Defects In Protein Trafficking In Venous Malformations: A Novel Disease Model
Emma Iaconetti, BA1, Noa Shapiro-Franklin, BS2, Ajit Muley, PhD3, Carrie J. Shawber, PhD3, June K. Wu, MD2.
1Columbia University Vagelos College of Physicians & Surgeons, New York, NY, USA, 2Columbia University Irving Medical Center Department of Surgery, Division of Plastic and Reconstructive Surgery, New York, NY, USA, 3Columbia University Irving Medical Canter Department of Obstetrics and Gynecology, New York, NY, USA.
Purpose: Venous malformations (VMs) are congenital defects that arise due to disordered development of the venous system. Recently discovered germline and somatic mutations in VM endothelial cells (VMECs) result in hyperactivation of PI3K/AKT/mTOR and/or RAS/RAF/MAPK signaling pathways and thus hyperproliferation of VMECs. Due to the similarities of these mutations to those found in cancer, current disease models and treatment strategies have focused on the repurposing of anti-proliferative, anti-cancer drugs such as sirolimus (mTOR inhibitor), alpelisib (PI3K inhibitor), and trametinib (MEK inhibitor) to inhibit VMs. However, these therapies have had only limited and anecdotal success, highlighting the need for the development of new pharmacotherapies for treatment of VMs. Previous work in our lab has shown that junctional proteins are punctate and misexpressed on VM endothelium. We hypothesize that VM pathobiology involves the mislocalization of critical junctional proteins.
Methods: We isolated patient-derived VMECs by selecting for the progenitor EC marker, CD133, and confirmed endothelial identity with FACS analysis and IHC staining. FACS analysis of VMECs was used to assess cell surface protein expression of junctional proteins (CD31, VECADHERIN, CD146), EC markers (VEGFR1, VEGFR2, VEGFR3), and a progenitor marker (CD90). VMECs were stained for cell surface adhesion molecule CD31 and markers of cellular organelles: Calnexin (endoplasmic reticulum); GOLPH4 (Golgi bodies); EEA1 (early endosome); and LAMP2 (lysosome). Cells were treated with 10nM sirolimus, 10ÁM alpelisib, or vehicle (DMSO) in order to assess changes in protein localization and/or expression resulting from drug treatment.
Results: FACS analysis demonstrated that cell surface CD31 and VECADHERIN were lower in VMECs compared to control human microvascular ECs (HMVECs). Fluorescent staining for CD31 and markers of cellular organelles revealed that CD31 was erroneously trafficked to early endosomes and lysosomes rather than to the cell surface of VMECs. Treatment of VMECs with sirolimus (mTOR inhibitor) and alpelisib (PI3K inhibitor) with subsequent analysis of cell surface protein expression by FACS demonstrated changes in level of protein expression. The surface expression of CD31 was increased with sirolimus and alpelisib treatment while surface expression of VEGFR1, VEGFR2, and VEGFR3 were decreased. Treatment of VMECs with sirolimus and subsequent IHC staining similarly increased localization of CD31 to the cell surface relative to vehicle-treated VMECs.
Conclusions: Our results demonstrated protein trafficking defects in VMECs, which is a previously unappreciated pathology of VMs. Treatment with sirolimus and alpelisib were shown to affect protein localization and trafficking, with higher levels of CD31 appropriately trafficking to the cell surface. These results may represent an additional mechanism of action of sirolimus and alpelisib in addition to their known anti-proliferative effect which contributes to the partial clinical efficacy of these drugs. Future research may further describe this model and exploit these defects as a novel therapeutic target. Pharmacotherapies that can correct protein mislocalization may be more efficacious in the treatment of VMs than current therapies.
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