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.
Unlike its application for atherosclerotic plaque analysis, Raman microspectroscopy was sporadically used to check the sole nature of bioapatite deposits in stenotic aortic valves, neglecting the involvement of accumulated lipids/lipoproteins in the calcific process. Here, Raman microspectroscopy was employed for examination of stenotic aortic valve leaflets to add information on nature and distribution of accumulated lipids and their correlation with mineralization in the light of its potential precocious diagnostic use. Cryosections from surgically explanted stenotic aortic valves (n=4) were studied matching Raman maps against specific histological patterns. Raman maps revealed the presence of phospholipids/triglycerides and cholesterol, which showed spatial overlapping with one another and Raman-identified hydroxyapatite. Moreover, the Raman patterns correlated with those displayed by both von-Kossa-calcium- and Nile-blue-stained serial cryosections. Raman analysis also provided the first identification of carotenoids, which co-localized with the identified lipid moieties. Additional fit concerned the distribution of collagen and elastin. The good correlation of Raman maps with high-affinity staining patterns proved that Raman microspectroscopy is a reliable tool in evaluating calcification degree, alteration/displacement of extracellular matrix components, and accumulation rate of different lipid forms in calcified heart valves. In addition, the novel identification of carotenoids supports the concept that valve stenosis is an atherosclerosis-like valve lesion, consistently with their previous Raman microspectroscopical identification inside atherosclerotic plaques.
Spinal motoneurons and locomotor networks are regulated by monoamines, among which, the contribution of histamine has yet to be fully addressed. The present study investigates histaminergic regulation of spinal activity, combining intra- and extracellular electrophysiological recordings from neonatal rat spinal cord in vitro preparations. Histamine dose-dependently and reversibly generated motoneuron depolarization and action potential firing. Histamine (20 µM) halved the area of dorsal root reflexes and always depolarized motoneurons. The majority of cells showed a transitory repolarization, while 37% showed a sustained depolarization maintained with intense firing. Extracellularly, histamine depolarized ventral roots (VRs), regardless of blockage of ionotropic glutamate receptors. Initial, transient glutamate-mediated bursting was synchronous among VRs, with some bouts of locomotor activity in a subgroup of preparations. After washout, the amplitude of spontaneous tonic discharges increased. No desensitization or tachyphylaxis appeared after long perfusion or serial applications of histamine. On the other hand, histamine induced single motoneuron and VR depolarization, even in the presence of tetrodotoxin (TTX). During chemically induced fictive locomotion (FL), histamine depolarized VRs. Histamine dose-dependently increased rhythm periodicity and reduced cycle amplitude until near suppression. This study demonstrates that histamine induces direct motoneuron membrane depolarization and modulation of locomotor output, indicating new potential targets for locomotor neurorehabilitation.
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