exist: bosentan and macitentan, which are nonselective ET-1 receptor antagonists, and ambrisentan, which specifically inhibits ETA R. Bosentan has been shown to improve exercise capacity, pulmonary hemodynamics, and disease progression, and improves survival in pulmonary arterial hypertension (29, 143, 188). Similar results were achieved with macitentan, which also improved survival as compared to controls (178). Trials with the selective agent, ambrisentan, have been similarly positive, yet unexpectedly, do not seem to offer significant benefit above that seen with nonselective ET-1 receptor antagonism (77).

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An additional treatment for pulmonary hypertension that stems from our knowledge of a secreted EC mediator is the class of prostanoid compounds, which are related to prostacyclin, and as such, are potent pulmonary vasodilators and inhibitors of platelet aggregation (96, 163). Several formulations exist, and can be given by inhaled or intravenous routes. While they are mainstays of treatment for advanced disease, some preparations suffer from ultrashort half-lives, and heterogeneous patient response (96). The best studied of these drugs is epoprostenol, which decreases pulmonary vascular resistance and pulmonary arterial pressure, increases exercise capacity and quality life, and enhances survival in patients with severe pulmonary arterial hypertension (11). As such, it is often a first line therapy for severe disease. Similar benefits have been shown for pulmonary hypertension stemming from other causes including Eisenmenger syndrome (120), although small sample sizes have precluded any inferences regarding potential survival benefits. Taken together, these examples demonstrate the fundamental physiological importance of mediators secreted by pulmonary ECs. Knowledge of these molecules, and their downstream signaling pathways, has informed fundamental therapeutic advances, and will continue to do so in the future.

Factors Presented by the Endothelium Adhesion molecules for leukocytes The EC represents the point of egress for leukocytes migration to sites of inflammation, and as such, must be capable of expressing markers for their identification as (a) ECs, and (b) sites of inflammation. Therefore, surface marker expression by the activated EC must be tightly regulated spatially and temporally. Additionally, several important differences exist in EC-leukocyte interactions between the systemic and pulmonary circulations [reviewed in (51)]. While beyond the scope of this review, three key contrasts warrant mention: First, the predominant site of leukocyte transmigration in the pulmonary vascular bed is the capillary network, not the postcapillary venule. Second, leukocyte rolling, which is a prominent feature of the inflammatory reaction in the systemic circulation, is thought to not occur in the lung, since the small size of pulmonary capillaries makes this geometrically impossible. Lastly, at baseline, neutrophils spend a disproportionate amount of time in the pulmonary circulation, and this increases during lung inflammation, causing a relative systemic neutropenia. Again, this is thought to be due to the geometric considerations involved in moving a neutrophil through the pulmonary capillary network, which requires multiple cytoskeletal rearrangements on the part of the neutrophil (see Fig. 2). The resulting population of neutrophils sequestered in the pulmonary circulation has been termed the “marginated pool” (122). Initiation of neutrophil transmigration relies on the recognition of the activated EC by the neutrophil. In the systemic circulation, this process almost always relies on ICAM-1

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binding to leukocyte CD18; however, only some inflammatory stimuli require CD18 to elicit neutrophil emigration in the lung (151). As stated above, leukocyte rolling—which depends upon EC expression of P-selectin and E-selectin— does not occur in the pulmonary circulation. In fact, knock out of both P- and E-selectin does not inhibit neutrophil transmigration into the alveolus during Streptococcus pneumonia pneumonia (152). However, P-selectin inhibition has been shown to reduce ischemia-reperfusion injury in the lung (156). Additionally, L-selectin has been shown to prevent the rapid decrease in the number of circulating neutrophils after formylMet-Leu-Phe- (fMLP)-induced lung injury (164), and to prevent neutrophil accumulation and hemodynamic alterations in LPS-induced acute lung injury (121). Taken together, these data suggest an important role for selectins in neutrophil function in the pulmonary vasculature that is likely to be separate from their traditional role in the systemic circulation. While demonstrating that selectins are not essential in the lung, and that CD18 is not always required, these data do not make ICAM-1 unnecessary. ICAM-1 is expressed by the pulmonary EC (14), and is upregulated by certain stimuli, including LPS and some gram-negative bacteria. Pharmacologic or genetic disruption of ICAM-1 inhibits leukocyte transmigration in response to LPS or Pseudomonas, indicating an important role for this EC surface protein in the clearing of gramnegative infections (124,180). Binding of neutrophil receptors to ICAM-1 triggers critical signaling events in the EC, many of which converge around EC cytoskeletal rearrangements to facilitate neutrophil movement to the EC border for eventual transmigration (50). The above data clearly demonstrate that fundamental differences exist in the nature of leukocyte-EC interaction between the systemic and pulmonary circulations. While seminal work has been done to identify these contrasts, our knowledge of the precise mechanisms of neutrophil egress to the alveolus remains limited, and is the focus of ongoing research.

Adhesion molecules for platelets In addition to their known role in clotting, platelets have also recently been recognized to be important immune cells. The earliest phases of the coagulation cascade begin with platelet binding to extracellular matrix components in the exposed subendothelium of an injured vessel wall. However, important interactions also occur between platelets and the EC itself. Much of our knowledge of these interactions comes from work investigating atherogenesis and plaque disruption, but several important points with respect to the lung endothelium can be extrapolated from that literature. In resting vessels, platelets are constantly rolling on the EC monolayer, but firm adhesion is rare (72, 219). The lack of adhesion is due in part to a lack of integrin activation on the resting platelet, but is also critically influenced by constitutive secretion of NO and prostacyclin by the EC, both of which inhibit platelet activation (242). Activation of the EC, as simulated in experiments using a Ca2+ ionophore (72), or


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by experimental ischemia-reperfusion injury (141), increases platelet adhesion to the EC by fourfold. Adhesion between platelets and EC is thought to follow a similar sequence as ECleukocyte interaction, with rolling preceding tethering, which ultimately leads to stable adhesion [reviewed in (219)]. Transient interactions between selectins mediate tethering, while integrins on the surface of activated platelets undergo conformational changes that allow them to bind the EC surface with high affinity, leading to stable adhesion. Stable association of platelets and EC results in releases of proinflammatory mediators from the platelet, triggering further activation and phenotypic changes in the EC. Key differences in this process exist in the pulmonary circulation versus that of the systemic vasculature. As stated earlier, pulmonary capillary EC lack WPB, and perhaps also lack functional P-selectin. As such, P-selectin-mediated interactions are unlikely to be the primary trigger of EC binding by platelets in pulmonary vessels. Notably, ICAM-1 expression is high in the pulmonary circulation; reaching levels of 2- to 20-fold compared to those found in other vascular beds (170). Similarly, αν β3 integrin expression is highest in the lung vasculature (208). It is possible, therefore, that platelet retention in the lungs is mediated by the constitutive expression of these two adhesion molecules by the pulmonary EC. Activation of the platelet—which is a requirement for firm adhesion to the EC—results in a conformational change in platelet gpIIb/IIIa that increases its affinity for fibronectin, which can then act as a bridge to EC αν β3 integrin. Coupled with the low flow, low shear stress state of the lung, this integrin-mediated interaction may be sufficient for platelet binding to the lung EC. Stimulation of platelets with thrombin increases their affinity for the EC, which adds further strength to a model whereby platelet activation alone is sufficient for enhanced binding to constitutively expressed pulmonary EC receptors (219).

Transcytosis and presentation of chemokines The EC comprises the cellular barrier between the vessel lumen and the extravascular space. During tissue inflammation, leukocytes must traverse that barrier to migrate to the site of inflammation. This migration is thought to follow a soluble chemokine gradient. Most chemokines are secreted by extravascular cells, so chemokines must also be capable of crossing the EC monolayer, albeit in the opposite direction traveled by leukocytes. This system presents several problems, including: (a) how do chemokines cross the EC in a manner that maintains their spatial distribution and concentration gradient? (b) How are chemokine gradients maintained in the face of blood flow in the intravascular space? Current data suggest that the EC is able to transport chemokines from their basolateral surface to their apical one via a transcytotic pathway. Once at the apical membrane, surface receptors immobilize the chemokines so that they can be presented to circulating leukocytes (148, 149). Three-dimensional reconstructions of electron micrographs has shown that intradermally injected IL-8 is taken up


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Transcytosis of interleukin-8 by endothelial cells. Stacked and reconstructed electron micrographs of human skin venular endothelial cells, with gold labeling of interleukin-8 (IL-8). (A) IL-8 binding and uptake at the basolateral (abluminal) surface of the venular EC. (B) and (C) demonstrate IL-8 in vesicles below the plasma membrane. (D) and (E) demonstrate IL-8 now subsequently appearing at the luminal surface of the EC. (F) Colocalization of IL-8 (10 nm gold) and caveolin-1 (5 nm gold). Reproduced, with permission, from (148).

via caveolae at the basolateral surface of the EC, and is subsequently presented on the luminal side (148) (Fig. 10). This same process has been shown to exist for other chemokines as well (149). Additionally, chemokines produced by the EC itself, such as fractalkine, are transported to the luminal surface and presented in a similar fashion. Furthermore, IL-8, which is a component of WPB, is similarly translocated to the EC plasma membrane after exocytosis of the WPB (219). In order for this process to occur, a receptor for chemokines must exist on the EC membrane. An important chemokine binding partner on the EC is glycosaminoglycan (GAG), which is a highly negatively charged polysaccharide, often conjugated to a core protein to form a proteoglycan (125). The predominant GAG on the EC is heparan sulfate. Heparan sulfate can associate with high affinity to positively

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charged chemokines. The role of GAGs in presenting chemokines, and therefore, in facilitating neutrophil transmigration has been demonstrated in several systems, including the lung [reviewed in (149)]. Additionally, genetic depletion of an enzyme responsible for heparan sulfate synthesis results in impaired neutrophil chemotaxis in a mouse model of inflammation (240), and surface GAGs increase local concentrations of chemokines and promote their presentation to leukocytes (125). Finally, binding of IL-8 to GAG prevents IL-8 unfolding, thereby promoting its stability in the circulation (83). The above data have allowed Middleton’s group to present a model (149) whereby interstitially secreted chemokines are taken up by caveolae at the abluminal surface of the EC, and are transported to the luminal membrane via transcytosis. The bound chemokine can then be presented to circulating leukocytes, which will then traverse the EC using either a trans- or paracellular pathway. This model highlights the importance of the EC as an effector of inflammation and leukocyte migration beyond its involvement in the secretion of inflammatory mediators (149). One could speculate that such a system may be particularly effective in the lung, where both the alveolar epithelium and the capillary endothelium are specifically rich in caveolae, allowing for rapid chemokine crosstalk between the alveolar and vascular compartments that would be expected to allow for a rapid and localized inflammatory response to airspace invasion by pathogens.


endothelial adhesion molecules, which only become accessible to circulating cells after surface layer degradation. Such degradation can occur, for example, by ROS at sites of inflammation, providing another means by which inflammatory cells are locally recruited to sites of inflammation (177) or by LPS during experimental models of sepsis (197) (Figs. 11 and 12). Another intriguing function ascribed to the glycocalyx is that of sensing mechanical stress and transducing that signal to the EC interior [reviewed in (35)]. The sensing of mechanical stress may provide the basis by which the pulmonary endothelium responds to changes in airway and vascular pressures, and may be involved in the pathogenesis of conditions marked by perturbations in these pressures, such as pulmonary hypertension secondary to left heart disease or mechanical overventilation of lungs. Work done in several model systems has identified syndecans—a major heparan sulfate component of the glycocalyx, and the only one to span the EC plasma membrane—as potential sources of mechanical information for the EC interior (35). Syndecans are linked to the EC actin cytoskeleton, which suggests they may have a role in outside-in signaling to the EC cytoskeleton (224). Recent reviews have summarized data showing that shear stress, acting through syndecans, is capable of activating eNOS, resulting in increased EC permeability (35, 224). This postulated role for the glycocalyx as a mechanotransducer is an intriguing one, and may link EC dysfunction in a variety of disease states with the observation that the glycocalyx is disrupted in those same conditions (35).

The endothelial glycocalyx GAG function in vascular biology goes beyond their role in presenting chemokines. GAGs are a major component of the endothelial glycocalyx; a coating of glycoproteins, proteoglycans, and glycolipids that are bound to the EC surface. An important distinction in the nomenclature of the endothelial surface should be made: The glycocalyx comprises the molecules that directly bind to, or are constituents of, the EC plasma membrane. The much thicker coating on top of the glycocalyx by GAGs, heparan sulfate, hyaluronan, and other molecules is termed the endothelial surface layer [reviewed in (177)]. The precise structure and composition of the glycocalyx has been reviewed extensively elsewhere (185, 246). The endothelial glycocalyx forms a dynamic layer at the luminal surface of the EC that excludes circulating blood cells, as well as many plasma proteins, and forms an integral component of the endothelial barrier (35). In fact, the glycocalyx is thought to form a plasma protein-free microenvironment that regulates the oncotic pressure sensed by the EC, thereby helping to regulate fluid flux across the EC membrane (236). Disruption of the glycocalyx by enzymatic digestion or by charge neutralization increases the permeability of the EC monolayer, highlighting the role played by the glycocalyx in EC barrier function (1, 230). Notably, there is also an important role for the endothelial surface layer in the regulation of interactions between the EC and leukocytes or platelets. In the resting state, the endothelial surface layer will shield

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Factors Metabolized by the Endothelial Cell Angiotensin-converting enzyme and Angiotensin II The renin-angiotensin system (RAS) is a well-characterized endocrine axis that regulates a multiplicity of cellular functions. In its simplest form, renin (produced in the kidney) cleaves angiotensinogen (produced in the liver) to form angiotensin I (AngI). AngI is a decapeptide, which is subsequently cleaved by the angiotensin-converting enzyme (ACE) into the biologically active octapeptide, Angiotensin II (AngII). ACE exists in its highest concentration on the luminal membrane of pulmonary ECs. In reality, the majority of AngI production is mediated by the enzyme chymase (22), which is highly expressed in the lung, especially by resident mast cells (173). AngII, by binding its cognate receptors AT1 and AT2 , then exerts its effects on various organ systems, including its best described effect, which is to stimulate aldosterone secretion from the adrenal cortex, promoting sodium and water retention in the kidney, thereby increasing intravascular volume and systemic blood pressure. In reality, both the makeup of this hormonal axis and its end effects are far more complex than this simple picture suggests, with various “branches” existing in the above scheme, and a vast number of additional effector sites for angiotensin signaling. The RAS is


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Structure of the endothelial glycocalyx and endothelial surface layer (ESL). At the luminal surface of the pulmonary EC, the glycocalyx comprises a thin layer of plasma membrane glycoproteins and glycolipids. External to that is a thicker layer of glycosaminoglycans (GAG), heparan sulfate, hyaluronan, and other molecules, termed the ESL. These layers (A) separate the EC surface from the circulating plasma, (B) allow for mechanosensation and outside-in signaling to the EC, (C) performs a critical role in maintaining the endothelial barrier, and (D) can be importantly degraded and modified during periods of inflammation.

responsible for far more than the acute regulation of systemic blood pressure; in fact, it has been shown to play a role in chronic hypertension, the progression of diabetic complications, atherogenesis, and adverse myocardial remodeling in response to hypertension (reviewed in (22)] (Fig. 13). The distinct effects of AT1 and AT2 receptors, as well as their differential tissue expression, can partially explain the multiplicity of responses to AngII. The AT1 receptor is G-protein coupled, and activation of the receptor results in phospholipase C activation, Ca2+ release, and changes in gene transcription (22). Furthermore, activation of Rac and Rho by AT1 activation has been associated with vascular smooth muscle cell contraction and Ca2+ sensitization (93). In the EC specifically, AT1 signaling has been demonstrated to favor eNOS uncoupling (153). AT1 is expressed in nearly all vascular beds, and its activation results in the responses classically assigned to AngII activity, namely vasoconstriction, elevation of systemic blood pressure, increased myocardial contractility, aldosterone secretion from the adrenal gland, and increased glomerular filtration (107). AT1 ligation also stimulates ROS production, cell proliferation, vascular remodeling, coagulation, and extracellular matrix deposition (105). The majority of pharmacologically available angiotensin receptor blockers (ARBs) specifically target the AT1 receptor subtype, and as such, many of the cardiovascular benefits of angiotensin signaling blockade are attributed to signaling through this receptor. A landmark paper by Imai and colleagues (98) demonstrated that activation of the


classical pathway via ACE and AT1 is a critical component of the development of acute lung injury following either acid aspiration or sepsis due to cecal ligation and puncture in mice. Conversely, activation of ACE2 and the AT2 receptor were both protective. Our understanding of this interplay has been deepened by multiple subsequent studies demonstrating the same effect (87,250). These data highlight both the fundamental role played by angiotensin signaling in acute lung injury, and the differential effects of AT1 and AT2 receptor signaling. The AT2 receptor, in general, acts in opposition to AT1 . AT2 expression is high during fetal development, and decreases sharply after birth, suggesting a developmental role for the receptor (107). Expression of AT2 persists, however, in the lung, brain, heart, and adrenal medulla (107, 202). AT2 expression is also induced by cardiovascular injury or wound healing (130), and receptor activation is responsible for vasodilation, and is both antiproliferative and antihypertrophic (28, 209). Unfortunately, current data regarding the specific role of AT2 in the lung vasculature are lacking, and should be a future area of research. The EC surface is a critical site of ACE activity; in fact, the majority of ACE is found at the surface of the pulmonary EC [reviewed in (173)]. Therefore, the bulk of conversion of AngI to AngII, as well as inactivation of bradykinin, occurs at the EC luminal membrane. One important functional implication of the pulmonary EC-localization of ACE activity is that during lung injury, the appropriate presentation of ACE to circulating AngI may be disrupted, thereby upsetting proper

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(A) Endothelial cell

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The endothelial glycocalyx in vivo, and its degradation by LPS. (A) Schematic representation of the glycocalyx and endothelial surface layer (ESL). Note that the glycocalyx is not permeable to large molecules such as fluorescent dextrans. Therefore, measurement of vessel diameter by visible light microscopy versus fluorescence should allow for the measurement of the ESL. The images in the right of the panel show mouse sub-pleural microvessels (MV) imaged with visible light (DIC) or fluorescence (FITC). The difference between the DIC and FITC width represents the ESL. (A) Alveolus. (B) Group data of calculated ESL thickness as in (A), taken from mice treated with LPS or TNF-α, demonstrating how inflammatory stimuli can degrade the ESL. (C) These effects are not seen in mice lacking functional TNF receptors. Reproduced, with permission, from (197).

AngII homeostasis in the whole organism. Data exist to support this hypothesis, detailing the effects of acute lung injury (165), pulmonary arterial hypertension (127), and chronic thrombotic lung disease (166) on EC surface ACE activity. It is clear that disease states that alter lung vascular function and EC activation can also result in alterations of angiotensin metabolism at the EC surface, and that this phenomenon can upset the balance of angiotensin signaling, leading to the deleterious outcomes detailed above.

ACE2 and Angiotensin-(1-7) A more recently described metabolic pathway for AngII processing involves the enzyme ACE2 (61). ACE2 can act upon AngII to produce the biologically active peptide Angiotensin(1-7) [Ang-(1-7)]. A second pathway also exists, whereby ACE2 acts directly upon AngI to produce Angiotensin 1-9, which is then cleaved by ACE to form Ang-(1-7) [reviewed in (105)]. Ang-(1-7) is the biological ligand of the receptor Mas, which is a G protein-coupled receptor that is highly expressed in lung, brain, heart, and kidney (195).

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The discovery of the Ang-(1-7)-Mas axis generated a great deal of interest since the effects of this pathway were thought to antagonize AngII (62, 145). In a model of bleomycininduced pulmonary fibrosis, rats were protected from fibrosis by infusion of Ang-(1-7) (145). Furthermore, ACE2 deficient mice have been shown to develop significantly worse acute lung injury in response to acid inhalation or cecal ligation and puncture when compared to controls (98). Furthermore, this effect could be rescued by the administration of exogenous ACE2 (98). The authors of this landmark paper went on to show that AT1 R ligation by AngII was a key driver of acute lung injury. Together, these findings indicate that a critical balance exists between AngII and Ang-(1-7) signaling, and that modulation of this balance may be a fundamental mechanism in pulmonary health and disease. Indeed, a search for Ang-(1-7)-based therapeutics is underway. The basis for such a search is strengthened by our own work performed in three animal models of acute lung injury, namely acid inhalation, ventilated-induced lung injury, and oleic acid instillation (110). These experiments indicated that delivery of exogenous Ang-(1-7) protected rodents from pulmonary


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Pulmonary endothelial angiotensin signaling. The majority of conversion of AngI to AngII is carried out by angiotensin converting enzyme (ACE), bound to the EC surface. AngII can then bind to AT1 R or AT2 R (left side). Signaling through AT1 R results in G-protein-mediated release of IP3 and DAG, as well as stimulation of MAP kinases. The downstream effects include PASMC contraction and migration, as well as EC ROS production and vascular remodeling. Signaling via the AT2 R is incompletely understood in the lung, but in general antagonizes the effects of AT1 R signaling, leading to PASMC relaxation, and protection from adverse remodeling. The cell on the right depicts further metabolism of AngII by ACE2, producing Ang(1-7), which subsequently binds the Mas receptor. Mas has a highly protective effect during states of acute lung injury and inflammation, decreasing ROS production, enhancing NO release, and protecting against thrombosis and EC apoptosis.

edema (110). Ang-(1-7)-treated animals also displayed less histological evidence of lung injury, less myeloperoxidase activity in lung samples, and lower pulmonary artery resistance as compared to nontreated animals with experimental lung injury (110). These data suggest that Ang-(1-7)-based therapies could be promising tools for the prevention and treatment of acute lung injury from a variety of stimuli. Additionally, Ang-(1-7) appears to play a key role in the pathophysiology of pulmonary hypertension. In a cohort study of patients with pulmonary hypertension due to congenital heart disease, patients with PH were found to have lower levels of circulating Ang-(1-7) as compared to controls, and Ang-(17) levels inversely correlated with elevations in PA pressure (38). Furthermore, in experimentally induced PH, treatment with diminazine—an antimicrobial that incidentally increases ACE2 activity—protected rats from the development of PH in response to monocrotaline or chronic hypoxia (204). Further data implicating Ang-(1-7) as a protective factor in PH have been reviewed previously (205), and exploitation of ACE2 activity remains an exciting field in PH research. The mechanism by which Ang-(1-7) acts under such conditions is beginning to be understood, and involves a close


connection with NO signaling. Most notably, Ang1-7 has been shown to stimulate endothelial NO (110). The interplay between Ang-(1-7) and NO extends into platelets and clotting pathways as well. Ang-(1-7) enhances NO production, and inhibits platelet aggregation (117,182,183). This antiaggregative effect is mediated by Ang-(1-7)-induced stimulation of NO release from platelets (71). In in vitro models, Ang-(1-7) protects cultured ECs from apoptosis as a result of bleomycin or AngII stimulation (231). This effect was mediated by inhibition of JNK (231). Finally, Ang-(1-7) has also been shown to inhibit signalling through MAPK and NF-κB (145).

Endothelial Signal Propagation Connexins in the pulmonary endothelium Up to this point, this review has dealt with the role of individual ECs in lung vascular tone, inflammation coagulation. In the whole vessel and whole organism, however, ECs do not function in isolation. In fact, they are an interconnected monolayer of cells that are capable of direct communication

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Figure 14 The conducted response in hypoxic pulmonary vasoconstriction. (A) Intravital microscopic images taken from wild-type (top panels) or Cx40−/− mice (bottom panels) during normoxic or hypoxic ventilation, as indicated. Endothelial cells have been loaded with the voltage-sensitive fluorophore, di-8-ANEPPS. Arteriolar vessel margins are indicated by dotted lines. Capillary endothelial cells are circled, while arteriolar EC are indicated by squares. Not that in wild-type mice, depolarization in response to hypoxia occurs in both the capillary and arteriolar EC. However, in Cx40−/− mice, only the capillary EC depolarize, indicating the requirement of gap junction communication for the conducted response. (C) and (D) show group data describing the above. In (D), depolarization in response to hypoxia is not seen in the pulmonary arteriolar EC in Cx40−/− mice, indicative of the requirement for gap junctional communication. Reproduced, with permission, from (241).

between neighbors. Such direct communication between ECs has long been recognized as an important mechanism of propagation for intracellular signals in both the nervous and cardiovascular systems (64). The important structural mediators of this functional network are gap junctions, which are comprised of connexin proteins. In forming a gap junction, six connexin monomers assemble to form a connexon hemichannel within the plasma membrane of a single cell (84). This hemichannel then associates with a similar connexon in the adjacent cell. The multiplicity of connexin subtypes allows for a huge number of potential connexin channel combinations; there are over 20 connexin types, and neighboring connexons can form homo- or heterotypic associations in making a mature channel (84). These channels are involved in diverse cellular functions, including neuronal communication,

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neurovascular coupling, the spread of muscle depolarization, and—importantly in the context of the present review—EC communication (64, 84) (Fig. 14). In the vasculature, gap junctions contain combinations of connexin (Cx)-37, -40, -43, and Cx45 (64). While Cx45 is restricted to the smooth muscle, Cx40 is predominantly expressed in the EC (64). Cx40 is highly expressed in the pulmonary EC, and is downregulated during acute inflammation (186). Additionally, Cx43 is expressed in pulmonary capillaries (172). The first evidence for a functional role of gap junctions in the lung was derived from real-time imaging of lung ECs in situ, which revealed the existence of spontaneous endothelial Ca2+ oscillations that were propagated between neighboring ECs as interendothelial Ca2+ waves and could be blocked by administration of gap junctional uncouplers such


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as halothane or heptanol (123, 258). These observations provided the basis for further work into this phenomenon, which revealed novel insights into endothelial communication during physiological responses.

Connexins and the conducted response The search for a mechanism that would allow for the conduction of a vasomotor response along a vessel wall revealed the importance of connexins in the systemic circulation. Several important observations regarding this system were made which pointed to gap junctions as the required portal for EC communication: first, the direction of vasomotor signal propagation was opposite to the direction of blood flow (44). Stimulation of systemic vessels with acetylcholine, bradykinin, or potassium led to changes in vascular tone upstream of the initial stimulus. This meant that the messenger involved in this process could not be secreted into the blood stream. Second, the propagation of this signal was lost in Cx40−/− mice (44). Third, only signals that required the EC were affected by loss of Cx40, demonstrating that Cx40 is required for direct communication between ECs (44). Together, these observations indicated a critical role for Cx40 in the conduction of the vasomotor response in systemic ECs. Additionally, gap junctions exist between the EC and the SMC, and these junctions are involved in signaling from the EC to the effector of the vasomotor response, which is the SMC (64) (Fig. 15). The presence of a similar systemic in the pulmonary vasculature had not been demonstrated until recently. It has long been thought that the lung vascular response to hypoxia

(HPV) was entirely housed within the PASMC. While these data suited in vitro observations, they did not fit with the anatomical arrangement of the lung vasculature. The ideal site for sensing of alveolar oxygenation would clearly be the alveolar capillary, since it is in close proximity to the airspace, and the diffusion barrier between the capillary and alveolus is by definition easily traversed by oxygen. However, capillaries are devoid of smooth muscle. Therefore, there must be a spatial separation between the alveolar capillary and the site of HPV, which is the medium-sized precapillary arteriole of at least 30 μm in diameter (220). These same vessels are also the smallest to contain SMC (95, 168). These anatomical considerations provided the basis for investigations into the existence of the conducted response in the lung, since such a spatial arrangement necessitates a means by which to propagate a hypoxic signal from the capillary to the arteriole against the direction for blood flow. Cx40-containing gap junctions are an ideally placed conduit for such a response due to their high expression in the lung EC (241). Cx40−/− mice do, indeed, display a severely attenuated HPV response, and exhibit enhanced arterial oxygen desaturation in response to small airway obstruction (241). Intravital microscopy and ratiometric imaging of cell membrane potential in mouse pulmonary arterioles revealed that in Cx40−/− mice, alveolar capillary EC depolarization is unimpaired, but that this depolarization is not conducted to the arteriolar EC (241). The downstream signaling events involved in this response involved phospholipase A2 (cPLA2) and EETs (241). Furthermore, as has been observed in the systemic circulation, there may be an additional role for myoendothelial gap junctions in

Hypoxia O2 sensitive K+ channel

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Figure 15 Schematic diagram representing the conducted response. It is hypothesized that hypoxia inhibits oxygen-sensitive K+ channels, leading to EC depolarization at the alveolar capillary level. This depolarization spreads between EC in a retrograde fashion to the muscularized arteriolar EC via Cx40-containing gap junctions. At this point, communication with the PASMC can occur, resulting in vasoconstriction. A contribution of myoendothelial gap junctions containing Cx43 and Cx40 is likely involved in this process.


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this system as well. Notably, as aforementioned, Cx43 inhibition also had an inhibitory effect on HPV, which was additive to that of Cx40 inhibition alone. Along the same lines, it has also been demonstrated that gap junction uncouplers are also capable of inhibiting the vasoconstrictive response to hypoxia in isolated pulmonary artery rings (109). This finding is significant because an isolated ring does not require a conducted EC response for vasoconstriction, since all EC in the ring have direct access to the hypoxic stimulus. Taken together, these data demonstrate the importance of another type of gap junction in this response, which must be the myoendothelial junction. These findings have shed considerable light on a process of fundamental physiological importance. These results also emphasize the importance of contextualizing findings outside of the model system in which they were collected, and considering the integration of such findings into the whole organism. Several other key aspects of connexin-mediated signalling in the pulmonary vasculature bear mention. First, Cx43 appears to have a role in the regulation of endothelial permeability as well as vasomotor responses. Genetic deletion of Cx43 resulted in the loss of Ca2+ wave propagation in response to a local inflammatory stimulus, and Cx43 blockade abrogated the increase in endothelial permeability in response to thrombin (172). Second, intact EC-to-cell communication via connexins seems to be a prerequisite for normal parenchymal homeostasis in the intact lung. Strikingly, a double knockout mouse specifically lacking endothelial Cx43 and Cx40 displays a phenotype of lung parenchymal fibrosis and alveolar abnormalities (113). Intriguingly, single knockout mice of either Cx40 or Cx43 alone do not develop such abnormalities. These findings suggest that interendothelial communication plays an important role in lung homeostasis. Lastly, endothelial connexins may play an important role in inflammatory responses, beyond the findings related above with respect to Cx43. Neutrophils have been shown to communicate with ECs directly via connexins (259). This interaction was shown to be essential for subsequent resealing of the endothelial monolayer following leukocyte transmigration. In all, connexin-mediated interendothelial communication is a fascinating topic, with demonstrated importance in both vasomotor and inflammatory signaling, as well as in overall organ homeostasis and development. Since the lung is such an important site of intercellular interaction in all of these domains, connexins will continue to be an important research focus going forward.

Emerging Areas of Inquiry Many new areas are arising in pulmonary vascular biology, which involve the lung endothelium. Due to space limitations, we are unable to highlight all of the fascinating new fields that have emerged in lung vascular research. We will, however, focus on two that are of particular interest.

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Substrate stiffness and contextual biology One such line of inquiry seeks to explore mechanosensation by the EC, and how the stiffness of the substrate influences endothelial biology. While much of this work to date has been carried out in vascular beds outside the lung, we can extrapolate these data into pulmonary biology and make important inferences. In a fascinating manner, when considering mechanical inputs, the lung is a truly unique environment, since such forces can be applied to the EC from many sources, including the basement membrane, vascular shear forces, alveolar pressure, and others. The integration of these forces by the EC likely results in the net response of the cell to its environment, and provides a physical context for EC populations in different lung regions. In the systemic circulation, changes in substrate stiffness and EC mechansosensation have been linked to fundamental pathological processes, including atherogenesis (88). In such a model, the shear stress provided by turbulent blood flow is transduced to the cell interior, resulting in the phenotypic cell changes associated with atherosclerosis (88). While the receptors responsible for sensing stiffness and shear forces have not been fully elucidated, several candidates exist, including integrins (251), cellular adhesion molecules (88), the transient receptor potential channel, TRPV4 (226), and others. Most of these receptors are capable of associating with the cytoskeleton, which is likely a key requirement for a mechanosensor. Further downstream, multiple signaling molecules involved in transducing mechanical information have been identified, including the Hippo pathway effector, Yap/Taz, which shuttles between the nucleus and cytosol in a stiffness-dependent fashion (89), the guanine nucleotide exchange factor GEF-H1 (140), and others. Future work will focus on further identification of relevant players in mechanosensation and transduction of such signals. In the lung, substrate stiffness can similarly be expected to present an important modulator of EC phenotype, although less work has been published on contextual biology in the pulmonary as compared to the systemic circulation. In physiological and pathological states, lung stiffness can be increased by many factors, including pulmonary hypertension (adding stiffness to the luminal face of the EC), interstitial fibrosis (which would stiffen the basal aspect of EC), high ventilation pressures, pulmonary edema, and infiltrative processes. In fact, one major hurdle in the exploration of the role of stiffness in the lung is manipulating and measuring stiffness in a whole organ that is subject to so many mechanical inputs, with such a high degree of regional variability. In spite of this difficulty, some key observations have been made. In cultured pulmonary artery EC and pulmonary microvascular EC, increasing substrate stiffness leads to enhanced sensitivity to barrier-disruptive agents, and concurrent rearrangements of the actin cytoskeleton, resulting in the formation of intracellular gaps (19). This echoes findings in the systemic circulation, where intimal stiffening leads to enhanced vascular leak and transmigration of immune cells (97), and may


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simultaneously underlie the poor reproducibility of in vivo endothelial permeability data in in vitro assays which commonly plate cells on substrates that are magnitudes stiffer than those observed in the intact lung (232). Furthermore, substrate stiffening enhanced the EC inflammatory phenotype seen after LPS treatment of cultured cells (133). Stiffening in the lung has also been shown to modulate PGE2 signaling, and causes fibroblasts to adopt a proliferative, fibrogenic phenotype (140). It is possible that substrate stiffness lies at the core of several pathologic pulmonary processes, including fibrosis, ventilator-induced lung injury, and others. Future work in this exciting field, combined with the development of improved whole organ and in vivo models of the lung in this context, are sure to yield fascinating developments in the field of pulmonary EC physiology.

Gender differences in the pulmonary endothelial cell Research into gender differences in disease, and appropriate representation of gender in human and animal studies, have been highlighted by the American Physiological Society (210). Several pulmonary disease entities have been reported to have gender differences in both disease incidence and severity, including ARDS and pulmonary hypertension. Mortality rates from ARDS has been shown to be worse for males (158), while the incidence of familial or idiopathic pulmonary hypertension favors females (45). These factors, along with a high level of interest in the differences in cardiovascular risk and disease burden between males and females, have led to an increasing interest in the basic mechanism behind these observed variations. Furthermore, a recent call from the NIH for research focusing on gender-related issues in health and disease has helped strengthen interest in this field (32). The presence of gender related differences in cardiovascular disease burden has led to research into the role of sex hormones in endothelial function. Estrogen receptors have been identified in both vascular smooth muscle and ECs [reviewed in (192)]. Estrogen is known to upregulate eNOS, which, as discussed above, is a fundamental mediator of EC function (92), as well as prostacyclin synthase and VEGF (192). A key component of the observed gender differences in endothelial biology is the renin-angiotensin system. Angiotensinogen gene transcription is enhanced (57), while renin, ACE, and the AT1 R are down-regulated by estrogen (65). Complex feedback mechanisms make the net result of estrogen on the renin-angiotensin difficult to tease out in total, but in summary, estrogen dampens angiotensin II activity (65). In contrast, components of the alternate pathway for angiotensin signaling are upregulated in females, including the Mas receptor, the AT2 R, and ACE2, which is encoded on the X chromosome, resulting in enhanced signaling through the ACE2/Ang-(1-7)/Mas axis (193). These changes in angiotensin signaling dynamics are an intense focus of research in vascular differences between males and females, and will likely yield key insights in the


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lung vasculature as well. Further investigation into such gender differences may help explain the differing burdens of pulmonary disease between genders, and perhaps could result in tailored therapies.

Conclusion Pulmonary ECs are uniquely positioned at an interface between several critical biological systems. The lining of the pulmonary vessels is in close structural and functional proximity to the airways, circulatory system, and the interstitium. As such, it is not surprising that the pulmonary endothelial monolayer plays critical regulatory roles in processes affecting all of these sites; including inflammation, coagulation, and the control of vascular tone. The research outlined above into the importance of gap junctions demonstrates the most direct manifestation of this concept, whereby ECs communicate directly with one another, as well as with vascular smooth muscle and cells of the innate immune system. On a similar conceptual level is autocrine signaling among ECs, during which mediators secreted by the EC—such as ET1—then act upon endothelial surface receptors to regulate cellular function. Paracrine signals again take advantage of the unique milieu of the pulmonary endothelium, since factors secreted by the pulmonary EC can influence vascular smooth muscle, immune cells, and platelets. In turn, factors secreted by blood cells, or presented on their surface, can then bind the pulmonary EC to shape the interaction between these two important tissues. Without question, the pulmonary EC monolayer is a gatekeeper of lung function. Ongoing research will continue to elucidate the role of the EC in regulating lung barrier function, protein metabolism, and both the local and systemic inflammatory response. A deeper understanding of pulmonary EC biology will help us to better understand the pathophysiology of acute and chronic lung diseases, including Acute lung injury (ALI) (198), pulmonary hypertension (25), Chronic obstructive pulmonary disease (COPD) (31), and fibrotic lung diseases (56). Our current knowledge base has allowed researchers to take great strides in the treatment of important clinical entities such as pulmonary hypertension. Further understanding of pulmonary endothelial biology will hopefully lead to novel therapies and biomarkers for devastating conditions for which targeted treatments continue to elude us, such as acute lung injury.

Acknowledgements Supported by the Canadian Institutes of Health Research (CIHR) and the Deutsche Forschungsgemeinschaft (DFG).

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Endothelial regulation of vascular tone.

An endothelial cell model for the investigation of the molecular regulation of fetal vascular tone.

Normal and pathophysiologic considerations of endothelial regulation of vascular tone and their relevance to nitrate therapy.

Impact of endothelial microparticles on coagulation, inflammation, and angiogenesis in age-related vascular diseases.

Role of endothelium-derived relaxing factor in regulation of vascular tone and remodeling. Update on humoral regulation of vascular tone.

Hemorrhagic shock primes for lung vascular endothelial cell pyroptosis: role in pulmonary inflammation following LPS.

Nitric oxide and regulation of coronary vascular tone.

Adrenergic and cholinergic regulation of bronchial vascular tone.

Role of endothelial cell-derived angptl2 in vascular inflammation leading to endothelial dysfunction and atherosclerosis progression.

Regulation of fetal vascular tone in the human placenta.

Endothelial microparticles mediate inflammation-induced vascular calcification.

Reduced contribution of endothelin to the regulation of systemic and pulmonary vascular tone in severe familial hypercholesterolaemia.

Regulation of pulmonary inflammation by mesenchymal cells.

Role of smooth muscle cell mineralocorticoid receptor in vascular tone.

Vascular endothelial growth factor signaling in hypoxia and inflammation.

Endothelial modulation of vascular tone: relevance to coronary angioplasty and restenosis.

Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis.

The pulmonary endothelial cell.

PKG-1α leucine zipper domain defect increases pulmonary vascular tone: implications in hypoxic pulmonary hypertension.

Transcription factor MEF2C suppresses endothelial cell inflammation via regulation of NF-κB and KLF2.

Endothelial Cell Junctional Adhesion Molecules: Role and Regulation of Expression in Inflammation.

Effect of Rho-kinase Inhibitor, Y27632, on Porcine Corneal Endothelial Cell Culture, Inflammation and Immune Regulation.

Platelets in inflammation: regulation of leukocyte activities and vascular repair.

A model of cytosolic calcium regulation and autacoids production in vascular endothelial cell.