a novel therapeutic approach for corneal vascularization in veterinary ophthalmology

PhD thesis - University of Veterinary Medicine Vienna - 2021 The full text is only available to university members. Please log in!

Corneal vascularization is a major challenge in veterinary ophthalmology and is associated with a variety of adverse effects, including visual impairment due to corneal edema, deposition of pigments and lipids, persistent corneal inflammation, and loss of the corneal immune privilege (Dana and Streilein 1996, Andrew et al. 1998, Murphy et al. 2001, Andrew 2008, Dean and Meunier 2013, Lassaline et al. 2015, Plummer 2015, Hindley et al. 2016, Ledbetter et al. 2016, Krecny et al. 2018). Normally, the mammalian cornea is free of blood and lymph vessels, which is one of the prerequisites for corneal transparency (Crispin and Barnett 1983, Werkmeister et al. 2017). Corneal avascularity is regulated by a balance of pro- and antiangiogenic mechanisms (Cursiefen et al. 2004b, Ambati et al. 2006, Cursiefen et al. 2006, Matsui et al. 2012, Zakaria et al. 2012). Stimuli such as hypoxia, inflammation, and infection can tip the scales to the angiogenic side that cause ingrowth of blood vessels into the cornea (Mastyugin et al. 2001, Gan et al. 2004, Goyal et al. 2010, Shi et al. 2010, Lutty et al. 2011, Chen et al. 2012, Park et al. 2018). One of the key mediators of angiogenesis is the vascular endothelial growth factor (VEGF)-A, which is involved in both physiological and pathological vascularization (Shalaby et al. 1995, Amano et al. 1998, Gerhardt et al. 2003, Gan et al. 2004, Jia et al. 2004, Eming and Krieg 2006, Pieh et al. 2008). Vascular endothelial growth factor A is a glycoprotein that exerts its effect through two tyrosine kinase cell surface receptors, VEGF receptor (VEGFR)-1 and VEGFR-2. (Gerhardt et al. 2003, Jia et al. 2004). A causative function of VEGF-A and VEGFR-2 during corneal angiogenesis has been demonstrated in experimental animal models of corneal vascularization and in humans with naturally occurring corneal angiogenesis (Cursiefen et al. 2000, Mastyugin et al. 2001, Gan et al. 2004, Chen et al. 2012, Yun et al. 2020). Nonetheless, the function of VEGF in ocular diseases in animal patients is largely unknown (Zarfoss et al. 2010, Binder et al. 2012, Sandberg et al. 2012, Balicki and Sobczyńska-Rak 2014). There are few studies indicating a possible role of VEGF in a number of canine ocular diseases, such as chronic superficial keratitis and pre-iridal fibrovascular membranes (Zarfoss et al. 2010, Sandberg et al. 2012, Balicki and Sobczyńska-Rak 2014). In experimental canine models of retinopathy of prematurity, the usage of therapeutic VEGF inhibitors led to an efficient reduction of retinal vascularization (Lutty et al. 2011). In horses affected by equine recurrent uveitis, a causal role of VEGF has been postulated (Deeg et al. 2006, 2007, Curto et al. 2016). Although studies in dogs, cats, and horses are sparse, it can be speculated that VEGF-driven mechanisms are causally involved in corneal vascularization in these species, too. The discovery of drugs that inhibit VEGF-A and its effectiveness in neovascular disorders has revolutionized human ophthalmology, in particular in retinal neovascular disorders (Avery et al. 2006, Schmidt-Erfurth et al. 2014, Dugel et al. 2020, Sahni et al. 2020). Two of the most intensively studied VEGF inhibitors are bevacizumab and aflibercept (Presta et al. 1997, Holash et al. 2002). Bevacizumab is a humanized murine anti-VEGF monoclonal antibody that binds to human VEGF-A (Presta et al. 1997). Aflibercept is a fusion protein, consisting of regions of VEGFR-1, VEGFR-2 and human IgG1, that binds to human VEGF-A, VEGF-B and placenta growth factor (PIGF), and to VEGF-A and PIGF of mice (Holash et al. 2002, Papadopoulos et al. 2012). The use of bevacizumab and aflibercept is considered a promising approach to suppress corneal vascularization in humans (Dastjerdi et al. 2009, Aksoy 2019, Cholak et al. 2020) and a clinical applicability in dogs, cats, and horses is conceivable. This is of great interest in veterinary ophthalmology, as the incidence of corneal vascularization is high, persistent corneal blood vessels can result in vision-threatening complications, and existing standard therapies may be insufficient or carry a high risk of adverse events (Slatter et al. 1977, Bedford and Longstaff 1979, Kaswan and Salisbury 1990, Tolar et al. 2006, Bock et al. 2014, Plummer 2015, Dowling et al. 2016, Hindley et al. 2016, Rigas et al. 2020, Villar et al. 2020). Bevacizumab and aflibercept were designed to bind specifically to human VEGF-A (Presta et al. 1997, Holash et al. 2002, Papadopoulos et al. 2012). The pharmacological binding properties to canine, feline, and equine VEGF are unknown to date. In addition, there is no knowledge about the in-vivo effects of topical usage in these species. Therefore, the aim of this PhD study was to evaluate the binding properties of bevacizumab and aflibercept to canine, feline, and equine VEGF and to further investigate the effects of topical bevacizumab in-vivo in dogs. Amino acid sequence comparisons revealed a high homology and an identical sequence of the bevacizumab binding region of canine, feline, and equine VEGF compared to human VEGF. Using ELISA- and Western blot analyses, the binding ability of both bevacizumab and aflibercept to canine VEGF was demonstrated. However, the results indicated that feline and equine VEGF may not be bound by aflibercept and bevacizumab or bind in a nonspecific manner only. Subsequently, we set out to investigate bevacizumab eye drops in healthy dogs, derived from commercially available bevacizumab for intravenous use and diluted in sterile saline. The results showed that topical bevacizumab at a concentration of 2.5 mg/ml, administered twice a day over four weeks, was well tolerated. This finding allowed us to use topical bevacizumab in dogs with naturally occurring corneal vascularization refractory to standard therapy. A reduction in corneal vascularization, most notably as a significant decrease in vessel diameter, a reduction in distal vessel branching, an improvement in corneal edema and inflammatory cell infiltration, and a marked improvement in corneal clarity was observed. However, a French bulldog developed a corneal erosion and two Chihuahuas suffering from preexisting mitral valve insufficiency died during the treatment-free observation period. A causal relationship with the drug was considered unlikely. Nevertheless, dogs prone to corneal erosions and neurotrophic disease, as well as preexisting cardiovascular disorders, should be selected with caution (Kim et al. 2008, Yu et al. 2008a, Dong et al. 2017). This PhD work demonstrated the capability of bevacizumab and aflibercept to bind canine VEGF and the potential of topical bevacizumab to reduce persistent corneal vascularization in dogs. Overall, this PhD project provided an important step for future clinical use of therapeutic VEGF inhibitors in neovascular ocular disorders in animals.

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