Localization of collagen XVIII and the endostatin portion of collagen XVIII in aged human control eyes and eyes with age-related macular degeneration

PURPOSE: Endostatin, a C-terminal fragment of collagen XVIII (coll XVIII) formed by proteolysis, specifically inhibits endothelial cell migration and proliferation in vitro and potently inhibits angiogenesis and tumor growth in vivo. The purpose of this study was to examine the immunolocalization of endostatin and coll XVIII in the retina and choroid of human donor tissue sections from aged control donor eyes and to determine whether the localization or relative levels are changed in age-related macular degeneration (AMD). METHODS: Ocular tissues were obtained from six aged control donors (age range, 75-86 years; mean age, 80.5 years) without evidence or history of chorioretinal disease and from nine donors with AMD (age range, 74-105 years; mean age, 88.6 years). Tissues were cryopreserved, and streptavidin alkaline phosphatase immunohistochemistry was performed with goat anti-human and mouse anti-human endostatin antibodies and rabbit anti-mouse coll XVIII. Blood vessels were identified with mouse anti-human CD-34 antibody in adjacent sections. Pigment in RPE and choroidal melanocytes was bleached. Three independent observers scored the immunohistochemical reaction product. RESULTS: In aged control eyes, coll XVIII and endostatin (the endostatin portion of coll XVIII) immunoreactivity was observed in large retinal blood vessels and in capillaries in some individuals, but the internal limiting membrane (ILM) had the most intense retinal immunostaining. There was no significant difference in immunoreactivity to both antibodies in retinal blood vessels in aged control eyes. In the choroid, endostatin and coll XVIII were localized to blood vessels, Bruch's membrane, and RPE basal lamina. AMD retina and choroid had a similar pattern and intensity of coll XVIII immunostaining, as observed in control eyes but reaction product was more diffuse in the choroid. Endostatin immunoreactivity was significantly higher in ILM (P = 0.037) in AMD retina and significantly lower in the choriocapillaris, Bruch's membrane, and RPE basal lamina of AMD choroids (P < 0.05) and completely negative in some areas of AMD choroids. CONCLUSIONS: These data suggest that reduced levels of the endostatin portion of coll XVIII in Bruch's membrane, RPE basal lamina, intercapillary septa, and choriocapillaris in eyes with AMD may be permissive for choroidal neovascularization.     

Matrix metalloproteinases in human choroidal neovascular membranes excised following verteporfin photodynamic therapy

AIM: To evaluate expression of proangiogenic matrix metalloproteinases (MMP) 2 and 9 at distinct intervals after verteporfin photodynamic therapy (PDT) in human choroidal neovascular membranes (CNV) secondary to age-related macular degeneration (AMD). METHODS: Retrospective review of an interventional case series of 49 patients who underwent removal of CNV. Twenty-six patients were treated with PDT 3 to 383 days prior to surgery. Twenty-three CNV without previous treatment were used as controls. CNV were stained for CD34, cytokeratin 18, endostatin, MMP-2 and MMP-9 by immunohistochemistry. RESULTS: CNV without previous therapy disclosed MMP-2, MMP-9 in RPE-Bruch's membrane, vessels and stroma in different intensities. Three days after PDT, MMP-9 expression was significantly weaker in stroma (p = 0.0019). Endostatin was significantly reduced in vessels (p<0.001). At longer post-PDT intervals, a significant increase of MMP-9 in stroma (p = 0.037) and of endostatin in RPE-Bruch's membrane (p = 0.02), vessels (p = 0.005) and stroma (p<0.001) were disclosed. No significant changes in MMP-2 expression were detected. CONCLUSIONS: PDT induced an early, temporary decrease in MMP-9 and endostatin expression. At longer intervals, MMP-9 increase is possibly associated with the angiogenic process responsible for recurrence after PDT. MMP-9, however, acts as a double-edged sword by concomitant induction of endostatin, an endogenous inhibitor of angiogenesis.     

Insulin-like growth factor-I and its receptor in neovascular age-related macular degeneration

PURPOSE: The insulin-like growth factor (IGF)-I protein is a growth-promoting polypeptide that can act as an angiogenic agent in the eye. The purpose of the current study was to localize the expression of IGF-I and its receptor (IGF-IR) mRNA and IGF-IR protein in situ in the normal human eye and to examine the presence of expression in eyes with neovascular age-related macular degeneration (AMD). METHODS: Formalin-fixed, paraffin-embedded slides of 4 normal control eyes and 14 eyes with choroidal neovascularization (CNV) secondary to AMD were examined. Three eyes with proliferative diabetic retinopathy were studied as the positive control. IGF-I and IGF-IR mRNA was detected by in situ hybridization with digoxigenin-labeled RNA probes. IGF-IR protein was studied by immunohistochemistry. RESULTS: In the normal retina, IGF-I and IGF-IR mRNA expression was found throughout the neuroretinal layers, in the retinal pigment epithelium (RPE), and in some choriocapillary and retinal capillary endothelial cells. In eyes with CNV we found IGF and IGF-IR mRNA in capillary endothelial cells, some transdifferentiated RPE, and fibroblast-like cells. IGF-IR protein was found in normal eyes in all neuroretinal layers, in the RPE, and in the choroidal vessels. In eyes with CNV, IGF-IR protein was present in the RPE monolayer, in transdifferentiated RPE, and in newly formed vessels. CONCLUSIONS: The colocalization of protein and receptor indicates an autocrine function of IGF-I in the normal human retina. Because IGF-I participates in ocular neovascularization, synthesis of IGF-IR and IGF-I in endothelial cells, RPE cells, and fibroblast-like cells in CNV may point toward a role for this growth factor in the pathogenesis of neovascular AMD.     

Impaired expression of thrombospondin-1 in eyes with age related macular degeneration

AIMS: This study investigated the expression and localisation of thrombospondin-1 (TSP-1), a known anti-angiogenic extracellular matrix protein, in normal aged control human eyes and eyes with age related macular degeneration (AMD). METHODS: Immunohistochemical analysis with mouse anti-human TSP-1 antibody and mouse anti-human CD 34 antibody, as a blood vessel marker, was performed on frozen sections from macular and peripheral blocks of aged control donor eyes (n = 12; mean age 78.8 years), and eyes with AMD (n = 12; mean age 83.9 years). Pigment in retinal pigment epithelium (RPE) and choroidal melanocytes was bleached. Three independent observers scored the immunohistochemical reaction product. RESULTS: In the macular region, TSP-1 expression was observed intensely in Bruch's membrane and weakly in RPE basement membrane, choriocapillaris, and the wall of large choroidal blood vessels in the aged control eyes. In eyes with AMD, TSP-1 immunoreactivity was significantly lower in all structures except RPE basement membrane (p<0.01). There was significantly lower TSP-1 in the far periphery than the equator and submacular regions in all eyes. TSP-1 immunoreactivity was low in choroidal neovascularisation (CNV), but it was high and diffuse in adjacent scar tissue. CONCLUSION: These findings suggest that decreased TSP-1 in Bruch's membrane and choroidal vessels during AMD may permit the formation of CNV.     

Verteporfin photodynamic therapy induced apoptosis in choroidal neovascular membranes

AIM: To evaluate the impact of verteporfin photodynamic therapy (PDT) on the induction of apoptosis in choroidal neovascular membranes (CNV) secondary to age related macular degeneration. METHODS: Retrospective review of 22 surgically excised CNV. 12 of these patients had been treated with PDT 3-146 days previously. Apoptotic cells were detected with the TUNEL technique and compared to the expression of CD34 (endothelial cells, EC), CD105 (activated endothelial cells), Ki-67 (proliferation marker), and cytokeratin18 (retinal pigment epithelial cells, RPE). RESULTS: CNV excised 3 days after PDT were characterised both by collapsed and patent vessels. The EC displayed a statistical significant positive TUNEL reaction when compared to the remaining treated CNV (p < 0.001) and untreated CNV (P = 0.002). The proliferative activity was reduced. CNV excised 1-5 months after PDT displayed a patent vascularisation and high proliferative activity. All membranes either treated or untreated disclosed only sporadic TUNEL positive cells within the stroma and the RPE. CONCLUSIONS: Verteporfin PDT leads to selective and effective damage of EC within CNV. Both patent and occluded vessels were lined by apoptotic EC. This finding and the increased expression of proliferation marker at later time points suggest that revascularisation after PDT is caused by angiogenesis rather than recanalisation.     

Scavenger receptors for oxidized lipoprotein in age-related macular degeneration

PURPOSE: The accumulation of macrophages is known to be involved in the pathogenesis of age-related macular degeneration (AMD), but the reasons why macrophages accumulate in AMD lesions have not been determined. Because the histopathology of AMD has some factors common with those of atherosclerosis, the authors hypothesized that macrophages accumulate to take up oxidized lipoproteins in the eyes of patients with AMD, as has been demonstrated in atherosclerosis. METHODS: Immunohistochemistry was performed on 10 surgically excised choroidal neovascular (CNV) membranes from eyes with AMD. An antibody against oxidized lipoprotein and antibodies against the scavenger receptors SR-PSOX and LOX-1 were used. Antibodies against cytokeratin, CD68, and von Willebrand factor were used to identify retinal pigment epithelium (RPE), macrophages, and vascular endothelial cells, respectively. RT-PCR was performed to detect the mRNAs of the scavenger receptors in the CNV membranes. RESULTS: Oxidized lipoproteins were immunohistochemically detected in the CNV membranes. Intense immunostaining was observed at the surface of the CNV membranes with the SR-PSOX antibody, whereas LOX-1 immunostaining was weak. Cells expressing scavenger receptors were found to be predominantly macrophages with a minority of RPE. Both SR-PSOX and LOX-1 mRNAs were detected in CNV membranes. CONCLUSIONS: Oxidized lipoproteins are present in AMD lesions. Macrophages and RPE in the CNV membranes express cell surface scavenger receptors for oxidized lipoproteins. These findings suggest that macrophages may accumulate to take up oxidized lipoproteins in AMD and that the control of oxidative stress and macrophage responses may therefore be potential treatments for AMD.     

Expression of leukocyte adhesion molecules in human subfoveal choroidal neovascular membranes treated with and without photodynamic therapy

PURPOSE: The purposes of this study were to investigate the immunostaining of the leukocyte adhesion molecules intercellular adhesion molecule (ICAM)-1 and E-selectin in subfoveal choroidal neovascular membranes (CNVMs) surgically excised from patients with age-related macular degeneration (AMD) and to determine whether prior photodynamic therapy (PDT) alters their immunostaining. METHODS: The localization of ICAM-1 and E-selectin in 10 subfoveal CNVMs was determined by immunohistochemistry. Membranes were also immunostained for CD31 to assess vascularity. RESULTS: Significantly higher numbers of CD31-staining vessels per unit membrane area were found in the peripheral regions of the membranes compared with the central regions (P = 0.05). ICAM-1 immunoreactivity in the CNVMs was found predominantly on RPE cells, but also on small vessels in the periphery. ICAM-1 staining was significantly more intense in the peripheral, more cellular areas of the membranes than in the central, more fibrotic regions (P = 0.04). ICAM-1 staining in the periphery of the CNVMs was greater than that in choroidal vessels and the RPE of the normal control eye. ICAM-1 immunostaining grade in peripheral regions of the CNVMs decreased with the increasing number of PDT treatments (P = 0.05). Some of the CNVMs also stained for E-selectin in RPE cells and small vessels in the periphery. CONCLUSIONS: In subfoveal CNVMs from patients with AMD, there is increased immunostaining for leukocyte adhesion molecules, particularly in the peripheral, more cellular regions where angiogenesis may be ongoing. Increasing numbers of PDT treatments may be associated with decreased ICAM-1 immunostaining in the proliferating edges of the CNVMs.     

45岁以上中心性浆液性脉络膜视网膜病变与渗出型老年性黄斑变性眼底血管造影对比分析

目的 观察45岁以上的中心性浆液性脉络膜视网膜病变（CSC）与渗出型老年性黄斑变性（AMD）患者眼底血管造影图像特征的异同。 方法 回顾分析45岁以上CSC患者32例39只眼和渗出型AMD患者20例22只眼的眼底彩色像、荧光素眼底血管造影（FFA）和吲哚青绿血管造影（ICGA）检查资料。 结果 39只CSC眼中，FFA显示典型CSC 11只眼，占282％；扩散性视网膜色素上皮病变（DRPE）28只眼，占718％。ICGA显示39只眼早期脉络膜充盈迟缓及随后的脉络膜血管扩张，占100％；中期均有多灶性脉络膜血管通透性增强，占100％，16只眼中后期呈现渗漏点强荧光，占41.0％；后期5只眼见脉络膜大血管负影，占12.8％。22只未伴明显出血的渗出型AMD眼中，ICGA显示焦点状脉络新生血管（CNV） 13只眼，占59.1％；斑状CNV 8只眼，占36.4％；结合型CNV 1只眼，占4.5％。造影早期5只眼黄斑区及周围脉络膜血管代偿性扩张，占22.7％；中期均未见脉络膜血管通透性增强，后期均未见脉络膜大血管负影。 结论 45岁以上的CSC与渗出型AMD不同的ICGA特征为局灶性或多灶性的RPE渗漏点强荧光；多灶性脉络膜血管通透性增强；造影后期脉络膜血管负影；造影期间无焦点状或斑状CNV性强荧光。     

Angiographic-histological correlation of late exudative age-related macular degeneration

AIMS: To correlate the fluorescein angiographic (FAG) classification in age-related macular degeneration (AMD) with the histology of surgically excised choroidal neovascularization (CNV) membrane specimens. METHODS: 36 subretinal lesions surgically excised during macular translocation were investigated by light microscopy. Histologic results were correlated with fluorescein angiographic findings. RESULTS: Classic CNV lesions showed a subretinal fibrovascular membrane on histological examination. About 50% of the investigated classic CNV lesions had additional subpigment epithelial components, too. Occult CNV lesions showed a subpigment epithelial fibrovascular membrane on histological examination. A common histological component in lesions of exudative age-related macula degeneration was the appearance of diffuse deposits at the choroidal side of the retinal pigment epithelium. CONCLUSION: Different FAG characteristics in exudative AMD lesions were comparable with the histological results. In correspondence with FAG classification the CNV membranes were localized in different positions in relation to the RPE.     

Reduced foveolar choroidal blood flow in eyes with increasing AMD severity

PURPOSE: In an earlier study, the authors reported that foveolar choroidal blood flow (ChBFlow) decreases in patients with AMD and drusen. To explore further the choroidal circulatory changes in patients with AMD, the relationship between ChBFlow and fundus features associated with increased risk of choroidal neovascularization (CNV) were investigated. METHODS: The study included 26 control eyes of 17 normal subjects and 163 eyes with early AMD characteristics of 123 patients with AMD. The AMD study eyes were divided into three groups according to increasing risk for development of CNV: (1) drusen > or =63 microm, no RPE hyperpigmentary changes in the study eye, and no CNV in the fellow eye; (2) drusen > or =63 microm, RPE hyperpigmentary changes in the study eye, and no CNV in the fellow eye; and (3) eyes with CNV in the fellow eye. Laser Doppler flowmetry was used to assess relative foveolar choroidal blood velocity (ChBVel), volume (ChBVol), and flow (ChBFlow). Differences in the mean circulatory parameters were assessed by analysis of variance (ANOVA) and test of linear trend. RESULTS: Mean ChBVel, ChBVol, and ChBFlow decreased with increased risk for CNV (linear trend, P < 0.05). The lowest circulatory parameters were observed in the eyes with the highest risk for CNV development. Trends for ChBVel and ChBFlow were still significant after adjustment for multiple factors. CONCLUSIONS: There is a systematic decrease in choroidal circulatory parameters with an increase in the severity of AMD features associated with risk for the development of CNV, suggesting a role for ischemia in the development of CNV.     

The presence of AC133-positive cells suggests a possible role of endothelial progenitor cells in the formation of choroidal neovascularization

PURPOSE: Recent evidence suggests that vasculogenesis as well as angiogenesis occurs throughout the body during neovascularization. The recruitment of circulating stem cells is a key feature of vasculogenesis. The purpose of the present study was to determine whether markers of endothelial progenitor cells (EPCs) are present in choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD). METHODS: Surgically excised CNV (n = 9) membranes from patients with AMD were probed with immunohistochemical techniques using the following monoclonal antibodies: AC133 a putative marker of EPCs and hematopoietic stem cells (HSCs); the endothelial cells markers CD31, CD34, and von Willebrand factor (vWF); and cytokeratins and CD68, markers for retinal pigment epithelium (RPE) and macrophages, respectively. After secondary antibody amplification, reactions were visualized with fast red substrate. RESULTS: Six of nine specimens demonstrated cells positive for AC133 that were all found within predominantly cellular regions of the specimens. In the avascular fibrous stromal core of all specimens, the predominant cells were RPE cells and macrophages. The peripheral component of all CNV membranes was highly vascular and showed varying immunoreactivity for all endothelial markers. The greatest immunoreactivity for endothelial markers was observed with CD34 and vWF and least for CD31. CONCLUSIONS: These findings support animal studies that vasculogenesis, in addition to angiogenesis, may contribute to the neovascularization that occurs in AMD.     

Expression of pigment epithelium derived factor and vascular endothelial growth factor in choroidal neovascular membranes and polypoidal choroidal vasculopathy

AIMS: To determine whether pigment epithelium derived factor (PEDF), a protein that inhibits angiogenesis, is expressed in human choroidal neovascular membranes (CNVMs) and in tissues from an eye with polypoidal choroidal vasculopathy (PCV). In addition, to compare the expression of PEDF with that of vascular endothelial growth factor (VEGF), a known stimulator of angiogenesis, in these tissues. METHODS: CNVMs, associated with age related macular degeneration (AMD), angioid streaks, and PCV, were obtained during surgery. The expression of PEDF and VEGF in the excised subretinal fibrovascular membranes was determined by immunohistochemistry. RESULTS: PEDF and VEGF were strongly expressed in the vascular endothelial cells and retinal pigment epithelial (RPE) cells in the CNVMs where numerous new vessels were prominent (clinically active CNVMs). On the other hand, immunoreactivity for PEDF and VEGF was weak in the new vessels where fibrosis was prominent (clinically quiescent CNVMs). However, the RPE cells were still positive for PEDF and VEGF. The specimens from the eye with PCV also showed strong expression of PEDF and VEGF in the vascular endothelial cells and the RPE cells. CONCLUSION: Because PEDF is an inhibitor of ocular angiogenesis and an inhibitor of ocular cell proliferation, our results suggest that PEDF along with VEGF may modulate the formation of subfoveal fibrovascular membranes.     

Molecular mechanism of choroidal neovascularization in age-related macular degeneration

The exudative form of age-related macular degeneration (AMD) is characterized by choroidal neovascularization (CNV). Retinal pigment epithelial cells (RPE) secrete various angiogenesis-related factors, especially vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). The imbalance between the VEGF and PEDF secreted by RPE is a key contributor to the development of CNV in AMD. The earliest clinical hallmark of AMD is the presence of drusen. Although drusen are an epidemiological risk factor for the development of CNV, the mechanism of how drusen induce the development of CNV remains unclear. Recent proteome analysis demonstrated that amyloid beta (Abeta) deposition was specific to drusen from eyes with AMD. We focused on Abeta and investigated the effect of Abeta on cultured human RPE cells as well as ocular findings in neprilysin gene-disrupted mice, which leads to an increased deposition of Abeta. Our study demonstrates that Abeta accumulation affects the balance between VEGF and PEDF in the RPE, and reproduces features characteristic of human AMD, such as RPE atrophy and basal deposit formation in neprilysin gene-disrupted mice.     

内皮抑素抑制脉络膜新生血管的实验研究

目的　探讨内皮抑素蛋白抑制激光诱导的大鼠脉络膜新生血管 (CNV)的效果。方法采用基因重组方法获得内皮抑素蛋白 ,取BrownNorway大鼠 30只 (6 0只眼 ) ,用激光光凝方式建立CNV模型 ,随机分为内皮抑素组、生理盐水组及对照组 ,每组 10只 ,分别给予内皮抑素 2 0 μl(5g/L) ,生理盐水 2 0 μl视网膜下注射 ,对照组光凝后不做处理 ,观察期为 14d。应用激光共焦显微镜下脉络膜血管平铺法测量各组CNV面积 ,以荧光素眼底血管造影后荧光素渗漏程度、光镜下观察结果、CD10 5及因子Ⅷ免疫组化染色结果作为观察指标。结果　内皮抑素组CNV面积小于生理盐水组及对照组 (F=30 7 35 1,P <0 0 0 1) ;内皮抑素组各光凝斑荧光渗漏程度评分与生理盐水组及对照组间比较 ,差异有显著意义 (χ2 =13 711,P <0 0 0 1) ;光凝后 14d ,生理盐水组和对照组光凝区脉络膜层增厚 ,脉络膜毛细血管层内皮细胞增殖 ,新生血管增生 ,内皮抑素组内皮细胞增生数量及新生血管明显少于前两组 ;CD10 5、因子Ⅷ的阳性表达量明显降低 ;各组视网膜内核层及神经纤维层内皮细胞均无CD10 5阳性表达。结论　内皮抑素可以有效抑制CNV的形成 ,脉络膜血管平铺及CD10 5检测对于CNV的定性、定量评估具有一定意义。     

Pigment epithelium-derived factor is deficient in the vitreous of patients with choroidal neovascularization due to age-related macular degeneration

PURPOSE: Pigment epithelium-derived growth factor (PEDF) is a potent inhibitor of angiogenesis that is found in the normal eye. The purpose of this study is to report decreased levels of PEDF in the vitreous of eyes with choroidal neovascularization (CNV) due to age-related macular degeneration (AMD). DESIGN: Prospective case-control study. METHODS: In a prospective case-control study, undiluted vitreous was collected from nine eyes of nine patients with CNV due to AMD and from an age-matched control group of 12 eyes of 12 patients with retinal disorders not involving neovascularization. Vitreous PEDF and vascular endothelial growth factor (VEGF) concentrations were determined by Western blot analyses and enzyme-linked immunosorbent assay (ELISA), respectively. Angiogenic activities of the vitreous samples were assessed in vitro using an endothelial cell chemotaxis assay. RESULTS: In vitreous samples from nine eyes with CNV due to AMD the mean +/- SD PEDF level was 2.8 ng/microl +/- 1.3 ng/microl. In vitreous samples from 12 age-matched control eyes the mean +/- SD PEDF level was 16.4 ng/microl +/- 7.1 ng/microl. The difference between the two groups was statistically significant (P =.00003). No significant difference in vitreous VEGF concentration was seen between CNV/AMD samples and control samples (P =.23). All CNV/AMD vitreous samples induced endothelial cell migration in vitro. No sample from age-matched non-age-related macular degeneration controls could induce endothelial cell migration, and 11 of 12 were able to block VEGF-induced migration in vitro. This inhibitory activity required active PEDF. CONCLUSION: The vitreous of patients with CNV due to AMD contained lower levels of PEDF and lacked the antiangiogenic activity of vitreous from age-matched controls. This suggests that loss of PEDF creates a permissive environment for CNV patients with AMD.     

引起黄斑下脉络膜新生血管的几种常见疾病的 光相干断层扫描图像特征分析

目的观察引起黄斑下脉络膜新生血管(CNV)的几种常见疾病的光相干断层扫描（OCT）图像特征并对其分类，为CNV的鉴别诊断和治疗提供依据。方法回顾分析经常规眼底检查以及荧光素眼底血管造影（FFA）检查确诊的老年性黄斑变性(AMD)、病理性近视、中心性渗出性脉络膜视网膜炎(CEC)和特发性脉络膜新生血管(ICNV)患者165例187只眼的OCT检查资料，结合FFA检查结果对OCT图像进行分类并总结分析各类图像特征。结果可确定边界的CNV 60只眼，表现为边界清楚的视网膜色素上皮(RPE)层和脉络膜毛细血管层的梭形增厚；不易确定边界的CNV 101只眼，表现为弥散的脉络膜反向散射增强；浆液性RPE脱离19只眼，表现为RPE下的光学暗区；出血性RPE脱离11只眼，表现为RPE下的高反向散射区域，迅速衰减；纤维血管性RPE脱离10只眼，表现为RPE和脉络膜之间的轻至中度无反向散射区；神经上皮脱离45只眼，表现为神经上皮层与RPE分离，其间为光学暗区。结论引起黄斑下CNV的几种常见疾病的OCT图像可以分为6类，分析OCT的图像特征有助于CNV的鉴别诊断与治疗。(中华眼底病杂志,2005,21:69-73)      

Aqueous humor levels of vascular endothelial growth factor and pigment epithelium-derived factor in polypoidal choroidal vasculopathy and choroidal neovascularization

PURPOSE: To determine the aqueous levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) in patients with active polypoidal choroidal vasculopathy (PCV) and choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD) and pathologic myopia. DESIGN: Prospective, comparative control study. METHODS: Aqueous humors were collected from 32 eyes of 32 patients for either active PCV or CNV. Among them, 11 eyes had active and symptomatic PCV, 12 eyes had active CNV secondary to AMD, and nine eyes had active CNV of pathologic myopia. Levels of VEGF and PEDF were determined by commercially available enzyme-linked immunosorbent assay kits. A group of 10 aqueous samples from 10 patients who underwent cataract surgery without other ocular or systemic diseases comprised the controls. RESULTS: VEGF concentrations in aqueous humor were markedly increased in patients with PCV, CNV of AMD, and CNV of myopia when compared with the controls (analysis of variance [ANOVA], P < .001). VEGF levels in eyes with PCV were, however, significantly lower than those of exudative AMD (P = .045). The PEDF levels were also significantly different among the groups (ANOVA, P = .001), and we observed increased levels in PCV, CNV of AMD, and CNV of myopia. CONCLUSIONS: VEGF and PEDF factors were coexpressed and increased with positive correlation in aqueous humor of eyes with active PCV. The different levels of both factors in eyes of PCV and AMD might suggest distinct clinical entities or different angiogenesis courses between PCV and AMD.     

Mecamylamine suppresses Basal and nicotine-stimulated choroidal neovascularization

PURPOSE: Nicotinic acetylcholine receptors (nAChR) are best known for their role in neurotransmission, but they have recently been demonstrated on vascular endothelial cells. Acetylcholine is their endogenous ligand, but they are also stimulated by nicotine. By stimulating nAChR, nicotine promotes tumor angiogenesis as well as atherosclerotic plaque neovascularization. In this study, the authors investigated the role of nAChR in the pathogenesis of choroidal neovascularization (CNV). METHODS: The effect of the nonselective nAChR antagonist mecamylamine was tested on human retinal and choroidal endothelial cells in vitro and in a murine model of CNV. RESULTS: Several nAChR isoforms were identified in retinal and choroidal microvascular endothelial cells, and the ability of these cells to form tubules when grown in growth factor-reduced basement membrane matrix and supplemented with VEGF was suppressed by the nAChR antagonist mecamylamine. Supplementation of the drinking water of mice with nicotine increased the size of CNV lesions at Bruch membrane rupture sites, an effect that was blocked by subcutaneous administration of mecamylamine (50 mg/kg/d) by an osmotic pump. In the absence of nicotine, CNV formation was suppressed by the infusion of 50 mg/kg/d mecamylamine or by topical application 0.1 or 1% mecamylamine to the cornea. CONCLUSIONS: These data suggest that endogenous activation of nAChR promotes CNV and that activation of nAChR by nicotine may contribute to the increased incidence of CNV seen in smokers with age-related macular degeneration (AMD). Topically administered mecamylamine could provide an appealing new treatment approach for CNV.