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

Abstract

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.

Critical Involvement of Calcium-Dependent Cytosolic Phospholipase A2α in Aortic Valve Interstitial Cell Calcification

“…Evidence that high-phosphate-mediated inflammation can cause vascular cell calcification [ 33 , 34 ] leads to the assumption that cPLA2α over-expression by calcifying AVICs may be due to a possible pro-inflammatory effect of high phosphate, boosting that exerted by the employed pro-inflammatory stimuli. Occurrence of cPLA2α over-expression in the employed pro-calcific cultures was also consistent with failure of enzyme cleavage by caspase-3 [ 35 , 36 ], supporting our previous finding that apoptosis is not involved in the induced cell calcification processes [ 28 ]. Despite cPLA2α over-expression being associated with cell osteoblastic differentiation in vitro [ 37 , 38 ], it is worth noting that, in the present study as well as our previous ones on AVIC calcification in vitro [ 26 , 27 , 28 ], cells exhibiting osteoblast-like features were never encountered at the ultrastructural level, with the sole cell fate consisting in the described process of lipid-release-associated pro-calcific cell death.…”

“…In particular, enzyme involvement in calcific aortic valve stenosis is not exhaustively elucidated [ 16 , 17 ], although some attention was devoted to the role of non-cytosolic PLA2s in this valvulopathy [ 18 , 19 , 20 ]. In our previous studies carried out under transmission electron microscopy, the same degenerative patterns characterizing valve mineralization after in vivo calcific induction [ 21 , 22 , 23 , 24 , 25 ] were reproduced in vitro using original pro-calcific models [ 25 , 26 , 27 , 28 ]. Namely, primarily cultured aortic valve interstitial cells (AVICs) were subjected to either metastatic- or severe- dystrophic-like calcification treating cells with LPS, conditioned medium (CM) from LPS-stimulated macrophages, and 3.0 mM inorganic phosphate (Pi) or 2.0 mM Pi, respectively, in accordance with the well-known Pi values referred to these two types of calcification.…”

Assessing Calcification Onset in Aortic Valve Interstitial Cells

“…The process of aortic valve cell calcification is thought to involve multiple mechanisms and pathways, one of which is through ALP [17][18][19]. ALPs are membrane-bound metalloenzymes suggested to play an active role in calcium deposition by decreasing levels of pyrophosphate, a calcification inhibitor 8 , and increasing levels of inorganic phosphate, which promotes mineralization [20].…”

Phospholipids accumulation and calcification in cultured primary human aortic valve interstitial cells: New insights revealed by confocal Raman imaging

“…It is hypothesized that mineralization involves transdifferentiation of VICs into the osteoblast phenotype . Alternatively, it is suggested that valvular calcification is not always an osteogenic process and that apart from “bone‐like” calcification mechanism involving osteoid cells, accumulation of amorphous calcium nodules via cellular apoptosis and necrosis, autophagocytosis, and also non‐apoptotic, lipid‐release‐associated procalcific cell degeneration and death is possible. The ongoing discussion if interstitial cells can indeed adopt an osteoblast phenotype is often based on in vitro studies.…”

“…Calcification in the form of hydroxyapatite was obtained, however, in normal human aortic valve interstitial cells upon incubation with lipoprotein(a) that also significantly increased alkaline phosphatase activity, matrix vesicle formation, and induced apoptosis . Other research have shown that mineralization can be triggered by the elevated serum phosphate levels alone and enhanced after lipopolysaccharides supplementation and proinflammatory mediators derived from co‐cultures of LPS‐stimulated macrophages . Recently, it was proposed that matrix vesicles erroneously trapped in ECM were potential nucleation sites for extracellular calcification .…”