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(Circulation Research. 1999;84:867-875.)
© 1999 American Heart Association, Inc.

Original Contribution

Liposomal Delivery of Purified Inhibitory-B Inhibits Tumor Necrosis Factor-–Induced Human Vascular Smooth Muscle Proliferation

Craig H. Selzman, Brian D. Shames, Leonid L. Reznikov, Stephanie A. Miller, Xianzhong Meng, Hazel A. Barton, Ariel Werman, Alden H. Harken, Charles A. Dinarello, Anirban Banerjee

From the Departments of Surgery (C.H.S., B.D.S., S.A.M., X.M., H.A.B., A.H.H., A.B.) and Medicine (L.L.R., A.W., C.A.D.), University of Colorado Health Sciences Center, Denver, Colo.

Correspondence to Craig H. Selzman, MD, Department of Surgery, Campus Box C-320, University of Colorado Health Sciences Center, 4200 East Ninth Ave, Denver, CO 80262. E-mail craig.selzman{at}uchsc.edu

	Abstract

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Abstract—Vessel injury results in the elaboration of various cytokines, including tumor necrosis factor- (TNF-), which may influence vascular smooth muscle cell (VSMC) function and contribute to atherogenesis. We tested the hypothesis that TNF-–induced VSMC proliferation requires activation of the transcription factor nuclear factor-B (NF-B), which could be prevented by delivery of the NF-B inhibitory peptide, IB. TNF- induced concentration-dependent human VSMC proliferation, and neutralizing antibody to interleukin-6 reduced TNF-–induced VSMC proliferation by 65%. In TNF-–stimulated VSMCs, there was a 3-fold increase in NF-B–dependent luciferase reporter activity that was associated with degradation of IB. To determine an essential role for NF-B in TNF-–induced VSMC proliferation, recombinant IB was introduced into VSMCs via liposomal delivery. Under these conditions, TNF-–induced NF-B nuclear translocation and DNA binding were inhibited, NF-B–dependent luciferase activity was reduced by 50%, there was no degradation of native IB detected, interleukin-6 production was reduced by 54%, and VSMC proliferation was decreased by 60%. In conclusion, the mitogenic effect of TNF- on human arterial VSMCs is dependent on NF-B activation and may be prevented by exogenously delivered IB. Furthermore, liposomal delivery of endogenous inhibitory proteins may represent a novel, therapeutically accessible method for selective transcriptional suppression in the response to vascular injury.

Key Words: tumor necrosis factor • nuclear factor-B • inhibitory-B • vascular smooth muscle • cationic liposome

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tumor necrosis factor- (TNF-) is a pleiotropic cytokine that is abundantly expressed in atherosclerotic lesions.¹ In response to vascular injury, inflammatory cytokines and growth factors are released and influence vascular smooth muscle cell (VSMC) phenotype and growth. Although VSMC proliferation and migration are fundamental features of intimal hyperplasia and atherogenesis, ligand binding of TNF- receptors usually triggers cellular apoptosis. Thus, the presence of TNF- in atherosclerotic lesions raises important questions about its mechanistic role in the process. Studies to date have established that TNF- is produced in several vascular cells, including VSMCs themselves, and may influence VSMC production of cytokines and expression of adhesion molecules, as well as promote VSMC migration.^{2 3 4} Several investigators have reported that TNF- itself has no effect on VSMC apoptosis or growth.^{5 6} In contrast, other investigators have reported that TNF- induces modest (20%) increases in VSMC proliferation or stimulates growth such that detection was possible only after 6 days.^{7 8} As such, the relationship between TNF- and VSMC growth remains obscure.

Examination into the signaling pathways distal to TNF- receptor activation indicates that, in addition to proapoptotic cascades, TNF- receptors can also engage pathways that activate the transcription factor nuclear factor-B (NF-B). Activated NF-B has been identified in atherosclerotic lesions but not in normal vessels.⁹ NF-B is often viewed as a global regulator of cytokines that promotes gene transcription of mitogenic products, including interleukin (IL)-1ß, IL-2, IL-6, and IL-8, as well as adhesion molecules, acute-phase proteins, immunoreceptors, and TNF- itself.¹⁰ Accumulating evidence, however, suggests that NF-B has an important role in the signals that control VSMC proliferation. NF-B activity has been demonstrated constitutively in VSMCs in vitro,¹¹ as well as in atherosclerotic VSMCs in vivo.⁹ Additionally, NF-B activity appears to be important for serum- and thrombin-stimulated VSMC growth.^{12 13}

Regulation of NF-B is dependent, in part, on activation of proximal, sequential kinase cascades.¹⁴ When bound by its inhibitory protein, IB, classic NF-B (p65/p50) exists in the cytoplasm as an inactive dimer. On stimulation, phosphorylation of IB identifies it for ubiquitination and subsequent degradation. Released from the NF-B:IB complex, NF-B is free to translocate to the nucleus and engage DNA. Once activated, NF-B promotes the gene transcription of IB itself, thus creating an inducible autoregulatory system.¹⁵ In addition to binding cytoplasmic p65/p50 heterodimers, newly synthesized nuclear IB may also disrupt NF-B binding to DNA¹⁶ and promote NF-B translocation from the nucleus to the cytoplasm.¹⁷ Therefore, manipulation of IB levels represents an attractive strategy for modifying NF-B activity. Although TNF- induces NF-B DNA binding in VSMCs,⁹ the ability of IB to regulate NF-B activity and VSMC proliferation has yet to be examined. In the present study, we demonstrate that TNF- activation of NF-B is essential for the potent mitogenic effect of TNF- on VSMCs. Furthermore, TNF-–induced VSMC proliferation may be inhibited by strategies aimed at maintaining or elevating levels of IB. In particular, purified IB may be delivered by liposomes to VSMCs and inhibit both NF-B activity and VSMC proliferation.

	Materials and Methods

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Cell Culture and Proliferation Assay
Human VSMCs were isolated from segments of thoracic aortae harvested from transplant donors as previously described.¹⁸ Procurement of human VSMCs was approved by the Colorado Multiple Institutional Review Board (approval No. 96-161). Purity of isolation was demonstrated by the typical "hill and valley" morphology revealed by phase-contrast microscopy and, immunohistochemically, with uniform phallodin staining for F-actin and –smooth muscle actin (Sigma), as well as lack of staining for the endothelial cell surface antigen von Willebrand factor. Cultures were routinely screened and consistently remained >95% pure. VSMCs were trypsinized and plated at a density of 2500 cells per well on 1% gelatin-coated 96-well microtiter plates with a "complete medium" containing DMEM (Sigma), 5% each of FBS (Summit Biotechnology) and human cord serum (graciously provided by Dr Lawrence Horwitz, University of Colorado, Denver, Colo), 0.01% MEM vitamins (Sigma), 10 000 U/mL penicillin G, 10 000 mg/mL streptomycin sulfate, and 25 mg/mL amphotericin (GIBCO-BRL). After 8 hours, the medium was removed and replaced with serum-free medium for 48 hours to allow for growth arrest. Twenty-four hours after the substitution of medium with the experimental agent, rates of proliferation were determined using a nonradioactive cell proliferation assay (Promega). Other investigators have demonstrated this technique to be equivalent to cell counting and thymidine uptake.^{19 20} In addition, we have validated this method in our model of human VSMC proliferation. We have previously demonstrated that this assay accurately correlates with direct cell counting when accessing mitogen-induced VSMC proliferation.¹⁸ After the addition of 20 µL of methyltetrazolium salt/phenazine ethosulfate, plates were incubated at 37°C for 90 minutes. Absorbance was then recorded at 490 nm with a microtiter plate reader (Bio-Rad). Results, reported as optical densities, represent experiments done in quadruplicate from 3 separate donors during passages 1 through 4.

Liposome Preparation
Liposomal delivery of recombinant IB–glutathione S-transferase (GST) fusion protein was performed as a modification of a previously described technique.²¹ A lipid solution composed of 2.0 mg of egg L--phosphatidylcholine, 0.5 mg of cholesterol, 0.5 mg of 1,2-dioleoyl-3-trimethylammonium-propane, and 0.5 mg of dioleoyl phosphatidylethanolamine (Avanti Polar Lipids, Inc) was dissolved in chloroform and was dried in a chloroform-pretreated 12x75–mm glass tube by rotation in a vacuum. Human IB-GST fusion protein (Santa Cruz Biotechnology) was dissolved (50 µg) in 100 µL of 50 mmol/L Tris-HCl (pH 7.5) and was added to the dried lipids and agitated by alternate cycles of sonication (10 seconds) and vortex (20 seconds). Liposomes with the GST moiety alone were prepared in a similar manner, but IB-GST fusion protein was substituted with an equimolar concentration of recombinant GST. Control liposomes contained 100 µL of 50 mmol/L Tris-HCl buffer. The liposome mixture was extruded for 20 passes through a 0.1 mm membrane with the aid of an ethanol-pretreated extrusion device (LiposoFast, Avestin, Inc) and mixed with DMEM/5% FBS medium.

Immunohistochemistry
VSMCs were plated on chambered tissue culture slides (Becton Dickinson) at a density of 2000 cells per well in complete medium. After growth arrest in serum-free medium, VSMCs were incubated in experimental medium for 1, 2, or 4 hours. Slides were washed with cold PBS and fixed with a 70% methanol/30% acetone solution for 10 minutes. After air drying, slides were washed 3 times with PBS for 5 minutes and blocked in 10% goat serum for 25 minutes at room temperature. Subsequently, cells were incubated at 4°C overnight with rabbit polyclonal anti–NF-B p65 antibody (Santa Cruz Biotechnology), 1:40 dilution with PBS/1% BSA, and mouse monoclonal anti-GST antibody (Santa Cruz Biotechnology), 1:40 dilution. After 3 washes with PBS, cells were incubated in Cy3-labeled goat anti-rabbit IgG, 1:250 dilution, and Alexa-green goat anti-mouse IgG (Molecular Probes), 1:250 dilution, for 45 minutes in the dark at room temperature. After washing, nuclei were stained with bis-benzimide (2.5 µg/mL). Fluorescent images were observed with appropriate filter cubes and photographed using an automated Leica confocal microscope under full software control (Intelligent Image Innovations).

Cell Lysates
VSMCs were cultured to 50% to 70% confluence in 35-mm plates. After growth arrest for 48 hours, cells were incubated in new medium for 30, 60, or 120 minutes before harvesting the cell lysates. VSMCs were washed twice with cold PBS and incubated on ice for 30 minutes with 250 µL of a lysis buffer containing (in mmol/L) Tris 50, NaCl 100, EDTA 2, EGTA 2, and DTT 1, and protease inhibitor tablets (Boehringer Mannheim). Cells were scraped into Eppendorf tubes and centrifuged at 13 000g for 15 minutes. The resultant supernatant representing the cytosolic fraction was aliquoted and frozen at -70°C. The nuclear pellet was resuspended in 50 µL of a nuclear extraction buffer containing (in mmol/L) HEPES (pH 7.9) 20, EGTA 1, and DTT 1; 0.4 mol/L NaCl; and protease inhibitor tablets. The tube was then placed on ice for 30 minutes with gentle vortexing every 10 minutes. The nuclear extract was then centrifuged at 12 000g for 5 minutes at 4°C. The supernatant (nuclear fraction) was collected and stored at -70°C. Protein was quantified in both cytosolic and nuclear extracts with the Coomassie Plus protein assay (Pierce).

Western Blots
Cytosolic lysates were thawed, mixed with equal volumes of sample buffer (Bio-Rad), boiled for 10 minutes, and loaded at 15 µg of protein/lane. Electrophoresis was performed on 4% to 20% linear gradient SDS-polyacrylamide gels (Bio-Rad). After transfer to a nitrocellulose membrane (Bio-Rad), membranes were Ponceau stained (Bio-Rad) and digitally scanned to assure equivalent protein transfer between lanes. Subsequently, membranes were blocked in 5% nonfat milk at room temperature for 1 hour. The primary polyclonal rabbit anti-IB (Santa Cruz Biotechnology), 1:200, was added at room temperature and mixed for 1 hour. After sequential washing in 0.1% Tween-20 in PBS, membranes were incubated in horseradish peroxidase linked to a goat anti-rabbit secondary antibody (R&D Systems), 1:5000, for 45 minutes and detected using an enhanced chemiluminescence (ECL) system (Amersham).

Electrophoretic Mobility Shift Assay
Electrophoretic gel shift assays were performed on nuclear extracts as previously described.²² NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3') or activator protein-1 (AP-1; 5'-CGCTTGATGAGTCAGCCG-GAA-3') (Promega) was 5' end-labeled with [-³²P]ATP using T4 polynucleotide kinase. Unincorporated nucleotide was removed using a probe purification column (NucTrap, Stratagene). Five micrograms of nuclear protein was incubated with labeled oligonucleotide (100 000 to 200 000 cpm) in binding buffer ([in mmol/L] Tris-HCl 10 [pH 7.5], NaCl 50, EDTA 0.5, and MgCl₂ 1; 0.5 µg poly(dI-dC)-poly(dI-dC); 1% NP-40; and 4% glycerol) for 25 minutes at room temperature in a final volume of 25 µL. To demonstrate specificity of binding, 100-fold excess of unlabeled oligonucleotide (both NF-B and AP-1) was used as a specific competitor. Subsequently, the free oligonucleotide and oligonucleotide bound proteins were separated by electrophoresis on a native 4% polyacrylamide gel. The gel was then dried and exposed to an x-ray film with intensifying screens overnight at -70°C.

NF-B–Induced Luciferase Promoter Activity
As a surrogate for NF-B–dependent gene transcriptional activity, transfections were performed using a luciferase reporter construct containing a thymidine kinase promoter with 5 upstream tandem NF-B binding sites (kindly provided by Werner Falk, PhD, University of Regensburg, Germany). VSMCs were seeded at a density of 1x10⁵ cells per well in 35-mm tissue culture plates with complete medium and allowed to adhere overnight. A transfection mixture of 0.35 µg/well plasmid DNA, 1 µL/well LipofectAMINE (Life Technologies, Inc), and 200 µL/well serum-free DMEM was incubated for 40 minutes. After addition of 800 µL/well of serum-free medium, 1 mL of transfection mixture was added with 1 mL of DMEM/20% FBS in each well. VSMCs were placed in the incubator for 12 hours. Subsequently, the cells were washed with PBS, and experimental agents were added.

After 10 hours of stimulation, supernatants were removed and luciferase production was determined using a commercial kit (Promega). Cells were washed with PBS and incubated with 200 µL/well of reporter lysis buffer for 10 minutes. Cells were scraped, transferred into Eppendorf tubes, and vortexed for 15 seconds. Cells were subjected to 1 freeze-thaw cycle, vortexed, and centrifuged at 12 000g for 15 seconds. Twenty microliters of supernatant was mixed with the luciferase substrate reagent, and absorbencies (expressed as light units) were determined by a fluorimeter (Lumat LB 9501, Berthold). Transfection efficiency was routinely surveyed by cotransfection with a LacZ-reporter construct (Invitrogen) driven by a cytomegalovirus upstream promoter (0.2 µg DNA/well). ß-Galactosidase (ß-Gal) activity was determined by standard assay (Invitrogen). In general, ß-Gal activity remained invariant throughout the various experimental conditions. Luciferase data are corrected for ß-Gal activity and are reported as percentage activity compared with control cells. To create an NF-B–deleted plasmid, the NF-B consensus binding base pairs were cut using HindIII and BglII, filled in with the Klenow fragment of DNA polymerase 1, and ligated with T4 DNA ligase (GIBCO-BRL).

Cytokine Assays
Cytokines were measured in cell supernatants by an ECL method.²³ Briefly, polyclonal goat anti-human IL-6 (R&D Systems) was labeled with biotin (Igen Inc). The biotinylated antibody was diluted to a final concentration of 1 mg/mL in ECL buffer that contained PBS, pH 7.4, with 0.25% BSA, 0.5% Tween-20, and 0.01% azide. Biotinylated antibodies were incubated with 1 mg/mL of streptavidin-coated paramagnetic beads (Dynal Corp) for 30 minutes at room temperature with vigorous shaking. Subsequently, it was combined with cell supernatants (25 µL) and goat monoclonal anti-human IL-6 (R&D Systems) previously labeled with ruthenium (Igen). This mixture was shaken vigorously for an additional 3 hours. The reaction was quenched with 200 mL of ECL buffer, and the chemiluminescence was determined using an Origen Analyzer (Igen). The detection limit was 40 pg/mL.

Statistical Analysis
Data are presented as mean±SEM. ANOVA with Bonferroni-Dunn post hoc analysis was used to analyze differences between experimental groups. Statistical significance was accepted within 95% confidence limits.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TNF- Stimulates Human VSMC Proliferation
After growth arrest, human aortic VSMCs were stimulated with human TNF- (R&D Systems) for 24 hours. As shown in Figure 1, TNF- induced concentration-dependent human VSMC growth. Compared with unstimulated control cultures, TNF- induced VSMC proliferation in concentrations as low as 100 pg/mL (0.67±0.09 versus 0.42±0.05, P<0.002). Maximal TNF- stimulation was observed at 10 ng/mL (0.81±0.03, P<0.002 versus control). On the basis of these results, a concentration of 10 ng/mL of TNF- was used in subsequent experiments.

View larger version (18K): [in this window] [in a new window]   Figure 1. TNF-–induced vascular smooth muscle proliferation. VSMCs were incubated in control medium (Cont) or increasing concentrations of TNF- for 24 hours. Results represent experiments done in quadruplicate from 2 individual donors on 3 separate occasions. *P<0.002, compared with control.

TNF- Translocates and Activates NF-B
To determine the influence of TNF- on NF-B activation in VSMCs, we examined the ability of TNF- to promote NF-B nuclear translocation, DNA binding, and functional gene transcription. Immunohistochemistry revealed that, in control VSMCs, NF-B staining in the cytoplasm was significantly more intense than in the nucleus (Figure 2A). In the cytoplasm, NF-B was present both in the lamellopodia and in the cell body. After stimulation with TNF-, red fluorescent pixels in the cytoplasm appeared to have gathered into coarser structures, compared with the fine pattern in controls, suggesting increased density of NF-B at subcellular sites (Figure 2B). Additionally, colocalization of red fluorescences (NF-B) at blue pixels (nuclei) led to more purple pixels, suggesting the intranuclear presence of translocated NF-B. Within the nucleus, NF-B showed fine, punctate patterns, as well as diffuse segments. In many cells, NF-B appeared to be concentrated at only 1 or both opposite poles of the nucleus.

View larger version (89K): [in this window] [in a new window]   Figure 2. Immunohistochemical localization of NF-B in human VSMCs. Cells stained for NF-B (red) and nuclei (blue) were examined with fluorescent microscopy (x95). Compared with control cells (A), 1 hour of TNF- exposure (10 ng/mL) resulted in nuclear translocation of NF-B as visualized by intranuclear purple staining (B). TNF-–treated VSMCs were incubated simultaneously with liposomal IB and additionally stained against GST-fusion protein (green). Liposomal delivery of the IB-GST complex successfully entered the cells (C) and effectively inhibited TNF-–induced NF-B translocation (D).

To determine whether the translocated NF-B was competent and able to bind DNA, electromobility shift assays were performed. After TNF- stimulation of VSMCs, considerably more NF-B binding to DNA was observed (Figure 3). To verify that DNA binding resulted in functional gene transcription, we performed transient transfections with a luciferase reporter construct. Our preliminary experiments demonstrated a fairly modest increase in luciferase activity after stimulation (3-fold). Initially, several different methods of transfection were used (calcium phosphate, lipofection, and dendrameres), and all demonstrated a similar fold increase. In addition to determining efficiency of transfection with ß-Gal activity and to validate this 3-fold response, we performed a series (n=9) of experiments in which cell density and transfection exposure were varied. The scattergram depicted in Figure 4 demonstrates that, although a given condition may increase luciferase activity in control cells, the corresponding increase in TNF-–stimulated cells remained consistent. Indeed, there was a high degree of correlation between transfection efficiency with luciferase activity (r²=0.99). As represented by the slope of the regression curve (y=2.93x–128), the addition of TNF- consistently induced a near 3-fold elevation of luciferase activity compared with control. To verify that these effects were truly related to TNF- treatment and NF-B activity, the NF-B consensus sequence was excised from the plasmid by restriction enzymes and religated. After transfection and TNF- stimulation, luciferase activity was at the level of control (252±12 versus 246±9, respectively). From these results, we infer that TNF- not only promotes NF-B nuclear translocation and DNA binding but promotes NF-B–dependent gene transcription.

View larger version (40K): [in this window] [in a new window]   Figure 3. Electrophoretic mobility shift assay for NF-B DNA binding. VSMCs were incubated for 1 hour with various combinations of TNF-, liposomal IB-GST protein (IB/GST), or liposome containing the GST moiety alone (GST). Nuclear extracts were obtained, and 2 µg of labeled nuclear protein was loaded in each lane. TNF- increased NF-B DNA binding, which was inhibited with liposomal IB-GST. The liposome with the GST moiety alone had no effect on TNF-–induced NF-B DNA binding activity.

View larger version (14K): [in this window] [in a new window]   Figure 4. TNF- induction of NF-B–dependent luciferase activity in human VSMCs. Data are from 9 consecutive experiments using 3 individual donors with varying cell numbers and transfection exposure. Results of luciferase activity in control and TNF-–treated (10 ng/mL) cells are recorded in light units. At any level of luciferase activity in control cells, TNF- stimulation resulted in consistent luciferase production. Regression analysis of individual data points demonstrates a close correlation of TNF-–induced NF-B activity compared with control cells (r2=0.99). As represented by the slope of the regression line (y=2.93x–128), TNF- stimulation reproducibly resulted in a 3-fold increase in NF-B–driven luciferase activity.

TNF- Stimulates IB Degradation
Having demonstrated that TNF- induces NF-B nuclear translocation and gene transcription, we sought to determine the mechanism of this activation. TNF- is thought to induce NF-B translocation by promoting degradation of IB, thus allowing NF-B to freely translocate into the nucleus and initiate transcription. Western blots of whole-cell lysates of TNF-–treated VSMCs were performed to examine the relative amounts of IB (Figure 5A). When VSMCs were incubated with control medium for 30, 60, and 120 minutes, no differences in the levels of IB were observed (30-minute time point is shown). However, treatment with TNF- (10 ng/mL) essentially depleted cellular IB within 30 minutes of stimulation. Within 90 minutes, IB levels returned to the level of control. These results suggest that TNF- induces an early degradation of IB, thus allowing NF-B to translocate into the nucleus.

View larger version (30K): [in this window] [in a new window]   Figure 5. Western immunoblot for IB. VSMCs were stimulated for either 30, 60, or 120 minutes, cell lysates were extracted, and 15 µg of protein was loaded in each lane of SDS-polyacrylamide gels. Representative gels were reproduced on 5 occasions with 2 separate cell lines. A, Cont indicates IB levels in control cells 30 minutes after delivery of control medium. Control cells had similar levels of IB at 60 and 120 minutes (data not shown). After stimulation with TNF-, native 37-kDa IB protein was rapidly degraded. B, Concomitant stimulation with TNF- and liposomal delivery of the 65-kDa IB-GST fusion protein inhibits TNF-–induced degradation of the native IB protein.

IB Regulates TNF-–Induced NF-B Translocation and Activity
Because TNF- promotes degradation of IB, we next examined whether maintenance of IB levels could dominantly inhibit NF-B activation. We used 2 strategies to investigate this question. In several cell lines, preventing IB degradation with agents such as calpain inhibitors or inducing IB levels with dexamethasone precludes the release of active NF-B.^{24 25} These 2 inhibitors of NF-B activation were added to human VSMCs concomitantly with TNF- stimulation. As shown in Figure 6, TNF- induced NF-B–dependent luciferase production (319%±32.5, P<0.001). Compared with control, neither calpain inhibitor-1 (100 µg/mL, Calbiochem) or dexamethasone (1 µmol/L, Sigma) had an effect on luciferase activity when added to unstimulated cells. However, in the presence of either dexamethasone or calpain inhibitor-1, TNF-–induced NF-B–dependent luciferase activity was reduced by 66% and 74%, respectively (P<0.001).

View larger version (17K): [in this window] [in a new window]   Figure 6. Effect of dexamethasone (Dex) and calpain inhibitor-1 (CI-1) on NF-B activity. Vertical axis depicts percentage change in luciferase activity with control (Cont) set at 100%. Data represent 3 separate experiments done in duplicate from 2 individual donors. Control medium, dexamethasone, and calpain inhibitor-1 had no effect on unstimulated NF-B activity. Compared with control, TNF- stimulated a 3-fold increase in luciferase activity (P<0.001). Both dexamethasone and calpain inhibitor-1 inhibited TNF-–induced NF-B–dependent luciferase activity (*P<0.001, compared with TNF-).

Calpain inhibitors and glucocorticoids are nonspecific inhibitors of NF-B activation; they may act to effect proteolysis and signaling of several other transduction intermediates, not just IB. Therefore, we sought to achieve an acute elevation of IB levels by direct delivery of the recombinant protein. Preliminary experiments using VSMC proliferation and cell necrosis as outcome variables demonstrated that the optimal dose of purified IB was a final concentration of 20 µg/mL, and as such, this concentration was used for all ensuing experiments (data not shown). VSMCs were concomitantly treated with TNF- (10 ng/mL) and liposome-encapsulated IB-GST fusion protein for 1 hour and subsequently immunohistochemically stained for NF-B and GST protein (Figure 2C). The number of VSMCs with intracellular staining for GST protein (green) were counted over 10 high-power fields. More than 95% of VSMCs had uptake of the stain, suggesting that delivery of the liposomal IB-GST fusion protein complex was not only successful but also efficient. The GST-tagged IB was observed restricted to the cytoplasm and perinuclear membranes. Superposition of green pixels (IB-GST) with red pixels (NF-B) produces a yellow color. Indeed, more pixels are green-yellow than green, especially at the nuclear membranes and within the cell body. This observation suggests that delivered IB is colocalized with cytoplasmic NF-B. Indeed, compared with TNF-–treated VSMCs (Figure 2B), liposomal IB appears to actively inhibit TNF-–induced NF-B nuclear translocation (Figure 2D). Immunohistochemical staining after treatment with empty liposome or liposome containing the GST moiety alone demonstrated no effect on TNF-–induced NF-B translocation (data not shown).

We corroborated these immunohistochemical observations with Western blots. After simultaneous delivery of liposomal IB and TNF- to VSMCs, Western immunoanalysis for IB demonstrated a strong band at 65 kDa, corresponding to an excess of exogenous IB-GST fusion protein (Figure 5B). Whereas TNF- treatment resulted in the degradation of IB at 30 minutes (Figure 5A), concurrent delivery of liposomal IB appeared to maintain the 37-kDa IB band at this early time point. These results demonstrate that liposomal delivery of IB protein was sufficient to prevent TNF-–induced proteolysis of native IB.

To further validate the influence of liposomal IB on NF-B activity, we performed gel-shift assays on TNF-–stimulated VSMCs. As depicted in Figure 3, TNF- increased NF-B DNA binding. Simultaneous delivery of liposomal IB abrogated the TNF-–induced increase in NF-B DNA binding. The control liposome containing the GST moiety alone had no effect on TNF-–induced NF-B DNA binding. Binding specificity was confirmed by including excess of unlabeled consensus oligonucleotide, which resulted in the obliteration of the NF-B band. To verify that the liposomal IB specifically influenced the NF-B signaling system, we performed parallel gel-shift assays for another inflammatory transcription factor, AP-1. As demonstrated in Figure 7, the TNF-–induced increase in AP-1 DNA binding was not influenced by either liposomal IB or the liposome containing the GST moiety alone.

View larger version (34K): [in this window] [in a new window]   Figure 7. Electrophoretic mobility shift assay for AP-1 DNA binding. VSMCs were incubated for 1 hour with various combinations of TNF-, liposomal IB-GST protein (IB/GST), or liposome containing the GST moiety alone (GST). Nuclear extracts were obtained, and 2 µg of labeled nuclear protein was loaded in each lane. TNF- increased AP-1 DNA binding, which was not influenced by concurrent treatment with liposomal IB-GST. In addition, the liposome with the GST moiety alone had no effect on TNF-–induced AP-1 DNA binding activity. Therefore, liposomal IB appears to specifically intervene in the NF-B signaling pathway.

Although liposomal IB clearly inhibited TNF-–induced nuclear translocation of NF-B, confirmation of functional NF-B–driven gene transcriptional activity was needed. As shown in Figure 8, in unstimulated cells, liposome-introduced IB had little effect on luciferase production compared with control. When incubated simultaneously with TNF-, liposomal IB inhibited TNF-–induced NF-B–dependent luciferase activity by >50%. Delivery of liposome vehicle as well as liposomes with recombinant GST moiety alone with or without TNF- had no effect on luciferase production. These latter results suggest that our observations appear to be directly related to the IB protein.

View larger version (18K): [in this window] [in a new window]   Figure 8. Effect of liposomal delivery of IB on NF-B activity. Vertical axis depicts percentage change in luciferase activity with control (Cont) set at 100%. Data represent 3 separate experiments done in duplicate from 2 individual donors. Compared with control, TNF- induced a 3-fold increase in luciferase activity (P<0.001). Delivery of liposomal IB (IB), empty liposome (Lipo), or liposome containing the GST moiety alone (GST) had no effect on unstimulated NF-B activity. However, concurrent delivery of liposomal IB with TNF- inhibited TNF-–induced NF-B-dependent luciferase activity (*P<0.001, compared with TNF-). Neither empty liposome or liposome with GST alone influenced TNF-–induced NF-B activity.

IB Inhibits TNF-–Induced VSMC Proliferation
To determine the requirement for NF-B activation and TNF-–induced VSMC proliferation, we examined the effect of IB on VSMC proliferation. Unstimulated VSMCs treated with dexamethasone, calpain inhibitor-1, and liposomal IB proliferated at the same rate as VSMCs without added inhibitors. In addition, neither control liposomes nor liposomes with recombinant GST moiety alone affected unstimulated or TNF-–stimulated VSMC proliferation. As depicted in Figure 9, TNF- resulted in a 2.7-fold increase in VSMC proliferation compared with control (P<0.001). In the presence of calpain inhibitor-1 or dexamethasone, TNF-–induced VSMC proliferation was reduced by 50% and 42%, respectively (P<0.001). Furthermore, direct delivery of liposomal IB also inhibited TNF–induced VSMC proliferation by 61% (P<0.001). Calpain inhibitor-1, dexamethasone, and liposomal IB-treated VSMCs all remained >95% viable by trypan blue exclusion.

View larger version (19K): [in this window] [in a new window]   Figure 9. Effect of IB on VSMC proliferation. Results represent experiments done in quadruplicate from 2 individual donors on 3 separate occasions. Neither liposomal IB (IB) nor liposome containing the GST moiety alone (GST) influenced unstimulated VSMC proliferation. Compared with control, TNF- (10 ng/mL) induced a 2.7-fold increase in VSMC proliferation (P<0.001). Nonspecific inhibition of NF-B activity with dexamethasone (Dex) and calpain inhibitor-1 (CI-1), as well as specific inhibition of NF-B activity with liposomal IB, abrogated TNF-–induced VSMC proliferation (*P<0.001, compared with TNF-). The liposome with the GST moiety alone had no effect on TNF-–induced VSMC proliferation.

Influence of IL-6 on TNF-–Mediated VSMC Proliferation
NF-B regulates transcriptional activity of a wide array of mitogenic genes, including IL-6.¹⁰ To mechanistically link the effects of TNF-–induced NF-B activation and subsequent VSMC proliferation, we examined the influence of TNF-–induced IL-6 release on VSMC growth (Figure 10). After TNF- stimulation, IL-6 production increased nearly 40-fold (3104 pg/mL±159 versus 75 pg/mL±12, P<0.01). Concomitant liposomal delivery of IB decreased IL-6 release by 54% (1426 pg/mL±155 versus TNF-, P<0.01). To determine the influence of TNF-–induced IL-6 release on VSMC proliferation, cells were stimulated with TNF- in the presence of a monoclonal antibody to IL-6 (kindly provided by Dr Daniela Novick, The Weizman Institute of Science, Rehovot, Israel). TNF- stimulated VSMC proliferation (0.54±0.06 versus control, 0.14±0.01, P<0.01). The mitogenic response of VSMCs to TNF- was attenuated with neutralization of IL-6 by 65% (0.24±0.02 versus TNF-, P<0.01). Delivery of a similar antibody isotype (IgG₁) had no effect on TNF-–induced mitogenicity.

View larger version (16K): [in this window] [in a new window]   Figure 10. Effect of IL-6 on TNF-–induced mitogenicity. A, Compared with control, TNF- induced IL-6 release (P<0.001), which was reduced by 50% with concurrent liposomal delivery of IB (*P<0.01 vs TNF-; P<0.01 vs control). Data represent 3 separate experiments done in duplicate from 2 individual donors. B, Compared with control, TNF- induced VSMC proliferation (P<0.001), which was reduced with concomitant delivery of a monoclonal antibody (mAb; 1 µg/mL) against IL-6 (*P<0.001 compared with TNF-). Data represent 3 separate experiments done in quadruplicate from 2 individual donors.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Whereas the present study demonstrates that TNF- induces a nearly 3-fold increase in VSMC proliferation, other investigators have reported that TNF- has little effect on VSMC growth. Geng et al⁶ observed no effect on VSMC viability or apoptosis in human VSMCs when treated with TNF- alone. However, in that study, concentrations of TNF- in excess of 100 ng/mL were used. Indeed, our data are consistent with those results in that we observed less proliferation at our highest dose of 100 ng/mL (Figure 1). Using comparable concentrations of TNF-, Morisake et al⁵ observed no influence of TNF- on rabbit aortic smooth muscle cells. This disparity might be related to species differences. Importantly, the later study demonstrated that TNF- pretreatment transformed VSMCs into a synthetic phenotype.

The location of NF-B in resting smooth muscle cells and its translocation after stimulation has not been imaged to our knowledge (Figure 2). The results confirm the familiar paradigm of nuclear translocation but also suggest unexpected detail, including the density and distribution of newly translocated NF-B. Although other investigators have reported TNF-–induced NF-B DNA binding activity in VSMCs,⁹ few have mechanistically linked this activation with cellular proliferation. Current evidence supporting the proliferative mechanisms of TNF- is contingent on the cell line examined. In U-937 cells, TNF-–induced activation of NF-B is not sufficient to influence cellular proliferation.²⁶ Conversely, TNF-–driven NF-B activity induces proliferation in HuT 78 cells.²⁷ The relationship between TNF- and VSMC growth is dependent, in part, on the TNF- receptor engaged. Although the TNF- p55 receptor directs signals directed toward apoptosis, it may also, along with the TNF- p75 receptor, promote activation of sequential kinases, ultimately resulting in nuclear translocation of NF-B.^{14 28} In addition, the TNF- p55 receptor may activate the sphingomyelin pathway, thus inducing ceramidase-dependent NF-B activation.²⁹ The relative contributions of signaling from these 2 receptors in VSMCs remain unknown. Although TNF- programs apoptotic signals in several cell lines, our observations suggest that in human VSMCs, a low concentration of TNF- promotes signals that favor proliferative, rather than apoptotic, events.

Although the present study demonstrates that NF-B activation is essential for TNF-–induced VSMC proliferation, the mechanism of NF-B–driven VSMC proliferation remains speculative. On one hand, evidence suggests that NF-B can suppress TNF-–induced apoptosis in several cell lines.³⁰ As such, activation of NF-B provides a survival pathway that may balance TNF-–mediated signals in favor of cell growth. Alternatively, TNF-–induced activation of NF-B results in the production of several well-known mitogens, including platelet-derived growth factor, IL-1ß, IL-6, IL-8, and TNF- itself.³¹ IL-6 is 1 NF-B–dependent gene product that is produced in VSMCs and can be a potent growth factor for VSMCs.³² As such, we tested the concept that TNF-–induced mitogenicity is, in part, related to NF-B–dependent IL-6 production. TNF- results in increased IL-6 production, which is inhibited by lipofection with IB (Figure 9). The promoter for the IL-6 gene involves several transcription factors including NF-B and nuclear factor–IL-6. Indeed, delivery of IB to TNF-–stimulated cells fails to inhibit IL-6 production completely. On the other hand, the degree of inhibition of TNF-–induced VSMC proliferation afforded by concurrent treatment with a neutralizing monoclonal antibody to IL-6 was 65% and may be greater at higher concentrations of anti-IL-6. Although IL-6 is 1 of many TNF- and NF-B–induced genes, these data suggest that TNF- induction of IL-6 has an important influence on VSMC proliferation.

Because of its central role in the transcription of stress genes, NF-B is an attractive therapeutic target for inflammatory disorders. Several experimental approaches have been implemented to inhibit NF-B activity, including transdominant IB mutants, antisense p65 oligonucleotides, microinjection of IB, protease inhibitors, and anti-inflammatory and immunosuppressive drugs.³³ However, these strategies can be limited by their nonspecific effects or clinical inaccessibility. Although dexamethasone and calpain inhibitors prevent NF-B activation and inhibit TNF-–induced VSMC proliferation, both are nonspecific inhibitors and may influence other intracellular events. Therefore, direct delivery of the specific natural inhibitor, IB, is an engaging strategy. Currently, there are 6 known members of the IB family (, ß, , , , and Bcl-3). The present study focuses on IB, but the relative importance of these different isoforms of IB in coordinating NF-B activity remains uncertain. The interaction between IB and NF-B is, however, the best understood of the isoforms. Furthermore, it appears that unlike IL-1ß, TNF- has little effect on IBß degradation in VSMCs.⁹

Cationic liposomes have typically been used as a method to introduce DNA intracellularly. Few studies demonstrate successful delivery of polypeptide proteins by liposomes. Several different liposomal preparations exist. Recently, Scott-Burden et al³⁴ reported that in rat and bovine aortic VSMCs, liposomes alone stimulated inducible nitric oxide synthase expression. Nonspecific lipid effects and nitric oxide production by the liposomal preparation are potentially troublesome, as they may alter VSMC proliferation itself. In the present study, the empty liposomal preparation, as well as the liposome with recombinant GST moiety alone, had no effect on NF-B DNA binding, NF-B–dependent luciferase activity, or VSMC proliferation. These results suggest that the empty submicrometer liposome preparations used in the present study lack detectable independent biological activity in our model of human VSMCs.

Although liposomal gene transfer has been used both in vivo and in vitro, nonviral vectors of gene transfer have been limited by low transfection efficiency.³⁵ In the present study, recombinant IB was attached to a GST tag that could be detected by anti-GST antibodies. Immunohistochemistry with a fluorescently labeled secondary antibody revealed that the liposomally delivered protein complex entered >95% of VSMCs. Furthermore, electromobility shift assays demonstrated that liposomal IB specifically and effectively inhibited NF-B DNA binding. In addition, Western immunoblots for IB demonstrated large amounts of recombinant IB protein inside the cell. Interestingly, simultaneous delivery of TNF- and exogenous IB resulted in the maintenance of native 37-kDa IB levels. TNF- signals result in the phosphorylation of IB by activating kinases that program its degradation, thus allowing NF-B translocation.²⁴ Although this cascade of NF-B activation is well recognized, some evidence suggests that NF-B activation may occur without proteolysis of IB by a second parallel pathway,³⁶ perhaps by direct tyrosine phosphorylation of IB itself.³⁷ These alternative pathways may explain, in part, the inability of liposomally delivered IB to return TNF-–induced luciferase activity completely to the level of control. Nonetheless, one may speculate that delivery of excessive IB overwhelmed the TNF-–induced proteolytic cascade, thus preventing degradation of the native peptide and resultant NF-B activation.

We examined the effect of TNF- alone on VSMC proliferation. Recognizing the diverse cytokine milieu associated with vascular injury, the effects of TNF- ultimately may be determined by its interactions with other inflammatory mediators and the stage of vessel remodeling. Indeed, when delivered with interferon-, TNF- appears to promote VSMC apoptosis and nitric oxide production.⁶ Although VSMC proliferation is important in intimal hyperplasia, VSMCs in advanced lesions are often quiescent.³⁸ Quite possibly, TNF- activates intracellular signals that depend, in part, on the phenotype of the stimulated VSMCs. As such, one conceivable explanation for these diverse observations is that in the early response to vascular injury, TNF- promotes VSMC proliferation and the development of neointimal hyperplasia. As the lesions mature, TNF- may act to promote apoptosis of VSMCs, thus influencing the integrity of the advanced atherosclerotic plaque. This latter effect may ultimately contribute to plaque instability and thrombosis associated with acute myocardial infarction.³⁹

Within the inflammatory paradigm of atherogenesis, we demonstrate that TNF- induces human VSMC proliferation that is dependent on activation of NF-B and is associated with IL-6 release. Furthermore, we demonstrate that TNF-–induced VSMC proliferation may be inhibited by strategies aimed at increasing intracellular levels of IB. In particular, we show that purified IB may be directly delivered to VSMCs by liposomes and inhibit both NF-B activity and VSMC proliferation. Direct administration of native inhibitory proteins, such as IB, may offer a novel, clinically accessible method of selective transcriptional regulation over signaling events important in the response to vascular injury.

	Acknowledgments

 
This work was supported in part by National Institutes of Health grants GM08315 (to C.H.S.), GM49222 (to A.H.H.), and AI15614 (to C.A.D.) and the Pacific Vascular Research Foundation (G.H.S.). We are grateful to Giamila Fantuzzi, PhD; Daniela Novick, PhD; and Sylene Johnson for their assistance.

Received July 14, 1998; accepted February 4, 1999.

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