 |
|
Expression of LOX-1, an Oxidized Low-Density Lipoprotein Receptor, in Choroidal Neovascularization
Arch Ophthalmol. 2004;122:1873-1876.
Subfoveal choroidal neovascularization of various macular diseases is one of the causes of severe blindness, including age-related macular degeneration (AMD). Several environmental risk factors have been elucidated in the pathogenesis of AMD, including smoking,1 atherosclerosis,2 increased levels of plasma fibrinogen,3 and low levels of antioxidant vitamins.4 Recent observations support the hypothesis that antioxidant and/or vitamin treatment may delay progression of AMD and vision loss.5 However, the exact cause of AMD remains to be determined.
Recently, Ikeda et al6 showed that increased plasma oxidized low-density lipoprotein (oxLDL) levels may be involved in the pathogenesis of AMD. Oxidized LDL has been implicated as having a major role in atherosclerosis, and many of the pathologic and biochemical features seen in choroidal neovascularization are analogous to those seen in advanced atherosclerosis, such as the infiltration of monocytes and macrophages and the overexpression of adhesion molecules, monocyte chemotactic proteins, growth factors, and cytokines within lesions.7-8 Lectinlike oxidized low-density lipoprotein receptor type 1 (LOX-1) is a recently identified oxLDL receptor that is abundantly expressed in vascular endothelial cells.9 Its messenger RNA has been shown to be expressed in atheromatous lesions,9 and LOX-1 up-regulation has been observed in several vascular lesions, including hypertensive remodeling lesions, diabetic vascular lesions, and macrophages.10 The observation of LOX-1 up-regulation in vascular lesions, the potential roles of oxLDL in the pathogenesis of AMD, and the possible similarity between the pathogenesis of atherosclerosis and that of AMD prompted us to examine LOX-1 expression in choroidal neovascularization. In addition, we sought to measure plasma cholesterol levels to investigate the relationship between LOX-1 expression and hyperlipidemia.
We examined LOX-1 localization in 13 surgically excised neovascular membranes, including 10 from patients with AMD, 1 from a patient with idiopathic choroidal neovascularization, and 2 from patients with myopic choroidal neovascularization. The membranes were frozen in liquid nitrogen within 30 minutes of excision. Multiple 8-µm cryosections from each membrane were air-dried, fixed in acetone for 5 minutes, washed with phosphate-buffered saline, and blocked for 30 minutes with 2% bovine serum albumin in phosphate-buffered saline. They were then incubated with primary antibody and washed 3 times for 5 minutes with phosphate-buffered saline. Bound antibody was detected with Cy3-biotin–conjugated secondary antibody. Polyclonal antibodies against human von Willebrand factor (DAKO Corp, Kyoto, Japan) were used to identify vascular endothelial cells. Antihuman LOX-1 monoclonal antibody (JTX92) was generated by immunizing BalbC/c mice with the CHO cell line that was transfected human LOX-1 complementary DNA (HLOX-1-CHO). Hybridomas from the splenocytes were prepared with the use of standard procedures and screened by means of the immunostaining of HLOX-1-CHO. The specificity of the antibody was determined by means of Western blot analysis and the immunostaining of HLOX-1-CHO (Figure 1). Immunohistochemical staining was repeated on cryosections of 10 choroidal neovascularization membranes, omitting the anti–LOX-1 primary antibody as controls. Additional control samples included immunohistochemical staining for LOX-1 of the posterior sclera, choroid, choriocapillaries, and retina of a normal donor eye.
|
|
|
|
Figure 1. Immunostaining (A) and Western blot analysis (B) results of human lectinlike oxidized low-density lipoprotein receptor type 1 (LOX-1) complementary DNA (hLOX-1-CHO). A, Immunostaining of hLOX-1-CHO was performed. The nonfixed CHO cells were incubated with the primary antibody JTX92, then the bound antibody was detected with fluorescein isothiocyanate conjugated–antihuman IgG. B, Western blot analysis was performed to determine the specificity of the antibody. M indicates molecular weight marker; W, wild-type CHO cells; and L, CHO cells expressing hLOX-1. The arrowhead points to the expected molecular weight.
|
|
|
The choroidal neovascular membranes ranged from moderately cellular with prominent neovascularization to paucicellular and fibrotic with few vascular channels. LOX-1 expression was detected in all, and most of the LOX-1 was localized to the endothelial cells (Figure 2). Staining for endothelial cells was seen in the LOX-1–positive cells. The LOX-1–positive profiles exceeded the number of von Willebrand factor–positive profiles, suggesting that LOX-1 was localized in nonvascular cells or within the stroma of neovascular membranes, as well as in the endothelial cells. The finding that the labeling of LOX-1 was not restricted to vascular endothelial cells is in line with recent observations in advanced atherosclerosis that LOX-1 is extensively expressed in the new blood vessels, macrophages, and smooth muscle cells of advanced atherosclerotic lesions.10-12
|
|
|
|
Figure 2. Fundus photograph and angiographic image (A) and immunostaining of surgically excised choroidal neovascular (CNV) membrane (B). A, Representative color fundus photograph (left) and angiographic image (right) of the fundus of a 55-year-old man with age-related macular degeneration (patient 1). The area of serous retinal detachment is indicated by arrows. B, panels a through f, A CNV membrane from patient 1. Panels a and d, Lectinlike oxidized low-density lipoprotein receptor type 1 (LOX-1) staining is seen in red, some of which is indicated by arrows. Panels b and e, von Willebrand factor (vWF) staining for vascular endothelial cells is seen in green, some of which is indicated by small arrowheads. Panels c and f, Confocal image of double staining for LOX-1 and vWF. LOX-1 expression and vWF localization were associated, some of which is indicated by large arrowheads. Panel g, Control staining of a CNV membrane from patient 1. Panel h, image of double staining for LOX-1 and vWF for panel g. von Willebrand factor staining for vascular endothelial cells is seen in green, some of which is indicated by small arrowheads. Panel i, A CNV membrane from a 37-year-old woman with idiopathic CNV (patient 13). LOX-1 is faintly stained and seen in red, some of which is indicated by arrows. Panels a through c, original magnification x200; d through i, original magnification x600.
|
|
|
We did not find LOX-1 within the posterior segment of normal eyes, including the choriocapillaries (Table). As is further summarized in the Table, greater LOX-1 staining was found in the membranes of the patients with AMD compared with those with idiopathic or myopic choroidal neovascularization, those with a relatively high plasma total cholesterol level, and those of patients with larger macular serous detachment.
|
|
|
|
Table. Clinical and Histological Characteristics
|
|
|
Our findings suggest that LOX-1 plays an active role in the pathogenesis of choroidal neovascularization, especially in AMD. However, further experiments are needed to determine whether LOX-1 plays a role in mediating the processes in AMD that are compatible with those in atherosclerosis.
AUTHOR INFORMATION
Correspondence: Dr Honjo (megumi{at}kuhp.kyoto-u.ac.jp).
Funding/Support: This study was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Tokyo (Drs Honjo, Sawamura, Honda, and Kiryu), and by the Ministry of Health, Labor, and Welfare of Japan, Tokyo (Dr Sawamura).
Megumi Honjo, MD, PhD;
Tatsuya Sawamura, MD, PhD;
Junichi Hinagata, PhD;
Kayo Nakamura, PhD;
Nobuhito Sanada, PhD;
Hidenobu Tanihara, MD, PhD;
Yoshihito Honda, MD, PhD;
Junichi Kiryu, MD, PhD
REFERENCES
|
1. Vingerling JR, Hofman A, Grobbee DE, de Jong PT. Age-related macular degeneration and smoking: the Rotterdam Study. Arch Ophthalmol. 1996;114:1193-1196.
FREE FULL TEXT
2. Friedman E. The role of the atherosclerotic process in the pathogenesis of age-related macular degeneration. Am J Ophthalmol. 2000;130:658-663.
FULL TEXT
|
ISI
| PUBMED
3. Smith W, Mitchell P, Leeder SR, Wang JJ. Plasma fibrinogen levels, other cardiovascular risk factors, and age-related maculopathy: the Blue Mountains Eye Study. Arch Ophthalmol. 1998;116:583-587.
FREE FULL TEXT
4. Smith W, Mitchell P, Webb K, Leeder SR. Dietary antioxidants and age-related maculopathy: the Blue Mountains Eye Study. Ophthalmology. 1999;106:761-767.
FULL TEXT
|
ISI
| PUBMED
5. Age-Related Eye Disease Study Research Group. A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8. Arch Ophthalmol. 2001;119:1417-1436.
FREE FULL TEXT
6. Ikeda T, Obayashi H, Hasegawa G, et al. Paraoxonase gene polymorphisms and plasma oxidized low-density lipoprotein level as possible risk factors for exudative age-related macular degeneration. Am J Ophthalmol. 2001;132:191-195.
FULL TEXT
|
ISI
| PUBMED
7. Penfold PL, Madigan MC, Gillies MC, Provis JM. Immunological and aetiological aspects of macular degeneration. Prog Retin Eye Res. 2001;20:385-414.
FULL TEXT
|
ISI
| PUBMED
8. Oh H, Takagi H, Takagi C, et al. The potential angiogenic role of macrophages in the formation of choroidal neovascular membranes. Invest Ophthalmol Vis Sci. 1999;40:1891-1898.
FREE FULL TEXT
9. Sawamura T, Kume N, Aoyama T, et al. An endothelial receptor for oxidized low-density lipoprotein. Nature. 1997;386:73-77.
FULL TEXT
| PUBMED
10. Nagase M, Abe J, Takahashi K, Ando J, Hirose S, Fujita T. Genomic organization and regulation of expression of the lectin-like oxidized low-density lipoprotein receptor (LOX-1) gene. J Biol Chem. 1998;273:33702-33707.
FREE FULL TEXT
11. Li DY, Chen HJ, Staples ED, et al. Oxidized low-density lipoprotein receptor LOX-1 and apoptosis in human atherosclerotic lesion. J Cardiovasc Pharmacol Ther. 2002;7:147-153.
FREE FULL TEXT
12. Kataoka H, Kume N, Miyamoto S, et al. Expression of lectinlike oxidized low-density lipoprotein receptor-1 in human atherosclerotic lesion. Circulation. 1999;99:3110-3117.
FREE FULL TEXT
SECTION EDITOR: W. RICHARD GREEN, MD
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati
What's this?
|
