Fibronectin-based isolation of valve interstitial cell subpopulations: relevance to valve disease.
Myxomatous mitral valves (MVs) contain elevated proportions of unique cell populations such as myofibroblasts. Without a reliable technique to isolate such cell populations, however, it has been difficult to study the role of these cells. The goal of this study was to use fibronectin (FN) to isolate distinct cell subpopulations from normal porcine MVs. Cells from porcine posterior MV leaflets were separated based on time-dependent adhesion to either tissue culture plastic (TCP) flasks or FN-coated flasks. The resultant "FAST" and "SLOW" adhering subpopulations from each technique were phenotyped using flow cytometry and immunocytochemistry to detect expression of myofibroblast markers, enzymes for collagen synthesis, and MAP kinases. Compared with FN SLOW, FN FAST showed significantly higher expression of prolyl 4-hydroxylase, heat shock protein-47 (HSP47), smooth muscle alpha-actin (SMalphaA), nonmuscle myosin (Smem), extracellular-related signaling kinase (ERK) 1, ERK2, and phosphorylated-ERK. In contrast, TCP FAST showed higher expression of only HSP47, SMalphaA, and Smem compared with TCP SLOW. In conclusion, differential adhesion to FN successfully separated a myofibroblast-like subpopulation from the posterior leaflet of the MV. This subpopulation may be useful in studying myxomatous MV disease, although additional studies remain to verify that this myofibroblast-like population resembles that observed in myxomatous MV disease.
Stephens
Elizabeth H
EH
Department of Bioengineering, Rice University, Houston, Texas 77005, USA.
Huynh
Thanh N
TN
Cieluch
Jennifer D
JD
Grande-Allen
K Jane
KJ
D000818:Animals; D002448:Cell Adhesion / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D005353:Fibronectins / Q000494:pharmacology*; D005434:Flow Cytometry; D005453:Fluorescence; D006349:Heart Valve Diseases / Q000473:pathology*; D006801:Humans; D007150:Immunohistochemistry; D008943:Mitral Valve / Q000166:cytology*; D010969:Plastics / Q000494:pharmacology; D015203:Reproducibility of Results; D034421:Sus scrofa; D013997:Time Factors; D046509:Tissue Culture Techniques
D005353:Fibronectins; D010969:Plastics