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(Investigative Ophthalmology and Visual Science. 2007;48:4741-4747.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-1534
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Photodynamic Therapy Induces Caspase-Dependent Apoptosis in Rat CNV Model

Akihisa Matsubara,1 Toru Nakazawa,1 Kosuke Noda,1 Haicheng She,1 Edward Connolly,1 Tara A. Young,1,2 Yuichiro Ogura,3 Evangelos S. Gragoudas,1 and Joan W. Miller1

1From the Angiogenesis and Laser Laboratories, Retina Service, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts; 2Retina Division, Jules Stein Eye Institute/UCLA, Los Angeles, California; and 3Department of Ophthalmology and Visual Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To investigate the mechanism of cell death in laser-induced choroidal neovascularization (CNV) after photodynamic therapy (PDT).

METHODS. PDT was performed in Brown-Norway rats using laser light at a wavelength of 689 nm, irradiance of 600 mW/cm2, and fluence of 25 J/cm2 after intravenous injection of verteporfin at the doses of 3, 6, and 12 mg/m2. Apoptotic cells in CNV were detected by TUNEL assay at 1, 3, 6, 15, 24, and 48 hours after PDT. Caspase activation at 1, 3, 6, 15, and 24 hours after PDT was determined by immunohistochemistry (IHC) with a cleaved caspase-3 or -9 antibody. Akt activity was determined by Western blot and IHC with a phosphorylated-Akt (pAkt) antibody. To investigate the roles of Akt in PDT-induced apoptosis, insulin-like growth factor (IGF)-1, an Akt activator, with or without wortmannin, an inhibitor of PI3K-Akt pathway, was injected into the vitreous before PDT.

RESULTS. The number of TUNEL-positive cells in CNV increased at 3 hours after PDT and peaked at 6 hours, showing a dose dependence of verteporfin. Caspase activation was detected in TUNEL-positive cells. Dephosphorylation of Akt in CNV occurred within 1 hour. IGF-1 significantly activated Akt and suppressed the number of TUNEL-positive cells in CNV, and the effects of IGF-1 were diminished by wortmannin.

CONCLUSIONS. PDT induced caspase-dependent apoptosis in CNV. These results suggest that PDT leads to dephosphorylation of Akt and subsequent activation of the caspase-dependent pathway. Understanding the intracellular signaling mechanisms of apoptosis in PDT may lead to more selective and effective treatment of CNV secondary to age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of blindness in people older than 50 in developed countries.1 The wet, or neovascular, form of AMD is characterized by choroidal neovascularization (CNV) and is responsible for severe vision loss from AMD.2 Although conventional laser photocoagulation may be appropriate in some patients, treatment at or close to the center of vision results in immediate loss of vision from thermal retinal damage. Several multicentered randomized clinical trials have demonstrated photodynamic therapy (PDT) to be effective in the treatment of subfoveal CNV.3 4 PDT involves the administration of light-sensitive dye followed by low-intensity light irradiation to the target lesion at a wavelength selectively absorbed by the dye. This form of treatment is relatively selective for CNV and results in occlusion of neovascular tissue while it produces minimal damage to the retina and choroidal tissues.

Although PDT is an advance over previous treatment modalities for CNV, the mechanisms behind this therapy remain incompletely understood. In general, the anticancer effects of PDT are thought to occur at two levels, directly through a lethal effect on tumor cells and indirectly through vascular occlusion, which limits blood supply to the region.5 PDT, using the photosensitizer verteporfin, catalyzes the formation of reactive oxygen intermediates and has been reported to rapidly induce apoptosis in a variety of cell types after light irradiation.6 7 8 9 10 In previous studies, we showed that PDT administered in vitro with another photosensitizer, lutetium texaphyrin, causes apoptosis and induces rapid caspase-dependent apoptosis in bovine retinal capillary endothelial cells.11 However, detailed apoptotic mechanisms after PDT administered in vivo for CNV remain to be elucidated.

Vascular endothelial growth factor (VEGF) is a key regulator of normal and abnormal neovascularization, including CNV. VEGF has various functions on endothelial cells, one of which is regulating endothelial cell survival through the phosphatidylinositol-3 kinase (PI3K)/Akt signal transduction pathway.12 13 A serine/threonine kinase, Akt/protein kinase B is a downstream effecter of PI3K; the active form of phosphorylated Akt (pAkt) promotes cell survival by inhibiting apoptotic signals such as caspase-9, Bad, forkhead family of transcription factors, and I{kappa}B kinase.14 Thus, Akt plays a critical role in controlling the balance between survival and apoptosis.15

In this study, we characterized PDT-induced cell death quantitatively using PDT of laser-induced CNV in a rat model, and we investigated the involvement of the caspase pathway and Akt in PDT-induced cell death. Clarifying the mechanisms of PDT-induced cell death may lead to improved therapies for CNV.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Induction of Choroidal Neovascular Membranes
All experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines established by the Animal Care Committee of the Massachusetts Eye and Ear Infirmary. Brown-Norway rats were anesthetized with a 0.1 to 0.2 mL of a 50:50 mixture of 100 mg/mL ketamine and 20 mg/mL xylazine. Pupils were dilated with a topical application of 5.0% phenylephrine and 0.8% tropicamide. CNVs were experimentally induced with an argon-dye–pulsed laser at 630 nm (model 920; Coherent Medical Laser, Palo Alto, CA) to disrupt Bruch membrane using a protocol similar to one previously described.16 A spot size of 100 µm was used, and the power delivered ranged from 130 to 150 mW, applied for 0.1 second. Four lesions were induced for immunohistochemistry and TUNEL assay, and 20 lesions were induced for Western blot analysis in the left eye of each animal. On occasion, the inducing laser burst created an extensive subretinal hemorrhage, and these spots were excluded from further treatment or analysis.

Photodynamic Therapy
PDT was performed as previously described.17 Two weeks after induction of CNV, anesthetized rats were immobilized on a stereotactic frame. Verteporfin (Visudyne; Novartis, Basel, Switzerland) was injected intravenously at doses of 3, 6, and 12 mg/m2 15 minutes before PDT. PDT was performed with a diode laser at a wavelength of 689 nm, irradiance of 600 mW/cm2, spot size of 800 µm, and fluence of 25 J/cm2 (Visulas; Carl Zeiss Meditec, Dublin, CA). Untreated rats were used as controls.

Immunohistochemistry
Eyes for IHC were enucleated at 1, 3, and 6 hours after PDT. Immunohistochemistry was performed as previously reported.18 19 The eyes were bisected behind the limbus and placed in 4% paraformaldehyde at 4°C overnight and then cryoprotected with PBS (0.1 M phosphate buffer, pH 7.4, 0.15 M NaCl) containing 20% sucrose. Cryosections (10 µm) were mounted onto slides and incubated with blocking buffer (PBS containing 10% goat serum, 0.5% gelatin, 3% BSA, and 0.2% Tween 20) and then were incubated with a primary antibody against pAkt or cleaved caspase-3 and -9 (dilution 1:100; Cell Signaling Technology, Inc., Beverly, MA). Normal rabbit serum (DAKO, Osaka, Japan) was used as a negative control. The sections were then incubated with fluorescence-conjugated secondary antibody diluted 1:200 in blocking buffer for 1 hour. These included goat anti-rabbit immunoglobulin G conjugated to fluorescent dye (Alexa TM 546; Molecular Probes, Eugene, OR). Sections were mounted with mounting media with DAPI (Vectashield; Vector Laboratories, Burlingame, CA). Photomicrographs were taken with fluorescence microscopy (Qfluoro System; Leica Microsystems, Wetzlar, Germany).

Immunofluorescence-positive cells in CNV were counted in the sections cut through the middle of the CNV lesions. The number of DAPI-positive cells in the CNV was defined as the number of total cells in the CNV per section. The rate of pAkt, cleaved caspase-3–positive, or cleaved caspase-9–positive cells in the CNV was measured by dividing the number of cells positive for these markers by the number of total cells in CNV. Cell counting was performed in a masked fashion.

Terminal dUTP Nick-End Labeling
TUNEL assay was performed as previously reported20 21 (ApoTag kit; Intergen, Purchase, NY) to detect cell death in CNV induced by PDT. Eyes were obtained 1, 3, 6, 15, 24, and 48 hours after PDT, and transverse sections with CNV were prepared as mentioned. After two washes with PBS, sections were incubated with TdT enzyme at 37°C for 1 hour. The sections were washed three times in PBS for 1 minute and incubated with anti-digoxigenin conjugate (rhodamine) in a humidified chamber for 30 minutes at room temperature (RT), followed by three rinses with PBS at RT. Sections were mounted with mounting media (Vectashield; Vector Laboratories) including DAPI for nuclear stain. The number of TUNEL positive cells was determined.

The number of TUNEL-positive cells in CNV was counted in the sections that were cut through the middle of the CNV lesions. DAPI-positive staining was used to define the total number of cells in the CNV per section. The rate of TUNEL-positive cells in the CNV was determined by dividing the number of TUNEL-positive cells by the total number of cells in the CNV.

Western Blot Analysis
Western blot analysis was performed as described.18 19 In brief, after the RPE–choroid complex was sonicated in lysis buffer, 30 µg total protein was used for SDS-PAGE and then was transferred to a polyvinyl fluoride membrane (Bio-Rad, Hercules, CA). After blocking, the membrane was incubated in a blocking buffer containing rabbit anti-pAkt antibody or total Akt antibody (dilution 1:1000; Cell Signaling Technology, Inc.) overnight at 4°C. Chemiluminescence was detected with an alkaline phosphatase–conjugated anti-rabbit IgG (dilution 1:20,000; Promega Corp., Madison, WI) and with a chemiluminescent substrate (CDP-Star; Amersham Pharmacia Biotech, Buckinghamshire, UK). For loading control, the membrane was incubated with mouse ß-actin (1:2000; Sigma-Aldrich, St. Louis, MO). The blot was exposed to medical x-ray film (RX-U; Fujifilm, Tokyo, Japan).

Intravitreous Injection Procedure
After rats were anesthetized and the pupils dilated, vitreous injections were performed as previously reported.18 19 Briefly, a 33-gauge needle tip was connected to the Hamilton syringe and inserted into the vitreous through a sclerotomy site 1 mm posterior to the corneal limbus of the eye. The tip of the needle was placed in the midvitreous, and 2 µL solution was slowly injected into the vitreous under direct observation with an operating microscope. Any eyes showing lens or retinal damage were excluded from the study.

Experimental Design
Apoptotic cells in CNV were detected by TUNEL assay at various time points after verteporfin PDT (verteporfin 3 mg/m2—6 and 24 hours; 6 mg/m2—1, 3, 6, 15, and 24 hours; verteporfin 12 mg/m2—1, 3, 6, 15, 24, and 48 hours) (n = 5 in each verteporfin dose at each time point).

Activation of caspase-3 and -9 in CNV was determined by IHC with antibody against cleaved caspase-specific antibodies at various time points (1, 3, 6, 15, and 24 hours) after PDT (verteporfin 12 mg/m2; n = 5 for each time point).

Akt activity in CNV was determined by IHC with a pAkt-specific antibody at various time points (1, 3, 6, 15, and 24 hours) after PDT (verteporfin 12 mg/m2; n = 5 for each time point).

Double staining for caspase-9 and TUNEL was performed at 3 hours after PDT. Double staining for pAkt and TUNEL was performed at 1 and 3 hours after PDT.

To investigate the role of Akt in PDT-induced apoptosis, 5 µg insulin-like growth factor (IGF)-1, an activator of Akt, with or without 12.5 µM wortmannin, an inhibitor of the PI3K-Akt pathway, was injected into the vitreous before PDT (verteporfin 12 mg/m2). IGF-1 was injected 1 hour before PDT, and wortmannin was injected 30 minutes before IGF-1 injection. Activation of Akt in RPE-choroid was determined by Western blot analysis with a pAkt-specific antibody 1 hour after PDT. Apoptotic cells in CNV were detected by TUNEL assay at 6 hours after PDT (n = 5 in each group). In TUNEL assay, 10-µg IGF-1 or 12.5-µM wortmannin injected groups were added.

Table 1 shows verteporfin dose and time after PDT for each aspect of the study.


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  		TABLE 1. Verteporfin Dose and Time after PDT for Each Phase of the Study

 
Statistical Analysis
All values are presented as mean ± SD. Paired groups were compared using the paired t-test. For three groups, data were compared by ANOVA followed by post hoc comparisons tested using the Bonferroni procedure (Statview version 5.0; SAS Institute, Cary, NC). P < 0.05 was considered statistically significant.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
TUNEL-Positive Cells in CNV after PDT
PDT was performed 2 weeks after laser-induced CNV. In control rats without PDT, TUNEL-positive cells were not seen in CNV. In PDT-treated rats, few TUNEL-positive cells could be recognized 1 hour after PDT. The rate of TUNEL-positive cells per total cells in CNV increased 3 hours after PDT, peaked at 6 hours, and then decreased by 48 hours, showing dose dependence with respect to verteporfin dose (Fig. 1) . Six hours after PDT, the rate of positive cells in CNV was 72.5% ± 12.5% at the doses of 12 mg/m2.


Figure 1
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  		FIGURE 1. TUNEL assay in CNV after PDT. TUNEL-positive cells are indicated by red fluorescence in contrast to blue fluorescence of DAPI-stained nuclei. (A) No TUNEL-positive cells were present in control CNV. (B–F) Representative pictures of CNV treated with PDT (verteporfin 12 mg/m2). (G) Time course of TUNEL-positive cells in CNV. The percentage of TUNEL-positive cells in CNV started to increase 3 hours after PDT, peaked at 6 hours, and decreased by 48 hours. Values are mean ± SD. Five different rats were used at each time point in each group. Dotted line: CNV lesion. Scale bar, 50 µm.

 
Cleaved Caspase-3 and -9 Immunoreactivity
To investigate whether caspase was involved in PDT-induced apoptosis, IHC with anti-cleaved caspase-3 and -9 was performed 1, 3, 6, 15, and 24 hours after PDT. In PDT-treated rats, several cleaved caspase-3–positive cells were recognized 1 hour after PDT. The number of positive cells in CNV peaked at 3 hours and then decreased by 24 hours (Figs. 2A 2B 2C 2D) . The rate of positive cells in CNV was 19.0% ± 2.4% at 3 hours after PDT. The number of cleaved caspase-9–positive cells in CNV also peaked at 3 hours after PDT and then decreased by 24 hours (Figs. 2E 2F 2G 2H) . The rate of positive cells in CNV was 26.8% ± 9.6% at 3 hours after PDT. These data suggest that caspases are activated after PDT and are earlier events preceding TUNEL positivity.


Figure 2
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  		FIGURE 2. Cleaved caspase-3 and -9 immunohistochemical staining in CNV. Positive signals are indicated by red fluorescence in contrast to blue fluorescence of DAPI-stained nuclei. (A–C) Representative pictures of cleaved caspase-3 immunohistochemical staining (verteporfin 12 mg/m2). (D) Time course of cleaved caspase-3–positive cells in CNV. Percentages of cleaved caspase-3–positive cells in CNV started to increase 1 hour after PDT, peaked at 3 hours, and then decreased by 24 hours. (E–G) Representative pictures of cleaved caspase-9 immunohistochemical staining (verteporfin 12 mg/m2). (D) Time course of cleaved caspase-9–positive cells in CNV. Percentages of cleaved caspase-9–positive cells in CNV started to increase 1 hour after PDT, peaked at 3 hours, and then decreased by 24 hours. Values are mean ± SD. Five different rats were used at each time point in each group. Dotted line: CNV lesion. Scale bar, 50 µm.

 
Double Staining of Cleaved Caspase-9 and TUNEL
To determine whether cleaved caspase-9–positive cells are committed to apoptosis, double staining of cleaved caspase-9 and TUNEL was performed 3 hours after PDT (verteporfin 12 mg/m2). Some cells stained by DAPI in CNV were characterized by the presence of condensed and fragmented nuclei (Fig. 3A , arrows). These cells colocalized with TUNEL-positive signal (Figs. 3B 3C) . Most of the cleaved caspase-9–positive cells were TUNEL positive (Fig. 3E) . These results suggest that PDT induced caspase-dependent apoptosis in CNV.


Figure 3
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  		FIGURE 3. Double staining of cleaved caspase-9 and TUNEL. (A) CNV stained with DAPI 3 hours after PDT. (B) TUNEL staining. (C) Merged images of TUNEL and DAPI nuclear staining. Condensed nuclei show TUNEL-positive. (D) Immunoreactivity of cleaved caspase-9. (E) Merged images of immunoreactivity of cleaved caspase-9 and TUNEL. Almost all cleaved caspase-9–positive cells merged with TUNEL-positive cells. (F) Merged images of immunoreactivity of cleaved caspase-9, TUNEL, and DAPI. Scale bar, 20 µm.

 
Double Staining of pAkt and TUNEL and Time Course of pAkt Immunoreactivity
To investigate further the signaling pathway after PDT, we examined whether the Akt signaling pathway was activated after PDT and served as an upstream regulator of the caspase pathway. Without PDT treatment, nearly all cells in CNV were immunoreactive for pAkt with no TUNEL positivity (Figs. 4A 4D 4G 4J) . One hour after PDT, the number of pAkt-positive cells was significantly reduced (P < 0.01; Fig. 4B ), and the rate of pAkt-positive cells in CNV was 50.3% ± 16.8% at 1 hour after PDT, suggesting that Akt was significantly dephosphorylated 1 hour after PDT in CNV. However, at this time point, there were few TUNEL-positive cells in CNV (Fig. 4E) . Three hours after PDT, though the number of TUNEL-positive cells increased, the number of pAkt-positive cells was still significantly reduced compared with controls (Fig. 4C) . Interestingly, TUNEL signals and pAkt signals were seldom colocalized in cells of the PDT-treated CNV (Fig. 4I) . These results strongly suggest that Akt is first dephosphorylated by 1 hour after PDT, followed by activation of caspase-9 and -3 by 3 hours after PDT, with eventual apoptosis. The rate of pAkt-positive cells in CNV reached a minimum (14.2% ± 11.5%) 6 hours after PDT and returned to 74.8% ± 13.8% by 48 hours (Fig. 4J) .


Figure 4
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  		FIGURE 4. Double staining of pAkt and TUNEL. (A, D, G) Control PDT. (B, E, H) One hour after PDT. (C, F, I) Three hours after PDT. (A) Almost all cells in CNV showed phosphorylated Akt-positive in controls. (D) There are no TUNEL-positive cells in controls. (G) Merged images of pAkt and TUNEL. (B) The number of phosphorylated Akt-positive cells was significantly reduced 1 hour after PDT. (E) There are few TUNEL-positive cells 1 hour after PDT. (H) Merged images of pAkt and TUNEL. (C) The number of pAkt-positive cells was still low 3 hours after PDT. (F) There are some TUNEL-positive cells in CNV at 3 hours after PDT. (I) Merged images of pAkt and TUNEL. pAkt-positive cells in CNV do not stain with TUNEL. (J) Time course of the number of pAkt and TUNEL-positive cells. *P < 0.01 compared with control (pAkt). Dotted line: CNV lesion. Scale bar, 50 µm.

 
Western Blot Analysis
To confirm quantitatively that Akt in RPE-choroid is dephosphorylated after PDT, the RPE–choroid complex was harvested 1 hour after PDT, and protein levels of pAkt and total Akt were examined by Western blot analysis. As shown in Figure 5A , the RPE–choroid complex treated with PDT had a sparser pAkt band (molecular weight, approximately 60 kDa) than controls, whereas the total Akt band showed a similar signal density for the two conditions. Thus, PDT changed the level of phosphorylation but not the amount of Akt protein. To investigate the role of the Akt pathway in PDT-induced apoptosis, we attempted to modify the phosphorylation of Akt in the RPE–choroid complex with intravitreal injection of IGF-1, an activator of Akt. One hour after PDT with IGF-1 injection, PDT-induced dephosphorylation of Akt was significantly suppressed (Fig. 5B) . The effect of IGF-1 was diminished by wortmannin, an inhibitor of the PI3K-Akt pathway (Fig. 5B) , without affecting the level of Akt protein. These data suggest that PDT-induced Akt dephosphorylation is reversed by intravitreal injection of IGF-1 through PI3K activation.


Figure 5
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  		FIGURE 5. Western blot analysis of phospho-Akt. PDT reduced the level of phosphorylation of Akt in RPE-choroid complex, whereas total Akt level was similar between them 1 hour after PDT (A). Injection of IGF-1 (5 µg) activated Akt in RPE-choroid 1 hour after PDT. Effect of IGF-1 on phosphorylation of Akt was diminished by wortmannin (B).

 
TUNEL Assay after PDT with Activation of Akt
Next, we performed TUNEL assay after PDT using the same experimental conditions (Fig. 5) . Rates of TUNEL-positive cells in IGF-1–treated rats were 40.9% and 31.4% (5 µg and 10 µg IGF-1, respectively) 6 hours after PDT (Figs. 6B 6C) . They were significantly lower than those of the PDT-only treated group (P < 0.01). The effect of IGF-1 (5 µg) on the rate of TUNEL-positive cells in CNV was significantly diminished by wortmannin (P < 0.05; Figs. 6D 6E ). However, there was no significant difference in the number of TUNEL positive cells between PDT-only treated rats and wortmannin-only injected rats 6 hours after PDT (Fig. 6E) . These results suggest that Akt activation reduced apoptosis in CNV after PDT.


Figure 6
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  		FIGURE 6. TUNEL staining in CNV 6 hours after PDT (A–D). Rate of TUNEL-positive cells in CNV (E). (A) PDT only. (B) IGF-1 (5 µg) + PDT. (C) IGF-1 (10 µg) + PDT. (D) Wortmannin (12.5 µM) + IGF-1 (5 µg) + PDT. IGF-1 significantly reduced the number of TUNEL-positive cells in CNV, and the effect of IGF-1 was diminished by wortmannin (E). *P < 0.01 compared with PDT-treated rats. {dagger}P < 0.05 compared with IGF-1 (5 µg) + PDT-treated rats. Dotted line: CNV lesion. Scale bar, 50 µm.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
PDT is a clinically effective treatment for AMD; however, the mechanism of PDT-induced cellular apoptosis is largely unknown. With the use of a rat model of PDT for laser-induced CNV, we characterized the signaling pathways leading to cell death after PDT. One hour after PDT, Akt in the cells of CNV was dephosphorylated; this was followed by caspase activation at 3 hours. The number of TUNEL-positive cells increased by 6 hours. Western blot analysis showed that the level of pAkt protein in the RPE–choroid complex was significantly decreased after PDT, indicating dephosphorylation but not degradation of Akt protein. Intravitreal injection of IGF-1 before PDT led to activation of the Akt protein in the RPE–choroid complex and reversed the PDT-induced dephosphorylation of Akt in the CNV through PI3K activation. Furthermore, IGF-1 suppressed the increase in TUNEL-positive cells in CNV after PDT. Thus, we have demonstrated that the suppression of Akt activity and the activation of caspase may play an important role in the death of cells in CNV after PDT.

To the best of our knowledge, an apoptosis time course after verteporfin PDT in rat laser-induced CNV has not been previously reported. Granville et al.6 reported that DNA fragmentation in cultured human aortic smooth muscle cells was observed by 1 hour after PDT with verteporfin and that more than 40% of cells exhibited DNA fragmentation by 5 hours after PDT. In this rat study, the number of TUNEL-positive cells showed an increase at 3 hours after PDT, peaked at 6 hours, and decreased by 48 hours. The variations in time course may be related to cell- and tissue-specific differences. It is well known that PDT causes direct activation of free radical–mediated cell injury and secondary formation of thrombi, leading to vessel occlusion. We examined TUNEL staining 3 and 7 days after PDT at the verteporfin doses of 12 mg/m2 in consideration of that point. Three days after PDT, there were few TUNEL-positive cells in CNV like the results 48 hours after PDT. No TUNEL-positive cells were observed in CNV 7 days after PDT. We speculate that the critical time is up to 2 days after PDT. Moreover, we have shown that PDT-induced thrombosis already occurred 24 hours after PDT.17 Although it is difficult to define which effect induces individual apoptosis, further studies defining specific cell types that undergo apoptosis and the time courses after PDT may clarify that point.

Generally, apoptotic cell death pathways are classified as caspase dependent or caspase independent.22 In the caspase-dependent pathway, cytochrome c is released from mitochondria after injury and results in the activation of caspase 9. Caspase 3, a downstream effector of caspase 9, executes cellular apoptosis. In the present study, we showed that peak caspase activation occurred earlier than peak TUNEL positivity in cells and that colocalization of TUNEL positivity and caspase signals in the same cells occurred after PDT. These data are supported by previous reports that PDT with verteporfin induces the activation of caspase-3 and -9 in other cells and tissues.6 9 10 Phosphorylated Akt strongly suppresses mitochondria-dependent apoptosis by inhibiting caspase-9 and Bad.14 23 We did not investigate whether a caspase inhibitor suppressed PDT-induced cell death in CNV; however, IGF-1, a specific upstream activator of the Akt signaling pathway, suppressed the immunoreactivity of cleaved caspase-9 in CNV (data not shown) and suppressed TUNEL-positive cells after PDT (Fig. 6) . Taken together, caspase activation appears to be the central mediator for verteporfin PDT-induced cellular apoptosis in CNV.

Many studies have demonstrated that VEGF is expressed in human and experimental CNV.24 25 VEGF regulates endothelial cell survival through the PI3K-Akt signaling pathway in angiogenesis.12 13 In this study, almost all cells in CNV showed pAkt positivity in control rats, suggesting that pAkt promotes cell survival in CNV by inhibiting apoptotic signals. The number of pAkt-positive cell numbers in CNV was significantly reduced 1 hour after PDT, followed by TUNEL-positive cells demonstrated 3 hours after PDT. These time-course data suggest that the dephosphorylation of Akt precedes apoptotic cell death. We also showed that pAkt-positive cells seldom colocalize with TUNEL-positive cells (Fig. 4I) and that the suppression of PDT-induced dephosphorylation of Akt by IGF-1 reduced the number of TUNEL-positive cells in CNV after PDT (Fig. 6E) . Furthermore, these IGF-1–induced responses were totally suppressed by wortmannin, an inhibitor of the PI3K-Akt pathway, strongly suggesting that Akt is an important determinant of cellular fate after PDT.

Akt is activated by various growth factors and cytokines, including VEGF, IL-1ß, and TNF-{alpha}.12 26 On the other hand, phosphatase and tensin homologue deleted on chromosome (PTEN) phosphatase is known as a major negative regulator of the PI3 kinase/Akt signaling pathway.27 PTEN inhibits PI3K-dependent activation of AKT, and deletion or inactivation of PTEN results in constitutive AKT activation. Akt plays a critical role in controlling survival and apoptosis by the balance of signals between kinase and phosphatase. Schmidt-Erfurth et al.4 reported that VEGF was detected in endothelial cells of the choriocapillaris after PDT. Solban et al.28 reported that subcurative PDT induced VEGF in prostate cancer cells, and this induction may contribute to tumor survival and regrowth and, therefore, may be responsible for limiting the efficacy of PDT for tumors. Recently, the suppression of PDT-induced inflammation using steroid appears to improve the therapeutic efficacy of PDT for AMD.29 30 In our study, Akt phosphorylation was significantly suppressed by PDT within 24 hours; however, the rate of pAkt-positive cells in CNV returned to the baseline level by 48 hours. Thus, if PDT for CNV induces inflammation- and inflammatory-related cytokines such as VEGF and TNF, it may affect Akt phosphorylation and may lead in turn to cell survival and to CNV recurrence. Further investigations are needed to seek the upstream factors that control PDT-induced dephosphorylation of Akt in CNV.

Our next interest is to define the specific cell types that undergo apoptosis in CNV after PDT. The main target of PDT for CNV is endothelial cells. In our other experiment using human umbilical vascular endothelial cells, p-Akt was dephosphorylated and apoptosis was induced after PDT. Hence, we speculate that vascular endothelial cells in CNV might induce caspase-dependent apoptosis after PDT. Further investigations will be needed to define the specific cell types and pathway that undergo apoptosis in CNV using immunohistochemical double staining in vivo.

In conclusion, we have begun to delineate the molecular mechanisms of PDT-induced cellular apoptosis in laser-induced CNV. Targeting Akt and caspase signaling pathways may improve the efficiency of PDT of CNV by increasing cellular apoptosis in CNV cells. These findings may yield new insight into the development of strategies for the treatment of PDT for patients with AMD.


   Footnotes
 
Supported by the Neovascular Research Fund, National Eye Institute Core Grants P30 EY014104 and NIH AI050775, Alcon Research Award, Massachusetts Lions Foundation, and Research to Prevent Blindness.

Submitted for publication December 26, 2006; revised May 26, 2007; accepted August 14, 2007.

Disclosure: A. Matsubara, None; T. Nakazawa, None; K. Noda, None; H. She, None; E. Connolly, None; T.A. Young, None; Y. Ogura, None; E.S. Gragoudas, None; J.W. Miller, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Joan W. Miller, Angiogenesis and Laser Laboratories, Retina Service, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA; joan_miller{at}meei.harvard.edu.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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