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(Circulation. 2003;107:911.)
© 2003 American Heart Association, Inc.

Basic Science Reports

Rapamycin Induces Heme Oxygenase-1 in Human Pulmonary Vascular Cells

Implications in the Antiproliferative Response to Rapamycin

Gary A. Visner, DO; Fuhua Lu, PhD; Hailan Zhou, PhD; Jun Liu, MS; Kristy Kazemfar, BS; Anupam Agarwal, MD

From the Departments of Pediatrics (G.A.V., F.L., H.Z., J.L., K.K.) and Medicine (A.A.), College of Medicine, University of Florida, Gainesville.

Correspondence to Dr Gary A. Visner, Department of Pediatrics, University of Florida, Box 100296, 1600 SW Archer, Gainesville, FL 32610. E-mail visnega{at}peds.ufl.edu

	Abstract

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Background— Rapamycin is an immunosuppressive agent with antiproliferative properties against not only lymphocytes but also vascular endothelial and smooth muscle cells, and it reduces the fibroproliferative response to vascular injury. Heme oxygenase-1 (HO-1) has also been shown to have graft protective effects and to inhibit vascular remodeling. In this study, we evaluated whether there is an interaction between rapamycin and HO-1.

Methods and Results— In human pulmonary artery endothelial or smooth muscle cells, HO-1 expression was evaluated in response to rapamycin or wortmannin, an inhibitor of the upstream modulator of mammalian target of rapamycin (mTOR) PI-3K. We also evaluated whether the inhibitory actions of rapamycin on platelet-derived growth factor–dependent proliferation was mediated by HO using the chemical inhibitor tin protoporphyrin. Rapamycin induced HO-1 expression in both pulmonary endothelial and smooth muscle cells, whereas no to little increase was seen in response to another immunosuppressive agent, cyclosporin A. HO-1 expression was also increased in response to wortmannin, suggesting that the PI-3K–mTOR pathway is required for this induction. Inhibition of HO activity resulted in a loss of the antiproliferative activity of rapamycin in growth factor–stimulated smooth muscle cells.

Conclusions— The induction of HO-1 expression by rapamycin and, more importantly, the effects of tin protoporphyrin, an inhibitor of HO activity, on the antiproliferative actions of rapamycin suggest that the effects of rapamycin may be, at least in part, modulated by its actions on HO-1.

Key Words: heme oxygenase • sirolimus • wortmannin • cells, endothelial • cells, muscle, smooth

	Introduction

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Rapamycin is a fungal product with potent antiproliferative activity, and its lymphopenic properties initially generated interest in using it as an immunosuppressive agent in transplantation.¹ The immunosuppressive properties of rapamycin are related to the inhibition of T-cell proliferation induced by cytokines.² In addition, rapamycin inhibits growth factor–stimulated proliferation or migration of endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts,^3–5 and these effects of rapamycin have led to its use to prevent vascular neointimal proliferation and restenosis^6,7

Even though rapamycin has a structure similar to that of FK506 and binds to FK-binding protein 12 (FKBP12), the rapamycin-FKBP12 complex has no effect on calcineurin phosphatase, so that the immunosuppressive effects of rapamycin are mediated differently from those of cyclosporin A (CSA) or FK506, which is a calcineurin-mediated pathway.⁸ The rapamycin-FKBP12 complex binds to mammalian targets of rapamycin (mTOR), and this leads to inhibition of both DNA and protein synthesis, resulting in cell cycle arrest, thereby inhibiting T-cell proliferation.⁸ The antiproliferative properties of rapamycin on mesenchymal cells and ECs are also mediated by the formation of the rapamycin-FKBP12 complex.⁴ Its different mechanism of action, lack of end-organ toxicity, synergistic effects with CSA, and antiproliferative effects on mesenchymal cells and ECs makes it a potentially very promising agent in transplantation.

Rejection is characterized by inflammatory cells with concomitant release of a variety of inflammatory mediators that contribute to the injury. There is mounting evidence that neutrophils, with the release of their products, including oxidants, play a critical role in the development of acute cellular rejection and chronic rejection.⁹ An indicator of oxidant stress¹⁰ and a cellular response to injury is the induction of heme oxygenase-1 (HO-1).^11,12 Our laboratory observed an increased HO-1 expression in transplanted lungs with evidence of rejection,¹³ and increased HO-1 expression has been linked to graft survival of cardiac xenografts^14,15 and liver transplants.¹⁶ In addition, RDP1278, an immunosuppressive peptide derived from the HLA class I heavy chain, prolongs allograft survival, and this is mediated in part by HO-1 induction.¹⁷

The protective effects of HO-1 may be related to the degradation of the pro-oxidant heme substrate¹⁸ or through the generation of its products, carbon monoxide (CO),¹⁹ biliverdin with subsequent generation of bilirubin,²⁰ or iron with the induction of ferritin.²¹ CO has properties similar to those of nitric oxide²² and alters the expression of inflammatory mediators, tumor necrosis factor, interleukin-1, and interleukin-10.¹⁹ CO also has a protective role against vascular injury, because it inhibits vascular SMC proliferation.^23,24 Adenovirus-mediated HO-1 gene transfer inhibits balloon injury–induced neointimal formation, potentially through the generation of CO.²⁴ Rapamycin also protects against vascular injury by inhibiting vascular SMC proliferation.^6,7

In this study, we examined the possibility that the cytoprotective activities of rapamycin may include the induction of HO-1. Human pulmonary artery ECs (HPAECs) and SMCs (HPASMCs) were evaluated for HO-1 expression in response to rapamycin and whether it mediates the antiproliferative activity of rapamycin.

	Methods

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Reagents
Rapamycin, wortmannin, platelet-derived growth factor (PDGF), and CSA were purchased from Sigma. Tin protoporphyrin (SnPP) was from Porphyrin Products, and the Cell Proliferation Kit II (XTT) was obtained from Boehringer Mannheim. BCA protein assay reagent and Supersignal Chemiluminescent substrate were purchased from Pierce.

Cell Culture
HPAECs were obtained as previously described,²⁵ and HPASMCs were obtained from Clonetics. The cells were grown in endothelial growth medium (EGM-MV) (Clonetics) plus 10% FBS (Atlanta Biologicals) at 37°C in room air/5% CO₂ and were passed 1:3 approximately every 4 days at confluence with 0.10% Trypsin and 0.02 mmol/L EDTA. U937 cells were purchased from ATCC and grown in RPMI (Sigma) plus 10% FBS.

RNA Isolation and Northern Analysis
Ten micrograms of total RNA were isolated by the guanidine–acid phenol method and purified with RNeasy mini kits (Qiagen). The RNA was fractionated by size on a 1% agarose/6% formaldehyde gel buffered by MOPS, electrotransferred to a nylon membrane (GeneScreen, NEN), and crosslinked with ultraviolet light. The RNA was hybridized to a ³²P-labeled human HO-1 cDNA and rehybridized with a radiolabeled human GAPDH cDNA. The membranes were subjected to autoradiography with an intensifying screen at -85°C, and densitometric analysis was performed using NIH image software.

Protein Isolation and Western Analysis
Cells were washed twice with ice-cold PBS, and 10⁶ cells/mL were lysed with Triton lysis buffer. Protein concentrations were determined, and 40 µg of total protein was separated by use of a 10% SDS–polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Immobilon-P, Millipore). The membranes were blocked overnight with 5% nonfat dry milk in 10 mmol/L Tris, pH 7.5, 100 mmol/L NaCl, and 0.05% Tween 20 and incubated for 1.5 hours with a 1:1000 dilution of the anti–HO-1 antibody (StressGen). The membrane was incubated with the secondary antibody, peroxidase-conjugated goat anti-rabbit IgG antibody, for 1 hour, and the labeled protein bands were examined by use of a chemiluminescence method according to the manufacturer’s recommendations (Pierce).

HO Activity Assay
HO activity was measured by bilirubin generation as previously described.²⁶ HPAECs or HPASMCs were grown to confluence in 10-cm tissue culture dishes. After treatment, HPA cells were washed, scraped with a rubber policeman, and pelleted at 3000g for 10 minutes. The pellet was resuspended in 0.1 mol/L KPO₄ and 2 mmol/L MgCl₂, and the cells were frozen (-80°C) and thawed 3 times to break up the cell membrane. The samples were sonicated on ice, and 10 µL was taken to determine protein concentration. The remaining sonicate was centrifuged at 12 000g at 4°C for 20 minutes, and the supernatant was added to the reaction mixture (400 µL) containing 3 mg rat liver cytosol, 20 µmol/L hemin, 2 mmol/L glucose 6-phosphate, 0.2 units glucose 6-phosphate dehydrogenase, and 0.8 mmol/L ß-NADPH and incubated at 37°C for 1 hour in the dark. One milliliter of chloroform was added to extract the bilirubin, and the change in optical density at 464-530 nm was measured. The concentration of bilirubin produced in 60 minutes was calculated using the extinction coefficient, 40 mmol/L^-1cm^-1 for bilirubin per mg protein.

Cell Proliferation
Proliferation/metabolic activity was evaluated with the tetrazolium-based assay XTT (Boehringer Mannheim) according to manufacturer’s instructions. HPASMCs (1x10⁴) were incubated in 96-well plates in cell culture media with or without PDGF (10 ng/mL), rapamycin (10.0 µmol/L), or SnPP (5 µmol/L). XTT at a concentration of 0.3 mg/mL was added to each well for 8 hours at 37°C and with 6.5% CO₂, and the absorbance of the samples was measured between 450 and 500 nm with a reference wavelength >650 nm.

Statistical Analysis
Results are expressed as mean±SEM and are representative of at least 3 independent experiments. HO-1 mRNA, protein, and activity levels and XTT cell proliferation assays for the various conditions were evaluated on the basis of 1-way ANOVA using GraphPad InStat software.

	Results

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To determine whether the cytoprotective effects of rapamycin are mediated by HO-1, we first evaluated HO-1 expression in response to rapamycin in HPAECs and HPASMCs. We observed an increase in HO-1 mRNA levels in response to rapamycin in both HPAECs and HPASMCs. Figure 1A illustrates a Northern analysis showing HO-1 mRNA levels in HPASMCs after 2 to 24 hours of exposure to 10 µmol/L rapamycin. We observed a statistically significant increase (P<0.05) in HO-1 mRNA levels, 6.7- and 4.6-fold in HPAECs and 7.1- and 3.0-fold in HPASMCs, after 4 and 24 hours of exposure to rapamycin. Figure 2 illustrates a Northern analysis of HPAECs exposed to increasing concentrations of rapamycin, 0.01 to 10.0 µmol/L for 4 hours. The peak induction was observed after 10.0 µmol/L rapamycin in HPAECs and in HPASMCs. Higher concentrations were not tested because 100.0 µmol/L of rapamycin resulted in cell injury as seen by phase-contrast microscopy (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 1. A, RNA was isolated from untreated HPASMCs (C) or exposed to vehicle methanol (M) for 4 hours or to rapamycin 10 µmol/L (R) for 2, 4, or 24 hours. An HO-1 and GAPDH mRNA was identified by Northern analysis. B, Graph showing relative fold induction of HO-1 mRNA levels in HPAECs or HPASMCs exposed to 10 µmol/L rapamycin compared with untreated cells, control. There were 2 to 8 independent experiments for various time points.

View larger version (26K): [in this window] [in a new window]   Figure 2. A, Northern analysis of HPAECs exposed to increasing concentrations of rapamycin (0 to 10.0 µmol/L) for 4 hours illustrating HO-1 and GAPDH mRNA levels. B, Graph of relative fold induction of HO-1 mRNA levels in HPAECs and HPASMCs exposed to increasing concentrations of rapamycin for 4 hours (n=2 to 8 independent experiments).

To determine whether HO-1 induction was a general response to immunosuppressive agents, we also examined the effect of CSA on HO-1 expression. Exposure up to 10.0 µmol/L CSA resulted in little if any increase in HO-1 mRNA levels in either HPAECs or HPASMCs (Figure 3, A and B); however, like rapamycin, CSA increased the expression of HO-1 in a monocytic cell line, U937 (Figure 3C). This demonstrates a differential response between rapamycin and CSA depending on the cell type.

View larger version (41K): [in this window] [in a new window]   Figure 3. Northern analysis of HPAECs (A), HPASMCs (B), or U937 cells (C) exposed to 10 µmol/L CSA or 10 µmol/L rapamycin (rap) for 4 and 24 hours, 5 µmol/L hemin (H) for 4 hours, or methanol (M) or untreated (C) for 24 hours. These are representative results of 3 independent experiments for each of the cell lines tested.

To verify that the increase in HO-1 expression in response to rapamycin also reflects an increase in HO-1 protein levels and, more importantly, activity level, we evaluated HO-1 protein and HO activity in response to rapamycin in both HPAECs and HPASMCs (Figure 4). HO-1 protein and activity were increased in both cell types after 24 hours of rapamycin exposure. HO-1 protein increased 6- to 11-fold in human pulmonary vascular cells, and activity was increased 4-fold, which was similar to hemin, a known stimulus for HO-1.

View larger version (22K): [in this window] [in a new window]   Figure 4. A and B, Western analysis of HPAECs (A) or HPASMCs (B) using an anti–HO-1 polyclonal antibody from untreated cells and cells exposed to 10 µmol/L rapamycin for 2, 4, or 24 hours. C, Graph of relative fold HO-1 protein induction in response to rapamycin in HPAECs and HPASMCs (n=2 to 5 independent experiments for each time point). D, Graph of relative fold induction of HO activity in response to rapamycin in HPAECs and HPASMCs (n=2 to 5).

Rapamycin binding to FKBP12 results in the inhibition of mTOR protein kinase activity.²⁷ TOR proteins are similar to the lipid kinases, especially phosphoinositide 3-kinase (PI-3K), belonging to a larger protein kinase family called the phosphoinositide kinase–related kinases.²⁸ PI-3K activity is critical in growth factor–dependent cell cycle progression²⁹ and mediates the activation of mTOR by extracellular stimuli.³⁰ To determine whether this pathway is involved in HO-1 induction, cells were treated with wortmannin, a relatively specific PI-3K inhibitor.³¹ Exposure of HPASMCs to wortmannin resulted in an increase in HO-1 mRNA levels similar to rapamycin exposure. Figure 5 illustrates HO-1 mRNA levels in HPASMCs after 2 to 24 hours of exposure to wortmannin and to increasing concentrations of wortmannin. HO-1 mRNA levels were increased 2 and 4 hours after exposure and responded to both low and higher concentrations (0.01 to 10.0 µmol/L). Similar results were also observed in HPAECs (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 5. A, Northern analysis of HPASMCs exposed to 10 µmol/L wortmannin (W) for 2, 4, or 24 hours and either untreated (C) or vehicle- or methanol-treated (M), representative of 3 independent experiments. B, Northern analysis of HPASMCs exposed to increasing concentrations of wortmannin (0, 0.01, 0.1, 1.0, or 10.0 µmol/L) for 4 hours and representative of 2 experiments.

Rapamycin inhibits growth factor–dependent proliferation of ECs, fibroblasts, and SMCs.^3,4 In addition, activity of HO-1 or one of its products, CO, has also been shown to suppress vascular SMC proliferation in response to an injury.^15,23,24 Like previous studies, we found that rapamycin inhibited PDGF-dependent human SMC proliferation (Figure 6). To determine whether the inhibitory actions of rapamycin on PDGF-dependent proliferation may be mediated by its induction of HO-1, we exposed cells to rapamycin, PDGF, and SnPP, an inhibitor of HO activity. The addition of SnPP resulted in a loss of the suppressive effects of rapamycin on PDGF-stimulated cell growth (Figure 6), suggesting that rapamycin-mediated increases in HO activity contribute to the antiproliferative effects of rapamycin.

View larger version (21K): [in this window] [in a new window]   Figure 6. Graph of relative increase of treated HPASMCs compared with control (exposed to vehicle) based on tetrazolium-based XTT assay. HPASMCs were incubated with or without PDGF (10 ng/mL), rapamycin (Rap, 10.0 µmol/L), or SnPP (5 µmol/L). Results are an average from 3 independent experiments, with each experiment having 6 individual samples for each condition, and are represented as mean±SEM. *P<0.05 vs control.

	Discussion

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Rapamycin is a new immunosuppressive agent with a unique mechanism of action compared with CSA or FK506.^1–4 In addition to its antiproliferative effects on lymphocytes, rapamycin has potent antiproliferative activity on a variety of cell types, including SMCs, ECs, and fibroblasts, compared with CSA or FK506.^3,4 Rapamycin inhibits the kinase mTOR, whereas CSA and FK506 are calcineurin inhibitors. Previous studies have compared the antiproliferative effects of rapamycin, CSA, and FK506 in vascular SMCs in vitro^3,4 and in models of neointimal proliferation.^32,33 These studies demonstrated that the antiproliferative effects of CSA on vascular cells are mediated indirectly through its immune effects on lymphocytes without any local effects attributed to CSA directly. Interestingly, FK506 antagonizes the antiproliferative properties of rapamycin in vascular SMCs, because both agents bind to the same cytosolic receptor, FKBP12.^2–4 Previous studies have also shown that although CSA attenuates neointimal proliferation in the setting of transplantation, it does not reduce neointimal proliferation after balloon injury.³² However, rapamycin has unique direct antiproliferative effects on vascular SMCs,^3,4 popularizing its use, rather than that of CSA or FK506, as an agent in stents to prevent vascular restenosis.^34,35

HO-1 was recently identified as a graft survival gene with protective actions against early complications such as reperfusion injury of liver isografts,¹⁶ chronic injury, and arteriosclerosis in a cardiac allograft model.¹⁵ In addition, HO-1 expression has been linked to the graft survival actions of the immunosuppressive peptide derived from the HLA class I heavy chain, with the protective effect of this peptide being abolished by inhibition of HO activity.¹⁷

In this study, we examined the possibility that the cytoprotective and/or antiproliferative properties of rapamycin may be, at least in part, mediated through the induction of HO-1. We found that rapamycin differentially induces the expression of HO-1 in HPAECs, HPASMCs, and U937 cells compared with CSA, which induces the gene in U937 cells but not in HPAECs or HPASMCs. Rapamycin exposure resulted in an increase in both HO-1 mRNA and protein levels, and the increase in HO activity in the HPA vascular cells was similar to that of the potent inducer hemin. This leads to the possibility that some of the actions of rapamycin may be mediated through the induction of HO-1. One such possibility is its antiproliferative activities on nonlymphocytic cells. Rapamycin differentially inhibits growth in both ECs and SMCs, and rapamycin increases HO-1 expression in these cell types compared with CSA.

Chronic rejection results from an abnormal fibroproliferative response with an increase in mesenchymal cells leading to obstruction.³⁶ Neointimal proliferation after vascular injury is another example of a pathological response to wound healing. The potent antiproliferative actions of rapamycin on vascular SMCs have led to studies evaluating the potential use of rapamycin in vascular injury. Oral rapamycin has been shown to reduce the neointimal formation after balloon-induced injury in an animal model,⁷ and implanted stents coated with rapamycin inhibited neointimal formation in animals and in human subjects undergoing coronary angioplasty.^6,33–35 Interestingly, HO-1 is induced in vascular injury, and this induction is believed to be a cytoprotective response against vascular injury, whether it is transplant arteriosclerosis¹⁵ or restenosis in a localized vascular injury.^23,24 We found that the antiproliferative effects of rapamycin against PDGF-stimulated cell growth was blocked with the chemical inhibitor of HO activity, SnPP. This raises the possibility that the antiproliferative effects of rapamycin against vascular injury and SMC proliferation may be mediated by HO-1 and is consistent with findings by Volti et al³⁷ that HO-1 induction inhibits vascular SMC proliferation.

The actions of rapamycin are mediated by the formation of a complex with FKBP12, and this complex inhibits the activity of mTOR.^2–4 The antiproliferative affects of rapamycin on SMCs is also dependent on its binding to FKBP12⁴ and therefore most likely through its inhibition of mTOR activity. Growth factor activation of mTOR is believed to be downstream to a wortmannin-sensitive PI-3K activity,²⁹ which is an important mediator in growth factor–dependent cell cycle progression.³⁰ We found that wortmannin also induced HO-1 expression, suggesting that the PI-3K-mTOR signaling pathway is involved in HO-1 induction. This is a critical pathway in growth factor–dependent proliferation, and HO-1 actions with respect to proliferation may also involve this pathway.

Rapamycin, an immunosuppressive agent, has unique properties distinct from those of the classic calcineurin inhibitors, such as a more potent antiproliferative activity on nonlymphocytic cells. We found that HO-1 is differentially expressed in human pulmonary vascular cells by rapamycin and that the antiproliferative effects may be mediated by this induction. These studies identify a potentially unique graft survival activity of rapamycin, the induction of HO-1, which has not been previously recognized.

	Acknowledgments

 
This work was supported by the American Lung Association of Florida and the Department of Health, Florida Biomedical Research Program (Dr Visner).

Received August 23, 2002; revision received October 30, 2002; accepted October 30, 2002.

	References

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