Metastatic calcification of cardiac valves is a common complication in patients affected by chronic renal failure. In this study, primary bovine aortic valve interstitial cells (AVICs) were subjected to pro-calcific treatments consisting in cell stimulation with (i) elevated inorganic phosphate (Pi ¼ 3 mM), to simulate hyperphosphatemic conditions; (ii) bacterial endotoxin lipopolysaccharide (LPS), simulating direct effects by microbial agents; and (iii) conditioned media (CM) derived from cultures of either LPS-stimulated heterogenic macrophages (commercial murine RAW264.7 cells) or LPS-stimulated fresh allogenic monocytes/macrophages (bCM), simulating consequent inflammatory responses, alone or combined. Compared to control cultures, spectrophotometric assays revealed shared treatment-dependent higher values of both calcium amounts and alkaline phosphatase activity for cultures involving the presence of elevated Pi. Ultrastructurally, shared peculiar pro-calcific degeneration patterns were exhibited by AVICs from these latter cultures irrespectively of the additional treatments. Disappearance of all cytomembranes and concurrent formation of material showing positivity to Cuprolinic Blue and co-localizing with silver precipitation were followed by the outcropping of such a material, which transformed in layers outlining the dead cells. Subsequent budding of these layers resulted in the formation of bubbling bodies and concentrically laminated calcospherulae mirroring those in actual soft tissue calcification. In conclusion, the in vitro models employed appear to be reliable tools for simulating metastatic calcification and indicate that hyperphosphatemic-like conditions could trigger valve calcification per se, with LPS and allogenic macrophage-derived secretory products acting as possible calcific enhancers via inflammatory responses.
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.
The involvement of calcium-dependent cytosolic phospholipase A2α (cPLA2α) in aortic valve calcification is not exhaustively elucidated. Here, cPLA2α expression in aortic valve interstitial cell (AVIC) pro-calcific cultures simulating either metastatic or dystrophic calcification was estimated by qPCR, Western blotting, and counting of cPLA2α-immunoreactive cells, with parallel ultrastructural examination of AVIC calcific degeneration. These evaluations also involved pro-calcific AVIC cultures treated with cPLA2α inhibitor dexamethasone. cPLA2α over-expression resulted for both types of pro-calcific AVIC cultures. Compared to controls, enzyme content was found to increase by up to 300% and 186% in metastatic and dystrophic calcification-like cultures, respectively. Increases in mRNA amounts were also observed, although they were not as striking as those in enzyme content. Moreover, cPLA2α increases were time-dependent and strictly associated with mineralization progression. Conversely, drastically lower levels of enzyme content resulted for the pro-calcific AVIC cultures supplemented with dexamethasone. In particular, cPLA2α amounts were found to decrease by almost 88% and 48% in metastatic and dystrophic calcification-like cultures, respectively, with mRNA amounts showing a similar trend. Interestingly, these drastic decreases in cPLA2α amounts were paralleled by drastic decreases in mineralization degrees, as revealed ultrastructurally. In conclusion, cPLA2α may be regarded as a crucial co-factor contributing to AVIC mineralization in vitro, thus being an attractive potential target for designing novel therapeutic strategies aimed to counteract onset or progression of calcific aortic valve diseases.
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