The Effect of High-Dose Aflibercept on Inflammatory, Apoptotic, and VEGF Gene Expression in HREC Cells In Vitro
Zahra Zia1 , M. Hossein Nowroozzadeh 1 , Fatemeh Sanie-Jahromi1 *
- Poostchi Ophthalmology Research Center, Department of Ophthalmology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
Abstract: Developed countries are facing the burden of age-related macular degeneration (AMD)and diabetic retinopathy as the leading causes of blindness. Aflibercept has become a very popular treatment recently, and a high-dose formation (8 mg) has recently been approved to target these conditions. However, the exact interaction of escalating dosages of aflibercept and human retinal microvascular endothelial cells (HREC) is not yet clear. This study aims to evaluate the impact of the higher concentration of aflibercept on inflammatory, apoptotic, and VEGF gene expression in HREC.
Methods: The neural retina layer containing HREC cells was isolated, cultured, and exposed to the different concentrations of aflibercept (500 µg/mL [x] and 2 mg/mL [4x], corresponding to 2 and 8 mg clinical dosages in 4 mL vitreous). The cellular metabolic activity was assessed by using the MTT test. Gene expression analysis using the primer sequences for BCL-2, Bax, IL-1b, IL-6, LIF, TGF-beta, and VEGF-A genes were measured with real-time PCR technique.
Results: Monolayer, flat, and polygonal HREC cells were observed in 3 groups (2 treatment groups plus control)with a reduction in cell number in the 4x aflibercept concentration group. The MTT test showed a reduction in the HREC cell viability in the 'x' (71.8%, p=0.006) and the '4x' (63.2%, p=0.002) aflibercept concentration groups compared to the control (set as 100%). There was an increase in antiapoptotic BCL-2 gene and proapoptotic Bax gene expression and a higher VEGF 2/Bax ratio at 4x concentration (1.83 ± 0.66), indicating the potential modulation of inflammatory pathways at the higher concentration.
Conclusion: This study enhances our understanding of the dose-dependent effects of aflibercept on HRECs, encompassing anti-proliferative, antiapoptotic, and potentially proinflammatory aspects. These findings contribute to the broader comprehension of aflibercept's mechanisms of action, emphasizing the importance of considering multiple cellular processes in the evaluation of anti-VEGF therapies for AMD.
