Substrate properties influence calcification in valvular interstitial cell culture

Abstract

Background and aim of the study: Valvular calcification is an active, cell-mediated process that results in significant morbidity and mortality. In standard culture, valvular interstitial cells (VICs) elicit significant calcification as a result of myofibroblast activation, and this limits their use in characterization studies. The study aim was to identify culturing substrates that would suppress atypical VIC calcification, and to investigate culture substrates representing a more physiological system.

Methods: Several culture platforms were selected to compare and contrast the influence of biochemical and mechanical properties on VIC calcification. Substrates investigated included: tissue culture polystyrene (TCPS), TCPS coated with either fibronectin or fibrin, and an elastic poly(ethylene glycol) (PEG) hydrogel, also with fibronectin or fibrin coupled to the surface. Experiments were repeated with profibrotic growth factor transforming growth factor-beta 1 (TGF-beta1). VIC calcification was characterized by calcific nodule formation, alkaline phosphatase activity and calcium accumulation. Gene and protein expression of alpha smooth muscle actin (aSMA) and core binding factor-1 (CBFa-1) were analyzed with qRT-PCR and immunostaining.

Results: Unmodified TCPS substrates had an innate ability to promote the markers of calcification studied. The addition of TGF-beta1 enhanced levels of all osteoblastic markers studied. When TCPS surfaces were modified with fibronectin, all markers for calcification were repressed, but alphaSMA - a marker for myofibroblastic activity was unchanged. Meanwhile, fibrin-modified TCPS surfaces enhanced calcification over unmodified TCPS substrates. On soft PEG hydrogels, all markers for calcification were repressed, regardless of the surface chemistry, while alphaSMA expression remained unaffected.

Conclusion: Collectively, VIC properties are highly linked to the culture microenvironment. Both, the biochemical and mechanical environment of tissue culture has an effect on the spontaneous calcification of VICs, and may also have a profound effect on their molecular properties, as related to an understanding of the disease process in vivo.

Figure 1

Characterization of VIC calcification cultured on TCPS substrates. (a) Calcific nodules per cm² culture area without (grey) and with 1 ng/mL TGF-β1 (black). (b) Images of nodule morphology and apoptotic staining without (i) & (ii) and with 1 ng/mL TGF-β1 (iii) & (iv). Cells positive for early apoptosis stained green with annexin v and cells positive for late apoptosis and necrosis stained positive with red staining for propidium iodide. Nodules were larger in the absence TGF-β1 but were more abundant on TGF-β1 supplemented surfaces (data not shown). (c) ALP activity and (d) Total calcium accumulation of VICs cultured on different TCPS coated substrates. All data was analyzed at day 6 of culture. Data was normalized to total protein content and is expressed as percent of control of the unmodified TCPS substrate without TGF-β1. (* denotes p ≤ 0.01 over the unmodified TCPS control, n = 5 for all samples)

Figure 2

Characterization of calcification for VICs cultured on PEG hydrogels modified with ECM proteins for six days. (a) Nodules per cm² culture area without (grey) and with 1 ng/mL TGF-β1(black). (b) ALP activity of cells on PEG culture platforms. (c) Total calcium uptake of VICs cultured on different TCPS substrates. The dashed line represents the unmodified TCPS substrate assay values from Figure 1 for comparison purposes. Data has been normalized to total protein content and is expressed as percent of control of the unmodified TCPS substrate without TGF-β1. All data was statistically significant compared to TCPS control. (p ≤ 0.01, n = 5 for all samples))

Figure 3

qRT-PCR analyses of mRNA expression of VIC myofibroblast and osteoblast markers. (a) αSMA mRNA expression on TCPS surfaces without (grey) and with 1 ng/mL TGF-β1 (black). (b) CBFa-1 expression on TCPS surfaces without and with 1 ng/mL TGF-β1. (c) αSMA expression on PEG hydrogels without and with 1 ng/mL TGF-β1 cultured. (d) CBFa-1 expression on PEG hydrogels without and with 1 ng/mL TGF-β1. All samples were cultured for six days. Results are normalized to GAPDH expression and fold change calculated using the Pfaffl method. All samples are expressed as percent of the unmodified TCPS surfaces. (* denotes p ≤ 0.01 over the unmodified TCPS control, n = 8 for all samples) The dashed line represents the unmodified TCPS surface levels as a reference.

Figure 4

Representative images of VICs cultured for 6 days and immunostained for VIC myofibroblast and osteoblast markers on TCPS or PEG hydrogel culture platforms. (a), (c), (e), (g), and (i) represent images of immunostaining for myofibroblastic marker αSMA, whereas (b), (d), (f), (h), and (j) represent immunostaining images for osteoblastic marker CBFa-1. For all images, the target protein (αSMA or CBFa-1) is stained green and cell nuclei counterstained blue (DAPI). DAPI images have been omitted for pictures with nodules present due to interference in visualizing the green stain. Scale bar represents 50 μm for all images.