As described for actual aortic valve mineralization,1 metastatic-like calcification of primarily cultured aortic valve interstitial cells (AVICs) was previously found to consist in intracellular release of acid-phospholipid-rich material (PPM) followed by its layering (PPLs) at the edges of dying cells and cell-derived debris, and acting as major hydroxyapatite nucleator.2 Since calcifying AVICs often showed electrondense particles resembling free ribosomes embedded within PPM, immunocytochemical and immunogold labelling detection of ribosomal ribonucleic acid (rRNA) was performed on AVICs untreated or treated for 3 up to 9 days with 3.0 mM inorganic phosphate, 100 ng/ml bacterial lipopolysaccharide (LPS), and 20% (v/v) conditioned medium derived from LPS-stimulated macrophages, so simulating metastatic calcification. Immunolocalization of rRNA was also performed on human aortic valve leaflets affected by severe calcific stenosis. Under light microscope, immunopositivity to rRNA resulted for both untreated cells and calcifying AVICs, with negligible differences depending on cell treatment or incubation times. In pro-calcific AVIC cultures, immunopositivity to rRNA was also appreciable at level of calcific nodules. Ultrastructurally, antibody-conjugated gold particles were found to decorate all ribosomes in untreated AVICs as well as PPM/PPLs and still recognizable ribosomes in calcifying AVICs. In stenotic aortic valve leaflets, immunopositivity to rRNA was clearly detectable around calcific nodules, with ultrastructural analysis revealing gold particles to be mainly localized at level of PPLs lining degenerating cells and cell-derived byproducts. As observed for cultured AVICs, free and membrane-bound ribosomes were additional immunopositive sites in suffering cells populating valve areas affected by incipient mineralization. In conclusion, these results suggest that rRNAs derived from ribosome degradation may contribute to aortic valve mineralization in both in vitro and in vivo conditions, with their acidic nature possibly enhancing PPM/PPL capacity to nucleate hydroxyapatite. The present results also strengthen the requirement of nucleic acid removal in preparing decellularized aortic valve scaffolds to attain calcification-free heart valve substitutes before surgical implantation.3 1. Kim KM Scanning Microsc 1995, 9:1137–78. 2. Bonetti A et al. Anat Rec 2012, 295:1117-27. 3. Iop L et al. PLoS One 2014, 9:e99593.

Involvement of ribosomal ribonucleic acids in mineralization of aortic valve interstitial cells in experimental and pathological conditions

BONETTI, Antonella;DELLA MORA, Alberto;ORTOLANI, Fulvia
2016

Abstract

As described for actual aortic valve mineralization,1 metastatic-like calcification of primarily cultured aortic valve interstitial cells (AVICs) was previously found to consist in intracellular release of acid-phospholipid-rich material (PPM) followed by its layering (PPLs) at the edges of dying cells and cell-derived debris, and acting as major hydroxyapatite nucleator.2 Since calcifying AVICs often showed electrondense particles resembling free ribosomes embedded within PPM, immunocytochemical and immunogold labelling detection of ribosomal ribonucleic acid (rRNA) was performed on AVICs untreated or treated for 3 up to 9 days with 3.0 mM inorganic phosphate, 100 ng/ml bacterial lipopolysaccharide (LPS), and 20% (v/v) conditioned medium derived from LPS-stimulated macrophages, so simulating metastatic calcification. Immunolocalization of rRNA was also performed on human aortic valve leaflets affected by severe calcific stenosis. Under light microscope, immunopositivity to rRNA resulted for both untreated cells and calcifying AVICs, with negligible differences depending on cell treatment or incubation times. In pro-calcific AVIC cultures, immunopositivity to rRNA was also appreciable at level of calcific nodules. Ultrastructurally, antibody-conjugated gold particles were found to decorate all ribosomes in untreated AVICs as well as PPM/PPLs and still recognizable ribosomes in calcifying AVICs. In stenotic aortic valve leaflets, immunopositivity to rRNA was clearly detectable around calcific nodules, with ultrastructural analysis revealing gold particles to be mainly localized at level of PPLs lining degenerating cells and cell-derived byproducts. As observed for cultured AVICs, free and membrane-bound ribosomes were additional immunopositive sites in suffering cells populating valve areas affected by incipient mineralization. In conclusion, these results suggest that rRNAs derived from ribosome degradation may contribute to aortic valve mineralization in both in vitro and in vivo conditions, with their acidic nature possibly enhancing PPM/PPL capacity to nucleate hydroxyapatite. The present results also strengthen the requirement of nucleic acid removal in preparing decellularized aortic valve scaffolds to attain calcification-free heart valve substitutes before surgical implantation.3 1. Kim KM Scanning Microsc 1995, 9:1137–78. 2. Bonetti A et al. Anat Rec 2012, 295:1117-27. 3. Iop L et al. PLoS One 2014, 9:e99593.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11390/1107074
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