The Cell and Molecular Biology of Glaucoma: Biomechanical Factors in Glaucoma

Glaucoma is a heterogeneous group of diseases that progressively damage the optic nerve, leading to visual impairment and blindness. One major risk factor for most forms of glaucoma is the pressure inside the eye (intraocular pressure [IOP]). IOP is involved in both the development and progression of glaucoma. It can directly affect pressure-sensitive cells and tissues in the eye, especially those cells involved in glaucomatous damage to the eye. These pressure-sensitive cells and tissues are (1) the trabecular meshwork (TM); (2) cells in the optic nerve head (ONH), including lamina cribrosa cells and optic nerve head astrocytes (ONHAs); (3) the peripapillary sclera around the ONH; (4) retinal ganglion cells (RGCs); and (5) RGC axons in the retinal nerve fiber layer (RNFL) and in the ONH (Fig. 1A). 

Figure 1.

 

Regions of the eye involved in glaucomatous damage and cellular elements involved in the response to biomechanical stress. (A) Cells and tissues of the eye involved in glaucomatous damage are mechanosensitive and respond to glaucomatous pressure changes within the eye. (B) There is an intimate connectivity between the ECM, cell membrane, cytoskeleton, nuclear membrane, and nuclear chromatin that rapidly respond to biomechanical stresses on the cell.

These biological cells and tissues experience and respond to pressure-induced mechanical deformation, such as stretching and compression (i.e., biomechanical stress). In addition, cells in certain regions of the TM are subjected to sheer stress from the flow of the aqueous humor, the nutritive fluid in the anterior portion of the eye, through the outflow pathway. The eye experiences normal physiological fluctuations in IOP caused by eye blinking and movement, eye rubbing, and the daily variations in the rate of aqueous humor production that do not damage the eye. However, the pressure-sensitive tissues of the eye are pathologically affected by the chronically elevated IOP and abnormal IOP spikes associated with glaucoma. In addition, there are molecular, morphologic, and physiological changes in the eye that occur with aging and may make the eye more susceptible to the biomechanical insults associated with glaucoma. 

Cellular Biomechanics

Various parts of the cell, including the extracellular matrix (ECM), cell membrane, cytoskeleton, and nucleus, are closely interconnected and respond together to biomechanical stress ¹ (Fig. 1B). The ECM consists of interconnected extracellular proteins to which cells are attached and that organize the cells. Cells bind to the ECM through receptors (such as integrins) embedded in the cell membrane. These ECM receptors are connected via linking proteins to the cytoskeleton, the internal scaffolding of the cell that regulates numerous cell functions. The cytoskeleton is attached to the nuclear membrane and can very rapidly (in millionths of a second) send signals to the nucleus to alter the expression of genes in an attempt to adapt to the biomechanical insult. This intricate connectivity allows signaling to occur from the outside to the inside (from the ECM to the nucleus) and from the inside to the outside (from the nucleus to the ECM) of cells. Therefore, biomechanical stress affects all four of these cellular compartments, which can respond in a healthy physiological manner or in a pathologic disease-associated manner, depending on the degree of stress and the overall state of the cell. 

There is now good evidence that the biomechanical properties of the TM and ONH are altered in glaucoma. In a recent study, the investigators used atomic force microscopy to measure the compliance (elasticity) of TM tissue isolated from normal and glaucomatous human donor eyes and showed that the glaucomatous TM is much stiffer (less compliant) than age-matched control TM tissue. ² The ONH also remodels and becomes less compliant in both human glaucoma and in a nonhuman primate model of glaucoma. ^3,4 Although initially compliant, with acute changes in IOP, the ONH becomes significantly and progressively less compliant (i.e., stiffer) after prolonged (weeks to months) IOP elevation in a nonhuman primate model of glaucoma. What are the molecular and cellular mechanisms responsible for this increased tissue stiffness in glaucoma? 

Useful Glaucoma Models

Several experimental models and insults have been used to study glaucomatous biomechanical damage to the eye. Normal and glaucomatous human donor eyes have been used to study morphologic and biochemical differences in the TM, the ONH, the RGCs, and the sclera. The anterior segments of human eyes have also been perfusion cultured to evaluate the TM ex vivo. The molecular and cellular responses to glaucomatous insults have been studied in cultured primary cells isolated from human donor eyes. The nonhuman primate model of glaucoma has provided important new insights into the biomechanics associated with pressure-induced damage to the ONH. ³ A variety of rodent models are being used to study glaucomatous damage and changes to the TM, RGCs, RGC axons, and ONH. 

Several agents that are directly related to and cause glaucoma are used as experimental tools to further dissect the molecular and cellular changes involved in glaucoma. Glucocorticoids are used therapeutically as anti-inflammatory agents, but these drugs can elevate IOP and cause glaucoma in some individuals. Levels of the cytokine transforming growth factor (TGF)-β2 are elevated in the aqueous humor, TM, and ONH of patients with glaucoma. Both glucocorticoids and TGFβ2 directly elevate IOP and cause many molecular, biochemical, and morphologic changes in the TM, similar to those in glaucoma. 

The Extracellular Matrix

There are distinct changes in the ECM in TM and ONH tissues of glaucomatous eyes. In the TM, there is increased expression of some ECM proteins, increased deposition of ECM material, decreased spaces between trabecular beams, and thickening of the trabecular beams, all of which are associated with elevated IOP. In addition to this increased deposition of ECM material in the TM, extracellular enzymes such as transglutaminase ⁵ and lysyl oxidase ⁶ are elevated in glaucoma and after TGFβ2 treatment. These enzymes covalently cross-link ECM proteins, making the ECM more rigid and less susceptible to degradation. TM cell behavior is heavily dependent on the very fine (nano) structure of the ECM. Changes in the nanostructure pattern of the cellular substrate and/or the stiffness of the substrate alter cell shape, cytoskeletal organization, and gene expression. ^7,8 The ONH ECM is remodeled in glaucoma, changing the ECM protein profile to stiffen this structure to make it more resistant to pressure-induced deformation. It seems apparent that a significant portion of the tissue stiffness in glaucoma is associated with these changes in the ECM. 

The Cytoskeleton

The internal scaffolding of cells, the cytoskeleton, plays a major role in regulating cell structure and function. It is composed of three structural elements: actin microfilaments, microtubules, and intermediate filaments. The cytoskeleton of TM cells, ONH cells, and RGCs is altered in glaucoma and glaucoma-like conditions. There is a dramatic reorganization of actin microfilaments to form cross-linked actin networks in glaucomatous TM cells and tissues ^9,10 and in glaucomatous ONH lamina cribrosa cells. ¹¹ Glaucoma-associated insults such as those caused by glucocorticoids and TGFβ2 also induce these cross-linked actin network structures in TM and ONH cells. The cytoskeletal changes are likely to make these cells stiffer and alter their overall biomechanical properties. The cytoskeleton also plays an important role in the structure and function of RGC axons, allowing long, thin extensions from the RGC body to target neurons in the visual axis of the brain. In addition, the axonal cytoskeleton actively participates in the trafficking of organelles and neurotrophic factors between the RGC body and the target neurons in the brain. In human glaucoma and animal models of glaucoma, actin microfilaments and neurofilaments of the axonal cytoskeleton are disturbed, ¹² and intracellular transport and RGC function are directly compromised. 

Stretch-Activated Membrane Receptors

Many cells that are mechanosensitive have specific receptor channels on their cell membranes that respond to cellular stretching. ¹³ For example, IOP elevation distends (stretches) TM and ONH tissues and distorts RGC bodies and axons. Each of these cells contains mechano-gated ion channels that alter intracellular ionic balance on receiving the stretch stimulus. There are maxi-K+ channels in TM and ONH cells ¹⁴ that initiate a signaling cascade when activated. Activation of the TRPV1 channels in RGCs in turn activates signaling pathways that can lead to apoptosis. ¹⁵ These changes in ions within the cells subsequently alter the activities of other ion channels and gene expression. Ionic imbalances, particularly calcium imbalances, associated with glaucoma have been reported in both TM cells ¹⁶ and ONH cells. ¹⁷  

Changes in Gene Expression

Cells often respond to changes in their environment by altering the expression of specific genes so that they can properly adapt to these changes, especially in TM and ONH cells exposed to stretching. Elevated IOP alters the expression of numerous genes in the TM, including those involved in ECM remodeling and cytoskeletal reorganization. ¹⁸ In ONH cells, stretching increases the expression of many ECM genes and growth factors, including TGFβ2. ¹⁹ These results are consistent with the ECM remodeling and increased expression of TGFβ2 seen in glaucomatous ONH tissues, suggesting that stretching initiates the pathogenic changes associated with glaucoma. 

Summary

The TM, ONH, and RGCs are exposed to biomechanical stress in glaucoma. TM cells, ONH cells, and RGCs are mechanosensitive, and biomechanical stress is transduced through intimate interactions among the ECM, membrane, cytoskeleton, and nucleus. It is known that the biomechanical properties of the TM and the ONH are altered in glaucoma, but there must be a better understanding of the molecular mechanisms involved in biomechanical stress in the disease process. How do cells in the TM, ONH, and retina sense biomechanical stress, and what signaling pathways are involved? To answer this question, the cellular/tissue biomechanics in normal versus glaucomatous TM and ONH must be characterized. Are glaucoma-related cytoskeletal changes in the TM and the ONH responsible for altered cell and tissue stiffness, and are these changes directly involved in glaucoma pathogenesis? Is ECM cross-linking associated with glaucoma pathogenesis in the TM and the ONH? Do stretch channels in the TM, the ONH, and/or RGCs play a role in glaucoma pathogenesis? Answering these questions will give us an opportunity not only to know more about the etiology of glaucoma but also to significantly improve glaucoma therapy in the future. 

Footnotes

 Disclosure: A.F. Clark, Alcon Research, Ltd. (F)