Inorganic-phosphate-dependent autophagocytosis derangement and calcific events. Responsivity of aortic valve interstitial cells in cultures at different normophosphatemic-like conditions

UNDERGROUND - Calcific aortic valve stenosis is the third cause of cardiovascular disease in the developed world with surgical valve replacement still being unavoidable because of poor prognostic capability and therapeutic effectiveness. The calcific process can take place in hyperphosphatemic conditions, metastatic calcification, as well as in normophosphatemic ones, dystrophic calcification. The conventional threshold values ascribed to inorganic phosphate concentration ([Pi]) in diagnosing normophosphatemia range between 0.8mM and 1.45mM to 2.0mM [Pi]. In primary cultures mimicking metastatic calcification ([Pi]=3.0mM) a (critical) major role was found to be played by [Pi] in priming a procalcific cell degeneration of bovine aortic valve interstitial cells (bAVICs), with mineralization enhancing subsequent to superstimulation with bacterial lipopolysaccharide (LPS) plus conditioned medium (CM) from cultured LPS-stimulated macrophages [1]. Here, bAVIC cultures containing different final [Pi] (0.8mM, 1.3mM, and 2.0mM) were used, thus mimicking dystrophic calcification and including borderline concentrations on respect to hypophosphatemic- and hyperphosphatemic-like conditions. RESULTS - Under the inverted microscope, neither cell death signs nor appearance of calcific nodules were observed for bAVIC cultures except for those containing 2.0mM Pi. At 3-day-long incubation, immunoreactivity to the specific marker of mature autophagosomes MAP1-LC3A was higher for control bAVICs with decrease for both Pi-cultures and PI-LPS-CM-cultures containing 0.8mM, 1.3mM and 2.0mM Pi in the order. For all cases there was a superimposing time-dependent decrease. Parallel immunohistochemical detection of apoptosis showed low positivity to caspase-8 and almost unreactivity for Caspase-9, caspase-3 and annexin-V. For Pi-cultures and Pi-LPS-CM-cultures containing 0.8mM Pi and, at greater extent, those containing 1.3mM Pi, bAVICs showed prominent autophagocytosis to have started and atypically progressed, with (i) a progressive RER enlargement, thereby causing cytoplasm compartimentalization into hollows or canalicular spaces which confined altered organules, and (ii) concurrent loss of autophagosomes. Conversely, in all 2.0mM-Pi-cultures most bAVICs were affected by degenerative events as described for severe metastatic-like calcification, such as the appearance of phthalocyanin-positive material outcropping at cell surface and acting as hydroxyapatite nucleator, besides being source of real calcospherulae. Quantitative spectrophotometric estimations of calcium amounts and alkaline phosphatase activity were consistent with the morphological data, with (i) similar values for Pi-LPS-CM-cultures versus Pi-cultures and control cultures, at 0.8mM Pi and 1.3mM Pi, and (ii) significantly higher values for Pi-LPS-CM-cultures versus Pi-cultures and these latter versus controls, at 2.0mM Pi. CONCLUSIONS - The differential reactivity to MAP1-LC3A suggested autophagocytosis to be an epiphenomenon which is simply related to cell survival mechanisms. The restriction of immunopositivity to caspase-8 suggests apoptosis to be not significant in promoting the calcific process. Although the finding of atypical features of autophagocytosis, no relation with calcification seems to exist, unless this process derangement may occur very fastly in the first incubation hours for 2.0mM-Pi-cultures. Moreover, bacterial-derived inflammation seems to be regarded as an effective trigger for the higher normophosphatemic [Pi]. Interestingly, the propensity of bAVICs to undergo procalcific degeneration resulted to correlate with [Pi] in such a way that a differential discrimination of this parameter within the conventional normophosphatemic range is mandatory for a proper evaluation of the risk for dystrophic valve calcification. References [1] Bonetti A., Della Mora A., Contin M., Tubaro F., Marchini M., Ortolani F. (2012). Anat. Rec. 295: 1117-1127.