Abstract
purpose. To investigate the effects of cholesterol-lowering statin drugs on trabecular meshwork cellular properties and aqueous humor outflow.
methods. Primary cell cultures of porcine trabecular meshwork (PTM) and ciliary body (PCB) were treated with either lovastatin or compactin, to determine the effects of statins on cell shape, actin cytoskeletal organization, and cell–extracellular matrix interactions (focal adhesions) by immunofluorescence staining. Changes in myosin light-chain (MLC) phosphorylation were evaluated by Western blot analysis. Changes in Rho GTPase content of membrane fractions from lovastatin-treated PTM cells were assessed by Western blot analysis. A constant-flow, organ-culture perfusion system was used to measure the effects of statins on aqueous humor outflow facility in the anterior segments of porcine eyes.
results. PTM and PCB cells treated with lovastatin or compactin exhibited dramatic changes in cell shape and cytoskeletal organization within 24 hours, consisting of cell rounding, actin depolymerization, and decreased focal adhesions. These effects were found to be reversible on supplementation with geranylgeranyl pyrophosphate. Both lovastatin and compactin decreased MLC phosphorylation in PTM and PCB cells. PTM cells treated with lovastatin exhibited marked decreases in membrane-bound Rho GTPase. In addition, perfusion of organ-cultured porcine eye anterior segments with 100 μM lovastatin for 96 hours caused a significant increase in aqueous humor outflow facility (110%) compared with control eyes, in a reversible manner.
conclusions. This study demonstrates that the statin drugs lovastatin and compactin induce changes in cell shape and actin cytoskeletal organization and decrease MLC phosphorylation in PTM and PCB cells, all of which are events that are likely to lead to cellular and tissue relaxation. In addition, these effects of the statins appear to be mediated by inhibition of isoprenylation of the small GTP-binding proteins such as Rho GTPase. An important finding is that statins exert an ocular hypotensive response in an organ-culture perfusion model, indicating the potential for this class of drugs in glaucoma therapy.
To provide more specific medical treatment for glaucoma, a blinding disease, we need a better understanding of the cellular mechanisms that control aqueous humor outflow. Increased intraocular pressure (IOP) is a major risk factor for primary open-angle glaucoma (POAG),
1 2 and lowering the IOP is the only current treatment available for the management of POAG. The conventional route of aqueous humor outflow through trabecular meshwork (TM) and Schlemm’s canal (SC) is generally thought to be the major pathway for the drainage of aqueous humor from the eye.
1 Impaired drainage through the conventional outflow pathway is believed to be responsible for the increased IOP in POAG.
1 2 Therefore, it would seem both necessary and critical to understand the normal regulation of aqueous outflow through TM and SC.
Our recent studies and those from other laboratories have demonstrated that the morphologic integrity of TM and SC may influence aqueous outflow through the conventional pathway. Perfusion of various pharmacological agents, particularly compounds known to affect actin cytoskeletal organization and cell–cell junctions, increase aqueous outflow and decrease IOP in both in vivo and in vitro systems.
3 4 5 6 7 8 9 10 11 Selective inhibitors of the Rho/Rho kinase pathway, protein kinase C (PKC) or myosin light-chain kinase (MLCK), have all been documented to influence aqueous outflow facility.
5 6 12 13 14 15 In many of these studies, the increased outflow facility correlated well with observed changes noted in TM cell shape, decreased myosin light-chain (MLC) phosphorylation, and altered actomyosin cytoskeletal organization.
12 13 14 15 Collectively, these studies have revealed the importance of cellular tension (contraction and relaxation) and the state of the cell–cell junctions, as well as cell–extracellular matrix (ECM) interactions in the modulation of aqueous outflow through TM tissue and that the activity of Rho/Rho kinase pathway may be an important intracellular “switch” regulating these effects on outflow function of TM cells.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Statin drugs are widely used medications for the treatment of hypercholesterolemia and the prevention of coronary artery disease and stroke.
16 17 18 19 20 21 Statins have also been demonstrated to be beneficial for many other human diseases,
22 including macular degeneration
23 and multiple sclerosis.
24 Statins are selective inhibitors of HMG-CoA reductase, which decrease the biosynthesis of cholesterol and thereby reduce serum cholesterol levels in humans.
25 However, statins not only decrease cholesterol biosynthesis, they also reduce the levels of various intermediary products of cholesterol biosynthesis. For example, statins decrease the synthesis of isoprenoids, including farnesyl pyrophosphate and geranylgeranyl pyrophosphate.
26 27 Isoprenoids play an important role in basic cell biology by regulating the activities of various cellular proteins, including small GTP-binding proteins, heterotrimeric G-proteins, and laminins.
26 28 29 By decreasing the production of isoprenoids, statins may have other beneficial effects in addition to lowering cholesterol.
17 30 31 32 33 34 35 36 The Rho subfamily of small GTP-binding proteins, including Rho, Rac, and CDC42, all of which play a critical role in regulating cellular contraction, actin cytoskeletal organization, and cell–cell and cell–ECM interactions, are covalently modified by isoprenylation at the C-terminal end of the protein.
27 29 37 38 39 This enzymatically regulated posttranslational modification is essential for the activities and functionality of the Rho GTPases in vital cellular processes.
22 29
In a preliminary human study, patients who were administered statin drugs had a reduced risk of glaucoma.
40 To understand the possible mechanistic basis of these observations and evaluate the role of the Rho/Rho kinase signaling pathway in aqueous humor outflow function through the TM, we investigated the effects of statins on aqueous humor outflow facility. Using lovastatin and compactin, we demonstrated that statins induce changes in porcine TM and CB cell morphology, decrease MLC phosphorylation, and alter actomyosin cytoskeletal organization, similar to the effects of Rho kinase inhibitors.
12 All the observed cellular changes induced by statins were associated with increased aqueous outflow facility in an organ-cultured porcine eye anterior segment perfusion model.
Lovastatin (Mevacor) was generously provided as a gift by Merck Pharmaceuticals (Rahway, NJ). Horseradish peroxidase (HRP), rhodamine-phalloidin, vinculin monoclonal antibody, compactin, and geranylgeranyl pyrophosphate were purchased from Sigma-Aldrich (St. Louis, MO); cell culture medium and fetal bovine serum (FBS) from Invitrogen-Gibco Corp. (Carlsbad, CA); phosphospecific MLC polyclonal antibody from Cell Signaling Technology, Inc. (Beverly, MA); monoclonal anti-RhoA GTPase antibody from Upstate Cell Signaling Solutions (Lake Placid, NY); and Enhanced chemiluminescence (ECL) detection reagents from Amersham Pharmacia Biotech (Piscataway, NJ). All chemicals were of analytical grade.
Porcine eyes (obtained fresh from a local abattoir) were placed in culture according to published techniques.
43 Anterior segments from paired eyes were prepared by dissecting the eyes at the equator; removing the lens, iris, and vitreous; and rinsing thoroughly with culture medium before clamping them to a two-cannulae, modified Petri-dish, as described by Vittitow et al.
44 To avoid disturbing the TM tissue, anterior segments were left with some ciliary body and iris. Anterior segments were perfused with high-glucose DMEM containing 0.1% FBS, penicillin (100 U/mL), streptomycin (100 μg/mL), gentamicin (36 μg/mL), and amphotericin (0.25 μg/mL) at a constant flow (3 μL/min), using microinfusion pumps (Harvard Bioscience, South Natick, MA), under 5% CO
2 at 37°C. Perfusion pressure was monitored continuously with a pressure transducer connected to the second cannula and recorded with an automated computerized system. After the initial baseline aqueous outflow facility was recorded for 24 hours, the anterior segments of the test eyes were transferred into, and perfused with, medium containing lovastatin (100 μM) for 96 hours. The contralateral fellow eyes were perfused with medium containing vehicle alone.
Data were recorded continuously at 1-minute intervals over 5 days. The outflow facility at the initial 20-hour perfusion before exposure of eyes to drug was taken as baseline facility. The percentage of change in the aqueous outflow facility from the baseline values was computed for both, drug- and sham-perfused eyes. At each 12-hour interval, the significance of differences in median percentage of change in outflow facility was assessed with the Wilcoxon signed ranks test.
In another set of perfusion experiments, we perfused the organ cultured eyes, first with 100 μM lovastatin for 72 hours. Then, the pumps were stopped and the anterior chambers were exchanged with medium, supplemented with 10 μM geranylgeranyl pyrophosphate without lovastatin. Perfusion was continued for another 48 hours with medium supplemented with geranylgeranyl pyrophosphate, and the changes in aqueous outflow were monitored.
Effects of Statins on Cell Morphology and Cytoskeletal Organization in PTM and PCB cells
Reversal of Lovastatin-Induced Morphologic and Cytoskeletal Changes by Isoprenoid Supplementation of PTM Cells
Lovastatin-Induced Changes in Localization of Rho GTPase to the TM Cell Membrane Fraction
Histology of Aqueous Outflow Pathway in Lovastatin-Perfused, Organ-Cultured Eye Anterior Segments
In this study, we sought to determine the effects of statin drugs on the function of the aqueous humor outflow pathway. Statins are selective inhibitors of HMG-CoA reductase and are widely used in the clinical setting for cholesterol reduction and the treatment of myocardial infarction.
16 17 18 19 20 21 The results of our study demonstrate that lovastatin increases aqueous outflow facility in the organ-cultured anterior eye segment perfusion model. This effect of the statins was associated with induced changes in cell shape, actomyosin cytoskeletal reorganization, and impaired membrane localization of Rho GTPase, as well as decreased MLC phosphorylation in both PTM and PCB cells in vitro.
TM tissue is known to possess smooth-muscle–like properties, and the contractile characteristics of TM and CB are thought to influence aqueous outflow through the TM.
3 4 5 6 7 8 9 10 12 13 Further, based on several perfusion studies, induced changes in TM cell morphology, intercellular junctions, and focal adhesions correlated with observed changes in aqueous humor outflow facility through the TM.
3 4 5 7 8 10 12 13 45
The small GTPase Rho is believed to have an important role in the regulation of actin cytoskeletal organization and formation of intercellular junctions and focal adhesions in many cell types.
28 46 47 48 Activated Rho GTPase, in turn, influences myosin II–mediated cellular contraction,
49 50 51 through the sequential activation of Rho-kinase, which serves as the downstream effector of Rho GTPase. It is noteworthy that the statins have been shown to affect directly the activity of Rho GTPase while inhibiting cholesterol biosynthesis.
22 27 38 52 53 54 Posttranslational modification of the Rho GTPases involves geranylgeranyl isoprenylation, a covalent lipid modification essential for biological activity of this class of small GTPases, including Rho, Rac, and CDC42, all of which play critical role(s) in both cell migration and actomyosin cytoskeletal organization.
29 Statins can impair this posttranslational modification by decreasing the levels of isoprenoids including farnesyl pyrophosphate and geranylgeranyl pyrophosphate which are the intermediary products of cholesterol biosynthesis.
26 27
Both PTM and PCB primary cell cultures exhibited dramatic changes in cell morphology on treatment with lovastatin and compactin
(Fig. 1) , and this effect on cell morphology was also associated with an observed decrease in actin stress fiber and focal adhesion staining
(Fig. 2) . These morphologic and cytoskeletal changes were found to be completely reversible after supplementation of cell culture media with geranylgeranyl pyrophosphate
(Fig. 4) . This effect of geranylgeranyl pyrophosphate supplementation implicates the potential involvement of Rho GTPases activity in the observed lovastatin-induced changes in cell morphology and cytoskeletal integrity.
38 52 53 In support of this finding, we also observed reduced levels of Rho GTPase in membrane fractions from lovastatin-treated PTM cells
(Fig. 5) . Thus, the geranylgeranyl supplementation data and the changes observed in Rho GTPase distribution in the membrane fraction of lovastatin-treated PTM cells collectively demonstrate the involvement of Rho GTPases in statin-induced effects on PTM cell morphology and contractile properties.
In addition to inducing changes in cell morphology and cytoskeletal integrity, we observed that lovastatin and compactin also substantially reduced MLC phosphorylation levels in both TM and CB cells
(Fig. 3) . The phosphorylation status of MLC is a key determinant of cellular contraction, and decreased MLC phosphorylation implies cellular relaxation.
49 50 51 55 TM and CB tissues both express Rho GTPase and Rho kinase.
12 15 49 56 In earlier studies, these two tissues have been reported to exhibit distinct contractile responses when treated with external factors or agonists of G-protein coupled receptors.
10 In this study, however, both the TM and the CB exhibited similar responses in cell morphology, cytoskeletal organization, and MLC phosphorylation.
As has been reported previously in a study of Rho kinase inhibitors,
12 we found a strong relationship between the decreased MLC phosphorylation induced by statins and the observed increase in aqueous outflow facility in response to perfusion with lovastatin. Perfusion of organ-cultured porcine eye anterior segments with lovastatin resulted in increased aqueous humor outflow facility
(Fig. 6) . We also confirmed that increased aqueous humor outflow facility is reversed by supplementation with lovastatin with geranylgeranyl pyrophosphate. Whereas in the cell culture system, both TM and CB cells exhibited dramatic changes in cell morphology in a reversible manner
(Fig. 1) , we did not find alterations in TM cell morphology in lovastatin-perfused eyes
(Fig. 8) . However, the TM facing the anterior chamber exhibited widened space between the trabecular beams
(Fig. 7) , indicating possible influence on the geometry of TM. In this study, we used the organ-cultured anterior segment perfusion model instead of whole-eye perfusion, because statins typically require at least 18 to 24 hours to demonstrate detectable effects on cell morphology. To reduce the isoprenoid levels below normal, we had to perfuse the samples with statins for more than 24 hours.
Statins are widely used in clinical practice and exhibit widely different potencies for lowering cholesterol levels.
16 17 18 19 20 21 Therefore, in the future, it is important to compare and contrast the effects of different classes of statins on aqueous outflow facility and IOP. Because the use of statins generally results in minimal adverse effects,
57 their potential use for lowering IOP is obvious. It should be noted, however, that the concentrations of statins used in this study were much higher (100-fold) than the serum levels of statins in patients who are treated with statins.
16 17 18 19 20 21 22 The results of our study may provide additional support to the recently reported beneficial effect of statins in decreasing glaucoma risk in patients.
40 Alternatively, the latter finding relate more importantly to possible neuroprotective mechanisms, rather than IOP lowering from systemic statin administration. It is noteworthy that statins have been observed to increase retinal ganglion cell axonal growth through inhibition of Rho GTPase and Rho kinase.
58 In addition, inhibitors of Rho kinase have also been reported to stimulate axonal growth in retinal ganglion cells.
59
In conclusion, this study demonstrates significantly increased aqueous outflow facility with lovastatin in an organ cultured porcine anterior eye segment perfusion model. This effect of statins on aqueous outflow facility appears to be associated with relaxation of TM and CB muscle induced by decreased MLC phosphorylation. The statins appear to exert these changes by affecting the activity of regulatory proteins involved in actomyosin cytoskeletal integrity, such as the Rho GTPases. Although this is an experimental in vitro study and the levels of statins used in this study far exceed those noted in the serum of patients who are on statin regimens, our findings suggest that these compounds could lend themselves to a novel treatment for glaucoma, since the statins are used widely in clinical practice with minimal adverse effects on a long-term basis. Further studies involving both living animals and human subjects seem well justified, to evaluate the therapeutic potential of statins in the treatment of glaucoma.
Presented in part at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, April 2004.
Ophthalmology Times has summarized this work in one of the recent issues.
Supported by research grants from Fight for Sight; National Eye Institute Grants EY13573, EY12202 (PVR), EY01894 (DLE), P30-EY05772; and Research to Prevent Blindness.
Submitted for publication July 1, 2004; revised November 17, 2004, and March 16, 2005; accepted March 25, 2005.
Disclosure:
J. Song, None;
P.-F. Deng, None;
S.S. Stinnett, None;
D.L. Epstein, (P);
P.V. Rao, (P)
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “
advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: P. Vasantha Rao, Duke University Medical Center, Box 3802, Durham, NC 27710;
[email protected].
Table 1. Lovastatin-Induced Changes in Aqueous Humor Outflow Facility in Organ-Cultured Anterior Segments of Porcine Eyes
Table 1. Lovastatin-Induced Changes in Aqueous Humor Outflow Facility in Organ-Cultured Anterior Segments of Porcine Eyes
Drug Perfusion Time (h) | Control | Drug | P * |
12 | −4.5 ± 7.0 | 3.1 ± 7.5 | 0.383 |
| (−1.8) | (8.4) | |
24 | 2.6 ± 12.9 | 34.6 ± 22.5 | 0.312 |
| (−2.5) | (18.0) | |
36 | 3.7 ± 14.3 | 56.7 ± 31.1 | 0.195 |
| (−3.3) | (33.5) | |
48 | 13.8 ± 13.4 | 82.7 ± 31.9 | 0.055 |
| (3.0) | (46.4) | |
60 | 22.2 ± 17.6 | 83.3 ± 30.4 | 0.078 |
| (1.5) | (39.1) | |
72 | 21.4 ± 18.2 | 109.0 ± 37.2 | 0.039 |
| (2.5) | (78.3) | |
84 | 27.3 ± 18.7 | 117.2 ± 43.5 | 0.109 |
| (1.6) | (99.4) | |
96* , † | 36.1 ± 25.4 | 152.3 ± 55.3 | 0.016 |
| (3.2) | (217.9) | |
The authors thank Wenxiu Zhang for assistance with histology.
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