Ebook The pulmonary endothelium: Part 2

(BQ) Part 2 book The pulmonary endothelium has contents: Hypoxia and the pulmonary endothelium, hypoxia and the pulmonary endothelium, therapeutic strategies to limit lung endothelial cell permeability, targeted delivery of biotherapeutics to the pulmonary endothelium,... and other contents.

18 Hypoxia and the Pulmonary Endothelium Matthew Jankowich, Gaurav Choudhary and Sharon Rounds Vascular Research Laboratory, Alpert Medical School of Brown University, Providence VA Medical Center, Providence, RI, USA INTRODUCTION Cellular responses to oxygen are critical for normal energy metabolism, mediator release, proliferation, and survival The lung has three sources of oxygen – from inspired gas, from the bronchial circulation, and from systemic venous blood returned to the lung via the right ventricle The endothelia of conduit pulmonary arteries and veins are not exposed to oxygen in alveoli and the endothelium of small pulmonary blood vessels does not benefit from the bronchial circulation The lung has unique responses to hypoxia – arterial vasoconstriction (hypoxic pulmonary vasoconstriction, see Chapter 12), vascular remodeling (see Chapters 11 and 27), and increased fluid flux into tissues (pulmonary edema, see Chapters 8, 21, and 24) Owing to these unique pulmonary physiologic responses to hypoxia, in this chapter we focus on the effects of hypoxia on pulmonary microvascular and arterial endothelium Less is known about effects of hypoxia on pulmonary venous endothelium and endothelium of bronchial vessels (see Chapter 14) Hypoxia is generally defined as a pO2 less than 60 torr or blood oxygen saturation less than 90%, based on the sigmoid shape of the oxyhemoglobin desaturation curve However, in the lung, endothelium of large pulmonary arteries is “normally” exposed to oxygen from mixed venous blood with a pO2 about 40 torr, while microvascular endothelial cells (ECs) are exposed to both mixed venous oxygen and alveolar pO2 of about 100 torr at sea level Thus, it is not surprising that there is heterogeneity in the response of lung vascular endothelium to hypoxia, depending upon the type of blood vessel Studies utilizing cultured pulmonary ECs have been very important in understanding responses to hypoxia However, studies of cultured cells are confounded by The Pulmonary Endothelium: Function in health and disease © 2009 John Wiley & Sons, Ltd ISBN: 978-0-470-72361-6 the fact that tissue culture media not have the same oxygen carrying capacity as hemoglobin, oxygen can diffuse through tissue culture plastic, and long-term studies of hypoxia may necessitate intermittent return of cultures to room air conditions when the medium is changed Indeed, while intact lungs display hypoxic vasoconstriction with ventilation by gas of FI O2 of 12%, it may be necessary to expose cultured ECs to oxygen concentrations of 3% or less to achieve a tissue culture media pO2 of less than 60 torr In addition, it is likely that intermittent hypoxia has more profound effects on reactive oxygen species (ROS) than sustained hypoxia [1] Thus, interpretation of cultured cell studies requires careful consideration of experimental conditions HYPOXIA AND PULMONARY EC METABOLISM, VIABILITY, AND PROLIFERATION In an early study from Una Ryan’s laboratory, Cummiskey et al compared responses to hypoxia of bovine pulmonary artery ECs (BPAECs) and bovine aortic ECs (BAECs) with respect to bioenergetic enzyme activities (pyruvate kinase, phosphofructokinase, and cytochrome oxidase) [2] They noted increased glycolytic enzyme activity upon exposure to pO2 15 torr for 48–96 h, but found no differences between the two cultured cell types They noted increased glycolytic enzyme activity in freshly isolated intimal strips from bovine pulmonary artery when compared to aorta strips, suggesting that increased glycolysis is also seen under hypoxic conditions in vivo Lee and Fanburg reported that BPAECs exposed to or 0% oxygen for up to 72 h displayed decreased cell proliferation and increased lactate release, but no change in ATP content, indicating a capacity to respond Edited by Norbert F Voelkel and Sharon Rounds 288 HYPOXIA AND THE PULMONARY ENDOTHELIUM to hypoxia with glycolysis [3] Tretyakov and Farber compared hypoxia-tolerant BPAECs to immortalized opossum renal tubular ECs, which are more sensitive to hypoxia [4] They found that the pulmonary artery cells exposed to 0% oxygen for up to 18 h were not damaged, displayed increased adenosine and guanosine uptake, and did not decrease cell ATP levels over 18 h hypoxic exposure Farber et al have further demonstrated that hypoxia-tolerant cultured main pulmonary artery endothelial cells express a specific set of stress proteins [5], including glyceraldehyde 3-phosphate dehydrogenase [6], non-neuronal enolase [7], and protein disulfide isomerase [8] Thus, it is apparent that PAECs that are exposed to low environmental oxygen in vivo are tolerant of hypoxia and can upregulate enzymes that enhance glycolytic capacity and activity of the transcription factor hypoxia-inducible factor (HIF)-1α [9] Farber et al have demonstrated that BPAECs and BAECs are both capable of proliferation and retain responsiveness to hypoxic stimuli when cultured long-term (5 days to 16 weeks) under hypoxic conditions [4, 10, 11] However, the rate of cell proliferation is slowed by hypoxia Interestingly, lung microvascular ECs have recently been reported to have a proproliferative and vasculogenic phenotype [12, 13] Since ECs from lungs of patients with pulmonary artery hypertension also replicate rapidly and display enhanced glycolytic capacity [14], it will be interesting to determine if there is a correlation between EC proliferative capacity and bioenergetics In summary, ECs from conduit pulmonary arteries are tolerant of hypoxia, and are able to enhance glycolysis and proliferate under hypoxic conditions Further research is needed to determine if there is heterogeneity in these responses among ECs from different parts of the pulmonary vasculature (see Chapter 9) HYPOXIA SENSOR(S) The pulmonary EC sensor(s) for hypoxia are not well described The pulmonary microvascular EC is appropriately positioned to sense alveolar hypoxia, thereby stimulating hypoxic pulmonary vasoconstriction of precapillary vessels of 60–100 μm internal diameter However, it is now generally accepted that pulmonary vascular smooth muscle is the primary sensor cell for hypoxic vasoconstriction, while the EC is capable of modulating the vasoconstrictor response by mediator release (see Chapters and 12) [15, 16] Nevertheless, it is useful to review the various hypoxia sensors that have been proposed since it is possible that these sensors also function in lung ECs (Table 18.1) Ward has categorized putative oxygen sensors as bioenergetic oxygen sensing mechanisms and biosynthetic oxygen sensing mechanisms [17] Among the Table 18.1 Candidates for hypoxia sensors Bioenergetic sensing mechanisms Mitochondrial ROS ATP production Redox state Biosynthetic sensing mechanisms ROS from NOXs CO from heme oxygenases H2 S from cystathione β-synthase and cystathione γ-ligase Cytochrome P450 monooxygenases HIF-1α bioenergetic sensors are mitochondrial ROS production, ATP production, and redox state (see Chapter 17) There is controversy as to whether hypoxia is sensed via increased mitochondrial ROS production from electron transport [18] or via decreased mitochondrial ROS production resulting in a more reduced redox state and inhibition of O2 -sensitive Kv channels [16] Previously investigators used chemical inhibitors of oxidative phosphorylation to assess the role of ATP production in oxygen sensing [19] However, the moderate degrees of hypoxia that elicit physiologically significant responses are not sufficient to suppress mitochondrial ATP production Thus, mitochondrial ATP production is probably not an important sensor of hypoxia in vivo Among the biosynthetic sensing mechanisms are NADPH oxidases (NOXs), inhibition of which could result in decreased ROS production However, mice deficient in the gp91phox -containing NOX, NOX2, had decreased ROS production, but preserved pulmonary hypoxic vasoconstriction [16] Pulmonary EC NOXs are similar to phagocyte NOXs and have been shown to play a role in ROS production in a variety of circumstances, such as inflammation and ischemia–reperfusion injury (see Chapter 17) However, there is no evidence that EC NOXs are important in sensing of hypoxia Heme oxygenases, HO-1, -2, and -3, have been suggested as oxygen sensors since they degrade heme to CO and biliverdin and Fe(II) in the presence of oxygen and NADPH [17], and since HO-1 and -2 are expressed in pulmonary arteries [20] In rat PAECs, HO-1 has been localized to plasma membrane caveolae in association with caveolin-1 [21] Thus, EC caveolae may act as a functional unit for HO-1 activity with modulation by caveolin-1 It is possible that HOs modulate pulmonary vasoconstrictor hypoxic responses via the product CO stimulating production of vasoconstrictor, endothelin [20] However, knockdown or inhibition of HO-1 and -2 did not prevent hypoxic vasoconstriction of pulmonary arteries [20] OTHER EFFECTS OF HYPOXIA ON PULMONARY ENDOTHELIUM 289 Cytochrome P450 monooxygenases include a large number of oxygen sensitive enzymes Most attention has been paid to those metabolizing arachidonic acid Among the products of ω-hydroxylases are hydroxyeicosatetraenoic acids (19- and 20-hydroxyeicosatetraenoic acidHETE) and of epoxygenases are cis-epoxyeicosatrienoic acids (EETs) However, arachidonic acid availability, rather than oxygen tension may be rate-limiting for these enzymes In elegant studies from the laboratory of Elizabeth Jacobs, cytochrome P450 4A (CYP4A) protein and mRNA have been localized in PAECs which also possess the capacity to synthesize the pulmonary vasodilator, 20-HETE [22] Hydrogen sulfide is another possible oxygen sensor [23] Like nitric oxide (NO) and CO, it is a gaseous molecule, soluble in tissues, and it is enzymatically generated in blood vessels in an oxygen-dependent manner H2 S is generated from cysteine via cystathione β-synthase and cystathione γ-lyase H2 S is a systemic vasodilator, like NO [24] The effects of H2 S may be mediated by ATP-sensitive K+ channels, by interaction with heme proteins such as cyclooxygenase, or by interactions with NO [25] Pulmonary artery ECs respond to H2 S generation with increased NOX activity [26], suggesting that the pulmonary endothelium is capable of responding to H2 S included oxidoreductases, collagens/modifying enzymes, cytokines/growth factors, receptors, signal transduction proteins, and transcription factors Genes suppressed by hypoxia in PAECs included those involved with cell proliferation, RNA binding and metabolism, and protein ubiquitination and proteosomal degradation Using serial analysis of gene expression (SAGE), Choi et al have assessed the effects of short-term hypoxia (1% oxygen for and 24 h) on human pulmonary artery and aortic ECs derived from a single donor and maintained in tissue culture under identical conditions [33] They found that hypoxia increased expression of stress-response genes, proapoptotic genes, and genes encoding extracellular matrix factors Surprisingly, hypoxia increased expression of genes encoding antiproliferative factors in pulmonary artery endothelium SAGE analysis demonstrated differences between human aortic and PAEC responses to hypoxia For example, hypoxia decreased expression of pulmonary endothelial genes encoding proteins involved in oxidative energy production, such as ATP synthase, and decreased transcription of a transcriptional regulator of glycolytic genes This is consistent with studies indicating increased glycolysis in hypoxic PAECs described above HYPOXIA AND GENE TRANSCRIPTION OTHER EFFECTS OF HYPOXIA ON PULMONARY ENDOTHELIUM The transcription factor HIF-1α induces expression of genes involved in erythropoiesis, angiogenesis, and ion channel expression [27] The mechanism of oxygen sensing by HIF-1α involves oxygen control of degradation of HIF-1α HIF-1α is ubiquinated and degraded in proteosomes when bound to von Hippel–Lindau tumor suppressor protein, which requires proline hydroxylation Pro564 and Pro402 of HIF-1α are hydroxylated by oxygen-dependent prolyl-hydroxylase-1 to -3 with Km for O2 slightly above atmospheric concentrations [28] The Asp803 of HIF-1α is hydroxylated also in an O2 -dependent manner by factor-inhibiting HIF-1 Thus, hypoxia prevents degradation of HIF-1α and thereby facilitates gene transcription Via HIF-1α action, hypoxia induces endothelial gene expression of vasoactive and angiogenic factors, including endothelin [29], platelet-derived growth factor (PDGF) [30], inducible (type II) NO synthase (nitric oxide synthaseNOS) [31], and thrombospondin [32] Among the angiogenic factors are vascular endothelial growth factor (VEGF), angiopoietin-1 and -2, placental growth factor, and PDGFβ Manalo et al have investigated gene expression (transcriptome) induced by hypoxia and/or by overexpression of HIF-1α in PAECs [9] Remarkably, they found that more than 2% of all genes in human ECs are regulated by HIF-1α The induced genes Hypoxic exposure changes the cellular morphology of pulmonary ECs Bernal et al have reported that rat pulmonary microvascular ECs contract reversibly when exposed to anoxic gas which reduced the medium pO2 to 13 ± torr [34] These results suggest that the EC cytoskeleton contracts in response to acute hypoxia and that this contractility may contribute to hypoxic constriction of partially muscularized or nonmuscularized small pulmonary vessels Exposure of PAECs to more sustained hypoxia (1.5% v/v oxygen for days) caused enlargement (megalocytosis) of cultured PAECs with enlargement of the Golgi [35] These changes were accompanied by the loss of cell surface endothelial NOS (endothelial nitric oxide synthaseeNOS) and appearance of eNOS in the cytoplasmic compartment in Golgi and endoplasmic reticulum, and loss of NO production at the cell surface Furthermore, eNOS colocalized with Golgi tethers and SNARES Similar changes were seen with senescent cultured ECs and with cells treated with monocrotaline – an agent causing pulmonary hypertension in animal models Similarly, Murata et al described loss of eNOS from the cell membrane in “atrophied” PAECs from rats exposed to week of hypoxia [36] Owing to these changes and reported ultrastructural changes in pulmonary ECs in pulmonary 290 HYPOXIA AND THE PULMONARY ENDOTHELIUM hypertension, it has been suggested that dysfunctional intracellular trafficking of eNOS in pulmonary ECs might contribute to the pathogenesis of pulmonary hypertension [37] Since optimal function of eNOS and vasodilator NO production requires appropriate protein–protein interactions (see Chapter 6), it is possible that reduced NO synthesis by hypoxic pulmonary ECs is due to effects of hypoxia on eNOS intracellular trafficking Farber et al have described interesting differences in responses to hypoxia between cultured systemic ECs and PAECs ECs from bovine systemic arteries responded to exposure to 10% oxygen (pO2 85 torr) and 3% oxygen (pO2 51 torr) with secretion of lipid-derived neutrophil chemoattractant activity, while main PAECs were less sensitive, requiring 0% oxygen (pO2 32 torr) [38] Similarly, PAECs were less responsive to hypoxia than aortic ECs in induction of lipid bodies [39] Lipid bodies are non-membrane-bound, lipid-rich cytoplasmic inclusions that are an intracellular store of fatty acids and may be a nonmembrane site of eicosanoid formation Finally, Farber has reported that cultured PAECs are slower than aortic ECs in synthesis of prostacyclin and thromboxane in response to acute hypoxia [40] These studies suggested that main PAECs (that are exposed to lower pO2 in vivo) are less responsive to hypoxic stimuli than ECs from the systemic vasculature, supporting the concept of heterogeneity of endothelium, depending upon the vascular bed (see Chapter 9) Hypoxia regulates production of polyamines by PAECs [41] The polyamines, putrescine, spermidine, and spermine, are low-molecular-weight compounds that are required for cell growth and differentiation, and may modulate other cell activities Lung polyamine contents are increased in hypoxia PAECs increase polyamine uptake with hypoxic exposure, although there is a decrease in the activity of the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase Hypoxia also modulates the production of heparan sulfates by PAECs [42, 43] Heparan sulfates are cell surface-associated proteoglycans that help maintain an antithrombotic EC surface by catalyzing thrombin inactivation by antithrombin III Karlinsky et al reported that hypoxic exposure (3% oxygen for 24 h) decreased heparan sulfate production by both pulmonary artery and aortic ECs [43] INTERMITTENT VERSUS SUSTAINED HYPOXIA AND PULMONARY ENDOTHELIAL CELLS Sustained hypoxia complicates high-altitude exposure and lung diseases, such as chronic obstructive pulmonary disease and interstitial pulmonary fibrosis Chronic intermittent hypoxia is seen in the common condition, obstructive sleep apnea, in which brief apneas or hypopneas during sleep result in frequent, intermittent decreases in oxygen saturation Sustained hypoxia causes pulmonary hypertension and right ventricular failure, but does not increase systemic blood pressure On the other hand, intermittent hypoxia results in more modest degrees of pulmonary hypertension, but sustained systemic hypertension, myocardial ischemia, and neuronal injury [1] Studies of non-ECs indicate that the degree of oxidative stress and inflammation may be greater with intermittent hypoxia, as compared to sustained hypoxia [1] Studies of gene transcription in rat lungs showed that intermittent hypoxia induced genes involved in ion transport and homeostasis, neurological processes, and steroid hormone receptor activity [44], while sustained hypoxia induced genes principally participating in immune responses Transcriptional responses to chronic intermittent hypoxia [45] and post-translational protein modifications during chronic intermittent hypoxia [46] are just beginning to be understood For example, intermittent hypoxia has been shown to increase HIF-1α phosphorylation in cultured ECs via protein kinase A [47] Little is known regarding effects of intermittent hypoxia on pulmonary ECs HYPOXIA AND PULMONARY VASCULAR PERMEABILITY Pulmonary ECs can modulate vasoconstriction and the proliferation of adjacent vascular smooth muscle The effects of the hypoxic pulmonary endothelium on vasoreactivity are described in Chapter 12, while effects on pulmonary vascular remodeling are described in Chapter 11 In this chapter we focus on hypoxia effects on pulmonary endothelium that result in changes in lung vascular permeability The effect of hypoxia on permeability of the pulmonary endothelial barrier has been a topic of controversy for decades A variety of experimental models, ranging from in vivo animal studies to isolated perfused lung models, to studies of cultured endothelial monolayer permeability, have attempted to address the question of whether hypoxia alone directly alters pulmonary endothelial barrier function This question is most directly relevant to the study of the pathogenesis of high-altitude pulmonary edema (HAPE) – the most common situation in which global alveolar hypoxia occurs and a condition in which altered vascular permeability is implicated In addition, in 1942, Madeline Warren and Cecil Drinker, pioneers in the study of hypoxic pulmonary vascular permeability, postulated that pulmonary edema caused by regional hypoxia could be conceived to contribute to “a vicious circle” of regional hypoxia leading to localized CELL SIGNALING AND PULMONARY ENDOTHELIAL PERMEABILITY pulmonary edema, resulting in further impairment of gas exchange and worsening of hypoxemia [48] EFFECTS OF HYPOXIA ON CULTURED PULMONARY EC MONOLAYER PERMEABILITY Understanding of molecular pathways involved in endothelial permeability has expanded tremendously in recent years In vitro studies using cultured ECs have demonstrated alterations in endothelial monolayer permeability under controlled hypoxic conditions and tentative elucidation of the mechanisms involved Alterations in endothelial monolayer permeability with hypoxia were initially demonstrated in vitro using cultured BAECs [49] In this study, permeability of the endothelial monolayer to radiolabeled macromolecules was increased after 24 h in hypoxia The relative increase in permeability was dependent on both the duration and the degree of hypoxia, and was reversible within 48 h of restoration of normoxia The permeability changes were associated histologically with the formation of intercellular gaps and alterations in the actin cytoskeleton (see Chapter 8) A mild increase in monolayer permeability to albumin was demonstrated after only 90 of exposure to a similar level of hypoxia in another study using BPAECs [50] In this study, reoxygenation worsened barrier function, an effect prevented by antioxidants Increased monolayer permeability to dextran was seen within h of exposure to hypoxia in experiments with porcine PAECs [51] Other work utilizing bovine pulmonary microvascular ECs demonstrated that ECs derived from the pulmonary microcirculation also responded to hypoxia with increased permeability after h of hypoxia, associated with the formation of intercellular gaps and stress fiber formation However, after 24 h of hypoxic incubation there was restoration of barrier function and resolution of intercellular gaps [52] In this study, the oxygen content of the tissue culture medium at 24 h was greater than at h, raising the question of whether the improvement in permeability with more prolonged hypoxic EC incubation was related to the apparent increase in available environmental oxygen Pulmonary ECs derived from animals exposed to chronic hypoxia after birth displayed increased monolayer permeability even under normoxic conditions, suggesting that chronic hypoxic exposure induced persistent effects on endothelial permeability [51] In summary, studies of cultured pulmonary ECs have established that endothelial monolayer barrier function is impaired by hypoxia alone in a dose–response relationship and that monolayer permeability changes following acute hypoxia were generally reversible following a return to normoxia These 291 principles derived from tissue culture experiments are helpful in interpreting the results of in vivo experiments of pulmonary vascular permeability using widely varying levels of hypoxia and conducted over various time courses CELL SIGNALING AND PULMONARY ENDOTHELIAL PERMEABILITY Molecular transport across the endothelial barrier can occur via paracellular and transcellular pathways [53] (see Chapter 8) Most attention in hypoxia-induced endothelial permeability signaling has focused on paracellular transport involving signaling pathways which cause cell rounding and intercellular junction disassembly via regulation of the actin–myosin apparatus and cell junction stability Morphologic changes in the actin cytoskeleton are seen following exposure of pulmonary arterial and microvascular ECs to hypoxia, with disassembly of the cortical actin band and formation of intracellular stress fibers [51, 52, 54], mediating changes in EC shape during hypoxia Intercellular junctions are dispersed during hypoxia [51], allowing intercellular gaps to form [54] These cytoskeletal rearrangements, well recognized following endothelial exposure to other permeability enhancing agonists such as thrombin, allow for increased paracellular permeability of the EC monolayer to small and large molecules under hypoxic conditions Multiple intracellular signaling pathways influence endothelial barrier maintenance and permeability, including signaling via cAMP, small GTPases, p38 mitogenactivated protein kinase (MAPK) and ROS; many of these systems have been demonstrated to influence endothelial permeability in hypoxia Hypoxia-induced BPAEC monolayer permeability was associated with decreases in cAMP and adenylate cyclase activity, and cAMP analogs or activators of adenylate cyclase could restore barrier function [54] Dexamethasone prevented the increase in monolayer permeability if given before or at the time of exposure to hypoxia, and prevented the decrease in cAMP seen with hypoxia exposure, but could not completely restore barrier function if given after exposure to hypoxia for 12 h or more [54] In homogenized lung tissue preparations exposed to hypoxia, no decrease in cAMP content was observed compared with normoxic lung preparations, but hypoxic perfused lung preparations showed decreased ability to synthesize cAMP in response to terbutaline, as measured by lung perfusate cAMP levels [55] These results support a role for cAMP second messenger signaling in the maintenance of the pulmonary vascular barrier in normoxia, whereas decreases in adenylate cyclase activity and secondarily cAMP result in hypoxia-induced alterations in barrier permeability 292 HYPOXIA AND THE PULMONARY ENDOTHELIUM The mechanisms leading from hypoxia to altered adenylate cyclase activity in hypoxia likely involve Ca2+ – an inhibitor of selected adenylate cyclase isoforms (Figure 18.1) Hypoxia leads to a transient spike in intracellular calcium content in BPAECs, followed by a higher baseline calcium level [56] Hypoxia induces an increase in cytosolic calcium in human umbilical vein ECs (HUVECs) as well [57] Increases in intracellular calcium have been shown to inhibit BPAEC cAMP production; BPAECs express a Ca2+ -inhibitable isoform of adenyl cyclase [58] Intracellular Ca2+ levels and cAMP activity are inversely related in ECs, and normoxic monolayer permeability results from increased intracellular Ca2+ via decreased cAMP [58] Intracellular Ca2+ concentration changes induced by hypoxia are likely involved in mediating the decrease in EC adenylate cyclase activity observed in hypoxia (see Chapters and regarding pulmonary EC calcium) Regulators of actin have been implicated in hypoxia-induced increases in endothelial monolayer permeability The p38 MAPK is activated in hypoxic rat microvascular ECs [110] A substrate of p38, MK2, a protein kinase activated by hypoxia, appears to regulate actin redistribution in hypoxic pulmonary microvascular ECs [111] Inhibition of p38 MAPK attenuates the permeability changes induced by hypoxia in both microand macrovascular pulmonary ECs [59] Overexpression of the p38 substrate MK2 leads to analogous cytoskeletal changes to those seen in hypoxia and expression of dominant-negative MK2 blocks hypoxia-induced actin reorganization Heat shock protein HSP27 appears to mediate the interaction between MK2 and the actin cytoskeleton [111] Thus, the p38 pathway appears to regulate cytoskeletal alterations mediating microvascular endothelial monolayer permeability in hypoxia (Figure 18.1) Rho GTPases are also among the key regulators of the actin cytoskeleton [60] In hypoxia, activity of the small GTPase Rac1 falls while conversely RhoA activity increases in PAECs [61] (Figure 18.1) Inhibitors of RhoA and its downstream effector, RhoA kinase, prevent actin redistribution seen with hypoxia, while Rac1 inhibitors prevent recovery of barrier function following reoxygenation, suggesting differential roles of these interrelated small GTPases in barrier regulation [61] ROS produced via the NOX pathway appear to be critical regulators of small GTPase activity in lung ECs [61] The role of small GTPases in regulating the cytoskeletal response of ECs to hypoxia is analogous to their role in regulating cytoskeletal rearrangements leading to permeability changes induced by inflammatory stimuli The role of ROS in hypoxia-induced signaling cascades associated with endothelial monolayer permeability changes is incompletely understood Antioxidants can prevent the increase in permeability of monolayers of HUVECs associated with hypoxia [62] as well as reoxygenation [50] Endothelial-derived interleukin (IL)-6, via autocrine and paracrine pathways, acts downstream of ROS to effect changes in HUVEC monolayer permeability in a finely tuned mechanism sensitive to interleukin-6 levels [62] However, IL-6 production seems unlikely to represent the sole effector mechanism in permeability changes induced by ROS in hypoxia There is in fact contradictory evidence regarding the effects of hypoxia on free radical production in ECs In ECs derived from porcine pulmonary arteries, ROS are decreased in the setting of hypoxia (3% O2 ) of h duration [51], whereas in HUVECs, ROS formation is increased by hypoxia (1% O2 ) within h [62] Both decreased ROS production and increased ROS production have been implicated in initiating different intracellular signaling pathways involved in endothelial barrier function changes These differing observations may be related to species differences, EC vascular bed/tissue origin differences, or the specific experimental conditions and techniques employed Further work is needed to better comprehend ROS signaling as related to endothelial permeability changes in hypoxia Potential extracellular stimulants of hypoxia-induced pulmonary vascular permeability include VEGF and inflammatory cytokines such as tumor necrosis factor (TNF)-α (Figure 18.1) TNF is a well-recognized vascular permeability agonist [53] Hypoxia induces TNF-α production by pulmonary ECs, especially microvascular ECs, which may result in autocrine or paracrine effects on endothelial permeability [59], amplifying the permeability effect of hypoxia As noted above, IL-6 is another proinflammatory cytokine that has been implicated in hypoxia-induced permeability and ROS produced by inflammatory cells recruited to hypoxic endothelium may provoke endothelial permeability alterations as well There is considerable overlap between the EC molecular and phenotypic responses to hypoxia and to inflammation, and shared signaling pathways are likely involved in the increased vascular permeability seen in both conditions There is emerging evidence that the principal transcriptional pathways in inflammation, governed by nuclear factor-κB (NF-κB), and in hypoxia, governed by HIF-1α, are linked by molecular cross-talk [63] Evidence from non-EC models suggests a dependence of HIF-1α transcription on NF-κB [64] as well the ability of HIF-1α to induce NF-κB expression [65, 66] If these findings are extended to ECs, this would help explain the characteristic induction of inflammatory responses, including permeability alterations, by hypoxic ECs VEGF also increases vascular permeability [67] through effects on endothelial barrier function [68] CELL SIGNALING AND PULMONARY ENDOTHELIAL PERMEABILITY Indeed, VEGF was originally called “vascular permeability factor” VEGF signaling has been implicated in the increased vascular permeability edema seen in ischemia–reperfusion lung injury [69] VEGF expression is upregulated by hypoxia via HIF-1α in many cell types, including cultured HUVECs and lung epithelial cells, and in vivo [70–72] This suggests the possibility of paracrine stimulation of lung ECs by VEGF leading to increased vascular permeability in the setting of hypoxia The role of autocrine stimulation has been challenged as lung ECs were not seen to produce VEGF in vivo [73], although the capability of lung microvascular ECs to produce VEGF has been demonstrated [74] The relevance of VEGF to increased vascular permeability in vivo is questionable, as hypoxia upregulates VEGF receptor (vascular endothelial growth factor receptorVEGFR)-1 expression in lung ECs [73], but VEGFR-2 signaling seems most relevant to vascular permeability, with VEGFR-2 stimulation resulting in alterations in the integrity of adherens junctions [75] In vivo, serum venous [76] and capillary [77] VEGF levels not increase in hypobaric hypoxia, even in human subjects with altitude sickness, though these values may not reflect local lung expression levels of VEGF While VEGF is a plausible candidate molecule for affecting vascular permeability changes in hypoxia, the role played by VEGF signaling in inducing increased lung endothelial permeability in hypoxia is at present uncertain Bradykinin induces vascular permeability through pathways not involving Rho GTPase or myosin light chain kinase [53] Bradykinin does not potentiate aortic endothelial monolayer permeability induced by hypoxia [78] However, neprilysin, an enzyme present in the lung which degrades bradykinin, is downregulated by hypoxia in rats; hypoxic exposure of rats was associated with increased lung vascular permeability; the increased lung vascular permeability of hypoxia correlated with the decrease in neprilysin expression [79] This suggests a possible role for unopposed bradykinin and/or substance P (a related neuropeptide also degraded by neprilysin) signaling in hypoxia-induced lung vascular permeability (Figure 18.1) In summary, exposure to environmental hypoxia induces alterations in cytoskeletal arrangement and intracellular junction disassembly in lung and other ECs, leading to increased paracellular permeability to small and large molecules This suggests that increased permeability pulmonary edema in vivo, induced by hypoxia alone, is indeed plausible Mechanisms involved in mediating monolayer permeability changes have been examined in some detail; parallels to signaling pathways involved in vascular permeability induced by other agonists have been noted The requirement for prolonged duration of hypoxia (hours) suggests that increased monolayer permeability requires protein expression Endothelial Monolayer HYPOXIA HYPOXIA Endothelial Cell p38 HYPOXIA HIF-1a Ca2+ RhoA Neprilysin NF-κB Rac VEGF cAMP Bradykinin Stress Fiber Formation 293 TNF-a , IL-6, Pro-inflammatory mediators Increased Paracellular Permeability Figure 18.1 Signaling of hypoxia-induced increases in pulmonary endothelial paracellular permeability 294 HYPOXIA AND THE PULMONARY ENDOTHELIUM EFFECT OF HYPOXIA ON VASCULAR PERMEABILITY OF ISOLATED PERFUSED LUNGS Isolated perfused lung models of hypoxia-induced vascular permeability edema are helpful in that the isolated perfused lung model allows manipulation of the perfusing pressure – a factor which confounds most in vivo studies given the hypoxic vasoconstrictive response Most [55, 80, 81], but not all [82, 83], have demonstrated alterations in pulmonary vascular permeability or lung extravascular water content in isolated perfused lung preparations exposed to alveolar hypoxia Kinasewitz et al utilized isolated blood-perfused canine lungs, encased in a water impermeable membrane, and measured fluid and protein filtration into the artificial pleural space created by the membrane in the presence and absence of hypoxia They utilized a calcium channel blocker to prevent hypoxic vasoconstriction; hydrostatic pressures were similar in the hypoxic and normoxic lung preparations In this study, the hydraulic conductivity doubled and the diffusional permeability of protein tripled under hypoxic conditions (0% O2 ) Greater protein concentration was measured in the pleural fluid collected from hypoxic preparations, consistent with increased permeability of the pleural capillary endothelium [80] In this study, xanthine oxidase inhibition prevented the increased permeability associated with hypoxia, implying a role for free radicals in inducing the permeability change [80] Parker et al demonstrated an increase in the pulmonary capillary filtration coefficient in hypoxic isolated perfused dog lungs; in this study, perfusion pressures were maintained constant and no increase in capillary hydrostatic pressures occurred during hypoxia [81] The authors attributed the increase in filtration coefficient to increased vascular permeability, as an increase in surface area for exchange seemed unlikely in the constant pressure system Dehler et al utilized isolated perfused rat lungs perfused at constant pressures and exposed to varying levels of oxygen (1.5–35%) They measured lung edema formation by changes in weight, and observed an earlier weight gain in lungs exposed to hypoxia [55] Bronchoalveolar lavage of hypoxic lung preparations demonstrated 2.5-fold greater protein content in the bronchoalveolar lavage fluid in hypoxia compared with normoxia The authors interpreted their findings as being consistent with an increase in vascular permeability caused by hypoxia In contrast, a study by Aarseth et al demonstrated no change in the water content of hypoxic isolated rat lung preparations compared with control lungs exposed to normoxia [82] The contradictory results may be related to the brief (4-min) hypoxic exposures utilized by Aarseth et al., which, based on in vitro and in vivo data, may have been too short to allow for permeability changes and significant increased fluid filtration to occur Overall, the data from isolated lung preparations are consistent with the notion that hypoxia increases lung vascular permeability and causes lung edema These studies are consistent with studies of cultured pulmonary endothelial monolayers which also display increased permeability under hypoxic conditions Isolated lung preparations are helpful, in that perfusate hydrostatic forces can be maintained at constant levels, thus allowing the examination of permeability changes in isolation from the hypoxia-induced changes in hydrostatic forces which occur in vivo Isolated lung preparations may be limited in that isolation of the heart and lungs is associated with some delay in perfusion which may cause tissue injury via ischemia–reperfusion and potentially accentuate the effects of subsequent exposure to hypoxia EFFECT OF HYPOXIA ON LUNG VASCULAR PERMEABILITY IN ANIMALS Animal models have generated the most controversy in the study of pulmonary vascular permeability in hypoxia Initial studies in anesthetized, ventilated dogs by Warren and Drinker utilized the rate of lymphatic outflow from the lungs as a surrogate for the measurement of lung fluid filtration They demonstrated rapid increases in lymphatic flow from the lungs following exposure to hypoxia (8.6% O2 ), concluding that “the pulmonary capillaries are peculiarly susceptible to oxygen lack as a cause of increased permeability” [48] Their hemodynamic data were limited, although in a subsequent study they demonstrated a fall in cardiac output with hypoxia, concluding that increased flow was not a cause of the increased lymphatic production observed during hypoxia [84] Many animal studies examining lung permeability changes in hypoxia would follow Warren and Drinker’s seminal work, with conflicting and confusing results A number of studies have demonstrated no alteration in pulmonary vascular barrier function in hypoxia [85–87]; other studies suggested that hypoxia only produced or exacerbated pulmonary edema due to an increase in hydrostatic forces and did not increase permeability per se [88, 89] These studies raise the question of whether increased permeability edema due to hypoxia exists in vivo However, many other experiments in animals have demonstrated an increase in pulmonary vascular permeability with hypoxia, including experiments in puppies [90], dogs [91], and rats [79, 92] Rats clearly develop histological evidence of pulmonary edema with hypoxia, with initial perivascular edema after exposures of less than h [93], which then progresses to frank alveolar edema accompanied by inflammation with longer exposure times [94] Furthermore, pulmonary edema occurs HAPE AND ALTERED LUNG VASCULAR PERMEABILITY IN HYPOXIC HUMANS in humans at altitude in the setting of hypobaric hypoxia and is associated with increased permeability of the pulmonary vascular barrier [95], although other factors, including altered hydrostatic forces, are clearly involved in this disease [96] The balance of evidence suggests that in some species, including humans, exposure to hypoxia is associated with increased pulmonary vascular permeability and pulmonary edema The animal studies that have not shown evidence of hypoxia-induced increased permeability may be due in part to genetically determined species differences For example, sheep are particularly resistant to vascular permeability changes caused by hypoxia, whereas rats appear more vulnerable [92] This is plausible, given that different species, such as domestic cattle and yaks, have genetically determined differences in pulmonary circulatory responses to hypoxia, as well as a different morphology of ECs [97] The exposure time in experiments that have failed to demonstrate pulmonary edema with hypoxia in vivo may have been too short; in rats, hypoxia-induced pulmonary edema is most prominent after 16 h of exposure [94] This is intriguing, in that most humans with HAPE develop symptoms 12 h or more after ascent to altitude Alterations in endothelial barrier function are not necessarily immediate, in some experiments taking h [52] or more [49] to develop This suggests that short exposure times [86, 87] may have been too brief to allow significant alterations in endothelial barrier function to occur However, it is also likely that some species are resistant to the development of pulmonary edema with hypoxia even after relatively prolonged exposure times For example, Bland et al exposed three sheep to 10% O2 for 48 h without finding any evidence of pulmonary edema on postmortem examination [85] While a number of studies support the concept that hypoxia can alter pulmonary vascular permeability in vivo, there is a relative paucity of data to exclude the hemodynamic consequences of hypoxia as a cause of this increase in permeability Stelzner et al was able to show that a short term elevation of the pulmonary artery pressure caused by hypoxic pulmonary vasoconstriction did not affect the protein leak index in rats, whereas the measured protein leak index as well as gravimetric lung water did increase after 24–48 h of exposure to hypoxia [92] In this study, dexamethasone reduced transvascular protein leak without affecting pulmonary hemodynamics, while adrenalectomy exacerbated the pulmonary vascular permeability The authors concluded that increased hydrostatic pressures alone not explain the vascular permeability induced by hypoxia This is in keeping with the observations of hypoxia-induced increased vascular permeability in isolated perfused lung models in which perfusion pressures were kept constant [80, 81] Therefore hemodynamic forces are not likely to be the sole 295 determinants of increased pulmonary vascular permeability in hypoxia In summary, the balance of evidence from animal models, coupled with observations of pulmonary edema due to hypobaric hypoxia in humans, suggests that hypoxia can stimulate pulmonary edema formation in at least some species The evidence from cultured cell studies and experiments with isolated perfused lung models coupled with observations in animal models demonstrates that, in susceptible species, hypoxia induces alterations in endothelial barrier function, even in the absence of alterations in hydrostatic forces, which lead to increased paracellular protein and solute leak, manifesting as increased permeability pulmonary edema This paracellular leak does not manifest immediately, as demonstrated in vitro, requiring hours to occur and being seen in vivo following several hours of exposure to hypoxia HAPE AND ALTERED LUNG VASCULAR PERMEABILITY IN HYPOXIC HUMANS Lung histology and protein content of bronchoalveolar lavage indicate that HAPE is an increased permeability type of pulmonary edema [95, 98, 99] As the main site of hypoxic pulmonary vasoconstriction is known to be the precapillary arterioles, relating pulmonary edema to altered hydrostatic forces at the capillary level was conceptually difficult Early hemodynamic data did not support elevation of the pulmonary capillary wedge pressure in patients afflicted with HAPE [100] However, lowering of elevated pulmonary arterial pressures using vasodilator therapy improves oxygenation in this condition [101], suggesting a role for hydrostatic pressures in the pathogenesis of the pulmonary edema Elevated pulmonary capillary pressures may occur in HAPE-susceptible subjects as measured by pulmonary artery pressure decay curves, even in the absence of elevations in the pulmonary capillary wedge pressure [102] Reconciling precapillary vasoconstriction, which would protect the pulmonary capillaries from elevated hydrostatic pressures, with the pulmonary edema that occurs in HAPE has been accomplished through the hypothesis of heterogeneous vasoconstriction as initially postulated by Hultgren, discussed in Bartsch 96 Heterogeneous pulmonary vasoconstriction in response to hypoxia would cause regional elevations in pulmonary capillary pressures in vessels not protected by vasoconstriction It is possible that capillary mechanical stress failure subsequently occurs in those unprotected capillaries, thereby explaining the increased permeability edema seen in this disorder [96] Ischemia–reperfusion injury could also potentially occur in this setting as regions of lung with low perfusion subsequently become reperfused as regional vasoconstriction lessens Defects in alveolar fluid clearance have also been 296 HYPOXIA AND THE PULMONARY ENDOTHELIUM proposed as an adjunctive mechanism, as heterogeneous pulmonary vasoconstriction alone may be insufficient to induce this disorder [103] A role for altered endothelial permeability due to hypoxia in the pathogenesis of HAPE, regulated by cytoskeletal changes occurring at the EC, is attractive for several reasons Increased vascular permeability caused by hypoxia takes hours to occur in most cultured cell and in vivo models, consistent with the observed delay in onset of HAPE for hours or even days after exposure to hypobaric hypoxia In contrast, capillary stress failure due to increased pressures occurs within a few minutes of exposure to elevated hydrostatic pressures [104, 113] Other conditions in which pulmonary capillary stress failure has been postulated to occur, such as neurogenic pulmonary edema and the pulmonary edema occurring with extreme exercise [113], are of rapid onset, consistent with the time course of capillary stress failure The transmural pressures associated with capillary stress failure in animal models [104] are much higher than the presumed transmural forces suggested by the capillary pressures recorded in humans with HAPE [102] As reviewed above (Section “Effects of Hypoxia on Cultured Pulmonary EC Monolayer Permeability” and “Cell Signaling and Pulmonary Endothelial Permeability”), altered endothelial barrier function induced by hypoxia is rapidly reversible upon exposure to normoxia, consistent with the reversibility of HAPE with oxygen or return to lower altitudes Ready reversibility seems incompatible with the tissue breaks observed in animals exposed to high transmural pressures resulting in capillary stress failure However, Elliot et al have shown that exposure to low transmural pressures following exposure to high transmural pressures did result in fewer apparent stress breaks, suggesting that stress failure is reversible [105] Disruptions of the alveolar-capillary barrier have been demonstrated in an animal model of HAPE [113], although it is not clear that the ultrastructural changes seen in this model are incompatible with the occurrence of increased permeability due to regulated cell–cell junction and membrane alterations in cells induced by hypoxia In summary, current data does not exclude a role for altered endothelial permeability due to hypoxia in HAPE; the time course of altered endothelial permeability regulated by cytoskeletal and junctional changes induced by hypoxia is more consistent with the time course observed in the development of HAPE in humans than the stress failure hypothesis Hypoxia-induced, cytoskeletally regulated endothelial permeability changes would potentially explain the occurrence of HAPE in humans at relatively low capillary pressures and hypoxia-induced edema in isolated perfused lung models under conditions of controlled hydrostatic pressures (see Chapter 20 for pressure/flow-induced changes in pulmonary endothelial function) Altered endothelial permeability due to hypoxia is compatible with the finding that lowering pulmonary artery pressures results in improvements in clinical parameters in HAPE Increases in either intravascular hydrostatic forces and/or membrane permeability favor fluid filtration out of the vascular space per the Starling equation, and improvements in either or both of these parameters would result in decreased fluid filtration across the alveolar–capillary barrier Vasoactive agents including inhaled NO [101] and the phosphodiesterase inhibitor tadalafil [106] are effective in treatment or prevention of HAPE, confirming, but not proving, a role for elevated hydrostatic forces in the formation of HAPE Intriguingly, increases in cGMP mediated by NO and sildenafil may decrease endothelial barrier dysfunction induced by hypoxia in vitro, suggesting that these agents may have vasomotor tone-independent in vivo [107] Dexamethasone, not conventionally regarded as a vasoactive agent, is effective in prophylaxis against the respiratory symptoms of acute mountain sickness [108] and in preventing HAPE [106] Dexamethasone minimized the increase in pulmonary artery pressures occurring with exposure to hypobaric hypoxia [106] and has other effects in vivo, including the regulation of gene expression, anti-inflammatory properties, and effects on barrier function The mechanism of action of dexamethasone in preventing HAPE remains incompletely understood; alterations in cGMP levels, inflammatory mediators, and vascular barrier function are all possible [106] Agents effective at preventing HAPE may have pleiotropic effects in addition to their beneficial effects on pulmonary hemodynamics that contribute to their usefulness in this condition Improved understanding of the mechanisms of altered endothelial permeability in HAPE holds the potential for novel prophylactic agents and treatments of HAPE that may contribute to the role of vasoactive agents in this condition CONCLUSIONS AND PERSPECTIVES Pulmonary ECs respond to hypoxia and these responses may be important in modulating lung vascular responses to hypoxia Although the EC sensor(s) for hypoxia are poorly defined, it is likely that they are similar to those demonstrated in other tissues, including pulmonary vascular smooth muscle Since there are differences between ECs from pulmonary conduit arteries and systemic arteries, it is likely that ECs within the lung circulation will also differ in response to hypoxia, dependent upon the INDEX neovascularization 219–20 overview 217 physiological function 218–23 progenitor cells 223 structural features 217–18 bronchiolitis obliterans syndrome (BOS) 417, 426, 428–9 bronchopulmonary dysplasia (BPD) 15, 210–12 c-FLIP protein 251 c-Jun N-terminal kinase (JNK) 246, 267 c-Met 405–6 C/EBP homologous protein (CHOP) 246–7 Ca2+ channels 73–88 bronchial vasculature 222 calcium entry 75–83 calcium release 73–5 disease pathogenesis 418 EC heterogeneity 130–8 endoplasmic reticulum stores 73, 74–5, 83 endothelial function 34, 39, 41 endothelial permeability 84–5, 117, 119, 121–2 hypoxia 292 lung endothelial phenotypes 129–42 mechanical forces 312–14, 316–22, 326 nitric oxide 94 oxidant-mediated signaling 265, 273 phospholipase C 73–4 potassium/sodium ions 76, 85–6 pulmonary circulation 130–1, 139, 389–90 regulation 80–1 T-type calcium channels 79–80 therapeutic strategies 338 transient receptor potential channels 74, 75–9, 81–6 vasomotor control 186, 191, 192–6 Ca2+ -activated Cl− channels 195 Ca2+ -activated K+ channels 191, 194–5 Ca2+ -permeable nonselective cation channels 193 CAD see caspase-activated DNase cadherins adherens junctions 33, 35, 43 anatomy of the PE 28 Ca2+ channels 74 cytosketal role 35 E-cadherin dynamics 35–6 endothelial function 33–7, 40 endothelial permeability 114, 117, 119, 122 function 36–7 GTPases 36 interactions among junctional proteins 41–3 mesenchymal–endothelial interactions 177 oxidant-mediated signaling 271 phosphorylation 34–5 protein mapping 229 subtypes 33–4 surface metabolic functions 109 therapeutic strategies 343, 346, 348 thromboembolism 477 CAFs see carcinoma-associated fibroblasts 497 calmodulin endothelial permeability 119 mechanical forces 317, 321 nitric oxide 94 oxidant-mediated signaling 265 therapeutic strategies 338 calmodulin protein kinase (CaMK) 39, 75, 82 cAMP see cyclic AMP canalicular stage 10 cancer see malignancies capillaries anatomy of the PE 26–9 Ca2+ channels 130–1, 139 leukocyte–endothelial interactions 154–6 mechanical forces 315–16 vasculogenesis carbon monoxide 437 carcinoma-associated fibroblasts (CAFs) 170 cardiogenic pulmonary edema 323–4 caspase-activated DNase (CAD) 245, 248 CAT-1 see cationic amino acid transporter catalase 266 catenins endothelial function 34–6 endothelial permeability 118–19 surface metabolic functions 109 therapeutic strategies 343 cationic amino acid transporter (CAT-1) 90–2, 95, 97–8 cavaolae-mediated transcytosis 120, 123 caveolae anatomy of the PE 26–7 endothelial permeability 115–17, 120 protein mapping 233–4 caveolar transcytosis 361 caveolin-1 Ca2+ channels 80–1 endothelial permeability 120, 122 mechanical forces 314 protein mapping 233–4 vasomotor control 189 caveolin-enriched microdomains (CEMs) 346, 405–6 CC chemokine ligand (CCL2) 420 CD34 antigen 11 cdk see cyclin-dependent kinase CECs see circulating endothelial cells cell death 243–60 apoptosis 243–55, 438–9, 441–2, 451, 464 autophagy 243–4, 249 ceramide 251 collagen vascular diseases 464 control 247 detection methods 247–9 disease pathways 252–5 emphysema 438–9, 441–2 ER stress-induced pathways 246–7, 249–50 growth factor signaling 250–2 lipopolysaccharide 251, 253 lungs 245, 252–5 498 cell death (continued ) mediation 249–52 mitochondrial DNA damage 251–2 necrosis 243–5, 249 overview 243–4 oxidant-mediated signaling 271–2 pulmonary hypertension 254, 451 signaling pathways 244–7 cell distention 274 cell distortion 310–11 cell–ECM interactions 51–72 angiogenesis 53, 60, 64 basement membrane 51–3 cell cycle regulation 59–60 components 51–9 DG contacts 55, 57–9 dysregulated ECM 52–3 ECM remodeling 52 fibrillar adhesions 53–9 focal adhesions 53–9 focal contacts 53–9 functional effect 59–64 hemidesmosomes 55, 57, 59 junction types 53–9 PE barrier function maintenance 60–4 podosomes 54, 57–8 pulmonary disease 52–3 pulmonary vasculature 59–64 CEMs see caveolin-enriched microdomains CEPs see circulating endothelial progenitor cells ceramide 251 CF see cystic fibrosis CFU see colony-forming unit cGMP see cyclic GMP CGP see circulating granulocyte pool chemical poisoning 275 chemokine receptors (CXCR) 400–2, 404, 426 chemokines 221, 474 chemotherapeutic agents 275 CHF see chronic heart failure CHOP see C/EBP homologous protein chronic heart failure (CHF) 324 chronic obstructive pulmonary disease (COPD) bronchial vasculature 219 disease pathogenesis 427, 429 endothelial cell–ECM interactions 53, 60 leukocyte–endothelial interactions 157 nitric oxide 98 pulmonary hypertension 449, 455 chronic pressure stresses 319, 326 chronic thromboembolic pulmonary hypertension (CTEPH) 471, 475–7, 479 chronic viral infections 303–5 cigarette smoke extract (CSE) 439 cigarette smoking 437 circulating endothelial cells (CECs) 465 circulating endothelial progenitor cells (CEPs) 203–5, 212, 223, 465 circulating granulocyte pool (CGP) 145 INDEX citrulline 388 Cl− channels 195 clathrin-coated pits 117 claudins 33, 43, 114, 118 clot clearance 473, 475–6 CMV see cytomegalovirus coagulation 471–2 cofilin 342 collagen 51–2, 62 collagen vascular diseases 461–9 cell death 464 circulating ECs 465 EC injury mechanisms 461–4 immune dysfunction 464 mediators of endothelial dysfunction 464–5 vascular lesions 461–4 colloidal silica nanoparticles 233 colony-forming unit (CFU)-Hill cells 204–5 computed tomography (CT) emphysema 437–8 pulmonary hypertension 454 targeted delivery 362 connective tissue growth factor (CTGF) 475 connexins adherens junctions 40 endothelial function 33, 37–43 endothelial–leukocyte communication 40–1 gap junctions 33, 37–42 inflammation 41–3 interactions among junctional proteins 41–3 subtypes 38–9 trafficking 39–40 tumor cell metastasis 40 COPD see chronic obstructive pulmonary disease cortactin 342 COX see cyclooxygenase CREST 461, 463–5 cross-talk 16–17 CSE see cigarette smoke extract CT see computed tomography CTEPH see chronic thromboembolic pulmonary hypertension CTGF see connective tissue growth factor Cx see connexins CXCR see chemokine receptors cyclic AMP (cAMP) Ca2+ channels 134 endothelial function 34, 39 hypoxia 291–2 mechanical forces 316 pulmonary circulation 382, 386–90 therapeutic strategies 340, 346 vasomotor control 190, 195 cyclic GMP (cGMP) hypoxia 296 mechanical forces 316, 319, 322 pulmonary circulation 382, 384, 385–90 pulmonary hypertension 453 therapeutic strategies 348 cyclin-dependent kinase (cdk) 207 INDEX cyclooxygenase (COX) disease pathogenesis 419 oxidant-mediated signaling 263 pulmonary circulation 385–6, 389 vasomotor control 188, 189–90, 192 cystic fibrosis (CF) 219, 421 cytochrome P450 hypoxia 289 oxidant-mediated signaling 264–5 vasomotor control 185, 190–1 cytokine receptors 73–4 cytomegalovirus (CMV) 305 DAG see diacylglycerol DCs see dendritic cells death-inducing signaling complex (DISC) 245 deep vein thrombosis (DVT) 471–4 dendritic cells (DCs) 420, 426, 429, 442 dexamethasone 296–7 DG see dystrophin-associated glycoprotein diacylglycerol (DAG) 73–4, 77, 189 dipeptidyl peptidase (DPP-IV) 486 DISC see death-inducing signaling complex disease pathogenesis 417–36 adaptive immunity 418, 424–9 antibodies 427–9 B cells 427–9 immune cells interactions with PE 417 inflammation 417–18, 422–3, 430 innate immunity 418, 419–24 leukocyte transmigration 419 mast cells/eosinophils/basophils 420–1 monocytes/macrophages/dendritic cells 420, 426, 429 natural killer cells 420 neutrophils 418–19, 421, 424 platelets 421–4 quiescent endothelium 418 T cells 423, 424–7, 429–30 DNA oxidation 269–70 DPP-IV see dipeptidyl peptidase DVT see deep vein thrombosis dynamin-2 122, 233–4 dystrophin-associated glycoprotein (DG) contacts 55, 57–9 E-cadherin anatomy of the PE 28 endothelial function 33–7 mesenchymal–endothelial interactions 177 protein mapping 229 E-selectin disease pathogenesis 418–19 leukocyte–endothelial interactions 150, 155–6, 158 targeted delivery of biotherapeutics 365 thromboembolism 478–9 ECE-1 see endothelin-converting enzyme ECFCs see endothelial colony-forming cells ECL see extracellular loop ECM see extracellular matrix ECs see endothelial cells 499 EDCF see endothelium-derived constricting factor edema see pulmonary edema EDHF see endothelium-derived hyperpolarizing factor EDNO see endothelium-derived nitric oxide EDRF see endothelium-derived relaxing factor EET see epoxyeicosatrienoic acid efferocytosis 243 Eisenmeiger’s syndrome 450 elastase-induced emphysematous lung injury 212 elastin 51–2 electron microscopy (EM) anatomy of the PE 25–8 bronchial vasculature 218 cell death 244 endothelial permeability 115–16 mechanical forces 314–15, 323 protein mapping 230 targeted delivery of biotherapeutics 363 electron transport chain (ETC) 262, 264 EM see electron microscopy EMAP see endothelial-monocyte activating polypeptide embryonic stage 10 emphysema 437–47, 492 cell death 252, 254, 438–9, 441–2 immune mechanisms 442 lung structure maintenance program 438 overview 437–8 pathogenesis 439–40 VEGF/VEGFR 438–43 viral infections 304 emphysemagenesis 442 EMTs see epithelial–mesenchymal transitions ENaCs see epithelial Na+ channels endoplasmic reticulum (ER) Ca2+ channels 73–5, 83, 130, 132, 134 cell death 246–7, 249–50 emphysema 441–3 hypoxia 289 oxidant-mediated signaling 264 stores 73–5, 83 vasomotor control 193 endothelial barrier regulation 399–415 angiotensin-converting enzyme 399–400, 401–3 barrier-regulatory agonist receptors 404–6 chemokine receptors 400–2, 404 cytoskeletal protein targets 406–7 genetic insights 399–415 growth arrest DNA damage-inducible 400–2, 409 hepatocyte growth factor 400–1, 405–6 interleukins 402, 403–4, 408 macrophage-migration inhibitory factor 400–1, 407 mechanosensitive genes 400–2, 407–9 myosin light chain kinase 400–1, 406–7 overview 399–401 pre-B cell colony-enhancing factor 400–2, 407–10 sphingosine 1-phosphate 400–1, 404–5 tumor necrosis factor 400–1, 403 vascular endothelial growth factor 400–1, 404 500 endothelial cells (ECs) anatomy of the PE 26–9 bronchial vasculature 217–24 Ca2+ channels 73–88, 129–42 cadherins 34, 36–7 cell cycle regulation 59–60 cell death 243–60 cell–ECM interactions 51–72 collagen vascular diseases 461–5 connexins 41 disease pathogenesis 417–30 emphysema 437–43 fetal pulmonary circulation 381–2 hypoxia 287–97 interactions with PE 417 leukocyte–endothelial interactions 143, 146–52, 154–6, 158–9 malignancies 485–8 mechanical forces 309–14, 319–20, 325–6 mesenchymal–endothelial interactions 169, 173–8 nitric oxide 92–3 oxidant-mediated signaling 261 permeability 113–27, 337–49 protein mapping 229–40 pulmonary hypertension 449–56 surface metabolic functions 105–12 targeted delivery of biotherapeutics 355–6, 358–61, 365–6 therapeutic strategies 337–49 thromboembolism 471–9 vascular barrier function 73–88 vasculogenesis 3, 4–8, 10–12, 15, 17 vasomotor control 185–8, 193–5 viral infections 303–5 see also endothelial progenitor cells endothelial colony-forming cells (ECFCs) 204–6, 212 endothelial-dependent vasodilation 219 endothelial ion channels 192–6 endothelial–leukocyte communication 40–1 endothelial-monocyte activating polypeptide (EMAP) 16 endothelial nitric oxide synthase (eNOS) 89–98, 492 collagen vascular diseases 465 endothelial permeability 117 hypoxia 289–90 mechanical forces 314, 317–19, 322, 324 oxidant-mediated signaling 265 protein mapping 234 pulmonary circulation 383–4, 385, 388–9 pulmonary hypertension 453 thromboembolism 473, 477 vasomotor control 186–9 endothelial permeability 113–27 basal lung 117–18 Ca2+ channels 84–5 caveolae 115–17, 120 cell–cell junction disruption 119 characteristics 113–17 extracellular matrix 119 focal adhesion kinase 119–20 hypoxia 291–3 INDEX inflammation 115, 121–2 junction-related proteins 115 lungs 113–27, 271, 337–54 mechanical forces 316 metabolite transport 118 overview 113 paracellular 118–20 properties 117 regulation of oncotic pressure 118 structural features 114–17 therapeutic strategies 337–54 transcellular 120 endothelial phenotypes 129–42 endothelial progenitor cells (EPCs) 203–16 bronchial vasculature 223 bronchopulmonary dysplasia 210–12 cell death 254 circulating 203–5, 212, 223 clinical disorders 209–12 collagen vascular diseases 465 developmental heterogeneity 206 emphysema 442–3 in vitro regulation 209 lungs 209–12 macrovascular proliferation 206–7 microvascular proliferation 206–9, 210 proliferation potential 204–5, 206–7 pulmonary hypertension 452 resident 205–9, 210 therapeutic potential 211–12 vascular growth 204, 210–11 endothelial protein C receptor (EPCR) 347 endothelial-specific growth factors 15 endothelial surface layer (ESL) 311 endothelin (ET-1) collagen vascular diseases 464–5 disease pathogenesis 421 mechanical forces 324 mesenchymal–endothelial interactions 173 pulmonary circulation 381, 382, 387–90 thromboembolism 473, 477–8 vasomotor control 185–7, 192, 196 endothelin-converting enzyme (ECE-1) 382, 389 endothelium-derived constricting factor (EDCF) 192 endothelium-derived hyperpolarizing factor (EDHF) 190, 191–2, 194 endothelium-derived nitric oxide (EDNO) 382–9 endothelium-derived relaxing factor (EDRF) 186, 192 endotoxin 145 eNOS see endothelial nitric oxide synthase environmental toxins 275 enzyme replacement therapies 364 eosinophils 420–1 EPCR see endothelial protein C receptor ephrins 7, 230 epinephrine 145, 154–5 epithelial Na+ channels (ENaCs) 195 epithelial–mesenchymal transitions (EMTs) 171, 176–7 epithelial/mesenchymal interface 12–13 INDEX epoxyeicosatrienoic acid (EET) Ca2+ channels 78, 135–6 hypoxia 289, 290 vasomotor control 190, 191 ER see endoplasmic reticulum ERK see extracellular signal-regulated mitogen-activated protein kinase ESL see endothelial surface layer ET-1 see endothelin ETC see electron transport chain Evan’s blue dye extravasation 343–4 extracellular domains (EXDs) 34 extracellular loop (ECL) 38 extracellular matrix (ECM) angiogenesis endothelial cell–ECM interactions 51–72 endothelial permeability 119 mesenchymal–endothelial interactions 167, 169–70, 173 vasculogenesis 3, 10, 14, 15–16 extracellular signal-regulated kinase (ERK) mechanical forces 314 mesenchymal–endothelial interactions 174–5 oxidant-mediated signaling 267 therapeutic strategies 346 extrapulmonary capillaries 29 F-actin 347 factor VIII 476 FADD see Fas-associated death domain FAK see focal adhesion kinase Fas-associated death domain (FADD) 247, 251 FAT see focal adhesion target fetal pulmonary circulation 381–5 FGF see fibroblast growth factor fibrillar adhesions 53–9 fibrinolysis 472–3 fibroblast growth factor (FGF) 13, 15, 418, 424 fibroblasts 169, 170–5 fibronectin 51–2, 172, 175 fibrosis 491 bronchial vasculature 219 cell death 253–4 endothelial cell–ECM interactions 53 mesenchymal–endothelial interactions 167–8, 170–3, 177–8 filipin 234 flavin mononucleotide (FMN) 264 flavoproteins 262–3 fluorescence microscopy 146–7 FMN see flavin mononucleotide focal adhesion kinase (FAK) cadherins 36 cell death 245 endothelial cell–ECM interactions 55–8, 60–1, 63 endothelial permeability 119–20 mechanical forces 312 surface metabolic functions 109 therapeutic strategies 343 focal adhesion target (FAT) 57 focal adhesions 53–9 501 focal contacts 53–9 free iron 264, 272, 275 free radicals see reactive oxygen species FTY720 343–4, 349 fumagillin 16 G-protein-coupled receptors (GPCRs) 73–4, 107, 337 GADD45α see growth arrest DNA damage-inducible gamma-scintigraphy 362 gap junctions (GJs) cadherins 33 connexins 37–42 endothelial permeability 116–17 surface metabolic functions 109 gap-junctional intercellular communication (GJIC) 487 GBMs see glomerular basement membranes GE see gel electrophoresis GEF see guanine nucleotide exchange factor gel electrophoresis (GE) 233, 235 genomic analyses 231 GJIC see gap-junctional intercellular communication GJs see gap junctions glomerular basement membranes (GBMs) 428 glucose oxidase (GOX) 362–3 glutathione (GSH) 266, 269, 270 glycocalyx 312, 356 glycoproteins 361 Golgi apparatus 289 Goodpasture’s syndrome 428 GOX see glucose oxidase GPCRs see G-protein-coupled receptors GPx enzymes 266 granulocytes 145 growth arrest DNA damage-inducible (GADD45α) 400–2, 409 growth factor receptors 73–4 GRP94 249–50 GSH see glutathione GTPases Ca2+ channels 79–80, 83–4 connexins 36 endothelial cell–ECM interactions 57, 60 hypoxia 292 mechanical forces 320 protein mapping 234 therapeutic strategies 337, 340–4, 348 guanine nucleotide exchange factor (GEF) 74, 345, 348–9 H5N1 virus 303 HAECs see human aortic endothelial cells Hanta viruses 303 HAPE see high-altitude pulmonary edema heat shock proteins (HSP) 388 hemangioblasts hemangiomas 487–8 heme oxygenases 288 hemidesmosomes 55, 57, 59 heparan sulfates 290 hepatitis virus 305 hepatocyte growth factor (HGF) 340–2, 346–7, 400–1, 405–6 502 20-HETE see hydroxyeicosatetraenoic acid HGF see hepatocyte growth factor HHV-8 see human herpesvirus HIF see hypoxia-inducible factor high performance liquid chromatography (HPLC) 233 high-altitude pulmonary edema (HAPE) hypoxia 290–1, 295–7 mechanical forces 309, 315, 324–5 high-mobility group box (HMGB1) 249 histamine 148, 158 HIV/AIDS 304, 305 collagen vascular diseases 461, 463 emphysema 437 pulmonary hypertension 451, 455 HMG-CoA reductase inhibitors 344–5 HMGB1 see high-mobility group box HPLC see high performance liquid chromatography HPV see hypoxic pulmonary vasoconstriction HPV-16 see human papilloma virus HSP see heat shock proteins 5-HT see serotonin human aortic endothelial cells (HAECs) 205 human herpesvirus (HHV-8) 303, 304–5, 456 human immunodeficiency virus see HIV/AIDS human papilloma virus (HPV-16) 303 human umbilical vein endothelial cells (HUVECs) cadherins 34 connexins 40 emphysema 439 endothelial progenitor cells 204–5 hypoxia 292 leukocyte–endothelial interactions 148, 150 hydrolytic proteins 105–7 hydroxyeicosatetraenoic acid (20-HETE) 190–1 hyperoxia 272, 275 hypersensitivity pneumonitis 437 hypertension see pulmonary arterial hypertension; pulmonary hypertension hypoxia 287–302 cell signaling 291–3 emphysema 440 endothelial permeability 290–3, 294–7 gene transcription 289 in vitro studies 291 inflammation 296–7 intermittent/sustained 290 isolated perfused lung models 294 mesenchymal–endothelial interactions 171–2 metabolism, viability and proliferation 287–8 nitric oxide 92, 96 physiological responses 289–90, 296–7 pulmonary circulation 382, 389 pulmonary edema 290–1, 294–7 pulmonary hypertension 167–9, 172, 188, 192, 289–90, 454 sensors 288–9 vasomotor control 185, 188–9, 192, 195 hypoxia-inducible factor (HIF) 288–90, 292–3, 297 collagen vascular diseases 464 endothelial progenitor cells 211 INDEX gene transcription 289 oxidant-mediated signaling 270 pulmonary hypertension 449, 451, 452 vasculogenesis 7, 15 viral infections 305 hypoxic pulmonary vasoconstriction (HPV) 188, 190, 192 IAP see inhibitor of apoptosis proteins ICAD see inhibitor of caspase-activated DNase ICAM see intercellular adhesion molecule ICMT see isoprenylcysteine-O-carboxyl methyltransferase idiopathic pulmonary arterial hypertension (IPAH) 449–55, 461, 465 idiopathic pulmonary fibrosis (IPF) 253 IFs see intermediate filaments IL see interleukins; intracellular loop imaging agents 362 imatinib 456 immune cells 417 immunofluorescence 42, 342 immunoprecipitation 231 inducible nitric oxide synthase (iNOS) 89 oxidant-mediated signaling 265 pulmonary circulation 383–4, 385 targeted delivery of biotherapeutics 364 inflammation bronchial vasculature 219–20 connexins 41–3 disease pathogenesis 417–18, 422–3, 430 endothelial barrier regulation 399–410 endothelial permeability 115, 121–2 hypoxia 296–7 leukocyte–endothelial interactions 151, 155–9 mechanical forces 325 mesenchymal–endothelial interactions 167, 169–77 nitric oxide 93 oxidant-mediated signaling 271, 272 targeted delivery of biotherapeutics 358, 361 therapeutic strategies 343 thromboembolism 474 inhibitor of apoptosis proteins (IAP) 245, 251 inhibitor of caspase-activated DNase (ICAD) 245 innate immunity 418, 419–24 iNOS see inducible nitric oxide synthase inositol-requiring enzyme (IRE) 246, 441 integrins disease pathogenesis 421, 423 endothelial cell–ECM interactions 60 leukocyte–endothelial interactions 146, 151, 156–7, 159 malignancies 486 therapeutic strategies 338 interalveolar septa 28–9 intercellular adhesion molecule (ICAM) collagen vascular diseases 464 disease pathogenesis 418–19, 421, 424–5 endothelial permeability 115, 121–2 leukocyte–endothelial interactions 151, 156 malignancies 486 INDEX oxidant-mediated signaling 271 targeted delivery of biotherapeutics 357, 359–62, 364, 366 interendothelial cell contacts 118–19 interferons (IFN) 41, 424–5 interleukins (IL) collagen vascular diseases 464 disease pathogenesis 419, 422, 425 endothelial barrier regulation 402, 403–4, 408 leukocyte–endothelial interactions 150–1 malignancies 486 mesenchymal–endothelial interactions 175 oxidant-mediated signaling 263 therapeutic strategies 343 viral infections 303 intermediate filaments (IFs) 59 internal ribosomal entry sequence (IRES) 92 intracellular loop (IL) 38 intrapulmonary capillaries 29 intravital microscopy 146–7 inward rectifier K+ channels 194 IPAH see idiopathic pulmonary arterial hypertension IPF see idiopathic pulmonary fibrosis IRE see inositol-requiring enzyme IRES see internal ribosomal entry sequence iron 264, 272, 275 ischemia 244, 273–4, 321 ischemia–reperfusion injury 491 cell death 249, 253–4 emphysema 440 endothelial cell–ECM interactions 62 hypoxia 294, 295 therapeutic strategies 343 isolated perfused lung models 294 isoprenylcysteine-O-carboxyl methyltransferase (ICMT) 249–50 JAMs see junctional adhesion molecules JNK see c-Jun N-terminal kinase junction-related proteins 115 junctional adhesion molecules (JAMs) 33, 114, 152 K+ channels 85–6 hypoxia 289 mechanical forces 312, 321 oxidant-mediated signaling 274, 276 pulmonary hypertension 452 vasomotor control 191, 194–5 Kaposi sarcoma 303 kinase insert domain-containing receptor (KDR) 439 L-arginine 90–3 L-selectin 150, 152, 156–8, 317 LAD see leukocyte adhesion deficiency lamellipodia 340–1 laminin 51–2, 62 laser capture microdissection 232 left pulmonary artery obstruction 220–1 leukocyte adhesion deficiency (LAD) 146–7, 149–50, 156 leukocyte recruitment 222–3 503 leukocyte sequestration 156–9 leukocyte–endothelial interactions 143–66 cellular and molecular influences 152–5 human model 143–6, 161 inflammation 151, 155–9 leukocyte sequestration 156–9 marginated granulocyte pool 145–6, 152–6 multistep paradigm 147, 149–52 nitric oxide 90 overview 143 physiologic/adhesive margination 154–6 platelets 160 polymorphonuclear neutrophils 144–8, 149–61 surface metabolic functions 107 surrogate experimental systems 146–9 leukopenia 145 leukotrienes (LTs) 186, 190, 263 LIGHT 423 linoleic acids 263 lipid peroxidation 269 lipopolysaccharide (LPS) Ca2+ channels 83 cell death 251, 253 disease pathogenesis 424 endothelial barrier regulation 405–6, 409 endothelial cell–ECM interactions 52 endothelial progenitor cells 204, 211 leukocyte–endothelial interactions 145, 150, 157–8 targeted delivery of biotherapeutics 362 therapeutic strategies 343, 345–9 lipoprotein lipase 107 lipoxygenase (LOX) 190, 263 LPS see lipopolysaccharide LSMP see lung structure maintenance program LTs see leukotrienes lung structure maintenance program (LSMP) 438, 442 lungs anatomy of the PE 25–9 angiogenesis 11–13 Ca2+ channels 73–88 cell death 245, 252–5 development stages 10 disease pathogenesis 417, 421, 424 emphysema 437–43 endothelial barrier regulation 399–410 endothelial permeability 113–27, 271, 337–54 endothelial phenotypes 129–42 endothelial progenitor cells 209–12 fetal pulmonary circulation 383–4 growth factors 13–15 hypoxia 287, 291–2, 294–7 leukocyte–endothelial interactions 143, 145, 152–9 malignancies 485–7 mechanical forces 320–1, 322–6 neovascularization 10–11 nitric oxide 89–90, 96 origins 9–10 oxidant-mediated signaling 261, 271, 275–6 protein mapping 229, 234–5 504 lungs (continued ) therapeutic strategies 337–54 thromboembolism 473, 475–6 transplantation 276 vascular barrier function 73–88 vasculogenesis 9–15 lupus see systemic lupus erythematosus macrophage-migration inhibitory factor (MIF) 400–1, 407 macrophages 420, 440, 474 magnetic resonance imaging (MRI) 362 major histocompatibility complex (MHC) 418, 420, 425 malignancies 485–90 bronchial vasculature 219 cancer cell–EC interactions 486–7 cell adhesion molecules 485–6 endothelial cell–ECM interactions 53 lungs 485–7 organotropism 485–6 pulmonary endothelium 487–8 targeted delivery of biotherapeutics 362–3 mammalian target of rapamycin (mTOR) 244 MAPK see mitogen-activated protein kinase marginated granulocyte pool (MGP) 145–6, 152–6 mass spectrometry (MS) 231–2 mast cells 420–1 matrix metalloproteinases (MMPs) cell death 252 endothelial cell–ECM interactions 52–3, 60, 62 malignancies 486 mesenchymal–endothelial interactions 178 oxidant-mediated signaling 268 pulmonary hypertension 455 thromboembolism 474 viral infections 305 MCTD see mixed connective tissue disease mechanical forces 309–35 acute pressure stresses 314–19 blood flow effects 319–22 chronic pressure stresses 319, 326 decentralization 311–12 lung disease 322–6 mechanotransduction 311–14 overview 309–11 oxidant-mediated signaling 273–4 shear stress 273–4, 309–10, 319–22, 384, 390 strain 311 stretch 274, 310–11 mechanosensitive genes 400–2, 407–9 mechanotransduction 234, 311–14 melanoma cell adhesion molecule (MelCAM) 486 mesenchymal–endothelial interactions 167–83 adventitial stromal cells 169–77 angiogenesis 167, 169–77 endothelial cells 169, 173–8 epithelial–mesenchymal transitions 171, 176–7 fibroblasts 169, 170–3 fibrosis 167–8, 170–3 inflammation 167, 169–77 INDEX pulmonary hypertension 167–9 stromal cell intermediates 175–6 metastatic tumors 485–7 methylnaltrexone (MNTX) 348–9 MGP see marginated granulocytes pool MHC see major histocompatibility complex microarray analysis 61–2 microspheres 356 MIF see macrophage-migration inhibitory factor mitochondrial DNA damage 251–2 mitochondrial electron transport chain (ETC) 264 mitochondrial outer membrane permeabilization (MOMP) 245, 247 mitogen-activated protein kinase (MAPK) endothelial barrier regulation 409 hypoxia 291–2 oxidant-mediated signaling 267–8, 274 protein expression 234 therapeutic strategies 340, 346 mixed connective tissue disease (MCTD) 461 MLC see myosin light chain MLCK see myosin light chain kinase MLCP see myosin light chain phosphatase MMPs see matrix metalloproteinases MNTX see methylnaltrexone MOMP see mitochondrial outer membrane permeabilization monoclonal antibodies 362 monocytes 420 monocytes–platelet aggregates (MPAs) 479 mOP-R see mu opioid receptor MPAs see monocytes–platelet aggregates MRI see magnetic resonance imaging MS see mass spectrometry mTOR see mammalian target of rapamycin mu opioid receptor (mOP-R) 348–9 multidimensional protein identification technology (MudPIT) 233 multistep paradigm 147, 149–52 myeloid differentiation factor (MyD88) 251 myocardial infarction 319 myosin light chain kinase (MLCK) Ca2+ channels 80 endothelial barrier regulation 400–1, 406–7 endothelial permeability 119, 122, 338–43, 346 myosin light chain phosphatase (MLCP) 382, 389–90 myosin phosphatase target subunit (MYPT1) 389 myristate acylation 95 N-cadherin anatomy of the PE 28 endothelial function 33–4, 37 endothelial permeability 117 protein mapping 229 N-ethylmaleimide-sensitive fusion protein (NSF) 233–4 Na+ channels 195, 312 Na+ /Ca2+ exchanger (NCX) 76, 85–6 Na+ /H+ exchanger regulatory factor (NHERF) 76, 85 INDEX NADPH oxidase (NOX) hypoxia 288 mechanical forces 321 oxidant-mediated signaling 262–3, 267, 272, 274, 276 pulmonary circulation 388 targeted delivery of biotherapeutics 363 therapeutic strategies 344 NADPH-cytochrome P450 reductase 265 NAP-1 see nucleosome assembly protein natural killer (NK) cells 420 NCX see Na+ /Ca2+ exchanger necrosis detection methods 249 disease pathways 254 overview 243–4 oxidant-mediated signaling 271–2 signaling pathways 245 nef gene 304 neoplastic disease 462 neovascularization 5, 10–11 bronchial vasculature 219–20 collagen vascular diseases 465 endothelial progenitor cells 208 mesenchymal–endothelial interactions 167 neprilysin 293 neurogenic pulmonary edema (NPE) 325 neuronal nitric oxide synthase (nNOS) 89 oxidant-mediated signaling 265 pulmonary circulation 383–4, 385 neutrophil–endothelial communication 40 neutrophils Ca2+ channels 138 disease pathogenesis 418–19, 421, 424 emphysema 437 endothelial barrier regulation 408 endothelial permeability 122 leukocyte–endothelial interactions 144–8, 149–61 malignancies 486 oxidant-mediated signaling 261–3, 271–2, 275–6 newborn pulmonary circulation 385–9 NF-κB see nuclear factor NHERF see Na+ /H+ exchanger regulatory factor Niemann–Pick disease (NPD) 364 nitric oxide (NO) 89–104, 491–2 angiogenesis 89–90 biological fate 96 blood gas transport 90 Ca2+ channels 77, 84, 129–30 chronic obstructive pulmonary disease 98 collagen vascular diseases 465 disease pathogenesis 421 endothelial permeability 117–18, 121 fatty acylation 95 hypoxia 289–90, 296 L-arginine 90–3 leukocyte–and platelet–endothelial interactions 90 leukocyte–endothelial interactions 152 lungs 89–90, 96 mechanical forces 313–14, 317–19, 321–2 505 oxidant-mediated signaling 263, 265 phosphorylation 95 post-transcriptional regulation of eNOS 94 post-translational regulation of eNOS 94–6 protein–protein interactions 94–5 pulmonary arterial hypertension 93, 96–8 pulmonary circulation 381–90 pulmonary hypertension 453 pulmonary vascular tone 89 pulmonary vessels 90 S-nitrosylation 95–6 thromboembolism 473, 477 transcriptional regulation of eNOS 93–4 vasculogenesis 89–90 vasomotor control 185, 186–9, 195 ventilation/perfusion matching 90 NK see natural killer nNOS see neuronal nitric oxide synthase NO see nitric oxide nocadozole 80 nonendothelial-specific growth factors 15 nonselective cation channels 193, 195 NOX see NADPH oxidase NPD see Niemann–Pick disease NPE see neurogenic pulmonary edema Nrf2 see nuclear factor-erythroid 2-related factor NSF see N-ethylmaleimide-sensitive fusion protein nuclear factor (NF-κB) cell death 247, 251 endothelial barrier regulation 401 hypoxia 292 oxidant-mediated signaling 268 nuclear factor-erythroid 2-related factor (Nrf2) 268 nucleosome assembly protein (NAP-1) 209, 212 OAG see 1-oleoyl-2-acetyl-sn-glycerol OB-cadherin 40 occludins 33, 42–3, 109, 114 occupational health 437 1-oleoyl-2-acetyl-sn-glycerol (OAG) 77, 79, 83 onionskinning 462–3 organotropism 485–6 oxidant-mediated signaling 261–85 altered mechanical forces 273–4 ancillary antioxidants 267 antioxidants 265–7 biomolecule oxidation/nitration 268–70 blood-borne antioxidants 267 cell death 271–2 cell distention 274 cellular manifestations of oxidative stress 268–72 endothelial function 270–1 enzymatic antioxidants 265–6 inflammation 271, 272 mitogen-activated protein kinases 267–8 nonenzymatic antioxidants 266–7 pathological mechanisms 272–4 physiological roles of ROS 267–8 pulmonary syndromes 274–6 506 oxidant-mediated signaling (continued ) reactive nitrogen species 261, 265, 268–70, 276 reactive oxygen species 261–5, 267–76 signaling pathways 267 transcription factors 268 oxidized phospholipids 347–8 oxygen free radicals see reactive oxygen species oxygen tension 15 oxygen toxicity 275 P-selectin bronchial vasculature 222 Ca2+ channels 80, 137, 139 disease pathogenesis 418–19, 421–4 leukocyte–endothelial interactions 150–2, 155–6, 158–9 mechanical forces 317, 322 protein mapping 229 thromboembolism 479 P-selectin glycoprotein (PSGL-1) 150–1 p21-activated kinase (PAK) 55, 245 p21-associated serine/threonine kinase (PAK) 342 p38 292, 340, 409 p53 170 pacemaker cells 73 PAECs see pulmonary artery endothelial cells PAF see platelet-activating factor PAH see pulmonary arterial hypertension PAI-1 see plasminogen activator inhibitor PAK see p21-activated kinase; p21-associated serine/threonine kinase palmitate acylation 95 pancreatic endoplasmic reticulum-like kinase (PERK) 246 PAR-1 see protease-activating receptor paracellular permeability 118–20 PARP see poly(ADP-ribose) polymerase paxillin 58, 343 PBEF see pre-B cell colony-enhancing factor 4α-PDD see 4α-phorbol 12,13-didecanoate PDEs see phosphodiesterases PDGF see platelet-derived growth factor PE see pulmonary embolism PECAM see platelet-endothelial cell adhesion molecule PEEP see positive end-expiratory pressure PEG see polyethylene glycol pericytes 14 PERK see pancreatic endoplasmic reticulum-like kinase; protein kinase R-like ER kinase perlecan 52 permeability see endothelial permeability persistent pulmonary hypertension of the newborn (PPHN) 381, 387–9 pertussis toxin (PTX) 340–1, 344 PET see positron emission tomography PG see prostaglandins phage libraries 231 pharmacokinetics 355–6 4α-phorbol 12,13-didecanoate (4α-PDD) 135–7 phosphatidylinositols Ca2+ channels 73–5, 79–80, 84, 132–3 INDEX cell death 244 endothelial cell–ECM interactions 55–8 mechanical forces 317, 321 vasomotor control 193 phosphodiesterases (PDEs) 384, 386, 388 phospholipases 73–4, 86, 132–3, 189 phosphorylation cadherins 34–5 nitric oxide 95 oxidant-mediated signaling 273 therapeutic strategies 338–40, 342, 347–8 vasomotor control 187 PIGF see placental growth factor PKA see protein kinase A PKC see protein kinase C PKG see protein kinase G placental growth factor (PIGF) PLAs see platelet–leukocyte aggregates plasminogen activator inhibitor (PAI-1) 402, 404, 423–4, 472–6 plasminogen activators 365–7 platelet-activating factor (PAF) disease pathogenesis 419, 422, 425 leukocyte–endothelial interactions 151, 160 pulmonary circulation 382–3, 387, 390 platelet-derived growth factor (PDGF) collagen vascular diseases 464 disease pathogenesis 422 hypoxia 289 mesenchymal–endothelial interactions 178 platelet-endothelial cell adhesion molecule (PECAM) 11, 16 disease pathogenesis 419, 421 mesenchymal–endothelial interactions 168, 176–7 targeted delivery of biotherapeutics 357–61, 364–7 platelet–endothelial interactions 90, 160 disease pathogenesis 421–4 thromboembolism 478–9 platelet–leukocyte aggregates (PLAs) 479 PM/DM see polymyositis/dermatomyositis PMNs see polymorphonuclear neutrophils PMVECs see pulmonary microvascular endothelial cells podosomes 54, 57–8 poly(ADP-ribose) polymerase (PARP) 245 polyamines 290 polyethylene glycol (PEG) carriers 355–6 polymorphonuclear neutrophils (PMNs) disease pathogenesis 421, 424 endothelial barrier regulation 408 endothelial permeability 122 leukocyte–endothelial interactions 144–8, 149–61 malignancies 486 oxidant-mediated signaling 261–3, 271–2, 275–6 polymyositis/dermatomyositis (PM/DM) 461 positive end-expiratory pressure (PEEP) 222 positron emission tomography (PET) 362, 453 postcapillary segment 29 PPHN see persistent pulmonary hypertension of the newborn Prdx enzymes 266 pre-B cell colony-enhancing factor (PBEF) 400–2, 407–10 precapillary segment 26 INDEX prednisone 145 primary pulmonary vascular plexus 12–13 progenitor cells see endothelial progenitor cells proliferation potential 204–5, 206–7 proline-rich tyrosine kinase-2 (Pyk2) 34–5 propidium iodide 247 prostacyclin Ca2+ channels 129–30 collagen vascular diseases 465 hypoxia 290 leukocyte–endothelial interactions 152 pulmonary circulation 384–5, 388, 390 vasomotor control 185, 187, 189–90 prostaglandins (PG) collagen vascular diseases 465 oxidant-mediated signaling 263 pulmonary circulation 384–5, 388, 390 therapeutic strategies 341, 348 vasomotor control 185–7, 189–90 protease-activating receptor (PAR-1) 83 protein kinase A (PKA) 39, 388–90 protein kinase C (PKC) 62–3 Ca2+ channels 75, 77, 84 endothelial permeability 121–2 nitric oxide 92 therapeutic strategies 339–40, 346 protein kinase G (PKG) 384, 385–90 protein kinase R-like ER kinase (PERK) 441 protein mapping 229–40 caveolae 233–4 cell culture 230–1 chemical labeling of surface proteins 232 colloidal silica nanoparticles 233 comprehensive identification 233 in vivo studies 231–2 large-scale approaches 231–2 laser capture microdissection 232 lung-specific proteins 234–5 mechanotransduction 234 overview 229 phage libraries 231 purification of ECs 232 segmental differences 229–30 study problems 230 therapeutic implications 236 transport vesicles 234 protein nitration 270 protein oxidation 269 protein scaffolds 53 pruning pseudoglandular stage 10, 11 PSGL-1 see P-selectin glycoprotein PTE see pulmonary thromboendarterectomy PTX see pertussis toxin pulmonary arterial hypertension (PAH) 60, 449 cell death 254 collagen vascular diseases 461–5 disease pathogenesis 417, 424, 425–9 mesenchymal–endothelial interactions 169, 171 507 nitric oxide 93, 96–8 viral infections 303, 304 pulmonary artery endothelial cells (PAECs) Ca2+ channels 85, 130–2, 134–5 cell death 254 endothelial cell–ECM interactions 52, 61 endothelial progenitor cells 206–8 hypoxia 287–90, 291 mesenchymal–endothelial interactions 173–5, 177 pulmonary circulation 383 therapeutic strategies 343, 347 vasomotor control 188, 195 pulmonary atresia 219 pulmonary circulation 3–24, 381–97 anatomy of the PE 25–9 angiogenesis 5–7, 11–13 antiangiogenic factors 16–17 arterial/venous differentiation 7–8, 12 Ca2+ channels 130–1, 139 cross-talk 16–17 cyclic GMP 382, 384, 385–7 endothelial regulation 381–90 endothelial-specific factors 15 endothelin 381, 382, 387–90 endothelium-derived nitric oxide 382–9 endothelium-derived vasoconstrictors 382–3 environmental influences 15 epithelial/mesenchymal interface 12–13 fetal 381–5 growth factors 4, 7–9, 13–17 lung morphogenesis 9–15 mesenchymal–endothelial interactions 167–83 newborn 385–9 nonendothelial-specific growth factors 15 ontogeny of vascular cells 4–7 persistent pulmonary hypertension of the newborn 381, 387–9 platelet activating factor 382–3, 387, 390 prostanoids 384–5, 390 pulmonary vasculature 3, receptor-mediated vasodilation/vasoconstriction 387 suppressed endothelium-dependent vasodilators 383–5 transitional 385–7 vasculogenesis 3–17 pulmonary edema 271 hypoxia 290–1, 294–7 mechanical forces 309, 319, 323–5 pulmonary embolism (PE) 471, 474–5 pulmonary fibrosis see fibrosis pulmonary hypertension 449–60, 492 angiogenesis 450–2 bone marrow 453–5 cell death 254, 451 EC dysfunction 455–6 EC proliferation 450–2 epithelial progenitor cells 452 hypoxia 167–9, 172, 188, 192, 289–90, 454 malignancies 488 mechanical forces 326 508 pulmonary hypertension (continued ) mesenchymal–endothelial interactions 167–9 metabolic shift 452–3 newborn 381, 387–9 overview 449 oxidant-mediated signaling 276 viral infections 303–5 see also pulmonary arterial hypertension pulmonary microvascular endothelial cells (PMVECs) 131, 134–5, 138, 206–9 pulmonary thromboendarterectomy (PTE) 253–4 pulmonary vasculature 3, 9, 59–64 pulmonary vein endothelial cells (PVECs) 130–2 Pyk2 see proline-rich tyrosine kinase-2 quinones 264–5, 275 RA see rheumatoid arthritis Rac GTPases 340–3, 346, 348 radioiodination 232 radioisotope therapies 362–3 RANTES 422 RBCs see red blood cells reactive nitrogen species (RNS) 261, 265, 268–70, 276 reactive oxygen species (ROS) Ca2+ channels 73–4, 85 cell death 245, 253 cellular manifestations of oxidative stress 268–72 endothelial permeability 121–2 extraendothelial sources 265 generation from endogenous enzymes 262–3 generation from nonenzymatic sources 264–5 hypoxia 287, 288, 291–2 mechanical forces 320–1 nitric oxide 89 oxidant-mediated signaling 261–5, 267–76 pathological mechanisms for oxidative stress 272–4 physiological roles 267–8 pulmonary syndromes 274–6 sources 262–5 targeted delivery of biotherapeutics 358–9 vasomotor control 185, 187–8, 196 receptor-mediated barrier protection 340–1 receptor-operated channels (ROCs) 76–9, 83, 193 red blood cells (RBCs) 143, 153–4, 158–9 remodeling angiogenesis 5, 6–7 renin–angiotensin–aldosterone system 106 reoxygenation after anoxia 272–3 resident endothelial progenitor cells (EPCs) 205–9 resident microvascular endothelial progenitor cells (RMEPCs) 207–12 rheumatoid arthritis (RA) 169, 170, 178 Rho GTPases 36 Ca2+ channels 79–80, 83–4 endothelial cell–ECM interactions 57 hypoxia 292 Rho kinase 418 RhoA-Rho kinase (ROCK) 55, 389–90, 404 INDEX RMEPCs see resident microvascular endothelial progenitor cells RNS see reactive nitrogen species ROCK see RhoA-Rho kinase ROCs see receptor-operated channels rolipram 134 ROS see reactive oxygen species ryanodine receptors 75 S-nitrosothiols (SNOs) 90 S-nitrosylation 95–6 S1P see sphingosine 1-phosphate saccular stage 10 SAGE see serial analysis of gene expression sarco/endoplasmic reticulum calcium ATPase (SERCA) 75, 132–3 SARS see severe acute respiratory syndrome scanning electron microscopy (SEM) 26, 28, 218 scleroderma 429, 461 SDS-PAGE 235 secreted protein, acidic and rich in cysteine (SPARC) 52, 486 selectins bronchial vasculature 222 Ca2+ channels 80, 137, 139 disease pathogenesis 418–19, 421–4 leukocyte–endothelial interactions 150–2, 155–9 mechanical forces 317, 322 protein mapping 229 targeted delivery of biotherapeutics 361, 365 thromboembolism 478–9 SERCA see sarco/endoplasmic reticulum calcium ATPase serial analysis of gene expression (SAGE) 289 serotonin (5-HT) Ca2+ channels 139 oxidant-mediated signaling 270, 272 surface metabolic functions 105–6, 109 serotonin transporter (SERT) 105, 107 SERT see serotonin transporter severe acute respiratory syndrome (SARS) 303 sGC see soluble guanylyl cyclase shear stress 309–10, 319–22 oxidant-mediated signaling 273–4 pulmonary circulation 384, 390 shear stress response element (SSRE) 93 sickle cell disease 96–7 signal transducer and activator of transcription (STAT-3) 452–3 simvastatin 340, 344–5 single nucleotide polymorphisms (SNPs) 400–1, 403–9 single photon emission computer tomography (SPECT) 362 siRNA see small interfering RNA SLE see systemic lupus erythematosus small interfering RNA (siRNA) 83 smooth muscle cells (SMCs) Ca2+ channels 77, 85, 132 cell death 254 emphysema 443 endothelial permeability 115 mesenchymal–endothelial interactions 170, 176 nitric oxide 89 INDEX pulmonary circulation 381–3, 386, 390 pulmonary hypertension 449, 452–3, 455–6 surface metabolic functions 105–9 targeted delivery of biotherapeutics 358 thromboembolism 475–7 vasculogenesis 12, 14 vasomotor control 185, 190–2, 194 SNAP see soluble NSF attachment protein SNARE see soluble NSF receptor SNOs see S-nitrosothiols SNPs see single nucleotide polymorphisms SOCs see store-operated Ca2+ channels sodium decylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 235 SODs see superoxide dismutases solid tumors 236 soluble guanylyl cyclase (sGC) 384, 388 soluble NSF attachment protein (SNAP) 233–4 soluble NSF receptor (SNARE) 233–4 sonic hedgehog signaling 13 sorafenib 456 Spanish toxic oil syndrome 463 SPARC see secreted protein, acidic and rich in cysteine SPECT see single photon emission computer tomography sphingosine 1-phosphate (S1P) Ca2+ channels 73, 81, 84, 86 cell death 249 endothelial barrier regulation 400–1, 404–5 hypoxia 297 therapeutic strategies 339, 340–4, 348 sphingosine kinase (SPHK) 84 sprouting angiogenesis 5, SSc see systemic sclerosis SSRE see shear stress response element STAT-3 see signal transducer and activator of transcription statins 52 stem cells 454 STIM see stromal interacting molecule store-operated Ca2+ channels (SOCs) 76–8, 80–1, 83–5, 132–4, 193 strain 311 stretch 274, 310–11 stromal cells adventitial 169–77 intermediates 175–6 sources 176–7 stromal interacting molecule (STIM) 81 superoxide dismutases (SODs) 265–6, 360, 363–4 surface metabolic functions 105–12 active transport 107 barrier regulation 109 binding properties 107–9 hydrolytic proteins 105–7 intercellular communication 109 syndecan 52 systemic lupus erythematosus (SLE) 428–9, 461, 465 systemic sclerosis (SSc) 305, 461, 465 509 T cells 423, 424–7, 429–30, 442 T-type Ca2+ channels 79–80 bronchial vasculature 222 lung endothelial phenotypes 136–8 vasomotor control 193–4 TAFI see thrombin-activatable fibrinolysis inhibitor targeted delivery of biotherapeutics 355–77 angiotensin-converting enzyme 355, 357 antibodies 361–3 antioxidants 360, 363–4 antithrombotic agents 365–6 antitumor agents 362–3 cell adhesion molecules 357–62, 364 enzyme replacement therapies 364 genetic materials 365 glucose oxidase 362–3 imaging agents 362 overview 355 plasminogen activators 365–7 radioisotope therapies 362–3 selectins 361 specific applications 361–6 thrombomodulin 355, 357, 363 transmembrane glycoproteins 357 vascular pharmacokinetics 355–6 vascular targeting to PE 356 TEM see transmission electron microscopy tenascin-C 16, 52 tensin 58 TER see transendothelial electrical resistance TGF-β see transforming growth factor TGN see trans-Golgi network thalidomide 16 thapsigargin 76, 132–7, 206 therapeutic strategies 337–54 activated protein C 347–8 adenosine triphosphate 345–6 barrier restoration 337–40 cytoskeleton EC signaling 337–40 endothelial permeability 337–49 hepatocyte growth factor 340–2, 346–7 methylnaltrexone 348–9 oxidized phospholipids 347–8 prostaglandins 341, 348 receptor-mediated barrier protection 340–1 simvastatin 340, 344–5 sphingosine 1-phosphate 339, 340–4 targeted delivery of biotherapeutics 355–77 thrombin bronchial vasculature 222 Ca2+ channels 77, 79, 81–2 disease pathogenesis 422 endothelial permeability 121–2 hypoxia 291 leukocyte–endothelial interactions 148 mesenchymal–endothelial interactions 172 oxidant-mediated signaling 263 therapeutic strategies 344–5, 348 510 thrombin-activatable fibrinolysis inhibitor (TAFI) 472 thromboembolism 471–83 angiogenesis 477 candidate gene expression 476, 477 clot clearance 473, 475–6 coagulation 471–2 cross-talk 478–9 deep vein thrombosis 471–4 endothelium roles 472 fibrinolysis 472–3 in situ thrombosis 475–6 inflammation 474 lungs 473, 475–6 platelets 478–9 pulmonary embolism 471, 474–5 rate/sequence of thrombus organization 474 targeted delivery of biotherapeutics 365–6 vascular remodeling 474–5 vascular tone regulation 477–8 thrombomodulin (TM) 355, 357, 363, 472 thromboxanes 290, 422, 424 Tie2 receptors 11 tight junctions (TJs) cadherins 33 connexins 41–3 endothelial permeability 113, 114, 116, 118–19 therapeutic strategies 337–8, 346 tissue inhibitors of metalloproteinases (TIMPs) 52–3 tissue-type plasminogen activator (tPA) 472–4, 476 TJs see tight junctions TLRs see toll-like receptors TM see thrombomodulin TNF-α see tumor necrosis factor tocopherols 266 toll-like receptors (TLRs) 442 tPA see tissue-type plasminogen activator trans-Golgi network (TGN) 36, 39 transcellular permeability 120 transendothelial electrical resistance (TER) 84 transforming growth factor (TGF-β) cell death 250–1, 253 collagen vascular diseases 464–5 emphysema 440 endothelial barrier regulation 402, 404 mesenchymal–endothelial interactions 170–2, 177 nitric oxide 94, 98 pulmonary hypertension 450–1, 455 thromboembolism 473, 474, 478 transient receptor potential (TRP) Ca2+ channels 74, 75–9, 81–6, 135–9 mechanical forces 313–14, 316–19, 321–2, 325–6 vasomotor control 193, 195 transitional pulmonary circulation 385–7 transmission electron microscopy (TEM) anatomy of the PE 27–8 cell death 244 endothelial permeability 115–16 targeted delivery of biotherapeutics 363 transplant rejection 417 INDEX transport vesicles 234 TRP see transient receptor potential Trypan blue 247 tryptophan 34 tubulogenesis tumor cell metastasis 40 tumor necrosis factor (TNF-α) cell death 245, 247, 250, 253 disease pathogenesis 419–20, 422, 424–5 endothelial barrier regulation 400–1, 403 endothelial cell–ECM interactions 52, 59 endothelial function 34, 41 endothelial permeability 118, 121 hypoxia 292 leukocyte–endothelial interactions 150, 157–8 oxidant-mediated signaling 263 therapeutic strategies 338–9, 341, 349 viral infections 304 tumors see malignancies TUNEL staining 248–9 two-dimensional HPLC 233 UAPCs see utrophin-associated protein complexes ubiquinol 264 ubiquinone 264 unfolded protein response (UPR) 246, 441 urokinase-type plasminogen activator (uPA) 472, 474–5 utrophin-associated protein complexes (UAPCs) 59 VALI see ventilator-associated lung injury Valsalva maneuver 144 vanishing lung syndrome see emphysema vasa vasorum endothelial cells (VVECs) 169, 171–6 vascular barrier function 73–88 vascular cell adhesion molecule (VCAM-1) 361, 418–19, 420, 486 vascular endothelial growth factor receptors (VEGFR) 438–9 cell death 250–2 disease pathogenesis 426 endothelial cell–ECM interactions 59 endothelial progenitor cells 204, 210 hypoxia 293 vasculogenesis 4, 9, 11, 13–17 vascular endothelial growth factor (VEGF) angiogenesis bronchial vasculature 219–20 Ca2+ channels 77, 81 cell death 250–2 collagen vascular diseases 464 disease pathogenesis 418, 424–6 emphysema 438–43 endothelial barrier regulation 400–1, 404 endothelial cell–ECM interactions 57, 59 endothelial permeability 115, 121 endothelial progenitor cells 204, 210–11 hypoxia 289, 292–3 mesenchymal–endothelial interactions 170–5, 178 nitric oxide 94 pulmonary hypertension 451–2, 455–6 INDEX targeted delivery of biotherapeutics 365 therapeutic strategies 338–9, 349 thromboembolism 477–8 vasculogenesis 4, 8–9, 16–17 viral infections 303, 304 vascular lesions 461–4 vascular permeability 290–1, 294–7 vascular pharmacokinetics 355–6 vascular smooth muscle cells (VSMCs) 254 vasculogenesis angiogenesis 5–7, 11–13 antiangiogenic factors 16–17 arterial/venous differentiation 7–8, 12 blood islands cellular mechanisms cross-talk 16–17 endothelial cells 3, 4–8, 10–12, 15, 17 endothelial-specific growth factors 15 environmental influences 15 epithelial/mesenchymal interface 12–13 extracellular matrix 3, 10, 14, 15–16 growth factors 4, 7–9, 13–17 hemangioblasts key moments 8–9 lung morphogenesis 9–15 nitric oxide 89–90 nonendothelial-specific growth factors 15 ontogeny of vascular cells 4–7 overview 3–4 vasoactive amines 270–1 vasodilator-stimulated phosphoprotein (VASP) 55 vasomotor control 185–202 arachidonic acid metabolites 189–90, 191, 196 Ca2+ entry 186, 191, 192–6 COX pathway 188, 189–90, 192 cytochrome P450 pathway 185, 190–1 endothelial ion channels 192–6 endothelin 185–7, 192 endothelium-derived hyperpolarizing factor 190, 191–2 lipoxygenase pathway 190 membrane potential 192–6 nitric oxide 185, 186–9, 195 overview 185 prostacyclin 185, 187, 189–90 vasoactive substances 185–92 VASP see vasodilator-stimulated phosphoprotein VCAM-1 see vascular cell adhesion molecule VE-cadherin anatomy of the PE 28 endothelial function 33–7 endothelial permeability 114, 117, 119, 122 oxidant-mediated signaling 271 protein mapping 229 surface metabolic functions 109 therapeutic strategies 343, 346, 348 511 thromboembolism 477 VEGF see vascular endothelial growth factor VEGFR see vascular endothelial growth factor receptors venous system anatomy of the PE 29 Ca2+ channels 130–1 vasculogenesis 7–8, 12 venous thromboembolism see thromboembolism ventilation/perfusion matching 90 ventilator-associated lung injury (VALI) 400, 404 ventilator-induced lung injury (VILI) endothelial barrier regulation 400, 408–10 endothelial permeability 339, 343, 347–8 mechanical forces 309, 325–6 venules 154–6 VGCCs see voltage-gated Ca2+ channels VILI see ventilator-induced lung injury vinculin 58 viral infections 303–7 vitamins A/C/E 266–7 voltage-gated Ca2+ channels (VGCCs) 193–4 voltage-gated K+ channels 452 volume-regulated anion channels (VRACs) 195 von Willebrand factor bronchial vasculature 222 Ca2+ channels 80, 130, 137 protein mapping 229 VRACs see volume-regulated anion channels VSMCs see vascular smooth muscle cells VVECs see vasa vasorum endothelial cells Warburg effect 453 WASP see Wiskott–Aldrich syndrome protein WBCs see white blood cells Weibel–Palade bodies bronchial vasculature 222 Ca2+ channels 130, 137, 139 disease pathogenesis 419 mechanical forces 317, 322 platelet–endothelial interactions 150 protein mapping 229 thromboembolism 478 Western blots 231, 233, 324 white blood cells (WBCs) leukocyte–endothelial interactions 143–4, 146, 149, 155–7 targeted delivery of biotherapeutics 358–9 Wiskott–Aldrich syndrome protein (WASP) 55, 57 X box-binding protein (XBP) 246, 441 xanthine oxidase 263 xenobiotics 265 zyxin 58 ... et al (20 08) Lung microvascular endothelium is enriched with progenitor cells that exhibit vasculogenic capacity 29 8 13 14 15 16 17 18 19 20 21 22 23 24 25 HYPOXIA AND THE PULMONARY ENDOTHELIUM... 20 .8 and 20 .9) Pressure-induced exocytotic EFFECTS OF ACUTE PRESSURE STRESS ON THE PULMONARY ENDOTHELIUM 317 PLA (cmH2O) time (s) 20 20 PLA (cmH2O) 25 0 control 20 0 [Ca2+]i (nM) 15 150 EC [Ca2+]i... EC [Ca2+]i (nM) 20 0 elevated 100 10 μm PLA (cmH2O) 10 20 0 EC [Ca2+]i (nM) 180 TRPV4+/+ TRPV4−/− 160 140 120 100 80 10 20 30 time (min) 40 50 Figure 20 .6 Role of TRPV4 in the pulmonary endothelial