Kinetics of Corneal Epithelium Turnover In Vivo - IOVS

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Content, Distribution, and Activity of Corneal. 3-Hydroxy-3-Methylglutaryl Coenzyme A. (HMG-CoA) Reductase. The HMG-CoA reductase concentration was esti-.
Investigative Ophthalmology & Visual Science, Vol. 31, No. 10, October 1990 Copyright © Association for Research in Vision and Ophthalmology

Kinetics of Corneal Epithelium Turnover In Vivo Studies of Lovasrarin Richard J. Cenedella and Charles R. Fleschner The authors developed a direct chemical approach for estimating the rate of turnover of the corneal epithelium in vivo. The method was used to examine the effects of lovastatin, a potent inhibitor of cholesterol biosynthesis, on proliferation and turnover of the epithelium. Corneal DNA was labeled by pulse injection (IP) of the rat with 3H-thymidine, and 3H-labeled DNA was recovered from peripheral and central corneas over the next 15 days. Only the epithelium became labeled, and the loss of label by cell desquamation began 3 days after injection. The loss of 3H-DNA from the cornea (peripheral plus central region) followed first-order kinetics. The half-life of the disappearance was about 3 days. The peripheral cornea became more highly labeled than the central cornea and began to lose 3H-DNA before the central cornea. These observations support the possibility of a higher mitotic rate in the peripheral region and the centripetal movement of a population of peripheral epithelial cells in the normal cornea. The half-lives of the disappearance of 3H-DNA from peripheral and central corneas measured between days 5 and 15 postinjection were identical, both at 3 days. Complete turnover of the corneal epithelium would, therefore, require about 2 weeks (4-5 half-lives). Treatment of the rat with lovastatin had no obvious effects upon the proliferation or turnover of the corneal epithelium. Although lovastatin inhibited corneal 3-hydroxy-3-methylglutaryl coenzyme A reductase, the key regulatory enzyme of cholesterol synthesis, the cornea compensated by induction of this enzyme so that there was no net inhibition of cholesterol synthesis in the cornea. Invest Ophthalmol Vis Sci 31:1957-1962,1990

The corneal epithelium undergoes dynamic turnover due to sustained proliferation of basal epithelial cells. These basal cells are then displaced outward by the next generation of mitotic cells and eventually lost by desquamation.1'2 A slow centripetal movement of peripheral epithelial cells also contributes to the renewal of the epithelium.3-4 Net turnover of the epithelium has been estimated in vitro by measuring the rate of labeling of the basal cells from 3H-thymidine1'2-5 and in vivo by following the movement of dye-tagged cells.3 Both experimental approaches provide data which suggest that the corneal epithelium is completely replaced in about 2 weeks. We are interested in relationships between membrane synthesis in epithelial cell populations and their turnover. Rapid membrane growth is required for cells to proceed through the cell cycle.6"9 Because

cholesterol is required for membrane formation and because the cornea appears obligated to synthesize most of the cholesterol which it requires for cell growth,10 we were interested in the possibility that inhibition of cholesterol synthesis in the cornea might affect proliferation and turnover of the corneal epithelium in normal and wounded states. To investigate these possibilities, we developed a chemical method for directly and quantitatively estimating rates of proliferation and turnover of the corneal epithelium in vivo. The approach basically involves pulse labeling proliferative corneal epithelial cells from a single intraperitoneal (IP) injection of the rat with 3H-thymidine and then measuring the concentration and distribution of 3H-DNA in the cornea with time after injection. Kinetic analysis of the data permitted calculation of the net rate of epithelium turnover of both the central and peripheral cornea and the time required for mitotic cells to desquamate. Information on the possible migration of labeled epithelial cells from peripheral to central cornea was also obtained. This method was used to test the influence of lovastatin, the prototype of the new generation of hypocholesterolemic drugs," on proliferation and turnover of the corneal epithelium. We observed pre-

From the Department of Biochemistry, Kirksville College ofOsteopathic Medicine, Kirksville, Missouri. Supported in part by NIH grant EY02568 and by the Kirksville College of Osteopathic Medicine. Reprint requests: Richard J. Cenedella, Department of Biochemistry, Kirksville College of Osteopathic Medicine, 800 West Jefferson, Kirksville, MO 63501.

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viously that lovastatin could block proliferation of lens epithelial cells in culture and that the block was completely reversed by addition of serum low-density lipoproteins to the media.12 Materials and Methods Animals

Animal use was approved by the institutional animal care committee, and all experimental procedures reported here conformed to the ARVO Resolution on the Use of Animals in Research. Male SpragueDawley rats (Hilltop, Scottdale, PA), 150 g, were fed Purina rat chow (Ralston Purina, St. Louis, MO) or ground chow containing 0.1% lovastatin for 2 weeks. Cornea Epithelium Turnover

The rats (control or lovastatin treated) were injected IP with 2 nCi/g of body weight of 3H-(methyl) thymidine (2 Ci/mmol; New England Nuclear, Boston, MA). They were killed by carbon dioxide inhalation at various times over the next 15 days. The corneas were removed and freed of attached scleral tissue by dissection. The central and peripheral corneas were separated with a 3-mm diameter trephine. Peripheral cornea accounted for an average 61 % and 60% of the total cornea wet weight in control and treated rats, respectively. In some cases, corneas were physically separated into epithelial and stromal fractions as done previously.10 Corneal fractions from individual rats were separately homogenized in 1.0 ml of saline containing 0.4 mg of carrier DNA (salmon, type III; Sigma, St. Louis, MO) using a 2-ml, ground glass-ground glass hand homogenizer (Radnoti Glass, Monrovia, CA). The homogenates were combined with two separate 1-ml saline washes of the homogenizer, protein was digested with proteinase K, and the DNA was recovered and assayed for tritium content as described previously.13 The 3H-DNA content of the central, peripheral, and whole cornea was calculated. The distribution of substrate in the cornea for DNA synthesis 1 and 6 hr after injection of 3H-thymidine was measured by treating homogenized corneal fractions with trichloroacetic acid (TCA). After centrifugation, the supernatant was collected and the TCA extracted with ether. The extracted supernatant was quick frozen and lyophilized. The residue was redissolved in water and counted. We showed previously that the TCA-soluble, nonvolatile radioactivity present in the ocular lens after IP injection of 3 H-thymidine was 3H-thymidine.14 The TCA-soluble, volatile radioactivity was 3H2O.

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Content, Distribution, and Activity of Corneal 3-Hydroxy-3-Methylglutaryl Coenzyme A (HMG-CoA) Reductase

The HMG-CoA reductase concentration was estimated by immunofluorescence microscopy;15 it was purified from rat liver,16 and antibodies to the purified enzyme were raised in rabbits.17 Frozen sections of rat cornea were incubated with rabbit anti-rat HMG-CoA reductase diluted 1:100 in 0.1% albumin, 0.1 M potassium phosphate, 0.9% NaCl, pH 7.8 (albumin-PBS). After extensive washing with albuminPBS, the sections were incubated with fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin G (Sigma) diluted 1:40 with albumin-PBS. The stained sections were examined and photographed using an Olympus BH-2 microscope (Jacobs Instrument Co., Overland Park, KS) with fluorescence attachment. The HMG-CoA reductase activity was assayed according to Panini et al18 as previously described.10 One unit of reductase activity was defined as 1 pmol of mevalonate formed per min at 37°C. Corneal Sterol Synthesis In Vivo

The rats (control or treated with lovastatin, 0.1% in the diet for 14 days) were injected IP with 50 mCi/kg body weight of 3H2O (25 mCi/ml, New England Nuclear) between 1 and 4 PM. A blood sample (20-40 /ul) was collected from the tail 2 and 24 hr after injection to measure the 3H2O content of the plasma. The mean specific activity over this interval was calculated for each rat by averaging the 2- and 24-hr values. Between 2 and 24 hr, the specific activity of plasma water (thus, also body water) decreased by about 25%. The rats were killed 24 hr after injection. Corneas, lenses, and about 200 mg of liver were collected from each rat, weighed, and saponified in ethanolic KOH. The digitonin precipitable sterols (DPS) were prepared, recovered, and assayed for 3H-content as described previously.1019 All samples (including background and blanks) were counted to a 2-a error of 2.5% or less. The results were expressed as nmol of 3 H of 3H2O incorporated into DPS per mg of tissue. Results The epithelium accounted for essentially all of the H-thymidine incorporated into DNA by the cornea (Table 1). Furthermore, on a unit-weight basis, the peripheral cornea contained a significantly higher concentration of 3H-labeled DNA than the central cornea soon after injection (Table 2). No differences were seen on day 5 or later. The lower 3H-DNA concentration in the central cornea was apparently not due to a lesser availability of substrate for DNA syn3

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Table 3. Corneal distribution of substrate for DNA synthesis with time after injection of 3H-thymidine

Table 1. Corneal epithelial versus stromal concentration of 3H-DNA

TCA-soluble: nonvolatile 3H (dpm/mg cornea)

DPM/mg fraction* Time (days) postinjection 1 2

Epithelium

Stroma

1895 1271 1313 1079

6 8 12 27

* Each set of values is for a pair of divided corneas from an individual rat injected 1 or 2 days earlier with 2 /iCi/g body water of 3 H-thymidine. The stromal fraction also contained the endothelium.

thesis in this region. In fact, the concentration of TCA-soluble, nonvolatile radioactivity (3H-thymidine) was higher in the central cornea 1 hr after injection (Table 3). By 6 hr postinjection no differences were seen between central and peripheral cornea. It is apparent from Table 3 that substrate for 3H-DNA synthesis was available for only a few hours after injection, since the concentration of TCA-soluble, nonvolatile 3H-label greatly decreased between 1 and 6 hr in both peripheral and central corneas. The 3H-DNA content of the peripheral cornea remained constant until day 3 after injection but then decreased by an apparent first-order process (Fig. 1). The half-life was about 3.3 days. The 3H-DNA content of the central cornea remained constant or slightly increased until day 5 after injection-. Then, as with the peripheral cornea, the 3H-DNA content disappeared by first-order kinetics with a half-life of 3.1 days (Fig. 1). The onset of 3H-DNA loss from the whole cornea (peripheral plus central fractions) resembled that of the peripheral cornea; ie, loss began between days 3-5 postinjection. The slope of the curve describing the loss of 3H-DNA from the whole cornea was indistinguishable from those of the peripheral or central cornea.

Corneal* fraction Peripheral (P) Central (C)

p/ct

+7 hr

+6 hr

177 ±34 291 ±67 0.62 ±0.05

47 ± 2 43 ± 4 1.10 ±0.07

* Six rats were injected intraperitoneally with 3 H-thymidine (2 /xCi/g body water) and killed in groups of three at 1 and 6 hours later. Values are mean ±SEM. t Mean (± SEM) ratio in peripheral/central cornea. f\X) < 0.05 of difference between hour 1 and 6.

The level of incorporation of 3H-thymidine into DNA and the distribution of the labeled DNA in the cornea of lovastatin-treated rats were very similar to that in corneas of control rats (Table 2 and Fig. 2). Also like controls, the 3H-DNA content of the peripheral and whole cornea of treated rats began to decrease after day 3. The half-lives of the curves describing the disappearance of 3H-DNA from all epithelial fractions of treated rats were essentially equal to those of the control animals (Figs. 1, 2).

10,000

1

1

1

1

T I / 2 (Days)

1

3 5 7 9 12 15

Lovastatin-treated (peripheral/central)

.65 ± 0.04 .68 ± 0.08 .17 ± 0.13 ().97 ± 0.04 .07 ± 0.07 .22 ±0.11 .18 ±0.06

1.77 ±0.14 1.40 ±0.07 1.10 ±0.03 1.06 ±0.07 1.25 ±0.14 1.08 ±0.05 .05 ± 0.09

-

—o PERIPHERAL 3.34 3.12 • — -••• CENTRAL

N

\

\

1000-

#•*•"

x \\ i \ X

\ ^

_

\

\

\

K

\ \\KNx > X

K

100

* Each value is the mean ± SEM of four or five rats (exception was day 12 control, 3 rats). The peripheral fraction was 61% of total cornea in control (range, 59-63%) and 60% in control (range, 56-64%).

3.22

o-

DPM/mg fraction* Control (peripheral/central)

i

—» TOTAL

5OOO-

Table 2. Peripheral versus central corneal concentration of 3H-DNA

Time (days) postinjection

1

i

1

1

1

i

1

i

DAYS POSTINJECTION

Fig. 1. Disappearance of 3H-DNA from the cornea of control rats. The concentration of 3H-DNA was measured in peripheral and central corneas at 1 to 15 days after injecting (intraperitoneally) 2 fiC\/g body water of 3H-thymidine. Peripheral cornea comprised 61% of total cornea wet weight. Disappearance curves were fitted for day 3 to 15 or day 5 to 15 by computer. All correlation coefficients (r) were 0.99.

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10,000

1

1

i

1

1

1

i

TI/2 (Days) * TOTAL

5000-

o

3.08 o PERIPHERAL 3.20



• CENTRAL

-

3.08

\ T

X \

N

1000\

500-

100

i

i

1

i

i

3

5

7

9

i

DAYS POSTINJECTION

Fig. 2. Disappearance of 3H-DNA from the cornea of lovastatintreated rats. Rats were treated for 14 days with 0.1% lovastatin in the diet. Peripheral cornea comprised 60% of total cornea wet weight.

The concentration of HMG-CoA reductase protein in the corneal epithelium of lovastatin-treated rats was shown by immunofluorescence analysis to be clearly increased relative to that of controls (Fig. 3). Also, HMG-CoA reductase activity measured in microsomes from corneas of control and treated rats was 61 and 209 units/mg protein, respectively. Lovastatin is washed out of the microsomes during their preparation, and thus the activity of the increased enzyme became apparent. The total cholesterol synthesized over a 24-hour period by the cornea was similar for control and treated rats (Table 4). In contrast, the 24-hour cholesterol synthesis by the liver of treated rats was almost double that of controls and synthesis by lenses of treated rats was significantly less than that of controls (Table 4). Discussion Brief exposure in vivo of corneal epithelial cells to H-thymidine should pulse label cells undergoing mitosis at this time. The fate of this discrete population of labeled cells was followed by measuring the time required for the onset of loss of 3H-DNA from the cornea and the sustained rate of loss. Epithelial cells leave the cornea by desquamation. Labeled cells (3H-DNA) started to disappear from the whole cor3

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nea approximately 3 days after injecting 3H-thymidine. This indicated that, once an epithelial cell was displaced from the basal layer, about 3 days were required for it to reach the corneal surface and desquamate. We found that the rate of loss of labeled cells followed apparent first-order kinetics with a half-life of about 3 days. Thus, 50% of the basal epithelial cells should be replaced about every 3 days. These results show that the corneal epithelium of the rat should completely turnover in 12-15 days (4-5 half-lives). This conclusion agrees well with earlier results using autoradiographic methods.1'2'5 Interesting differences were observed between the labeling of the peripheral and central cornea. In our experiments, the peripheral cornea comprised about 60% of the cornea's total mass. Early after exposure to 3 H-thymidine (days 1-3), the concentration of 3HDNA (dpm/mg tissue) was greater in the peripheral cornea. Assuming that the 3H-DNA content per labeled cell is constant, this suggested that the density of mitotically active epithelial cells was greater in this region, a possibility consistent with earlier suggestions.4'20 Several observations also support the possibility that a population of epithelial cells migrated from peripheral to central cornea. The higher concentration of 3H-DNA in the peripheral cornea disappeared by day 5 after epithelial cell labeling. Thus, some labeled cells could have moved from the periphery to the center between days 3-5. Also, the total 3 H-DNA content of the peripheral cornea began to decrease before it did in the central cornea (day 3 versus day 5, respectively) even though the turnover rates of the peripheral and central cornea appeared identical (T1/2 = 3 days). These observations are consistent with the possibility that between days 3-5 after injection, the central cornea was gaining labeled cells from the peripheral cornea at about the same rate that it was losing labeled cells by desquamation. The central cornea should have lost about 400 dpm of 3 H-DNA during this period, a level equal to about 15% of the labeled peripheral cells present at day 3 (about 2900 dpm of 3H-DNA). A sustained migration of labeled cells from peripheral to central regions throughout the experimental period is inconsistent with the observation that the net rates of turnover of the central and peripheral cornea were the same. If the peripheral cornea was losing labeled cells between days 5-15 due to migration and desquamation, then the rate of turnover of the peripheral epithelium should have been significantly greater than that of the central cornea where cell loss occurred only by desquamation. Lovastatin is a potent competitive inhibitor of the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase.21 After oral dosing it becomes

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I

Fig. 3. Immunofluorescence of HMG-CoA reductase in corneas from normal and lovastatin-treated rats. Frozen sections of rat corneas were examined for HMG-CoA reductase content by immunofluorescence microscopy using rabbit anti-rat HMG-CoA reductase as described in the text. (A, left) control rats. (B, right) rats treated with lovastatin (0.1%, w/w in ground chow) for 13 days. Bar= 10.7 ^m. The HMG-CoA reductase-spectfic activity of control rat corneas was 61.2 U/mg microsomal protein and of treated rat corneas was 209.0 U/mg microsomal protein.

widely distributed and inhibits cholesterol synthesis in many tissues.22'23 Treatment of the rat with lovastatin produced clear effects on the concentration and activity of HMG-CoA reductase in the corneal epithelium. Since the cornea appears dependent on onsite synthesis to furnish the cholesterol required for membrane growth,10 why did lovastatin not alter turnover of the epithelium? The answer could be that regardless of the drug's presence net cholesterol synthesis in the cornea was not inhibited. When cholesTable 4. Effects of lovastatin on 24-hr cholesterol synthesis in vivo by cornea, liver, and lens nmol iH o[3H2O incorporated into DPS/mg/24 hr Group* Control Treated

No. of rats

Cornea

Liver

Lens

9.22 ± 0.42 8.01 ±0.75

31.6 ±2.9 57.0 ±8.8

0.591 ±0.036 0.474 ± 0.026f

• Rats, control, and lovastatin-treated (0.1% in the diet for 14 days), were injected with 3 H 2 O, 50 mCi/kg BW. The specific activity of body water was determined for each rat. Cholesterol as digitonin-precipitable sterol (DPS) was recovered at 24 hr after injection and assayed for tritium content. t P(l) of difference from control < 0.05 (student t-test).

terol synthesis and tissue levels decrease due to inhibition of HMG-CoA reductase, cellular HMG-CoA reductase protein concentrations can dramatically increase through derepression of transcription of the mRNA for the enzyme.24 In the rat cornea, the concentration of HMG-CoA reductase protein apparently increased adequately to compensate for the direct inhibitory effects of the drug. The net result was no inhibition of cholesterol synthesis, In the liver, which is extremely sensitive to feedback induction of the enzyme,23 total 24-hour cholesterol synthesis almost doubled in the lovastatin-treated group. In contrast to both the liver and cornea, the 24-hour level of cholesterol synthesis in the ocular lens of treated rats was significantly decreased. During formation of cortical fiber cells, the site of cholesterol synthesis in the lens,25 the cell's nucleus and DNA undergo disintegration.26 This could limit the capacity of the lens to compensate for inhibition of HMG-CoA reductase by induction of the enzyme. Treatment of the rat with lovastatin had no clear effects on the proliferation or rate of turnover of the corneal epithelium. Because of possible interspecies differences in response to lovastatin, one should be

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cautious in extrapolating these findings to other species. For example, Kornbrust et al27 recently reported that HMG-CoA reductase activity was induced 180fold in rat liver after 7 days of treatment but only twofold in rabbit liver after 10 days of treatment with triple the dose. The authors suggest that the rabbit liver has little capacity to induce HMG-CoA reductase. Whether this applies to other rabbit tissues is unknown. Key words: cornea, epithelium, turnover, lovastatin, cholesterol

Acknowledgments The authors thank Mrs. Nancy Waletzko for her excellent technical assistance.

References 1. Hanna C and O'Brien JE: Cell production and migration in the epithelial layer of the cornea. Arch Ophthalmol 64:536, 1960. 2. Hanna C, Bicknell DS, and O'Brien JE: Cell turnover in the adult human eye. Arch Ophthalmol 65:695, 1961. 3. Buck RC: Measurement of centripetal migration of normal corneal epithelial cells in the mouse. Invest Ophthalmol Vis Sci 26:1296, 1985. 4. Sharma A and Coles WH: Kinetics of corneal epithelial maintenance and graft loss: A population balance model. Invest Ophthalmol Vis Sci 30:1962, 1989. 5. Shapiro MS, Thoft RA, Friend J, Parrish RK, and Gressel MG: 5-Fluorouracil toxicity to the ocular surface epithelium. Invest Ophthalmol Vis Sci 26:580, 1985. 6. Blueminck JG, van Maurik PA, Tertoolen LG, van der Saag PT, and de Laat SW: Ultrastructural aspects of rapid plasma membrane growth in mitotic neuroblastoma cells. Eur J Cell Biol 32:7, 1983. 7. Chen HW, Heiniger HJ, and Kandutsch AA: Relationship between sterol synthesis and DNA synthesis in phytohemagglutin-stimulated mouse lymphocytes. Proc Natl Acad Sci USA 72:1950, 1975. 8. Cornell RB and Honvitz AF: Apparent coordination of the biosynthesis of lipids in cultured cells: Its relationship to the regulation of the membrane sterohphospholipid ratio and cell cycling. J Cell Biol 86:810, 1980. 9. El-Sayed GN and Cenedella RJ: Relationship of cholesterolgenesis to DNA synthesis and proliferation by lens epithelial cells in culture. Exp Eye Res 45:443, 1987. 10. Cenedella RJ and Fleschner CR: Cholesterol biosynthesis by the cornea: Comparison of rates of sterol synthesis with accumulation during early development. J Lipid Res 30:1079, 1989. 11. The Lovastatin Study Group II: Therapeutic response to lovastatin (mevinolin) in nonfamilial hypercholesterolemia: A multicenter study. JAMA 256:2829, 1986.

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12. El-Sayed GN and Cenedella RJ: Relationship of cholesterolgenesis to DNA synthesis and proliferation by lens epithelial cells in culture. Exp Eye Res 45:443, 1987. 13. Cenedella RJ: Aging and rates of lens-cell differentiation in vivo, measured by a chemical approach. Invest Ophthalmol Vis Sci 30:575, 1989. 14. Cenedella RJ: Direct chemical measurement of DNA synthesis and net rates of differentiation of rat lens epithelial cells in vivo: Applied to the selenium cataract. Exp Eye Res 44:677, 1987. 15. Singer II, Kawka DW, Kazazis DM, Alberts AW, Chen JS, Huff JW, and Ness GC: Hydroxymethylglutaryl-coenzyme A reductase-containing hepatocytes are distributed periportally in normal and mevinolin-treated rat livers. Proc Natl Acad Sci USA 81:5556, 1984. 16. Edwards PA, Lemongello D, and Fogelman AM: Purification and properties of rat liver 3-hydroxy-3-methlglutaryl coenzyme A reductase. Biochim Biophys Acta 574:123, 1979. 17. Hillam RP, Tengerdy RP, and Brown GL: Local antibody production against the murine toxin of Yersinia pestis in golf ball-induced granuloma. Infect Immunol 10:458, 1974. 18. Panini SR, Sexton RC, and Rudney H: Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by oxysterol by-products of cholesterol biosynthesis: Possible mediators of low density lipoprotein action. J Biol Chem 259:7767, 1984. 19. Cenedella RJ: Sterol synthesis by the ocular lens of the rat during postnatal development. J Lipid Res 23:619, 1982. 20. Thoft RA and Friend J: The X, Y, Z hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci 24:1442, 1983. 21. Alberts AW, Chen J, Kuron G, Hunt V, Huff J, Hoffman C, Rothrock J, Lopez M, Joshua H, Harris E, Patchett A, Monaghan R, Currie S, Stapley E, Albers-Schonberg G, Henses O, Hirshfield J, Hoogsteen K, Liesch J, and Springer J: Mevinolin: A highly potent competive inhibitor of hydroxymethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent. Proc Natl Acad Sci USA 77:3957, 1980. 22. Li AC, Tanaka RD, Callaway K, Fogelman AM, and Edwards PA: Localization of 3-hydroxy-3-methylglutaryl CoA reductase and 3-hydroxy-3-methylglutaryl CoA synthase in the rat liver and intestine is affected by cholestyramine and mevinolin. J Lipid Res 29:781, 1988. 23. Mosley ST, Kalinowski SS, Schafer BL, and Tanaka RD: Tissue-selective acute effects of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase on cholesterol biosythesis in lens. J Lipid Res 30:1411, 1989. 24. Luskey KL, Faust JR, Chin DJ, Brown MS, and Goldstein JL: Amplification of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, but not for the 53-kDa protein, in UT-1 cells. J Biol Chem 258:8462, 1983. 25. Cenedella RJ: Regional distribution of sterol and fatty acid synthesis in the ocular lens. Exp Eye Res 38:95, 1984. 26. Appleby DW and Modak SP: DNA degradation in terminally differentiating lens fiber cells from chick embryos. Proc Nat Acad Sci USA 74:5579, 1977. 27. Kornbrust DJ, MacDonald JS, Peter CP, Duchai DM, Stubbs J, Germershausen JI, and Alberts AW: Toxicity of the HMG CoA coenzyme A reductase inhibitor, lovastatin, to rabbits. J Pharmacol Exp Ther 248:498, 1989.