March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Statins and Dominant-negative RhoA Inhibit Dexamethasone-induced Calcification in Human Trabecular Meshwork Cells
Author Affiliations & Notes
  • Juan Carabana
    Ophthalmology, UNC at Chapel Hill, Chapel Hill, North Carolina
  • Wei Xue
    Ophthalmology, UNC at Chapel Hill, Chapel Hill, North Carolina
  • Teresa Borras
    Ophthalmology, UNC at Chapel Hill, Chapel Hill, North Carolina
  • Footnotes
    Commercial Relationships  Juan Carabana, None; Wei Xue, None; Teresa Borras, None
  • Footnotes
    Support  NIH Grants EY11906, EY13126, and a RPB unrestricted grant to the UNC Dept. of Ophthalmology
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 3247. doi:
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      Juan Carabana, Wei Xue, Teresa Borras; Statins and Dominant-negative RhoA Inhibit Dexamethasone-induced Calcification in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3247.

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

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Purpose: : We previously showed that the trabecular meshwork (TM) undergoes a calcification process under glaucomatous conditions. Statins inhibit the pathway leading to the formation of cholesterol and activation of RhoA. In the vascular system, statins inhibit calcification, with RhoA as a possible mediator. In glaucoma, statins and RhoA pathway inhibitors are being pursued as potential regulators of elevated IOP. Thus, we investigated the effect of these two candidate molecules in the calcification of primary human TM cells (HTM)

Methods: : HTM cell lines were generated from nonglaucomatous human donors. Statin toxicity to HTM cells was evaluated by LDH enzyme release. A dominant-negative RhoA adenovirus (AdhRhoA2) was previously constructed/characterized to inhibit RhoA pathway. HTM cell calcification was induced by treatment with 0.1 µM DEX for 5 d, or infection with an Ad virus encoding human BMP2 (Ad5BMP2) for 48 h. For the statins, cells were treated with 0.1 µM DEX or AdhBMP2 in the presence and absence of nontoxic (0.1 µM) concentrations of lovastatin or simvastatin. For the RhoA experiments, cells were treated with 0.1 µM DEX and infected with 1-10x107 viral particles of AdhRhoA2 or vehicle. Untreated, and single treated (lovastatin, simvastatin, DEX, Ad5BMP2, AdhRhoA2) cells were used as controls. Calcification was assayed by the calcification marker Alkaline Phosphatase (ALP) using a kit lacking endogenous ALP inhibitors; values were normalized to cell’s DNA. All experiments were done in triplicate in two HTM cell lines.

Results: : A toxicity titration curve of the statins (0.01 µM to 100 µM) showed 95% cell viability between 0.01 and 1 µM. DEX-treated cells increased their endogenous ALP 7.4±0.8-fold over untreated controls. Both, lovastatin and simvastatin added to DEX reverted ALP levels to 2.3±0.2 and 2.6±0.2-fold over untreated (p=0.001, 0.002). Similarly, both statins reduce significantly the ALP induced by Ad5BMP2 from 10±4-fold to 1.7±0.2-fold and 1.6±0.6-fold (p=0.09, 0.09) over untreated cells. In parallel, inhibition of RhoA (by overexpression of dominant-negative RhoA) reverted the DEX-induced ALP in a dose-dependent manner, from 4.8 ±0.9-fold to 1.9±0.4-fold and 0.8± 0.2-fold respectively (p=0.03, 0.005). Statins and AdhRhoA2 controls, without DEX or BMP2, had a minimal effect on ALP levels.

Conclusions: : Statin treatment and inhibition of the RhoA reverted calcification induced by DEX and BMP2 in HTM cells. Since statins inhibit RhoA, these finding suggest that statin-dependent inhibition of calcification could be mediated by its inhibition of RhoA. Inhibition of calcification might be a therapeutic mechanism by which statins and the RhoA pathway act to lower IOP.

Keywords: trabecular meshwork • intraocular pressure • drug toxicity/drug effects 

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