Fibrosis and diseases of the eye
`
`Martin Friedlander
`
`J Clin Invest. 2007;117(3):576-586. https://doi.org/10.1172/JCI31030.
`
`Review Series
`
`Most diseases that cause catastrophic loss of vision do so as a result of abnormal
`angiogenesis and wound healing, often in response to tissue ischemia or inflammation.
`Disruption of the highly ordered tissue architecture in the eye caused by vascular leakage,
`hemorrhage, and concomitant fibrosis can lead to mechanical disruption of the visual axis
`and/or biological malfunctioning. An increased understanding of inflammation, wound
`healing, and angiogenesis has led to the development of drugs effective in modulating
`these biological processes and, in certain circumstances, the preservation of vision.
`Unfortunately, such pharmacological interventions often are too little, too late, and
`progression of vision loss frequently occurs. The recent development of progenitor and/or
`stem cell technologies holds promise for the treatment of currently incurable ocular
`diseases.
`
`Find the latest version:
`
`http://jci.me/31030-pdf
`
`Lassen - Exhibit 1018, p. 1
`
`

`

`Review series
`
`Fibrosis and diseases of the eye
`
`Martin Friedlander
`
`Department of Cell Biology, The Scripps Research Institute, and Division of Ophthalmology, Scripps Clinic, La Jolla, California, USA.
`
`Most diseases that cause catastrophic loss of vision do so as a result of abnormal angiogenesis and wound healing,
`often in response to tissue ischemia or inflammation. Disruption of the highly ordered tissue architecture in the
`eye caused by vascular leakage, hemorrhage, and concomitant fibrosis can lead to mechanical disruption of the
`visual axis and/or biological malfunctioning. An increased understanding of inflammation, wound healing, and
`angiogenesis has led to the development of drugs effective in modulating these biological processes and, in certain
`circumstances, the preservation of vision. Unfortunately, such pharmacological interventions often are too little,
`too late, and progression of vision loss frequently occurs. The recent development of progenitor and/or stem cell
`technologies holds promise for the treatment of currently incurable ocular diseases.
`
`Introduction
`To see well, we must maintain a clear visual axis and normally func-
`tioning cellular phototransduction. Light entering the eye passes 
`through the cornea (the major refractive surface), the lens, the vitre-
`ous (gel in the posterior chamber of the eye), the inner retina, and, 
`finally, into the photoreceptors of the outer retina (Figure 1). These 
`photoreceptors are the site at which photons of light are converted 
`into electrical signals that are transmitted to the visual cortex of the 
`brain by a complex series of synaptic transmissions (Figure 1). To 
`maintain a visual axis through which light can pass undisturbed, 
`a highly ordered tissue structure is required. Any disturbance in 
`normal cell-cell relationships can lead to biological malfunctioning 
`and/or diffraction, absorbance, or reflection of photons, resulting 
`in disturbed or diminished vision.
`Homeostasis of the eye, as in tissues elsewhere in the body, 
`depends on the presence of normal vasculature, ECM, and vari-
`ous cell types. If homeostasis is disturbed by infection, inflam-
`mation, or metabolic disease, visual function becomes impaired. 
`The end result of these conditions is often fibrosis. In the CNS, 
`of which the retina is a part, such wound-healing responses and 
`associated fibrosis are mediated by glial cells, which perform 
`functions in the CNS similar to those performed by fibroblasts 
`in  the  rest  of  the  body.  Therefore,  gliosis  is  frequently  used 
`to  describe  the  glial  cell–mediated  wound-healing  response 
`observed in the CNS, much as fibrosis (which is fibroblast medi-
`ated) is used to describe similar processes in non-CNS tissues. In 
`the skin, fibrosis can lead to a cosmetic blemish in the form of a 
`scar; in the eye this can have disastrous consequences for vision 
`— mechanically disrupting the visual axis or sufficiently disturb-
`ing the tissue microenvironment such that proper cellular func-
`tioning is no longer possible. For example, fibrosis of the cornea 
`can occur after a viral infection, leading to corneal opacification 
`and thereby loss of vision. In the posterior segment of the eye 
`(Figure 1), uncontrolled retinal vascular proliferation, as a result 
`of diabetes-associated retinal hypoxia, can lead to fibrosis and 
`traction retinal detachment, a dreaded complication of advanced 
`diabetic retinopathy (DR). Under the retina, similar fibrosis can 
`
`Nonstandard abbreviations used: ARMD, age-related macular degeneration; 
`CNTF, ciliary neurotrophic factor; DR, diabetic retinopathy; EPC, endothelial  
`progenitor cell; PEX, carboxyterminal, noncatalytic domain of MMP-2; ROP,  
`retinopathy of prematurity; RPE, retinal pigmented epithelium; TIMP, tissue  
`inhibitor of metalloproteinases.
`Conflict of interest: The author has declared that no conflict of interest exists.
`Citation for this article: J. Clin. Invest. 117:576–586 (2007). doi:10.1172/JCI31030.
`
`occur subsequent to subretinal hemorrhage associated with neo-
`vascular age-related macular degeneration (ARMD).
`Collectively, these conditions of fibrosis in the eye lead to vision 
`loss in millions of individuals worldwide. In this Review, I discuss 
`the cellular pathophysiology associated with fibrosis in the anterior 
`and posterior segments of the eye (Figure 1), with a focus on the 
`latter. Therapeutic approaches for treating these disorders, based 
`on advances in our understanding of the biological mechanisms 
`underlying these conditions, are reviewed and then discussed in the 
`context of recent novel advances in the area of cell-based therapies.
`
`Fibrosis in the eye: general considerations
`Fibrosis commonly refers to the response of a tissue to injury. 
`The injury can occur as a result of a mechanical wound or various 
`metabolic malfunctions, including responses to inflammation, 
`ischemia, and degenerative disease. The local response to such 
`injuries includes infiltration by inflammatory cells, neovascular-
`ization, altered vascular permeability, proliferation of fibroblasts 
`and fibroblast-like cells, modification of the ECM, and, ultimately, 
`some sort of resolution of the damaged tissue. The CNS is highly 
`specialized in many ways, including the types of inflammatory 
`and wound-healing cells present. Since the retina is part of the 
`CNS, its response to injury utilizes mechanisms very similar to 
`those observed in the rest of the brain; this is true not only for the 
`wound-healing response but also for utilization of migratory cues 
`functional during development of the neuronal and vascular com-
`ponents of this highly organized tissue (1, 2). As discussed below, 
`the response of the anterior segment of the eye to wound healing 
`more closely resembles the response of non-CNS tissues than do 
`such events in the posterior segment or the eye. Therefore, I refer 
`to such wound-healing events in the anterior segment as fibrosis, 
`whereas comparable events in the retina are referred to as gliosis. 
`Although such distinction is somewhat artificial, it does serve to 
`differentiate between the fibroblasts and glial cells that effect the 
`wound-healing and scar-formation events.
`
`Anterior segment fibrotic diseases of the eye
`Two major diseases of the anterior segment of the eye leading to 
`visual loss are corneal opacification and glaucoma. In glaucoma, 
`there is progressive loss of ganglion cells of the nerve fiber layer; 
`this results in degeneration of the neuronal tracts through which 
`efferent signals travel from the retina to the visual cortex (3). Typi-
`cally associated with increased intraocular pressure, this disease 
`can lead to progressive constriction of the visual fields and, even-
`
`576
`
`The Journal of Clinical Investigation      http://www.jci.org      Volume 117      Number 3      March 2007
`
`Lassen - Exhibit 1018, p. 2
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`

`

`review series
`
`Figure 1
`Schematic representation of the eye and principal types of retinal neovascularization and fibrosis/gliosis. (A) The anterior segment of the eye,
`consisting primarily of the cornea and iris, is separated from the posterior segment by the lens. The posterior segment consists primarily of the
`vitreous and the retina. (B) The retina is a highly ordered, multilayered structure that is richly vascularized. Ischemic retinopathies, such as DR,
`can lead to ischemia and neovascularization on the surface of the retina. (C) In extreme cases, associated gliosis can lead to tractional retinal
`detachments. Reproduced with permission from the American Academy of Ophthalmology (122). (D) ARMD can be associated with subretinal
`neovascularization originating from the choriocapillaris, and this can lead to subretinal hemorrhage and fibrosis (E).
`
`tually, complete loss of vision. Although increased intraocular 
`pressure can occur from either increased production of intraocu-
`lar fluid or increased resistance to outflow, it is more commonly 
`believed that progressive fibrosis of the tracts through which the 
`intraocular fluid leaves the eye (called the trabecular meshwork) 
`accounts for most of the damage that causes glaucoma. Increased 
`understanding of the molecular basis for malfunctioning of the 
`trabecular meshwork (4) (in particular, the aberrant production 
`of ECM components) and of the fibrosis associated with increased 
`resistance to outflow, holds promise for developing therapeutics 
`for this relentlessly progressive disease (5).
`Although there are those who consider the cornea simply a “dust-
`cover for the retina,” it in fact is a highly organized tissue through 
`
`which light must pass before entering the rest of the eye. The cor-
`nea is covered externally by a stratified nonkeratinizing epithelium 
`and internally by a single layer of transporting endothelium with 
`multiple orthogonal arrays of collagen in between. It is normally 
`avascular due to the high concentration of soluble VEGFR-1 (6) 
`and is surrounded by a transitional margin, the corneal limbus, 
`within which resides nascent endothelium and corneal epithelial 
`stem cells (7), which have high potential for therapeutic value (8). 
`Diseases of the cornea can be genetic (e.g., inherited dystrophies) or 
`acquired secondary to infection (e.g., herpetic keratitis) or inflam-
`mation (e.g., pterygia). Elastoid degeneration of the conjunctiva, 
`resulting in pingueculae and pterygia (fibrovascular growths on 
`the surface of the cornea), can lead to visual loss secondary to 
`
`
`
`The Journal of Clinical Investigation      http://www.jci.org      Volume 117      Number 3      March 2007 
`
`577
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`Lassen - Exhibit 1018, p. 3
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`review series
`
`induced astigmatism and/or obstruction of the visual axis and 
`would be amenable to topically applied inhibitors of fibrosis 
`and/or angiogenesis (9). The final common events in all of these 
`diseases are often inflammatory changes associated with neovas-
`cularization, tissue edema, and, ultimately, fibrosis of the corneal 
`stroma, which leads to opacification and decreased vision (10). 
`Nearly 20 years ago, penetrating keratoplasty (or corneal trans-
`plants) changed the uniformly dismal prognosis for patients with 
`opacified or failed corneas; in a substantial percentage of patients 
`undergoing this procedure, if there are no other associated abnor-
`malities, the visual axis is cleared and vision is restored. Despite 
`advances in the use of antiinflammatory drugs, antibiotics, and 
`hypertonic solutions to reduce corneal edema associated with the 
`immune response to the transplant, there is a substantial failure 
`rate, typically due to recurrent opacification. Recent advances in 
`corneal limbal stem cell biology hold the promise of reducing the 
`failure rate for this procedure (11).
`
`Posterior segment fibrotic diseases of the eye
`General comments. The posterior segment of the eye consists of 
`structures behind the lens; the interior of the back of the eye is 
`filled with vitreous, a viscoelastic material consisting largely of 
`water, collagen, and hyaluronic acid (12). The vitreous serves as 
`a shock absorber, among other things, for the retina, the most 
`posterior tissue in the eye. In addition, the vitreous can provide 
`scaffolding over which glial and endothelial cells migrate from 
`their normal intraretinal position anteriorly over the retinal sur-
`face and/or into the vitreous in certain disease states (e.g., diabetes, 
`proliferative vitreoretinopathy, retinopathy of prematurity [ROP]). 
`The retina consists of multiple layers of neurons, blood vessels, 
`ECM, and various resident and transient cells such as glial cells 
`and monocytes. The vascular supply of the retina consists of the 
`retinal blood vessels (found in three layers on the innermost por-
`tion of the retina) and the choriocapillaris (a rich vascular plexus 
`found in the outermost portion of the retina). The photoreceptors 
`are in the outermost portion of the neurosensory retina and rest 
`on a monolayer of cells, the retinal pigmented epithelium (RPE), 
`discussed further below. The RPE rests on a collagenous basement 
`membrane (Bruch membrane), and directly beneath this structure 
`flows the choriocapillaris, providing blood supply for the outer 
`third of the retina. Although there is a blood-retina barrier and 
`relative immune privilege in this part of the eye, normal inflam-
`matory responses to irritation and hypoxia can be quite robust 
`and can lead to much of the pathology observed in diseases that 
`decrease vision (Figure 1).
`Most diseases that lead to vision loss in industrialized nations 
`do  so  as  a  result  of  abnormalities  in  the  retinal  or  choroidal 
`vasculature. These diseases, characterized by macula edema, reti-
`nal and vitreous hemorrhage, and fibrovascular scarring, include 
`ARMD, DR, ROP, and neovascular glaucoma. The final common 
`pathophysiological denominator in all of these diseases is the 
`retinal response to injury, with chronic wound healing leading to 
`fibrosis. Although the underlying principles of wound healing in 
`other tissues apply to this process in the eye, it is the uniqueness 
`of the cellular composition and anatomical structure of the retina 
`that makes this normal biological process so potentially devastat-
`ing to vision. The photoreceptors are located in the outermost 
`portion of the neurosensory retina, just anterior to the RPE and 
`choriocapillaris. Overlying these cells are the vitreous, nerve fiber 
`layer, inner nuclear layer, numerous capillary plexuses, and Muel-
`
`ler-glial cells and their processes (Figure 1). For light to hit the 
`photoreceptors in an undisturbed manner such that visual images 
`can be formed, it is important that the highly organized architec-
`ture of the retina is preserved. When abnormal blood vessels form 
`in response to inflammatory or hypoxic stimuli, they can leak 
`fluid, causing retinal thickening and edema, and/or bleed, leading 
`to fibrovascular proliferation and tractional retinal detachment. 
`The following discussion focuses on the unique aspects of wound 
`healing, fibrosis, and scar formation as it occurs in the posterior 
`segment of the eye.
`Fibrovascular scarring and gliosis in the retina. In simplest terms, 
`fibrovascular scarring is a consequence of the underlying inflam-
`matory or hypoxia-driven neovascularization and its associated 
`fibrosis. Therefore, prevention of the primary vascular abnormality 
`is the most appropriate therapeutic target to preserve retinal struc-
`ture and function. To understand fibrosis and its consequences in 
`the back of the eye, understanding the unique aspects of retinal 
`fibrosis is necessary. Glial cells are the CNS counterparts of periph-
`eral fibroblasts, with several key distinctions, and are therefore the 
`primary participants in the formation of fibrotic scars in response 
`to retinal injury and disease. In addition to their fibrotic tenden-
`cies, glial cells also perform a myriad of supportive functions for 
`the neurons with which they are intimately associated. In the reti-
`na, this trophic relationship to neurons is extended to the vascular 
`endothelium, with which certain glia are intimately associated in 
`both developing and mature tissue. For example, activated astro-
`cytes form the template over which retinal vascular endothelial 
`cells migrate during formation of the superficial vascular plexus 
`(1, 13); disturbances in the number or distribution of these cells 
`disrupts the normal development of the retinal vasculature (14). 
`Glial cells of the retina include the resident immune cells, microg-
`lial cells, and two types of macroglial cell, the astrocyte and the 
`retina-specific Mueller-glial cell (15). Two broad categories of dis-
`ease account for most of the conditions that lead to fibrovascular 
`scarring in the retina and its associated vision loss — inflammatory 
`diseases (e.g., ARMD) and ischemic diseases (e.g., DR).
`Subretinal fibrosis: ARMD. The leading cause of vision loss in 
`Americans over the age of 65 is ARMD; 12–15 million Americans 
`over the age of 65 have this disease and 10%–15% of them will lose 
`central vision as a direct effect of choroidal (subretinal) neovascu-
`larization and fibrosis. Clinically, most of these individuals develop 
`atrophic changes in the RPE, which performs a myriad of functions 
`associated with normal photoreceptor functioning (16) and is the 
`cellular interface between the underlying choriocapillaris and the 
`outermost portion of the neurosensory retina, the photoreceptors. 
`As the RPE ages or becomes diseased, it can function improperly, 
`and a build-up of subretinal deposits, called drusen, accumulate. 
`These drusen contain, among other things, angiogenic lipids and 
`damaged proteins (17). RPE dysfunction and the accumulation of 
`drusen can lead to thickening of Bruch membrane, and the accu-
`mulation of angiogenic drusen associated with this fibrosis can 
`lead to decreased diffusion of oxygen from the choriocapillaris to 
`the photoreceptors, further exacerbating conditions that can lead 
`to choroidal neovascularization. Once these new abnormal blood 
`vessels begin to grow in the subretinal space, they often hemor-
`rhage, leading to further wound-healing responses and, ultimately, 
`to subretinal fibrosis (Figure 1, D and E). Needless to say, local 
`destruction of photoreceptors, the RPE, and choroidal blood ves-
`sels leads to permanent reduction in macular function and vision. 
`Efforts to develop animal models to study this process have been 
`
`578
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`The Journal of Clinical Investigation      http://www.jci.org      Volume 117      Number 3      March 2007
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`Table 1
`Molecules with angiogenic activity in the eye
`
`Name
`Angiogenin
`
`Angiopoietin-1
`
`Angiopoietin-2
`
`FGFs
`
`IGF-1
`
`
`Integrins
`
`
`IL-8
`
`PlGF
`
`PDGF-BB
`
`TGF-β
`
`
`TNF-α
`
`
`VE-cadherin
`
`
`VEGF
`
`
`General angiogenic activity
`Increases EC proliferation
`Promotes tubular organization in vitro
`Stabilizes neovessels
`Matures neovessels
`Can be angiogenic or angiostatic (depends
` on cofactors)
`Increases angiogenesis in vitro and in vivo
`
`Expression correlates with angiogenesis
`Expression correlates with tumor metastasis
`
`Some (e.g., αvβ3, and αvβ5) are critical
` for vessel growth and survival
`
`Increases EC proliferation
`Increases angiogenesis
`Specific modulator of EC response to VEGF
` during angiogenesis
`Induces VEGF expression
`Increases angiogenesis
`Low doses increase angiogenesis
`High doses decrease angiogenesis
`Inflammatory
`Increases EC proliferation
`Increases growth factor effects
`High doses decrease angiogenesis
`Critical for EC intracellular adhesion
`Modulates VEGF activity
`
`Critical proangiogenic growth factor
`
`
`Angiogenic activity in the eye
`Increased in vitreous of patients
` with PDR and PVR
`Important role during development
`Important role in pathological NV
`Can increase ischemia-induced NV
`Increased in patients with PDR
`Associated with choroidal and retinal NV
`
`Mediates VEGF-induced NV in ischemic
` retinopathies
`
`αvβ3 mediates basic FGF-increased
` angiogenesis
`αvβ5 mediates VEGF-increased angiogenesis
`Associated with ischemic retinal NV
`Associated with inflammation
`Increased in human CNV
`Inhibition increased NV in mouse
`Might mediate pericyte recruitment
`Might mediate vascular stabilization
`Increases vascular permeability in the retina
` by increased MMP9
`
`Clinical use
`Possible tumor prognostic
` marker
`Might prevent vessel
` permeability in the eye
`Under evaluation for
` potential clinical use
`Under evaluation for
` potential clinical use
`Somatostatin analogs in
` clinical trials to treat diabetic
` retinopathy
`Integrin antagonists are being
` tested as potent angiostatics
`
`Under evaluation for potential
` clinical use
`Under evaluation for potential
` clinical use
`Under evaluation for potential
` clinical use
`Under evaluation for potential
` clinical use
`
`Associated with various ocular diseases
` with related NV
`
`Infliximab (TNF-α–specific
` antibody)
`
`Retinal vascular development
`
`
`Vascular development, pathological NV
`
`
`Inhibitory antibodies and
` T2-TrpRS are antiangiogenics
` for tumors or ocular diseases
`Multiple anti-VEGF treatments
` in the clinic or clinical trials
`
`NV, neovascularization; PlGF, placental growth factor; PDR proliferative DR; PVR, proliferative vitreoretinopathy, VE-cadherin, vascular endothelial cadherin.
`
`hampered by the fact that rodents do not seem to faithfully mimic 
`the human disease, although transgenic mice have provided some 
`use in this regard (18).
`Advances in therapeutic options available to treat neovascular 
`ARMD have provided some benefit to small subsets of patients 
`with this disease (19, 20). Most drugs currently in clinical trials or 
`approved for treating ARMD-associated choroidal neovasculariza-
`tion are directed at inhibiting promoters of angiogenesis, such as 
`VEGF. There is extensive literature covering these approaches, and 
`I refer the reader to several excellent recent reviews (refs. 16, 19). 
`Unfortunately, inhibiting angiogenic cytokines does not address 
`the underlying pathophysiology — ischemia and inflammatory 
`stimuli. Efforts to minimize sub- and epiretinal fibrosis have met 
`with limited success and, in any event, would represent a thera-
`peutic intervention occurring too late to rescue vision, since such 
`scarring would have already led to photoreceptor death.
`Epiretinal fibrosis: DR. The leading cause of visual loss for Ameri-
`cans  under  the  age  of  65  is  diabetes;  6%–8%  of  the  American 
`population is diabetic, and 40,000 patients each year suffer visual 
`loss from complications of the disease, often as a result of reti-
`nal edema or neovascularization (21). Virtually every diabetic has 
`some form of DR after 20 years of the disease (21). Ischemia occurs 
`as a result of the diabetic microvasculopathy that includes peri-
`
`cyte cell death, microaneurysms, intraretinal microvascular abnor-
`malities, altered vascular permeability, and macular edema (22). 
`As the hypoxia increases, neovascularization can occur, leading to 
`intraretinal, subhyaloid (between the retinal surface and posterior 
`vitreous base) and vitreous hemorrhage (Figure 1B). These prolif-
`erating blood vessels are accompanied by fibrosis that occurs as a 
`consequence of glial cell activation and proliferation (gliosis) (Fig-
`ure 1C). As abnormal vessels continue to proliferate on the retinal 
`surface, they can extend into the vitreous and contract, causing 
`traction on the retinal surface and leading to retinal detachment, a 
`dreaded complication of proliferative DR. Retinal neovasculariza-
`tion and associated gliosis and fibrosis are also observed in ROP 
`(23) and as a complication of surgery to treat retinal detachment 
`(24, 25). Surgical intervention and laser obliteration of the periph-
`eral retina (to decrease the metabolic demand and thereby match 
`up supply and demand) are the current treatments and are of 
`limited benefit. Although animal models of ischemic retinopathy 
`have been very useful in helping to develop a better understanding 
`of factors that control retinal vascular proliferation (24, 26), the 
`rodent does not develop the associated preretinal fibrosis, limiting 
`its utility in studying the gliosis observed in the human condition. 
`Given that abnormal vascular proliferation serves as the stimulus 
`for pathological fibrotic responses in these diseases, the following 
`
`
`
`The Journal of Clinical Investigation      http://www.jci.org      Volume 117      Number 3      March 2007 
`
`579
`
`Lassen - Exhibit 1018, p. 5
`
`

`

`review series
`
`discussion focuses on known basic molecular and/or biological 
`pathways of angiogenesis and rational approaches to therapeutic 
`interventions based on this knowledge.
`
`Neovascularization of the retina leads to gliosis
`and fibrous scarring
`General considerations. The ocular response to hypoxia and inflam-
`matory insults typically leads to retinal or choroidal neovascu-
`larization. During development, this process is highly regulat-
`ed and leads to the establishment of a well organized, mature 
`vasculature (1). In the adult eye, this is often not the case, and 
`associated glial cells (e.g., astrocytes, microglia, and Mueller-glial 
`cells) proliferate with the endothelial cells, leading to fibrosis and 
`scar formation. To understand this gliotic response, it is impor-
`tant to understand angiogenesis.
`In the normal adult, angiogenesis (defined as the growth of 
`new blood vessels from preexisting ones) is tightly regulated and 
`limited to wound healing, pregnancy, and uterine cycling. Our 
`understanding of the molecular events involved in the angiogenic 
`process has advanced substantially since the purification of the 
`first angiogenic molecules nearly two decades ago (27). This pro-
`cess, under physiologic conditions, can be activated by specific 
`angiogenic molecules (Table 1), such as basic and acidic FGF (28), 
`VEGF (29), angiogenin (30), TGF-β (31), IFN-β (32), TNF-α (33), 
`and PDGF (34). Angiogenesis can also be suppressed by inhibitory 
`molecules (Table 2), such as IFN-α (35), thalidomide (36), throm-
`bospondin-1 (37), angiostatin (38), endostatin (39), a naturally 
`occurring form of the carboxyterminal, noncatalytic domain of 
`MMP-2 (PEX) (40), transfer RNA (tRNA) synthetases (41, 42), and 
`pigment epithelium–derived factor (43). It is the balance of these 
`naturally occurring stimulators and inhibitors of angiogenesis 
`that is thought to tightly control the normally quiescent capillary 
`vasculature (44). When this balance is upset, as in certain disease 
`states (e.g. DR), capillary endothelial cells are induced to prolifer-
`ate, migrate, and differentiate.
`The role of cell adhesion molecules, such as integrins, in regulat-
`ing the relationship between proliferating vascular cells and their 
`environment, has been the focus of many studies (45). At least three 
`cytokine-dependent pathways of angiogenesis have been described 
`and defined by their dependency on distinct vascular cell integrins, 
`αvβ3 (46), αvβ5 (47), and αvβ1 (48). Cell migration through the ECM 
`also depends on proteolysis of the matrix. Integrins (32, 49, 50), 
`MMPs (51–53), and tissue inhibitors of metalloproteinases (TIMPs) 
`(54) are found throughout the eye, where they interact with each 
`other (40) to maintain a quiescent vasculature until the balance is 
`upset, resulting in pathological angiogenesis. Signaling molecules, 
`including SRC tyrosine kinases (55), modify endothelial cell behav-
`ioral responses to changes in the microenvironment, and similar 
`pathways are operational in migrating neurons, differentiating 
`progenitor cells, and glial cells (56). Vascular endothelial cells are 
`protected from apoptotic stimuli by αv integrin subunit interaction 
`with RAF kinase (57). This response is differentially regulated by 
`two distinct pathways, one involving FGF and the other involving 
`VEGF-stimulated endothelial cell apoptosis (47).
`Ocular angiogenesis. Several reports suggest that VEGF is the domi-
`nant angiogenic stimulus in experimentally induced iris neovascu-
`larization (58, 59) as well as endogenous neovascular retinopathies 
`(60–62). Although there clearly is a direct correlation between intra-
`ocular VEGF levels and ischemic retinopathic ocular neovasculariza-
`tion, a role for FGF cannot be ruled out (63–65). Substantial inhibi-
`
`tion of retinal vascular proliferation in a mouse model of hypoxic 
`retinopathy is observed with antibody (66) and aptamer (67) antag-
`onists of VEGF as well as with selective PKC antagonists (68).
`Although normal human ocular blood vessels do not ordinar-
`ily display αvβ3 or αvβ5 integrins, only αvβ3 has been consistently 
`observed in ocular tissue from patients with ARMD, whereas both 
`αvβ3 and αvβ5 were present in tissues from patients with prolif-
`erative DR (49). Systemically administered peptide antagonists 
`of both integrins blocked new blood vessel formation in a mouse 
`model of retinal angiogenesis (49, 69, 70), reinforcing the con-
`cept that both integrins might have a role in active fibrovascu-
`lar proliferation of the type seen in proliferative DR. Consistent 
`with the concept that integrins such as αvβ3 and αvβ5 are required 
`for proliferating endothelial cells to successfully navigate the 
`extracellular milieu is the observation that these integrins can 
`selectively bind MMPs, including PEX (40). Small-molecule PEX 
`mimetics also bind αvβ3, preventing binding of MMP2, and there-
`by mimicking the action of PEX (71, 72). Clearly, interactions 
`between developing vasculature and the ECM are critical during 
`normal and abnormal angiogenesis (73).
`The  antiangiogenic  activity  of  several  compounds  exhibits 
`strain-related differences in various animal models of angiogen-
`esis (74). Steroids (75), in particular an angiostatic steroid devoid 
`of glucocorticoid activity (anecortave acetate) (76, 77), pigment 
`epithelium–derived factor (43), MMP antagonists (78), and soma-
`tostatin analogs (79–81) have demonstrated potent antiangiogen-
`ic activity in various animal models of ocular neovascularization. 
`Laser photocoagulation has been effective in preventing severe 
`visual loss in subgroups of high-risk diabetic patients but, with 
`few exceptions, has not been effective at preventing visual loss 
`in patients with choroidal neovascularization due to ARMD or 
`inflammatory eye diseases such as ocular histoplasmosis. Photo-
`dynamic therapy using nonthermal lasers to activate photoacti-
`vateable dyes reduces severe vision loss in a small subset of ARMD 
`patients (82). Very recent substantial advances in the use of anti-
`angiogenic monotherapy, principally VEGF antagonists, have led 
`to the reduction of severe vision loss in a subset of patients with 
`ARMD (83). Although slowing, or even minimally improving, 
`vision loss in some patients, this approach does not offer relief 
`for the underlying condition or the ischemia driving the neovas-
`cularization. Thus, a substantial challenge in developing effective 
`treatments for these diseases remains the relief of retinal isch-
`emia. Combination therapy holds great promise in this area (84) 
`but remains relatively unexplored.
`Retinal neovascularization leads to gliosis. Retinal glial cells, and 
`astrocytes in particular, have an important role in establishing and 
`maintaining the highly ordered retinal vasculature (1, 14). Retinal 
`injury due to hypoxia or inflammatory changes, similar to injuries 
`in other tissues, is typically associated with neovascularization and 
`fibrosis (85). Reactive gliosis can be both neuro- and vasculopro-
`tective but can also directly contribute to scar formation and trac-
`tional lesions that lead to vision loss. Therefore, if we are to better 
`control the retinal response to injury, a better understanding of 
`the regulation of glial cell proliferation is necessary. A number of 
`studies have examined the control of Mueller-glial cell prolifera-
`tion and activation after retinal injury, and a role for p27Kip1 has 
`been demonstrated in the regulation of Mueller-glial cell prolifera-
`tion during injury-associated gliosis (86). These cells not only par-
`ticipate in glial scar formation after injury but can also upregulate 
`the production of trophic factors that facilitate neuronal survival 
`
`580
`
`The Journal of Clinical Investigation      http://www.jci.org      Volume 117      Number 3      March 2007
`
`Lassen - Exhibit 1018, p. 6
`
`

`

`review series
`
`Table 2
`Molecules with antiangiogenic activity in the eye
`
`Name
`
`General antiangiogenic activity
`
`Antiangiogenic activity in the eye
`
`ECM-derived molecules
`Ef