Cross talk between the actin cytoskeleton and the microtubule (MT) network plays a critical role in regulation of endothelial permeability. We have previously demonstrated that MT disruption by nocodazole results in increases in MLC phosphorylation, actomyosin contraction, cell retraction, and paracellular gap formation, cardinal features of endothelial barrier dysfunction (Verin AD, Birukova A, Wang P, Liu F, Becker P, Birukov K, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 281: L565–L574, 2001; Birukova AA, Smurova K, Birukov KG, Usatyuk P, Liu F, Kaibuchi K, Ricks-Cord A, Natarajan V, Alieva A, Garcia JG, and Verin AD. J Cell Physiol. In press.). Although activation of PKA opposes barrier-disrupting effects of edemagenic agents on confluent EC monolayers, information about the molecular mechanisms of PKA-mediated EC barrier protection is limited. Our results suggest that MT disassembly alters neither intracellular cAMP levels nor PKA enzymatic activity; however, elevation of cAMP levels and PKA activation by either cholera toxin or forskolin dramatically attenuates the decline in transendothelial electrical resistance induced by nocodazole in human pulmonary EC. Barrier-protective effects of PKA on EC were associated with PKA-mediated inhibition of nocodazole-induced stress fiber formation, Rho activation, phosphorylation of myosin phosphatase regulatory subunit at Thr696, and decreased MLC phosphorylation. In addition, forskolin pretreatment attenuated MT disassembly induced by nocodazole. These results suggest a critical role for PKA activity in stabilization of MT cytoskeleton and provide a novel mechanism for cAMP-mediated regulation of Rho-induced actin cytoskeletal remodeling, actomyosin contraction, and EC barrier dysfunction induced by MT disassembly.
- pulmonary endothelium
- myosin phosphatase
- Rho-associated kinase
the vascular endothelium functions as a semiselective barrier for mass transport through the vessel wall, and compromise of endothelial cell (EC) barrier integrity increases vascular permeability, a cardinal feature of acute lung injury (9, 13, 15). Regulation of endothelial barrier is achieved via balance between actomyosin-driven contractile events regulated through myosin light chain (MLC) phosphorylation by MLC kinase (MLCK)-dependent and -independent mechanisms and tethering forces implied by cell-adhesive structures and cortical actin cytoskeletal network (9, 13, 29, 32). For example, edemagenic agonist thrombin induces EC barrier dysfunction primarily via microfilament reorganization, stress fiber formation, and actomyosin-driven contraction, which is triggered by Ca2+/calmodulin-dependent MLCK and Rho-associated kinase (Rho-kinase) (2, 13, 14, 33).
Microtubules (MT) represent another key component of the cytoskeleton and are intimately involved in many cellular processes such as mitosis, locomotion, and protein and organelle transport (25, 35). MT respond to a number of signaling molecules by altering dynamics of assembly-disassembly and by spatial rearrangements. Conversely, changes in MT dynamics modulate intracellular signal transduction (16). Several reports demonstrate that MT disruption increases cellular mechanical tension and contractility via Rho-dependent mechanisms in a variety of cell types (6, 10, 32). In EC, MT depolymerization is associated with dissolution of the cortical actin cytoskeleton, increased MLC phosphorylation, stress fiber formation, contraction, and EC barrier dysfunction (5, 32). MLC phosphorylation observed during MT depolymerization is a result of activation of small GTPase Rho and its effector Rho-kinase (5, 32). Rho-kinase-induced MLC phosphorylation occurs as result of Rho-kinase-mediated phosphorylation of the myosin-associated phosphatase type 1 (PPase 1) myosin-binding subunit (myosin phosphatase 1 or MYPT1) at Thr695, Ser894, and Thr850, which leads to PPase 1 inactivation and dissociation from myosin (12). In addition, Rho-kinase may directly phosphorylate MLC and activate actomyosin ATPase activity (12).
The second messenger cAMP mediates relaxation of smooth muscle and promotes barrier integrity in the endothelium (25, 35). Several studies have shown the protective role of elevated cAMP levels and consequent activation of cAMP-dependent protein kinase (PKA) in a vascular leakage induced by inflammatory mediators, such as thrombin, phorbol myristate acetate, pertussis toxin, and bacterial wall lipopolysaccharide (11, 13, 23, 26). Activated PKA phosphorylates endothelial MLCK, thereby reducing its activity, leading to decreased basal MLC phosphorylation (13). Elevation of intracellular cAMP levels and activation of PKA stimulate phosphorylation of the actin-binding proteins filamin and adductin (19, 34) and focal adhesion proteins paxillin and focal adhesion kinase, as well as the disappearance of stress fibers and filamentous actin (F-actin) accumulation in the membrane ruffles (17, 31). PKA-mediated modulation of Rho GTPase activity is another potentially important mechanism for regulation of actin cytoskeletal organization (8, 22). Elevation of intracellular cAMP and increased PKA activity attenuates RhoA activation via RhoA phosphorylation at Ser188 (22), which decreases Rho association with Rho-kinase (8). PKA activation also increases interaction of RhoA with Rho-GDP dissociation inhibitor (Rho-GDI) and translocation of RhoA from the membrane to the cytosol (22, 27, 30). Thus the overall effect of PKA on RhoA is the inhibition of RhoA activity and stabilization of cortical actin cytoskeleton, which may promote EC barrier enhancement, whereas MT depolymerization exhibits barrier-disruptive effects. In this work, we investigated the effects of elevated cAMP levels and PKA activation on EC cytoskeletal changes and barrier dysfunction induced by MT depolymerization and tested the potential mechanism of PKA-mediated barrier protection, which includes PKA-mediated stimulation of myosin-associated PPase 1 activity.
MATERIALS AND METHODS
Nocodazole, vinblastine, paclitaxel, and forskolin were purchased from Sigma (St. Louis, MO); cholera toxin and PKA- inhibitory peptide (PKI) were purchased from Calbiochem (La Jolla, CA); PKA assay kit was purchased from Promega (Madison, WI); Rho activation kit was purchased from Upstate Biotechnology (Lake Placid, NY); and anti-Rho-kinase antibody was purchased from BD Biosciences-Transduction Laboratories (Lexington, KY). MLC antibody was produced in rabbit against baculovirus-expressed and purified smooth muscle MLC by Biodesign International (Kennebunk, ME). All reagents used for immunofluorescent staining were purchased from Molecular Probes (Eugene, OR). Unless specified, all other reagents were obtained from Sigma Chemical.
Bovine pulmonary artery EC were obtained frozen at 16th passage from American Type Culture Collection (culture line-CCL 209; Rockville, MD) and were utilized at passages 19–24 as previously described (13, 32 ). Cells were cultured in M-199 medium (GIBCO), supplemented with 20% (vol/vol) colostrums-free bovine serum (Irvine Scientific, Santa Ana, CA), 15 μg/ml of EC growth supplement (Collaborative Research, Bedford, MA), 1% antibiotic and antimycotic (10,000 U/ml penicillin, 10 μg/ml streptomycin, and 25 μg/ml amphotericin B; K. C. Biologicals, Lenexa, KS), and 0.1 mM nonessential amino acids (GIBCO), and maintained at 37°C in a humidified atmosphere of 5% CO2-95% air. The EC grew to contact-inhibited monolayers with the typical cobblestone morphology. Human pulmonary artery EC were obtained from Clonetics (BioWhittaker, Frederick, MD), propagated in culture medium EGM-2, and used at passages 6–10.
Measurement of endothelial monolayer electrical resistance.
Cellular barrier properties were measured using the highly sensitive biophysical assay with an electrical cell-substrate impedance sensing system (Applied Biophysics, Troy, NY) described previously (13, 32).
MLC phosphorylation assay.
Protein extracts were separated by SDS-PAGE on 10% gels, transferred to nitrocellulose membrane (30 V for 18 h or 90 V for 2 h), and probed with specific antibodies as previously described (32). Immunoreactive proteins were detected using an enhanced chemiluminescent detection system according to the manufacturer's protocol (ECL; Amersham, Little Chalfont, UK). The relative intensities of the protein in the bands were quantified by scanning densitometry.
Determination of PKA activity.
PKA activity was measured using Signa TECT PKA Assay System (Promega) according to manufacturer's recommendations with minor modification. Confluent EC grown in 100-mm dishes were treated with agonists at 37°C. The cells were lysed on ice in extraction buffer (25 mM Tris, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, and 0.1% 2-mercaptoethanol) including 1:500 diluted protease inhibitor cocktail (200 μM 4-(2-aminoethyl)benzenesulfonyl fluoride, 160 nM aprotinin, 10 μM bestatin, 3 μM E-64, 4 μM leupeptin, and 2 μM pepstatin A; Calbiochem). Cell lysates were homogenized by sonication. Cell debris was removed by a 5-min centrifugation at 14,000 g (4°C), and the supernatants were incubated for 5 min at 30°C with PKA biotinylated peptide substrate kemptide (100 μM) in the kinase assay buffer containing 40 mM Tris, pH 7.4, 20 mM MgCl2, 0.5 mg/ml BSA, ATP, and [γ-32P]ATP. The radiolabeled substrate was allowed to bind to SAM2 Biotin Capture Membrane, and the radioactivity per minute was measured in a scintillation counter.
Determination of RhoA activity.
RhoA activity was measured using a Rho activation assay kit (Cytoskeleton, Denver, CO) according to manufacturer's recommendations with minor modification. EC (70–80% confluent) grown in 100-mm dishes were treated with agonists at 37°C. The cells were lysed on ice in lysis buffer (50 mM Tris, pH 7.5, 10 mM MgCl2, 0.5 M NaCl, 0.1% Triton X-100, and 0.1% SDS) including 1:100 diluted protease inhibitor cocktail (10 μg/ml leupeptin, 10 μg/ml aprotinin, and 500 μg/ml tosyl arginine methyl ester). Cell lysates were homogenized by sonication. Cell debris was removed by a 5-min centrifugation at 12,000 g (4°C), and the supernatants were incubated with rhotekin-RBD beads for 1 h at 4°C on a rotator. Beads were washed, and the immunoprecipitated complex was resuspended in 2× SDS sample buffer and subjected to 15% SDS-PAGE, followed by Western blotting.
EC grown on glass coverslips were fixed after agonist treatment in 3.7% formaldehyde solution in PBS for 10 min at 4°C, washed three times with PBS, permeabilized with 0.2% Triton X-100 in PBS-Tween (PBST) for 30 min at room temperature, and blocked with 2% BSA in PBST for 30 min. Incubation with antibody of interest was performed in blocking solution for 1 h at room temperature followed by staining with either Alexa 488- or Alexa 594-conjugated secondary antibodies (Molecular Probes). Actin filaments were stained with Texas red-conjugated phalloidin (Molecular Probes) for 1 h at room temperature. After immunostaining, the glass slides were analyzed using a Nikon video-imaging system (Nikon Instech) consisting of a phase-contrast inverted microscope (Nikon Eclipse TE2000) connected to a digital camera and image processor (Hamamatsu Photonics). The images were recorded and processed using the Adobe Photoshop 7.0 program, using a Pentium III PC.
Image analysis of stress fiber formation and assembled MT.
Texas red-stained EC monolayers were pretreated with forskolin, stimulated with either nocodazole or vehicle, viewed under a microscope using a 20/0.4 objective, and images were captured as described above. The 16-bit images were analyzed using MetaVue 4.6 (Universal Imaging, Downington, PA). Actin fibers or MT were marked out, and the ratio to the cell area covered by stress fibers or assembled MT to the whole cell area was determined. The values were statistically processed using Sigma Plot 7.1 (SPSS Science, Chicago, IL) software.
Results are expressed as means ± SD of three to five independent experiments. Stimulated samples were compared with controls by unpaired Student's t-test. For multiple-group comparisons, ANOVA followed by the post hoc Fisher's test were used. P < 0.05 was considered statistically significant.
cAMP elevation and PKA activation attenuate EC barrier dysfunction induced by MT disruption.
It has recently been reported that MT disruption caused by nocodazole or vinblastine decreases transendothelial resistance (TER) in a dose-dependent manner, reflective of increased permeability and barrier failure (5, 32). To test the hypothesis that cAMP-mediated PKA activation prevents EC barrier failure evoked by MT dissolution, we incubated EC monolayers with forskolin (50 μM, 1 h) before treatment with nocodazole (0.2 μM, 5 min). As shown in Fig. 1A, EC pretreatment with forskolin significantly attenuated nocodazole-induced increases in permeability compared with EC treated with nocodazole alone. Figure 1B summarizes the results of TER measurements and demonstrates that stimulation of PKA activity by either forskolin or cholera toxin (1 μg/ml) significantly attenuated the reduction of TER induced by the MT inhibitors nocodazole and vinblastine.
To examine the effects of MT inhibitors and forskolin on PKA activation, we performed PKA activity assays. As shown in Fig. 2A, MT disruption by nocodazole did not affect the maximal PKA in vitro activation in the presence of exogenous cAMP, compared with vehicle controls. EC were next treated with either MT inhibitor (nocodazole), MT stabilizer (taxol), cAMP-elevating agent (forskolin), or with combinations (forskolin plus nocodazole and nocodazole plus taxol; Fig. 2B). In these experiments, maximal PKA activation was achieved by adding exogenous cAMP to the in vitro PKA enzymatic reactions. In the next series of experiments, PKA kinase assays were performed in the absence of exogenous cAMP. This modification in the kinase assay conditions allowed us to detect changes in PKA activation induced by intracellular factors. EC treatment with forskolin induced a 2.5-fold increase in PKA activity, which was not affected by nocodazole treatment. Nocodazole and taxol did not affect PKA activity compared with vehicle controls. Interestingly, EC pretreatment with taxol followed by nocodazole stimulation was also without effect on PKA activity. In the previous studies, we have demonstrated complete inhibition of nocodazole-induced barrier disruption by taxol (5, 32). Collectively, these results suggest that elevations in cAMP/PKA are unlikely to be involved in taxol-induced EC barrier protection against barrier failure induced by MT inhibitors.
Effect of forskolin on actin reorganization induced by MT disruption.
EC barrier dysfunction triggered by MT disassembly is accompanied by F-actin cortical ring dissolution, stress fiber, and intracellular gap formation, which are closely associated with increased MLC phosphorylation (5, 32). Figure 3A shows that forskolin completely abolishes nocodazole-induced stress fiber and gap formation, results highly consistent with the TER measurements depicted in Fig. 1A. Quantitative analysis of F-actin in control and forskolin- and nocodazole-treated cells shown in Fig. 3B suggests that forskolin pretreatment significantly attenuates nocodazole-induced stress fiber formation (43 ± 7% of actin fiber area per cell in nocodazole-treated cells compared with 17 ± 4% of actin stress fiber area per cell in forskolin-pretreated cells followed by nocodazole stimulation). These results strongly suggest the involvement of actin rearrangement in protective effect of intracellular cAMP elevation and PKA activation on nocodazole-induced cytoskeletal remodeling and barrier compromise.
Previous reports, including our own, suggest a key role for MLC phosphorylation in activation of contraction and permeability induced by MT disruption (5, 21, 32). In this study, we hypothesized that cAMP/PKA may attenuate EC contraction and barrier dysfunction via inhibition of MLC phosphorylation. To test this hypothesis, we pretreated EC monolayers with forskolin or cholera toxin followed by nocodazole challenge and separated unphosphorylated and mono- and diphosphorylated MLC species by urea gel electrophoresis (Fig. 4A). Alternatively, diphospho-MLC was detected using anti-diphospho-MLC antibodies (Fig. 4B). Increased cAMP levels and PKA activity significantly decreased the levels of monophosphorylated and diphosphorylated MLC in nocodazole-stimulated EC. Similar inhibitory effects of cholera toxin-induced cAMP elevation and PKA activation on MLC phosphorylation were reported in EC stimulated with thrombin (26). Recent studies by Qiao et al. (27) have shown that protective effects of elevated cAMP levels on agonist-induced EC barrier dysfunction are mediated predominantly through PKA-dependent mechanisms. In the next series of experiments, EC cultures were preincubated with cell-permeable PKI alone or in combination with forskolin for 1 h before nocodazole treatment. Because nocodazole-induced MLC phosphorylation is MLCK independent and solely mediated by Rho-kinase (5, 32), we assessed PKA-dependent regulation of Rho activity in nocodazole-stimulated EC by monitoring the levels of MLC phosphorylation. Treatment of EC with PKA inhibitor and forskolin did not abolish nocodazole-induced increases in phospho-MLC, whereas forskolin without PKA inhibition possessed inhibitory effect (Fig. 4C). Consistent with our biochemical data, immunofluorescent staining of human pulmonary EC with anti-diphospho-MLC antibody revealed dramatic reduction of dihospho-MLC immunoreactivity in forskolin-treated cells (Fig. 4D).
Effect of forskolin on Rho activity.
Our results suggest that MLC phosphorylation status plays a critical role in the modulation of nocodazole-induced EC barrier dysfunction by cAMP and PKA, and the net MLC phosphorylation is determined by the balance between MLCK and MYPT1 activities (5, 32). We have previously reported that MT inhibitors failed to alter MLCK activity, as MLC phosphorylation was not affected by MLCK inhibitors (5, 32). However, inhibition of Rho GTPase completely abolished increases in MLC phosphorylation induced by MT disassembly, and inhibition of Rho-kinase markedly attenuated TER decreases induced by MT inhibitors (5, 32). Therefore, we next investigated effects of PKA activation on nocodazole-induced RhoA activation. As shown in Fig. 5, stimulation of PKA activity by forskolin significantly attenuated Rho activation in nonstimulated cells and completely abolished Rho activation in response to MT depolymerization. In our recent publication (5), we showed that Rho activation is observed as early as 15 min after nocodazole stimulation. This study (Fig. 5) shows that nocodazole-induced Rho activation was also observed after 30 min of stimulation and was inhibited by forskolin-induced activation of PKA. In addition, forskolin-induced attenuation of Rho activation was also observed after 15 min of nocodazole treatment (data not shown), which was consistent with the maximal effect of nocodazole on barrier disruption (Fig. 1).
Effect of PKA on nocodazole-induced MYPT1 phosphorylation.
The level of MLC phosphorylation in EC is regulated by MLCK- and Rho-dependent mechanisms (2, 13). Besides the potential for direct phosphorylation of MLC, Rho-activated kinase inhibits MYPT1 activity via phosphorylation of myosin-binding subunit, which leads to its dissociation from myosin (12). We have previously shown that MT disruption by nocodazole induces MYPT1 phosphorylation at Thr696 (5). Next, we examined the effect of PKA activation on MYPT1 phosphorylation in the pulmonary endothelium. As shown in Fig. 6, pretreatment of cells with forskolin significantly attenuated nocodazole-induced MYPT1 phosphorylation at Thr696.
Interaction of PKA and MT.
Immunofluorescent analysis of intracellular MT arrangements suggests that forskolin did not affect MT structure in pulmonary EC (Fig. 7A). Surprisingly, EC pretreatment with forskolin before nocodazole stimulation partially prevented MT dissolution induced by nocodazole (Fig. 7A). Quantitative analysis of MT content in control and nocodazole-stimulated EC (Fig. 7B) demonstrates the statistically significant protective effect of PKA activation on MT structure in the EC treated with nocodazole (0.2 μM).
We and others (23, 26) have noted that PKA plays an important role in vascular EC barrier regulation by modulating the balance between EC contractile and tethering forces. Recent studies indicate that cAMP elevation not only activates PKA but increases the activity of several other targets, including the small GTPase Rap1 (7). However, we and others (24, 26, 27) have previously shown that protective effects of cAMP on agonist-induced EC barrier dysfunction are likely mediated predominantly through PKA-dependent mechanisms. Consistent with this conclusion, inhibition of either cAMP (by cAMP antagonist Rp diastereomer of adenosine 3′,5′-cyclic monophosphorothioate) or PKA activities (by PKI inhibitor) significantly increases stress fiber formation as well as the formation of paracellular gaps indicating barrier compromise (23). MT disruption initiates specific signaling pathways that couple to microfilament network, resulting in EC contractility and barrier dysfunction (5, 32). In the present study, we examined the role of PKA activity in barrier alteration induced by MT disassembly.
Results of this study suggest a novel mechanism for PKA-mediated EC barrier protection that involves stabilization of the MT network and inhibition of small GTPase Rho and its downstream target, Rho-kinase, which prevents MLC phosphorylation, actomyosin contraction, and paracellular permeability. Quantitative analysis of MT content in control and nocodazole-stimulated EC (Fig. 7A) demonstrates a statistically significant protective effect of PKA activation on MT structure in EC treated with nocodazole (0.2 μM). Furthermore, nocodazole at this concentration caused statistically significant increases in permeability across human pulmonary EC, which was blocked by forskolin pretreatment. Our results are in a good agreement with the report by Qiao et al. (27) who have recently demonstrated that PKA activation inhibited thrombin-induced Rho activation and increases in EC permeability. Our results suggest several potential mechanisms of PKA-mediated EC barrier protection. One mechanism may involve direct RhoA inactivation by PKA-mediated phosphorylation of RhoA at Ser188 (22). Another mechanism of RhoA regulation may be linked to the MT network. Recent reports suggest an important role for MT network in functional regulation of small GTPases via MT-associated guanine nucleotide exchange factors (GEFs). GEFs control the cycling of Rho family GTPases (Rac, Rho, and Cdc42) between inactive GDP-bound form and activated GTP-bound form (35). Several MT-bound GEFs, including Rho-GEF-p190, GEF-H1, and Lfc have been recently characterized, and GEF-H1 activation by its dissociation from MT has been described (25). Among RhoA-specific exchange factors, Rho-GEF-p190 colocalizes with MT (25). Although the regulation of Rho-GEF-p190 is not completely clear, dissociation from MT may also modulate Rho-GEF-p190 activity. Thus MT dynamics are critical for MT-dependent regulation of Rho activity, and PKA-mediated mechanisms of MT stabilization may serve as a negative regulator of Rho function. Previous reports demonstrate association of PKA catalytic subunit with MT and actin in cultured hippocampal neurons (28). MT stability is controlled by a number of MT-binding proteins. Phosphorylation of the MT-associated protein Tau by PKA plays an important role in neuron development (3). MT-associated protein 2, also known as MAP2, is essential for MT stability and organization and also functions as a PKA-anchoring protein (1). Another MT-stabilizing protein, stathmin, is regulated by PKA-mediated phosphorylation (20). Although the complete set of MT-stabilizing proteins in EC has not been yet determined, we speculate that PKA-mediated phosphorylation of MT-associated proteins may be an important mechanism for regulation of MT stability.
It was previously demonstrated that the increase in MLC phosphorylation elicited by MT disruption is mediated by the small GTPase Rho and its effector Rho kinase rather than by MLCK activity (5, 32). We and others (2, 4, 5, 12) have also shown that activation of Rho-kinase induced MYPT phosphorylation at Thr696 and Thr850 and inhibited MYPT1 phosphatase activity in endothelium. Results of this study indicate that PKA activation attenuated MYPT1 phosphorylation as a result of nocodazole-induced activation of the Rho-Rho-kinase cascade. Whether PKA can directly affect MYPT1 activity in the endothelium is currently under investigation. Although PKA-specific phosphorylation sites within MYPT1 have not yet been identified, the functional role of MYPT1 phosphorylation by PKA has been established. It is believed that phosphorylation of MYPT1 by PKA alters its cellular localization, which is a key determinant of substrate-specific phosphatase activity (18). Thus changes in MYPT1 localization or protein conformation induced by PKA-mediated phosphorylation may prevent MYPT1 phosphorylation by Rho-kinase at Thr696 and Thr850, which is critical for MYPT1 activation.
In summary, on the basis of results of this study and previous reports, we propose a mechanism for PKA-dependent stabilization of MT network and actin cytoskeleton and regulation of EC permeability. Nocodazole-induced disruption of MT network may cause release of activated Rho-GEFs, which stimulate Rho and its effector Rho-kinase, leading to stress fiber formation, MLC phosphorylation, and EC barrier disruption. Stimulation of PKA by forskolin opposes these effects of nocodazole and attenuates nocodazole-induced Rho activation, either directly via PKA-mediated Rho phosphorylation or indirectly via modulation of Rho-specific GEF activities. In addition, PKA may stabilize the MT network by phosphorylation of MT-associated proteins, which prevents release of activated Rho-GEFs and Rho stimulation. As a result, PKA-mediated Rho inhibition inactivates Rho-kinase, which results in MLC dephosphorylation, inhibition of stress fiber assembly, and EC barrier protection.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-67307, HL-68062, and HL-58064.
The authors thank Nurgul Moldobaeva for superb technical assistance and Maria Birukova for technical assistance in preparation of the manuscript.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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