Robust Generation of Quiescent Porcine Valvular Interstitial Cell Cultures

Abstract

Background: Valvular interstitial cells (VICs) in the healthy aortic valve leaflet exhibit a quiescent phenotype, with <5% of VICs exhibiting an activated phenotype. Yet, in vitro culture of VICs on tissue culture polystyrene surfaces in standard growth medium results in rapid transformation to an activated phenotype in >90% of cells. The inability to preserve a healthy VIC phenotype during in vitro studies has hampered the elucidation of mechanisms involved in calcific aortic valve disease. This study describes the generation of quiescent populations of porcine VICs in 2-dimensional in vitro culture and their utility in studying valve pathobiology.

Methods and results: Within 4 days of isolation from fresh porcine hearts, VICs cultured in standard growth conditions were predominantly myofibroblastic (activated VICs). This myofibroblastic phenotype was partially reversed within 4 days, and fully reversed within 9 days, following application of a combination of a fibroblast media formulation with culture on collagen coatings. Specifically, culture in this combination significantly reduced several markers of VIC activation, including proliferation, apoptosis, α-smooth muscle actin expression, and matrix production, relative to standard growth conditions. Moreover, VICs raised in a fibroblast media formulation with culture on collagen coatings exhibited dramatically increased sensitivity to treatment with transforming growth factor β1, a known pathological stimulus, compared with VICs raised in either standard culture or medium with a fibroblast media formulation.

Conclusions: The approach using a fibroblast media formulation with culture on collagen coatings generates quiescent VICs that more accurately mimic a healthy VIC population and thus has the potential to transform the study of the mechanisms of VIC activation and dysfunction involved in the early stages of calcific aortic valve disease.

Keywords: aortic valve; calcific aortic valve disease; differentiation; myofibroblasts; quiescence; valvular interstitial cell.

Figure 1

Immunohistochemical staining for activated VIC markers in passage 0 VIC cultures. VICs expressed vimentin and the myofibroblastic markers αSMA, calponin, and SM22 after culture in control conditions. Detected proteins are stained red, and nuclei are stained blue. Scale bar represents 100 μm. αSMA indicates α‐smooth muscle actin; SM22, smooth muscle protein 22‐α; VIC, valvular interstitial cell.

Figure 2

VICs cultured in FIB or FIB‐Coll conditions exhibit a more elongated and spindled morphology compared with control VICs. A, Staining for phalloidin (red), nuclei are counterstained blue. Scale bar represents 100 μm. Quantification of (B) cell shape and (C) aspect ratio for VICs cultured in control, FIB, or FIB‐Coll conditions. *P<0.01 compared with control VICs at the same time point. ^{^} P<0.01 compared with day 4 VICs cultured in the same condition. n=6. FIB indicates fibroblast media formulation; FIB‐Coll, fibroblast media formulation with culture on collagen coatings; VIC, valvular interstitial cell.

Figure 3

VIC proliferation and apoptosis decrease after culture in FIB‐Coll. A, Quantification of the percentage of proliferating cells after 8 hours of incubation with EdU. B, Evaluation of apoptosis levels through a caspase 3/7 activity assay. *P<0.01 compared with control VICs at the same time point. ^{^} P<0.01 compared with day 4 VICs cultured in the same condition. ^# P<0.01 for comparisons indicated on graph. n=6 to 8. FIB indicates fibroblast media formulation; FIB‐Coll, fibroblast media formulation with culture on collagen coatings; VIC, valvular interstitial cell.

Figure 4

Expression of myofibroblastic markers decreases dramatically after culture in FIB and FIB‐Coll. A through C, Gene expression analysis of (A) ACTA2, (B) CNN2, and (C) SM22 after 4, 9, and 14 days of culture in control, FIB, or FIB‐Coll conditions. D through F, Evaluation of protein expression for the same markers: (D) αSMA, (E) calponin, and (F) SM22. MFI levels were obtained through flow cytometry. G, Immunostaining for myofibroblastic markers. Scale bar represents 100 μm. *P<0.01 compared with control VICs at the same time point. ^{^} P<0.01 compared with day 4 VICs cultured in the same condition. ^# P<0.01 for comparison indicated on graph. n=6. αSMA indicates α‐smooth muscle actin; FIB, fibroblast media formulation; FIB‐Coll, fibroblast media formulation with culture on collagen coatings; MFI, mean fluorescence intensity; SM22, smooth muscle protein 22‐α; VIC, valvular interstitial cell.

Figure 5

Extracellular matrix production decreases after culture in FIB and FIB‐Coll. A, COL1A1 gene expression was evaluated after 4, 9, and 14 days of culture in control, FIB, or FIB‐Coll conditions. Fibronectin (B) gene and (C) protein expression were also assessed. *P<0.01 compared with control VICs at the same time point. ^{^} P<0.01 compared with day 4 VICs cultured in the same condition. ^# P<0.01 for comparison indicated on graph. n=6. FIB indicates fibroblast media formulation; FIB‐Coll, fibroblast media formulation with culture on collagen coatings; VIC, valvular interstitial cell.

Figure 6

TGF‐β1 induces changes in cell morphology, proliferation, and apoptosis in VICs generated via FIB‐Coll culture. VICs raised in control, FIB, or FIB‐Coll culture were treated with 0, 0.25, or 1 ng/mL TGF‐β1 for 5 days on tissue culture polystyrene. A, Staining for phalloidin (green), nuclei are counterstained blue. Scale bar represents 100 μm. Quantification of (B) cell shape, (C) aspect ratio, (D) proliferation, and (E) apoptosis. *P<0.01 compared with 0 ng/mL in the same condition (control, FIB, or FIB‐Coll). ^{^} P<0.01 compared with control VICs treated with the same amount of TGF‐β1. n=6 to 8. FIB indicates fibroblast media formulation; FIB‐Coll, fibroblast media formulation with culture on collagen coatings; TGF‐β1, transforming growth factor β1; VIC, valvular interstitial cell.

Figure 7

VICs raised in FIB‐Coll culture respond more strongly to TGF‐β1 treatment than VICs raised in FIB and control culture. Myofibroblastic marker and ECM expression were also analyzed in response to TGF‐β1 treatment. The extent of VIC activation was evaluated through gene expression analysis of (A) ACTA2 and (B) SM22 and (C) immunofluorescent staining for αSMA and SM22. Red indicates positive staining for each marker; nuclei are counterstained blue. Scale bar represents 100 μm. ECM production was assessed through quantitative reverse transcriptase–polymerase chain reaction for (D) COL1A1 and (E) FN, as well as an in situ ELISA for (F) fibronectin protein deposition. *P<0.01 compared with 0 ng/mL in the same condition (control, FIB, or FIB‐Coll). ^# P<0.01 for comparison indicated on graph. n=6. αSMA indicates α‐smooth muscle actin; ECM, extracellular matrix; FIB, fibroblast media formulation; FIB‐Coll, fibroblast media formulation with culture on collagen coatings; SM22, smooth muscle protein 22‐α; TGF‐β1, transforming growth factor β1; VIC, valvular interstitial cell.