Lung Microvascular Endothelium Research Articles

α1-Acid glycoprotein disrupts capillary-like tube formation of human lung microvascular endothelia

ABSTRACTPurpose: The acute phase protein, α1-acid glycoprotein, is expressed in the lung, and influences endothelial cell function. We asked whether it might regulate angiogenesis in human lung microvascular endothelia. Materials and Methods: α1-acid glycoprotein was isolated from human serum by HPLC ion exchange chromatography. Its effects on endothelial cell functions including capillary-like tube formation on Matrigel, migration in a wounding assay, chemotaxis in a modified Boyden chamber, adhesion, and transendothelial flux of the permeability tracer, 14C-albumin, were tested. Results: α1-acid glycoprotein dose-dependently inhibited capillary-like tube formation without loss of cell viability. At ≥0.50 mg/mL, it inhibited tube formation >70%, and at 0.75 mg/mL, >97%. α1-acid glycoprotein dose- and time-dependently restrained EC migration into a wound as early as 2 hours, and in washout studies, did so reversibly. It was inhibitory against vascular endothelial growth factor-A and fibroblast growth factor-2-driven migration but failed to inhibit chemotactic responsiveness. When α1-acid glycoprotein was added to preformed tubes, it provoked their almost immediate disassembly. As early as 15 minutes, it induced tube network collapse without endothelial cell–cell disruption. It exerted a biphasic effect on cell adhesion to the Matrigel substrate. At lower concentrations (0.05–0.25 mg/mL), it increased cell adhesion, whereas at higher concentrations (≥0.75 mg/mL) decreased adhesion. In contrast, it had no effect on transendothelial 14C-albumin flux. Conclusion: α1-acid glycoprotein, at concentrations found under physiological conditions, rapidly inhibits endothelial cell capillary-like tube formation that may be explained through diminished cell adhesion to the underlying matrix and/or reversibly decreased cell migration.

The lung microvascular endothelium as a therapeutic target in severe influenza

Severe infections with influenza virus are characterized by acute respiratory distress syndrome (ARDS), a life-threatening disorder in which the alveolocapillary membrane in the lung becomes leaky. This leads to alveolar flooding, hypoxemia and respiratory failure. Recent data suggest that influenza virus can exert both direct and indirect effects on the lung endothelium, activating it and inducing microvascular leak. These findings raise the possibility that enhancing lung endothelial barrier integrity or modulating lung endothelial activation may prove therapeutically useful for severe influenza. In this paper, we review evidence that lung endothelial activation and vascular leak are a “final common pathway” in severe influenza, as has been reported in bacterial sepsis, and that enhancing endothelial barrier function may improve the outcome of illness. We describe a number of experimental therapies that have shown promise in preventing or reversing increased vascular leak in animal models of sepsis or influenza.

Influenza Infects Lung Microvascular Endothelium Leading to Microvascular Leak: Role of Apoptosis and Claudin-5

Severe influenza infections are complicated by acute lung injury, a syndrome of pulmonary microvascular leak. The pathogenesis of this complication is unclear. We hypothesized that human influenza could directly infect the lung microvascular endothelium, leading to loss of endothelial barrier function. We infected human lung microvascular endothelium with both clinical and laboratory strains of human influenza. Permeability of endothelial monolayers was assessed by spectrofluorimetry and by measurement of the transendothelial electrical resistance. We determined the molecular mechanisms of flu-induced endothelial permeability and developed a mouse model of severe influenza. We found that both clinical and laboratory strains of human influenza can infect and replicate in human pulmonary microvascular endothelium, leading to a marked increase in permeability. This was caused by apoptosis of the lung endothelium, since inhibition of caspases greatly attenuated influenza-induced endothelial leak. Remarkably, replication-deficient virus also caused a significant degree of endothelial permeability, despite displaying no cytotoxic effects to the endothelium. Instead, replication-deficient virus induced degradation of the tight junction protein claudin-5; the adherens junction protein VE-cadherin and the actin cytoskeleton were unaffected. Over-expression of claudin-5 was sufficient to prevent replication-deficient virus-induced permeability. The barrier-protective agent formoterol was able to markedly attenuate flu-induced leak in association with dose-dependent induction of claudin-5. Finally, mice infected with human influenza developed pulmonary edema that was abrogated by parenteral treatment with formoterol. Thus, we describe two distinct mechanisms by which human influenza can induce pulmonary microvascular leak. Our findings have implications for the pathogenesis and treatment of acute lung injury from severe influenza.

Inhibition of pre-B cell colony-enhancing factor attenuates inflammation and apoptosis induced by pandemic H1N1 2009 in lung endothelium

The recent pandemic influenza A (H1N1 2009) virus infection has caused acute lung injury in susceptible population resulting in high mortality in ICU patients. In this report, we observed the effect of pre-B cell colony-enhancing factor (PBEF) on the inflammation and apoptosis in H1N1-infected human pulmonary microvascular endothelial cells (HPMECs). We constructed an in vitro HPMEC monolayer model. The results showed that H1N1 2009 induced the increased expression of inflammatory cytokines (IL-6/IL-8/TNF-α/IP-10) and apoptosis factors (FasL/TRAIL) in infected HPMECs. However, PBEF silencing with siRNA inhibited the expression of some inflammatory cytokines and decreased the apoptosis mediated by FasL. We conclude that PBEF might be partially responsible for the localized inflammatory response to H1N1 2009 in the lung microvascular endothelium and the H1N1-induced endothelial cell apoptosis probably through the FasL-mediated pathway.

Activation of TNFR1 ectodomain shedding by mitochondrial Ca2+ determines the severity of inflammation in mouse lung microvessels

Shedding of the extracellular domain of cytokine receptors allows the diffusion of soluble receptors into the extracellular space; these then bind and neutralize their cytokine ligands, thus dampening inflammatory responses. The molecular mechanisms that control this process, and the extent to which shedding regulates cytokine-induced microvascular inflammation, are not well defined. Here, we used real-time confocal microscopy of mouse lung microvascular endothelium to demonstrate that mitochondria are key regulators of this process. The proinflammatory cytokine soluble TNF-α (sTNF-α) increased mitochondrial Ca2+, and the purinergic receptor P2Y2 prolonged the response. Concomitantly, the proinflammatory receptor TNF-α receptor-1 (TNFR1) was shed from the endothelial surface. Inhibiting the mitochondrial Ca2+ increase blocked the shedding and augmented inflammation, as denoted by increases in endothelial expression of the leukocyte adhesion receptor E-selectin and in microvascular leukocyte recruitment. The shedding was also blocked in microvessels after knockdown of a complex III component and after mitochondria-targeted catalase overexpression. Endothelial deletion of the TNF-α converting enzyme (TACE) prevented the TNF-α receptor shedding response, which suggests that exposure of microvascular endothelium to sTNF-α induced a Ca2+-dependent increase of mitochondrial H2O2 that caused TNFR1 shedding through TACE activation. These findings provide what we believe to be the first evidence that endothelial mitochondria regulate TNFR1 shedding and thereby determine the severity of sTNF-α-induced microvascular inflammation.

TNFR1 shedding by mitochondrial RISP in lung microvascular endothelium

In sepsis, ligation of the endothelial (EC) TNFα receptor 1 (TNFR1) tumor necrosis factor alpha (TNFα) promotes lung inflammation. However, the role of EC TNFR1 shedding in lung inflammation is unknown. To determine real‐time, in‐situ TNFR1 dynamics and inflammation, we imaged ECs of single microvessels of inflated isolated, blood‐perfused mouse lungs by live confocal microscopy. By micropuncture, we infused microvessels with a fluorophore‐conjugated TNFR1 or E‐selectin mAb followed by buffer wash. At baseline, TNFR1 expression was stable for >1h. Following 10 min infusion of TNFα (1 ng/ml), EC fluorescence decreased by 72±3% (P<0.05 n=3) in 30 min. TNFR1 shedding was blocked in lungs from mice injected with siRNA to mitochondrial complex III Rieske Iron‐Sulphur protein (siRISP). Infusion of TNFα for 2h increased EC E‐selectin expression and leukocyte adhesion two‐fold (P<0.05). By contrast, TNFα infusion in lungs from siRISP‐mice resulted in a three‐fold increase in E‐selectin expression and leukocyte adhesion relative to control (P<0.05). To determine shedding in sepsis, we instilled mice with lipopolysaccharide (LPS 1 mg/kg, 4h). TNFR1 shedding was inhibited >50% (P<0.05). We interpret, TNFR1 ligation promotes RISP‐dependent TNFR1 shedding, possibly limiting inflammation. In sepsis, mitochondrial dysfunction may impair TNFR1 shedding thus exacerbating inflammation (support: HL57556).

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

BackgroundHighly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.AimTo study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.MethodsWe use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.ResultsWe demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.ConclusionThe predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.

Rap1 mediates protective effects of iloprost against ventilator-induced lung injury

Prostaglandin I(2) (PGI(2)) has been shown to attenuate vascular constriction, hyperpermeability, inflammation, and acute lung injury. However, molecular mechanisms of PGI(2) protective effects on pulmonary endothelial cells (EC) are not well understood. We tested a role of cAMP-activated Epac-Rap1 pathway in the barrier protective effects of PGI(2) analog iloprost in the murine model of ventilator-induced lung injury. Mice were treated with iloprost (2 microg/kg) after onset of high tidal volume ventilation (30 ml/kg, 4 h). Bronchoalveolar lavage, histological analysis, and measurements of Evans blue accumulation were performed. In vitro, microvascular EC barrier function was assessed by morphological analysis of agonist-induced gap formation and monitoring of Rho pathway activation and EC permeability. Iloprost reduced bronchoalveolar lavage protein content, neutrophil accumulation, capillary filtration coefficient, and Evans blue albumin extravasation caused by high tidal volume ventilation. Small-interfering RNA-based Rap1 knockdown inhibited protective effects of iloprost. In vitro, iloprost increased barrier properties of lung microvascular endothelium and alleviated thrombin-induced EC barrier disruption. In line with in vivo results, Rap1 depletion attenuated protective effects of iloprost in the thrombin model of EC permeability. These data describe for the first time protective effects for Rap1-dependent signaling against ventilator-induced lung injury and pulmonary endothelial barrier dysfunction.

REDUCED LUNG WATER TRANSPORT RATE ASSOCIATED WITH DOWNREGULATION OF AQUAPORIN‐1 AND AQUAPORIN‐5 IN AGED MICE

1. The purpose of the present study was to examine lung water transport properties and the expression and regulation of the alveolar endothelial water channel aquaporin (AQP)-1 and the epithelial water channel AQP-5 in aged mouse lung using gene expression analysis and water permeability measurements. 2. In aged (20-24-month-old) mice, AQP-1 and AQP-5 mRNA expression decreased by 55.5 and 50.3%, respectively, compared with that in young (8-10-week-old) mice (P < 0.01). In addition, AQP-1 and AQP-5 protein expression decreased in aged mice by 36.9 and 44.6%, respectively, compared with that in young mice (P < 0.01). 3. The osmotically driven water transport rate between the airspace and capillary compartments was reduced by 31.7% in aged mice compared with young mice (2.8 +/- 0.3 vs 4.1 +/- 0.3 mg/s, respectively; P < 0.01). The hydrostatically driven lung water accumulation rate in response to a 10 cmH(2)O increase in pulmonary artery pressure was also reduced in aged mice by 21.9% compared with young mice (0.32 +/- 0.06 vs 0.41 +/- 0.04 mg/s, respectively; P < 0.01). 4. There was a 62.7% decrease in serum glucocorticoids in aged mice compared with young mice (67.6 +/- 26.8 vs 181.3 +/- 44.4 nmol/L, respectively; P < 0.01). In vivo administration of dexamethasone (4 mg/kg) for 5 consecutive days to aged mice increased lung AQP-1 mRNA and protein expression by 2.1 +/- 0.1 fold (P < 0.01) and 1.8 +/- 0.2 fold (P < 0.01), respectively. Accordingly, osmotically and hydrostatically driven water transport rates increased by 35.6% (P < 0.01) and 31.2% (P < 0.01), respectively. 5. The present study provides the first evidence of altered lung water transport associated with downregulation of AQPs in aged lung. Blood glucocorticoid hormone levels are important to maintain normal AQP-1 expression in the lung microvascular endothelium. Corticosteroid-induced AQP-1 upregulation may contribute to the role of corticosteroids in accelerating oedema clearance in aged lung.

Recent Advances in Imaging of the Lung

Lung has a complex cellular, physical and physiological architecture. To fully appreciate the normal and the altered cellular and physiological phenotype of the lung, it is critical to acquire cell‐specific and quantifiable information in intact lungs or isolated lung cells using relevant imaging methods. The integration of sound principles of morphometry with advanced light and ultrastructural imaging systems has provided quantification of cellular and molecular alterations in the lung. Recent use of intravital imaging has provided novel data on the interactions of leukocytes with lung microvascular endothelium and the responses of microvascular endothelium. Synchrotron radiation is also emerging as a tool to image changes in the living lung. This talk will provide an overview of the established and newly emerging imaging methods and their applications to the lung.

Rap1 mediates protective effects of iloprost against lung endothelial dysfunction and vascular leak

Prostaglandin I2 has been implicated in the regulation of vascular function, inflammation, and acute lung injury. However molecular mechanisms of prostaglandin I2 protective effects on pulmonary endothelial cells (EC) are not well understood. We tested a role of novel cAMP‐activated Epac‐Rap1 pathway in barrier protective effects of synthetic analogue of prostaglandin I2 iloprost using in vivo and in vitro models of lung vascular leak. Iloprost dramatically reduced bronchoalveolar lavage (BAL) protein content, neutrophil accumulation in BAL and lung tissue, capillary filtration coefficient, and Evans blue albumin extravasation caused by high tidal volume ventilation (HTV, 30 ml/kg, 4 hrs). SiRNA‐based Rap1 knockdown inhibited protective effects of iloprost against HTV‐induced lung injury. In vitro, iloprost induced sustained TER increase in lung microvascular endothelium, enhanced intercellular junctions and formation of actin cortical rim, and attenuated thrombin‐induced EC barrier disruption. Consistent with in vivo experiments, Rap1 depletion attenuated protective effects of iloprost in the thrombin model of EC permeability. These data demonstrate that Rap1‐dependent signaling is a major contributor to protective effects of iloprost against lung endothelial barrier dysfunction and vascular leak.HL87823, HL58064

The allergic inflammatory reaction in neonatal streptozotocininduced diabetic rats: evidence of insulin resistance and microvascular dysfunction

To investigate the allergic reaction in neonatal streptozotocin (nSTZ)-induced diabetes mellitus. Male newborn Wistar rats were made diabetic by the injection of streptozotocin (160 mg/kg, i. p.) and used 8 weeks thereafter. Animals were sensitized against ovalbumin (OA, 50 microg and Al(OH)3, 5 mg, s. c.) and challenged 14 or 21 days thereafter. OA-induced airway inflammation and OA-induced pleurisy models were used to investigate leukocyte migration (total and differential leukocyte counts) and lung vascular permeability (Evans blue dye extravasation). nSTZ-diabetic rats presented glucose intolerance and insulin resistance. Relative to controls, nSTZ rats exhibited a 30% to 50% reduction in lung vascular permeability. Leukocyte infiltration in both models of allergen-induced inflammation, and number of pleural mast cells did not differ between groups. Data suggest that the reduction of allergic inflammatory reactions in nSTZ rats is restricted to microvascular dysfunctions and associated, probably, with insulin resistance in lung microvascular endothelium.

Endothelial-derived microparticles (EMPs) demonstrate preferential binding to lung microvascular endothelium

Bezilla Peterson, Danielle MD; Kaul, Sushma MS; Gourlay, David M. MD; Sander, Tara PhD; Pritchard, Kirkwood A. Jr PhD; Oldham, Keith T. MD, FACS Author Information

Red blood cells induce hypoxic lung inflammation

AbstractHypoxia, which commonly associates with respiratory and cardiovascular diseases, provokes an acute inflammatory response. However, underlying mechanisms are not well understood. Here we report that red blood cells (RBCs) induce hypoxic inflammation by producing reactive oxygen species (ROS) that diffuse to endothelial cells of adjoining blood vessels. Real-time fluorescence imaging of rat and mouse lungs revealed that in the presence of RBC-containing vascular perfusion, hypoxia increased microvascular ROS, and cytosolic Ca2+, leading to P-selectin–dependent leukocyte recruitment. However, in the presence of RBC-free perfusion, all hypoxia-induced responses were completely inhibited. Because hemoglobin (Hb) autoxidation causes RBC superoxide formation that readily dismutates to H2O2, hypoxia-induced responses were lost when we inhibited Hb autoxidation with CO or nitrite, or when the H2O2 inhibitor, catalase was added to the infusion to neutralize the RBC-derived ROS. By contrast, perfusion with RBCs from BERK-trait mice that are more susceptible to Hb autoxidation and to hypoxia-induced superoxide production enhanced the hypoxia-induced responses. We conclude that in hypoxia, increased Hb autoxidation augments superoxide production in RBCs. Consequently, RBCs release H2O2 that diffuses to the lung microvascular endothelium, thereby initiating Ca2+-dependent leukocyte recruitment. These findings are the first evidence that RBCs contribute to hypoxia-induced inflammation.

Time Course for Expression of VEGF and Its Receptor and Regulator Levels of Contraction and Relaxation in Increased Vascular Permeability of Lung Induced by Phosgene

Acute lung injury (ALI) induced by phosgene increases risk of serious edema and mortality. Increased permeability of the microvascular endothelium is implicated in the progression of ALI, but the processing interaction and time course activity of the vascular regulators in exudation are still not understood. The main aim of this study was to investigate the time course and potential role for vascular endothelial growth factor (VEGF), its receptors, and some vascular function regulators related to increased vascular permeability of lung induced by phosgene. Sprague Dawley rats were randomly divided into seven groups according to time post phosgene exposure (control, and 1, 3, 6, 12, 24, and 48 h groups). Lung tissue was removed to evaluate VEGF isoforms, fms-like tyrosine kinase receptor 1 (Flt-1), and kinase insert domain containing region (KDR/Flk-1) by reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Blood samples were collected for measurement of plasma endothelin-1 (ET-1) and nitric oxide (NO) level. The results showed that the mRNA and protein expression profile of the VEGF system after phosgene exposure was time dependent. The VEGF system expression in lung tissue was related closely to the level of ET-1 and NO. In conclusion, increased permeability of the lung microvascular endothelium induced by phosgene was primarily a result of differential expression of VEGF and its receptors, and was related to the level of ET-1 and NO. The results suggest that the cooperation of VEGF system, ET-1, and NO plays a critical role, and all those parameters emerge as time dependent in the early phase of the permeability process induced by phosgene exposure.

Lung microvascular endothelium is enriched with progenitor cells that exhibit vasculogenic capacity

Endothelial progenitor cells (EPCs) have been isolated postnatally from bone marrow, blood, and both the intima and adventitia of conduit vessels. However, it is unknown whether EPCs can be isolated from the lung microcirculation. Thus we sought to determine whether the microvasculature possesses EPCs capable of de novo vasculogenesis. Rat pulmonary artery (PAEC) and microvascular (PMVEC) endothelial cells were isolated and selected by using a single-cell clonogenic assay. Whereas the majority of PAECs (approximately 60%) were fully differentiated, the majority of PMVECs (approximately 75%) divided, with approximately 50% of the single cells giving rise to large colonies (>2,000 cells/colony). These highly proliferative cells exhibited the capacity to reconstitute the entire proliferative hierarchy of PMVECs, unveiling the existence of resident microvascular endothelial progenitor cells (RMEPCs). RMEPCs expressed endothelial cell markers (CD31, CD144, endothelial nitric oxide synthase, and von Willenbrand factor) and progenitor cell antigens (CD34 and CD309) but did not express the leukocyte marker CD45. Consistent with their origin, RMEPCs interacted with Griffonia simplicifolia and displayed restrictive barrier properties. In vitro and in vivo Matrigel assays revealed that RMEPCs possess vasculogenic capacity, forming ultrastructurally normal de novo vessels. Thus the pulmonary microcirculation is enriched with EPCs that display vasculogenic competence while maintaining functional endothelial microvascular specificity.

Src-family kinase–dependent disruption of endothelial barrier function by Plasmodium falciparum merozoite proteins

AbstractPulmonary complication in severe Plasmodium falciparum malaria is manifested as a prolonged impairment of gas transfer or the more severe acute respiratory distress syndrome (ARDS). In either clinical presentation, vascular permeability is a major component of the pathologic process. In this report, we examined the effect of clinical P falciparum isolates on barrier function of primary dermal and lung microvascular endothelium in vitro. We showed that parasite sonicates but not intact infected erythrocytes disrupted endothelial barrier function in a Src-family kinase–dependent manner. The abnormalities were manifested both as discontinuous immunofluorescence staining of the junctional proteins ZO-1, claudin 5, and VE-cadherin and the formation of interendothelial gaps in monolayers. These changes were associated with a loss in total protein content of claudin 5 and redistribution of ZO-1 from the cytoskeleton to the membrane and the cytosolic and nuclear fractions. There was minimal evidence of a proinflammatory response or direct cellular cytotoxicity or cell death. The active component in sonicates appeared to be a merozoite-associated protein. Increased permeability was also induced by P falciparum glycophosphatidylinositols (GPIs) and food vacuoles. These results demonstrate that parasite components can alter endothelial barrier function and thus contribute to the pathogenesis of severe falciparum malaria.

Depolarization Evokes Calcium Entry Through Ca V 3.1 Channels in Lung Microvascular Endothelium in situ .

Pulmonary microvascular endothelial cells (PMVECs) express a voltage-gated T-type Ca2+ channel CaV3.1 subunit that has been proposed to contribute to agonist-induced Ca2+ entry. Ca2+ entry via CaV3.1 channel occurs when PMVEC membrane potential is within the CaV3.1 “window current” range of voltages that is predicted to be −60 to −30 mV. In the present study, we performed experiments to directly detect Ca2+ influx within the window current range of voltages. Utilizing whole-cell patch-clamp recordings, cultured PMVECs were depolarized at potentials of −45 mV and 0 mV for 1.5 s, and then the inward Ca2+ current was evaluated by measuring the deactivation tail current by transitioning to −120 mV. The tail current measurement revealed a PMVEC Ca2+ conductance in the T-type window current range of voltages. We further performed in situ subpleural microvascular fluorescence microscopy in isolated perfused rat lungs to examine whether infusion of KCl at high concentration (40 mM) results in an elevation of cytosolic Ca2+ ([Ca2+]i) in lung microvascular endothelium. KCl reproducibly induced a slowly developing, sustained increase in [Ca2+]i from 101±2 nM to 138±4 nM in microvascular endothelium (P <0.05), an effect that was completely inhibited by the T-type Ca2+ channel blocker mibefradil (10 μM; n=5 each, P <0.05). Together, these results suggest that CaV3.1 T-type Ca2+ channel is expressed in pulmonary microvascular endothelial cells and mediates depolarization-induced [Ca2+]i transitions both in culture and in situ. Supported by HL074116 and Kaiserin-Friedrich Foundation

Early glycation products of endothelial plasma membrane proteins in experimental diabetes

The participation of glucose and two intermediates of glucose metabolism: glucose-6-phosphate (G6P) and glyceraldehyde-3-phosphate (Gald3P) to the formation of early glycation products was comparatively evaluated in the endothelial plasma membrane of streptozotocin-induced diabetic rats. Antibodies risen to a carrier protein reductively glycated by each of the sugars mentioned above were used to probe by immunoblotting the proteins of the lung microvascular endothelium plasmalemma purified from normal and diabetic rats. The amount of glycated endothelial plasma membrane proteins was below the limit of detection in normoglycemic animals but increased dramatically in diabetic animals for glucose and G6P. In contrast, no signal was found in diabetic rats for Gald3P, indicating that either the contribution of this phosphotriose to the glycation of intracellular proteins is negligible in vivo, or the Schiff base generated by this sugar transforms very rapidly into products of advanced glycation. Globally, the endothelial plasma membrane proteins bound on average 300 times more glucose than G6P proving that, in spite of its low in vitro potency as glycating agent, glucose represents the main contributor to the intracellular formation of early glycation products. The most abundant glycated proteins of the lung endothelial plasma membrane were separated by two dimensional electrophoresis and identified by mass spectrometry.

Expression of chemokines on the surface of different human endothelia

Expression of chemokines at the endothelial surface depends on their rate of synthesis, the capacity of the endothelium to bind chemokines and the rate of clearance from the surface. The aim of this study was to establish how these factors depend on the chemokine and the tissue of origin of the endothelium. Human lung and dermal microvascular endothelium, saphenous and umbilical vein endothelium, and a bone marrow endothelial line were assayed in vitro. Chemokine expression, localization and transport was measured by immunoassay and confocal microscopy. All endothelia bound CCL3 (MIP-1alpha), CCL5 (RANTES) and CXCL10 (IP-10). CCL3 and CCL5 bound at high levels, and CXCL10 bound less strongly. However, the profile of chemokine expression varied between endothelia, and different chemokines were shown to bind to the endothelial surface by distinct mechanisms. The half-life of CCL3 and CCL5 at the cell surface was approximately 30 min and chemokines were cleared primarily by endocytosis into caveolae. Endothelia from different tissues synthesize distinctive sets of chemokines, but the profile of surface-expressed chemokines also depends on the distinctive characteristics of each endothelia. These two mechanisms may contribute to the differential recruitment of leucocyte subsets to different tissues.