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ORIGINAL ARTICLE
Caveolae and Caveolae Constituents in Mechanosensing Effect of Modeled Microgravity on Cultured Human Endothelial Cells
Enzo Spisni,1 Mattia Toni,1 Antonio Strillacci,1 Grazia Galleri,2 Spartaco Santi,3 Cristiana Griffoni,1 and Vittorio Tomasi1,* 1Department
of Experimental Biology, University of Bologna, Bologna, Italy; 2Department of Physiological, Biochemical and Cellular Science, University of Sassari, Sassari, Italy; and 3Institute of Organ Transplant and Immunocytology (ITOI), Bologna Unit – CNR c/o I.O.R, Via di Barbiano 1/10, I-40138 Bologna, Italy
Abstract Studies in modeled microgravity or during orbital space flights have clearly demonstrated that endothelial cell physiology is strongly affected by the reduction of gravity. Nevertheless, the molecular mechanisms by which endothelial cells may sense gravity force remain unclear. We previously hypothesized that endothelial cell caveolae could be a mechanosensing system involved in hypergravity adaptation of human endothelial cells. In this study, we analyzed the effect on the physiology of human umbilical vein endothelial cell monolayers of short exposure to modeled microgravity (24–48 h) obtained by clinorotation. For this purpose, we evaluated the levels of compounds, such as nitric oxide and prostacyclin, involved in vascular tone regulation and synthesized starting from caveolae-related enzymes. Furthermore, we examined posttranslational modifications of Caveolin (Cav)-1 induced by simulated microgravity. The results we collected clearly indicated that short microgravity exposure strongly affected endothelial nitric oxide synthase activity associated with Cav-1 (Tyr 14) phosphorylation, without modifying the angiogenic response of human umbilical vein endothelial cells. We propose here that one of the early molecular mechanisms responsible for gravity sensing of endothelium involves endothelial cell caveolae and Cav-1 phosphorylation. Index Entries: Microgravity; endothelial cells; nitric oxide; Caveolin-1; caveolae.
basic cellular and molecular mechanisms underlying this insufficient vasoconstrictor responsiveness are not fully understood. Hind-limb unloading (HU) in rodents has been extensively used as a model to simulate cardiovascular deconditioning in humans. This model exhibits many of the known cardiovascular consequences of microgravity in humans. The HU rodents model has clearly shown that, among the multiple factors influencing arterial functions, the increased release of vasoactive substances from the endothelium, particularly nitric oxide (NO), is a major mechanism deregulating vascular tone (5). Sangha and coworkers (6) provided functional evidence that NO synthesis was strongly enhanced in HU rodents. It has been reported that both activity and expression of endothelial nitric oxide synthase (eNOS)
INTRODUCTION The physiological responses that are impaired in microgravity-adapted individuals involve multiple mechanisms including hypovolemia, attenuated baroreflex sensitivity, cardiovascular structural changes, and neurohumoral vascular tone regulation (1). Evidence from bed rest and postflight human studies indicates that inadequate vasoconstrictor responsiveness is an important factor in orthostatic intolerance (2). Yet, this aspect would require further investigation to be elucidated (3,4). Even after several decades of study, the
*Author to whom all correspondence and reprint requests should be addressed. E-mail:
[email protected] Cell Biochemistry and Biophysics
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and inducible NO synthase (iNOS) are increased in the carotid artery and thoracic aorta of HU rodents (7). Instead, in other vascular districts, different results have been collected; for example, the expression of eNOS and iNOS in femoral arteries did not change during HU (7). The endothelium is a highly heterogeneous and disseminated organ with a wide variety of functional proprieties, including vascular homeostasis and vascular tone control. Endothelium may adapt its functional phenotype on the basis of various pathophysiological factors, such as hormones, growth factors, cytokines, and biomechanical stimuli. It is well known that endothelium can respond to mechanical deformations resulting from strain associated with vessel stretch and from shear stress (8). Nevertheless, the mechanism by which endothelial cells recognize mechanical stimuli (mechanosensing) is not well understood. Several potential mechanosensing systems have been proposed, including cytoskeleton (9,10), ion channels (11,12), junction proteins (13,14), G proteins (15), and caveolae (16–18). Many independent studies clearly indicate that caveolae are involved in endothelial response to shear stress (19,20). Boyd and collaborators (19) found an increased caveolae formation in endothelial cells exposed to chronic shear stress. They also found that caveolin-1 (Cav-1), the main organizer of caveolae, dramatically changes its distribution after 24 h of laminar shear. Rizzo and colleagues (17) have shown an increased expression of Cav-1 and eNOS in shear-adapted bovine aortic endothelial cells associated with an increased density of caveolae at the luminal plasma membrane. Other evidence of the involvement of caveolae in mechanotransduction comes from studies based on cholesterol depletion disassembling of rafts and caveolae. Ferraro and collaborators (21) demonstrated that cholesterol depletion strongly decreased mechanotransduction in osteoblast cultures, whereas Lungu and collaborators (22) showed that a decreased cholesterol content in caveolae, induced by Cyclosporin A, was associated with an inhibition of the flow-mediated activation of eNOS. In our laboratory, it has been shown that the modulations of Cav-1 and eNOS are among the main mechanisms involved in the hypergravity adaptation of endothelial cells (18). Therefore, we have also proposed that endothelium may directly detect gravity force by using mechanisms involving endothelial caveolae, because we recorded several modifications involving the expression and the activity of enzymes, mainly compartmentalized into endothelial cell caveolae, such as eNOS, Cav-1, cyclooxygenase 2, and prostacyclin synthase (PGIS) in human umbilical vein endothelial (HUVE) cells exposed to mild hypergravity conditions (3g). It is well known that HUVE cells are very sensitive to gravitational unloading (23). In the attempt to elucidate if caveolae and caveolae constituents could also be Cell Biochemistry and Biophysics
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involved in early events after microgravity adaptation, we have grown HUVE cell monolayers for 24–48 h in modeled microgravity obtained by using a three-dimensional (3D) clinostat. Our findings are consistent with the hypothesis that caveolae and Cav-1 are involved in the adaptation of endothelium to microgravity.
MATERIALS AND METHODS Cell Culture HUVE cells were isolated from recently collected unfrozen umbilical cords. Cells were grown in M199 medium supplemented with 20% fetal calf serum, 2 mM L-glutamine, 100 µg/mL endothelial cell growth supplements (ECGS), 5 U/mL heparin, and antibiotics (penicillin 100 U/mL and streptomycin 100 µg/mL), as previously described (18). Cells were maintained at 37ºC in a 5% CO2 incubator and used for experiments at third and fourth passages. Cells were subcultured using 0.05% trypsin, 0.02% ethylenediaminetetraacetic acid (EDTA) solution. Cell viability was assessed by using the trypanblue dye exclusion assay. In this study, microgravity was generated by a 3D clinostat positioned in a thermostatic room, and particular attention was taken to exclude the possibility of false-positive results. The effects of vibration and gradient accumulation or depletion of nutrients were almost completely eliminated by conducting control experiments. Modeled microgravity conditions (named here as microgravity or 0g) were achieved by Random Position Machine (RPM, Dutch-Space). The RPM reproduces gravity acceleration between 10–4 and 10–3. Cell cultures exposed to modeled microgravity were positioned close to the center of the frame. This frame rotates within a second rotating frame. Both frames are driven by separate mechanisms. Rotation of each frame is random, autonomous, and regulated by computer software. The rotation velocity of the frames was 60°/s–1. Static control cultures (named here as 1g) were positioned on the basement of the RPM (24). HUVE cells were grown on both sides of OptiCells (BioCristall Ltd.) flasks, without any coating. OptiCells were then completely filled (to avoid shear stress) with 10 mL of complete medium. OptiCells generate a closed cellular environment with high cellular density (100 cm2/10 mL) in which the O2 and CO2 levels into the medium are maintained through the pores of the growth membranes. As a result, oxygenation of the medium was not required. At confluence, cells were exposed to modeled microgravity for 24–48 h. Both exposed and control OptiCells were treated identically until the start of clinorotation. The temperature and the pH of the culture medium were measured and kept constant to ascertain that the experiments were conducted under identical conditions. Scrupulous attention was made to eliminate the formation of bubbles into the Volume 46, 2006
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OptiCells flasks during clinorotation. Immediately after microgravity exposure, cells were lysed for protein expression or detached and used for angiogenesis assay in normal gravity. Conditioned media were collected and frozen at –80ºC before NO and PGI2 analysis.
Precleared supernatants were immunoprecipitated using anti Cav-1 polyclonal antibody (3 µg/mL) and then incubated with protein A Sepharose. After washings, immunoprecipitated samples were processed by Western blotting to detect eNOS and PGIS.
Antibodies and Reagents
Fluorescence Microscopy and Colocalization Analysis
The primary antibodies used for immunofluorescence studies were a monoclonal anti–Cav-1 (BD Bioscience), and a polyclonal antibody against eNOS (Santa Cruz Biotechnology). The primary antibodies used for Western blotting were a polyclonal anti–Cav-1 (BD Bioscience), a polyclonal anti-phospho-Cav-1 (Tyr 14) (Cell Signalling), a polyclonal anti-phospho-eNOS (Ser 1177) (Santa Cruz Biotechnology), a polyclonal anti-PGI2 synthase (Cayman), and polyclonal antibodies against eNOS and iNOS (Santa Cruz Biotechnology). Secondary antibodies for immunofluorescence (FITC or Cy3 conjugated) and for Western blotting (horseradish peroxide-conjugated) were purchased from Sigma. Medium 199, L-glutamine, antibiotics, heparin, bovine serum albumin (BSA), 1,4-diazabicyclo-(2,2,2)-octane (DABCO) were purchased from Sigma; fetal calf serum was from Life Technologies. ECL reagents were from Amersham, UK.
Western Blotting For Western blotting analysis, confluent cells were scraped from the OptiCells and lysed in a buffer containing Tris-HCl 50 mM, pH 7.5; EDTA 2 mM, NaCl 100 mM, NP 40 1%, and protease inhibitors (10 mg/mL aprotinin, 10 mg/mL leupeptin, 10 mg/mL soybean trypsin inhibitor). Samples were sonicated three times for 10 s on ice and centrifuged at 10,000g to discard the pellet-containing nucleus and cell debris. A small amount of the supernatants was used for Lowry protein assay. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) buffer was added to the supernatant collected and, after boiling, 40 µg of denatured proteins were separated in 12% SDS-PAGE and then transferred to nitrocellulose papers (Hybond-C Extra, Amersham). After blotting, nitrocellulose papers were incubated with antibodies. Detection was performed by using the ECL procedure developed by Amersham. Quantization of the bands was performed by using densitometric image analysis software (Image Master, Pharmacia Biotech) and normalization was made against β-actin expression.
Immunoprecipitation Assay Immunoprecipitation of Cav-1 complexes was carried out essentially as previously described (25). Briefly, HUVE cells exposed to micro or normal gravity were lysed in 500 µL of a buffer containing 10 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.4% SDS, 0.5% NP40, 10% glycerol, and protease inhibitors. Cell Biochemistry and Biophysics
To visualize the colocalization between Cav-1 and eNOS, HUVE cells, grown on OptiCells, were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with PBS-Triton X100 0.05% and then incubated with anti-Cav-1 and anti-eNOS primary antibodies diluted 1:200 in PBSBSA 10 mg/mL. After washing, cells were incubated with anti-mouse FITC-conjugated and anti-rabbit CY3coniugated secondary antibodies diluted 1:50 in PBSBSA 10 mg/mL. Finally, cuts of OptiCells growth membranes were mounted directly on coverslips in glycerol-PBS medium containing 50 mg/mL DABCO. The confocal imaging was performed on a Radiance 2000 confocal laser scanning microscope (BioRad Laboratories), equipped with Nikon ×40 and ×63 oil immersion objectives and with an argon/krypton laser. For FITC and CY3 double detection, the samples were simultaneously excited with the 488- and 568-nm lines of the argon/krypton laser. Optical sections were obtained at increments of 0.1 µm in the Z-axis and were digitized with a scanning mode format of 512 × 512 pixels and 256 gray levels. The image processing and the volume rendering were performed using the ImageSpace software. Negative controls consisted of samples not incubated with the primary antibody. The double-labeling immunofluorescence experiments were carried out avoiding cross-reactions between primary and secondary antibodies. In addition, different controls were performed to ensure antibody specificity. The two-dimensional scatter plot diagram of each section was analyzed to evaluate the spatial colocalization of the fluorochromes. For each scatter plot diagram, pixels with highly colocalized fluorochromes (i.e., with intensity values greater than 150 gray levels on a scale from 0 to 255) for both detectors, were selected to calculate the colocalization maps and create a binary image. Moreover, Pearson coefficients (27) were calculated on acquired optical sections. This coefficient was calculated on 50 sections for each sample, as previously described (28).
PGI2 Synthase Activity Determination The evaluation of PGIS enzymatic activity was assayed by using the 6-Κeto prostaglandin F1α kit (Cayman) and revealed by enzyme-linked immunosorbent assay. For each sample, the 6-keto PGF1α production was related to Volume 46, 2006
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the cell number evaluated by using the acidic phosphatase method.
NO Production Analysis NO production was measured in conditioned media of HUVE cells by using the Griess method for nitrate quantification. The colorimetric Griess reaction (29) for nitrite was applied to measure the level of NO. The samples were diluted 1:2 in H2O mQ. Because the main amount of NOx in biological fluids is found in nitrate, they were exposed to nitrate reductase (250 mU/mL) and NADPH (100 µM) for 30 min at 37°C to reduce nitrate to nitrite. The nitrite-containing samples were treated with L-glutamine dehydrogenase (670 mU/mL), 2-oxoglutaric acid (4 mM), and NH4Cl (100 mM) for 10 min at 37°C to consume any residual NADPH. Finally, the samples were mixed with an equal volume of freshly prepared Griess reagent (0.05% N-[1-naphthyl] ethylenediamine dihydrochloride and 0.5% sulfanilamide in 2.5% ortho-phosphoric acid) for 10 min at 37ºC. The absorbance of each colored sample was measured at 540 nm. Concentrations of NO in the samples were determined using a calibration curve generated with standard NaNO2 solutions (0,1–100 µM). The sensitivity of the methods was 0.05 µM. Data were normalized against the cell number, evaluated by using the acidic phosphatase method.
3D Angiogenesis Test To evaluate the effect of microgravity on in vitro angiogenesis, 80,000 cells detached from OptiCell after clinorotation were seeded on a 3D collagen gel. The gel was prepared as previously described (25) and jellied into single wells of a 24-multiwell plate. Capillary formation was stimulated for 24 h by using human recombinant fibroblast growth factor (bFGF) at 0.5 ng/mL. Quantitative measurement of angiogenesis was obtained by counting the number of capillary-like structures formed by HUVE cells into the gel 24 h after bFGF stimulation. For each well, six images, covering almost all the well surface, were recorded and the number of capillary-like ring structures was calculated.
Statistical Analysis All results are expressed as mean ± SEM. Differences were analyzed by Student t-test and considered statistically significant at p < 0.05 and p < 0.01.
Fig. 1. (A) Nitric oxide (NO) production was evaluated in the conditioned media, after Griess reaction, as nitrate concentration. Each point represents the mean ± SEM of 18 determinations obtained starting from the medium collected from six different OptiCells for each treatment (sample in triplicate, n = 18). # indicates that the difference was statistically significant (p < 0.01). (B) Analysis of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) proteins expression in human umbilical vein endothelial cells exposed for 24 h to normal gravity (1g) or microgravity (0g) by Western blotting, starting from three different OptiCells for each treatment. The images showed are representative of the results obtained in two different experiments. (C) Densitometric analysis of eNOS and iNOS expression obtained by band quantification. Densitometry was performed on six different bands for each treatment (n = 6). Both eNOS and iNOS were normalized against β-actin expression. Band quantification results are shown setting equal to 100 the mean value obtained from the quantification and normalization of the bands obtained starting from normal gravity control cells.
RESULTS NO Synthesis is Increased in Microgravity The first interesting datum we collected was an increased NO synthesis in HUVE cells grown in microgravity. NO increased 2.5-fold (Fig. 1A) after 24–48 h of Cell Biochemistry and Biophysics
microgravity. To verify if an increased expression of NO synthases was responsible for NO increase, we analyzed by Western blotting the levels of iNOS and eNOS in endothelial cells monolayers exposed to normal gravVolume 46, 2006
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ity or to microgravity. We found that iNOS expression was weak both in HUVE cells cultured in normal gravity and in microgravity. On the contrary, eNOS was strongly expressed in both the experimental conditions tested, but with no significant difference between 1g and 0g (Fig. 1B,C). This is particularly interesting considering that a different mechanism—increasing eNOS mRNA expression—is involved in shear stress adaptation of cultured endothelial cells (30). Our results are in agreement with data collected from other researchers, showing that eNOS activity is increased by HU in rodents (31).
Regulation of eNOS Activity A well-described molecular mechanism regulating eNOS activity in endothelium is its localization into caveolae and its protein–protein interaction with Cav-1 (32). In vivo and in vitro studies have clearly shown that Cav-1 is the major negative regulatory protein for eNOS and that Cav-1 expression is inversely correlated to NO production (33). Moreover, these findings are supported by genetic data showing that Cav-1 knockout mice have increased endothelium-dependent relaxations and NO levels in blood (34,35). Thus we analyzed if the increased NO production was linked to a decreased Cav-1 expression in HUVE cell monolayers cultured in microgravity for 24 h (Fig. 2A). Results confirmed that the increased NO synthesis did not correlate with decreased Cav-1 expression. The regulation of eNOS by phosphorylation is poorly understood. Among the different known phosphorylation sites on eNOS, Ser-1179 in bovine (Ser-1177 in humans) has been characterized most extensively (36). Ser-1177 phosphorylation is stimulatory for both basal and agonist-mediated NO release and seems to be the major site for Akt phosphorylation (36). We found that in HUVE cells, Ser-1177 phosphorylation is not affected after 24 h of microgravity treatment (Fig. 2A,B). Cav-1 was first identified as a substrate for the v-Src tyrosine kinase, which phosphorylates Cav-1 on Tyr 14. Moreover, it has been hypothesized that Cav1 phosphorylation at Tyr14 may override or downregulate the activity of the caveolin-scaffolding domain (37), the region of Cav-1 involved in eNOS binding and inhibition. Interestingly, after 24 h, microgravity augmented Cav-1 Tyr14 phosphorylation in HUVE cells (Fig. 2A). Our densitometric analysis results indicate that Cav-1 phosphorylation increased 1.8-fold after 24 h of clinorotation (Fig. 2B). Other caveolar enzymes, such as PGIS, are involved in the regulation of vascular tone and are also bound to Cav-1. To verify if microgravity may affect the release of other vasoactive substances, we measured PGI2 production in conditioned media of HUVE cell monolayers after clinorotation. Results (Fig. 2C) indicated that only Cell Biochemistry and Biophysics
Fig. 2. (A) Western blotting analysis of Cav-1, Ser 1177 phospho eNOS, Tyr 14 phospho Cav-1 expressions in human umbilical vein endothelial cells grown for 24 h under normal (1g) or microgravity (0g) conditions. Images are representative of the results obtained from two independent experiments, consisting of three different OptiCells for each treatment (n = 6). (B) Densitometric analysis of the Western blotting results obtained. p-Cav-1 was normalized against Cav-1, whereas p-eNOS was normalized against β-actin expression. Densitometry was performed on six different bands for each treatment (n = 6). Band quantification results are shown setting equal to 100 the mean value obtained from the quantification and normalization of the bands obtained from normal gravity control cells. (C) Prostacyclin (PGI2) production was evaluated in the conditioned media as 6-keto PGF1α concentration, by using enzyme-linked immunosorbent assay. Normalization of the values shown was made relating the 6-keto PGF1α concentration to the total number of cells in each Opticell, evaluated by using the acidic phosphatase method. Each point represents the mean ± SEM of 18 determinations obtained starting from the supernatants collected from six different OptiCells dosed in triplicate. *Indicates that difference was statistically significant (p < 0.05).
NO synthesis was affected after 24 h of microgravity exposure. To verify the hypothesis that Cav-1 Tyr 14 phosphorylation could inactivate the CSD activity and in particular the coupling between Cav-1 and eNOS, we Volume 46, 2006
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Fig. 3. Confocal microscopy localization of Cav-1 (FITC, represented in green) and endothelial nitric oxide synthase (eNOS) (Cy3, represented in red) in human umbilical vein endothelial cells exposed for 24 h to normal gravity (1g, A–H) or to microgravity (0g, I–P). Color images result from integrating different optical sections. (A–D) and (I–L) were obtained by using the ×40 oil immersion objectives, whereas (E–H) and (M–P) were obtained by using the ×63 oil immersion objectives. Whereas in (A–D) and (I–L) a wide number of cells are shown, (E–H) and (M–P) show single-cell images. Superposition of the green (Cav-1) and red (eNOS) signals is shown (C,G,K,O); yellow indicates that eNOS and Cav-1 colocalize. Scatter plot colocalization maps (D,H,L,P) were obtained as described under Materials and Methods and represent the colocalization levels in a binary black and white image (bar = 5 µm).
performed confocal microscopy analysis of Cav1/eNOS colocalization. Figure 3 shows that 24 h after microgravity exposure the colocalization between eNOS and Cav-1 at the plasma membrane level was clearly diminished (compare panels D and H with panels L and P, respectively). Analysis on 50 different sections indicates that Pearson’s correlation coefficient decreases from 0.78 to 0.59 in modeled microgravity conditions. The results of the colocalization analysis Cell Biochemistry and Biophysics
were confirmed also by the immunoprecipitation experiments (Fig. 4A,B), showing that the amount of eNOS recovered after Cav-1 immunoprecipitation was reduced in HUVE cells after 24 h of microgravity exposure. The selectivity of this uncoupling phenomenon has been revealed by the examination of PGIS/Cav-1 coimmunoprecipitation, because it is known that also PGIS is an enzyme that bind and coimmunoprecipitate with Cav-1 (Fig. 4A). Volume 46, 2006
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Fig. 4. (A) Immunoprecipitation of Cav-1 complexes in human umbilical vein endothelial cells exposed to normal gravity (1g) or microgravity (0g) for 24 or 48 h. Cell lysates were immunoprecipitated (IP) with polyclonal antibody to Cav-1, and the resulting immunocomplexes were analyzed for the presence of endothelial nitric oxide synthase (eNOS, upper lanes) or prostacyclin synthase (PGIS) (lower lanes) by Western blotting. Image in (A) is representative of two different sets of experiments, consisting of three different OptiCells for each treatment. (B) Densitometric analysis of the eNOS and PGIS bands obtained by Western blotting after Cav-1 immunoprecipitation. Quantification was performed on six different bands for each treatment (n = 6). Numerical data were obtained setting equal to 100 the mean value obtained from the quantification of the bands obtained after the immunoprecipitation of normal gravity control cells lysates. *Indicates that the difference was statistically significant (p < 0.05).
Microgravity and Angiogenesis Because NO has been described as an angiogenic factor, the angiogenic response of HUVE cells was examined, after 24 h of microgravity, by using an in vitro 3D angiogenesis test based on capillary-like structures formation into a collagen gel (Fig. 5A,B). We decided to stimulate the angiogenic response by using bFGF to exclude the effects of the reciprocal regulation between NO and vascular endothelial growth factor during angiogenesis (38). Results of the count of the number of capillary-like ring structures formed in each well are shown in Fig. 5C, indicating that the formation of capilCell Biochemistry and Biophysics
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Fig. 5. Effect of microgravity on in vitro angiogenesis, evaluated by using three-dimensional collagen gel angiogenesis test. After 24 h of normal gravity or microgravity conditioning, HUVE cells were seeded on three-dimensional collagen gel and stimulated with 0.5 ng/mL bFGF for 24 h (A,B). After 24 h, human umbilical vein endothelial (HUVE) cells started the formation of capillary-like ring structures (indicated by arrows) into the gel. Quantitative analysis of the angiogenic response of HUVE cells (C) was made counting the number of capillary-like ring structures, formed in each well. For each condition tested, 12 wells were analyzed (n = 12). Data are expressed as the mean ± SEM (bar = 60 µM).
lary-like structures, after 24 h of stimulation by bFGF, was not affected by microgravity exposure.
DISCUSSION It is well known that changes in gravity force may deeply influence eukaryotic cell behavior. One of the aims of this study was to verify which molecular mechanisms are involved in the endothelial cells early adaptation to the decreased gravity. We have recently shown that caveolae and caveolar enzymes are involved in the endothelial cell adaptation to hypergravity conditions (18), and we have hypothesized that caveolae may have a gravisensing role, at least in these cells. The strong increase in NO synthesis we observed, in the experimental conditions we adopted, is a piece of data generally confirmed by literature in most in vitro and in vivo models for microgravity simulation. Nevertheless, different mechanisms may regulate eNOS activity and NO synthesis in endothelial cells. Among them, the modulation of eNOS expression, the posttranslational regulation of the enzyme and its association with regulative proteins are the well-described regulatory mechanisms (39,40). Among them, the Ser 1177 phosphorylation of Volume 46, 2006
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eNOS and the binding of Cav-1 scaffolding domain to the reductase domain of eNOS are believed to be the major posttranslational mechanisms modulating the enzyme activity (38). Our results showed that, although Ser 1177 phosphorylation was not affected, eNOS inhibitory binding to Cav-1 was significantly reduced by microgravity. The Tyr 14 Cav-1 phosphorylation, probably induced by Src kinase, seems to be the molecular key of Cav-1/eNOS complex dissociation. It has been hypothesized that Tyr 14-phosphorylated Cav-1 may function like a growth factor receptor that recruits SH2 domain-containing proteins to the plasma membrane (41). Moreover, Colonna and Podestà (42) have recently observed that Cav-1 Tyr 14 phosphorylation is associated with a cytoskeleton rearrangement in Y1 adrenal cells. A marked cytoskeletal reorganization has been also observed in HUVEC, by Carlsson and collaborators (23) in response to modeled microgravity obtained by using the rotating wall vessel bioreactor. Yet, after 24 h of clinorotation, we could not detect any cytoskeleton modification (data not shown). Even if the increased NO synthesis seems to be a common feature of microgravity adaptation, the molecular mechanisms involved in this upregulation could be different in different cell types. Indeed, although modeled microgravity caused an increased expression of eNOS mRNA in microvascular endothelial cells (43), in HUVE cells, the increased NO synthesis mainly acted on posttranslational mechanisms. Our data clearly showed that the colocalization between Cav-1 and eNOS is significantly decreased after 24 h of clinorotation. Immunoprecipitation assay confirms that the uncoupling of Cav-1 and eNOS may be the cause of the increased eNOS activity we measured in microgravity. Thus it is very likely that Cav-1 phosphorylation on Tyr 14 may be the molecular event causing eNOS dissociation. Interestingly, we observed that the binding between Cav-1 and PGIS (also a caveolar enzyme bound to Cav-1 scaffolding domain) is not affected by microgravity-induced Cav-1 phosphorylation. This is not surprising because we have previously shown that the scaffolding domain of Cav-1 has structural features in agreement with the hypothesis of a multiple functional domain (44). Moreover, whereas for PGIS the binding to Cav-1 seems to be mainly linked to its subcellular compartmentalization and does not change its activity (25), for eNOS the binding to Cav-1 is a fundamental regulative step leading to its inactivation. Our hypothesis that caveolae could behave as endothelial cell mechanosensors and gravisensors is substantiated by the early Cav-1 Tyr-14 phosphorylation we have observed during microgravity adaptation. This hypothesis is also confirmed by findings showing that cholesterol depletion inhibited eNOS activation induced Cell Biochemistry and Biophysics
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by shear stress (22) and reduced mechanotransduction pathways induced by hydrostatic pressure (21). NO is a vasoactive compound considered to be a proangiogenic factor. The evaluation of the angiogenic response of endothelial cells grown in microgravity is of great interest for space medicine and bioengineering, especially for the generation of vascularized tissues by using bioreactors (45). Moreover, the effect of microgravity on the angiogenic response is still debated. For example, although Carlsson and collaborators (23) found an increased proliferation in HUVE cells exposed to microgravity, Morbidelli and coauthors (46) observed that microgravity strongly decreased proliferation in porcine aortic endothelial cells. Our results clearly indicate that microgravity, obtained by clinorotation, does not modify the angiogenic competence of HUVE cells. It is likely that the increased angiogenesis stimulation resulting from the increased NO synthesis was counterbalanced by the modification of other signaling pathways induced by microgravity. How cells may couple gravity force changes to signaling mechanisms is still an open question. Our results demonstrate that both Cav-1 Tyr 14 phosphorylation and eNOS activation are caveolar mechanisms involved in early microgravity adaptation of HUVE cells. We believe that caveolae can behave as gravity sensors, at least in endothelial cells. Our studies open up the possibility that caveolae and Cav-1 could be deeply involved in the impairment of vascular functions observed in the microgravity adaptation of mammals. Yet, we can not exclude that other signaling pathways, dependent or independent from Cav-1, also play a role in microgravity impairment of vascular tone. Our conclusion is that, in the conditions we tested, Cav-1 and caveolae are responsible for the early activation of the signalling mechanisms involved in endothelial cells adaptation to the decreased gravity force.
ACKNOWLEDGMENTS We thank Mr. Gavino Campus and Prof. Proto Pippia for technical support in the use of three-dimensional clinostat. Supported by DCMC project (2006) from Agenzia Spaziale Italiana (Roma) to VT.
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