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Control of cAMP in lung endothelial cell phenotypes. Implications for control of barrier function
Abstract
Pulmonary microvascular endothelial cells (PMVECs) form a more restrictive barrier to macromolecular flux than pulmonary arterial endothelial cells (PAECs); however, the mechanisms responsible for this intrinsic feature of PMVECs are unknown. Because cAMP improves endothelial barrier function, we hypothesized that differences in enzyme regulation of cAMP synthesis and/or degradation uniquely establish an elevated content in PMVECs. PMVECs possessed 20% higher basal cAMP concentrations than did PAECs; however, increased content was accompanied by 93% lower ATP-to-cAMP conversion rates. In PMVECs, responsiveness to beta-adrenergic agonist (isoproterenol) or direct adenylyl cyclase (forskolin) activation was attenuated and responsiveness to phosphodiesterase inhibition (rolipram) was increased compared with those in PAECs. Although both types of endothelial cells express calcium-inhibited adenylyl cyclase, constitutive PMVEC cAMP accumulation was not inhibited by physiological rises in cytosolic calcium, whereas PAEC cAMP accumulation was inhibited 30% by calcium. Increasing either PMVEC calcium entry by maximal activation of store-operated calcium entry or ATP-to-cAMP conversion with rolipram unmasked calcium inhibition of adenylyl cyclase. These data indicate that suppressed calcium entry and low ATP-to-cAMP conversion intrinsically influence calcium sensitivity. Adenylyl cyclase-to-cAMP phosphodiesterase ratios regulate cAMP at elevated levels compared with PAECs, which likely contribute to enhanced microvascular barrier function.
... Cell culture-Primary cultures of rat pulmonary microvascular endothelial cells ( 19,20 :PMVECs) were used for production of cytotoxic supernatants and analysis of cytotoxicity. The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum and maintained at 37°C in 5% CO 2 as detailed previously. ...
... PKA activation-PKA was activated pharmacologically by treatment with forskolin and rolipram. 19,24 Briefly, rolipram (10 μM) and forskolin (100 μM) were added to PMVECs, and the cells were cultured for up to 9 h. Control cells were treated with an equal volume of vehicle alone (100% EtOH). ...
... Finally, when supernatants from rolipram-and forskolin-treated cells were added to cultures of PMVECs, cytotoxic activity could not be detected. Collectively, these results indicate that even the maximal stimulation of transmembrane adenylyl cyclases and inhibition of phosphodiesterases, 19,24 which increases cAMP and activates PKA is not sufficient for PMVECs to generate cytotoxic oligomeric tau, illustrating the privileged intracellular location of ExoY activity. ...
Patients who recover from hospital‐acquired pneumonia exhibit a high incidence of end‐organ dysfunction following hospital discharge, including cognitive deficits. We have previously demonstrated that pneumonia induces the production and release of cytotoxic oligomeric tau from pulmonary endothelial cells, and these tau oligomers can enter the circulation and may be a cause of long‐term morbidities. Endothelial‐derived oligomeric tau is hyperphosphorylated during infection. The purpose of these studies was to determine whether Ser‐214 phosphorylation of tau is a necessary stimulus for generation of cytotoxic tau variants. The results of these studies demonstrate that Ser‐214 phosphorylation is critical for the cytotoxic properties of infection‐induced oligomeric tau. In the lung, Ser‐214 phosphorylated tau contributes to disruption of the alveolar–capillary barrier, resulting in increased permeability. However, in the brain, both the Ser‐214 phosphorylated tau and the mutant Ser‐214‐Ala tau, which cannot be phosphorylated, disrupted hippocampal long‐term potentiation suggesting that inhibition of long‐term potentiation was relatively insensitive to the phosphorylation status of Ser‐214. Nonetheless, phosphorylation of tau is essential to its cytotoxicity since global dephosphorylation of the infection‐induced cytotoxic tau variants rescued long‐term potentiation. Collectively, these data demonstrate that multiple forms of oligomeric tau are generated during infectious pneumonia, with different forms of oligomeric tau being responsible for dysfunction of distinct end‐organs during pneumonia.
... The strength of this calcium signal is amplified by calcium inhibition of trans-membrane adenylyl cyclases . Endothelial cells express the type 6 adenylyl cyclase (AC6), which is inhibited by submicromolar increases in calcium, especially by calcium influx through store operated calcium entry channels (Cioffi et al. 2002;Creighton et al. 2003;Stevens et al. 1999). The sensitivity of calcium inhibition is most evident when measuring the ATP-to-cAMP turnover rates in plasma membrane fractions, although it can be detected by radioimmunoassay in whole cell lysates as well. ...
... Development of Fluorescence Resonance Energy Transfer (FRET) approaches to measure cAMP also enables visualization of AC6 calcium inhibition in single living cells (Werthmann et al. 2009). Depending upon the endothelial cell phenotype studied, the magnitude of calcium inhibition can range from 20 to nearly 90% of the membrane cyclase activity (Stevens et al. 1999), bringing into question the importance of this crosstalk mechanism in controlling endothelial cell barrier integrity. ...
Exoenzyme Y (ExoY) was identified as a component of the Pseudomonas aeruginosa type 3 secretion system secretome in 1998. It is a common contributor to the arsenal of type 3 secretion system effectors, as it is present in approximately 90% of Pseudomonas isolates. ExoY has adenylyl cyclase activity that is dependent upon its association with a host cell cofactor. However, recent evidence indicates that ExoY is not just an adenylyl cyclase; rather, it is a promiscuous cyclase capable of generating purine and pyrimidine cyclic nucleotide monophosphates. ExoY’s enzymatic activity causes a characteristic rounding of mammalian cells, due to microtubule breakdown. In endothelium, this cell rounding disrupts cell-to-cell junctions, leading to loss of barrier integrity and an increase in tissue edema. Microtubule breakdown seems to depend upon tau phosphorylation, where the elevation of cyclic nucleotide monophosphates activates protein kinases A and G and causes phosphorylation of endothelial microtubule associated protein tau. Phosphorylation is a stimulus for tau release from microtubules, leading to microtubule instability. Phosphorylated tau accumulates inside endothelium as a high molecular weight, oligomeric form, and is then released from the cell. Extracellular high molecular weight tau causes a transmissible cytotoxicity that significantly hinders cellular repair following infection. Thus, ExoY may contribute to bacterial virulence in at least two ways; first, by microtubule breakdown leading to loss of endothelial cell barrier integrity, and second, by promoting release of a high molecular weight tau cytotoxin that impairs cellular recovery following infection.
... L'AMPc intracellulaire augmente la phosphorylation, par la protéine kinase A (PKA), de la MLCK (kinase des chaînes légères de myosine), ce qui diminue la tension cellulaire, relâche les cellules endothéliales et accélère la fermeture des interstices intercellulaires qui s'étaient créés au cours de l'inflammation à cause d'une rupture d'adhésion entre les cellules et d'une déformation du cytosquelette (1,40,133,184,185). ...
... Elles hydrolysent l'AMPc et le GMPc et leur action est variable selon le tissu concerné et le sous-groupe considéré(189). Les traitements inhibant la PDE-4 ont fait la preuve de leur efficacité pour réduire l'inflammation pulmonaire chronique notamment dans l'asthme et la broncho-pneumopathie obstructive.L'augmentation du taux d'AMPc par les inhibiteurs de PDE-4 est aussi efficace, (voir plus efficace) que la stimulation de l'adénylate cyclase par les ß2-agonistes(45,184,204). Ces médicaments ont surtout démontré une très forte synergie avec les ß2-agonistes pour augmenter le taux intracellulaire d'AMPc permettant d'espérer un gain thérapeutique via l'association de ces deux classes de médicaments(1,192).Dans la dernière partie de notre étude nous avons donc souhaité tester l'efficacité d'une telle association médicamenteuse dans l'espoir d'une potentialisation des effets thérapeutiques sur la perméabilité aux protéines et d'une modération des effets sur les pressions et le débit sanguin dans la circulation pulmonaire. ...
We developed a new modality of broncho-alveolar lavage technique to measure repeatedly (every 15 min. during 4 hours), the capillary-alveolar permeability to a macromolecule (FITC-dextran). After an oleic acid lung injury, capillary-alveolar permeability to FITC-dextran reaches a peak within 30 minutes. Thereafter the permeability decreases slowly until the end of the experiment.
We assessed the effects of terbutaline, a β2-agonist assumed to reduce in vitro the microvascular permeability in acute lung injury. Terbutaline infusion started 10 min. after oleic acid injury did not change the time course of permeability. Terbutaline infusion started 90 min. after injury interrupted the recovery with an aggravation in permeability. As cardiac index and pulmonary capillary pressure increased with terbutaline infusion, we speculate that terbutaline recruits leaky capillaries and increases FITC-dextran permeability during late recovery from oleic acid injury.
... The C × t-dependent depletion of nucleophilic antioxidants and peptides may then lead to disruption of enzymatic functions and membrane barrier functions. Concurrent with this hypothesis is that the prophylactic administration of strong nucleophiles markedly increased the tolerance to phosgene (Diller, 1980;Stevens et al., 1999). The self-tolerance to edema formation following pre-phosgene-exposed rats (Ghio and Hatch, 1996;Hatch et al., 2001) does not support the notion that hydrolysis and HCl-related tissue injury occur to any appreciable extent or are the principal causes of lung edema. ...
... The permeability and fenestration of the capillary endothelium is possibly affected by changes in (neuro)endocrine/paracrine control. Furthermore, lung microvascular endothelial cells possess an intrinsic capacity to preserve intracellular cyclic adenosine monophosphate (cAMP), which enhances their barrier function (Stevens et al., 1999). Increased cAMP levels are necessary to keep smooth muscle cell tight junctions intact to prevent or impede interstitial and/or alveolar edema. ...
... Mouse pulmonary ECs were isolated as previously described, and validated by staining for vascular endothelial cadherin and CD31, and their response to VEGF [19]. Mouse ECs were maintained in Dulbecco Modified Eagle Medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Omega Scientific, Tarzana, CA, USA). ...
While sustained nuclear factor-κB (NF-κB) activation is critical for proin-flammatory molecule expression, regulators of NF-κB activity during chronic inflammation are not known. We investigated the role of focal adhesion kinase (FAK) on sustained NF-κB activation in tumor necrosis factor-α (TNF-α)-stimulated endothelial cells (ECs) both in vitro and in vivo. We found that FAK inhibition abolished TNF-α-mediated sustained NF-κB activity in ECs by disrupting formation of TNF-α receptor complex-I (TNFRC-I). Additionally, FAK inhibition diminished recruitment of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and the inhibitor of NF-κB (IκB) kinase (IKK) complex to TNFRC-I, resulting in elevated stability of IκBα protein. In mice given TNF-α, pharmacological and genetic FAK inhibition blocked TNF-α-induced IKK-NF-κB activation in aortic ECs. Mechanistically, TNF-α activated and redistributed FAK from the nucleus to the cytoplasm, causing elevated IKK-NF-κB activation. On the other hand, FAK inhibition trapped FAK in the nucleus of ECs even upon TNF-α stimulation, leading to reduced IKK-NF-κB activity. Together, these findings support a potential use for FAK inhibitors in treating chronic inflammatory diseases. KEY WORDS: FAK; TNF-α; RIPK1; NF-κB; IκB; IKK. James M. Murphy and Kyuho Jeong contributed equally to this work.
... Procedures for isolation of rat PMVECs were approved by the University of South Alabama Institutional Animal Care and Use Committee. PMVECs were isolated from male Sprague Dawley rats as previously described (19). ...
KD025 is a ROCK2 inhibitor currently being tested in clinical trials for treatment of fibrotic lung diseases. The therapeutic effects of KD025 are partly due to its inhibition of profibrotic pathways and fat metabolism. However, whether KD025 affects pulmonary microvascular endothelial cell (PMVEC) function is unknown, despite evidence that alveolar capillary membrane disruption constitutes major causes of death in fibrotic lung diseases. We hypothesized that KD025 regulates PMVEC metabolism, pH, migration and survival, a series of inter-related functional characteristics that determine pulmonary barrier integrity. We used PMVECs isolated from Sprague Dawley rats. KD025 dose-dependently decreased lactate production and glucose consumption. The inhibitory effect of KD025 was more potent compared to other metabolic modifiers, including 2-deoxy-glucose (2DG), extracellular acidosis, dichloroacetate and remogliflozin. Interestingly, KD025 increased oxidative phosphorylation, while 2DG did not. KD025 also decreased intracellular pH and induced a compensatory increase in anion exchanger 2. KD025 inhibited PMVEC migration, but fasudil (non-specific ROCK inhibitor) did not. We tested endothelial permeability in vivo using Evans Blue dye in the bleomycin pulmonary fibrosis model. Baseline permeability was decreased in KD025-treated animals independent of bleomycin treatment. Under hypoxia, KD025 increased PMVEC necrosis as indicated by increased LDH release and propidium iodide uptake and decreased ATP; it did not affect Annexin V binding. ROCK2 knockdown had no effect on PMVEC metabolism, pH and migration, but it increased non-apoptotic caspase-3 activity. Together, we report that KD025 promotes oxidative phosphorylation, decreases glycolysis, intracellular pH and migration, and strengthens pulmonary barrier integrity in a ROCK2 independent manner.
... Rat pulmonary microvascular endothelial cells (PMVECs) were isolated as described previously (14) by the cell culture core of the Center for Lung Biology at University of South Alabama. PMVECs were maintained in Dulbecco's modified Eagles medium (DMEM, Life Technologies Inc.) supplemented with 10% v/v fetal bovine serum (Gemini), 100 μg/ml streptomycin, and 100 U/ml penicillin, pH 7.0. ...
Ca2+ and cAMP are ubiquitous second messengers known to differentially regulate a variety of cellular functions over a wide range of timescales. Studies from a variety of groups support the hypothesis that these signals can be localized to discrete locations within cells, and that this subcellular localization is a critical component of signaling specificity. However, to date, it has been difficult to track second messenger signals at multiple locations within a single cell. This difficulty is largely due to the inability to measure multiplexed florescence signals in real time. To overcome this limitation, we have utilized both emission scan- and excitation scan-based hyperspectral imaging approaches to track second messenger signals as well as labeled cellular structures and/or proteins in the same cell. We have previously reported that hyperspectral imaging techniques improve the signal-to-noise ratios of both fluorescence and FRET measurements, and are thus well suited for the measurement of localized second messenger signals. Using these approaches, we have measured near plasma membrane and near nuclear membrane cAMP signals, as well as distributed signals within the cytosol, in several cell types including airway smooth muscle, pulmonary endothelial, and HEK-293 cells. We have also measured cAMP and Ca2+ signals near autofluorescent structures that appear to be golgi. Our data demonstrate that hyperspectral imaging approaches provide unique insight into the spatial and kinetic distributions of cAMP and Ca2+ signals in single cells.
... Procedures for isolation of rat endothelial cells were approved by the University of South Alabama Institutional Animal Care and Use Committee. PMVECs and PAECs were isolated from male Sprague-Dawley rats as previously described (52). ...
... Pulmonary artery endothelial cells were isolated from Sprague-Dawley rats and characterized as described 17 ...
Pulmonary artery endothelial cells (PAECs) express a cation current, ISOC (store-operated calcium entry current), which when activated permits calcium entry leading to inter-endothelial cell gap formation. The large molecular weight immunophilin FKBP51 inhibits ISOC but not other calcium entry pathways in PAECs. However, it is unknown whether FKBP51-mediated inhibition of ISOC is sufficient to protect the endothelial barrier from calcium entry-induced disruption. The major objective of this study was to determine whether FKBP51-mediated inhibition of ISOC leads to decreased calcium entry-induced inter-endothelial gap formation and thus preservation of the endothelial barrier. Here, we measured the effects of thapsigargin-induced ISOC on the endothelial barrier in control and FKBP51 overexpressing PAECs. FKBP51 overexpression decreased actin stress fiber and inter-endothelial cell gap formation in addition to attenuating the decrease in resistance observed with control cells using electric cell-substrate impedance sensing. Finally, the thapsigargin-induced increase in dextran flux was abolished in FKBP51 overexpressing PAECs. We then measured endothelial permeability in perfused lungs of FKBP51 knockout (FKBP51-/-) mice and observed increased calcium entry-induced permeability compared to wild-type mice. To begin to dissect the mechanism underlying the FKBP51-mediated inhibition of ISOC, a second goal of this study was to determine the role of the microtubule network. We observed that FKBP51 overexpressing PAECs exhibited increased microtubule polymerization that is critical for inhibition of ISOC by FKBP51. Overall, we have identified FKBP51 as a novel regulator of endothelial barrier integrity, and these findings are significant as they reveal a protective mechanism for endothelium against calcium entry-induced disruption.
... Procedures for isolation of rat endothelial cells were approved by the University of South Alabama Institutional Animal Care and Use Committee. Pulmonary microvascular and artery endothelial cells were isolated from male Sprague Dawley rats as previously described (10,37). Whereas PMVECs are isolated from vessels ≤25 µm in diameter, mostly reflective of capillaries, PAECs are isolated from the main pulmonary artery and two to three additional vessel branches (14,21,27,36). ...
Pulmonary artery, capillary, and vein endothelial cells possess distinctive structures and functions, which represent a form of vascular segment specific macroheterogeneity. However, within each of these segmental populations, individual cell functional variability represents a poorly characterized microheterogeneity. Here, we hypothesized that single cell clonogenic assays would reveal microheterogeneity among the parent cell population and enable isolation of highly representative cells with committed parental characteristics. To test this hypothesis, pulmonary microvascular endothelial cells (PMVECs) and pulmonary arterial endothelial cells (PAECs) were isolated from different Sprague Dawley rats. Serum stimulated proliferation of endothelial populations and single cell clonogenic potential were evaluated. In vitro Matrigel assays were utilized to analyze angiogenic potential and the Seahorse assay was used to evaluate bioenergetic profiles. PMVEC populations grew faster and had a higher proliferative potential than PAEC populations. Fewer PMVECs were needed to form networks on Matrigel when compared with PAECs. PMVECs primarily utilized aerobic glycolysis, while PAECs relied more heavily on oxidative phosphorylation, to support bioenergetic demands. Repeated single cell cloning and expansion of PAEC colonies generated homogeneous first-generation clones that were highly reflective of the parental population in terms of growth, angiogenic potential, and bioenergetic profiles. Repeated single cell cloning of the first-generation clones generated second-generation clones with increased proliferative potential while maintaining other parental characteristics. Second-generation clones were highly homogeneous populations. Thus, single cell cloning reveals microheterogeneity among the parent cell population and enables isolation of highly representative cells with parental characteristics.
... Rat pulmonary microvascular endothelial cells (PMVECs) were isolated as described previously 14 by the Cell Culture Core of the Center for Lung Biology at University of South Alabama. PMVECs were maintained in Dulbecco's modified Eagles medium (DMEM, Life Technologies Inc.) supplemented with 10% v/v fetal bovine serum (Gemini), 100 μg/ml streptomycin, and 100 U/ml penicillin, pH 7.0. ...
Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions. Several lines of evidence suggest that the distribution of cAMP within cells is not uniform. However, to date, no studies have measured the kinetics of 3D cAMP distributions within cells. This is largely due to the low signal-to-noise ratio of FRET-based probes. We previously reported that hyperspectral imaging improves the signal-to-noise ratio of FRET measurements. Here we utilized hyperspectral imaging approaches to measure FRET signals in five dimensions (5D) - three spatial (x, y, z), wavelength (λ), and time (t) - allowing us to visualize cAMP gradients in pulmonary endothelial cells. cAMP levels were measured using a FRET-based sensor (H188) comprised of a cAMP binding domain sandwiched between FRET donor and acceptor - Turquoise and Venus fluorescent proteins. We observed cAMP gradients in response to 0.1 or 1 μM isoproterenol, 0.1 or 1 μM PGE1, or 50 μM forskolin. Forskolin- and isoproterenol-induced cAMP gradients formed from the apical (high cAMP) to basolateral (low cAMP) face of cells. In contrast, PGE1-induced cAMP gradients originated from both the basolateral and apical faces of cells. Data suggest that 2D (x,y) studies of cAMP compartmentalization may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D (x,y,z) studies are required to assess mechanisms of signaling specificity. Results demonstrate that 5D imaging technologies are powerful tools for measuring biochemical processes in discrete subcellular domains. This work was supported by NIH P01HL066299, R01HL058506, S10RR027535, AHA 16PRE27130004 and the Abraham Mitchell Cancer Research Fund.
... Rat pulmonary microvascular endothelial cells (PMVECs) were isolated as described previously 14 by the Cell Culture Core of the Center for Lung Biology at University of South Alabama. PMVECs were maintained in Dulbecco's modified Eagles medium (DMEM, Life Technologies Inc.) supplemented with 10% v/v fetal bovine serum (Gemini), 100 µg/ml streptomycin, and 100 U/ml penicillin, pH 7.0. ...
... To determine whether S1P induces proliferation of cultured rat PAECs and PASMCs, the cells were harvested from normal adult male Sprague-Dawley rats and cultured as described elsewhere. 26 Both cell types were cultured in low-serum medium (Dulbecco's modified Eagle medium [DMEM] plus 2% fetal bovine serum [FBS]) for 24 hours prior to drug treatment. Low-serum medium was replaced with growth-arrest medium (DMEM plus 0.1% FBS for PAECs and DMEM-F12 50 ∶ 50 for PASMCs) for 24 hours. ...
Despite several advances in the pathobiology of pulmonary arterial hypertension (PAH), its pathogenesis is not completely understood. Current therapy improves symptoms but has disappointing effects on survival. Sphingosine-1-phosphate (S1P) is a lysophospholipid synthesized by sphingosine kinase 1 (SphK1) and SphK2. Considering the regulatory roles of S1P in several tissues leading to vasoconstriction, inflammation, proliferation, and fibrosis, we investigated whether S1P plays a role in the pathogenesis of PAH. To test this hypothesis, we used plasma samples and lung tissue from patients with idiopathic PAH (IPAH) and the Sugen5416/hypoxia/normoxia rat model of occlusive PAH. Our study revealed an increase in the plasma concentration of S1P in patients with IPAH and in early and late stages of PAH in rats. We observed increased expression of both SphK1 and SphK2 in the remodeled pulmonary arteries of patients with IPAH and PAH rats. Exogenous S1P stimulated the proliferation of cultured rat pulmonary arterial endothelial and smooth-muscle cells. We also found that 3 weeks of treatment of late-stage PAH rats with an SphK1 inhibitor reduced the increased plasma levels of S1P and the occlusive pulmonary arteriopathy. Although inhibition of SphK1 improved cardiac index and the total pulmonary artery resistance index, it did not reduce right ventricular systolic pressure or right ventricular hypertrophy. Our study supports that S1P is involved in the pathogenesis of occlusive arteriopathy in PAH and provides further evidence that S1P signaling may be a novel therapeutic target.
... Rat pulmonary microvascular endothelial cells (PMVECs) were isolated as described previously (12) by the cell culture core of the Center for Lung Biology at University of South Alabama. PMVECs were maintained in Dulbecco's modified Eagles medium (DMEM, Life Technologies Inc.) supplemented with 10% v/v fetal bovine serum (Gemini), 100 μg/ml streptomycin, and 100 U/ml penicillin, pH 7.0. ...
Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions over a wide range of timescales. Several lines of evidence have suggested that the distribution of cAMP within cells is not uniform, and that cAMP compartmentalization is largely responsible for signaling specificity within the cAMP signaling pathway. However, to date, no studies have experimentally measured three dimensional (3D) cAMP distributions within cells. Here we use both 2D and 3D hyperspectral microscopy to visualize cAMP gradients in endothelial cells from the pulmonary microvasculature (PMVECs). cAMP levels were measured using a FRETbased cAMP sensor comprised of a cAMP binding domain from EPAC sandwiched between FRET donors and acceptors — Turquoise and Venus fluorescent proteins. Data were acquired using either a Nikon A1R spectral confocal microscope or custom spectral microscopy system. Analysis of hyperspectral image stacks from a single confocal slice or from summed images of all slices (2D analysis) indicated little or no cAMP gradients were formed within PMVECs under basal conditions or following agonist treatment. However, analysis of hyperspectral image stacks from 3D cellular geometries (z stacks) demonstrate marked cAMP gradients from the apical to basolateral membrane of PMVECs. These results strongly suggest that 2D imaging studies of cAMP compartmentalization — whether epifluorescence or confocal microscopy — may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D studies are required to assess mechanisms of signaling specificity.
... PMVECs were obtained from the Cell Culture Core at the University of South Alabama Center for Lung Biology. Isolation and characterization of these cells under the approval of the Animal Care and Use Committee of the University of South Alabama are described elsewhere in detail (25,48). Cells were cultured in endothelial cell medium (high-glucose DMEM, 10% fetal bovine serum, and 1% penicillin-streptomycin) at 37°C in 21% O 2 and 5% CO2. ...
Bacterial induced sepsis is a common cause of pulmonary endothelial barrier dysfunction and can progress toward acute respiratory distress syndrome (ARDS). Elevations in intracellular cAMP tightly regulate pulmonary endothelial barrier integrity; however, cAMP signals are highly compartmentalized and it depends on which compartment the signal is generated - plasma membrane versus cytosolic - as to whether it is barrier protective or disruptive, respectively. The mammalian soluble adenylyl cyclase (AC) isoform 10 (AC10 or sAC) is uniquely stimulated by bicarbonate and is expressed in pulmonary microvascular endothelial cells (PMVECs). Elevated extracellular bicarbonate increases cAMP in PMVECs to disrupt the endothelial barrier and increase the filtration coefficient (Kf) in the isolated lung. Herein, we tested the hypothesis that sepsis-induced endothelial barrier disruption and increased permeability is dependent upon extracellular bicarbonate and activation of AC10. Our findings reveal that LPS-induced endothelial barrier disruption is dependent upon extracellular bicarbonate: LPS-induced barrier failure and increased permeability is exacerbated in elevated bicarbonate compared to low extracellular bicarbonate. The AC10 inhibitor, KH7, attenuated the bicarbonate-dependent LPS-induced barrier disruption. In the isolated lung, LPS failed to increase the Kf in the presence of minimal perfusate bicarbonate. When perfusate bicarbonate was increased to the physiological range (24 mM) this revealed the LPS-induced increase in the Kf, which was attenuated by KH7. Further, when PMVECs were treated with LPS for 6-hours there was a dose-dependent increase in AC10 expression. Thus, these findings reveal that LPS-induced pulmonary endothelial barrier failure requires bicarbonate activation of AC10.
... In fact, the major site of emigration within the distal lung tissue is the pulmonary capillaries. Although technical difficulties may preclude use of microvascular cells, many differences in phenotype between endothelial cells from different sites have been identified (27,28). For example, differences in shape between pulmonary arterial, capillary, and venous endothelial cells have been described (29). ...
... Witzenrath et al.' s study supported the hypothesis that TNFincreases endothelial permeability by inducing endothelial PDE2 expression, through activation of p38 MAPK pathway, and leading to decreases in both cyclic nucleotides (cAMP and cGMP) accumulation [167,168]. Reciprocally, overexpression of PDE2 is linked to destabilization of endothelial barrier function with a subsequent rise in endothelial permeability in HUVECs (human umbilical vein endothelial cells, a model system for the study of the function and pathology of endothelial cells). In isolated mice lungs, PDE2 inhibition was effective in preventing thrombin-induced lung edema, as shown with a reduction in both lung wet-to-dry ratio and human serum albumin (HSA) flux from the vascular to bronchoalveolar compartment. ...
Phosphodiesterase 2 (PDE2) is a ubiquitous enzyme whose major role is to hydrolyze the important second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). In the central nervous system, pharmacological inhibition of PDE2 results in boosted cAMP and/or cGMP signaling, which is responsible for series of changes in protein expression relevant to psychiatric and learning and memory disorders, such as depression, anxiety, and cognition deficits in Alzheimer's disease. In the periphery, inhibition of PDE2 exhibits beneficial effects in the diseased cardiovascular system, the respiratory system, skeletal muscles and Candida albicans-caused systemic infections. Even though blood-brain barrier penetration properties and selectivity of currently available PDE2 inhibitors have hindered them from entering clinical trials, PDE2 is still of great potential therapeutic values in different categories of diseases, and there is demand for development of new generation drugs targeting PDE2 for treatment of diseases in central nervous and peripheral systems.
... Primary rat lung microvascular endothelial cells (RLMVEC), a kind gift from Dr. Troy Stevens (University of South Alabama, Mobile, AL), were maintained in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and treated at 37uC in 5% CO 2 [25]. Rat lung epithelial cells were from ATCC (Manassas, VA) and maintained in F12 medium containing 10% fetal bovine serum. ...
The homeostatic lung protective effects of alpha-1 antitrypsin (A1AT) may require the transport of circulating proteinase inhibitor across an intact lung endothelial barrier. We hypothesized that uninjured pulmonary endothelial cells transport A1AT to lung epithelial cells. Purified human A1AT was rapidly taken up by confluent primary rat pulmonary endothelial cell monolayers, was secreted extracellularly, both apically and basolaterally, and was taken up by adjacent rat lung epithelial cells co-cultured on polarized transwells. Similarly, polarized primary human lung epithelial cells took up basolaterally-, but not apically-supplied A1AT, followed by apical secretion. Evidence of A1AT transcytosis across lung microcirculation was confirmed in vivo by two-photon intravital microscopy in mice. Time-lapse confocal microscopy indicated that A1AT co-localized with Golgi in the endothelium whilst inhibition of the classical secretory pathway with tunicamycin significantly increased intracellular retention of A1AT. However, inhibition of Golgi secretion promoted non-classical A1AT secretion, associated with microparticle release. Polymerized A1AT or A1AT supplied to endothelial cells exposed to soluble cigarette smoke extract had decreased transcytosis. These results suggest previously unappreciated pathways of A1AT bidirectional uptake and secretion from lung endothelial cells towards the alveolar epithelium and airspaces. A1AT trafficking may determine its functional bioavailablity in the lung, which could be impaired in individuals exposed to smoking or in those with A1AT deficiency.
... Rat pulmonary microvascular endothelial cells (RPMEC) were purchased from VEC Technologies (VEC Technologies, Rensselaer, NY, USA) and cultured in MCDB-131 complete medium (VEC Technologies). Rat pulmonary artery endothelial cells (RPAEC), isolated [27] and donated by Dr. Troy Stevens (University of South Alabama, Tuscaloosa, AL), were cultured in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Carlsbad, CA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Carlsbad, CA, USA). RPMEC and RPAEC were incubated at 37°C in humidified atmosphere with 21% O 2 and 5% CO 2 . ...
Although a critical role for caveolae-mediated albumin transcytosis in pulmonary endothelium is well established, considerably less is known about caveolae-independent pathways. In this current study, we confirmed that cultured rat pulmonary microvascular (RPMEC) and pulmonary artery (RPAEC) endothelium endocytosed Alexa488-labeled albumin in a saturable, temperature-sensitive mode and internalization resulted in co-localization by fluorescence microscopy with cholera B toxin and caveolin-1. Although siRNA to caveolin-1 (cav-1) in RPAEC significantly inhibited albumin uptake, a remnant portion of albumin uptake was cav-1-independent, suggesting alternative pathways for albumin uptake. Thus, we isolated and cultured mouse lung endothelial cells (MLEC) from wild type and cav-1(-/-) mice and noted that ~ 65% of albumin uptake, as determined by confocal imaging or live cell total internal reflectance fluorescence microscopy (TIRF), persisted in total absence of cav-1. Uptake of colloidal gold labeled albumin was evaluated by electron microscopy and demonstrated that albumin uptake in MLEC from cav-1(-/-) mice was through caveolae-independent pathway(s) including clathrin-coated pits that resulted in endosomal accumulation of albumin. Finally, we noted that albumin uptake in RPMEC was in part sensitive to pharmacological agents (amiloride [sodium transport inhibitor], Gö6976 [protein kinase C inhibitor], and cytochalasin D [inhibitor of actin polymerization]) consistent with a macropinocytosis-like process. The amiloride sensitivity accounting for macropinocytosis also exists in albumin uptake by both wild type and cav-1(-/-) MLEC. We conclude from these studies that in addition to the well described caveolar-dependent pulmonary endothelial cell endocytosis of albumin, a portion of overall uptake in pulmonary endothelial cells is cav-1 insensitive and appears to involve clathrin-mediated endocytosis and macropinocytosis-like process.
... Rat pulmonary microvascular endothelial cells (PMVECs) were isolated, characterized, and maintained using methods that have been reported previously [29]. Animal procedures were in accordance to NIH Guidelines and procedures were reviewed and approved by the University of South Alabama Institutional Animal Care and Use Committee (protocol 278237). ...
Pseudomonas aeruginosa uses a type III secretion system to introduce the adenylyl and guanylyl cyclase exotoxin Y (ExoY) into the cytoplasm of endothelial cells. ExoY induces Tau hyperphosphorylation and insolubility, microtubule breakdown, barrier disruption and edema, although the mechanism(s) responsible for microtubule breakdown remain poorly understood. Here we investigated both microtubule behavior and centrosome activity to test the hypothesis that ExoY disrupts microtubule dynamics. Fluorescence microscopy determined that infected pulmonary microvascular endothelial cells contained fewer microtubules than control cells, and further studies demonstrated that the microtubule-associated protein Tau was hyperphosphorylated following infection and dissociated from microtubules. Disassembly/reassembly studies determined that microtubule assembly was disrupted in infected cells, with no detectable effects on either microtubule disassembly or microtubule nucleation by centrosomes. This effect of ExoY on microtubules was abolished when the cAMP-dependent kinase phosphorylation site (Ser-214) on Tau was mutated to a non-phosphorylatable form. These studies identify Tau in microvascular endothelial cells as the target of ExoY in control of microtubule architecture following pulmonary infection by Pseudomonas aeruginosa and demonstrate that phosphorylation of tau following infection decreases microtubule assembly.
... Thus, from data currently available it appears that both endothelial cells from different organs and their inflammatory activation (acute versus chronic inflammation) may considerably affect their inducible chemokine/cytokine profiles and related monocyte adhesion events. Against the background that HUVEC are often considered as a "standard" endothelial cell type, it is particularily relevant that there are actually many differences in phenotype between endothelial cells from different vascular beds (22,26). Future studies with macro-and microvascular endothelial cells isolated from different organs should lead to a better understanding of endothelium-derived chemokine dependent leukocyte arrest under flow conditions. ...
... Isolation and culture of rat PMVECs and pulmonary artery endothelial cells. Isolation of rat PMVECs and pulmonary artery endothelial cells (PAECs) has been described in detail elsewhere (54) under the approval of the Animal Care and Use Committee of the University of South Alabama. Cell cultures where used from passage 8 through passage 15 and were maintained in EC media (high-glucose DMEM, 10% fetal bovine serum, 100 U/ml penicillin, and 100 g/ml streptomycin) at 5% CO 2. ...
It is becoming increasingly apparent that cAMP signals within the pulmonary endothelium are highly compartmentalized, and this compartmentalization is critical to maintaining endothelial barrier integrity. Studies demonstrate that the exogenous soluble bacterial toxin, ExoY, and heterologous expression of the forskolin-stimulated soluble mammalian adenylyl cyclase (AC) chimera, sACI/II, elevate cytosolic cAMP and disrupt the pulmonary microvascular endothelial barrier. The barrier disruptive effects of cytosolic cAMP generated by exogenous soluble ACs are in contrast to the barrier protective effects of subplasma membrane cAMP generated by transmembrane AC, which strengthens endothelial barrier integrity. Endogenous soluble AC isoform 10 (AC10 or commonly known as sAC) lacks transmembrane domains and localizes within the cytosolic compartment. AC10 is uniquely activated by bicarbonate to generate cytosolic cAMP, yet its role in regulation of endothelial barrier integrity has not been addressed. Here we demonstrate that within the pulmonary circulation, AC10 is expressed in pulmonary microvascular endothelial cells (PMVECs) and pulmonary artery endothelial cells (PAECs), yet expression in PAECs is lower. Furthermore, pulmonary endothelial cells selectively express bicarbonate cotransporters. While extracellular bicarbonate generates a phosphodiesterase 4 sensitive cAMP pool in PMVECs, no such cAMP response is detected in PAECs. Finally, addition of extracellular bicarbonate decreases resistance across the PMVEC monolayer and increases the filtration coefficient in the isolated perfused lung above osmolality controls. Collectively, these findings suggest that PMVECs have a bicarbonate-sensitive cytosolic cAMP pool that disrupts endothelial barrier integrity. These studies could provide an alternative mechanism for the controversial effects of bicarbonate correction of acidosis of ARDS patients.
... The cAMP/PKA signaling pathway was shown to decrease isometric tension development, intercellular gap formation, and vascular permeability in multiple experimental preparations (27,(40)(41)(42)(43). Our recent study showed that genistein activates the cAMP signaling system in ECs (28). ...
The soy-derived phytoestrogen genistein has received attention for its potential to improve vascular function, but its mechanism remains unclear. Here, we report that genistein at physiologically relevant concentrations (0.1-10 μM) significantly inhibited thrombin-induced increase in endothelial monolayer permeability. Genistein also reduced the formation of stress fibers by thrombin and suppressed thrombin-induced phosphorylation of myosin light chain (MLC) on Ser(19)/Thr(18) in endothelial cells (ECs). Genistein had no effect on resting intracellular [Ca(2+)] or thrombin-induced increase in Ca(2+) mobilization. Addition of the inhibitors of endothelial nitric oxide synthase or estrogen receptor did not alter the protective effect of genistein. RhoA is a small GTPase that plays an important role in actin-myosin contraction and endothelial barrier dysfunction. RhoA inhibitor blocked the protective effect of genistein on endothelial permeability and also ablated thrombin-induced MLC-phosphorylation in ECs. Inhibition of PKA significantly attenuated the effect of genistein on thrombin-induced EC permeability, MLC phosphorylation, and RhoA membrane translocation in ECs. Furthermore, thrombin diminished cAMP production in ECs, which were prevented by treatment with genistein. These findings demonstrated that genistein improves thrombin-induced endothelial barrier dysfunction in ECs through PKA-mediated suppression of RhoA signaling.
... PMVECs have high phosphodiesterase 4 activity so that cAMP accumulation is tightly regulated. 31,40 We sought to determine whether phosphodiesterase 4 inhibition in the Figure 1. Extracellular protein profiles and expression analysis of P aeruginosa strains used in this study. ...
Mammalian transmembrane adenylyl cyclases synthesize a restricted plasmalemmal cAMP pool that is intensely endothelial barrier protective. Bacteria have devised mechanisms of transferring eukaryotic factor-dependent adenylyl cyclases into mammalian cells. Pseudomonas aeruginosa ExoY is one such enzyme that catalyzes cytosolic cAMP synthesis, with unknown function. Pseudomonas aeruginosa genetically modified to introduce only the ExoY toxin elevated cAMP 800-fold in pulmonary microvascular endothelial cells over 4 hours, whereas a catalytically deficient (ExoY(K81M)) strain did not increase cAMP. ExoY-derived cAMP was localized to a cytosolic microdomain not regulated by phosphodiesterase activity. In contrast to the barrier-enhancing actions of plasmalemmal cAMP, the ExoY cytosolic cAMP pool induced endothelial gap formation and increased the filtration coefficient in the isolated perfused lung. These findings collectively illustrate a previously unrecognized mechanism of hyperpermeability induced by rises in cytosolic cAMP.
The pulmonary artery endothelium forms a semipermeable barrier that limits macromolecular flux through intercellular junctions. This barrier is maintained by an intrinsic forward protrusion of the interacting membranes between adjacent cells. However, dynamic interactions of these membranes have been incompletely quantified. Here, we present a novel technique to quantify motion of the peripheral membrane of cells, called paracellular morphological fluctuations (PMFs), and to assess the impact of substrate stiffness on PMFs. Substrate stiffness impacted large length scale morphological changes such as cell size and motion. Cell size was larger on stiffer substrates, whereas the speed of cell movement was decreased on hydrogels with stiffness either larger or smaller than 1.25 kPa, consistent with cells approaching a jammed state. Pulmonary artery endothelial cells moved fastest on 1.25 kPa hydrogel, stiffness consistent with a healthy pulmonary artery. Unlike these large length scale morphological changes, the baseline of PMFs was largely insensitive to substrate stiffness on which cells were cultured. Activation of store-operated calcium channels using thapsigargin treatment triggered a transient increase in PMFs beyond control treatment. However, in hypocalcemic conditions, such an increase in PMFs was absent on 1.25 kPa hydrogel but was present on 30 kPa hydrogel - a stiffness consistent with that of a hypertensive pulmonary artery. These findings indicate: (i) PMFs occur in cultured endothelial cell clusters, irrespective of the substrate stiffness; (ii) PMFs increase in response to calcium influx through store-operated calcium entry channels; and (iii) stiffer substrate promotes PMFs through a mechanism that does not require calcium influx.
The lungs of patients with acute respiratory distress syndrome (ARDS) have hyperpermeable capillaries that must undergo repair in an acidic microenvironment. Pulmonary microvascular endothelial cells (PMVECs) have an acid-resistant phenotype, in part due to carbonic anhydrase IX (CA IX). CA IX also facilitates PMVEC repair by promoting aerobic glycolysis, migration, and network formation. Molecular mechanisms of how CA IX performs such a wide range of functions are unknown. CA IX is comprised of four domains known as the proteoglycan-like (PG), catalytic (CA), transmembrane (TM), and intracellular (IC) domains. We hypothesized that the PG and CA domains mediate PMVEC pH homeostasis and repair, and the IC domain regulates aerobic glycolysis and PI3k/Akt signaling. The functions of each CA IX domain were investigated using PMVEC cell lines that express either a full-length CA IX protein or a CA IX protein harboring a domain deletion. We found that the PG domain promotes intracellular pH homeostasis, migration, and network formation. The CA and IC domains mediate Akt activation but negatively regulate aerobic glycolysis. The IC domain also supports migration while inhibiting network formation. Finally, we show that exposure to acidosis suppresses aerobic glycolysis and migration, even though intracellular pH is maintained in PMVECs. Thus, we report that 1) The PG and IC domains mediate PMVEC migration and network formation, 2) the CA and IC domains support PI3K/Akt signaling, and 3) acidosis impairs PMVEC metabolism and migration independent of intracellular pH homeostasis.
Sodium-hydrogen exchangers (NHEs) tightly regulate intracellular pH (pH i ), proliferation, migration and cell volume. Heterogeneity exists between pulmonary endothelial cells derived from different vascular segments, yet the activity and isoform expression of NHEs between these vascular segments has not been fully examined. Utilizing the ammonium-prepulse and recovery from acidification technique in a buffer lacking bicarbonate, pulmonary microvascular and pulmonary artery endothelial cells exhibited unique recovery rates from the acid load dependent upon the concentration of the sodium transport inhibitor, amiloride; further, pulmonary artery endothelial cells required a higher dose of amiloride to inhibit sodium-dependent acid recovery compared to pulmonary microvascular endothelial cells, suggesting a unique complement of NHEs between the different endothelial cell types. While NHE1 has been described in pulmonary endothelial cells, all NHE isoforms have not been accounted for. To address NHE expression in endothelial cells, qPCR was performed. Using a two-gene normalization approach, Sdha and Ywhag were identified for qPCR normalization and analysis of NHE isoforms between pulmonary microvascular and pulmonary artery endothelial cells. NHE1 and NHE8 mRNA were equally expressed between the two cell types, but NHE5 expression was significantly higher in pulmonary microvascular versus pulmonary artery endothelial cells, which was confirmed at the protein level. Thus, pulmonary microvascular and pulmonary artery endothelial cells exhibit unique NHE isoform expression and have a unique response to acid load revealed through recovery from cellular acidification.
A ubiquitous second messenger molecule, cAMP is responsible for orchestrating many different cellular functions through a variety of pathways. Fӧrster resonance energy transfer (FRET) probes have been used to visualize cAMP spatial gradients in pulmonary microvascular endothelial cells (PMVECs). However, FRET probes have inherently low signal-to-noise ratios; multiple sources of noise can obscure accurate visualization of cAMP gradients using a hyperspectral imaging system. FRET probes have also been used to measure cAMP gradients in 3D; however, it can be difficult to differentiate between true FRET signals and noise. To further understand the effects of noise on experimental data, a model was developed to simulate cAMP gradients under experimental conditions. The model uses a theoretical cAMP heatmap generated using finite element analysis. This heatmap was converted to simulate the FRET probe signal that would be detected experimentally with a hyperspectral imaging system. The signal was mapped onto an image of unlabeled PMVECs. The result was a time lapse model of cAMP gradients obscured by autofluorescence, as visualized with FRET probes. Additionally, the model allowed the simulated expression level of FRET signal to be varied. This allowed accurate attribution of signal to FRET and autofluorescence. Comparing experimental data to the model results at different levels of FRET efficiency has allowed improved understanding of FRET signal specificity and how autofluorescence interferes with FRET signal detection. In conclusion, this model can more accurately determine cAMP gradients in PMVECs. This work was supported by NIH award P01HL066299, R01HL58506 and NSF award 1725937.
Pulmonary microvascular endothelial cells (PMVECs) uniquely express an α 1G -subtype of voltage-gated T-type Ca ²⁺ channel. We have previously revealed that the α 1G channel functions as a background Ca ²⁺ entry pathway that is critical for the cell proliferation, migration, and angiogenic potential of PMVECs, a novel function attributed to the coupling between α 1G -mediate Ca ²⁺ entry and constitutive Akt phosphorylation and activation. Despite this significance, mechanism(s) that link the α 1G -mediated Ca ²⁺ entry to Akt phosphorylation remain incompletely understood. In the present study, we demonstrate that Ca ²⁺ /calmodulin-dependent protein kinase (CaMK) 4 serves as a downstream effector of the α 1G -mediated Ca ²⁺ entry to promote the angiogenic potential of PMVECs. Notably, CaMK2 and CaMK4 are both expressed in PMVECs. Pharmacological blockade or genetic knockdown of the α 1G channel led to a significant reduction in the phosphorylation level of CaMK4 but not the phosphorylation level of CaMK2. Pharmacological inhibition as well as genetic knockdown of CaMK4 significantly decreased cell proliferation, migration, and network formation capacity in PMVECs. However, CaMK4 inhibition or knockdown did not alter Akt phosphorylation status in PMVECs, indicating that α 1G /Ca ²⁺ /CaMK4 is independent of the α 1G /Ca ²⁺ /Akt pathway in sustaining the cells' angiogenic potential. Altogether, these findings suggest a novel α 1G -CaMK4 signaling complex that regulates the Ca ²⁺ -dominated angiogenic potential in PMVECs.
Since the totemic achievement of human-induced pluripotent stem cells (iPSCs) in the stem cell field, we have witnessed a remarkable expansion in publications related to this discovery that could have central relevance to health and disease. One example of an offshoot from this achievement was the differentiation of iPSCs utilizing 3D in vitro models that resulted in the formation of organoids, such as lung organoids. These fascinating miniature organs are robustly generated through the process of self-assembly in response to morphogenetic cues. Organoids beautifully resemble the architecture and function of their in vivo counterparts. The marriage of this system and genome-editing techniques has powerfully advanced our knowledge of human lung biology, disease modeling, and drug discoveries, particularly where animal models are limited. Presently, human iPSC–derived lung organoids are developmentally nascent compared to their adult tissues. A deeper comprehension of the principles governing lung organogenesis combined with “lung-on-a-chip” technology could reconcile diverse cellular, vascular, and matrix strategies to reconstruct a complete niche in vitro. Ultimately, future progress is dependent on the convergence of multiple disciplines of biomedical science. The epochal impact of iPSC-lung organoid studies heralds their coming-of-age in creating novel, personalized therapeutics for regenerative medicine.
Low tidal volume ventilation protects the lung in mechanically ventilated patients. The impact of the accompanying permissive hypoxemia and hypercapnia on endothelial cell recovery from injury is poorly understood. Carbonic anhydrase IX (CA IX) is expressed in pulmonary microvascular endothelial cells (PMVECs), where it contributes to CO2 and pH homeostasis, bioenergetics and angiogenesis. We hypothesized that CA IX is important for PMVEC survival, and CA IX expression and release from PMVECs are increased during infection. While plasma CA IX was unchanged in human and rat pneumonia, there was a trend towards increasing CA IX in bronchoalveolar fluid of mechanically ventilated critically ill pneumonia patients and a significant increase in CA IX in lung tissue lysate of rat pneumonia. To investigate functional implications of the lung CA IX increase, we generated PMVEC cell lines harboring domain-specific CA IX mutations. Using these cells, we found that infection promotes intracellular expression, release and metalloproteinase-mediated extracellular cleavage of CA IX in PMVECs. Intracellular domain deletion uniquely impaired CA IX membrane localization. Loss of the CA IX intracellular domain promoted cell death following infection, suggesting the important role of intracellular domain in PMVEC survival. We also found that hypoxia improves survival, whereas hypercapnia reverses the protective effect of hypoxia, during infection. Thus, we report that: (1) CA IX increases in rat pneumonia lung; and, (2) the CA IX intracellular domain and hypoxia promote PMVEC survival during infection.
Aims
Sepsis is a systemic inflammatory complication, which is the common cause of death in critical patients. This study aimed to evaluate the potential regulatory mechanisms of miR-150 in lipopolysaccharide (LPS)-challenged HUVECs and cecal ligation and puncture (CLP)-induced septic mice.
Materials and methods
Human umbilical vein endothelial cells (HUVECs) were challenged with LPS. Pulmonary arterial endothelial cells (PAECs) were isolated from CLP-induced septic mice. The mRNA and protein levels of target molecules were detected by RT-qPCR and Western blotting. Apoptosis of HUVECs was determined by Annexin V/PI staining on a flow cytometry. The interaction between miR-150 and MALAT1 was assessed by luciferase reporter assay, RIP and RNA pull-down assay.
Key findings
MiR-150 was downregulated in LPS-induced HUVECs. MiR-150 mimics restrained LPS-induced inflammatory response by reducing TNF-α and IL-6 levels, but increasing IL-10 level. Moreover, miR-150 mimics downregulated endoplasmic reticulum (ER) stress-related proteins, GRP78 and CHOP levels in LPS-exposed HUVECs. Additionally, LPS-induced apoptosis was suppressed by miR-150 mimics via decreasing cleaved caspase-3 and Bax levels, while enhancing Bcl-2 level. Mechanistically, MALAT1 could competitively bind to miR-150. LPS-induced apoptosis, ER stress and inflammation were promoted by MALAT1 overexpression, but reversed by siMALAT1. Furthermore, miR-150 inhibitor strengthened LPS-induced apoptosis, ER stress and inflammation, which could be attenuated by siMALAT1 via regulating NF-κB pathway. Finally, agomiR-150 repressed ER stress and inflammatory response in PAECs isolated from septic mice via decreasing MALAT1 level.
Significance
Our findings suggest that miR-150 affects sepsis-induced endothelial injury by regulating ER stress and inflammation via MALAT1-mediated NF-κB pathway.
Activation of the inflammasome-caspase-1 axis in lung endothelial cells is emerging as a novel arm of the innate immune response to pneumonia and sepsis caused by Pseudomonas aeruginosa. Increased levels of circulating autacoids are hallmarks of pneumonia and sepsis, and induce physiological responses via cAMP signaling in targeted cells. However, it is unknown whether cAMP affects other functions such as P. aeruginosa-induced caspase-1 activation. Herein, we describe the effects of cAMP signaling on caspase-1 activation using a single cell flow cytometry-based assay. P. aeruginosa infection of cultured lung endothelial cells caused caspase-1 activation in a distinct population of cells. Unexpectedly, pharmacological cAMP elevation increased the total number lung endothelial cells with activated caspase-1. Interestingly, the addition of cAMP agonists augmented P. aeruginosa infection of lung endothelial cells as a partial explanation underlying cAMP priming of caspase-1 activation. The cAMP effect(s) appeared to function as a priming signal, because the addition of cAMP agonists was required either prior to, or early during the onset of infection. However, absolute cAMP levels measured by ELISA were not predictive of cAMP priming effects. Importantly, inhibition of de novo cAMP synthesis decreased the number of lung endothelial cells with activated caspase-1 during infection. Collectively, our data suggest lung endothelial cells rely on cAMP signaling to prime caspase-1 activation during P. aeruginosa infection.
Acidosis is common among critically ill patients, but current approaches to correct pH do not improve disease outcomes. During systemic acidosis, cells are either passively exposed to extracellular acidosis that other cells have generated (extrinsic acidosis) or they are exposed to acid which they generate and export into the extracellular space (intrinsic acidosis). Although endothelial repair following intrinsic acidosis has been studied, the impact of extrinsic acidosis on migration and angiogenesis is unclear. We hypothesized that extrinsic acidosis inhibits metabolism and migration but promotes capillary-like network formation in pulmonary microvascular endothelial cells (PMVECs). Extrinsic acidosis was modeled by titrating media pH. Two types of intrinsic acidosis were compared; including increasing cellular metabolism by chemically inhibiting carbonic anhydrases (CAs) IX and XII (SLC-0111) and with hypoxia. PMVECs maintained baseline intracellular pH for 24 hours with both extrinsic and intrinsic acidosis. Whole cell CA IX protein expression was decreased by extrinsic acidosis, but not affected by hypoxia. When extracellular pH was equally acidic, extrinsic acidosis suppressed glycolysis, whereas intrinsic acidosis did not. Extrinsic acidosis suppressed migration, but increased Matrigel network master junction and total segment length. CRISPR-Cas9 CA IX knockout PMVECs revealed an independent role of CA IX in promoting glycolysis, as loss of CA IX alone was accompanied by decreased hexokinase I and pyruvate dehydrogenase E1α expression and decreasing migration. 2-deoxy-D-glucose had no effect on migration, but profoundly inhibited network formation and increased N-cadherin expression. Thus, we report that while extrinsic acidosis suppresses endothelial glycolysis and migration, it promotes network formation.
The second messenger, cAMP, is highly compartmentalized to facilitate signaling specificity. Extracellular vesicles (EVs) are submicron, intact vesicles released from many cell types that can act as biomarkers or be involved in cell-to-cell communication. Although it is well recognized that EVs encapsulate functional proteins and RNAs/miRNAs, currently it is unclear whether cyclic nucleotides are encapsulated within EVs to provide an additional second messenger compartment. Using ultracentrifugation, EVs were isolated from the culture medium of unstimulated systemic and pulmonary endothelial cells. EVs were also isolated from pulmonary microvascular endothelial cells (PMVECs) following stimulation of transmembrane adenylyl cyclase (AC) in the presence or absence of the phosphodiesterase 4 inhibitor rolipram over time. Whereas cAMP was detected in EVs isolated from endothelial cells derived from different vascular beds, it was highest in EVs isolated from PMVECs. Treatment of PMVECs with agents that increase near-membrane cAMP led to an increase in cAMP within corresponding EVs, yet there was no increase in EV number. Elevated cell cAMP, measured by whole cell measurements, peaked 15 min after treatment, yet in EVs the peak increase in cAMP was delayed until 60 min after cell stimulation. Cyclic AMP was also increased in EVs collected from the perfusate of isolated rat lungs stimulated with isoproterenol and rolipram, thus corroborating cell culture findings. When added to unperturbed confluent PMVECs, EVs containing elevated cAMP were not barrier disruptive like cytosolic cAMP but maintained monolayer resistance. In conclusion, PMVECs release EVs containing cAMP, providing an additional compartment to cAMP signaling.
It has been long established that the pathobiology of acute lung injury (ALI) includes increased alveolar permeability due to altered integrity of intracellular adhesion molecules. More recently, it has been recognized that other alveolar proteins (e.g. antioxidants, solute transport or surfactant proteins) may also be compromised in ALI. Up to now, no avenues existed to restore the function of these proteins. Gene therapy and its potential to increase protein function has created the possibility of restoring or even augmenting alveolar function in the setting of ALI and as such offers tremendous promise for new treatments of ALI.
Endothelial barrier function is an essential and tightly regulated process that ensures proper compartmentalization of the vascular and interstitial space, while allowing for the diffusive exchange of small molecules and the controlled trafficking of macromolecules and immune cells. Failure to control endothelial barrier integrity results in excessive leakage of fluid and proteins from the vasculature that can rapidly become fatal in scenarios such as sepsis or the acute respiratory distress syndrome. Here, we highlight recent advances in our understanding on the regulation of endothelial permeability, with a specific focus on the endothelial glycocalyx and endothelial scaffolds, regulatory intracellular signaling cascades, as well as triggers and mediators that either disrupt or enhance endothelial barrier integrity, and provide our perspective as to areas of seeming controversy and knowledge gaps, respectively.
This paper addresses the mechanisms behind selective endothelial permeability and their regulations. The singular properties of each of the seven blood-tissues barriers. Then, it further revisits the physical, quantitative meaning of permeability, and the way it should be measured based on sound physical chemistry reasoning and methodologies. Despite the relevance of permeability studies one often comes across inaccurate determinations, mostly from oversimplified data analyses. To worsen matters, the exact meaning of permeability is being lost along with this loss of accuracy. The importance of proper permeability calculation is illustrated with a family of derivatives of kyotorphin, an analgesic dipeptide.
Pulmonary endothelial dysfunction plays an integral role in the pathogenesis and development of pulmonary hypertension. It is difficult and inconvenient to obtain pulmonary arterial endothelial cells (PAECs) from humans and large animals. Some methods for the isolation of PAECs from rats require complex equipment and expensive reagents. In this study, we describe a new method of obtaining cultures of PAECs isolated from rat pulmonary arteries with Chinese acupuncture needles. We acquired PAECs in 5 steps. These were: the isolation of pulmonary arteries, exposure of endothelium, enzymatic digestion, concentration of resuspended pellets and incubation. PAECs were characterized by morphological activity and by immunostaining for von Willebrand factor, CD31 and CD34, but not for α-smooth muscle actin, smooth muscle myosin heavy chain or CD90/Thy-1. Furthermore, transmission electron microscopy was carried out, confirming the presence of Weibel-Palade bodies that are characteristic ultrastructures of vascular endothelial cells. In conclusion, we established a simple and economical technique to isolate and culture PAECs from rat pulmonary arteries. These PAECs exhibit features consistent with vascular endothelial cells, and they could subsequently be used to study pathophysiological mechanisms involving the pulmonary arterial endothelium. © 2013 S. Karger AG, Basel.
The syndrome of acute lung injury (ALI), known in its most severe form as the acute respiratory distress syndrome (ARDS),
is characterized by increased alveolar-capillary membrane permeability and subsequent pulmonary edema. ARDS can be initiated
by any one of an extensive and heterogeneous list of pulmonary or systemic insults (Fig. 1), the most frequent of which is sepsis [1]. What these inciting factors have in common, is the ability to initiate activation of an acute inflammatory response, leading
to dysfunction of multiple organs including the lung, heart, kidneys, and liver. Polymorphonuclear leukocytes (PMN, neutrophils),
that normally pass through the microcirculation of the lung and other tissues relatively unimpeded, are sequestered and activated
in the microvasculature during the genesis of an inflammatory response. The activation of neutrophils leads to the release
of cytotoxic products that, when released in an unregulated manner, may damage cells in proximity, leading to organ injury
and dysfunction.
Fig. 1. Leukocyte-mediated tissue injury in systemic inflammation
The vascular endothelium can be considered as an organ/tissue which comprises a monolayer of endothelial cells which serve
as a semipermeable cellular barrier separating the inner space of blood vessels from its surrounding tissue and to control
the exchange of fluids and cells between the two compartments. Since the pulmonary circulation receives the entire cardiac
output, the large surface area of the lung microvasculature is well suited for sensing mechanical, chemical, and cellular
injury by inhaled or circulating substances. This endothelial barrier is dynamically regulated through exposure to these various
stimuli of physiological and pathological origin and serves to regulate multiple key biological processes (including lung
fluid balance and solute transport between vascular compartments). For example, an increase in vascular permeability is a
necessary feature of the body’s defense mechanism to provide injured tissues with access to leucocytes, resulting in tissue
edema due to fluid extravasation. However, during conditions of intense lung inflammation such as observed in acute lung injury
or its severer form of acute respiratory distress syndrome, the large surface area becomes a liability and provides the opportunity
for profound vascular permeability resulting in massive fluid accumulation in the alveolar space and progressively leading
to pulmonary failure. Alterations in vascular permeability occur not only in acute inflammatory lung disorders primarily caused
by sepsis, pneumonia, and trauma which result in high rates of patient morbidity and mortality, but are an attractive target
for therapeutic intervention in subacute lung inflammatory disorders such as ischemia–reperfusion injury, radiation lung injury,
and asthma. Thus, understanding the mechanisms of endothelial barrier dysfunction is vital for the management and treatment
of key and enigmatic pulmonary disorders.
At sites of ongoing inflammation, polymorphonuclear leukocytes (PMN, neutrophils) migrate across vascular endothelia, and such transmigration has the potential to disturb barrier properties and can result in intravascular fluid loss and edema. It was recently appreciated that endogenous pathways exist to dampen barrier disruption during such episodes and may provide an important anti-inflammatory link. For example, during transmigration, PMN-derived adenosine activates endothelial adenosine receptors and induces a cAMP-dependent resealing of endothelial barrier function. In our study reported here, we sought to understand the link between cyclic nucleotide elevation and increased endothelial barrier function. Initial studies revealed that adenosine-induced barrier function is tightly linked to activation of protein kinase A (PKA). Because PKA selectively phosphorylates serine and threonine residues, we screened zonula occludens-1 (ZO-1) immunoprecipitates for the existence of such phosphorylated proteins as targets for barrier regulation. This analysis revealed a dominantly phosphorylated band at 50 kDa. Microsequencing identified this protein as vasodilator-stimulated phosphoprotein (VASP), an actin binding protein with multiple serine/threonine phosphorylation sites. Immunofluorescent microscopy revealed that VASP localizes to endothelial junctional complexes and colocalizes with ZO-1, occludin, and junctional adhesion molecule-1 (JAM-1). To address the role of phospho-VASP in regulation of barrier function, we generated a phosphospecific VASP antibody targeting the Ser157 residue phosphorylation site, the site preferred by PKA. Immunolocalization studies with this antibody revealed that upon PKA activation, phospho-VASP appears at cell-cell junctions. Transient transfection of truncated VASP fragments revealed a parallel increase in barrier function. Taken together, these studies reveal a central role for phospho-VASP in the coordination of PKA-regulated barrier function, such as occurs during episodes of inflammation.
cAMP was the first second messenger discovered. Since its original description,elements of the cAMP signal transduction cascade have grown in complexity. The idea that cAMP quickly and evenly disperses within the cytosolic compartment to achieve a uniform cell signal has slowly faded. The challenge before us is to understand how CAMP signaling is achieved with spatio-temporal fidelity. This fidelity is cell-type specific and in accordance with any cell's unique function. To fully accomplish such a high level of understanding, identifying the molecular anatomy of the cAMP signaling scaffold must be put in context with a cell's specialized phenotype. Pulmonary microvacular endothelial cells must maintain a tight barrier function to limit fluid, solute and macromolecular flux, and preserve alveolar gas exchange. Therefore, membrane cAMP concentrations are maintained at a high level, and CAMP spillover into the bulk cytosol is limited by high, membrane-associated phosphodiesterase activity. This arrangement allows CAMP to achieve concentrations high enough to activate localized effectors that promote barrier function, while maintaining bulk cytosolic cAMP concentrations below a threshold required to activate effectors that disrupt barrier function. Calcium inhibition of membrane cAMP allows transient gap formation. Generation of cAMP outside the membrane compartment produces a sustained increase in microvascular endothelial cell permeability. In this context, pulmonary microvascular endothelium has developed a highly ordered and specialized mechanism for regulating cAMP production to dynamically control alveolar-capillary stability on a moment-to-moment basis.
Phosgene gas is a lower respiratory tract irritant. As such, it stimulates nociceptive vagal C-fiber related reflexes in a dose-rate and concentration x exposure duration (Cxt)-dependent manner. In rats this reflex is characterized by extended apnea time periods, bradycardia, and hypothermia. While inhalation exposures at non-lethal Cxt products show rapid reversibility of reflexively-induced changes in respiratory patterns, lethal Cxt products seem to cause pro-longed stimulation after discontinued exposure to phosgene. This observation has been taken as indirect evidence that phosgene-induced lethal lung edema is likely to be associated with a dysfunctional neurogenic control of cardiopulmonary and microvascular physiology. In order to verify this hypothesis, data from respiratory function measurements during and after the inhalation exposure to phosgene gas were compared with time-course measurements of cardiac function over 20 hours post-phosgene exposure. These data were complemented by time-course analyses of nitric oxide (NO(e)) and carbon dioxide in exhaled breath, including time-dependent changes of extravasated protein in bronchoalveolar lavage fluid (BALF) and hemoglobin in blood. The nitric oxidase synthetase inhibitors L-NAME and L-NIL were used to further elucidate the role of NO(e) in this type of acute lung injury and whether its analysis can serve as an early biomarker of pulmonary injury. Collectively, the sequence and time-course of pathological events in phosgene-induced lung edema appear to suggest that over-stimulated, continued sensorimotor vagal reflexes affect cardiopulmonary hemodynamics. A continued parasympathetic tone appears to be involved in this etiopathology.
This chapter focuses on the heterogeneity of pulmonary endothelium. The heterogeneity of endothelial cells has become increasingly more apparent and more appreciated. The phenotypic variation is characterized by differences in function, antigenic composition, metabolic properties, and in their response to growth factors. Continuous endothelium is characteristic of brain, retina, and muscular capillaries. While endothelial cells in brain exhibit close junctional apposition to one another and a tight permeability barrier to fluid and solutes (i.e., the blood/brain barrier), fenestrated endothelium is found in endocrine glands and the kidney. The endothelium of the glomeruli is loosely connected and readily permits the transendothelial passage of relatively large solutes. Moreover, within an organ, the endothelial cells vary with the size, function, and location of the vessel and can even vary within discrete segments of a single microcirculatory loop. As the lungs receive both right heart pulmonary circulation and left heart bronchial circulation, the endothelium of these respective systems might be expected to exhibit diversity. Because of the large capillary surface area of pulmonary circulation and the extremely fine alveolar–capillary membrane, formed by juxtaposition of the thin endothelial cell and alveolar type I epithelial cells, there is a critical necessity for maintenance of barrier integrity in the pulmonary circulation. Therefore, the function of pulmonary endothelium has received considerable investigative attention.
Neutrophil adherence to tumor necrosis factor-alpha (TNF-alpha)-treated human pulmonary microvascular endothelial cells (PMECs) induces cytoskeletal changes in endothelial cells that require intercellular adhesion molecule-1 (ICAM-1)-dependent signaling events. This study determined whether similar changes occurred in rat PMECs and whether rat pulmonary arterial endothelial cells (PAECs) responded differently. Neutrophil adherence induced an increase in the formation of F-actin and in the apparent stiffness of TNF-alpha-treated rat PMECs. These responses, however, were absent in PAECs. To determine the mechanisms underlying these differences, ICAM-1-mediated signaling events were compared. Upregulation of ICAM-1 by TNF-alpha and redistribution of ICAM-1 induced by cross-linking antibodies were similar in both cell types. However, neutrophil adherence induced production of reactive oxygen species only in PMECs and not in PAECs. Moreover, phosphorylation of p38 mitogen-activated protein kinase induced by ICAM-1 cross-linking occurred only in PMECs and not in PAECs. This increase in p38 phosphorylation in PMECs was inhibited by allopurinol, a xanthine oxidase inhibitor. These data demonstrated that whereas TNF-alpha upregulated ICAM-1 and ICAM-1 cross-linking induced a similar redistribution of ICAM-1 on the endothelial cell surface, ICAM-1 ligation initiated p38 activation and cytoskeletal rearrangements only in PMECs and not in PAECs. Thus, neutrophil adhesion through ICAM-1 induced signaling events leading to cytoskeletal changes only in PMECs, the site of neutrophil emigration and edema formation, and not in PAECs.
Cyclic AMP signals encode information required to differentially regulate a wide variety of cellular responses; yet it is not well understood how information is encrypted within these signals. An emerging concept is that compartmentalization underlies specificity within the cAMP signaling pathway. This concept is based on a series of observations indicating that cAMP levels are distinct in different regions of the cell. One such observation is that cAMP production at the plasma membrane increases pulmonary microvascular endothelial barrier integrity, whereas cAMP production in the cytosol disrupts barrier integrity. To better understand how cAMP signals might be compartmentalized, we have developed mathematical models in which cellular geometry as well as total adenylyl cyclase and phosphodiesterase activities were constrained to approximate values measured in pulmonary microvascular endothelial cells. These simulations suggest that the subcellular localizations of adenylyl cyclase and phosphodiesterase activities are by themselves insufficient to generate physiologically relevant cAMP gradients. Thus, the assembly of adenylyl cyclase, phosphodiesterase, and protein kinase A onto protein scaffolds is by itself unlikely to ensure signal specificity. Rather, our simulations suggest that reductions in the effective cAMP diffusion coefficient may facilitate the formation of substantial cAMP gradients. We conclude that reductions in the effective rate of cAMP diffusion due to buffers, structural impediments, and local changes in viscosity greatly facilitate the ability of signaling complexes to impart specificity within the cAMP signaling pathway.
In recent years there has been an increasing appreciation of the functional heterogeneity that exists between extraalveolar and alveolar endothelial cells. One of the most striking features of pulmonary microvascular endothelial cells is that they possess a highly impermeable barrier with respect to pulmonary artery or vein endothelial cells. This cellular feature is observed in culture and in the intact microcirculation, prompting a reevaluation of the key physiological principles that control permeability and the fate of fluid (or exudate) once it leaves the circulation. Pulmonary microvascular endothelial cells express calcium channels not found in extraalveolar endothelial cells, including the vanilloid family transient receptor potential 4 channel and the α1G T-type calcium channel. Whereas activation of the TRPV4 channel causes alveolar flooding, activation of the α1G T-type calcium channel promotes P-selectin surface translocation, events specific to the microcirculation. Although endothelium is an attractive therapeutic target in acute lung injury and other vascular disorders, the growing awareness of pulmonary endothelial cell heterogeneity increasingly suggests that a panendothelial cell approach is suboptimal. Rather, development of novel therapeutics based upon anatomically restricted expression of molecular signatures may be developed to better combat vascular disease.
We studied the effects of intravenous infusion of recombinant human tumor necrosis factor type alpha (rTNF-alpha; 12 micrograms/kg) on lung fluid balance in sheep prepared with chronic lung lymph fistulas. The role of neutrophils was examined in sheep made neutropenic with hydroxyurea (200 mg/kg for 4 or 5 days) before receiving rTNF-alpha. Infusion of rTNF-alpha resulted in respiratory distress and 3-fold increases in pulmonary arterial pressure and pulmonary vascular resistance within 15 min, indicating intense pulmonary vasoconstriction. Pulmonary lymph flow (i.e., net transvascular fluid filtration rate) and transvascular protein clearance rate (a measure of vascular permeability to protein) increased 2-fold within 30 min. The increased permeability was associated with leukopenia and neutropenia. The pulmonary hypertension and vasoconstriction subsided but fluid filtration and vascular permeability continued to increase. Sheep made neutropenic had similar increases in pulmonary transvascular fluid filtration and vascular permeability. rTNF-alpha also produced concentration-dependent increases in permeability of 125I-labeled albumin across ovine endothelial cell monolayers in the absence of neutrophils or other inflammatory mediators. The results indicate that rTNF-alpha increases pulmonary vascular permeability to protein by an effect on the endothelium.
VASP (vasodilator-stimulated phosphoprotein), an established substrate of cAMP- and cGMP-dependent protein kinases in vitro and in living cells, is associated with focal adhesions, microfilaments, and membrane regions of high dynamic activity. Here, the identification of an 83-kDa protein (p83) that specifically binds VASP in blot overlays of different cell homogenates is reported. With VASP overlays as a detection tool, p83 was purified from porcine platelets and used to generate monospecific polyclonal antibodies. VASP binding to purified p83 in solid-phase binding assays and the closely matching subcellular localization in double-label immunofluorescence analyses demonstrated that both proteins also directly interact as native proteins in vitro and possibly in living cells. The subcellular distribution, the biochemical properties, as well as microsequencing data revealed that porcine platelet p83 is related to chicken gizzard zyxin and most likely represents the mammalian equivalent of the chicken protein. The VASP-p83 interaction may contribute to the targeting of VASP to focal adhesions, microfilaments, and dynamic membrane regions. Together with our recent identification of VASP as a natural ligand of the profilin poly-(L-proline) binding site, our present results suggest that, by linking profilin to zyxin/p83, VASP may participate in spatially confined profilin-regulated F-actin formation.
Maintenance by the endothelium of a semi-permeable barrier is critically important in the exchange of oxygen and carbon dioxide in the lung. Intracellular free Ca2+ ([Ca2+]i) and cAMP are principal determinants of endothelial cell barrier function through their mutually opposing actions on endothelial retraction. However, details of the mechanisms of this antagonism are lacking. The recent discovery that certain adenylyl cyclases (EC 4.6.1.1) could be acutely inhibited by Ca2+ in the intracellular concentration range provided one possible mechanism whereby elevated [Ca2+]i could decrease cAMP content. This possibility was explored in pulmonary artery endothelial cells. The results indicate that a type VI Ca(2+)-inhibitable adenylyl cyclase exists in pulmonary artery endothelial cells and is modulated by physiological changes in [Ca2+]i. Furthermore, the results suggest the inverse relationship between [Ca2+]i and cAMP that is established by Ca(2+)-inhibitable adenylyl cyclase plays a critical role in modulating pulmonary artery endothelial cell permeability. These data provide evidence that susceptibility to inhibition of adenylyl cyclase by Ca2+ can be exploited in modulating a central physiological process.
The vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP- and cGMP-dependent protein kinases in vitro and in intact cells, is associated with actin filaments, focal adhesions and dynamic membrane regions. VASP, cloned here from human HL-60 and canine MDCK cells, is organized into three distinct domains. A central proline-rich domain contains a GPPPPP motif as a single copy and as a 3-fold tandem repeat, as well as three conserved phosphorylation sites for cyclic nucleotide-dependent protein kinases. A C-terminal domain contains a repetitive mixed-charge cluster which is predicted to form an alpha-helix. The hydrodynamic properties of purified human VASP together with the calculated molecular mass of cloned VASP suggest that the native protein is a homotetramer with an elongated structure. VASP over-expressed in transiently transfected BHK21 cells was predominantly detected at stress fibres, at focal adhesions and in F-actin-containing cell surface protrusions, whereas truncated VASP lacking the C-terminal domain was no longer concentrated at focal adhesions. These data indicate that the C-terminal domain is required for anchoring VASP at focal adhesion sites, whereas the central domain is suggested to mediate VASP interaction with profilin. Our results provide evidence for the structural basis by which VASP, both a target of the cAMP and cGMP signal transduction pathways and a component of the actin-based cytoskeleton, including the cytoskeleton-membrane interface, may be able to exchange signals between these networks.
Cyclic AMP induces corticosteroid production, differential gene transcription, and cell cycle arrest in adrenal cortex-derived Y1 cells. These responses follow a cAMP-controlled transformation in Y1 cell morphology: the conversion of flat epithelial cells into rounded, highly refractile cells with short processes. Little is known about effector proteins and mechanisms that link activated protein kinase A to the alteration in cell shape. We now report that cAMP causes rapid (</=1 min) and selective tyrosine dephosphorylation of paxillin, a focal adhesion protein. Paxillin is maximally dephosphorylated before other physiological effects of cAMP are detected in Y1 cells. Dephosphopaxillin translocates from focal adhesions to the cytoplasm as stress fibers vanish and F-actin accumulates in membrane ruffles and cytoplasmic aggregates. Remnants of focal adhesion complexes dissociate from the cell cortex and coalesce into large structures that contain aggregated F-actin. Pervanadate, an inhibitor of protein-tyrosine phosphatases, abrogates all effects of cAMP. Conversely, genistein-sensitive protein-tyrosine kinase activity is essential for establishing epithelial morphology and reversing effects of cAMP in Y1 cells. Thus, cAMP/protein kinase A (PKA) actions are initially targeted to focal adhesions and cortical actin cytoskeleton; paxillin is an early and unexpected downstream target in a PKA-mediated signaling pathway, and protein-tyrosine phosphatase activity provides an essential link between PKA activation and the control of cell shape.
Forskolin- and Gsα-stimulated adenylyl cyclase activity is observed after mixture of two independently-synthesized ∼25-kDa cytosolic fragments
derived from mammalian adenylyl cyclases (native M
r ∼ 120,000). The C1a domain from type V adenylyl cyclase (VC1) and the C2 domain from type II adenylyl cyclase (IIC2) can both be expressed in large quantities and purified to homogeneity. When mixed, their maximally stimulated specific activity,
150 μmol/min/mg protein, substantially exceeds values observed previously with the intact enzyme. A soluble, high-affinity
complex containing one molecule each of VC1, IIC2, and guanosine 5′-O-(3-thiotriphosphate) (GTPγS)-Gsα is responsible for the observed enzymatic activity and can be isolated. In addition, GTPγS-Gsαinteracts with homodimers of IIC2 to form a heterodimeric complex (one molecule each of Gsα and IIC2) but not detectably with homodimers of VC1. Nevertheless, Gsα can be cross-linked to VC1 in the activated heterotrimeric complex of VC1, IIC2, and Gsα, indicating its proximity to both components of the enzyme that are required for efficient catalysis. These results and those
in the accompanying report (Dessauer, C. W., Scully, T. T., and Gilman, A. G. (1997) J. Biol. Chem. 272, 22272–22277) suggest that activators of adenylyl cyclase facilitate formation of a single, high-activity catalytic site
at the interface between C1 and C2.
Exposure of cultured human umbilical vein endothelial cells to the cAMP agonists theophylline and forskolin decreased constitutive isometric tension of a confluent monolayer inoculated on a collagen membrane, but it did not prevent increased tension in cells exposed to thrombin. The inability of cAMP agonists to prevent tension development correlated with an inability of cAMP stimulation to prevent increased 20-kDa myosin light chain (MLC20) phosphorylation in response to thrombin. Although cAMP did not prevent tension development or increased MLC20 phosphorylation, cAMP attenuated the effect of thrombin on transendothelial electrical resistance across a confluent monolayer inoculated on a gold microelectrode. Activation of cAMP-dependent signal transduction did not prevent a decline in resistance in thrombin-treated cells, but it more promptly restored transendothelial resistance to initial basal levels (10 min) compared with thrombin only (60 min). ML-7, an MLC kinase antagonist, at doses that attenuate increased MLC20 phosphorylation and tension development, did not prevent a decline in resistance in thrombin-treated cells. Yet, ML-7 also restored transendothelial resistance more rapidly than thrombin alone (20 min) but at a slower rate than cAMP. These data demonstrate that activation of cAMP-dependent signal transduction protects barrier function independent of inhibition of MLC20-dependent tension development.
Cytosolic Ca2+ concentration ([Ca2+]i) plays an important role in control of pulmonary vascular endothelial cell (ECs) barrier function. In this study, we investigated whether thapsigargin- and ionomycin-induced changes in cytosolic Ca2+ induce permeability in rat pulmonary microvascular (RPMV) versus macrovascular (RPA) ECs. In Transwell cultures, RPMVECs formed a tighter, more restrictive barrier than RPAECs to 12,000-, 72,000-, and 150,000-molecular-weight FITC-labeled dextrans. Thapsigargin (1 microM) produced higher [Ca2+]i levels in RPAECs than in RPMVECs and increased permeability in RPAEC but not in RPMVEC monolayers. Due to the attenuated [Ca2+]i response in RPMVECs, we investigated whether reduced activation of store-operated Ca2+ entry was responsible for the insensitivity to thapsigargin. Addition of the drug in media containing 100 nM extracellular Ca2+ followed by readdition media with 2 mM extracellular Ca2+ increased RPMVEC [Ca2+]i to a level higher than that in RPAECs. Under these conditions, RPMVEC permeability was not increased, suggesting that [Ca2+]i in RPMVECs does not initiate barrier disruption. Also, ionomycin (1.4 microM) did not alter RPMVEC permeability, but the protein phosphatase inhibitor calyculin A (100 nM) induced permeability in RPMVECs. These data indicate that, whereas increased [Ca2+]i promotes permeability in RPAECs, it is not sufficient in RPMVECs, which show an apparent uncoupling of [Ca2+]i signaling pathways or dominant Ca(2+)-independent mechanisms from controlling cellular gap formation and permeability.
Activation of Ca2+ entry is known to produce endothelial cell shape change, leading to increased permeability, leukocyte migration, and initiation of angiogenesis in conduit-vessel endothelial cells. The mode of Ca2+ entry regulating cell shape is unknown. We hypothesized that activation of store-operated Ca2+ channels (SOCs) is sufficient to promote cell shape change necessary for these processes. SOC activation in rat pulmonary arterial endothelial cells increased free cytosolic Ca2+ that was dependent on a membrane current having a net inward component of 5.45 +/- 0.90 pA/pF at -80 mV. Changes in endothelial cell shape accompanied SOC activation and were dependent on Ca2+ entry-induced reconfiguration of peripheral (cortical) filamentous actin (F-actin). Because the identity of pulmonary endothelial SOCs is unknown, but mammalian homologues of the Drosophila melanogaster transient receptor potential (trp) gene have been proposed to form Ca2+ entry channels in nonexcitable cells, we performed RT-PCR using Trp oligonucleotide primers in both rat and human pulmonary arterial endothelial cells. Both cell types were found to express Trp1, but neither expressed Trp3 nor Trp6. Our study indicates that 1) Ca2+ entry in pulmonary endothelial cells through SOCs produces cell shape change that is dependent on site-specific rearrangement of the microfilamentous cytoskeleton and 2) Trp1 may be a component of pulmonary endothelial SOCs.
An intact endothelial cell barrier maintains normal gas exchange in the lung, and inflammatory conditions result in barrier disruption that produces life-threatening hypoxemia. Activation of store-operated Ca2+ (SOC) entry increases the capillary filtration coefficient (Kf,c) in the isolated rat lung; however, activation of SOC entry does not promote permeability in cultured rat pulmonary microvascular endothelial cells. Therefore, current studies tested whether activation of SOC entry increases macro- and/or microvascular permeability in the intact rat lung circulation. Activation of SOC entry by the administration of thapsigargin induced perivascular edema in pre- and postcapillary vessels, with apparent sparing of the microcirculation as evaluated by light microscopy. Scanning and transmission electron microscopy revealed that the leak was due to gaps in vessels >/= 100 micrometer, consistent with the idea that activation of SOC entry influences macrovascular but not microvascular endothelial cell shape. In contrast, ischemia and reperfusion induced microvascular endothelial cell disruption independent of Ca2+ entry, which similarly increased Kf,c. These data suggest that 1) activation of SOC entry is sufficient to promote macrovascular barrier disruption and 2) unique mechanisms regulate pulmonary micro- and macrovascular endothelial barrier functions.
The increase in endothelial permeability in response to inflammatory mediators such as alpha-thrombin and histamine is accompanied by cell rounding and interendothelial gap formation, implicating that the predominant transport pathway is a diffusive one [i.e., via cellular junctions (paracellular transport)]. However, the possible contribution by vesicle-mediated transport (i.e., via albumin binding protein gp60) to the overall permeability increase needs investigation. Regulation of paracellular transport in endothelial cells is associated with modulation of actin-based systems which anchor the cell to its neighbor or extracellular matrix, thus maintaining endothelial integrity. At the cell-cell junctions, actin is linked indirectly to the plasma membrane by linking proteins (e.g., vinculin, catenins, alpha-actinin) to cadherins, which function in homophilic intercellular adhesion. Cadherins may also play a role in regulating the formation of tight junctions, which also may be associated with actin. At endothelial focal contacts, the transmembrane receptors (integrins) for matrix proteins are linked to actin via linking proteins (i.e., vinculin, talin, alpha-actinin). In response to inflammatory mediators, second messengers signal two regulatory pathways which modulate the actin-based systems, which may lead to impairment of the endothelial barrier integrity. One pathway is based on protein kinase C (PKC) isozyme-specific phosphorylation of linking proteins at the cell-cell and cell-matrix junctions. The increased phosphorylation is associated with actin reorganization, cell rounding, and increased paracellular transport. The other is the activation of myosin light-chain kinase, (MLCK), which causes an actin-myosin-based contraction that may lead to a centripetal retraction of endothelial cells. Current research is in the identification of protein substrates of PKC isozymes, the specific role of their phosphorylation in barrier function, and determining the precise role of MLCK in modulation of endothelial barrier function.
This unit presents two basic protocols to determine adenylate cyclase and guanylate cyclase activity in tissue and cell homogenates, permeabilized cells, or subcellular fractions. Two parts are presented for each method. First, the enzyme reaction that causes the formation of the labeled cyclic nucleotide is performed. Second, the separation of cyclic nucleotide products from unreacted nucleotide triphosphates and metabolites is performed using Dowex 50 resin and aluminum oxide chromatographies. In the case of guanylate cyclase, alternative separation protocols are also provided. Additionally, support protocols are provided that describe both the preparation of columns used in the assays and the preparation of tissue or cells to be assayed.
Surface proteins were compared in endothelial cells (EC) obtained from bovine peripheral lung, pulmonary artery and vein,
and dorsal aorta using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Galactose-containing glycoproteins
[molecular weight (Mr) 160–220 and 40 kDa] binding to theRicinus communis agglutinin (RCA) and peanut agglutinin (PNA) were selectively observed on pulmonary microvessel EC as compared to EC from
pulmonary artery, pulmonary vein, and dorsal aorta. The unique RCA- and PNA-binding profiles of EC from the pulmonary artery
and microvessels may be important in characterizing EC from different sites in the pulmonary circulation. The pulmonary microvessel
EC monolayer was also 15-fold more restrictive to transendothelial flux of [14C]sucrose (Mr=342 Da) than the pulmonary artery EC monolayer. In contrast, the microvessel EC were only six- and twofold more restrictive
to the flux of larger tracer molecules, ovalbumin (Mr 43 kDa) and albumin (Mr=69 kDa) than pulmonary artery EC. The greater restrictiveness of pulmonary microvessel EC monolayer indicates a major phenotypic
difference in the cultured pulmonary microvessel EC barrier function.
Despite the importance of anaphylaxis, little information is available on its clinical features.
To evaluate the clinical and allergologic features of anaphylaxis in children referred to the allergology and immunology unit of A. Meyer Children's Hospital (Florence, Italy) from 1994 to 1996.
Ninety-five episodes of anaphylaxis occurred in 76 children (50 boys and 26 girls). Sixty-six children (87%) had only one episode of anaphylaxis, while 10 (13%) had two or more episodes. Sixty-two (82%) of the 76 patients had a personal history of atopic symptoms, although 14 (18%) did not. Sixty (79%) of the 76 children studied had at least one positive skin prick test to one or more of the common inhalant and/or food allergens. Children with venom-induced anaphylaxis usually had negative skin tests to the allergens tested. A younger age and eczema were more frequent among children with food-dependent anaphylaxis, whereas an older age together with urticaria-angioedema were common among those with exercise-induced anaphylaxis. The mean latent period (+/-SD) of the anaphylaxis episodes was 15.4 +/- 27.5 minutes. Skin and respiratory manifestations had an earlier onset and were more common than the gastrointestinal and cardiovascular ones. The most frequent clinical manifestation in children with food anaphylaxis was gastrointestinal symptoms, whereas cardiovascular symptoms were rare. The most probable causative agents in the 95 episodes described were foods (57%), drugs (11%), hymenoptera venom (12%), exercise (9%), additives (1%), specific immunotherapy (1%), latex (1%), and vaccines (2%), but in 6 cases (6%) the agent was never determined. Among the foods, seafood and milk were the most frequently involved. As for location, 57% of the anaphylactic events occurred in the home (54/95), 12% outdoors (11/95); 5% in restaurants (5/95); 3% in the doctor's office (3/95); 3% in hospitals (3/95); 3% on football fields (3/95); 2% on the beach (2/95); 1% in the gym (1/95); 1% at school (1/95); and 1% in the operating room (1/95). In the remaining 12% of cases (11/95) the site remained unknown. Sixty-two percent of the patients (59/95) were treated in an emergency room or hospital, while 32% (30/95) were not (this information is lacking in 6% of the cases [6/95]). Patients were treated with corticosteroids in 72% of the cases (68/95), with antihistamines in 20% (19/95), with epinephrine in 18% (17/95), with beta2-agonists in 5% (5/95), and with oxygen in 4% (4/95).
In our area, foods, particularly seafood and milk, seem to be the most important etiologic factors triggering anaphylaxis. Food-induced anaphylaxis often occurs in younger children with a severe food allergy, whereas exercise-induced anaphylaxis occurs more often in older children with a history of urticaria-angioedema. The venom-induced variant usually presents itself in nonatopic subjects. Given the fact that most of the children had only one anaphylactic reaction, prevention is almost impossible. Epinephrine, although it is the first-choice treatment of anaphylaxis, often goes unused, even in hospitals and doctors' offices.
We infused Escherichia coli endotoxin, 0.07-1.33 microgram/kg, intravenously into chronically instrumented unanesthetized sheep and measured pulmonary arterial and left atrial pressures, lung lymph flow, lymph and blood plasma protein concentrations, and arterial blood gases. Endotoxin caused a biphasic reaction: an early phase of pulmonary hypertension and a long late phase of steady state increased pulmonary vascular permeability during which pulmonary arterial and left atrial pressures were not increased significantly and lung lymph flow was 5 times the baseline value. Lymph: plasma total protein concentration ratio during the late phase (0.76 +/- 0.04) was significantly (P less than 0.05) higher than during baseline (0.66 +/- 0.03). The lymph response was reproducible. Lung lymph clearance of endogenous proteins with molecular radii (r) 35.5 to 96 A was increased during the steady state late phase of the reaction, but, as during baseline, clearance decreased as r increased. The endotoxin reaction was similar to the reaction to infusing whole Pseudomonas bacteria, except that endotoxin had less effect on pressures during the steady state response and caused a relatively larger increase in lymph clearance of large proteins. We conclude that E. coli endotoxin in sheep causes a long period of increased lung vascular permeability and may have a greater effect on large solute pathways across microvessels than do Pseudomonas bacteria.
Ischemia-reperfusion (IR) is a form of oxidant injury known to increase microvascular permeability in the lung. Agents that increase adenosine 3',5'-cyclic monophosphate (cAMP) levels have been shown to have beneficial effects in several models of oxidant lung injury associated with increased microvascular permeability. We investigated the role of adenylate cyclase activation with isoproterenol (ISO) or forskolin (FSK) in reversing the increased microvascular permeability associated with IR. ISO or FSK administered after 45 min of ischemia and 46 min of reperfusion caused a reduction in the capillary filtration coefficient (Kfc) from 1.25 +/- 0.13 to 0.53 +/- 0.08 and 0.55 +/- 0.10 ml.min-1.cmH2O-1.100 g tissue-1, respectively, at 90 min of reperfusion. This reduction in Kfc was accompanied by a rise in perfusate cAMP levels from 16.5 +/- 4.9 and 31.2 +/- 11.9 pmol/ml at 45 min of reperfusion to 444.2 +/- 147.8 and 276.1 +/- 91.0 pmol/ml at 105 min of reperfusion in lungs treated with ISO or FSK, respectively, at 46 min of reperfusion. Dibutyryl cAMP (DBcAMP), a membrane-permeable cAMP analogue, mimicked the permeability effect by reducing Kfc to 0.67 +/- 0.15 at 90 min of reperfusion. Significant hemodynamic changes occurred but were small and cannot explain the observed effect on Kfc. Photomicrographs from lungs treated with ISO or FSK revealed a reversal of the morphological manifestations of increased microvascular permeability. We conclude that the increased microvascular permeability associated with IR can be reversed by ISO, FSK, and DBcAMP and that cAMP produced by the lung contributes to the observed reversal.
This study evaluated the physiological effects of compounds that increase adenosine 3',5'-cyclic monophosphate (cAMP) on changes in pulmonary capillary permeability and vascular resistance induced by ischemia-reperfusion (I-R) in isolated blood-perfused rabbit lungs. cAMP was elevated by 1) beta-adrenergic stimulation with isoproterenol (ISO, 10(-5) M), 2) post-beta-receptor stimulation of adenylate cyclase with forskolin (FSK, 10(-5) M), 3) and dibutyryl cAMP (DBcAMP, 1 mM), a cAMP analogue. Vascular permeability was assessed by determining the capillary filtration coefficient (Kf,c), and capillary pressure was measured using the double occlusion technique. The total, arterial, and venous vascular resistances were calculated from measured pulmonary arterial, venous, and capillary pressures and blood flow. Reperfusion after 2 h of ischemia significantly (P less than 0.05) increased Kf,c (from 0.115 +/- 0.028 to 0.224 +/- 0.040 ml.min-1.cmH2O-1.100 g-1). These I-R-induced changes in capillary permeability were prevented when ISO, FSK, or DBcAMP was added to the perfusate at reperfusion (0.110 +/- 0.022 and 0.103 +/- 0.021, 0.123 +/- 0.029 and 0.164 +/- 0.024, and 0.153 +/- 0.030 and 0.170 +/- 0.027 ml.min-1.cmH2O-1.100 g-1, respectively). I-R significantly increased total, arterial, and venous vascular resistances. These increases in vascular resistance were also blocked by ISO, FSK, and DBcAMP. These data suggest that beta-adrenergic stimulation, post-beta-receptor activation of adenylate cyclase, and DBcAMP prevent the changes in pulmonary vascular permeability and vascular resistances caused by I-R in isolated rabbit lungs through a mechanism involving an increase in intracellular levels of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)
Surface proteins were compared in endothelial cells (EC) obtained from bovine peripheral lung, pulmonary artery and vein, and dorsal aorta using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Galactose-containing glycoproteins [molecular weight (M(r)) 160-220 and 40 kDa] binding to the Ricinus communis agglutinin (RCA) and peanut agglutinin (PNA) were selectively observed on pulmonary microvessel EC as compared to EC from pulmonary artery, pulmonary vein, and dorsal aorta. The unique RCA- and PNA-binding profiles of EC from the pulmonary artery and microvessels may be important in characterizing EC from different sites in the pulmonary circulation. The pulmonary microvessel EC monolayer was also 15-fold more restrictive to transendothelial flux of [14C]sucrose (M(r) = 342 Da) than the pulmonary artery EC monolayer. In contrast, the microvessel EC were only six- and twofold more restrictive to the flux of larger tracer molecules, ovalbumin (M(r) 43 kDa) and albumin (M(r) = 69 kDa) than pulmonary artery EC. The greater restrictiveness of pulmonary microvessel EC monolayer indicates a major phenotypic difference in the cultured pulmonary microvessel EC barrier function.
Experiments were carried out in order to isolate and characterize the cyclic nucleotide phosphodiesterase activities in primary and low passages of cultured bovine aortic endothelial cells. The subcellular characterization of the cyclic nucleotide hydrolytic activity showed that both cAMP and cGMP hydrolytic activities were predominant in the cytosolic rather than the particulate fraction of the endothelial cell homogenate. At a low substrate concentration (0.25 microM), the major hydrolytic activity was for cAMP while at a high concentration (20 microM) it was for both cAMP and cGMP. Both cAMP and cGMP hydrolytic activities were insensitive to calmodulin. Cytosolic cyclic nucleotide phosphodiesterase activity was resolved into two distinct phosphodiesterase forms using HPLC. The first eluted form was designated cGS-PDE: it hydrolysed both cAMP and cGMP and its cAMP hydrolytic activity was markedly enhanced by the presence of cGMP. The second form was designated cAMP-PDE: it selectively hydrolysed cAMP. The cytosolic cAMP-PDE was inhibited by micromolar concentrations of cAMP-PDE inhibitors such as trequinsin, rolipram, dipyridamole or papaverine. The cGS-PDE was inhibited by micromolar concentrations of trequinsin, dipyridamole and papaverine and was insensitive to rolipram, except for the hydrolysis of cAMP which was inhibited in the micromolar range. Both the cAMP-PDE and the cGS-PDE were relatively insensitive to the selective cGMP-PDE inhibitor, zaprinast which was about 750-fold less potent on endothelial PDEs than on smooth muscle cGMP-PDE. The identification of selective and specific PDE inhibitors of the different PDE forms may allow a better understanding of the regulation and the role of cyclic nucleotides in endothelial cells.
Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cells, a cyclic GMP-stimulated PDE and a cyclic AMP PDE. Cyclic GMP-stimulated PDE had Km values of 367 microM for cyclic AMP and 24 microM for cyclic GMP, and low concentrations (1 microM) of cyclic GMP increased the affinity of the enzyme for cyclic AMP (Km = 13 microM) without changing the Vmax. This isoenzyme was inhibited by trequinsin [IC50 (concn. giving 50% inhibition of substrate hydrolysis) = 0.6 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 0.6 microM for cyclic GMP hydrolysis] and dipyridamole (IC50 = 5 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 3 microM for cyclic GMP hydrolysis). Cyclic AMP PDE exhibited a Km of 2 microM for cyclic AMP and did not hydrolyse cyclic GMP. This activity was inhibited by trequinsin (IC50 = 0.2 microM), dipyridamole (IC50 = 6 microM) and, selectively, by rolipram (IC50 = 3 microM). Inhibitors of cyclic GMP PDE (M&B 22948) and of low Km (Type III) cyclic AMP PDE (SK&F 94120) only weakly inhibited the two endothelial PDEs. Incubation of intact cells with trequinsin and dipyridamole induced large increases in cyclic GMP, which were completely blocked by LY-83583. Rolipram, SK&F 94120 and M&B 22948 did not significantly influence cyclic GMP accumulation. Dipyridamole enhanced the increase in cyclic GMP induced by sodium nitroprusside. Cyclic AMP accumulation was stimulated by dipyridamole and trequinsin with and without forskolin. Rolipram, although without effect alone, increased cyclic AMP in the presence of forskolin, whereas M&B 22948 and SK&F 94120 had no effects on resting or forskolin-stimulated levels. These results suggest that cyclic GMP-stimulated PDE regulates cyclic GMP levels and that both endothelial PDE isoenzymes contribute to the control of cyclic AMP.
The effect of glass-bead microemboli (diameter 100 micron, range 77-125 micron) in the absence of fibrinolysis inhibition on pulmonary hemodynamics and microvascular permeability was determined in anesthetized, microfilaria-free dogs acutely prepared for the collection of lung lymph. Pulmonary vascular resistance, pulmonary capillary pressure (Pc), lymph flow (QL), and the ratio of lymph (CL) to plasma (Cp) protein concentrations were measured after 0.2 (n = 4), 0.4 (n = 6), or 0.6 (n = 3) g/kg beads. In all cases, emboli increased resistance and QL severalfold (P less than 0.05), while CL/Cp remained unchanged. In part, the increase in QL could be attributed to an increase in Pc compared with control (12.4 +/- 2.2 vs. 6.7 +/- 0.6 mmHg, P less than 0.05). Furthermore, since the solvent-drag reflection coefficient (sigma f) for total proteins approaches the osmotic reflection coefficient (sigma d) at high QL, sigma d was estimated under these conditions with sigma f approximately equal to sigma d approximately equal to 1 - (CL/Cp)min. The sigma d was decreased (P less than 0.05) after 0.4 and 0.6 g/kg beads to 0.55 +/- 0.03 and 0.50 +/- 0.07, respectively, when compared with that in control lungs (sigma d = 0.62 +/- 0.02; Parker et al., Circ. Res. 48: 549-561, 1981). A pore-stripping analysis demonstrated that after emboli the pulmonary endothelial barrier could be described by a population of small (80 A) and large (350 A) pores. However, the number of large to small pores was 1:1,195, compared with 1:195 in control lungs, suggesting an increased contribution of extra-alveolar vessels upstream of the emboli.(ABSTRACT TRUNCATED AT 250 WORDS)
Corticosteroids are widely used as therapy for the adult respiratory distress syndrome (ARDS) without proof of efficacy. We conducted a prospective, randomized, double-blind, placebo-controlled trial of methylprednisolone therapy in 99 patients with refractory hypoxemia, diffuse bilateral infiltrates on chest radiography and absence of congestive heart failure documented by pulmonary-artery catheterization. The causes of ARDS included sepsis (27 percent), aspiration pneumonia (18 percent), pancreatitis (4 percent), shock (2 percent), fat emboli (1 percent), and miscellaneous causes or more than one cause (42 percent). Fifty patients received methylprednisolone (30 mg per kilogram of body weight every six hours for 24 hours), and 49 received placebo according to the same schedule. Serial measurements were made of pulmonary shunting, the ratio of partial pressure of arterial oxygen to partial pressure of alveolar oxygen, the chest radiograph severity score, total thoracic compliance, and pulmonary-artery pressure. We observed no statistical differences between groups in these characteristics upon entry or during the five days after entry. Forty-five days after entry there were no differences between the methylprednisolone and placebo groups in mortality (respectively, 30 of 50 [60 percent; 95 percent confidence interval, 46 to 74] and 31 of 49 [63 percent; 95 percent confidence interval, 49 to 77]; P = 0.74) or in the reversal of ARDS (18 of 50 [36 percent] vs. 19 of 49 [39 percent]; P = 0.77). However, the relatively wide confidence intervals in the mortality data make it impossible to exclude a small effect of treatment. Infectious complications were similar in the methylprednisolone group (8 of 50 [16 percent]) and the placebo group (5 of 49 [10 percent]; P = 0.60). Our data suggest that in patients with established ARDS due to sepsis, aspiration, or a mixed cause, high-dose methylprednisolone does not affect outcome.
Microcarriers of known diameter can be used to collect endothelial cells from microvessels of the same or slightly smaller internal diameter. The procedure is illustrated by collection of endothelial cells from rabbit pulmonary pre-capillary vessels. The lungs are perfused free of blood with physiological saline and then cold vessels. The lungs are perfused free of blood with physiological saline and then cold (4 degrees C) saline (containing EDTA, 0.02%, and microcarriers 600/ml; 40-60 micrometers diameter) is perfused via the pulmonary artery. The direction of flow is reversed periodically to collect the bead-cell harvest from the arterial side. Cold shock and EDTA cause the endothelial cells to detach from the vessels under conditions such that the cells remain attached to the microcarriers. The selective attachment to microcarriers is apparently aided by the tight fit of the beads within vessels of the same diameter. Beads do not emerge on the venous side, all being trapped at the pre-capillary level. Electron microscopic examination of lungs fixed during the perfusion shows that the beads lodge in terminal arterioles and pre-capillary vessels (approximately 40-60 micrometers in diameter, with one, sometimes incomplete, muscle layer). Endothelial cells recovered on microcarriers can be allowed to migrate on to flasks and back on to beads. The resultant cultures have an endothelial morphology and possess high levels of angiotensin coverting enzyme and carboxypeptidase N activity.
Previous studies have reported that endothelial cells isolated from large vessels compared with microvessels from the same or distinct organs showed considerable phenotypic and biochemical heterogeneity. In the present study we extend these findings by comparison of the effects of 8-bromo-cyclic adenosine monophosphate (8Br-cAMP), human alpha-thrombin, 8Br-cAMP followed 5 min later by thrombin or no treatment (control) on the equivalent "pore" radii (rp) of endothelial monolayers isolated from the main bovine pulmonary artery (BPAEC) compared with lung microvessels (BLMV). BLMV, isolated from a 1-cm peripheral segment of the lung, were significantly larger than those obtained from large vessels (1602 +/- 142 microns2 vs 398 +/- microns2, respectively). In addition, BLMV monolayers formed a heteroporous barrier with less size-selectivity compared with BPAEC monolayers. 8Br-cAMP caused monolayers of both cell types to close their large "pores" which completely restricted the passage of solute molecular radii > 35-60 A across these barriers, consistent with a rp of approximately 75-100 A. This effect was due to a reduction in the area available for solute exchange (Ap) and/or an increase in the path length of the transport pathway (delta X). Human alpha-thrombin produced an increase in the Ap/delta X consistent with the formation of large open areas between adjacent cells that exposed the approximately 2000 A pore radius of the filter support. Since this effect was more marked in microvessel compared with large vessel monolayers, microvessel endothelial cells appear to be more sensitive to the effects of thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenylyl cyclase is the prototypical second messenger generator. Nearly all of the eight cloned adenylyl cyclases are regulated by one or other arm of the phospholipase C pathway. Functional and ultrastructural investigations have shown that adenylyl cyclases are intimately associated with sites of calcium ion entry into the cell. Oscillations in cellular cyclic AMP levels are predicted to arise because of feedback inhibition of adenylyl cyclase by Ca2+. Such findings inextricably intertwine cellular signalling by cAMP and internal Ca2+ and extend the known regulatory modes available to cAMP.
Endothelial cells produce a variety of vasoactive substances including prostacyclin (PGI2) and endothelium-derived relaxing factor (EDRF/NO) which are potent inhibitors of platelet adhesion/aggregation and vascular smooth muscle cell contraction/proliferation. PGI2 and EDRF elevate cAMP or cGMP, respectively, in vascular cells and other targets. The intracellular effects of cAMP and cGMP in vascular smooth muscle cells and platelets are primarily mediated by the family of cAMP- and cGMP-dependent protein kinases and their substrates. Important effector systems include enzymes, channels and regulatory proteins responsible for the regulation of intracellular Ca++. Other evidence suggests that VASP, a focal adhesion protein phosphorylated in platelets and smooth muscle cells in response to PGI2 and EDRF, is important for the regulation of integrins and cell-matrix interactions.
Investigation of the regulation of permeability properties of the endothelium has yielded evidence to support the concept of a dual regulation of EC gap formation and barrier function. In this model, the primary determinants of EC permeability are tethering/adhesive properties (Figure 1) and tensile centripetal force generation (Figure 2). The importance of actin-myosin interactions and active cellular contraction and force generation has been reviewed. In the model of thrombin-induced EC barrier dysfunction, there is a strong shift in the MLC species from the unphosphorylated to the diphosphorylated form, indicating activation of MLCK, a key enzyme whose importance in EC contraction has been well established. Although important differences between EC and SMC exist, endothelial cell gap formation involves actomyosin-dependent contractile mechanisms similar to SMC, a cellular system in which MLC phosphorylation correlates with the initial rate of tension development. The increase in MLC phosphorylation and isometric tension is consistent with the hypothesis that activation of signal transduction mediates an increase in isometric tension to a new level of "latch state" through the cytoskeleton. Thus, the available evidence implicates a strong role for cellular force generation and contraction in the evolution of thrombin-induced barrier dysfunction. Accumulating evidence also indicates that modulation of tethering properties, primarily those involving cell-matrix and cell-cell adhesion, is also a key determinant of basal EC barrier properties as well as agonist-mediated barrier dysfunction. Because each of these focal adhesion constituents may be involved in establishing tethering properties in endothelium, they each may be involved in determining barrier permeability and may be involved in the evolution of agonist-mediated barrier dysfunction. Therefore, in addition to MLCK-dependent active tensile force generation, agonist-induced barrier dysfunction may occur via MLCK-independent pathways that rely on basal levels of MLC phosphorylation or by affecting proteins involved in tethering properties of endothelium that contribute to barrier function. Further examination of tethering force properties, combined with elucidation of EC relaxation via MLC dephosphorylation may yield clues as to how this important vascular barrier is maintained and restored after vascular insult.
Isoproterenol (ISO) and forskolin, agents that increase adenosine 3',5'-cyclic monophosphate (cAMP) via adenylyl cyclase activation, reverse lung injury associated with increased microvascular permeability. We studied the role of rolipram, a relatively isozyme-selective cAMP phosphodiesterase (PDE) inhibitor, in reversing increased capillary permeability due to ischemia-reperfusion (I/R), a form of oxidant injury in the lung, by using the isolated perfused rat lung model. Rolipram (2 microM) administered after 45 min of ischemia and 45 min of reperfusion reduced I/R-increased permeability as measured by the capillary filtration coefficient to control lung values. Computer image analysis of air space edema and perivascular cuffing, as well as wet-to-dry weight ratios, confirms the permeability reversal by rolipram administration. Rolipram inhibition of cAMP PDE in the lung was assessed by using [3H]adenine prelabeling adapted for the whole lung and perfusate [3H]cAMP accumulation. Rolipram failed to increase perfusate cAMP alone but dramatically increased perfusate cAMP above ISO alone. Dose-response relationships of ISO or rolipram show a close correlation of the half-maximal effective dose (ED50) for injury reversal and perfusate cAMP production. The combination of rolipram and ISO produced synergistic reversal of I/R injury. We conclude that reversal of I/R-induced increased microvascular permeability can be achieved with rolipram and that the mechanism of action of rolipram is probably through PDE isozyme-selective inhibition. The similarity of the ED50 values for cAMP efflux and reversal of permeability increases also supports a close coupling between cAMP accumulation and endothelial cell permeability.
Type IV phosphodiesterases (PDE IV) specifically hydrolyze cAMP and are inhibited by rolipram. RT-PCR was applied to analyze the expression patterns of mRNAs for four cloned human and rat phosphodiesterase type IV isogenes (PDE IV-A, -B, -C and -D). Although these patterns were mostly coincident for the human and rat PDE IV genes, some differences were found between the two species. PDE IV-A expression was detectable in human blood but not in rat blood, suggesting a species-specific difference in the expression of this PDE IV isogene. PDE IV-C was neither detected in human or rat blood nor in different cell populations of the human immune system. It is further demonstrated that the PDE IV isogene expression is differentially regulated by cAMP in different cell types.
The barrier function, surface biochemistry, and morphology of confluent monolayers of endothelial cells isolated from different segments of the bovine lung vasculature [microvessels (BLMVEC), vein (BPVEC) and artery (BPAEC)] were grown in culture and compared. A number of common cell surface proteins were identified along with two proteins of 46 and 48 kDa found exclusively on BPVEC. Lectin affinity chromatography revealed multiple glycosylation differences. The lectins, Arachis hypogaea (AHA) and Lycopersicum esculentum (LEA) agglutinins, interacted with several glycoproteins of BLMVEC but not of BPAEC. Bandeiraea simplicifolia (BS-1) and Caragana arborescens (CAA) agglutinins recognized several glycoproteins of BPVEC and BPAEC but not BLMVEC. Permeabilities were much lower for BLMVEC than BPAEC or BPVEC monolayers, with a range of about 16-fold less for sucrose to 2-fold less for albumin. Electron microscopy revealed that BLMVEC have a greater surface density of plasmalemmal vesicles (approximately 4-fold) and more extensively developed intercellular junctions with more focal membrane adhesion sites per junction (approximately 9-fold) than the other cells. We conclude that: i) BLMVEC monolayers form a much more restrictive barrier to molecular transport as a result of the tighter junctional formation; and ii) endothelial surface glycoproteins may be differentially glycosylated depending on their segmental location within the vasculature.
After many years of increasing morbidity and mortality, several avenues of scientific investigation now appear to be converging to offer an explanation for the asthma paradox and indicate that regular or long-term use of short-acting inhaled beta-agonist drugs is inappropriate. Pharmacoepidemiologic studies indicate a strong association between increased beta-agonist use and asthma deaths, which does not appear entirely related to confounding by severity. Clinical data, although still limited, show little evidence for symptomatic or functional improvement during long-term beta-agonist therapy and, in many instances, reveal significant adverse effects. Related investigations offer evidence of potential plausible mechanisms, notably increased bronchial responsiveness to inhaled allergen, to explain these findings. A radical revision of the therapeutic use of these drugs in asthma has been prompted by these findings. Beta-agonist drugs remain essential for the management of acute severe attacks. They are also useful on demand for the relief of breakthrough symptoms and for prophylaxis of exercise-induced symptoms. In chronic asthma, however, adequate anti-inflammatory therapy is the treatment of choice. Long-term treatment with short-acting beta-agonist, even in the presence of seemingly adequate anti-inflammatory therapy, may be associated with deterioration of asthma over the long-term. The effects of long-acting beta-agonists remain under review. To date, there are no data that clearly indicate a deleterious effect, and many clinical trials show benefits in symptom control and improved lung function associated with their regular use. The significance of tachyphylaxis remains to be defined. Their current role is still somewhat unclear, but they have been successfully used in subjects in whom, despite the use of moderate doses of inhaled corticosteroid, short-acting bronchodilator is still frequently required. The use of twice-daily long-acting beta-agonist appears preferable to frequent use of short-acting beta-agonists.
Molecular cloning has permitted identification of several novel isoforms of mammalian adenylyl cyclase; these proteins now comprise a family of at least 10. All of the membrane-bound enzymes are activated by the alpha subunit of G alpha, a receptor-regulated, heterotrimeric guanine nucleotide-binding protein, and by the diterpene forskolin. Certain cyclases are also activated by Ca(2+)-calmodulin, while some are inhibited by the alpha subunits of the three Gi proteins. The discovery of new isoforms has also revealed unanticipated mechanisms of regulation, including activation or inhibition by the G-protein beta gamma subunit complex, inhibition by G(o) alpha, inhibition by Ca2+, and phosphorylation by protein kinases C and A. The effects of activators are often highly synergistic or conditional, suggesting function of these enyzmes as coincidence detectors. The plethora of receptors, G proteins, and adenylyl cyclases permits assembly of very complex signaling systems with a wide variety of integrative characteristics.
Exercise-induced urticaria and anaphylaxis have become increasingly recognized during the past 2 decades as more people participate in physical activities. These syndromes can be categorized as cholinergic urticaria or exercise-induced anaphylaxis based on the clinical manifestation. Newer subsets such as food-dependent and familial exercise-induced anaphylaxis have also been recognized. Further studies are needed to characterize the variables involved in mast cell activation and mast cell mediator release in these syndromes. The management strategy for patients who have exercise-induced syndromes with skin manifestations only differs from the management for those with systemic symptoms. Currently, antihistamines, as a single agent or in combination with other agents, may be helpful prophylactically in both groups. Avoidance of precipitating factors, modification of exercise, and use of a self-injectable epinephrine kit are recommended for patients with anaphylaxis.
Pulmonary microvascular endothelium forms a tighter barrier than does pulmonary artery endothelium; the mechanism of this important phenotypic difference is uncertain. We examined two regulators of endothelial permeability, cytosolic Ca2+ concentration ([Ca2+]i) and adenosine 3',5'-cyclic monophosphate (cAMP), in microvascular (PMVEC) and pulmonary conduit artery (PAEC) endothelium. Both resting and stimulated [Ca2+]i were lower in PMVEC compared with PAEC (resting [Ca2+]i, 94 +/- 7 vs. 123 +/- 8 nM; ATP-stimulated peak, 1.04 +/- 0.14 vs. 1.98 +/- 0.13 microM). Sustained Ca2+ transients in response to either ATP or thapsigargin were reduced in PMVEC compared with PAEC (ATP, 199 +/- 22 vs. 411 +/- 43 nM; thapsigargin, 195 +/- 13 vs. 527 +/- 65 nM), suggesting reduced Ca2+ influx in PMVEC. Reduced Ca2+ influx in PMVEC was confirmed by Mn2+ quenching and patch-clamp experiments. mRNA for Ca(2+)-inhibitable and protein kinase C-stimulated adenylyl cyclases was detected in both cell types. Whereas ATP caused a [Ca2+]i-mediated decrease in cAMP in PAEC, ATP caused a protein kinase C-mediated increase in cAMP in PMVEC. We conclude that PMVEC express a unique phenotype that favors enhanced barrier function through attenuated Ca2+ influx and preservation of cAMP content.
The flux of multisized fluorescein-isothiocyanate-labeled hydroxy ethyl starch (FITC-HES) macromolecules was used to assess changes in barrier function of rat pulmonary microvascular endothelial cell (RPMVEC) monolayers exposed to protein phosphatase (PP) inhibitors or cGMP analogs and atriopeptin (ANF). Two potent PP inhibitors, calyculin A (CalA) and okadaic acid (OA), increased RPMVEC permeability in a dose- and time-dependent manner, and CalA had a higher intrinsic activity than OA. In contrast, ANF and potent cGMP analogs had no effect on basal RPMVEC permeability. The phosphohistone PP activity contained in RPMVEC sonicates was inhibited by OA with an inhibition profile that suggested at least two components were present, with PP2A accounting for approximately 70% of the OA-inhibitable phosphohistone phosphatase activity. Following separation with heparin-Sepharose chromatography, PP activity exhibited equipotent inhibition by CalA and differential inhibition by OA. Differential inhibition of PP1 and PP2A by OA suggested that PP1 is involved in regulating RPMVEC barrier function. Permeabilized RPMVEC showed increased phosphorylation of several proteins in the presence of phosphatase inhibitors. Treatment with KT 5926, a myosin light chain (MLC) kinase (MLCK) inhibitor, or rolipram, a phosphodiesterase inhibitor, decreased 32P incorporation into immunoprecipitated MLC by CalA and OA. However, this effect did not abolish either the CalA- or OA-induced decrease in the RPMVEC barrier function. Localization of filamentous (F) actin was at the periphery as well as in the cytoplasm and perinuclear region, whereas nonmuscle myosin was seen in the perinuclear region. Neither of these patterns was changed in the presence of CalA. Thus, cGMP does not alter RPMVEC permeability, but inhibition of PP activity results in loss of barrier function by a mechanism independent from MLC phosphorylation.
Myosin light chain (MLC) phosphorylation catalyzed by the Ca(2+)- calmodulin-dependent MLC kinase (MLCK) is critical to thrombin-mediated endothelial cell gap formation and barrier dysfunction. We have tested the hypothesis that the Ca2+ ionophore ionomycin stimulates MLCK-dependent endothelial cell contraction and permeability. Ionomycin significantly increased albumin clearance and decreased electrical resistance across confluent bovine pulmonary microvascular and macrovascular endothelial cell monolayers in a concentration-dependent manner that was temporally similar to that produced by thrombin. In contrast, however, ionomycin produced a significant Ca(2+)-dependent reduction in the levels of phosphorylated MLC with evidence of serine/threonine phosphatase activation. Potential MLCK-independent mechanisms of endothelial cell permeability were examined with little evidence to support a role for stimulated nitric oxide synthase or phospholipase A2 activities. Importantly, ionomycin produced 1) reductions in the activities of the barrier protective adenylate cyclase and the adenosine 3',5'-cyclic monophosphate-dependent protein kinase A, 2) dramatic dose- and time-dependent inhibition of endothelial cell tyrosine kinase activities, and 3) marked decreases in the phosphotyrosine content of the p125 focal adhesion kinase. These data indicate that ionomycin produces endothelial cell barrier dysfunction by mechanisms that are independent of MLCK activation and may involve reductions in endothelial cell tethering forces via inhibition of protein kinase A and tyrosine kinase activities, especially the p125 focal adhesion kinase.
Intracellular mechanisms responsible for endothelial cell disruption are unknown, although either elevated cytosolic Ca2+ ([Ca2+]i) or decreased adenosine 3',5'-cyclic monophosphate (cAMP) promotes permeability. Recent identification that Ca(2+)-inhibitable adenylyl cyclase establishes an inverse relationship between [Ca2+]i and cAMP in macrovascular endothelial cells provided a possible mechanism of development of permeability. However, these data utilized an in vitro model; lacking was evidence supporting 1) expression of Ca(2+)-inhibitable adenylyl cyclase in pulmonary microvascular endothelium and 2) Ca2+ inhibition of adenylyl cyclase and cAMP content as a paradigm for inflammatory mediator-induced permeability in the intact circulation. We therefore addressed these issues in microvascular endothelial cells derived from rat lung and in an isolated perfused rat lung preparation. Results demonstrate expression of a Ca(2+)-inhibitable adenylyl cyclase in microvascular endothelial cells. Furthermore, data suggest that Ca2+ inhibition of adenylyl cyclase is necessary for development of microvascular permeability in the intact circulation. We conclude Ca2+ inhibition of cAMP represents a critical step in genesis of microvascular permeability in the intact pulmonary circulation.
Acute asthma exacerbations are frequent presentations to the emergency department. Those patients who deteriorate despite nebulized beta-agonist therapy and intravenous steroids are experiencing a life-threatening event. They pose a significant management challenge to the emergency physician. This review describes the pharmacologic options and techniques for mechanical ventilation which are available in resuscitating these patients.
Generally favorable literature was published regarding the contributions of bronchodilators to asthma therapy. Analysis of asthma mortality data suggested that the risk of death from asthma was primarily associated with the use of fenoterol rather than beta-adrenergic bronchodilators as a class, or reflected the severity of the underlying asthma, which resulted in increasing beta-agonist use. Salmeterol was shown to outperform oral beta-adrenergic agonists and individual dose-titrated theophylline in controlling asthma symptoms while causing fewer adverse effects. When salmeterol was added to low-dose inhaled corticosteroids, the combination outperformed moderate-dose inhaled corticosteroids alone. Regular use of salmeterol, but not albuterol, improved the quality of life for patients with asthma to a clinically significant degree. Finally, theophylline, at relatively low blood levels, was clearly shown to improve asthma control, even in patients receiving moderately high-dose inhaled corticosteroids. More importantly, this symptomatic response was accompanied by decreases in activated lymphocytes, eosinophils, and proinflammatory cytokines in bronchial biopsy results.
Endothelial cell (EC) gap formation and barrier function are subject to dual regulation by (1) axial contractile forces, regulated by myosin light chain kinase activity, and (2) tethering forces, represented by cell-cell and cell-substratum adhesions. We examined whether focal adhesion plaque proteins (vinculin and talin) and focal adhesion kinase, p125FAK (FAK), represent target regulatory sites involved in thrombin-mediated EC barrier dysfunction. Histologically, thrombin produced dramatic rearrangement of EC actin, vinculin, and FAK in parallel with the evolution of gap formation and barrier dysfunction. Vinculin and talin were in vitro substrates for phosphorylation by EC PKC, a key effector enzyme involved in thrombin-induced EC barrier dysfunction. Although vinculin and talin were phosphorylated in situ under basal conditions in 32P-labeled EC, thrombin failed to alter the basal level of phosphorylation of these proteins. Phosphotyrosine immunoblotting showed that neither vinculin nor talin was significantly phosphorylated in situ on tyrosine residues in unstimulated ECs, and this was not further increased after thrombin. In contrast, both thrombin and the thrombin receptor-activating peptide (TRAP) produced an increase in FAK phosphotyrosine levels (corrected for immunoreactive FAK content) present in EC immunoprecipitates. Ionomycin, which produces EC barrier dysfunction in a myosin light chain kinase-independent manner, was used to increase intracellular Ca2+ and evaluate the Ca2+ sensitivity of this observation. In contrast to thrombin, ionomycin effected a dramatic decrease in the phosphotyrosine-to-immunoreactive FAK ratios, suggesting distinct effects of the two agents on FAK phosphorylation and function. These data indicate that modulation of cell tethering via phosphorylation of focal adhesion proteins is complex, agonist-specific, and may be a relevant mechanism of EC barrier dysfunction in permeability models that do not depend on an increase in myosin 20-kD regulatory light chain phosphorylation.
Primary cultures of bovine aortic endothelial cells (BAEC) express cyclic nucleotide phosphodiesterase (CN PDE) isozymes of the PDE2, PDE4 and PDE5 gene families. We report here that the isozyme profiles of CN PDE and the amounts of each vary with the passage number of BAEC cultures. Characterization by anion-exchange chromatography and pharmacological criteria were used to study CN PDE in early (4-6), intermediate (6-10), and late (> 17) passages of purified BAEC. PDE2 and a minor fraction of PDE5 accounted for cyclic GMP hydrolysis in early passages, but both isozymes were lost with cell passage. Cyclic AMP was hydrolyzed by both PDE2 and PDE4 isozymes in early passage endothelial cells, but PDE4 was increased dramatically in higher passage cells. Also appearing in the higher passage cells were prominent PDE1 and minor PDE3 activities. The ratios of cytosolic to particulate activities were similar at all passages. BAEC PDE isoforms in intact cells assessed by [3H]-adenine prelabeling showed that atriopeptin II decreased isoproterenol-induced cyclic AMP accumulation in early but not later passage cells, consistent with the loss of PDE2 expression. Enhancement of isoproterenol-induced cyclic AMP accumulation by rolipram, a PDE4 inhibitor, was also greatly diminished during culture passages. Changes in CN PDE isoform expression and consequent cyclic AMP turnover validate the importance of considering cell passage number when cultures of BAEC are used to study the regulation of endothelial cell cyclic nucleotide metabolism and processes mediated by cyclic nucleotides in this model system.
Pulmonary endothelium forms a semiselective barrier that regulates fluid balance and leukocyte trafficking. During the course of lung inflammation, neurohumoral mediators and oxidants act on endothelial cells to induce intercellular gaps permissive for transudation of proteinaceous fluid from blood into the interstitium. Intracellular signals activated by neurohumoral mediators and oxidants that evoke intercellular gap formation are incompletely understood. Cytosolic Ca2+ concentration ([Ca2+]i) and cAMP are two signals that importantly dictate cell-cell apposition. Although increased [Ca2+]i promotes disruption of the macrovascular endothelial cell barrier, increased cAMP enhances endothelial barrier function. Furthermore, during the course of inflammation, elevated endothelial cell [Ca2+]i decreases cAMP to facilitate intercellular gap formation. Given the significance of both [Ca2+]i and cAMP in mediating cell-cell apposition, this review addresses potential sites of cross talk between these two intracellular signaling pathways. Emerging data also indicate that endothelial cells derived from different vascular sites within the pulmonary circulation exhibit distinct sensitivities to permeability-inducing stimuli; that is, elevated [Ca2+]i promotes macrovascular but not microvascular barrier disruption. Thus this review also considers the roles of [Ca2+]i and cAMP in mediating site-specific alterations in endothelial permeability.
We tested the hypothesis that increased endothelial cell adenosine 3',5'-cyclic monophosphate (cAMP) decreases microvascular permeability in vivo. The effects of cAMP-specific phosphodiesterase type IV inhibition and adenylate cyclase activation on microvascular hydraulic conductivity (Lp) were investigated in intact individual capillaries and postcapillary venules in mesentery of pithed frogs (Rana pipiens). Treatment with rolipram (10 microM) and forskolin (5 microM) for 25 min decreased Lp to 37% of control. Rolipram alone also significantly decreased Lp. Isoproterenol (10 microM) decreased Lp to 27% of control within 20 min. A subgroup of eight vessels treated with rolipram and forskolin, in which mean Lp fell to 25% of control, was examined with transmission electron microscopy. The mean number of tight junctions in the treated vessels was 2.2 per cleft (303 clefts), significantly higher than in a matched control group (192 clefts), which was 1.7 per cleft. The results indicate that microvascular Lp can be modulated by intracellular cAMP and that one of the structural end points of stimulated cAMP levels is an increase in the mean number of tight-junction strands between endothelial cells.
Mechanisms of Ischemia-Reperfusion Injury in the Lung
- T Moore
- P Khimenko
- A Taylor
Moore, T., P. Khimenko, and A. Taylor. Mechanisms of Ischemia-Reperfusion Injury in the Lung. New York: Futura, 1998.
- T Moore
- A Taylor
Moore, T., and A. Taylor. Pathophysiology. Oxford, UK: Oxford
University Press, 1998.