Detection and Quantitation of mRNA of Matrix Metalloproteinase Enzymes in Cultured Human Trabecular Meshwork and Ciliary Body Treated with Latanoprost.

Abstract

Abstract: : Purpose: We set out to determine the expression pattern of all the known matrix metalloproteinases (MMPs) in human trabecular meshwork (TM) as well as ciliary body (CB) and to examine their transcriptional response to incubation with latanoprost. Methods: The presence of MMPs was examined, using RT–PCR, in human TM and CB tissues as well as cultures of TM endothelial and CB smooth muscle (CBSM) cells. The PCR products were sequenced and compared against the Genbank database. The transcriptional response to latanoprost was assessed using quantitative RT–PCR in control and latanoprost–treated cultures of TM endothelial and CBSM cells. The cultures were incubated with 30 ng/µl, 300 ng/µl, and 3 µg/µl of latanoprost for 24 hours. The comparative C_T method was used to quantify target genes relative to GAPDH as a reference. Results: There was no difference in the mRNA expression pattern of MMPs between TM and CB. MMP–11, –12, –14, –15, –16, –17, –19, –24 as well as MMP–1, –2 , –3 were transcribed in both tissues and cultures of TM and CBSM cells. MMP–25 was transcribed in both TM and CB tissues, but not in cultures of TM and CBSM. In response to latanoprost incubation at therapeutic level (30 ng/µl), cultures of TM showed that MMP–11 and –12 were up–regulated, while MMP–16, –17 and –19 were down–regulated, and MMP–14 and –15 were not changed. In CBSM cultures, MMP–3, –11, –15 and –17 were up–regulated, while MMP–14 and –19 were down–regulated. MMP–16 is unchanged. Variability among three individuals was noted. Conclusions: The results showed that MMP–1, –2, –3, –11, –12, –19 and all membrane–type MMPs (MT–MMPs) except MMP–25 (MT6–MMP) are transcribed with no difference in the pattern of expression between TM and CB, and also that incubation with latanoprost may induce transcriptional changes of MMPs in both of tissues. These results suggest that MMPs –15 and –17 may play a role in latanoprost–mediated increased uveoscleral outflow.

Keywords: outflow: trabecular meshwork • outflow: ciliary muscle • gene/expression