Photochemistry and Photobiology, 2004, 79(6): 494-498

Rapid Communication

Angiogenesis Induced by Photodynamic Therapy in Normal Rat Brain?

Feng Jiang', Zheng Gang Zhang', Mark Katakowskiii2, Adam M Robin', Michelle Faber', Fan Zhang' and Michael Choppy<'?' 'Heniy Ford Health Sciences Center, Neurology Department, Detroit, MI and *Oakland University, Physics Department, Rochester, MI

Received 19 November 2003; accepted 8 March 2004

ABSTRACT

Angiogenesis promotes tumor growth and invasiveness in brain. Because brain injury often induces expression of angiogelaic-pronncPtillg molecniles, we hypothesize that oxidative insult induced by photodynamic therapy (PDT) could lead to an endogenous angiogenic response, possibly diminishing the efficacy of PDT treatment of tumors. Therefore, we sought to establish whether PDT induced an angiogenic response within the nontumored brain. PDT using Photofrin as a sensitizer at an optical dose of 140 J/cm2 was performed on normal rat brain (n = 30). Animals were sacsificed at 24 h, and 1,2,3 and 6 weeks after PDT treatment. Fluorescein isothiocyanate- dextran perfusion was performed, and brains were fixed for immnnohistological study. Immunostaining revealed that vascular endothelial growth factor (VEGF) expression in- creased within the PDT-treated hemisphere 1 week after treatment and remained elevated for 6 weeks. Three-dimen- sional morphologic analysis of vasculature within PDT-treated and contralateral brain demonstrated PDT-induced angio- genesis, as indicated by a significant increase in vessel con- nectivity (P < 0.001) concomitant with decreased (P < 0.05) mean segment length compared with vessels within the contralateral hemisphere. Volumetric measurement of angio- genic regions indicate that neovascular expansion continued for 4 weeks after PDr. These data demonstrate that PDT induces VEGF expression and neovascularimtion within normal brain. Because angiogenesis promotes growth and invasiveness of tumor, antagonizing this endogenous angio- genic response to PDT may present a practical means to enhance the efficacy of PDT.

qPosted on the website on 25 March 2004. *To whom correspondence should be addressed: 25 March 2004:

Neurology Research Building, Room 3056, Henry Ford Health Sciences Center, 2799 West Grand Boulevard, Detroit, MI 48202, USA. Fax: 313- 916-13 18; e-mail: chopp@neuro.hfi.edu

Abbreviations ' CNS, central nervous system; FITC, fluorescein isothio- cyanate; HIF-la, hypoxia-inducible factor-1 alpha; LSCM, laser- scanning confocal microscopy; PDT, photodynamic therapy; VEGF, vascular endothelial growth factor. 0 2004 American Society for Photobiology 0031-8655/04 $5.00+0.00

INTRODUCTION Vascular endothelial growth factor (VEGF) and its receptors Flk and Flt are strong promoters of endothelial cell-mediated angiogenesis. Angiogenesis i s considered essential for tumor growth beyond s few millimeters in diameter (1) . Proangiogenic factors that me most specific to endothelium include VEGF, angiopoietin-1 and angiopoietin-2. Both cultured glioma cells and tissues express large amounts of angiogenic factors, mostly VEGF (2,3).

Photodynamic therapy (PDT) has been used to treat glioma, with mixed results. Treatment of solid malignancies with PDT involves tissue-penetrating laser light exposure aftei: systemic admiiaistra- tion of a tumor-localizing photosensitizer (4). The properties of photosensitizer retention in tumor tissue and the photochemical production of reactive oxygen species are combined with the precise delivery of laser-generated light to produce a treatment that offers localized tunioricidal activity, while minimizing insult to normal tissue. Clinical studies suggest improved survival time with PDT and demonstrate the effectiveness of PDT in producing necrosis of solid malignant gliomas (5-7). However, the efficacy of PDT treatment of central neilious system (CNS) neoplasms remains inconclusive. Currently, PDT is undergoing clinical evaluations for treatment of brain, head and neck, lung 'and other malignancies (8-10). We hypothesize that a reason for the failure of PDT as an effective treatment of glioma may be attributed to enhanced angiogenesis powered by PDT induction of VEGF. The relationship between PDT and angiogenesis was first proposed by Gomer and colleagues (1 I). Their studies indicate that Photofrin PDT induced expression of Iiypoxia-inducible factor-1 alpha (IllF-la) and also increased protein levels of the HIF-1 target gene, VEGF in tumors. Tumor-bearing mice treated wit11 combined antiangiogenic therapy (IM862 or EMAP-II) 'and PD?' had improved turnoricidal respomes compared with individual treatments. In addition, PDT-induced VEGF expression in tumors decreased with combination antiangio- genic and PDT therapy ( I 1). The expression of VEGF in areas surrounding tumor necrosis initially raised suggestions that hypoxia plays a major angiogenic role within tumors (12,13). PDT produces significant increases in VEGF within treated lesions (11). Both tumor angiogenesis and recurrence may therefore be mediated by PDT via the enhancement of VEGF expression within the treated tumor mass (1 3).

Although the oxidative effects of PDT occur preferentially within the targeted tumor tissue, endogenous boundary tissue is not wholly

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Photochemistry and Photobiology, 2004, 79(6) 495

excluded (8). One common response of the normal CNS to insult in a variety of paradigms is characterized by il transient upregulation of angiogenic factors including VEGF (14,15). The growth and invasiveness of malignant glioina is largely dependent on angio- genesis; therefore, induction of angiogenesis witbin the nontumor lesion periphery may support tumor survival, reducing the efficacy of PDT. Therefore, we applied PDT to normal brain tissue to test whether this therapy promoted an angiogenic response.

MATERIALS AND METHODS All experimental procedures have been approved by the Institutional Animal Care and Use Committee of Henry Ford Hospital.

Light delivery. An argon-dye laser system (Coherent, model INNOVA- 70 and CR-500, Palo Alto, CA) provided the light (632 nm) for the PDT treatment and optical measurement. The light was coupled into a 400 pit4 optical fiber with a distal microlens (PDT, Santa Barbara, CA) for a 5 mm diameter, uniform spot for supdicial irradiation. The power at the distal end of the fiber was adjusted to 20 mW and was measured before and after each treatment with a power meter (Photodyne, Westlake Village, CA) with a 1‘ integrating sphere detector head. The irradiation power p v e d stable in a l l experiments.

PUT treatment. Thirty Fisher rats (200-220 g) were used in this ex- periment. Photofrii (Quaddogic Technologies, Vancouver, BC, Canada) dissolved in dextrose solution was administered to the rats (innapmi- toneally) at a dose of 12.5 mg/kg. Animals were treated with laser at 24 h after drug adminisnation. Animals Were anesthetized with ketamine (80 m a g ) and xylazine (13 mgkg). Once fixed in a stereotaxic device, the scalp was remcted and the cranium exposed, and an incision was ma& directly down the midline. Using a drill, a 5 mm craniectomy waa made on the right hemisphem anterior to the c m n l suture. Laser light was delivered through the 5 mm craniectomy. The optical dose used in this experiment was 140 J/ cm’, at a fluence rate of 100 mW/cm’, consistent with nn optical dose used in our previous studies (16-18). The craniectomy was covered wirh a film of polyvinyl chloride glued to the surrounding intact bone, and the incision was closed with 4-0 silk suture (Ethicon, SomerviUe, NJ) after surgery.

Tissue preparation. Fluorescein isothiocyanate (FlTC)dextran (2 X lo6 molecular weight, Sigma, St. Louis, M O 0.1 mL of 50 mg/mL) was administered intravenously to the rats subjected to PDT at 1 day, 1,2,3 and 6 weeks (n = 6 rats per group) after the light treatment. FlTC-dextran remains dissolved and free in plasma. Two minutes after the injection of PITC- dextsan, the animals were sacrificed and the brains were rapidly removed from the severed heads and placed in 4% paraformaldehyde at 4°C for 48 h. Coronal sectiom (100 pan) wnr? cut on a vibratome.

Measurement of the v o l w of cerebral tissue with changed vascular srrucrure induced by PDT. In two-dimensional imaging of vibratome sections, abnormal vascular structures induced by PDT are visible to the naked eye and can be described and identified as regions of high vessel density or the presence of abnormally large vessels (or both) (Fig. la,b) as

Figure 1. a: FITC-dextran-petfused vessels within the PDT-treated area. Abnormally high vessel density and large vessels are indicative of angiogenesis ( m w ) , bar is 20 pm. b: Normal ruts were treated with 140 J/cm2, 12.5 m a g PhotoErin PDT, perfused with FlTC- dextran and sacrificed at 24. h, 1, 2, 3 ,4 and 6 weeks after PDT. The figure shows a qmsentative coronal section at 2 weeks after PDT. The red outline indicates the area of abnormal vascula- ture. Bar is 100 pm.

compared with a contralateral counterpart. Tlie volume of tissue with this abnormal structure was measured using a Global Lab Image analysis program (Data Translation, Marlborn, MA). The areas containing the changed vascular stnicture (mm2) were calcnlated by tracing in M S imaging software (Imaging Research, St. Catharines, ON, Canada). The volume (mm3) was determined by multiplying the appropriate area by the section interval thickness.

Three-dimensional image acquisition and analysis. To examine dynamic changes in cerebral blood vessels after PDT treatment, we performed morphologic analysis of vessels of the area surrounding the lesion as weU as the contralateral hemisphere of rat brain in three dimensions at different time points after PDT. Our 3-D quantitative analysis program has features to measure numbers of vessels, numbers of branch points, segment length, and vessel diameters (19,20). The vibratome sections were analyzed with a Rio- Rad MRC 1024 (argon and krypton) laser-scanning confocal imaging system mounted onto a Zeiss rnicmscope (Bio-Rad, Cambridge, MA), as previously described (21). Every 100 prn thick vibratome coronal section fmm the start to the end of the treatment area from each animal injected with FTTC-dextran was selected. These regions were scanned in 512 X 512 pixel (279 X 279 p2) format in the x-y direction using a 4X fixme-scan average and 25 optical sections along the z-axis with a 1 pm step-size were acquired under a 40X objective. Vascular branch points, segment length and diameter were measured. Abnormal vasculamt was identified in thxe-dimensional analysis by significant increases in mean vessel diameter mid number of vessel branch points, coiiicidcnt with a signiiicaut decrease ki ineari vessel length (19,20).

Immpnohistochemisby and quantifrcation. A goat polyclonal ‘antibody against VEGF (Santa Cruz Biotechnology, Santa Cruz, CA) was used at a titer of k200 to assess VEGF. Vibratome sections (100 pm thick) were incubated with primary antibodies against VEGF (1:lOO) and with Cy-5- conjugated secondary antibodies (Vector, Burlingame, CA). For semi- quantification of VBGP imrnunoreactivity, five VEGF irmnunostained coronal sections at 100 prn intervals were andyyzed from each brain. Eight fields of view froin the boundary regions of necrosis and contralateral hemisphere were imaged at e:rch time poilit. The riecrotic boundary wtts defined as tissue adjacent to the lesion area lacking FITC-dextran perfusion.

Table 1. The measurement of the volume of tissue with abnormal vasclllature in the Fisher rat brain at different time points after PDT treatment

~ ~~~

1 Week 2Weeks 3 Weeks 4Weeks 6W&s

Volum of tiisue with abnormal vasculature (nun3) 1.4+ 0.1 2.5 ?0.9 3 . 3 f 1.1 3.6 f 1.6 3.1 f 1.2

496 Feng Jiang eta/,

Figure 2. LSCM images of HTC-dex- tran-perfused vessels within normal brain 6 weeks after PDT treatment, ipsilateral to PDT (B), contralateral homologous tissue (A). Increased vessel density within the PDT-treated hemi- sphere is evident. Image size is 276 X 276 X 30 pm.

Imaged sections were digitized under a 20X objective (Olympus BX40) using a 3-CCD color video camera (Sony DXC-970MD) interfaced with an MCID image analysis system (Imaging Research). The area of tissue positive for VEGF compared with the total image area was calculated to provide a semiquantifiable density measurement. All values were presented as a percentage of the maximum value obtained.

RESULTS In all rats subjected to Photofrin PDT, a lesion and abnormal vascular structure area surrounding the lesion were detected in the treated hemisphere on coronal sections (Fig. la). The demarcation of cerebral tissues containing this abnormal vascular structure after treatment is illustrated in Fig. 1 b. These data demonstrate that PDT changes the structure of cerebral blood vessels, and this alteration occurs in a post-PDT time-dependent manner. The volumes of these changed vascular structure tissues obtained by image analysis are summarized in Table 1 .

Figure 2 shows reconstructed 3-D cerebral microvessels of the PDT-treated area and the contralateral hemisphere derived from the original images obtained from laser-scanning confocal microscopy (LSCM). Green and red colors in Fig. 3 code for diameters of blood vessels smaller than 7.5 pm and larger than 7.5 pm, respectively. Three-dimensional analysis of cerebral vessels in the boundary regions of necrosis and vessels in homologous area of the contralateral hemisphere in 30 rats at different times after PDT treatment was performed using software developed in our laboratory (19,20). Many cerebral vessels in the PDT-treated brain became extensively branched, and these vessels had different diameters compared with vessels in the contralateral hemisphere. Quantitative data reveals that (Table 2), at 3 and 6 weeks after PDT, vessel branching significantly (P < 0.001) increased in the treated brain compared with vessels in the homologous tissue of the contralateral

hemisphere. Furthermore, at 2 weeks or longer after PDT treatment, segments of capillaries were significantly (P < 0.05) shorter in the PDT-treated brain tissues than in the contralateral hemisphere. These data indicate that new small vessels are generated by PDT.

Immunostaining for endogenous rat VEGF showed increases in VEGF immunoreactivity within the necrosis boundary zone in rats treated with PDT (Fig. 4) at 1 week or longer after PDT treatment. Semiquantitative analysis revealed that VEGF immunoreactivity was significantly (P < 0.05) increased in rats treated with PDT compared with the immunoreactivity in the contralateral brain at 1 week or longer after PDT treatment (Fig. 5).

DISCUSSION VEGF is a potent mediator of endothelial function and a strong promoter of angiogenesis (22,23). Here, we demonstrate that normal brain exposed to PDT responds via a sustained upregulation of VEGF production local to the treated volume. The morphological pattern of VEGF reactivity in PDT-treated brain suggests astrocytic expression of VEGF, which we have previously established within angiogenic areas of stroke brain (20). VEGF receptors are highly expressed in a variety of neoplasms (24-26), and an overwhelming majority of tumors respond to VEGF with increased proliferation and survivability. Thus, the presence of VEGF could antagonize the localized tumorcidal activity of PDT.

Enlarged thin-walled vessels are termed “mother” vessels and have been found in pathological angiogenesis (27). Mother vessels can develop into small vessels by sprouting, by invaginating or by forming transluminal endothelial bridges to structure smaller caliber daughter vessels and glomeruloid bodies (27). In this study, we have found that enlarged blood vessels localized to the lesion boundary and that some of these vessels sprout at the boundary of the PDT-

Figure 3. LSCM images of vasculature within normal brain 6 weeks after PDT treatment. Image B was obtained from the hemisphere ipsilateral to treatment. Image A was obtained from contralateral homologous tissue. Color coding high- lights vessel diameters. Image size is 276 X 276 X 30 pm.

Photochemistry and Photobiology, 2004, 79(6) 497

Figure 5. VEGF immunoreactivity in rat brain at different time points after PDT treatment compared with contralateral untreated brain (averaged over all time points).

induced lesion. These data indicate that neovascularization occurs in the PDT-treated brain.

PDT of brain tumor has been preclinically and clinically evaluated in many laboratories and clinical centers (5-7). But to our knowledge, there has been no investigation of angiogenesis induced by PDT in normal brain tissues. The strength of PDT lies in its ability to preferentially target tumor within a matrix of endogenous tis- sue. This selective antitumor activity makes PDT especially at- tractive when fragmented neoplastic cellular invasion results in an ill-defined tumor-brain boundary. However, PDT does not wholly exclude normal tissue, and insult to brain tissue is often characterized by an induction of angiogenic factors. Because neo- vascularization has been demonstrated to support tumor survival, growth and likely invasiveness, we hypothesize that PDT could induce angiogenesis in the tissue adjacent to the tumor, which sub- sequently contributes to tumor regrowth. As part of a systematic investigation for testing this hypothesis, we evaluated angiogenesis induced by PDT within normal brain. We chose to investigate PDT- induced angiogenesis in brain without tumor for two primary

Figure 4. Immunostaining for VEGF within the PDT-treated brain (A) and contralateral homologous tissue (B). In- creased VEGF immunoreactivity is ap- parent in the PDT-treated area. Bar is 20 wn.

reasons. One objective was to exclude influence of the established proangiogenic response of tumor to necrosis. Second, PDT is often used postoperatively targeting the BAT where the majority of the tissue is endogenous; hence, the response to PDT in this scenario would likely be dominated by the normal brain. Here we demonstrate that PDT induces production of VEGF and promotes angiogenesis within the normal brain. Because angiogenesis and its promoting factors are known to enhance tumor proliferation and survival (28), we suggest that the efficacy of PDTmay be diminished by this response.

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Table 2. Quantitative three-dimensional data indicating vessel connectivity and vessel length

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Length (pm) 40.1 i 2.8 39.4 2 3.2 31.3 5 4.4* 30.8 ? 3.1* 26.7 ? 1.7* 21.9 ? 3.4* No. of branch points 28.3 2 1.9 27.3 i 3.4 36.2 2 4.1 40.1 2 7.1* 39.2 2 2.6* 44.2 2 4.3*

*P < 0.05 compared with contralateral.

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