**7. Oxidative injury to the RPE**

**6. Hypertension and age-related macular degeneration**

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

signal transduction pathways.

early AMD to CNV.

in AMD, and its properties will be described below.

Even though age is the major determinant for developing AMD, clinical and epidemiolog‐ ic studies have suggested systemic vascular disease, especially hypertension, as an impor‐ tant risk factor for AMD [3,119] and a correlation between AMD and aging and hypertension [3, 120]. Aging is the risk factor with the greatest correlation with incidence, prevalence and severity of AMD, but the age-related susceptibility factors remain completely unknown [3,120]. However, increased sensitivity to the injurious effects of hypertension and to oxidantinjury are all more severe in the elderly than in the young [121-123]. For exemple, the interaction of Angiotensin II (Ang II) (the most important hormone associated with hyperten‐ sion) with AT1 receptors, produces ROS, and with advancing age, oxidative alterations accumulate in cellular components, including those in the antioxidative defense systems, due to disrupted redox regulation during aging can influence the gene transcription and

Hypertension is of particular interest among the systemic risk factors, due to its increasing incidence in Western societies. In several cross sectional and case control studies, systemic hypertension was associated with increased prevalence and progression of the severity and incidence of drusen [124] and with the development of wet AMD [125-127]. Interestingly, a recent study provides a strong association between hypertension and the development of wet AMD in the presence of early AMD [128]. Despite of the apparent link between AMD and hypertension, these studies make no mention of the mechanism(s) by which hyperten‐ sion may induce or contribute to the pathogenesis of dry AMD and its progression from

Traditionally, hypertension is believed to contribute to chronic diseases by at least two distinct mechanisms: hemodynamic injury and humoral factors [129-131]. Hemodynamic injury refers to mechanical damage induced by flow turbulence in large vessels or stretch‐ ing of capillaries induced by increased blood pressure. Humoral factors refer to cellular activities induced by hormones or growth factors associated with hypertension [129,130] shuch as Ang II, which is upregulated in hypertension, present in the blood of many hypertensive patients and has been demonstrated to activate specific receptors to induce various cellular functions. Ang II as the molecular surrogate for the effects of hypertension

The classical view of the renin-angiotensin system (RAS) as a systemic regulator of blood pressure has been extended, and a substantial number of studies have highlighted the importance of local RAS in a variety of extra-renal tissues, including adrenal glands [132], thymus [133] and recently in the eye [134,135]. In the eye, Ang II, and Ang II type 1 and type 2 receptors (AT1R, AT2R) have been found in the retina, particularly in the retinal pigment epithelium (RPE) [59,60,136-139]. Similarly, studies in rat retinal tissues also suggested local synthesis of both renin and angiotensin convertingenzyme (ACE) [140]. Along this line, Milenkovic et al. (2010) demonstrated that the RPE expresses renin and secretes it towards the retinal side. The presence of the most important RAS components in the retina implies a physiological function of RAS within the eye. However, despite the considerable evidence for As mentioned previously, a substantial body of literature suggests a role for oxidant injury to the RPE as a putative mechanism in the pathogenesis of AMD and addresses the protective actions of anti-oxidant. Although intuitively obvious, oxidant injury can induce either lethal responses, leading to cell death, or nonlethal responses inducing a functional change from baseline compatible with continued life of the cell but leading to dysfunction of the tissue or organ. Most studies focus on oxidant-mediated death of RPE [150-153]. Yet, RPE death (socalled geographic atrophy) is a very late stage of dry AMD, resulting from a very chronic and progressive process. Subretinal deposits and thickening of BrM, the hallmarks of early AMD, develop decades before the RPE cells actually die. Therefore, nonlethal cellular responses to RPE oxidant injury must contribute to early AMD.

Oxidative modifications in key cellular molecules such as DNA, carbohydrates, cellular proteins and cell membranes can often produce a cytotoxic chain reaction that contributes to the pathogenesis of many diseases [154-156]. However, we believe that oxidative damage to cell membranes and cellular proteins is more important in AMD.

RPE cell membranes are highly susceptible to lipid peroxidation. Phospholipids in RPE and photoreceptor membranes are especially rich in polyunsaturated fatty acid (PUFAs), including the ω-3 fatty acid docosahexanoic acid and the ω-6 fatty acid arachadonic acid. The presence of both types of PUFAs in phagocytosed photoreceptor outer segments renders the RPE cell membrane especially susceptible to lipid peroxidation and blebbing [157,158]. Cellular proteins are also important targets of oxidant-induced modification. Typical chemical modi‐ fications include breakdown of disulfide bonds, tyrosylation, acetylation and many other biochemical changes that can alter function of the molecule [159]. Accordingly, several welldescribed pathways exist to remove these damage biomolecules [160]. Although any cellular protein is potentially susceptible, the actin cytoskeleton is especially vulnerable to oxidantinduced damage.

Although oxidants derived from blue-light exposure, inflammation, or endogenous metabo‐ lism are more frequently implicated in RPE injury, we have also hypothesized that environ‐ mental toxicants and various hormones and other plasma-derived molecules related to systemic health cofactors serve as oxidants to contribute to deposit formation. We embrace the pathogenic paradigm based on the response-to-injury hypothesis which proposes that sub-RPE deposits originate from RPE-derived cell membrane blebs and dysregulation of ECM turnover induced by chronic nonlethal injury to the RPE in response to oxidative damage and propose that cigarette smoking-related hydroquinone and Ang II-induced hypertension contributes to the pathogeneis of AMD by causing oxidative damage to the RPE.

**Figure 5.** Plasma membrane microvesicles (blebs).

White arrowheads show the presence of membrane blebs.

**Figure 6.** (a) Induction of membrane blebs in ARPE-19 cells by hydroquinone-induced cellular stress. ARPE-19 cells ex‐ pressing GFP at the plasma membrane (A) were exposed to HQ (100 μM) for 6 h (B). Cells were observed immediately under epifluorescence microscope (magnification, ×40). The figure shows GFP localized to the membrane and the presence of membrane blebs (white arrowheads) after HQ treatment. A detailed view of the blebs that accumulated in the conditioned medium after HQ treatment is shown (C). (b) Induction of cellular changes in ARPE-19 cells by Ang II. Fluorescent GFP-ARPE-19 derived blebs before and after exposure to Ang II (100 nM) alone or in combination with a specific Ang II receptor antagonists AT1 (CD, 100 nM) or AT2 (PD 123319, 100 nM) for 24 hours. Cells were treated and observed immediately under epifluorescent microscope. White arrows show GFP localized to the membrane.

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#### **7.1. Nonlethal cell derived microparticles**

Cell derived microparticles are considered to be microvesicles released through the process of exocytic budding of the plasma membrane following stimulation of different cell types [161, 162]. There are two well-known cellular processes that can lead to the formation of micropar‐ ticles: chemicals and physical cell activation, and apoptosis [163,164]. Mild injuries inflected to the retina elicit a cellular response in the RPE consisting in pinching off small areas of the plasma membrane, which renders small microvesicles called blebs [165] (Fig. 5). The reason(s) behind membrane blebbing remains unknown, although it has been postulated to be an attempt to discard damaged cellular constituents by the RPE cell [14]. Nonlethal cell membrane blebbing was first introduced 30 years ago as a possible pathogenic mechanism in drusen formation [38, 166,167]. Blebbing is an early morphologic sign of cell injury which occurs immediately after exposure to a wide variety of toxic agents. However this process is different from lethal blebbing and apoptosis [168-170]. As mentioned previously, under prolonged injury, blebs may accumulate between the RPE and the basal lamina underneath this cell monolayer. Based on this concept, a plausible role for blebs in the pathogenesis of dry AMD has been suggested as a likely contributor to build-up of the sub-RPE deposits, which are characteristic of the early stages of this disorder [14].

Cigarette Smoking and Hypertension Two Risk Factors for Age-Related Macular Degeneration http://dx.doi.org/10.5772/53958 51

**Figure 5.** Plasma membrane microvesicles (blebs).

the pathogenesis of many diseases [154-156]. However, we believe that oxidative damage to

RPE cell membranes are highly susceptible to lipid peroxidation. Phospholipids in RPE and photoreceptor membranes are especially rich in polyunsaturated fatty acid (PUFAs), including the ω-3 fatty acid docosahexanoic acid and the ω-6 fatty acid arachadonic acid. The presence of both types of PUFAs in phagocytosed photoreceptor outer segments renders the RPE cell membrane especially susceptible to lipid peroxidation and blebbing [157,158]. Cellular proteins are also important targets of oxidant-induced modification. Typical chemical modi‐ fications include breakdown of disulfide bonds, tyrosylation, acetylation and many other biochemical changes that can alter function of the molecule [159]. Accordingly, several welldescribed pathways exist to remove these damage biomolecules [160]. Although any cellular protein is potentially susceptible, the actin cytoskeleton is especially vulnerable to oxidant-

Although oxidants derived from blue-light exposure, inflammation, or endogenous metabo‐ lism are more frequently implicated in RPE injury, we have also hypothesized that environ‐ mental toxicants and various hormones and other plasma-derived molecules related to systemic health cofactors serve as oxidants to contribute to deposit formation. We embrace the pathogenic paradigm based on the response-to-injury hypothesis which proposes that sub-RPE deposits originate from RPE-derived cell membrane blebs and dysregulation of ECM turnover induced by chronic nonlethal injury to the RPE in response to oxidative damage and propose that cigarette smoking-related hydroquinone and Ang II-induced hypertension

Cell derived microparticles are considered to be microvesicles released through the process of exocytic budding of the plasma membrane following stimulation of different cell types [161, 162]. There are two well-known cellular processes that can lead to the formation of micropar‐ ticles: chemicals and physical cell activation, and apoptosis [163,164]. Mild injuries inflected to the retina elicit a cellular response in the RPE consisting in pinching off small areas of the plasma membrane, which renders small microvesicles called blebs [165] (Fig. 5). The reason(s) behind membrane blebbing remains unknown, although it has been postulated to be an attempt to discard damaged cellular constituents by the RPE cell [14]. Nonlethal cell membrane blebbing was first introduced 30 years ago as a possible pathogenic mechanism in drusen formation [38, 166,167]. Blebbing is an early morphologic sign of cell injury which occurs immediately after exposure to a wide variety of toxic agents. However this process is different from lethal blebbing and apoptosis [168-170]. As mentioned previously, under prolonged injury, blebs may accumulate between the RPE and the basal lamina underneath this cell monolayer. Based on this concept, a plausible role for blebs in the pathogenesis of dry AMD has been suggested as a likely contributor to build-up of the sub-RPE deposits, which are

contributes to the pathogeneis of AMD by causing oxidative damage to the RPE.

cell membranes and cellular proteins is more important in AMD.

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

induced damage.

**7.1. Nonlethal cell derived microparticles**

characteristic of the early stages of this disorder [14].

**Figure 6.** (a) Induction of membrane blebs in ARPE-19 cells by hydroquinone-induced cellular stress. ARPE-19 cells ex‐ pressing GFP at the plasma membrane (A) were exposed to HQ (100 μM) for 6 h (B). Cells were observed immediately under epifluorescence microscope (magnification, ×40). The figure shows GFP localized to the membrane and the presence of membrane blebs (white arrowheads) after HQ treatment. A detailed view of the blebs that accumulated in the conditioned medium after HQ treatment is shown (C). (b) Induction of cellular changes in ARPE-19 cells by Ang II. Fluorescent GFP-ARPE-19 derived blebs before and after exposure to Ang II (100 nM) alone or in combination with a specific Ang II receptor antagonists AT1 (CD, 100 nM) or AT2 (PD 123319, 100 nM) for 24 hours. Cells were treated and observed immediately under epifluorescent microscope. White arrows show GFP localized to the membrane. White arrowheads show the presence of membrane blebs.

Using a genetically modified human RPE cell line containing fluorescent protein anchored to the inner leaflet of the plasma membrane, we demonstrated nonlethal blebbing in response to oxidative stress. Bebbing can be induced by oxidant injury with blue light, myeloperoxidase (MPO), hydroquinone (Monroy D., et al., IOVS, 2001, 42(4), ARVO Abstract, 4060; Suner I.J., et al. IOVS 2004; ARVO E-Abstract 1810) [56,171], or Ang II (Fig. 6). Today, however, RPE bleb composition and potential functions remain largely unexplored.

cytoskeleton turnover, presumably secondary to direct oxidative changes of disulfide bonds within actin filaments [180, 189-192]. The best characterized inhibitor/ stabilizer of cytoskeleton turnover is heat shock protein 25/27 (Hsp25/27). This molecule belongs to the family of small heat shock proteins (which includes α-β crystallins) and serves a dual role as cytoskeleton

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Oxidant injury can induce a rapid upregulation of Hsp25/27 protein through the activation of presynthesized heat shock factors (hsf-1,-2,-3) which upon oxidation form trimers that become very potent transcription factors [184-189]. Upon phosphorylation, Hsp25/27 acts as an F-actin cap-binding protein, binding to the active edge of a growing microfilament, causing a transient delay in actin cytoskeleton reassembly and repair [180,181,194,195]. Transient delay in reassembly allows the unsupported plasma membrane to be forced outward by the hydrostatic forces of the cytoplasm, and a vesicle or bleb forms if cortical cytoskeleton and microfilaments do not completely reform within the outpouched membrane. Phosphorylation of Hsp25/27 is mediated through the MAP kinase cascade, but is directly phosphorylated by MAPKAP 2 kinase (MK2), which in turn is phosphorylated by p38 kinase (Figure 4) [180, 196]. Thus, oxidative stress induces profound rearrangements of the actin cytoskeleton [197, 189, 198] leading to membrane blebbing through the activation of p38 mitogen activated protein kinase

In response to stress, phosphorylated Hsp27 undergoes conformational changes and reorgan‐ izes into dimeric units [198-202]. Phosphorylated Hsp27 regulates actin filaments dynamics by repressing the ability of Hsp27 to block actin polymerization [201]. Hsp27 phosphorylation has been abundantly described in several human diseases [203]. Yet, there is a complete lack of information regarding the possible association between phosphorylated Hsp27 and AMD. Increased Hsp27 protein content along with evidence of cellular oxidative stress was reported in human eyes with AMD, but Hsp27 phosphorylation was not investigated [204]. Therefore, we believe that Hsp27 is an important mediator of RPE response to hydroquinone-induced oxidative damage, which may contribute to injury-induced actin rearrangement and blebbing. We studied the phosphorylation of Hsp27 in RPE from human eye donors and found that the RPE constitutively express phosphorylated Hsp27, and that its expression is increased in patients with dry AMD (Fig. 8) [205] providing novel evidence that phosphorylated Hsp27

We reported that human RPE cells constitutively express high levels of Hsp27 which were regulated by oxidative-mediated injury and that Hsp27 is expressed in extruded blebs confirming that Hsp27 plays a role in actin filaments dynamics [205, 206]. We also addressed the question whether hydroquinone-induced oxidative injury can activate different MAPK pathways of any posttranslational modifications such as dimerization or phosphorylation of Hsp27 known to regulate F-actin polymerization. Our previous observations were extended by showing that exposure of RPE cells to cigarette smoke-derived hydroquinone non lethal injury induces the transcriptional activation of Hsp27, accumulation of Hsp27 dimers and a rapid phosphorylation of Hsp27 as well as actin rearrangements inton aggregates and membrane blebbing (Fig. 9) [205]. These results together with the observation that SB203580, a specific pharmacologic inhibitor of p38 kinase activity [207], efficiently blocked Hsp27

stabilizer and chaperone protein [191-193].

(MAPK)//Heat shock protein 27 (Hsp27) pathway [190,198].

may play a major role in the pathogenesis of AMD.

Membrane bleb or microvesicle production stimulated by a variety of stress has been exten‐ sively described in many different cell types [172–178]. To gain a better understanding of the functional relevance of blebs in general and the pathogenic mechanism(s) involved in early AMD in particular, we investigated the identity of proteins carried by human RPE blebs. In our study, we showed the proteomics characterization of stress-induced blebs in RPE cells from human retina. We report identification of several proteins, some of them potentially involved in MMP activation, membrane lipid raft formation, and immunogenic processes (Fig. 7) [179]. In our study, we intended to gain some insight into the functional characterization of blebs to unravel some of the biological consequences of cell membrane blebbing in disease.

**Figure 7.** Isolation of blebs (left). *A,* scheme for bleb isolation. ARPE-19 cells were treated with HQ (100 μM) for 6 h. Culture medium was collected and centrifuged at 100 × g for 15 min at 4 °C. The resulting pellet was washed twice with PBS and resuspended. The resuspended pellet was centrifuged at 100 × g for 15 min at 4 °C, and the supernatant was removed. Blebs were collected and used for protein extraction. *B*, representative one-dimensional gel showing the Coomassie Blue and silver stainings of resolved proteins present in ARPE-19 blebs. Functional characterization of proteins identified in hydroquinone-induced blebs (right). The distribution profile of the proteins identified in hydro‐ quinone-induced blebs is depicted according to functional categories. The KEGG database number and its corre‐ sponding metabolic pathway are shown. *TCA*, tricarboxylic acid cycle.

Blebbing are closely interrelated with the actin cytoskeleton [180-188]. The cytoskeleton is a dynamic structure undergoing continuous turnover by disassembly and reassembly of G-actin

monomers, which are added to the active edge of a growing filament. Cytoskeletal turnover is mediated by actin polymerizing protein system (APP), which is a large system of interacting proteins. The identity of these proteins is a rapidly growing field, and includes kinases, phosphatases, cleavage proteins, and elongating proteins. Their individual specific function and regulatory interactions remain essentially unknown. However, in general, the APP functions as a coordinated system such that individual molecular regulators of cytoskeleton dynamics exert their actions on the entire APP in coordinated fashion [180-188].

Very little information is available regarding the molecules that regulate the APP system and disease pathogenesis. Interestingly, oxidant injury is an effective activator of increased cytoskeleton turnover, presumably secondary to direct oxidative changes of disulfide bonds within actin filaments [180, 189-192]. The best characterized inhibitor/ stabilizer of cytoskeleton turnover is heat shock protein 25/27 (Hsp25/27). This molecule belongs to the family of small heat shock proteins (which includes α-β crystallins) and serves a dual role as cytoskeleton stabilizer and chaperone protein [191-193].

Using a genetically modified human RPE cell line containing fluorescent protein anchored to the inner leaflet of the plasma membrane, we demonstrated nonlethal blebbing in response to oxidative stress. Bebbing can be induced by oxidant injury with blue light, myeloperoxidase (MPO), hydroquinone (Monroy D., et al., IOVS, 2001, 42(4), ARVO Abstract, 4060; Suner I.J., et al. IOVS 2004; ARVO E-Abstract 1810) [56,171], or Ang II (Fig. 6). Today, however, RPE bleb

Membrane bleb or microvesicle production stimulated by a variety of stress has been exten‐ sively described in many different cell types [172–178]. To gain a better understanding of the functional relevance of blebs in general and the pathogenic mechanism(s) involved in early AMD in particular, we investigated the identity of proteins carried by human RPE blebs. In our study, we showed the proteomics characterization of stress-induced blebs in RPE cells from human retina. We report identification of several proteins, some of them potentially involved in MMP activation, membrane lipid raft formation, and immunogenic processes (Fig. 7) [179]. In our study, we intended to gain some insight into the functional characterization of blebs to unravel some of the biological consequences of cell membrane blebbing in disease.

**Figure 7.** Isolation of blebs (left). *A,* scheme for bleb isolation. ARPE-19 cells were treated with HQ (100 μM) for 6 h. Culture medium was collected and centrifuged at 100 × g for 15 min at 4 °C. The resulting pellet was washed twice with PBS and resuspended. The resuspended pellet was centrifuged at 100 × g for 15 min at 4 °C, and the supernatant was removed. Blebs were collected and used for protein extraction. *B*, representative one-dimensional gel showing the Coomassie Blue and silver stainings of resolved proteins present in ARPE-19 blebs. Functional characterization of proteins identified in hydroquinone-induced blebs (right). The distribution profile of the proteins identified in hydro‐ quinone-induced blebs is depicted according to functional categories. The KEGG database number and its corre‐

Blebbing are closely interrelated with the actin cytoskeleton [180-188]. The cytoskeleton is a dynamic structure undergoing continuous turnover by disassembly and reassembly of G-actin

monomers, which are added to the active edge of a growing filament. Cytoskeletal turnover is mediated by actin polymerizing protein system (APP), which is a large system of interacting proteins. The identity of these proteins is a rapidly growing field, and includes kinases, phosphatases, cleavage proteins, and elongating proteins. Their individual specific function and regulatory interactions remain essentially unknown. However, in general, the APP functions as a coordinated system such that individual molecular regulators of cytoskeleton

Very little information is available regarding the molecules that regulate the APP system and disease pathogenesis. Interestingly, oxidant injury is an effective activator of increased

dynamics exert their actions on the entire APP in coordinated fashion [180-188].

composition and potential functions remain largely unexplored.

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

sponding metabolic pathway are shown. *TCA*, tricarboxylic acid cycle.

Oxidant injury can induce a rapid upregulation of Hsp25/27 protein through the activation of presynthesized heat shock factors (hsf-1,-2,-3) which upon oxidation form trimers that become very potent transcription factors [184-189]. Upon phosphorylation, Hsp25/27 acts as an F-actin cap-binding protein, binding to the active edge of a growing microfilament, causing a transient delay in actin cytoskeleton reassembly and repair [180,181,194,195]. Transient delay in reassembly allows the unsupported plasma membrane to be forced outward by the hydrostatic forces of the cytoplasm, and a vesicle or bleb forms if cortical cytoskeleton and microfilaments do not completely reform within the outpouched membrane. Phosphorylation of Hsp25/27 is mediated through the MAP kinase cascade, but is directly phosphorylated by MAPKAP 2 kinase (MK2), which in turn is phosphorylated by p38 kinase (Figure 4) [180, 196]. Thus, oxidative stress induces profound rearrangements of the actin cytoskeleton [197, 189, 198] leading to membrane blebbing through the activation of p38 mitogen activated protein kinase (MAPK)//Heat shock protein 27 (Hsp27) pathway [190,198].

In response to stress, phosphorylated Hsp27 undergoes conformational changes and reorgan‐ izes into dimeric units [198-202]. Phosphorylated Hsp27 regulates actin filaments dynamics by repressing the ability of Hsp27 to block actin polymerization [201]. Hsp27 phosphorylation has been abundantly described in several human diseases [203]. Yet, there is a complete lack of information regarding the possible association between phosphorylated Hsp27 and AMD. Increased Hsp27 protein content along with evidence of cellular oxidative stress was reported in human eyes with AMD, but Hsp27 phosphorylation was not investigated [204]. Therefore, we believe that Hsp27 is an important mediator of RPE response to hydroquinone-induced oxidative damage, which may contribute to injury-induced actin rearrangement and blebbing. We studied the phosphorylation of Hsp27 in RPE from human eye donors and found that the RPE constitutively express phosphorylated Hsp27, and that its expression is increased in patients with dry AMD (Fig. 8) [205] providing novel evidence that phosphorylated Hsp27 may play a major role in the pathogenesis of AMD.

We reported that human RPE cells constitutively express high levels of Hsp27 which were regulated by oxidative-mediated injury and that Hsp27 is expressed in extruded blebs confirming that Hsp27 plays a role in actin filaments dynamics [205, 206]. We also addressed the question whether hydroquinone-induced oxidative injury can activate different MAPK pathways of any posttranslational modifications such as dimerization or phosphorylation of Hsp27 known to regulate F-actin polymerization. Our previous observations were extended by showing that exposure of RPE cells to cigarette smoke-derived hydroquinone non lethal injury induces the transcriptional activation of Hsp27, accumulation of Hsp27 dimers and a rapid phosphorylation of Hsp27 as well as actin rearrangements inton aggregates and membrane blebbing (Fig. 9) [205]. These results together with the observation that SB203580, a specific pharmacologic inhibitor of p38 kinase activity [207], efficiently blocked Hsp27

phosphorylation as well as actin cytoskeleton remodeling and blebs formation in response to hydroquinone in RPE cells, strongly suggest that p38 MAPK pathways activation by hydro‐ quinone modulates F-actin aggregates formation and membrane blebbing through Hsp27 phosphorylation. Our findings are in agreement with prior studies reporting p38 MAPK signaling pathway as an upstream mediator in oxidative stress-induced actin reorganization

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**Figure 9.** Inhibition of ERK MAPK pathway blocks hydroquinone (HQ)-induced Hsp27 phosphorylation and focal ag‐ gregates formation. A: Abrogation of HQ-induced Hsp27 phosphorylation by PD98059. Confluent serum-starved ARPE-19 cells were pretreated for 1 hour with 40μM of ERK inhibitor PD98059 (PD), and then exposed to 100μM HQ for 5 min. Total Hsp27, p-Hsp27 and GAPDH protein expression was evaluated by Western blot analysis. Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons (kD). p-Hsp27 protein expression was normalized to total Hsp27 protein. Bottom: average densitometry results of three inde‐ pendent experiments run in duplicate. Data are expressed as percentage of control and are means ± SE. \*\*p< 0.01 versus control; ##p< 0.01 versus HQ alone. B: Decreased formation of F-actin aggregates showed by staining for Factin in ARPE-19 cells pretreated for 1 hour with or without 40μM of PD and then exposed to 100μM HQ for 6 hours. Cells were stained with rhodamine-phalloidin and examined by confocal microscopy using magnification x40. White arrowheads show formation of focal aggregates. C: Quantification of F-acting aggregates from three independent ex‐ periments run in duplicate. Data are expressed as percentage of HQ-treated cells and are means ± SE. \*\*\*p < 0.001

and Hsp27 phosphorylation [190, 198-200, 208].

versus HQ-treated cells.

**Figure 8.** Increased phosphorylated Hsp27 (p-Hsp27) expression in human RPE from patient donors with dry AMD. A: Representative Western blot for p-Hsp27, total Hsp27 and GAPDH on RPE lysates from 3 donors with dry AMD and 3 controls with no known history of eye disease. B-G: Representative immunofluorescent double staining of p-Hsp27 (green) and nuclei (bleu) in retina sections from human donor eyes with no known eye disease (D), and human donor eyes with dry AMD (E). Negative controls were generated by omission of the primary antibody (B, C). Higher magnifi‐ cation showing RPE and BrM in control (F) and AMD (G) patients. Sections were analyzed using confocal microscopy (magnification x40 and x400). INL, inner nuclear layer; ONL, outer nuclear layer; PIS, photoreceptor inner segments; POS, photoreceptor outer segments; RPE, retinal pigment epithelium; Ch, choroid; BrM, Bruch's membrane.

phosphorylation as well as actin cytoskeleton remodeling and blebs formation in response to hydroquinone in RPE cells, strongly suggest that p38 MAPK pathways activation by hydro‐ quinone modulates F-actin aggregates formation and membrane blebbing through Hsp27 phosphorylation. Our findings are in agreement with prior studies reporting p38 MAPK signaling pathway as an upstream mediator in oxidative stress-induced actin reorganization and Hsp27 phosphorylation [190, 198-200, 208].

**Figure 9.** Inhibition of ERK MAPK pathway blocks hydroquinone (HQ)-induced Hsp27 phosphorylation and focal ag‐ gregates formation. A: Abrogation of HQ-induced Hsp27 phosphorylation by PD98059. Confluent serum-starved ARPE-19 cells were pretreated for 1 hour with 40μM of ERK inhibitor PD98059 (PD), and then exposed to 100μM HQ for 5 min. Total Hsp27, p-Hsp27 and GAPDH protein expression was evaluated by Western blot analysis. Top: Western blot from a representative experiment. Numbers on the left represent protein molecular mass in kilodaltons (kD). p-Hsp27 protein expression was normalized to total Hsp27 protein. Bottom: average densitometry results of three inde‐ pendent experiments run in duplicate. Data are expressed as percentage of control and are means ± SE. \*\*p< 0.01 versus control; ##p< 0.01 versus HQ alone. B: Decreased formation of F-actin aggregates showed by staining for Factin in ARPE-19 cells pretreated for 1 hour with or without 40μM of PD and then exposed to 100μM HQ for 6 hours. Cells were stained with rhodamine-phalloidin and examined by confocal microscopy using magnification x40. White arrowheads show formation of focal aggregates. C: Quantification of F-acting aggregates from three independent ex‐ periments run in duplicate. Data are expressed as percentage of HQ-treated cells and are means ± SE. \*\*\*p < 0.001 versus HQ-treated cells.

**Figure 8.** Increased phosphorylated Hsp27 (p-Hsp27) expression in human RPE from patient donors with dry AMD. A: Representative Western blot for p-Hsp27, total Hsp27 and GAPDH on RPE lysates from 3 donors with dry AMD and 3 controls with no known history of eye disease. B-G: Representative immunofluorescent double staining of p-Hsp27 (green) and nuclei (bleu) in retina sections from human donor eyes with no known eye disease (D), and human donor eyes with dry AMD (E). Negative controls were generated by omission of the primary antibody (B, C). Higher magnifi‐ cation showing RPE and BrM in control (F) and AMD (G) patients. Sections were analyzed using confocal microscopy (magnification x40 and x400). INL, inner nuclear layer; ONL, outer nuclear layer; PIS, photoreceptor inner segments;

POS, photoreceptor outer segments; RPE, retinal pigment epithelium; Ch, choroid; BrM, Bruch's membrane.

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

Several reports have shown that PP2A is involved in Hsp27 dephosphorylation [209, 210] and that oxidative stress causes extracellular-signal-regulated kinases (ERK) phosphorylation and reorganization of actin cytoskeleton in RPE cells [211]. Using okadaic acid as an inhibitor of Hsp27 dephosphorylation by PP1 and 2A, we observed an increase in Hsp27 phosphorylation in hydroquinone-stimulated RPE cells as well as F-actin reorganization and blebs formation [205]. Moreover, we demonstrate that Hsp27 phosphorylation and F-actin aggregates forma‐ tion is almost completely abolished in cells transfected with siRNA against Hsp27 following treatment with hydroquinone [205].

to play a role in changes in actin cytoskeletal reorganization. RhoA acts through ROCK to form stress fibers [240], and ROCK has been shown to be involved in cell contraction induced by Ang II [222]. mDia1 is a member of the ubiquitous formin protein family. These proteins are activated by interaction with Rho GTPases and are then able to mediate actin polymerization [223]. Overexpression of the GTP-Rho binding domain of mDia1 causes spontaneous mem‐

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Ang II-stimulated membrane bleb formation involves RhoA. Furthermore, membrane blebbing activated by Ang II through AT1 receptor activation is attenuated in the presence of the β-arrestin amino-terminal domain, Ral GDP dissociation stimulator (RalGDS) β-arrestin binding domain, and short interfering RNA (siRNA) depletion of β-arrestin2 [225]. In addition, the inhibition of the downstream RhoA effectors ROCK and MLCK effectively attenuated AT1 receptor-mediated membrane blebbing. Thus, membrane blebbing in response to AT1 receptor signaling was dependent on β-arrestin2 and was mediated by a RhoA/ROCK/MLCK-depend‐ ent pathway providing evidence that agonist stimulation of AT1 receptor leads to plasma membrane blebbing responses by activation of RhoA and subsequent coupling to the ROCK/

Cytoplasmic actin aggregates were observed after nonlethal blebbing, but not with lethal oxidant injury. Interestingly, activation of the AT1 receptor with Ang II resulted in the rapid formation of ROS and membrane blebs at early time points of Ang II stimulation that cease within 60 min of Ang II stimulation (Marin-Castano., et al., IOVS, 2001, 42(4), ARVO Abstract, 4060). Further, Ang II-enhanced ROS production and blebbing was prevented by pre-treat‐ ment of RPE with the AT1 receptor blocker, which attenuates oxidative stress [226]. However, RPE derived blebs after Ang II were bigger than those seem when RPE cells were exposed to blue light, MPO, or hydroquinone. Moreover, formation of RPE-membrane blebs induced by Ang II occurred earlier than with the other oxidants we studied. Therefore, we believe that Rho-kinase pathway could be implicated as an important mediator of RPE response to Ang

In conclusion, characterization of the molecular mechanism(s) by which hydroquinone and the AT1 receptor regulates the actin cytoskeleton and plasma membrane dynamics will be essential for modulating the degree of blebbing in vitro and deposit formation in vivo for

Another injury response relevant to early AMD is imbalance in ECM turnover. The normal anatomy and physiology of ECM in most tissues requires continuous turnover of matrix components by a tightly regulated balance in the production of matrix molecules like collagen, laminin, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) [227,228]. Matrix metalloproteinases (MMPs) are a family of at least 20 zinc endopep‐ tidases that take part in the regulation of cell matrix composition by cleaving basal lamina and ECM proteins. MMPs can be secretory or cell surface bound. Under normal conditions, MMP activity is required for tissue remodeling, but altered MMP activity has been reported in disease. Most MMPs are secreted as inactive pro-proteins but get activated when cleaved by extracellular proteinases. Interestingly, one of the targets of MMP activity is the ECM mole‐

II-induced oxidative damage instead of Hsp27/Hsp25/p38/ERK pathway.

brane blebbing [224].

MLCK pathway [225].

potential therapeutic strategies.

cules in BrM.

As mentioned above ERK cascade participates in numerous intracellular signaling pathways in response to environmental stimuli, such as oxidative stress [211]. Our study also show that treatment with hydroquinone led to a robust activation of ERK signaling pathway in ARPE-19 cells as well as in mice. These results together with the observation that PD98059, a specific pharmacologic inhibitor of MEK, completely abolished Hsp27 phosphorylation as well as actin cytoskeleton remodeling in response to hydroquinone, strongly suggest that ERK is also a key upstream activator of hydroquinone-induced Hsp27 phosphorylation in RPE cells [205] (Fig 9). Our results not only showed that kinetics of p38 and ERK phosphorylation correlated well with that of Hsp27, but also that Hsp27 phosphorylation and F-actin aggregates formation were decreased after inhibition of either p38 or ERK signaling cascades. These observations suggest that p38 as well as ERK MAPK pathways are required for the optimal activation of Hsp27 leading to F-actin rearrangement and bleb formation in RPE cells in response to hydroquinone. Taken together,these data establish; a) a direct correlation between levels of phosphorylated Hsp27 and actin cytoskeleton reorganization in response to hydroquinoneinduced oxidative injury in human RPE cells; b) present ERK as a novel upstream positive regulator of Hsp27 and actin aggregates formation in response to hydroquinone-induced oxidative injury in RPE cells; and c) give support to a key role of phosphorylated Hsp27 in the regulation of F-actin filaments dynamics and blebs formation following hydroquinoneinduced oxidative stress in RPE cells. Given that there is no effective treatment for dry AMD, this study highlights Hsp27 as a potential, disease-related protein as well as biochemical pathways for potential therapeutic strategies.

In addition to Hsp27/Hsp25, other important molecules such as small GTPases protein superfamily (RAS and Ral) have been involved in the regulation of the actin polymerizing protein system and oxidative stress [212,213]. Membrane blebbing is RhoA-, Rho kinase (ROCK)-, and myosin light chain kinase (MLCK)-dependent, and blebs are devoid of actin, mDia1 and Arp2/3 [214]. Rals are small G proteins that cycle between an active GTP-bound state and an inactive GDP-bound state [215]. Ral GDP dissociation stimulator (RalGDS) was found to be an effector of Ras [216-218] and highly specific for RalA and RalB, whereby it facilitates the exchange of GDP for GTP on Rals [216,219,220]. Moreover, it has been demon‐ strated that RalGDS forms a cytosolic complex with β-arrestin, and that in response to formyl-Met-Leu-Phe (fMLP) receptor stimulation, RalGDS is released from β-arrestin and translocates to the plasma membrane.

RhoA is an additional small G protein that is implicated in regulating plasma membrane dynamics. RhoA is activated by Ang II and is necessary for AT1 receptor-induced stress fiber formation [221]. The proteins ROCK and mDia1 are both RhoA effectors and have been shown to play a role in changes in actin cytoskeletal reorganization. RhoA acts through ROCK to form stress fibers [240], and ROCK has been shown to be involved in cell contraction induced by Ang II [222]. mDia1 is a member of the ubiquitous formin protein family. These proteins are activated by interaction with Rho GTPases and are then able to mediate actin polymerization [223]. Overexpression of the GTP-Rho binding domain of mDia1 causes spontaneous mem‐ brane blebbing [224].

Several reports have shown that PP2A is involved in Hsp27 dephosphorylation [209, 210] and that oxidative stress causes extracellular-signal-regulated kinases (ERK) phosphorylation and reorganization of actin cytoskeleton in RPE cells [211]. Using okadaic acid as an inhibitor of Hsp27 dephosphorylation by PP1 and 2A, we observed an increase in Hsp27 phosphorylation in hydroquinone-stimulated RPE cells as well as F-actin reorganization and blebs formation [205]. Moreover, we demonstrate that Hsp27 phosphorylation and F-actin aggregates forma‐ tion is almost completely abolished in cells transfected with siRNA against Hsp27 following

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

As mentioned above ERK cascade participates in numerous intracellular signaling pathways in response to environmental stimuli, such as oxidative stress [211]. Our study also show that treatment with hydroquinone led to a robust activation of ERK signaling pathway in ARPE-19 cells as well as in mice. These results together with the observation that PD98059, a specific pharmacologic inhibitor of MEK, completely abolished Hsp27 phosphorylation as well as actin cytoskeleton remodeling in response to hydroquinone, strongly suggest that ERK is also a key upstream activator of hydroquinone-induced Hsp27 phosphorylation in RPE cells [205] (Fig 9). Our results not only showed that kinetics of p38 and ERK phosphorylation correlated well with that of Hsp27, but also that Hsp27 phosphorylation and F-actin aggregates formation were decreased after inhibition of either p38 or ERK signaling cascades. These observations suggest that p38 as well as ERK MAPK pathways are required for the optimal activation of Hsp27 leading to F-actin rearrangement and bleb formation in RPE cells in response to hydroquinone. Taken together,these data establish; a) a direct correlation between levels of phosphorylated Hsp27 and actin cytoskeleton reorganization in response to hydroquinoneinduced oxidative injury in human RPE cells; b) present ERK as a novel upstream positive regulator of Hsp27 and actin aggregates formation in response to hydroquinone-induced oxidative injury in RPE cells; and c) give support to a key role of phosphorylated Hsp27 in the regulation of F-actin filaments dynamics and blebs formation following hydroquinoneinduced oxidative stress in RPE cells. Given that there is no effective treatment for dry AMD, this study highlights Hsp27 as a potential, disease-related protein as well as biochemical

In addition to Hsp27/Hsp25, other important molecules such as small GTPases protein superfamily (RAS and Ral) have been involved in the regulation of the actin polymerizing protein system and oxidative stress [212,213]. Membrane blebbing is RhoA-, Rho kinase (ROCK)-, and myosin light chain kinase (MLCK)-dependent, and blebs are devoid of actin, mDia1 and Arp2/3 [214]. Rals are small G proteins that cycle between an active GTP-bound state and an inactive GDP-bound state [215]. Ral GDP dissociation stimulator (RalGDS) was found to be an effector of Ras [216-218] and highly specific for RalA and RalB, whereby it facilitates the exchange of GDP for GTP on Rals [216,219,220]. Moreover, it has been demon‐ strated that RalGDS forms a cytosolic complex with β-arrestin, and that in response to formyl-Met-Leu-Phe (fMLP) receptor stimulation, RalGDS is released from β-arrestin and translocates

RhoA is an additional small G protein that is implicated in regulating plasma membrane dynamics. RhoA is activated by Ang II and is necessary for AT1 receptor-induced stress fiber formation [221]. The proteins ROCK and mDia1 are both RhoA effectors and have been shown

treatment with hydroquinone [205].

pathways for potential therapeutic strategies.

to the plasma membrane.

Ang II-stimulated membrane bleb formation involves RhoA. Furthermore, membrane blebbing activated by Ang II through AT1 receptor activation is attenuated in the presence of the β-arrestin amino-terminal domain, Ral GDP dissociation stimulator (RalGDS) β-arrestin binding domain, and short interfering RNA (siRNA) depletion of β-arrestin2 [225]. In addition, the inhibition of the downstream RhoA effectors ROCK and MLCK effectively attenuated AT1 receptor-mediated membrane blebbing. Thus, membrane blebbing in response to AT1 receptor signaling was dependent on β-arrestin2 and was mediated by a RhoA/ROCK/MLCK-depend‐ ent pathway providing evidence that agonist stimulation of AT1 receptor leads to plasma membrane blebbing responses by activation of RhoA and subsequent coupling to the ROCK/ MLCK pathway [225].

Cytoplasmic actin aggregates were observed after nonlethal blebbing, but not with lethal oxidant injury. Interestingly, activation of the AT1 receptor with Ang II resulted in the rapid formation of ROS and membrane blebs at early time points of Ang II stimulation that cease within 60 min of Ang II stimulation (Marin-Castano., et al., IOVS, 2001, 42(4), ARVO Abstract, 4060). Further, Ang II-enhanced ROS production and blebbing was prevented by pre-treat‐ ment of RPE with the AT1 receptor blocker, which attenuates oxidative stress [226]. However, RPE derived blebs after Ang II were bigger than those seem when RPE cells were exposed to blue light, MPO, or hydroquinone. Moreover, formation of RPE-membrane blebs induced by Ang II occurred earlier than with the other oxidants we studied. Therefore, we believe that Rho-kinase pathway could be implicated as an important mediator of RPE response to Ang II-induced oxidative damage instead of Hsp27/Hsp25/p38/ERK pathway.

In conclusion, characterization of the molecular mechanism(s) by which hydroquinone and the AT1 receptor regulates the actin cytoskeleton and plasma membrane dynamics will be essential for modulating the degree of blebbing in vitro and deposit formation in vivo for potential therapeutic strategies.

Another injury response relevant to early AMD is imbalance in ECM turnover. The normal anatomy and physiology of ECM in most tissues requires continuous turnover of matrix components by a tightly regulated balance in the production of matrix molecules like collagen, laminin, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) [227,228]. Matrix metalloproteinases (MMPs) are a family of at least 20 zinc endopep‐ tidases that take part in the regulation of cell matrix composition by cleaving basal lamina and ECM proteins. MMPs can be secretory or cell surface bound. Under normal conditions, MMP activity is required for tissue remodeling, but altered MMP activity has been reported in disease. Most MMPs are secreted as inactive pro-proteins but get activated when cleaved by extracellular proteinases. Interestingly, one of the targets of MMP activity is the ECM mole‐ cules in BrM.

It has been shown that relatively small dysregulation in the relative production of MMPs, TIMPs, and collagen types I and IV [227,228] may lead to net changes in the ECM, including thickening and deposit formation [228,231]. Accordingly, dysregulated turnover of ECM is a major mechanism of disease pathogenesis in many tissue sites, including renal disease, atherosclerosis, lung disease, and others [228-231]. Unfortunately, minimal information is available concerning normal turnover in healthy BrM or imbalanced turnover in AMD.

glycoprotein [236,239]. The basigin gene (BSG) encodes for a 29 kDa protein which is prone to glycosylation increasing its molecular weight between 35 and 65 kDa [238-240]. Induction of matrix MMPs constitutes the most relevant function of basigin [240, 242-244]. A clear require‐ ment for basigin to be glycosylated exists for MMP induction [245-247]. The fact that basigin is a prominent MMP inducer provides this protein with a putative role in normal tissue remodeling physiology and ECM pathologies other than cancer. In particular, glycosylated basigin has been reported to be necessary for maturation of the retina photoreceptor cells [248]. Within the mature eye, basigin is expressed in the RPE, Mueller cells, and in endothelial cells of blood vessels [249-252]. A known mechanism by which basigin increases the MMPs occurs at transcriptional level [253, 254]. However, shedding of basigin from the plasma membrane constitutes an additional regulatory mechanism recently proposed where basigin is transport‐ ed in microvesicles which are later on degraded, releasing soluble, active basigin [255]. This proposed mechanism may permit basigin to exert its actions at distant sites. MMP-14 is an interesting candidate responsible for the basigin shedding [256-258]. Based on the cited evidence, we hypothesize a mechanistic model by which basigin and MMP-14 are carried to distal sites from the RPE where they exert their actions promoting MMP-2 activity. In addition, MMP-14 may interact with basigin releasing fully functional "soluble" basigin (Fig. 10).

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**Figure 10.** Mechanistic model by which basigin and MMP-14 are carried to distal sites from RPE where they exert their

We reported the presence of glycosylated basigin and MMP-14 in blebs, which was con‐ firmed by Western blot and immunofluorescence staining in human control and AMD retinas (Fig. 11) [179]. Basigin and MMP-14 were confined to the RPE in normal retina, while it was widely expressed in AMD retina. In addition, our data showed that RPE cells incubated with blebs exhibit increased active MMP-2. MMP-2 activity returned to basal levels after incuba‐ tion with anti-basigin and MMP-14 antibodies (Figure 10), suggesting that both proteins may play a pivotal role determining MMP-2 activity [179]. Thus, blebs accumulated under the RPE will stimulate ECM turnover increasing active MMP-2 through the action of two bleb-

actions promoting MMP-2 activity.

Ample evidence supports the idea that MMPs and their tissue inhibitors TIMPs play an important role in AMD. The RPE is capable of producing many ECM components such as MMP-2, MMP-14, Basigin, also known as EMMPRIN (extracellular matrix metalloproteinase inducer) or CD147, collagen, and TIMP-2 [56,171,232, 233]. MMP-2 and MMP-14 are the major RPE enzymes synthesized for the degradation of matrix type IV and I collagens, laminin, and fibronectin, which are components found in BrM [232,233]. MMP-2 is the key enzyme for ECM turnover in BrM and is synthesized as an inactive zymogen pro form (pro-MMP-2) [227]. The transmembrane metalloproteinase MMP-14 is well known to activate MMP-2 in a specific manner [233]. On the other hand, TIMP-2 may inhibit the cleavage of pro-MMP-2 into MMP-2 [228]. Based on these data our research focuses on MMP-2, MMP-14, basigin, and TIMP-2 and propose that dysregulation of MMP-2 is the primary cause for the accumulation of sub-RPE deposits that become BLD and drusen. Thus, preservation of RPE-derived MMP-2 function will prevent these events.

As mentioned before, oxidative injury to the RPE may not only produce blebbing but also dysregulated ECM turnover [5,46,47,206,234,235]. Our group has demonstrated that nonlethal oxidant injury to the RPE with hydroquinone induces a wide range of changes in gene expression, especially for those genes involved in regulation of ECM [47], and that blebs form and accumulate in the absence of activated MMP-2, which otherwise induce breakdown of types I and IV collagen present in the basement membrane of the RPE [235]. Sustained oxidant injury with hydroquinone is capable of downregulating MMP-2 activity in an in vitro system of RPE cells, correlating with an increase in bleb levels [56,171]. Also, type IV collagen accumulation, the main component of the RPE basement membrane, correlated with the absence of MMP-2 activity [56]. Accordingly, we have shown in vivo [235] that reducing MMP-2 activity leads to an increase in deposit formation by RPE cells. This allows the blebs to become trapped between the RPE cell membrane and the basement membrane to form BLD.

For the BLD deposits to progress into linear deposits ECM upregulation with MMP-2 activa‐ tion is necessary for collagen and BLD deposit degradation. This will ultimately displace sub-RPE deposits into the inner layer of BrM where BLinD and drusen are histologically found. As proposed in our hypothetical model fro dry AMD, RPE-derived blebs accumulated as BLD could stimulate RPE basement membrane breakdown allowing the migration of BLD and buildup of BLiD or drusen. We postulate that various plasma-derived molecules related to systemic health cofactors (i.e., Ang II) are implicated in this stage.

As an attempt to better understand the mechanisms involved in early AMD, we investigated the proteomic profile of RPE-derived blebs induced by hydroquinone. Interestingly MMP-14 and basigin were identified in the RPE blebs [236]. Both proteins are of special relevance to the AMD pathology as they promote MMP-2 activation. Basigin, is a pleiotropic transmembrane glycoprotein [236,239]. The basigin gene (BSG) encodes for a 29 kDa protein which is prone to glycosylation increasing its molecular weight between 35 and 65 kDa [238-240]. Induction of matrix MMPs constitutes the most relevant function of basigin [240, 242-244]. A clear require‐ ment for basigin to be glycosylated exists for MMP induction [245-247]. The fact that basigin is a prominent MMP inducer provides this protein with a putative role in normal tissue remodeling physiology and ECM pathologies other than cancer. In particular, glycosylated basigin has been reported to be necessary for maturation of the retina photoreceptor cells [248]. Within the mature eye, basigin is expressed in the RPE, Mueller cells, and in endothelial cells of blood vessels [249-252]. A known mechanism by which basigin increases the MMPs occurs at transcriptional level [253, 254]. However, shedding of basigin from the plasma membrane constitutes an additional regulatory mechanism recently proposed where basigin is transport‐ ed in microvesicles which are later on degraded, releasing soluble, active basigin [255]. This proposed mechanism may permit basigin to exert its actions at distant sites. MMP-14 is an interesting candidate responsible for the basigin shedding [256-258]. Based on the cited evidence, we hypothesize a mechanistic model by which basigin and MMP-14 are carried to distal sites from the RPE where they exert their actions promoting MMP-2 activity. In addition, MMP-14 may interact with basigin releasing fully functional "soluble" basigin (Fig. 10).

It has been shown that relatively small dysregulation in the relative production of MMPs, TIMPs, and collagen types I and IV [227,228] may lead to net changes in the ECM, including thickening and deposit formation [228,231]. Accordingly, dysregulated turnover of ECM is a major mechanism of disease pathogenesis in many tissue sites, including renal disease, atherosclerosis, lung disease, and others [228-231]. Unfortunately, minimal information is available concerning normal turnover in healthy BrM or imbalanced turnover in AMD.

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

Ample evidence supports the idea that MMPs and their tissue inhibitors TIMPs play an important role in AMD. The RPE is capable of producing many ECM components such as MMP-2, MMP-14, Basigin, also known as EMMPRIN (extracellular matrix metalloproteinase inducer) or CD147, collagen, and TIMP-2 [56,171,232, 233]. MMP-2 and MMP-14 are the major RPE enzymes synthesized for the degradation of matrix type IV and I collagens, laminin, and fibronectin, which are components found in BrM [232,233]. MMP-2 is the key enzyme for ECM turnover in BrM and is synthesized as an inactive zymogen pro form (pro-MMP-2) [227]. The transmembrane metalloproteinase MMP-14 is well known to activate MMP-2 in a specific manner [233]. On the other hand, TIMP-2 may inhibit the cleavage of pro-MMP-2 into MMP-2 [228]. Based on these data our research focuses on MMP-2, MMP-14, basigin, and TIMP-2 and propose that dysregulation of MMP-2 is the primary cause for the accumulation of sub-RPE deposits that become BLD and drusen. Thus, preservation of RPE-derived MMP-2 function

As mentioned before, oxidative injury to the RPE may not only produce blebbing but also dysregulated ECM turnover [5,46,47,206,234,235]. Our group has demonstrated that nonlethal oxidant injury to the RPE with hydroquinone induces a wide range of changes in gene expression, especially for those genes involved in regulation of ECM [47], and that blebs form and accumulate in the absence of activated MMP-2, which otherwise induce breakdown of types I and IV collagen present in the basement membrane of the RPE [235]. Sustained oxidant injury with hydroquinone is capable of downregulating MMP-2 activity in an in vitro system of RPE cells, correlating with an increase in bleb levels [56,171]. Also, type IV collagen accumulation, the main component of the RPE basement membrane, correlated with the absence of MMP-2 activity [56]. Accordingly, we have shown in vivo [235] that reducing MMP-2 activity leads to an increase in deposit formation by RPE cells. This allows the blebs to become trapped between the RPE cell membrane and the basement membrane to form BLD. For the BLD deposits to progress into linear deposits ECM upregulation with MMP-2 activa‐ tion is necessary for collagen and BLD deposit degradation. This will ultimately displace sub-RPE deposits into the inner layer of BrM where BLinD and drusen are histologically found. As proposed in our hypothetical model fro dry AMD, RPE-derived blebs accumulated as BLD could stimulate RPE basement membrane breakdown allowing the migration of BLD and buildup of BLiD or drusen. We postulate that various plasma-derived molecules related to

As an attempt to better understand the mechanisms involved in early AMD, we investigated the proteomic profile of RPE-derived blebs induced by hydroquinone. Interestingly MMP-14 and basigin were identified in the RPE blebs [236]. Both proteins are of special relevance to the AMD pathology as they promote MMP-2 activation. Basigin, is a pleiotropic transmembrane

systemic health cofactors (i.e., Ang II) are implicated in this stage.

will prevent these events.

**Figure 10.** Mechanistic model by which basigin and MMP-14 are carried to distal sites from RPE where they exert their actions promoting MMP-2 activity.

We reported the presence of glycosylated basigin and MMP-14 in blebs, which was con‐ firmed by Western blot and immunofluorescence staining in human control and AMD retinas (Fig. 11) [179]. Basigin and MMP-14 were confined to the RPE in normal retina, while it was widely expressed in AMD retina. In addition, our data showed that RPE cells incubated with blebs exhibit increased active MMP-2. MMP-2 activity returned to basal levels after incuba‐ tion with anti-basigin and MMP-14 antibodies (Figure 10), suggesting that both proteins may play a pivotal role determining MMP-2 activity [179]. Thus, blebs accumulated under the RPE will stimulate ECM turnover increasing active MMP-2 through the action of two bleb-

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 health cofactors are implicated in this stage.

**8. Inflammation and angiogenesis**

receptors [50,265,266].

other role in the generation of drusen.

**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

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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

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

**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‐ toreceptor outer segments; Ch, choroi.
