**8. Inflammation and angiogenesis**

carried proteins, basigin and MMP-14. Therefore, we speculate that blebs may play an important role for sub-RPE to traverse RPE basement membrane. RPE are subsequently stimulated to increase synthesis of collagens and other molecules responsible for ECM turnover, affecting both RPE basement membrane and BrM. This process leads to the formation of new basement membrane under the RPE to trap these deposits within BrM. We postulate that various hormones and other plasma-derived molecules related to systemic

60 Age-Related Macular Degeneration - Etiology, Diagnosis and Management - A Glance at the Future

**Figure 11.** Immunohistochemical analysis of basigin and MMP-14 in human retina. Retina sections from human donor eyes with no known eye disease (A, C, and E) or from human donor eyes with dry AMD (B, C, and F) were stained with either mouse polyclonal anti-basigin (C and D) or mouse monoclonal anti-MMP-14 (E and F) as indicated. Negative controls were generated by omission of the primary antibody (A and B). Secondary antibodies were coupled to Alexa Fluor 488. Nuclei were stained with 4,6-diamidino-2-phenylindole dihydrochloride. Sections were analyzed under a confocal microscope. INL, inner nuclear layer; ONL, outer nuclear layer; PIS, photoreceptor inner segments; POS, pho‐

health cofactors are implicated in this stage.

toreceptor outer segments; Ch, choroi.

#### **8.1. Inflammation: Role of cigarette smoke and angiotensin II**

Another potential oxidative injury stimulus in AMD may occur during inflammation. Histopathology of AMD demonstrates that all stages of the disease, including drusen, geographic atrophy and CNV, are associated with inflammatory cells, especially macrophages [24,25,37,259,260]. One well-characterized inflammatory oxidant is myeloperoxidase (MPO), a heme protein secreted by neutrophils and macrophages that converts its substrate hydrogen peroxide into an active oxidant [206]. RPE metabolism results in high quantities of hydrogen peroxide synthesis, which by itself is a weak oxidant and is neutralized by catalase and other anti-oxidant enzyme systems [261-263]. However, in the setting of MPO release, RPE-derived hydrogen peroxide can become a powerful oxidant [171]. Macrophage-derived MPO will remain extracellular, but may initiate or potentiate the RPE injury response by catalyzing hydrogen peroxide into the formation of powerful oxidants such as hydroxyl radicals, hydroperoxides, hypochlorous acid, and tyrosyl radicals [50,264]. Among their actions, MPOderived oxidants induce injury to the cell membrane and modify cell surface proteins and receptors [50,265,266].

Data from a number of laboratories provide compelling evidence that inflammatory and/or immune-mediated events may participate in the development of sub-RPE deposits formation and/or progression to CNV [12,24, 267-269]. Based upon available data, a new paradigm has been introduced for sub-RPE formation and its relationship to AMD. This integrated hypoth‐ esis is based largely upon the dynamic interactions between those factors that induce and sustain chronic local inflammation at the level of the RPE-BrM-choroidal interface, and those mechanisms that attenuate it. Complement and immune complexes have been identified in drusen, but their pathogenic role has not been defined. This information has been recently reviewed [24-27]. Other investigators have observed that choroidal monocytes/ macrophages are present in human specimens of both early and late AMD [24,25,27,37,259,270]. Macro‐ phages have been detected along the choriocapillaris-side of BrM underlying areas of thick drusen or other deposits [10, 260-273] and processes from choroidal monocytes have been noted to insert into BrM deposits [260] Moreover, dentritic cells are often observed in the sub-RPE space in association with whole, or portions of, RPE cells that have been shunted into BrM, prior to the time that drusen are detectable. Therefore, macrophages and choroidal dentritic cells may be activated and recruited by locally damaged and/or sublethaly injured RPE cells. This idea is consistent with the data showing that macrophages and/or dentritic cells, and thus the innate immune system, can be activated by microenvironmental tissue damage [14,274,275]. However it remains to be determined whether drusen-associated macrophages and dentriric cells initiate a classical immune response involving T helper cells, secreted cytokines, elicit an inflammatory or complement-mediated response, or play some other role in the generation of drusen.

While it is largely recognized that macrophages accumulate in AMD lesions, there is ambiguity surrounding their role in the disease process with conflicting evidence regarding whether they might be helpful by scavenging accumulated debris and therefore protecting against CNV or harmful by stimulating CNV [100]. This might be due to the largely observational nature of human samples but also probably reflects different functions macrophages serve during distinct phases of the disease.

MCP-1 production by aging RPE cells may impair recruitment of macrophages/dentritic cells essential for scavenging debris which may lead to drusen formation and accumulation in AMD patients. It can be speculated that declining RPE-derived MCP-1 production resulting from cumulative exposure to oxidative damage may be an important factor that could accelerate and promote the formation of sub-RPE deposits in smokers. This theory is put forth bearing in mind the complexity of underlying cellular and molecular mechanisms involved in the inflammatory response and with the acknowledgment that numerous AMD genotypes may exist. Therefore, we fully recognize that only some aspects of the proposed hypothesis may be

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Based on the above hypothesis, we have evaluated the possibility that hydroquinone-induced oxidative stress might regulate MCP-1 expression in the RPE. We showed that prolonged exposure to hydroquinone-induced oxidative injury downregulated MCP-1 production by ARPE-19 cells and RPE/choroids from C57BL/6 mice [294]. An earlier study by Joly et al is in line with our observations showing a decline in MCP-1 gene expression in retinas of mice exposed to light-induced oxidative damage for several days [294]. Our observations suggest that sustained exposure to hydroquinone might impair RPE-derived MCP-1-mediated scavenging macrophages and dentritic cells recruitment and phagocytosis which might lead to incomplete clearance of proinflammatory debris trapped between the RPE and its BrM. On the other hand, our preliminary date show that RPE-derived blebs activate RPE MCP-1 production (unpublished data), suggesting that when significant BLD will have already formed, RPE-blebs and other debris will activate MCP-1 secretion by RPE leading to infiltration of proangiogenic macrophages to sub-RPE deposit areas, where they will scavenge, relate more cytokines and mediators, and amplify the process leading to progression of drusen to CNV in

Angiotensin II is not only a potent vasoconstrictor which elevates arterial blood pressure, but also a powerful pro-inflammatory cytokine, chemokine and growth factor [276, 283-285,295], which mediates the activation of inflammatory mechanisms involved in age-related diseases [296,297]. There is accumulating evidence that Ang II can cause target organ damage by facilitating inflammatory and growth responses through activation of NFκB [276, 283-285], the key nuclear transcription factor in inflammatory and fibrotic diseases. Activation of NFκB by Ang II may stimulate transcription of numerous inflammatory genes, including MCP-1, RANTES (Regulated on Active Normal T cell Expressed and Secreted) and interleukin (IL)-6, TNF-α and TGF-β [276,283-285]. The view that MCP-1 is one of the most important chemokines in Ang II-induced inflammatory responses is supported by numerous studies, although the mechanisms by which Ang II increases MCP-1 expression and production are still not well understood [279,283-285]. In a rabbit model of atherosclerosis, ACE1 inhibitor quinapril inhibited NFκB activity, expression and production of MCP-1 and neointimal macrophage infiltration at the injured sites [283]. In Ang II-induced hypertensive rats, vascular MCP-1 mRNA expression increased almost four-fold, which was significantly reduced by normali‐ zation of hypertension by the non-specific vasodilator hydralazine, but the effects of AT1 receptor blockade were not studied [301]. However, in a different study, Ang II enhanced expression of MCP-1 mRNA and protein production in rat vascular smooth muscle cells in a dose- and time-dependent fashion, and these effects were mediated by AT1 receptors involv‐ ing the Rho-kinase pathway [298]. In mice, Wu et al. [299] showed that the AT1 receptor

involved in any given AMD genotype.

smoker patients with AMD.

Inflammation is a complex process that involves local secretion of pro-inflammatory cytokines (leukocyte adhesion molecules, ICAM-1 and VCAM-1) [276,277], MCP-1 [278-282] NFκB [283-285], and growth factors (tumour necrosis factor, TNF-α, TGF-β). The key inflammatory molecule in initiating inflammatory responses may be MCP-1 [278-284] a powerful chemokine, expressed at sites of injury. Indeed, the importance of MCP-1 in inflammatory diseases is highlighted by a careful MEDLINE search for MCP-1, which readily returns more than 3000 citations following its characterization in the late 1980s. NFκB appears to control expression of MCP-1 [283-285]. After release, it activates the CCR2 chemotactic receptor to induce chemotactic responses that mediate monocyte and macrophage migration into sites of active inflammation in various diseases [286,287].

Based upon the assumption that injured RPE may serve as stimulatory factors that initiate macrophages recruitment and activation, it was determined if monocyte populations from individual AMD patients exhibited heterogeneity in terms of cytokine production in culture or in mRNA expression of freshly isolated cells, and if high levels served as a biomarker of risk for progression into neovascular AMD [51]. TNF-α expression, a potent cytotoxic and proangiogenic cytokine for wet AMD. Typically human macrophages in culture, after stimulation with RPE derived blebs produced an increase in TNF-α. However, extremely high variability in baseline TNF-α expression from isolated macrophages was observed among different subjects [51]. These results are consistent with the hypothesis that the pre-existing macrophage activation state, defined as level of cytokine or mediator expression of the circulating monocyte, might determine the negative or positive consequence of macrophage recruitment as a disease modifier. Macrophages with low expression might remove deposits safely whereas macrophages with high expression might produce mediators that contribute to disease progression.

As mentioned above, aberrant expression of chemokines occurs in a variety of diseases that have an inflammatory component. Highly specialized RPE cells play a pivotal role in the maintenance of the outer retina by secreting several cytokines including monocyte chemoat‐ tractant protein-1 (MCP-1) [288,289] which has been suggested to be implicated in the pathogenesis of AMD [290,291]. RPE cells can secrete MCP-1 in the direction of choroidal blood vessels during inflammatory responses therefore suggesting that RPE cells might promote macrophage recruitment to the choroid from circulating monocytes [292]. It was reported that MCP-1 is regulated in injured ARPE-19 cells and that free radicals might be immunostimula‐ tory [293] providing support for the notion that injured RPE cells may induce monocyte migration.

We have investigated MCP-1 expression in RPE from patients with AMD as well as the regulation of RPE-derived MCP-1 expression following cigarette hydroquinone-mediated oxidative injury. Our data report for the first time that MCP-1 expression is markedly de‐ creased in RPE from smoker patients with AMD [294] thereby pointing to a critical role for MCP-1 in the pathogenesis of the disease. We acknowledge that due to the nature of our study, it cannot be determined whether the altered expression of MCP-1 in human RPE lysates is a cause or consequence of the disease. However, our current findings suggest that declining MCP-1 production by aging RPE cells may impair recruitment of macrophages/dentritic cells essential for scavenging debris which may lead to drusen formation and accumulation in AMD patients. It can be speculated that declining RPE-derived MCP-1 production resulting from cumulative exposure to oxidative damage may be an important factor that could accelerate and promote the formation of sub-RPE deposits in smokers. This theory is put forth bearing in mind the complexity of underlying cellular and molecular mechanisms involved in the inflammatory response and with the acknowledgment that numerous AMD genotypes may exist. Therefore, we fully recognize that only some aspects of the proposed hypothesis may be involved in any given AMD genotype.

human samples but also probably reflects different functions macrophages serve during

62 Age-Related Macular Degeneration - Etiology, Diagnosis and Management - A Glance at the Future

Inflammation is a complex process that involves local secretion of pro-inflammatory cytokines (leukocyte adhesion molecules, ICAM-1 and VCAM-1) [276,277], MCP-1 [278-282] NFκB [283-285], and growth factors (tumour necrosis factor, TNF-α, TGF-β). The key inflammatory molecule in initiating inflammatory responses may be MCP-1 [278-284] a powerful chemokine, expressed at sites of injury. Indeed, the importance of MCP-1 in inflammatory diseases is highlighted by a careful MEDLINE search for MCP-1, which readily returns more than 3000 citations following its characterization in the late 1980s. NFκB appears to control expression of MCP-1 [283-285]. After release, it activates the CCR2 chemotactic receptor to induce chemotactic responses that mediate monocyte and macrophage migration into sites of active

Based upon the assumption that injured RPE may serve as stimulatory factors that initiate macrophages recruitment and activation, it was determined if monocyte populations from individual AMD patients exhibited heterogeneity in terms of cytokine production in culture or in mRNA expression of freshly isolated cells, and if high levels served as a biomarker of risk for progression into neovascular AMD [51]. TNF-α expression, a potent cytotoxic and proangiogenic cytokine for wet AMD. Typically human macrophages in culture, after stimulation with RPE derived blebs produced an increase in TNF-α. However, extremely high variability in baseline TNF-α expression from isolated macrophages was observed among different subjects [51]. These results are consistent with the hypothesis that the pre-existing macrophage activation state, defined as level of cytokine or mediator expression of the circulating monocyte, might determine the negative or positive consequence of macrophage recruitment as a disease modifier. Macrophages with low expression might remove deposits safely whereas macrophages with high expression might produce mediators that contribute

As mentioned above, aberrant expression of chemokines occurs in a variety of diseases that have an inflammatory component. Highly specialized RPE cells play a pivotal role in the maintenance of the outer retina by secreting several cytokines including monocyte chemoat‐ tractant protein-1 (MCP-1) [288,289] which has been suggested to be implicated in the pathogenesis of AMD [290,291]. RPE cells can secrete MCP-1 in the direction of choroidal blood vessels during inflammatory responses therefore suggesting that RPE cells might promote macrophage recruitment to the choroid from circulating monocytes [292]. It was reported that MCP-1 is regulated in injured ARPE-19 cells and that free radicals might be immunostimula‐ tory [293] providing support for the notion that injured RPE cells may induce monocyte

We have investigated MCP-1 expression in RPE from patients with AMD as well as the regulation of RPE-derived MCP-1 expression following cigarette hydroquinone-mediated oxidative injury. Our data report for the first time that MCP-1 expression is markedly de‐ creased in RPE from smoker patients with AMD [294] thereby pointing to a critical role for MCP-1 in the pathogenesis of the disease. We acknowledge that due to the nature of our study, it cannot be determined whether the altered expression of MCP-1 in human RPE lysates is a cause or consequence of the disease. However, our current findings suggest that declining

distinct phases of the disease.

to disease progression.

migration.

inflammation in various diseases [286,287].

Based on the above hypothesis, we have evaluated the possibility that hydroquinone-induced oxidative stress might regulate MCP-1 expression in the RPE. We showed that prolonged exposure to hydroquinone-induced oxidative injury downregulated MCP-1 production by ARPE-19 cells and RPE/choroids from C57BL/6 mice [294]. An earlier study by Joly et al is in line with our observations showing a decline in MCP-1 gene expression in retinas of mice exposed to light-induced oxidative damage for several days [294]. Our observations suggest that sustained exposure to hydroquinone might impair RPE-derived MCP-1-mediated scavenging macrophages and dentritic cells recruitment and phagocytosis which might lead to incomplete clearance of proinflammatory debris trapped between the RPE and its BrM. On the other hand, our preliminary date show that RPE-derived blebs activate RPE MCP-1 production (unpublished data), suggesting that when significant BLD will have already formed, RPE-blebs and other debris will activate MCP-1 secretion by RPE leading to infiltration of proangiogenic macrophages to sub-RPE deposit areas, where they will scavenge, relate more cytokines and mediators, and amplify the process leading to progression of drusen to CNV in smoker patients with AMD.

Angiotensin II is not only a potent vasoconstrictor which elevates arterial blood pressure, but also a powerful pro-inflammatory cytokine, chemokine and growth factor [276, 283-285,295], which mediates the activation of inflammatory mechanisms involved in age-related diseases [296,297]. There is accumulating evidence that Ang II can cause target organ damage by facilitating inflammatory and growth responses through activation of NFκB [276, 283-285], the key nuclear transcription factor in inflammatory and fibrotic diseases. Activation of NFκB by Ang II may stimulate transcription of numerous inflammatory genes, including MCP-1, RANTES (Regulated on Active Normal T cell Expressed and Secreted) and interleukin (IL)-6, TNF-α and TGF-β [276,283-285]. The view that MCP-1 is one of the most important chemokines in Ang II-induced inflammatory responses is supported by numerous studies, although the mechanisms by which Ang II increases MCP-1 expression and production are still not well understood [279,283-285]. In a rabbit model of atherosclerosis, ACE1 inhibitor quinapril inhibited NFκB activity, expression and production of MCP-1 and neointimal macrophage infiltration at the injured sites [283]. In Ang II-induced hypertensive rats, vascular MCP-1 mRNA expression increased almost four-fold, which was significantly reduced by normali‐ zation of hypertension by the non-specific vasodilator hydralazine, but the effects of AT1 receptor blockade were not studied [301]. However, in a different study, Ang II enhanced expression of MCP-1 mRNA and protein production in rat vascular smooth muscle cells in a dose- and time-dependent fashion, and these effects were mediated by AT1 receptors involv‐ ing the Rho-kinase pathway [298]. In mice, Wu et al. [299] showed that the AT1 receptor antagonist valsartan, at a dose that did not influence systolic blood pressure, significantly reduced the expression of MCP-1 along with other inflammatory genes such as TNF-α, IL-6, IL-1β and monocyte/macrophage infiltration in injured vessels. Interestingly, the effects of valsartan on MCP-1 expression were attenuated in AT2 receptor-deficient mice, suggesting that both AT1 and AT2 receptors are involved.The advantage of using a lower dose of AT1 receptor blockers is that the treatment does not reduce systolic blood pressure to the normo‐ tensive level, but retains clinical efficacy in inhibiting MCP-1 expression and improving cardiac function and mortality. Accordingly, the beneficial effects of the AT1 receptor blockers may be explained by a mechanism other than high blood pressure. A direct effect of Ang II on MCP-1 expression and production may be implicated [295].

Future studies addressing whether or not Ang II directly or indirectly activates NFκB and the signalling pathways could improve our understanding of the potential role of Ang II in the

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**Figure 12.** Ang II upregulated MCP-1 protein expression (A) and secretion (B) through Ang II receptor 1 (AT1) activa‐ tion in RPE cells. Human ARPE-19 cells were incubated with Ang II alone (100 nM) for 24 hours or in combination with candesartan (CD, 100 nM), an AT1 receptor antagonist, or PD123319 (PD, 100 nM) an Ang II type 2 receptor antago‐ nist for 30 min before Ang II stimulation, then washed with PBS and incubated in assay medium (0.1% FBS) for 24 hours. Supernatants and cell homogenates were collected to assess MCP-1 mRNA expression by real-time PCR and protein secretion by ELISA. Results are expressed as mean±SEM. \*\*P<0.01, statistically significant difference compared

progression of dry AMD to CNV.

with the control

Finally, the signalling mechanisms by which Ang II increases MCP-1 expression and produc‐ tion and induces end-organ damage remain to be elucidated. As mentioned previously, there is mounting evidence that NFκB may be one of the most important nuclear transcription factors that mediates Ang II-stimulated MCP-1 expression and production [276,283-285]. However, the signalling pathways by which Ang II directly or indirectly activates NFκB, which is then translocated into the nucleus to mediate MCP-1 transcription and synthesis, remain largely unknown. A local RAS may be activated in most, if not all, diseases with consequently increased tissue or intracellular Ang II. Binding of extracellular Ang II to cell surface AT1 receptors may stimulate MCP-1 mRNA expression through activation of different intracellular signalling cascades, likely involving protein kinase C-activated intracellular calcium mobili‐ zation [300], tyrosine kinase and mitogen-activated protein kinase [301], phospholipase A2 [302] and redox-sensitive NADH/NAD(P)H oxidase [301]. Future studies further addressing these important issues could improve our understanding of the potential role of pro-inflam‐ matory cytokines and chemokines in mediating Ang II-induced target organ damage and assist in further development of novel drugs to prevent and treat these diseases.

Even if it has been suggested that increased MCP-1 expression may be a key mediator between Ang II and retinal damage in hypertensive patients, MCP-1 expression has not been investi‐ gated in RPE from hypertensive patients with AMD nor has been the regulation of RPE-derived MCP-1 expression following Ang II-mediated injury.

On the basis of our preliminary data and those of others, we have proposed a molecular inflammatory hypothesis for AMD that describes a central role for the redox-sensitive NF-κB in modulating the gene expression of MCP-1. Many studies have provided experimental evidence indicating that NF-κB can be activated by oxidative stress [303-305] and that the antioxidant may have beneficial effects on vascular inflammation that occurs in other age-related diseases [306,307]. Thus, we proposed that Ang II will active RPE MCP-1 production leading to recruitment of macrophages to sub-RPE deposit areas, where they will scavenge, release more cytokines and mediators, and amplify the process promoting complications, especially CNV formation. Our preliminary data indicate that expression of MCP-1, key mediators pertinent to inflammation is markedly increased in cultured human RPE cells in response to Ang II (Fig. 12). Moreover, our data indicate that the increase in MCP-1 mRNA and protein secretion by RPE was through AT1 receptors activation (Fig. 12), highlighting such proinflammatory role of Ang II and their mechanism. These observations may have strong implications for the drusen progression to CNV in hypertensive patients with dry AMD. Future studies addressing whether or not Ang II directly or indirectly activates NFκB and the signalling pathways could improve our understanding of the potential role of Ang II in the progression of dry AMD to CNV.

antagonist valsartan, at a dose that did not influence systolic blood pressure, significantly reduced the expression of MCP-1 along with other inflammatory genes such as TNF-α, IL-6, IL-1β and monocyte/macrophage infiltration in injured vessels. Interestingly, the effects of valsartan on MCP-1 expression were attenuated in AT2 receptor-deficient mice, suggesting that both AT1 and AT2 receptors are involved.The advantage of using a lower dose of AT1 receptor blockers is that the treatment does not reduce systolic blood pressure to the normo‐ tensive level, but retains clinical efficacy in inhibiting MCP-1 expression and improving cardiac function and mortality. Accordingly, the beneficial effects of the AT1 receptor blockers may be explained by a mechanism other than high blood pressure. A direct effect of Ang II on

64 Age-Related Macular Degeneration - Etiology, Diagnosis and Management - A Glance at the Future

Finally, the signalling mechanisms by which Ang II increases MCP-1 expression and produc‐ tion and induces end-organ damage remain to be elucidated. As mentioned previously, there is mounting evidence that NFκB may be one of the most important nuclear transcription factors that mediates Ang II-stimulated MCP-1 expression and production [276,283-285]. However, the signalling pathways by which Ang II directly or indirectly activates NFκB, which is then translocated into the nucleus to mediate MCP-1 transcription and synthesis, remain largely unknown. A local RAS may be activated in most, if not all, diseases with consequently increased tissue or intracellular Ang II. Binding of extracellular Ang II to cell surface AT1 receptors may stimulate MCP-1 mRNA expression through activation of different intracellular signalling cascades, likely involving protein kinase C-activated intracellular calcium mobili‐ zation [300], tyrosine kinase and mitogen-activated protein kinase [301], phospholipase A2 [302] and redox-sensitive NADH/NAD(P)H oxidase [301]. Future studies further addressing these important issues could improve our understanding of the potential role of pro-inflam‐ matory cytokines and chemokines in mediating Ang II-induced target organ damage and assist

Even if it has been suggested that increased MCP-1 expression may be a key mediator between Ang II and retinal damage in hypertensive patients, MCP-1 expression has not been investi‐ gated in RPE from hypertensive patients with AMD nor has been the regulation of RPE-derived

On the basis of our preliminary data and those of others, we have proposed a molecular inflammatory hypothesis for AMD that describes a central role for the redox-sensitive NF-κB in modulating the gene expression of MCP-1. Many studies have provided experimental evidence indicating that NF-κB can be activated by oxidative stress [303-305] and that the antioxidant may have beneficial effects on vascular inflammation that occurs in other age-related diseases [306,307]. Thus, we proposed that Ang II will active RPE MCP-1 production leading to recruitment of macrophages to sub-RPE deposit areas, where they will scavenge, release more cytokines and mediators, and amplify the process promoting complications, especially CNV formation. Our preliminary data indicate that expression of MCP-1, key mediators pertinent to inflammation is markedly increased in cultured human RPE cells in response to Ang II (Fig. 12). Moreover, our data indicate that the increase in MCP-1 mRNA and protein secretion by RPE was through AT1 receptors activation (Fig. 12), highlighting such proinflammatory role of Ang II and their mechanism. These observations may have strong implications for the drusen progression to CNV in hypertensive patients with dry AMD.

MCP-1 expression and production may be implicated [295].

in further development of novel drugs to prevent and treat these diseases.

MCP-1 expression following Ang II-mediated injury.

**Figure 12.** Ang II upregulated MCP-1 protein expression (A) and secretion (B) through Ang II receptor 1 (AT1) activa‐ tion in RPE cells. Human ARPE-19 cells were incubated with Ang II alone (100 nM) for 24 hours or in combination with candesartan (CD, 100 nM), an AT1 receptor antagonist, or PD123319 (PD, 100 nM) an Ang II type 2 receptor antago‐ nist for 30 min before Ang II stimulation, then washed with PBS and incubated in assay medium (0.1% FBS) for 24 hours. Supernatants and cell homogenates were collected to assess MCP-1 mRNA expression by real-time PCR and protein secretion by ELISA. Results are expressed as mean±SEM. \*\*P<0.01, statistically significant difference compared with the control

Because hypertension has been unequivocally linked to the pathogenesis of AMD, it can be speculated that increase in RPE-derived MCP-1 production resulting from exposure to Ang II may be an important factor that could accelerate and promote the progression of early AMD to CNV. This theory is put forth bearing in mind the complexity of underlying cellular and molecular mechanisms involved in the inflammatory response.

smoke-derived hydroquinone and nicotine might also regulate VEGF and PEDF expression in

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We report that VEGF expression is increased and PEDF expression is decreased in RPE from smoker patients with AMD resulting in an increased VEGF-to-PEDF ratio [282]. A disruption in the critical balance of these opposing stimuli may be permissive for the development of wet AMD. Our findings are consistent with clinical observations describing dysregulated expres‐ sion of VEGF and PEDF [315-317,321,322,324] in eyes with AMD. Oxidant-mediated RPE damage might promote abnormal angiogenesis [309]. In vitro, although oxidative injury declined the production of PEDF without significantly changing VEGF expression in ARPE-19 cells regardless of dose and duration of exposure to hydroquinone, we observed an increased VEGF-to-PEDF ratio which may favor angiogenesis [282]. These results suggest that cigarette smoke-related HQ-induced oxidative stress might impair the delicate balance between VEGF and PEDF that controls angiogenic homeostasis in the retina. Other previous reports showed that cigarette smoke extract induces VEGF expression in ARPE-19 cells [104] and that H2O2 induced oxidative stress increased the production of VEGF in human RPE cells [309]. The discrepancy between our in vitro findings and those earlier observations with regards to VEGF might reflect differences in cellular responses in the setting of different types of oxidant-

The endogenous angiogenic inhibitors are believed to be essential for maintaining the homeostasis of angiogenesis in the retina. Given the evidence that PEDF is an important negative regulator of angiogenesis, lower levels of PEDF is strongly suggestive of a decreased anti-angiogenic activity that may lead to the initiation of angiogenesis in response to hydro‐ quinone-induced oxidative stress. However, we do not rule out the possibility that a decreased level of inhibitory factor PEDF by itself may not be sufficient for inducing the angiogenic switch leading to CNV. Reciprocal increase in stimulatory VEGF might also be needed. In fact, a longer more sustained exposure to hydroquinone might be necessary to induce VEGF expression in ARPE-19 cells. Furthermore, angiogenesis is a highly complex and tightly orchestrated multistep process involving extensive interplay between multiple angiogenic factors. It is therefore possible that several other molecules besides VEGF and PEDF regulated by hydro‐ quinone might permit the development of abnormal angiogenesis. In vivo, we observed elevated expression of VEGF and PEDF protein in RPE/choroids from HQ-treated mice which translated into an enhanced VEGF-to-PEDF ratio. As stated earlier, important species-specific differences may also account for the discrepancy between human cells and mice results. In addition, one has to keep in mind that inherent in vitro and in vivo differences might explain this disparity. PEDF has multiple dose-dependent biological functions. Interestingly, it has been reported that low doses of PEDF are inhibitory but high doses can increase the develop‐ ment of CNV induced by laser in mice [331]. In addition, a study showed that RPE-derived VEGF upregulates PEDF expression via VEGF receptor-1 in an autocrine manner [332], therefore highlighting regulatory interactions between these two counterbalancing systems of angiogenic stimulators and inhibitors. In any case, our in vivo findings confirm that hydro‐ quinone-induced oxidative damage is unequivocally associated with an imbalance between

RPE cells.

mediated injury.

VEGF and PEDF in the RPE.

#### **8.2. Angiogenesis: Role of cigarette smoke and angiotensin II**

Angiogenesis is a highly complex biological process that involves a delicate balance between numerous stimulators and inhibitors, each regulated by multiple control systems. CNV-related angiogenesis requires an alteration in the concentration of molecules that stimulate or inhibit growth of new blood vessels [308,309]. Vascular endothelial growth factor (VEGF) constitu‐ tively produced and secreted by RPE in culture [310-318], is a major angiogenic cytokine central to the development of wet AMD [312-315].VEGF regulates endothelial cells proliferation, migration and survival [315]. Interestingly, secretion of VEGF by RPE cells is polarized towards BrM [250]. There is ample clinical evidence that VEGF expression is increased in surgically excised AMD-associated choroidal neovascular membranes [315-317]. Eyes with early forms of AMD have increased expression of VEGF in the RPE and the vitreous of eyes with CNV have increased concentration of VEGF [315]. Similar observations have been made in animal models of CNV [311,320]. Furthermore, Reich et al reported that subretinal injection of VEGF siRNA significantly inhibited the growth of laser-induced CNV in a mouse model [318]. PEDF, a potent angiogenic inhibitor [321] secreted by RPE cells [322-324], counterbalances the effects of VEGF and modulates the formation of CNV [322,323]. A decrease in PEDF expression has been reported in eyes with AMD, therefore disrupting the critical balance between VEGF and PEDF that may lead to pathological angiogenesis and be permissive for the development of CNV [322]. PEDF levels decline in the vitreous of patients with CNV [324].

Interestinly, TNFSF15, a cytokine that belongs to the TNF superfamily, which originally was reported to be expressed exclusively in endothelial cells, and more recently in other several cell types in inflammatory diseases [325-328], could be implicated in the development of CNV. TNFSF15 is a potent inhibitor of endothelial cell proliferation, angiogenesis, and tumor growth [329] which has been involved in atherogenesis and neovascularization. Our preliminary data has shown constitutive TNFSF15 production and secretion by RPE in vitro and in vivo (Marin-Castano, unpublished results). Moreover, there is recent evidence showing down-regulation of TNFSF15 by VEGF in endothelial cells, therefore disrupting the critical balance between TNFSF15 and VEGF and leading to development of neovascularization [330]. Based on this recent evidence, it is conceivable that the balance between TNFSF15 and VEGF could be of great importance in CNV development. However, until now, there is no report in the literature examining VEGF, PEDF, and TNFSF15 expression in RPE from AMD patients or evaluating whether or not cigarette smoke-related hydroquinone and nicotine and Ang II have the potential to dysregulate the VEGF/PEDF and VEGF/TNFSF15 balance in RPE cells.

Given the critical role of VEGF and PEDF in AMD and that oxidative damage to the RPE and angiogenesis appear to be central in the pathogenesis of the disease; we studied the expression of these chemokines in RPE from smoker patients diagnosed with AMD and whether cigarette smoke-derived hydroquinone and nicotine might also regulate VEGF and PEDF expression in RPE cells.

Because hypertension has been unequivocally linked to the pathogenesis of AMD, it can be speculated that increase in RPE-derived MCP-1 production resulting from exposure to Ang II may be an important factor that could accelerate and promote the progression of early AMD to CNV. This theory is put forth bearing in mind the complexity of underlying cellular and

Angiogenesis is a highly complex biological process that involves a delicate balance between numerous stimulators and inhibitors, each regulated by multiple control systems. CNV-related angiogenesis requires an alteration in the concentration of molecules that stimulate or inhibit growth of new blood vessels [308,309]. Vascular endothelial growth factor (VEGF) constitu‐ tively produced and secreted by RPE in culture [310-318], is a major angiogenic cytokine central to the development of wet AMD [312-315].VEGF regulates endothelial cells proliferation, migration and survival [315]. Interestingly, secretion of VEGF by RPE cells is polarized towards BrM [250]. There is ample clinical evidence that VEGF expression is increased in surgically excised AMD-associated choroidal neovascular membranes [315-317]. Eyes with early forms of AMD have increased expression of VEGF in the RPE and the vitreous of eyes with CNV have increased concentration of VEGF [315]. Similar observations have been made in animal models of CNV [311,320]. Furthermore, Reich et al reported that subretinal injection of VEGF siRNA significantly inhibited the growth of laser-induced CNV in a mouse model [318]. PEDF, a potent angiogenic inhibitor [321] secreted by RPE cells [322-324], counterbalances the effects of VEGF and modulates the formation of CNV [322,323]. A decrease in PEDF expression has been reported in eyes with AMD, therefore disrupting the critical balance between VEGF and PEDF that may lead to pathological angiogenesis and be permissive for the development of

molecular mechanisms involved in the inflammatory response.

66 Age-Related Macular Degeneration - Etiology, Diagnosis and Management - A Glance at the Future

**8.2. Angiogenesis: Role of cigarette smoke and angiotensin II**

CNV [322]. PEDF levels decline in the vitreous of patients with CNV [324].

potential to dysregulate the VEGF/PEDF and VEGF/TNFSF15 balance in RPE cells.

Given the critical role of VEGF and PEDF in AMD and that oxidative damage to the RPE and angiogenesis appear to be central in the pathogenesis of the disease; we studied the expression of these chemokines in RPE from smoker patients diagnosed with AMD and whether cigarette

Interestinly, TNFSF15, a cytokine that belongs to the TNF superfamily, which originally was reported to be expressed exclusively in endothelial cells, and more recently in other several cell types in inflammatory diseases [325-328], could be implicated in the development of CNV. TNFSF15 is a potent inhibitor of endothelial cell proliferation, angiogenesis, and tumor growth [329] which has been involved in atherogenesis and neovascularization. Our preliminary data has shown constitutive TNFSF15 production and secretion by RPE in vitro and in vivo (Marin-Castano, unpublished results). Moreover, there is recent evidence showing down-regulation of TNFSF15 by VEGF in endothelial cells, therefore disrupting the critical balance between TNFSF15 and VEGF and leading to development of neovascularization [330]. Based on this recent evidence, it is conceivable that the balance between TNFSF15 and VEGF could be of great importance in CNV development. However, until now, there is no report in the literature examining VEGF, PEDF, and TNFSF15 expression in RPE from AMD patients or evaluating whether or not cigarette smoke-related hydroquinone and nicotine and Ang II have the

We report that VEGF expression is increased and PEDF expression is decreased in RPE from smoker patients with AMD resulting in an increased VEGF-to-PEDF ratio [282]. A disruption in the critical balance of these opposing stimuli may be permissive for the development of wet AMD. Our findings are consistent with clinical observations describing dysregulated expres‐ sion of VEGF and PEDF [315-317,321,322,324] in eyes with AMD. Oxidant-mediated RPE damage might promote abnormal angiogenesis [309]. In vitro, although oxidative injury declined the production of PEDF without significantly changing VEGF expression in ARPE-19 cells regardless of dose and duration of exposure to hydroquinone, we observed an increased VEGF-to-PEDF ratio which may favor angiogenesis [282]. These results suggest that cigarette smoke-related HQ-induced oxidative stress might impair the delicate balance between VEGF and PEDF that controls angiogenic homeostasis in the retina. Other previous reports showed that cigarette smoke extract induces VEGF expression in ARPE-19 cells [104] and that H2O2 induced oxidative stress increased the production of VEGF in human RPE cells [309]. The discrepancy between our in vitro findings and those earlier observations with regards to VEGF might reflect differences in cellular responses in the setting of different types of oxidantmediated injury.

The endogenous angiogenic inhibitors are believed to be essential for maintaining the homeostasis of angiogenesis in the retina. Given the evidence that PEDF is an important negative regulator of angiogenesis, lower levels of PEDF is strongly suggestive of a decreased anti-angiogenic activity that may lead to the initiation of angiogenesis in response to hydro‐ quinone-induced oxidative stress. However, we do not rule out the possibility that a decreased level of inhibitory factor PEDF by itself may not be sufficient for inducing the angiogenic switch leading to CNV. Reciprocal increase in stimulatory VEGF might also be needed. In fact, a longer more sustained exposure to hydroquinone might be necessary to induce VEGF expression in ARPE-19 cells. Furthermore, angiogenesis is a highly complex and tightly orchestrated multistep process involving extensive interplay between multiple angiogenic factors. It is therefore possible that several other molecules besides VEGF and PEDF regulated by hydro‐ quinone might permit the development of abnormal angiogenesis. In vivo, we observed elevated expression of VEGF and PEDF protein in RPE/choroids from HQ-treated mice which translated into an enhanced VEGF-to-PEDF ratio. As stated earlier, important species-specific differences may also account for the discrepancy between human cells and mice results. In addition, one has to keep in mind that inherent in vitro and in vivo differences might explain this disparity. PEDF has multiple dose-dependent biological functions. Interestingly, it has been reported that low doses of PEDF are inhibitory but high doses can increase the develop‐ ment of CNV induced by laser in mice [331]. In addition, a study showed that RPE-derived VEGF upregulates PEDF expression via VEGF receptor-1 in an autocrine manner [332], therefore highlighting regulatory interactions between these two counterbalancing systems of angiogenic stimulators and inhibitors. In any case, our in vivo findings confirm that hydro‐ quinone-induced oxidative damage is unequivocally associated with an imbalance between VEGF and PEDF in the RPE.

We also studied whether Nicotine (NT), a potent angiogenic agent abundant in second hand smoke, 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 the RPE and determine the effects of NT on RPE-derived VEGF and PEDF expression in the context of passive smoking. We demonstrated that cultured RPE cells constitutively expressed nAchR α3, α10 and β1 subunits, β1 being most prevalent (Fig. 13). nAchR α4, α5, α7 and β2 subunits were detected in RPE sheets from rats, among which α4 is the predominant subtype (Fig. 14). NT which did not, induced β1 nAchR, upregulated VEGF and downregulated PEDF expression through nAChR in ARPE-19 cells (Fig. 15). Moreover, transcriptional activation of nAchR α4 subunit and nAChR-mediated upregulation of VEGF and PEDF were observed in RPE from rats exposed to NT [333]. Our findings confirm that NT is associated with an increased VEGF-to-PEDF ratio in RPE through nAchR in vitro and in vivo which may play a key role in the progression to wet AMD in passive smokers [333].Taken together, these data provide strong support for a key role played by hydroquinone and NT-injured RPE cells in the progression of dry AMD to CNV. We demonstrated that RPE dysfunction might lead to dysregulation of macrophage clearance function and angiogenic homeostasis as a result of oxidative damage which may trigger progression towards CNV in smoker patients with dry AMD.

As mentined previously, hypertension has been associated with the development of wet AMD in the presence of early AMD [128]. These studies make no mention of the mechanism(s) by which hypertension may induce or contribute to the progression from early AMD to CNV. Some investigators have shown that Ang II contributes to to pathological conditions such as neovascularization, atherosclerosis, and tumor [334-340]. Moreover, it has been shown that Ang II-induced angiogenesis is mediated by VEGF receptor-1 [341] and that Ang II type 2 receptor inhibits VEGF-induced migration and in vitro tube formation of human endothelial cells [342]. However, nothing is known about the regulation of TNFSF15 by Ang II in any of the tissues where it is expressed. Therefore, investigating the regulation of TNFSF15 by Ang II helped us to understand how regulation of this cytokine by Ang II could participate in the development of neovascularization in wet AMD patients with HTN. We hypothesize that hypertension through Ang II will alter the secretion of TNFSF15 release by the RPE, which may contribute to an imbalance between VEGF and TNFSF15 leading to CNV development. Our preliminary data showed that Ang II diminishes release of TNFSF15 by RPE cells through activation of both Ang II receptor subtypes and increases release of VEGF by RPE cells through activation of the AT2 Ang II receptor subtype (Marin-Castano, unpublished data), which might permit the development of abnormal angiogenesis contributing to CNV. Our study may result in the identification of TNFSF-15 as an important target to inhibit the initiation of CNV and in Ang II receptors blockade as therapeutic preventive strategy

#### **9. Animal model for dry AMD**

Animal models are extremely useful in preclinical testing of theories of disease pathogenesis and can serve to question current hypotheses and predict outcomes of therapeutic interven‐ tions. Our laboratory and others have used the C57BL/6 mouse model to evaluate mechanisms for age, gender, diet, environmental toxins, and to text hypothesis [46,55,60,343]. Until recently, no animal models for dry AMD were available. The only available model for dry AMD and

**Figure 13.** Human RPE cells constitutively express α3, α10 and β1 nAchR subunits. Real-time PCR demonstrated the presence of nAchR α3, α10 and β1 subunits transcripts and the prevalence of β1 subtype in confluent serum-starved (A, B, E, G, H) ARPE-19 and (C, D, E, G, H) human primary RPE cells. GAPDH was used as housekeeping gene. Real-time PCR for (E) β1, (G) α3 and (H) α10 nAchR was followed by ethidium bromide-stained agarose gel electrophoresis to visualize the products in ARPE-19 and primary RPE cells. Shown is a representative gel. Number on the left represents size of transcript in base pairs (bp). (F) Western blot analysis demonstrated the expression of the most prevalent nAchR β1 subunits in confluent serum-starved ARPE-19 and primary RPE cells. Shown is a representative gel. Number

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hypertension was reported by Jonas et al in 2003 [124].

on the left represents protein molecular weight in kilodaltons (kDa).

We also studied whether Nicotine (NT), a potent angiogenic agent abundant in second hand smoke, 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 the RPE and determine the effects of NT on RPE-derived VEGF and PEDF expression in the context of passive smoking. We demonstrated that cultured RPE cells constitutively expressed nAchR α3, α10 and β1 subunits, β1 being most prevalent (Fig. 13). nAchR α4, α5, α7 and β2 subunits were detected in RPE sheets from rats, among which α4 is the predominant subtype (Fig. 14). NT which did not, induced β1 nAchR, upregulated VEGF and downregulated PEDF expression through nAChR in ARPE-19 cells (Fig. 15). Moreover, transcriptional activation of nAchR α4 subunit and nAChR-mediated upregulation of VEGF and PEDF were observed in RPE from rats exposed to NT [333]. Our findings confirm that NT is associated with an increased VEGF-to-PEDF ratio in RPE through nAchR in vitro and in vivo which may play a key role in the progression to wet AMD in passive smokers [333].Taken together, these data provide strong support for a key role played by hydroquinone and NT-injured RPE cells in the progression of dry AMD to CNV. We demonstrated that RPE dysfunction might lead to dysregulation of macrophage clearance function and angiogenic homeostasis as a result of oxidative damage

68 Age-Related Macular Degeneration - Etiology, Diagnosis and Management - A Glance at the Future

which may trigger progression towards CNV in smoker patients with dry AMD.

Ang II receptors blockade as therapeutic preventive strategy

**9. Animal model for dry AMD**

As mentined previously, hypertension has been associated with the development of wet AMD in the presence of early AMD [128]. These studies make no mention of the mechanism(s) by which hypertension may induce or contribute to the progression from early AMD to CNV. Some investigators have shown that Ang II contributes to to pathological conditions such as neovascularization, atherosclerosis, and tumor [334-340]. Moreover, it has been shown that Ang II-induced angiogenesis is mediated by VEGF receptor-1 [341] and that Ang II type 2 receptor inhibits VEGF-induced migration and in vitro tube formation of human endothelial cells [342]. However, nothing is known about the regulation of TNFSF15 by Ang II in any of the tissues where it is expressed. Therefore, investigating the regulation of TNFSF15 by Ang II helped us to understand how regulation of this cytokine by Ang II could participate in the development of neovascularization in wet AMD patients with HTN. We hypothesize that hypertension through Ang II will alter the secretion of TNFSF15 release by the RPE, which may contribute to an imbalance between VEGF and TNFSF15 leading to CNV development. Our preliminary data showed that Ang II diminishes release of TNFSF15 by RPE cells through activation of both Ang II receptor subtypes and increases release of VEGF by RPE cells through activation of the AT2 Ang II receptor subtype (Marin-Castano, unpublished data), which might permit the development of abnormal angiogenesis contributing to CNV. Our study may result in the identification of TNFSF-15 as an important target to inhibit the initiation of CNV and in

Animal models are extremely useful in preclinical testing of theories of disease pathogenesis and can serve to question current hypotheses and predict outcomes of therapeutic interven‐ tions. Our laboratory and others have used the C57BL/6 mouse model to evaluate mechanisms

**Figure 13.** Human RPE cells constitutively express α3, α10 and β1 nAchR subunits. Real-time PCR demonstrated the presence of nAchR α3, α10 and β1 subunits transcripts and the prevalence of β1 subtype in confluent serum-starved (A, B, E, G, H) ARPE-19 and (C, D, E, G, H) human primary RPE cells. GAPDH was used as housekeeping gene. Real-time PCR for (E) β1, (G) α3 and (H) α10 nAchR was followed by ethidium bromide-stained agarose gel electrophoresis to visualize the products in ARPE-19 and primary RPE cells. Shown is a representative gel. Number on the left represents size of transcript in base pairs (bp). (F) Western blot analysis demonstrated the expression of the most prevalent nAchR β1 subunits in confluent serum-starved ARPE-19 and primary RPE cells. Shown is a representative gel. Number on the left represents protein molecular weight in kilodaltons (kDa).

for age, gender, diet, environmental toxins, and to text hypothesis [46,55,60,343]. Until recently, no animal models for dry AMD were available. The only available model for dry AMD and hypertension was reported by Jonas et al in 2003 [124].

**Figure 14.** RPE from rats constitutively express α4, α5, α7 and β2 nAchR subunits. Real-time PCR demonstrated the presence of nAchR α4, α5, α7 and β2 subunits transcripts (A-D) and the prevalence of α4 subtype (B-D) in RPE from Sprague-Dawley rats (pooled RNA from 5 rats/lane). Real-time PCR for α4, α5, α7 and β2 nAchR isoforms was followed by ethidium bromide-stained agarose gel electrophoresis to visualize the products (A). Shown are a representative gels. Number on the left represents size of transcript in base pairs (bp). \*\*\* is p<0.0001.

Based on the idea that hydroquinone and arterial hypertension might influence the develop‐ ment and severity of drusen, we extended our in vitro data to a more physiological environ‐ ment using; a) the 16-month-old or b) the 9-month-old C57BL/6 mouse model for dry AMD published by our laboratory [46,55,60,343], but providing an alternative source of oxidant stimulus by replacing exposure to blue light with exposure to hydroquinone in food [55] or drinking water [56] for 4.5 months or with Ang II alone or in combination with the AT1 receptor antagonist (candesartan) or the AT2 receptor antagonist (PD123319) for 4 weeks or 3.5 months. In addition, all mice received a regular fat diet instead of high-fat diet. We evaluated the impact of these compounds on the development of sub-RPE deposits, by using TEM.

group demonstrated moderate BLD. Animals exposed to hydroquinone showed pathologic changes in the RPE and BrM characterized by moderate BLD [56]. Approximately a 83% of eyes exhibited moderate BLD. The BrM was thickened, with coated vesicles, membranous profiles, and banded structures, Figs. 16B, 16C ), typical of those described in some human AMD specimens [46]. Findings were of a magnitude similar to those previously observed in mice exposed to high-fat diet plus blue green light [62]. Interestingly, animals showed blebs

duplicate.\* is p<0.05 and \*\*\* is p<0.0001 versus control; # is p<0.01 and & is p<0.05 versus NT alone.

**Figure 15.** NT increased VEGF expression and decreased PEDF expression through nAchR in ARPE-19 cells. NT (A) in‐ creased VEGF and (C) decreased PEDF mRNA expression in ARPE-19 cells. Confluent serum-starved ARPE-19 cells were treated with NT 10-8M for 72 hours. Total RNA was extracted to assess VEGF and PEDF mRNA expression by real-time PCR. GAPDH was used as housekeeping gene. NT (B) increased VEGF and (D) decreased PEDF protein expression. Con‐ centration of VEGF and PEDF secreted in supernatants of confluent serum-starved ARPE-19 cells treated with NT 10-8M for 72 hours was assessed by ELISA. (E) NT increased VEGF/PEDF protein ratio. NT-induced (F) upregulation of VEGF and (G) downregulation of PEDF mRNA expression was abolished by hexamethonium (HXM). Confluent serumstarved ARPE-19 cells were pre-incubated with HXM 10-5M for 1 hour then exposed to NT 10-8M for 72 hours. Total RNA was extracted to assess VEGF and PEDF mRNA expression by real-time PCR. GAPDH was used as housekeeping gene. Data are expressed as means ± SE and represent the average results of 3 to 4 independent experiments run in

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As published previuosly, hydroquinone-treated mice had increased blood levels of hydroqui‐ none relative to control mice that showed non detectable levels [56]. Mice not exposed to hydroquinone showed normal morphology of the RPE, BrM, and choriocapillaris endothelium (Fig. 16A). Some specimens demonstrated mild frequency of any BLD. None of the eyes in this

Cigarette Smoking and Hypertension Two Risk Factors for Age-Related Macular Degeneration http://dx.doi.org/10.5772/53958 71

**Figure 15.** NT increased VEGF expression and decreased PEDF expression through nAchR in ARPE-19 cells. NT (A) in‐ creased VEGF and (C) decreased PEDF mRNA expression in ARPE-19 cells. Confluent serum-starved ARPE-19 cells were treated with NT 10-8M for 72 hours. Total RNA was extracted to assess VEGF and PEDF mRNA expression by real-time PCR. GAPDH was used as housekeeping gene. NT (B) increased VEGF and (D) decreased PEDF protein expression. Con‐ centration of VEGF and PEDF secreted in supernatants of confluent serum-starved ARPE-19 cells treated with NT 10-8M for 72 hours was assessed by ELISA. (E) NT increased VEGF/PEDF protein ratio. NT-induced (F) upregulation of VEGF and (G) downregulation of PEDF mRNA expression was abolished by hexamethonium (HXM). Confluent serumstarved ARPE-19 cells were pre-incubated with HXM 10-5M for 1 hour then exposed to NT 10-8M for 72 hours. Total RNA was extracted to assess VEGF and PEDF mRNA expression by real-time PCR. GAPDH was used as housekeeping gene. Data are expressed as means ± SE and represent the average results of 3 to 4 independent experiments run in duplicate.\* is p<0.05 and \*\*\* is p<0.0001 versus control; # is p<0.01 and & is p<0.05 versus NT alone.

Based on the idea that hydroquinone and arterial hypertension might influence the develop‐ ment and severity of drusen, we extended our in vitro data to a more physiological environ‐ ment using; a) the 16-month-old or b) the 9-month-old C57BL/6 mouse model for dry AMD published by our laboratory [46,55,60,343], but providing an alternative source of oxidant stimulus by replacing exposure to blue light with exposure to hydroquinone in food [55] or drinking water [56] for 4.5 months or with Ang II alone or in combination with the AT1 receptor antagonist (candesartan) or the AT2 receptor antagonist (PD123319) for 4 weeks or 3.5 months. In addition, all mice received a regular fat diet instead of high-fat diet. We evaluated the impact

**Figure 14.** RPE from rats constitutively express α4, α5, α7 and β2 nAchR subunits. Real-time PCR demonstrated the presence of nAchR α4, α5, α7 and β2 subunits transcripts (A-D) and the prevalence of α4 subtype (B-D) in RPE from Sprague-Dawley rats (pooled RNA from 5 rats/lane). Real-time PCR for α4, α5, α7 and β2 nAchR isoforms was followed by ethidium bromide-stained agarose gel electrophoresis to visualize the products (A). Shown are a representative

As published previuosly, hydroquinone-treated mice had increased blood levels of hydroqui‐ none relative to control mice that showed non detectable levels [56]. Mice not exposed to hydroquinone showed normal morphology of the RPE, BrM, and choriocapillaris endothelium (Fig. 16A). Some specimens demonstrated mild frequency of any BLD. None of the eyes in this

of these compounds on the development of sub-RPE deposits, by using TEM.

gels. Number on the left represents size of transcript in base pairs (bp). \*\*\* is p<0.0001.

70 Age-Related Macular Degeneration - Etiology, Diagnosis and Management - A Glance at the Future

group demonstrated moderate BLD. Animals exposed to hydroquinone showed pathologic changes in the RPE and BrM characterized by moderate BLD [56]. Approximately a 83% of eyes exhibited moderate BLD. The BrM was thickened, with coated vesicles, membranous profiles, and banded structures, Figs. 16B, 16C ), typical of those described in some human AMD specimens [46]. Findings were of a magnitude similar to those previously observed in mice exposed to high-fat diet plus blue green light [62]. Interestingly, animals showed blebs (Fig. 16C). In preliminary experiments, we have also demonstrated that mice receiving subconjunctival injections of hydroquinone exhibeted a rudimentary form of BLD, often demonstrating small vesicular vesicles bleblike structures (Reinoso, et al. IOVS 2005;46:ARVO E-Abstract 3010). In addition, RPE from mice exposed to hydroquinone in drinking water showed increased levels of phosphorylated Hsp25, p38 and ERK [205], suggesting that phosphorylated Hsp25 might be a key mediator in early cellular events associated with actin reorganization and bleb formation involved in sub-RPE deposits formation.

Animals exposed to Ang II for 3.5 months revealed moderate BLD deposits. Sub-RPE changes were characterized by accumulation of moderately dense homogeneous material between the RPE and its basement membrane (Marin-Castano, unpublished data). Given that the Ang II receptors in rodents are similar to human Ang II receptors, our study help to elucidate the mechanism(s) by which Ang II receptor blockers may prevent these ECM changes important for early AMD development and provide a potential future clinical tool for the prevention of AMD. Moreover, our observations indicate that Ang II may induce the development of BLD. Thus, the results suggest the role for Ang II in ECM turnover and sub-RPE formation and propose Ang II-induced hypertension as an injury stimulus to the RPE, which may serve to

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Taken together, these observations indicate that different oxidant stimuli (i.e., blue light, hydroquinone, and Ang II) may induce a common response in the RPE and that a high-fat diet is not an absolute requirement for the development of BLD. Thus, the results suggest the role for blebs in sub-RPE formation and propose hydroquinone and Ang II another oxidative injury stimulus to the RPE, which may serve to explain the mechanisms that underlie pathologic BLD

In summary, we postulate that cigarette smoke-related and Ang II play a role in the develop‐ ment of dry AMD and its progression to wet AMD. Although our hypothesis remains to be proven, we have proposed new ideas and suggested different mechanisms highlighting Hsp27, MMP-2, basigin, MMP-14, MCP-1, MAPK, and TNFSF15 as potential disease-related proteins as well as biochemical pathways for potential therapeutic strategies, which might result in prevention of more severe and irreversible late stages of this dreadful disease. Our goal is to intervene promptly in the early stages of the disease so that progression to the more severe late forms of AMD can be prevented. In this respect, RPE-derived MMPs and blebs formation are potential target due to their pivotal role in stimulating drusen formation and progression into CNV. Levels of phosphorylated Hsp27, glycosilated basigin and MMP-14, as well as, MCP-1 and TNFSF15 could be markers, which may contribute and aid to the ophthal‐ mologic community in the management of the drusen. Moreover, AT1 receptor antagonists and p38/ERK1/2 MAPK blockers could be used successfully on the prevention of sub-RPE

Department of Ophthalmology, Bascom Palmer Eye institute, University of Miami, Miami,

deposits formation in selected high-risk AMD patients.

explain the mechanisms that underlie pathologic BLD deposits in early AMD.

deposits in early AMD.

**10. Conclusions**

**Author details**

FL, USA

Maria E. Marin-Castaño

**Figure 16.** Transmission electron microscopy of the outer retina and choroid from a 16-month-old female mouse fed with a regular fat diet for 4 months. (A) Outer retina and choroid of a mouse fed a regular diet without oxidant showed a normal RPE, BrM, and choriocapillaris. (B) Outer retina and choroid of a mouse fed a regular diet and ex‐ posed to HQ (0.8% for 4 months) in drinking water revealed sub-RPE deposits characterized by accumulation of mod‐ erately severe BLD with dense granular material between the RPE and its basement membrane (\*), compatible with a high mean severity score. The specimen, also, shows the abnormal choriocapillaris endothelium with increased thick‐ ening and loss of fenestration. (C) TEM of the outer retina and choroids from another 16-month-old female mouse fed a regular diet and exposed to HQ. The specimen shows moderately thick BLD with bandes structures (\*) and occasion‐ al blebs (black arrows). CC, choriocapillaris, Magnification: (A, C) x25,000; (B) x7,200.

We used Ang II to determine whether hypertension-associated Ang II was important for ECM regulation in RPE and the development of sub-RPE deposits. We reported that Ang II-treated mice had increased blood pressure as well as plasma and ocular levels of Ang II relative to control mice [60]. Ang II also regulated AT1a and AT1b receptor mRNA expression, and the intracellular concentration of calcium [Ca2+]I, showing that Ang II AT1 receptor is functional. In addition, MMP-2 activity, and type IV collagen accumulation were regulated by Ang II. 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. In addition, Ang II stimulates MMP-14, basigin, and phosphorylation of ERK, p38, and JNK in RPE sheets from mice. These effects were mediated by Ang II type 1 receptors [344].

Animals exposed to Ang II for 3.5 months revealed moderate BLD deposits. Sub-RPE changes were characterized by accumulation of moderately dense homogeneous material between the RPE and its basement membrane (Marin-Castano, unpublished data). Given that the Ang II receptors in rodents are similar to human Ang II receptors, our study help to elucidate the mechanism(s) by which Ang II receptor blockers may prevent these ECM changes important for early AMD development and provide a potential future clinical tool for the prevention of AMD. Moreover, our observations indicate that Ang II may induce the development of BLD. Thus, the results suggest the role for Ang II in ECM turnover and sub-RPE formation and propose Ang II-induced hypertension as an injury stimulus to the RPE, which may serve to explain the mechanisms that underlie pathologic BLD deposits in early AMD.

Taken together, these observations indicate that different oxidant stimuli (i.e., blue light, hydroquinone, and Ang II) may induce a common response in the RPE and that a high-fat diet is not an absolute requirement for the development of BLD. Thus, the results suggest the role for blebs in sub-RPE formation and propose hydroquinone and Ang II another oxidative injury stimulus to the RPE, which may serve to explain the mechanisms that underlie pathologic BLD deposits in early AMD.
