Anti-VEGF agents were found to have clinical implications for the successful treatment of vascular-driven diseases in humans. In this study, a detailed biological characterization of bevacizumab in a variety of in vitro assays was carried out to determine the effect of bevacizumab on equine umbilical vein endothelial cells (EqUVEC). EqUVECs were harvested from umbilical cords of clinically healthy horses and exposed to different concentrations (1, 2, 4, 6, 8 mg/mL) of bevacizumab (Avastin^®). Assays concerning the drug’s safety (cell viability and proliferation assay) and efficacy (cell tube formation assay, cell migration assay, and Vascular endothelial growth factor (VEGF) expression) were carried out reflecting multiple cellular processes. Bevacizumab significantly decreased VEGF expression at all concentrations over a 72 h period. No cytotoxic effect of bevacizumab on EqUVECs was observed at concentrations of 4 mg/mL bevacizumab or lower. Incubated endothelial cells showed delayed tube formation and bevacizumab efficiently inhibited cell migration in a dose-dependent manner. Bevacizumab potently inhibits VEGF-induced cellular processes and could be a promising therapeutic approach in vascular-driven diseases in horses.

Angiogenesis plays a crucial role in various physiological and pathological conditions. However, research in equine angiogenesis is relative limited, necessitating the development of suitable in-vitro models. To effectively analyze angiogenesis in-vitro, it is essential to target the specific cells responsible for this process, namely endothelial cells. Human umbilical vein endothelial cells (HUVECs) are one of the most used in vitro models for studying angiogenesis in humans. Serving as an equivalent to HUVECs, we present a comprehensive isolation protocol for equine umbilical vein endothelial cells (EqUVECs) with relatively minimal requirements, thereby enhancing accessibility for researchers. Umbilical cords obtained from five foals were used to isolate endothelial cells, followed by morphological and immunohistochemical identification. Performance of the cells in various assays commonly used in angiogenesis research was studied. Additionally, EqUVEC expression of vascular endothelial growth factor (VEGF) was assessed using ELISA. EqUVECs exhibited endothelial characteristics, forming a homogeneous monolayer with distinctive morphology. Immunohistochemical staining confirmed positive expression of key endothelial markers including von Willebrand factor (vWF), CD31, and vascular endothelial growth factor receptor-2 (VEGFR-2). Furthermore, performance assessments in in-vitro assays demonstrated the viability, proliferation, migration, tube formation and VEGF-expression capabilities of EqUVECs. The findings suggest that EqUVECs are a promising in-vitro model for studying equine angiogenesis, offering a foundation for further investigations into equine-specific vascular processes and therapeutic interventions.

Monoclonal antibodies have made an immense contribution to the treatment of various human diseases. We aimed at investigating an affordable treatment option for veterinary patients with corneal neovascularization by adding the preservative benzalkonium chloride (BAC) to bevacizumab (Avastin®) for usage in multidose containers. A comprehensive analytical similarity assessment of preserved and unpreserved bevacizumab after dilution and storage was carried out. Diluted and preserved bevacizumab was analysed at different time points for a 4-week period and compared with unpreserved bevacizumab at the same concentrations at each time point. Native-PAGE, immunoblotting and HP-SEC were used to observe aggregation and degradation. DLS provided information about particle size and dispersity. Bevacizumab quantified by ELISA was conducted to determine its biological activity. Dose response curves and cell migration assays were performed to detect possible toxic effects and determine biological activity and efficacy of the drug using HUVECs. Native-PAGE, immunoblotting and HP-SEC analysis did not show any changes or degradation products in the presence of BAC and after storage compared to unpreserved bevacizumab. The overlapping intensity-based particle size distribution obtained from DLS showed similarity in all tested groups and homogeneity was maintained. ELISA accurately detected bevacizumab at different concentrations. HUVECs incubated with preserved or unpreserved bevacizumab showed a comparable effect on cell migration. No decrease in cell viability was detected. Equivalence tests demonstrated that bevacizumab is stable after dilution, storage and preservation with BAC. Our study shows that preserved bevacizumab applied in mutidose containers can be considered as a cost-effective alternative to the otherwise single-dose treatments.

Corneal neovascularization (CNV) in dogs causes chronic ocular surface inflammation, which may disrupt tear film stability and meibomian gland function. As bevacizumab inhibits abnormal vessel growth and affects tear composition, it may benefit dogs with ocular surface disorders. The aim of this pilot study was to evaluate the effects of bevacizumab eye drops on the ocular surface in dogs with chronic CNV. Fourteen eyes from nine client-owned dogs were included. Dogs received either preserved (B_BAC-group) or unpreserved (B-group) 0.25% bevacizumab eye drops twice daily for 28 days. Re-examinations occurred one week after treatment initiation, at the end of treatment, and three months later. An ocular surface analyzer was used to assess tear composition and meibomian glands via interferometry, tear meniscus height, and meibography. Ocular and systemic toxicities were monitored, and clinical signs were scored. Serum VEGF levels were measured via ELISA before and after treatment. No statistically significant changes in intraocular pressure, tear production, or systemic VEGF levels were observed. Statistically significant improvements were noted in conjunctival hyperemia and chemosis in both groups, ocular discharge in the B_BAC-group, and fluorescein uptake in the B-group. Both formulations were safe and well tolerated. These preliminary findings suggest that topical bevacizumab may have potential benefits for CNV and ocular surface disorders in dogs, although larger studies are needed.