Modulation of Human Valve Interstitial Cell Phenotype and Function Using a Fibroblast Growth Factor 2 Formulation

Abstract

Valve interstitial cells (VICs) are fibroblastic in nature however in culture it is widely accepted that they differentiate into a myofibroblastic phenotype. This study assessed a fibroblast culture media formulation for its ability to maintain the phenotype and function of VICs as in the intact healthy valve. Normal human VICs were cultured separately in standard DMEM and in fibroblast media consisting of FGF2 (10ng/ml), insulin (50ng/ml) and 2% FCS for at least a week. Cell morphology, aspect ratio, size, levels and distribution of protein expression, proliferation, cell cycle, contraction and migration were assessed. Some VICs and some valve endothelial cells expressed FGF2 in valve tissue and this expression was increased in calcified valves. VICs in DMEM exhibited large, spread cells whereas VICs in fibroblast media were smaller, elongated and spindly. Aspect ratio and size were both significantly higher in DMEM (p<0.01). The level of expression of α-SMA was significantly reduced in fibroblast media at day 2 after isolation (p<0.01) and the expression of α-SMA, SM22 and EDA-fibronectin was significantly reduced in fibroblast media at days 7 and 12 post-isolation (p<0.01). Expression of cytoskeletal proteins, bone marker proteins and extracellular matrix proteins was reduced in fibroblast media. Proliferation of VICs in fibroblast media was significantly reduced at weeks 1 (p<0.05) and 2 (p<0.01). Collagen gel contraction was significantly reduced in fibroblast media (p<0.05). VICs were found to have significantly fewer and smaller focal adhesions in fibroblast media (p<0.01) with significantly fewer supermature focal adhesions in fibroblast media (p<0.001). Ultrastructurally, VICs in fibroblast media resembled native VICs from intact valves. VICs in fibroblast media demonstrated a slower migratory ability after wounding at 72 hours (p<0.01). Treatment of human VICs with this fibroblast media formulation has the ability to maintain and to dedifferentiate the VICs back to a fibroblastic phenotype with phenotypic and functional characteristics ascribed to cells in the intact valve. This methodology is fundamental in the study of normal valve biology, pathology and in the field of tissue engineering.

Robust Generation of Quiescent Porcine Valvular Interstitial Cell Cultures

“…Latif et al published a full rationale for the selection of the cocktail present in FIB media. 11 Briefly, supplementation with 2% FBS was selected over the traditional 10% concentration to reduce the amount of growth factors present in the medium. In addition, insulin and FGF-2 were selected for their reported ability to regulate growth 18 and decrease VIC aSMA expression, 19 respectively.…”

“…14,20,21 Our experiments found that collagen coating alone can decrease the expression of contractile proteins but significantly increases VIC proliferation and thus is unable to yield a qVIC phenotype ( Figure S6); others have similarly found that collagen is not sufficient to yield qVIC cultures. 11,14 We hypothesized, however, that the combination of a collagen coating with soluble factors (ie, FIB-Coll condition) might provide additional deactivation cues. This hypothesis was confirmed by the data: Although the FIB formulation alone was sufficient to generate significant reductions in myofibroblast markers, culture in FIB-Coll achieved the same results, sometimes sooner (4 versus 9 days for ECM production) and at other times more dramatically (eg, proliferation and apoptosis), compared with FIB alone.…”

“…Aortic valve leaflets were excised from male porcine hearts, and VICs were immediately isolated via collagenase digestion, as described previously. 11,13 The resulting passage 0 VICs were seeded and expanded on TCPS in low-glucose DMEM (Sigma-Aldrich) supplemented with 10% heat-inactivated FCS (Sigma-Aldrich), 150 U/mL penicillin/streptomycin (Sigma-Aldrich), and 2 mmol/L L-glutamine (Sigma-Aldrich). At 4 days after isolation, VICs were examined via immunocytochemistry and flow cytometry for expression of myofibroblastic markers.…”

Shape-Specific Nanoceria Mitigate Oxidative Stress-Induced Calcification in Primary Human Valvular Interstitial Cell Culture

“…We further characterized the differences between normal and diseased hVICs by quantifying mRNA expression of key VIC markers including VIM (qui escent VIC marker 43 ), α - SMA (activated VIC marker 43 ), RUNX - 2 and OPN (osteoblastic VIC marker 44 ). Compared to the normal hVICs, both diseased hVICs demonstrated higher RUNX - 2 and OPN levels, while showing similar mRNA levels of VIM (Fig.…”

Fibroblast Growth Factor 2 as an Antifibrotic: Antagonism of Myofibroblast Differentiation and Suppression of Pro-Fibrotic Gene Expression

“…TGF-β3 has demonstrated antifibrotic activity in vivo [89], and elevated TGF-β3/TGF-β1 ratios have been associated with scarless healing phenotypes including those of oral mucosa [90] and those induced by tattooing [91]. In human valvular interstitial cells, which are of mesenchymal origin and are largely myofibroblastic in phenotype, addition of exogenous FGF2 reduced cell area, contractile activity, and a host of myofibroblast markers and deposition of ECM proteins [92]. Valvular interstitial cells demonstrate a classical activation response to TGF-β, and it has been reported that FGF2 has the ability to impede this TGF-β response in an ERK-dependent manner, likely via a decrease in SMAD-driven promoter activity through inhibition of SMAD3 nuclear accumulation [93].…”