March 2012
Volume 53, Issue 3
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Retina  |   March 2012
Age-Related Changes in Venous Endothelial Phenotype at Human Retinal Artery–Vein Crossing Points
Author Affiliations & Notes
  • Paula K. Yu
    From the Centre for Ophthalmology and Visual Science and
    the ARC (Australian Research Council) Centre of Excellence in Vision Science, The University of Western Australia, Perth, Australia.
  • Priscilla E. Z. Tan
    From the Centre for Ophthalmology and Visual Science and
    the ARC (Australian Research Council) Centre of Excellence in Vision Science, The University of Western Australia, Perth, Australia.
  • William H. Morgan
    From the Centre for Ophthalmology and Visual Science and
  • Stephen J. Cringle
    From the Centre for Ophthalmology and Visual Science and
    the ARC (Australian Research Council) Centre of Excellence in Vision Science, The University of Western Australia, Perth, Australia.
  • Ian L. McAllister
    From the Centre for Ophthalmology and Visual Science and
  • Dao-Yi Yu
    From the Centre for Ophthalmology and Visual Science and
    the ARC (Australian Research Council) Centre of Excellence in Vision Science, The University of Western Australia, Perth, Australia.
  • Corresponding author: Dao-Yi Yu, Centre for Ophthalmology and Visual Science and The ARC Centre of Excellence in Vision Science, The University of Western Australia, Nedlands, Western Australia 6009; dyyu@cyllene.uwa.edu.au
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 1108-1116. doi:10.1167/iovs.11-8865
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      Paula K. Yu, Priscilla E. Z. Tan, William H. Morgan, Stephen J. Cringle, Ian L. McAllister, Dao-Yi Yu; Age-Related Changes in Venous Endothelial Phenotype at Human Retinal Artery–Vein Crossing Points. Invest. Ophthalmol. Vis. Sci. 2012;53(3):1108-1116. doi: 10.1167/iovs.11-8865.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: To investigate the venous endothelial phenotype at retinal artery–vein (AV) crossings and age-related changes in human cadaveric eyes.

Methods.: Eighteen human donor eyes free of known ocular diseases were divided into two groups according to age (≤30 and >50 years). The central retinal artery was cannulated and perfused with oxygenated Ringer's solution with 1% bovine serum albumin. The perfusate solutions were switched to fixative, membrane permeabilizing solution, and selected labeling solutions for microfilament F-actin and nucleic acid in vascular endothelial cells and smooth muscle cells. The eyes were then immersion fixed and the retinas flat mounted. The venous endothelial cells were examined by confocal microscopy at the AV, pre-AV, and post-AV crossing regions.

Results.: There was no significant difference between the younger and older groups in endothelial cell length upstream or downstream from an AV crossing. At the AV crossing, the venous endothelial cells were shorter in the younger group and longer in the older group compared with those upstream or downstream from the AV crossing. Stress fibers were not frequently observed in the endothelial cells of younger donors. However, the older group had numerous stress fibers in their endothelia at AV crossing points.

Conclusions.: Age-related phenotype changes in venous endothelial cells have been identified in the region of AV crossings providing supportive evidence for the hypothesis of age-related and site-specific changes in the vascular endothelial cells as an important factor contributing to the pathogenesis of branch retinal vein occlusion.

Endothelial cells lining the vessel luminal surface of the vascular wall are highly sensitive to hemodynamic shear stress. 1,2 Variations of shear stress can functionally regulate the vascular tone and also induce structural endothelial cell changes by slow adaptive remodelling when the shear stress of altered blood flow is sustained. There is evidence that shear stress contributes to regional and focal heterogeneity of endothelial gene expression which is a factor in vascular disease. 3,4 For example, regions of flow disturbances near arterial branches, bifurcations, and high curvatures can induce complex spatiotemporal shear stress, and these characteristics can predict regional susceptibility to atherosclerosis. Changes in local vascular geometry can further modify shear stress characteristics at the endothelium. 5,6 Flow-induced endothelial cell responses are known as mechanotransduction and involve a repertoire of cell-signaling events ranging from instantaneous ion fluxes, biochemical pathways, and gene and protein expression. Endothelial cytoskeleton, stretch-activated ion molecules, glycocalyx, and cell junction proteins involved in shear stress transduction and signaling are localized to various endothelial compartments. These are not directly exposed to flow. The endothelial cytoskeleton is a natural candidate that can tie together all these shear stress receptors. 7 The cytoskeleton also has an important role in maintaining the normal structure and function of cells. The vascular endothelial cytoskeleton is known to be associated with modification in cell shape and adherence, in response to changes in hemodynamics in vitro, 8 10 ex vivo, 11 and in vivo. 12,13 Hemodynamic factors are known to influence the organization of endothelial cell cytoskeleton in situ. 14 Aging has been reported to be associated with reorganization of the actin cytoskeleton in endothelial cell culture as well as in animals. 15,16  
Retinal vein occlusion is the second commonest sight-threatening vascular disorder after diabetic retinopathy. Branch retinal vein occlusion (BRVO) is the commonest type of retinal vein occlusion, with a prevalence of 0.6%. 17 21 It is well established that age is a crucial factor, as most patients are older than 50 years. 22 BRVO typically occurs at an arteriovenous (AV) crossing and in most cases, the artery passes anterior to the vein. The pathogenic roles of systemic risk factors, hematologic disorders, and degenerative changes of vessel wall have been suggested. 23 However, the cause-and-consequence relationship in the pathogenesis of BRVO has not been defined. 23 26 BRVO appears to be a complex retinal vascular disorder with appropriate therapy still undetermined. 25 We have reported fluorescein leakage, presumed thrombi, and flow abnormalities within the vicinity of AV crossings in a retrospective review of 110 BRVO patients, suggesting that hemodynamic changes may induce endothelial damage and thrombus generation. 27 It has been proposed that turbulent blood flow occurs in the retinal vein at the AV crossing, producing injury to venous endothelial cells that ultimately leads to thrombus formation and occlusion of the vein. Local geometric changes in the retinal vein at the AV crossing may be caused by anatomic curvature or mechanical compression of the vein through the more rigid artery, causing compression of the underlying vein. 28 31  
It is clearly evident that BRVO is an age-related, site-specific, endothelium-relevant disease; however, its pathogenesis remains unclear. The purpose of this study was to investigate whether age-related endothelial cell changes are present in the retinal vein at the AV crossing point. We used our established perfusion and labeling technique in human donor eyes. 32 34 In this study, we examined the morphology and cytoskeleton in vascular endothelial cells of the venous part of AV crossings at three locations: pre-AV (upstream), at the AV crossing, and post-AV (downstream) regions in both young and older donor eyes. 
Materials and Methods
This study was approved by the human research ethics committee at the University of Western Australia. All human tissue was handled according to the Declaration of Helsinki. 
Human Donor Eyes
Eighteen donor eyes from 17 donors were studied and divided into two groups. Six eyes came from young donors with a mean age of 24.5 ± 1.9 years (range, 19–30). Twelve eyes came from older donors with an average age of 58.7 ± 2.5 years (range, 51–74). All eyes were obtained from the Lions Eye Bank of Western Australia or Donate West, the West Australian agency for organ donation. The corneal buttons were removed in all donor eyes by the eye bank for transplantation. None of the eyes used in the present study had a known history of eye disease. The demographic data, cause of death, and postmortem time to eye perfusion or fixation for each donor are presented in Table 1. Calculation of the average time from death to cannulation was 11.1 ± 1.69 hours in the older vascular group (12 eyes) and 16.7 ± 1.19 hours in the younger group (6 eyes). Subsequent R analysis did not show any significant association between postmortem time and any parameter measured (all P ≥ 0.10). 
Table 1.
 
Donor Details
Table 1.
 
Donor Details
Donor Age (y) Sex Cause of Death Time to Cannulation (h)
A 30 M MVA 16
B 19 F MVA 20
C 22 M MVA 15
D 27 M MVA 20
E 27 M MVA 14
F 22 M Suicide 15
G 58 F Pseudomyxoma 6
H 54 F Pulmonary embolism secondary to cancer 20
I 66 M Cancer 15
J 51 M Cancer 6
K 51 M IHD 9.5
L 51 M IHD 19
M 52 M IHD 17
N 74 M SAH 5
O 61 M IHD 10
P 62 M LCI 6
Q 66 M MI 14
Preparation
Details of the method of perfusion staining of retinal microvasculature have been published. 32 35 Briefly, the central retinal artery was cannulated and residual blood washed out with oxygenated Ringer's solution with 1% bovine serum albumin. After the 20-minute Ringer's wash, 4% paraformaldehyde in 0.1 M phosphate buffer was perfused for at least 1 hour for fixation. A dilute detergent of 0.1% Triton-X-100 in 0.1 M phosphate-buffered solution was then perfused for 5 to 7 minutes to aid in the permeation of endothelial cell membranes. The detergent was washed out by a further 30 minutes of 0.1 M phosphate-buffered perfusion. Then, the microfilaments and cell nuclei were labeled over the course of 2 hours, using a mixture of phalloidin conjugated to AlexaFluor 546 (30 U; Invitrogen, Carlsbad, CA) or Alexa Fluor 488 (30 U; Invitrogen) and bis benzamide H 33258 (1.2 μg/mL; Sigma-Aldrich, St. Louis, MO) or iodide dye (YO-PRO-1; 6.6 μM; Invitrogen). Residual label was cleared from the vasculature by further perfusion of 0.1 M phosphate buffer. The posterior chamber was then immersion fixed in 4% paraformaldehyde overnight. 
Flat Mounting of the Retina
The posterior globe was dissected at the equator to allow viewing of the posterior retina. The retina was carefully dissected, without inclusion of the optic disc region. A few cuts were made in the peripheral retina to enable the retina to lie flat. 
The whole-mount retinas were observed initially at low magnification by epifluorescence microscope (E800; Nikon, Tokyo, Japan) before confocal imaging. Figure 1A is an image of a fundus fluorescein angiograph in a normal subject as an example to illustrate the criteria of selecting AV crossing in this study. Figure 1B is a composite confocal image of the retinal vasculature from donor M and shows an AV crossing in the superotemporal quadrant, which is where most of the crossings were sampled. 
Figure 1.
 
(A) An image of a fundus fluorescence angiograph from a normal subject (not from one of the eyes studied) illustrating the selection criteria of AV crossing for this study and the locations of pre-AV, AV crossing, and post-AV regions for imaging venous endothelial cells. There are several AV crossings (arrows) between the artery (A) and vein (V) in the superotemporal quadrant. The AV crossing in the boxed region would be preferred for study, as the artery overlies the vein and the angle between the artery, and the vein allows the adjacent pre- and post-AV segments of the vein to be studied. Inset: an enlarged image from the highlighted region showing pre-AV, AV, and post-AV regions. (B) A composite image of low-magnification confocal images of retinal vasculature of a donor eye from a 52-year-old man. Inset: a higher magnification confocal image of the AV crossing outlined by the dotted lines. The image composite was taken from the superotemporal region of the retina 3 to 6 mm from the center of the macula. Retinal artery (A) labeled much more intensively than the retinal vein (V), because of the greater amount of F-actin in the arterial smooth muscle cells. In the pre-AV, AV, and post-AV regions, the retinal vein can be seen crossing underneath a retinal artery. The capillary-free zone is evidenced along with the retinal artery. Scale bar, 200 μm, low magnification composite; 168 μm, high-magnification inset.
Figure 1.
 
(A) An image of a fundus fluorescence angiograph from a normal subject (not from one of the eyes studied) illustrating the selection criteria of AV crossing for this study and the locations of pre-AV, AV crossing, and post-AV regions for imaging venous endothelial cells. There are several AV crossings (arrows) between the artery (A) and vein (V) in the superotemporal quadrant. The AV crossing in the boxed region would be preferred for study, as the artery overlies the vein and the angle between the artery, and the vein allows the adjacent pre- and post-AV segments of the vein to be studied. Inset: an enlarged image from the highlighted region showing pre-AV, AV, and post-AV regions. (B) A composite image of low-magnification confocal images of retinal vasculature of a donor eye from a 52-year-old man. Inset: a higher magnification confocal image of the AV crossing outlined by the dotted lines. The image composite was taken from the superotemporal region of the retina 3 to 6 mm from the center of the macula. Retinal artery (A) labeled much more intensively than the retinal vein (V), because of the greater amount of F-actin in the arterial smooth muscle cells. In the pre-AV, AV, and post-AV regions, the retinal vein can be seen crossing underneath a retinal artery. The capillary-free zone is evidenced along with the retinal artery. Scale bar, 200 μm, low magnification composite; 168 μm, high-magnification inset.
AV crossings were mostly sampled from the superotemporal quadrant. Although there were usually several AV crossings available, we selected at least one crossing from each eye. Selection was based on the presence of a branch retinal vein passing underneath an overlying retinal artery of comparable branch order (mostly first- and second-order branches) and size (generally 70–150 μm in diameter) and at a sufficiently large angle to allow access to pre- and post-AV sections of vein close to the AV crossing point (Fig. 1). 
Confocal Imaging
A with three-laser unit (405, 488, and 532 nm) was used in conjunction with a fluorescence microscope with imaging software (model E800 microscope and EZ-C1 ver. 3.20; Nikon). Confocal imaging was performed simultaneously for the different wavelengths with emission signals separated into different channels. Imaging began at low magnification. In this study, high-quality staining of the intracellular cytoskeleton in the retinal vein was necessary for detailed imaging. The AV crossing and the pre- and post-AV regions were examined using higher power objective lenses (×40 plan apochromatic oil-immersion lenses). A z-series of images was taken as far as the scleral side of the venous endothelial cells. In analyzing the endothelial cells, the most scleral side of the vessel was used to avoid interference from the overlying artery when present. This method also helped ensure that the region of vein studied was aligned with the plane of the retina. The vein's diameter was measured from the combined z series. 
Detailed Study of Venous Endothelial Cells
The venous endothelial cells at the three regions were examined in detail, with measurements taken for endothelial cell length and width, endothelial nuclei length and width, and the relative position of the upstream pole of the nuclei to the upstream pole of the cell. As far as possible, data were included only if all measurements could be taken from the same endothelium. Sketches of endothelial cells were made to outline the cell shape and nucleus position. 
Statistical Analysis
All data were tested with a statistical-analysis package (R; R Foundation for Statistical Computing, Vienna, Austria). 36 We tested for differences in measurements between venous regions (pre-AV, AV, and post-AV) or between age groups (older or young) as independent factors in the analysis. All one-way analysis of variance (ANOVA) included eye donor as a random effect using linear mixed modeling to test measurement differences between vessel regions. The assignment of eye donor as a random effect was used to account for the effects of intraeye correlation because multiple AV crossings were analyzed in some eyes. Where old to young age group comparisons were performed, P < 0.05 was significant. Where pre-AV, AV, and post-AV regions were compared, two comparisons for each region were required, and so P < 0.025 was significant. Data are shown as the mean ± SE. 
Results
Retinal Veins at an AV Crossing
Confocal microscopy showed the relationship between the retinal artery and vein at AV crossing points, both topographically and in sections. Figure 2 shows the representative images of AV crossings from a young and an older donor (22 and 66 years old, respectively). The image intensity of retinal veins was weaker than that of retinal arteries because of the subarterial position of the retinal veins and differences in microfilament content of the vessel walls. The veins meandered deeper into the retinal layers at AV crossings, and the lumens remained patent in both groups. Longitudinal folds appeared to be more common in the veins of older donors and often accompanied the deformation of the cylindrical shape of the vein directly underneath the artery. 
Figure 2.
 
Composite confocal images of AV crossings from a 22-year-old (A, B) and a 66-year-old (CD) donor. (A, C) Confocal image stacks of AV crossings; (B, D) respective cross sections of the stacks at the position of the yellow dotted line. Both specimens were labeled with phalloidin conjugated with AlexaFluor 546, and the nuclei were counterstained. In both cases, the veins dived at the AV crossings at various angles, and the venous lumen was wide open during passage under the artery. Scale bar, 50 μm.
Figure 2.
 
Composite confocal images of AV crossings from a 22-year-old (A, B) and a 66-year-old (CD) donor. (A, C) Confocal image stacks of AV crossings; (B, D) respective cross sections of the stacks at the position of the yellow dotted line. Both specimens were labeled with phalloidin conjugated with AlexaFluor 546, and the nuclei were counterstained. In both cases, the veins dived at the AV crossings at various angles, and the venous lumen was wide open during passage under the artery. Scale bar, 50 μm.
Table 2 shows venous diameter at AV crossings. Although pressurized perfusion fixation and staining were used for each donor eye, deformation and collapse of the vein wall were present in some eyes. However, none demonstrated significant narrowing at the AV crossing. The diameter of the retinal veins was comparable at each region (P = 0.8340, compared across regions in the young group; P = 0.681, compared across regions in the older group). 
Table 2.
 
Venous Width in the Three Regions
Table 2.
 
Venous Width in the Three Regions
Group Pre-AV AV Post-AV
Younger (n = 7) 102.8 ± 16.5 97.8 ± 16.7 106.9 ± 17.5
Older (n = 6) 128.0 ± 10.3 118.0 ± 8.5 134.5 ± 12.3
Endothelial Cells at an AV Crossing
Figure 3 shows low-magnification confocal images that illustrate the shape of the venous endothelial cells of the younger and older groups in pre-AV, AV crossing, and post-AV regions. The shape of the endothelial cells is indicated by peripheral border microfilament staining. Unlike the cells in the retinal artery, the venous endothelial cells appeared to be smaller, shorter, and more irregular, with some spindle shaped and some more rectangular. Few smooth muscle cells were found in the veins. 
Figure 3.
 
Confocal images of retinal venous endothelial cells from the pre-AV, AV, and post-AV crossing regions of the younger (AC) and older groups (DF). The F-actin microfilaments of the endothelia and the vascular smooth muscle cells were labeled with phalloidin (red), and their nuclei were counterstained with Hoechst (blue). Individual endothelial cells from each region of each group are outlined along with their nuclei and displayed under the images. The endothelial cells in the AV region of the younger group (B) were shorter, whereas those from the same region of the older group (E) tended to be more elongated and variable in shape. Arrows: flow direction. Scale bar, 50 μm.
Figure 3.
 
Confocal images of retinal venous endothelial cells from the pre-AV, AV, and post-AV crossing regions of the younger (AC) and older groups (DF). The F-actin microfilaments of the endothelia and the vascular smooth muscle cells were labeled with phalloidin (red), and their nuclei were counterstained with Hoechst (blue). Individual endothelial cells from each region of each group are outlined along with their nuclei and displayed under the images. The endothelial cells in the AV region of the younger group (B) were shorter, whereas those from the same region of the older group (E) tended to be more elongated and variable in shape. Arrows: flow direction. Scale bar, 50 μm.
In a comparison of the age groups, the most striking difference was the significantly longer endothelial cell in the AV region of the older eyes (Table 3). The venous endothelial cells from the older group measured 86.9 ± 5.2 μm versus 59.5 ± 2.0 μm from the younger group (P = 0.003). This difference was also reflected in the significantly greater endothelial cell length-to-width aspect ratio (Table 4) in the AV region in the older group than in the younger group (4.5 ± 0.42 vs. 3.0 ± 0.12; P = 0.019). 
Table 3.
 
Endothelial Dimension in the Three Regions
Table 3.
 
Endothelial Dimension in the Three Regions
Age Group Length Width
Pre-AV AV Post-AV Pre-AV AV Post-AV
Younger 72.0 ± 2.2 (28) 59.5 ± 2.0 (21)† 68.4 ± 2.1 (29) 21.7 ± 1.2 (28) 20.4 ± 1.0 (21) 21.9 ± 0.9 (29)
Older 75.8 ± 2.4 (40) 86.9 ± 5.2 (17)* 75.4 ± 2.6 (40)‡ 23.5 ± 1.0 (39) 21.1 ± 1.6 (17) 23.8 ± 0.8 (40)
Table 4.
 
Endothelial Cell Length-to-Width Aspect Ratio
Table 4.
 
Endothelial Cell Length-to-Width Aspect Ratio
Age Group Pre AV AV Post AV
Younger 3.5 ± 0.16 (28) 3.0 ± 0.12 (21) 3.2 ± 0.14 (29)
Older 3.5 ± 0.21 (39) 4.5 ± 0.42 (17)* † 3.3 ± 0.16 (38)
Comparison between the three regions (Table 3) within the younger group found significantly shorter endothelial cells in the AV region compared with both pre-AV (P < 0.001) and post-AV regions (P = 0.002). However, this result was not reflected in the aspect ratio (Table 4) comparison with pre-AV (P = 0.028) or post-AV (P = 0.212) regions, indicating that smaller endothelial cells of similar aspect ratio are present in the AV region of younger eyes. No statistically significant difference was identified in the other parameters measured across the three regions of the younger group. 
Comparison between the three regions (Table 3) in the older group showed significantly longer endothelia in the AV region (P = 0.014) than in the post-AV region. This difference was also reflected in the significantly greater length-to-width aspect ratio (Table 4), indicating more elongated cells when compared to the pre-AV (P = 0.016) and post-AV (P = 0.001) regions of this group. None of the other parameters measured showed a significant difference across the three regions in the older group. 
Endothelial Nuclei Dimensions and Intracellular Position
Some interesting data were noted in the dimension of the endothelial nuclei across the two age groups (Table 5). A comparison found significantly longer (P = 0.034) endothelial cell nuclei in the AV region of the older group than in the younger eyes, whereas the width was significantly greater in the older group in the pre-AV (P = 0.046) and post-AV regions (P = 0.033), when compared with the corresponding regions of the younger group. However, no statistically significant difference was noted when the aspect ratios of the two age groups were compared (pre-AV, P = 0.951; AV, P = 0.117; and post-AV, P = 0.079). 
Table 5.
 
Dimensions of Endothelial Nuclei in Three Regions
Table 5.
 
Dimensions of Endothelial Nuclei in Three Regions
Age Group Length Width
Pre AV AV Post AV Pre AV AV Post AV
Younger 12.7 ± 0.5 (28) 14.0 ± 0.7 (20) 13.4 ± 0.6 (29) 11.1 ± 0.4 (28) 10.4 ± 0.7 (20) 10.7 ± 0.5 (29)
Older 14.3 ± 0.5 (39) 16.4 ± 0.9 (17)* † 13.1 ± 0.4 (40) 12.4 ± 0.4 (39)* 10.9 ± 1.2 (17)† 12.9 ± 0.4 (40)*
In young donor eyes, the mean endothelial nuclear length and width were comparable across the three regions (Table 5, all P > 0.120). Similarly, no statistically significant difference was found in the aspect ratios of the nuclei across any of the three regions. 
In the older group, however, the endothelial nuclei at the AV crossing were significantly longer (16.4 ± 0.9 μm, all P < 0.004) and narrower (10.9 μm, all P < 0.006) than in the pre- and post-AV regions. The nuclei were broader in the pre-AV (P = 0.045) and post-AV (P = 0.033) regions of older subjects than in the younger group. The endothelial cell nuclear aspect ratio in the AV region was larger than in the pre- and post-AV regions (both P < 0.001). The nuclear width in the AV region was similar (P = 0.823) between the two age groups; however, the nuclear length was longer in the older group (P = 0.034). 
The venous endothelial nuclei were normally situated approximately one third of the way from the upstream pole of the cell (mean, 30.7% ± 0.89%; n = 130). No significant difference was noted in this positional ratio measurement between the two age groups, nor was there any significant difference between the three regions within either age group (Table 6). 
Table 6.
 
Position of the Endothelial Nuclei
Table 6.
 
Position of the Endothelial Nuclei
Age Group Pre AV AV Post AV
Younger 31.4 ± 2.1 (27) 23.5 ± 2.0 (21) 29.8 ± 1.5 (29)
Older 31.7 ± 1.8 (38) 35.7 ± 4.1 (12) 29.8 ± 1.7 (36)
Intracellular Microfilaments
Figure 4 contains high-magnification confocal images of venous endothelial cells of the younger and older groups, to illustrate the intracellular microfilaments from the regions distant from AV crossings, as well as the pre-AV, AV crossing, and post-AV areas. In general, the cytoplasm in retinal venous endothelia of young donors was relatively faint because of the reduced presence of cytoplasmic microfilaments (Figs. 4A–C). However, there was an overall increased presence of microfilaments throughout the cytoplasm in the venous endothelia of older donors, as shown on the sketches below Figures 4D to 4F. Microfilament lengths across the three regions of older donor eyes were not significantly different (Table 7). 
Figure 4.
 
High-magnification confocal images of individual venous endothelial cells from the pre-AV, AV, and post-AV crossing regions of the younger (AC) and older groups (DF). Sketches of individual endothelial cells of the corresponding regions shown beneath the images were drawn based on the peripheral border and intracellular location of F-actin microfilaments and Hoechst staining of their nuclei. F-actin microfilaments or stress fibers bundles were relatively scarce in the venous endothelia of the younger donor, as indicated by the confocal images and sketches. In contrast, F-actin microfilaments and stress fibers bundles were frequently observed in the venous endothelia of the older donors, as indicated by the confocal images and sketches. The stress fibers were short and were either present throughout the cytoplasm of the older venous endothelial cells or were associated with their nuclei. Scale bar, 20 μm. Arrows: direction of flow.
Figure 4.
 
High-magnification confocal images of individual venous endothelial cells from the pre-AV, AV, and post-AV crossing regions of the younger (AC) and older groups (DF). Sketches of individual endothelial cells of the corresponding regions shown beneath the images were drawn based on the peripheral border and intracellular location of F-actin microfilaments and Hoechst staining of their nuclei. F-actin microfilaments or stress fibers bundles were relatively scarce in the venous endothelia of the younger donor, as indicated by the confocal images and sketches. In contrast, F-actin microfilaments and stress fibers bundles were frequently observed in the venous endothelia of the older donors, as indicated by the confocal images and sketches. The stress fibers were short and were either present throughout the cytoplasm of the older venous endothelial cells or were associated with their nuclei. Scale bar, 20 μm. Arrows: direction of flow.
Table 7.
 
Length of Microfilament Fibers in Venous Endothelia of Older Donor Eyes
Table 7.
 
Length of Microfilament Fibers in Venous Endothelia of Older Donor Eyes
Length Pre AV AV Post AV
Older 8.4 ± 0.45 (92) 7.8 ± 0.58 (58) 8.6 ± 0.68 (86)
Discussion
Our results demonstrate that the venous endothelial cell shape and intracellular structure exhibit age-related changes in the AV-crossing regions. The venous endothelia at the AV crossing of the older group are significantly elongated when compared with those in the pre- and post-AV regions in the same group. In addition, there is an overall increased presence of cytoskeleton throughout the cytoplasm in venous endothelia of older donors, whereas the cytoplasm in venous endothelia of young donors has a relatively faint presence or absence of stained cytoplasmic microfilaments. Our results clearly suggest that alteration of endothelial cell phenotypes occurs at the AV crossings of older eyes. 
It is generally accepted that the phenotypic alterations in endothelial cell senescence plays a key role in age-associated vascular diseases. 15,16 It has also been demonstrated that microvascular endothelial cells have more dramatic aging changes when compared with large vessels. 15 The phenotypic changes in vascular endothelial cells have mostly been studied in the artery, with the focus on atherosclerosis. 37 In the eye, changes in the endothelial cell cytoskeleton in the cornea with aging and diabetes and at the aqueous outflow tract of normal and glaucomatous eyes have been reported. 38,39 As far as we know, phenotypic changes in venous endothelial cells at the AV crossing have not been reported. 
It should be noted that many of the donors in the older age group died of vascular diseases, whereas most of the younger donors died in motor vehicle accidents. The differences in cause of death are an unavoidable consequence of dealing with a limited number of donor eyes and make it difficult to separate age-related changes from those associated with systemic vascular disease. We cannot assess the influence of vascular disease, since the duration and severity of vascular disease in individual donors is not known. 
An interesting question raised by this study is how the venous endothelial cells respond to the site-specific hemodynamic environment at the AV crossings. As the veins approached the AV crossing, they dived below the artery. In all donor eyes studied, there was no significant narrowing and the vein diameters were comparable in the pre-AV, AV crossing, and post-AV regions. However, the geometric changes of the retinal vein in the AV crossing would probably chronically alter the local hemodynamics. Vascular endothelial cells are known to respond to hemodynamic changes by modifying their morphologic appearance along with variation in gene and protein expressions. Three different mechanical forces have been identified that can induce changes in endothelial morphology and include shear stress, hydrostatic pressure, and cyclic stretch pressure. 1 Endothelial cell shape and cytoskeleton are also known to be modulated by the flow condition and adopt varied phenotypes under no-flow, one-way, reciprocating, or alternating orthogonal flow conditions after various time durations. 1 In general, arterial endothelial cells that are chronically exposed to high shear stress forces tend to become long, spindle-shaped cells containing stress fibers closely aligned with the flow direction, whereas retinal vein endothelial cells are exposed to much lower shear stress forces and appear to be shorter in length; more irregular in shape, ranging from spindle to more rectangular; and contain almost no stress fibers. 33,34 Endothelial cells from different anatomic locations also demonstrate remarkable heterogeneity in both structure and function under normal and pathologic conditions. Changes observed in endothelial cell phenotypes at AV crossings appear to be a localized phenomenon perhaps in response to chronic exposure to a localized change in hemodynamics. It is generally presumed that a turbulent blood flow condition occurs in the retinal vein at AV crossings. 31 The endothelial cell shape and intracellular cytoskeleton changes shown in our results are thus likely to reflect the complex interactions of biomechanical forces and the signaling pathways of vascular endothelial cells yet to be defined. 
Another interesting question is what the implications of our findings are for the pathogenesis of BRVO. As the occurrence of BRVO is far more frequent in AV crossings and is much more frequent in older individuals with a higher prevalence of some systemic diseases, such as diabetes, obesity, and hypertension, 22,40 it has been hypothesized that age-related and site-specific changes in vascular endothelial cells may be an important factor contributing to BRVO pathogenesis. Information from our study supports this hypothesis. However, to prove that the hypothesis needs further study and clinicopathologic correlation, the most important approach to determine the primary cause for BRVO. Pathologic changes observed at AV crossings can certainly help to understand clinical observations made with the ophthalmoscope much more accurately. However, this can be achieved only if one performs a detailed histopathologic examination of eyes that have previously undergone careful clinical examination. Only in this way can a direct correlation be established between clinical and histopathologic findings. Information from these cases is extremely important, but only a few cases have been reported, with no evidence shown for the expected venous compression at these crossings. 28 30,41 The clinicopathologic sequelae were described in an eye with BRVO a few hours after onset showing that the endothelial cells were damaged. 24 The so-called crossing phenomena have been studied by observations comparing ophthalmoscopic and histologic findings at the AV crossing. Using numerous serial sections, venous compression was not found at the AV crossing region, even though the blood column in the underlying vein appears to become narrow toward the crossing and appears to remain attenuated after the crossing, only to reach its full calibre at some distance from the crossing. The crossing phenomena, or compressing signs, are actually caused by the thickened and dense perivascular connective tissue sheaths. 24 We did not find significant narrowing of the vein at AV crossing points, which is in agreement with the observations of Seitz and Blodi. 24 Arguably, phenotypic changes in the venous endothelial cells are a critical pathogenic factor in BRVO. Our results showing the presence of age-related changes in venous endothelial cells at AV crossings in human donor eyes may support the hypothesis that age-related changes in vascular endothelial cells are an important factor contributing to BRVO pathogenesis. This hypothesis could link other proposed hypotheses regarding hemodynamic changes, degenerative changes of the vessel wall, and blood thrombus formation. 
In summary, this study provides important information regarding the existence of age-related phenotype venous endothelial cells changes at AV crossing points. The cause-and-consequence relationship between these phenotype changes and BRVO could be critical for clarifying the pathogenesis of BRVO. Fortunately, the physiological or pathologic relevance of the interrelation between shear forces and the vascular endothelial cells has been extensively investigated. 7 New knowledge is rapidly accumulating with data emerging from studies on the endothelial flow-induced cell-signaling repertoire, collectively known as mechanotransduction, which ranges from instantaneous ion fluxes and biochemical pathways to gene and protein expression. A spatially decentralized mechanism of endothelial mechanotransduction is dominant, in which deformation at the cell surface induced by shear stress is transmitted as cytoskeletal tension changes to sites that are mechanically coupled to the cytoskeleton. Attempts have been made to delineate whether shear stress receptors or shear stress responsive genes can serve as novel targets for the design of diagnostic and therapeutic modalities in vascular pathologies. 7,37  
Footnotes
 Supported by the National Health and Medical Research Council of Australia and the Australian Research Council Centre of Excellence in Vision Science.
Footnotes
 Disclosure: P.K. Yu, None; P.E.Z. Tan, None; W.H. Morgan, None; S.J. Cringle, None; I.L. McAllister, None; D.-Y. Yu, None
The authors thank the staff of the Lions Eye Bank of Western Australia, Lions Eye Institute for providing human donor eyes; the staff of DonateWest, the Western Australian agency for organ and tissue donation, who facilitated the recruitment of donors into the study by referral and completion of consent processes; and Dean Darcey for expert technical assistance. 
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Figure 1.
 
(A) An image of a fundus fluorescence angiograph from a normal subject (not from one of the eyes studied) illustrating the selection criteria of AV crossing for this study and the locations of pre-AV, AV crossing, and post-AV regions for imaging venous endothelial cells. There are several AV crossings (arrows) between the artery (A) and vein (V) in the superotemporal quadrant. The AV crossing in the boxed region would be preferred for study, as the artery overlies the vein and the angle between the artery, and the vein allows the adjacent pre- and post-AV segments of the vein to be studied. Inset: an enlarged image from the highlighted region showing pre-AV, AV, and post-AV regions. (B) A composite image of low-magnification confocal images of retinal vasculature of a donor eye from a 52-year-old man. Inset: a higher magnification confocal image of the AV crossing outlined by the dotted lines. The image composite was taken from the superotemporal region of the retina 3 to 6 mm from the center of the macula. Retinal artery (A) labeled much more intensively than the retinal vein (V), because of the greater amount of F-actin in the arterial smooth muscle cells. In the pre-AV, AV, and post-AV regions, the retinal vein can be seen crossing underneath a retinal artery. The capillary-free zone is evidenced along with the retinal artery. Scale bar, 200 μm, low magnification composite; 168 μm, high-magnification inset.
Figure 1.
 
(A) An image of a fundus fluorescence angiograph from a normal subject (not from one of the eyes studied) illustrating the selection criteria of AV crossing for this study and the locations of pre-AV, AV crossing, and post-AV regions for imaging venous endothelial cells. There are several AV crossings (arrows) between the artery (A) and vein (V) in the superotemporal quadrant. The AV crossing in the boxed region would be preferred for study, as the artery overlies the vein and the angle between the artery, and the vein allows the adjacent pre- and post-AV segments of the vein to be studied. Inset: an enlarged image from the highlighted region showing pre-AV, AV, and post-AV regions. (B) A composite image of low-magnification confocal images of retinal vasculature of a donor eye from a 52-year-old man. Inset: a higher magnification confocal image of the AV crossing outlined by the dotted lines. The image composite was taken from the superotemporal region of the retina 3 to 6 mm from the center of the macula. Retinal artery (A) labeled much more intensively than the retinal vein (V), because of the greater amount of F-actin in the arterial smooth muscle cells. In the pre-AV, AV, and post-AV regions, the retinal vein can be seen crossing underneath a retinal artery. The capillary-free zone is evidenced along with the retinal artery. Scale bar, 200 μm, low magnification composite; 168 μm, high-magnification inset.
Figure 2.
 
Composite confocal images of AV crossings from a 22-year-old (A, B) and a 66-year-old (CD) donor. (A, C) Confocal image stacks of AV crossings; (B, D) respective cross sections of the stacks at the position of the yellow dotted line. Both specimens were labeled with phalloidin conjugated with AlexaFluor 546, and the nuclei were counterstained. In both cases, the veins dived at the AV crossings at various angles, and the venous lumen was wide open during passage under the artery. Scale bar, 50 μm.
Figure 2.
 
Composite confocal images of AV crossings from a 22-year-old (A, B) and a 66-year-old (CD) donor. (A, C) Confocal image stacks of AV crossings; (B, D) respective cross sections of the stacks at the position of the yellow dotted line. Both specimens were labeled with phalloidin conjugated with AlexaFluor 546, and the nuclei were counterstained. In both cases, the veins dived at the AV crossings at various angles, and the venous lumen was wide open during passage under the artery. Scale bar, 50 μm.
Figure 3.
 
Confocal images of retinal venous endothelial cells from the pre-AV, AV, and post-AV crossing regions of the younger (AC) and older groups (DF). The F-actin microfilaments of the endothelia and the vascular smooth muscle cells were labeled with phalloidin (red), and their nuclei were counterstained with Hoechst (blue). Individual endothelial cells from each region of each group are outlined along with their nuclei and displayed under the images. The endothelial cells in the AV region of the younger group (B) were shorter, whereas those from the same region of the older group (E) tended to be more elongated and variable in shape. Arrows: flow direction. Scale bar, 50 μm.
Figure 3.
 
Confocal images of retinal venous endothelial cells from the pre-AV, AV, and post-AV crossing regions of the younger (AC) and older groups (DF). The F-actin microfilaments of the endothelia and the vascular smooth muscle cells were labeled with phalloidin (red), and their nuclei were counterstained with Hoechst (blue). Individual endothelial cells from each region of each group are outlined along with their nuclei and displayed under the images. The endothelial cells in the AV region of the younger group (B) were shorter, whereas those from the same region of the older group (E) tended to be more elongated and variable in shape. Arrows: flow direction. Scale bar, 50 μm.
Figure 4.
 
High-magnification confocal images of individual venous endothelial cells from the pre-AV, AV, and post-AV crossing regions of the younger (AC) and older groups (DF). Sketches of individual endothelial cells of the corresponding regions shown beneath the images were drawn based on the peripheral border and intracellular location of F-actin microfilaments and Hoechst staining of their nuclei. F-actin microfilaments or stress fibers bundles were relatively scarce in the venous endothelia of the younger donor, as indicated by the confocal images and sketches. In contrast, F-actin microfilaments and stress fibers bundles were frequently observed in the venous endothelia of the older donors, as indicated by the confocal images and sketches. The stress fibers were short and were either present throughout the cytoplasm of the older venous endothelial cells or were associated with their nuclei. Scale bar, 20 μm. Arrows: direction of flow.
Figure 4.
 
High-magnification confocal images of individual venous endothelial cells from the pre-AV, AV, and post-AV crossing regions of the younger (AC) and older groups (DF). Sketches of individual endothelial cells of the corresponding regions shown beneath the images were drawn based on the peripheral border and intracellular location of F-actin microfilaments and Hoechst staining of their nuclei. F-actin microfilaments or stress fibers bundles were relatively scarce in the venous endothelia of the younger donor, as indicated by the confocal images and sketches. In contrast, F-actin microfilaments and stress fibers bundles were frequently observed in the venous endothelia of the older donors, as indicated by the confocal images and sketches. The stress fibers were short and were either present throughout the cytoplasm of the older venous endothelial cells or were associated with their nuclei. Scale bar, 20 μm. Arrows: direction of flow.
Table 1.
 
Donor Details
Table 1.
 
Donor Details
Donor Age (y) Sex Cause of Death Time to Cannulation (h)
A 30 M MVA 16
B 19 F MVA 20
C 22 M MVA 15
D 27 M MVA 20
E 27 M MVA 14
F 22 M Suicide 15
G 58 F Pseudomyxoma 6
H 54 F Pulmonary embolism secondary to cancer 20
I 66 M Cancer 15
J 51 M Cancer 6
K 51 M IHD 9.5
L 51 M IHD 19
M 52 M IHD 17
N 74 M SAH 5
O 61 M IHD 10
P 62 M LCI 6
Q 66 M MI 14
Table 2.
 
Venous Width in the Three Regions
Table 2.
 
Venous Width in the Three Regions
Group Pre-AV AV Post-AV
Younger (n = 7) 102.8 ± 16.5 97.8 ± 16.7 106.9 ± 17.5
Older (n = 6) 128.0 ± 10.3 118.0 ± 8.5 134.5 ± 12.3
Table 3.
 
Endothelial Dimension in the Three Regions
Table 3.
 
Endothelial Dimension in the Three Regions
Age Group Length Width
Pre-AV AV Post-AV Pre-AV AV Post-AV
Younger 72.0 ± 2.2 (28) 59.5 ± 2.0 (21)† 68.4 ± 2.1 (29) 21.7 ± 1.2 (28) 20.4 ± 1.0 (21) 21.9 ± 0.9 (29)
Older 75.8 ± 2.4 (40) 86.9 ± 5.2 (17)* 75.4 ± 2.6 (40)‡ 23.5 ± 1.0 (39) 21.1 ± 1.6 (17) 23.8 ± 0.8 (40)
Table 4.
 
Endothelial Cell Length-to-Width Aspect Ratio
Table 4.
 
Endothelial Cell Length-to-Width Aspect Ratio
Age Group Pre AV AV Post AV
Younger 3.5 ± 0.16 (28) 3.0 ± 0.12 (21) 3.2 ± 0.14 (29)
Older 3.5 ± 0.21 (39) 4.5 ± 0.42 (17)* † 3.3 ± 0.16 (38)
Table 5.
 
Dimensions of Endothelial Nuclei in Three Regions
Table 5.
 
Dimensions of Endothelial Nuclei in Three Regions
Age Group Length Width
Pre AV AV Post AV Pre AV AV Post AV
Younger 12.7 ± 0.5 (28) 14.0 ± 0.7 (20) 13.4 ± 0.6 (29) 11.1 ± 0.4 (28) 10.4 ± 0.7 (20) 10.7 ± 0.5 (29)
Older 14.3 ± 0.5 (39) 16.4 ± 0.9 (17)* † 13.1 ± 0.4 (40) 12.4 ± 0.4 (39)* 10.9 ± 1.2 (17)† 12.9 ± 0.4 (40)*
Table 6.
 
Position of the Endothelial Nuclei
Table 6.
 
Position of the Endothelial Nuclei
Age Group Pre AV AV Post AV
Younger 31.4 ± 2.1 (27) 23.5 ± 2.0 (21) 29.8 ± 1.5 (29)
Older 31.7 ± 1.8 (38) 35.7 ± 4.1 (12) 29.8 ± 1.7 (36)
Table 7.
 
Length of Microfilament Fibers in Venous Endothelia of Older Donor Eyes
Table 7.
 
Length of Microfilament Fibers in Venous Endothelia of Older Donor Eyes
Length Pre AV AV Post AV
Older 8.4 ± 0.45 (92) 7.8 ± 0.58 (58) 8.6 ± 0.68 (86)
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