Loss of CD34 Expression in Aging Human Choriocapillaris Endothelial Cells

Structural and gene expression changes in the microvasculature of the human choroid occur during normal aging and age-related macular degeneration (AMD). In this study, we sought to determine the impact of aging and AMD on expression of the endothelial cell glycoprotein CD34. Sections from 58 human donor eyes were categorized as either young (under age 40), age-matched controls (> age 60 without AMD), or AMD affected (>age 60 with early AMD, geographic atrophy, or choroidal neovascularization). Dual labeling of sections with Ulex europaeus agglutinin-I lectin (UEA-I) and CD34 antibodies was performed, and the percentage of capillaries labeled with UEA-I but negative for anti-CD34 was determined. In addition, published databases of mouse and human retinal pigment epithelium-choroid were evaluated and CD34 expression compared between young and old eyes. Immunohistochemical studies revealed that while CD34 and UEA-I were colocalized in young eyes, there was variable loss of CD34 immunoreactivity in older donor eyes. While differences between normal aging and AMD were not significant, the percentage of CD34 negative capillaries in old eyes, compared to young eyes, was highly significant (p = 3.8×10⁻⁶). Endothelial cells in neovascular membranes were invariably CD34 positive. Published databases show either a significant decrease in Cd34 (mouse) or a trend toward decreased CD34 (human) in aging. These findings suggest that UEA-I and endogenous alkaline phosphatase activity are more consistent markers of aging endothelial cells in the choroid, and suggest a possible mechanism for the increased inflammatory milieu in the aging choroid.     

An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice

The study and treatment of age-related macular degeneration (AMD), a leading cause of blindness, has been hampered by a lack of animal models. Here we report that mice deficient either in monocyte chemoattractant protein-1 (Ccl-2; also known as MCP-1) or its cognate C-C chemokine receptor-2 (Ccr-2) develop cardinal features of AMD, including accumulation of lipofuscin in and drusen beneath the retinal pigmented epithelium (RPE), photoreceptor atrophy and choroidal neovascularization (CNV). Complement and IgG deposition in RPE and choroid accompanies senescence in this model, as in human AMD. RPE or choroidal endothelial production of Ccl-2 induced by complement C5a and IgG may mediate choroidal macrophage infiltration into aged wild-type choroids. Wild-type choroidal macrophages degrade C5 and IgG in eye sections of Ccl2(-/-) or Ccr2(-/-) mice. Impaired macrophage recruitment may allow accumulation of C5a and IgG, which induces vascular endothelial growth factor (VEGF) production by RPE, possibly mediating development of CNV. These models implicate macrophage dysfunction in AMD pathogenesis and may be useful as a platform for validating therapies.     

Microglia in the Mouse Retina Alter the Structure and Function of Retinal Pigmented Epithelial Cells: A Potential Cellular Interaction Relevant to AMD

Background

Age-related macular degeneration (AMD) is a leading cause of legal blindness in the elderly in the industrialized word. While the immune system in the retina is likely to be important in AMD pathogenesis, the cell biology underlying the disease is incompletely understood. Clinical and basic science studies have implicated alterations in the retinal pigment epithelium (RPE) layer as a locus of early change. Also, retinal microglia, the resident immune cells of the retina, have been observed to translocate from their normal position in the inner retina to accumulate in the subretinal space close to the RPE layer in AMD eyes and in animal models of AMD.

Methodology/Principal Findings

In this study, we examined the effects of retinal microglia on RPE cells using 1) an in vitro model where activated retinal microglia are co-cultured with primary RPE cells, and 2) an in vivo mouse model where retinal microglia are transplanted into the subretinal space. We found that retinal microglia induced in RPE cells 1) changes in RPE structure and distribution, 2) increased expression and secretion of pro-inflammatory, chemotactic, and pro-angiogenic molecules, and 3) increased extent of in vivo choroidal neovascularization in the subretinal space.

Conclusions/Significance

These findings share similarities with important pathological features found in AMD and suggest the relevance of microglia-RPE interactions in AMD pathogenesis. We speculate that the migration of retinal microglia into the subretinal space in early stages of the disease induces significant changes in RPE cells that perpetuate further microglial accumulation, increase inflammation in the outer retina, and fosters an environment conducive for the formation of neovascular changes responsible for much of vision loss in advanced AMD.     

Transforming growth factor-beta induces expression of vascular endothelial growth factor in human retinal pigment epithelial cells: involvement of mitogen-activated protein kinases

Vascular endothelial growth factor (VEGF) is a major agent in choroidal and retinal neovascularization, events associated with age-related macular degeneration (AMD) and diabetic retinopathy. Retinal pigment epithelium (RPE), strategically located between retina and choroid, plays a critical role in retinal disorders. We have examined the effects of various growth factors on the expression and secretion of VEGF by human retinal pigment epithelial cell cultures (HRPE). RT-PCR analyses revealed the presence of three isoforms of mRNA corresponding to VEGF 121, 165, and 189 that were up regulated by TGF-beta1. TGF-beta1, beta2, and beta3 were the potent inducers of VEGF secretion by HRPE cells whereas bFGF, PDGF, TGF-alpha, and GM-CSF had no effects. TGF-beta receptor type II antibody significantly reversed induction of VEGF secretion by TGF-beta. In contrast activin, inhibin and BMP, members of TGF-beta super family, had no effects on VEGF expression in HRPE. VEGF mRNA levels and protein secretion induced by TGF-beta were significantly inhibited by SB203580 and U0126, inhibitors of MAP kinases, but not by staurosporine and PDTC, protein kinase C and NF-kappaB pathway inhibitors, respectively. TGF-beta also induced VEGF expression by fibroblasts derived from human choroid of eye. TGF-beta induction of VEGF secretion by RPE and choroid cells may play a significant role in choroidal neovascularization (CNV) in AMD. Since the secretion of VEGF by HRPE is regulated by MAP kinase pathways, MAP kinase inhibitors may have potential use as therapeutic agents for CNV in AMD.     

Intraocular microablation of choroidal tissue by a 308 nm AIDA excimer laser for RPE-transplantation in patients with age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the western nations beyond 50 years of age. The most frequent cause for severe visual loss is the growth of neovascular membrances from the choroid into the subretinal space. This usually results in irreversible degeneration of the overlying retina. Surgical removal of the membrane is feasible, however, usually results in functional loss of apposing retinal photoreceptors since retinal pigment epithelial (RPE) cells are removed concurrently due to their tight adherence to the neovascular complex. Therefore, various attempts have been undertaken to fill the resulting RPE cell defect with either heterologous or autologous RPE cell transplants. So far cell survival, function and subsequent visual function has been disappointing. To minimize trauma and resulting dedifferentiation harvesting in the eye and transplantation in whole sheets and without temporary removal from the eyes would be desirable. This may be achieved by isolating grafts consisting of choroid, Bruch's membrance and RPE cells from the peripheral retina and transplantation of this graft under the neurosensory retina after removal of the choroidal neovascularization. However, the choroidal component of such a graft would be expected to interfere with diffusion of metabolites to and from the retina. Therefore, outcome would be expected to be better if the choroidal tissue would be removed before translocation. In preclinical experiments we used a 308 nm UV AIDA excimer laser to microablate choroidal tissue from such a graft in human donor eyes.     

Bevacizumab cured age-related macular degeneration (AMD) via down-regulate TLR2 pathway

AMD is the main cause of visual impairment in people over 50 years of age and the most common cause of blindness. In recent years, the use of bevacizumab to treat neovascular AMD has become a preferred treatment in the United States. However, whether bevacozumab is available for RPE or AMD patients is unknown. We firstly indicate that Pam3CSK4 (P3C) activates TLR2 pathway during ARPE-19 apoptosis as determined by western blotting. And then, the expression of MyD88, NF-κB, p-IKK in primary RPE cells from AMD patients is significantly down-regulated after treatment with 50 µg L^?1 Bevacizumab. Therefore, our data shows that MyD88 is involved in the TLR2 pathway in ARPE-19 cell apoptosis resulting from Pam3CSK4 (P3C). And more importantly, our findings suggested that Bevacizumab cured age-related macular degeneration (AMD) via down-regulate Toll—like receptor 2 (TLR2) pathway in RPE from AMD patients.     

CD36 deficiency leads to choroidal involution via COX2 down-regulation in rodents

Background

In the Western world, a major cause of blindness is age-related macular degeneration (AMD). Recent research in angiogenesis has furthered the understanding of choroidal neovascularization, which occurs in the “wet” form of AMD. In contrast, very little is known about the mechanisms of the predominant, “dry” form of AMD, which is characterized by retinal atrophy and choroidal involution. The aim of this study is to elucidate the possible implication of the scavenger receptor CD36 in retinal degeneration and choroidal involution, the cardinal features of the dry form of AMD.

Methods and Findings

We here show that deficiency of CD36, which participates in outer segment (OS) phagocytosis by the retinal pigment epithelium (RPE) in vitro, leads to significant progressive age-related photoreceptor degeneration evaluated histologically at different ages in two rodent models of CD36 invalidation in vivo (Spontaneous hypertensive rats (SHR) and CD36^−/− mice). Furthermore, these animals developed significant age related choroidal involution reflected in a 100%–300% increase in the avascular area of the choriocapillaries measured on vascular corrosion casts of aged animals. We also show that proangiogenic COX2 expression in RPE is stimulated by CD36 activating antibody and that CD36-deficient RPE cells from SHR rats fail to induce COX2 and subsequent vascular endothelial growth factor (VEGF) expression upon OS or antibody stimulation in vitro. CD36^−/− mice express reduced levels of COX2 and VEGF in vivo, and COX2^−/− mice develop progressive choroidal degeneration similar to what is seen in CD36 deficiency.

Conclusions

CD36 deficiency leads to choroidal involution via COX2 down-regulation in the RPE. These results show a novel molecular mechanism of choroidal degeneration, a key feature of dry AMD. These findings unveil a pathogenic process, to our knowledge previously undescribed, with important implications for the development of new therapies.     

VEGF-Production by CCR2-Dependent Macrophages Contributes to Laser-Induced Choroidal Neovascularization

Age-related macular degeneration (AMD) is the most prevalent cause of blindness in the elderly, and its exsudative subtype critically depends on local production of vascular endothelial growth factor A (VEGF). Mononuclear phagocytes, such as macrophages and microglia cells, can produce VEGF. Their precursors, for example monocytes, can be recruited to sites of inflammation by the chemokine receptor CCR2, and this has been proposed to be important in AMD. To investigate the role of macrophages and CCR2 in AMD, we studied intracellular VEGF content in a laser-induced murine model of choroidal neovascularisation. To this end, we established a technique to quantify the VEGF content in cell subsets from the laser-treated retina and choroid separately. 3 days after laser, macrophage numbers and their VEGF content were substantially elevated in the choroid. Macrophage accumulation was CCR2-dependent, indicating recruitment from the circulation. In the retina, microglia cells were the main VEGF⁺ phagocyte type. A greater proportion of microglia cells contained VEGF after laser, and this was CCR2-independent. On day 6, VEGF-expressing macrophage numbers had already declined, whereas numbers of VEGF⁺ microglia cells remained increased. Other sources of VEGF detectable by flow cytometry included in dendritic cells and endothelial cells in both retina and choroid, and Müller cells/astrocytes in the retina. However, their VEGF content was not increased after laser. When we analyzed flatmounts of laser-treated eyes, CCR2-deficient mice showed reduced neovascular areas after 2 weeks, but this difference was not evident 3 weeks after laser. In summary, CCR2-dependent influx of macrophages causes a transient VEGF increase in the choroid. However, macrophages augmented choroidal neovascularization only initially, presumably because VEGF production by CCR2-independent eye cells prevailed at later time points. These findings identify macrophages as a relevant source of VEGF in laser-induced choroidal neovascularization but suggest that the therapeutic efficacy of CCR2-inhibition might be limited.     

TNF-α Decreases VEGF Secretion in Highly Polarized RPE Cells but Increases It in Non-Polarized RPE Cells Related to Crosstalk between JNK and NF-κB Pathways

Asymmetrical secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells in situ is critical for maintaining the homeostasis of the retina and choroid. VEGF is also involved in the development and progression of age-related macular degeneration (AMD). We studied the effect of tumor necrosis factor-α (TNF-α) on the secretion of VEGF in polarized and non-polarized RPE cells (P-RPE cells and N-RPE cells, respectively) in culture and in situ in rats. A subretinal injection of TNF-α caused a decrease in VEGF expression and choroidal atrophy. Porcine RPE cells were seeded on Transwell™ filters, and their maturation and polarization were confirmed by the asymmetrical VEGF secretion and trans electrical resistance. Exposure to TNF-α decreased the VEGF secretion in P-RPE cells but increased it in N-RPE cells in culture. TNF-α inactivated JNK in P-RPE cells but activated it in N-RPE cells, and TNF-α activated NF-κB in P-RPE cells but not in N-RPE cells. Inhibition of NF-κB activated JNK in both types of RPE cells indicating crosstalk between JNK and NF-κB. TNF-α induced the inhibitory effects of NF-κB on JNK in P-RPE cells because NF-κB is continuously inactivated. In N-RPE cells, however, it was not evident because NF-κB was already activated. The basic activation pattern of JNK and NF-κB and their crosstalk led to opposing responses of RPE cells to TNF-α. These results suggest that VEGF secretion under inflammatory conditions depends on cellular polarization, and the TNF-α-induced VEGF down-regulation may result in choroidal atrophy in polarized physiological RPE cells. TNF-α-induced VEGF up-regulation may cause neovascularization by non-polarized or non-physiological RPE cells.     

Role of hypoxia and extracellular matrix-integrin binding in the modulation of angiogenic growth factors secretion by retinal pigmented epithelial cells.

The retinal pigmented epithelium (RPE) is a monolayer of polarized cells located between retinal photoreceptors and blood vessels of the choroid. The basal surface of RPE cells rests on Bruch's membrane, a complex extracellular matrix structure which becomes abnormal in several disease processes, including age-related macular degeneration (AMD). Ruptures or abnormalities in Bruch's membrane are frequently accompanied by choroidal neovascularization. Disturbed interaction of RPE cells with their extracellular matrix (ECM) could play a role in this process. The present study was undertaken to examine the complex interactions between hypoxia, integrin, and ECM in the regulation of RPE functions. Antibody blocking experiments demonstrated that RPE cell adhesion to vitronectin is mediated primarily through alphavbeta5 and adhesion to fibronectin occurs through alpha5beta1. RPE adhesion to immobilized laminin demonstrated highest level of non-RGD-mediated adhesion as compared to that with collagen IV or the RGD matrices such as vitronectin (alphavalpha5) , fibronectin (alpha5beta1), or thrombospondin (alpha5beta1 + alphavbeta5). Addition of soluble vitronectin, or fibrinogen to RPE cell cultures resulted in a small to moderate increase in VEGF and FGF2 in the media, while each of these growth factors was dramatically increased after addition of thrombospondin 1 (TSP1). In contrast, soluble fibronectin resulted in differential upregulation of VEGF but not FGF2. Similarly, immobilized TSP1 resulted in differential greater upregulation in VEGF but not FGF2 release from RPE as compared to other ECMs under either normoxic or hypoxic conditions. Additionally, hypoxia resulted in a time-dependent increase in VEGF, but not FGF2 release in the media. RPE cells grown on TSP1-coated plates showed increased VEGF and FGF2 in their media compared to cells grown on plates coated with type IV collagen, laminin, vitronectin, or fibronectin. The TSP1-induced increase in secretion of growth factors was partially blocked by anti-alpha5beta1, anti-alphavbeta3, and anti-alphavbeta5 antibodies indicating that it may be mediated in part by TSP1 binding to those integrins. These data suggest that alterations in oxygen levels (hypoxia/ischemia) and ECM of RPE cells, a prominent feature of AMD, can cause increased secretion of angiogenic growth factors that might contribute to the development of choroidal neovascularization. These data also suggest the potential modulatory role of VEGF release from RPE by ECM and alphavbeta5 and alpha5beta1 integrins.     

The COX-2-Selective Antagonist (NS-398) Inhibits Choroidal Neovascularization and Subretinal Fibrosis

Choroidal neovascularization (CNV) is an important pathologic component of neovascular age-related macular degeneration (AMD), and CNV lesions later develop into fibrous scars, which contribute to the loss of central vision. Nowadays, the precise molecular and cellular mechanisms underlying CNV and subretinal fibrosis have yet to be fully elucidated. Cyclooxygenase-2 (COX-2) has previously been implicated in angiogenesis and fibrosis. However, the role of COX-2 in the pathogenesis of CNV and subretinal fibrosis is poorly understood. The present study reveals several important findings concerning the relationship of COX-2 signaling with CNV and subretinal fibrosis. Experimental CNV lesions were attenuated by the administration of NS-398, a COX-2-selective antagonist. NS-398-induced CNV suppression was found to be mediated by the attenuation of macrophage infiltration and down-regulation of VEGF in the retinal pigment epithelium–choroid complex. Additionally, NS-398 attenuated subretinal fibrosis, in an experimental model of subretinal scarring observed in neovascular AMD, by down-regulation of TGF-β₂ in the retinal pigment epithelium–choroid complex. Moreover, we cultured mouse RPE cells and found that NS-398 decreased the secretion of VEGF and TGF-β₂ in mouse RPE cells. The results of the present study provide new findings regarding the molecular basis of CNV and subretinal fibrosis, and provide a proof-of-concept approach for the efficacy of COX-2 inhibition in treating subretinal fibrosis.     

Topical Application of PPADS Inhibits Complement Activation and Choroidal Neovascularization in a Model of Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly. AMD patients have elevated levels of membrane attack complex (MAC) in their choroidal blood vessels and retinal pigment epithelium (RPE). MAC forms pores in cell membranes. Low levels of MAC result in an elevation of cytokine release such as vascular endothelial growth factor (VEGF) that promotes the formation of choroidal neovascularization (CNV). High levels of MAC result in cell lysis and RPE degeneration is a hallmark of advanced AMD. The current standard of care for CNV associated with wet AMD is intravitreal injection of anti-VEGF molecules every 4 to 12 weeks. Such injections have significant side effects. Recently, it has been found that membrane pore-forming proteins such as α-haemolysin can mediate their toxic effects through auto- and paracrine signaling and that complement-induced lysis is amplified through ATP release followed by P2X receptor activation. We hypothesized that attenuation of P2X receptor activation may lead to a reduction in MAC deposition and consequent formation of CNV. Hence, in this study we investigated topical application of the purinergic P2X antagonist Pyridoxalphosphate-6-azophenyl-2'',4''-disulphonic acid (PPADS) as a potential treatment for AMD. We found that 4.17 µM PPADS inhibited formation of HUVEC master junctions and master segments by 74.7%. In a human complement mediated cell lysis assay, 104 µM PPADS enabled almost complete protection of Hepa1c1c7 cells from 1% normal human serum mediated cell lysis. Daily topical application of 4.17 mM PPADS for 3 days attenuated the progression of laser induced CNV in mice by 41.8% and attenuated the deposition of MAC at the site of the laser injury by 19.7%. Our data have implications for the future treatment of AMD and potentially other ocular disorders involving CNV such as angioid streaks, choroidal rupture and high myopia.     

Cigarette smoke-related hydroquinone dysregulates MCP-1, VEGF and PEDF expression in retinal pigment epithelium in vitro and in vivo

Background

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly population. Debris (termed drusen) below the retinal pigment epithelium (RPE) have been recognized as a risk factor for dry AMD and its progression to wet AMD, which is characterized by choroidal neovascularization (CNV). The underlying mechanism of how drusen might elicit CNV remains undefined. Cigarette smoking, oxidative damage to the RPE and inflammation are postulated to be involved in the pathophysiology of the disease. To better understand the cellular mechanism(s) linking oxidative stress and inflammation to AMD, we examined the expression of pro-inflammatory monocyte chemoattractant protein-1 (MCP-1), pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial derived factor (PEDF) in RPE from smoker patients with AMD. We also evaluated the effects of hydroquinone (HQ), a major pro-oxidant in cigarette smoke on MCP-1, VEGF and PEDF expression in cultured ARPE-19 cells and RPE/choroids from C57BL/6 mice.

Principal Findings

MCP-1, VEGF and PEDF expression was examined by real-time PCR, Western blot, and ELISA. Low levels of MCP-1 protein were detected in RPE from AMD smoker patients relative to controls. Both MCP-1 mRNA and protein were downregulated in ARPE-19 cells and RPE/choroids from C57BL/6 mice after 5 days and 3 weeks of exposure to HQ-induced oxidative injury. VEGF protein expression was increased and PEDF protein expression was decreased in RPE from smoker patients with AMD versus controls resulting in increased VEGF/PEDF ratio. Treatment with HQ for 5 days and 3 weeks increased the VEGF/PEDF ratio in vitro and in vivo.

Conclusion

We propose that impaired RPE-derived MCP-1-mediated scavenging macrophages recruitment and phagocytosis might lead to incomplete clearance of proinflammatory debris and infiltration of proangiogenic macrophages which along with increased VEGF/PEDF ratio favoring angiogenesis might promote drusen accumulation and progression to CNV in smoker patients with dry AMD.     

Complement Factor H Expressed by Retinal Pigment Epithelium Cells Can Suppress Neovascularization of Human Umbilical Vein Endothelial Cells: An in vitro Study

Complement factor H (CFH) is one of the most important soluble complement regulatory proteins and is closely associated with age-related macular degeneration (AMD), the leading cause of irreversible central vision loss in the elderly population in developed countries. Our study searches to investigate whether CFH expression is changed in oxidative damaged retinal pigment epithelium (RPE) cells and the role of CFH in the in vitro neovascularization. First, it was confirmed by immunofluorescence staining that CFH was expressed by ARPE-19 cells. CFH mRNA and protein in oxidative (H₂O₂) damaged ARPE-19 cells were both reduced, as determined by Real-time PCR and Western blotting analysis. Enzyme-linked immunosorbent assay (ELISA) also showed that ARPE-19 cells treated with H₂O₂ caused an increase in C3a content, which indicates complement activation. Then, wound assays were performed to show that CFH expression suppression promoted human umbilical vein endothelial cell (HUVECs) migration. Thereafter, ARPE-19 cells were transfected with CFH-specific siRNA and CFH knockdown was confirmed with the aid of Real-time PCR, immunofluorescence staining and Western blotting. The ELISA results showed that specific CFH knockdown in ARPE-19 cells activated the complement system. Finally, in vitro matrigel tube formation assay was performed to determine whether change of CFH expression in RPE would affect tube formation by HUVECs. More tubes were formed by HUVECs co-cultured with ARPE-19 cells transfected with CFH specific-siRNA when compared with controls. Our results suggested that RPE cells might be the local CFH source, and RPE cell injuries (such as oxidative stress) may cause CFH expression suppression, which in turn may lead to complement activation and promotion of tube formation by HUVECs. This finding is of importance in elucidating the role of complement in the pathogenesis of ocular neovascularization including choroidal neovascularization.     

Resveratrol prevents the development of choroidal neovascularization by modulating AMP-activated protein kinase in macrophages and other cell types

The development of choroidal neovascularization (CNV) is a critical step in the pathogenesis of age-related macular degeneration (AMD), a vision-threatening disease. In this study, we used a mouse model of AMD to study the protective effects of resveratrol (RSV) supplementation against CNV as well as the underlying molecular mechanisms. Mice were orally pretreated with RSV daily for 5 days. On the fifth day, the mice underwent laser photocoagulation to induce CNV. One week after laser treatment, CNV volume was significantly lower in the RSV-treated mice compared with vehicle-treated animals. In addition, RSV treatment significantly inhibited macrophage infiltration into the retinal pigment epithelium (RPE)-choroid and suppressed the expression of inflammatory and angiogenic molecules, including vascular endothelial growth factor, monocyte chemotactic protein-1 and intercellular adhesion molecule-1. Importantly, RSV prevented the CNV-induced decrease in activated AMP-activated protein kinase and increase in activated nuclear factor-κB in the RPE-choroid complex. The regulatory effects of RSV on these molecules were confirmed in RPE, microvascular endothelial and macrophage cell lines. Inhibition of macrophage infiltration by RSV was confirmed by in vitro scratch and migration assays. RSV suppressed CNV development, reducing the levels of multiple cytokines secreted from several cell types and inhibiting macrophage migration. The direct effects of RSV on each cell type were confirmed in vitro. Although further studies are needed, RSV could potentially be applied in the clinic to prevent CNV development in AMD.     

Regulation of angiotensin II receptors and extracellular matrix turnover in human retinal pigment epithelium: role of angiotensin II

The early stage of age-related macular degeneration (AMD) is characterized by the formation of subretinal pigment epithelium (RPE) deposits as a result of the dysregulation in the turnover of extracellular matrix (ECM) molecules. However, the mechanism involved remains unclear. Hypertension (HTN) is an important risk factor for AMD, and angiotensin II (ANG II) is the most important hormone associated with HTN. However, the relevance of ANG II receptors and ANG II effects on RPE have not been investigated yet. Therefore, the expression and regulation of ANG II receptors as well as the ECM turnover were studied in human RPE. ANG II receptors were expressed and upregulated by ANG II in human RPE. This regulation resulted in functional receptor expression, since an increase in intracellular concentration of calcium was observed upon ANG II stimulation. ANG II also increased matrix metalloproteinase (MMP)-2 activity and MMP-14 at the mRNA and protein levels as well as type IV collagen degradation. These ANG II effects were abolished in the presence of the ANG II receptor subtype 1 (AT1) receptor antagonist candesartan. In contrast, ANG II decreased type IV collagen via both AT1 and AT2 receptors, suggesting a synergistic effect of the two receptor subtypes. In conclusion, we have confirmed the presence of ANG II receptors in human RPE and their regulation by ANG II as well as the regulation of ECM molecules via ANG II receptors. Our data support the hypothesis that ANG II may exert biological function in RPE through ANG II receptors and that ANG II may cause dysregulation of molecules that play a major role in the turnover of ECM in RPE basement membrane and Bruch's membrane, suggesting a pathogenic mechanism to explain the link between HTN and AMD.