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Rapamycin Inhibits Release of Tumor Necrosis Factor-[Alpha] From Human Vascular Smooth Muscle Cells

Posted on: Tuesday, 1 June 2004, 06:00 CDT

Neointimal proliferation with plaque formation is the principal cause of coronary artery disease. In the neointima, inflammatory cytokines like tumor necrosis factor-[alpha] (TNF-[alpha]) are expressed by vascular smooth muscle cells (VSMCs). These cytokines stimulate proliferation and migration of VSMCs, events that are crucial to neointima formation. Stents, liberating rapamycin, have been shown to reduce neointima formation in human coronary arteries. The purpose of this study was to determine if rapamycin could inhibit the production of TNF-[alpha] by VSMCs. With institutional review board approval, VSMCs were cultured from saphenous vein segments obtained from five patients. Cells were identified as VSMC by immunostaining for smooth muscle [alpha]-actin. Cells were exposed to bacterial lipopolysaccharide (LPS), LPS plus rapamycin, or LPS plus isoproterenol for 24 hours. Cells with no treatment served as controls. The culture medium was then removed and analyzed for TNF-[alpha]. Additionally, the effect of treatment on viability was determined by assay of mitochondrial activity. TNF-[alpha] released into the culture medium is expressed as pg TNF-[alpha]/mg cell protein. Statistical analysis was by ANOVA. In control cells, TNF-[alpha] was undetectable in the culture medium. The addition of LPS (10 g/mL) increased TNF-[alpha] release to 4312 705 pg/mg at 24 hours. The addition of 1 ng/mL rapamycin with LPS reduced TNF- [alpha] production 50 per cent (P < 0.01 vs LPS alone). A similar reduction of TNF-[alpha] release was seen with 1 M isoproterenol. LPS, rapamycin, or isoproterenol did not affect cell viability. These data show that rapamycin effectively inhibits the release of TNF-[alpha] from VSMCs stimulated with inflammatory mediators like LPS. Rapamycin is as effective as agents that raise intracellular cyclic AMP (e.g., isoproterenol). Therefore, a potential mechanism for the effectiveness of rapamycin-releasing stents is reduction of inflammatory cytokine expression by VSMCs.

DURING THE PAST 10 YEARS, treatment of occlusive lesions in coronary arteries with percutaneous placement of metallic stents within the lumen of the vessel has increased. These metallic stents act as foreign bodies triggering an inflammatory response with consequent multiplication and migration of vascular smooth muscle cells (VSMCs). Proliferation and migration of these cells forms a neointima that results in a reocclusion rate in the stented vessel of 20 per cent to 40 per cent per year. In an attempt to reduce reocclusion, stents have been designed with a polymer coating that produces a sustained release of a pharmacological agent directly at the lesion site. These agents include the anticoagulants heparin and hirudin, antiinflammatory steroids, and antimitotic agents such as paclitaxel and rapamycin (sirolimulus). (For a recent review of coated stents in animal and human studies, see Refs. 1 and 2.) In ongoing randomized clinical trials of coated versus uncoated stents, only stents releasing rapamycin have been shown to reduce significantly the incidence of major coronary events and the incidence of restenosis. For instance, in a study incorporating 238 patients followed for 12 months, there was 0 per cent restenosis in the rapamycin group and 27 per cent in the group with uncoated stents.3 Likewise, rapamycin stents were associated with 6 per cent major cardiac events during the 12 months, whereas uncoated stents were associated with 29 per cent. These studies suggest a significant inhibitory effect of rapamycin on proliferation and migration of VSMCs to reduce neointima formation.

In cultured smooth muscle cells, rapamycin has been shown to inhibit proliferation and reduce migration.4, 5 Inflammatory cytokines such as tumor necrosis factor-[alpha] (TNF-[alpha]) and interleukin-6 (IL-6) are expressed by vascular smooth muscle cells within the neointima.6, 7 These cytokines can stimulate VSMC migration and proliferation.8, 9 Therefore, we tested the hypothesis that rapamycin could inhibit release of TNF-[alpha] by VSMCs.

Methods

Culture of Vascular Smooth Muscle Cells

With institutional review board approval and patient consent, segments of saphenous vein unused in coronary bypass were obtained from five patients. The segments were stripped of adventitia, opened longitudinally, and the endothelium removed. Expiants (2 2 mm) were placed luminal side down in a Petri dish and incubated with Dulbecco's Modified Eagle's Medium (DMEM) plus 20 per cent fetal bovine serum (FBS) and antibiotics (penicillin, 100 IU/mL, and streptomycin, 100 g/mL) at 37C in room air plus 5 per cent CO2. After 1 to 2 weeks, the tissue was removed, and the cells that had exited were cultured to confluence passed with 0.25 per cent trypsin and subcultured in DMEM plus 10 per cent FBS and antibiotics. Cells from each patient were grown to confluence and then stained with a monoclonal antibody to smooth muscle alpha actin. In experiments reported here, cells were from 5 patients and used in subcultures 4 through 8.

Cells were seeded into 24-well culture dishes and allowed to grow to confluence. The medium was removed, and fresh medium was added containing 10 g/mL bacterial lipopolysaccharide (LPS), LPS plus 1 ng/ mL rapamycin, or LPS plus 1 M isoproterenol. Cells without these treatments served as controls. The cells were incubated for 24 hours and the culture medium removed for assay of TNF-[alpha]. Then, 500 L of 0.4 N NaOH was added to the cells to solubilize protein.

Determination of Cell Protein

Cell protein in each well of the 24-well plate was determined by the method of Bradford.10

Assay for Tumor Necrosis Factor-[alpha]

Biologically active TNF-[alpha] in the cell culture medium was measured by a cell cytotoxicity assay using murine fibroblast L929 cells as described previously.11 L929 cells were seeded into 96- well plates at an initial density of 5.0 10^sup 4^ cells per well in 100 L of DMEM plus 5 per cent fetal bovine serum and antibiotics and incubated for 24 hours at 37C in 5 per cent CO2. After incubation, the medium was removed, and 50 L of DMEM with 5 per cent FBS containing 20 g/mL actinomycin D (final concentration 5 g/ mL) was added to all wells. In each plate, a standard curve was determined with serial dilutions of human recombinant TNF-[alpha]. Then, 150 L of test culture medium was added to the remaining wells in each 96-well plate. The plates were incubated for 18 hours, and the medium was decanted. The remaining viable L929 cells were stained with 50 L per well of 0.1 per cent crystal violet in 20 per cent ethanol, rinsed with phosphate-buffered saline and air-dried. Then, 100 L of methanol were added to each well 5 minutes prior to reading in order to solubilize the dye. The optical density of each well was then read on an automated microplate reader Elx 800 (Bio- Tek Instrument, Inc., Winooski, VT) at 595 nm. TNF-[alpha] data are expressed as pg/mg cell protein. All test agents, LPS, rapamycin, and isoproterenol were added directly into the L929 cell assay. All were without effect on the assay.

Cell Viability Assay

Cell viability was assessed by the mitochondrial-dependent reduction of methyltetrazolium (MTT) to formazan as described.12 Briefly, VSMCs were seeded into 96-well plates and allowed to grow to confluence. Cells were exposed to LPS (10 g/mL), rapamycin (1 ng/ mL), and rapamycin plus LPS for 24 hours. At the end of the experiment, cells in 96-well plates were incubated with MTT (0.5 mg/ mL) dissolved in culture medium for 4 hours. Formazan reduced from MTT was extracted with DMSO and was then quantitated by measurement of optical density by ELISA reader at 595 nm.

Statistical Analysis

Data were analyzed by ANOVA. Values are mean SE. P < 0.05 was considered significantly different.

Results

The cells cultured from the saphenous veins grew in the characteristic "hill-and-valley" pattern of VSMCs in culture and were uniformly stained for smooth muscle alpha actin. Additionally, staining for the endothelial cell marker, factor VIII related antigen, was negative. These findings indicate that the cells in culture were indeed vascular smooth muscle.

As shown in Fig. 1, the addition of 10 g/mL of LPS for 24 hours caused TNF-[alpha] release into the medium to increase to 4311 705 pg/mg cell protein. Incubation of cells with LPS plus 1 ng/mL rapamycin reduced release of TNF-[alpha] by about 50 per cent to 2105 681 pg/mg at 24 hours (P < 0.01 vs LPS alone). The addition of 1 M isoproterenol with LPS inhibited release to 2300 657 pg/mg (P < 0.01 vs LPS alone and not significantly different from LPS plus rapamycin). Rapamycin or isoproterenol, alone, had no effect on TNF- [alpha] release. Furthermore, VSMCs incubated in medium alone for 24 hours as controls had no detectable TNF-[alpha] in the culture medium (data not shown).

FIG. 1. Inhibition of tumor necrosis factor-[alpha] (TNF) release from human vascular smooth muscle cells by rapamycin and isoproterenol. Cultured human vascular smooth muscle cells were incubated with bacterial lipopolysaccharide (LPS), LPS plus rapamycin (Rap), or LPS plus isoproterenol (Lsop) for 24 hours. TNF released into the culture medium was analyzed by L929 cell cytotoxic\ity assay. Values are mean SE for experiments in cells cultured from five patients. *P < 0.05 versus LPS alone. In cells cultured for 24 hours without additions, no TNF was detected in the culture medium.

Figure 2 shows the results of the MTT assay for cell viability. Incubation of cells with LPS, rapamycin, or LPS plus rapamycin for 24 hours had no significant effect on cell viability.

Discussion

In these studies, we found that rapamycin significantly inhibited the production of TNF-[alpha] by human vascular smooth muscle cells. This effect of rapamycin on inflammatory cytokine production has not been previously reported. In these experiments, we used a rapamycin concentration of 1 ng/mL. This concentration is similar to the plasma level of rapamycin, approximately 1.5 ng/mL, found in minipigs in which rapamycin reduced neointimal formation in balloon- injured carotid arteries.13 Inhibition of LPS-stimulated TNF- [alpha] release from VSMC by rapamycin was as effective as 1 M isoproterenol. We have previously shown that [beta]-adrenergic agonists like isoproterenol inhibit the production of inflammatory cytokines through a cyclic AMP dependent mechanism that involves inhibition of cytokine gene expression.14 The mechanism by which rapamycin inhibited LPS-stimulated TNF-[alpha] production was not determined in this experiment. We did eliminate a reduction in cell viability induced by rapamycin as a potential mechanism, as MTT staining was not affected (Fig. 2).

FIG. 2. Effect of LPS, rapamycin, and LPS plus rapamycin on viability of human vascular smooth muscle cells. Viability was determined with the MTT assay (see "Methods") and expressed as spectrophotometric absorbance. Values arc mean SE for experiments in cells cultured from five patients.

As shown in the introductory part of this paper, rapamycin eluting stents appear to be effective treatment for stenotic arteries. The mechanism of this effect is unknown. In previous studies, rapamycin has been shown to inhibit two key events in plaque and neointima formation, proliferation and migration of VSMC.4, 5 Injury to an artery stimulates smooth muscle cells to proliferate, migrate, form extracellular matrix, release proteases, growth factors and cytokines which further contribute to the migration and proliferation. Our data suggest that part of the mechanism by which rapamycin inhibits this process of neointimal formation is by inhibition of inflammatory cytokine production.

DISCUSSION

DR. GRACE S. ROZYCKI (Atlanta, GA): Although the insertion of coronary artery stents is an acceptable treatment for symptomatic coronary artery disease, the high rates of re-stenosis have challenged investigators to improve the patency of these stents. In this elegant work, Dr. Adkins and his colleagues have developed a cell culture model to determine if the inflammatory cytokine, TNF- [alpha], stimulates the migration and proliferation of vascular smooth muscle cells within the neointima, the process believed to be responsible for the reocclusion of these stents.

I have the following questions for the authors:

1. Please explain the rationale for the comparison of rapamycin to isoproterenol on the production of TNF-[alpha].

2. The cells that were used as controls were not exposed to lipopolysaccharide, yet the data are not delineated in the manuscript. Would you comment on these data?

3. Why was rapamycin selected? Are there other agents that could have been used?

4. If the dose of rapamycin used in the study reduces TNF- [alpha] production by 50 per cent, why not increase the dose to improve the outcome? Did you do a dose-response curve to demonstrate the effects?

5. Does rapamycin decrease the production of other cytokines, for example IL-6?

I would like to congratulate Dr. Adkins on an excellent presentation and a well-designed experimental model to answer a question that has important implications for the practice of cardiac, vascular, and trauma surgery. I encourage them to continue their investigations in this area and look forward to the results of other outstanding contributions in this area.

DR. J. PATRICK O'LEARY (New Orleans, LA): You have shown, and I am willing to believe that there is an effect of rapamycin when coated on the stint and you're ascribing that to TNF alpha. The rapamycin may also have many other effects. It may affect metalloproteinases. It may affect cytokines like interleukin-6 or others. Why have you chosen TNF alpha as the culprit when it could have been some other culprit?

DR. JONATHAN ADKINS (Macon, GA): I'll start with Dr. Rozycki's question, why did we use isoproterenol? Isoproterenol is an agent that we are familiar working with in the lab and is a known inhibitor of tumor necrosis factor. So we wanted to compare rapamycin with a known inhibitor.

Regarding the control data, you are right, we did not put the controls in the results graph. This was due to the fact that lipopolysaccharide was not added to the controls and so no tumor necrosis factor was produced, which would have made it "0" on our results graph.

Why use rapamycin whereas other agents could be used? The stints are being coated with different agents such as corticosteroids, heparin, paclitaxel, and many others. So far, rapamycin has shown to be the most effective in preventing re-stenosis rates. The dose of rapamycin? In this study, we did use 1 ng/mL. We tried a dose 10 times higher than this and it showed effects of killing the cells. So a higher dose was not used, but maybe an incremental dose that was less than 10 the amount could be tried as far as preventing TNF. This goes along with Dr. O'Leary's question regarding the other cytokines involved. There, obviously, are many cytokines involved in the inflammatory response that results in neointimal proliferation: Interleukin-6, metalloproteinases, and as you mentioned, many others. These are all worth investigating. This study specifically just looked at tumor necrosis factor. As we know, it is one of the many cytokines involved, but there obviously could be others. Rapamycin could inhibit these others. That is research that would be worth doing in the future.

REFERENCES

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8. Newman WH, Castresana MR, Webb JG, Wang Z. Cyclic AMP inhibits production of interleukin-6 and migration in human vascular smooth muscle cells. J Surg Res 2003;57:57-61.

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13. Suzuki T, Kopia G, Hayashi S, et al. Stent-based sirolimus reduces neointimal formation in a porcine coronary model. Circulation 2001;104:1188-93.

14. Newman WH, Castresana MR, Webb JG, et al. Isoproterenol inhibits transcription of cardiac cytokine genes induced by reactive oxygen intermediates. Anesthesiology 2002;96:947-54.

JONATHAN R. ADKINS, M.D.,* MANUEL R. CASTRESANA, M.D.,[dagger] ZHONGBIAO WANG, M.D., PH.D.,[double dagger] WALTER H. NEWMAN, PH.D.[double dagger]

From the * Department of Surgery and [double dagger] Division of Basic Sciences, Mercer University School of Medicine, Macon, Georgia; and [dagger] Department of Anesthesiology and Perioperative Medicine, Medical College of Georgia, Augusta, Georgia

Supported by grants from the MedCen Foundation and the Clinical Research Center of the Medical Center of Central Georgia, Macon, Georgia.

Presented at the Annual Scientific Meeting and Postgraduate Course Program, Southeastern Surgical Congress, Atlanta, Georgia, January 31-February 3, 2004.

Address correspondence and reprint requests to Jonathan Adkins, M.D., 777 Hemlock Street, HB 140, Macon, GA 31201.

Copyright The Southeastern Surgical Congress May 2004

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