|Molecular Vision 2007;
Received 1 June 2007 | Accepted 18 October 2007 | Published 15 November 2007
Ocular clusterin expression in von Hippel-Lindau disease
Min Zhou,1,2 Defen Shen,1 James E. Head,1
Emily Y. Chew,1 Patricia Chévez-Barrios,3 W. Richard
Green,4 Chi-Chao Chan1
1National Eye Institute, National Institutes of Health, Bethesda, MD, 2Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Shanghai, China, 3Ophthalmic Pathology Program, Department of Pathology, Methodist Hospital, Houston, TX, 4Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD
Correspondence to: Chi-Chao Chan, Bldg. 10, Rm. 10N103, NIH/NEI, 10 Center Drive, Bethesda, MD 20892-1857; Phone: (301) 496-0417; FAX: (301) 402-8664; email: email@example.com
Purpose: Clusterin is a multifunctional glycoprotein. Its mRNA is ubiquitously expressed, with high levels in von Hippel-Lindau (VHL) target organs such as the brain, liver, kidney, and adrenal medulla. Decreased clusterin secretion has been reported in renal cell carcinoma associated with VHL disease. The purpose of this study was to investigate ocular clusterin expression in VHL disease.
Methods: This retrospective case series included nine eyes with retinal hemangioblastoma/hemangioma associated with VHL disease, one eye from a patient with a history of VHL disease and central nervous system hemangioblastomas but without ocular lesions, one surgically-excised optic nerve with optic nerve hemangioblastoma/hemangioma, and three normal control eyes. Ocular specimens were evaluated by routine histology, immunohistochemistry for clusterin expression, and molecular detection of clusterin transcripts within ocular VHL hemangioblastomas compared with normal tissue from the same eye using microdissection and quantitative real-time PCR.
Results: All retinal hemangioblastoma were composed of typical VHL tumor cells admixed with small vascular channels as well as glial cells. Marked decrease of clusterin immunoreactivity was detected in all retinal hemangioblastoma and the optic nerve hemangioblastoma, whereas positive clusterin reactivity of the vascular and glial components was similar to that of normal retina. Quantitative real-time PCR analysis confirmed the decrease of clusterin mRNA in the VHL associated retinal hemangioblastoma and optic nerve hemangioblastoma in five cases.
Conclusions: Clusterin shows possible important functions in tumor suppression by the VHL gene product (pVHL) and the potential to be a novel biomarker in retinal hemangioblastoma associated VHL disease. Further investigation of clusterin may provide better understanding of retinal hemangioblastoma associated with VHL disease.
von Hippel-Lindau (VHL) disease is an autosomal dominantly inherited, multi-systemic, familial cancer syndrome caused by germline mutations in the VHL tumor suppressor gene [1-4]. The birth incidence of VHL disease is 1 in 36,000 . The penetrance is age-dependent, being nearly by age 65 [5,6]. The clinical features of VHL are extremely variable. About 40 different lesions in 14 different organ systems, including the eye, central nervous system (CNS), and kidney, have been described . The diagnosis of VHL disease can be made in an individual with either multiple tumors or with isolated tumors in the presence of a positive family history [4,8]. The diagnosis can also be made by genetic analysis for mutant alleles as well as sequence variations of the VHL gene . This disease is of particular relevance to the ophthalmologist because retinal hemangioblastoma/hemangioma is the most frequent and often the earliest clinical manifestation of VHL disease . Although retinal hemangioblastoma associated with VHL disease usually presents at a relatively young age (median 25 years) [9-11], the cumulative probability of developing a retinal hemangioblastoma increases during each decade of life, reaching 80% by the eighth decade . The majority of initial retinal hemangioblastoma associated with VHL disease are located in the peripheral retina, while peripapillary lesions are only reported in up to 10% of patients with the disease. It is important to note that the complications of retinal hemangioblastoma, even in optimally treated cases, are visually significant . The probability of visual loss is age-dependent. The lifetime cumulative probability of permanent visual loss is 60%, with most of the risk (43%) falling within the first 30 years of life .
Recent advances in molecular genetics have provided insight into the molecular mechanisms of tumorigenesis in VHL disease. The VHL gene, which is localized and cloned at chromosome 3p25-26, is a tumor suppressor [13,14]. Extensive clinical and genetic studies suggest that tumor formation in VHL disease is consistent with the Knudson "two-hit" model seen in retinoblastoma, which states that tumors develop only after both copies of the suppressor gene in a cell are damaged [15,16]. The best known function of the VHL gene product (pVHL) relates to its ability to target the transcription factor hypoxia-inducible factor (HIF) for polyubiquitination and hence destruction [17-19]. In cells that are hypoxic, or lack pVHL, HIF escapes destruction and is free to activate HIF target genes such as vascular endothelial growth factor, platelet derived growth factor, transforming growth factor, and erythropoietin.
Clusterin (also termed apolipoprotein J) is a multifunctional glycoprotein constitutively produced and secreted by almost all cell types and is found in all body fluids, with especially high levels of expression in VHL target organs such as the brain, liver, kidney, and adrenal medulla [20,21]. In the eye, clusterin has been shown to be present in the cornea, lens, ciliary body, retina, aqueous, and vitreous [22-25]. A number of physiologic functions have been proposed for clusterin, including roles in apoptosis and complement regulation, protection of cell membranes, stabilization of cell-cell and cell-matrix interactions, and inhibition of stress-induced precipitation and aggregation of misfolded proteins through its action as an extracellular chaperone [25-27]. In addition, the potential role of clusterin in DNA repair and carcinogenesis has been demonstrated, though it is still unclear as to the relationship between clusterin gene expression, apoptosis, and other cell functions [21,28,29].
A recent study by Nakamura indicates that clusterin plays an important role in VHL tumorigenesis and induction of clusterin reflects a HIF-independent pVHL function that may be important for tumor suppression . Cells lacking wild-type pVHL are defective in the expression and secretion of clusterin, which does not behave like a HIF target. To our knowledge, the expression of clusterin in VHL-associated retinal hemangioblastoma and optic nerve hemangioblastoma/hemangioma has not been previously reported. Herein, we investigate the expression of clusterin in retinal hemangioblastoma and optic nerve hemangioblastoma associated with VHL disease and evaluate the correlation of clusterin and VHL genes.
This study was approved by the National Eye Institute (NEI) Institutional Review Board for human subjects and informed consent was obtained from all patients. This study was conducted at the NEI and followed the tenets stated in the Declaration of Helsinki.
The study consisted of nine eyes from eight patients with retinal hemangioblastoma and one surgically excised partial optic nerve with optic nerve hemangioblastoma associated with VHL disease, one eye from a patient with a history of systemic VHL disease and CNS hemangioblastomas but without VHL ocular lesions, one surgically-excised optic nerve with VHL-associated hemangioblastoma, and three normal control eyes obtained from the archives of the NEI. Case 9 was enucleated in Texas and processed by Dr. Chevez-Barrios, who sent a paraffin block to the NEI where the study was conducted. Three of the nine VHL ocular tissues were frozen sections; the remaining eight specimens were formalin-fixed, paraffin-embedded tissues. Vail's classification was used in these cases. Vail's classification includes the following: stage I, early stage with dilation of feeding artery and draining vein and angioma formation; stage II, development of hemorrhages and exudation; stage III, massive exudation and retinal detachment; stage IV, uveitis, absolute glaucoma, and loss of the eye.
Immunohistochemical analysis using the avidin-biotin complex immunoperoxidase technique was performed as described previously [30-32]. Formalin-fixed, paraffin-embedded tissue blocks were cut into 5 μm thin sections, transferred to glass slides, and microwave treated in boiling 10 mmol/l citrate buffer (pH 6.0; KD Medicine, Columbia, MD) before incubation with primary antibody. The primary antibodies against clusterin were diluted to 1:500 (monoclonal antibody, clusterin-b: sc-5289, [33,34] Santa Cruz Biotechnology, Santa Cruz, CA), applied to the tissue sections, and allowed to incubate for 1 h at room temperature. The secondary antibodies were biotinylated horse anti-mouse antibodies (Vector Laboratories, Burlingame, CA). Detection was carried out using avidin-biotin-peroxidase complex (Vector Laboratories) with 3,3'-diaminobenzidine as the chromogen. Control procedures included isotype-matched murine immunoglobulin G (I-2000; Vector Laboratories) of irrelevant specificity. Normal eye tissue was used as the positive control for the applied primary antibodies.
Frozen sections and formalin-fixed, paraffin-embedded sections were stained, without a cover slide, according to the instructions of PicopureTM RNA Isolation Kit (Arcturus Bioscience, Mountain View, CA) .Under the microscope, retinal hemangioblastoma cells or normal retina cells were selected from their respective samples and microdissected.
Quantitative real-time reverse transcriptase polymerase chain reaction
Total RNA was isolated from frozen sections using the PicopureTM RNA Isolation Kit (Arcturus Bioscience) and from formalin-fixed, paraffin-embedded sections using the ParadizeTM Sample Quality Assessment Kit (Arcturus Bioscience). First strand cDNA synthesis was performed using Invitrogen SuperScriptTM II reverse transcriptase (Invitrogen, Carlsbad, CA). A standard β-actin-based quantitative RT-PCR protocol was used for the study. mRNA levels were measured by real-time quantitative PCR method performed on the ABI Prism 7500 (Applied Biosystems, Foster City, CA). This system employs fluorescent-based PCR chemistries to provide quantitative detection of nucleic acid sequences using real-time analysis and qualitative detection of nucleic acid sequences with end-point and dissociation-curve analysis. For each treatment, two distinct amplifications were conducted in parallel to amplify clusterin and β-actin cDNA. The amplification reactions were performed in 25 μl volumes containing 1 μl of first-strand cDNA per treatment, 12.5 μl of 2x TaqMan Universal PCR Master Mix (Applied Biosystems), and 1.25 μl of 20x TaqMan Gene Expression Assays for clusterin (Hs00156548_m1; Applied Biosystems), and β-actin (Hs99999903_m1; Applied Biosystems). Clusterin mRNA levels from each treatment were normalized to the corresponding amount of β-actin mRNA levels. Water controls and samples without PCR mixtures were established to eliminate the possibility of significant DNA contamination.
Clinical data of von Hippel-Lindau patients
Clinical characteristics of nine eyes from nine VHL patients with retinal hemangioblastoma (cases 1-9), one optic nerve with VHL associated optic nerve hemangioblastoma (case 11), and one eye from a VHL patient without retinal hemangioblastoma (case 10) are summarized in Table 1. There were three females and six males in the VHL with retinal hemangioblastoma group. The average age of the patients was 39±13.8 years, while the average age at the time of initial VHL symptom development was 19 years. Multiple retinal hemangioblastoma lesions were identified in seven cases, among which three cases also showed peripapillary retinal hemangioblastoma. Using Vail's classification for the 10 cases with ocular lesions, we identified three of the cases as having early-stage retinal hemangioblastoma, while the remaining seven cases had end-stage retinal hemangioblastoma .
All cases in the retinal hemangioblastoma and the optic nerve hemangioblastoma associated with VHL group illustrated typical hemangioblastoma lesions characterized by an anastomosing network of thin vascular capillary-like channels lined by endothelial cells and separated by masses of plump, heavily lipidized, foamy "stromal (VHL) cells" that have been well described in the literature (Figure 1A,C, Figure 2A,C) [32,36,37]. Most of the advanced tumor tissues were confluent, merging indistinguishably into sheets of glial tissue (Figure 3). In addition, phthisis bulbi, iris neovascularization, cataracts, retinal detachment, choroidal retinal pigment epithelium bone metaplasia, and optic nerve atrophy were present in the seven end-stage cases. The VHL tumor cells in each case demonstrated loss of heterozygosity of the VHL gene, as has previously been described [32,37].
Immunoreactivity against clusterin in ocular von Hippel-Lindau lesions
In normal control retina, clusterin was confined primarily to the ganglion cell and inner nuclear layers of the neuroretina (Figure 4). A similar pattern of immunoreactivity was observed in the normal retina tissues from VHL cases either with or without retinal/optic nerve hemangioblastoma. Lack of clusterin immunoreactivity was noted in the tumor ("stromal" or VHL) cells of both early and end-stage retinal hemangioblastoma lesions and the optic nerve hemangioblastoma (Figure 1B,D, Figure 2B,D), whereas vascular and glial components within or surrounding the lesions exhibited positive, stronger clusterin signals.
Clusterin transcripts in ocular von Hippel-Lindau tissues
Sufficient tissue for clusterin mRNA expression analysis was obtained from five cases. Clusterin mRNA levels from each case, which included: VHL tumor cells alone and normal retinal cells without VHL cellular component, were normalized to the corresponding amount of β-actin transcripts. The levels from each case were compared to the normal retinal samples of the three normal control eyes. Expression of clusterin mRNA within the retinal hemangioblastomas was much lower than that of the normal retinal tissue obtained from the same eye (Table 2). The remaining retinal hemangioblastoma cases and the optic nerve hemangioblastoma demonstrated low-to-below detectable levels of clusterin mRNA by TaqMan® RT-PCR analysis.
This study utilized immunohistochemistry and quantitative real-time PCR to demonstrate a marked decrease in clusterin expression in tumor cells (stromal cells) within retinal hemangioblastoma and optic nerve hemangioblastoma associated with VHL as compared to the surrounding normal retinal tissue. Conversely, vascular and glial components within or surrounding these VHL lesions exhibited more immunopositive clusterin signals by immunohistochemistry. To our knowledge, there is no previous report of clusterin expression in VHL-associated retinal or CNS hemangioblastomas.
A recent study indicates that clusterin may contribute to tumor suppression by actions through, pVHL . Additionally, clusterin has pro-apoptotic activity in that its loss promotes survival of hypoxic neurons, which relates to the role of pVHL in oxygen sensing and suggests that loss of clusterin might promote survival within the hypoxic zones of pVHL-defective solid tumors . Another observation is that clusterin inhibits nuclear factor B activity through stabilization of IkB [38-40]. Therefore, loss of clusterin expression may be one explanation for why pVHL-defective tumor cells exhibit increased nuclear factor B signaling and resistance to tumor necrosis factor. Clusterin has also been shown to decrease neuroblastoma cell invasion in vitro .
As mentioned previously, it has been shown that renal carcinoma cells lacking wild-type pVHL are deficient with respect to secretion of clusterin, suggesting that the regulation of clusterin expression is both oxygen- and HIF-independent in these cells . The mechanisms whereby clusterin may contribute to tumor suppression through interactions with pVHL are unclear, but the presence of mutant pVHL in tumors from patients with this disease has been documented in hemangioblastomas and renal cell carcinomas. In our VHL cases, which demonstrated classical hemangioblastomas occurring in retina and CNS, the finding of low levels of clusterin expression is consistent with prior studies of VHL-associated tumors including renal cell carcinoma and pheochromocytoma .
This finding should be contrasted with the role of clusterin in several other human malignancies. Recent studies have reported clusterin up-regulation in association with neoplasms of the bladder, kidney, prostate, colon, breast, and lung . In addition, clusterin immunoreactivity may have prognostic significance in breast cancer . In prostate, breast, and colorectal cancers, clusterin is associated with anti- or pro-apoptotic activity, regulated by calcium homeostasis. Reports suggest "two faces" of clusterin activity: the calcium-dependent cellular retention of clusterin positively correlates with cell survival, whereas nuclear translocation of this protein promotes cell death in calcium-deprived cells . Furthermore, decreases in clusterin protein or transcripts have been documented in various stages of neoplasms of the pancreas, prostate, as well as in neuroblastoma .
Grossniklaus and colleagues recognized that there are three cell types present in the retinal hemangioblastoma: endothelial cells, pericytes, and stromal cells . However, it is interesting that in our study only stromal cells exhibited deficiency or extremely low levels of clusterin expression. To date, the precise origin of the stromal cells is still an enigma, but it is believed that they are the true neoplastic components of retinal and optic nerve hemangioblastomas associated with VHL disease and might arise from aberrant stem cells . First, Chan et al. reported expression of vascular endothelial growth factor limited to only the stromal component of the retinal hemangioblastoma . Moreover, loss of heterozygosity involving VHL gene has been demonstrated in the stromal cells but not in vascular or glial cells or normal tissues [37,44,45]. A similar pattern was confirmed in renal cell carcinoma without wild-type VHL gene . The study of clusterin expression may provide further evidence to suggest that stromal cells are the true neoplastic components of the retinal hemangioblastoma.
In addition, clusterin may serve as a potential marker for VHL gene status and, thereby, pVHL integrity. Our previous study of VHL genotyping in VHL-associated retinal hemangioblastoma and optic nerve hemangioblastoma demonstrated that VHL gene deletion or loss of heterozygosity is limited to only stromal cells, while heterozygosity is retained in vascular and normal tissues [3,32,37,46]. This finding is consistent with the deficiency of clusterin expression in VHL-associated retinal and optic nerve hemangioblastomas. In the study of renal cell carcinoma, the potential function of clusterin as a marker for VHL gene status has been disclosed .
In conclusion, clusterin shows a potentially important function in tumor suppression by pVHL. Low levels of clusterin expression have now been demonstrated in retinal and optic nerve hemangioblastomas associated with VHL disease, clearly showing that this decrease in expression is compatible with other previously studied VHL-associated tumors. Of particular importance, clusterin may represent a new biomarker in retinal hemangioblastoma- and optic nerve hemangioblastomas associated VHL disease in that a marked decrease in clusterin could provide an important clue as to the identity of the tumor cells and serve as a reflection of VHL gene status. Further investigation to identify novel mechanistic insights into tumor suppression by pVHL, particularly to further our understanding of the possible regulatory interactions between pVHL and clusterin, is warranted.
We would like to thank the NEI Intramural Research Program for support. A portion of these findings was presented at the American Academy of Ophthalmology/American Association of Ophthalmic Pathologists Symposium on November 10, 2006. J.E.H. is a fellow in the 2006-07 Clinical Research Training Program, a public-private partnership supported jointly by the NIH and Pfizer, Inc. (via a grant to the Foundation for the NIH from Pfizer, Inc.).
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