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Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:759-764

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:759.)
© 2001 American Heart Association, Inc.

Vascular Biology

Photodynamic Therapy With Motexafin Lutetium Induces Redox-Sensitive Apoptosis of Vascular Cells

Zhiping Chen; Kathryn W. Woodburn; Can Shi; Daniel C. Adelman; Campbell Rogers; Daniel I. Simon

From the Cardiovascular Division (Z.C., C.S., C.R., D.I.S.), Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass; Harvard-M.I.T. Division of Health Sciences and Technology (C.R.), Massachusetts Institute of Technology, Cambridge, Mass; and Pharmacyclics, Inc (K.W.W., D.C.A.), Sunnyvale, Calif.

Correspondence to Daniel I. Simon, MD, Cardiovascular Division, Brigham and Women’s Hospital, 75 Francis St, Tower 3, Boston, MA 02115. E-mail dsimon{at}rics.bwh.harvard.edu


*    Abstract
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Abstract—Motexafin lutetium is a photosensitizer that accumulates in atherosclerotic plaque and, after activation by far-red light, produces cytotoxic singlet oxygen. The combination of photosensitizer and illumination, known as photodynamic therapy (PDT), has been shown to reduce atheroma formation in animal models and is under clinical investigation. However, the effects of PDT with motexafin lutetium on isolated vascular cells are unknown. This study was designed to characterize the effects of PDT on vascular cell viability and to define the cell-death pathway for this agent. Fluorescence microscopy of RAW macrophages and human vascular smooth muscle cells revealed time-dependent uptake of motexafin lutetium. Illumination of motexafin lutetium–loaded cells with 732-nm light (2 J/cm2) impaired cellular viability and growth (IC50 5 to 20 µmol/L). Depletion of intracellular glutathione potentiated (P=0.035) and the addition of antioxidant N-acetylcysteine attenuated (P=0.002) cell death, suggesting that the intracellular redox state influences motexafin lutetium action. PDT was associated with the loss of mitochondrial membrane potential, mitochondrial release of cytochrome c, and caspase activation. PDT promoted phosphatidylserine externalization and induced apoptotic DNA fragmentation, with the number of apoptotic cells increasing from 7±2% to 34±3% of total cells. Reducing plaque cellularity by the induction of apoptosis may be one mechanism by which PDT reduces plaque burden, possibly modulates plaque vulnerability, and inhibits restenosis in vivo.


Key Words: apoptosis • photodynamic therapy • vascular cells • reactive oxygen species • atherosclerosis


*    Introduction
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More than 1 million percutaneous coronary intervention procedures are performed annually worldwide to relieve the symptoms of coronary artery disease. Restenosis of the treated segment occurs in 32% to 57% of patients undergoing balloon angioplasty within 6 months of the procedure.1 In the United States, {approx}150 000 cases of restenosis occur annually and account for one fourth of the total percutaneous coronary intervention procedures performed, at a cost of >$3 billion.2 By providing rigid scaffolding, endovascular stents reduce restenosis rates in select lesions.3 However, in-stent restenosis remains a recognized clinical problem and can be expected to increase in incidence as stenting increases in frequency (>80% of procedures) and is applied to small vessels (ie, <2.7-mm diameter) and long lesions and in patients with diabetes mellitus.

Radiation therapy with ionizing (ie, ß- and {gamma}-radiation) and nonionizing (ie, photon) radiation is under active preclinical and clinical investigation for the prevention and treatment of restenosis after percutaneous coronary intervention. Despite consistent and compelling clinical trial evidence that intracoronary radiation with ß or {gamma} sources is capable of effectively treating in-stent restenosis,4 significant adverse effects (excessive vascular damage producing pseudoaneurysms,5 total vessel occlusion,5 or atherosclerosis6 ; delayed stent thrombosis7 ; and the possible risk of neoplasm in surrounding tissues) may limit its widespread application.

The use of nonionizing light energy may provide an alternative to intracoronary radiation. Photoangioplasty (PA) involves the combined use of a photosensitizing agent that accumulates in the target tissue and endovascular illumination to produce cytotoxic singlet oxygen8 9 for the treatment of primary atherosclerosis (ie, regression and plaque stabilization) and for the prevention and treatment of restenosis. PA is capable of inducing cell death with a variety of photosensitizing agents, including porphycene derivatives, chloroaluminum sulfonated phthalocyanine, photofrin II, 5-amino-levulinic acid, and motexafin lutetium.9 10 11 12 13 14 15 However, the relative contributions of apoptosis and necrosis are dependent on the cell line or target tissue, photosensitizing agent, and experimental conditions. PA is an experimental therapy with unproven clinical benefit that is currently under phase I and II clinical investigation.

We have identified an apoptotic cell-death pathway promoted by photodynamic therapy (PDT) with motexafin lutetium in macrophages and smooth muscle cells (SMCs). This redox-sensitive pathway involves loss of mitochondrial membrane potential ({Delta}{Psi}m) and the release of cytochrome c from mitochondria to the cytosol, thereby triggering caspase activation and initiation of the apoptotic program.


*    Methods
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Materials
Motexafin lutetium16 formulated in 5% mannitol at a concentration of 2 mmol/L was obtained from Pharmacyclics, Inc. WST-1 cell viability reagent was obtained from Roche Molecular Biochemicals. 5,5',6,6'-Tetrachloro-1,1'3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) and fluorescent probes for staining mitochondria (MitoTracker Green), lysosomes (LysoTracker red), endoplasmic reticulum (rhodamine B, hexyl ester), and the nucleus (Hoechst 33342) were purchased from Molecular Probes. zVAD-fmk was purchased from Enzyme Systems, Inc. Antibody to cytochrome c was from Santa Cruz Biotechnology, Inc. All other reagents were obtained from Sigma Chemical Co.

Cell Lines and Culture Conditions
Murine macrophages (RAW cells, American Type Culture Collection) and low-passage (passages 2 to 4) human saphenous vein SMCs were maintained in DMEM supplemented with 10% FBS, 2 mmol/L glutamine, 25 mmol/L HEPES, penicillin, and streptomycin.

Cellular Motexafin Lutetium Loading and Illumination
RAW cells and SMCs were grown to confluence, washed with serum-free medium, and then incubated with motexafin lutetium (0 to 100 µmol/L) or 5% mannitol vehicle diluted in serum-free medium containing 0.5% BSA in 5% CO2 at 37°C. In experiments examining the effect of intracellular antioxidants on viability, L-buthionine-[S,R]-sulfoximine (BSO, 10 to 100 µmol/L) or N-acetylcysteine (NAC, 10 mmol/L) was added 18 hours before illumination or at the time of addition of exogenous ceramide. After 18 hours, motexafin lutetium–containing or vehicle-containing media were removed, and cells were washed with sterile PBS and then illuminated in a darkened room with a 732-nm diode laser (Diomed) and microlens (Pioneer Optics) at a fluence rate of 5 mW/cm2 to achieve a total fluence of 2 J/cm2. After illumination, PBS was removed, and serum-containing medium was added.

Fluorescence Microscopy
Motexafin lutetium (10 µmol/L) was added to the cells and incubated for 2 to 72 hours. Fluorescence uptake analysis and colocalization studies using specific organelle probes were performed as previously described.17

Viability Assays
Cellular viability and growth were assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche Molecular Biochemicals) according to the manufacturer’s protocol. Briefly, RAW macrophages and SMCs (3x104 cells per well) seeded in 96- and 24-well tissue culture plates, respectively, were incubated with motexafin lutetium overnight in 5% CO2 at 37°C. Ceramide-induced apoptosis was assessed by incubating cells overnight with the synthetic compound C2-ceramide (40 µmol/L). Twenty-four hours after illumination, WST-1 solution was added to cells (1:10 [vol/vol]) and incubated in 5% CO2 at 37°C for 1 hour. An aliquot (100 µL) was removed to measure optical density at 450 nm. In untreated cells, optical density at 450 nm represented 100% viability; color formation of WST-1 added to medium alone represented 0% viability. Percent viability for the indicated treatment groups was calculated by fitting a linear regression line between these values.

Flow Cytometric Analysis of Cell Cycle Status and Apoptosis
DNA fragmentation was used as an indicator of apoptosis. Cellular DNA content was quantified by using propidium iodide (PI) staining of ethanol-fixed RAW cells and flow cytometry (FACScan, Becton-Dickinson), as previously described.18

Apoptosis Assessment by Annexin V Staining
Apoptosis was also assessed by use of annexin V staining.19 At indicated times after illumination, cells were washed in PBS and resuspended in staining solution containing fluorescein annexin V and PI (Apoptosis Detection Kit, Alexis Biochemicals), according to the manufacturer’s protocol. In experiments performed to evaluate the role of caspases in cell death induced by PA with motexafin lutetium, the broad-spectrum caspase inhibitor zVAD-fmk was added to the cells 1 hour before illumination. Cells were analyzed by flow cytometry, and staining was expressed as percent positive cells.

Measurement of {Delta}{Psi}m by Flow Cytometry
{Delta}{Psi}m was measured by incubating macrophages (106/mL) with 10 µg/mL JC-1 in culture medium at 37°C for 10 minutes in the dark, as previously described.20 Samples were treated in parallel with the 50 µmol/L carbonyl cyanide m-chlorophenylhydrazone (mClCCP), added 15 minutes before JC-1, to depolarize {Delta}{Psi}m as a positive control. The percentage of cells with JC-1 aggregates and monomers was calculated by quadrant statistical analysis of FL1 and FL2, respectively. Quadrant boundaries were set with reference to a parallel sample stained in the presence of mClCCP.

Cytochrome c Release
Release of cytochrome c from mitochondria was assessed as previously described.21 Samples (30 µg protein per lane) were subjected to 15% SDS-PAGE under reducing conditions and then blotted onto nitrocellulose. Cytochrome c was detected with a polyclonal antibody from Santa Cruz Biotechnology, and tubulin was detected with a monoclonal antibody from Sigma.

Statistical Analysis
All data are presented as the mean±SD. Groups were compared by the nonpaired t test. A value of P<0.05 was considered significant.


*    Results
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Cellular Accumulation and Subcellular Localization of Motexafin Lutetium
We assessed the accumulation and subcellular localization of motexafin lutetium by using fluorescence microscopy. The signature fluorescence emission band of motexafin lutetium is centered at 750 nm and was used to monitor real-time biolocalization within RAW macrophages and human SMCs. A time-dependent increase in intracellular motexafin lutetium fluorescence was seen in both cell types. The temporal uptake of macrophages is shown in online FigureDown I (please see www.ahajournals.org).



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  		Figure 1. Cellular viability after PDT. The effect of PDT with motexafin lutetium (ML, 0 to 100 µmol/L; 18-hour incubation) on macrophage viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent. Redox sensitivity was examined by incubating cells with the antioxidant NAC (10 mmol/L) or by depleting intracellular glutathione stores by use of BSO (10 to 100 µmol/L, insert). Data are expressed as percent control (Ctl) viability (n=3 or 4).

Subcellular localization of motexafin lutetium in macrophages and SMCs was determined by using organelle-specific fluorescent probes. Online FigureUp I illustrates typical localization patterns for SMCs after incubation with motexafin lutetium for 24 hours. Colocalization of motexafin lutetium within intracellular organelles was determined by using interferometric Fourier spectroscopy. Fluorescence spectra were acquired, with fluorescence emission profiles obtained at every pixel. Intracellular biolocalization showed overlay of the photosensitizer (750 nm) with the emission maxima of the organelle fluorophore for lysosomes (600 nm), endoplasmic reticulum (586 nm), and mitochondria (532 nm); see online FigureUp I. Motexafin lutetium was mainly localized within the lysosomes and endoplasmic reticulum and, to a lesser extent, within the mitochondria.

Effect of Motexafin Lutetium on Cellular Viability and Growth
Illumination of motexafin lutetium–loaded cells with 732-nm light, delivered at a fluence rate of 5 mW/cm2 to achieve a total fluence of 2 J/cm2, resulted in significant morphological changes in macrophages and SMCs (online FigureUp II; see www.ahajournals.org) and impaired macrophage viability and growth (IC50 {approx}20 µmol/L), reducing viability by up to 90% at 100 µmol/L (Figure 1Up). PDT also reduced human SMC viability (IC50 {approx}5 µmol/L); see online FigureUp III (www.ahajournals.org). Induction of cell death required the combination of motexafin lutetium and light, inasmuch as neither drug nor light alone had significant effects on cellular viability.

Intracellular Redox State Influences Effect of Motexafin Lutetium on Cellular Viability
Because atherosclerosis is associated with enhanced oxidative stress and the depletion of intracellular antioxidants,22 23 we explored the effect of altering the intracellular redox state on PDT-induced cell death by depleting intracellular glutathione stores with the use of BSO. BSO is a specific inhibitor of {gamma}-glutamyl cysteine synthetase, and treatment of cells with this agent results in glutathione depletion.24 BSO potentiated the effect of motexafin lutetium on macrophage viability (IC50 1 µmol/L, Figure 1Up). BSO alone had no effect on macrophage viability. Moreover, treatment of the cells with the antioxidant NAC significantly reduced cell death induced by PDT (Figure 1Up, insert). We compared the effects of BSO and NAC in a non-PDT apoptotic pathway. Ceramide may act as a second messenger in signaling for apoptosis induced by tumor necrosis factor-{alpha} and Fas.25 In contrast to PDT, macrophage apoptosis induced by exogenous C2-ceramide was largely unaffected by treatment with BSO or NAC (online FigureUp IV; see www.ahajournals.org). Taken together, these observations suggest that apoptosis initiated by PDT is redox sensitive and that distinct signaling cascades may be operative in PDT compared with certain non-PDT pathways.

Motexafin Lutetium–Induced Apoptotic Cell Death in Macrophages and SMCs
The mechanism of cell death induced by PDT with motexafin lutetium was examined by using annexin V staining of macrophages and SMCs. Annexin V binds membrane-associated phosphatidylserine (PS), which is located in the inner phospholipid bilayer but is externalized rapidly to the cell surface (ie, outer lipid bilayer) in the apoptotic process. Because translocation/exposure of PS also occurs during necrosis, annexin V–FITC is used in conjunction with PI to distinguish apoptotic (annexin V–FITC positive, PI negative) from necrotic cells (annexin V–FITC positive, PI positive). PDT increased the number of apoptotic macrophages 4.2±1.2-fold (mean±SD, n=4; Figure 2Down) and the number of apoptotic SMCs 4.0±1.9-fold (n=3). The percentage of necrotic cells did not increase from baseline after PDT (Figure 2Down).



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  		Figure 2. PS externalization as an indicator of apoptosis (apopt) after PDT. Externalization of PS after PDT was detected by staining macrophages with annexin V–FITC (FL1). Cells were then analyzed by flow cytometry, and the percentage of cells with PS externalization is indicated. Because exposure of PS also occurs in necrosis, annexin V–FITC is used in conjunction with PI staining (FL2) to distinguish apoptotic (annexin V–FITC positive, PI negative; bottom right quadrant of each panel) from necrotic (annexin V positive, PI positive; top right quadrant of each panel) cells. Fluorescence intensity for annexin V–FITC is plotted on the x-axis, and PI is plotted on the y-axis. The effect of caspase activation on PS exposure was examined by incubating cells with the broad-spectrum caspase inhibitor zVAD-fmk. Data are representative of 3 separate experiments.

Induction of apoptosis was also confirmed by staining the cells with PI and analyzing DNA content by flow cytometry. PDT with motexafin lutetium induced apoptotic DNA fragmentation, with the number of apoptotic cells increasing from 7±2% to 34±3% of total cells (Figure 3Down).



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  		Figure 3. DNA fragmentation as an indicator of apoptosis. Cellular DNA content of macrophages after PDT was quantified by PI staining (FL2) of ethanol-fixed macrophages and flow cytometry. The percent of total cells with DNA fragmentation (MI) as an indicator of apoptosis is quantified. Veh indicates vehicle. Data are representative of 2 independent experiments.

Mitochondrial Membrane Depolarization and Cytochrome c Release Is Associated With Motexafin Lutetium Action
The very short lifetime of singlet oxygen generated by PDT and our observation that motexafin lutetium accumulates in the mitochondria led us to consider the possibility that cytotoxicity to mitochondria was likely an upstream event in motexafin lutetium action. {Delta}{Psi}m is necessary for the supply of energy by the mitochondrion, and loss of {Delta}{Psi}m is associated with cells undergoing apoptosis. To investigate whether PDT with motexafin lutetium resulted in a loss of {Delta}{Psi}m, macrophages were incubated with the potential sensitive fluorescent probe JC-1, which undergoes a molecular aggregation and shift in fluorescence from green to red-orange at high membrane potentials. Loss of {Delta}{Psi}m results in a decrease in red-orange fluorescence, as visualized in online FigureUp V (see www.ahajournals.org), by incubating macrophages with the mClCCP as a positive control (83% depolarized cells). PDT with motexafin lutetium resulted in a time-dependent loss of {Delta}{Psi}m (20% depolarized cells at 30 minutes, 47% at 60 minutes).

Cytochrome c, a component of the mitochondrial electron-transfer chain that is present in the intermembrane space, is released into the cytosol during the early phases of apoptosis.26 Therefore, we assayed the accumulation of mitochondrial cytochrome c into the cytosol after PDT by Western blot analysis of cytosolic extracts prepared under conditions that keep mitochondria intact. As shown in Figure 4Down, cytosol from untreated macrophages or macrophages loaded with motexafin lutetium, but not illuminated, contained no cytochrome c. In contrast, cytochrome c accumulated in the cytosol of macrophages after PDT with motexafin lutetium.



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  		Figure 4. Release of cytochrome c from mitochondria during apoptosis. Macrophage cytosolic content of cytochrome c was determined by Western blot by using an anti–cytochrome c antibody after indicated treatments. Mitochondrial pellet serves as a positive control. Equal protein loading was verified by using an anti-tubulin antibody. Data are representative of 2 separate experiments.

In many apoptotic systems, release of cytochrome c into the cytosol results in the activation of the executioner caspases of apoptosis. To determine whether caspase activation is needed for the apoptotic program after PDT with motexafin lutetium, the effect of the broad-spectrum caspase inhibitor zVAD-fmk on PS externalization was examined. zVAD-fmk reduced dose-dependently the percentage of apoptotic macrophages, as determined by annexin V staining after PDT with motexafin lutetium, from 49% to 23% (25 µmol/L zVAD-fmk) and 11% (50 µmol/L zVAD-fmk) (Figure 2Up).


*    Discussion
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In the present study, we have identified a cell-death pathway induced by PDT with motexafin lutetium in vascular cells. This redox-sensitive pathway involves the loss of {Delta}{Psi}m, release of cytochrome c from mitochondria to the cytosol, and activation of caspases that trigger apoptosis. This mode of cell death may be central to the proposed clinical use of PDT to prevent and treat restenosis or as a primary atherosclerotic plaque–ablating therapy, allowing for vascular cell dropout without promoting an inflammatory response.

Several groups have reported that PDT can induce cell death with a variety of photosensitizing agents, including porphycene derivatives, chloroaluminum sulfonated phthalocyanine, photofrin II, and 5-amino-levulinic acid.10 11 12 13 14 15 However, the relative contributions of apoptosis and necrosis are dependent on the cell line, photosensitizing agent, and/or experimental conditions. We demonstrated that singlet oxygen–generating motexafin lutetium induced apoptotic cell death, that glutathione depletion potentiated cell death, and that the antioxidant NAC attenuated cell death. Taken together, these findings suggest that PDT with motexafin lutetium induced apoptosis in a redox-sensitive manner. There is mounting evidence that many agents that induce apoptosis act either as oxidants or stimulators of cellular oxidative metabolism.27 28 29 30 Reactive oxygen species (ROS) serve as important signal transduction molecules in apoptosis induced by UV light,31 ionizing radiation,32 anthracyclines,33 and arsenic.18 Scavenging ROS, either by antioxidants34 or overexpression of phospholipid hydroperoxide glutathione peroxidase,35 suppress apoptosis, providing additional evidence that ROS act as signaling intermediates in programmed cell death.

Mitochondria play a central role in programmed cell death.36 Mitochondrial respiration generates a major physiological source of ROS, and activators of apoptosis (eg, caspase-2, caspase-9, cytochrome c, and apoptosis-inducing factor) reside in mitochondria. Mitochondria-derived ROS may signal apoptosis by modifying membrane proteins, such as a large-conductance channel known as permeability transition pore, to modulate {Delta}{Psi}m37 or by activating downstream targets such as stress-activated protein kinase cascades.38 Mitochondria also contain proteins that regulate apoptosis, such as Bcl-2, Bcl-xL, Bax, and Bad, which can prevent or accelerate programmed cell death. In the present study, we have shown that PDT with motexafin lutetium results in the loss of {Delta}{Psi}m, release of cytochrome c from mitochondria to the cytosol, and activation of caspases that trigger apoptosis. The precise mechanism by which PDT with motexafin lutetium is linked to these mitochondrial events remains to be determined and is the focus of ongoing studies.

The influence of redox state on apoptosis may have important implications for the clinical use of motexafin lutetium. Motexafin lutetium and other photosensitizers accumulate in atherosclerotic vessels more than in normal vessels, probably secondary to photosensitizer binding to lipoproteins.39 40 41 42 Atherosclerotic vessels are associated with oxidative stress22 43 and with the depletion of key intracellular antioxidants such as glutathione.23 In the present study, treatment of macrophages with the glutathione-depleting agent BSO24 potentiated the effect of motexafin lutetium on macrophage viability {approx}20-fold. Therefore, enhanced susceptibility of oxidatively stressed tissues may provide an additional mechanism by which the actions of motexafin lutetium may be relatively selective for atherosclerotic compared with normal vessels.

It is important to note that the vascular effects of PDT are not limited to apoptosis but extend to other important biological processes, including SMC migration, proliferation, and extracellular matrix production.9 PDT has been shown to modulate cytokine release or activation44 and growth factor responses45 that promote vascular growth. Numerous studies reporting the effectiveness of PDT in inhibiting neointimal hyperplasia after experimental angioplasty13 46 47 48 49 attest to the importance of these nonapoptotic effects of PDT, given prior observations that neointimal SMCs may be more resistant to apoptosis than medial SMCs secondary to neointimal upregulation of antiapoptotic genes.38

Study Limitations
Although we have demonstrated that mitochondria-dependent apoptosis is a mode of cell death after light treatment of motexafin lutetium–loaded cells, the significance of motexafin lutetium accumulation and singlet oxygen generation within lysosomes and endoplasmic reticulum is unknown. ROS have been implicated in the translocation of Bax and Bad from the cytosol to the mitochondria, where these factors form heterodimers with Bcl-2 and induce cytochrome c release.50 The experimental conditions for cellular motexafin lutetium exposure and illumination in the present in vitro study have been designed intentionally to mimic the clinical PA procedure under phase I and II investigations with respect to drug and light doses. Nonetheless, we cannot rule out alternative cell-death pathways (ie, necrosis) in vivo that are due to complex issues relating to drug distribution/localization, light delivery, and tissue penetration. A blood field is not expected to attenuate the tissue ablative capacity and clinical efficacy of PA with motexafin lutetium because it is activated by blood- and tissue-penetrating far-red (732 nm) light.9


*    Conclusions
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We have identified a redox-sensitive apoptotic cell-death pathway promoted by PA with motexafin lutetium in vascular cells. PA is an experimental therapy with unproven clinical benefit. Further studies clarifying the mechanisms of PA action will help to guide photosensitizer and light treatment–delivery protocols to optimize clinical efficacy.


*    Acknowledgments
 
This work was supported in part by grants from the National Institutes of Health (HL-57506 and DK-55656 to D.I.S. and HL-03104 to C.R.) and by unrestricted research grants from Pharmacyclics, Inc, to D.I.S. and C.R. D.I.S. and C.R. serve as scientific consultants to Pharmacyclics, Inc. The authors would like to thank Dr Wai-Fung Cheong for expert assistance in the endovascular illumination protocol.

Received January 3, 2001; accepted January 18, 2001.


*    References
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 *References
 

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