|Molecular Vision 2006;
Received 18 November 2005 | Accepted 20 March 2006 | Published 20 July 2006
Transpupillary thermotherapy-induced modification of angiogenesis- and coagulation-related gene expression in the rat posterior fundus
Yoko N. Ito,1 Mitsuteru Ito,1 Hiroyasu
Takita,1 Shin Yoneya,1 Gholam A. Peyman,2 Peter L.
1Department of Ophthalmology, Saitama Medical University, Iruma, Saitama, Japan; 2Department of Ophthalmology, Tulane University Medical Center, New Orleans, LA; 3Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD
Correspondence to: Keisuke Mori, MD, PhD, Department of Ophthalmology, Saitama Medical University, 38 Morohongo, Moroyama, Iruma, Saitama, 350-0495, Japan; Phone: +81 (492) 76-1250; FAX: +81 (492) 95-8002; email: email@example.com
Purpose: To study gene expression changes in the rat retina and choroid following transpupillary thermotherapy (TTT) and to identify molecular mechanisms that may enhance treatment of choroidal neovascularization, complicating age-related macular degeneration.
Methods: One fundus of Brown Norway rats was treated with an 810 nm diode laser while the contralateral fundus received no treatment. The mRNA was extracted and processed for cDNA microarray analysis. Genes with increased expression were validated by semiquantitative reverse transcription polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR).
Results: Of the 14,815 cDNA elements on the array, 12 genes were up-regulated in TTT treated eyes. Upregulation of eight of these 12 genes could be verified by semiquantitative RT-PCR. The eight verified genes were EPCR, IL-1β, MCP-1, TSP-1, Fgl, Asns, MT-2, and NMDMC, which included 4 angiogenesis- and coagulation-related genes.
Conclusions: This study demonstrates upregulation of angiogenesis- and coagulation-related genes following TTT. The response profile and its temporal relationships provide insight into the molecular mechanisms that lead to vascular occlusion and antiangiogenesis induced by TTT.
Therapeutic hyperthermia is reported to decrease blood flow in tumors to a greater extent than in normal tissue and to influence tumor angiogenesis . The threshold for thermal damage in normal tissue is higher than that in tumors [1,2]. Addition of hyperthermia to radiotherapy improves survival in patients with advanced pelvic tumors, breast cancer, head and neck tumors, and glioblastoma [3-6]. The differential susceptibility to hyperthermia is attributed in part, to both abnormalities in tumor vessels and to inhibition of tumor-associated angiogenesis [7,8]. The biological mechanisms that lead to clinical efficacy following hyperthermic treatment in the eye are not yet fully known .
Because of optical clarity, the interior of the eye is easily accessible to laser therapy. Hyperthermic laser therapy in the eye is referred to as transpupillary thermotherapy (TTT). TTT was first used to treat choroidal melanoma [10-12]. Subsequent applications of TTT included treatment of choroidal neovascularization (CNV) complicating age-related macular degeneration (AMD) [13,14].
Histological examination of eyes with choroidal melanoma following TTT reveals occlusion of tumor vessels by thrombi . Angiographic changes following TTT for CNV in AMD suggest endothelial cell damage and thrombus formation leading to microvascular occlusion in CNV . In an in vitro model of human fibrosarcoma, hyperthermic treatment results in reduced gene expression and diminished production of tumor-derived vascular endothelial growth factor (VEGF), resulting in inhibition of the angiogenic responses of human umbilical vein endothelial cells . Gene expression profiles of endothelial cells subjected to heat shock demonstrate upregulation of plasminogen activator inhibitor 1 (PAI-1), an antiangiogenic protein involved in the control of extracellular matrix degradation .
In this study we examine rat retinal and choroidal tissues following TTT and measure mRNA expression using cDNA microarray and semiquantitative reverse transcription-polymerase chain reaction (PCR) in order to generate a TTT modified, retinal and choroidal gene expression profile. The profile is used to describe possible molecular mechanisms for TTT mediated treatment effects. In addition, we examined temporal expression patterns using quantitative real-time PCR (qRT-PCR).
Pigmented Brown Norway rats, aged 6 to 8 weeks, were anesthetized by intramuscular injections of 80 mg/kg ketamine hydrochloride. The pupils were dilated with 0.5% tropicamide and 0.5% phenylephrine hydrochloride. Topical anesthesia was achieved by 0.5% proparacaine hydrochloride. All procedures were conducted in accordance with Public Health Service Policy on Humane Care and Use of Laboratory Animals.
A diode laser (Iris Medical Instruments, Mountain View, CA) emitting at 810 nm wavelength mounted on a modified Haag-Streit slit lamp was used. After test studies to determine the laser power setting needed for subthreshold lesions in the rat, laser was applied at 50 mW for 60 s, using a 3 mm fixed spot size of this delivery system. The calculated area of laser irradiation using a handheld cover slide as a contact lens in the rat retina is 1.15 mm in diameter if the distance from corneal surface to the retina in the rat eye is 5.0 mm. Based on this estimate, the total fluence was calculated to 285 J/cm2.
Six nonoverlapping laser lesions were delivered to the fundus of each eye covering the visible posterior pole. Five eyes of four rats were examined immediately following TTT by funduscopy and fluorescein angiography and then again at eight days following TTT. For microarray analysis, 10 rats received TTT in one eye and no treatment in contralateral eye. For semiquantitative RT-PCR analysis, TTT was performed on the fundus of seven eyes of four rats, and eyes of both rat as a control. Two h after TTT, all animals were sacrificed, and their eyes were enucleated for RNA extraction. The anterior segment, lens, and vitreous were carefully removed. Samples from each experimental group were pooled and immediately homogenized in TRIzol (Invitrogen, Carlsbad, CA).
The eyes from 16 rats were used for qRT-PCR to quantify gene expression changes over time. Each animals received left eye TTT, while the right eye served as an untreated control. The animals were sacrificed, and their eyes were enucleated at 2, 6, 12, 24, and 72 h after TTT.
RNA extraction and purification
For microarray analysis and semiquantitative RT-PCR analysis, total RNA was isolated by the acid guanidine thiocyanate-phenol-chloroform extraction method using TRIzol (Invitrogen). For microarray analysis, the extracted total RNA was quantified, and 75 μg was used in the purification procedure (Dynabeads mRNA Purification Kit: DYNAL BIOTECH, Oslo, Norway) for both the TTT-treated and control groups. For qRT-PCR analysis, total RNA was extracted with RNeasy Mini (Qiagen, Valencia, CA).
Dye-labeled cDNA was synthesized from 10 μg of poly(A)+RNA with Direct label Kit (Agilent Technologies, Palo Alto, CA). Two sets of cDNA with two different dye-labeled dCTP (Cyanine-3, and Cyanine-5-dCTP) were made for each TTT-treated and control group. These dye-labeled cDNA were purified with QIA quick PCR Purification Kit (Qiagen) and then hybridized with rat cDNA microarray (G4105A, Agilent Technologies) which contained spots for 14,743 genes and ESTs. The microarray plates were scanned by a Microarray Scanner (Agilent Technologies) to detect and quantify the dye-signal intensity of each well. Microarray analyses were duplicated by a two-color dye swapping method to confirm the reproducibility of the experiments. The scanned microarray data was analyzed using Feature Extraction Software (Agilent Technologies) to reflect the probability of a significant change in expression (p<0.001).
Semiquantitative reverse transcription-polymerase chain reaction
Reverse transcription-polymerase chain reaction (RT-PCR) was performed in a semiquantitative manner with the 12 candidate genes obtained from the microarray analysis. Five μg of total RNA was reverse-transcribed to cDNA with a first-strand cDNA synthesis kit (Invitrogen), following DNA digestion by DNaseI (RNase-free; Takara Bio Inc., Ohtsu, Japan). The PCR was carried out by adding 1 U of Takara Taq DNA polymerase Hot Start version (Takara Bio Inc.). Then PCR was performed in a thermalcycler (GeneAmp PCR system 9700: Applied Biosystems, Foster City, CA) under the following conditions: initial denaturation step of 94 °C for 5 min, cycling step of denaturation at 94 °C for 30 s, annealing for 30 s and extension at 72 °C for 30 s. Annealing temperature, PCR cycling numbers, and primers for each gene are listed in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping control gene. To confirm the origin of PCR products, each product was either digested with three restriction enzymes or nucleotide sequencing of amplicon was carried out.
Quantitative real-time PCR
We utilized both the RNase-Free DNase Set (Qiagen) and DNaseI (RNase-free; Takara Bio Inc.) to remove genomic DNA contamination. Two hundred nanograms of total RNA was applied to RT with 25 U SuperScript II reverses transcriptase (Invitrogen) in a thermalcycler (GeneAmp PCR system 9700, Applied Biosystems) to generate cDNA. qRT-PCR was carried out with 4 ng of cDNA using Assay-on-DemandTM Gene Expression (Asns, Fgl, IL-1β, MCP-1) or Assay-by-DesignSM Service-Gene Expression products (EPCR, MT-2, NMDMC, TSP-1; Applied Biosystems). The design for primers and probes for Assay-by-DesignTM Service-Gene Expression products of four genes are listed in Table 2. We used TaqMan® Rodent GAPDH Control Reagents (Applied Biosystems) as an endogenous control gene. TaqMan® Universal PCR Master Mix (Applied Biosystems) was applied as DNA polymerase with uracil-N-glycosylase (UNG). qRT-PCR was performed in triplicate for each sample with a commercial system (ABI PRISM® 7900HT Sequence Detection System, Applied Biosystems). Reaction conditions were as follows: 50 °C for 2 min for UNG activation and 95 °C for 10 min for Taq DNA polymerase activation, followed by 40 cycles of 95 °C for 15 s for denaturation, alternating with 60 °C for 1 min for annealing and extension, were carried out. The expression level of each gene was assigned arbitrary units (relative to baseline samples) using the comparative Ct methods [19,20]. All qRT-PCR experiments were performed following the guidelines supplied by Applied Biosystems. Statistical analysis was performed using a Student's unpaired t-test. A p value of less than 0.05 was prospectively assigned as the level at which a finding would be considered statistically significant.
Rat transpupillary thermotherapy parameters
In accordance with our previously published methods for determining threshold parameters of TTT in experimental animals [21,22], TTT was administered at powers of 50, 60, 100, and 200 mW for 60 s using a spot size 3 mm in diameter. Immediately following treatment, lesions using a power of 50 mW showed no visible retinal alteration by funduscopy and no significant change by fluorescein angiography. All lesions created with 100 and 200 mW irradiation demonstrated immediate retinal and choroidal thermal burns (whitening). Eight days after treatment, none of the 18 retinal areas irradiated with a power of 50 mW had visible evidence of thermal burn on funduscopic examination. However, 8 out of 18 lesions had subtle hyperfluorescence evident during the late phase of fluorescein angiography (Table 3). As no retinal whitening occurred at a power setting of 50 mW and retinal whitening was observed early in evaluation of the 60 mW setting, a power setting of no greater than 50 mW was targeted for study in rats. In the clinical setting for treatment of subfoveal choroidal neovascularization, the retinal appearance at treatment endpoint ranged from no visible color change, to a light-gray appearance [13,14]. A 50 mW irradiation was therefore used to model clinical TTT in rats. Each treatment was followed by fundus photography and fluorescein angiography to document that the appropriate level of treatment was provided.
The molecular mechanisms that lead to blood vessel closure in response to TTT are examined in this study using microarray. The cDNA microarrays used contained 14,743 rat genes and ESTs that were hybridized to cDNA targets in duplicate. The samples tested were synthesized from rat retinochoroidal RNAs derived from TTT-treated and untreated naive eyes. Twelve genes showed greater than threefold differential expression in TTT-treated retinal and choroidal tissues on duplicate microarray testing. These genes are asparagines synthetase (Asns), c-jun oncogene for transcription factor AP-1 (c-jun), endothelial cell activated protein C receptor (EPCR), fibrinogen-like protein (Fgl), interleukin-1β (IL-1β), monocyte chemoattractant protein-1 (MCP-1), macrophage inhibiting cytokine-1 (MIC-1), metallothionein-1 and β2 (MT-1 and MT-2), NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC), p53 regulated PA26-T3 nuclear protein (sestrin1) and thrombospondin-1 (TSP-1; Table 4). The list of all 2,576 genes differentially expressed is available at GEO (GSE4601, GSM102888, GSM102889).
Semiquantitative reverse transcriptase polymerase chain reaction
To verify increased expression of candidate genes obtained from microarray analysis, semiquantitative RT-PCR was performed on the 12 listed genes. Since the microarray used contained human, mouse, and rat cDNA as well as genomic DNA, some genes were hybridized with human or mouse cDNA rather than rat cDNA. EPCR, Fgl, NMDMC, and TSP-1 were identified with mouse cDNA, sestrin1 with human cDNA, and the other genes were identified with rat cDNA (Table 4). To determine the primer sequences for the five candidate genes that matched rat cDNA, we referred to the NCBI accession number (Table 4) and found homology in the rat genome using a BLAST database. Homologues for other species were also referenced. We then designed the primers specifically for rat genome. MT-1 and MT-2 were identified together as a genomic DNA in the microarray. Primers for MT-1 and MT-2 were designed for each. The RT-PCR primers used are listed in Table 1.
Although differences in amplification of c-jun, MIC-1, MT-1 and sestrin1 were present in TTT-treated eyes they did not reach significance by prospective criteria. The remaining eight genes however were confirmed to have more than a 2 fold increase in expression in TTT-treated eyes as compared to untreated controls (Figure 1). The results indicated that MT-2, but not MT-1, expression was significantly elevated following TTT treatment. Restriction-enzyme digestion of all amplicons was performed to confirm their origin. All amplicons except for EPCR were of the expected size and were thus confirmed to be derived from the target cDNA. Nucleotide sequencing of EPCR was then carried out with the RT-PCR product for EPCR. The results indicated that the amplicon was a rat EPCR sequence. The sequencing also showed that the primer for EPCR (designed in our laboratory) was not completely homologous to the rat genome.
Quantitative real time polymerase chain reaction
qRT-PCR was performed for Asns, EPCR, Fgl, IL-1β, MCP-1, MT-2, NMDMC, and TSP-1 to verify the microarray and semi-quantitative RT-PCR findings. qRT-PCR was also used to demonstrate the temporal expression relationships for the verified genes for the period extending from 2 h to 72 h after TTT treatment (Table 5). As significant upregulation of c-jun, MCP-1, MT-1 and sestrin1 was not verified by semiquantitative RT-PCR, qRT-PCR was not performed.
At 2 h following TTT, EPCR, Fgl, MCP-1, and TSP-1 exhibited peak expression and then gradually declined (significance at 2 h, as compared to baseline): EPCR (p=0.04), Fgl (p=0.04), MCP-1(p=0.04), MT-2 (p=0.005), NMDMC (p=0.003), TSP-1 (p=0.004). Asns, MT-2, and NMDMC reached peak expression at 6 h after TTT (significance at 6 h, as compared to baseline); Asns (p<0.001), MT-2 (p=0.003), NMDMC (p=0.04). Note that Asns expression was near baseline at 2 h after treatment but increased sharply to approximately 5 times baseline by 6 h. Thereafter Asns and NMDMC rapidly decreased in their expression. The largest response was noted for NMDMC, which expressed 7 fold above baseline at 2 h following TTT and then returned to baseline. Significant elevation of MT-2 expression above baseline (p=0.03), was present at 12 h after TTT but not at 24 or 72 h.
This study demonstrates that normal retinal and choroidal tissue responds to TTT with gene expression changes involving angiogenesis- and blood clotting-related genes, such as IL-1β, EPCR, MCP-1, and TSP-1. These in vivo changes were discovered by comparing microarray data from TTT-treated and untreated retinochoroidal tissues. The findings were confirmed by repeat microarray, semiquantitative RT-PCR and qRT-PCR. The eight genes that were successfully verified and their temporal expression patterns provide insight into the molecular and biological basis of treatment effects following TTT in patients with choroidal neovascularization and ocular tumors.
Thrombospondin-1 (TSP-1) was first described as the major β granule protein of human platelets. It binds to the activated platelet surface upon platelet stimulation for platelet aggregation and clotting processes . TSP-1 is also an inhibitor of angiogenesis that is produced and secreted by numerous tumor types [24-27]. TSP-1 inhibits tumor angiogenesis by binding to the transmembrane receptor CD36 inducing apoptosis in endothelial cells . Retinal pigment epithelium (RPE) cells produce and release TSP-1 in vitro, and TSP-1 accumulates in the cytoplasm of RPE cells and Bruch's membrane . Our data indicate that TTT stimulation of retinal and choroidal tissue upregulates TSP1 expression. Clinical application of TTT may also result in enhanced production of TSP-1, potentially inducing apoptosis of endothelial cells and augmenting clotting processes that may contribute to a treatment effect in choroidal neovascularization that complicate age-related macular degeneration [23,27].
EPCR is also believed to act in the physiologic regulation of coagulation . EPCR is a transmembrane glycoprotein homologous to the major histocompatibility complex class I/CD1 family of proteins [31-33]. Soluble EPCR has been reported in plasma . Activated protein C reduces thrombin formation. Plasma EPCR inhibits the anticoagulant activity of activated protein C by blocking its binding to phospholipids, resulting in reduced thrombin formation  Patients with the A3 haplotype of the EPCR gene are reported to be at increased risk for thrombosis when plasma levels of soluble EPCR are elevated . TTT-induced upregulation of the EPCR gene in rat retinal and choroidal tissues may contribute to clot formation observed in the microvasculature of treated choroidal neovascular networks. Further studies will be required to determine the temporal and spatial distribution of EPCR and its relationship to choroidal neovascular response to TTT.
In contrast to TSP-1, IL-1β and MCP-1 are known inducers of angiogenesis. IL-1β is a pro-inflammatory agent associated with angiogenesis and increased vascular permeability . In various experimental models, IL-1 increased tumor invasiveness and metastasis, which may be mediated by induction of angiogenesis [36-40]. Mechanistically, IL-1 induces VEGF production, by selective induction of hypoxia inducible factor-1β (HIF-1β) . IL-1 also stimulates proliferation of endothelial cells, adhesion molecule expression, production of cytokines and production of small/inflammatory mediator molecules . Local tumor invasiveness, metastases and angiogenesis in B16 melanoma, DA/3 mammary and prostate cancer cell models was significantly diminished in IL-1β knockout mice, as compared to wild-type mice.
MCP-1 is an indirect inducer of angiogenesis that is associated with macrophage recruitment [41-43]. MCP-1 has been suggested to play a role in ocular angiogenesis [44-47]. MCP-1 also upregulates HIF-1β gene expression in human aortic endothelial cells, which induces VEGF-A expression . Therefore, upregulation of IL-1β and MCP-1 may have regulatory effects that are counter to those resulting from TSP-1. Recently, results  from the TTT4CNV clinical trial revealed no significant difference between TTT-treated and sham-treated eyes in the combined study group. Subgroup analysis of eyes with poorer baseline visual acuity, however, indicated a statistically significant treatment benefit. Our study of the molecular and biological response to TTT suggests that potential benefit from TTT-induced upregulation of inhibitors of angiogenesis may, in part, be countered by simultaneous induction of stimulators of angiogenesis.
Mainster and Reichel have hypothesized that TTT modifies inflammatory responses and may also alter heat shock protein (Hsp) expression . Hsps are ubiquitous proteins that are upregulated in response to various cellular stresses including hyperthermia, free radicals, inflammation, and ischemia [50,51]. The stress-induced functions of Hsps serve to stabilize and protect the cell, and include protein refolding, translocation, and degradation. Hsps also act as chaperones that stabilize cell structure and function . Hyperexpression of Hsps may enhance survival of surrounding retinochoroidal tissues following hyperthermic stress induced by TTT. Using immunocytochemical preparation of pigmented rabbit fundi, Desmettre et al.  have shown that TTT induces Hsp hyperexpression in the choroid. In their report, immunostaining for Hsp 70 was strongest at the fluence of 532 J/cm2 but was significantly decreased at the fluence of 450 J/cm2. No Hsp immunoreactivity was detected at a fluence of 386 J/cm2 . Our in vivo study detected no significant increase in Hsp expression. This may relate to a number of factors including the laser fluence used in our experimental, 285 J/cm2 (less than 386 J/cm2) and differences in Hsp response associated with different strains of experimental animal.
Although this preliminary in vivo study has identified several potentially relevant angiogenesis- and blood clotting-related genes in the retina and choroid, other genes, in other in vitro experimental settings of different cell types, have been suggested to contribute to the molecular basis of antiangiogenic response, following thermal therapy. In human fibrosarcoma HT-1080 cells, 4 h of heat shock suppressed gene expression of VEGF, resulting in inhibition of tumor cell-induced proliferation and MMP production in endothelial cells . In vitro gene expression profiles of endothelial cells subjected to heat shock have demonstrated that PAI-1, an antiangiogenic protein involved in the control of extracellular matrix degradation, is specifically upregulated . In this in vivo study of retinal and choroidal tissue, gene expression of VEGF, PAI-1 and other angiogenic/antiangiogenic factors in retinal and choroidal tissues was not significantly modified by TTT. These findings suggest that thermal therapy induces different gene expression profiles in different tissues; in vitro studies may not necessarily reflect the full complexity of the response seen in vivo and that potential therapeutic effects resulting from TTT appear to derive from differential activation of multiple induced molecular pathways acting in concert. Further studies are also needed to demonstrate the significance of thermal therapy in modification of gene expression of various angiogenic or antiangiogenic factors, at various times after treatment, in experimental models of ocular neovascularization.
These sutdies were supported by grants-in-aid for scientific research (14571685 and 90251090 to KM) and (1377041 to YNI) from the Ministry of Education, Culture and Science in Japan, Research to Prevent Blindness (PG), and the Maruki Memorial Scholarship for Special Research (KM).
The authors thank Yasuo Kato of Topcon Co. and Masayuki Takasu, Myutec Ltd. for the calculation of retinal irradiance in the rat eye.
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