Cell Surface Indicators of Vascular Senescence

National Institute On Aging

Investigators

Abstract

Senescent cells contribute greatly to the development of vascular diseases including atherosclerosis, an age-associated disease without cure, making senescent vascular cells important therapeutic targets. While senolytics have demonstrated moderate effectiveness in animal models, the need for increasingly selective and specific therapeutics remains. We hypothesize that senescent vascular cells display unique cell surface proteins (surfaceome) compared to proliferating cells. We further hypothesize that antibody-drug conjugates directed at senescent cell surface proteins may be an effective therapeutic intervention in mouse models of atherosclerosis. We will perform mass spectrometry on cell surface proteins isolated from senescent vascular cells (Objective 1) and further investigate the transcriptomes associated with newly identified cell surface proteins using CITE-seq (Objective 2). Lastly, we will determine the conservation of senescent cell surface proteins in mouse vascular cells and test antibody-drug conjugates (ADCs) directed at our shared protein targets in murine atherosclerosis (Objective 3). In sum, we propose to generate vascular senescent cell-specific ADCs capable of selectively removing senescent cells to improve outcomes associated with atherosclerosis. In the preliminary phase of Objective 1, cell surface capture and mass spectrometry of proliferating human (h)VSMCs identified 4760 peptides and 1753 unique proteins following. To confirm the proteins identified are indeed cell surface proteins, we conducted gene ontology analysis. The results of the analysis revealed 84% of the proteins were membrane proteins and 28% were considered cell surface proteins. We performed the same experiment in proliferating human aortic ECs (hAECs) once again identifying 2589 peptides and 1064 unique proteins. Through gene ontology analysis, we confirmed 86% of proteins were considered membrane proteins and 33.8% were specifically cell surface proteins. Interestingly, 266 proteins were shared among the proliferating hVSMCs and hAECs such as ICAM1, ICAM2 and VEGFR, all proteins related to atherosclerosis. In the next phase of the project, we performed the same experiment and include both proliferating and senescent VSMCs and ECs to determine the unique cell surface proteome of senescent vascular cells. Results of this study identified 913 deamidated proteins in ECs, with 247 upregulated and 614 downregulated during senescence. Similarly, found 913 deamidated proteins in hVSMCs, with 150 upregulated and 625 downregulated during senescence. A closer look at the surfaceome of senescent ECs that selected only statistically significant annotated surface proteins recognized 17 proteins increased on senescent ECs and 49 proteins lost with senescence. Among these proteins were those involved in cytokine-receptor signaling, cell adhesion, metabolic processes, and lysosomal activity. Next, we will validate these 17 proteins by flow cytometry or cell-surface western blot for application in CITE-seq on senescent ECs (Objective 2).