Expression of LOX-1, an Oxidized Low-Density Lipoprotein Receptor,in Choroidal Neovascularization

Subfoveal choroidal neovascularization of various macular diseases isone of the causes of severe blindness, including age-related macular degeneration(AMD). Several environmental risk factors have been elucidated in the pathogenesisof AMD, including smoking,¹ atherosclerosis,² increased levels of plasma fibrinogen,³ andlow levels of antioxidant vitamins.⁴ Recentobservations support the hypothesis that antioxidant and/or vitamin treatmentmay delay progression of AMD and vision loss.⁵ However,the exact cause of AMD remains to be determined.

Recently, Ikeda et al⁶ showed that increasedplasma oxidized low-density lipoprotein (oxLDL) levels may be involved inthe pathogenesis of AMD. Oxidized LDL has been implicated as having a majorrole in atherosclerosis, and many of the pathologic and biochemical featuresseen in choroidal neovascularization are analogous to those seen in advancedatherosclerosis, such as the infiltration of monocytes and macrophages andthe overexpression of adhesion molecules, monocyte chemotactic proteins, growthfactors, and cytokines within lesions.^7,8 Lectinlikeoxidized low-density lipoprotein receptor type 1 (LOX-1) is a recently identifiedoxLDL receptor that is abundantly expressed in vascular endothelial cells.⁹ Its messenger RNA has been shown to be expressed inatheromatous lesions,⁹ and LOX-1 up-regulationhas been observed in several vascular lesions, including hypertensive remodelinglesions, diabetic vascular lesions, and macrophages.¹⁰ Theobservation of LOX-1 up-regulation in vascular lesions, the potential rolesof oxLDL in the pathogenesis of AMD, and the possible similarity between thepathogenesis of atherosclerosis and that of AMD prompted us to examine LOX-1expression in choroidal neovascularization. In addition, we sought to measureplasma cholesterol levels to investigate the relationship between LOX-1 expressionand hyperlipidemia.

We examined LOX-1 localization in 13 surgically excised neovascularmembranes, including 10 from patients with AMD, 1 from a patient with idiopathicchoroidal 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 inacetone for 5 minutes, washed with phosphate-buffered saline, and blockedfor 30 minutes with 2% bovine serum albumin in phosphate-buffered saline.They were then incubated with primary antibody and washed 3 times for 5 minuteswith phosphate-buffered saline. Bound antibody was detected with Cy3-biotin–conjugatedsecondary 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/cmice with the CHO cell line that was transfected human LOX-1 complementaryDNA (HLOX-1-CHO). Hybridomas from the splenocytes were prepared with the useof 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 analysisand the immunostaining of HLOX-1-CHO (Figure 1). Immunohistochemical staining was repeated on cryosections of10 choroidal neovascularization membranes, omitting the anti–LOX-1 primaryantibody as controls. Additional control samples included immunohistochemicalstaining for LOX-1 of the posterior sclera, choroid, choriocapillaries, andretina of a normal donor eye.

Figure 1. 

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Immunostaining (A) and Western blotanalysis (B) results of human lectinlike oxidized low-density lipoproteinreceptor type 1 (LOX-1) complementary DNA (hLOX-1-CHO). A, Immunostainingof hLOX-1-CHO was performed. The nonfixed CHO cells were incubated with theprimary antibody JTX92, then the bound antibody was detected with fluoresceinisothiocyanate conjugated–antihuman IgG. B, Western blot analysis wasperformed to determine the specificity of the antibody. M indicates molecularweight 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 cellularwith prominent neovascularization to paucicellular and fibrotic with few vascularchannels. LOX-1 expression was detected in all, and most of the LOX-1 waslocalized 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–positiveprofiles, suggesting that LOX-1 was localized in nonvascular cells or withinthe stroma of neovascular membranes, as well as in the endothelial cells.The finding that the labeling of LOX-1 was not restricted to vascular endothelialcells is in line with recent observations in advanced atherosclerosis thatLOX-1 is extensively expressed in the new blood vessels, macrophages, andsmooth muscle cells of advanced atherosclerotic lesions.^10-12

Figure 2. 

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Fundus photograph and angiographicimage (A) and immunostaining of surgically excised choroidal neovascular (CNV)membrane (B). A, Representative color fundus photograph (left) and angiographicimage (right) of the fundus of a 55-year-old man with age-related maculardegeneration (patient 1). The area of serous retinal detachment is indicatedby arrows. B, panels a through f, A CNV membrane from patient 1. Panels a and d, Lectinlike oxidized low-density lipoprotein receptortype 1 (LOX-1) staining is seen in red, some of which is indicated by arrows.Panels b and e, von Willebrandfactor (vWF) staining for vascular endothelial cells is seen in green, someof 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 indicatedby large arrowheads. Panel g, Control staining ofa CNV membrane from patient 1. Panel h, image ofdouble staining for LOX-1 and vWF for panel g. vonWillebrand 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 (patient13). LOX-1 is faintly stained and seen in red, some of which is indicatedby arrows. Panels a through c,original magnification ×200; d through i, original magnification ×600.

We did not find LOX-1 within the posterior segment of normal eyes, includingthe choriocapillaries (Table). As isfurther summarized in the Table, greaterLOX-1 staining was found in the membranes of the patients with AMD comparedwith those with idiopathic or myopic choroidal neovascularization, those witha relatively high plasma total cholesterol level, and those of patients withlarger macular serous detachment.

Table. 

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Clinical and Histological Characteristics

Our findings suggest that LOX-1 plays an active role in the pathogenesisof choroidal neovascularization, especially in AMD. However, further experimentsare needed to determine whether LOX-1 plays a role in mediating the processesin AMD that are compatible with those in atherosclerosis.

Correspondence: Dr Honjo (megumi@kuhp.kyoto-u.ac.jp).

Funding/Support: This study was supported inpart 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).