Tracing G-Protein-Mediated Contraction and Relaxation in Vascular Smooth Muscle Cell Spheroids

Abstract

Analyses of G-protein-mediated contraction and relaxation of vascular smooth muscle cells (VSMCs) are usually hampered by a rigid growth surface and culture conditions promoting cell proliferation and a less contractile phenotype. Our studies indicated that mouse aortic VSMCs cultured in three-dimensional spheroids acquire a quiescent contractile status while decreasing the baseline G-protein-dependent inositolphosphate formation and increasing the expression of endothelin receptor type A (Ednra). Endothelin-1 (ET-1) promoted inositolphosphate formation in VSMC spheroids, but not in VSMCs cultured under standard conditions. To trace ET-1-mediated contraction of VSMC spheroids, we developed an assay by adhering them to collagen hydrogels and recording structural changes by time-lapse microscopy. Under these conditions, mouse and human VSMC spheroids contracted upon treatment with ET-1 and potassium chloride or relaxed in response to caffeine and the prostacyclin analogue Iloprost. ET-1 activated AKT-, MKK1-, and MKK3/6-dependent signaling cascades, which were inhibited by an overexpressing regulator of G-protein signaling 5 (Rgs5) to terminate the activity of Gα subunits. In summary, culture of VSMCs in three-dimensional spheroids lowers baseline G-protein activity and enables analyses of both contraction and relaxation of mouse and human VSMCs. This model serves as a simple and versatile tool for drug testing and investigating G-protein-depending signaling.

Characterisation of Lipoma-Preferred Partner as a Novel Mechanotransducer in Vascular Smooth Muscle Cells

“…To study whether the loss of LPP affects the contractile and dilatory capacity of the VSMCs in a 3D environment, we employed a spheroid contraction assay [ 16 ]. After testing several contractile agents, we opted for potassium chloride (KCl), which, by way of depolarization and hence activation of voltage-dependent L-type calcium channels (LTCC, not shown), produced a stable reproducible contraction of VSMCs grown in spheroids.…”

“…For the spheroid contraction assay , spheroids were also generated by the hanging drop method as described above and harvested in cell culture medium with 15% FCS, but they were plated on top of the collagen gel matrices, employing a recently established protocol [ 16 ]. Briefly, for the preparation of collagen gels, all ingredients were held on ice.…”