Molecular Vision 2008; 14:983-991 <>
Received 30 March 2008 | Accepted 10 May 2008 | Published 28 May 2008

Nitric oxide, a survival factor for lens epithelial cells

Coral G. Chamberlain, Kylie J. Mansfield, Anna Cerra

School of Medical Sciences and Bosch Institute, University of Sydney, Sydney, Australia

Correspondence to: Dr. Coral G. Chamberlain, Anatomy and Histology (F13), University of Sydney, Sydney, NSW 2006, Australia; Phone: +61 2-9351-5169; FAX: +612-9351-2817; email:

Dr. Mansfield is now at the Division of Basic Sciences, Graduate School of Medicine, University of Wollongong, Wollongong, NSW 2522, Australia


Purpose: Nitric oxide (NO) is capable of promoting either cell death or cell survival depending on cell type and experimental conditions. In this study, the possible effects of NO on the viability of lens epithelial cells were investigated in an explant model used previously to identify cellular changes associated with posterior capsule opacification following cataract surgery.

Methods: Rat lens epithelial explants prepared from weanling rats were cultured in a serum-free medium for five days with or without the addition of the nitric oxide synthase inhibitor, L-Nω-nitro-L-arginine methyl ester (L-NAME), using the inactive enantiomer D-NAME as a control. Alternatively, explants were cultured for nine days with or without the NO donor, sodium nitroprusside. Explants were assessed morphologically and immunohistochemically or by determining DNA content.

Results: In the presence of L-NAME but not in controls, progressive rounding up and detachment of cells from the lens capsule occurred, leading to extensive cell loss. Affected cells showed apoptosis-like cell-surface blebbing and nuclear fragmentation. Conversely, inclusion of sodium nitroprusside suppressed the morphological changes and spontaneous cell loss that occurred when sparsely covered explants were cultured for nine days, increased cell coverage fourfold during that period, and prevented the expression of the transdifferentiation markers α-smooth muscle actin and fibronectin. In addition, whereas L-NAME exacerbated cell loss induced by culturing with 50 pg/ml transforming growth factor-β2, sodium nitroprusside offered protection.

Conclusions: This study points to a previously unidentified role for NO as an endogenously produced survival factor for lens epithelial cells, raising the possibility of using NO deprivation as a means of removing residual lens cells following cataract surgery and thereby preventing posterior capsule opacification.


Many cells have the capacity to synthesize nitric oxide (NO), a readily diffusible, short-lived molecule that is produced by the action of nitric oxide synthase (NOS) on L-arginine. Two of three known cytoplasmic isoforms of NOS, endothelial cell NOS (eNOS/NOS-1) and neuronal NOS (nNOS/NOS-3), are expressed constitutively while the third, inducible NOS (iNOS/NOS-2), is generally expressed in response to immunological challenge or some other pathophysiological stimulus [1,2]. Transient stimulation of constitutive NOS activity results in relatively low levels of NO production whereas iNOS activity can produce much larger amounts of NO over several days [3,4]. A wide variety of biological functions is served by controlled production of NO, which can act both intracellularly as a second messenger and extracellularly as a conveyor of information between cells. However, excessive NO production can result in cellular damage via various mechanisms, which include the formation of highly reactive free radicals such as peroxynitrite [4].

Normal ocular tissues including the retina, ciliary body, iris, conjunctiva, and cornea express NOS [5,6], and NO is normally present at a low concentration in the aqueous humor that bathes the lens [7,8]. Although there is some evidence that constitutive levels of NO production contribute in positive ways to normal ocular function, overproduction of NO in response to induction of iNOS is generally regarded as deleterious. For example, induction of iNOS and abnormal production of NO occur in uveitis, retinitis, and glaucoma [5,6] and in certain animal models of cataract [9-11]. Moreover, the concentration of NO in the aqueous humor is known to be elevated in endotoxin-induced uveitis and traumatic cataract [8,12] and to increase with age in senile cataract patients or following cataract surgery in the rabbit [7,12,13]. A role for NO in the etiology of cataract has been proposed because of its ability to modify lens proteins and/or cause or exacerbate oxidative damage to lens cells or predispose them to such damage [6,14-17].

Lens cells themselves appear to express NOS. NADP-diaphorase activity, which is indicative of NOS activity, has been detected in the rat lens epithelium [18]. In addition, iNOS has been detected at low levels in the normal rat lens by western blot analysis and RT–PCR and shown to be upregulated in the lenses of rats treated with selenite in vivo [9]. iNOS mRNA is also upregulated in human lens epithelial cells cultured with a combination of lipopolysaccharide and interferon-γ [11,19]. Furthermore, it has been shown that opacification of the rat lens in selenite and hereditary cataract models is accompanied by and apparently dependent upon the induction of iNOS in the lens [9,10]. However, little is known about the regulation and biological significance of the synthesis of NO by lens cells under normal conditions.

In other cell types, NO has been shown to affect cell viability in profound and often paradoxical ways. NO may promote either cell death or cell survival in vivo and in vitro depending upon experimental conditions and the tissue or cell type involved. For example, it may either promote apoptosis or protect against apoptosis induced by various means including exposure to transforming growth factor-β (TGFβ) or activation of the Fas pathway [4,20-25]. While progress has been made in elucidating the diverse mechanisms involved in determining cell fate in response to NO, the issues are complex and not yet fully understood. Here, we report the effects of NO on the viability of lens epithelial cells.


Lens explants were prepared from 20-day-old to 21-day-old Wistar rats as described previously [26,27]. The medium used in all experiments (control medium) was serum-free medium M199 supplemented with 0.1% BSA and antibiotics [28]. All experimental procedures were in accordance with the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research and approved by the Animal Ethics Committee, University of Sydney (Sydney, Australia). HEPES at a final concentration of 20 mM was added to the control medium during initial pinning out of explants. After replacing the HEPES-containing medium with 1 ml of the control medium, explants were examined by phase contrast microscopy and given a ‘cell coverage’ grade ranging from 0%–100%. This was based on an estimate of the percentage of the total explant surface covered by patches of attached, confluent cells [27,29]. Cell coverage immediately after setting up the explants was 57%±3% (mean±SEM). Explants were then assigned to treatment groups matched with respect to initial cell coverage and precultured for one day to ensure uniformly high coverage in all treatment groups at the start of the experiment (day 0; Figure 1). On day 0, the medium was replaced with control medium or with medium containing 5 mM L-Nω-nitro-L-arginine methyl ester (L-NAME), a widely used NOS inhibitor, or its inactive enantiomer, D-NAME (both from Sigma, St Louis, MO), readjusted to the control medium pH with 0.5 M NaOH. Previous studies indicate that all NOS isoforms would be inhibited at the concentration of L-NAME used, especially given that lens epithelial cells express esterases capable of converting L-NAME to the more potent inhibitor Nω-nitro-L-arginine [30,31]. Explants were cultured at 37 °C in 5% CO2/95% air for five days. Cell coverage and morphological changes were monitored daily by phase contrast microscopy. On day 5, explants were fixed as whole mounts in 10% neutral buffered formalin, stained with methylene blue-hematoxylin, and counterstained with Hoechst dye (H33342; Sigma).

Experiments were also performed to test the effects of a classical NO donor, sodium nitroprusside (SNP), on cell survival. Explants with initial cell coverage of 20% or less were used for these experiments because previous studies had shown that sparsely covered explants were susceptible to spontaneous cell loss during culture in the control medium [27]. The explants were assigned to two matched groups and precultured for 24 h as described above and then cultured for nine days with or without the addition of SNP (Calbiochem, La Jolla, CA) at a final concentration of 50 μM. The medium was replaced on day 5 of culture with or without the addition of SNP as appropriate, and explants were monitored daily by phase contrast microscopy. At the end of the culture period, representative explants were fixed as whole mounts in Carnoy’s fixative (3:1 ethanol:acetic acid), and α-smooth muscle actin (αSMA) and fibronectin were immunolocalized by a double-labeling technique with Hoechst counterstaining of nuclei [32].

In some experiments, recombinant TGFβ2 (R&D Systems, Minneapolis, MN) was included in the medium on day 0 with or without L-NAME or SNP and explants were cultured for two days. The final concentration of TGFβ2 was 50 pg/ml, a concentration shown previously to induce rapid loss of cells from rat lens epithelial explants [26,27]. In some experiments, explants were lysed at the end of the culture period and the DNA content was determined by the PicoGreen method as described [33].


Over the first three days of culture, cell coverage was virtually 100% in explants cultured in the control medium or in the medium containing L-NAME or D-NAME (Figure 1). However, beyond three days of culture there was a dramatic decrease in cell coverage in explants cultured with L-NAME (Figure 1 and Figure 2B,E). In contrast, explants cultured in the control medium or with D-NAME remained completely covered with a monolayer of cells in cobblestone arrays throughout the entire five-day culture period (Figure 1 and Figure 2A,C,D,F).

The progressive denuding of the lens capsule that occurred in the presence of L-NAME was accompanied by an extensive loss of cells from the explant. Indeed, 2 of the 11 explants in the L-NAME-treated group were completely devoid of cells by day 5 of culture. During culture, large numbers of cells were observed rounding up, becoming highly refractile, and detaching from the lens capsule. This process was accompanied by cell-surface blebbing, especially at cellular margins abutting denuded regions of capsule (Figure 3A). Nuclear fragmentation, of the type observed in cells undergoing caspase-dependent apoptosis [34,35], was observed in marginal cells in L-NAME-treated explants (Figure 2E) but not in untreated controls or D-NAME-treated explants (Figure 2D,F). The cell loss observed in L-NAME-treated explants (Figure 3A) was comparable in some respects to that observed in explants cultured with 50 pg/ml TGFβ2, although cell loss induced by TGFβ began at an earlier stage of culture and was accompanied by more extensive cell-surface blebbing (see Figure 3A,B). In addition, the capsular wrinkling characteristically induced by TGFβ (Figure 3B) [26,27,32] was not apparent in L-NAME-treated explants (Figure 2B and Figure 3A).

The above experiment suggested that suppressing NOS activity had a negative effect on cell survival. In analogous experiments designed to determine whether the addition of an NO donor promoted cell survival, inclusion of SNP not only promoted cell survival but also permitted the repopulation of the denuded lens capsule. At the end of the culture period, all SNP-treated explants were 50%–100% covered with confluent cells in cobblestone arrays (Table 1; Figure 3D) including an explant with very low initial cell coverage (<5%). In contrast, most explants in the control group cultured without SNP showed extensive and progressive cell loss over the same period (Table 1; Figure 3C).

Including SNP in the medium also suppressed the capsular wrinkling and cell-surface blebbing typically associated with cell detachment under these conditions (Table 1). In the control group, cells abutting the denuded lens capsule were particularly prone to surface-blebbing and detachment (Figure 3C) whereas corresponding cells in SNP-treated explants generally remained bleb-free and extended lamellipodia onto the lens capsule at cellular margins (not shown). Furthermore, residual cells in explants that were cultured in the control medium for nine days exhibited specific reactivity for transdifferentiation markers, αSMA and fibronectin (Figure 4A-C), a finding consistent with previous studies [29,32], whereas reactivity for these markers was negligible in corresponding explants cultured with SNP (Figure 4D-F).

In experiments in which TGFβ2 was included in the medium on day 0, L-NAME appeared to exacerbate TGFβ-induced cell loss during the two days of culture (Table 2A) whereas SNP again exhibited a positive effect on cell survival (Table 2B). The latter was evidenced by the significantly higher cell coverage and DNA content of explants cultured with TGFβ plus SNP compared with those cultured with TGFβ alone (Table 2B). However, the cell-surface blebbing and capsular wrinkling that generally precede cell loss were visible in at least some regions of all explants cultured with TGFβ plus SNP by day 2 of culture, and all these explants became virtually devoid of cells by day 6 (not shown).


The results of this study demonstrate that suppressing NO synthesis by including an NOS inhibitor led to an extensive loss of cells from the lens capsule in initially well covered lens epithelial explants and also exacerbated TGFβ-induced cell loss. On the other hand, inclusion of an NO donor enhanced cell survival in sparsely covered explants under conditions that typically result in spontaneous loss of cells during extended culture and also afforded partial protection against TGFβ-induced cell loss. Therefore, the results point to a previously unrecognized role for NO as a survival factor for lens epithelial cells.

Detachment of cells from rat lens epithelial explants cultured with TGFβ is preceded by changes typical of apoptosis including DNA fragmentation, cell-surface blebbing, nuclear pyknosis, and nuclear fragmentation [36,37]. Cell-surface blebbing and nuclear fragmentation were observed in well covered explants undergoing cell loss in response to L-NAME. They were also observed in sparsely covered, untreated explants undergoing spontaneous cell loss, and in the latter case, these changes were suppressed by including SNP in the medium. Therefore, it is likely that NO was, at least in part, regulating lens epithelial cell survival in these explants by influencing apoptotic pathways as is known to occur for many other cell types [4,20,21,38].

Several growth factors and cytokines have been shown previously to serve as survival factors for lens epithelial cells and/or protect them from apoptosis induced by serum starvation or exposure to agents such as TGFβ and staurosporine. These include FGF [32,39-41], lens epithelium-derived growth factor [42], transferrin [43], insulin-like growth factor-1 [44], and growth arrest-specific gene 6 [45]. It has also been reported previously by others that culturing sparsely covered rat lens epithelial explants in serum-free medium is associated with poor cell survival [46], a finding consistent with the present study (Table 1 and Figure 3C) and a previous study in this laboratory [27]. The data of Ishizaki et al. [45] indicated that lack of diffusible survival factor(s) produced by the lens cells themselves contributed to cell death when cell numbers were low. They also showed that FGF-2 was ineffective as a substitute for this survival factor. Furthermore, it has recently been reported that lens epithelial cells are capable of producing unidentified diffusible factor(s) that protect them against Fas-induced apoptosis provided that they remain attached to the lens capsule or a collagen substratum [47].

The finding of the present study that cell loss is largely prevented in sparsely populated lens epithelial explants by including a source of NO strongly suggests that cell death may occur because of depletion of endogenously produced NO when few lens epithelial cells are present. Moreover, because lens epithelial cells express TGFβ and experience autocrine TGFβ stimulation following wounding of the epithelium [48,49], cell death that is initiated by NO deficiency in lens epithelial explants may be linked with or augmented by TGFβ-induced apoptosis. In lens epithelial cells, the latter process is known to be associated with myofibroblastic/fibroblastic transdifferentiation [50-52]. Thus, the finding that spontaneous cell loss during long-term culture of sparsely covered explants resulted in the induction of transdifferentiation markers is consistent with the suggestion that stimulation by endogenous TGFβ accompanies loss of cells due to NO deprivation. Furthermore, transdifferentiation as well as cell loss were suppressed by including an NO donor.

NO has been shown to have the potential to exert either harmful or beneficial effects in other cellular systems via a variety of mechanisms [4,20,21,53]. These include both pro-apoptotic and anti-apoptotic effects. The outcome depends not only on the concentration of NO to which the cells are exposed but also on factors such as their capacity to deal with oxidative stress and the relative abundance of other free radicals in their environment. Generally, anti-apoptotic effects are observed at relatively low concentrations of an NO donor (30–300 μM SNP) whereas the induction of apoptosis requires higher concentrations (1–4 mM SNP) [54-60], although exceptions to this general rule have been noted (see reference [61], for example). At high concentrations and under conditions that lead to the generation of large amounts of peroxynitrate, NO is cytotoxic and may induce cell death by necrosis if not by apoptosis [38]. The paradoxical anti-apoptotic and pro-apoptotic effects of NO at low and high concentrations, respectively, can occur in the same cell type [54-57].

Oxidative damage to the lens, especially the lens epithelium, is thought to be a triggering factor in the etiology of several forms of cataract, and NO is regarded as an agent capable of contributing to such damage [6,14-17]. Consistent with the latter suggestion, changes that are indicative of oxidative stress and therefore potentially cataractogenic were noted in a study in which rat lenses were exposed in vitro to an NO donor at a high concentration (1 mM) [17]. However, consistent with findings for other cellular systems cited above, exposing explanted lens epithelial cells to an NO donor at a low concentration (50 μM) in the present study promoted lens epithelial cell survival. Furthermore, the low concentration of SNP used in the present study also prevented fibroblastic transdifferentiation, an early event in the etiology of certain forms of cataract and the sight-threatening condition known as posterior capsule opacification, a sequela of cataract surgery [36,50,52,62].

It is not known whether a low level of stimulation by NO is required in the eye in situ to maintain a healthy lens epithelium and protect against cataractous transdifferentiation under physiological or adverse conditions. However, NO is known to be present in the lens environment under physiological conditions. A low concentration of NO is present in the aqueous that bathes the lens anterior. Furthermore, the lens epithelium and neighboring ocular tissues express NOS in situ and therefore represent potential sources of NO ( [5,7-9,18] and see Introduction).

In the experiments reported here, the NOS activity inhibited by L-NAME may have been baseline activity associated with the lens epithelium in situ. However, some damage to the lens epithelium inevitably occurs during the preparation of explants such as those used in the present study, and it is not clear whether the rate of NO production was influenced by such damage. Irrespective of this, the present study has clearly identified a new role for NO as an endogenously produced survival factor for lens cells with the potential to play a role in maintaining the integrity of the lens epithelium.

Human cataract surgery results in extensive damage to the lens epithelium. Many lens epithelial cells are removed by excision of the central anterior capsule and adhering lens epithelium during this procedure. However, variable numbers of lens epithelial cells remain in situ, attached to the annulus of anterior lens capsule that is retained. These cells have the potential to undergo fibroblastic transdifferentiation and migrate posteriorly into the visual axis causing posterior capsule opacification [49,62,63]. The present study raises the possibility of inducing the lens epithelial cells that remain after cataract surgery to die by restricting NO availability through means such as inhibiting NOS production or activity or sequestering NO. Thus, it offers a new perspective on preventing posterior capsule opacification.


We thank Joel Symonds for providing expert assistance. This project was supported by funding from the National Health and Medical Research Council of Australia, Canberra, ACT and iCare Pty Ltd, Sydney, NSW. We thank the University of Sydney’s Electron Microscope Unit (Sydney, Australia) for the use of its facilities.


  1. Hofseth LJ, Robles AI, Espey MG, Harris CC. Nitric oxide is a signaling molecule that regulates gene expression. Methods Enzymol. 2005; 396:326-40. [PMID: 16291242]
  2. Kleinert H, Pautz A, Linker K, Schwarz PM. Regulation of the expression of inducible nitric oxide synthase. Eur J Pharmacol. 2004; 500:255-66. [PMID: 15464038]
  3. Boucher JL, Moali C, Tenu JP. Nitric oxide biosynthesis, nitric oxide synthase inhibitors and arginase competition for L-arginine utilization. Cell Mol Life Sci. 1999; 55:1015-28. [PMID: 10484661]
  4. Li CQ, Wogan GN. Nitric oxide as a modulator of apoptosis. Cancer Lett. 2005; 226:1-15. [PMID: 16004928]
  5. Becquet F, Courtois Y, Goureau O. Nitric oxide in the eye: multifaceted roles and diverse outcomes. Surv Ophthalmol. 1997; 42:71-82. [PMID: 9265703]
  6. Chiou GC. Review: effects of nitric oxide on eye diseases and their treatment. J Ocul Pharmacol Ther. 2001; 17:189-98. [PMID: 11324986]
  7. Er H, Gunduz A, Turkoz Y, Cigli A, Isci N. Effects of NG-nitro L-arginine and corticosteroids on aqueous humor levels of nitric oxide and cytokines after cataract surgery. J Cataract Refract Surg. 1999; 25:795-9. [PMID: 10374159]
  8. Goureau O, Bellot J, Thillaye B, Courtois Y, de Kozak Y. Increased nitric oxide production in endotoxin-induced uveitis. Reduction of uveitis by an inhibitor of nitric oxide synthase. J Immunol. 1995; 154:6518-23. [PMID: 7539024]
  9. Inomata M, Hayashi M, Shumiya S, Kawashima S, Ito Y. Involvement of inducible nitric oxide synthase in cataract formation in Shumiya cataract rat (SCR). Curr Eye Res. 2001; 23:307-11. [PMID: 11852433]
  10. Ito Y, Nabekura T, Takeda M, Nakao M, Terao M, Hori R, Tomohiro M. Nitric oxide participates in cataract development in selenite-treated rats. Curr Eye Res. 2001; 22:215-20. [PMID: 11462158]
  11. Wang S, Li D, Ito Y, Liu X, Zhang J, Wu C. An ocular drug delivery system containing zinc diethyldithiocarbamate and HPbetaCD inclusion complex—corneal permeability, anti-cataract effects and mechanism studies. J Pharm Pharmacol. 2004; 56:1251-7. [PMID: 15482639]
  12. Kao CL, Chou CK, Tsai DC, Hsu WM, Liu JH, Wang CS, Lin JC, Wu CC, Peng CH, Chang CJ, Kao CL, Chiou SH. Nitric oxide levels in the aqueous humor in cataract patients. J Cataract Refract Surg. 2002; 28:507-12. [PMID: 11973099]
  13. Jurowski P, Gos R, Piasecka G. Nitric oxide levels in aqueous humor after lens extraction and poly(methyl methacrylate) and foldable acrylic intraocular lens implantation in rabbit eyes. J Cataract Refract Surg. 2002; 28:2188-92. [PMID: 12498858]
  14. Lou MF. Redox regulation in the lens. Prog Retin Eye Res. 2003; 22:657-82. [PMID: 12892645]
  15. Ornek K, Karel F, Buyukbingol Z. May nitric oxide molecule have a role in the pathogenesis of human cataract? Exp Eye Res. 2003; 76:23-7. [PMID: 12589772]
  16. Spector A. Oxidative stress-induced cataract: mechanism of action. FASEB J. 1995; 9:1173-82. [PMID: 7672510]
  17. Varma SD, Hegde KR. Susceptibility of the ocular lens to nitric oxide: implications in cataractogenesis. J Ocul Pharmacol Ther. 2007; 23:188-95. [PMID: 17444807]
  18. Yamamoto R, Bredt DS, Snyder SH, Stone RA. The localization of nitric oxide synthase in the rat eye and related cranial ganglia. Neuroscience. 1993; 54:189-200. [PMID: 7685860]
  19. Nagai N, Liu Y, Fukuhata T, Ito Y. Inhibitors of inducible nitric oxide synthase prevent damage to human lens epithelial cells induced by interferon-gamma and lipopolysaccharide. Biol Pharm Bull. 2006; 29:2077-81. [PMID: 17015954]
  20. Brune B, von Knethen A, Sandau KB. Nitric oxide and its role in apoptosis. Eur J Pharmacol. 1998; 351:261-72. [PMID: 9721017]
  21. Chung HT, Pae HO, Choi BM, Billiar TR, Kim YM. Nitric oxide as a bioregulator of apoptosis. Biochem Biophys Res Commun. 2001; 282:1075-9. [PMID: 11302723]
  22. Dilworth C, Bigot-Lasserre D, Bars R. Spontaneous nitric oxide in hepatocyte monolayers and inhibition of compound-induced apoptosis. Toxicol In Vitro. 2001; 15:623-30. [PMID: 11698161]
  23. Kim YM, Bombeck CA, Billiar TR. Nitric oxide as a bifunctional regulator of apoptosis. Circ Res. 1999; 84:253-6. [PMID: 10024298]
  24. Mannick JB, Miao XQ, Stamler JS. Nitric oxide inhibits Fas-induced apoptosis. J Biol Chem. 1997; 272:24125-8. [PMID: 9305857]
  25. Weller R, Schwentker A, Billiar TR, Vodovotz Y. Autologous nitric oxide protects mouse and human keratinocytes from ultraviolet B radiation-induced apoptosis. Am J Physiol Cell Physiol. 2003; 284:C1140-8. [PMID: 12676653]
  26. Schulz MW, Chamberlain CG, McAvoy JW. Inhibition of transforming growth factor-beta-induced cataractous changes in lens explants by ocular media and alpha 2-macroglobulin. Invest Ophthalmol Vis Sci. 1996; 37:1509-19. [PMID: 8675393]
  27. Symonds JG, Lovicu FJ, Chamberlain CG. Posterior capsule opacification-like changes in rat lens explants cultured with TGFbeta and FGF: effects of cell coverage and regional differences. Exp Eye Res. 2006; 82:693-9. [PMID: 16359663]
  28. Hales AM, Chamberlain CG, McAvoy JW. Susceptibility to TGFbeta2-induced cataract increases with aging in the rat. Invest Ophthalmol Vis Sci. 2000; 41:3544-51. [PMID: 11006250]
  29. Symonds JG, Lovicu FJ, Chamberlain CG. Differing effects of dexamethasone and diclofenac on posterior capsule opacification-like changes in a rat lens explant model. Exp Eye Res. 2006; 83:771-82. [PMID: 16713596]
  30. Griffith OW, Kilbourn RG. Nitric oxide synthase inhibitors: amino acids. Methods Enzymol. 1996; 268:375-92. [PMID: 8782604]
  31. Roelfzema H, Broekhuyse RM, Veerkamp JH. Subcellular distribution of sphingomyelinase and other acid hydrolases in different parts of the calf lens. Exp Eye Res. 1974; 18:579-94. [PMID: 4369246]
  32. Mansfield KJ, Cerra A, Chamberlain CG. FGF-2 counteracts loss of TGFbeta affected cells from rat lens explants: implications for PCO (after cataract). Mol Vis. 2004; 10:521-32. [PMID: 15303087]
  33. Cerra A, Mansfield KJ, Chamberlain CG. Exacerbation of TGF-beta-induced cataract by FGF-2 in cultured rat lenses. Mol Vis. 2003; 9:689-700. [PMID: 14685144]
  34. Chen Q, Yano T, Matsumi H, Osuga Y, Yano N, Xu J, Wada O, Koga K, Fujiwara T, Kugu K, Taketani Y. Cross-talk between Fas/Fas ligand system and nitric oxide in the pathway subserving granulosa cell apoptosis: a possible regulatory mechanism for ovarian follicle atresia. Endocrinology. 2005; 146:808-15. [PMID: 15528299]
  35. Huerta S, Goulet EJ, Huerta-Yepez S, Livingston EH. Screening and detection of apoptosis. J Surg Res. 2007; 139:143-56. [PMID: 17257621]
  36. Liu J, Hales AM, Chamberlain CG, McAvoy JW. Induction of cataract-like changes in rat lens epithelial explants by transforming growth factor beta. Invest Ophthalmol Vis Sci. 1994; 35:388-401. [PMID: 8112986]
  37. Maruno KA, Lovicu FJ, Chamberlain CG, McAvoy JW. Apoptosis is a feature of TGF beta-induced cataract. Clin Exp Optom. 2002; 85:76-82. [PMID: 11952402]
  38. Choi BM, Pae HO, Jang SI, Kim YM, Chung HT. Nitric oxide as a pro-apoptotic as well as anti-apoptotic modulator. J Biochem Mol Biol. 2002; 35:116-26. [PMID: 16248976]
  39. Garcia CM, Yu K, Zhao H, Ashery-Padan R, Ornitz DM, Robinson ML, Beebe DC. Signaling through FGF receptor-2 is required for lens cell survival and for withdrawal from the cell cycle during lens fiber cell differentiation. Dev Dyn. 2005; 233:516-27. [PMID: 15778993]
  40. Renaud F, Oliver L, Desset S, Tassin J, Romquin N, Courtois Y, Laurent M. Up-regulation of aFGF expression in quiescent cells is related to cell survival. J Cell Physiol. 1994; 158:435-43. [PMID: 7510293]
  41. Wang Y, He H, Zigler JS, , Jr Iwata T, Ibaraki N, Reddy VN, Carper D. bFGF suppresses serum-deprivation-induced apoptosis in a human lens epithelial cell line. Exp Cell Res. 1999; 249:123-30. [PMID: 10328960]
  42. Singh DP, Ohguro N, Kikuchi T, Sueno T, Reddy VN, Yuge K, Chylack LT, , Jr Shinohara T. Lens epithelium-derived growth factor: effects on growth and survival of lens epithelial cells, keratinocytes, and fibroblasts. Biochem Biophys Res Commun. 2000; 267:373-81. [PMID: 10623627]
  43. Davidson MG, Harned J, Grimes AM, Duncan G, Wormstone IM, McGahan MC. Transferrin in after-cataract and as a survival factor for lens epithelium. Exp Eye Res. 1998; 66:207-15. [PMID: 9533846]
  44. Chandrasekher G, Sailaja D. Phosphatidylinositol 3-kinase (PI-3K)/Akt but not PI-3K/p70 S6 kinase signaling mediates IGF-1-promoted lens epithelial cell survival. Invest Ophthalmol Vis Sci. 2004; 45:3577-88. [PMID: 15452065]
  45. Valverde P, Obin MS, Taylor A. Role of Gas6/Axl signaling in lens epithelial cell proliferation and survival. Exp Eye Res. 2004; 78:27-37. [PMID: 14667825]
  46. Ishizaki Y, Voyvodic JT, Burne JF, Raff MC. Control of lens epithelial cell survival. J Cell Biol. 1993; 121:899-908. [PMID: 8491781]
  47. Futter CE, Crowston JG, Allan BD. Interaction with collagen IV protects lens epithelial cells from Fas-dependent apoptosis by stimulating the production of soluble survival factors. Invest Ophthalmol Vis Sci. 2005; 46:3256-62. [PMID: 16123427]
  48. Gordon-Thomson C, de Iongh RU, Hales AM, Chamberlain CG, McAvoy JW. Differential cataractogenic potency of TGF-beta1, -beta2, and -beta3 and their expression in the postnatal rat eye. Invest Ophthalmol Vis Sci. 1998; 39:1399-409. [PMID: 9660488]
  49. Saika S. Relationship between posterior capsule opacification and intraocular lens biocompatibility. Prog Retin Eye Res. 2004; 23:283-305. [PMID: 15177204]
  50. Dawes LJ, Elliott RM, Reddan JR, Wormstone YM, Wormstone IM. Oligonucleotide microarray analysis of human lens epithelial cells: TGFbeta regulated gene expression. Mol Vis. 2007; 13:1181-97. [PMID: 17679943]
  51. Hales AM, Schulz MW, Chamberlain CG, McAvoy JW. TGF-beta 1 induces lens cells to accumulate alpha-smooth muscle actin, a marker for subcapsular cataracts. Curr Eye Res. 1994; 13:885-90. [PMID: 7720396]
  52. Lovicu FJ, Schulz MW, Hales AM, Vincent LN, Overbeek PA, Chamberlain CG, McAvoy JW. TGFbeta induces morphological and molecular changes similar to human anterior subcapsular cataract. Br J Ophthalmol. 2002; 86:220-6. [PMID: 11815351]
  53. Kroncke KD, Fehsel K, Suschek C, Kolb-Bachofen V. Inducible nitric oxide synthase-derived nitric oxide in gene regulation, cell death and cell survival. Int Immunopharmacol. 2001; 1:1407-20. [PMID: 11515808]
  54. Antonova GN, Snead CM, Antonov AS, Dimitropoulou C, Venema RC, Catravas JD. Nitric oxide preconditioning regulates endothelial monolayer integrity via the heat shock protein 90-soluble guanylate cyclase pathway. Am J Physiol Heart Circ Physiol. 2007; 292:H893-903. [PMID: 17012359]
  55. Kwak HJ, Park KM, Lee S, Lim HJ, Go SH, Eom SM, Park HY. Preconditioning with low concentration NO attenuates subsequent NO-induced apoptosis in vascular smooth muscle cells via HO-1-dependent mitochondrial death pathway. Toxicol Appl Pharmacol. 2006; 217:176-84. [PMID: 17027882]
  56. Tai YT, Cherng YG, Chang CC, Hwang YP, Chen JT, Chen RM. Pretreatment with low nitric oxide protects osteoblasts from high nitric oxide-induced apoptotic insults through regulation of c-Jun N-terminal kinase/c-Jun-mediated Bcl-2 gene expression and protein translocation. J Orthop Res. 2007; 25:625-35. [PMID: 17262823]
  57. Yoshioka Y, Yamamuro A, Maeda S. Nitric oxide/cGMP signaling pathway protects RAW264 cells against nitric oxide-induced apoptosis by inhibiting the activation of p38 mitogen-activated protein kinase. J Pharmacol Sci. 2006; 101:126-34. [PMID: 16778361]
  58. Brynczka C, Merrick BA. Nerve growth factor potentiates p53 DNA binding but inhibits nitric oxide-induced apoptosis in neuronal PC12 cells. Neurochem Res. 2007; 32:1573-85. [PMID: 17592775]
  59. Pan SL, Guh JH, Chang YL, Kuo SC, Lee FY, Teng CM. YC-1 prevents sodium nitroprusside-mediated apoptosis in vascular smooth muscle cells. Cardiovasc Res. 2004; 61:152-8. [PMID: 14732212]
  60. Yoshioka Y, Kitao T, Kishino T, Yamamuro A, Maeda S. Nitric oxide protects macrophages from hydrogen peroxide-induced apoptosis by inducing the formation of catalase. J Immunol. 2006; 176:4675-81. [PMID: 16585560]
  61. Du C, Guan Q, Diao H, Yin Z, Jevnikar AM. Nitric oxide induces apoptosis in renal tubular epithelial cells through activation of caspase-8. Am J Physiol Renal Physiol. 2006; 290:F1044-54. [PMID: 16352744]
  62. Marcantonio JM, Vrensen GF. Cell biology of posterior capsular opacification. Eye. 1999; 13:484-8. [PMID: 10627829]
  63. Wormstone IM. Posterior capsule opacification: a cell biological perspective. Exp Eye Res. 2002; 74:337-47. [PMID: 12014915]