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Effects of monocrotaline pyrrole and thrombin on pulmonary endothelial cell junction and matrix adhesion proteins
Toxicology 184 (2003) 227 /240 www.elsevier.com/locate/toxicol
Effects of monocrotaline pyrrole and thrombin on pulmonary endothelial cell junction and matrix adhesion proteins Debra W. Taylor a, Michael W. Lame´ b, Lynn S. Nakayama b, H.J. Segall b, Dennis W. Wilson a, a
Departments of Veterinary, Pathology, Microbiology and Immunology, University of California-Davis, 2150 Haring Hall, Davis, CA 95616, USA b Department of Molecular Biosciences, University of California-Davis, Davis, CA 95616, USA Received 14 June 2002; received in revised form 26 August 2002; accepted 24 September 2002
Abstract Previous work in our laboratory has shown that monocrotaline pyrrole (MCTP) interacts with actin and potentiates thrombin-mediated endothelial barrier permeability through increasing the overall surface area of intercellular gaps. To better characterize endothelial barrier leak in this model, we examined the effects of MCTP and thrombin on the localization and structure of three adhesion associated proteins that directly or indirectly interact with actin in regulating barrier function: cell /cell occludens junction molecule (ZO-1), the cell /cell adherens junction linker, ßcatenin, and the cell /matrix intermediary signaling protein, focal adhesion kinase (FAK). Immunohistochemistry demonstrated that thrombin treatment resulted in radial reorganization of focal adhesions and broader distribution of adherens and occludins junctions at the cell border suggestive of membrane stretching in contracture. MCTP pretreatment resulted in fewer and more disorganized focal adhesions and marked thinning of occludins and adherens junctions. MCTP pretreatment also interfered with thrombin stimulated junctional reorganization. Western blot analysis showed thrombin stimulated catalysis of ZO-1 and FAK while MCTP pretreatment resulted in FAK fragmentation similar to previous reports for apoptosis. We conclude that both MCTP and thrombin alter critical endothelial cell adhesion molecules and this may be an underlying mechanism for the potentiating effect MCTP has on thrombin induced vascular permeability in vitro. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Monocrotaline pyrrole; Thrombin; Pulmonary hypertension; Adhesion molecules; ZO-1; ß-Catenin; pp125FAK; Vascular permeability; Endothelial cells
1. Introduction
Corresponding author. Tel.: /1-530-752-0158; fax: /1530-752-3349 E-mail address: [email protected] (D.W. Wilson).
Monocrotaline (MCT), or its metabolite monocrotaline pyrrole (MCTP), causes delayed, progressive pulmonary hypertension in Sprague / Dawley rats (Mattocks, 1986; Pan et al., 1993;
0300-483X/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 2 ) 0 0 5 8 2 - 6
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Reindel et al., 1990). Pulmonary vascular changes have been reported to begin in less than 24 h and possibly as early as 4 h post intravenous injection. Changes are manifested as a mild sub-intimal edema in vessels of the interalveolar septa and walls of alveolar ducts (Roth and Reindel, 1991; Valdivia et al., 1967). These changes progress to subsequent proliferative vasculitis, cor pulmonale, and pulmonary hypertension (Merkow and Kleinerman, 1966; Meyrick et al., 1980; Reindel et al., 1990; Rosenberg and Rabinovitch, 1988; Roth et al., 1981; Roth and Reindel, 1991). We have shown that MCTP, but not MCT, nor the glutathione metabolite GSH-DHP1, causes a delayed onset of progressive changes in cultured monolayers of bovine pulmonary artery endothelial cells (BPAECs, Taylor et al., 1997). These lesions include megalocytosis, increased membrane permeability and altered function (indicated by increased extracellular LDH and increased prostacyclin production) similar to those reported in vivo (Reindel and Roth, 1991; Taylor et al., 1997). Other effects of MCTP include cell-cycle derangements and induction of apoptosis in BPAEC monolayers (Thomas et al., 1998a,b, 1996). Apoptosis has also been reported to occur in the pulmonary arteries of MCT treated rats (Jones and Rabinovitch, 1996). Vascular permeability has been cited as one of the most important early lesions preceding the onset of MCT induced pulmonary hypertension (Meyrick, 1990). Endothelial cells play a critical role in maintaining physical and functional barrier integrity, particularly in smaller vessels where they comprise a larger portion of the vessel wall. Recent acute in vivo studies in our laboratory show platelet accumulation and degranulation in the pulmonary microvasculature as early as 4 h post MCTP treatment. These changes were associated with loss of endothelial cell junction integrity and matrix adhesion in association with microvascular leak in arterioles and venules (unpublished observations). Endothelial cells form an effective polarized, yet dynamic barrier to the movement of fluids and 1
6,7-Dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine.
solutes between the blood compartment and extracellular matrix (ECM) through cell /cell junctions and cell-to-ECM attachments (via focal adhesion plaques, Barry et al., 1995; Lum and Malik, 1996). Modulation of endothelial barrier function related to development of intercellular gaps can be regulated through receptor mediated signaling pathways. One pathway acts through phosphorylation of linking proteins at cell/cell junctions (Garcia et al., 1996) resulting in dissociation of junction protein complexes and degradation of linking proteins (Kevil et al., 2000; Ukropec et al., 2000; Wachtel et al., 1999). A second pathway occurs through cytoskeletal (actin/myosin) contraction of endothelial cells (Lum and Malik, 1994). Tight junctions (zonula occludens), are a complex network of transmembrane proteins (Dejana et al., 1995; Dejana and Del Maschio, 1995; Telo` et al., 1997) that occur at the apical surface of epithelial and endothelial cells. ZO-1 is a 225 kDa peripheral membrane protein that links to the intercellular adhesion protein occludin (Wachtel et al., 1999; Willott et al., 1992). Adherens junctions also participate in maintaining barrier integrity in endothelial cells. Adherns junctions are composed of the intercellular adhesion molecule VE cadherin which is linked to the cytoskeleton through intermediate proteins in the catenin family (Dejana, 1997; Ukropec et al., 2000). Focal adhesion plaques are integrin based transmembrane adhesion structures located on the basal aspect of the cell where they attach to the basement membrane and underlying matrix. These plaques mediate attachment of the cytoskeleton to the ECM and may participate in signaling that affects cytoskeleton organization. This process is critical to modulation of endothelial permeability, particularly in the thrombin model (Schaphorst et al., 1997). One of the signaling proteins in the cytoplasmic domain of focal adhesion plaques is a tyrosine kinase, focal adhesion kinase (FAK; Gilmore and Romer, 1996). FAK signaling has been shown to play a role in initiating apoptosis in anchorage-dependent cells that are prevented from forming normal attachments to a substrate (van de Water et al., 1999; Xiong and Parsons, 1997). Loss of survival signals
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through ECM components such as fibronectin may signal apoptosis in normal cells (Ilic et al., 1997; Steller, 1995; Thompson, 1995) by signal transduction through FAK (Ilic et al., 1998). Growth factor deprivation and caspase induction result in fragmentation of FAK into characteristic subunits (Levkau et al., 1998). Previously, we characterized an in vitro model of endothelial monolayer permeability using thrombin-mediated stimulus in MCTP pretreated BPAECs. This model is predicated on in vivo evidence of persistent microvascular leak and platelet accumulation in MCT treated rats. In our in vitro model, there is no morphologic evidence of cell death or loss from the monolayer within the first 24 h of exposure. Although there is a small percentage (ca. 5 vs. 2.2% in controls) of cells in the early stages of apoptosis (Thomas et al., 1998a), the MCTP pretreated monolayers appear morphologically intact. Pretreatment of BPAEC monolayers with MCTP potentiates thrombin induced monolayer permeability as measured by transudation of Evans-Blue albumin across monolayers grown on semi-permeable membrane inserts (Wilson et al., 1998). MCTP treatment results in changes in stress fiber arrangement and apparent derangement of actin polymerization (Wilson et al., 1998 and unpublished observations). The mechanism of monolayer permeability appears to relate to regulation of intercellular junctions since MCTP pretreated monolayers develop a markedly increased surface area of intercellular space in response to thrombin stimulation. The combination of in vivo and in vitro evidence suggests a hypothesis that MCTP treatment results in endothelial cell responses leading to platelet activation and a stimulus for microvascular permeability that is abnormally prolonged by persistent alteration in endothelial cell fluid barrier maintenance. The purpose of the present study was to characterize the effects of MCTP pretreatment on thrombin induced changes in cell /cell and cell /matrix adhesions. Our objectives were (1) to determine whether MCTP treatment had a morphological effect on organization of adhesion structures involved in cell /cell contact in confluent BPAEC monolayers; (2) to determine whether thrombin or MCTP treatment altered the expres-
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sion or composition of membrane adhesion proteins; and (3) to evaluate the potential interaction of MCTP treatment with thrombin induced changes in adhesion molecule reorganization. We examined biochemical characteristics and localization of marker proteins from each type of cell adhesion zone: ZO-1 for occludens junctions, ß-catenin for adherens junctions and FAK for ECM attachments in cells treated with MCTP, thrombin, dually with MCTP/thrombin, or a vehicle as a control.
2. Methods 2.1. Cell isolation, culture, characterization Endothelial cells were isolated non-enzymatically (Ryan et al., 1978) from bovine pulmonary arteries obtained from an abattoir and characterized as previously described (Taylor et al., 1997). Pure cultures of BPAECs were scraped from culture dishes and seeded into tissue culture flasks, expanded, then collected, frozen and stored in liquid N2 until thawed for use. Passages 11 /16 were used for these experiments. 2.2. Synthesis of MCTP MCTP was synthesized from MCT according to the method of Mattocks et al. (1989) and recrystallized from anhydrous ethyl ether as previously described (Taylor et al., 1997). Using fast atom bombardment mass spectrometry the conversion of MCT to MCTP was found to be complete; daughter spectra contained ions characteristic of the pyrrole (Lame´ et al., 2000; Pan et al., 1993). MCTP was stored in N ,N -dimethylformamide at /80 8C until just prior to use. 2.3. Immunocytochemistry BPAECs were grown to confluence on fibronectin coated 25 mm round coverslips placed in six well Costar† medical grade polystyrene plates. The experimental design replicated previous experiments demonstrating synergistic augmentation of monolayer permeability (Wilson et al., 1998).
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Replicate experiments began with a 24 h pretreatment with MCTP (10 mg/ml; final concentration 0.031 mM) in 1 ml/ml dimethyformamide (DMF). Concurrently, control cells were treated with DMF. One hour prior to the end of the 24 h MCTP treatment, half of these coverslips were exposed to thrombin (1 mM). At the conclusion of the 24 h period cells on coverslips from all groups were fixed in 1% para-formaldehyde in PBS (PFM) for 10 min at room temperature, followed by two 5 min washes with PBS, permeabilized and blocked with appropriate normal serum in 0.1% Triton-X-100 in PBS for 5 min. Fixed, permeabilized cells were incubated with monoclonal antibodies to either pp125FAK (1:1000; Upstate Biotechnology, Lake Placid, NY), ß-catenin (1:1000) ZYMED Laboratories, So. San Francisco, CA), or with a polyclonal antibody to ZO-1 (1:150) (ZYMED Laboratories) in 0.1% BSA for 1 h. Fluorescene isothiocyanate (FITC) labeled secondary antibodies to mouse or goat IgG were used to label bound primaries followed by enhancement of label by anti-FITC IgG conjugated to ALEXA 568 dye according to the manufacturer’s directions (Molecular Probes, Eugene, OR). Background controls consisted of monolayers processed the same as the positive slides, but stained either with FITC-conjugated secondary antibodies or FITC-conjugated secondary plus the ALEXA anti-FITC dye. The cells were examined with an MRC-1024 krypton/argon laser confocal scanning microscope (Bio-Rad Laboratories, Hercules, CA) equipped with a 3 mm aperture and a 60 / oil immersion objective lens. Alexa-568 staining was observed using an excitation of 568 nm. Emissions were recorded in a series of 0.5 mm optical sections with a photomultiplier tube (PMT) and 585 nm long pass filter. Digital images were recorded using Lasersharp 3.2 acquisition and processing software (Bio-Rad). 2.4. Cell density/cell size Digitized images were also captured using an Olympus Provis fluorescent microscope equipped with 40 / objective lens and 568 /584 nm excitation filter (Chroma Technology, Brattleboro, VT)
from monolayers treated and stained as described above. These images were used to count the total number of cells in each of 12 high-powered microscopic fields per treatment group (four fields from each group fluorescently stained for adhesion molecule markers). A general linear model analysis of variance was used to determine statistical significance of treatment group effects on number of cells per field. Least squares difference (LSD) post hoc analysis was used to compare treatment groups (P B/0.05). Data Desk Statistical Software (Data Description, Inc., Ithaca, NY) was used for statistical calculations. 2.5. Western blotting Cells were grown to confluence in 75 cm2 tissue culture flasks (Corning). Treatment groups and concentrations of DMF, MCTP, Thrombin and MCTP/thrombin, were as previously described. Confluent cells were exposed for 24 h to MCTP (10 mg/ml; 0.031 mM in DMF, while control cells were treated with an equivalent volume of DMF (1 ml/ml). Cells were exposed for 1 h to thrombin (1 mM) alone or with MCTP pretreatment. Media was aspirated and cells were lysed immediately with 200 ml cold RIPA buffer (50 mM Tris /HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mg/ml each of aprotinin, leupeptin, pepstatin, 1 mM Na3VO4 1 mM NaF). Lysed cells were scraped from the bottom of the flask and homogenized on an iceslurry using an ultrasonicator (Sonicator† Ultrasonic Processor XL, Misonix, Inc.) for 15 /20 s. Protein determination was performed using the bicinchoninic acid assay (Pierce Laboratories, Rockford, IL). Protein (100 mg) from each treatment group was separated according to the sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protocol of Laemmli (1970). The running gel (7.5% T, 2.7% C or 11% T, 2.7% C) was overlaid with a 4% stacking gel and run at 4 8C on a Hoefer SE-600 model vertical tank chamber (Hoefer Scientific Instruments, San Francisco, CA) at 10 mA per gel for an average of 5.5 h. Gels were transferred onto 0.2 mm polyvinylidene difluoride (PVDF) membranes (BioRad Laboratories). The PVDF membrane was
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blocked with 3% non-fat milk (NFM; Bio-Rad Laboratories) and probed with ZO-1 (polyclonal rabbit, 2 mg/ml, Zymed Laboratories) or ß-catenin (mouse monoclonal, 1 mg/ml, Zymed Laboratories) by incubating with antibody overnight at 4 8C with gentle rocking. Membranes were developed with either alkaline phosphatase (AP) conjugated goat anti-rabbit or anti-mouse, respectively, according to manufacturer directions (Bio-Rad). Membrane proteins were visualized by incubating the membrane for 90 min at room temperature with an AP substrate kit according to the manufacture’s specifications (Bio-Rad Laboratories). Membranes probed with mouse monoclonal pp125FAK (1/1000, Transduction Labs, Lexington, KY) or rabbit polyclonal antibodies, N-terminal (1/1000) or C-terminal (1/200) pp125FAK (Santa Cruz Biotechnology, Santa Cruz, CA) followed the same protocol with the exception that horseradish peroxidase (HRP) conjugated secondary (1/10 000) anti-mouse or antirabbit antibodies (Amerscham Pharmacia Biotech, Piscatway, NJ) were employed. Blots were developed with the ECL/ detection system according to manufacturer’s specifications (Amersham Pharmacia). Membranes were digitally scanned using an Arcus II scanner (Agfa, Gevaert NV) and ADOBE PHOTOSHOP. Densitometric analysis of major gel bands was done with NIH image (ver. 1.62, National Institutes of Health) using unaltered images and gel electrophoresis macros included with the program. Gels from three replicate experiments for each probe were analyzed. Integrated areal band densities were compared between treatment groups with statistical analysis done by general linear models ANOVA to determine significance of treatment effects followed by LSD post hoc testing (P B/0.05) to compare treatment groups.
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significant decrease in cell density in the MCTP groups (Fig. 1). In cells fluorescently stained for FAK (Fig. 2), control cells were close spaced and had a stippled, irregular distribution of positive fluorescence that appeared to radiate in double or triple concentric rows from the center of the cells (Fig. 2a). Cells exhibited a uniform appearance throughout the entire field. Intercellular spaces were very narrow and cells were in close apposition to one another. The cell density range was 11/14 cells per 1.4 /104 mm2 area. MCTP treated cells stained for FAK had an overall decrease in staining. Some cells appeared to have fewer focal adhesions. Cells had an irregular, clustered staining distribution pattern within the cytoplasm of individual cells (Fig. 2b). Some cells had a linear distribution of FAK, while other cells appeared to have an eccentric ‘starburst’ staining pattern. The cell density was lower than that of the other groups (B/7 cells per 1.4 /l04 mm2 area) and intercellular gaps were variable in size. Thrombin treated cells had a staining distribution pattern within the cytoplasm that gave a corona-like appearance to the affected cells (Fig. 2c). The distribution of FAK was around the periphery of
3. Results 3.1. Immunocytochemistry Cells from cultures treated with MCTP with or without thrombin were larger than those in control or thrombin treated cultures. This resulted in a
Fig. 1. Graph compares the average number of cells in 40 / high-powered microscopic fields for (vehicle) controls, MCTP treated, thrombin treated, and MCTP/thrombin treated BPAEC monolayers. Bars represent standard errors. 1, Significant difference from control and thrombin treated cells; 1, 2, significant difference from control and thrombin treated cells and also significantly different from MCTP treated cells.
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Fig. 2. Thru-view of confocal z-series of BPAEC monolayers probed fluorescently with anti-FAK antibody in vehicle control (a), MCTP treated (b), thrombin treated (c), and dual MCTP/thrombin treated (d), cells. Photomicrographs were taken at the same magnification (bar /25 mm). While thrombin treatment caused rearrangement of FAK to a peripheral radiating corona, MCTP treated cells had fewer and disorganized focal adhesions. Dually treated cells have more disorderly peripheral aggregates of FAK.
the cell in a single row outlining cell boundaries. Cell density was similar to that of control group (10 /14 cells per 1.4 /l04 mm2 area). Gaps were uniformly dispersed and consistently sized. The cells first treated with MCTP and subsequently with thrombin had a combination of features present in cells treated with MCTP and thrombin separately (Fig. 2d). The fluorescent staining showed an open corona-like or starburst staining
pattern reminiscent of the thrombin treated group, with irregular eccentric aggregates of stellar projections of positive staining in the cytoplasm of affected cells similar to the MCTP treated cells. Scattered cells had a more linear staining pattern through the cytoplasm. Cell density was intermediate between the MCTP cells and the thrombin treated cells (8 /9 cells per 1.4 /104 mm2 area). There was more variability in gap size and
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distribution than in the thrombin treated monolayers and less variability than in the MCTP treated monolayers. The effects of MCTP and thrombin treatment on ZO-1 staining were examined in BPAEC monolayers probed with fluorescently labeled polyclonal anti-ZO-1 antibody. Control cells stained with anti-ZO-1 antibody showed a thin band of fluorescence around the periphery of the membrane in adjacent cells in a pattern typical for this protein in normal endothelial monolayers (Fig. 3a). In areas where three cells adjoined, there was a slight thickening or infolding of the stained material. There were occasional gaps between cells. Compared with controls, MCTP lacked the thickening and infolding at tri-cellular corners. Cells were larger and the staining sharper and thinner. There were scattered gaps between cells, sometimes evidenced by narrow breaks in ZO-1 staining (Fig. 3b). Thrombin treatment of BPAEC monolayers had a very different ZO-1 staining pattern characterized by an overall granular thickening of the entire cell junctional membrane (Fig. 3c) instead of a distinct narrow band of fluorescence around the perimeter of the cells typical of the ZO-1 staining pattern seen in control cultures. Cells treated with both MCTP/thrombin were large, similar to MCTP treated cells, with slight thickening of the peripheral band of ZO-1 staining caused by an accordion like infolding of the membrane around the entire periphery (Fig. 3d). Gaps between cells were variably sized. BPAEC monolayers were also examined for the effects of MCTP and thrombin treatment on the adherens junction linker protein, ß-catenin, labeled with FITC-conjugated anti-monoclonal antibody. Staining of control cells showed well demarcated cell borders with a relatively thick fibrillar network of positive staining which was thicker at some tri-cellular junctions (Fig. 4a). MCTP treated monolayers had crisp, markedly thinner, sharper staining membranes with mild folding of membranes in areas of tri-cellular junctions (Fig. 4b). Thrombin treated monolayers had slightly less intense staining of ß-catenin than the controls and MCTP treated cells. The cell /cell junctions of the thrombin treated cultures had sharply irregular infoldings of the thinned catenin
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network (Fig. 4c). The MCTP/thrombin treated monolayers had both attenuated catenin networks as well as prominent infoldings. Intercellular gaps lacked membrane catenin staining (Fig. 4d). 3.2. Western blotting The effects of MCTP and thrombin treatment on BPAEC junctional proteins were compared with western blots. Membranes probed for FAK showed differences in band patterns between the different treatment groups and vehicle control (Fig. 5). Membranes probed with a mouse monoclonal antibody for FAK had a prominent band at approximately 125 kDa consistent with the molecular weight of FAK in lysates from control cells. This band was also present in MCTP treated cells but was significantly decreased in lysates from both thrombin and dual treated cultures (34 and 15% of control, respectively). Thrombin treatment was also associated with an increased intensity in a band at approximately 100 kDa (Fig. 5). While the monoclonal antibody did not recognize lower molecular weight proteins, polyclonal antibodies raised to the C or N-terminal portions of FAK had distinctive smaller molecular weight bands (Fig. 6). Thrombin treated cultures had intense C-terminal fragments at 100, 90 and 32 kDa as well as an intense N-terminal fragment at 90 kDa. MCTP treated cultures had prominent 100 and 90 kDa Cterminal fragments as well as an additional fragment at 19 kDa that was not present in control or thrombin only cultures. Probing with the Nterminal antibody showed that both the 125 kDa whole protein and 100 kDa fragment had decreased intensity in MCTP treated cultures compared with controls. Conversely, the N-terminal antibody recognized a more intense 100 kDa band in thrombin treated cultures. As shown in Fig. 7, major bands staining with anti-ZO-1 antibody spanned from 210 to 110 kDa in both MCTP treated and control cells. In the thrombin and MCTP/thrombin treated lanes, the larger molecular weight bands were significantly reduced (4 and 5% of control, respectively) but there were bands in the 160/110 kDa range. Two additional bands at 84 and 60 kDa was also present in lanes from thrombin treated cells.
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Fig. 3. Thru-view of confocal z-series of ZO-1 junctional protein in BPAEC monolayers probed fluorescently with anti-ZO-1 antibody subsequent to exposure to vehicle control (a), MCTP treatment (b), thrombin treatment (c), and dual MCTP/thrombin treatment (d), (bar /25 mm). MCTP treated cells have thinner but distinct discontinuous peripheral bands of ZO-1 (example delimited by arrowheads) while thrombin treatment resulted in a thicker more dispersed expression of ZO-1 (example delimited by arrowheads). Dual treatment attenuated the formation of the thicker peripheral bands that were evident in cells treated with thrombin alone.
Western blots of whole cell lysates from cells treated with MCTP, thrombin and MCTP/thrombin probed for ß-catenin showed a band at 96 kDa that was not significantly altered in any treatment group compared with controls (Fig. 8).
4. Discussion Previous studies in this laboratory have shown a synergistic effect of dual MCTP/thrombin treatment on albumin transudation in endothelial cell
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Fig. 4. Thru-view of confocal z-series of BPAEC monolayers probed fluorescently with antibody to ß-catenin after treatment with vehicle control (a), MCTP (b), thrombin (c), and MCTP/thrombin (d), (bar /25 mm). MCTP treated cells had thin but still well demarcated expression of catenin at cell borders compared with controls. Peripheral bands of catenin had less intensity and prominent infoldings in thrombin treated cells with similar changes in dual treated cells. Larger gaps in dual treated cells (examples delimited by arrowheads) were evident because they lacked catenin staining.
monolayers (Wilson et al., 1998). The current experiments show that both MCTP and thrombin treatment of BPAEC monolayers alter the location and structure of intracellular adhesion molecules. MCTP treatment changed the cellular distribution of FAK in BPAEC monolayers. While MCTP treatment did not alter the location or apparent
protein structure of ZO-1 or ß-catenin (compared with controls); it did induce electrophoretically recognizable changes in FAK. Thrombin changed the morphologic distribution of ZO-1, FAK and ß-catenin in a pattern consistent with cellular contraction. Thrombin treatment also altered electrophoretic migration of both ZO-1 and
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Fig. 5. Western blot of membrane probed with monoclonal antibody for FAK. Note band at 90 kDa in MCTP treated lane and the lack of the higher weight bands in the lanes containing lysates from thrombin treated cells.
FAK in a manner consistent with cleavage of these proteins. Visualization and localization of adhesion molecules via immunofluorescence showed that MCTP interfered with thrombin induced retraction of occludins junctions and reorganization of focal adhesion contacts. MCTP treatment also increased cell size as estimated by cell density. FAK is critical in signaling many adhesion related functions in the cell, including motility, replication, permeability and survival. Forces
modulating endothelial barrier function and permeability that act via FAK may occur via either an increase in phosphorylation or a decrease in phosphorylation in an agonist specific way (Schaphorst et al., 1997). Thrombin has been shown to increase FAK phosphorylation as it increases endothelial barrier permeability (Schaphorst et al., 1997). While antibodies in the present study do not discriminate between FAK phosphorylation states, fragmentation patterns of FAK differed between MCTP and thrombin treatments. While the major 100 kDa fragment induced in MCTP treated cultures appeared to represent a large C-terminal fragment, thrombin treated cultures had a large N-terminal fragment as well. The presence of 100, 90 and 19 kDa fragments of FAK in MCTP treated cultures is similar to previous descriptions of caspase induced changes of FAK in apoptosis (van de Water et al., 1999). This is consistent with our previous work demonstrating progressive apoptosis in MCTP treated cells (Thomas et al., 1998a). The differing pattern seen in thrombin treated cells suggests an alternative
Fig. 6. Western blots of endothelial cell lysates probed with antibodies for either c-terminal or n-terminal portions of FAK. C-terminal fragments at 32 kDa are present in all treatment groups and a fragment at 19 kDa occurs in MCTP treated cells. N-terminal antibodies showed almost no full molecular weight protein in thrombin treated cultures and demonstrate a thrombin specific N-terminal fragment at 90 kDa.
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Fig. 7. Western blot of membrane probed with antibody to ZO-1. Lysates from thrombin treated groups have lost the bands corresponding to the complete protein and have smaller molecular weight fragments not present in control cells.
Fig. 8. Western blot of membrane probed with antibody to ßcatenin adherens junction protein marker showed no change in protein migration in thrombin treated cells compared with controls and MCTP treated groups.
pathway for loss of FAK protein. Whether the apparent FAK proteolysis represents thrombinmediated catalysis or signal transduction mediated degradation of FAK through alternate cellular pathways is the subject of ongoing studies. While some loss of FAK expression was subjectively evident by confocal microscopy in MCTP treated cultures, the principle morphologic change
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was a disorderly arrangement of focal adhesion complexes in MCTP treated cells and an interference with their circumferential rearrangement induced by thrombin in MCTP pretreated cultures. Since focal adhesion complexes were readily evident in thrombin treated cells using anti-FAK antibodies, the dissolution of FAK seen by Western blotting must not result in its dislocation from adhesion complexes. The presence of apoptosis associated FAK fragments in MCTP treated cells suggests a role for this process in the mechanism of MCTP induced microvascular permeability. Whether this effect is due to MCTP direct crosslinking of proteins in the focal adhesion complex or, alternatively, is due to secondary effects subsequent to DNA cross-linking is not clear. It is possible that MCTP derangements of the focal adhesion complex, whether direct or indirect, may contribute to the apoptosis seen in MCTP treated cells and exacerbation of thrombin effects on permeability. Zonula occludens are important in maintaining endothelial barrier function, integrity and cell-tocell contact. ZO-1 expression has been shown to be affected by a variety of factors including, but not limited to, diabetes, wound healing and repair, and changes in vascular shear stress (Schnittler, 1998). In these experiments, pretreatment of cells with MCTP appeared to have little effect on the expression of ZO-1. While the distribution of ZO-1 in a linear pattern at the periphery of the cells was not changed by MCTP treatment, there was an evident discontinuity in this barrier that was accentuated by thrombin stimulus. The decreased expression of ZO-1 by Western blot analysis in thrombin treated cultures suggests that this treatment leads to its degradation. Current models of occludens junctions suggest that a permeability signal results in phosphorylation of occludin with subsequent dissociation from ZO-1. Occludin is then targeted for degradation by metalloproteases while ZO-1 remains attached to actin (Wachtel et al., 1999). The redistribution of ZO-1 in thrombin stimulated cultures is suggestive of actin contractility withdrawing ZO-1 from the cell periphery and would be consistent with its dissociation from occludin. The breakdown of ZO-1 evident on western blots of thrombin treated
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cells suggests that proteolysis of this protein also occurs. This has not been previously described for the thrombin signal transduction pathway and the mechanisms are currently under investigation. While MCTP treatment did not alter either expression or location of ZO-1, the redistribution of ZO-1 by thrombin was inhibited in MCTP pretreated cells. This suggests that there may be functional significance to the MCTP-actin interactions we have previously described (Wilson et al., 1998). Although adherens junctions are not the primary molecule for maintaining tight contact between cells, they are, nonetheless, important in maintaining endothelial barrier integrity and regulating intercellular gap formation (Lum and Malik, 1996). Vascular endothelial-cadherin and associated catenins have been reported to show marked alterations in distribution when treated with agents that increase vascular permeability and gap formation (Rabiet et al., 1996). In contrast to ZO-1, ß-catenin remains attached to the junctional protein VE cadherin when the endothelial cell is stimulated by thrombin. Signal transduction leads to phosphorylation of ß-catenin and its dissociation from a-catenin, which remains attached to the actin cytoskeleton. In the present study, neither the location or expression of ßcatenin by Western blot showed significant responses to thrombin or MCTP treatment. The only morphologic change evident was loss of ßcatenin staining in membranes adjacent to intercellular gaps. This suggests either focal downregulation of junctional protein or that the loss of actin tethering allows movement of the cadherin/ catenin complex within the membrane to form non-adherent spaces. MCTP treatment alone can cause derangement or perturbation of function in the actin cytoskeleton. Recent evidence from our laboratory has shown that MCTP binds to actin and the actinbinding protein, tropomyosin (Lame´ et al., 2000). Tropomyosin interacts with actin in formation of stress fibers (Adam et al., 2000). We have previously demonstrated that stress fibers are altered by MCTP treatment and the formation of stress fibers in response to thrombin is inhibited by MCTP pretreatment (Wilson et al., 1998). Actin is
associated and interacts with membrane adhesion proteins. The present study suggests that the cytoskeletal effects of MCTP may be compounded by alterations in intercellular junctional proteins and/or their interaction with actin. Vascular insult resulting from MCT toxicity has a bi-phasic development. Early biochemical changes in endothelial cells are subtle and increase in severity over time. Lesions occur early as an inflammatory phase and latently as effects of DNA and protein binding (Coulombe et al., 1999, 1992; Lame´ et al., 2000) alter endothelial responses that drive remodeling of the vascular wall. Early inflammatory lesions include the presence of platelet thrombi in small vessels (Lalich et al., 1977; Turner and Lalich, 1965) and microvascular leak in arteriolar walls (unpublished observations). Activation of platelets would result in thrombin release and activation of the coagulation cascade. While normal permeability responses to thrombin are relatively transient, MCTP effects persisted for 24 h in this study suggesting a mechanism by which thrombin induced permeability could be prolonged in vivo. Failure to maintain intercellular junctions may also result in vascular leak at abnormal sites in the vascular tree. Persistent leak of large molecular weight, potentially biologically active, serum components provides a plausible mechanism for the interactive effects of MCTP and thrombin on adhesion proteins to stimulate vascular remodeling.
Acknowledgements Dr Taylor has been a recipient of an NIEHS Training Grant (ES07055) and an NIH Supplemental award to NIH Grant HL4841. This work was supported by NIH Grant HL48411.
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Steller, H., 1995. Mechanisms and genes of cellular suicide. Science 267, 1445 /1449. Taylor, D.W., Wilson, D.W., Lame´, M.W., Dunston, S.D., Jones, A.D., Segall, H.J., 1997. Comparative cytotoxicity of monocrotaline and its metabolites in cultured pulmonary artery endothelial cells. Toxicol. Appl. Pharmacol. 143, 196 /204. Telo`, P., Lostaglio, S., Dejana, E., 1997. Structure of intercellular junctions in the endothelium. Therapie 52, 395 /398. Thomas, H.C., Lame´, M.W., Wilson, D.W., Segall, H.J., 1996. Cell cycle alterations associated with covalent binding of monocrotaline pyrrole to pulmonary artery endothelial cell DNA. Toxicol. Appl. Pharmacol. 141, 319 /329. Thomas, H.C., Lame´, M.W., Dunston, S.K., Segall, H.J., Wilson, D.W., 1998a. Monocrotaline pyrrole induces apoptosis in pulmonary artery endothelial cells. Toxicol. Appl. Pharmacol. 151, 236 /244. Thomas, H.C., Lame´, M.W., Morin, D., Wilson, D.W., Segall, H.J., 1998b. Prolonged cell-cycle arrest associated with altered cdc2 kinase in monocrotaline pyrrole-treated pulmonary artery endothelial cells. Am. J. Respir. Cell Mol. Biol. 19, 129 /142. Thompson, C.B., 1995. Apoptosis in the pathogenesis and treatment of disease. Science 267, 1456 /1462. Turner, J.H., Lalich, I.J., 1965. Experimental cor pulmonale in the rat. Arch. Pathol. 79, 409 /410. Ukropec, J.A., Hollinger, M.K., Salva, S.M., Woolkalis, M.J., 2000. SHP2 association with VE-cadherin complexes in
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Effects of monocrotaline pyrrole and thrombin on pulmonary endothelial cell junction and matrix adhesion proteins Debra W. Taylor a, Michael W. Lame´ b, Lynn S. Nakayama b, H.J. Segall b, Dennis W. Wilson a, a
Departments of Veterinary, Pathology, Microbiology and Immunology, University of California-Davis, 2150 Haring Hall, Davis, CA 95616, USA b Department of Molecular Biosciences, University of California-Davis, Davis, CA 95616, USA Received 14 June 2002; received in revised form 26 August 2002; accepted 24 September 2002
Abstract Previous work in our laboratory has shown that monocrotaline pyrrole (MCTP) interacts with actin and potentiates thrombin-mediated endothelial barrier permeability through increasing the overall surface area of intercellular gaps. To better characterize endothelial barrier leak in this model, we examined the effects of MCTP and thrombin on the localization and structure of three adhesion associated proteins that directly or indirectly interact with actin in regulating barrier function: cell /cell occludens junction molecule (ZO-1), the cell /cell adherens junction linker, ßcatenin, and the cell /matrix intermediary signaling protein, focal adhesion kinase (FAK). Immunohistochemistry demonstrated that thrombin treatment resulted in radial reorganization of focal adhesions and broader distribution of adherens and occludins junctions at the cell border suggestive of membrane stretching in contracture. MCTP pretreatment resulted in fewer and more disorganized focal adhesions and marked thinning of occludins and adherens junctions. MCTP pretreatment also interfered with thrombin stimulated junctional reorganization. Western blot analysis showed thrombin stimulated catalysis of ZO-1 and FAK while MCTP pretreatment resulted in FAK fragmentation similar to previous reports for apoptosis. We conclude that both MCTP and thrombin alter critical endothelial cell adhesion molecules and this may be an underlying mechanism for the potentiating effect MCTP has on thrombin induced vascular permeability in vitro. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Monocrotaline pyrrole; Thrombin; Pulmonary hypertension; Adhesion molecules; ZO-1; ß-Catenin; pp125FAK; Vascular permeability; Endothelial cells
1. Introduction
Corresponding author. Tel.: /1-530-752-0158; fax: /1530-752-3349 E-mail address: [email protected] (D.W. Wilson).
Monocrotaline (MCT), or its metabolite monocrotaline pyrrole (MCTP), causes delayed, progressive pulmonary hypertension in Sprague / Dawley rats (Mattocks, 1986; Pan et al., 1993;
0300-483X/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 2 ) 0 0 5 8 2 - 6
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Reindel et al., 1990). Pulmonary vascular changes have been reported to begin in less than 24 h and possibly as early as 4 h post intravenous injection. Changes are manifested as a mild sub-intimal edema in vessels of the interalveolar septa and walls of alveolar ducts (Roth and Reindel, 1991; Valdivia et al., 1967). These changes progress to subsequent proliferative vasculitis, cor pulmonale, and pulmonary hypertension (Merkow and Kleinerman, 1966; Meyrick et al., 1980; Reindel et al., 1990; Rosenberg and Rabinovitch, 1988; Roth et al., 1981; Roth and Reindel, 1991). We have shown that MCTP, but not MCT, nor the glutathione metabolite GSH-DHP1, causes a delayed onset of progressive changes in cultured monolayers of bovine pulmonary artery endothelial cells (BPAECs, Taylor et al., 1997). These lesions include megalocytosis, increased membrane permeability and altered function (indicated by increased extracellular LDH and increased prostacyclin production) similar to those reported in vivo (Reindel and Roth, 1991; Taylor et al., 1997). Other effects of MCTP include cell-cycle derangements and induction of apoptosis in BPAEC monolayers (Thomas et al., 1998a,b, 1996). Apoptosis has also been reported to occur in the pulmonary arteries of MCT treated rats (Jones and Rabinovitch, 1996). Vascular permeability has been cited as one of the most important early lesions preceding the onset of MCT induced pulmonary hypertension (Meyrick, 1990). Endothelial cells play a critical role in maintaining physical and functional barrier integrity, particularly in smaller vessels where they comprise a larger portion of the vessel wall. Recent acute in vivo studies in our laboratory show platelet accumulation and degranulation in the pulmonary microvasculature as early as 4 h post MCTP treatment. These changes were associated with loss of endothelial cell junction integrity and matrix adhesion in association with microvascular leak in arterioles and venules (unpublished observations). Endothelial cells form an effective polarized, yet dynamic barrier to the movement of fluids and 1
6,7-Dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine.
solutes between the blood compartment and extracellular matrix (ECM) through cell /cell junctions and cell-to-ECM attachments (via focal adhesion plaques, Barry et al., 1995; Lum and Malik, 1996). Modulation of endothelial barrier function related to development of intercellular gaps can be regulated through receptor mediated signaling pathways. One pathway acts through phosphorylation of linking proteins at cell/cell junctions (Garcia et al., 1996) resulting in dissociation of junction protein complexes and degradation of linking proteins (Kevil et al., 2000; Ukropec et al., 2000; Wachtel et al., 1999). A second pathway occurs through cytoskeletal (actin/myosin) contraction of endothelial cells (Lum and Malik, 1994). Tight junctions (zonula occludens), are a complex network of transmembrane proteins (Dejana et al., 1995; Dejana and Del Maschio, 1995; Telo` et al., 1997) that occur at the apical surface of epithelial and endothelial cells. ZO-1 is a 225 kDa peripheral membrane protein that links to the intercellular adhesion protein occludin (Wachtel et al., 1999; Willott et al., 1992). Adherens junctions also participate in maintaining barrier integrity in endothelial cells. Adherns junctions are composed of the intercellular adhesion molecule VE cadherin which is linked to the cytoskeleton through intermediate proteins in the catenin family (Dejana, 1997; Ukropec et al., 2000). Focal adhesion plaques are integrin based transmembrane adhesion structures located on the basal aspect of the cell where they attach to the basement membrane and underlying matrix. These plaques mediate attachment of the cytoskeleton to the ECM and may participate in signaling that affects cytoskeleton organization. This process is critical to modulation of endothelial permeability, particularly in the thrombin model (Schaphorst et al., 1997). One of the signaling proteins in the cytoplasmic domain of focal adhesion plaques is a tyrosine kinase, focal adhesion kinase (FAK; Gilmore and Romer, 1996). FAK signaling has been shown to play a role in initiating apoptosis in anchorage-dependent cells that are prevented from forming normal attachments to a substrate (van de Water et al., 1999; Xiong and Parsons, 1997). Loss of survival signals
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through ECM components such as fibronectin may signal apoptosis in normal cells (Ilic et al., 1997; Steller, 1995; Thompson, 1995) by signal transduction through FAK (Ilic et al., 1998). Growth factor deprivation and caspase induction result in fragmentation of FAK into characteristic subunits (Levkau et al., 1998). Previously, we characterized an in vitro model of endothelial monolayer permeability using thrombin-mediated stimulus in MCTP pretreated BPAECs. This model is predicated on in vivo evidence of persistent microvascular leak and platelet accumulation in MCT treated rats. In our in vitro model, there is no morphologic evidence of cell death or loss from the monolayer within the first 24 h of exposure. Although there is a small percentage (ca. 5 vs. 2.2% in controls) of cells in the early stages of apoptosis (Thomas et al., 1998a), the MCTP pretreated monolayers appear morphologically intact. Pretreatment of BPAEC monolayers with MCTP potentiates thrombin induced monolayer permeability as measured by transudation of Evans-Blue albumin across monolayers grown on semi-permeable membrane inserts (Wilson et al., 1998). MCTP treatment results in changes in stress fiber arrangement and apparent derangement of actin polymerization (Wilson et al., 1998 and unpublished observations). The mechanism of monolayer permeability appears to relate to regulation of intercellular junctions since MCTP pretreated monolayers develop a markedly increased surface area of intercellular space in response to thrombin stimulation. The combination of in vivo and in vitro evidence suggests a hypothesis that MCTP treatment results in endothelial cell responses leading to platelet activation and a stimulus for microvascular permeability that is abnormally prolonged by persistent alteration in endothelial cell fluid barrier maintenance. The purpose of the present study was to characterize the effects of MCTP pretreatment on thrombin induced changes in cell /cell and cell /matrix adhesions. Our objectives were (1) to determine whether MCTP treatment had a morphological effect on organization of adhesion structures involved in cell /cell contact in confluent BPAEC monolayers; (2) to determine whether thrombin or MCTP treatment altered the expres-
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sion or composition of membrane adhesion proteins; and (3) to evaluate the potential interaction of MCTP treatment with thrombin induced changes in adhesion molecule reorganization. We examined biochemical characteristics and localization of marker proteins from each type of cell adhesion zone: ZO-1 for occludens junctions, ß-catenin for adherens junctions and FAK for ECM attachments in cells treated with MCTP, thrombin, dually with MCTP/thrombin, or a vehicle as a control.
2. Methods 2.1. Cell isolation, culture, characterization Endothelial cells were isolated non-enzymatically (Ryan et al., 1978) from bovine pulmonary arteries obtained from an abattoir and characterized as previously described (Taylor et al., 1997). Pure cultures of BPAECs were scraped from culture dishes and seeded into tissue culture flasks, expanded, then collected, frozen and stored in liquid N2 until thawed for use. Passages 11 /16 were used for these experiments. 2.2. Synthesis of MCTP MCTP was synthesized from MCT according to the method of Mattocks et al. (1989) and recrystallized from anhydrous ethyl ether as previously described (Taylor et al., 1997). Using fast atom bombardment mass spectrometry the conversion of MCT to MCTP was found to be complete; daughter spectra contained ions characteristic of the pyrrole (Lame´ et al., 2000; Pan et al., 1993). MCTP was stored in N ,N -dimethylformamide at /80 8C until just prior to use. 2.3. Immunocytochemistry BPAECs were grown to confluence on fibronectin coated 25 mm round coverslips placed in six well Costar† medical grade polystyrene plates. The experimental design replicated previous experiments demonstrating synergistic augmentation of monolayer permeability (Wilson et al., 1998).
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Replicate experiments began with a 24 h pretreatment with MCTP (10 mg/ml; final concentration 0.031 mM) in 1 ml/ml dimethyformamide (DMF). Concurrently, control cells were treated with DMF. One hour prior to the end of the 24 h MCTP treatment, half of these coverslips were exposed to thrombin (1 mM). At the conclusion of the 24 h period cells on coverslips from all groups were fixed in 1% para-formaldehyde in PBS (PFM) for 10 min at room temperature, followed by two 5 min washes with PBS, permeabilized and blocked with appropriate normal serum in 0.1% Triton-X-100 in PBS for 5 min. Fixed, permeabilized cells were incubated with monoclonal antibodies to either pp125FAK (1:1000; Upstate Biotechnology, Lake Placid, NY), ß-catenin (1:1000) ZYMED Laboratories, So. San Francisco, CA), or with a polyclonal antibody to ZO-1 (1:150) (ZYMED Laboratories) in 0.1% BSA for 1 h. Fluorescene isothiocyanate (FITC) labeled secondary antibodies to mouse or goat IgG were used to label bound primaries followed by enhancement of label by anti-FITC IgG conjugated to ALEXA 568 dye according to the manufacturer’s directions (Molecular Probes, Eugene, OR). Background controls consisted of monolayers processed the same as the positive slides, but stained either with FITC-conjugated secondary antibodies or FITC-conjugated secondary plus the ALEXA anti-FITC dye. The cells were examined with an MRC-1024 krypton/argon laser confocal scanning microscope (Bio-Rad Laboratories, Hercules, CA) equipped with a 3 mm aperture and a 60 / oil immersion objective lens. Alexa-568 staining was observed using an excitation of 568 nm. Emissions were recorded in a series of 0.5 mm optical sections with a photomultiplier tube (PMT) and 585 nm long pass filter. Digital images were recorded using Lasersharp 3.2 acquisition and processing software (Bio-Rad). 2.4. Cell density/cell size Digitized images were also captured using an Olympus Provis fluorescent microscope equipped with 40 / objective lens and 568 /584 nm excitation filter (Chroma Technology, Brattleboro, VT)
from monolayers treated and stained as described above. These images were used to count the total number of cells in each of 12 high-powered microscopic fields per treatment group (four fields from each group fluorescently stained for adhesion molecule markers). A general linear model analysis of variance was used to determine statistical significance of treatment group effects on number of cells per field. Least squares difference (LSD) post hoc analysis was used to compare treatment groups (P B/0.05). Data Desk Statistical Software (Data Description, Inc., Ithaca, NY) was used for statistical calculations. 2.5. Western blotting Cells were grown to confluence in 75 cm2 tissue culture flasks (Corning). Treatment groups and concentrations of DMF, MCTP, Thrombin and MCTP/thrombin, were as previously described. Confluent cells were exposed for 24 h to MCTP (10 mg/ml; 0.031 mM in DMF, while control cells were treated with an equivalent volume of DMF (1 ml/ml). Cells were exposed for 1 h to thrombin (1 mM) alone or with MCTP pretreatment. Media was aspirated and cells were lysed immediately with 200 ml cold RIPA buffer (50 mM Tris /HCl, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mg/ml each of aprotinin, leupeptin, pepstatin, 1 mM Na3VO4 1 mM NaF). Lysed cells were scraped from the bottom of the flask and homogenized on an iceslurry using an ultrasonicator (Sonicator† Ultrasonic Processor XL, Misonix, Inc.) for 15 /20 s. Protein determination was performed using the bicinchoninic acid assay (Pierce Laboratories, Rockford, IL). Protein (100 mg) from each treatment group was separated according to the sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protocol of Laemmli (1970). The running gel (7.5% T, 2.7% C or 11% T, 2.7% C) was overlaid with a 4% stacking gel and run at 4 8C on a Hoefer SE-600 model vertical tank chamber (Hoefer Scientific Instruments, San Francisco, CA) at 10 mA per gel for an average of 5.5 h. Gels were transferred onto 0.2 mm polyvinylidene difluoride (PVDF) membranes (BioRad Laboratories). The PVDF membrane was
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blocked with 3% non-fat milk (NFM; Bio-Rad Laboratories) and probed with ZO-1 (polyclonal rabbit, 2 mg/ml, Zymed Laboratories) or ß-catenin (mouse monoclonal, 1 mg/ml, Zymed Laboratories) by incubating with antibody overnight at 4 8C with gentle rocking. Membranes were developed with either alkaline phosphatase (AP) conjugated goat anti-rabbit or anti-mouse, respectively, according to manufacturer directions (Bio-Rad). Membrane proteins were visualized by incubating the membrane for 90 min at room temperature with an AP substrate kit according to the manufacture’s specifications (Bio-Rad Laboratories). Membranes probed with mouse monoclonal pp125FAK (1/1000, Transduction Labs, Lexington, KY) or rabbit polyclonal antibodies, N-terminal (1/1000) or C-terminal (1/200) pp125FAK (Santa Cruz Biotechnology, Santa Cruz, CA) followed the same protocol with the exception that horseradish peroxidase (HRP) conjugated secondary (1/10 000) anti-mouse or antirabbit antibodies (Amerscham Pharmacia Biotech, Piscatway, NJ) were employed. Blots were developed with the ECL/ detection system according to manufacturer’s specifications (Amersham Pharmacia). Membranes were digitally scanned using an Arcus II scanner (Agfa, Gevaert NV) and ADOBE PHOTOSHOP. Densitometric analysis of major gel bands was done with NIH image (ver. 1.62, National Institutes of Health) using unaltered images and gel electrophoresis macros included with the program. Gels from three replicate experiments for each probe were analyzed. Integrated areal band densities were compared between treatment groups with statistical analysis done by general linear models ANOVA to determine significance of treatment effects followed by LSD post hoc testing (P B/0.05) to compare treatment groups.
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significant decrease in cell density in the MCTP groups (Fig. 1). In cells fluorescently stained for FAK (Fig. 2), control cells were close spaced and had a stippled, irregular distribution of positive fluorescence that appeared to radiate in double or triple concentric rows from the center of the cells (Fig. 2a). Cells exhibited a uniform appearance throughout the entire field. Intercellular spaces were very narrow and cells were in close apposition to one another. The cell density range was 11/14 cells per 1.4 /104 mm2 area. MCTP treated cells stained for FAK had an overall decrease in staining. Some cells appeared to have fewer focal adhesions. Cells had an irregular, clustered staining distribution pattern within the cytoplasm of individual cells (Fig. 2b). Some cells had a linear distribution of FAK, while other cells appeared to have an eccentric ‘starburst’ staining pattern. The cell density was lower than that of the other groups (B/7 cells per 1.4 /l04 mm2 area) and intercellular gaps were variable in size. Thrombin treated cells had a staining distribution pattern within the cytoplasm that gave a corona-like appearance to the affected cells (Fig. 2c). The distribution of FAK was around the periphery of
3. Results 3.1. Immunocytochemistry Cells from cultures treated with MCTP with or without thrombin were larger than those in control or thrombin treated cultures. This resulted in a
Fig. 1. Graph compares the average number of cells in 40 / high-powered microscopic fields for (vehicle) controls, MCTP treated, thrombin treated, and MCTP/thrombin treated BPAEC monolayers. Bars represent standard errors. 1, Significant difference from control and thrombin treated cells; 1, 2, significant difference from control and thrombin treated cells and also significantly different from MCTP treated cells.
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Fig. 2. Thru-view of confocal z-series of BPAEC monolayers probed fluorescently with anti-FAK antibody in vehicle control (a), MCTP treated (b), thrombin treated (c), and dual MCTP/thrombin treated (d), cells. Photomicrographs were taken at the same magnification (bar /25 mm). While thrombin treatment caused rearrangement of FAK to a peripheral radiating corona, MCTP treated cells had fewer and disorganized focal adhesions. Dually treated cells have more disorderly peripheral aggregates of FAK.
the cell in a single row outlining cell boundaries. Cell density was similar to that of control group (10 /14 cells per 1.4 /l04 mm2 area). Gaps were uniformly dispersed and consistently sized. The cells first treated with MCTP and subsequently with thrombin had a combination of features present in cells treated with MCTP and thrombin separately (Fig. 2d). The fluorescent staining showed an open corona-like or starburst staining
pattern reminiscent of the thrombin treated group, with irregular eccentric aggregates of stellar projections of positive staining in the cytoplasm of affected cells similar to the MCTP treated cells. Scattered cells had a more linear staining pattern through the cytoplasm. Cell density was intermediate between the MCTP cells and the thrombin treated cells (8 /9 cells per 1.4 /104 mm2 area). There was more variability in gap size and
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distribution than in the thrombin treated monolayers and less variability than in the MCTP treated monolayers. The effects of MCTP and thrombin treatment on ZO-1 staining were examined in BPAEC monolayers probed with fluorescently labeled polyclonal anti-ZO-1 antibody. Control cells stained with anti-ZO-1 antibody showed a thin band of fluorescence around the periphery of the membrane in adjacent cells in a pattern typical for this protein in normal endothelial monolayers (Fig. 3a). In areas where three cells adjoined, there was a slight thickening or infolding of the stained material. There were occasional gaps between cells. Compared with controls, MCTP lacked the thickening and infolding at tri-cellular corners. Cells were larger and the staining sharper and thinner. There were scattered gaps between cells, sometimes evidenced by narrow breaks in ZO-1 staining (Fig. 3b). Thrombin treatment of BPAEC monolayers had a very different ZO-1 staining pattern characterized by an overall granular thickening of the entire cell junctional membrane (Fig. 3c) instead of a distinct narrow band of fluorescence around the perimeter of the cells typical of the ZO-1 staining pattern seen in control cultures. Cells treated with both MCTP/thrombin were large, similar to MCTP treated cells, with slight thickening of the peripheral band of ZO-1 staining caused by an accordion like infolding of the membrane around the entire periphery (Fig. 3d). Gaps between cells were variably sized. BPAEC monolayers were also examined for the effects of MCTP and thrombin treatment on the adherens junction linker protein, ß-catenin, labeled with FITC-conjugated anti-monoclonal antibody. Staining of control cells showed well demarcated cell borders with a relatively thick fibrillar network of positive staining which was thicker at some tri-cellular junctions (Fig. 4a). MCTP treated monolayers had crisp, markedly thinner, sharper staining membranes with mild folding of membranes in areas of tri-cellular junctions (Fig. 4b). Thrombin treated monolayers had slightly less intense staining of ß-catenin than the controls and MCTP treated cells. The cell /cell junctions of the thrombin treated cultures had sharply irregular infoldings of the thinned catenin
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network (Fig. 4c). The MCTP/thrombin treated monolayers had both attenuated catenin networks as well as prominent infoldings. Intercellular gaps lacked membrane catenin staining (Fig. 4d). 3.2. Western blotting The effects of MCTP and thrombin treatment on BPAEC junctional proteins were compared with western blots. Membranes probed for FAK showed differences in band patterns between the different treatment groups and vehicle control (Fig. 5). Membranes probed with a mouse monoclonal antibody for FAK had a prominent band at approximately 125 kDa consistent with the molecular weight of FAK in lysates from control cells. This band was also present in MCTP treated cells but was significantly decreased in lysates from both thrombin and dual treated cultures (34 and 15% of control, respectively). Thrombin treatment was also associated with an increased intensity in a band at approximately 100 kDa (Fig. 5). While the monoclonal antibody did not recognize lower molecular weight proteins, polyclonal antibodies raised to the C or N-terminal portions of FAK had distinctive smaller molecular weight bands (Fig. 6). Thrombin treated cultures had intense C-terminal fragments at 100, 90 and 32 kDa as well as an intense N-terminal fragment at 90 kDa. MCTP treated cultures had prominent 100 and 90 kDa Cterminal fragments as well as an additional fragment at 19 kDa that was not present in control or thrombin only cultures. Probing with the Nterminal antibody showed that both the 125 kDa whole protein and 100 kDa fragment had decreased intensity in MCTP treated cultures compared with controls. Conversely, the N-terminal antibody recognized a more intense 100 kDa band in thrombin treated cultures. As shown in Fig. 7, major bands staining with anti-ZO-1 antibody spanned from 210 to 110 kDa in both MCTP treated and control cells. In the thrombin and MCTP/thrombin treated lanes, the larger molecular weight bands were significantly reduced (4 and 5% of control, respectively) but there were bands in the 160/110 kDa range. Two additional bands at 84 and 60 kDa was also present in lanes from thrombin treated cells.
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Fig. 3. Thru-view of confocal z-series of ZO-1 junctional protein in BPAEC monolayers probed fluorescently with anti-ZO-1 antibody subsequent to exposure to vehicle control (a), MCTP treatment (b), thrombin treatment (c), and dual MCTP/thrombin treatment (d), (bar /25 mm). MCTP treated cells have thinner but distinct discontinuous peripheral bands of ZO-1 (example delimited by arrowheads) while thrombin treatment resulted in a thicker more dispersed expression of ZO-1 (example delimited by arrowheads). Dual treatment attenuated the formation of the thicker peripheral bands that were evident in cells treated with thrombin alone.
Western blots of whole cell lysates from cells treated with MCTP, thrombin and MCTP/thrombin probed for ß-catenin showed a band at 96 kDa that was not significantly altered in any treatment group compared with controls (Fig. 8).
4. Discussion Previous studies in this laboratory have shown a synergistic effect of dual MCTP/thrombin treatment on albumin transudation in endothelial cell
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Fig. 4. Thru-view of confocal z-series of BPAEC monolayers probed fluorescently with antibody to ß-catenin after treatment with vehicle control (a), MCTP (b), thrombin (c), and MCTP/thrombin (d), (bar /25 mm). MCTP treated cells had thin but still well demarcated expression of catenin at cell borders compared with controls. Peripheral bands of catenin had less intensity and prominent infoldings in thrombin treated cells with similar changes in dual treated cells. Larger gaps in dual treated cells (examples delimited by arrowheads) were evident because they lacked catenin staining.
monolayers (Wilson et al., 1998). The current experiments show that both MCTP and thrombin treatment of BPAEC monolayers alter the location and structure of intracellular adhesion molecules. MCTP treatment changed the cellular distribution of FAK in BPAEC monolayers. While MCTP treatment did not alter the location or apparent
protein structure of ZO-1 or ß-catenin (compared with controls); it did induce electrophoretically recognizable changes in FAK. Thrombin changed the morphologic distribution of ZO-1, FAK and ß-catenin in a pattern consistent with cellular contraction. Thrombin treatment also altered electrophoretic migration of both ZO-1 and
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Fig. 5. Western blot of membrane probed with monoclonal antibody for FAK. Note band at 90 kDa in MCTP treated lane and the lack of the higher weight bands in the lanes containing lysates from thrombin treated cells.
FAK in a manner consistent with cleavage of these proteins. Visualization and localization of adhesion molecules via immunofluorescence showed that MCTP interfered with thrombin induced retraction of occludins junctions and reorganization of focal adhesion contacts. MCTP treatment also increased cell size as estimated by cell density. FAK is critical in signaling many adhesion related functions in the cell, including motility, replication, permeability and survival. Forces
modulating endothelial barrier function and permeability that act via FAK may occur via either an increase in phosphorylation or a decrease in phosphorylation in an agonist specific way (Schaphorst et al., 1997). Thrombin has been shown to increase FAK phosphorylation as it increases endothelial barrier permeability (Schaphorst et al., 1997). While antibodies in the present study do not discriminate between FAK phosphorylation states, fragmentation patterns of FAK differed between MCTP and thrombin treatments. While the major 100 kDa fragment induced in MCTP treated cultures appeared to represent a large C-terminal fragment, thrombin treated cultures had a large N-terminal fragment as well. The presence of 100, 90 and 19 kDa fragments of FAK in MCTP treated cultures is similar to previous descriptions of caspase induced changes of FAK in apoptosis (van de Water et al., 1999). This is consistent with our previous work demonstrating progressive apoptosis in MCTP treated cells (Thomas et al., 1998a). The differing pattern seen in thrombin treated cells suggests an alternative
Fig. 6. Western blots of endothelial cell lysates probed with antibodies for either c-terminal or n-terminal portions of FAK. C-terminal fragments at 32 kDa are present in all treatment groups and a fragment at 19 kDa occurs in MCTP treated cells. N-terminal antibodies showed almost no full molecular weight protein in thrombin treated cultures and demonstrate a thrombin specific N-terminal fragment at 90 kDa.
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Fig. 7. Western blot of membrane probed with antibody to ZO-1. Lysates from thrombin treated groups have lost the bands corresponding to the complete protein and have smaller molecular weight fragments not present in control cells.
Fig. 8. Western blot of membrane probed with antibody to ßcatenin adherens junction protein marker showed no change in protein migration in thrombin treated cells compared with controls and MCTP treated groups.
pathway for loss of FAK protein. Whether the apparent FAK proteolysis represents thrombinmediated catalysis or signal transduction mediated degradation of FAK through alternate cellular pathways is the subject of ongoing studies. While some loss of FAK expression was subjectively evident by confocal microscopy in MCTP treated cultures, the principle morphologic change
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was a disorderly arrangement of focal adhesion complexes in MCTP treated cells and an interference with their circumferential rearrangement induced by thrombin in MCTP pretreated cultures. Since focal adhesion complexes were readily evident in thrombin treated cells using anti-FAK antibodies, the dissolution of FAK seen by Western blotting must not result in its dislocation from adhesion complexes. The presence of apoptosis associated FAK fragments in MCTP treated cells suggests a role for this process in the mechanism of MCTP induced microvascular permeability. Whether this effect is due to MCTP direct crosslinking of proteins in the focal adhesion complex or, alternatively, is due to secondary effects subsequent to DNA cross-linking is not clear. It is possible that MCTP derangements of the focal adhesion complex, whether direct or indirect, may contribute to the apoptosis seen in MCTP treated cells and exacerbation of thrombin effects on permeability. Zonula occludens are important in maintaining endothelial barrier function, integrity and cell-tocell contact. ZO-1 expression has been shown to be affected by a variety of factors including, but not limited to, diabetes, wound healing and repair, and changes in vascular shear stress (Schnittler, 1998). In these experiments, pretreatment of cells with MCTP appeared to have little effect on the expression of ZO-1. While the distribution of ZO-1 in a linear pattern at the periphery of the cells was not changed by MCTP treatment, there was an evident discontinuity in this barrier that was accentuated by thrombin stimulus. The decreased expression of ZO-1 by Western blot analysis in thrombin treated cultures suggests that this treatment leads to its degradation. Current models of occludens junctions suggest that a permeability signal results in phosphorylation of occludin with subsequent dissociation from ZO-1. Occludin is then targeted for degradation by metalloproteases while ZO-1 remains attached to actin (Wachtel et al., 1999). The redistribution of ZO-1 in thrombin stimulated cultures is suggestive of actin contractility withdrawing ZO-1 from the cell periphery and would be consistent with its dissociation from occludin. The breakdown of ZO-1 evident on western blots of thrombin treated
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cells suggests that proteolysis of this protein also occurs. This has not been previously described for the thrombin signal transduction pathway and the mechanisms are currently under investigation. While MCTP treatment did not alter either expression or location of ZO-1, the redistribution of ZO-1 by thrombin was inhibited in MCTP pretreated cells. This suggests that there may be functional significance to the MCTP-actin interactions we have previously described (Wilson et al., 1998). Although adherens junctions are not the primary molecule for maintaining tight contact between cells, they are, nonetheless, important in maintaining endothelial barrier integrity and regulating intercellular gap formation (Lum and Malik, 1996). Vascular endothelial-cadherin and associated catenins have been reported to show marked alterations in distribution when treated with agents that increase vascular permeability and gap formation (Rabiet et al., 1996). In contrast to ZO-1, ß-catenin remains attached to the junctional protein VE cadherin when the endothelial cell is stimulated by thrombin. Signal transduction leads to phosphorylation of ß-catenin and its dissociation from a-catenin, which remains attached to the actin cytoskeleton. In the present study, neither the location or expression of ßcatenin by Western blot showed significant responses to thrombin or MCTP treatment. The only morphologic change evident was loss of ßcatenin staining in membranes adjacent to intercellular gaps. This suggests either focal downregulation of junctional protein or that the loss of actin tethering allows movement of the cadherin/ catenin complex within the membrane to form non-adherent spaces. MCTP treatment alone can cause derangement or perturbation of function in the actin cytoskeleton. Recent evidence from our laboratory has shown that MCTP binds to actin and the actinbinding protein, tropomyosin (Lame´ et al., 2000). Tropomyosin interacts with actin in formation of stress fibers (Adam et al., 2000). We have previously demonstrated that stress fibers are altered by MCTP treatment and the formation of stress fibers in response to thrombin is inhibited by MCTP pretreatment (Wilson et al., 1998). Actin is
associated and interacts with membrane adhesion proteins. The present study suggests that the cytoskeletal effects of MCTP may be compounded by alterations in intercellular junctional proteins and/or their interaction with actin. Vascular insult resulting from MCT toxicity has a bi-phasic development. Early biochemical changes in endothelial cells are subtle and increase in severity over time. Lesions occur early as an inflammatory phase and latently as effects of DNA and protein binding (Coulombe et al., 1999, 1992; Lame´ et al., 2000) alter endothelial responses that drive remodeling of the vascular wall. Early inflammatory lesions include the presence of platelet thrombi in small vessels (Lalich et al., 1977; Turner and Lalich, 1965) and microvascular leak in arteriolar walls (unpublished observations). Activation of platelets would result in thrombin release and activation of the coagulation cascade. While normal permeability responses to thrombin are relatively transient, MCTP effects persisted for 24 h in this study suggesting a mechanism by which thrombin induced permeability could be prolonged in vivo. Failure to maintain intercellular junctions may also result in vascular leak at abnormal sites in the vascular tree. Persistent leak of large molecular weight, potentially biologically active, serum components provides a plausible mechanism for the interactive effects of MCTP and thrombin on adhesion proteins to stimulate vascular remodeling.
Acknowledgements Dr Taylor has been a recipient of an NIEHS Training Grant (ES07055) and an NIH Supplemental award to NIH Grant HL4841. This work was supported by NIH Grant HL48411.
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