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MD Silverman, ML DePriest, Y Pan, SR Planck, JT Rosenbaum; Gene Array Profiling of Inflammatory Mediators in Human Iris and Retinal Endothelium . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1561.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In ocular inflammation, endothelial cells (EC) orchestrate leukocyte extravasation into affected tissues. We investigated potential molecular mediators of uveitis and retinitis by developing comprehensive gene expression profiles in cultured human iris and retinal EC (HIEC and HREC), in constitutive and TNFα-activated scenarios. Methods: Pure HIEC and HREC cultures were isolated from collagenase-digested tissues with anti-PECAM-1 antibody-conjugated magnetic beads. Radiolabeled cDNA probes were prepared with total RNA extracted from control and activated cells, and were used to screen for the simultaneous expression of over 1000 known genes using cDNA arrays (AtlasArrays, Clontech, Inc.). Baseline and time-dependent (to 24 hrs) TNFα-induced expression levels of various cell adhesion molecules, cytokines, chemokines, and other inflammatory mediators were confirmed and further characterized by RT-PCR, ELISA, western blotting, and/or functional assays. Results: Gene expression profiles of HIEC and HREC revealed the constitutive and/or cytokine-activated expression of a variety of adhesion molecules (e.g., CD34, CD44, CD81, ICAM-1, ICAM-2, VCAM, fractalkine, selectins, integrins) chemokines (MCP-1, MCP-3, MIP2α, RANTES, IL-8), cytokines (IL-1, IL-6), and growth factors & receptors (FLT3 ligand, TGFß, Axl, FGF8, CD105). Furthermore, TNFα appeared to modulate some of these molecules differently in donor-matched HIEC versus HREC (e.g., IL-8 higher in HIEC; MCP-1 higher in HREC). Conclusion: The vascular endothelia elaborate a plethora of molecules that coordinately regulate inflammation in the eye. Gene array profiling of ocular EC provides a powerful means to rapidly screen for the expression of a large number of molecules that may play previously unsuspected roles in ocular inflammatory diseases. Furthermore, this approach holds potential for revealing tissue-specific differences in gene expression amongst the iris and retinal microvasculatures, and these insights might prove useful for developing novel tissue-specific anti-inflammatory regimens.
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