Responsivity of aortic valve interstitial cells in cultures at different normophosphatemic-like conditions. Calcific events versus autophagocytosis and apoptosis

To mimick dystrophic calcification, occurring in normophosphatemic conditions, bovine aortic valve interstitial cell (bAVIC) cultures containing different Pi concentrations (0.8, 1.3, and 2.0mM) were accomplished, including borderline ones on respect to hypo- and hyperphosphatemic-like conditions. Neither cell death signs nor appearance of calcific nodules were observed except for bAVIC cultures containing 2.0mM Pi. At 3-day-long incubation, immunoreactivity to specific marker of mature autophagosomes MAP1-LC3A was higher for control bAVICs and decreased for cultures containing 0.8, 1.3, and 2.0mM Pi in the order. Moreover, there was a time-dependent decrease for all cases. On detecting apoptosis occurrence, low positivity resulted to caspase-8 and almost unreactivity to caspase-9, caspase-3, and annexin-V. For 0.8mM-Pi-cultures and, at greater extent, 1.3 ones, bAVICs showed autophagocytosis to have started and atypically progressed, with (i) a progressive RER enlargement causing cytoplasm compartimentalization into hollows/canalicular spaces, which confined altered organules, and (ii) parallel autophagosome loss. Conversely, most bAVICs in 2.0mM-Pi-cultures were affected by degenerative events, with appearance of phthalocyanin-positive material outcropping at cell surface and acting as hydroxyapatite nucleator, besides being source of real calcospherulae. Spectrophotometric estimations of calcium amounts and alkaline phosphatase activity were consistent with the morphological data, resulting (i) similar values between control cultures and those containing 0.8 and 1.3mM Pi and (ii) significantly higher values for 2.0mM Pi containing cultures versus those as at point (i). In conclusion, the immunohistochemical data suggest that (i) autophagocytosis might be an epiphenomenon simply related to cell survival mechanisms and (ii) apoptosis to be not significant in promoting the calcific process. The ultrastructural detection of abnormal autophagocytosis suggests a derangement of this process but without apparent correlation with calcification. Interestingly, the propensity of bAVICs to undergo procalcific degeneration resulted to correlate with Pi concentration in such a way that a differential discrimination of this parameter within the conventional normophosphatemic range seems to be mandatory for a proper evaluation of dystrophic valve calcification risk.