Maria E. Marin-Castaño

Nicotine increases the VEGF/PEDF ratio in retinal pigment epithelium: a possible mechanism for CNV in passive smokers with AMD.

PURPOSE Cigarette smoking is the strongest environmental risk factor for wet age-related macular degeneration (AMD). Inappropriate expression of proangiogenic vascular endothelial growth factor (VEGF) and antiangiogenic pigment epithelium derived factor (PEDF) may cause choroidal neovascularization (CNV), a key event in wet AMD, resulting in vision loss. Nicotine (NT), a potent angiogenic agent abundant in second-hand smoke, may play a major role in the pathogenesis of wet AMD. The purpose of this study was to evaluate the expression of nicotinic acetylcholine receptors (nAchR) in retinal pigment epithelium (RPE) and determine the effects of NT on RPE-derived VEGF and PEDF expression in the context of passive smoking. METHODS Human RPE cells were treated with NT (10(-8) M), with or without the nAchR-nonspecific antagonist hexamethonium (HXM) (10(-5) M) for 72 hours. RPE sheets were microdissected from rats exposed to NT in drinking water (100 μg/mL), with or without HXM (40 mg/kg/d, intraperitoneally), for 72 hours. Cell death was determined by cell count and proliferation by Western blot for proliferating cell nuclear antigen (PCNA). nAchR expression was examined by real-time PCR and Western blot. ERK activation was evaluated by Western blot analysis. VEGF and PEDF expression was assessed by ELISA, Western blot, and real-time PCR. RESULTS Cultured RPE cells constitutively expressed the nAchR α3, α10, and β1 subunits, with β1 being the most prevalent. The nAchR α4, α5, α7, and β2 subunits were detected in RPE sheets from rats, among which α4 is the predominant subtype. NT, which did not result in either cell death or proliferation, induced β1 nAchR, upregulated VEGF, and downregulated PEDF expression through nAChR in ARPE-19 cells. Transcriptional activation of the nAchR α4 subunit and nAChR-mediated upregulation of VEGF and PEDF were observed in RPE from rats exposed to NT. CONCLUSIONS NT increased the VEGF-to-PEDF ratio in the RPE through nAchR in vitro and in vivo. This alteration in the ratio may play a key role in the progression to wet AMD in passive smokers.

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.

Angiotensin II–Induced MMP-2 Activity and MMP-14 and Basigin Protein Expression Are Mediated via the Angiotensin II Receptor Type 1–Mitogen-Activated Protein Kinase 1 Pathway in Retinal Pigment Epithelium: Implications for Age-Related Macular Degeneration

Accumulation of various lipid-rich extracellular matrix (ECM) deposits under the retinal pigment epithelium (RPE) has been observed in eyes with age-related macular degeneration (AMD). RPE-derived matrix metalloproteinase (MMP)-2, MMP-14, and basigin (BSG) are major enzymes involved in the maintenance of ECM turnover. Hypertension (HTN) is a systemic risk factor for AMD. It has previously been reported that angiotensin II (Ang II), one of the most important hormones associated with HTN, increases MMP-2 activity and its key regulator, MMP-14, in RPE, inducing breakdown of the RPE basement membrane, which may lead to progression of sub-RPE deposits. Ang II exerts most of its actions by activating the mitogen-activated protein kinase (MAPK) signaling pathway. Herein is explored the MAPK signaling pathway as a potential key intracellular modulator of Ang II-induced increase in MMP-2 activity and MMP-14 and BSG protein expression. It was observed that Ang II stimulates phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAPK in RPE cells and ERK/p38 and Jun N-terminal kinase (JNK) in mice. These effects were mediated by Ang II type 1 receptors. Blockade of ERK or p38 MAPK abrogated the increase in MMP-2 activity and MMP-14 and BSG proteins in ARPE-19 cells. A better understanding of the molecular events by which Ang II induces ECM dysregulation is of critical importance to further define its contribution to the progression of sub-RPE deposits in AMD patients with HTN.

Proteomics Characterization of Cell Membrane Blebs in Human Retinal Pigment Epithelium Cells

Age-related macular degeneration (AMD) is the leading cause of legal blindness among the elderly population in the industrialized world, affecting about 14 million people in the United States alone. Smoking is a major environmental risk factor for AMD, and hydroquinone is a major component in cigarette smoke. Hydroquinone induces the formation of cell membrane blebs in human retinal pigment epithelium (RPE). Blebs may accumulate and eventually contribute first to sub-RPE deposits and then drusen formation, which is a prominent histopathologic feature in eyes with AMD. As an attempt to better understand the mechanisms involved in early AMD, we sought to investigate the proteomic profile of RPE blebs. Isolated blebs were subjected to SDS-PAGE fractionation, and in-gel trypsin-digested peptides were analyzed by LC-MS/MS that lead to the identification of a total of 314 proteins. Identified proteins were predominantly involved in oxidative phosphorylation, cell junction, focal adhesion, cytoskeleton regulation, and immunogenic processes. Importantly basigin and matrix metalloproteinase-14, key proteins involved in extracellular matrix remodeling, were identified in RPE blebs and shown to be more prevalent in AMD patients. Altogether our findings suggest, for the first time, the potential involvement of RPE blebs in eye disease and shed light on the implication of cell-derived microvesicles in human pathology.

Cigarette Smoke-Related Hydroquinone Induces Filamentous Actin Reorganization and Heat Shock Protein 27 Phosphorylation through p38 and Extracellular Signal-Regulated Kinase 1/2 in Retinal Pigment Epithelium : Implications for Age-Related Macular Degeneration

Retinal pigment epithelium (RPE)-derived membranous debris named blebs, may accumulate and contribute to sub-RPE deposit formation, which is the earliest sign of age-related macular degeneration (AMD). Oxidative injury to the RPE might play a significant role in AMD. However, the underlying mechanisms are unknown. We previously reported that hydroquinone (HQ), a major pro-oxidant in cigarette smoke, foodstuff, and atmospheric pollutants, induces actin rearrangement and membrane blebbing in RPE cells as well as sub-RPE deposits in mice. Here, we show for the first time that phosphorylated Heat shock protein 27 (Hsp27), a key regulator of actin filaments dynamics, is up-regulated in RPE from patients with AMD. Also, HQ-induced nonlethal oxidative injury led to Hsp27mRNA up-regulation, dimer formation, and Hsp27 phosphorylation in ARPE-19 cells. Furthermore, we found that a cross talk between p38 and extracellular signal-regulated kinase (ERK) mediates HQ-induced Hsp27 phosphorylation and actin aggregate formation, revealing ERK as a novel upstream mediator of Hsp27 phosphorylation. Finally, we demonstrated that Hsp25, p38, and ERK phosphorylation are increased in aging C57BL/6 mice chronically exposed to HQ, whereas Hsp25 expression is decreased. Our data suggest that phosphorylated Hsp27 might be a key mediator in AMD and HQ-induced oxidative injury to the RPE, which may provide helpful insights into the early cellular events associated with actin reorganization and bleb formation involved in sub-RPE deposits formation relevant to the pathogenesis of 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 Bruchs membrane, suggesting a pathogenic mechanism to explain the link between HTN and AMD.

Pro)renin receptor is expressed in human retinal pigment epithelium and participates in extracellular matrix remodeling

The (pro)renin receptor (PRR) is believed to potentiate the renin-angiotensin system (RAS), conferring to prorenin, a likely pathological role at tissue level. The PRR has been identified in the microvascular endothelial cells of the retina, in which it seems to be involved in pathological neovascularization processes. In the present study, we sought to explore PRR expression and prorenin action in human retinal pigment epithelium (RPE) cells, as well as its potential implication in extracellular matrix (ECM) turnover. Isolated RPE cells from donor human eyes as well as freshly isolated human retinas demonstrated expression of PRR at mRNA and protein levels. Moreover, we demonstrate that PRR expressed in the RPE cells is functional, as shown by prorenin-induced increases in Erk1/2 phosphorylation. PRR expression was also shown to be regulated by its main physiological agonist prorenin. We found evidence that the PRR may be involved in ECM-remodeling processes through a prorenin-induced upregulation of type I collagen. Immunostaining analysis of human retinas revealed higher PRR and type I collagen expression in the RPE of eye donors with dry age-related macular degeneration (AMD) and hypertension, supporting the in vitro findings using human-isolated RPE cells. Taken together, the present study demonstrates for the first time that the PRR is expressed in human RPE and suggests a molecular mechanism by which hypertension may exacerbate the pathology of dry AMD.

Angiotensin II-induced hypertension regulates AT1 receptor subtypes and extracellular matrix turnover in mouse retinal pigment epithelium.

Accumulation of specific deposits and extracellular molecules under the retinal pigment epithelium (RPE) has been previously observed in eyes with age-related macular degeneration (AMD) and may play a role in the pathogenesis of AMD. Even though age is the major determinant for developing AMD, clinical studies have revealed hypertension (HTN) as another systemic risk factor. Angiotensin II (Ang II) is considered the most important hormone associated with HTN. To evaluate the relationship of Ang II to AMD, we studied whether mouse RPE expresses functional Ang II receptor subtypes and whether HTN-induced Ang II regulates expression of these receptors as well as critical ECM molecules (MMP-2 and type IV collagen) involved in ECM turnover in RPE. We used 9-month-old C57BL/6 male mice infused with Ang II alone or Ang II in combination with the AT1 receptor antagonist candesartan or the AT2 receptor antagonist PD123319 for 4 weeks to determine whether HTN-associated Ang II was important for ECM regulation in RPE. We found that mouse RPE expressed both Ang II receptor subtypes at the mRNA and protein levels. Infusion with Ang II induced HTN and elevated plasma and ocular Ang II levels. Ang II also regulated AT1a and AT1b receptor mRNA expression, the intracellular concentration of calcium [Ca(2+)](i), MMP-2 activity, and type IV collagen accumulation. Concurrent administration of Ang II with the AT1 receptor blocker prevented the increase in blood pressure and rise in ocular Ang II levels, as well as the calcium and MMP-2 responses. In contrast, the type IV collagen response to Ang II was prevented by blockade of AT2 receptors, but not AT1 receptors. Plasma Ang II levels were not modified by the AT1 or AT2 receptor blockade. Since the effects of Ang II on MMP-2 and type IV collagen require inhibition of both Ang II receptor subtypes, these receptors may play a role as a potential therapeutic targets to prevent ECM turnover dysregulation in the RPE basement membrane, suggesting a pathogenic mechanism to explain the link between HTN and AMD.

Retinal Pigmented Epithelial Cells Cytotoxicity and Apoptosis through Activation of the Mitochondrial Intrinsic Pathway: Role Of Indocyanine Green, Brilliant Blue and Implications for Chromovitrectomy

Purpose To investigate the in vitro effect of four vital dyes on toxicity and apoptosis in a human retinal pigment epithelial (RPE) cell line. Methods ARPE-19 cells were exposed to brilliant blue (BriB), methyl blue (MetB), acid violet (AcV) and indocyanine green (ICG). Balanced salt solution was used as control. Five different concentrations of each dye (1, 0.5, 0.25, 0.05 and 0.005 mg/mL) and two exposure times (3 and 30 min) were tested. Cell viability was determined by cell count and MTS assay and cell toxicity by LDH assay. Real-time PCR and Western blotting were used to access the apoptosis process. Results ICG significantly reduced cell viability after 3 minutes of exposure at all concentrations (p<0.01). BriB was safe at concentrations up to 0.25 mg/mL and MetB at concentrations up to 0.5 mg/mL, while AcV was safe up to 0.05 mg/ml, after 3 minutes of exposure. Toxicity was higher, when the cells were treated for 30 minutes. Expression of Bax, cytochrome c and caspase-9 was upregulated at the mRNA and protein level after ICG exposure, while Bcl-2 was downregulated. AcV and MetB were similar to control. However, BriB resulted in upregulation of Bcl-2, an antiapoptotic protein. Conclusions The safest dye used on RPE cells was MetB followed by BriB and AcV. ICG was toxic at all concentrations and exposure times tested. Moreover, ICG was the only dye that induced apoptosis in ARPE-19 cells. BriB significantly increased Bcl-2 protein levels, which might protect against the apoptosis process.

Effect of Vital Dyes on Retinal Pigmented Epithelial Cell Viability and Apoptosis: Implications for Chromovitrectomy

Purpose: To investigate the in vitro effect of vital dyes on toxicity and apoptosis in a human retinal pigment epithelial cell line. Methods: ARPE-19 cells were exposed to brilliant blue (BBG), Evans Blue (EB), bromophenol blue (BroB), indocyanine green (ICG), infracyanine green (IfCG), light green (LG), fast green (FG), indigo carmine (IC) and Congo red (CR). Balanced salt solution was used as the control. Five different concentrations and 2 exposure times were tested. Cell viability was determined by the MTS (1-solution methyl thiazolyl tetrazolium) assay and apoptosis by Bax expression on Western blot. Results: All dyes significantly reduced cell viability after 3 min of exposure at all concentrations (p < 0.01), except for BBG that was safe at concentrations up to 0.25 mg/ml and CR up to 0.05 mg/ml, while LG was safe at all concentrations. Toxicity was higher after 30 min of exposure. Expression of Bax was upregulated after all dye exposures, except BBG; ICG had the highest Bax expression (p < 0.01). Conclusions: Overall the safest dye was BBG followed by LG, IfCG, FG, CR, IC, BroB, EB and ICG. ICG was toxic at all concentrations and exposure times tested. Moreover, BBG was the only dye that did not induce apoptosis in ARPE-19 cells.