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

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.

Number and Distribution of Intraganglionic Laminar Endings in the Mouse Esophagus as Demonstrated with Two Different Immunohistochemical Markers

Intraganglionic laminar endings (IGLEs) represent the only vagal mechanosensory terminals in the tunica muscularis of the esophagus. Two specific markers for IGLEs were recently described in mouse: the purinergic P2X2 receptor and the vesicular glutamate transporter 2 (VGLUT2). This study aimed at comparing both markers with respect to their suitability for quantitative analysis. We counted IGLEs immunostained for VGLUT2 and P2X2, respectively, and mapped their distribution in esophageal wholemounts of C57Bl/6 mice. Numbers and distribution of IGLEs were compared with those of myenteric ganglia as demonstrated by cuprolinic blue histochemistry. Whereas the distribution of VGLUT2-immunopositive IGLEs closely matched that of myenteric ganglia, P2X2-immunopositive IGLEs were rarely found in upper and middle esophagus but increasingly in its lower parts. P2X2 stained only half the number of IGLEs found with VGLUT2 immunostaining. We also investigated the correlation between anterograde tracing and immunohistochemistry for identifying IGLEs. Confocal microscopy revealed colocalization of all three markers in ∼50% of IGLEs. The remaining IGLEs showed only tracer and VGLUT2 labeling but no P2X2 immunoreactivity. Thus, VGLUT2 and P2X2 represent two specific markers for qualitative demonstration of esophageal IGLEs. However, VGLUT2 may be superior to P2X2 as a quantitative marker for IGLEs in the esophagus of C57Bl/6 mice.