Fibronectin-based isolation of valve interstitial cell subpopulations: relevance to valve disease

Abstract

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.

Fig. 1

Schematic illustrating the process of isolating subpopulations from plastic flasks. The same method was used for subpopulations isolated from fibronectin-coated flasks, except with different time increments.

Fig. 2

A) Adhesion to TCP over time using subpopulations previously separated using TCP. B) Adhesion to TCP over time using subpopulations previously separated using FN-coated flasks. Our laboratory has previously reported that such VIC subpopulations retain their adhesive properties over multiple passages.

Fig. 3

A) Flow cytometry marker fluorescence of TCP differential adhesion subpopulations. Error bars indicate standard error of the mean, as the graph includes data from 3 independent experiments. *=p<0.05. B) Flow cytometry marker fluorescence of FN differential adhesion subpopulations. Error bars indicate standard error of the mean, as the graph includes data from 3 independent experiments. *=p<0.05, ^=p<0.075.

Fig. 4

A) Ratio of FN FAST to FN SLOW flow cytometry marker mean fluorescence for human myxomatous VIC subpopulations. Error bars indicate standard error of the mean. B) Immunohistochemical staining illustrates the presence of fibronectin in both normal (left, 79 year-old male) and myxomatous heart valves (right, 70 year-old male). Scale bar represents 200 µm.

Fig. 5

A) Immunocytochemistry marker integrated optical density per cell of TCP differential adhesion subpopulations. Error bars indicate standard deviation, as the graph includes data from one representative experiment. Data between experiments showed the same differences between cell subpopulations, but varied in integrated optical density. *=p<0.05. B) Immunocytochemistry marker integrated optical density per cell of FN differential adhesion subpopulations. Error bars indicate standard deviation, as the graph includes data from one representative experiment. Data between experiments showed the same differences between cell subpopulations, but varied in integrated optical density. *=p<0.05.

Fig. 6

Representative images of immunocytochemistry for given markers on TCP differential adhesion subpopulations. Images were captured using 10× objective. Scale bar represents 100 µm and applies to all images.