Professor/Advisor Lehigh University, United States
Introduction:: Abdominal Aortic Aneurysms (AAAs) are localized, rupture-prone expansions of the aorta wall primarily afflicting elderly male smokers. In AAAs, an initial insult to the aorta wall triggers chronic inflammation. Inflammatory cells recruited to the injury site cause proteolytic breakdown of the structural extracellular matrix (ECM) of the injured aortal segment. Loss of elastic fibers that enable vessel stretch and recoil leads to irreversible vessel expansion and fatal rupture. There are no non-surgical treatments for small (< 5.5 cm max. diameter) AAAs. Reversing AAA pathophysiology is challenged by the current lack of approaches to stimulate naturally poor and impaired elastic fiber assembly by adult and diseased vascular smooth muscle cells (SMCs), critical to restoring elastin homeostasis. We recently showed that activation of the epidermal growth factor receptor-mitogen-activated protein kinase (EGFR-MAPK) signaling pathway causes downstream elastic matrix degeneration in aneurysmal SMC cultures1. In this work, we investigate potential matrix regenerative benefits of an FDA-approved EGFR inhibiting drug; Afatinib. Afatinib has been extensively used in cancer research, however its potential use in other disease scenarios, such as AAAs, remains unelucidated till date. Our goal is to investigate if blocking EGFR activity in aneurysmal SMCs via Afatinib is a useful therapeutic approach to downregulate MAPK (particularly extracellular signal-regulated kinase (ERK1/2)) towards stimulating both downstream elastogenesis and anti-matrix metalloprotease (MMP) effects. This work can potentially lead to addition of a new drug in the pipeline for treatment of AAAs, which can further lead to an EGFR-targeted regenerative nanotherapy for AAA wall repair.
Materials and Methods:: Aneurysmal rat aortic smooth muscle cells (EaRASMCs) were isolated from male Sprague-Dawley rats induced with AAAs via elastase infusion-mediated aortal injury. Aneurysmal aortae were harvested 14 days postoperative. Cells were isolated from 3 separate animals and pooled prior to propagation and passaging (P3-8). EaRASMCs were seeded at 30 K-50 K cells/well in a 6-well plate and cultured in DMEM-F-12 medium containing 10% v/v fetal bovine serum (FBS) and 1% v/v penicillin–streptomycin (ThermoFisher Scientific, Waltham, MA) for 14 days. Cells were serum starved for 3 hours in 1% v/v FBS DMEM-F-12 and treated with 0.1 nM-10 nM Afatinib (Cayman Chemical, Ann Arbor, MI) for 1 hour prior to stimulation with 5 μg/ml of human leucocyte elastase (Elastin Product Company, Owensville, MO) for 30 min. Cells were harvested for western blot samples in RIPA lysis buffer and protein levels in the samples were quantified using bicinchoninic acid assay. Proteins were run on 4-12% SDS-PAGE gels and then were transferred to nitrocellulose membranes using iBlot 2 gel transfer device (Thermo Fisher), followed by antibody incubation. The protein band intensities on the membrane were normalized against the β-actin (served as the housekeeping protein) band intensity. Elastic matrix density and ultrastructure were analyzed by transmission electron microscopy (TEM) (0.5, 1 nM drug doses).
Results, Conclusions, and Discussions:: Results/Discussions: To evaluate the effect of Afatinib on EGFR related protein inhibition, a dose optimization study was conducted. Results indicate that ≥ 50% inhibition is achieved in EGFR and p-ERK1/2 levels (p < 0.05) at an optimal dose of 0.5 nM and 1 nM, while these trends reverse as the dose increases to 10 nM. Total ERK1/2 levels remained unchanged post Afatinib treatment. Although p-EGFR levels did not show any significant differences vs. control, a decreasing trend in the expression level was observed with dose dependent EGFR inhibition via Afatinib. TEM showed robust maturing fibers with coalescing tropoelastin deposits in Afatinib-treated cultures, but not in treatment controls.
Conclusion: Engagement of EGFR on the SMC surface within the AAA tissue milieu has critical implications in its pathogenesis. EGFR exerts its negative effects via activation of the MAPK pathway. Our studies suggest that Afatinib has the potential to provide inhibitory effects in EaRASMCs by blocking the EGFR-MAPK pathway. Afatinib doses of 0.5 nM and 1 nM provide effective suppression (≥ 50% vs. control) in EGFR and p-ERK1/2 levels. This suggests that Afatinib has the capability to serve as a novel drug for repair of small AAAs, which can help to prevent elastolysis and improve elastogenesis in AAAs. Future work will focus on the development of nanotherapeutics for elastic matrix repair in AAAs by targeting EGFR inhibition on aneurysmal SMCs.
Acknowledgements (Optional): :
References (Optional): : 1. Dayal, S. et al. Targeting Epidermal Growth Factor Receptor to Stimulate Elastic Matrix Regenerative Repair. Tissue Eng. Part A (2023)
