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L. Bellner, L. Martinelli, N. Puri, K. A. Patil, M. W. Dunn, N. G. Abraham, M. Laniado Schwartzman; HO-2 Deletion Causes Endothelial Activation via Alteration in the VEGF Pathway. Invest. Ophthalmol. Vis. Sci. 2009;50(13):66.
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The heme oxygenase system (HO-1 and HO-2) represents an intrinsic cytoprotective and anti-inflammatory pathway based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins via its products biliverdin/bilirubin and carbon monoxide (CO). We have shown that epithelial injury in HO-2 null mice leads to impaired healing and chronic inflammatory complications including ulceration, perforation and neovascularization. We explored possible mechanisms underlying the increased susceptibility of the HO-2 null mice to neovascularization by examining whether HO-2 deletion impacts the phenotype of endothelial cells, the major player in the neovascular response.
Aortic endothelial cells (mAEC) were isolated from wild type (WT) and HO-2 null mice and cultured in the absence and presence of inflammatory stimuli and the inflammatory response was monitored. The angiogenic response was assessed by measuring capillary-like tube formation of mAEC cultured on Matrigel in the absence or presence of angiogenic stimuli. The expression of inflammatory markers was assessed by Western blot and by real-time PCR. Expression of proinflammatory cytokines was assessed using the Pierce SearchLight mouse microarray.
Under basal conditions, the HO-2 null mAECs showed a 3-fold higher angiogenic response when cultured on Matrigel, as compared to WT with the tubule length (mm) being 17.70±0.78 vs 4.95±0.58 (n=8, p<0.05). The HO-2 null mAECs demonstrated decreased HO-1 mRNA levels and 2-fold reduced HO-activity as compared to the WT. Assessment of superoxide levels (oxidative stress) by DHE staining indicated significantly higher levels in HO-2 null mAECs as compared to WT. Western blot results showed a 2-fold increase in the NF-kappaB activity in the HO-2 null endothelial cells. RT-PCR results showed a 3-fold increase in the VEGFR-1 expression and an 8-fold decrease in the VEGFR-3 expression in the HO-2 null mAECs.
HO-2 deletion transforms endothelial cells from "normal" to an "activated" phenotype characterized by increased inflammatory, oxidative and angiogenic factors. This switch may be the result of reduced HO activity and the associated cytoprotective signals including carbon monoxide and biliverdin. Such transformation may explain the massive injury-induced corneal neovascularization in the HO-2 null mouse and further underscores the importance of the HO system in the regulation of cell homeostasis.
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