|Molecular Vision 2007;
Received 10 April 2007 | Accepted 19 July 2007 | Published 23 July 2007
Age and topographical comparison of telomere lengths in human corneal endothelial cells
Nancy C. Joyce1
1Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA; 2Department of Ophthalmology, Shizuoka Red Cross Hospital, Shizuoka, Japan
Correspondence to: Nancy C. Joyce, Ph.D., Schepens Eye Research Institute, 20 Staniford Street, Boston, MA, 02114; Phone: (617) 912-0265; FAX: (617) 912-0144; email: firstname.lastname@example.org
Purpose: Human corneal endothelium exhibits both age-related and topographical differences in relative proliferative capacity and in senescence characteristics. The purpose of these studies was to compare telomere lengths in human corneal endothelial cells (HCEC) from the central and peripheral areas of corneas from young and older donors to determine whether these changes may be due to replicative senescence or to stress-induced premature senescence.
Methods: Pairs of corneas from five young (<30 years old) and six older donors (>65 years old) were separated into central and peripheral areas using a 9.5 mm diameter trephine to remove scleral tissue and a 6.0 mm diameter trephine to mark the central-peripheral boundary. One of the pair of corneas was cut into quarters and stained with a peptide nucleic acid (PNA)/fluorescein isothiocyanate (PNA/FITC) probe that specifically binds to telomere repeats. HCEC from the central (0 - 6.0 mm) and peripheral areas (6.0 - 9.5 mm) were isolated from the second cornea, mounted on slides by Cytospin, and stained with the PNA/FITC probe. Fluorescence confocal microscopy was used to obtain digital images. The average FITC intensity of nuclei was compared between the central and peripheral areas within and between the two age groups. Ccl185 and 1301 cells were analyzed as controls. Student's unpaired t-test was used to determine the statistical significance of the data.
Results: Average FITC intensity from the central endothelium was 205.8±4.2 (younger) and 194.2±10.5 (older) and from the peripheral endothelium was 208.1±9.3 (younger) and 195.9±10.8 (older). Average intensity of single cells isolated from central endothelium was 113.9±31.1 (younger) and 107.9±26.1 (older) and from the periphery was 109.9±12.0 (younger) and 106.9±32.4 (older). Average FITC intensity of Ccl185 cells and 1301 cells was 50.5±5.0 and 206.9±19.4, respectively. Comparison of the results indicates no statistically significant difference between the central and peripheral areas within each group or between the young and older age group.
Conclusions: Results indicate that the age-related and topographical reduction in relative proliferative capacity and senescence characteristics observed in HCEC are not due to replicative senescence caused by critically short telomeres but implicate stress-induced premature senescence as a cause of these clinically important changes.
Human corneal endothelial cells (HCEC) are normally nonproliferative in vivo and this inability to replace dead or injured cells results in an age-related decrease in cell density [1-3]. As a result, HCEC are thought to compensate for cell loss by migration and enlargement instead of by proliferation [2,4]. Studies from this laboratory have demonstrated that HCEC in vivo are inhibited in the G1-phase of the cell cycle [5,6] but that HCEC in ex vivo corneas  and in culture [8,9] can divide under appropriate growth-promoting conditions. These findings indicate that HCEC retain the ability to proliferate; however, HCEC from young donors (<30 years old) proliferate more readily than those from older donors (>50 years old). G1-phase of the cell cycle is negatively regulated by cyclin-dependent kinase inhibitors (CKIs) including p27kip1 , p21cip1 , and p16INK4a . Western blot analysis indicates that HCEC from young and older donors express similar levels of p27kip1 . The CKI, p27kip1, mediates inhibition of proliferation resulting from the formation of cell-cell contacts and exposure to TGF-β [14,15], both of which appear to play a role in maintaining mitotic quiescence in HCEC [16-19]. Interestingly, decreasing p27kip1 protein levels using siRNA technology promoted proliferation in HCEC from young donors but did not promote cell division in HCEC from older donors . Further, western blot analysis has demonstrated that HCEC from older donors express higher levels of p21cip1 and p16INK4a than cells from younger donors , strongly suggesting that the cell cycle in HCEC from older donors is under stronger negative regulation than cells from young donors.
Our studies have also documented topographical differences in relative proliferative capacity and senescence characteristics. Using both ex vivo corneas  and cultured cells , we found that HCEC from the peripheral area (6.0 - 9.0 mm rim) consistently exhibit greater competence to proliferate than cells from the central area (6.0 mm diameter) and that this difference is most marked in HCEC from older donors. In corneas from older donors, the central area also stains most strongly for senescence-associated β-galactosidase (SA-beta-Gal) , a marker of cell senescence . Together, accumulating data suggests that the age- and topographically-dependent decrease in relative proliferative capacity exhibited by HCEC is due to cellular senescence.
"Senescence" is a viable state of growth arrest that is distinct from mitotic quiescence and acts as an important tumor suppressive mechanism . Senescent cells typically exhibit decreased saturation density and slower cell cycle kinetics, become stably arrested with a G1-phase DNA content, and express higher protein levels of p21Cip1 and p16INK4a. Cells also stain positively for SA-beta-Gal, reflecting an increase in lysosomal content . Although the nomenclature remains somewhat confusing, researchers in the field of aging have begun to distinguish between two forms of cellular senescence: replicative senescence and stress-induced premature senescence. "Replicative senescence" results from the successive shortening of telomeres that occurs during DNA replication . Telomeres are hexametric repeats at the end of every chromosome and stabilize chromosome ends, preventing end-to-end fusions. Normal human telomeres can be up to 15 Kb in length and 50-100 bp are lost in each somatic cell division [27-29]. Once telomeres have eroded to a critically short length, the senescence program is activated and cells become irreversibly inhibited from dividing. "Stress-induced premature senescence" (SIPS) is caused by exposure of cells to certain environmental stresses [30,31]. SIPS is considered to be "premature" because cells lose the ability to proliferate prior to telomere exhaustion. Thus, in SIPS, cells retain proliferative potential based on telomere length but stop dividing due to inhibitory mechanisms activated by stress-induced damage.
The current study compared relative telomere length in the central and peripheral endothelium from young (<30 years old) and older donors (>50 years old) to help distinguish the type of cellular senescence exhibited by HCEC. It should be noted that these are not the first studies to measure telomere length in HCEC. Egan, et al.  and Amano  have demonstrated that HCEC retain sufficient telomere length to permit cell division. However, in both studies, only the average telomere restriction fragment length of HCEC recovered from whole corneas was determined. In the current study, we used quantitative fluorescence in situ hybridization (Q-FISH) analysis to investigate whether there is a difference in telomere lengths in HCEC located in the central versus the peripheral area in cells from both young and older donors.
Hanks' Balanced Salt Solution (HBSS) and trypsin/ ethylenediaminetetraacetic acid (EDTA) solution were purchased from Invitrogen Life Technologies (Carlsbad, CA). 0.02% EDTA disodium salt was purchased from Sigma (St. Louis, MO). Barron Donor Cornea Punches (6.00 mm, 9.5 mm) were purchased from Katena Products, Inc. (Denville, NJ). Peptide nucleic acid (PNA) probe/fluorescein isothiocyanate (FITC) was from Dako Cytomation (West Grove, PA). Vectashield mounting medium with propidium iodide was from Vector Laboratories, Inc. (Burlingame, CA). CCl185 cells were purchased from American Type Culture Collection (Manassas, VA) and 1301 cells were from European Collection of Cell Cultures (Wiltshire, UK).
Donor Human Corneas
Donor corneas were obtained from the National Disease Research Interchange (NDRI), Philadelphia, PA, and stored in Optisol-GS at 4 °C. Handling of donor information by the source eyebank, NDRI, and this laboratory adhered to the tenets of the Declaration of Helsinki in protecting donor confidentiality. Table 1 provides information regarding the corneas used for this study. All corneas received from NDRI were considered to be unsuitable for transplantation. Donor ages ranged from 7-76 years old. Exclusion criteria for donor corneas were the same as previously reported .
Peptide nucleic acid staining of corneal tissue and isolated human corneal endothelial cells
Corneas were removed from Optisol-GS and washed three times with HBSS before being placed in a Barron Donor Cornea Punch. One of a pair of corneas were cut with a trephine at 9.5 mm diameter, marked at 6.00 mm diameter with a second trephine, then rinsed with HBSS. Each cornea was then cut in half and then halved again to form four equal quarters similar to that described previously . Each quarter was placed in one chamber of a four-chamber slide. A Telomere PNA Kit/FITC (DAKO Cytomation) was used to specifically stain telomeres. For preparation of corneal quarters, 600 μl of PNA probe/FITC solution was added to each slide chamber. Slides were placed on a hot plate at 82 °C for 10 min and then left overnight at room temperature. The solution was gently decanted, 600 μl of wash solution was added to each chamber and then slides were placed in a pre-warmed heating block adjusted to 40 °C for 15 min. Chambers were washed as previously described. Each corneal quarter was placed on a glass slide with the endothelial side up. To secure the tissue on the slide, eight small cover glasses were placed on each side of the corneal quarter with glue. Mounting medium containing propidium iodide (PI) was applied to each quarter. A cover glass was placed on top and fixed with glue to flatten the tissue.
From the second cornea, HCEC were isolated from the central (0-6.0 mm) and peripheral areas (6.0-9.5 mm rim) according to published protocols [8,9] and immediately placed on slides using a Cytospin centrifuge. Briefly, slides were coated with 50 μl of fetal bovine serum (FBS) at 800 rpm for one minute. Then, 200 μl of cell suspension was centrifuged at 800 rpm for three min. The attached cells were stained with 40 μl of PNA probe/FITC solution at 82 °C for 10 min, washed twice, and mounted in a medium containing PI. Hybridization solution without PNA probe acted as a negative control for all studies. Ccl185 cells, which have short telomeres (approximately 6 kb)  and 1301 cells, which have long telomeres [35,36], were used as internal controls. Cells were applied to slides by Cytospin and stained with PNA probe/FITC using the same described procedures.
For all slides, digital images were taken 12 h after preparation and all microscope settings remained constant. Digitized images of the nuclei were first obtained using a Leica TSC-SP2 confocal microscope with excitation at 488 nm for FITC and 543 nm for PI. All images were acquired as 8-bit TIFF files. A Z-series through the cells was captured with a step size of 0.36 μm per image. Ten Z-series images were collapsed onto a single image plane by projecting the maximal pixel intensity of the images. The red color images of PI were converted to blue images for image analysis.
Telomere image analysis
Telomere FISH analysis was performed using methods reported by O'Sullivan . Image analysis software was a kind gift of Peter S. Rabinovitch, Ph.D., Department of Pathology, University of Washington, Seattle, WA. The dimmest 20% of green nuclear pixels was taken as a representative of non-labeled nuclear background and the mean intensity of this background was subtracted from the average of the brightest 5% of green pixels in that same nucleus. The resulting integer was considered to be the absolute telomere intensity. The average FITC intensity was then calculated from at least 100 nuclei from five images per sample. These intensities were compared between the central and peripheral areas within each age group. The average intensity of the young group was also compared to the older group. For analysis of isolated cells, the FITC intensity in Ccl185 and 1301 cells were analyzed as relative controls. Unpaired Student's t-test was used to determine the statistical significance of the data. p<0.05 was considered significant.
Telomere lengths of human corneal endothelial cells in ex vivo corneas
Data were obtained from five younger and six older donors. Figure 1A-E shows representative images of the central and peripheral areas of the endothelium in corneal quarters obtained from an 18-year-old donor and a 69-year-old donor. Bright dots of PNA probe/FITC-stained telomeres were observed in nuclei while no staining was seen in the cytoplasm. In both areas of the endothelium, cells in which the nucleus was not stained with this probe were rarely seen. Also, there was no apparent heterogeneity in the staining pattern of HCEC in either area. The graph in Figure 1F shows average FITC intensity. The average FITC intensity from the central endothelial cells within the corneal tissue was 205.8±4.2 (younger) and 194.2±10.5 (older) while the average intensity from the peripheral area was 208.1±9.3 (younger) and 195.9±10.8 (older). No significant difference (younger: p=0.449, older: p=0.802) was observed between the central and peripheral area within either age group.
Telomere length of human corneal endothelial cells directly isolated from corneal tissue
Results of the telomere analysis in corneal whole mounts were confirmed by isolating central and peripheral endothelial cells from corneas, by immediately recovering the cells by centrifugation, and by staining with the PNA probe. Figure 2A-F shows representative images of single cells recovered by cytospin from the central and peripheral area of the endothelium from the same 18-year-old and 69-year-old donors as shown in Figure 1. Images of Ccl185 cells and 1301 cells are shown for comparison. Some PNA probe/FITC dots were located outside of the nuclei in a few cells, suggesting that some damage might have occurred during the isolation procedures. These cells were not counted for image analysis. Most cells retained their normal intracellular integrity and almost every nucleus was stained with PNA probe/FITC as seen in HCEC in ex vivo corneas. The graph in Figure 2G shows the average FITC intensity of cells from both areas and age groups. The average intensity of single cells from the central endothelial area was 113.9±31.1 (younger) and 107.9±26.1 (older) while the average from the peripheral area was 109.9±12.0 (younger) and 106.9±32.4 (older). The average intensity of Ccl185 cells and 1301 cells was 50.5±5.0 and 206.9±19.4, respectively. There was no statistical difference observed in the average intensity of cells between the central and peripheral areas in either age group (younger group: p=0.828, older group: p=0.955). Figure 3 compares the data obtained from the two previous studies to show that there was also no statistical difference in average FITC intensity of HCEC within the whole endothelium between the younger and older group when cells were either analyzed in ex vivo corneas (p=0.067) or analyzed following isolation and cytospin (p=0.770).
Southern blot analysis has been the conventional method  used to measure telomere length. This method was chosen by Egan, et al.  and Amano  for their analyses of telomere length in HCEC isolated from the entire cornea. For topographic analysis of telomere length in HCEC, the volume of DNA obtained from each area is often too small to obtain reliable data by Southern blotting. To perform Southern blotting, 1 μg of DNA is normally needed for analysis and it was very difficult to consistently obtain that amount of DNA from HCEC isolated from both the central and peripheral areas. In addition, this method cannot be used to directly measure telomere length in cells within a tissue. Measurement of terminal restriction fragment (TRF) length with Southern blot analysis can overestimate the true telomere length because subtelomeric sequences are included . In contrast, the PNA probe used with the Q-FISH technique does not hybridize to subtelomeric sequences, since these sequences are not composed of the hexametric repeats recognized by the probe. The Q-FISH method can be applied to isolated cells by imaging  or flow cytometry  as well as to intact tissues and this type of image analysis shows excellent correlation with TRF estimates obtained by Southern analysis [35,41]. In addition, telomere lengths of single cells can be theoretically obtained using this type of analysis. For these reasons, the Q-FISH technique was applied in the current study to determine whether there are age-related or topographic differences in relative telomere lengths in HCEC that might underlie the observed differences in replication competence and development of senescence characteristics in these cells.
Results from the Q-FISH image analysis of HCEC in ex vivo corneas and of isolated single cells were similar and showed no significant difference in telomere length between cells in the central and peripheral area among either age group or between age groups. The average telomere length of HCEC has been reported to be 10-15 kb [30,31]. Although our results only indicate relative telomere lengths, the values for HCEC were between those of CCL185 cells and 1301 cells, suggesting that their telomere lengths are not critically short. The method used in our current studies would not have been sufficiently sensitive to detect small changes in telomere length in individual cells and the sample size may not have been large enough to demonstrate statistical significance.
Egan, et al.  investigated the hypothesis that critically short telomeres limit the division of adult human corneal endothelium in vivo but demonstrated that adult HCEC retained long telomeres, indicating that the lack of proliferation was not due to critically short telomeres. Amano  obtained similar results. However, in both studies, average telomere lengths were measured in HCEC recovered from whole cornea and did not exclude the potential for heterogeneity in telomere length within the HCEC population. Studies by Schimmelpfennig  and Amann, et al.  have demonstrated the existence of topographical differences in the endothelium by showing that the density of HCEC in central endothelium is lower than in the periphery. Our laboratory has expanded the information regarding topographical differences in the endothelium by finding that central HCEC exhibit reduced proliferative capacity and increased senescence characteristics compared with peripheral cells, particularly in corneas from older donors. The fact that human corneal endothelium exhibits both age-associated and topographic differences related to proliferative capacity and senescence led us to further examine whether critically short telomeres might underlie the cause for these observed differences. The current studies provide important new information indicating that the age-related and topographic differences observed in HCEC are not due to replicative senescence caused by critically short telomeres. Together, the current results strongly suggest that these changes are due to stress-induced premature senescence (SIPS). Thus, in SIPS, cells retain proliferative potential based on telomere length but stop dividing due to inhibitory mechanisms activated by stress-induced damage. Ectopic expression of telomerase (hTERT) can prevent entrance of cells into replicative senescence by preventing the formation of critically short telomeres; however, SIPS cannot be overcome by ectopic hTERT expression [30,31]. Importantly, if the stress or stresses leading to SIPS in HCEC can be identified, it may be possible to develop treatments to prevent or reverse their effects, thus increasing the relative proliferative capacity of HCEC.
The specific reason why HCEC retain relatively long telomeres is currently unknown. It is possible that telomere length is maintained through the activity of telomerase; however, in their studies, Egan, et al. did not find evidence of telomerase activity in either human corneal endothelial cells or epithelial cells, or in endothelial cells transformed with HPV-16 E6/E7. It is also possible that central HCEC have not divided significantly more than have peripheral cells. The studies by Schimmelphennig and Amann, et al. suggest that HCEC in the periphery may act as a "reservoir of cells" and that peripheral cells slowly migrate centripetally to replace central cells that have died. This idea is supported by the findings of Amann, et al. that the relative density of peripheral cells decreases with donor age. Some studies have indicated that a subpopulation of cells in the peripheral area exhibit characteristics of precursor or stem cells [44,45]. For example, Whikehart, et al.  detected telomerase activity in a subpopulation of cells in a region of the posterior limbus between the peripheral endothelium and trabecular meshwork. It is possible that these cells may act as a slowly renewing population within the periphery, but that significant cell division ceases once cells begin to migrate centripetally to form differentiated endothelium.
In conclusion, the current results confirm and extend those of previous studies and indicate that human corneal endothelial cells in the central cornea have similar telomere lengths to cells in the periphery, regardless of donor age. This suggests that the age-related and topographic decrease in relative proliferative capacity observed in HCEC is not due to replicative senescence caused by critically short telomeres and leads us to hypothesize that these changes are due to stress-induced premature senescence. Studies are currently underway to identify the source of this stress and to test whether preventing this stress or reversing its effects could lead to increased proliferative capacity in HCEC.
The authors wish to thank Randy Huang of the Flow Cytometry Core Facility at Schepens Eye Research Institute for his excellent assistance and Peter S. Rabinovitch, Ph.D., Department of Pathology, University of Washington, Seattle, WA, for providing the image analysis software. Funding for the study was from grant NEI R01 EY05767 (N.C.J.).
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