Ординатура / Офтальмология / Английские материалы / Basic Sciences in Ophthalmology_Velayutham_2009

Alpha crystallin function as molecular chaperones i.e., they help to maintain the normal molecular conformation of other lens crystallins by binding to them and prevent the crystallins aggregation that may lead to cataract formation. Light scattering is a phenomenon associated with beta crystallin aggregation and this is inhibited by alpha crystallin.

Molecular Biology of Crystallins

Crystallins are related to heat shock proteins (heat shock proteins are those made in response to an unusual temperature rise in organism to protect the organism by solubilising and refolding any heat denatuated proteins).

The genetic patterns of crystallins are given below:

Transcription of gene is initiated by binding of certain specific nuclear protein (alpha ACRYBP1) to the promoter region in the DNA. Another transcription factor PAX6, helps to control the hn RNA and hence, the mRNA formation. DNA damage by UV radiation may produce cataract of the lens.

Genetic Defects of Crystallin

Any chemical or physical process that produces a defective gene and causes it to be transmitted or causes any normal gene to be over / under transcribed will result in some pathological process or disease. So, there will be production of either abnormal protein or normal protein in abnormal amounts resulting in congenital cataract e.g., given in table.

Fig 13.15B: Cotical cataract

conformational changes in the other oxidizable groups resulting in oxidation and aggregate formation.

H2O2 in the aqueous may induce progressive oxidation. Glutathione protects crystallins from cross linking by binding to such exposed groups. Function of glutathione may be overwhelmed in cataract formation.

Nuclear cataract

Radiation in the range of 300400 nm (including sunlight) has the potential of being cataractogenic since the lens absorbs these wavelengths.

The UV light oxidises tryptophan in the crystallin protein to N - formyl kynureniune (responsible for the dark colour of cataract), 3 hydroxy kynurenine, anthranilate, betacarboline etc., leading to non-sulfide cross linking of HM4 aggregates. 3 hydroxykymurenine can form reactive intermediates that covalently bind to the lens protein.

Other inherited disorders resulting in impairment of lens function are as follows:

Autosomal Dominant disorders

1.Marfans - Dislocation of lens (upwards) due to defect in collagen cross linking.

2.Ehler's Danlos syndromeEctopia lentis due to defective Type II collagen from lysyl hydroxylase gene mutation.

3.Myotonic dystrophy - Cataract due to mutation in chromosome 19.

Autosomal recessive:

1.Homocystinuria - dislocation of lens due to the enzyme cystathionine synthase deficiency. Sulfite deficiency - ectopia lentis.

2.Hyperlysinemia - deficiency of the enzyme lysine dehydrogenase resulting in microspherophakia.

3.Alport's syndrome - anterior lenticonus, cataract due to mutation in COL4A5 gene.

Lens Metabolism

Carbohydrates

Energy demands in the lens are lower compared to cornea as there is slower production of lens fibre cells after birth. Glucose is the source of energy and it is derived from aqueous humor. Glucose enters the cells by facilitated transport independent of insulin. Due to the low oxygen tension in the lens; only about 3% of lens glucose passes through TCA cycle and also there is loss of subcellular organelles necessary for TCA cycle in the lens fibre cells. So, in the lens, the more active pathway is anaerobic glycolysis. This can maintain the ion pump and the amino acid pump activators of the lens and also the mitotic activity of the epithelial cells. Actual O2 consumption rate is only 0.5 µl/ mg of lens tissue / hour. In the lens epithelial cells, 81% of glucose is metabolized through anaerobic glycolysis and only 4 % through aerobic glycolysis, 15% glucose enters pentose phosphate pathway to produce NADPH needed for detoxification and to nullify the oxidative stress created by the H2O2 present in aqueous.

In lens fibre cells, 83 % of glucose enters anaerobic glycolysis, 2 % aerobic and 15 % HMP shunt. So, the products of ATP in lens epithelial cells are 228 ATP totally from 81 x 2 =162 anaerobically and 2 x 36= 72 aerobic glycolysis. Normally < 5 % of glucose enters polyol pathway. The polyol pathway is induced only when there is excess glucose as in diabetes.

Diabetic Lens: Cataract

As the lens cells are not dependent on insulin for glucose uptake, toxic levels of glucose enter lens fibre cells in diabetes mellitus and activate aldose reductase which converts glucose to sorbitol. Due to the high Km of polyol dehydrogenase to sorbitol, and also due to the poor permeability of lens to sorbitol, there is retention of sorbitol in the lens. The accumulated sorbitol generates high intracellular osmotic pressure that is sufficient to burst the lens cells. The cellular debris generated by this damage becomes the manifestation of cataract. Apart from this, protein denaturing effects of glycation changing the 3 dimensional structure of protein and oxidative stress also lead to cataract formation. When the blood glucose level increases, lysine residues of the protein are glycated. This leads to increased susceptibility for sulfhydryl oxidation and consequent aggregation of protein resulting in opalescence and cataract. Protein aggregates with molecular weight > 50 million will produce light scatter. Protein metabolism: Synthesis of new protein causes with lens fibre cell formation and all changes that occur to lens protein, after this stage are posttranslational modification only. Phosphorylation of many proteins occur including crystallins, cytoskeletal proteins and MIP26.

Lipid Metabolism

Cholesterol is present in the lens fibre membrane and it imparts rigidity to the lens membrane along with sphingomyelin. It makes up 50 - 60 % of lipids and its concentration increases with age.

Redox system in the lens microenvironment:

Lens is constantly exposed to attack by oxidative agents. There is high level of H2O2 in normal aqueous and peroxidase activity is also present in lens. The other enzymes to neutralize the effect of oxidants in the lens epithelium are catalase, superoxide dismutase, glutathione peroxidase and glutathione S transferase. The concentration of glutathione in lens is high with highest concentration in lens epithelium for detoxification via mercapturic pathway and for the protection of thiol groups in protein especially cation - transport membrane protein (Figs 13.16A, B, C).

Figs 13.16A,B,C

VITREOUS

The vitreous body is a specialized connective tissue containing collagen and glycosaminoglycans and cells. It is 98 % water, 1% macromolecules and the rest solutes and low molecular weight materials. Thus, it's a mixture of fluid and gel, the ratio of which varies with age. It begins as 80 % gel and 20% fluid and gradually changes to 40% gel and 60% fluid. Gel portion is stiff but a semirigid precipitate retaining the liquid.

The peculiar characteristics of vitreous are due to the presence of Type II collagen and other collagens, as well as proteoglycans. Collagen imparts the gel structure and hyaluronic acid contributes to the viscosity of vitreous. The gel like nature of vitreous gives it rheological (deformation) properties that it is able to cushion the eye from shock by absorbing exterior forces placed on it. This is known as viscoelasticity and it prevents mechanical shock 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. Vitreous gel also has resilience (the ability to reform its original shape after deformation) and some flow (movement without stored energy)

Components of vitreous gel:

Matrix or the gel has interacting GAG and collagen. The collagen of vitreous is termed vitrosin and it consists of type II and IX. The proteoglycans are versican and agrin. The GAGs are chondroitin sulfate and predominantly hyaluronic acid and some proteins, opticin.

Type II collagen is the principal, structural collagen and accounts for 75% of the total vitreous collagen. It has more of galactosylglucose side chain and higher content of alanine. Its presence is essential for gel formation. Combined Type V, XI collagen represent ~ 10 % of total collagen.

Type IX collagen is a non-fibrillar collagen with 2 domains of importance. Collagenous and non-collagenous. Chondroitin sulfate is attached to the alpha2 chain of noncollagenous domain (NC3) (Type IX collagen is classified also as a proteoglycan due to the attachment of the GAG). The collagenous domain COL2 has sites for cross linking to Type II collagen (Fig 13.17).

Thus, type IX collagen serves as a linking molecule between Type II collagen and GAG chondroitin sulfate. Some completely, non-collagenous proteins are also present e.g., opticin (Fig. 13.18)

Fig 13.17

Fig 13.18

Opticin is an extracellular matrix protein with leucine rich repeats (ECM, LRR protein) that binds non-covalently to collagen and prevents the fusion or aggregation of collagen fibrils.

Versicon

It is the principal proteoglycan found in the vitreous. The protein component of this versicon consists of 3 functional domains:

1.A C-terminal domain for binding to non GAG sugars.

2.A central domain where GAGs - chondroitin sulfate bind.

3.A N-terminal domain that binds to hyaluron.

Hyaluron

It is hyaluronate, the negatively charged version of hyaluronic acid, not linked to a core protein in vitreous. It may form a twofold helix in solution and stabilized by non-collagenous protein there. It is a GAG with repeating unit of glucuronic acid and N - acetyl glucosamine linked by beta 1, 3 glycosidic bond. The repeating units are linked by beta 1, 4 glycosidic bonds. At physiological pH, it occurs as sodium hyaluronate, which is spheroidal and can hold an extremely large volume of water relative to its weight. This hyaluronin binds to versicon. It increases the mechanical stability of vitreous. The concentration varies between 100 - 400 µg/ml combination of these molecules represents the vitreous.

Vitreous Cells

Vitreous contains a monolayer of cells called hyalocytes that line the adult vitreous cortex. They are responsible for production of hyaluronic acid in the gel. These cells are two types 1) Fibrocyte like 2) Macrophage like.

Vitreous transmits light by the same mechanism as cornea i.e., its collagen fibrils (10 - 20 nm) are thinner than half the wavelength of light and the interfibrilllar space is filled sith GAGs (hyaluronic acid) at intervals that reduce the effect of diffraction in the system.

Vitreous retards the bulkflow of fluid and diffusion of small molecules. due to the presence of hyaluronic acid. Thus, flow of aqueous from posterior chamber towards the retina is slower in young eyes.

Comparison of some component of blood with vitreous

This table reveals that ascorbate is more in vitreous probably due to the high level present in aqueous fluid which dissipates into the vitreous.

Molecular Effects of Aging and Liquefaction

As age advances, a process called syneresis occurs. In this process, hyaluronic acid molecules are degraded to smaller moieties and the collagen fibril conglomerate to form larger fibrils, becoming visible as floaters.

Liquefaction is already at 20 % around the age of 18 and progress to greater than 50 % by 80th age. As liquefaction increases GAGs become equally divided between the gel and liquid portion of the vitreous while collagen is only associated with gel portion. With aging, vitreous collagen fibres collapse or contract resulting in decreasing volume of gel and pocket of fluid form in the vitreous. These small, central pockets of water then coalesce and may cause posterior vitreal detachment (PVD) Fig. 13.19. In time, this could bring about a retinal detachment also. PVD occurs in 25 % population. Thus, age related

Fig 13.19