Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

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I. INTRODUCTION

The ciliary body (CB), the site of aqueous humor secretion, is comprised of a bilayered secretory polarized neuroepithelium, the pigmented (PE) and the non-pigmented (NPE) ciliary epithelium (CE), overlapping the ciliary muscle and stroma containing blood vessels. Both epithelial cell layers of the CE and the iris share a common embryological origin with the multiple cell layers of the retina and with the retinal pigment epithelium (RPE). These tissues derive from the optic cup during development as four

distinct structures: the iris, the CB, the retina and the RPE (Beebe, 1986; Napier and Kidson, 2006). The CE is formed at the rim of the optic cup, whereas the retina and RPE are formed around the optic cup. The PE cell layer of the CE is continuous with the RPE, and the NPE cell layer is related to the neural retina. The anatomical configuration of the CE is unique in the sense that the PE and NPE cell layers appose each other by their apical plasma membranes, and their basal plasma membranes face different environments. The basal plasma membrane of PE faces the stroma

containing vasculature and the underlying ciliary muscle; on the other hand the basal membrane of NPE cells faces the aqueous humor side of the anterior segment.

The secretion of aqueous humor is an attribute of the CE and it is mostly dependent on the unidirectional transcellular movement from the stromal interstitial fluid to the aqueous humor side, by a complex transport system along the PE and NPE plasma membranes (Civan, 1998). It has not been totally ruled out that reabsorption of aqueous humor back to the stroma may occur, but this possibility has not yet been tested. At least three regulated steps have been suggested to be involved in the unidirectional transfer of solutes and water from the stroma towards the aqueous side: (1) the uptake of Na , K and Cl by PE cells by the Na /K /2Cl symport, and the Na /H and Cl /HCO3 antiports; (2) the transfer from PE to NPE through gap junctions; and (3) the transfer of solute and water by the NPE cells into the aqueous by the Na /K –ATPase, K , and Cl channels, H –ATPase and water channels (Civan, 1998). The presence of tight junctions between NPE cells forms a blood–aqueous barrier that prevents the free passage of macromolecules through the paracellular space between NPE cells, from the stroma to the aqueous humor side. However, plasma proteins in aqueous humor represent less than 1% of the concentration found in plasma, suggesting possible alternative routes in their transfer including the root of the iris (Freddo et al., 1990, Freddo, 2001). Aqueous humor exits the anterior segment of the eye through routes that are either dependent or independent of pressure by mechanisms not yet understood. The main pressure-dependent route of aqueous humor drainage is through the trabecular meshwork (TM) and the Schlemm’s canal, and the main pressure-insensitive route is through the interstitial space of the ciliary muscle and choroid. This alternative pathway of aqueous drainage is also known as uveoscleral

outflow, and it could account for more than 50% of the total aqueous drainage in young healthy humans (Bill, 1971) and higher in patients with Primary Open Angle Glaucoma (POG) (Toris et al., 1999; Gabelt and Kaufman, 2005). In POG, the most prevalent form of glaucoma, an increase ( 14mmHg) in intraocular pressure (IOP) is a risk factor in the progression of the disease, and the most effective way so far to lower IOP in glaucoma is by reducing the rate of secretion of aqueous humor.

A. Neuroendocrine Functions

IOP reflects the balance between inflow and outflow of aqueous humor. Work at the cellular and molecular levels has suggested that modulators released at the inflow may establish cross-talk communication with the tissues at the outflow pathways of aqueous humor. This endocrine view of inflow– outflow communication is supported by the biological nature of many of the signal factors (i.e. neuropeptides, peptide-hormones) release by the ciliary epithelium into the aqueous humor. Earlier studies, aimed to analyze the pattern of transcriptional expression in the human CB, revealed that this tissue actually expresses the mRNA for many of the secretory proteins found in the aqueous humor fluid, including: proteases, neuropeptides, hormones, neurotrophic factors, growth factors and plasma proteins (Escribano et al., 1995; Escribano and CocaPrados, 2002; National Eye Institute Bank (NEIBank) http://neibank.nei.nih.gov).

Thus, for example, mRNA has been detected in the human CB for plasma proteins including: β2-macroglobulin (Escribano et al., 1995); transferrin (Bertazolli-Filho et al., 2003); transthyretin (Kawaji et al., 2005); and ceruloplasmin (Bertazolli-Filho et al., 2006). Additional studies have also shown expression of proteases including cathepsins D and O (Ortego and Coca-Prados, 1997a); neuropeptides (i.e. neurotensin) (Ortego et al., 2002); hormone-peptides (i.e. natriuretic peptides) (Ortego and Coca-Prados,

1999); angiotensin (Savaskan et al., 2004), and the anti-angiogenic proteins (i.e. pigment epithelium-derived factor (PEDF)) (Ortego et al., 1996a,b) and chondromodu- lin-I (Funaki et al., 2001). Most recently, the extracellular matrix proteins including myocilin (Ortego et al., 1997b); steroid-converting enzymes (Coca-Prados et al., 2003); and growth factors (i.e. EGF) (unpublished results), have been also found. When verified by in situ hybridization or by immunocytochemistry the cellular distribution of specific mRNA or proteins within the CB revealed their restricted localization within the ciliary muscle (i.e. myocilin) (Huang et al., 2000), the vascular endothelium (i.e. natriuretic peptide C-type) (Fidzinski et al., 2004), the PE cells (i.e. cellular retinaldehyde binding protein) (Martin-Alonso et al., 1993; Salvador-Silva et al., 2005), or NPE cells (i.e. somatostatin) (Ghosh et al., 2006).

The expression of neuroendocrine peptides in the CE is of particular interest, since many of them usually are restricted to neuroendocrine cells and tissues around the body. Neuropeptides can function as neurotransmitters, neurohormones, or neuromodulators depending of the distance and effect mediated by the neuropeptide. Their expression in the CE has been confirmed by the detection of their mRNA, by conventional RT-PCR or by Northern blotting analysis, and by radioimmunoassay. Further analysis by indirect immunofluorescence had also helped to verify their cellular distribution along the CE.

Neuropeptides are synthesized as large pre-pro-peptides, with the presequence encoding an endoplasmic reticulum targeting signal sequence, and targeted into either the constitutive secretory pathway (CSP) or the regulated secretory pathway (RSP). In this process there is a multistep cascade involving endoand exo-proteolytic cleavages into smaller biologically active peptides (i.e. three to 40 amino acid residues). Peptides and processing enzymes are packaged and stored in dense core vesicles for regulated release. Among the

endopeptidases involved in the endoproteolytic cleavage of pro-peptides figure the prohormone convertases (PCs). These enzymes cleave the pro-peptide on the C-terminal of basic residue pairs (i.e. Lys-Arg). The basic residues are then removed by a carboxypeptidase E (i.e. CPE) resulting in a mature peptide. However, peptides that have a glycine residue preceding the basic cleavage site can be further modified by the removal of the glycine residue, by the action of the peptidyl-glycine-β-amidating monooxigenase (PAM) generating an amidated carboxyl group. Approximately half of the biologically active neuropeptides are amidated. In the CE, the expression and localization of PCs has been shown, including furin, PC1, PC2, and the chaperone neuroendocrine peptide 7B2, and the exopeptidases CPE and PAM (Ortego et al., 1997a,b; Ortego et al., 2002; Ghosh et al., 2006). Neuropeptides, upon synthesis and release by the CE, are expected to act on target cells expressing specific receptors. Because neuropeptides and their cognate receptors are co-expressed in the CE, it is believed that an autocrine feedback mechanism is involved in their mechanism of action. This is the case, for example, for neuropeptides expressed in the CE, including neurotensin (Ortego and Coca-Prados, 1997c; Ortego et al., 2002), galanin (Ortego and Coca-Prados, 1998) and somatostatin (Klisovic et al., 2001; Ghosh et al., 2006), for which cognate receptors are also co-expressed in the same tissue. On the other hand, emerging information indicates that cells at the conventional aqueous humor pathway express cognate receptors for many of the neuropeptides and hormones released by the CE, suggesting also an endocrine/paracrine mechanism of action. This is the case, for example, for natriuretic peptides (NPs) released by the CE (Coca-Prados et al., 1999; Ortego et al., 2002; Fidinski et al., 2004; Fernandez-Durango et al., 1995) and NPRs expressed in the trabecular meshwork (Chang et al., 1996; Pang et al., 1996). The finding that neuropeptides

expressed by the CE are detected in the aqueous humor, in high levels, indicates that they derive from the secretory activity of the NPE cells, rather than from diffusion, for example from sympathetic, parasympathetic or sensory terminal nerve endings in the ciliary body.

Among the neuropeptides and neuro- peptide-processing enzymes found in the CE, usually restricted to neuroendocrine cells and tissues, were somatostatin and the prohormone convertases PC1, PC2 and the 7B2 chaperon (Ghosh et al., 2006). Somatostatin is a neuropeptide found in neuroendocrine cells in the brain, gastrointestinal track and endocrine cells (Patel, 1999). The prohormone comvertases PC1 and PC2 are abundant in neuropeptiderich regions as in the hypothalamus, hippocampus and cerebral cortex (Seidah and Chrétien, 1999). The expression of these enzymes in the CE indicates that they are distinctly distributed along the PE or NPE cell layer, suggesting that same-substrate proneuropeptide may result in different biologically active end products. Overall these studies support that the CE contains a distinctive neuroendocrine phenotype.

Although we do not know whether neuropeptides and hormones released by the CE are involved in inflow and outflow of aqueous humor, several of the peptides and hormones identified in aqueous humor have been extensively studied in the cardiovascular system because of their hypotensive biological activity in lowering blood pressure. Thus, for example, natriuretic peptides (NPs) which are expressed in the ciliary processes (Fernandez-Durango et al., 1991), when injected in the eye elicit a hypotensive effect by lowering IOP (Goldmann and Waubke, 1989). Although the molecular mechanism by which this is accomplished is not yet known, recent studies have suggested that the hypotensive activity by NPs may be mediated by a reduction in aqueous humor secretion as a consequence of the attenuation of the Na – H -exchanger activity in the bilayered CE

(Fidzinski et al., 2004). This interpretation is consistent with the hypotensive effect of amiloride derivatives in lowering IOP in mice (Avila et al., 2002).

Neuropeptides, hormones and growth factors released by the CE in the aqueous humor could serve as messengers to communicate with surrounding tissues in the anterior segment of the eye, in particular tissues localized in the conventional outflow pathway (i.e. trabecular meshwork (TM)) and in the uveoscleral pathway (i.e. ciliary muscle). This is also consistent with the expression of receptors in the cells of the conventional outflow pathway for peptides and hormones released by the CE.

II. CILIARY EPITHELIAL PEPTIDES AND AQUEOUS FLOW

Why does the CE secrete peptides into the aqueous humor? Do these released substances exert their action elsewhere in the anterior chamber? These active agents, some with short half-lives, can interact with their corresponding receptors and exert either autocrine and/or paracrine responses once released from the CE (Figure 4.1). Perhaps the best studied of these agents released by the CE are the natriuretic peptides. There are three common natriuretic peptides: atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) (formerly known as brain natriuretic peptide) and C-type natriuretic peptide (CNP). Each of the natriuretic peptides (NPs) is encoded by a distinct gene, their expression varies among tissues and their level changes in response to physiological as well as pathological challenges. Like most hormone peptides NPs are expressed in tissues as pre-pro-hormones and the mature peptide is produced from a series of proteolytic processing (Potter et al., 2006). In the eye, basal levels of ANP, BNP and CNP have been measured in aqueous humor of rabbits and it was found that BNP had the highest level (Potter et al., 2004). Similarly,

STROMA PE

Peptide

receptors Autocrine Aqueous veins action

IOP

it was found that BNP levels were the highest in human aqueous humor (Salzmann et al., 1998). It should also be noted that the concentrations in the aqueous humor are higher than that of plasma, indicating that there is a secretion of these peptides into the aqueous humor. Such a finding suggests that these peptides may exert their effects either downstream of their source, perhaps on the trabecular meshwork, or directly on the cells from which they originate, ciliary epithelial cells. In fact, it has been found that both ANP and CNP can decrease the intraocular pressure in rabbits (Sugrue and Viader, 1986; Stone and Glembotski, 1986; Mittag et al., 1987; Takashima et al., 1998), monkeys (Samuelsson-Almen et al., 1991) and humans (Goldmann and Waubke, 1989).

The lowering of IOP by NPs appears to be through actions on outflow facility (Takashima et al., 1996, 1998), although direct actions on the ciliary epithelial transport systems to decrease aqueous humor formation has also been suggested (Mittag et al., 1987; Chang et al., 1996; Pang et al., 1996; Fidzinski et al., 2004). The latter mechanism relates to the actions of NPs on ion transport (Kourie and Rive, 1999), including effects on Na and K channels (Beltowski and Wojcicka, 2002; Hirsch et al., 1999), Na /H exchanger

(NHE) (Fidzinski et al., 2004) as well as on aquaporins (Han et al., 1998). NPs have also been shown to influence Na /K /2Cl co-transport (Kourie and Rive, 1999). Such an action would favor effects on both ciliary epithelial transport (Civan, 1998; To et al., 2002) and actions on the trabecular meshwork (Brandt and O’Donnell, 1999; Mitchell et al., 2002). It is not clear if these effects account for the IOP lowering actions of NPs, but there are enough data on other agents that affect this transport system that also reduce IOP. For instance, derivatives of amiloride, which are inhibitors of NHE activity, also reduce IOP when applied topically to the eye (Avila et al., 2002, 2003) and agents that alter ion transport in the TM also appear to influence outflow (Mitchell et al., 2002; Soto et al., 2004). Thus NPs could be influencing both inflow and outflow to bring about a decrease in IOP. It still remains uncertain if endogenous NPs serve a homeostatic function in regulating inflow and outflow of aqueous humor. The fact that various stimuli modulate NP secretion indicates that NPs may play a role in pathologies and/or normal physiological processes that are involved in regulating cell volume and fluid transport or perhaps in affecting smooth muscle activity and cell contraction. The actions of NPs are derived from their interactions with their