* Department of Aeronautical and Mechanical Engineering, and Department of Cell Biology and Human Anatomy, University of California, Davis, California 95616
Received March 3, 2004; accepted July 1, 2004
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ABSTRACT |
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Key Words: migration; pregnancy; uterus.
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INTRODUCTION |
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The ability of endothelial cells to migrate is important for successful re-endothelialization following vessel wall injury and is also important during angiogenesis. Failure to repair endothelial damage results in platelet and leukocyte attachment to exposed sub-endothelial matrix and promotes the development of vascular disease (Gotlieb and Lee, 1999). Endothelial cell migration is accompanied by changes in the distribution and/or expression of several adhesion molecules and cell junction proteins (Cao et al., 2002
; Carlevaro et al., 1997
; Kiosses et al., 2001
; Noria et al., 1999
; Urbich et al., 2002
). It was reported (Shen et al., 1996
) that cigarette smoke constituents upregulate endothelial cell adhesion molecule expression (ICAM-1, ELAM-1, and VCAM-1) and another study (Snajdar et al., 2001
) showed that cigarette smoke condensate reduced human umbilical vein endothelial cell (HUVEC) migration. In contrast, a study using rats suggested increased re-endothelialization following balloon injury in animals exposed to cigarette smoke (Sarkar et al., 1999
). We are unaware of studies on the effects of cigarette smoke on endothelial junctional protein expression.
Another important factor that is missing from the above studies on the effect of cigarette smoke components on adhesion molecule expression and endothelial cell migration is fluid flow-derived shear stress. Shear stress has been shown to intricately regulate endothelial function by altering gene expression, adhesion molecule expression, and cell migration (Barakat and Davies, 1998; Barbee et al., 1994
; Davies, 1995
; Gosgnach et al., 2000
; Jalali et al., 2001
; Langille, 2001
; Morigi et al., 1995
; Papadaki and Eskin, 1997
). Of more direct relevance to the present study, recent data suggest interplay between shear stress and cytokines in modulating the expression of adhesion molecule genes in endothelial cells (Chiu et al., 2004
). We hypothesized that such interplay also exists between shear stress and other factors that impact endothelial function including cigarette smoke-derived factors.
Exposure to cigarette smoke during pregnancy is associated with low birth weight, premature delivery, early pregnancy loss, perinatal mortality, and ectopic pregnancy (Shiverick and Salafia, 1999). It is possible that cigarette smoke component-induced uterine endothelial dysfunction is related to these adverse pregnancy outcomes. We are interested in understanding the effects of cigarette smoke constituents on uterine endothelial function during early pregnancy when placental trophoblast cells are invading uterine blood vessels and when vessel walls are being remodeled. We are particularly interested in endothelial cell migration and the role of platelet endothelial cell adhesion molecule-1 (PECAM-1). PECAM-1 is an adhesion molecule expressed by endothelial cells and is involved in both homotypic and heterotypic cell-cell interactions (Jackson, 2003
; Piali et al., 1995
; Sun et al., 1996
; Watt et al., 1995
; Wong et al., 2000
). Beyond its suggested role in facilitating the transmigration of leukocytes (Jackson, 2003
; Kalra et al., 1996
; Liao et al., 1995
; Su et al., 2002
; Zocchi et al., 1996
), studies have implicated PECAM-1 in endothelial cell migration (Cao et al., 2002
; Ji et al., 2002
). PECAM-1 may also influence endothelial migration through its function as a signaling molecule that plays a role in integrin activation (Famiglietti et al., 1997
; Gurubhagavatula et al., 1998
; Jackson, 2003
; Jackson et al., 1997
; Newman, 1997
). Recent work also shows that PECAM-1 can function as a non-selective cation channel in endothelial cells (Ji et al., 2002
; O'Brien et al., 2001
). Apart from a report that cigarette smoke condensate caused phosphorylation of PECAM-1 in HUVECs (Shen et al., 1996
), nothing is known about the effects of tobacco smoke components on PECAM-1 expression under either static or shear stress conditions. In the present article we have shown that soluble components of tobacco smoke alter the expression and cellular localization of PECAM-1 in uterine microvascular endothelial cells under physiological shear stress conditions. These results along with other data showing that CSM perturbs endothelial cell migration provide new information concerning the possible mechanisms by which cigarette smoke-derived factors induce endothelial dysfunction during pregnancy.
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MATERIALS AND METHODS |
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Endothelial cells. Human uterine microvascular endothelial cells (UtMVEC, passage 3) were purchased from Clonetics Corporation (San Diego, CA) and maintained in Endothelial Growth Medium (EGM). This growth medium consisted of Endothelial Basal Medium-2 (Clonetics) supplemented with human recombinant epidermal growth factor, human fibroblast growth factor, vascular endothelial growth factor, ascorbic acid, hydrocortisone, human recombinant insulin-like growth factor, heparin, gentamicin, amphotercin, and 5% fetal bovine serum. Cells were used between passages 47 and plated onto a collagen-coated permanox slide that was placed in a parallel plate chamber. The slides were incubated at 37°C in humidified 95% air and 5% CO2 for 23 days to allow formation of a confluent UtMVEC monolayer. The cells were then exposed to cigarette smoke-conditioned medium and/or shear stress as described below.
Exposure to shear stress. Confluent endothelial cells were exposed to 24 h of steady fluid shear stress in a standard parallel plate flow chamber using protocols that have been previously described. Briefly, cell culture medium, gently gassed with CO2, was drawn from a reservoir using a peristaltic flow pump (Cole-Parmer Instruments, Chicago, IL) and passed through two smaller buffer reservoirs inserted between the pump and the flow chamber to dampen pulsatility. All reservoirs were maintained at 37°C by placing them in a temperature controlled water bath. Flow was recirculated back into the feed reservoir. The flow rate in the recirculating flow loop was adjusted to provide a shear stress at the endothelial cell surface of either 7.5 or 15 dyne/cm2. Once assembled, the flow chamber was placed on the stage of an inverted phase contrast microscope (Nikon TE300, Tokyo, Japan) equipped with a digital CCD camera (QImaging, Retiga 1300, Canada) and interfaced with a computer and software for image acquisition. Parallel control experiments were carried out where endothelial cells were maintained inside the closed flow chamber under static (no flow) conditions for 24 h.
Immunocytochemistry and image analysis. Cells in culture chambers were fixed and permeabilized in ice-cold methanol or fixed in 4% paraformaldehyde (without permeabilization) then incubated with a monoclonal antibody against PECAM-1 (CD31) (DakoCytomation, Carpentaria, CA). The primary antibody was detected using an Alex Fluor-488-labeled goat anti-mouse Ig (Molecular Probes, Eugene OR). Antibody controls in which cells were incubated with isotype-matched mouse Ig followed by Alexa Fluor 488-labeled secondary antibody, were also included. The stained cells were examined using a Nikon Eclipse E800 epifluorescence microscope. For image analysis (see below) multiple digital images from random fields were captured using a Spot RT CCD camera and Spot RT software. Identical exposure and brightness level settings were used for test and control samples.
PECAM-1 immunofluorescence was often found to form prominent bands in regions of cell-cell contact. To quantify changes in the thickness of these junctional bands in response to the various treatments, several bands were examined, and the lengths of five randomly selected lines drawn orthogonal to the major axis of each band were determined using Scion Image version beta 3 (www.scioncorp.com).
The intensity of overall (i.e., cell junction-associated and non cell junction-associated) PECAM-1 immunofluorescence was quantified using Image Pro software (Media Cybernetics, Silver Spring, MD). Relative fluorescence intensity was measured by reference to a fluorescence standard curve obtained using fluorescence calibration beads (Molecular Probes, Eugene, OR). Images from at least three random fields per well viewed using a 40X objective were analyzed for each experimental condition.
Analysis of endothelial cell motility. he flow chamber was positioned on the stage of an inverted phase microscope (Nikon TE300, Tokyo, Japan), and the cells were subjected to the desired shear stress levels. Endothelial cell images were acquired within the selected field of view every 15 min for 24 h using a digital CCD camera (QImaging, Retiga 1300, Canada), interfaced with a computer. Similar recordings were performed on control cells under static conditions. To measure cell motion, the contours of endothelial cells were traced using an electronic graphics tablet and pen (artZII, Wacom, Vancouver, WA), and the data were quantitatively analyzed using Scion image analysis software. For each cell, the image analysis involved quantitation of cell area and perimeter as well as the x- and y-coordinates of the center of gravity (cog) at the end of each hour during the 24-h recording period. Cell activity, which provides an assessment of average cell velocity regardless of movement direction, was determined as the total distance traversed by the cell (determined from the changes in the x- and y-coordinates of the cog) during the 24 h recording period divided by 24. Absolute cell displacement in the flow direction (x-direction) was determined from the absolute value of the difference between the x-coordinates of the cell cog at the zero and 24 h time points.
Western blotting. For Western blot analysis, endothelial cells on permanox slides were washed with ice-cold PBS containing Ca2+ and Mg2+. The cells were then solubilized on ice by the addition of ice-cold lysis buffer containing 1% protease inhibitor cocktail P-8340 (Sigma). After 5 min, the cells were scraped, aspirated, and stored at 20°C. A modified Lowry assay was used to determine the protein concentration of each extract using BSA as the standard. The extracts were then mixed with an equal volume of Laemmli sample buffer (BioRad) containing 2-mercaptoethanol and heated to 100°C for 5 min. Aliquots of the boiled lysate (containing equal amounts of protein) were run on 8% SDS-polyacrylamide gels and then electrophoretically transferred to polyvinylidene difluoride (PVDF) membrane. After blocking with 1% non-fat milk in TBS containing 0.1% Tween 20, the membrane was incubated overnight with a monoclonal anti-tubulin antibody (used as an internal loading standard) followed by incubation with monoclonal anti-PECAM-1 antibody (DakoCytomation) for 1 h. The membrane was washed and incubated with a secondary goat anti-mouse Ig antibody labeled with horseradish peroxidase. Immunoreactive bands were detected using the SuperSignal West Pico chemiluminescence kit (Pierce, Rockford, IL).
Immunoprecipitation. Estimation of cell-surface PECAM-1 was carried out by a slight modification of the procedure described by Goldberger et al. (1994) and Ochi et al. (1998)
. Cells were incubated in the presence or absence of flow and in the presence or absence of CSM for 24 h as described above. The cells were immediately cooled to 4°C, washed with ice-cold PBS and then incubated (again on ice) with anti-PECAM-1 antibody (20 µg/ml) for 1 h. The cells were then lysed by the addition of ice-cold lysis buffer as described in the previous section. Protein G-Sepharose beads (Pierce, Rockford IL) were added to the lysates and incubated overnight at 4°C. The beads were washed six times and then mixed with an equal volume of Laemmli sample buffer containing 2-mercaptoethanol and heated to 100°C for 5 min. After centrifugation, the supernatants were run on 8% polyacrylamide gels and transferred to PVDF membrane as described above. The blots were incubated with the anti-PECAM-1 antibody or with an anti-phosphotyrosine antibody (Upstate Biotechnology, Lake Placid NY; clone 4G10). Primary antibodies were detected and the bands visualized as described under Western Blotting. Densitometric analysis of protein bands was carried out using Kodak 1D software (Kodak Scientific Imaging Systems, New Haven, CT).
Statistical analyses. Experiments were repeated at least three times. Statistical analyses were performed by ANOVA followed by Tukey-Kramer multiple comparison and linear trend post-tests using the Instat software program (GraphPad Inc., San Diego, CA). Differences in means were considered significant if p < 0.05.
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RESULTS |
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When endothelial cells were exposed to a shear stress of 15 dyne/cm2 in the absence of CSM, the PECAM-1 staining pattern changed with time (Fig. 2). The morphology of the cells became more elongated and the cells were aligned parallel to the direction of fluid flow. Similar changes in morphology have been noted for large vessel endothelial cells (Girard and Nerem, 1995; Levesque and Nerem, 1985
). After 2 h exposure to flow (Fig. 2A), bands of PECAM-1 staining at cell-cell junctions in methanol-fixed cultures were thinner than non-flow controls and there was some evidence of cell separation. Quantitative assessment of thinning of the PECAM-1 bands was provided by image analysis of fluorescence micrographs from several experiments (see below). Many cells showed weak staining over the nucleus. This perinuclear fluorescence was not observed in paraformaldehyde-fixed cells (Fig. 2B), suggesting that it was cytoplasmic. After 24 h, the effects of shear stress were more marked with cell separation and generalized loss of the junctional PECAM-1 staining pattern in methanol-fixed cultures (Fig. 2C). However, areas of thin junctional staining were occasionally found where cell-cell contact was maintained. A diffuse, punctate staining pattern, which was often quite bright, was seen over the cytoplasm. Paraformaldehyde-fixed cultures at 24 h also showed a diffuse fluorescence over the cytoplasm (Fig. 2D).
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To provide a more objective assessment of PECAM-1 band thickness, multiple images from three separate experiments similar to those in Figures 1 and 2 were subjected to quantitative image analysis. The results of this analysis of PECAM-1 band thickness under different culture conditions are shown in Figure 3 and confirm the visual impression that the band thickness was significantly decreased in the presence of shear stress alone or CSM alone, or shear stress plus CSM compared to the static control. The effect was more pronounced after 24 h. Also, the mean PECAM-1 band thickness in cells exposed to both shear stress of 15 dyne/cm2 and CSM for 24 h was significantly smaller than that of cells exposed to shear stress alone or to CSM alone.
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Taken together, Figures 36 suggest that exposure of uterine endothelial cells to either flow alone or CSM alone causes PECAM-1 redistribution from intercellular junctions to the cytoplasmic compartment and apical surface and also increases total PECAM-1 expression. The combined effect of flow and CSM leads to recruitment of PECAM-1 predominantly to the cell surface.
Effect of CSM and Flow on Cell Motility
We analyzed the dynamic response of confluent uterine endothelial cells to shear stress using time lapse videomicroscopy. Under static conditions, the cells were highly dynamic presenting continuous and complex positional shifts. On exposure to shear stress, cell activity (as defined in Methods) increased with the level of shear stress applied. Mean (± SEM) cell activities of 6.51 ± 0.67 and 8.74 ± 0.54 µm/h were found for the 7.5 and 15 dyne/cm2 shear stress levels, respectively. In comparison, the activity value for the cells under static conditions was 4.96 ± 0.14 µm/h. Cell activity at 15 dyne/cm2 was significantly different (p < 0.0001) from both control cultures and cultures exposed to 7.5 dyne/cm2 (Fig. 7A). Similarly, measurement of the absolute cell displacement in the direction of flow (as defined in Methods) showed a statistically significant increase (p = 0.0002) upon exposure to a shear stress of either 7.5 or 15 dyne/cm2 relative to control cells in static culture (Fig. 7B). These flow-induced (15 dyne/cm2) increases in activity and displacement were completely abrogated when the cells were incubated in CSM (Figs. 7A and 7B).
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DISCUSSION |
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Cells exposed to shear stress also exhibited more PECAM-1 staining over the cytoplasm. More than half of this staining was intracellular based on quantitative comparison of permeabilized and non-permeabilized cells. It is unclear at this time whether the appearance of the punctate cytoplasmic PECAM-1 pattern represents a redistribution of existing PECAM-1 from the reticular compartment or whether it represents a block in the intracellular processing of PECAM-1 to the reticular compartment. The fact that the reticular network is significantly diminished under shear stress conditions raises questions about the physiological relevance of this compartment. We are unaware of other studies that have investigated the effects of shear stress on endothelial PECAM-1 distribution in vitro. However, a study of perfusion-fixed intact endothelium revealed that PECAM-1 was evenly distributed over the cell surface and did not show concentration at cell-cell junctions (Scholz and Schaper, 1997). These authors speculated that the classic junctional staining pattern frequently reported for PECAM-1 in cultured cells was illusory and due to overlapping of cells. Immunohistochemical staining for PECAM-1 in cross sections of various human and rodent tissues is consistent with the idea that PECAM-1 is evenly distributed on the endothelial cell surface (Feng et al., 2004
; Francis et al., 2002
; Lubeseder-Martellato et al., 2002
; Muller et al., 2002
). The results of a previous immunohistochemical examination of PECAM-1 expression in the macaque uterus (Blankenship and Enders, 1997
) also support this view. The results presented in the present article may shed further light on this issue since the prominent junctional staining pattern seen in static cultures was greatly diminished when the cells were subjected to shear stress. These data may therefore be the in vitro correlate of the findings reported by Scholz et al. (Scholz and Schaper, 1997
) for intact endothelium. We speculate that the absence of prominent junctional staining in intact endothelium results, at least in part, from the effects of physiological levels of shear stress on PECAM-1 distribution. Other endothelial cell junctional proteins show a varied response to shear stress. Catenins redistribute away from cell junctions whereas VE-cadherin remains junction-associated, even in subconfluent cultures (Noria et al., 1999
).
The diminution of junctional PECAM-1 staining and appearance of more prominent cytoplasmic and apical surface staining was also seen when endothelial cells were incubated in cigarette smoke-conditioned medium in the absence of flow. The effect was greater when cells were exposed to both CSM and flow for 24 h. Western blot analyses of total lysates of 24 h cultures showed increased amounts of PECAM-1 and suggest that CSM and flow, either alone or in combination, increase PECAM-1 synthesis (or decrease PECAM-1 degradation) in endothelial cells. To our knowledge nothing is known about the effects of cigarette smoke constituents on endothelial PECAM-1 distribution/expression. However, in agreement with the results reported here, a study by Sho et al. (2003) using a rabbit arteriovenous fistula model showed that arterial PECAM-1 expression was upregulated after 24 h exposure to high flow. Shear stress is also reported to induce PECAM-1 phosphorylation (Osawa et al., 2002
). Cigarette smoke constituents upregulate the expression of endothelial cell adhesion molecules (ICAM-1, ELAM-1, and VCAM-1) and increase the phosphorylation of PECAM-1 (Shen et al., 1996
). The results presented here confirm that shear stress and CSM increase the tyrosine phosphorylation of cell surface PECAM-1 in uterine endothelial cells. Cigarette smoke-conditioned cultured medium has been used for numerous in vitro studies and has been shown to have a wide range of effects on cultured cells (Raza et al., 1999
; Yin et al., 2000
; Zappacosta et al., 2001
). Such media are used as a model for studying the effects of soluble cigarette smoke-derived components that are present in the bloodstream of individuals exposed to cigarette smoke. In many cases the in vitro effects are similar to those seen in vivo. However, it must be remembered that the identity and concentration of all the soluble smoke-derived components in the bloodstream are not known and may or may not relate to the composition of the smoke-conditioned culture media used in vitro.
In cells exposed to both flow and CSM, the diminution of junctional PECAM-1 staining was accompanied by a significant increase in the appearance of PECAM-1 at the cell surface. In comparison to cells exposed to shear stress alone, where PECAM-1 was equally distributed between the cell surface and intracellular compartments, cells exposed to both shear stress and CSM expressed PECAM-1 predominantly on the surface. The immunoprecipitation data substantiate the immunofluorescence data and show that the combination of shear stress and CSM causes an increase in the expression of PECAM-1 at the cell surface. PECAM-1 is reported to play a role in maintaining endothelial adhesion and stabilizing lateral cell-cell junctions (Bird et al., 1999; Jackson, 2003
). There is also evidence that PECAM-1 plays a role in controlling vascular permeability (Graesser et al., 2002
; Turegun et al., 1999
) and endothelial permeability is increased when endothelial cells are exposed to tobacco smoke constituents (Holden et al., 1989
). Taken together, these results and the data presented here suggest that alterations in PECAM-1 distribution brought about by exposure to as yet unidentified soluble component(s) of cigarette smoke may contribute to the mechanism of smoke component-associated increases in vascular permeability. Redistribution of PECAM-1 away from lateral junctions to the apical surface has been reported for endothelial cells exposed to verotoxin-1 (Morigi et al., 2001
) and histamine (Leach et al., 1995
) and in the former case it was speculated that this could lead to increased platelet adhesion and thrombus formation. Similar adverse affects may result from the effects of cigarette smoke components on endothelium. The redistribution of PECAM-1 reported here may also have implications for the ability of PECAM-1 to function as a signaling molecule. O'Brien et al. (2001)
suggested that surface-disposed PECAM-1 represents the signaling pool whereas junctional PECAM-1 is relatively quiescent.
It was also found that cigarette smoke-conditioned medium inhibited the shear stress-induced migration of uterine endothelial cells. This suggests that physiological events such as wound healing and angiogenesis that are dependent on the ability of endothelial cells to migrate could be compromised in individuals exposed to tobacco smoke. Uterine angiogenesis occurs during the normal estrus cycle and during pregnancy (Ma et al., 2001; Torry and Rongish, 1992
; Yasuda et al., 1998
). Uterine endothelium is also remodeled by invading endovascular cytotrophoblast cells during pregnancy. Disruption of uterine endothelial migration resulting from exposure to cigarette smoke constituents could adversely affect these normal processes and lead to the pregnancy complications associated with cigarette smoke exposure during pregnancy. Using a different in vitro assay system (blade injury), Snajdar et al. (2001)
showed that the migration of HUVECs was reduced in the presence of cigarette smoke condensate. Several studies have implicated PECAM-1 in the control of endothelial migratory activity (Cao et al., 2002
; Gratzinger et al., 2003
; Kim et al., 1998
). If PECAM-1 is involved in uterine endothelial cell migration, the inhibitory effects of CSM on cell motility may be related to the abnormal redistribution of PECAM-1 from cell junctions to the cell surface described above. The precise relationship between these various events merit future investigation.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at Department of Cell Biology and Human Anatomy, School of Medicine, Tupper Hall, One Shields Ave., University of California, Davis, CA 95616-8643. Fax: (530) 752-8520. E-mail: gcdouglas{at}ucdavis.edu.
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