Ultrastructural and spectrophotometric study on the effects of putative triggers on aortic valve interstitial cells in in vitro models simulating metastatic calcification.

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 = 3mM), in order 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 allogeneic 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 the same cultures irrespectively of the applied treatment. 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 allogeneic macrophage-derived secretory products acting as possible calcific enhancers via inflammatory responses

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

Ultrastructural characterization of calcification onset and progression in subdermally implanted aortic valves. Histochemical and spectrometric data

Detailed characterization of the subdermal model is a significant tool for better understanding of calcification mechanisms occurring in heart valves. In previous ultrastructural investigation on six-week-implantated aortic valve leaflets, modified pre-embedding glutaraldehyde-cuprolinic-blue reactions (GA-CB) enabled sample decalcification with concurrent retention/staining of lipid-containing polyanionic material, which lined cells and cell-derived matrix-vesicle-like bodies (phthalocyanin-positive layers: PPLs) co-localizing with the earliest apatite nucleation sites. Additional post-embedding silver staining (GA-CB-S) revealed PPLs to contain calcium-binding sites. This investigation concerns valve leaflets subjected to shorter implantation times to shed light on the modifications associated with PPLs generation and calcification onset/progression. Spectrometric estimations revealed time-dependent calcium increase, for unreacted samples, and copper modifications indicating an increase in acidic, non-glycanic material, for GA-CB-reacted samples. Two-day-implant thin sections showed emission and subsequent reabsorption of lamellipodium-like protrusions by cells, originating ECM-containing vacuoles, and/or degeneration stages characterized by the appearance of GA-CB-S-reactive, organule-derived dense bodies and progressive dissolution of all cell membranes. In one-week-implants, the first PPL-lined cells were found to co-exist with cells where GA-CB-S-reactive material accumulated, or exudated towards their edges, or outcropped at the ECM milieu, so acquiring PPL features. PPL-derived material was observed increasingly to affect the ECM on thin sections of one-week- to six-week-implants. These results show an endogenous source for PPLs and reveal that a peculiar cascade of cell degenerative steps is associated with valve mineralization in the subdermal model, providing new useful parameters for more reliable comparison of this experimental calcification process versus the physiological and pathological processes

Involvement of cytosolic phospholipase A2 alpha in pathological and experimental cardiovascular mineralization

Cytosolic phospholipase A2 alpha (cPLA2a) is a calcium-dependent enzyme constitutively expressed by most human cells catalyzing the hydrolysis of membrane glycerophospholipids bearing arachidonic acid at the sn-2 position with production of downstream pro-inflammatory lipid mediators (Murakami and Kudo, 2002). Although cPLA2a seems to facilitate the release of pro-calcific matrix vesicles by hypertrophic chondrocytes during ossification (Wuthier et al., 1977), its involvement in pathological biomineralization has not yet been elucidated. Here, cPLA2a expression was assessed in the context of both pathological and experimentally induced mineralization affecting cardiovascular tissues and cultured aortic valve interstitial cells (AVICs). cPLA2a resulted to be expressed by fibroblasts, smooth muscle cells, macrophages, and activated endothelium populating both calcified aortic valves and atherosclerotic aorta walls. cPLA2a was also expressed by cultured AVICs, with enzyme expression rate correlating with mineralization rate, being enhanced by inflammation and high phosphate concentrations. For all calcific contexts, ultrastructural examination revealed mineralization to depend on progressive accumulation and release of acidic lipids, acting as major hydroxyapatite nucleators, followed by cell disgregation into a multitude of particles having calcium nucleation capability, according to peculiar degenerative patterns as those previously described (Ortolani et al., 2010). In conclusion, enzyme expression and ultrastructural patterns being shared by both pathological and experimental calcific conditions suggests that cPLA2a might be actually involved in the etiopathogenesis of cardiovascular mineralization, besides representing a potential target for novel therapeutic strategies aimed to counteract the progression of cardiovascular calcific diseases

Involvement of cytosolic phospholipase A2 alpha in pathological and experimental cardiovascular mineralization

Cytosolic phospholipase A2 alpha (cPLA2a) is a calcium-dependent enzyme constitutively expressed by most human cells catalyzing the hydrolysis of membrane glycerophospholipids bearing arachidonic acid at the sn-2 position with production of downstream pro-inflammatory lipid mediators (Murakami and Kudo, 2002). Although cPLA2a seems to facilitate the release of pro-calcific matrix vesicles by hypertrophic chondrocytes during ossification (Wuthier et al., 1977), its involvement in pathological biomineralization has not yet been elucidated. Here, cPLA2a expression was assessed in the context of both pathological and experimentally induced mineralization affecting cardiovascular tissues and cultured aortic valve interstitial cells (AVICs). cPLA2a resulted to be expressed by fibroblasts, smooth muscle cells, macrophages, and activated endothelium populating both calcified aortic valves and atherosclerotic aorta walls. cPLA2a was also expressed by cultured AVICs, with enzyme expression rate correlating with mineralization rate, being enhanced by inflammation and high phosphate concentrations. For all calcific contexts, ultrastructural examination revealed mineralization to depend on progressive accumulation and release of acidic lipids, acting as major hydroxyapatite nucleators, followed by cell disgregation into a multitude of particles having calcium nucleation capability, according to peculiar degenerative patterns as those previously described (Ortolani et al., 2010). In conclusion, enzyme expression and ultrastructural patterns being shared by both pathological and experimental calcific conditions suggests that cPLA2a might be actually involved in the etiopathogenesis of cardiovascular mineralization, besides representing a potential target for novel therapeutic strategies aimed to counteract the progression of cardiovascular calcific diseases

Critical effects of inorganic phosphate at threshold concentrations on cultured aortic valve interstitial cells. Macroautophagocytosis versus procalcific cell degeneration

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 cultures mimicking metastatic calcification ([Pi]=3.0mM) a major role was found to be played by [Pi] (Pi-cultures) 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 from cultured LPS-stimulated macrophages (Pi-LPS-CM-cultures) [1]. Here, bAVIC primary cultures were carried out which contained different [Pi] (0.4, 0.6, and 1.3mM in added solutions, i.e. 0.8, 1.3, and 2.0mM in final cultures), so including border concentrations on respect to hypophosphatemic- and hyperphosphatemic-like conditions. At 0.8mM and 1.3 [Pi] and for each incubation time (3, 9, 15, 21, and 28 days), bAVICs from Pi-cultures and Pi-LPS-CMcultures shared common ultrastructural features showing prominent macroautophagocytosis to occur, consistently with the immunohistochemical detection of the specific marker of mature autophagosomes MAP1-LC3A. Neither cell death signs nor appearance of calcific nodules were observed. At 2.0 [Pi], most bAVICs were affected by degenerative fragmentation as described for severe metastatic-like calcifcation, i.e. the appearence of phthalocianin- positive material outcropping at cell surface, acting as hydroxyapatite nucleator and being source of real calcospherulae. Quantitative spectrophotometric estimation of calcium amounts and alkaline phosphatase activity were consistent with the ultrastructural data, with (i) similar values for Pi-LPS-CM-cultures versus Pi-cultures and control cultures, at 0.8 and 1.3mM [Pi], and (ii) significantly higher values for Pi-LPS-CM-cultures versus Picultures and these latter versus controls, at 2.0mM [Pi]. Restriction of immunopositivity to caspase-8 to very few cells and complete immunonegativity to annexin-V suggested apoptosis to be a negligible epiphenomenon. In conclusion, the propensity of bAVICs to undergo procalcific degeneration resulted to correlate with [Pi] in such a manner that a differential discrimination of this parameter within the conventional normophosphatemic range is suggested for a proper evaluation of the risk for dystrophic valve calcification. Moreover, bacterial-derived inflammation seems to be regarded as an effective trigger for the higher normophosphatemic [Pi]

Pro-calcific responses by aortic valve interstitial cells in a novel in vitro model simulating dystrophic calcification

Etiopathogenetic mechanisms in calcific aortic valve stenosis are still poorly understood despite this being the third major cause of heart disease in western world. In prior in vitro cultures simulating metastatic calcification, pro­calcific effects on aortic valve interstitial cells (AVICs) resulted by adding bacterial endotoxin lipopolysaccharide (LPS) at high inorganic phosphate (Pi) levels. Here we accomplished improved in vitro models simulating either metastatic (Pi = 2.6 mM) or dystrophic calcification (Pi = 1.3 mM), in which LPS­stimulated bovine AVICs underwent extra-stimulation with macrophage-cytokine-containing media derived from paral­ lel cultures of allogeneic monocyte/macrophages in turn stimulated with LPS. In dystrophic calcification-like cultures, lower calcium amount was spectrometrically assessed with parallel reduced alkaline phosphatase activity with respect to metastatic calcification­like cultures, with an about three­fold slower progression of mineralization. Hydroxyapatite crystal precipitation was ultrastructurally found to correlate with AVIC degeneration processes culminating with the formation of phthalocyanin-positive lipidic layers (PPLs) at the surface of cells and cell-derived matrix-vesicle-like bodies, acting as calcium nucleators according to a pattern mirroring those we had previously found in in vivo conditions. In conclusion, an in vitro model has been devel­ oped enabling reliable simulations of the effects exerted on AVICs by putatively pro- or anti-calcific agents

Identification of secondary targets of N-containing bisphosphonates in mammalian cells via parallel competition analysis of the barcoded yeast deletion collection

Growth competition assays using barcoded yeast deletion-mutants reveal the molecular targets of nitrogen containing bisphosphonates used for the treatment of bone cancers and osteoporosis

In vitro reproduction and ultrastructural detection of the genesis of calcifying cell-derived structures identical to actual hydroxyapatite nucleators in calcific aortic valve stenosis

Unlike fairly good characterization gained for the major hydroxyapatite nucleators involved in physiological mineralization, i.e. matrix vesicles and apoptotic bodies, no details are available about source and genesis of so called “thick walled cell-derived-products” (CDPs) and “concentrically laminated calcospherulae” (CLCs), which are primary calcium deposition foci in ectopic calcification, including calcific aortic valve stenosis. Here, generation of these structures was successfully reproduced in vitro using an original model simulating either metastatic or dystrophic calcification, subsequent to differential inorganic phosphate supplementations to primary cultures of interstitial cells from bovine aortic valve cusps (AVICs). Superimposed bacterial infectious effects were simulated by additional stimulation with bacterial endotoxin lipopolysaccharide (LPS) and superstimulation with conditioned medium derived from cultures of bovine LPS-stimulated native monocytes/macrophages. At reverted microscope monitoring, AVICs were observed to undergo fragmentation giving rise to irregular debris and/or sporulation-like processes resulting in the formation of barely appreciable punctate bodies, concurrently with specific increase in spectrophotometrically assayed calcium amounts and alkaline phosphatase activity. Ultrastructurally, these cell-derived products showed features comparable to those characterizing CDPs and CLCs in ex vivo samples. The thick wall of CDPs was found to depend on a peculiar AVIC degenerative process culminating with outcropping of multilaminated lipid-containing phthalocyanin-positive layers (PPLs), as revealed by histochemical reactions with Cuprolinic blue. PPLs represented major calcium nucleators, as revealed by co-precipitation of hydroxyapatite needle-like crystals and co-localization of metallic silver deposition after von Kossa staining applied to electron microscopy. In addition, multilaminated PPLs of both degenerating AVICs and CDPs underwent sporulation-like budding and pinching off, so generating a lot of spherical CLC-like bodies, with many being superimposed by near radially oriented hydroxyapatite crystals. In conclusion, the way by which CDPs and CLCs form was ascertained. In addition, in-vitro reproduction of pro-calcific structures identical to those characterizing actual samples from calcified aortic valve cusps suggests the developed in vitro models to be a reliable tool for testing pro-calcific effects exterted by putative etiopathologic agents as well as anti-calcific by therapeutic compounds

Different propensity of cultured aortic valve interstitial cells to uptake native or aggregated low density lipoprotein

Atherosclerosis is a progressive disease characterized by modified low density lipoprotein (LDL) accumulation in the large artery walls, with subsequent phagocytosis by macrophages, transforming into foam cells, and vascular smooth muscle cells (VSMCs). In particular, native LDL (nLDL) can modify joining to each other giving rise to aggregated LDL (agLDL), which were reported to be internalized more avidly by VSMCs in in vitro conditions (1). Here, primarily cultured aortic valve interstitial cells (AVICs) were treated for 3 up to 21 days with 50 mg/ml blood-derived nLDL or agLDL, alone or combined with pro-calcific culture media, to ascertain whether (i) agLDL are taken up more rapidly than nLDL also by AVICs and (ii) treatment with LDL at low, normolipidemic-like concentration can influence AVIC mineralization. Ultrastructurally, LDL uptake was observed exclusively for agLDL-treated AVICs, which were characterized by a lot of endocytic vesicles and intracytoplasmic vacuoles entrapping agLDL particles. Consistently, treatment with agLDL resulted in a significant increase in intracellular amounts of both esterified cholesterol and triglycerides, as chromatographically assayed. Ultrastructural analyses also revealed pro-calcific AVIC degenerative process to occur, consisting in intracellular release of lipidic material and its layering at cell edges, there acting as major hydroxyapatite nucleator (2,3). The extent of pro-calcific effects resulted to be LDL-dependent, being mitigated in the presence of nLDL and exacerbated in the presence of agLDL. These findings were consistent with spectrophotometrical analyses showing decreased calcium amounts in AVIC cultures superstimulated with nLDL and increased mineral content in the counterpart superstimulated with agLDL. Since aortic valve stenosis is considered as a valve atherosclerotic lesion, these preliminary data suggest that avid uptake of agLDL by AVICs might strongly contribute to lipid accumulation within aortic valves, with agLDL possibly affecting subsequent valve tissue mineralization even at concentrations comprised within the normolipidemic range