Single-Cell Analysis of Smooth Muscle Cells

Smooth muscle cells (SMCs) have been heavily implicated in the progression of vascular disease: aberrant proliferation of SMCs leads to narrowing of the blood vessel, and deposition of ectopic calcified deposits compromises the structural integrity of the vessel wall. Since the 1960s, scientists have characterized SMCs as a largely inactive homogeneous population that dedifferentiates to become proliferative and migratory upon vascular injury. However, other studies have suggested that the tunica media layer is comprised of separate subpopulations of SMCs that are not interchangeable. Furthermore, the source of SMCs in atherosclerotic plaques and neointima formation has been proven to be oligoclonal, or derived from a few cells; dedifferentiation of SMCs, which experts describe as a widespread and escalating process undergone by SMCs, should result in a distinctly polyclonal origin of SMCs in neointima and plaques. To investigate heterogeneity among SMCs, single-cell analysis was necessary. For our experiments, RFP+ SMCs were dissociated from the aorta of SMMHC-CreERT2/LoxP-tdTomato transgenic mice and immediately used for strict primary culture or lysis of cell contents. Single-cell analysis of functional contractility showed that although all SMCs were contractile, the level of contractile force was heterogeneous among SMCs. Moreover, the levels of cell traction force and contractile force after exposure to a vasoconstrictor peptide did not correlate with the expression level of essential contractile proteins, α-SMA, CNN-1, or SMMHC. These results indicated that the common practice of characterizing phenotypes of SMCs based on contractile state may be misguided, and direct assessment of the pathogenic behavior of SMCs, such as proliferation and differentiation, may be more appropriate to defining subpopulations of SMCs. To optimize the efficiency of obtaining single-cell clones, we integrated optoelectronic tweezers (OET) with a micropatterned substrate designed for clonal culture. Using the light-induced dielectrophoretic force of OET, single SMCs were selected and positioned in an array of ECM-conjugated islands surrounded by PEG. Through behavioral analysis of single-cell clonal colonies, two subpopulations of SMCs were distinguished: proliferative and migratory SMCs that were capable of osteogenic differentiation and calcium-phosphate deposition as well as non-proliferative SMCs with extensive cell spreading and no differentiation potential. Additionally, the protein expression of SMCs from the native vessel and from primary culture were compared through immunostaining of cultured clones and single-cell Western blotting of cells that were dissociated from tissue and directly analyzed. From the protein expression profiles of SMMHC and α-SMA, we determined that clustering individual SMCs based on proliferative potential, protein expression of SMCs from normal vasculature, or protein expression of SMCs in primary culture all outlined the same subpopulations of SMCs. Therefore, SMCs are heterogeneous within the normal blood vessel wall. Our body of evidence suggests that only a minority subpopulation of SMCs is capable of undergoing dedifferentiation to proliferate or osteogenic differentiation to deposit ectopic calcium. Consequently, upon perturbation to the vessel wall, these phenotypically plastic SMCs may launch the response of proliferation and differentiation that leads to the progression of vascular disease.

Advisor: Li, Song