November 2009
Volume 50, Issue 11
Free
Glaucoma  |   November 2009
Effect of Bimatoprost, Latanoprost, and Unoprostone on Matrix Metalloproteinases and Their Inhibitors in Human Ciliary Body Smooth Muscle Cells
Author Notes
  • From the Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts. 
  • Corresponding author: Douglas J. Rhee, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114; dougrhee@aol.com
Investigative Ophthalmology & Visual Science November 2009, Vol.50, 5259-5265. doi:10.1167/iovs.08-3356
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Yen Hoong Ooi, Dong-Jin Oh, Douglas J. Rhee; Effect of Bimatoprost, Latanoprost, and Unoprostone on Matrix Metalloproteinases and Their Inhibitors in Human Ciliary Body Smooth Muscle Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(11):5259-5265. doi: 10.1167/iovs.08-3356.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose.: Matrix metalloproteinase (MMP)-mediated turnover of extracellular matrix (ECM) affects outflow resistance in the uveoscleral pathway. The balance of MMPs and tissue inhibitors of metalloproteinases (TIMPs) governs the rate of ECM turnover in many tissues. The hypothesis was that a differential effect on MMPs and TIMPs in ciliary body smooth muscle (CBSM) cells would relate to the relative intraocular pressure–lowering effectiveness of the prostaglandin analogues (PGAs) bimatoprost, latanoprost, and unoprostone.

Methods.: Human CBSM cells isolated from donor corneoscleral rims were incubated for 24 hours with control (0.015% ethanol in DMEM) or the free acid forms of bimatoprost (0.01 or 0.1 μg/mL), latanoprost (0.03 or 0.3 μg/mL), or unoprostone (0.145 or 1.45 μg/mL). Western blot analysis determined the relative protein concentrations of MMP-1, -2, -3. -9, and -24 as well as TIMP-1 through -4. Zymography measured the relative activity levels of MMP-1, -2, -3, and -9.

Results.: All PGAs increased MMP-1, -3, and -9. Bimatoprost and latanoprost did not change MMP-2. Unoprostone decreased MMP-2 (21% ± 3%). On zymography, MMP-1 and -2 did not change. Bimatoprost and latanoprost increased MMP-9 activity by 75% ± 27% and 75% ± 24%, respectively. MMP-3 activity was not detected on zymography. All PGAs increased TIMP-3, but only unoprostone increased TIMPs1 and -4 by 100% ± 20% and 61% ± 11%, respectively. TIMP-2 was unchanged by bimatoprost and latanoprost, but decreased by unoprostone (35% ± 8%).

Conclusions.: Decreased MMP-2 with concurrent increases of TIMP-1 and -4 by unoprostone may explain the lower clinical efficacy of unoprostone. The MMP/TIMP balance relates to the observed intraocular pressure–lowering effectiveness in clinical studies with PGAs.

Elevated intraocular pressure (IOP) is a major risk factor in the pathogenesis of glaucoma. Aqueous humor drains through the conventional pathway (trabecular meshwork, Schlemm's canal, collecting channels, and episcleral venous system) and alternative pathway (ciliary body face, suprachoroidal space with diffusion through sclera, and larger molecules via vortex venous system). 
Within the ciliary body stroma portion of the uveoscleral tract, outflow resistance can be modulated by both ciliary smooth muscle cell tone 14 and enhanced turnover of extracellular matrix (ECM) by matrix metalloproteinases (MMPs). 512 We previously determined that the mRNAs of MMP-1, -2, -3, -11, -12, -14, -15, -16, -17, -19, and -24 as well as tissue inhibitors of metalloproteinase (TIMP)-1 through -4 are present in ciliary body and ciliary body smooth muscle (CBSM) cells. 12 In other human tissues, the ratio of MMPs to TIMPs correlates to the rate of ECM turnover. 1321  
PGAs, such as bimatoprost, latanoprost, and unoprostone, treat glaucoma by lowering intraocular pressure. They affect aqueous drainage, but vary in the degree to which uveoscleral or conventional pathways are affected. 2225 All three are prostaglandin F (FP) receptor agonists. 2630 Numerous studies have found a greater IOP-lowering ability of latanoprost than that of unoprostone. 3137 Some studies have found bimatoprost to have a greater IOP-lowering effect than latanoprost, 38,39 but others have found them to be equivalent. 40  
We hypothesized that the different PGAs would have differing effects on the MMP/TIMP balance in CBSM cells that relate to their relative effectiveness in lowering IOP. 
Methods
Tissue and Explant Culture
CBSM cells were cultured according to previously published protocols. 12,41 Briefly, all ciliary body (CB) tissue was dissected from human donor corneoscleral buttons from the Massachusetts Eye and Ear Infirmary within 6 hours of corneal transplant surgery; these corneoscleral buttons contain significant CBSM cells. 42 Information provided by the eye bank indicated the donors' age and nonglaucomatous status. CBSM cell cultures were generated from CB isolated from 13 separate individuals, ages 23 to 65 years. The cells' identity was confirmed by labeling with anti-desimin and anti-smooth muscle actin antibodies, as originally described by Weinreb et al., 41 and confirmed that >99% of cells in culture were CBSM cells. Furthermore, the cells had the usual morphologic appearance with individual cells having a spindle shape. Once confluent, CBSM cells grow in bundles mimicking a muscular pattern. The cultures were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen-Gibco, Grand Island, NY) containing 20% fetal bovine serum, 1% l-glutamine (2 mM), and gentamicin (0.1 mg/mL) at 37°C in a 10% CO2 atmosphere. All the cells used were from passage-4 and -5 cultures. 
Bimatoprost, Latanoprost, and Unoprostone Incubation
The free acid forms of bimatoprost, latanoprost, and unoprostone (Cayman Chemical, Ann Arbor, MI) were prepared in ethanol and diluted to experimental concentrations with serum-free medium. CBSM cells (1 × 106) were plated into T-75 flasks and allowed to grow to confluence with medium changes every 3 to 5 days. Once confluent, the cultures were trypsinized into seven 60-mm dishes and allowed to grow to confluence. The cells were then incubated with serum-free medium for 24 hours and exposed to vehicle control (0.015% ethanol), 0.01 or 0.1 μg/mL free acid bimatoprost, 0.03 or 0.3 μg/mL free acid latanoprost, or 0.145 or 1.45 μg/mL unoprostone, for 24 hours. The concentrations of these prostaglandin analogues (PGAs) were chosen based on the peak aqueous concentrations of these drugs after topical administration in humans (0.009 μg/mL, 0.028 μg/mL, and 0.145 μg/mL for bimatoprost, latanoprost, and unoprostone, respectively). 4345 In our previous work with latanoprost, we have found that 0.3 μg/mL (or 10 times the peak aqueous concentration) can help confirm the trend seen at 0.03 μg/mL, but a higher concentration of 30 μg/mL did not provide further useful information. 12  
Cell Lysate and Conditioned Medium Preparation
Cultures of CBSM cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, 150 mM NaCl, 1% Igepal CA-630, 0.5% sodium deoxycholate, and 0.1% SDS) with protease inhibitors (10 μL/mL aprotinin, 0.1 mg/mL phenylmethylsulfonyl fluoride (PMSF), 1 mM EGTA, 1 μg/mL leupeptin, and 1 mM sodium orthovanadate to a final volume of 1 mL with RIPA buffer). A 21-gauge needle was used to shear the cells thoroughly and an additional 10 of 10 mg/mL PMSF was added before incubation on ice for 30 to 60 minutes. After incubation, the cell lysate was centrifuged at 14,000g for 10 minutes at 4°C. The supernatant was collected as whole-cell lysate. 
The conditioned culture media were collected every 24 hours after incubation with vehicle control and PGAs and concentrated 30-fold (Amicon Ultra-4, 10K; Millipore, Milford, MA). The protein concentrations were determined according to a protein assay protocol (BioMate; Thermo Spectronic, Rochester, NY). Ten microliters of each sample and RIPA with inhibitors as control were measured and compared with a previously established bovine serum albumin (BSA) curve at a 655-nm wavelength. The sample was aliquoted and stored frozen at −80°C until it was analyzed. 
Western Blot Analysis
Equal amounts of protein from whole-cell lysates or concentrated conditioned media were electrophoresed into 11% SDS-PAGE gels (XCell SureLock Mini-Cell; Invitrogen). The separated proteins were blotted onto a nitrocellulose membrane with 0.45 μm pore size (Invitrogen). The membrane was blocked in blocking buffer (Rockland, Gilbertsville, PA) and 1× Tris-buffered saline (TBS) followed by primary antibody (Table 1) incubation overnight at 4°C. The following day, they were washed with TBS/T, the membranes were incubated with IRDye 800-conjugated affinity purified anti-mouse or anti-rabbit IgG (Rockland, Gilbertsville, PA) diluted 1:10,000 for 1 hour. The membranes were washed with 1× TBS with 0.1% Tween 20 (TBS/T) and scanned (Odyssey Infrared Imaging System; Li-Cor; Lincoln, NE). The bands were quantified by using densitometric software (Odyssey; Li-Cor). 
Table 1.
 
Primary Antibodies and Their Dilutions Used for Immunoblot Analysis
Table 1.
 
Primary Antibodies and Their Dilutions Used for Immunoblot Analysis
Antibodies Manufacturer* Dilution Identified Band Size(s)
MMP–1 (M) R&D Systems 1:1,000 52 and 57 kDa
MMP–2 (M) R&D Systems 1:1,000 72 kDa
MMP–3 (P) Chemicon 1:1,000 57 and 59 kDa
MMP–9 (P) Chemicon 1:1,000 92 kDa
MMP–24 (P) Chemicon 1:1,000 64 kDa
TIMP–1 (P) Chemicon 1:20,000 28 kDa
TIMP–2 (P) Chemicon 1:2,000 21 kDa
TIMP–3 (M) Calbiochem 1:200 27 kDa
TIMP–4 (P) R&D Systems 1:7,000 29 kDa
GAPDH (P) R&D Systems 1:20,000–40,000 36 kDa
Zymographic Analysis
Zymography analyzes the ability of MMP-2 and -9 to degrade gelatin and MMP-1 and -3 to degrade casein as a measure of enzymatic activity. Gelatin (0.1%) or β-casein (0.1%) was mixed into liquid acrylamide when casting polyacrylamide gels. Concentrated conditioned medium, mixed with 2× Tris-glycine-SDS zymography sample buffer at 1:10 ratio, was loaded into 10% SDS-PAGE gels. The samples were electrophoresed at 130 V in tank buffer. The gels were washed at room temperature with 2.5% Triton X-100 (renaturing buffer), then transferred to development buffer overnight at 37°C. The resultant gels were stained with 0.1% Coomassie brilliant blue G-250 (Bio-Rad, Hercules, CA) solution for at least 3 hours, then destained with fixing/destaining solution until clear bands were visible and contrasted well with the blue background. The gels were scanned and analyzed for relative densities (Odyssey Infrared Imaging System; Li-Cor). All the MMPs were identified based on their molecular weights resolved by zymography and were confirmed by purified MMP-1, -2, -3, and -9 (Chemicon, Temecula, CA) as positive controls. 
Determination of Change
We determined the variability of our Western blot by repeating the immunoblot analysis nine separate times, which revealed an average difference of 10.05% ± 9.45%. Thus, any change greater than 10% was defined as an elevation or decrease for the individual sample. According to published criteria, the effect of the PGA was determined by the trend of the majority (>60%) of the samples. 7,12,46,47  
Results
Effect on MMP and TIMP Levels
MMPs -1, -2, -3, and -9 are secreted proteins and were identified in conditioned media; we were not able to detect them from cell scrapings. Conversely, MMP-24 (MT5-MMP) was membrane bound and found in cell scrapings, but was not detectable in conditioned media. Results are reported for the three PGAs in alphabetical order, bimatoprost (BIM), latanoprost (LAT), and unoprostone (UNO), and grouped by MMP subfamilies of collagenases, gelatinases, stromelysins, and membrane bound. Pharmacologic doses for BIM, LAT, and UNO were 0.01, 0.03, and 0.145 μg/mL, respectively, while suprapharmacologic doses were 10 times the peak pharmacologic concentrations. 
Pro-MMPs -1, -3, and -9 were all increased by BIM, LAT, and UNO. Pro-MMP-1 (collagenase-1) increased an average of 24% ± 6% in four of five donors, 20% ± 3% in three of five donors, and 23% ± 4% in three of five in donors in response to BIM, LAT, and UNO (Table 2, Fig. 1A), respectively. At supratherapeutic doses of BIM, collagenase-1 increased an average of 27% ± 15% in three of five donors and did not change in the other two donors. Collagenase-1 did not change in three of five donors in response to supratherapeutic doses of LAT and UNO. 
Table 2.
 
Summary of the Effect of PGAs on the Expression of MMPs and TIMPs from the Conditioned Media of Human CBSM Cells
Table 2.
 
Summary of the Effect of PGAs on the Expression of MMPs and TIMPs from the Conditioned Media of Human CBSM Cells
MMPs/TIMPs* Bimatoprost Latanoprost Unoprostone
MMPs
    Pro–MMP–1 ↑ 4/1/0 (24 ± 6) (20 ± 7) ↑ 3/2/0 (20 ± 4) (16 ± 4) ↑ 3/2/0 (23 ± 4) (13 ± 7)
    Pro–MMP–2 ↔ 0/4/1 (−2 ± 4) ↔ 0/4/1 (−3 ± 5) ↓ 0/2/3 (−21 ± 3) (−12 ± 6)
    Pro–MMP–3 ↑ 3/2/0 (63 ± 10) (35 ± 18) ↑ 3/2/0 (30 ± 5) (16 ± 9) ↑ 3/2/0 (65 ± 18) (35 ± 22)
    Pro–MMP–9 ↑ 3/1/1 (75 ± 27) (43 ± 28) ↑ 3/2/0 (76 ± 24) (45 ± 26) ↑ 3/0/2 (107 ± 53) (51 ± 50)
    MMP–24 (CL) ↔ 0/3/2 (−9 ± 5) ↔ 0/5/0 (−5 ± 3) ↔ 0/3/2 (−9 ± 3)
Active MMPs
    Inter MMP–1 ↔ 1/4/0 (7 ± 2) ↔ 1/4/0 (6 ± 2) ↔ 1/4/0 (5 ± 2)
    Active MMP–2 ↔ 0/5/0 (5 ± 1) ↔ 0/5/0 (3 ± 1) ↔ 0/5/0 (−1 ± 1)
    Active MMP–9 ↑ 3/2/0 (18 ± 3) (8 ± 5) ↑ 4/1/0 (19 ± 4) (13 ± 6) Ind 2/1/2 (0 ± 7)
TIMPs
    TIMP–1 Ind 1/2/2 (−8 ± 21) ↓ 1/1/3 (−35 ± 16) (1 ± 31) ↑ 3/0/2 (100 ± 20) (37 ± 41)
    TIMP–2 ↔ 0/3/2 (3 ± 11) ↔ 0/3/2 (−11 ± 8) ↓ 0/2/3 (−35 ± 8) (−24 ± 9)
    TIMP–3 ↑ 3/0/2 (57 ± 23) (21 ± 28) ↑ 4/1/0 (69 ± 15) (55 ± 19) ↑ 3/1/1 (57 ± 9) (24 ± 21)
    TIMP–4 ind 1/2/2 (2 ± 14) ↔ 1/3/1 (1 ± 12) ↑ 4/0/1 (61 ± 11) (42 ± 21)
    TIMP–4 (CL) ↓ 2/0/3 (−24 ± 4) (9 ± 21) ↑ 3/2/0 (19 ± 7) (11 ± 6) ind 2/1/2 (38 ± 36)
Figure 1.
 
Representative Western blots for MMP-1 (A), -2 (B), -3 (C), -9 (D), and -24 (E) from the conditioned media of human CBSM cells incubated with bimatoprost, latanoprost, and unoprostone for 24 hours. C, control; 1× and 10×, samples incubated with the peak aqueous concentrations and 10 times the peak aqueous concentration of these drugs, respectively, for 24 hours.
Figure 1.
 
Representative Western blots for MMP-1 (A), -2 (B), -3 (C), -9 (D), and -24 (E) from the conditioned media of human CBSM cells incubated with bimatoprost, latanoprost, and unoprostone for 24 hours. C, control; 1× and 10×, samples incubated with the peak aqueous concentrations and 10 times the peak aqueous concentration of these drugs, respectively, for 24 hours.
BIM and LAT did not change pro-MMP-2 (gelatinase A) in four of five donors (Table 2, Fig. 1B). UNO decreased pro-MMP-2 an average of 21% ± 3% at pharmacologic doses in three of five donors. Suprapharmacologic concentrations did not alter pro-MMP-2 in response to any of the PGAs in all five donors. 
Pro-MMP-3 (stromelysin-1) increased 63% ± 10%, 30% ± 5%, and 65% ± 18% in three of five donors in response to BIM, LAT, and UNO (Table 2, Fig. 1C), respectively. At suprapharmacologic doses of BIM and UNO, stromelysin-1 did not alter the protein level in three of five donors. Stromelysin-1 increased an average of 31% ± 6% in three of five donors in response to suprapharmacologic doses of LAT and did not alter in the other two donors. 
Pro-MMP-9 (gelatinase B) increased 75% ± 27%, 76% ± 24%, and 107% ± 53% in three of five donors in response to BIM, LAT, and UNO, respectively (Table 2, Fig. 1D). At suprapharmacologic doses, BIM increased gelatinase B by 51% ± 15% in three of five donors but did not alter the protein level in two donors; LAT increased pro-MMP-9 by 47% ± 25% in four of five donors, whereas UNO decreased pro-MMP-9 by 33% ± 5% in three of five donors. 
Pharmacologic doses of BIM, LAT, and UNO, did not alter MMP-24 (MT5-MMP) in three of five, five of five, and three of five donors, respectively (Table 2, Fig. 1E). At suprapharmacologic doses, LAT decreased MMP-24 by 17% ± 1% in three of five donors; BIM and UNO did not change MMP-24 levels in four of five and three of five donors, respectively. 
All TIMPs were found in both conditioned media and cell lysates. However in cell lysates, the basal levels of TIMP-1, -2, and -3 are consistently close to the lowest limit of detection; PGAs did not significantly change the levels of the aforementioned TIMPs in the cell lysates. In cell lysates, TIMP-4 decreased an average of 24% ± 4% in three of five donors in response to BIM. LAT increased TIMP-4 an average of 19% ± 7% in three of five donors. UNO increased TIMP-4 by 125% ± 4% in two donors, decreased it 29% ± 5% in another two donors, and had no effect on it in the fifth donor; given no clear majority, the effect of UNO on TIMP-4 in cell lysates was deemed indeterminate. At suprapharmacologic doses, BIM increased TIMP-4 an average of 69% ± 48% in three of five donors but decreased it 18% ± 2% in response to LAT in three of five donors. Suprapharmacologic doses of UNO increased TIMP-4 by 70% ± 37% of in three donors but had no effect on it in the other two. 
In conditioned media with pharmacologic concentrations, BIM decreased TIMP-1 an average of 55% ± 6% in two of five donors and had no effect on it in the other three. Given no clear majority, the effect of BIM was recorded as indeterminate. LAT decreased TIMP-1 (Table 2, Fig. 2A) an average of 35% ± 16% in three of five donors. UNO increased TIMP-1 an average of 100% ± 20% in three of five donors. TIMP-2 (Table 2, Fig. 2B) was not altered by BIM or LAT in three of five donors, but decreased an average of 35% ± 8% in three of five donors in response to UNO. TIMP-3 (Table 2, Fig. 2C) increased 57% ± 23% in three of five donors, 70% ± 15% in four of five donors, and 57% ± 9% in three of five donors in response to BIM, LAT, and UNO, respectively. TIMP-4 was not changed in two of five donors, decreased an average of 23% ± 10% in two donors, and increased in the remaining donor in response to BIM, which was an indeterminate result, given the lack of a majority response. LAT did not alter it in three of five donors (Table 2, Fig. 2D), but it increased 61% ± 11% in four of five donors in response to UNO. 
Figure 2.
 
Representative Western blots of TIMP-1 (A), -2 (B), -3 (C), and -4 (D) from conditioned media in human CBSM cells incubated with bimatoprost, latanoprost, and unoprostone for 24 hours. C, control; 1× and 10× represent samples incubated with the peak aqueous concentrations and 10 times the peak aqueous concentration of these drugs, respectively, for 24 hours.
Figure 2.
 
Representative Western blots of TIMP-1 (A), -2 (B), -3 (C), and -4 (D) from conditioned media in human CBSM cells incubated with bimatoprost, latanoprost, and unoprostone for 24 hours. C, control; 1× and 10× represent samples incubated with the peak aqueous concentrations and 10 times the peak aqueous concentration of these drugs, respectively, for 24 hours.
At suprapharmacologic doses in conditioned media, BIM increased TIMP-1 in three of five donors by 70% ± 31%. LAT did not change TIMP-1 in three of five donors. UNO increased TIMP-1 an average of 82% ± 40% in two donors, decreased it by 61% ± 2% in two donors, and had no effect in the remaining one. With no clear majority, the effect was defined as indeterminate. BIM did not change TIMP-2 in three of five donors and decreased it an average of 28% ± 4% in the other two. LAT increased TIMP-2 an average of 39% ± 9%, whereas UNO decreased it an average of 30% ± 2% in three of five donors. BIM and UNO increased TIMP-3 by 35% ± 9% and 325% ± 158% in three of five donors, respectively. LAT did not change TIMP-3 in two of five donors, decreased it an average of 37% ± 5% in two others, and had no effect on the level in the fifth one. Thus, the effect of LAT on TIMP-3 was indeterminate. TIMP-4 decreased an average of 40% ± 7% in all five donors in response to BIM. LAT decreased an average of 30% ± 4% in three of five donors; UNO increased TIMP-4 an average of 28% ± 8% in three of five donors. 
In summary, all three PGAs had similar effects on MMPs, although UNO decreased MMP-2 compared to BIM and LAT. However, UNO upregulated more of the TIMPs than either LAT or BIM. 
Effect on MMP Activity
Enzymatic activity of intermediate MMP-1 and -2 remained within 10% of control at pharmacologic and suprapharmacologic doses of all studied PGAs in four of five donors (Table 2, Figs. 3A, 3B). At pharmacologic doses, BIM increased MMP-9 activity by 18% ± 3% in three of five donors; LAT increased it an average of 19% ± 4% in four of five donors (Table 2, Fig. 3C). UNO did not change MMP-9 activity in two of five donors, decreased it by 16% ± 1% in two others, and had no effect in the fifth donor; thus, the result was indeterminate. At suprapharmacologic doses, MMP-9 activity increased by 18% ± 3% and 18% ± 4% in four of five donors in response to BIM and LAT, respectively, but did not change in four of five donors in response to UNO. 
Figure 3.
 
Representative zymograms for pro- and active forms of MMP-1 (A), -2 (B), and -9 (C) in human CBSM cells incubated with bimatoprost, latanoprost, and unoprostone for 24 hours. Active form of MMP-3 was not detected. C, control; 1× and 10× represent samples incubated at the peak aqueous concentrations and 10 times the peak aqueous concentration of these drugs, respectively, for 24 hours.
Figure 3.
 
Representative zymograms for pro- and active forms of MMP-1 (A), -2 (B), and -9 (C) in human CBSM cells incubated with bimatoprost, latanoprost, and unoprostone for 24 hours. Active form of MMP-3 was not detected. C, control; 1× and 10× represent samples incubated at the peak aqueous concentrations and 10 times the peak aqueous concentration of these drugs, respectively, for 24 hours.
Discussion
Our results indicate that the MMP and TIMP balance relates to the degree of IOP lowering exhibited by these three PGAs. All three agents lower IOP; the literature indicates that bimatoprost and latanoprost have at least an equivalent relationship with regard to lowering IOP. 3840 There are some studies that support a greater lowering of IOP by bimatoprost. 38,39 The literature strongly supports a greater effectiveness of latanoprost compared with unoprostone. 3137 Our findings implicate that the difference is due to a differential effect on TIMPs. Given the similar response of MMPs among the three PGAs, the different response of TIMPs would cause a different balance of MMPs and TIMPs. In general, MMPs are associated with increased ECM turnover. A higher proportion of TIMPs, the kinetic inhibitors of MMPs, may result in less ECM turnover and thus worse outflow facility through the uveoscleral pathway. 
With latanoprost, we found a high correlation between MMP and TIMP protein expression and our previously published findings on mRNA levels in CBSM cells. 12 This finding is consistent with those in prior studies indicating that many MMPs are regulated at the level of transcription. 48 The exceptions were MMP-1, where the mRNA level was decreased whereas pro-MMP-1 protein expression was increased, and TIMP-1, where the mRNA was equivalent to baseline, but the protein level was increased. The reasons for the differences are unknown but may be the result of compensatory feedback inhibition. 
The increase in pro-MMP-1, -3, -9 and the enzymatic activity of MMP-9 suggests a prominent role of these MMPs in PGA-mediated ECM turnover. Other groups have found increases in MMP-3 and -9 in response to latanoprost at the transcription, protein, and/or enzymatic levels. 7,9,10,47 Notably, MMP-9 has been found diffusely distributed in ciliary body 9 and mediates basement membrane degradation. 48 In contrast, we found that MMP-2 is unchanged by latanoprost or other PGA in transcription, 12 protein level, or enzymatic activity. However, the transcription and enzymatic activity of MMP-2 have been reported to be increased by latanoprost by other investigators. 7,47 A limitation of our study of MMP activity is the absence of a detailed time course. The enzymatic activity may have been altered earlier than 24 hours. However, we found a very consistent response between the mRNA, protein, and kinetic activities of MMP-2 and -9. Gelatin zymography proved useful in the study of gelatinases, but casein had limited utility for collagenase-1 (MMP-1) and stromelysin-1 (MMP-3). 
In patients and human tissue, variability of response between individuals is commonly noted with PGAs, both in clinical IOP response and the in vitro effect on MMPs, respectively. With regard to lowering IOP, the response rate (arbitrarily defined as >15% reduction from baseline) to latanoprost is approximately 85%. 49 However, among those who respond, the level of response varies. For example, Camras and Hedman 49 found the approximately 75% of patients had >20% IOP reduction but approximately 30% had >30% lowering of IOP. Bimatoprost has demonstrated similar variability of IOP reduction between individuals. 50 Similar to the clinical situation, our laboratory and others have noted variability in the response between cell lines from different donors in response to prostaglandins. 7,12,46,47  
It has been reported that the size of pro-MMP-1 released by various human cell types ranges from 52 to 62 kDa, depending on glycosylation. 7,5153 The size of active MMP-1 has been reported to be between 41 to 47 kDa. 5457 We found two bands at approximately 52 and 48 kDa. Therefore, we postulate that the 48-kDa form is the intermediate form of MMP-1, similar to findings in human umbilical vein endothelial cells. 58 We could not detect the expression of active MMP-3 in control and treated samples by zymography. Casein zymography has a detection limit at least two orders of magnitude lower than that of gelatin zymography. 59,60 In addition, MMP-3 (stromelysin-1) does not have high affinity to casein, further limiting its detection by casein zymography. 
With regard to TIMPs, we have reported that the amount of mRNA of TIMP-1 is unchanged in response to latanoprost, but in this present study, we found that the protein level decreased. 12 Our findings are in contrast to those of Anthony et al. 11 who found TIMP-1 to be increased both at the mRNA and protein levels. Our results agree with theirs on the effect on TIMP-2. TIMP-3 was increased by all three PGAs which is consistent with the increase at the mRNA level caused by latanoprost. 12 TIMP-3 has some unique qualities; it is the only TIMP that is sequestered in ECM to heparin-sulfate and chondroitin-sulfate containing proteoglycans. 6163 TIMP-3 strongly inhibits not only MMPs but several ADAMs (a disintegrin and metalloproteinases), ADAMTS (ADAM with thrombospondin motifs), and tumor necrosis factor (TNF)-α–converting enzyme (TACE). 6166 Upregulation of TIMP-4 by unoprostone other than bimatoprost and latanoprost is likely to mediate downregulation of MMP-2 because TIMP-4 has a relatively higher potency of MMP-2 inhibition than other MMPs. 67  
The exact mechanism of the relative differences in MMP/TIMP balance is unknown. It is possible that receptor binding affinity plays a role. Although there is disagreement on the relative binding affinity strength between bimatoprost and latanoprost, all studies show lower binding affinity of unoprostone to the FP receptor. 27,29,30,68,69 In most tissues, the MMP/TIMP balance determines the rate of ECM turnover. 15,19,21 Therefore, the decrease in MMP-2 and increases in TIMP-1 and -4 by unoprostone compared to latanoprost and bimatoprost may explain the lower clinical efficacy of unoprostone in lowering IOP compared with latanoprost and bimatoprost. 3437,40,7076 Our findings may have some benefit in future drug development, as forthcoming PGAs may be screened inexpensively by testing their effects on MMPs and TIMPs. 
Footnotes
 Supported by Allergan, American Glaucoma Society Clinician-Scientist Fellowship, Massachusetts Lions Eye Research Foundation, Pfizer Ophthalmics, and EY 14104 (MEEI Vision-Core Grant).
Footnotes
 Disclosure: Y.H. Ooi, None; D.-J. Oh, None; D.J. Rhee, Allergan (F, R), Pfizer Ophthalmic (F, R)
Footnotes
 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.
The authors thank Kathryn A. Colby, MD, PhD, and Roberto Pineda, MD (both at Massachusetts Eye and Ear Infirmary), for the generous gifts of donor corneoscleral rims. 
References
Barany EH Rohen JW . Localized contraction and relaxation within the ciliary muscle of the vervet monkey (Cercopithecus ethiops), In: Rohen JW ed. The Structure of the Eye, Second Symposium. Stuttgart, Germany: FK Schattauer Verlag; 1965:287–311.
Bill A . Effects of atropine and pilocarpine on aqueous humour dynamics in cynomolgus monkeys (Macaca irus). Exp Eye Res. 1967;6:120–125. [CrossRef] [PubMed]
Townsend DJ Brubaker RF . Immediate effect of epinephrine on aqueous formation in the normal human eye as measured by fluorophotometry. Invest Ophthalmol Vis Sci. 1980;19:256–266. [PubMed]
Schenker JI Yablonski ME Podos SM Linder L . Fluorophotometric study of epinephrine and timolol in human subjects. Arch Ophthalmol. 1981;99:1212–1216. [CrossRef] [PubMed]
Lütjen-Drecoll E Tamm E . Morphological study of the anterior segment of cynomolgus monkey eyes following treatment with prostaglandin F . Exp Eye Res. 1988;47:761–769. [CrossRef] [PubMed]
Lütjen-Drecoll E Tamm E . The effects of ocular hypotensive doses of PGF F-1-isopropylester on anterior segment morphology. In: Bito LZ Stjernschantz J eds. The ocular effects of prostaglandins and other eicosanoids. New York: Allan R. Liss, Inc. 1989:737–776.
Weinreb RN Kashiwagi K Kashiwagi F Tuskahara S Lindsey JD . Prostaglandins increase matrix metalloproteinase release from human ciliary smooth muscle cells. Invest Ophthalmol Vis Sci. 1997;38:2772–2780. [PubMed]
Ocklind A . Effect of latanoprost on the extracellular matrix of the ciliary muscle: a study on cultured cells and tissue sections. Exp Eye Res. 1998;67:179–191. [CrossRef] [PubMed]
El-Shabrawi Y Eckhardt M Berghold A . Synthesis pattern of matrix metalloproteinases (MMPs) and inhibitors (TIMPs) in human explant organ cultures after treatment with latanoprost and dexamethasone. Eye. 2000;14:375–383. [CrossRef] [PubMed]
Lindsey JD Kashiwagi K Boyle D Kashiwagi F Firestein GS Weinreb RN . Prostaglandins increase proMMP-1 and proMMP-3 secretion by human ciliary smooth muscle cells. Curr Eye Res. 1996;15:869–875. [CrossRef] [PubMed]
Anthony TL Lindsey JD Weinreb RN . Latanoprost's effects on TIMP-1 and TIMP-2 expression in human ciliary muscle cells. Invest Ophthalmol Vis Sci. 2002;43:3705–3711. [PubMed]
Oh DJ Martin JL Williams AJ . Analysis of expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human ciliary body following latanoprost. Invest Ophthalmol Vis Sci. 2006;47:953–963. [CrossRef] [PubMed]
Butler GS Will H Atkinson SJ Murphy G . Membrane-type-2 matrix metalloproteinase can initiate the processing of progelatinase A and is regulated by the tissue inhibitors of metalloproteinases. Eur J Biochem. 1997;244:653–657. [CrossRef] [PubMed]
Butler GS Butler MJ Atkinson SJ . The TIMP2 membrane type 1 metalloproteinase “receptor” regulates the concentration and efficient activation of progelatinase A; a kinetic study. J Biol Chem. 1998;273:871–880. [CrossRef] [PubMed]
Khasigov PZ Podobed OV Gracheva TS Salbiev KD Grachev SV Berezov TT . Role of matrix metalloproteinases and their inhibitors in tumor invasion and metastasis. Biochemistry (Mosc). 2003;68:711–717. [CrossRef] [PubMed]
Curran S Dundas SR Buxton J Leeman MF Ramsay R Murray GI . Matrix metalloproteinase/tissue inhibitors of matrix metalloproteinase phenotype identifies poor prognosis colorectal cancers. Clin Cancer Res. 2004;10:8229–8234. [CrossRef] [PubMed]
Zhou X Hovell CJ Pawley S . Expression of matrix metalloproteinase-2 and -14 persists during early resolution of experimental liver fibrosis and might contribute to fibrinolysis. Liver Int. 2004;24:492–501. [CrossRef] [PubMed]
Maata M Tervahartiala T Harju M Airaksinen J Autio-Harmainen H Sorsa T . Matrix metalloproteinases and their tissue inhibitors in aqueous humor of patients with primary open-angle glaucoma, exfoliation syndrome, and exfoliation glaucoma. J Glaucoma. 2005;14:64–69. [CrossRef] [PubMed]
Steinmetz EF Buckley C Shames ML . Treatment with simvastatin suppresses the development of experimental abdominal aortic aneurysms in normal and hypercholesterolemic mice. Ann Surg. 2005;241:92–101. [PubMed]
Ahmed Z Dent RG Leadbeater WE Smith C Berry M Logan A . Matrix metalloproteinases: degredation of the inhibitory environment of the transected optic nerve and the scar by regenerating axons. Mol Cell Neurosci. 2005;28:64–78. [CrossRef] [PubMed]
Pauschinger M Chandrasekharan K Schultheiss JP . Myocardial remodeling in viral heart disease: possible interactions between inflammatory mediators and MMP-TIMP system. Heart Fail Rev. 2004;9:21–31. [CrossRef] [PubMed]
Nilsson SFE Samuelsson M Bill A Stjernschantz J . Increased uveoscleral outflow as a possible mechanism of ocular hypotension caused by prostaglandin F-1-isopropylester in the Cynomolgus monkey. Exp Eye Res. 1989;48:707–716. [CrossRef] [PubMed]
Brubaker RF Schoff EO Nau CB Carpenter SP Chen K Vandenburgh AM . Effects of AGN 192024, a new ocular hypotensive agent, on aqueous dynamics. Am J Ophthalmol. 2001;131:19–24. [CrossRef] [PubMed]
Sakurai M Araie M Oshika T . Effects of topical application of UF-021, a novel prostaglandin derivative, on aqueous humor dynamics in normal human eyes. Jpn J Ophthalmol. 1991;35:156–165. [PubMed]
Lim KS Nau CB O'Byrne MM . Mechanism of action of bimatoprost, latanoprost, and travoprost in healthy subjects. Ophthalmology. 2008;115:790–795. [CrossRef] [PubMed]
Ota T Aihara M Narumiya S Araie M . The effects of prostaglandin analogues on IOP in prostanoid FP-receptor-deficient mice. Invest Ophthalmol Vis Sci. 2005;46:4159–4163. [CrossRef] [PubMed]
Sharif NA Kelly CR Crider JY Williams GW Xu SX . Ocular hypotensive FP prostaglandin (PG) analogs: PG receptor subtype binding affinities and selectivities, and agonist potencies at FP and other PG receptors in cultured cells. J Ocul Pharmacol Ther. 2003;19:501–515. [CrossRef] [PubMed]
Sharif NA Crider JY Husain S Kaddour-Djebbar I Ansari HR Abdel-Latif AA . Human ciliary muscle cell responses to FP-class prostaglandin analogs: phosphoinositide hydrolysis, intracellular Ca2+ mobilization and MAP kinase activation. J Ocul Pharmacol Ther. 2003;19:437–455. [CrossRef] [PubMed]
Sharif NA Kelly CR Crider JY . Human trabecular meshwork cell responses induced by bimatoprost, travoprost, unoprostone, and other FP prostaglandin receptor agonist analogues. Invest Ophthalmol Vis Sci. 2003;44:715–721. [CrossRef] [PubMed]
Sharif NA Kelly CR Crider JY . Agonist activity of bimatoprost, travoprost, latanoprost, unoprostone isopropyl ester and other prostaglandin analogs at the cloned human ciliary body FP prostaglandin receptor. J Ocul Pharmacol Ther. 2002;18:313–324. [CrossRef] [PubMed]
Susanna RJr Medeiros FA Vessani RM Giampani JJr Borges AS Jordao ML . Intraocular pressure fluctuations in response to the water-drinking provocative test in patients using latanoprost versus unoprostone. J Ocul Pharmacol Ther. 2004;20:401–410. [CrossRef] [PubMed]
Takahashi I Tanaka M . Switching to latanoprost monotherapy for 24 weeks in glaucoma patients. Eur J Ophthalmol. 2004;14:401–406. [PubMed]
Tsukamoto H Mishima HK Kitazawa Y Araie M Abe H Negi A . Glaucoma Study Group. A comparative clinical study of latanoprost and isopropyl unoprostone in Japanese patients with primary open-angle glaucoma and ocular hypertension. J Glaucoma. 2002;11:497–501. [CrossRef] [PubMed]
Jampel HD Bacharach J Sheu WP Wohl LG Solish AM Christie W . Latanoprost/Unoprostone Study Group. Randomized clinical trial of latanoprost and unoprostone in patients with elevated intraocular pressure. Am J Ophthalmol. 2002;134:863–871. [CrossRef] [PubMed]
Sponsel WE Paris G Trigo Y Pena M . Comparative effects of latanoprost (Xalatan) and unoprostone (Rescula) in patients with open-angle glaucoma and suspected glaucoma. Am J Ophthalmol. 2002;134:552–559. [CrossRef] [PubMed]
Aung T Chew PT Oen FT . Additive effect of unoprostone and latanoprost in patients with elevated intraocular pressure (published correction in Br J Ophthalmol. 2002;86:707). Br J Ophthalmol. 2002;86:75–79. [CrossRef] [PubMed]
Susanna RJr Giampani JJr Borges AS Vessani RM Jordao ML . A double-masked, randomized clinical trial comparing latanoprost with unoprostone in patients with open-angle glaucoma or ocular hypertension. Ophthalmology. 2001;108:259–263. [CrossRef] [PubMed]
Simmons ST Dirks MS Noecker RJ . Bimatoprost versus latanoprost in lowering intraocular pressure in glaucoma and ocular hypertension: results from parallel-group comparison trials. Adv Ther. 2004;21:247–262. [CrossRef] [PubMed]
Noecker RS Dirks MS Choplin NT Bernstein P Batoosingh AL Whitcup SM . Bimatoprost/Latanoprost Study Group. A six-month randomized clinical trial comparing the intraocular pressure-lowering efficacy of bimatoprost and latanoprost in patients with ocular hypertension or glaucoma. Am J Ophthalmol. 2003;135:55–63. [CrossRef] [PubMed]
Parrish RK Palmberg P Sheu WP . XLT Study Group. A comparison of latanoprost, bimatoprost, and travoprost in patients with elevated intraocular pressure: a 12-week, randomized, masked-evaluator multicenter study. Am J Ophthalmol. 2003;135:688–703. [CrossRef] [PubMed]
Weinreb RN Kim D-M Lindsey JD . Propagation of ciliary smooth muscle cells in vitro and effects of prostaglandin F on calcium efflux. Invest Ophthalmol Vis Sci. 1992;33:2679–2686. [PubMed]
Rhee DJ Tamm ER Russell P . Donor corneoscleral buttons: a new source of trabecular meshwork for research. Exp Eye Res. 2003;77:749–756. [CrossRef] [PubMed]
Camras CB Toris CB Sjoquist B . Detection of the free acid of bimatoprost in aqueous humor samples from human eyes treated with bimatoprost before cataract surgery. Ophthalmology. 2004;111:2193–2198. [CrossRef] [PubMed]
Sjoquist B Stjernschantz J . Ocular and systemic pharmacokinetics of latanoprost in humans. Surv Ophthalmol. 2002;47(suppl 1):S6–S12. [CrossRef] [PubMed]
Numaga J Koseki N Kaburaki T Kawashima H Tomita G Araie M . Intraocular metabolites of isopropyl unoprostone. Curr Eye Res. 2005;30:909–913. [CrossRef] [PubMed]
Oh DJ Martin JL Williams AJ Russell P Birk DE Rhee DJ . Effect of latanoprost on the expression of matrix metalloproteinases and their tissue inhibitors in human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2006;47:3887–3895. [CrossRef] [PubMed]
Weinreb RN Lindsey JD . Metalloproteinase gene transcription in human ciliary muscle cells with latanoprost. Invest Ophthalmol Vis Sci. 2002;43:716–717. [PubMed]
Nagase H Woessner JF . Matrix metalloproteinases. J Biol Chem. 1999;274:21491–21494. [CrossRef] [PubMed]
Camras CB Hedman K . US Latanoprost Study Group. Rate of response to latanoprost or timolol in patients with ocular hypertension or glaucoma. J Glaucoma. 2003;12:466–469. [CrossRef] [PubMed]
Cantor LB Hoop J Morgan L WuDunn D Catoira Y . the bimatoprost-travoprost study group: intraocular pressure-lowering efficacy of bimatoprost 0.03% and travoprost 0.004% in patients with glaucoma or ocular hypertension. Br J Ophthalmol. 2006;90:1370–1373. [CrossRef] [PubMed]
Nagase H Suzuki K Morodomi T . Activation mechanisms of the precursors of matrix metalloproteinases 1, 2 and 3. Matrix Suppl. 1992;1:237–244. [PubMed]
Wang N Lindsey JD Angert M . Latanoprost and matrix metalloproteinase-1 in human choroid organ cultures. Curr Eye Res. 2001;22(3):198–207. [CrossRef] [PubMed]
Hinz B Rösch S Ramer R . Latanoprost induces matrix metalloproteinase-1 expression in human nonpigmented ciliary epithelial cells through a cyclooxygenase-2-dependent mechanism. FASEB J. 2005;19(13):1929–1931. [PubMed]
Mathisen B Loennechen T Gedde-Dahl T . Fibroblast heterogeneity in collagenolytic response to colchicines. Biochem Pharmacol. 2006;71(5):574–583. [CrossRef] [PubMed]
Yu WH Woessner JFJr . Heparin-enhanced zymographic detection of matrilysin and collagenases. Anal Biochem. 2001;293(1):38–42. [CrossRef] [PubMed]
Ciccocioppo R Di Sabatino A Bauer M . Matrix metalloproteinase pattern in celiac duodenal mucosa. Lab Invest. 2005;85(3):397–407. [CrossRef] [PubMed]
Zong W Zyczynski HM Meyn LA . Regulation of MMP-1 by sex steroid hormones in fibroblasts derived from the female pelvic floor. Am J Obstet Gynecol. 2007;196(4):349.e1–e11. [CrossRef]
Qian LW Xie J Ye F . Kaposi's sarcoma-associated herpesvirus infection promotes invasion of primary human umbilical vein endothelial cells by inducing matrix metalloproteinases. J Virol. 2007;81(13):7001–7010. [CrossRef] [PubMed]
Fernández-Resa P Mira E Quesada AR . Enhanced detection of casein zymography of matrix metalloproteinases. Anal Biochem. 1995;224(1):434–435. [CrossRef] [PubMed]
Manicourt DH Lefebvre V . An assay for matrix metalloproteinases and other proteases acting on proteoglycans, casein, or gelatin. Anal Biochem. 1993;215(2):171–179. [CrossRef] [PubMed]
Yu WH Yu S Meng Q Brew K Woessner JFJr . TIMP-3 binds to sulphated glycosaminoglycans of the extracellular matrix. J Biol Chem. 2000;275:31226–31232. [CrossRef] [PubMed]
Pavloff N Staskus PW Kishnani NS Hawkes SP . A new inhibitor of metalloproteinases from chicken: ChIMP-3: a third member of the TIMP family. J Biol Chem. 1992;267:17321–17326. [PubMed]
Yang TT Hawkes SP . Role of the 21-kDa protein TIMP-3 in oncogenic transformation of culture chicken embryo fibroblasts. Proc Natl Acad Sci USA. 1992;89:10676–10680. [CrossRef] [PubMed]
Kashiwagi M Tortorella M Nagase H Brew K . TIMP-3 is a potent inhibitor of aggrecanase 1 (ADAM-TS4) and aggrecanase 2 (ADAM-TS5). J Biol Chem. 2001;276:12501–12504. [CrossRef] [PubMed]
Nagase H Brew K . Designing TIMP (tissue inhibitor of metalloproteinases) variants that are selective metalloproteinase inhibitors. Biochem Soc Symp. 2003;70:201–212. [PubMed]
Amour A Slocombe PM Webster A . TNF-alpha converting enzyme (TACE) is inhibited by TIMP-3. FEBS Lett. 1998;435:39–44. [CrossRef] [PubMed]
Liu YE Wang M Greene J . Preparation and characterization of recombinant tissue inhibitor of metalloproteinase 4 (TIMP-4). J Biol Chem. 1997;272:20479–20483. [CrossRef] [PubMed]
Sharif NA Kaddour-Djebbar I Abdel-Latif AA . Cat iris sphincter smooth-muscle contraction: comparison of FP-class prostaglandin analog agonist activities. J Ocul Pharmacol Ther. 2008;24:152–163. [CrossRef] [PubMed]
Sharif NA . Synthetic F. P-prostaglandin-induced contraction of rat uterus smooth muscle in vitro. Prostaglandins Leukot Essent Fatty Acids. 2008;78:199–207. [CrossRef] [PubMed]
Enoki M Saito J Hara M Uchida T Sagara T Nishida T . Additional reduction in intraocular pressure achieved with latanoprost in normal-tension glaucoma patients previously treated with unoprostone. Jpn J Ophthalmol. 2006;50(4):334–337. [CrossRef] [PubMed]
Aung T Chew PT Yip CC . A randomized double-masked crossover study comparing latanoprost 0.005% with unoprostone 0.12% in patients with primary open-angle glaucoma and ocular hypertension. Am J Ophthalmol. 2001;131(5):636–642. [CrossRef] [PubMed]
Tsukamoto H Mishima HK Kitazawa Y Araie M Abe H Negi A . Glaucoma Study Group. A comparative clinical study of latanoprost and isopropyl unoprostone in Japanese patients with primary open-angle glaucoma and ocular hypertension. J Glaucoma. 2002;11(6):497–501. [CrossRef] [PubMed]
Kobayashi H Kobayashi K Okinami S . A comparison of intraocular pressure-lowering effect of prostaglandin F2 -alpha analogues, latanoprost, and unoprostone isopropyl. J Glaucoma. 2001;10(6):487–492. [CrossRef] [PubMed]
Noecker RS Dirks MS Choplin NT Bernstein P Batoosingh AL Whitcup SM . Bimatoprost/Latanoprost Study Group. A six-month randomized clinical trial comparing the intraocular pressure-lowering efficacy of bimatoprost and latanoprost in patients with ocular hypertension or glaucoma. Am J Ophthalmol. 2003;135(1):55–63. [CrossRef] [PubMed]
Dirks MS Noecker RJ Earl M Roh S Silverstein SM Williams RD . A 3-month clinical trial comparing the IOP-lowering efficacy of bimatoprost and latanoprost in patients with normal-tension glaucoma. Adv Ther. 2006;23(3):385–394. [CrossRef] [PubMed]
Choplin N Bernstein P Batoosingh AL Whitcup SM . Bimatoprost/Latanoprost Study Group. A randomized, investigator-masked comparison of diurnal responder rates with bimatoprost and latanoprost in the lowering of intraocular pressure. Surv Ophthalmol. 2004;49(Suppl 1):S19–S25. [CrossRef] [PubMed]
Figure 1.
 
Representative Western blots for MMP-1 (A), -2 (B), -3 (C), -9 (D), and -24 (E) from the conditioned media of human CBSM cells incubated with bimatoprost, latanoprost, and unoprostone for 24 hours. C, control; 1× and 10×, samples incubated with the peak aqueous concentrations and 10 times the peak aqueous concentration of these drugs, respectively, for 24 hours.
Figure 1.
 
Representative Western blots for MMP-1 (A), -2 (B), -3 (C), -9 (D), and -24 (E) from the conditioned media of human CBSM cells incubated with bimatoprost, latanoprost, and unoprostone for 24 hours. C, control; 1× and 10×, samples incubated with the peak aqueous concentrations and 10 times the peak aqueous concentration of these drugs, respectively, for 24 hours.
Figure 2.
 
Representative Western blots of TIMP-1 (A), -2 (B), -3 (C), and -4 (D) from conditioned media in human CBSM cells incubated with bimatoprost, latanoprost, and unoprostone for 24 hours. C, control; 1× and 10× represent samples incubated with the peak aqueous concentrations and 10 times the peak aqueous concentration of these drugs, respectively, for 24 hours.
Figure 2.
 
Representative Western blots of TIMP-1 (A), -2 (B), -3 (C), and -4 (D) from conditioned media in human CBSM cells incubated with bimatoprost, latanoprost, and unoprostone for 24 hours. C, control; 1× and 10× represent samples incubated with the peak aqueous concentrations and 10 times the peak aqueous concentration of these drugs, respectively, for 24 hours.
Figure 3.
 
Representative zymograms for pro- and active forms of MMP-1 (A), -2 (B), and -9 (C) in human CBSM cells incubated with bimatoprost, latanoprost, and unoprostone for 24 hours. Active form of MMP-3 was not detected. C, control; 1× and 10× represent samples incubated at the peak aqueous concentrations and 10 times the peak aqueous concentration of these drugs, respectively, for 24 hours.
Figure 3.
 
Representative zymograms for pro- and active forms of MMP-1 (A), -2 (B), and -9 (C) in human CBSM cells incubated with bimatoprost, latanoprost, and unoprostone for 24 hours. Active form of MMP-3 was not detected. C, control; 1× and 10× represent samples incubated at the peak aqueous concentrations and 10 times the peak aqueous concentration of these drugs, respectively, for 24 hours.
Table 1.
 
Primary Antibodies and Their Dilutions Used for Immunoblot Analysis
Table 1.
 
Primary Antibodies and Their Dilutions Used for Immunoblot Analysis
Antibodies Manufacturer* Dilution Identified Band Size(s)
MMP–1 (M) R&D Systems 1:1,000 52 and 57 kDa
MMP–2 (M) R&D Systems 1:1,000 72 kDa
MMP–3 (P) Chemicon 1:1,000 57 and 59 kDa
MMP–9 (P) Chemicon 1:1,000 92 kDa
MMP–24 (P) Chemicon 1:1,000 64 kDa
TIMP–1 (P) Chemicon 1:20,000 28 kDa
TIMP–2 (P) Chemicon 1:2,000 21 kDa
TIMP–3 (M) Calbiochem 1:200 27 kDa
TIMP–4 (P) R&D Systems 1:7,000 29 kDa
GAPDH (P) R&D Systems 1:20,000–40,000 36 kDa
Table 2.
 
Summary of the Effect of PGAs on the Expression of MMPs and TIMPs from the Conditioned Media of Human CBSM Cells
Table 2.
 
Summary of the Effect of PGAs on the Expression of MMPs and TIMPs from the Conditioned Media of Human CBSM Cells
MMPs/TIMPs* Bimatoprost Latanoprost Unoprostone
MMPs
    Pro–MMP–1 ↑ 4/1/0 (24 ± 6) (20 ± 7) ↑ 3/2/0 (20 ± 4) (16 ± 4) ↑ 3/2/0 (23 ± 4) (13 ± 7)
    Pro–MMP–2 ↔ 0/4/1 (−2 ± 4) ↔ 0/4/1 (−3 ± 5) ↓ 0/2/3 (−21 ± 3) (−12 ± 6)
    Pro–MMP–3 ↑ 3/2/0 (63 ± 10) (35 ± 18) ↑ 3/2/0 (30 ± 5) (16 ± 9) ↑ 3/2/0 (65 ± 18) (35 ± 22)
    Pro–MMP–9 ↑ 3/1/1 (75 ± 27) (43 ± 28) ↑ 3/2/0 (76 ± 24) (45 ± 26) ↑ 3/0/2 (107 ± 53) (51 ± 50)
    MMP–24 (CL) ↔ 0/3/2 (−9 ± 5) ↔ 0/5/0 (−5 ± 3) ↔ 0/3/2 (−9 ± 3)
Active MMPs
    Inter MMP–1 ↔ 1/4/0 (7 ± 2) ↔ 1/4/0 (6 ± 2) ↔ 1/4/0 (5 ± 2)
    Active MMP–2 ↔ 0/5/0 (5 ± 1) ↔ 0/5/0 (3 ± 1) ↔ 0/5/0 (−1 ± 1)
    Active MMP–9 ↑ 3/2/0 (18 ± 3) (8 ± 5) ↑ 4/1/0 (19 ± 4) (13 ± 6) Ind 2/1/2 (0 ± 7)
TIMPs
    TIMP–1 Ind 1/2/2 (−8 ± 21) ↓ 1/1/3 (−35 ± 16) (1 ± 31) ↑ 3/0/2 (100 ± 20) (37 ± 41)
    TIMP–2 ↔ 0/3/2 (3 ± 11) ↔ 0/3/2 (−11 ± 8) ↓ 0/2/3 (−35 ± 8) (−24 ± 9)
    TIMP–3 ↑ 3/0/2 (57 ± 23) (21 ± 28) ↑ 4/1/0 (69 ± 15) (55 ± 19) ↑ 3/1/1 (57 ± 9) (24 ± 21)
    TIMP–4 ind 1/2/2 (2 ± 14) ↔ 1/3/1 (1 ± 12) ↑ 4/0/1 (61 ± 11) (42 ± 21)
    TIMP–4 (CL) ↓ 2/0/3 (−24 ± 4) (9 ± 21) ↑ 3/2/0 (19 ± 7) (11 ± 6) ind 2/1/2 (38 ± 36)
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×