Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

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thus will not be amenable to gene therapy directed at the germinal line. In the absence of a well-defined genetic defect which gives rise to AMD, gene therapy will likely focus on somatic therapy using growth factors and anti-apoptosis therapy to prolong the survival of the RPE and retinal photoreceptors.

GROWTH FACTOR GENE THERAPY FOR AMD

There is experimental evidence that growth factors play an important role in maintaining the health of RPE cells and in enabling them to respond to injury. RPE cells express growth factors and their receptors, demonstrating the autocrine and paracrine functions of these substances. Theoretically, it may be possible to enhance RPE cell survival by somatic modulation of growth factor gene expression in patients with AMD. For instance, age-related phagocytic dysfunction and incomplete digestion of photoreceptors membranes by the RPE result in loss of RPE cells and in geographic atrophy, perhaps because of the cytotoxicity of these deposits on the surrounding cells. Enhancing phagocytic activity in aging RPE cells using gene therapy is a potential approach to the treatment of AMD. Basic-fibroblast growth factor has been shown to stimulate phagocytic activity and prolong retinal survival in animals models (3). An important number of other growth factors and secondary messengers of the intracellular-signaling pathways have demonstrated a neuroprotective effect on the retinal neurons in animal models of retinal degeneration. Gene transfer and expression of these growth factor proteins may similarly inhibit retinal degeneration by a neuroprotective effect in AMD (4).

The RPE synthesizes proteins that are antiangiogenic, such as tissue inhibitors of metaloproteinases and pigment epithelium-derived factor (PEDF). Thus, potential gene therapy applications to CNV include anti-angiogenic growth factor gene therapy, antisense or ribozyme therapy directed at angiogenic factors, and suicide gene therapy directed at neovascular tissue. Moreover, recently it was demonstrated that expression of angiostatin in experimental CNV significantly reduces the size of CNV lesions (5).

TRANSPLANTATION OF GENETICALLY MODIFIED IRIS PIGMENT EPITHELIAL CELLS

Submacular transplantation of autologous iris pigment epithelial (IPE) cells has been proposed to replace the damage RPE following surgical removal of the CNV (6). The IPE is anatomically continuous with the RPE and has the same embryonic origin. In vitro IPE cells share functional properties with the RPE cells such as phagocytosis, degradation of rod outer segments, and synthesis of trophic factors. However, autologous transplantation of IPE cells alone has not resulted in a prolonged improvement of the vision patients with AMD, potentially because the lack of expression of one or several factors that is an important part of RPE function. Semkova et al. (6) have suggested a treatment for AMD based on transplantation of genetically modified autologous IPE cells. The most significant findings are summarized as follows: first, IPE cells were readily transduced with a high-capacity adenovirus (HC-ad) vector. Second, IPE cells secreted functionally active PEDF after HC-ad-mediated gene transfer. Third, subretinal transplantation of PEDF-expressing IPE cells inhibited neovascularization in models of retinal neoangiogenesis, and prevent photoreceptor degeneration.

ADENOVIRAL VECTORS GENE THERAPY

Preclinical proof, using either recombinant adenovectors to carry the genes encoding PEDF and endostatin or recombinant adeno-associated viruses (AAVs) carrying the transgene encoding for angiostatin, has recently been published and demonstrated that significant inhibition of the CNV in various animals models. Recently the intravenous administration of an adenoviral construct carrying the murine endostatin gene in a murine model of CNV was tested and found an almost complete inhibition of the neovascular activity. Similarly, subcutaneous injection of an AAV carrying a truncated angiostatin gene resulted in significant inhibition of retinal neovascularization. These encouraging results with endostatin and angiostatin suggest a potential role of anti-angiogenesis in ocular disease.

Recently, PEDF received major attention. PEDF was first described in 1989 by Tombran-Tink in conditioned-medium form-cultured, fetal RPE cells as a potent neurotrophic factor (7). Subsequently, PEDF has been purified and cloned both from humans and mice (8). The gene is expressed as early as 17 wk in human fetal RPE cells, suggesting that PEDF is intimately involved in early neuronal development. PEDF attracted even more attention when Dawson et al. demonstrated that PEDF is one of the most potent natural inhibitors of angiogenesis (9). In addition, PEDF is an inhibitor of endothelial cell migration. The amount of inhibitory PEDF produced by retinal cells was positively correlated with oxygen concentrations, suggesting that its loss plays a permissive role in ischemia-driven retinal neovascularization. Moreover, a correlation between changes in the vascular endothelial growth factor (VEGF)/PEDF ratio and the degree of retinal neovascularization in a rat model was demonstrated.

The AdPEDF (11) is a replication deficient adenovirus vector designed to deliver the human PEDF gene. Intravitreous injection of AdPEDF resulted in increased expression of PEDF mRNA in the eye compared with AdNull (the same vector without the transgene) or with uninjected controls. PEDF trail was present not only in the retina, but also in other parts of the eye, including the iris, the lens, and the corneal epithelium. Afterward, the subretinal injection of AdPEDF was strongly detected in the RPE cells compared with other ocular structures (8).

PHARMACOLOGICAL INHIBITION OF THE VEGF

IN PATIENTS WITH WET AMD

Background

Ocular neovascularization is a key factor of the most common causes of blindness in humans in the developed word: AMD and proliferative diabetic retinopathy are two well-known examples. Prevention of ocular neovascularization by development of antiangiogenic drugs represents a rational and appealing therapeutic approach. However, because these are chronic diseases characterized by ongoing new vessel formation, long-term inhibition of the angiogenic stimuli is likely to be needed.

The development of new blood vessels from pre-existing ones, a process known as angiogenesis, is a physiological process that is fundamental to normal healing, reproduction, and embryonic development (10). Angiogenesis plays an important role in a variety of pathological processes including proliferative retinopathies, AMD, rheumatoid arthritis, psoriasis, and cancer.

Over the past decade, our understanding of the complex processes involved in new vessel development has lead to the isolation of a family of angiogenic stimulators known collectively as VEGF.

VEGF-A is a pivotal angiogenic stimulator that binds to VEGF receptors, promoting endothelial cell migration, proliferation, and increasing vascular permeability.

Recognition of the central role of VEGF-A in angiogenesis has led to the hypothesis that its inhibition may represent a novel and effective approach to the treatment of choroidal neovascular membranes in wet AMD and other conditions characterized by pathologic angiogenesis.

VEGF AND ITS RECEPTORS

There are currently six known members of the VEGF family: VEGF-A, placental growth factor, VEGF-B, VEGF-C, VEGF-D, and VEGF-E.

VEGF, also known as vascular permeability factor (VPF), is a diffusable endothelial cell-specific mitogen and a pro-angiogenic factor that regulates vascular permeability. VEGF is highly specific for vascular endothelium and its potent angiogenic action has been demonstrated in arteries, veins, and lymphatic vessels, but not in other cell lines (Fig. 1). In addition, there is also evidence that VEGF functions as an anti-apoptotic factor for endothelial cells in newly formed vessels. Several mechanisms may regulate VEGF expression, the most important of which may be hypoxia. Other factors of importance include cytokines, such as epidermal growth factor, transforming growth factor-β, keratinocytes growth factor, cell differentiation, and oncogenes (11).

The biological effects of VEGF are mediated by two receptor tyrosine kinases: VEGFR-1 and VEGFR-2 (12). The expression of these receptors is largely restricted to the vascular endothelium and it is assumed, but not proven, that all of the effects of VEGF on the vascular endothelium may be mediated by these receptors (13).

THE ANGIOGENESIS PROCESS: HOW DO NEW VESSELS GROW?

Physiological adaptation to hypoxia is a necessity for organisms having an oxygenbased metabolism. In mammals, these adjustments include vasodilation, angiogenesis, upregulation of glucose transport, activation of glycolysis, and apoptosis (programmed cell death) (14).

Angiogenesis is a multistep process that is regulated by a fine balance between proand anti-angiogenic growth factors released in response to hypoxia, hypoglycemia, mechanical stress, release of inflammatory proteins, and genetic alterations (Table 1). Some of these factors are highly specific for the endothelium (e.g., VEGF), whereas others have a wide range of activities (e.g., matrix metalloproteinase inhibitors [MMIs]).

A series of complex and interrelated steps are necessary for angiogenesis to take place in a tissue (Fig. 2). The initial step requires the injured cell to release pro-angiogenic growth factors (mainly VEGF) into the surrounding tissues. The released VEGF binds to healthy adjacent endothelial cells located in the walls of normal blood vessels. VEGF exerts its action on endothelial cells through the VEGFR-1 and VEGFR-2 receptors (12). VEGF induces genetic modifications in the endothelial cell that results in the intracellular synthesis of lytic enzymes (e.g., matrix metalloproteinases [MMPs]) responsible for

Fig. 2. Angiogenesis: the formation of new blood vessels. Source: The angiogenesis Foundation (www.angio.org).

vascular loops carry blood back to the initially injured tissue in an effort to reverse the initial hypoxic injury.

ROLE OF VEGF IN PHYSIOLOGICAL AND PATHOLOGICAL ANGIOGENESIS IN AMD

During embryogenesis, blood vessels are formed by two distinct processes: vasculogenesis and angiogenesis. Vasculogenesis involves the de novo differentiation of endothelial cells from mesodermal precursors, whereas angiogenesis involves the generation of new vessels from pre-existing ones. Vasculogenesis, which occurs only during embryonic development, leads to the formation of a primary vascular plexus.

VEGF is naturally expressed in retinal tissues with especially high levels concentrated in the RPE (Fig. 3). In the normal eye, VEGF may play a protective role in maintaining adequate blood flow to the RPE and photoreceptors (15). Deficiencies in blood flow to the choriocapillaris, oxidative stress, and alterations in Bruch’s membrane have all been demonstrated to trigger the initial over expression of pathologic levels of VEGF in the RPE and the retina (16).

As mentioned earlier, VEGF promotes proliferation, chemotaxis of endothelial cells, and increases vascular permeability. These vascular permeability changes result in an artificial increase in interstitial fluid pressure, which produces leakage of plasma proteins. The increased oncotic pressure in the extravascular spaces results in the

Fig. 3. The VEGF has a specific affinity for its receptors at the level of the RPE and endothelial cells. Overexpressed VEGF promotes angiogenesis, increased vascular permeability, and express proteases and other cytokines. Courtesy of Eyetech Pharmaceuticals and Pfizer, Inc.

Fig. 4. The VEGF cascade contributes to CNV formation with breakdown of the blood–retinal barrier and violation of the subretinal space. Courtesy of Eyetech Pharmaceuticals and Pfizer, Inc.

formation of a fibrin gel, which provides a substrate for endothelial cell growth and migration (Fig. 4).

Localization of high levels of VEGF in the choroid in patients with wet AMD strongly suggests its direct role in the progression of this disease (17).

CLASSIFICATION OF ANGIOGENESIS INHIBITORS

Several agents targeting angiogenesis have been developed and can be grouped into four categories based on their mechanisms of action (Table 2) (18).

Table 2

Inhibitors of the Angiogenesis

1.Matrix metalloproteinase inhibitors

a.Marimastat (BB2516)

b.Prinomastat (AG3340)

c.BMS 275291

d.BAY 12-9566

e.Neovastat (AE-941)

2.Drugs blocking endothelial cell signaling

a.RhuMAb VEGF

b.Inhibitors of the VEGF receptors

c.SU5416

d.SU6668

e.ZD6474

f.CP-547,632

g.Other tyrosine kinase inhibitors

3.Endogenous inhibitors of angiogenesis

a.Endostatin

b.Interferons

4.Novel agents inhibiting endothelial cells

a.Thalidomide

b.Squalamine

c.Celecoxib

d.ZD6126

e.Integrin antagonists

f.TNP-470

1.Matrix metalloproteinase inhibitors: Degradation of the extracellular matrix and basement membrane is one of the first steps in angiogenesis. The MMPs are a family of secreted zincdependent enzymes that are capable of degrading the components of the extracellular matrix and basement membrane. The MMP family includes four principal classes of molecules: collagenases, gelatinases, stromelysins, and membrane-type MMPs (19). Because the upregulation and activation of proteinases represents a common pathway in the process of retinal neovascularization, pharmacological intervention of this pathway may be an alternative therapeutic approach to neovascular diseases (20).

2.Drugs blocking endothelial cell signaling and migration: Once degradation of the extracellular membrane and basement membrane occurs, the next step in angiogenesis is endothelial cell migration, proliferation, and differentiation. Clearly, VEGF and its receptors play a critical role during the stimulation of the endothelial cells and is a logical target for antiangiogenic therapies (17,21,22).

3.Endogenous inhibitors of angiogenesis: Endostatin and inteferons are both endogenous inhibitors of angiogenesis. Endostatin, a C-terminal fragment of collagen XVIII formed by proteolysis, specifically inhibits endothelial cell migration and proliferation in vitro and potently inhibits angiogenesis and tumor growth in vivo. Reduced levels of endostatin in Bruch’s membrane, RPE basal lamina, intercapillary septa, and choriocapillaris in eyes with AMD may be permissive for choroidal neovascularization (23). The therapeutic implications of endostatin remain to be investigated. Inteferons were not better than the natural history of AMD when tested in a well-controlled clinical trial as an endogenous inhibitor of angiogenesis (24).

4.Other agents: Thalidomide, an immunomodulator and antiangiogenic drug, has been tested as a treatment for wet AMD. Although the enrollment of patients is finished, the results are not yet known (25). Squalamine, an aminosterol originally derived from the liver of the dogfish shark, has shown potent anti-angiogenesis effects both in vitro and in vivo (26).

ANTI-ANGIOGENESIS THERAPY FOR EXUDATIVE AMD

Many clinical trials have been performed with the hope of finding a safe and efficacious pharmacological treatments for exudative AMD. Most of these drugs are directed at interrupting neovascularization at various points along the angiogenic pathway as illustrated in Table 3.

The recent Food and Drug Administration approval of Macugen in December of 2004 in the United States for the treatment of wet AMD represents a very important addition to the armamentarium of vitreo-retinal specialist.

Macugen®: Pegaptanib Sodium (Eyetech Pharmaceuticals)

Macugen is an aptamer with a high affinity and specificity for the extracellular pathological isoform VEGF known as VEGF-165. Macugen is injected into the vitreous cavity every 6 wk. By selectively targeting VEGF-165, Macugen prevents VEGF uptake by endothelial cells receptors, inhibits new vessel formation and prevents leakage from existing new vessels (Fig. 5).

Preliminary results from phase I/II clinical trials on Macugen were quite promising (22,27). Patients were randomly assigned to 0.3, 1, or 3 mg of pegaptanib sodium or to sham treatment. Injections were given intravitreally, through the pars plana every 6 wk, in most cases, for a total of nine treatments.

The subjects were then followed for 54 wk. Macugen showed evidence of efficacy at all three dosage levels. The 0.3 mg dose was chosen as the lowest efficacious dose and, at this concentration, 87.5% of the patients treated with Macugen showed stabilization or improvement in vision during a period of 3 mo. A 60% three-line gain at 3 mo was noted in patients who received both the anti-VEGF aptamer and photodynamic therapy (22).

Ranibizumab: RhuFab (Lucentis, Genentech)

RhuFab is a recombinant humanized anti-VEGF antibody fragment designed to bind and inactivate all the VEGF isoforms. Like Macugen, it is injected into the vitreous cavity in an office-based procedure. Lucentis is administered every 4 wk, whereas Macugen is administered every 6 wk. The antibody fragment consists solely of the antigen-binding portion of the entire antibody, which facilitates RhuFab’s retinal penetration. Results of a phase I/II clinical trial at 6 mo have been reported. In early testing on 53 patients with subfoveal CNVM, 50 (94) had stable or improved vision at day 98 following intravitreal injection of Lucentis. The proportion of patients who had an improvement in vision equivalent to three or more lines of vision was 26%, a result almost identical to Macugen’s. Two different arms of a phase III trial are under evaluation. MARINA evaluates the efficacy of Lucentis in patients with wet AMD. ANCHOR compares Lucentis vs photodynamic therapy with Verterporfin in patients with wet AMD. The only adverse effect in the treated group has been the development of a transient, mild- to-moderate inflammatory reaction following injections with Lucentis.