Survival-Related Autophagic Activity Versus Procalcific Death in Cultured Aortic Valve Interstitial Cells Treated With Critical Normophosphatemic-Like Phosphate Concentrations

• Published in: The journal of histochemistry and cytochemistry Vol. 65; no. 3; pp. 125 - 138
• Main Authors: Bonetti, Antonella, Della Mora, Alberto, Contin, Magali, Gregoraci, Giorgia, Tubaro, Franco, Marchini, Maurizio, Ortolani, Fulvia
• Format: Journal Article
• Language: English
• Published: Los Angeles, CA SAGE Publications 01.03.2017
• Subjects: Alkaline Phosphatase - metabolism; Animals; Aortic Valve - cytology; Aortic Valve - metabolism; Aortic Valve - pathology; Aortic Valve - ultrastructure; Autophagy; Calcinosis - metabolism; Calcinosis - pathology; Calcium - metabolism; Cattle; Cell Survival; Cells, Cultured; Endoplasmic Reticulum - metabolism; Endoplasmic Reticulum - pathology; Endoplasmic Reticulum - ultrastructure; Phosphates - metabolism; calcific valve disease; electron microscopy; autophagy; aortic valve interstitial cells; ectopic calcification; phosphatemia
• ISSN: 0022-1554; 1551-5044
• DOI: 10.1369/0022155416687760

Valve dystrophic calcification is a common disorder affecting normophosphatemic subjects. Here, cultured aortic valve interstitial cells (AVICs) were treated 3 to 28 days with phosphate (Pi) concentrations spanning the normal range in humans (0.8, 1.3, and 2.0 mM) alone or supplemented with proinflammatory stimuli to assess possible priming of dystrophic-like calcification. Compared with controls, spectrophotometric analyses revealed marked increases in calcium amounts and alkaline phosphatase activity for 2.0-mM-Pi-containing cultures, with enhancing by proinflammatory mediators. Ultrastructurally, AVICs treated with low/middle Pi concentrations showed an enormous endoplasmic reticulum (ER) enclosing organelle debris, so apparently executing a survival-related atypical macroautophagocytosis, consistently with ultracytochemical demonstration of ER-associated acid phosphatase activity and decreases in autophagosomes and immunodetectable MAP1LC3. In contrast, AVICs cultured at 2.0-mM Pi underwent mineralization due to intracellular release and peripheral layering of phospholipid-rich material acting as hydroxyapatite nucleator, as revealed by Cuprolinic Blue and von Kossa ultracytochemical reactions. Lack of immunoblotted caspase-3 cleaved form indicated apoptosis absence for all cultures. In conclusion, fates of cultured AVICs were crucially driven by Pi concentration, suggesting that serum Pi levels just below the upper limit of normophosphatemia in humans may represent a critical watershed between macroautophagy-associated cell restoring and procalcific cell death.