Evaluation of focal damage in the retinal pigment epithelium layer in serous retinal pigment epithelium detachment.

The purpose of this study was to evaluate focal damage in the retinal pigment epithelium (RPE) layer in serous retinal pigment epithelium detachment (PED) with multi-contrast optical coherence tomography (OCT), which is capable of simultaneous measurement of OCT angiography, polarization-sensitive OCT and standard OCT images. We evaluated 37 eyes with age-related macular degeneration that had serous PED. Focal RPE damage was indicated by hyper-transmission beneath the RPE-Bruch's membrane band in standard OCT images. Distribution of RPE melanin was calculated using the dataset from multi-contrast OCT. Twenty-four points with hyper-transmission were detected in 21 of the 37 eyes. Standard OCT images failed to show disruption of the RPE-Bruch's membrane band at 5 of the 24 hyper-transmission points. Conversely, multi-contrast OCT images clearly showed melanin defects in the RPE-Bruch's membrane band at all points. Areas of melanin defects with disruption of the RPE-Bruch's membrane band were significantly larger than those without disruption. The volume of intraretinal hyper-reflective foci was significantly larger in eyes with hyper-transmission than that in eyes without hyper-transmission. Multi-contrast OCT is more sensitive than standard OCT for displaying changes at the RPE-Bruch's membrane band when there are small areas of RPE damage.

Complement activation and choriocapillaris loss in early AMD: implications for pathophysiology and therapy.

Age-related macular degeneration (AMD) is a common and devastating disease that can result in severe visual dysfunction. Over the last decade, great progress has been made in identifying genetic variants that contribute to AMD, many of which lie in genes involved in the complement cascade. In this review we discuss the significance of complement activation in AMD, particularly with respect to the formation of the membrane attack complex in the aging choriocapillaris. We review the clinical, histological and biochemical data that indicate that vascular loss in the choroid occurs very early in the pathogenesis of AMD, and discuss the potential impact of vascular dropout on the retinal pigment epithelium, Bruch's membrane and the photoreceptor cells. Finally, we present a hypothesis for the pathogenesis of early AMD and consider the implications of this model on the development of new therapies.

Tissue inhibitor of metalloproteinases-3 peptides inhibit angiogenesis and choroidal neovascularization in mice.

Tissue inhibitors of metalloproteinases (TIMPs) while originally characterized as inhibitors of matrix metalloproteinases (MMPs) have recently been shown to have a wide range of functions that are independent of their MMP inhibitory properties. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is a potent inhibitor of VEGF-mediated angiogenesis and neovascularization through its ability to block the binding of VEGF to its receptor VEGFR-2. To identify and characterize the anti-angiogenic domain of TIMP-3, structure function analyses and synthetic peptide studies were performed using VEGF-mediated receptor binding, signaling, migration and proliferation. In addition, the ability of TIMP-3 peptides to inhibit CNV in a mouse model was evaluated. We demonstrate that the anti-angiogenic property resides in the COOH-terminal domain of TIMP-3 protein which can block the binding of VEGF specifically to its receptor VEGFR-2, but not to VEGFR-1 similar to the full-length wild-type protein. Synthetic peptides corresponding to putative loop 6 and tail region of TIMP-3 have anti-angiogenic properties as determined by inhibition of VEGF binding to VEGFR-2, VEGF-induced phosphorylation of VEGFR-2 and downstream signaling pathways as well as endothelial cell proliferation and migration in response to VEGF. In addition, we show that intravitreal administration of TIMP-3 peptide could inhibit the size of laser-induced choroidal neovascularization lesions in mice. Thus, we have identified TIMP-3 peptides to be efficient inhibitors of angiogenesis and have a potential to be used therapeutically in diseases with increased neovascularization.

A rat model for studying the biological effects of circulating LDL in the choriocapillaris-BrM-RPE complex.

Retention of apolipoprotein B-containing lipoproteins in Bruch's membrane (BrM) is believed to be important in early age-related macular degeneration (AMD). The origin of the lipoproteins in BrM is a hot topic in AMD research. Some studies hypothesize an intraocular origin. BrM is in direct contact to the choriocapillaris; a plasma origin has also been suggested for the low-density lipoprotein (LDL) particles. We developed an animal model to study the biological effects of circulating LDL on the retina. After injection of LDL for 7 days, our results showed evidence of circulating apolipoprotein B100 retention in BrM and showed induction of early AMD-like alterations in the rat retina, such as thickening of BrM, photoreceptor TUNEL-positive cells, and inflammatory cell infiltration. In vitro assays showed that oxidized LDL (ox-LDL) treatment decreased ARPE-19 cell viability in a dose-dependent manner and that 10 mg/L ox-LDL induced marked apoptosis. The ratio of matrix metalloproteinase-2 to tissue inhibitors of metalloproteinase-3 was dysregulated after LDL and ox-LDL treatment in ARPE-19 cells, which can produce profound changes in the extracellular matrix, including thickening of and deposit formation in BrM. The observation that circulating LDL may be a significant, but not complete, origin of the lipoprotein in BrM suggests that these findings can be readily exploited for the development of new model systems and the future benefit of patients with AMD.

Heat treatment of retinal pigment epithelium induces production of elastic lamina components and antiangiogenic activity.

Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. In advanced AMD, new vessels from choriocapillaris (CC) invade through the Bruch's membrane (BrM) into the retina, forming choroidal neovascularization (CNV). BrM, an elastic lamina that is located between the retinal pigment epithelium (RPE) and CC, is thought to act as a physical and functional barrier against CNV. The BrM of patients with early AMD are characterized by decreased levels of antiangiogenic factors, including endostatin, thrombospondin-1 (TSP-1), and pigment epithelium-derived factor (PEDF), as well as by degeneration of the elastic layer. Motivated by a previous report that heat increases elastin expression in human skin, we examined the effect of heat on human ARPE-19 cell production of BrM components. Heat treatment stimulated the production of BrM components, including TSP-1, PEDF, and tropoelastin in vitro and increased the antiangiogenic activity of RPE measured in a mouse corneal pocket assay. The effect of heat on experimental CNV was investigated by pretreating the retina with heat via infrared diode laser prior to the induction of CNV. Heat treatment blocked the development of experimental CNV in vivo. These findings suggest that heat treatment may restore BrM integrity and barrier function against new vessel growth.

Agents that bind annexin A2 suppress ocular neovascularization.

TM601 is a synthetic polypeptide with sequence derived from the venom of the scorpion Leiurus quinquestriatus that has anti-neoplastic activity. It has recently been demonstrated to bind annexin A2 on cultured tumor and vascular endothelial cells and to suppress blood vessel growth on chick chorioallantoic membrane. In this study, we investigated the effects of TM601 in models of ocular neovascularization (NV). When administered by intraocular injection, intravenous injections, or periocular injections, TM601 significantly suppressed the development of choroidal NV at rupture sites in Bruch's membrane. Treatment of established choroidal NV with TM601 caused apoptosis of endothelial cells and regression of the NV. TM601 suppressed ischemia-induced and vascular endothelial growth factor-induced retinal NV and reduced excess vascular permeability induced by vascular endothelial growth factor. Immunostaining with an antibody directed against TM601 showed that after intraocular or periocular injection, TM601 selectively bound to choroidal or retinal NV and co-localized with annexin A2, which is undetectable in normal retinal and choroidal vessels, but is upregulated in endothelial cells participating in choroidal or retinal NV. Intraocular injection of plasminogen or tissue plasminogen activator, which like TM601 bind to annexin A2, also suppressed retinal NV. This study supports the hypothesis that annexin A2 is an important target for treatment of neovascular diseases and suggests that TM601, through its interaction with annexin A2, causes suppression and regression of ocular NV and reduces vascular leakage and thus may provide a new treatment for blinding diseases such as neovascular age-related macular degeneration and diabetic retinopathy.

Improved assessment of laser-induced choroidal neovascularization.

The primary objective of this study was to develop and evaluate new methods of analyzing laser-induced choroidal neovascularization (CNV), in order to make recommendations for improving the reporting of experimental CNV in the literature. Six laser burns of sufficient power to rupture Bruch's membrane were concentrically placed in each eye of 18 adult Norway rats. Eyes received intravitreal injections of either triamcinolone acetonide, ketorolac, or balanced salt solution (BSS). Fluorescein angiography (FA) was performed 2 and 3 weeks after injection, followed by choroidal flat mount preparation. Vascular leakage on FAs and vascular budding on choroidal mounts were quantified by measuring either the cross-sectional area of each CNV lesion contained within the best-fitting polygon using Adobe Photoshop (Lasso Technique or Quick Selection Technique), or the area of bright pixels within a lesion using Image-Pro Plus. On choroidal mounts, the Lasso Technique and Image-Pro Plus detected a significant difference in lesion size between either ketorolac or triamcinolone when compared to BSS, while the Quick Selection Technique did not (Lasso Technique, 0.78 and 0.64; Image-Pro Plus, 0.77 and 0.65). On FA, the Lasso Technique and Quick Selection Technique detected a significant difference in lesion size between either ketorolac or triamcinolone when compared to BSS, while Image-Pro Plus did not (Lasso Tool, 0.81 and 0.54; Quick Selection Tool, 0.76 and 0.57). Choroidal mounts and FA are both valuable for imaging experimental CNV. Adobe Photoshop and Image-Pro Plus are both able to detect subtle differences in CNV lesion size, when images are not manipulated. The combination of choroidal mounts and FA provides a more comprehensive assessment of CNV anatomy and physiology.

Hematopoietic stem cells provide repair functions after laser-induced Bruch's membrane rupture model of choroidal neovascularization.

Vascular repair by adult hematopoietic stem cells (HSCs) is well-appreciated because these cells are known for their plasticity. We have shown that adult HSCs differentiate into endothelial cells and participate in both retinal and choroidal neovascularization. We asked whether HSCs participated in the wounding response by forming astrocytes, retinal pigment epithelia (RPE), macrophages, and pericytes. Lethally irradiated C57BL6/J mice were reconstituted with HSCs from mice homozygous for green fluorescent protein (GFP) and then subjected to laser-induced rupture of Bruch's membrane. After immunohistochemical examination of ocular tissue, GFP(+) astrocytes were observed concentrated along the edge of the laser wound, where they and mural cells closely ensheathed the neovasculature. GFP(+) vascular endothelial cells and macrophages/microglia were also evident. Large irregularly shaped GFP(+) RPE cells constituted approximately 93% of RPE cells adjacent to the edge of the denuded RPE area. In regions farther away from the wound, GFP(+) RPE cells were integrated among the GFP(-) host RPE. Thus, postnatal HSCs can differentiate into cells expressing markers specific to astrocytes, macrophages/microglia, mural cells, or RPE. These studies suggest that HSCs could serve as a therapeutic source for long-term regeneration of injured retina and choroid in diseases such as age-related macular degeneration and retinitis pigmentosa.

Association of drusen deposition with choroidal intercapillary pillars in the aging human eye.

PURPOSE: To determine the pattern of drusen accumulation with age and to investigate the initial sites of deposition and their relationship to choroidal capillaries in human donor eyes from the eye bank of Moorfields Eye Hospital. METHODS: Wholemounted, hydrated preparations of the choriocapillaris and Bruch's membrane from donor eyes ranging from 42 to 95 years, with or without retinal pigment epithelium (RPE), were examined by conventional and confocal microscopy. Drusen were visualized by their autofluorescence. RESULTS: In all age groups studied autofluorescent drusen were present at the equator but were not found centrally where the vascular architecture is different, being tubular rather than a honeycomb pattern. Autofluorescing drusen were strongly associated with the lateral walls of the choriocapillaris (an area commonly known as the intercapillary pillars of the choriocapillaris (P = 0.028; Wilcoxon signed ranks test). Nonfluorescing drusen were occasionally seen centrally, but were not easily identified, and because of their large size, their localization with respect to capillary walls was not possible. CONCLUSIONS: These results strongly support the notion that autofluorescent drusen are not randomly distributed and have a specific spatial relationship to choroidal vessel walls. That equatorial drusen fluoresce, whereas central drusen do not, suggests that they may have different chemical compositions at the two sites and possibly different significance in age-related macular disease.

Influence of laser photocoagulation on choroidal capillary cytoarchitecture.

AIM: To identify if laser photocoagulation induces morphological changes specifically related to the choroidal capillary endothelial processes that protrude into Bruch's membrane. METHODS: Two human eyes and one adult macaque monkey eye received retinal laser photocoagulation that was just suprathreshold, before enucleation or exenteration. They were examined by electron microscopy to determine the length of the endothelial processes emanating from the choroidal capillaries in the region around the laser burn. One human and two monkey untreated eyes were used for comparison. RESULTS: In human eyes, there was no increase in the number of processes 15 hours after laser treatment but at 5 days the processes were more numerous and longer within 400-500 microm of the burn than in the untreated half of the same eye. The processes were longer 9 days after photocoagulation in the monkey, when compared with untreated monkeys, and some breached the elastic lamina, a phenomenon not seen in the untreated eyes. Qualitative differences were also noted in the endothelial cell processes following photocoagulation. Neovascularisation was not observed. CONCLUSIONS: Protrusion of choroidal endothelial cell processes into Bruch's membrane is a normal anatomical feature but the number, length, and morphology of the processes change following mild photocoagulation. It is plausible that these processes may play a part in the clearance of debris from Bruch's membrane, and represent an early stage of angiogenesis. If the latter is true prophylactic laser photocoagulation at just suprathreshold levels may carry a risk of inducing choroidal neovascularisation.

Fas ligand (CD95 ligand) controls angiogenesis beneath the retina.

A principal cause of blindness is subretinal neovascularization associated with age-related macular degeneration. Excised neovascular membranes from patients with age-related macular degeneration demonstrated a pattern of Fas+ new vessels in the center of the vascular complex, surrounded by FasL+ retinal pigment epithelial cells. In a murine model, Fas (CD95)-deficient (Ipr) and FasL-defective (gld) mice had a significantly increased incidence of neovascularization compared with normal mice. Furthermore, in gld mice there is massive subretinal neovascularization with uncontrolled growth of vessels. We found that cultured choroidal endothelial cells were induced to undergo apoptosis by retinal pigment epithelial cells through a Fas-FasL interaction. In addition, antibody against Fas prevented vascular tube formation of choroidal endothelial cells derived from the eye in a three-dimensional in vitro assay. Thus, FasL expressed on retinal pigment epithelial cells may control the growth and development of new subretinal vessels that can damage vision.

Capillaries are present in Bruch's membrane at the ora serrata in the human eye.

PURPOSE: Because earlier studies indicate that the choroid close to the ora serrata may have unique anatomic features such as wandering cells, blood vessels in Bruch's membrane, and accumulated pigment in the retinal pigment epithelium (RPE), the morphology of the normal human eye at the ora serrata region was investigated. METHODS: Specimens from the ora serrata region of two normal human eyes (male donors, 48 and 52 years old) were investigated by light and electron microscope. Specimens from all quadrants were studied in one eye. RESULTS: The elastic layer of Bruch's membrane extended as far as 15 microm into the peripheral choroid; capillaries were included between the elastin layer and the RPE. Nasally, from the anterior end to 2 mm posterior of the ora serrata, the RPE cells contained more melanin than did those in the adjacent posterior region. Melanin granules in the RPE cells close to the ora either formed large clusters or appeared unusually small because of fragmentation. A unique, fine lamellar, membranous material with a fingerprint-like structure was found between the basal folds of the RPE. This material is also found within the extracellular matrix of the choroid and in association with red blood cells. CONCLUSIONS: The morphology of Bruch's membrane is varied near the ora serrata because capillaries and wandering cells are present in its outer collagenous layer. Unique, fine lamellar, fingerprint-like structures are extruded from the RPE and are removed from the eye together with red blood cells. Capillaries within the inner collagenous region of Bruch's membrane at the ora serrata may not necessarily represent a pathologic response but may be a normal characteristic of thick regions of Bruch's membrane.

Retinal epithelial fine structure in the southern fiddler ray (Trygonorhina fasciata).

The morphology of the retinal epithelium (RPE), choriocapillaris and Bruch's membrane (complexus basalis) has been investigated by light and electron microscopy in an elasmobranch, the southern fiddler ray or guitarfish (Trygonorhina fasciata). The RPE consists of a single layer of cuboidal cells which display basal (scleral) infoldings as well as numerous apical (vitreal) finger-like processes which interdigitate with the photoreceptor outer segments. The lateral cell borders are relatively smooth and are joined in the mid-region by a series of tight junctions. Internally the RPE nucleus is large, vesicular and centrally located. Smooth endoplasmic reticulum (SER) is abundant while rough endoplasmic reticulum (RER) is scarce. Polysomes are however widespread and mitochondria are plentiful. Two unusual organelles are also noted. One consists of a membrane bound array of tubules while the other is a membrane bound structure consisting of a granular matrix with again an internal tubular array. This species possesses a choroidally located tapetum lucidum in the superior fundus and over this tapetal area, melanosomes are absent from the RPE cells. In non-tapetal locations a few melanosomes are present that do not appear to undergo photomechanical movements. Bruch's membrane is a pentalaminate structure with an almost continuous central elastic layer (lamina densa). The choriocapillaris forms a single layer of capillaries with a thin but only minimally fenestrated endothelium facing Bruch's membrane.