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

Geanina Voicu,Cristina Ana Constantinescu,Manuela Calin,Cristina Mariana Uritu,Ionel Droc,Elena Valeria Fuior,Daniela Rebleanu,Maya Simionescu,Letitia Ciortan,Mariana Pinteala,Ileana Manduteanu

doi:10.3390/pharmaceutics12060507

Geanina Voicu, Cristina Ana Constantinescu + Show 9 more

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https://doi.org/10.3390/pharmaceutics12060507

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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.

Highlights

Calcific aortic valve disease (CAVD) is a common disorder that increases in prevalence with age or in pathological conditions
SHCLND-NM_004348) were from SIGMA-Aldrich (Merck KGaA, Darmstadt, Germany), fetal bovine serum (FBS), penicillin and streptomycin from Gibco (ThermoFisher Scientific, Waltham, MA, USA); cell culture dishes were from TPP® (Trasadingen, Switzerland); 2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide (XTT), rabbit polyclonal antibodies against Runx2, bone morphogenic protein 4 (BMP4), goat polyclonal antibody against α-smooth muscle actin (SMA), SuperSignal West Dura were from Thermo Fisher Scientific (Waltham, MA, USA); goat polyclonal antibodies against bone sialoprotein (BSP) and alkaline phosphatase (ALP)were from R&D Systems (Minneapolis, MN, USA); rabbit polyclonal antibody against osteopontin was from Novus Biologicals (Centennial, CO, USA); DAPI was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); X-tremeGene 9 was from Roche (Basel, Switzerland); Cy3-labeled plasmid was from Mirus Bio (Madison, WI, USA)
The average hydrodynamic diameter and ζ-potential of nano-polyplexes C60-PEI/short hairpin RNAs (shRNAs) plasmid formed at different N/P ratio were measured after 1:1000 dilution in distilled water

Summary

Introduction

Calcific aortic valve disease (CAVD) is a common disorder that increases in prevalence with age or in pathological conditions. The pathology is characterized by the thickening of the valve leaflets, due to fibrosis and calcification processes, causing impairment of valvular motion and obstruction of left ventricular blood outflow, leading to cardiac hypertrophy and heart failure [2]. The pathogenesis of this disease includes genes and proteins implicated in the subendothelial accumulation of atherogenic lipoproteins, chronic inflammation, fibrosis, neovascularization and ectopic calcification [3,4]. There are numerous similarities between CAVD and atherosclerosis, the main treatment of the latter, namely the administration of statins, has no effect on CAVD progression [6].

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