Smooth muscle cell spheroids as 3D model of phenotypic plasticity and matrix deposition revealed by 2D–3D proteomics

Vascular smooth muscle cells (VSMCs) are regulators of vascular homeostasis and play a role in cardiovascular diseases, including atherosclerosis and aortic aneurysms. Together with endothelial cells (ECs), they control vascular remodeling by producing extracellular matrix (ECM), regulating the expression of adhesion molecules, and releasing signaling factors. Since changes in the ECM composition critically determine disease progression, models that reproduce authentic cell-ECM interactions are indispensable. Two-dimensional (2D) cultures fail to capture the mechanical integration of vascular cells within their ECM environment. We established a scaffold-free three-dimensional (3D) spheroid model of murine and human aortic VSMCs derived from heart failure patients to mimic their multicellular organization, cell plasticity, and interactions with the ECM. Using mass spectrometry, we provided to our knowledge the first comprehensive proteomic profiling of relevant 3D-cultured VSMCs in comparison to conventional 2D culture. VSMCs formed compact spheroids with a protein signature indicative of a synthetic phenotype, characterized by active ECM organization, adhesion, and energy homeostasis pathways. We demonstrated that the spheroid protocol can also be used to generate EC spheroids, offering opportunities to study VSMC-EC crosstalk. Our findings show that VSMC 3D culture promotes the transition from contractile to synthetic VSMC phenotypes with ECM deposition, providing a translational in vitro system to investigate VSMC-mediated repair mechanisms and vascular remodeling in cardiovascular diseases.