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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 11, 2005 as doi:10.1096/fj.04-2733fje. |
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Departments of
* Biomedical Sciences and
Ophthalmology, University of Aberdeen, Aberdeen, U.K.
1 Corresponding author: Biomedical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, Scotland, UK. E-mail: m.zhao{at}abdn.ac.uk
SPECIFIC AIMS
Cataract, the most common form of blindness, is treatable by surgery. Unfortunately, many patients develop the complication of posterior capsule opacification (PCO), or secondary cataract. This arises from stimulated cell growth within the lens capsule and can greatly impair vision. The mechanisms underlying this aberrant growth of lens cells after surgery are not fully understood. We propose and show that cataract surgery might remove an important inhibitory factor for lens cell growth: electric fields. The lens generates a unique pattern of electric currents; we show that cutting and removing part of the anterior capsule as in cataract surgery significantly decreases the equatorial electric current. In addition, application of electric fields to human lens epithelial cells (LEC) in culture inhibits cell proliferation.
We set out to test the hypothesis that the unique physiological electric current pattern around the ocular lens is a control mechanisms of LEC proliferation and to determine the underlying molecular mechanisms.
PRINCIPAL FINDINGS
1. Removal of part of anterior capsule disrupts lens electric currents
Using vibrating probe techniques, we have shown that removal of a large part of the anterior capsule induced significant changes in lens electric current pattern. Normal lenses had a small inward current across the anterior surface (0.22–0.38 µA/cm2; Fig. 1
C). Removal of part of the anterior capsule and the lens nucleus resulted in larger outward currents (1.8–2.6 µA/cm2). The difference was highly significant (P<0.004).
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The equatorial electrical currents could not be measured in situ, so we measured equatorial currents in isolated lenses. A small cut (capsulotomy) 
1 mm in length significantly decreased outward equatorial currents to 56% of original size (Fig. 1F
) (P =0.01). Removing a larger piece of anterior capsule (capsulorrhexis) 1 mm in diameter rendered equatorial currents to almost zero (0.40±0.56µA/cm2). Opening and removing the anterior lens capsule by means of a continuous curvilinear capsulorrhexis, as performed in conventional cataract surgery, significantly decreased endogenous electric currents at the equator.
2. Electric fields (EF) inhibit proliferation of LECs
In control cultures (no EF exposure), density of LECs increased steadily with time, but when cultured in electric fields the density increased only marginally over 48 h. Same-field time lapse imaging showed significant quantitative differences between control culture and the culture in electric fields. Field exposure reduced the mean cell growth rate of LECs.
We further investigated proliferation with a mitotic index. The mitotic index of LECs decreased significantly after EF exposure. Mitotic indices of EF-exposed cells were 0.68% (12 h) and 0.48% (24 h). These are significantly lower than the mitotic indices of 2.01% (12 h) and 2.24% (24 h) in control culture (P<0.02 and P<0.01, respectively). There was no significant difference in mitotic indices between 12 and 24 h EF-exposed LECs.
3. Electric fields induce cell cycle arrest by regulation of cell cycle regulators
Flow cytometry showed that the EF exposure inhibited cell cycle progression through G1/S transition. Compared with control cells, the percentage of the cell population in the S and G2/M phases in EF-exposed cells decreased significantly whereas the proportion of the cell population in G1 phase increased significantly (Fig. 2
A, B). A sub-G1 peak, which indicates apoptosis, was not found in the DNA content histogram in EF-exposed LECs (Fig. 2A, B
). Thus, EF-induced inhibition of LEC proliferation resulted from cell cycle arrest or G1 block.
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Western blot analysis of the expression levels of selected key cell cycle regulatory molecules showed that, compared with control, EF exposure induced a significant decrease in the expression of cyclin E, but this field exposure did not influence expression of Cdk2. In contrast, EF exposure increased significantly the expression of p27kip1, an inhibitor of the cyclin E/Cdk2 complex (Fig. 2C
).
4. In vivo capsulorrhexis induced irregular proliferation of lens epithelial cells
We have shown that immediately after extracapsular lens extraction (ECLE) using continuous curvilinear capsulorrhexis, only LECs under the anterior lens capsule and at the lens bow remained in the capsular bag. However, 24 h after surgery LEC proliferation was evident and appeared to be maximal only 3 days after the procedure. At the center of the capsular bag, where no anterior lens capsule was present, irregular proliferation of LEC and capsular wrinkling were noted. Early fiber differentiation was evident 1 wk after capsulorrhexis.
CONCLUSIONS AND SIGNIFICANCE
A remarkable physiological characteristic of the ocular lens its unique pattern of circulating electric current (Fig. 1A
). It has been proposed that these currents serve as an internal circulatory system for the avascular lens and they seem to be essential to maintain clarity. Thus, LECs in vivo proliferate and migrate in the constant presence of electric fields (EF), and EFs have been shown to have significant effects on cell behavior. Since lens cell proliferation is important in the etiology of secondary cataract (PCO) and electric currents may be a neglected control mechanism for lens cell proliferation, we studied the effects of the attenuated/absence of electric current on lens cell proliferation, mimicking the clinical situation after cataract surgery. In this report, we show that 1) removal of part of anterior capsule and the lens nucleus, as in cataract surgery, significantly changed the electric current profile of the lens and decreased or removed the equatorial electric currents; 2) exposure to EFs inhibited proliferation of human LECs; 3) EF exposure decreased expression of G1-specific cell cycle protein cyclin E and increased the expression of cyclin-Cdk complex inhibitor p27kip1. Restoring key electrical features of the lens may contribute to the control of aberrant proliferation and migration of LECs.
An EF inhibits cell proliferation by cell cycle arrest
In the present study, an applied DC EF of 200 mV/mm inhibited significantly proliferation of cultured LECs. Many anti-proliferative factors act by triggering apoptosis. But the present study with LECs did not indicate that EF-induced inhibition of LEC proliferation was mediated by apoptosis (as shown by the lack of specific sub-G1 peak for apoptosis on the DNA content histogram after cell cycle analysis with flow cytometry). Instead, this inhibitory effect of EF on LEC proliferation resulted from cell cycle arrest (Fig. 2)
.
Cyclin E, a G1-specific cyclin, is necessary and rate-limiting for the passage of mammalian cells through the G1 phase. In EF-exposed LECs, cyclin E level decreased markedly but the expression level of Cdk2 did not change. Without their cyclin partners, the cyclin-dependent kinases (Cdks) are inactive. Therefore, reduced expression of cyclin E will inactivate the Cdk2-cyclin E complexes and prevent passage through G1. p27kip1 activity increases in response to growth inhibitory signals. In quiescent cells, the level of p27kip1 is relatively high. p27kip1 inhibits cyclin E/Cdk2 activity and induces G1 arrest. Inhibition of LEC proliferation by a physiological EF was accompanied by a significant increase in p27kip1 expression. We have shown that the physiological electric fields inhibited proliferation of LECs by arresting the cell cycle at G1 phase and preventing G1/S phase transition through selective up-regulation of p27kip1 expression and down-regulation of cyclin E expression.
Clinical importance
After in vivo ECLE in rodents, maximal LEC proliferation occurred in the first few days after the procedure, giving rise to PCO. This is the most common complication of modern cataract surgery with intraocular lens (IOL) implantation. Because cataract surgery greatly reduces the normal EFs in the lens, it is possible that the aberrant proliferation of LECs leading to PCO results at least partly from the loss of the regulatory control of endogenous EF in the lens. Experiments have shown that a voltage of 86 mV is needed to nullify net potassium currents in the rat lens. This voltage divided by normal cuboidal epithelial cells of 20 µm would give rise to endogenous electric fields of 86 mV/20 µm = 4300 mV/mm, an upper limit of the electric fields around the lens. Opening the capsule increases mitoses in the germinal areas near the equator. Therefore, disruption of physiological EF after cataract surgery is a strong candidate to contribute to stimulated cell growth after surgery.
PCO severely compromises vision in many patients undergoing cataract surgery. There is as yet no completely effective method for preventing PCO. It was shown that using IOL coated with thapsigargin, which kills aberrantly migrating LECs, prevents PCO ex vivo. Proliferation and directed migration of LECs are key events in PCO. Since an EF can direct the migration of LECs, prevent the healing of lens epithelial monolayer wounds, and inhibit LEC proliferation, an applied EF could regulate the behaviors of aberrant LECs that induce PCO. Taken together, these results point to a possible new controlling mechanism for the migration and proliferation of lens epithelial cells. A novel approach to controlling both proliferation and migration of lens epithelial cells using electric fields combined with other controlling mechanisms may be more effective in preventing and treating PCO.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2733fje;
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