Exacerbated corneal inflammation and neovascularization in the HO-2 null mice is ameliorated by biliverdin

Abstract

Heme oxygenase (HO-1 and HO-2) represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins. HO-2 deletion leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration, perforation and neovascularization. We examined the consequences of HO-2 deletion on hemangiogenesis and lymphangiogenesis in the model of suture-induced inflammatory neovascularization. A 8.0 silk suture was placed at the corneal apex of wild type and HO-2 null mice. Neovascularization was assessed by vital microscopy and quantified by image analysis. Hemangiogenesis and lymphangiogenesis were determined by immunofluorescence staining using anti-CD31 and anti-LYVE-1 antibodies, respectively. Inflammation was quantified by histology and myeloperoxidase activity. The levels of HO-1 expression and inflammatory cytokines were determined by real time PCR and ELISA, respectively. Corneal sutures produced a consistent inflammatory response and a time-dependent neovascularization. The response in HO-2 null mice was associated with a greater increase compared to the wild type in the number of leukocytes (827600±129000 vs. 294500±57510; p<0.05), neovessels measured by vital microscopy (21.91±1.05 vs. 12.77±1.55 mm; p<0.001) 4 days after suture placement. Hemangiogenesis but not lymphangiogenesis was more pronounced in HO-2 null mice compared to wild type mice. Induction of HO-1 in sutured corneas was greatly attenuated in HO-2 null corneas and treatment with biliverdin diminished the exaggerated inflammatory and neovascular response in HO-2 null mice. The demonstration that the inflammatory responses, including expression of proinflammatory proteins, inflammatory cell influx and hemangiogenesis are exaggerated in HO-2 knockout mice strongly supports the notion that the HO system is critical for controlling the inflammatory and neovascular response in the cornea. Hence, pharmacological amplification of this system may constitute a novel therapeutic strategy for the treatment of corneal disorders associated with excessive inflammation and neovascularization.

Heme Oxygenase-2 Deletion Causes Endothelial Cell Activation Marked by Oxidative Stress, Inflammation, and Angiogenesis

“…HO-1 is proangiogenic in VEGF-driven, inflammation-independent angiogenesis, but it blocks inflammatory angiogenesis (Bussolati et al, 2004), suggesting that a deficiency in HO activity can promote inflammation-driven angiogenesis. Our laboratory clearly showed that a deficiency in HO activity in HO-2(Ϫ/Ϫ) mice promotes massive inflammation-induced neovascularization of the cornea (Seta et al, 2006;Bellner et al, 2008). This study provides additional evidence to support a role for the HO system in the control of inflammatory angiogenesis.…”

“…Highly angiogenic endothelial cells isolated from the aorta of HO-2(Ϫ/Ϫ) mice experienced a 2.7-fold reduction in HO activity compared with corresponding cells from WT mice. However, the angiogenic activity of the HO-2(Ϫ/Ϫ) cells was significantly attenuated after addition of biliverdin, indicating that deficiency in HO activity in the absence of the HO-2 gene contributes to angiogenesis in vitro and neovascularization in vivo (Seta et al, 2006;Bellner et al, 2008).…”

“…In summary, deletion of the HO-2 gene is associated with oxidative stress, inflammation, and angiogenesis in vivo (Seta et al, 2006;Bellner et al, 2008) and in vitro in endothelial cells. These three processes have been linked to the HO system through the anti-inflammatory and antioxidative properties of its metabolites, CO and biliverdin/bilirubin.…”

“…Reverse transcription reaction of total RNA (400 ng) was performed using the Superscript III first-strand synthesis system (Invitrogen) according to the manufacturer's instructions. Quantitative real-time PCR was performed using QuantiTect SYBR Green PCR Kit (QIAGEN) and the Mx3000 real-time PCR system (Stratagene, La Jolla, CA) as described previously (Bellner et al, 2008). Specific primers were designed based on published sequences (GenBank).…”

“…In recent reports, we have showed that deletion of the HO-2 gene markedly impairs the inflammatory and reparative response of the cornea to injury and leads to unresolved corneal inflammation and chronic inflammatory complications, including ulceration, perforation, and neovascularization (Seta et al, 2006;Bellner et al, 2008). In the current study, we explore possible mechanisms underlying the increased susceptibility of the HO-2 null [HO-2(Ϫ/Ϫ)] mice to neovascularization by examining whether HO-2 deletion affects the phenotype of endothelial cells, the major player in the neovascular response.…”