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

receptors and these are NPR-A, NPR-B and NPR-C. They are also sometimes referred to as GC-A and GC-B for the analogous receptors and the clearance receptor for the NPR-C receptor (Potter et al., 2006). All three receptor subtypes appear to be localized to the ciliary epithelia (Ortego and Coca-Prados, 1999; Fernadez-Durango et al., 1999) and NPR-B appears to be present in trabecular meshwork (Pang et al., 1996). NPR-A and NPR-B are representative of the family of transmembrane guanylyl cyclases identified in humans (Potter, 2005). These receptor cyclases convert guanosine triphosphate into cyclic 3 ,5 -guanosine monophosphate, cGMP (Garbers and Lowe, 1994). It is the increase in cGMP that affects signaling systems downstream of the receptor that are ultimately responsible for the cellular functions that are expressed. For example, both ANP and CNP increased the accumulation of cGMP in isolated cultured trabecular meshwork (TM) cells and in ciliary muscle cells. This increase in cGMP was correlated with the suppression of carbachol-induced calcium mobilization in the cell (Pang et al., 1996). Such actions would result in a diminished responsiveness to carbachol, an acetylcholine mimetic that causes TM cells and ciliary muscle to contract. These authors concluded that the natriuretic receptor (NPR-B) when activated in TM or CM would alter the contractile response of these tissues and affect aqueous humor outflow and IOP.

Thus release of NPs from the CE into the aqueous humor could have both direct actions on the CE to regulate ion transport systems and ultimately aqueous humor flow and/or affect downstream tissues, such as the TM and perhaps CM to alter their contractile mechanisms and effect outflow. Do NPs play a role in aqueous humor dynamics? This remains unclear. However, there have been reports of changes in aqueous NP levels following elevation of IOP (Fernandez-Durango et al., 1990, 1991), and several growth factors that stimulate cell growth or proliferation tend to antagonize

the actions of NPs by regulating their receptor expression (Potter et al., 2006). This occurs through activation of cellular signaling systems that activate protein kinase C and include angiotensin II, vasopressin and endothelin (Potter and Hunter, 2000; Abbey and Potter, 2003; Jaiswal, 1992). Consistent with this has been the finding that angiotensin II (Inoue et al., 2001) and a vasopressin analog, desmopressin (Wallace et al., 1988), increase IOP in rabbits. However, the effects of endothelin on IOP have been varied and most reports demonstrate a significant decrease in IOP (Erickson-Lamy et al., 1991; Taniguchi et al., 1994), whereas it had no effect on IOP in another study, but this tested only one dose (Millar et al., 1998).

However, endothelin (ET) represents another example of a CE peptide that is released into the aqueous humor and appears to have both autocrine and paracrine effects. In fact, ETs concentration in the aqueous is twoto three-fold higher than in the plasma (Lepple-Wienhues et al., 1992). This finding along with the observation that intravitreal administration of ET-1 resulted in an ocular hypotensive effect in rabbits, producing a rather prolonged reduction in IOP (MacCumber et al., 1991), suggested that ET may play some role in regulating aqueous humor dynamics. The decrease in IOP was attributed to an increase in outflow, as it was thought to occur as a result of contraction of the ciliary muscle (Erickson-Lamy et al., 1991). Sugiyama et al. (1995) also showed a prolonged decrease in IOP from intravitreal injection of ET-1 in rabbits, but these authors further identified the ETB receptor as responsible for this action. ET exerts its actions through G-protein coupled receptors, ETA (EDNRA) and ETB (EDNRB). Besides actions on the outflow pathway, ET-1 inhibits Na / K -ATPase activity in the ciliary nonpigmented epithelial cells acting through ETB receptors (Prasanna et al., 2001). Such an action could result in a decrease in aqueous humor formation and contribute to the

IOP-lowering effects seen following ET-1 administration. ET-1 also produces contraction of the isolated bovine and human trabecular meshwork strips (Cellini et al., 2005, 2006; Choritz et al., 2005) and this action could account for the decrease in outflow seen in the isolated bovine eye (Wiederholt et al., 1995). This decrease in outflow is in direct conflict with other studies (Erickson-Lamy, 1991) that showed an increase in the facility of outflow. The difference might be due to species and the relationship between the ciliary muscle and the trabecular meshwork. If ET effects on the muscle are more pronounced than the effects on the trabecular meshwork, an increase in facility may be realized. In addition, the relative expression of ETA versus ETB receptors could also account for differences in observed responses. ETA receptor activation appears to result in contraction, whereas activation of ETB receptors results in relaxation. This difference in receptor expression was demonstrated recently when Zhang et al. (2003) showed that dexamethasone (DEX), a synthetic glucocorticoid, increased the release of ET-1 from human non-pigmented epithelial cells, a source of ET-1 in the anterior chamber, while decreasing the expression of functional ETB receptors on trabecular meshwork (TM) cells. This combined effect would favor contraction of the TM and a decrease in conventional aqueous outflow resulting in an increase in intraocular pressure that occurs following ocular glucocorticoid administration. This glucocorticoid mechanism is an example of how a protein from the CE can influence the functional activity of downstream tissues such as the trabecular meshwork. This demonstrates that the ciliary epithelium can communicate with other tissues of the eye through the release of active peptides. Natriuretic peptides and endothelin are just two examples of how such peptide messengers can be released by the CE and exert important regulatory actions on tissues that are responsible for aqueous humor (AH) inflow and outflow.

A. Steroidogenic Functions

In addition to neuroendocrine function, recent studies have suggested that the CB may exhibit also steroidogenic activities. This refers to the ability to mediate the local synthesis of sex steroid hormones from cholesterol. Although this is usually an activity restricted to the gonads, a large proportion of androgens in men and estrogens in women before menopause are synthesized in peripheral hormone-target tissues from circulating precursor steroids where enzymes involved in the formation of these sex steroid hormones are expressed (Labrie, 2003). In this process, a complex array of many enzymatic steps is involved. One group of enzymes, the 17β- hydroxysteroid dehydrogenases (17βHSDs) (Coca-Prados et al., 2003) involved in the last step of sex steroid synthesis and inactivation, have been found expressed in the human CB and in cultured ciliary NPE cells. The 17βHSDs are a relatively large family of steroidogenic enzymes and they are believed to be critical in many physiological processes. The 17βHSD types 1, 3, 5 and 7 catalyze the conversion of weaker steroids into more biologically active steroids, whereas 2, 4, 6 and 8 are oxidative enzymes involved in the conversion of more active steroids into less biologically active steroids. So far 17βHSD subtypes 2, 4, 5 and 7 have been found in the ciliary body (Coca-Prados et al., 2003; Kobayashi et al., 2004). The locally produced bioactive sex steroid hormones exert their action by a mechanism known as intracrine, that is, within the cells where synthesis occurs and without the need to release them extracellularly. Estrogen, androgen and progesterone receptors are expressed in the ciliary body (Ogueta et al., 1999; Wickham et al., 2000) and cultured human ciliary NPE cells and NPE cells are capable of metabolizing estrogen, androgen and progesterone which is mediated by the 17βHSD types 2, 5 and 7 (Coca-Prados et al., 2003). Although it is not yet known what exact

function 17βHSDs play in the ciliary body, 17βHSD2 plays a major role in the inactivation of potent steroid hormones oxidizing estradiol and testosterone to estrone and androstenedione, respectively.

Rauz et al. (2003) documented the expression of 11βHSD type 1 in the human CE. This enzyme catalyzes the generation of active cortisol from inactive cortisone. Cortisol is detected in the AH in a much higher level than cortisone and it is the main glucocorticoid in humans. The adrenal cortex shows pulsating secretion (8–12 episodes) over 24 h, with peak values 10 times higher during the morning (between 07:00 and 09:00) than around midnight, at its lowest level. It is not known whether the circadian rhythm secretion of cortisol in plasma is also circadian in the AH, but cortisol concentrations in this fluid exceed cortisone by approximately 14-fold (Rauz et al., 2001). Cortisol binds corticosteroid receptors (mineralocorticoid receptor and the glucocorticoid receptor) which regulate sodium transport via the epithelial sodium channel. Cortisol plays an important role in the cardiovascular system by decreasing, for example, the production of nitric oxide, a potent vasodilator involved in metabolic and immunologic homeostasis. Cortisol also inhibits almost every cell involved in inflammation, in part by inhibiting the production of pro-inflammatory substances (cytokines and prostaglandins). There is good evidence that the CE expresses enzymes in the synthesis of prostaglandins including cycloxygenase-2 (COX-2) (Maihofner et al., 2001) and prostaglandin D2 synthase (Escribano et al., 1995), and cytokines, including interleukin-1 beta (IL-1 beta) and IL-8 (unpublished results). These studies underline a potential crosstalk communication between neuroendocrine and immune systems in the CB.

The neuroendocrine and steroidogenic characteristics of the human CE suggest at least three possible endocrine loops: (i) intracrine; (ii) autocrine; and (iii) endocrine/paracrine. Since PE and NPE

co-express peptides and their cognate receptors, both cell layers of the CE can be targets of their own producing peptides. Thus, cells at the aqueous humor outflow pathways are targets of many of the endocrine signals released by the CE. The signals are carried by the AH and could target the TM cells, the main conventional type of cell in the outflow system. In contrast, the PE cells could release endocrine signals towards the stroma and target their cognate receptors in the vascular endothelium and ciliary muscle cells.

The steroidogenic functions of the CB could have an important role in the activity of two glaucoma-associated genes, MYOC and CYP1B1, which are highly abundant in the tissues associated with aqueous humor dynamics. The MYOC gene is responsive to prolonged periods of steroid treatment (Polansky, 1993), and the CYP1B1 capable to metabolize steroids (Zhang et al., 2000).

B. Anti-Microbial Functions

As the eye is inherently immune privileged, it has multiple defense mechanisms including those that operate via tears outside the eye and aqueous humor inside the eye. Defensins are naturally occurring peptides that regulate innate immunity and have a wide range of anti-microbial activities against gram-positive and gramnegative bacteria, as well as fungi and viruses (including HIV and HSV) (Lehrer et al., 1993; Nakashima et al., 1993; Daher et al., 1986). Both human alpha and beta defensins (HAD and HBD, respectively) are present in human cornea, tears and conjunctiva, and protect the ocular surface from infections. However, the vulnerability to severe intraocular infection is mitigated by the presence of HBD in the ciliary body (Haynes et al., 2000). Interestingly, only HBD-1 (mRNA expression) was present in human ciliary body while the inducible form HBD-2 mRNA expression was induced by combined treatment of human

ciliary body epithelial cultures with inter- leukin-1 and TNF-β. Therefore, cytokines could potentially stimulate HBD-2 in ciliary body. The inducible HBD-2 is a more potent anti-microbial protein than HBD-1. Human aqueous humor samples contained low levels of HBD-1 protein ( 16 ng/ml) (Haynes et al., 2000). Aqueous HBD-1 levels appear to be below the in vitro bacteriocidal concentrations of HBD. However, due to their high affinity to aggregate to mucus and epithelial tissues, HBD could be at high levels locally and be effective in maintaining low levels of microbial agents. Whether HBD-2 levels are increased in aqueous humor of patients with ocular inflammation or infection is not known. Defensins also play a key role in regulating corneal or other wound healing roles including fibrin formation and cell proliferation. Future use of purified defensins as therapeutic anti-infectives is a possibility since they are less susceptible to bacterial resistance and appear to be non-antigenic (Haynes et al., 2000).

C. Angiogenic and Anti-Angiogenic

Functions

The ciliary body is also unique in being a repository for several angiogenic and anti-angiogenic factors. Some of the angiogenic peptides identified in the ciliary body include angiotensin II (Ang II) (Savaskan et al., 2004) and secretoneurin (Troger et al., 2005). Ang II and components of the ren- nin–angiotensin system (RAS) are present in non-pigmented ciliary epithelial (NPE) cells and ciliary body while Ang II is present in aqueous humor indicating that it is actively secreted from various source tissues including the NPE (Savaskan et al., 2004; Danser et al., 1994). Ang II could potentially contribute either directly or indirectly to elevation of intraocular pressure (IOP) since it has been demonstrated that angiotensin receptor (AT1) antagonists lower IOP in humans and rabbits (Costagliola et al., 1999; Inoue et al., 2003).

However, Ang II also plays a role in promoting retinal angiogenesis, especially in diabetic retinopathy, and may be a key player in mediating retinal leukostasis (Chen et al., 2006). It is therefore possible that Ang II produced by the ciliary body and retina can affect retinal leukostasis in diabetic retinopathy.

The other angiogenic agent, secretoneurin (SN), is a 33 amino acid neuropeptide generated by proteolytic processing of secretogranin II (SgII) and belongs to the family of chromogranin C (Troger et al., 2005). SN exerts strong chemotactic effects to monocytes and eosinophils and is a very potent angiogenic agent comparable to VEGF (Kirchmair et al., 2004). In fact, SN has been shown to be a direct activator of corneal neovascularization in mice (Kirchmair et al., 2004). Also in the anterior segment of the eye, SN is closely associated with sensory innervation and plays a critical role in neurogenic inflammation, since topical application of formaldehyde elevates SN levels in rabbit aqueous humor (Kralinger et al., 2003; Troger et al., 2005). With regards to other roles, since SN is present in unmyelinated C fibers of the ciliary body it is thought to affect ciliary muscle tone and accommodation (Troger et al., 2005).

Some of the anti-angiogenic peptides and growth factors present in the ciliary body include endostatin, the C-terminal proteolytic fragment of collagen XVIII, pigmented epithelium derived factor (PEDF), and chondromodulin-1 (ChM-1). These antiangiogenic agents are thought to counteract the pro-angiogenic effects of bFGF, VEGF, and TGF-β, particularly in maintaining the avascularity of the cornea after corneal injury, infection, inflammation, and other trauma. ChM-1, a 25–32 kDa secreted glycoprotein which has been shown to prevent tube formation of vascular endothelial cells, is also localized in rat corneal epithelium and iris/ciliary body (Fukushima et al., 2003). The precise role of ChM-1 in the ciliary body is not quite clear but in

conjunction with endostatin localization along the rim of non-pigmented ciliary epithelium, lens epithelium, lens capsule and retinal inner limiting membrane clearly suggests that these anti-angiogenic agents form a barrier around the anterior and vitreous chambers against physiological angiogenesis (Ohlmann et al., 2005; Fukushima et al., 2003).

Patients with Knoblauch syndrome, an autosomal recessive characterized by vitreoretinal detachment, macular degeneration, myopia, and other ocular abnormalities, have mutations on the Col18A1 gene resulting in multiple collagen XVIII transcripts (see review by Marneros and Olsen, 2005). Abnormalities in Knoblauch syndrome in humans as well as in Col18A1 gene knock-out mice are restricted only to ocular tissues specifically associated with collagen XVIII. It is thought that endostatin may have additional roles of being involved in astrocyte migration, retinal vessel development and axon guidance. The C-terminal endostatin domain of collagen XVIII is thought to interact with basement membrane heparan sulfate proteoglycan (HSPG) and blunt FGF-2 mediated angiogenesis. Additionally, virally overexpressed endostatin has been shown to block VEGF-mediated retinal vascular permeability (mimicking diabetic retinopathy) and reduced laser-induced choroidal neovascularization (mimicking AMD) (Takahashi et al., 2003; Mori et al., 2001; Auricchio et al., 2002). However, the lack of retinal edema in patients with Knoblauch syndrome or in Col18A1 mice suggest that endostatin may not be a critical regulator of vascular permeability in vivo but may act as a contributor or a predisposing factor subjected to other genetic influences (Marneros and Olsen, 2005). In the ciliary body, it is suggested therefore that collagen XVIII/endostatin may have a normal function in promoting epithelial cell interaction with basement membrane, specifically pertaining to cytoskeletal changes.

D. Neuromodulatory Functions

As alluded to earlier under the neuroendocrine role of ciliary body, the presence of several neuromodulators including neuro- kinin-A (NKA), substance-P (SP), calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating peptide (PACAP), vasoactive intestinal peptide (VIP) and neuropeptide-Y (NPY) are indicative of intrinsic regulation of anterior chamber actions in the eye. For instance, ciliary nerve associated ganglion cells are immunoreactively labeled for several aforementioned peptides which localize as plexuses in the trabecular meshwork or colocalize with each other suggesting inter-regulation (e.g. VIP stained nerves are surrounded by SP nerve terminals in the choroid) (May et al., 2002). Interestingly in rats, NPY immunoreactivity in ciliary body appeared to oscillate in circadian fashion under dark– dark conditions similar to light–dark conditions (Otori et al., 1993). In fact, unilateral superior cervical ganglionectomy caused a significant decrease in NPY levels compared to intact eye, independent of lighting conditions. Both these latter findings indicate that there is an endogenous circadian rhythm involving NPY that is dependent on sympathetic input. PACAP has been found to participate in ocular inflammation and is colocalized with CGRP sensory nerves in the ciliary body. Specifically, aqueous humor levels of PACAP are elevated following noxious stimulation of the eye and capsaicin also stimulates both PACAP and CGRP release from iris/ciliary body further indicating a role for sensory nerves in ocular inflammation (Wang et al., 1996; Elsas et al., 1996).

E. Neuroprotection and

Neurodegeneration Functions

The ciliary body is a storehouse for several growth factors, particularly ciliary neurotrophic factor (CNTF) and brain derived neurotrophic factor (BDNF), which

support or promote neural growth, particularly after retinal injury and development (Thoenen et al., 1987; Bennett et al., 1999). Efforts to treat retinal degeneration caused either by diabetic retinopathy or optic nerve axotomy by topical delivery or long-term delivery via adenoviral vectors of CNTF have been attempted (Aizu et al., 2003; van Adel et al., 2003). Pigment epithelium derived factor (PEDF), another growth factor that is normally secreted by RPE, is also secreted by the ciliary body, particularly from non-pigmented ciliary epithelial cells (Figure 4.2). PEDF is known for its role as a potent anti-angiogenic agent, but also plays a crucial role in preventing retinal injury following peroxideinduced neuronal death (Cao et al., 1999) as well as photoreceptor damage following light exposure (Cao et al., 2001).

With regards to factors promoting neurodegeneration, endothelin-1 (ET-1), a potent vasoactive peptide, is associated with several CNS pathophysiological conditions (Schinelli, 2006). In the eye, ET-1 is synthesized and released from ciliary epithelium as well as from other ocular sources and

PEDF

FIGURE 4.2 Immunoblot analysis for PEDF in serum free cell culture media collected from either the insert with 75,000 human non-pigmented epithelial cells (HNPE) (lane 1) or the well seeded with 4000 RGC-5 (lanes 2 and 3) after a 24 hour period. Seven to ten micrograms of total protein (from culture media) were loaded per lane

is regulated by cytokines (Eicchorn and Lütjen-Drecoll, 1992; Prasanna et al., 1998; Osborne et al., 1993). ET-1 has been implicated to play an important role in promoting glaucomatous optic neuropathy and retinal/photoreceptor degeneration (see review by Yorio et al., 2002; Torbidoni et al., 2006; Rattner and Nathans, 2005); however, its role in diabetic retinopathy remains equivocal (Lam et al., 2003; Masuzawa et al., 2006; Roberts et al., 2006). It is known that ET-1 levels can increase in aqueous humor and optic nerve following elevation of intraocular pressure, a known risk factor for glaucoma (Prasanna et al., 2005; Kallberg et al., 2002). Whether the direct contribution of growth factors and vasoactive peptides released from ciliary body are responsible for glaucomatous optic neuropathy or other retinopathies is not known.

III. NEW FRONTIERS OF CILIARY BODY RESEARCH: RETINAL

PROGENITOR CELLS AND

OCULAR STEM CELLS

BOX 4.1

Recently, a lot of interest has been generated in the ophthalmology community following the identification of multipotent retinal stem cells or progenitor cells in the ciliary body (Ahmad et al., 2000; Tropepe et al., 2000; Das et al., 2005). These mitotically quiescent stem cells are found in the peripheral margin of the postnatal mammalian retina and in the ciliary epithelium. Due to their neuroepithelial origin, these progenitor cells appear to have similarities with early retinal neuronal progenitors since they express Pax6 and Chx10, homeodomain transcription factors seen in mature amacrine cells, RGCs and bipolar neurons (Abdouh and Bernier, 2006). Unlike lower vertebrates

(Continued)

BOX 4.1 (Continued)

that have the ciliary marginal zone (CMZ) located between neural retina and ciliary epithelium where retinal progenitors proliferate throughout life, higher vertebrates including mammals cease retinogenesis soon after birth. Specifically in rodents, the pigmented ciliary epithelium appears to harbor retinal stem cells capable of becoming rod photoreceptors, bipolar neurons and Muller glia (Ahmad et al., 2000). Stem cells either isolated from the ciliary epithelium or assessed in vivo have been shown to proliferate to form neurospheres in response to growth factors such as EGF, FGF-2, and insulin as well as express nestin, an intermediate filament protein and marker for neuroepithelial and glial stem cells (Das et al., 2005; Abdouh and Bernier, 2006). These ciliary epithelial stem cells appear to divide asymmetrically in vivo and preferentially generate RGCs in the presence of 1% FBS as assessed by expression of Brn3b, a POU-domain transcription factor mostly specific to RGCs and Thy-1 (Das et al., 2005; Abdouh and Bernier, 2006). However, Yanagi et al. (2006) observed that adult rat ciliary epithelial progenitors did not generate neurons expressing Thy-1, HPC-1 (amacrine marker) or O4 oligodendrocytes.

While the ciliary epithelial stem cells appear to express potential markers for early retinal neurons, they do not appear to differentiate to retinal neurons in vivo even when exposed to growth factors, indicating that the ciliary body may be inherently non-permissive for neurogenesis (Abdouh and Bernier, 2006). Additionally, general injury to the ciliary epithelium is also insufficient to trigger these stem cells to differentiate to retinal neurons. Another limitation of the ciliary epithelial stem cells could be their intrinsic inability to differentiate to adult retinal neurons in addition to inhibitory cues present in the ciliary body

(Abdouh and Bernier, 2006). Compared to fetal forebrain stem cells, ciliary epithelial stem cells form significantly fewer neurospheres with increasing passages (Yanagi et al., 2006). While Notch-signaling pathway molecules, characteristic of stem cells, are expressed initially in ciliary epithelial neurospheres, many of these molecules including Delta1, Notch1, and HES-5 gradually decrease with increasing passages indicating that undifferentiated cells within the neurospheres were becoming reduced (Yanagi et al., 2006). Another limitation is the potential to differentiate endogenous retinal progenitors from ciliary epithelial stem cells, although attempts to further characterize the two are under way (Das et al., 2005). Extensive research needs to be carried out in the following areas: to characterize these stem cells, to guide these stem cells to migrate to the retina when needed, and to promote their differentiation to new retinal neurons (Abdouh and Bernier, 2006). Despite these significant issues, the ciliary body continues to maintain a unique characteristic in possessing these ciliary stem cells which could be useful in the treatment of retinal degenerative diseases and retinal repair.

IV. ACKNOWLEDGMENTS

The authors are supported by National Eye Institute NIH grants EY04873, EY00785, Research to Prevent Blindness and The Connecticut Lions Foundation (to M.C.-P.), EY11979; EY016242, and the Texas Higher Education Coordinating Board to (T.Y.).

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