Abstract
Aim
To evaluate the impact of verteporfin photodynamic therapy (PDT) on the induction of apoptosis in choroidal neovascular membranes (CNV) secondary to age related macular degeneration.
Methods
Retrospective review of 22 surgically excised CNV. 12 of these patients had been treated with PDT 3–146 days previously. Apoptotic cells were detected with the TUNEL technique and compared to the expression of CD34 (endothelial cells, EC), CD105 (activated endothelial cells), Ki‐67 (proliferation marker), and cytokeratin18 (retinal pigment epithelial cells, RPE).
Results
CNV excised 3 days after PDT were characterised both by collapsed and patent vessels. The EC displayed a statistical significant positive TUNEL reaction when compared to the remaining treated CNV (p<0.001) and untreated CNV (P = 0.002). The proliferative activity was reduced. CNV excised 1–5 months after PDT displayed a patent vascularisation and high proliferative activity. All membranes either treated or untreated disclosed only sporadic TUNEL positive cells within the stroma and the RPE.
Conclusions
Verteporfin PDT leads to selective and effective damage of EC within CNV. Both patent and occluded vessels were lined by apoptotic EC. This finding and the increased expression of proliferation marker at later time points suggest that revascularisation after PDT is caused by angiogenesis rather than recanalisation.
Keywords: verteporfin, age related macular degeneration, apoptosis, choroidal neovascular membranes, photodynamic therapy
As a leading cause of legal blindness in the elderly, age related macular degeneration (AMD) accounts for most of the blindness in the industrialised world.1 The main cause of severe visual loss is the development of choroidal neovascular membranes (CNV).1 Photodynamic therapy (PDT) with verteporfin has been reported in several clinical trials to limit the disease and the progression of visual loss in subfoveal neovascular AMD.2,3,4,5,6 The therapy is based on a photosensitiser, which, activated by light irradiation, produces singlet oxygen that affects the exposed tissue. In neovascular AMD, the circulating photosensitiser is activated within the CNV in order to target the neovascular tissue without affecting the overlying neurosensory retina.7 Histopathological studies with light and electron microscopy emphasise this selectivity8 and recent clinicopathological studies evaluated the effect of PDT on CNV by light or electron microscopy.9,10,11,12,13
Histological findings of surgically excised CNV 3 days to 3 months after PDT have been published.9,10,11,12,13 In CNV 3 days after PDT endothelial damage without vascular occlusion as well as occluded vessels was noted.11,12 At later time points patent and perfused vessels with normal endothelium were detected.9,12 However, it was not clear whether the vasculature at later time points results from regeneration of damaged endothelium and recanalisation of the affected vessels or from angiogenesis and proliferation of endothelial cells.
In order to address this question we analysed surgically excised CNV for the presence of apoptosis and examined whether its expression could be correlated with the clinical picture and histological effects induced by PDT and the occurring changes thereafter.
Methods
Subjects and treatments
We retrospectively reviewed 22 eyes of 22 consecutive patients, in which surgery for CNV caused by AMD was performed (tables 1 and 2). Twelve of these patients underwent submacular surgery after verteporfin PDT (table 2). All patients had a complete ophthalmological examination including fundus photography and stereoscopic fluorescein angiography (FA). Patients receiving verteporfin PDT were examined by FA before treatment and thereafter on the day of surgery. CNV were classified according to the guidelines of the TAP and VIP studies.2,3,4,5,6 Therapeutic options, including observation, conventional thermal laser photocoagulation, PDT re‐treatment, macular translocation with 360° retinotomy, and CNV extraction were discussed with the patients. Surgical intervention and removal of the neovascular tissue were offered when (a) visual acuity was below 20/200, which is according to the TAP Investigation the minimum visual acuity to recommend the first PDT2,3,4,5,6; and (b) visual deterioration progressed after initial PDT.
Table 1 Clinical characteristics of patients without PDT treatment.
| Case | Eye | Age/sex | CNV type | Visual acuity | Time to surgery from onset/diagnosis of CNV |
|---|---|---|---|---|---|
| 1 | L | 70/M | predominantly classic | 0.6 | NA |
| 2 | L | 71/M | predominantly classic | 0.1 | 2 weeks |
| 3 | L | 71/F | occult | 0.025 | 4 months |
| 4 | L | 66/M | occult | 0.1 | 7 months |
| 5 | R | 63/M | classic | 0.05 | NA |
| 6 | L | 76/F | peripapillary occult | 0.05 | 8 months |
| 7 | R | 79/F | haemorrhagic | 0.1 | 4 months |
| 8 | L | 74/F | classic | 0.1 | 11 months |
| 9 | R | 83/M | classic | 0.1 | 3 months |
| 10 | L | 76/F | haemorrhagic | 0.1 | NA |
CNV, choroidal neovascular membrane; PDT, photodynamic therapy; NA, not available.
Table 2 Clinical characteristics of patients treated with PDT before surgical removal of the CNV.
| Case | Eye | Age/sex | CNV type | Visual acuity | PDT treatments | Time to surgery from PDT/ last PDT |
|---|---|---|---|---|---|---|
| 1 | L | 76/M | classic | 0.025 | 1 | 3 days |
| 2 | R | 78/F | classic | 0.02 | 1 | 3 days |
| 3 | L | 54/M | predominantly classic | 0.063 | 2 | 113/3 days |
| 4 | L | 84/M | classic | 0.025 | 1 | 3 days |
| 5 | L | 83/M | classic | 0.03 | 1 | 34 days |
| 6 | L | 85/F | classic | 0.03 | 1 | 37 days |
| 7 | R | 73/F | occult | 0.1 | 3 | 208/138/40 days |
| 8 | L | 79/M | classic | 0.1 | 1 | 55 days |
| 9 | L | 77/M | minimally classic | 0.6 | 1 | 84 days |
| 10 | L | 76/F | occult | 0.6 | 1 | 105 days |
| 11 | L | 81/M | classic | 0.08 | 2 | 213/131 days |
| 12 | L | 78/F | classic | 0.05 | 3 | 344/222/146 days |
CNV, choroidal neovascular membrane; PDT, photodynamic therapy.
Verteporfin PDT 3 days before macular surgery was performed in order to reduce the risk of bleeding as a result of the surgical extraction. Each patient gave written informed consent after the experimental nature of the treatment procedure and the risks and benefits of all options were discussed in detail. The study followed the guidelines of the declaration of Helsinki as revised in Tokyo and Venice. The study with histological analysis of the specimens was approved by the institutional review board.
Verteporfin PDT was applied according to the TAP study (6 mg/m2 body surface area; non‐thermal laser light (689 nm); 50 J/cm2; 600 mW/cm2; 83 seconds).3
Macular surgery including membrane extraction was performed via a standard three port vitrectomy.
Tissue preparation
Within minutes after surgery, excised CNV were fixed in 3.7% formalin and subsequently embedded in paraffin. Each specimen was serially sectioned into 5 μm sections and mounted on poly‐l‐lysine coated glass slides (Dako, Glostrup, Denmark) for immunohistochemical staining. Haematoxylin and eosin staining was performed to determine the histological orientation.
Immunohistology
After serial paraffin sections were deparaffinised with xylol and rehydrated with a graded series of alcohol, antigen retrieval, and immunohistochemical staining for CD 105, CD 34, Ki‐67, and cytokeratin 18 was performed with horseradish immunoperoxidase technique as previously described.14
For the detection of apoptosis sections were permeabilised with 20 μg/ml proteinase K in 10 mM TRIS/HCl pH 7.4 for 30 minutes at 37°C. The TUNEL (TdT mediated dUTP nick end labelling) method was performed according to the manufacturer̀s protocol (in situ cell death detection kit AP, Roche Diagnostics, Penzberg, Germany). To induce DNA strand breaks for the positive control, the control section was incubated with 1 mg/ml DNAse I in 50 mM TRIS/HCl, pH 7.5, 1 mM MgCl, 1 mg/ml bovine serum albumine for 30 minutes at 37°C. The negative control was incubated only with TUNEL label solution without TdT. For analysis by light microscopy the included Converter AP and the recommended fast red (Roche Diagnostics, Penzberg, Germany) were used. Counterstaining was performed with Mayer's haematoxylin.
Analysis
Apoptosis was evaluated by counting the numbers of TUNEL positive cells under ×200 magnification. Vascularisation was evaluated by analysis of the specimen with immunoperoxidase stain for CD 34 and CD 105 and counting the numbers of CD 34/CD 105 positive cells under ×200 magnification. Genuine proliferative activity was evaluated by counting the number of Ki‐67 positive nuclei in the area with the highest proliferation activity under ×400 magnification.
Staining for the different markers was determined independently by three masked observers (KP, OT, and SG). Inter‐observer and intra‐observer agreement was found in all cases.
Statistical analysis was performed with the software package JMP5.0.1.2 (SAS Institute, Cary, NC, USA). For statistical evaluation, with non‐parametric Fisher‐Pitman test was performed. The significance level was set to α = 0.05.
Results
Angiographic classification and characterisation
All cases were classified according to the TAP and VIP reports before treatment with PDT (tables 1 and 2). Depending on the post‐treatment interval, the angiographic features in the treated CNV differed. The four membranes extracted 3 days after PDT showed at the day of surgery a hypofluorescence in early phases of angiography, suggesting non‐perfusion of the irradiated area and the CNV. Late phases FA revealed leakage at the fovea consistent with choroidal ischaemia. In CNV extracted at longer post‐PDT intervals, on the day of surgery a hyperfluorescent membrane was seen in early phases of FA with leakage in late phases (data not shown).
Histological characterisation
All but one untreated CNV were vascularised as evidenced by CD 34 positive vessels. Fifty per cent of the specimens were strongly, 20% moderately, and 20% only weakly vascularised. Eighty per cent of the specimens displayed a concomitant expression of CD105 marking activated endothelial cells. One vascularised membrane, however, showed no CD105 expression at all. This specimen contained no proliferating Ki‐67 positive cells. In the other membranes, the number of Ki‐67 positive cells varied from 0 to 21 with a median number of 6.5 proliferating cells (table 3).
Table 3 Immunohistological findings in the CNV devoid of PDT.
| Case | TUNEL* | CD 34† | CD 105‡ | Ki‐67§ | ||
|---|---|---|---|---|---|---|
| RPE | Stroma | EC | ||||
| 1 | 0 | 0 | 0 | 47 | 42 | 7 |
| 2 | 1 | 1 | 0 | 183 | 176 | 21 |
| 3 | 1 | 0 | 1 | 17 | 28 | 0 |
| 4 | 0 | 0 | 0 | 398 | 411 | 10 |
| 5 | 0 | 2 | 0 | 193 | 178 | 5 |
| 6 | 0 | 5 | 0 | 15 | 37 | 10 |
| 7 | 0 | 0 | 0 | 0 | 0 | 1 |
| 8 | 0 | 0 | 0 | 71 | 68 | 4 |
| 9 | 0 | 2 | 0 | 89 | 92 | 6 |
| 10 | 3 | 0 | 0 | 25 | 11 | 0 |
*By analysis of the specimen with TUNEL technique, number of positive nuclei in the specimen under ×200 magnification.
†Immunoperoxidase stain for CD 34.
‡Immunoperoxidase stain for CD 105 (endoglin), number of positive cells under ×200 magnification.
§Immunoperoxidase stain for Ki‐67 counting the number of Ki‐67 positive nuclei in the area with the highest proliferation activity under ×400 magnification.
The number of apoptotic cells was low and inconsistent. Forty per cent of the specimens displayed no apoptosis at all. Three specimen contained 1–3 (median 1, range 0–3) apoptotic RPE cells. Only one CNV displayed one apoptotic endothelial cell. Whereas apoptotic cells were encountered within the stroma of four membranes with a varying low number of 1–5 (median 2, range 0–5) (table 3, fig 1).
Figure 1 Photomicrographs of a CNV devoid of PDT treatment. The specimen discloses patent vessels with healthy endothelial cells. Two cells within the stroma were TUNEL positive resulting in a red staining (arrows). The tissue was counterstained with haematoxylin. Scale bar: 100 μm.
All membranes previously treated by PDT were vascularised (table 3). The level of vascularisation appeared low at day 3 after PDT and most (mean 81.1%) of the vessels were occluded. A patent and strong vascularisation expressing CD105 could be observed 1–2 months after PDT. A moderate vascularisation was maintained at later time points. These characteristics were associated with an absence or a low number (median 0.5, range 0–6) of proliferating Ki‐67 positive cells 3 days after PDT, but increased levels (median 18, range 1–52) at later time points (table 4). Owing to the small number of cases, the increase of proliferating cells was barely not statistical significant (p = 0.091). But a larger number of examined specimens, comprising the presented ones, showed a statistical significant increase of Ki‐67 positive cells at later time points after PDT.15
Table 4 Immunohistological findings in the CNV treated by PDT.
| Case | TUNEL* | CD 34† | CD 105‡ | Ki‐67§ | Time to surgery from PDT/last PDT | ||
|---|---|---|---|---|---|---|---|
| RPE | Stroma | EC | |||||
| 1 | 2 | 6 | 13 | 12 | 15 | 0 | 3 days |
| 2 | 5 | 5 | 11 | 16 | 17 | 1 | 3 days |
| 3 | 12 | 5 | 74 | 72 | 77 | 0 | 113/3 days |
| 4 | 3 | 5 | 34 | 38 | 40 | 6 | 3 days |
| 5 | 2 | 15 | 0 | 183 | 179 | 37 | 34 days |
| 6 | 0 | 1 | 0 | 35 | 33 | 9 | 37 days |
| 7 | 0 | 3 | 0 | 371 | 365 | 52 | 208/138/40 days |
| 8 | 3 | 6 | 5 | 327 | 333 | 27 | 55 days |
| 9 | 0 | 0 | 0 | 15 | 13 | 3 | 84 days |
| 10 | 0 | 1 | 0 | 132 | 135 | 37 | 105 days |
| 11 | 9 | 1 | 2 | 209 | 165 | 6 | 213/131 days |
| 12 | 1 | 0 | 0 | 92 | 76 | 1 | 344/222/146 days |
*By analysis of the specimen with TUNEL technique, number of positive nuclei in the specimen under ×200 magnification.
†Immunoperoxidase stain for CD 34.
‡Immunoperoxidase stain for CD 105 (endoglin), number of positive cells under ×200 magnification.
§Immunoperoxidase stain for Ki‐67 counting the number of Ki‐67 positive nuclei in the area with the highest proliferation activity under ×400 magnification.
Three days after PDT the number of TUNEL positive apoptotic cells was prominent within the endothelium, while the number of apoptotic RPE cells and stromal cells were consistent or only modestly increased (table 4). Both collapsed and patent vessels disclosed apoptotic endothelial cells (fig 2).
Figure 2 Photomicrographs of CNV from case 1 (A) and 2 (B) in tables 2 and 4, extracted 3 days after PDT. Specimens are probed with TUNEL reaction kit for apoptosis. Collapsed vessels as well as patent appearing vessels were apoptotic. Scale bars: 50 μm (A) 100 μm (B).
At longer post‐treatment intervals, the number of apoptotic EC decreased significantly and was similar to the apoptosis rate in the untreated cases (tables 3 and 4, figs 3 and 4). The increase of apoptotic endothelial cells 3 days after PDT (median 23.5, range 11–74) was significant when compared to untreated CNV (p<0.001) and CNV at longer post‐treatment intervals (p = 0.002). In contrast, there was no statistical significance concerning the number of apoptotic RPE cells (median 4, range 2–12) and stromal cells (median 5, range 5–6) in CNV 3 days after PDT when compared to untreated CNV (RPE P = 0.727; stroma p = 0.501) or CNV at later time points after PDT (RPE P = 0.194; stroma p = 0.592) (tables 3 and 4, figs 3 and 4).
Figure 3 Photomicrographs of a CNV (case 7; tables 2 and 4) extracted 40 days after PDT (A) and a CNV (case 9; tables 2 and 4) 84 days after PDT (B). The specimen of case 7 discloses patent and vital looking vessels, only two apoptotic cells within the stroma are apoptotic. The specimen of case 9 discloses no apoptotic cell at all. Scale bars: 100 μm.
Figure 4 Photomicrograph depicting a CNV (case 11; tables 2 and 4) 131 days after PDT (A). The specimen discloses only one apoptotic cell within the stroma of the specimen, but at the edge of the CNV a small vessel with apoptotic endothelial cells was detected (B). Scale bars: 100 μm.
Discussion
The aim of PDT is to confine damage at the targeted site by avoiding a collateral damage to the neighbouring tissue. The photosensitiser, in this case verteporfin, absorbs light and induces the generation of reactive free radicals or excited singlet oxygen. The mechanisms of cell and tissue destruction are not yet fully understood. Cellular, vascular and even immunological mechanisms may be involved.14 According to Schmidt‐Erfurth and colleagues, damaged endothelial cells will release clotting factors that lead to a blood flow stasis and vascular occlusion.16
A few small case series have been published so far describing the histological findings of CNV excised at different time points after PDT. Ghazi et al examined one specimen and demonstrated vascular occlusion, endothelial cell damage, and thrombus formation in a CNV treated with PDT 4 weeks previously.10 Schnurrbusch et al found occluded vessels as well as vessels with normal endothelium containing intact red blood cells in two CNV examined 3 months after PDT.9 Moshfeghi et al examined eight specimens which were excised 3–152 days after PDT.12 The CNV excised 3 days after PDT showed a vascular occlusion which could not be detected at later time points after PDT. In previous histopathological studies we found that CNV excised 3 days after PDT contained both collapsed and patent vessels identified by CD 34 and CD 105 expression,11,14 even though FA showed a hypofluorescence of the treated area. There is a controversy in the interpretation of this hypofluorescence after PDT; a reduced blood flow of the choriocapillaris was shown in an experimental model.7,11,14 We therefore suppose that the reduction of the choroidal perfusion in the surrounding choroid explains the hypofluorescence seen 3 days after PDT. Specimens excised 34–146 days after PDT, in contrast, displayed a strong and healthy vascularisation, which showed a strong expression of CD 105. Accordingly, FA displayed a hyperfluorescence.
These observations led to the question whether the observed patent vessels at later time points after PDT result from regeneration and recanalisation of the treated vessels9,12 or from angiogenesis and proliferation of endothelial cells.10,14 In order to answer this question, the level of cellular damage expressed as apoptosis was assessed and compared to the vascularisation and proliferative activity of the specimen.
Apoptosis is a non‐inflammatory programmed cell death and has an important role in the homeostasis of normal tissue, wound healing processes and a wide array of diseases.17 Apoptosis seems to be also a regulating mechanism in choroidal neovascularisation. Previous studies demonstrated the presence of apoptotic retinal pigment epithelial cells, endothelial cells and macrophages in untreated CNV.18,19,20 We found the same sporadic pattern of expression in cases that were not treated by PDT previously. Wiezorrek and colleagues found apoptosis more frequent in recent CNV than in long standing lesions.19 Hinton and co‐workers found a high number of apoptotic cells in two of 10 membranes and suggested that apoptosis in active stages of CNV is related to the wound healing process.18 Additionally, it was postulated that apoptosis may be the mechanism that allows the non‐inflammatory resolution of the neovascular tissue into a fibrotic scar.18
Looking for apoptosis in previously treated membranes we disclosed that 3 days after PDT both collapsed and patent vessels were TUNEL positive. The presence of apoptotic endothelial cells was significantly higher compared to non‐treated specimens. Even though we found some apoptotic cells within the stroma and the RPE cell layer, there was no statistic significant increase in comparison with the non‐treated cases. Since the vessels, even the patent ones, are lined by apoptotic cells, while RPE and other cells within the stroma of CNV remain unaffected, we may conclude that PDT is in fact quite selective in targeting vascular endothelium. Apoptosis seems to be a major actor in the mechanism of PDT in CNV.
In contrast, specimens excised 1 month or later after PDT disclosed a strong vasculature with patent vessels lined by vital non‐apoptotic endothelial cells. The number and distribution of apoptotic cells within these CNV again were comparable to the histopathological findings in CNV without treatment of PDT.18,19 Our findings favour the theory that patent neovascularisation after PDT is hardly based on regeneration of existing vessels but on angiogenesis and proliferation of endothelial cells. The TUNEL reaction becomes positive in an intermediate stage of the programmed cell death and is based on labelling of DNA strand breaks.17,18,19 Thus, regeneration at this stage is not possible. Therefore, it is highly doubtful that apoptotic endothelial cells can regenerate.
Arroyo et al have suggested revascularisation to occur in larger calibre vessels which may be more resistant to the injurious effect of PDT.13 Capillaries with reduplication of basement membranes were thought to represent the endothelial revascularisation of the ghost vessel.10,13 However, we have detected apoptosis also in patent appearing vessels irrespective of size.
It is not excluded that existing vascular channels may be recanalised. The resurfacing of these vessels, however, is based on an angiogenic process. This assumption is supported in first line by the fact that apoptotic EC will not survive in recanalised vessels. Furthermore it is supported by the prominent expression of CD105 and the increase of Ki67,15 indicating a high proliferative activity at longer post‐treatment intervals. Another indication for the angiogenic rather than regenerative process may be the associated inflammatory infiltrate as already described by Moshfeghi et al.12
Verteporfin, when activated by light, has been shown to induce apoptosis in neoplastic tissues.21 However, we are unaware of previous reports analysing the expression of apoptosis in CNV after PDT. There is a potential for damaging retinal tissue if the treatment is not performed within the parameter guidelines. Electron microscopy of treated human retina without CNV showed an occlusion of the choriocapillaris and damaged endothelial cells but no retinal damage, when treated in within the parameter guidelines.8 Regarding our immunohistological results, PDT is selective for endothelial cells within CNV, since RPE cell layer and cells within the stroma remain unaffected, but we could not assess the overlying neurosensory retina.
The proper interpretation might be limited by the small number of cases. According to our results, treatment with PDT leads not only to an occlusion of most of the vessels, but induces also an acute and irreparable damage of the exposed endothelial cells. The vital EC composing the vessels at longer intervals after PDT are considered to result from angiogenesis. An adjuvant antiangiogenic therapy to counteract this rebound effect is being tested in pilot studies and will probably occupy a pivotal role in the routine treatment strategies of the near future.
Abbreviations
AMD - age related macular degeneration
CNV - choroidal neovascular membranes
EC - endothelial cells
FA - fluorescein angiography
PDT - photodynamic therapy
RPE - retinal pigment epithelium
TUNEL - TdT mediated dUTP nick end labelling
Footnotes
This work was supported by the Grimmke Foundation, Jung Foundation and Vision 100 Foundation.
The authors have no competing interests related to the manuscript.
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