Chronological Changes in Tip Cells during Sprouting Angiogenesis of Development of the Retinal Vasculature in Newborn Mice.

PURPOSE: To investigate a sequential chronological change in tip cells during the development of the retinal vasculature in newborn mice. MATERIALS AND METHODS: Newborn C57BL/6 mice were used for this study. To elucidate the patterns in the developing retinal vasculature, histology, and immunohistochemistry-antiplatelet endothelial cell adhesion molecule-1, anticollagen type IV, isolectin IB4-were performed on sections of mouse retina on postnatal days (P)-4, -8, and -12. Staining patterns of isolectin IB4-stained arterial and venous tip cells were compared in retinal wholemounts, in which the numbers and characteristics of tip cells were compared between arteries and veins on P-4, -6, and -8. In addition, vascular densities and branching patterns were compared between arterial and venous vascular forefront areas. RESULTS: Tip cells in the superficial vascular plexus were observed until P-8. The number of tip cells was highest on P-6, decreasing dramatically from P-6 to P-8 (P-4, 165.2 ± 10.1, n = 17; P-6, 183.8 ± 19.4, n = 15; P8, 21.4 ± 6.4, n = 15) (p < 0.05, respectively, t-test). There was a greater number of tip cells in veins versus arteries on P-4 and P-6 (P-4, 91.0 ± 9.2 veins versus 74.2 ± 10.4 arteries; P-6, 104.0 ± 10.2 veins versus 79.8 ± 11.3 arteries) (p < 0.05, respectively). Arterial tip cells had thinner and longer sprouts compared with venous tip cells (basal thickness: 15.7 ± 8.7 veins versus 9.9 ± 3.5 µm arteries) (length, 20.3 ± 9.1 veins versus 37.1 ± 13.2 µm arteries on P-4) (p < 0.05, respectively). Vessel areas and densities of vascular branch points were significantly higher around veins compared to arteries (vessel areas: 58.9 ± 1.2% veins versus 40.8 ± 1.9% arteries; vascular branch points, 1371.9 ± 136.7/mm2 veins versus 1046.7 ± 175.5/mm2 arteries) (p < 0.05, respectively). CONCLUSION: The number of tip cells increased to a greater extent in the superficial vascular plexus of veins versus arteries until P-6. Consequently, there are more vessel areas and vascular branch points near retinal veins versus arteries. Arterial tip cells are longer and thinner than the shorter and thicker venous tip cells.

Human Vision-Motivated Algorithm Allows Consistent Retinal Vessel Classification Based on Local Color Contrast for Advancing General Diagnostic Exams.

PURPOSE: Abnormalities of blood vessel anatomy, morphology, and ratio can serve as important diagnostic markers for retinal diseases such as AMD or diabetic retinopathy. Large cohort studies demand automated and quantitative image analysis of vascular abnormalities. Therefore, we developed an analytical software tool to enable automated standardized classification of blood vessels supporting clinical reading. METHODS: A dataset of 61 images was collected from a total of 33 women and 8 men with a median age of 38 years. The pupils were not dilated, and images were taken after dark adaption. In contrast to current methods in which classification is based on vessel profile intensity averages, and similar to human vision, local color contrast was chosen as a discriminator to allow artery vein discrimination and arterial-venous ratio (AVR) calculation without vessel tracking. RESULTS: With 83% ± 1 standard error of the mean for our dataset, we achieved best classification for weighted lightness information from a combination of the red, green, and blue channels. Tested on an independent dataset, our method reached 89% correct classification, which, when benchmarked against conventional ophthalmologic classification, shows significantly improved classification scores. CONCLUSIONS: Our study demonstrates that vessel classification based on local color contrast can cope with inter- or intraimage lightness variability and allows consistent AVR calculation. We offer an open-source implementation of this method upon request, which can be integrated into existing tool sets and applied to general diagnostic exams.

Effect of Robo4 on retinal endothelial permeability.

PURPOSE: To examine the role of Roundabout 4 (Robo4) in retinal endothelial permeability and analyze the structural events that lead to barrier disruption. MATERIALS AND METHODS: Small interfering RNA (siRNA) technology was used to knockdown Robo4 expression to study its effects on the permeability of human retinal vascular endothelial cells (HRVECs) in vitro. The endothelial cell permeability was detected by measuring the flux of rhodamine B isothiocyanate (RITC)-dextran across the HRVEC monolayers. The impact of Robo4 siRNA on the expression of tight junction (TJ) proteins and the activation of LIM-kinase (LIMK)/cofilin pathway were measured by western blotting. The change of actin cytoskeleton was detected using indirect immunofluorescence. RESULTS: Robo4 siRNA increased the permeability of HRVEC monolayers. The expression levels of TJ-associated proteins occludin and zonula occludens (ZO)-1 were suppressed in Robo4-depleted cells. In addition, there was rearrangement of F-actin in HRVECs. These processes were induced through increased activity in the LIMK/cofilin pathway which coincided with a disruption in the barrier properties of retinal endothelial monolayers. CONCLUSIONS: Knockdown of Robo4 expression in HRVECs induced endothelial hyperpermeability associated with the downregulation of ZO-1, occludin, and the rearrangement of F-actin and that LIM-kinase 1 (LIMK1)/cofilin signal transduction system may be involved in the modulating process.

Morphometric characteristics of central retinal artery and vein endothelium in the normal human optic nerve head.

PURPOSE: This study documents the morphometric features of arterial and venous endothelia in the different laminar regions of the normal human optic nerve head and speculates on the hemodynamic characteristics of the central retinal artery (CRA) and central retinal vein (CRV). METHODS: Twenty normal human eyes were used. Microcannulation techniques were used to label the cytoskeleton and nuclei of endothelial cells in the CRA and CRV, after which images were captured using confocal microscopy. Length, width, length-to-width ratio, and area measurements were obtained from endothelium and its nuclei. Nucleus position with respect to cell apex and direction of blood flow was also quantified. Comparisons were made between prelaminar, anterior lamina cribrosa, posterior lamina cribrosa, and retrolaminar regions. Venous and arterial endothelial cell morphology was also compared. RESULTS: There was significant variation in venous endothelial morphology across the different laminar regions; however, no differences were found in arterial endothelial characteristics (all P > 0.1065). Significant differences were found between arterial and venous endothelium in all laminar regions apart from the posterior lamina cribrosa, where only nuclear area (P = 0.0001) and nucleus position (P = 0.0088) were found to be different. CONCLUSIONS: Arterial-like appearance of venous endothelium in the posterior lamina cribrosa, where pressure gradient forces are predicted to be greatest and CRV luminal diameter is known to be narrowest, implicates this as a site of altered hemodynamic stress. Heterogeneity of venous endothelium may have relevance for understanding ocular vascular diseases such as central retinal vein occlusion.

[Morphometric analysis of retinal vascular endothelial cells in rats].

INTRODUCTION: In the present study, we observed the transformation of endothelial cells in the retinal vessel of normal rats and examined the relationship between morphometrical parameters and vessel calibers. MATERIALS AND METHODS: Retinal vessels of male Wistar-Kyoto rats were stained with an en face silver staining method and the vessel caliber, the axis of the cell oriented parallel to the longitudinal axis and to the transverse plane of the vessel, and the deviation angle of endothelial cells against the vessel axis were measured under a light microscope. RESULTS: As the vessel caliber increased, endothelial cells showed a tendency to extend along the longitudinal axis in the arterioles, but endothelial cells remained unchanged in the venules. The deviation angle of endothelial cells was nearly parallel to the vessel axis especially in the arterioles. CONCLUSIONS: Change in shape of endothelial cells in the retinal arterioles suggested a high shear stress in the large arterioles, and lower shear stress in the smaller arterioles with its decrease of caliber. In the venules, however, the cell shape was unchanged, and this suggests that the blood flow is fairly steady.

The relative distribution of retinal and choroidal blood in the human retina.

In this paper a model is presented to explain the relative distribution of retinal and choroidal blood to the human retina. According to this model the retina between the inner limiting membrane and the outer limiting membrane is metabolically controlled and nourished by the retinal vasculature, through Muller cells. The retina between the outer limiting membrane and Bruch's membrane is nourished by the choroidal vasculature, through the retinal pigment epithelium. Muller cells have the ability of metabolic uptake from the interphotoreceptor space as demonstrated in pathological situations suchs as central retinal artery occlusion, when both the Muller cell and the photoreceptor nuclei survive. This demonstrates an overlap existing between the two distributions. Neural elements in the retina depend on Muller cells and RPE for metabolic support. It is only reasonable to expect that the cleavage plane between the two blood supplies is the outer limiting membrane, the outer limit of the Muller cell. In this model both the photoreceptor nuclei (the outer nuclear layer) and the foveal retina are assumed to be supplied by retinal. and not choroidal, vasculature. One consequence is that after long standing retinal detachment the photoreceptor outer segments degenerate (since they become deprived of choroidal blood supply) but the outer nuclear layer, nourished by the Muller cell, continues to survive.

Basic fibroblast growth factor stimulates 3H-thymidine uptake in retinal venular and capillary endothelial cells in vivo.

Recent studies have found basic fibroblast growth factor (bFGF), an angiogenic peptide, in retina and have suggested that bFGF is responsible for retinal vascular proliferation. To test the hypothesis that bFGF stimulates 3H-thymidine uptake in retinal vascular cells in vivo, we injected bFGF (100 ng) into the vitreous cavity of six cats at 0 hr and again at 24 hr. Eight control eyes received boiled bFGF or no injection. After 46 hr, 3H-thymidine was injected into the vitreous cavity of all eyes and 2 hr later the eyes were enucleated. Intense 3H-thymidine uptake was seen in eyes with bFGF (56 +/- 20 SD positive cells per section) but not in control eyes (7-10 positive cells per section (P less than 0.001). Trypsin digest preparations showed that the thymidine uptake was predominantly in the venular (89%) and capillary (10%) endothelium and not in arterioles (1%) (P less than 0.001). The data suggest that retinal venular endothelial cells respond preferentially to exogenous bFGF, and in part may explain their prominent role in the neovascular process. In a second group of experiments to test the hypothesis that retinal ischemia releases a diffusable factor similar to bFGF that can cause 3H-thymidine uptake in retinal vascular cells, we created branch retinal vein occlusion in six cat eyes. The fellow eyes received no injections. In the eyes with branch vein occlusion there was an intense 3H-thymidine uptake within the distribution of the occluded vein (84 +/- 77 SD positive cells per section), but none in the areas outside the occluded vein (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Postnatal retinal vascular development of the puppy.

Retinal vascular development during the first three postnatal weeks was studied in 63 purebred beagle puppies. Use of a positive enzyme histochemical reaction for adenosine triphosphatase in the nuclei and nucleoli of vascular cells made visualization of the retinal vasculature possible. Animals were killed by decapitation. Thus, artifacts resulting from use of anesthetics or tracer substances were avoided. In general, this study demonstrates important similarities between canine and human retinal vascular development, and this gives further reason to use of the puppy retina as a superior model for studying retrolental fibroplasia pathogenesis. This staining technique demonstrates undifferentiated cells in the avascular retina that appear to be vascular precursors or angioblasts. Primordial vessels form by organization of differentiating angioblasts that exist in peripheral retinal cystic spaces at birth, or by addition of fully differentiated endothelium; they form unlike neovascularization. Müller cell processes appear to provide a structural matrix throughout the avascular puppy retina on which differentiated angioblasts organize into a vascular network. Arteries develop in beds of primordial capillaries lying near the leading edge of the developing vasculature. This precedes vein formation which occurs through a process involving coalescence of embryonic capillaries which themselves were derived from primordial capillaries. Preliminary examination of eight mongrel kitten retinas prepared by this method clearly indicates that the puppy retina is much more completely vascularized at birth than that of the newborn kitten. Moreover, the rate of postnatal retinal vascularization is significantly faster in the kitten. The kitten vasculature does appear to form by the organization of angioblasts as in the puppy, but kitten angioblasts have a different appearance from those in the puppy.

A morphologic and autoradiographic study of cell death and regeneration in the retinal microvasculature of normal and diabetic rats.

Cell loss and regeneration were investigated and compared in the retinal microvasculature of age- and sex-matched normal and streptozotocin diabetic rats. Selective pericyte loss in the diabetic rat was characterized by changes in the pericyte to endothelial cell ratio in retinal capillaries isolated for microscopy by the trypsin digest technique. A comparison of 3- and 9-month-old normal rats showed no significant change in the pericyte to endothelial cell ratio (1:2.7). In diabetic animals the ratio was reduced to 1:4.03, which was statistically significant (P less than .001). Premitotic retinal vascular cells in normal and diabetic rats were labelled with tritiated thymidine and the labelling indices calculated from cell counts of trypsin digest preparations. Methyl H3 thymidine was infused continuously over an eight-day period using osmotic mini pumps. The labelling index of endothelial cells (0.33%) in normal rats increased to 0.91% in diabetic animals (P less than .05). The labelling index of pericyte cells in normal animals (0.16%) did not increase significantly (P greater than .05) in diabetic animals (0.19%). A special stain was used to exclude labelled polymorphonuclear leukocytes from the cell counts.