Ординатура / Офтальмология / Английские материалы / Biochemistry of the Eye 2nd edition_Whikehart_2003

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transport proteins fail and this is followed by membrane destruction. The release of lysosomal enzymes then hastens the destruction of the remaining proteins, lipids, and nucleic acids.

Apoptosis, as a biochemically programmed feature of cell death, often occurs as a normal process to eliminate excess or unneeded cells during organizational development. This mechanism can, therefore, be used to control tissue size. However, in response to a pathological process, apoptosis may accompany the development of cancer, cellular immune reactions, and be initiated in the presence of toxins. This process has been described as metabolically active and is characterized by several anatomical and chemical features. Typically, an apoptotic cell decreases its size as its contents become more concentrated. The nucleus becomes fragmented and the cell separates into separate small bodies that are then phagocytosed without an inflammatory response. The gross anatomical changes for necrosis and apoptosis are shown in Figure 10–1.

Biochemically, the principal vehicle for cell destruction in apoptosis is the activation of caspases, which are cysteinyl aspartate-specific proteases. These are protein cleaving enzymes that require the presence of aspartate on the protein substrate and make use of cysteine at the enzyme’s active site (Zimmerman et al., 2001). The mechanism is complicated by the fact that the cell makes use of several caspases (7 of the 14 known enzymes in this family) as well as the fact that the enzyme will

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Fas receptor (death) protein. FasL is known to exist as a surface protein component of ocular cells (Wilson, 1999). However, in some cases, it is not apparent that the Fas/FasL system is the only signaling system. In the eye, apoptosis can occur in cells of the retina, cornea, lens, conjunctiva, and Tenon’s capsule.

In the cornea, the destructive processes of Fuch’s dystrophy results in the loss of both endothelial and stromal cells by apoptosis (Li et al., 2001). Both Fas and FasL were found to be increased (i.e., upregulated) in corneal cells afflicted with the dystrophy to indicate triggering of the death signal. It was also found that Bax protein mRNA was much higher in keratocyte cells compared to levels of Bcl protein mRNA. This did not seem to be the case for endothelial cells and indicated that while Bax promoted apoptosis in keratocytes, some other supplementation/ amplification process was at work to promote apoptosis in the endothelium.

In cataractous human lenses, epithelial cells have been found to express Fas, Bcl, and Bax (Nishi et al., 2001). Fas ligand was not detected. However, an antibody to Fas was able to induce apoptosis in these cells. It remains to be seen whether some other in vivo ligand may be able to induce apoptosis in these cells.

Much more work has been accomplished on mechanisms of apoptosis in the retina. For example, Sparrow and Cai (2001), have shown that A2E (a phosphatidyl-pyridinium bisretinoid a component of lipofuscin) can induce apoptosis in retinal pigment epithelial (RPE) cells. The structure of A2E is shown in Figure 10–3. The A2E related apoptotic mechanism that stimulates caspase 3 requires blue light (~480 nm) for activation. Therefore, this is apparently not a simple Fas-Fas ligand mechanism. A2E is a detergent with membrane solubilizing capacity and

Figure 10–3

Molecular structure of A2E (pyridinium bisretinoid). This is a component of lipofuscin, an autofluorescent agerelated pigment formed and deposited in older retinas. A2E is an undegraded fused ring form from two vitamin A molecules and has a role in initiating apoptosis in retinal pigment epithelial cells of the retina. See text for explanation. (Redrawn from Parrish CM, Chandler JW. Corneal trauma. In Kaufman HE, Barron BA, McDonald MB editors: The cornea. Boston, 1998, ButterworthHeinemann.)

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may possibly be released/activated itself by blue light. The membrane solubilizing capacity of A2E may be sufficient to activate the Fas receptors. The death of RPE cells by apoptosis has been related to Stargardt’s disease, Best’s disease, and some other forms of macular degeneration as well as some forms of retinitis pigmentosa.

An interesting discovery has been that insulin can spare retinal neurons from apoptosis by reducing the activation of caspase-3 (Barber et al., 2001). A cell line cultured from rat retina was deprived of serum for 24 hours to induce apoptosis. When these deprived cells were exposed to 10 nM insulin, it was found that caspase-3 activation was suppressed. The data suggests that insulin receptors prevent apoptosis by pathway activation of a serine/threonine kinase (Akt) that phosphorylates members of the apoptotic pathway and inhibits them. This would ultimately result in the inhibition of caspase-3.

Following glaucoma filtration surgery, for example, fibroblasts in Tenon’s capsule often develop to produce scar tissue (Crowston et al., 2002). These cells may be induced to die by apoptosis when treated with mitomycin-C. In this case mitomycin-C, a heterocyclic antibiotic, antitumor agent synthesized by Streptomyces caespitosus, acts as a death signal for tenon capsule fibroblasts to prevent the development of scar tissue.

Liquefaction of the Vitreous

The bulk of the volume of the eye is composed of a liquid/gel containing a few cells near its border with the retina. This liquid/gel is known as the vitreous or vitreous body. The vitreous referred to here is actually the secondary vitreous as the primary vitreous is only the embryological remnant of the hyaloid artery while the tertiary vitreous is more properly called the zonules. The vitreous, whose chemical composition is given in Table 1–4, is actually ~98% water (Mayne et al., 1997). The major nonaqueous biochemical components: collagen and glycosaminoglycans (GAGs) form the vitreous into a gel that has important viscoelastic properties. These properties prevent mechanical shock from being transmitted to the retina while, at the same time, exert continuous intraocular pressure to the retina preventing detachment of its delicate structure of functional neurons.

Here interest is focused on the gellike nature of the vitreous. This is so inasmuch as the gel content decreases with age as the propensity for vitreal and retinal detachment increases (Sebag, 1989). In the human, liquefaction is already at 20% (volume) by around age 18 and progresses to greater than 50% by the 80th decade (Bishop, 2000). As liquefaction increases, GAGs become equally divided between the gel and liquid portions of the vitreous, while collagen is only associated with the gel portion. The liquefaction begins as small central pockets and then coalesces (becomes unified). It may develop as a posterior vitreous detachment (Figure 10–4). In time this could bring about a retina detachment. Posterior vitreal detachments occur in about 25% of the population.

IMPORTANT VITREAL GEL COMPONENTS

In general, the existence of a gel in the vitreous depends upon critical interactions between GAGs and collagen (vitrosin). Bishop (2000), has reviewed important biochemical structural components of the vitreous. In additon to hyaluron (hyaluronate, the negatively charged version of

hyaluronic acid), other GAG components that are found in the vitreous include: Type IX collagen, versican, and possibly agrin. Type IX collagen will be discussed further on as it represents a complex of both GAGs and collagen. Versican is the principal GAG (as a proteoglycan, see Chapter 4) found in the vitreous and is composed of chondroitin sulfate. The protein component of this GAG (see Figure 4–45 for comparison with a typical cornea proteoglycan structure) consists of three functional domains: a C-terminal domain (for binding to non-GAG sugars), a central domain (where GAGs bind), and an N-terminal domain that binds to hyaluron. Facts about the latter domain will be addressed later. Agrin, which has been located in chick viteous, will not be discussed here (see Tsen et al., 1995).

Type II collagen accounts for 75% of the total vitreal collagen (see Chapter 2 for a review of structural collagen) and is the principal “structural” collagen present. That is, its presence is essential to gel formation. Combined types V/XI collagen represent ~10% of the collagen present. Currently, the exact role of this collagen hybrid has not been determined. Type IX collagen, previously referred to, is a true collagen of which two domains seem to be important. First, a chondroitin sulfate chain is attached to the α2 chain of noncollagenous domain 3 (NC3, Figure 10–5). Due to this, type IX collagen is also classified as a proteoglycan since it has chondroitin sulfate bound to it. Secondly, COL2 (see Figure 10–5) has sites for crosslinking to type II collagen. Therefore, type IX collagen is a “go between” molecule linking type II collagen with the GAG: chondroitin sulfate. Some completely, noncollagenous proteins are also present, for example: opticin. Opticin is an extracellular matrix, leucine-rich repeat protein (ECM, LRR protein) that binds noncovalently

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Figure 10–6

Representation of the molecular gel of the vitreous. Collagens types II and IX are represented by dark lines. Adhering chondroitin sulfate GAGs (attached to type IX collagen) and opticin are indicated by small spheres on each line. The GAGs allow the collagen fibers to maintain a flexible independence within the gel while the opticin prevents collagen aggregation. Gray areas represent molecular associations between collagen fibers that is facilitated by GAGs bound to type IX collagen. Hyaluronin fills the space between the fibers, but is not essential to gel formation. (Redrawn from Bishop PN: Structural macromolecules and supramolecular organization of the vitreous gel, Progress Ret Eye Res 19:323–344, 2000.)

to collagen and prevents the fusion of collagen fibrils. That is, the fibrils do not come together (aggregate), but remain independent.

So with all of these macromolecular components, one would wonder how the vitreous gel is assembled. Let’s recall again who the principal players are thought to be: hyaluronan, collagens types II and IX (proteoglycans), versican (also a proteoglycan), and opticin. This is easier to describe by eliminating one of the principal players: hyaluronin. Bishop et al. (1999), showed that hyaluronin is not essential for gel formation although it increases its mechanical stability. That is, it is a passive, unnecessary partner. Versican seems to be another passive partner that binds to hyaluronin. It is currently proposed that the collagens (i.e., essentially collagen II and IX) are: (1) held apart by GAGs located on type IX collagen while (2) opticin prevents fiber aggregation. The combination of these molecules represents the vitreous gel and is shown in Figure 10–6.

MOLECULAR AGING EFFECTS AND LIQUEFACTION

Age related liquefaction of the vitreous appears to be the result of the breakdown of the spacing mechanisms in vitreal collagen. Such a breakdown would allow the formation of noncollagenous volumes within the vitreous; i.e., liquid areas. Research in this direction has been directed toward genetic abnormalities of opticin. No significant results have been obtained (Takanosu et al., 2001) although there do seem to exist four sequence variations of this protein (Friedman et al., 2002). Therefore, detailed knowledge of the degeneration of the coating molecules on vitreal collagens is lacking.

Chemical Burns of the Cornea

Exposure of the human cornea to caustic agents is a serious event that requires immediate medical attention. Generally, both mineral acids and alkalis cause the most frequent chemical damage to corneal tissues. Investigations of these events have centered on damage caused by alkali such as sodium hydroxide.

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Exposure to alkali (depending on the concentration) will instantaneously change the cornea from a clear to a cloudy to white opaque tissue and represents the early and rapid chemical damage that is done. Subsequent events involve immune responses and even keratomalacia if the burn is sufficiently severe.

EARLY CHEMICAL DAMAGE

When alkali is exposed to the human cornea it produces the following effects: (1) destruction of corneal cells; (2) a two-stage reaction with corneal collagen; and (3) generally a single-stage reaction with proteoglycans (Whikehart, 1991). Generally, the cellular membrane damage to the cells (in sufficiently concentrated alkali) will bring about cell death upon saponification or lysis of lipid ester bonds in the cell membranes. The effects on collagen are twofold. Initially, alkali is sorbed onto the collagen macromolecules by the binding of hydroxide ions to basic amino acids and the formation of both van der Waals forces and hydrogen bonding. These events allow considerable swelling and distortion of collagen fibers to occur as water enters the enlarged spaces between the fibers. Secondarily, the peptide bonds of the exposed and distorted fibers can be hydrolyzed by the alkali (hydroxide groups) itself in what amounts to fiber polypeptide destruction. The proteoglycans are cleaved from their GAGs at the GAG-proteoglycan linkage (xylose-serine glycosidic bond) by β-elimination in the presence of the alkali (Neuberger et al., 1972). These reactions are shown in Figure 10–7. The destruction that occurs obliterates the regular collagen fiber-fiber distance and negates the mutual light interference that maintains a clear cornea.

IMMUNOCHEMICAL DAMAGE

When an alkali burn is severe (greater than 0.1 M alkali exposure), the initial chemical insult previously described is followed by a strong infiltration of polymorphonuclear lymphocytes (PMNs) to initiate the destructive elements of inflammation (Paterson et al., 1984). Some of this destructive process has already been described in Chapter 9 for bacterial and viral infections. However, in the case of alkali burns, the destruction is directed toward a continued degradation of corneal collagen with the possible perforation of the cornea. Pfister et al. (1995) and Haddix et al. (1999) found and characterized a degraded collagen peptide that acts as a chemoattractant for PMNs. This peptide is presumably generated by the early alkali protein lysis. It has been itdentified as the tripeptide: N-acetyl-Pro-Gly-Pro and its N-methylated analogue and was found only to be active when the N-terminal proline was blocked with a functional group such as acetate. How the chemoattraction occurs in vivo is not known, but is probably analogous to the events shown and described in Figure 9–14. Infiltration of PMNs occurs within the first two days of the burn. If the burn is severe, reparative processes cannot commence due to cellular destruction. Then ulceration of the tissue develops from the destructive activity of PMNs (Parrish, Chandler, 1998). Both the PMNs and any remaining undamaged cells produce and release matrix metalloproteinases (described in Chapter 3). These enzymes further lyse damaged corneal collagen and proteoglycan proteins. This is generally a severe and terminal situation for the cornea when the processes of protein degradation exceed the reparative synthesis of new collagen by any undamaged or newly formed cells from the periphery.