Gijs F.J.M. Vrensen

Vascular endothelial growth factors and angiogenesis in eye disease

The vascular endothelial growth factor (VEGF) family of growth factors controls pathological angiogenesis and increased vascular permeability in important eye diseases such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). The purpose of this review is to develop new insights into the cell biology of VEGFs and vascular cells in angiogenesis and vascular leakage in general, and to provide the rationale and possible pitfalls of inhibition of VEGFs as a therapy for ocular disease. From the literature it is clear that overexpression of VEGFs and their receptors VEGFR-1, VEGFR-2 and VEGFR-3 is causing increased microvascular permeability and angiogenesis in eye conditions such as DR and AMD. When we focus on the VEGF receptors, recent findings suggest a role of VEGFR-1 as a functional receptor for placenta growth factor (PlGF) and vascular endothelial growth factor-A (VEGF)-A in pericytes and vascular smooth muscle cells in vivo rather than in endothelial cells, and strongly suggest involvement of pericytes in early phases of angiogenesis. In addition, the evidence pointing to distinct functions of VEGFs in physiology in and outside the vasculature is reviewed. The cellular distribution of VEGFR-1, VEGFR-2 and VEGFR-3 suggests various specific functions of the VEGF family in normal retina, both in the retinal vasculature and in neuronal elements. Furthermore, we focus on recent findings that VEGFs secreted by epithelia, including the retinal pigment epithelium (RPE), are likely to mediate paracrine vascular survival signals for adjacent endothelia. In the choroid, derailment of this paracrine relation and overexpression of VEGF-A by RPE may explain the pathogenesis of subretinal neovascularisation in AMD. On the other hand, this paracrine relation and other physiological functions of VEGFs may be endangered by therapeutic VEGF inhibition, as is currently used in several clinical trials in DR and AMD.

VEGFR-3 in adult angiogenesis.

Vascular endothelial growth factor receptor 3 (VEGFR‐3, Flt‐4), the receptor for vascular endothelial growth factors (VEGFs) C and D, is expressed on lymphatic endothelium and may play a role in lymphangiogenesis. In embryonic life, VEGFR‐3 is essential for blood vessel development. The purpose of this study was to investigate whether VEGFR‐3 is also involved in blood vessel angiogenesis in the adult. This was studied in human tissues showing angiogenesis andin a model of VEGF‐A‐induced iris neovascularization in the monkey eye, by the use of immunohistochemistry at the light and electron microscopic level. VEGFR‐3 was expressed on endothelium of proliferating blood vessels in tumours. In granulation tissue, staining was observed in the proliferative superficial zone in plump blood vessel sprouts, in the intermediate zone in blood vessels and long lymphatic sprouts, and in the deeper fibrous zone in large lymphatics, in a pattern demonstrating that lymphangiogenesis follows behind blood vessel angiogenesis in granulation tissue formation. At the ultrastructural level, VEGFR‐3 was localized in the cytoplasm and on the cell membrane of endothelial cells of sprouting blood vessels and sprouting lymphatics. In monkey eyes injected with VEGF‐A, blood vessel sprouts on the anterior iris surface and pre‐existing blood vessels in the iris expressed VEGFR‐3. In conclusion, these results support a role for VEGFR‐3 and its ligands VEGF‐C and/or VEGF‐D in cell‐to‐cell signalling in adult blood vessel angiogenesis. The expression of VEGFR‐3 in VEGF‐A‐induced iris neovascularization and in pre‐existing blood vessels exposed to VEGF‐A suggests that this receptor and possibly its ligands are recruited in VEGF‐A‐driven angiogenesis. Copyright

Changes in the internal structure of the human crystalline lens with age and accommodation.

Scheimpflug images were made of the unaccommodated and accommodated right eye of 102 subjects ranging in age between 16 and 65 years. In contrast with earlier Scheimpflug studies, the images were corrected for distortion due to the geometry of the Scheimpflug camera and the refraction of the cornea and the lens itself. The different nuclear and cortical layers of the human crystalline lens were determined using densitometry and it was investigated how the thickness of these layers change with age and accommodation. The results show that, with age, the increase in thickness of the cortex is approximately 7 times greater than that of the nucleus. The increase in thickness of the anterior cortex was found to be 1.5 times greater than that of the posterior cortex. It was also found that specific parts of the cortex, known as C1 and C3, showed no significant change in thickness with age, and that the thickening of the cortex is entirely due to the increase in thickness of the C2 zone. With age, the distance between the sulcus (centre of the nucleus) and the cornea does not change. With accommodation, the nucleus becomes thicker, but the thickness of the cortex remains constant.

The pathology of after-cataract

Abstract After‐cataract can be defined as the retropupillary, non‐vitreal opacification of the lens remnants occurring after extracapsular cataract extraction (ECCE) leading to disturbance of transparency and impairment of vision. The synonymous secondary cataract is confusing, since it is also and more frequently used as cataract secondary to ocular diseases (Duke‐Elder 1969). The synonymous opacification of the posterior capsule is, in fact, a misnomer, since histological observations revealed that after‐cataract is not proven to be the result of changes in the remaining posterior capsule itself, but is caused by proliferation of lenticular epithelial cells, fibroblasts, macrophages and even iris‐derived pigment cells on the posterior capsule (Fig. 1).

Altered aggregation properties of mutant γ-crystallins cause inherited cataract

Protein inclusions are associated with a diverse group of human diseases ranging from localized neurological disorders through to systemic non‐neuropathic diseases. Here, we present evidence that the formation of intranuclear inclusions is a key event in cataract formation involving altered γ‐crystallins that are un likely to adopt their native fold. In three different inherited murine cataracts involving this type of γ‐crystallin mutation, large inclusions containing the altered γ‐crystallins were found in the nuclei of the primary lens fibre cells. Their formation preceded not only the first gross morphological changes in the lens, but also the first signs of cataract. The inclusions contained filamentous material that could be stained with the amyloid‐detecting dye, Congo red. In vitro, recombinant mutant γB‐crystallin readily formed amyloid fibrils under physiological buffer conditions, unlike wild‐type protein. These data suggest that this type of cataract is caused by a mechanism involving the nuclear targeting and deposition of amyloid‐like inclusions. The mutant γ‐crystallins initially disrupt nuclear function, but then this progresses to a full cataract phenotype.

Biological glass: structural determinants of eye lens transparency

The purpose of the lens is to project a sharply focused, undistorted image of the visual surround onto the neural retina. The first pre-requisite, therefore, is that the tissue should be transparent. Despite the presence of remarkably high levels of protein, the lens cytosol remains transparent as a result of short-range-order interactions between the proteins. At a cellular level, the programmed elimination of nuclei and other light-scattering organelles from cells located within the pupillary space contributes directly to tissue transparency. Scattering at the cell borders is minimized by the close apposition of lens fibre cells facilitated by a plethora of adhesive proteins, some expressed only in the lens. Similarly, refractive index matching between lens membranes and cytosol is believed to minimize scatter. Refractive index matching between the cytoplasm of adjacent cells is achieved through the formation of cellular fusions that allow the intermingling of proteins. Together, these structural adaptations serve to minimize light scatter and enable this living, cellular structure to function as ‘biological glass’.

Nuclear breakdown during terminal differentiation of primary lens fibres in mice: A transmission electron microscopic study

The pre and post-natal development of wild type mouse lenses was studied by transmission electron microscopy, with special emphasis on denucleation of primary lens fibres. Denucleation of primary fibres is characterized by nuclear accumulation of small granules, most likely nucleosomes, which are condensed to osmiophilic bodies in the nucleus and in the cytoplasm. The osmiophilic bodies are laid down in apposition to the fibre membrane and are invaded by vesicles and granules, which probably contain proteolytic enzymes. Part of the breakdown products are extruded into the extracellular space, transported to the anterior and posterior poles where they might be finally digested or discarded from the lens. The morphology of the denucleation process of primary fibres is different from the gradual fading of nuclei in secondary fibres as described by Kuwabara and Imaizumi (1974: Invest. Ophthalmol. Vis. Sci. 13, 973-81).

Local variation in absolute water content of human and rabbit eye lenses measured by Raman microspectroscopy

Raman spectra were obtained from fresh, fixed and sliced rabbit lenses and from human lens slices. For all lenses and lens slices the ratio R, defined as the Raman intensity at 3390 cm-1 divided by the Raman intensity at 2935 cm-1, was measured at different locations along the visual and equatorial axis. The ratios R were transformed to absolute water mass percentages by measuring solutions with known protein concentrations. It was shown that fixation and slicing have very little effect on the absolute water content of the lenses. The values obtained for the absolute water content are comparable to values given in literature. It was also shown that the water content in rabbit and human lenses rapidly decreases from the immediate anterior and posterior subsurface region to the deep superficial cortex and is relatively constant in the nucleus. Raman microspectroscopy appears to be a reliable method for the measurement of the absolute water content of small volumes on defined positions in the lens. This can be very useful when analyzing the possible relation between local variations in water content and the occurrence of opacities in the lens.

The peripheral and central projections of the Edinger-Westphal nucleus in the rat. A light and electron microscopic tracing study

The peripheral and central efferent projections of the rostral part of the Edinger-Westphal nucleus in the rat were investigated at the light and electron microscopic level by means of iontophoretic injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin and retrograde tracer injections of Fast blue and Nuclear yellow into the facial nucleus and into the principal olive. Two pathways leaving the rostral part of the Edinger-Westphal nucleus were studied, a peripheral and a central descending pathway. Fluorescent experiments demonstrated that the central pathway fibers originated from distinct individual Edinger-Westphal neurons. These neurons were mainly distributed throughout the rostral part of the Edinger-Westphal nucleus and had fusiform cell bodies. The neurons rarely form collateral projections. The central descending pathway left the Edinger-Westphal nucleus medially and terminated bilaterally in the principal olive, in the subnuclei A, B and C of the inferior olive and ipsilaterally in the medial accessory olive. The central pathway also terminated contralaterally in the lateral parabrachial nucleus, the facial nucleus, the trigeminal brainstem nuclear complex, the lateral reticular nucleus and the rostroventral reticular nucleus. The projection to the facial nucleus provides evidence for the existence of a polysynaptic loop forming the central part of the corneal blink reflex. Projections from the Edinger-Westphal nucleus to the cerebellar cortex or the deep nuclei, as described in cat and primate, could not be confirmed. The peripheral pathway left the Edinger-Westphal nucleus ventrally and terminated on dendrites of ciliary ganglion cells, along smooth muscle cells of ciliary ganglion associated arterioles and in the proximity of ciliary ganglion associated venules. The central and peripheral terminals that originate in the Edinger-Westphal nucleus all had similar ultrastructural features: clear, round vesicles and electron dense mitochondria. The terminals originating from the central descending pathway were often found to be arranged in glomerular-like structures. The central and peripheral terminals made asymmetric synaptic membrane specializations (Gray type one), except terminals innervating the ciliary ganglion associated vessels, which showed no synaptic contacts.

Efferent projections of the olivary pretectal nucleus in the albino rat subserving the pupillary light reflex and related reflexes a light microscopic tracing study

The olivary pretectal nucleus is a primary visual centre sensitive to luminance changes. It is involved in the pupillary light reflex, the consensual pupillary light reflex and related reflexes, such as the lid closure reflex whereby pupillary constriction takes place. Since the olivary pretectal nucleus is a small nucleus, previous studies using degeneration, horseradish peroxidase and radioactive amino acid tracing were limited regarding to the exclusiveness of the projections from the olivary pretectal nucleus. In the present study the position of the olivary pretectal nucleus in the rat was first localized by physiological recording of the neurons upon luminance stimulation. Subsequently, an anterograde tracer Phaseolus vulgaris leucoagglutinin was injected iontophoretically. This allows a much more precise localization of the olivary pretectal nucleus projections. Ascending and descending pathways originating from the olivary pretectal nucleus were observed. Ascending fibres project bilaterally to the intergeniculate leaflet, the ventral part of the lateral geniculate nucleus and ipsilaterally to the anterior pretectal nucleus. In addition, contralateral projections were observed to the zona incerta and the fields of Forel. Descending fibres project bilaterally to the periaqueductal gray, the nucleus of Darkschewitsch, the interstitial nucleus of Cajal, the Edinger-Westphal nucleus and the intermediate gray layer of the superior colliculus. Also a contralateral projection to the oculomotor nucleus and an ipsilateral projection to the pontine nucleus and the nucleus of the optic tract were found. Furthermore, the contralateral olivary pretectal nucleus received a small projection. Retrograde tracing experiments using two fluorescent dyes revealed that the fibres projecting to the contralateral olivary pretectal nucleus and to the contralateral interstitial nucleus of Cajal are collaterals. The projection from the olivary pretectal nucleus to the facial nucleus which has been described to receive an input in cats could not be confirmed for the rat. The fact that the Edinger-Westphal nucleus, the interstitial nucleus of Cajal and the superior colliculus receive an input from the olivary pretectal nucleus suggests that this primary visual centre is not only involved in the pupillary light reflex, but also in controlling eye and head position and saccadic eye movements. Although visual acuity largely depends on receptive field sizes of retinal ganglion cells and their central connections, the stronger sympathetic influence during the pupillary light reflex in animals with frontally placed eyes compared to animals with laterally placed eyes may also contribute to the higher visual acuity in animals with frontally placed eyes.