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Early effects of dexamethasone and anti-VEGF therapy in an inflammatory corneal neovascularization model

Significance statement

The present paper investigates the early, differential anti-angiogenic effects of steroidal and anti-VEGF (anti-Vascular Endothelial Growth Factor) therapy at cellular, tissue, and gene expression levels in an inflammatory neovascularization model. Angiogenesis is a well-known pathogenic mechanism of many serious systemic as well ocular diseases, among them corneal neovascularization. Both VEGF dependent and independent cascades are believed to play central roles; the latter are yet not well characterized. Current treatment of corneal neovascularization is based on steroids, which are effective but frequently associated with serious side effects, as secondary glaucoma, corneal thinning and perforation, cataract, infections. An alternative is anti-VEGF therapy, which is since recent years used in choroidal neovascularization and other exudative retinal diseases; anti-VEGF represents a more specific treatment with less side-effects than steroids, thus with lower efficacy. Our model is based on induction of neovascularization in rat cornea by intrastromal suture, followed by topical treatment with either dexamethasone, rat specific anti-VEGF or a control substance. The corneas were then studied longitudinally in vivo at different time points with slit lamp color photos and confocal microscopy (IVCM), which allows following tissue details in vivo in the same individual, details comparable to those seen by histology; further examinations were gene expression analysis (RT-PCR) and immuno-histochemistry. Since damage to the tissue may be inflicted before signs of new vessels appear, we focused on the early events of the process. We show that both treatments delay or inhibit early events such as limbal vessel dilation and inflammatory cell infiltration of the stroma. Both treatments down-regulate several major inflammatory cytokines. Furthermore, dexamethasone showed a much better inhibition of macrophage infiltration and new vessel formation. The differential effects of steroids and anti-VEGF treatment in suppressing neovascular growth could not be attributed to differential inhibition of several major angiogenic and inflammatory factors (IL-6, VEGF-A, FGF-2, TNF-{Alpha}, CCL2, CCL3, CXCL2, or DLL4) in the early pre-sprouting phase. This provides the motivation for further investigations in the search of factors influenced by steroids but not by anti-VEGF.

Figure Legend: Early migration of myeloid cells: Immunohistochemistry with anti-CD11b confirms the myeloid origin of the infiltrating cells that were seen on IVCM.

Early effects of dexamethasone and anti-VEGF therapy in an inflammatory corneal neovascularization model. Global Medical Discovery

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure legend: Table showing the expression of inflammatory and angiogenic genes by RT-PCR 24 hours after positioning of the inflammatory stimulus.

Early effects of dexamethasone and anti-VEGF therapy in an inflammatory corneal neovascularization model . Global Medical Discovery

 

 

 

 

 

 

 

 

 

 

Journal Reference

Mirabelli P1, Peebo BB2, Xeroudaki M1, Koulikovska M1, Lagali N1. Exp Eye Res. 2014 ;125:118-27.

1Department of Ophthalmology, Institute for Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, SE-581 83 Linköping, Sweden.

2Department of Ophthalmology, Institute for Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, SE-581 83 Linköping, Sweden. Electronic address: [email protected]

Abstract 

Inflammatory angiogenesis is the pathogenic mechanism of various sight-threatening eye diseases, among them corneal neovascularization. Current treatment options include steroids which have undesirable side effects, or anti-VEGF which has only limited efficacy. In an inflammatory environment, however, angiogenesis can be stimulated by numerous factors not directly targeted by anti-VEGF therapy. The aim of this study was to induce corneal inflammation leading to angiogenesis, and investigate the early, differential effects of steroid and anti-VEGF therapy at the cellular, tissue, and gene expression levels. Fifty-two Wistar rats received a single intrastromal corneal suture to induce a controlled inflammatory angiogenic response. Rats were subsequently treated with dexamethasone, rat specific anti-VEGF, or goat IgG (control), topically 4 times daily for 7 days. In vivo confocal microscopy of the cornea was performed longitudinally from 5 h up to 7 d to investigate morphology at the cellular and tissue-level. In vivo photographic vessel analysis and immunohistochemistry were also performed. RT-PCR for VEGF-A, FGF-2, IL-6, TNF-{Alpha}, CXCL2, CCL2, CCL3 and DLL4 was performed at 24 h, and for VEGF-A, IL-6, TNF-{Alpha}, FGF-2, CXCL2, CCL2, and CCL3 at 7 days. Early infiltration of CD11b + myeloid cells into the cornea at 5 h post-suture was delayed by both treatments relative to controls; however neither treatment was able to suppress accumulation of myeloid cells at day 2 or 7. Limbal vessel dilation was inhibited at 5 h by both treatments, but only dexamethasone showed sustained effect until day 2. Early macrophage recruitment was also suppressed by dexamethasone (but not by anti-VEGF) until day 2.Dexamethasone furthermore suppressed corneal neovascularization at day 7 by over 90%, whereas suppression by anti-VEGF was 14%. Despite differential suppression of vessel dilation, macrophage recruitment, and vascular invasion, anti-VEGF  and dexamethasone both down-regulated VEGF-A and IL-6 expression at 24 h with sustained effect to 7 d. They also both down regulated FGF-2 and TNF-{Alpha} at 24 h and CCL2 at 7 d. In conclusion, anti-angiogenic treatments influence early, pre-angiogenic tissue activity such as limbal vessel dilation, inflammatory cell infiltration of the stroma, and macrophage recruitment. Importantly, the differential effects of steroids and anti-VEGF treatment in suppressing neovascular growth could not be attributed to differential inhibition of several major angiogenic and inflammatory factors in the early pre-sprouting phase, including IL-6, VEGF-A, FGF-2, TNF-{Alpha}, CCL2, CCL3, CXCL2, or DLL4.

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