21.2.2  Neovascularization

The high metabolic rate of retinal tissues creates a continual need for nutrients such that even a small perturbation will promote the production of proangiogenic factors. For reasons that are still unclear, these new blood vessels grow in ways that disturb retinal structure. Furthermore, these new vessels are leaky and the resultant extracellular fluid greatly interferes with photoreceptor function. Pathological angiogenesis (neovascularization) is associated with many ocular diseases and thus is of fundamental importance. Ocular examples of diseases with major angiogenic components include diabetic retinopathy, AMD, neovascular glaucoma, retinal vein occlusions, ocular tumors, and retinopathy of prematurity (ROP) (Andreoli and Miller 2007; Penn et al. 2008).

In the healthy eye, resting vasculature remains in a state of quiescence through a balance between a host of endogenous angiogenic and antiangiogenic factors. Pathological ocular angiogenesis can begin with a hypoxic signal, activating the transcription and expression of growth factors such as vascular endothelial growth factor (VEGF) (Penn et al. 2008). Inflammation can also trigger angiogenesis by the induction of VEGF (Ramanathan et al. 2009). Although VEGF can be secreted by many different cell types, the predominant source of ocular VEGF is the Müller glial cell population (Pierce et al. 1995). VEGF-mediated signal transduction plays a role in virtually all aspects of angiogenesis (Cross et al. 2003). In part, this is mediated by a complex web of signal transduction pathways associated with the VEGF receptor (VEGFR2). Autophosphorylation of tyrosine residues upon engagement of VEGF promotes the association of numerous intracellular signaling proteins including phospholipase C gamma (PLCg) and phosphatidyl inositol-3-kinase (PI3K) (summarized in Fig. 21.3). PLCg cleaves components of the plasma membrane to create an activating ligand for protein kinase C (PKC). PKC, in turn activates the small G protein, Raf, and the MAP kinase cascade. Activation of the MAPK cascade promotes cellular proliferation as well as increased motility. Recently, the MAPK-mediated increase in EC motility was mapped to the regulation of Rho kinase (Mavria et al. 2006). PI3K, in turn, activates the kinase; Akt and the GTPase; Rac. PI3K provides survival signals and, via the activation of Rac, promotes an increase in vascular permeability (Eriksson et al. 2003). The combined effects of VEGF on the vascular endothelium results in a coordinated pattern of cellular differentiation and migration: a process termed sprouting.

Sprouting depends on the coordinated patterning of endothelial cells (EC) of which the explorative lead cell is referred to as the tip cell and the following cells as the stalk cells (Ruhrberg et al. 2002; Gerhardt et al. 2003). All of these cells express VEGF receptors. The tip cell is established through the secretion of delta-like 4 (Dll4) which binds to the Notch receptor on neighboring EC. Engagement of Notch on neighboring cells inhibits the expression of VEGF responsive genes that establish the tip cell differentiation program and thus keep neighboring stalk cells from differentiating.

VEGF

VEGF receptor 2

Fig. 21.3  VEGF receptor 2 signal transduction. Engagement of VEGF receptor 2 by VEGF results in the activation of phosphatidyl inositol 3 kinase (PI3K) and phospholipase C gamma (PLCg). PI3K, in turn activates the kinase, AKT and the GTPase, Rac. AKT activity results in the inhibition of apoptotic signaling thus promoting survival. Rac functions to decrease cellular adhesion and thus increase vascular permeability. PLCg promotes the production of diacylglycerol (DAG) from the membrane which activates protein kinase C (PKC). PKC, in turn, activates the map kinase (MAPK) cascade which further bifurcates to promote cell division (proliferation), and cytoskeletal reorganization (increased motility)

Migration requires the localized degradation of extracellular matrix (ECM) and selective interactions with integrins present on retinal cells. To this end VEGF induces the upregulation of factors such as urokinase plasminogen activator (uPA), which promote EC degradation and the exposure of “cryptic” binding sites on integrins. A diagram of these interactions is shown in Fig. 21.4. UPA binds to its receptor (uPAR), located on the leading edge of the migrating EC (Binder et al. 2007). The binding of uPA to its receptor induces the activation of plasmin which, in turn, cleaves and activates matrix metalloproteinases (MMPs) (Smith and Marshall 2010). MMPs then degrade numerous components of the ECM. The binding of uPA to uPAR also promotes clustering of uPAR and interactions with of uPAR complexes with vitronectin and with integrins promoting changes in integrin conformation. These changes activate a well-defined signal transduction cascade beginning with the activation of the focal adhesion kinase (FAK) as well as the Src kinase (Src) leading to actin assembly and the cytoskeletal modifications associated with migration (Binder et al. 2007; Streuli and Akhtar 2009). VEGF, by promoting vascular permeability, allows the exudation of plasma proteins which create an interim scaffold for migrating EC.

Ang2 were upregulated in ischemic disease while Ang1 remained constant (Takagi et al. 2003). Disruption of one copy of Ang 2 (resulting in a decreased gene dosage and decreased Ang 2 protein) decreases angiogenesis in the oxygen-induced retinopathy (OIR) mouse model (Feng et al. 2009). The complexity of angiogenesis has given rise to a plethora of potential targets. Some of these, such as anti-VEGF antibodies are currently in the clinic while others are in development. Other growth factors which might serve as targets include angiopoietin, FGF, hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), platelet derived growth factor B (PDGF-B), and placental growth factor (PIGF). Chemokines which might serve as targets include interleukin 8 (IL-8), stromal cell derived factor 1 (SDF1), and granulocyte-colony stimulating factor (G-CSF). Receptor systems might include CXCR1, FGF-R, PIGFR, PDGFR, and the Tie-receptors. Intracellular signaling molecules might include c-kit, PI3 kinase, PKC, and Src. Finally, extracellular mediators might include integrins, cadherins, MMPs, and peptides or protein fragments derived from the ECM. Given the vastness of this subject only the most important subset of these potential targets will be considered in subsequent sections.

21.2.3  Degeneration

The cells of the retina are neuronal in nature and are susceptible to degeneration through loss of a survival factor, the presence of a toxic factor, mechanical trauma, or finally, the activation of cellular stress. Neurons require a continuous source of survival factors. This requirement is likely a holdover from the developmental need to trim unnecessary connections via an activity dependent survival process (Kuczewski et al. 2009). Here, target tissues release neurotrophic factors which are taken up by active synapses. These factors are transported back along the axon to the cell body where they provide an antiapoptotic signal that balances a proapoptotic signal. Neurons that have not produced active circuits are thus removed and no longer take up valuable nutrients. Some forms of retinal degeneration may involve a constriction in the optic nerve leading to loss of neurotrophic factors. Degeneration may also be caused by perturbations in blood flow leading to the inappropriate release of toxic factors such as glutamate or excessive levels of nitric oxide (NO). Glaucoma, a disease involving anterior ocular structures, induces changes leading to retinal degeneration via both of these mechanisms. Neuron cell death is associated with several ocular diseases. Some of the more common ones include glaucoma and inflammatory optic neuropathies such as that caused by multiple sclerosis, consecutive optic atrophy, and ischemic optic neuropathy. In addition to these diseases, genetic conditions such as retinitis pigmentosa (RP) also result in an ocular pathology driven by cell death.

In general, besides mechanical trauma, neurons die either because of the presence of a toxic or proapoptotic compound or else because of the lack of a survival factor. This balance between neuronal death and survival is presumably a consequence of the mechanism by which connections are pruned within the CNS as described earlier.

Apoptosis, or programmed cell death, involves the ordered disassembly of the cell. This process is mediated by a family of cysteine aspartyl-specific proteases termed caspases (Earnshaw et al. 1999). These enzymes exist as zymogens, which are cleaved and thus activated in a cascade via either membrane receptors (the extrinsic pathway) or via mitochondrial factors (the intrinsic pathway) (Tempestini et al. 2003). The final (effector) caspases then target key proteins and DNA. The cell breaks into discrete vesicles which are quickly phagocytosed.

Glaucoma, while a disease of anterior structures, presents an excellent example of the interplay between these different forces leading ultimately to retinal degeneration. Ganglion cell axons which comprise the optic nerve pass out of the eyeball through a constriction called the lamina cribrosa. This sieve of lamellar connective tissue pores serves as a focal point for mechanical stress. Often, glaucoma is associated with IOP. This causes the constriction to tighten and thus cuts off the flow of material along the axons. Most important for this discussion, survival factors such as brain-derived growth factor (BDNF) and NGF are actively transported in the retrograde direction from synapses located in the lateral geniculate area of the cortex to the cell bodies located in the retina. Indeed a buildup of these factors has been observed at the lamina cribrosa in both humans and in animal models of glaucoma (Hollander et al. 1995; Pease et al. 2000; Quigley et al. 2000; Soto et al. 2008). Additionally, mitochondrial damage has also been observed (Ju et al. 2009; Osborne 2010). Increased ocular pressure also affects glial cells. These cells secrete cytokines such as TNFa (Yuan and Neufeld 2000; Tezel 2008), and NO (Neufeld 1999; Liu and Neufeld 2001), which function to activate inflammatory cells.

Control of increased ocular pressure does not always result in improved vision for glaucoma patients, indicating that other factors must be important. Separate from increased ocular pressure, glaucoma is also associated with a dysregulation in vascular perfusion. Vascular dysregulation has been divided into primary and secondary etiologies. Primary dysregulation, an inborn genetic trait, is associated with conditions such as Reynaud phenomena and migraine, and serves as a risk factor for glaucoma. Secondary dysregulation is associated with the onset of conditions that increase the vasoconstrictor protein; endothelin-1 (ET-1), such as rheumatoid arthritis and systemic lupus erythematosus but does not increase glaucoma risk (Grieshaber et al. 2007). Exogenous ET-1 does induce lamina cribrosa ischemia and RGC loss, however, strongly implicating it as a pathogenic factor causing RGC loss (Chauhan 2008). While a causal relationship has yet to be established between vascular dysregulation and RGC degeneration in human glaucoma, animal model data is consistent with this hypothesis (Lau et al. 2006; Krishnamoorthy et al. 2008; Munemasa et al. 2008). Decreased perfusion at the lamina cribrosa can result in increased MMP expression, increased NO production, and increased glutamate secretion; all of which may contribute to RGC apoptosis (Agarwal et al. 2009).

Protein misfolding can also be a cause of retinal neuron degeneration. A number of nascent protein chains are carefully guided to their final confirmation by a series of chaperone proteins (Surguchev and Surguchov 2010). These include large multisubunit enzymes and intrinsically unstructured proteins. Unfolded proteins tend to aggregate through associations among hydrophobic amino acid residues and inappropriate