REVIEW ARTICLE

Journal of

Cellular Physiology

Disease Pathways in Proliferative Vitreoretinopathy: An Ongoing Challenge GIAN MARCO TOSI,1* DAVIDE MARIGLIANI,1 NAPOLEONE ROMEO,1 1

Department of Medicine, Surgery and Neuroscience, University of Siena, Siena, Italy

2

Department of Medical Biotechnologies, University of Siena, Siena, Italy

AND

PAOLO TOTI2

Despite remarkable advances in vitreoretinal surgery, proliferative vitreoretinopathy (PVR) remains a common cause of severe visual loss or blindness. One of the critical reasons for PVR-induced blindness is tractional retinal detachment due to the formation of contractile preretinal fibrous membranes. This membrane formation is characterized by the proliferation and migration of cells and the excessive synthesis and deposition of extracellular matrix proteins. Herein we present the disease pathways of PVR, reviewing the role of both systemic and intraocular cells as well as molecular mediators. A chronological sequence of events leading to PVR is also hypothesized. Better understanding of the pathogenesis of PVR is needed in order to improve disease management. Efforts should be oriented towards greater cooperation between basic researchers and clinicians, aimed at matching the different clinical scenarios with the biological markers of the disease. J. Cell. Physiol. 229: 1577–1583, 2014. © 2014 Wiley Periodicals, Inc.

Proliferative vitreoretinopathy (PVR) can be defined as the growth and contraction of cellular membranes within the vitreous cavity and on both retinal surfaces (Ryan, 1985; Pastor et al., 2002). Several studies have confirmed the hypothesis that PVR occurs as a reparative process induced by retinal breaks and excessive inflammatory reaction (Pastor, 1998; Pastor et al., 2002). PVR can develop after longstanding primary retinal detachment (RD) and is the most common cause of the failed repair of rhegmatogenous RD. Risk factors for PVR are related to several well-known pre-, intra-, and post-operative clinical situations (Nagasaki et al., 1998; Pastor, 1998; Charteris et al., 2002; Pastor et al., 2002; Tseng et al., 2004). However, PVR must be considered as the end point of a number of intraocular diseases, rather than as a specific clinical entity. Retinal detachment and the associated vitreous alterations are indeed important factors, but not the only ones that need to be taken into consideration (Cardillo et al., 1997; Pastor, 1998; Pastor et al., 2002). Over the last 20 years vitreoretinal surgical techniques have evolved, greater emphasis has been placed the success of primary retinal detachment surgery to prevent PVR, case selection has been refined and the incidence of PVR might have been expected to decline. Yet the frequency of this condition remains largely unchanged, with a post-operative incidence of PVR ranging from 4% to 34% in prospective studies (Charteris et al., 2002; Heimann et al., 2007; Leiderman and Miller, 2009). Classification systems have been developed to describe the anatomical appearance of the epiretinal and subretinal membrane proliferations. In 1983, the Retina Society developed a PVR grading system to provide a more detailed description of the various stages of the disease. In 1989, as part of a multicenter, prospective, randomized, clinical trial examining the efficacy of extended gas versus silicone oil tamponade, a new classification system was devised to more accurately describe the types of contraction found in this disease (Lean et al., 1989; Nagasaki et al., 1998; Pastor, 1998). Subsequently, a committee appointed by the Retina Society published a revised classification system in 1991, based on the Silicone Study information and input from other investigations (Machemer et al., 1991) (Fig. 1). Anatomical Background to Pathogenesis

The posterior segment of the eyeball includes the anterior face of the vitreous and the structures behind it. Here we present © 2 0 1 4 W I L E Y P E R I O D I C A L S , I N C .

some brief anatomical notes regarding the vitreous, the choroid and the retina with its multiple layers, as the structural characteristics of these elements represent the basis for understanding PVR pathogenesis. The vitreous cavity is essentially a large extracellular space and the vitreous humor itself is composed of a collagenous (mainly type II) framework embedded in almost pure hyaluronate ground substance. The vitreous is virtually acellular, with only rare histiocytes (hyalocytes) evident in its cortex (Foos and Silverstein, 2004). Three regions are characterized by their firm attachment to the vitreous: the vitreous base, the papillary region and the lens. The vitreous base is normally 4–5 mm in width and straddles the oraserrata, extending from the ciliary epithelium to the neurosensory retina. The attachments within the posterior retinal portion of the base are related to the complex interdigitation of the vitreous with deep crypts in the retinal surface (Foos and Silverstein, 2004). The choroid can be divided into the following layers: suprachoroid, stroma, choriocapillaris, and Bruch’s membrane. The principal stromal cells of the choroid are fibrocytes and melanocytes, with occasional macrophages, mast cells, lymphocytes and plasma cells scattered throughout the stroma (Foos and Silverstein, 2004). The retina can be divided into the following layers, from its external to its internal structure: (1) the retinal pigment epithelium (RPE) (which differs from the others due to its distinct development, function, and pathologies) in a single layer, adhering to Bruch’s membrane, immediately above the capillaries; the photoreceptors rest on the RPE; (2) the outer

The authors have no conflict of interest to declare. *Correspondence to: Gian Marco Tosi, Department of Medicine, Surgery and Neuroscience, University of Siena, Viale Bracci 1, 53100 Siena, Italy. E-mail: [email protected] Manuscript Received: 3 January 2014 Manuscript Accepted: 16 January 2014 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 7 March 2014. DOI: 10.1002/jcp.24606

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Fig. 1. Fundus photograph showing anterior and posterior (confirmed during surgery) PVR creating a retinal detachment with funnel configuration.

and inner segments of the photoreceptors, together with villous processes of the pigment epithelium and Mueller’s cells, immersed in a matrix composed of acid mucopolysaccharides; (3) the outer limiting membrane, which consists of intercellular junctions that link the photoreceptors to the Mueller cells; (4) the outer nuclear layer, which contains the cell bodies of cones and rods; (5) the outer plexiform layer, which contains the photoreceptor axons, the dendrites of bipolar cells and the processes of horizontal cells; (6) the inner nuclear layer, containing the cell bodies of horizontal, bipolar, Mueller, and amacrine cells; (7) the inner plexiform layer with the interconnections between the processes of amacrine cells, bipolar cells and ganglion cells; (8) a layer of ganglion cells with scattered astrocytes; (9) the nerve fiber layer, that is, a layer of unmyelinated axons of ganglion cells; (10) the inner limiting membrane (ILM), composed of a lamina densa and lamina rara, which constitutes a sort of retinal basal lamina (Foos and Silverstein, 2004). The ILM extends from the periphery to the posterior pole and can be distinguished into a basal zone, an equatorial zone and a posterior zone. In the basal zone (at the level of the vitreous base) the dense membrane becomes very thin and adheres closely to the vitreous. In adults breaks in the ILM within the basal zone are common, with the ends of the interrupted lamina being directed towards the overlying vitreous. Cellular debris and occasional macrophages are usually present in these areas. In adults degenerative changes can also be more severe, even resulting in “degenerative remodeling,” when the Mueller cells become separated by deep clefts penetrated by sheets of vitreous (Foos and Silverstein, 2004). This degenerative remodeling appears to be the basis for the firm attachment of the vitreous to the retinal portion of the vitreous base. The ILM thickens progressively from the basal zone to the equatorial and posterior zones, with the exception of two sites in which it is very thin: where it overlies the retinal vessels and in the peripapillary area. In particular, in the peripapillary area the ILM decreases to a basal lamina of normal thickness (500 Å), which continues across the surface of the disc. In this area the firm attachment of the vitreous is mediated by intervening glial epipapillary membranes. Specifically, through focal gaps in the ILM glial cell processes continue on the overlying glial epipapillary JOURNAL OF CELLULAR PHYSIOLOGY

membranes, whose glial processes are closely connected to the overlying vitreous, forming strong vitreo-membranous adhesions (Foos and Silverstein, 2004). Although they do not constitute a definable retinal layer, the retinal glia merit consideration due to their role as major supportive cells of the retina, both structurally and metabolically. Two glial categories have been classified: Mueller cells and accessory glia. Mueller cells extend from the inner retina, where they expand as a mantle covering the surface under the inner limiting membrane, to the outer limiting membrane. Accessory glial cells resemble the astrocytes of the CNS: when stimulated by injury, they become actively phagocytic, proliferate and eventually take on the features of fibrous astrocytes, forming a scar in concert with the Mueller cells (Foos and Silverstein, 2004). The central retinal artery supplies blood to the inner half of the retina, while the ciliary circulation supplies blood to the outer half through the choriocapillaris. Two fundamental layers of retinal capillaries have been described: a deep, denser network at the outer aspect of the inner nuclear layer, and a superficial, less densenetwork in the nerve fiber layer. In the peripapillary region an additional layer of capillaries is found in the most superficial aspect of the nerve fiber layer, while in the pre-equatorial zone the deep capillary plexus progressively vanishes towards the periphery, with only superficial capillaries persisting (Foos and Silverstein, 2004). Pathogenesis

PVR consists in the formation of endovitreous, subretinal and epiretinal membranes, which derive from reparative, primary or post-operative fibrosis. Rhegmatogenous retinal detachment alters the composition of the vitreous and causes an interaction between the vitreous itself, the retinal cell population and other soluble mediators, as well as altering the interface between the external retina and RPE, with the possible translocation of the RPE, glial cells and macrophages in the vitreous and the whole posterior segment (Pastor et al., 2002). Morphological characteristics of the membranes

The term “membrane” is used improperly because the tissue comprises cellular components and extracellular matrix (ECM): it is not a sort of “sheet” with a cleavage plane towards the most internal part of the retina, but is often so intimately imbricated with the neurosensory retina that it cannot be surgically resected (Leiderman and Miller, 2009). RPE cells have been identified by light and electron microscopy and immunohistochemistry (Hiscott et al., 1984a,b). An experimental study has demonstrated the presence of proliferating RPE cells in an animal model of PVR, and that RPE cells within the eye may undergo metaplastic change to a macrophage or fibroblast-like morphology (Mandelcorn et al., 1975). They are thought to proliferate once a retinal break allows them to migrate into the ECM. This migration is facilitated by the cleavage of the collagen and elastin determined by metalloproteinases (MMP) (Glaser et al., 1993; Campochiaro, 1997). They also produce a growth factor (pigment epitheliumderived growth factor—PEDF), which contributes to retinal neovascularization (Berger et al., 2010) (Fig. 2). Glial cells have been shown to be present in PVR membranes (van Horn et al., 1977), although the cellular derivation of the glial component remains uncertain. Mueller’s cells, astrocytes, microglia, and perivascular glia have the potential to proliferate and contribute to periretinal membrane formation (Hiscott et al., 1984a,b). When activated, as in PVR, these cells synthesize greater quantities of glial fibrillary acidic protein (GFAP), form networks of processes (gliosis) and regulate the

PROLIFERATIVE VITREORETINOPATHY

Fig. 2. Proliferative vitreoretinopaty. A: Hematoxylin–eosin, original magnification 20. B: Hematoxylin–eosin, original magnification 40. Neovessels, scattered inflammatory cells and myofibroblasts in a very loose connective stroma. C: alfa-smooth muscle actin, original magnification 20. Myofibroblasts immersed in the connective stroma, sometimes boundering the newly formed vessels. D: alfa-smooth muscle actin, original magnification 40. An area particularly rich in myofibroblasts.

production of ECM glycoproteins (laminin, fibronectin, tenascin), as well as anti-adhesive molecules such as thrombospondin 1 (TSP1) and osteonectin (Secreted Protein Acidic and Rich in Cysteine –SPARC). Most studies of periretinal membranes in PVR have identified cells categorized as fibroblasts or fibrocytes. It has been argued that these represent transformed RPE, or that they may originate from vascular endothelial cells, glia, or hyalocytes. The fibroblastic cells may also contain myofibrils, and may therefore be responsible for the contraction of cellular membranes in PVR (Walshe et al., 1992) (Fig. 2). Inflammatory cells have consistently been identified as a component of periretinal membranes in PVR. Early morphological reports identified macrophages within membrane tissue, and immunohistochemical studies have subsequently confirmed their presence in PVR membranes, demonstrating that, in more severe intraocular proliferation, these macrophages tend to be of an acute inflammatory subtype (Baudouin et al., 1990; Esser et al., 1993). Both CD 4þ and CD 8þ T lymphocytes have also been found in PVR membranes (Charteris et al., 1992; Charteris, 1995). The JOURNAL OF CELLULAR PHYSIOLOGY

demonstration of the presence of macrophages and lymphocytes and the observation of MHC class II positive cells in PVR membranes (Elner et al., 1995) has led to increased interest in the role of the immune system in the development in PVR. Deposits of immunoglobulins and complement components have also been demonstrated on PVR epiretinal membranes and on pars plana biopsies taken from eyes with PVR. The cellular adhesion molecule ICAM-1 and LFA-1, which mediate the interaction of leucocytes with other cells and extracellular matrices, have also been found in PVR tissues (Van Zee et al., 1992). The ECM composition of periretinal membranes in PVR has also been analyzed immunohistochemically. Collagen is a characteristically prominent component of the fibrous part of epiretinal membranes (Jerdan et al., 1989): indeed, only a prominent collagen fiber component is capable of exerting the traction that causes detachment (Hiscott et al., 1984a,b; Kim and Arroyo, 2002). Many studies have demonstrated the consistent presence of interstitial collagen of types I and III and the variable presence of type II (vitreous associated) collagen (Morino et al., 1990). Type II collagen is mostly absent from, or

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only a minor element of, PVR epiretinal membranes. Other collagen subtypes, including I and III, are common to most contractile membranes and, as in healing wounds, the collagenous component of periretinal membranes tends to increase with time. It has been postulated that both RPE and glial cells may be responsible for the elaboration of collagen in periretinal membranes (Guidry et al., 1992). The presence of a small quantity of basal lamina proteins—type IV collagen and laminin—has also been demonstrated (Morino et al., 1990). Non-collagenous extracellular components of periretinal membranes include members of the elastic fiber family (though not usually mature elastic fibers) and a number of glycoproteins (Scheifforth et al., 1988). The cell attachment protein fibronectin has been identified as a significant component of PVR membranes. Fibronectin mRNA labeling and protein immunostaining have been demonstrated on retinal glia, RPE, and fibroblastic cells in PVR epiretinal membranes, suggesting an intrinsic production of fibronectin by PVR tissue. Fibronectin in periretinal membranes probably represents both plasma-derived and cellular fibronectins. Soluble fibronectin is chemotactic to many cell types, including RPE cells, and hence may be involved in recruiting cells to the retinal surfaces during early periretinal membrane formation. Moreover, fibronectins promote cell–cell and cell–substrate adhesion, and are responsible for providing early structural integrity in periretinal membranes and for the formation of a “contractile unit” (Hiscott et al., 1992). Proliferation, migration, and shape change require the partial detachment of cells from their substrate. The ECM in wounds contains anti-adhesive proteins, often belonging to a group of proteins known as “matricellular” proteins, which may facilitate cell detachment and hence permit cell proliferation and migration. Matricellular proteins include tenascin, TSP1 and TSP2, SPARC and osteopontin. Tenascin, TSP1 and SPARC have been described in PVR membranes and an association between TSP1 and RPE cells, as well as between SPARC and RPE cells, has been observed in these membranes (Hiscott et al., 2002). Pathogenetic events and factors

Based on the cellular and extracellular composition of the membrane and the fact that PVR is a fibrotic process, pathogenetic investigations tend to establish: (a) how the histological elements that constitute the posterior segment of the eye come to produce the membranes inside the vitreous cavity and on the retinal surfaces, and (b) how the membranes retract and exert traction on the retinal surface. It then remains to be understood what triggers and sustains the fibrotic process and maintains retraction over time. No repair process that replaces a loss of substance, like the healing of a wound by secondary intention (i.e., involving the intervention of granulation tissue), can take place in the absence of damage, such as a traumatic or spontaneous retinal tear, hypoxia (which, at worst, characterizes diabetic retinopathy), or a selective RPE lesion (e.g., neovascular agerelated macular degeneration) (Friedlander, 2007). Blood–retinal barrier breakdown is an important initiating factor in the development of PVR, as it determines cell migration and the proliferation of RPE, glia, fibroblasts, myofibroblasts, macrophages, lymphocytes and occasional polymorphonucleates (Nagasaki et al., 1998; Sadaka and Giuliari, 2012). Astrocytes and Mueller glial cells, fibroblasts and myofibroblasts, macrophages and hyalocytes, RPE cells and lymphocytes are the main participants in cicatricial repair in response to retinal damage (Newsome et al., 1981). Newly formed vessels (as occurs in all granulation tissue) act as a trophic substrate and as a source of blood cells (monocytes and JOURNAL OF CELLULAR PHYSIOLOGY

lymphocytes) (Friedlander, 2007). In adult life, angiogenesis is stimulated by vascular endothelial growth factor (VEGF), fibroblastic growth factor (FGF), angiogenin, transforming growth factor b (TGF-b), interferon b (IFN-b), TGF-a, and platelet derived growth factor (PDFG); it is inhibited by IFN-a, thalidomide, thrombospondin, angiostatin, endostatin, transfer RNA synthetase, and PEDF (Famiglietti et al., 2003; Friedlander, 2007). When, as in the case of PVR, angiogenetic factors prevail, capillary endothelial cells proliferate, migrate and differentiate, being protected by their integrins, by MMPs and by antiapoptotic molecules. In the eye, the dominant angiogenetic factor is VEGF, as demonstrated by anti-VEGF antibodies’ ability to block vascular proliferation in rabbits (Seo et al., 1999). VEGF and its receptors have been identified in the epiretinal membranes of patients with proliferative diabetic retinopathy, PVR and macular pucker (Chen et al., 1997; Toti et al., 1999). These data show that VEGF may be an autocrine and/or paracrine stimulator, which may contribute to the progression of vascular and avascular epiretinal membranes. The scarring process involves growth factors and cytokines that are found in the vitreous and promote an environment favorable to cell proliferation and migration, as well as to the production of ECM proteins (Wiedemann, 1992). Among the growth factors, PDGF is known to be an important factor in intercellular interaction between retinal cells (Fruttiger et al., 1996; Fruttiger, 2002), and its receptor (PDGFR-a) has been detected on the cellular membrane of RPE cells, glial cells and fibroblasts, as well as on the epiretinal membranes and in the vitreous of patients with PVR (Robbins et al., 1994; Moysidis et al., 2012). Of the five PDGF family members, only PDGF-C must undergo proteolytic processing for activation, and it is known that the vitreous contains the proteases capable of accomplishing this activation. The major vitreous PDGF-C processing protease is plasmin, and plasmin activity has been shown to be higher in rabbits with PVR than in control rabbits. Two genes encode PDGF receptors (PDGFR) subunits, which hetero- or homo-dimerize into three different PDGFRs: PDGFRa (aa homodimers), PDGFRb (bb homodimers) and PDGFRab (ab heterodimers). In rabbits, cells expressing PDGFR-a induce PVR much more effectively than cells expressing PDGFR-b, and cells expressing the heterodimer PDGFRab have intermediate potency in inducing PVR (Lei et al., 2010). These results are in agreement with the analysis of human specimens, demonstrating that a higher percentage of PDGFRa is activated in PVR. Non-PDGFs can also activate PDGFRa: for example, bFGF, epidermal growth factor (EGF), insulin, and hepatocyte growth factor (HGF) induce tyrosine phosphorylation of PDGFRa (Lei et al., 2009). Non-PDGFs activate their receptors resulting in an increase in intracellular reactive oxygen species (ROS) and in the activation of Src family kinase (SFK), which leads to the phosphorylation of PDGFRa. New evidence suggests that this indirect pathway, involving non-PDGFs as agonists of PDGFRa, is the primary pathway for activation of this receptor and plays an important role in the pathogenesis of PVR. While direct activation of PDGFRa results in rapid clearance of the receptor from the cell surface and subsequent degradation, indirect activation by non-PDGFs promotes persistent receptor signaling and induces the prolonged activation of phosphatidylinositol 3-kinase (PI3K)/Akt, which activates murine double minute (MDM2) to suppress p53 levels, driving processes intrinsic to PVR-survival, proliferation, and contraction (Lei et al., 2011a,b). A key role in the pathogenesis of PVR is played by TGF-b2, one of the three isoforms of TGF-b, a multifactorial cytokine that regulates the differentiation and migration, apoptosis and immune functions of cells, as well as the synthesis of ECM. TGFb2 has been found in significantly high concentrations in the vitreous of patients with PVR (Kawahara et al., 2008), and in

PROLIFERATIVE VITREORETINOPATHY quantities directly proportional to the extent of fibrosis (Connor et al., 1989). It is correlated with connective tissue growth factor (CTGF), which is also present in elevated concentrations in the vitreous of patients with PVR. TGFb2and CTGF are interdependently involved in the pathogenesis of PVR (Kita et al., 2007). In particular, TGF-b2 is closely correlated with the concentration of hyalocyte-containing collagen gels (Kita et al., 2008). Cytokines such as interleukin 1 (IL-1), interleukin1 b (IL-1b), interleukin 6 (IL-6), tumor necrosis factor a (TNFa), interferon g (IFNg), interleukin 8 (IL-8), interleukin 10 (IL-10), chemokines such as CCL3, CCL4, CCL5, growth factors such as FGF, and monocyte chemotactic protein 1 (MCP-1) have been detected in the vitreous in RNA encoding cells (detected by RTPCR), as well as in epiretinal membranes obtained from PVR patients, indicating a probable local production of these cytokines by vitreous and subretinal fluid cells and their role in the pathogenesis of the disease (Limb et al., 1994; El-Ghrably et al., 1999; Banerjee et al., 2007). There is still no definite answer to the question of what produces these growth factors and cytokines. Some Authors (Kampik et al., 1981; Hiscott et al., 1989; Hui et al., 1989; Jerdan et al., 1989; Baudouin et al., 1990; Vinores et al., 1995; Lin et al., 2011) have demonstrated the presence of macrophages in the periretinal membranes of patients with PVR and, experimentally, that macrophages (together with a few lymphocytes, neutrophils and erythrocytes) harvested from the peritoneal cavity and injected into the vitreous of rabbits caused the formation of epiretinal membranes after 4–9 weeks (Hui et al., 1987). Macrophages mediate the proteolysis of matrix proteins with their enzymes and mediate fibrosis though the synthesis of FGF. FGF is produced by macrophages that have acquired the phenotypical characteristics of fibroblasts (Hui et al., 1989). Moreover, macrophages are known to secrete a series of enzymes and growth factors, including PDGF, which, as pointed out above, has been detected in the vitreous and membranes in PVR. Are macrophages blood monocytes recalled into the vitreo-retinal district? Some Authors maintain that macrophages derive from the metaplasia of RPE cells (Mueller-Jensen et al., 1975). Others specify that RPE metaplasia does not stop, leading the macrophages to take on the immunophenotype of fibroblasts (Esser et al., 1993) and myofibroblasts (Umazume et al., 2012), while preserving the functional characteristics of an acute inflammatory subpopulation of macrophages. The hyalocytes in the cortical part of the vitreous (whose fibrinolytic activity maintains vitreous transparency) are morphologically comparable to macrophages as they originate from blood monocytes (Lazarus and Hageman, 1994; Sakamoto and Ishibashi, 2011), regulate the vitreous cavity immunology (vitreous cavity-associated immune deviation), synthesize ECM, produce cytokines and induce collagen contraction under stimulation from TGF-b2 mediated by Rho and Rho-kinase, and proliferate in response to PDGF (Sakamoto and Ishibashi, 2011). Theories indicating the crucial role of RPE cells in PVR are based on the fact that RPE cells represent the largest cellular component of the epiretinal membranes of patients with PVR (Machemer et al., 1978; Pastor et al., 2002). In conditions of quiescence, RPE cells adhere to the basal lamina, constituted principally of type IV collagen, on the innermost layer of Bruch’s membrane. In the initial phases of PVR, RPE cells are stimulated by TNF-a, which is found in PVR membranes and produced by macrophages and by RPE cells (Jin et al., 2000), and by PDGF to migrate towards the ECM, which is constituted of type I collagen and fibronectin, and to which the RPE cells adhere. TNF-a plays a key role in this migration and adhesion to the ECM by activating PDGF as a chemotactic factor. TNF-a also activates the a1 and a5 subunits of the integrins, which facilitate the migration of RPE cells and their adhesion to the JOURNAL OF CELLULAR PHYSIOLOGY

type I collagen and fibronectin that constitute the ECM, whereas it does not activate the a 3 subunits of the integrins, which would favor their adhesion to type IV collagen, an essential component of the basal lamina to which RPE cells adhere under physiological conditions. This has been demonstrated by the experimental evidence RPE cell migration is blocked by anti-integrin antibodies and by PD98059 which inhibits TNF-a through the block of the mitogen-activated protein kinase (MAPK) pathway (Jin et al., 2000). The adherence of RPE cells to Bruch’s membrane is countered by two matricellular proteins, TSP 1 and SPARC. These proteins, together with tenascin and osteopontin, are particularly strongly expressed in the repair of loss of substance. Thus they favor the migration of RPE cells towards the ECM, where the new fibrous membranes will be formed (Hiscott et al., 2002). It is known that in vitro RPE cells can dedifferentiate and take on the phenotype of fibroblasts and myofibroblasts (Kalluri and Weinberg, 2009; Tamiya et al., 2010), although it is not clear whether this can occur in vivo. PVR has been obtained by injecting RPE cells into the vitreous cavity of a pig, in which retinal detachment due to traction occurred on day 14. In this experiment, the RPE cells maintained their epithelial immunophenotype (they were positive for cytokeratins), but had acquired the morphology and positivity to vimentin typical of fibroblasts, and in some cases even the immunophenotype of myofibroblasts (positivity to a-SMA) (Umazume et al., 2012). Most of the contractile elements were RPE cells and their metaplastic, fibroblastic and myofibroblastic derivates, while astrocytes and Mueller glial cells were absent, perhaps due to the limited observation period of 14 days (Umazume et al., 2012). These data obtained from patients, animals and in vitro, show that RPE cells, with their migration, proliferation, fibroblastic and myofibroblastic transformation, and therefore their contractile activity, play a key role in PVR pathogenesis (an “Achilles heel” according to Lei et al., 2011a,b). Based on the finding of fibroblastic cells, some of which have been demonstrated to contain cytoplasmic myofilaments, it has been proposed that membrane shortening is mediated by the intrinsic contraction of these cells, thus producing the tractional forces responsible for clinical signs in PVR. An in vitro study has shown that both RPE and fibroblasts, but not retinal glia, are capable of mediating the contraction of a type I collagen matrix by cellular mobility and the subsequent attachment of collagen fibers (Mazure and Grierson, 1992). Another experimental study has suggested an alternative mechanism of membrane contraction, involving the interaction of RPE cells and collagen (Glaser et al., 1987). In this mechanism, collagen fibers are pulled by the RPE cells alternating the extension and retraction of their lamellipodia (fibronectin serving as a bridge between RPE and collagen) and collagen is piled up adjacent to the RPE cells, with subsequent tissue shortening. The migration and contractile activity of RPE cells is stimulated not only by PDGFR-a, in fact RPE cells maintain the capacity to induce PVR even when PDGFR-a expression is very low. An important role might be played by p53, which decreases in cells that overexpress PDGFR-a and contract. The question is whether or not p53 is a factor that prevents contraction, and therefore if its mutation or low expression can increase contraction (Lei et al., 2011a,b). It should be added that p53 can inhibit retinal angiogenesis through the activation of small MDM2 inhibitor molecules, such as Nutlin-3 (Chavala et al., 2013). Conclusions

Amacrine and ganglion cells in the normal adult retina, as well as retinal glial cells (Mueller cells in particular), provide continuous trophic support for their retinal blood supply, as documented by Famiglietti et al. (2003), who found VEGF synthesis and export from the abovementioned cells in normal

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adult retinas. When rhegmatogenous retinal detachment occurs two primary events become the initiating factors of the potential PVR cascade: the breakdown of blood–retinal barriers and retinal hypoxia (Nagasaki et al., 1998; Pastor et al., 2002; Friedlander, 2007). The breakdown of the internal blood–retinal barrier can be considered “traumatic” or “direct,” due to the presence of a retinal break and consequent anatomical disruption of the major retinal vessels and/or of the capillary plexa, while the breakdown of the external blood– retinal barrier can be considered “atraumatic” (no apparent break in the pigment epithelial cell layer) or “indirect.” As in granulation tissue (Pastor et al., 2002), this breakdown leads to an invasion of inflammatory cells (most likely macrophages from systemic circulation) together with protein leakage into the eye (Nagasaki et al., 1998; Pastor et al., 2002). This “intrusion” might initially be the consequence of the blood retinal barrier disruption and eventually be exacerbated by the neovascularization induced by retinal ischemia (Toti et al., 1999; Friedlander, 2007). Cytokines from systemic circulation and growth factors (PDGF) produced by blood macrophages and by activated hyalocytes (Sakamoto and Ishibashi, 2011) start an interaction with habitual intraocular cells (RPE cells and glial cells), culminating in PVR development. In particular, RPE cells migrating from their natural site are observed in the vitreous cavity (grade A PVR); a stiffness of the detached retina, most probably due to intraretinal fibrosis induced by the stimulated retinal glia, is observed (grade B PVR) and eventually definite epiretinal and subretinal membranes form (grade C PVR) due to the presence of fibroblasts and myofibroblasts. PVR consequent to retinal detachment is still a challenging scenario for clinicians and basic researchers; its incidence remains high compared to other conditions characterized by disruptions of the blood–retinal barrier (e.g., uveitis, aphakia, choroidal detachment). This could be due to the additional complete disruption of the normal cell–cell relationships (i.e., due to direct contact between the subretinal and supraretinal space), with the consequent disruption of ocular homeostasis. Future investigations should aim to confirm the pathogenetic pathway/mechanisms of the disease and better explore the clinical scenarios that have been less thoroughly studied to date (e.g., intraretinal and subretinal fibrosis). More frequent cooperation between clinicians and basic researchers would undoubtedly be beneficial. Literature Cited Banerjee S, Savant V, Scott RAH, Curnow J, Wallace GR, Murray PI. 2007. Multiplex bead analysis of vitreous humor of patients with vitreoretinal disorders. Invest Ophthalmol Vis Sci 48:2203–2207. Baudouin C, Fredj-Reygrobellet D, Gordon WC, Baudouin F, Peyman G, Lapalus P, Gastaud P, Bazan NG. 1990. Immunohistologic study of epiretinal membranes in proliferative vitreoretinopathy. Am J Ophthalmol 110:593–598. Berger W, Klockener-Gruissem B, Neidhardt J. 2010. The molecular basis of human retinal and vitreoretinal diseases. Prog Retin Eye Res 29:335–375. Campochiaro PA. 1997. Pathogenetic mechanisms in proliferative vitreoretinopathy. Arch Ophthalmol 115:237–241. Cardillo JA, Stout T, LaBree L, Azen S, Omphroy L, Cui ZJ, Kimura H, Hinton DR, Ryan SJ. 1997. Post-traumatic proliferative vitreoretinopathy: The epidemiologic profile, onset, risk factors, and visual outcome. Ophthalmology 104:1166–1173. Charteris DG. 1995. Proliferative vitreoretinopathy: Pathobiology, surgical management, and adjunctive treatment. Br J Ophthalmol 79:953–960. Charteris DG, Hiscott P, Grierson I, Lightman S. 1992. Proliferative vitreoretinopathy: Lymphocytes in epiretinal membranes. Ophthalmology 99:1364–1367. Charteris DG, Sethi CS, Lewis GP, Fisher SK. 2002. Proliferative vitreoretinopathy— Developments in adjunctive treatment and retinal pathology. Eye 16:369–374. Chavala SH, Kim Y, Tudisco L, Cicatiello V, Milde T, Kerur N, Claros N, Yanni S, Guaiquil H, Hauswirth WW, Penn JS, Rafii S, De Falco S, Lee TC, Ambati J. 2013. Retinal angiogenesis suppression through small molecule activation of p53. J Clin Invest 123:4170–4181. Chen YS, Hackett SF, Schoenfeld CL, Vinores MA, Vinores SA, Campochiaro PA. 1997. Localisation of vascular endothelial growth factor and its receptors to cells of vascular and avascular epiretinal membranes. Br J Ophthalmol 81:919–926. Connor TB , Jr. , Roberts AB, Sporn MB, Danielpour D, Dart LL, Michels RG, de Bustros S, Enger C, Kato H, Lansing M. 1989. Correlation of fibrosis and transforming growth factorbeta type 2 levels in the eye. J Clin Invest 83:1661–1666. El-Ghrably IA, Dua HS, Orr GM, Fischer D, TighePj.. 1999. Detection of cytokine mRNA production in infiltrating cells in proliferative vitreoretinopathy using reverse transcription polymerase chain reaction. Br J Ophthalmol 83:1296–1299.

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