Bovine Pulmonary Microvascular Endothelial Cells Research Articles

Effects of Extracellular ATP on Bovine Lung Endothelial and Epithelial Cell Monolayer Morphologies, Apoptoses, and Permeabilities

Pneumonia in cattle is an important disease both economically and in terms of animal welfare. Recent evidence in other species has shown ATP to be an important modulator of inflammation in the lung, where it is released by activated alveolar macrophages and damaged lung cells. Whether ATP serves a similar process during infection in the bovine lung is unknown. In the present study, we examined the effects of ATP treatment on the morphology, apoptosis, and permeability of bovine pulmonary epithelial (BPE) cells and bovine pulmonary microvascular endothelial cells (BPMEC). Monolayers of BPE cells underwent striking morphological changes when exposed to ATP that included separation of the cells. Neither BPE cells nor BPMEC exhibited increased apoptosis in response to ATP. BPE cell and BPMEC monolayers displayed virtually identical increases in permeability when exposed to ATP, with a 50% change occurring within the first hour of exposure. Both cell types contained mRNA for the P2X(7) receptor, a known receptor for ATP. In BPE cells, but not BPMEC, the change in permeability in response to ATP was reversed by the addition of a P2X(7) receptor antagonist. If similar permeability changes occur in vivo, they could be a factor in vascular leakage into lung airspaces during pneumonia.

Effects of the Mechanical Properties of Collagen Gel on the In Vitro Formation of Microvessel Networks by Endothelial Cells

Vascularization by endothelial cells (ECs) is an essential element in tissue-engineering of organoids. Morphogenesis of these cells is regulated not only by the biochemical properties of the extracellular matrix (ECM) but also by its mechanical properties. Here, we investigated the effect of substrate mechanical properties on the formation of capillary-like networks by ECs; in particular, we examined the three-dimensional (3D) configurations of the resulting networks. Bovine pulmonary microvascular ECs (BPMECs) were cultured on a series of collagen gels of different stiffness but the same collagen concentration. Imaging techniques revealed that cells cultured in rigid and flexible gels formed 3D networks via different processes; cells formed dense, thin networks in the flexible gel, whereas thicker and deeper networks were formed in the rigid gel. Cross-sections of the networks revealed that those formed within the rigid gel had large lumens composed of multiple cells, whereas those formed within the flexible gel had small, intracellular vacuoles. The expression of vinculin, a focal adhesion protein, appeared to change with the mechanical properties of collagen gel. Our results indicate that the mechanical properties of adhesion substrates play an important role in regulating 3D network formation.

Lysophosphatidylcholine increases endothelial permeability: role of PKCα and RhoA cross talk

Lysophosphatidylcholine (LPC) is a bioactive proinflammatory lipid that can be generated by pathological activities. We investigated the hypothesis that LPC signals increase in endothelial permeability. Stimulation of human dermal microvascular endothelial cells and bovine pulmonary microvascular endothelial cells with LPC (10-50 microM) induced decreases (within minutes) in transendothelial electrical resistance and increase of endothelial permeability. LPC activated (within 5 min) membrane-associated PKC phosphotransferase activity in the absence of translocation. Affinity-binding analysis indicated that LPC induced increases (also by 5 min) of GTP-bound RhoA, but not Rac1 or Cdc42. By 60 min, both signaling pathways decreased toward baseline. Inhibition of RhoA with C3 transferase inhibited approximately 50% of LPC-induced resistance decrease. Pretreatment with PKC inhibitor Gö-6983 (concentrations selective for classic PKC), PMA-induced depletion of PKCalpha, and transfection of antisense PKCalpha oligonucleotide each prevented 40-50% of the LPC-induced resistance decrease. Furthermore, these three PKC inhibition strategies inhibited 60-80% of the LPC-induced GTP-bound RhoA. These results show that LPC directly impairs the endothelial barrier function that was dependent, at least in part, on cross talk of PKCalpha and RhoA signals. The evidence indicates that elevated LPC levels can contribute to the activation of a proinflammatory endothelial phenotype.

Endothelial cells from bovine pulmonary microvasculature respond to Mycoplasma bovis preferentially with signals for mononuclear cell transmigration

Mycoplasma bovis can cause arthritis or mastitis following pneumonia and mycoplasmemia in cattle. Interactions with pulmonary vascular endothelium have been recorded as localized vasculitis, perivascular mononuclear cell infiltrations, and accumulation of inflammatory cells in lesions. We compared adhesion mediators and cytokine gene expression as well as cytotoxicity of cultured primary bovine aortic and bovine pulmonary microvascular endothelial cells (BPMEC) challenged with M. bovis. We also tested if abscess-forming ability of strains of M. bovis is associated with changes on endothelial cells. Increased VCAM-1 surface expression was found in both cell types, while only infected BPMEC increased MCP-1 transcription, both mediators specific for mononuclear cell transmigration. Given no induction of ICAM-1 mRNA in either cell type, induction of IL-8 mRNA by BPMEC suggested that neutrophil transmigration was signaled in microvascular areas. Infected BPMEC showed early induction of IL-1β and IL-6 mRNA. Excepting VCAM-1, differential strain effects were limited to BPMEC and not correlated with their abscess-forming capability. In addition, only strain DSA16 had minor cytotoxic effect on both cell types. We thus show that BPMEC are more susceptible than aortic cells to M. bovis-induced activation. Activation preferentially yielded signals for mononuclear cell transmigration, correlating well with in vivo observations of infiltrating cells at pulmonary sites.

Effect of shear stress on microvessel network formation of endothelial cells with in vitro three-dimensional model.

Shear stress stimulus is expected to enhance angiogenesis, the formation of microvessels. We determined the effect of shear stress stimulus on three-dimensional microvessel formation in vitro. Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor to make a microvessel formation model. We observed this model in detail using phase-contrast microscopy, confocal laser scanning microscopy, and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 h. Promotion of microvessel network formation was detectable after approximately 10 h in the flow chamber. After 48 h, the length of networks exposed to shear stress was 6.17 (+/-0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (+/-0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of microvessel network formation in the gel and expands the network because of repeated bifurcation and elongation.

In vitro evaluation of the role of platelet-activating factor and interleukin-8 in Mannheimia haemolytica-induced bovine pulmonary endothelial cell injury.

To develop an in vitro model of the bovine alveolar-capillary interface and to evaluate the roles of interleukin-8 (IL-8) and platelet-activating factor (PAF) in neutrophil-mediated endothelial injury induced by infection with Mannheimia haemolytica. Cultured bovine pulmonary microvascular endothelial cells, freshly isolated bovine neutrophils, and monocyte-derived bovine macrophages. A coculture system was developed in which endothelial cells were grown to confluence in tissue culture inserts, neutrophils were added to the inserts, and macrophages were added to tissue culture wells. Mannheimia haemolytica-derived lipopolysaccharide (LPS) or supernatant was added to activate macrophages, and inhibitors of PAF or IL-8 were added to the insert. Endothelial cell cytotoxicity and permeability (ie, albumin leakage) and neutrophil activation (ie, adhesion, degranulation [lactoferrin expression], and superoxide production) were assessed. The addition of M haemolytica-derived LPS to bovine macrophages in the coculture system resulted in significant increases in endothelial cell cytotoxicity and permeability and neutrophil degranulation and adhesion. Inhibition of IL-8 reduced endothelial cell permeability and neutrophil degranulation induced by exposure to M haemolytica-derived supernatant, whereas inhibition of PAF decreased superoxide release by neutrophils. In vitro activation of bovine macrophages by M haemolytica-derived LPS resulted in neutrophil activation and neutrophil-mediated endothelial damage. Neutrophil-mediated endothelial injury and neutrophil degranulation were, at least in part, mediated by IL8, whereas PAF promoted superoxide release by neutrophils in this in vitro system designed to mimic the in vivo events that occur during the early stages of bovine pneumonic pasteurellosis.

Human thrombospondin 2 inhibits proliferation of microvascular endothelial cells

This study was performed to characterize human thrombospondin 2 (TSP2). TSP2 has recently attracted attention as an endogenous negative regulator of angiogenesis in tumorigenesis. We cloned and transfected human TSP2 cDNA into the human colon cancer cell line SW-480. Stable transfectants (TSP2-1, TSP2-6) overexpressing TSP2 were established. Growth characteristics of TSP2-transfectants were investigated in vitro and in vivo. TSP2-transfectants showed similar growth properties to vector-transfectants and wild-type SW-480 cells. The overexpression of transfected human TSP2 cDNA did not affect proliferation of SW-480 cells. When the conditioned media of TSP2-transfectants were added to cultures of bovine pulmonary microvascular endothelial cells (BPMEC), the BPMEC proliferation was significantly inhibited. These results suggested that human TSP2 is a potential inhibitor of angiogenesis.

Protein phosphatase 2B inhibitor potentiates endothelial PKC activity and barrier dysfunction.

Serine/threonine (Ser/Thr) protein phosphatases (PPs) are implicated in the recovery from endothelial barrier dysfunction caused by inflammatory mediators. We hypothesized that Ser/Thr PPs may regulate protein kinase C (PKC), a critical signaling molecule in barrier dysfunction, in the promotion of barrier recovery. Western analysis indicated that bovine pulmonary microvascular endothelial cells (BPMECs) expressed the three major Ser/Thr PPs, PP1, PP2A, and PP2B. Pretreatment with 100 ng/ml of FK506 (a PP2B inhibitor) but not with the PP1 and PP2A inhibitors calyculin A or okadaic acid potentiated the thrombin-induced increase in PKC phosphotransferase activity. FK506 also potentiated thrombin-induced PKC-alpha but not PKC-beta phosphorylation. FK506 but not calyculin A or okadaic acid inhibited recovery from the thrombin-induced decrease in transendothelial resistance. Neither FK506 nor okadaic acid altered the thrombin-induced resistance decrease, whereas calyculin A potentiated the decrease. Downregulation of PKC with phorbol 12-myristate 13-acetate rescued the FK506-mediated inhibition of recovery, which was consistent with the finding that the thrombin-induced phosphorylation of PKC-alpha was reduced during the recovery phase. These results indicated that PP2B may play a physiologically important role in returning endothelial barrier dysfunction to normal through the regulation of PKC.

Role of MARCKS in regulating endothelial cell proliferation.

Myristoylated alanine-rich C kinase substrate (MARCKS), as a specific protein kinase C (PKC) substrate, mediates PKC signaling through its phosphorylation and subsequent modification of its association with filamentous actin (F-actin) and calmodulin (CaM). PKC has long been implicated in cell proliferation, and recent studies have suggested that MARCKS may function as a cell growth suppressor. Therefore, in the present study, we investigated MARCKS protein expression, distribution, and phosphorylation in preconfluent and confluent bovine pulmonary microvascular endothelial cells (BPMEC) in the presence or absence of the vascular endothelial growth factor (VEGF). In addition, we examined functional alterations of MARCKS in these cells by studying the association of MARCKS with F-actin and CaM-dependent myosin light chain (MLC) phosphorylation. Our results indicate that MARCKS protein is downregulated during BPMEC proliferation. Decreased MARCKS association with F-actin, increased actin polymerization, and CaM-dependent MLC phosphorylation appear to mediate cell shape changes and motility during BPMEC growth. In contrast, VEGF stimulated MARCKS phosphorylation without alteration of protein expression during BPMEC proliferation, which may result in reduced interaction between MARCKS and actin or CaM, leading to actin reorganization and MLC phosphorylation. Our data suggest a regulatory role of MARCKS during endothelial cell proliferation.

The Effect of Alkalosis on Intracellular Calcium Concentrations[Ca++]i in Pulmonary Microvascular Endothelial Cells • 1602

Background: The signaling mechanisms mediating alkalosis-induced pulmonary vasodilation are poorly understood. We have recently shown that an increase in pH from 7.4 to 7.7 stimulates cyclooxygenase (COX) and nitric oxide synthase (NOS) activity resulting in a 2-fold increase in cGMP and a 5-fold increase in prostacyclin production in pulmonary microvascular endothelial cells (EC) in culture. Objective: To test the hypothesis that alkalosis causes an increase in [Ca++]i by stimulating K+ channel activity resulting in membrane hyperpolarization. As EC do not have voltage-gated Ca++ channels, Ca++ entry in EC depends upon the electrochemical driving force. Methods: Bovine pulmonary microvascular EC were loaded for 1 hour at room temperature with 5 μM fura-2 AM and 0.01% Pluronic F127. Cultures were rinsed of free fura-2 and placed on the stage of an inverted microscope for measurement of[Ca++]i using a dual wavelength fluorescent imaging system(Intracellular Imaging, Inc.). Excitation wavelengths were altered between 340 and 380 nm and emission fluorescence was measured at 510 nm. Intracellular Ca++ concentrations were computed from Ca++ calibration curves generated from Ca++ standard solutions and the 340/380 ratios using the InCyt IM2 image acquisition and analysis software.Results: Basal [Ca++]i was 52 ± 22 nM. There was a pH-dependent increase in [Ca++]i: pH 9.0 caused an abrupt 7-fold increase, while pH 7.7 caused a 2.2-fold increase over basal levels. The pH-dependent increase in [Ca++]i was abolished in Ca++-free medium. TMB-8, an inhibitor of IP3-mediated Ca++ release, had no effect on the alkalosis-induced increase in[Ca++]i but abolished the Ca++ transient induced by bradykinin. Depolarization of membrane potential with an isotonic KCl buffer(50 mM) abolished the increase in [Ca++]i mediated by alkalosis. Pretreatment of EC for 15 min with tetraethylammonium (TEA) partially inhibited the alkalosis-induced increase in [Ca++]i.Conclusion: Alkalosis induces a pH-dependent increase in[Ca++]i in pulmonary microvascular EC, which is dependent upon the presence of extracellular Ca++. The alkalosis-induced increase in[Ca++]i is linked to K+ channel activation, as it is inhibited by membrane depolarization and partially blocked by TEA. These studies support the hypothesis that alkalosis mediates vasodilation by activating K+ channels, hyperpolarizing EC membrane potential, resulting in increased [Ca++]i, which ultimately activates Ca++- dependent enzymes, such as COX and NOS.

Localization and activation of type IV collagenase/gelatinase at endothelial focal contacts.

The cell-surface localization and site of activation of type IV collagenases/gelatinases (matrix metalloproteinases, MMP) in bovine pulmonary microvascular endothelial (BPMVE) cells was examined. Sucrose density centrifugation of plasma membranes and immunofluorescent staining of whole cells indicated association of 72 kDa (MMP-2) and 96 kDa (MMP-9) type IV collagenase/gelatinases with the plasma membrane. Incubation of the BPMVE cells with rhodaminated MMP-9 demonstrated colocalization with beta 1-integrin, indicating incorporation into the focal contacts. The focal contacts were extracted with saponin, and associated proteolytic activity was examined by zymography. The focal contacts contained latent MMP-2, and stimulation of the cells with cytochalasin D or with 8-bromoadenosine 3',5'-cyclic monophosphate with 3-isobutyl-1-methylxanthine increased both latent and activated MMP-9 in the focal contacts. Addition of these stimuli in unconditioned culture medium did not produce this effect, indicating that the MMP-9 in focal contact extracts was derived from previously secreted enzyme. The activated metalloproteinase degraded extracellular matrix collagens and was inhibited by 1,10-phenanthroline. These findings indicate that endothelial cells release MMP into the extracellular milieu and then concentrate and activate MMP-9 from medium at the focal contacts.

A 96-kDa gelatinase induced by TNF-alpha contributes to increased microvascular endothelial permeability

Tumor necrosis factor-alpha (TNF-alpha) may increase vascular endothelial permeability through alteration of the extracellular matrix (ECM). Incubation of bovine pulmonary microvascular endothelial (BPMVE) cells grown to confluence on microporous filters with 10(4) U/ml TNF-alpha for 24 h increased monolayer permeability to 125I-labeled albumin two- to threefold. TNF-alpha treatment also induced expression of a 96-kDa gelatinolytic metalloproteinase that was present in the medium and bound to the ECM. The induced 96-kDa metalloproteinase was purified from conditioned medium and found to cleave fibronectin, laminin, types IV and V collagens, and gelatins from types I and III collagens, suggesting identity as a type IV collagenase-gelatinase. Incubation of BPMVE cells with the 96-kDa gelatinase increased monolayer permeability, an effect prevented by inclusion of either tissue inhibitor of metalloproteinase (TIMP) or 1,10-phenanthroline. When BPMVE cells were incubated with the 96-kDa gelatinase or 10(4) U/ml TNF-alpha and then stripped from the filters, the remaining ECM displayed increased permeability to 125I-albumin compared with matrix from untreated BPMVE. The ECM extracts from both TNF-alpha- and enzyme-treated cells were found to contain less fibronectin, whereas their total protein contents were similar to those of untreated controls. These results suggest that the 96-kDa metalloproteinase induced by TNF-alpha contributes to increased vascular endothelial permeability through the degradation of specific extracellular matrix components.

Quantitation of specific binding of orosomucoid to cultured microvascular endothelium: role in capillary permeability

Orosomucoid (also known as alpha 1-acid glycoprotein) is a highly sialylated, polyanionic serum glycoprotein. Its presence in vascular perfusates alters microvascular permeability, presumably by binding to the endothelial glycocalyx, but the rest of its functions remains unknown. In this study, we assess the binding of bovine serum orosomucoid (BSO) to the surface of bovine pulmonary microvascular endothelial cells (BLMVEC) in culture at 4 degrees C. The binding of radioiodinated BSO (125I-BSO) reached equilibrium after 10 min of incubation, was not calcium dependent, and was reversible by greater than 80% in 30 min. The binding of 125I-BSO was competed with unlabeled BSO but not by transferrin, immunoglobulin, gelatin, ovalbumin, mannan, fucoidan, mucin, or asialomucin. In addition, binding was not affected by the presence of galactose, glucose, fucose, mannose, N-acetyl-D-galactosamine, or N-acetyl-D-glucosamine. This binding behavior is not consistent with carbohydrate recognition by lectin-like molecules such as the asialoglycoprotein receptor. Scatchard analysis of the BSO binding to BLMVEC produced a concave-upward shaped curve, which revealed a higher affinity binding component with an apparent equilibrium affinity constant (Kd) of 8.6 nM and a maximum receptor number of 40,000/cell and revealed a moderate affinity component with a Kd of 9.1 microM with a maximum binding of 10(7) binding sites per cell. Other blood vessel-associated cells clearly bound less BSO with a lower binding affinity than the BLMVEC monolayers. From the binding data, we estimated the total increase in the negative charge of the glycocalyx with orosomucoid binding to be approximately 17 meq/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary microvascular endothelial cells constitutively release xanthine oxidase

The generation of oxidants in reperfused ischemic tissues by xanthine oxidase (XO) may contribute to tissue damage. We exposed bovine pulmonary microvascular endothelial (BPMVE) cells to hypoxia and subsequent reoxygenation and examined alterations in intracellular and extracellular XO activities. BPMVE cells incubated 24 h under hypoxic conditions (<1% O 2) showed a twofold increase in intracellular xanthine dehydrogenase activity and a smaller increase in intracellular XO activity compared to normoxic BPMVE. Both normoxic and hypoxic BPMVE cells constitutively released XO activity into their culture media. Incubation of hypoxic or normoxic BPMVE cells with oxygenated medium (95% O 2) stimulated the release of XO activity into the extracellular medium within 5 min. The XO activity could not be detected in the oxygenated medium after 60 min incubation with 95% O 2. These results indicate that endothelial cells in culture constitutively release XO and that oxygenation rapidly enhances XO release. The released XO activity may play an important role in generation of oxidants in the extracellular milieu during reperfusion.

Tumor necrosis factor-alpha-mediated decrease in glutathione increases the sensitivity of pulmonary vascular endothelial cells to H2O2.

We examined the effects of tumor necrosis factor-alpha (TNF alpha) stimulation of endothelial cells on the increase in endothelial permeability induced by H2O2. Bovine pulmonary microvascular endothelial cells (BPMVEC) were grown to confluence on a microporous filter and the 125I-albumin clearance rate across the monolayer was determined. Pretreatment with TNF alpha (100 U/ml) for 6 h had no direct effect on transendothelial 125I-albumin permeability. However, TNF alpha pretreatment enhanced the susceptibility of BPMVEC to H2O2; that is, H2O2 (10 microM) alone had no direct effect, whereas H2O2 increased 125I-albumin permeability more than threefold when added to monolayers pretreated for 6 h with TNF alpha. Determination of lactate dehydrogenase release indicated that increased permeability was not due to cytolysis. We measured the intracellular contents of GSH and catalase to determine their possible role in mediating the increased susceptibility to H2O2. TNF alpha treatment (100 U/ml for 6 h) decreased total GSH content and concomitantly increased the oxidized GSH content, but did not alter the cellular catalase activity. The role of GSH was examined by pretreating endothelial cells with 2 mM GSH for 3 h, which produced an 80% increase in intracellular GSH content. GSH repletion inhibited the increased sensitivity of the TNF alpha-treated endothelial cells to H2O2. We tested the effects of xanthine oxidase (XO) inhibition since XO activation may be a source of oxidants responsible for the decrease in cellular GSH content. Pretreatment with 0.5 mM oxypurinol attenuated the synergistic effect of TNF alpha and H2O2 on endothelial permeability. The results indicate that decreased oxidant buffering capacity secondary to TNF alpha-induced reduction in intracellular GSH content mediates the increased susceptibility of endothelial cells to H2O2. This mechanism may contribute to oxidant-dependent vascular endothelial injury in septicemia associated with TNF alpha release.

Cytokeratin filament modulation in pulmonary microvessel endothelial cells by vasoactive agents and culture confluency

Recently, bovine pulmonary microvascular endothelial cells (PMV) were shown to contain cytokeratin 8 and 19 intermediate filaments (Patton et al., 1990). In this study, we examine the effect of culture contiguity and vasoactive agents on the content and assembly of cytokeratins in PMV. Immunofluorescent staining of PMV cultures show a progressive increase in cytokeratin filament assembly. In freshly plated PMV, keratin appears as hazy staining (<4 hr) and later organizes into keratin ‘plaques’ (4 days) associated with cell-cell contacts; post confluent (>7 days) PMV cultures contain fully assembled cytokeratin filaments which extend to the cell periphery and approach filaments in apposed cells. Vimentin filaments are also present in freshly plated PMV cultures but unlike cytokeratins, become less filamentous at confluency. This cell density-dependent modulation of cytokeratins is also demonstrated by densitometric analysis of autoradiographs of 35S-methionine labeled keratins in which PMV keratin content is elevated at high cell densities, while vimentin content remains constant. Desmoplakins I and II, components of desmosomes, could not be demonstrated in PMV by immunoblotting. PMV treated with permeability modulating agents (4 × 10 −3 M EGTA, 1 μM cytochalasin B, 1 μM bradykinin, 1 μM A23187, and 1 μM PMA) exhibit border retraction and altered keratin filament staining. From these studies we conclude: 1) cytokeratin 8 and 19 containing intermediate filaments are present in confluent PMV cultures with vimentin but without desmosomes, 2) the state of assembly of PMV cytokeratin and vimentin filaments appears to be oppositely affected by culture contiguity, and 3) treatment of monolayers with vasoactive agents alters the state of assembly of cytokeratin filaments. We speculate that modualtion of cytokeratin assembly in PMV may be involved in regulation of pulmonary microvascular structure and function.