Abstract Title

EFFECT OF LYSYL OXIDASE (LOX) AND TISSUE TRANSGLUTAMINASE (TGM2) GENES ON HUMAN TRABECULAR MESHWORK CELLS

Presenter Name

Kyle A Kirkland

Abstract

Purpose: Glaucoma is a leading cause of irreversible blindness in the world. TGF-β2 is elevated in glaucoma eyes and is an important factor in the extracellular matrix (ECM) metabolism of human trabecular meshwork (HTM) cells, leading to increased intraocular pressure (IOP). Both LOX and TGM2 enzymes are important in cross-linking the ECM in HTM cells. As TGF-β2 up regulates LOX and TGM2 expression, the aim of this experiment is to determine and quantify the amount of LOX- and TGM2-induced ECM crosslinking in glaucomatous trabecular meshwork cells (GTM). Methods: LOX and TGM2 cDNAs were obtained and amplified by PCR with specifically designed primers and isolated by gel electrophoresis. The cDNAs were ligated into the pGEM-T plasmid vector and cloned into E. coli gold cells. After sequencing, the genes were restriction digested and ligated to a pacAd5 vector to generate adenovirus expression vectors, which have a high selectivity for TM cells. The pacAd5 vectors will be used to transduce the GTM3 cell line. The plasmid expression vectors will also be transfected into GTM3 cells. Western immunoblot analysis will be utilized to evaluate the LOX and TGM2 protein expression and ECM crosslinking in GTM cells. Results: LOX and TGM2 plasmid vectors were successfully cloned and sequenced and are now being used to transfect GTM cells. LOX and TGM2 adenoviral vectors are being prepared. We expect that increased expression of LOX and TGM2 enzymes through both transduction and transfection will induce a greater amount of crosslinking in the GTM cells. Conclusions: TGF-β2 raises IOP by mechanisms that are still under investigation. One potential mechanism is increased ECM cross-linking via TGF-2 induction of LOX and TGM2 gene expression. Successful transduction and LOX and TGM2 expression in cultured GTM3 cells will allow us to directly test the roles of LOX and TGM2 on the regulation of IOP using an ex vivo bovine ocular perfusion culture model.

Purpose (a):

Glaucoma is a leading cause of irreversible blindness in the world. TGF-β2 is elevated in glaucoma eyes and is an important factor in the extracellular matrix (ECM) metabolism of human trabecular meshwork (HTM) cells, leading to increased intraocular pressure (IOP). Both LOX and TGM2 enzymes are important in cross-linking the ECM in HTM cells. As TGF-β2 up regulates LOX and TGM2 expression, the aim of this experiment is to determine and quantify the amount of LOX- and TGM2-induced ECM crosslinking in glaucomatous trabecular meshwork cells (GTM).

Methods (b):

LOX and TGM2 cDNAs were obtained and amplified by PCR with specifically designed primers and isolated by gel electrophoresis. The cDNAs were ligated into the pGEM-T plasmid vector and cloned into E. coli gold cells. After sequencing, the genes were restriction digested and ligated to a pacAd5 vector to generate adenovirus expression vectors, which have a high selectivity for TM cells. The pacAd5 vectors will be used to transduce the GTM3 cell line. The plasmid expression vectors will also be transfected into GTM3 cells. Western immunoblot analysis will be utilized to evaluate the LOX and TGM2 protein expression and ECM crosslinking in GTM cells.

Results (c):

LOX and TGM2 plasmid vectors were successfully cloned and sequenced and are now being used to transfect GTM cells. LOX and TGM2 adenoviral vectors are being prepared. We expect that increased expression of LOX and TGM2 enzymes through both transduction and transfection will induce a greater amount of crosslinking in the GTM cells.

Conclusions (d):

TGF-β2 raises IOP by mechanisms that are still under investigation. One potential mechanism is increased ECM cross-linking via TGF-b2 induction of LOX and TGM2 gene expression. Successful transduction and LOX and TGM2 expression in cultured GTM3 cells will allow us to directly test the roles of LOX and TGM2 on the regulation of IOP using an ex vivo bovine ocular perfusion culture model.

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EFFECT OF LYSYL OXIDASE (LOX) AND TISSUE TRANSGLUTAMINASE (TGM2) GENES ON HUMAN TRABECULAR MESHWORK CELLS

Purpose: Glaucoma is a leading cause of irreversible blindness in the world. TGF-β2 is elevated in glaucoma eyes and is an important factor in the extracellular matrix (ECM) metabolism of human trabecular meshwork (HTM) cells, leading to increased intraocular pressure (IOP). Both LOX and TGM2 enzymes are important in cross-linking the ECM in HTM cells. As TGF-β2 up regulates LOX and TGM2 expression, the aim of this experiment is to determine and quantify the amount of LOX- and TGM2-induced ECM crosslinking in glaucomatous trabecular meshwork cells (GTM). Methods: LOX and TGM2 cDNAs were obtained and amplified by PCR with specifically designed primers and isolated by gel electrophoresis. The cDNAs were ligated into the pGEM-T plasmid vector and cloned into E. coli gold cells. After sequencing, the genes were restriction digested and ligated to a pacAd5 vector to generate adenovirus expression vectors, which have a high selectivity for TM cells. The pacAd5 vectors will be used to transduce the GTM3 cell line. The plasmid expression vectors will also be transfected into GTM3 cells. Western immunoblot analysis will be utilized to evaluate the LOX and TGM2 protein expression and ECM crosslinking in GTM cells. Results: LOX and TGM2 plasmid vectors were successfully cloned and sequenced and are now being used to transfect GTM cells. LOX and TGM2 adenoviral vectors are being prepared. We expect that increased expression of LOX and TGM2 enzymes through both transduction and transfection will induce a greater amount of crosslinking in the GTM cells. Conclusions: TGF-β2 raises IOP by mechanisms that are still under investigation. One potential mechanism is increased ECM cross-linking via TGF-2 induction of LOX and TGM2 gene expression. Successful transduction and LOX and TGM2 expression in cultured GTM3 cells will allow us to directly test the roles of LOX and TGM2 on the regulation of IOP using an ex vivo bovine ocular perfusion culture model.