Rac1 Inhibition Prevents Tissue Contraction and MMP Mediated Matrix Remodeling in the Conjunctiva

Invest Ophthalmol Vis Sci. 2012 Jul 10;53(8):4682-91. doi: 10.1167/iovs.11-8577.

Abstract

Purpose: To evaluate the efficiency of Rac1 inhibition in preventing matrix contraction by Tenon's capsule fibroblasts.

Methods: The involvement of Rac1 in serum-stimulated matrix contraction by human Tenon's fibroblasts (HTFs) was investigated in a classic collagen contraction model and our ex vivo model of tissue contraction using immunocytochemistry, chemical inhibitors, and small interfering RNA (siRNA) technology. Matrix integrity was assessed using confocal reflection microscopy and Coomassie blue staining. Quantitative real-time polymerase chain reaction (QRT-PCR) and Western blot analysis were used to assess matrix metalloproteinase (MMP) expression.

Results: Serum induced Rac1 activation in HTF-populated collagen gels and stimulated HTFs to contract collagen matrices down to ~90% of their original size. Rac1 inhibition using NSC23766 or depletion using siRNA both significantly reduced HTF-mediated contraction. Early brief exposure to NSC23766 reduced HTF-mediated gel contraction by 70%, while transient treatment with the Rac1 inhibitor once a week decreased ex vivo tissue contraction down to serum-free levels. Transient exposure to NSC23766 prevented early cell protrusions, fiber alignment, and matrix degradation, as seen upon continuous exposure to broad-spectrum MMP inhibitor. However, unlike MMP inhibition, transient treatment with NSC23766 led to a significant reduction in MMP1 mRNA and protein expression during contraction, without increasing MMP2 and MMP14 expression.

Conclusions: Rac1 inhibition efficiently prevents conjunctival tissue and collagen matrix contraction and prevents matrix degradation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminoquinolines / pharmacology
  • Blotting, Western
  • Cells, Cultured
  • Collagen / metabolism
  • Conjunctiva / drug effects
  • Conjunctiva / metabolism*
  • Enzyme Inhibitors / pharmacology
  • Extracellular Matrix / drug effects
  • Extracellular Matrix / metabolism
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Humans
  • Matrix Metalloproteinases / metabolism*
  • Pyrimidines / pharmacology
  • RNA, Messenger / metabolism
  • Real-Time Polymerase Chain Reaction
  • rac1 GTP-Binding Protein / antagonists & inhibitors*
  • rac1 GTP-Binding Protein / metabolism

Substances

  • Aminoquinolines
  • Enzyme Inhibitors
  • NSC 23766
  • Pyrimidines
  • RAC1 protein, human
  • RNA, Messenger
  • Collagen
  • Matrix Metalloproteinases
  • rac1 GTP-Binding Protein