May 2006
Volume 47, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2006
Inhibition of LPS–Induced Nitric Oxide Secretion in Raw 264.7 Macrophages by TG100572– a Multi–Targeted Tyrosine Kinase Inhibitor
Author Affiliations & Notes
  • B. Tam
    Research & Development, TargeGen Inc., San Deigo, CA
  • J. Hood
    Research & Development, TargeGen Inc., San Deigo, CA
  • Z. Chen
    Research & Development, TargeGen Inc., San Deigo, CA
  • K. Barret
    Research & Development, TargeGen Inc., San Deigo, CA
  • S. Yee
    Research & Development, TargeGen Inc., San Deigo, CA
  • R.M. Soll
    Research & Development, TargeGen Inc., San Deigo, CA
  • Footnotes
    Commercial Relationships  B. Tam, TargeGen Inc., E; J. Hood, TargeGen Inc, E; Z. Chen, E, E; K. Barret, TargeGen Inc., E; S. Yee, TargeGen Inc., E; R.M. Soll, TargeGen Inc., E.
  • Footnotes
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Investigative Ophthalmology & Visual Science May 2006, Vol.47, 4554. doi:
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      B. Tam, J. Hood, Z. Chen, K. Barret, S. Yee, R.M. Soll; Inhibition of LPS–Induced Nitric Oxide Secretion in Raw 264.7 Macrophages by TG100572– a Multi–Targeted Tyrosine Kinase Inhibitor . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4554.

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

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Abstract

Purpose: : A number of immunological factors have been implicated in acute macular degeneration (AMD). The presence of immune cells and complement in drusen deposits formed in the macula preceding AMD are believed to further activate inflammatory pathways which contribute to the etiology of the disease. One such pathway is the recruitment and activation of macrophages which further aggravate inflammation in the eye and may contribute to choroidal neovascularization. Activation of macrophages leads to the production of nitric oxide (NO), and it has recently been reported that plasma NO levels are increased in AMD patients. These studies indicate that the macrophage may be an important target in treating AMD. Because Src family kinases are stimulated in macrophages in response to lipopolysaccharide (LPS), we evaluated the capacity of a small molecule VEGFR/src family kinase inhibitor, TG100572 to prevent macrophage activation in vitro as measured by NO release.

Methods: : 5 x 104 of Raw 264.7 macrophages were treated with LPS (12.3 ng/ml) in the absence or presence of compound (2 µM). After 14–16 h of incubation, supernatants were collected and the concentration of nitrite (µM) was determined with Griess reagent as described by the manufacturers (Cayman Chemicals). For Src phosphorylation studies, cells were stimulated with LPS for 5 minutes, and cell extracts were subjected to SDS–PAGE. Western blotting was performed using an anti–phospho–cSrc antibody (Santa Cruz Biotechnology).

Results: : TG100572 treatment reduced LPS–induced NO release by approximately 40% (p=7x10–7), while not affecting their proliferation. Additionally, TG100572 inhibited the activating phosphorylation of c–Src, which has recently been implicated as the key Src family kinase involved in LPS–stimulated nitric oxide secretion in macrophages. In contrast, compounds which selectively inhibit only VEGF did not inhibit macrophage activation.

Conclusions: : This work is consistent with the notion that TG100572 may have utility for the treatment of the complex etiology of back–of–the–eye diseases not offered by selective inhibitors of VEGF signaling.

Keywords: inflammation • immunomodulation/immunoregulation • endophthalmitis 
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