Nano-Polyplexes Mediated Transfection of Runx2-shRNA Mitigates the Osteodifferentiation of Human Valvular Interstitial Cells

Abstract

Calcific aortic valve disease (CAVD) is a progressive disorder that increases in prevalence with age. An important role in aortic valve calcification is played by valvular interstitial cells (VIC), that with age or in pathological conditions acquire an osteoblast-like phenotype that advances the disease. Therefore, pharmacological interventions aiming to stop or reverse the osteoblastic transition of VIC may represent a therapeutic option for CAVD. In this study, we aimed at developing a nanotherapeutic strategy able to prevent the phenotypic switch of human aortic VIC into osteoblast-like cells. We hypothesize that nanocarriers designed for silencing the Runt-related transcription factor 2 (Runx2) will stop the progress or reverse the osteodifferentiation of human VIC, induced by high glucose concentrations and pro-osteogenic factors. We report here the potential of fullerene (C60)-polyethyleneimine (PEI)/short hairpin (sh)RNA-Runx2 nano-polyplexes to efficiently down-regulate Runx2 mRNA and protein expression leading subsequently to a significant reduction in the expression of osteogenic proteins (i.e. ALP, BSP, OSP and BMP4) in osteoblast-committed VIC. The data suggest that the silencing of Runx2 could represent a novel strategy to impede the osteoblastic phenotypic shift of VIC and the ensuing progress of CAVD.

Keywords: Runx2; calcific aortic valve disease; nanocarriers; osteodifferentiation.; shRNA; valvular interstitial cells.

Figure 1

Characterization of fullerene (C60)-polyethyleneimine (PEI)/short hairpin (sh) RNA plasmid nano-polyplexes. (A) Average hydrodynamic diameter and ζ-potential of C60-PEI/shRNA plasmid polyplexes at different N/P ratios. Results are reported as mean ± S.D. for three individual measurements. (B) Agarose gel retardation assay performed for free shRNA plasmid and C60-PEI/shRNA plasmid polyplexes at different N/P ratios (200 ng shRNA plasmid/lane). (C) Viability of VIC exposed for 48 h to different N/P ratios of C60-PEI/shRNA plasmid nano-polyplexes. Data are presented as mean ± S.D. of three experiments made in three replicates (n = 9). * p < 0.05 and *** p < 0.001 versus control cells. (D,E) Uptake of C60-PEI/Cy3-labeled plasmid nano-polyplexes (N/P = 25) by VIC, observed by fluorescence microscopy (D) and by flow cytometry analysis (E). (F) Expression of fluorescent protein in VIC transfected with C60-PEI/pEYFP plasmid at N/P ratio of 15, 20 and 25 or with a commercial transfection reagent, as revealed at 48 h after transfection by fluorescence microscopy (scale bar 200 µm).

Figure 2

Time course expression of osteogenesis-related markers in valvular interstitial cells (VIC) exposed for 2, 7, 14 and 21 days to C (lane 1), high glucose concentration (HG) (lane 2), osteogenic medium (OM) (lane 3) or a combination of HG and OM (HGOM) (lane 4). The level of protein expression of α-smooth muscle actin (α-SMA) (A), runt-related transcription factor 2 (Runx2) (B), alkaline phosphatase (ALP) (C) and bone sialoprotein (BSP) (D) was determined by Western blot for different activation conditions, quantified by reporting to β-actin level at different periods and normalized to the day 2 values. Inserts show the level of protein expression at day 2 expressed as fold induction over control cells exposed to 5 mM glucose. Results represent the means ± S.D. of three independent experiments performed in duplicate (n = 6). Representative blots are shown above the graphs. * p < 0.05, ** p < 0.01, *** p < 0.001 versus values determined at day 2. & p < 0.05, && p < 0.01. &&& p < 0.001 versus values determined at day 7; Inserts: * p < 0.05, ** p < 0.01, *** p < 0.001 versus control.

Figure 3

Down-regulation of Runx2 mRNA (A) and protein (B) expression in HGOM-treated VIC and transfected with C60-PEI/shRNA-Runx2 plasmids polyplexes containing different shRNA sequences specific for Runx2 (sh_1, sh_2 and sh_3) and then investigated at 48 h after transfection. Results, normalized to β-actin, were expressed as mean ± S.D. of three experiments made in duplicate (n = 6) and represent fold change relative to HGOM condition (considered as 1). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with HGOM.

Figure 4

Down-regulation of Runx2 expression by C60-PEI/shRNA-Runx2 plasmids polyplexes reduces the expression of osteoblast differentiation markers ALP (A), OSP (B), BSP (C) and BMP-4 (D) in VIC exposed to HGOM. Results, normalized to β-actin, represent the means ± S.D. of three independent experiments performed in duplicate (n = 6) and represent fold change relative to HGOM condition (considered as 1). Representative blots are shown above the graphs. * p < 0.05, ** p < 0.01, *** p < 0.001 versus HGOM.

Figure 5

Down-regulation of Runx2 expression using C60-PEI/shRNA-Runx2 plasmids inhibits ALP activity in VIC exposed to HGOM. Quantitative assay at 7, 14 and 21 days (A) and histochemical detection at 14 days (B) of ALP activity (scale bar 100 µm). Data are presented as mean ± S.D. of two experiments made in three replicates (n = 6). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to HGOM; & p < 0.05, && p < 0.01 versus HGOM at day 7.