Selective regulation of MMP and TIMP mRNA levels in tree shrew sclera during minus lens compensation and recovery

Abstract

Purpose: In juvenile tree shrews, a minus-power lens placed in front of the eye produces increased axial elongation and a myopic shift in refractive state that compensates for the power of the lens. Scleral tissue remodeling and modulation of the mechanical properties of the sclera occur during lens compensation. In this study, the time course of changes in scleral mRNA levels of three MMPs and three TIMPs during compensation for a minus lens and during recovery was investigated, to determine which, if any, are temporally associated with changes in the mechanical properties of the sclera and the axial elongation rate.

Methods: Competitive RT-PCR was used to measure the levels of mRNA for MT1-MMP, MMP-2, MMP-3, TIMP-1, TIMP-2, and TIMP-3 in the scleras of tree shrews that had received either 1, 2, 4, or 11 days of monocular -5-D lens treatment, or 11 days of -5-D lens treatment followed by 2 or 4 days of recovery.

Results: Relative to their control eyes, treated eye MT1-MMP and MMP-2 mRNA levels were significantly higher, and TIMP-3 levels were lower by 1 to 4 days of minus lens treatment. These differential effects were absent by 11 days of treatment when the treated eyes had compensated for the lens. The levels of all three TIMPs spiked upward in both eyes after 2 days of recovery. The differential changes in MT1-MMP, MMP-2, and TIMP-3 mRNA levels were all restricted to the treated eye and were temporally associated with the differential changes in axial elongation, refractive state, and the previously measured changes in creep rate.

Conclusions: The observed changes in MT1-MMP, MMP-2, TIMP-2, and TIMP-3 mRNA are consistent with visually modulated MT1-MMP activation of MMP-2 and with MT1-MMP degradation of scleral extracellular matrix components. These data constitute further evidence that visual signals modulate gene expression of selected MMPs and TIMPs to control scleral remodeling, the mechanical properties of the sclera, axial elongation, and refractive state.

Figure 1

Regulation of the viscoelastic properties of the scleral tissue (measured as creep rate: an increase in length under constant tension) during monocular compensation for a −5-D lens (replotted from Siegwart and Norton.21) Creep rate was increased in the treated eyes, relative to control and normal eyes, after 2 days of −5-D lens wear, peaked at 4 days and had decreased by 11 days. Creep rate was measured in 3-mm wide strips of sclera under 3 g of constant tension. In the normal animals, left and right eyes were averaged. No creep rate measures are available for 2 or 4 days of recovery after 11 days of −5-D lens treatment. The creep rates shown at 2 days of recovery are after 11 days of MD. n = 3 in each group. Values are mean ± SEM.

Figure 2

Ocular refraction and vitreous chamber depth during −5-D lens treatment and recovery. Relative to their fellow control eyes, the treated eyes became increasingly myopic (A), and their vitreous chamber became increasingly deep (C) as a function of days of −5-D treatment. During recovery, the myopic progression and the increase in vitreous chamber depth abruptly reversed. Values from normal animals (average of left and right eyes) are shown for comparison. (B, D) The difference between the treated and control eyes. n = 5 in each group. Results are expressed as the mean ± SEM; *P < 0.05. Because of the small-eye artifact, a reading of 4 D of hyperopia is estimated to be emmetropia in tree shrews.,

Figure 3

mRNA levels in the treated and control eyes of animals with different durations of −5-D lens or −5-D/recovery and age-matched normal animals. All mRNA levels are expressed as copies per million copies of 18s. (A) MT1-MMP, (B) MMP-2, (C) MMP-3, (D) TIMP-1, (E) TIMP-2, and (F) TIMP-3. Treated and control eye error bars are 1 SEM. The average value and 95% confidence interval are shown for the normal animals. T vC, treated eyes different from control eyes (paired-test, P < 0.05); T v N, treated eyes compared with normal eyes (ANOVA, LSD, P < 0.05); C v N, control eyes compared with normal eyes (ANOVA, LSD, P < 0.05).

Figure 4

The percentage difference (treated − control eye) in the mRNA levels of the four genes that may be involved in the activation of pro-MMP-2 compared with the percentage difference in creep rate during minus lens compensation and recovery. (A–D) The percentage difference in the average mRNA copy number in the treated and control eyes from Figure 3. As computed, this difference has no associated error bars. *Statistical significance (P < 0.05), as shown in Figure 3. (E) The percentage difference in the average creep rate in the treated and control eyes from Figure 1, in which the animals underwent −5-D lens treatment for similar periods. No creep rate measures are available for 2 or 4 days of recovery after 11 days of −5-D lens treatment. Therefore, the creep rate at 2 days of recovery is after 11 days of MD.

Figure 5

Schematic showing the suggested relationship between MT1-MMP, TIMP-2, and MMP-2, as it relates to MT1-MMP activation of proMMP-2. MT1-MMP is a membrane-bound MMP that localizes the activation of pro-MMP-2 to the cell surface. TIMP-2 bound to MT1-MMP is thought to form a “receptor” for pro-MMP-2 that presents the proMMP-2 to another MT1-MMP that cleaves the pro-MMP-2 molecule to initiate the activation process. The relative amount of each of the molecules can alter the amount of pro-MMP-2 that is activated.