November 2006
Volume 47, Issue 11
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Cornea  |   November 2006
p27kip1 siRNA Induces Proliferation in Corneal Endothelial Cells from Young but Not Older Donors
Author Affiliations
  • Michiharu Kikuchi
    From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA, USA;
    Dokkyo University School of Medicine, Department of Ophthalmology, Tochigi, Japan.
  • Cheng Zhu
    From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA, USA;
  • Tadashi Senoo
    Dokkyo University School of Medicine, Department of Ophthalmology, Tochigi, Japan.
  • Yoshitaka Obara
    Dokkyo University School of Medicine, Department of Ophthalmology, Tochigi, Japan.
  • Nancy C. Joyce
    From the Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, MA, USA;
Investigative Ophthalmology & Visual Science November 2006, Vol.47, 4803-4809. doi:10.1167/iovs.06-0521
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      Michiharu Kikuchi, Cheng Zhu, Tadashi Senoo, Yoshitaka Obara, Nancy C. Joyce; p27kip1 siRNA Induces Proliferation in Corneal Endothelial Cells from Young but Not Older Donors. Invest. Ophthalmol. Vis. Sci. 2006;47(11):4803-4809. doi: 10.1167/iovs.06-0521.

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

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Abstract

purpose. To determine whether small interfering (si)RNA downregulation of the cyclin-dependent kinase inhibitor p27kip1 overcomes G1-phase arrest and promotes cell-cycle progression in human corneal endothelial cells (HCECs) from young (<30 years old) and older (>60 years old) donors.

methods. Transfection of siRNA was confirmed by incubating confluent cultures of HCECs with FITC-labeled nonsilencing siRNA. Confluent cultures were transfected for 48 hours with p27kip1 siRNA (2.5, 5, 25, or 100 nM) or nonsilencing siRNA, with a lipid transfection reagent. As a comparison, cultures were also transfected for 48 hours with p27kip1 antisense (AS) or missense (MS) oligonucleotides (oligo). At various times after transfection, cells were fixed for immunocytochemical localization of p27kip1 or extracted for Western blot analysis to assess relative p27kip1 protein levels. Cultures were also prepared for ZO-1 immunolocalization, to assess the effect of transfection on the morphology of the monolayer. The number of cells was counted at 0, 48, 96, 144, and 192 hours after incubation, and a cell-viability assay was performed.

results. A dose-dependent decrease in p27kip1 protein level was observed in Western blot analysis, and nuclear staining for p27kip1 was greatly reduced in HCECs incubated with p27kip1 siRNA. No change in p27kip1 levels or in nuclear staining was observed in the nonsilencing control. p27kip1 siRNA (25 nM) appeared to be quantitatively more efficient than antisense oligonucleotide (500 nM) in reducing p27kip1 protein levels. Viability was less affected by siRNA treatment than by AS oligo transfection. ZO-1 staining showed no effect on morphology of the monolayer. The number of HCECs from young donors (<30 years old) transfected with p27kip1 siRNA increased up to 144 hours after incubation, whereas no change in the number of cells was observed in HCECs transfected with nonsilencing siRNA. In contrast to the results from young donors, no change in the number of cells was observed at any time point tested in HCECs from older donors (>60 years old) after p27kip1 siRNA transfection.

conclusions. Transfection of p27kip1 siRNA was sufficient to promote proliferation in confluent cultures of HCECs from younger, but not older donors. These results suggest that inhibition of proliferation in older donors is regulated by other mechanisms in addition to p27kip1.

Corneal endothelium is the fragile monolayer of cells located at the posterior of the cornea that is responsible for maintaining corneal transparency through its barrier and ionic pump functions. 1 Human corneal endothelial cells (HCECs) are considered to be nonproliferative in vivo, since the rate of proliferation does not keep pace with the rate of cell loss. As a result, HCEC density decreases with age. 2 3 Maintenance of corneal clarity requires an intact endothelial monolayer, and transparency can be lost when endothelial cell density is reduced below a critical level. Although CECs in vivo exhibit limited mitotic activity, 4 they possess proliferative capacity. 
Corneal endothelial cells in vivo are arrested in the G1-phase of the cell cycle 5 6 and appear to be actively maintained in a nonproliferative state. The ability to progress through G1-phase of the cell cycle is dependent on the relative balance between the activity of stimulatory and inhibitory factors. Cells progress through the G1-phase when the balance favors the stimulatory factors. 7 The G1-phase arrest occurs when the cellular concentration and activity of inhibitory factors exceeds that of stimulatory factors. Cell-cycle progression is negatively regulated by the relative balance between the cellular concentration of cyclin-dependent kinase inhibitors (CKIs), such as members of the Cip/Kip and INK families, and that of cyclin/cyclin-dependent kinase (CDK) complexes, such as cyclin D1-CDK4 and cyclin E-CDK2. 8 On mitogenic stimulation, cyclins D and E are synthesized, increasing the concentration of positive stimulators relative to that of G1-phase inhibitors. 9 Once the overall concentration of cyclin/CDKs exceeds that of the inhibitors, active cyclin/CDK complexes will be formed, and the cell cycle will proceed. 10 11 12  
The Cip/Kip and INK families of proteins are cyclin-dependent kinase inhibitors and induce G1-phase arrest. 13 14 15 In many cell types, p27kip1, a member of the Cip/Kip family, helps mediate cell cycle arrest induced by cell–cell contact and TGF-β. 14 The level of p27kip1 protein expression is high in G0/G1 resting cells and declines as cells progress toward the S-phase. Overexpression of p27kip1 inhibits entry into the S-phase in normal and malignant cells. 16 17 In a study by Joyce et al. 18 , p27kip1 was shown to be important in maintaining cultured rat CECs in G1-phase arrest. This study demonstrated a correlation between high levels of p27kip1 protein and inhibition of proliferation in contact inhibited cells, providing evidence that p27kip1 is an important mediator of contact inhibition in rat corneal endothelium. Lee and Kay 19 and Kim et al. 20 have demonstrated that p27kip1 plays an important role in the negative regulation of proliferation in rabbit CECs. FGF-2 stimulates proliferation in these cells, mainly through the activity of the PI 3-kinase pathway. FGF-2 induced activation of this pathway results in specific phosphorylation of nuclear p27kip1 on Thr187, which in turn results in the proteolysis of p27kip1, reduction of p27kip1 protein levels, and subsequent reduction of inhibition of cell cycle progression. Downregulation of p27kip1 in knockout (−/−) mice results in unregulated cell growth and tumor formation. 21 In other cell types, downregulation of p27kip1 protein levels by antisense methods leads to increased proliferative activity. 22 In a previous study, we showed that lowering of p27kip1 protein levels using antisense methods promotes proliferation in confluent cultures of rat CECs. 23 Therefore, it would be expected that downregulation of p27kip1 protein expression using small interfering (si)RNA methods would overcome G1-phase arrest in HCECs. 
The long-term goal of our studies is to develop therapy that increases cell density in stressed corneal endothelium. Using the method of culturing HCECs that was established in this laboratory, we can consistently culture untransformed CECs from older donors that show normal polygonal morphology at confluence. 24 25 The present studies were designed to test the effect of lowering p27kip1 protein levels on induction of proliferation in HCECs cultured from young (<30 years old) and older (> 60 years old) donors. Viability studies comparing antisense and siRNA methods in cultured HCECs demonstrated that HCECs were much more sensitive to antisense treatment than rat corneal endothelium. As a result, siRNA technology was used to lower p27kip1 protein levels in HCECs. 26 27 28 29 siRNA has high specificity and the ability to inhibit the expression of biologically active proteins selectively and can produce complete inhibition of gene expression. 27 siRNA is also known to specifically block protein expression to a greater extent than antisense methods and it is possible to obtain the same blocking effect using smaller amounts of siRNA than of antisense oligonucleotides. 30 31 We hypothesized that HCEC proliferation would be stimulated by decreasing the cellular concentration of p27kip1 with siRNA methods. 
Materials and Methods
Human Corneal Tissue
Donor corneas were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) and stored in corneal preserving medium at 4°C (Optisol-GS; Bausch & Lomb, Tampa, FL). Exclusion criteria were the same as described by Joyce and Zhu. 32 Handling of donor information by the NDRI and this laboratory adhered to the tenets of the Declaration of Helsinki in protecting donor confidentiality. HCEC cultures were prepared as previously described. 24 25 Primary cultures were grown to confluence in culture medium (OptiMEM-I; Invitrogen Life Technologies, Carlsbad, CA), containing 8% fetal bovine serum (FBS: Hyclone, Logan, UT), 5 ng/mL epidermal growth factor (EGF: Upstate Biotechnology, Lake Placid, NY), 20 ng/mL nerve growth factor (NGF: Biomedical Technologies, Stoughton, MA), 100 μg/mL bovine pituitary extract (Biomedical Technologies), 20 μg/mL ascorbic acid (Sigma-Aldrich, St. Louis, MO), 200 mg/L calcium chloride (Sigma-Aldrich), 0.08% chondroitin sulfate (Sigma-Aldrich), 50 μg/mL gentamicin (Invitrogen-Life Technologies), and antibiotic-antimycotic solution diluted 1:100 (Invitrogen-Life Technologies). Depending on the experiment, confluent cells were subcultured onto sterile four-chamber slides (Laboratory Tek, Naperville, IL) or into T25 flasks that had been precoated with undiluted coating mix (FNC; Biological Faculty and Facility, Inc., Ijamsville, MD) and grown to confluence. The medium was changed every other day. All incubations were performed in a 5% CO2-95% air, humidified atmosphere at 37°C. 
p27kip1 siRNA and Antisense Transfection
p27kip1 siRNA (r[GUACGAGUGGCAAGAGGUG]dTT), nonsilencing siRNA (r[UUCUCCGAACGUGUCACGU]dTT), FITC-labeled nonsilencing siRNA, and transfection reagent (RNAiFect) were purchased from Qiagen (Valencia, CA). In an initial experiment, confluent cells were incubated for 18 hours with 5 nM FITC-labeled nonsilencing siRNA, to confirm transfection into HCECs. For the p27kip1 siRNA studies, the siRNA-to-lipid ratio was 1 μg to 6 μL. This complex was diluted in MEM-I (Hyclone, Inc.) with 8% FBS. Confluent cells were then transfected for 48 hours with p27kip1 siRNA (2.5, 5, 25, or 100 nM). Control cultures were incubated in one of the following conditions: (1) no siRNA or reagent, (2) reagent alone, or (3) reagent plus 25 nM nonsilencing siRNA. At specific time points after transfection, cells were fixed immediately for immunocytochemistry, extracted for Western blot analysis, or prepared for viability assay. 
Antisense (AS) and missense (MS) oligos for p27kip1 were obtained from Midland Certified Reagent Company (Midland, TX): antisense sequence, 5′ (PS)GCGTCTGCTCCACAG3′; mismatch sequence, 5′(PS)GCATCCCCTGTGCAG3′. 33 Transfection of p27kip1 AS or MS oligonucleotide (500 nM) was performed for 48 hours using a nonliposomal lipid transfection kit (Effectene; Qiagen), according to the manufacturer’s protocol. An oligo-to-transfection reagent ratio of 1:25 was used. The transfection complex was diluted in MEM-I with 8% FBS. Cells were incubated for 48 hours and either extracted for Western blot analysis or prepared for the cell-viability assay. Control cultures were incubated with no oligo or reagent. 
Immunocytochemistry
Endothelial cells were subcultured on four-chamber slides, grown to confluence, and transfected for 48 hours with p27kip1 siRNA or nonsilencing siRNA. The cells were then rinsed with phosphate-buffered saline (PBS; Invitrogen-Life Technologies) and fixed with 99.9% methanol for 10 minutes at −20°C. All further incubations were at room temperature. The slides were rinsed with PBS, and the cells were permeabilized for 10 minutes with 0.1% Triton X-100 (Sigma-Aldrich) in PBS. Nonspecific sites were blocked for 10 minutes using 4% bovine serum albumin (BSA) in PBS. The cells were incubated for 2 hours with rabbit polyclonal anti-p27kip1 (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:200 or with rabbit polyclonal anti-ZO-1 (Invitrogen) diluted 1:150. The slides were rinsed with PBS and incubated for 2 hours with fluorescein-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:100 in PBS. Control cultures were incubated with secondary antibody alone. Coverslips were mounted with antifade medium containing DAPI (4′6 diamidino-2-phenylindole; Vectashield; Vector Laboratories, Inc., Burlingame, CA) to stain the cell nuclei. Positive staining of cultured cells was visualized on a microscope (Eclipse E800, with a VFM Epi-Fluorescence Attachment; Nikon Inc., Melville, NY), equipped with a digital camera (Spot and Spot version 1.1 CE software; Diagnostic Instruments, Sterling Heights, MI). These experiments were repeated at least three times. 
Western Blot Analysis
After transfection for 48 hours with p27kip1 siRNA or nonsilencing siRNA, cultured cells were trypsinized and pelleted. Proteins were extracted by incubating cells for 30 minutes at 4°C in buffer containing 1% Triton X-100, 250 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl, 10 μg/mL leupeptin, and 1 mM phenylmethylsulfonyl fluoride (all from Sigma-Aldrich), followed by homogenization and centrifugation. Protein content was quantified by spectrophotometry. Equal protein was loaded on 4% to 12% Bis-Tris gels for SDS-PAGE. Peptides were then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA), and nonspecific binding was blocked by incubation overnight at 4°C in 5% nonfat milk diluted in PBS. Membranes were incubated for 2 hours with rabbit polyclonal anti-p27kip1 diluted 1:200 in blocking buffer. Blots were rinsed three times for 10 minutes with 0.1% Triton X-100, then reblocked and exposed for 1 hour to horseradish peroxidase (HRP)–conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) diluted 1:10,000 in blocking solution. The same blots were probed with rabbit anti-nonmuscle myosin (Biomedical Technologies, Inc.) diluted 1:200 to control for protein load. After a thorough wash, peptides were detected with a chemiluminescent substrate (SuperSignal West Pico; Pierce, Rockford, IL). For quantification, films were digitally scanned (BDS-Image; Biological Detection System, Pittsburgh, PA). Scans were analyzed with NIH Image software version 1.61 (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD), and protein content was normalized according to nonmuscle myosin protein content. These experiments were repeated at least three times. 
Quantification of Proliferating Cells
HCECs were cultured from five young donors (19, 22, 25, 27, and 28 years old) and from six older donors (60, 64, 67, 72, 72, and 75 years old). For all studies, passage 2 HCECs were used to test the effect of p27kip1 siRNA on cell proliferation. Cells were grown to confluence in 24-well culture plates, transfected for 48 hours with 25 nM p27kip1 siRNA or 25 nM nonsilencing siRNA, then postincubated for 0, 48, 96, 144, or 192 hours in the same culture medium described for primary culture. The medium was changed every other day. At each designated time point after transfection, cultures were trypsinized, and the number of cells was determined by cell counter (Coulter Electronics, Hialeah, FL). Cells from each well were counted three times, and at least three separate wells were counted per time point and condition. Results were averaged, and standard deviations were calculated. Statistical analysis using Student’s paired t-test was performed using Sigma Stat version 2.0 (SPSS, Chicago, IL). P < 0.05 was considered to be significant. 
Viability of HCECs
Viability was assessed (Live/Dead assay kit; Invitrogen-Molecular Probes, Eugene, OR) after 48 hours of incubation with 25 nM p27kip1 siRNA and 500 nM AS oligo, or incubation under control conditions. Staining was visualized by fluorescence microscopy using a microscope equipped with a digital camera (Eclipse YS100 with a Coolpix 995 camera; Nikon). The percentage of cell survival was calculated by dividing the number of healthy cells by the total number of cells. Results were compared with those of untreated control cultures in experiments repeated at least three times. 
Results
Confirmation of siRNA Transfection
Preliminary studies were conducted to confirm siRNA transfection using (RNAiFect Transfection Reagent; Qiagen). Confluent HCECs were incubated for 18 hours with transfection reagent alone (Fig. 1A)or with 5 nM FITC-labeled nonsilencing siRNA (Fig. 1B) . Cultures were then washed, and siRNA was visualized by fluorescence microscopy. The presence of FITC-positive cells indicated successful siRNA transfection. Results of the cell-viability assay demonstrated that viability was not significantly affected by siRNA transfection under these conditions (data not shown). 
Effect of siRNA Transfection on p27kip1 Protein Levels
The effect of siRNA transfection on p27kip1 levels was determined by Western blot analysis and immunocytochemistry (ICC). After transfection for 48 hours with increasing concentrations of p27kip1 siRNA or with transfection reagent without siRNA, cells were washed and either prepared for Western blot analysis or ICC. Figure 2Ademonstrates that transfection of confluent HCECs with p27kip1 siRNA reduced p27kip1 protein levels in a dose-dependent manner. Semiquantitative analysis of the blots (Fig. 2B)indicates that the protein level of p27kip1 was reduced with 2.5 nM p27kip1 siRNA after transfection and was suppressed to approximately 17% of control levels by 25nM siRNA. As a result of these studies, 25 nM p27kip1 siRNA was used in all subsequent studies. 
For ICC evaluation of the effect of p27kip1 siRNA transfection on p27kip1 levels, confluent cultures were incubated for 48 hours with 25 nM p27kip1 siRNA (Fig. 3A) , 25 nM nonsilencing siRNA (Fig. 3B) , transfection reagent alone (Fig. 3C) , or no siRNA or reagent (Fig. 3D) , and then processed for ICC of p27kip1. Staining for p27kip1 was greatly reduced in HCECs incubated with p27kip1 siRNA (Fig. 3A) . Images in Figures 3B 3C 3Dshow strong staining for p27kip1 in the nuclei with a low level of staining in the cytoplasm of HCECs, indicating no change in p27kip1 protein levels in these control cells. 
Comparison of the Effect of siRNA and Antisense Oligonucleotide Treatment on p27kip1 Protein Levels and Cell Viability
To compare the effect of siRNA and antisense oligonucleotide (oligo) treatment on p27kip1 protein levels and viability after transfection, HCEC cultures were incubated for 48 hours with 25 nM p27kip1 siRNA, 25 nM nonsilencing siRNA (Cont siRNA), no siRNA or reagent (Cont.), 500 nM p27kip1 antisense oligo (p27 AS), 500 nM missense oligo (p27 MS), or no oligo or reagent (Cont.) in the same conditions as just described and then processed for Western blot analysis (Figs. 4A 4B)and viability assay (Fig. 4C) . p27kip1 siRNA at 25 nM appeared to be quantitatively more efficient than antisense oligo at 500 nM (Figs. 4A 4B) . Viability was less affected by siRNA treatment than by antisense oligo (Fig. 4C)
Effect of p27kip1 siRNA on Monolayer Integrity
Confluent cultures of HCECs were immunostained for ZO-1, a tight junction-associated protein, to assess the effect of the transfection reagent and siRNA treatment on the morphology of the endothelial monolayer. Figure 5shows a normal ZO-1 pattern in p27kip1 siRNA-treated and control cultures, indicating that siRNA transfection did not have a deleterious effect on cell shape or monolayer integrity. 
Effect of p27kip1 siRNA on Proliferation of HCECs from Young and Old Donors
HCECs do not normally replicate in vivo and are arrested in the G1-phase, although they maintain proliferative capacity. 5 6 We questioned whether p27kip1 inhibition could overcome G1-phase arrest in a confluent monolayer of cells and promote proliferation. To study the effects of p27kip1 siRNA treatment on HCECs, confluent HCEC cultures were transfected for 48 hours with 25 nM p27kip1 siRNA or 25 nM nonsilencing siRNA; postincubated for 0, 48, 96, 144, or 192 hours; and processed for cell counting. Six corneas from older donors (>60 years old) and five corneas from young donors (<30 years old) were used for the study. HCECs cultured from older donors and transfected with p27kip1 siRNA showed no change in the number of cells when compared with control cells at any time-point tested (Fig. 6A) . In contrast, the relative number of HCECs from young donors that had been transfected with p27kip1 siRNA showed a statistically significant increase (to ∼130% of control levels) by 144 hours after incubation. After this time, the number of cells appeared to plateau (Fig. 6B)
Discussion
In many cell types, p27kip1 helps mediate inhibition of the cyclin-dependent kinase activity that is required for cell-cycle progression and is particularly active in mediating cell-cycle arrest induced by cell–cell contact and TGF-β. We have demonstrated that both cell–cell contact and TGF-β help maintain CECs in G1-phase arrest 19 34 and suggested that p27kip1 must play a role in inhibiting proliferation in CECs. To test this idea, we demonstrated that p27kip1 antisense oligo treatment could successfully decrease the cellular concentration of p27kip1 in cultured rat CECs and promote proliferation, as evidenced by the increased number of cells. 23  
In the current studies, we extended our inquiry to determine whether decreasing p27kip1 protein levels would promote proliferation of HCECs. We found that HCECs are much more sensitive to antisense oligo treatment than are rat CECs, resulting in an increased loss of cell viability. As a result, we tested p27kip1 siRNA as an alternative method to decrease p27kip1 protein levels in HCECs. Although there have been no reported studies using siRNA methods in human corneal endothelium to alter the synthesis of cell cycle regulatory proteins, we successfully used this technique to reduce p27kip1 protein levels and to promote proliferation in cultured HCECs. In recent years, siRNA techniques have been used for studying biological function and developing new therapeutic strategies in other cell types. 35 36 Recent studies of the downregulation of gene expression indicate that siRNA is an effective method for functional gene analysis. 28 29 It has been reported that siRNA methods are more effective and less harmful to cells than are antisense methods and that siRNA is generally more efficient at reducing protein levels. 30 31 The current studies indicate the feasibility of using siRNA, not only for controlling p27kip1 protein levels, but also for reducing the cellular concentration of other proteins of interest. 
Of particular interest, is the fact that p27kip1 siRNA treatment only promoted proliferation in cells cultured from young donors, whereas HCECs from older donors showed no change in the number of cells after similar treatment. These results strongly suggest that there is an age-related change in the molecular mechanisms that regulate proliferation in HCECs. Our laboratory has shown that HCECs from young donors are more efficient in responding to positive growth factors than are cells cultured from older donors. 25 32 37 We have also shown similar age-related differences in cell-cycle kinetics in an ex vivo wound model, thus demonstrating that this is not a culture artifact. 38 In addition, recent studies using cultured HCECs 39 indicate that there is an age-related increase in the relative expression of the cyclin-dependent kinase inhibitors p21cip1 and p16INK4a, whereas, there was little change in the relative expression of p27kip1 with donor age. Together, evidence strongly suggests that HCECs from younger donors have a higher proliferative capacity than do older HCECs. Among the underlying reasons for this observed age-related difference may be increased inhibition of cyclin-dependent kinase activity by p21cip1 and/or p16INK4a. The results obtained in the present study clearly suggest that p27kip1 must be an important barrier to proliferation in HCECs from younger donors; however, it appears that there are additional inhibitory control mechanisms in HCECs from older donors. 
Results of the current studies using HCECs appear to differ from those of previous studies from this laboratory that showed increased proliferation in rat CECs after p27kip1 antisense treatment. This discrepancy may be due to the relatively young rats used in those studies. Fitch et al. 40 demonstrated that there is a progressive decline in the number of cells and an increase in pleomorphism in rat corneal endothelium from age 6 months to 30 months, closely paralleling changes reported in human endothelium in individuals from 20 to 70 years old. Similar results were obtained by Meyer et al., 41 who demonstrated an age-related decrease in the number of cells and an increase in polymorphism and polymegathism in rat corneal endothelium by 34 weeks of age. The rats used in our p27kip1 AS oligo studies were 6 weeks of age. Thus, our rat model and the cellular response appear to reflect more closely the behavior of young human donor endothelium. 
In the past few years, studies have been conducted to determine the feasibility of transplanting the posterior cornea to replace endothelial cells 42 rather than transplanting full-thickness corneas. Another approach has been to culture HCECs on supports, such as amniotic membrane 43 and then transplant the construct to increase endothelial cell density. This second approach is hampered by the fact that HCECs are relatively difficult to culture. Cells from very young donors are most reliable for culture; however, these cells cannot be obtained in the quantities needed to make these new transplantation techniques widely available to patients. Our laboratory has successfully isolated and cultured HCECs from both young and older donors using reported techniques. 24 25 It is clear, however, that optimal culture of HCECs for transplantation purposes requires a deeper understanding of cell-cycle regulation. 
From the current data, we conclude that p27kip1 plays an important role in G1-phase arrest, at least in HCECs from young donors, and that decreasing its protein level using siRNA methods can promote proliferation in these cells. The induction of proliferation in this experiment occurred without apparent cell damage. Because this treatment might be applied to HCECs to increase cell density in individuals with abnormalities of endothelial cell density, it is important to understand the basis for the age-related difference observed in response to this treatment and to elucidate the specific mechanisms by which the lowering of p27kip1 promotes proliferation in HCECs. 
 
Figure 1.
 
Confirmation of siRNA transfection. HCEC cultures were incubated for 18 hours with transfection reagent alone (A) or with reagent plus FITC-labeled nonsilencing siRNA (B). Green fluorescence (FITC): transfected cells; blue fluorescence (DAPI): all nuclei. Magnification, ×60.
Figure 1.
 
Confirmation of siRNA transfection. HCEC cultures were incubated for 18 hours with transfection reagent alone (A) or with reagent plus FITC-labeled nonsilencing siRNA (B). Green fluorescence (FITC): transfected cells; blue fluorescence (DAPI): all nuclei. Magnification, ×60.
Figure 2.
 
(A) Western blot analysis comparing p27kip1 protein expression after 48-hour treatment with increasing concentrations of p27kip1 siRNA or transfection reagent without siRNA (Cont.). Nonmuscle myosin was used for normalization of protein load. (B) p27kip1 protein level standardized to the amount of nonmuscle myosin present in the same samples. Bars, SD.
Figure 2.
 
(A) Western blot analysis comparing p27kip1 protein expression after 48-hour treatment with increasing concentrations of p27kip1 siRNA or transfection reagent without siRNA (Cont.). Nonmuscle myosin was used for normalization of protein load. (B) p27kip1 protein level standardized to the amount of nonmuscle myosin present in the same samples. Bars, SD.
Figure 3.
 
p27kip1 immunostaining of HCEC cultures incubated for 48 hours with 25 nM p27kip1 siRNA (A), 25nM nonsilencing siRNA (B), transfection reagent (C), or no siRNA or reagent (D). Green fluorescence (FITC) localizes p27kip1; blue fluorescence (DAPI) stains all nuclei. Magnification, ×60.
Figure 3.
 
p27kip1 immunostaining of HCEC cultures incubated for 48 hours with 25 nM p27kip1 siRNA (A), 25nM nonsilencing siRNA (B), transfection reagent (C), or no siRNA or reagent (D). Green fluorescence (FITC) localizes p27kip1; blue fluorescence (DAPI) stains all nuclei. Magnification, ×60.
Figure 4.
 
Comparison of the inhibition of p27kip1 protein expression (A, B) and of the effect on viability (C) induced by siRNA and antisense oligo treatment. Confluent HCECs were incubated for 48 hours with 25 nM p27kip1 siRNA, 25 nM nonsilencing siRNA (Cont siRNA), no siRNA or reagent (Cont.), 500 nM p27kip1 antisense oligo (p27 AS), 500 nM missense oligo (p27 MS), or no oligo or reagent (Cont.) and then extracted for Western blot analysis (A). (B) p27kip1 protein levels standardized to the amount of nonmuscle myosin present in the same samples. (C) Percentage of live cells.
Figure 4.
 
Comparison of the inhibition of p27kip1 protein expression (A, B) and of the effect on viability (C) induced by siRNA and antisense oligo treatment. Confluent HCECs were incubated for 48 hours with 25 nM p27kip1 siRNA, 25 nM nonsilencing siRNA (Cont siRNA), no siRNA or reagent (Cont.), 500 nM p27kip1 antisense oligo (p27 AS), 500 nM missense oligo (p27 MS), or no oligo or reagent (Cont.) and then extracted for Western blot analysis (A). (B) p27kip1 protein levels standardized to the amount of nonmuscle myosin present in the same samples. (C) Percentage of live cells.
Figure 5.
 
Immunolocalization of ZO-1 in confluent cultures of HCECs incubated without transfection reagent or siRNA (A), or incubated with transfection reagent only (B), control siRNA (C), or p27kip1 siRNA (D). The normal disrupted, linear ZO-1 pattern is observed under all treatment conditions. Magnification: ×100.
Figure 5.
 
Immunolocalization of ZO-1 in confluent cultures of HCECs incubated without transfection reagent or siRNA (A), or incubated with transfection reagent only (B), control siRNA (C), or p27kip1 siRNA (D). The normal disrupted, linear ZO-1 pattern is observed under all treatment conditions. Magnification: ×100.
Figure 6.
 
Confluent HCECs, cultured from five young (19, 22, 25, 27, and 28 years old) and six older donors (60, 64, 67, 72, 72, and 75 years old), were transfected for 48 hours with p27kip1 siRNA or nonsilencing siRNA (cont.), transferred to fresh culture medium, and counted at 0, 48, 96, 144, or 192 hours after incubation. No change from control levels was observed at any time point tested in HCECs of older donors. Results are expressed as the mean ± SD. *P < 0.05.
Figure 6.
 
Confluent HCECs, cultured from five young (19, 22, 25, 27, and 28 years old) and six older donors (60, 64, 67, 72, 72, and 75 years old), were transfected for 48 hours with p27kip1 siRNA or nonsilencing siRNA (cont.), transferred to fresh culture medium, and counted at 0, 48, 96, 144, or 192 hours after incubation. No change from control levels was observed at any time point tested in HCECs of older donors. Results are expressed as the mean ± SD. *P < 0.05.
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Figure 1.
 
Confirmation of siRNA transfection. HCEC cultures were incubated for 18 hours with transfection reagent alone (A) or with reagent plus FITC-labeled nonsilencing siRNA (B). Green fluorescence (FITC): transfected cells; blue fluorescence (DAPI): all nuclei. Magnification, ×60.
Figure 1.
 
Confirmation of siRNA transfection. HCEC cultures were incubated for 18 hours with transfection reagent alone (A) or with reagent plus FITC-labeled nonsilencing siRNA (B). Green fluorescence (FITC): transfected cells; blue fluorescence (DAPI): all nuclei. Magnification, ×60.
Figure 2.
 
(A) Western blot analysis comparing p27kip1 protein expression after 48-hour treatment with increasing concentrations of p27kip1 siRNA or transfection reagent without siRNA (Cont.). Nonmuscle myosin was used for normalization of protein load. (B) p27kip1 protein level standardized to the amount of nonmuscle myosin present in the same samples. Bars, SD.
Figure 2.
 
(A) Western blot analysis comparing p27kip1 protein expression after 48-hour treatment with increasing concentrations of p27kip1 siRNA or transfection reagent without siRNA (Cont.). Nonmuscle myosin was used for normalization of protein load. (B) p27kip1 protein level standardized to the amount of nonmuscle myosin present in the same samples. Bars, SD.
Figure 3.
 
p27kip1 immunostaining of HCEC cultures incubated for 48 hours with 25 nM p27kip1 siRNA (A), 25nM nonsilencing siRNA (B), transfection reagent (C), or no siRNA or reagent (D). Green fluorescence (FITC) localizes p27kip1; blue fluorescence (DAPI) stains all nuclei. Magnification, ×60.
Figure 3.
 
p27kip1 immunostaining of HCEC cultures incubated for 48 hours with 25 nM p27kip1 siRNA (A), 25nM nonsilencing siRNA (B), transfection reagent (C), or no siRNA or reagent (D). Green fluorescence (FITC) localizes p27kip1; blue fluorescence (DAPI) stains all nuclei. Magnification, ×60.
Figure 4.
 
Comparison of the inhibition of p27kip1 protein expression (A, B) and of the effect on viability (C) induced by siRNA and antisense oligo treatment. Confluent HCECs were incubated for 48 hours with 25 nM p27kip1 siRNA, 25 nM nonsilencing siRNA (Cont siRNA), no siRNA or reagent (Cont.), 500 nM p27kip1 antisense oligo (p27 AS), 500 nM missense oligo (p27 MS), or no oligo or reagent (Cont.) and then extracted for Western blot analysis (A). (B) p27kip1 protein levels standardized to the amount of nonmuscle myosin present in the same samples. (C) Percentage of live cells.
Figure 4.
 
Comparison of the inhibition of p27kip1 protein expression (A, B) and of the effect on viability (C) induced by siRNA and antisense oligo treatment. Confluent HCECs were incubated for 48 hours with 25 nM p27kip1 siRNA, 25 nM nonsilencing siRNA (Cont siRNA), no siRNA or reagent (Cont.), 500 nM p27kip1 antisense oligo (p27 AS), 500 nM missense oligo (p27 MS), or no oligo or reagent (Cont.) and then extracted for Western blot analysis (A). (B) p27kip1 protein levels standardized to the amount of nonmuscle myosin present in the same samples. (C) Percentage of live cells.
Figure 5.
 
Immunolocalization of ZO-1 in confluent cultures of HCECs incubated without transfection reagent or siRNA (A), or incubated with transfection reagent only (B), control siRNA (C), or p27kip1 siRNA (D). The normal disrupted, linear ZO-1 pattern is observed under all treatment conditions. Magnification: ×100.
Figure 5.
 
Immunolocalization of ZO-1 in confluent cultures of HCECs incubated without transfection reagent or siRNA (A), or incubated with transfection reagent only (B), control siRNA (C), or p27kip1 siRNA (D). The normal disrupted, linear ZO-1 pattern is observed under all treatment conditions. Magnification: ×100.
Figure 6.
 
Confluent HCECs, cultured from five young (19, 22, 25, 27, and 28 years old) and six older donors (60, 64, 67, 72, 72, and 75 years old), were transfected for 48 hours with p27kip1 siRNA or nonsilencing siRNA (cont.), transferred to fresh culture medium, and counted at 0, 48, 96, 144, or 192 hours after incubation. No change from control levels was observed at any time point tested in HCECs of older donors. Results are expressed as the mean ± SD. *P < 0.05.
Figure 6.
 
Confluent HCECs, cultured from five young (19, 22, 25, 27, and 28 years old) and six older donors (60, 64, 67, 72, 72, and 75 years old), were transfected for 48 hours with p27kip1 siRNA or nonsilencing siRNA (cont.), transferred to fresh culture medium, and counted at 0, 48, 96, 144, or 192 hours after incubation. No change from control levels was observed at any time point tested in HCECs of older donors. Results are expressed as the mean ± SD. *P < 0.05.
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