Critical Involvement of Calcium-Dependent Cytosolic Phospholipase A2α in Aortic Valve Interstitial Cell Calcification

The involvement of calcium-dependent cytosolic phospholipase A2α (cPLA2α) in aortic valve calcification is not exhaustively elucidated. Here, cPLA2α expression in aortic valve interstitial cell (AVIC) pro-calcific cultures simulating either metastatic or dystrophic calcification was estimated by qPCR, Western blotting, and counting of cPLA2α-immunoreactive cells, with parallel ultrastructural examination of AVIC calcific degeneration. These evaluations also involved pro-calcific AVIC cultures treated with cPLA2α inhibitor dexamethasone. cPLA2α over-expression resulted for both types of pro-calcific AVIC cultures. Compared to controls, enzyme content was found to increase by up to 300% and 186% in metastatic and dystrophic calcification-like cultures, respectively. Increases in mRNA amounts were also observed, although they were not as striking as those in enzyme content. Moreover, cPLA2α increases were time-dependent and strictly associated with mineralization progression. Conversely, drastically lower levels of enzyme content resulted for the pro-calcific AVIC cultures supplemented with dexamethasone. In particular, cPLA2α amounts were found to decrease by almost 88% and 48% in metastatic and dystrophic calcification-like cultures, respectively, with mRNA amounts showing a similar trend. Interestingly, these drastic decreases in cPLA2α amounts were paralleled by drastic decreases in mineralization degrees, as revealed ultrastructurally. In conclusion, cPLA2α may be regarded as a crucial co-factor contributing to AVIC mineralization in vitro, thus being an attractive potential target for designing novel therapeutic strategies aimed to counteract onset or progression of calcific aortic valve diseases.


Introduction
Calcium-dependent cytosolic phospholipase A2α (cPLA2α), also known as group IVA PLA2 or PLA2G4A, is an enzyme belonging to the superfamily of PLAs that catalyses the hydrolysis of membrane phospholipids bearing arachidonic acid at the sn-2 position. Constitutively expressed by many cell types, cPLA2α is activated by calcium at submicromolar concentrations and phosphorylation of specific serine residues enabling enzyme translocation to endomembranes, with subsequent arachidonic acid release and production of downstream signalling molecules including lysophospholipids and eicosanoids [1,2]. In their turn, these pro-inflammatory lipid mediators are reported to act as autocrine signals enhancing cPLA2α expression [3,4], which can be additionally up-regulated by pro-inflammatory cytokines [5,6] and bacterial lipopolysaccharide (LPS) [7,8]. Consistently, increased cPLA2α expression was observed in several inflammatory diseases affecting different organs and tissues, including brain, bronchi, bones, cartilages, and atherosclerotic artery walls [9][10][11][12][13]. In contrast with these extensive studies, poor research exists on the role of cPLA2α in calcification [14,15]. In particular, enzyme involvement in calcific aortic valve stenosis is not exhaustively elucidated [16,17], although some attention was devoted to the role of non-cytosolic PLA2s in this valvulopathy [18][19][20]. In our previous studies carried out under transmission electron microscopy, the same degenerative patterns characterizing valve mineralization after in vivo calcific induction [21][22][23][24][25] were reproduced in vitro using original pro-calcific models [25][26][27][28]. Namely, primarily cultured aortic valve interstitial cells (AVICs) were subjected to either metastatic-or severe-dystrophic-like calcification treating cells with LPS, conditioned medium (CM) from LPS-stimulated macrophages, and 3.0 mM inorganic phosphate (Pi) or 2.0 mM Pi, respectively, in accordance with the well-known Pi values referred to these two types of calcification. High (≥ 2.0 mM) Pi was found to be sufficient per se to prime AVIC mineralization, and its pro-calcific effects were dose-dependent; thus, the higher the Pi concentration, the shorter the incubation time to obtain AVIC calcification. The observed pro-calcific degenerative process consisted in a progressive endomembrane dissolution with intracellular release of an acidic lipid material, referred to as PPM (phthalocyanine-positive material) because of its strong affinity for phthalocyanine cuprolinic blue. Subsequent PPM layering at AVIC edges was found to generate PPLs (phthalocyanine-positive layers) acting as major hydroxyapatite crystal nucleators together with PPL-derived multivesicular bodies and their further end products, consisting in paracrystallin calcospherulae. Raman microspectroscopy also revealed close co-localization between calcium salt precipitates and lipid deposits in aortic valve leaflets affected by actual calcific stenosis [29,30]. Due to its phospholipid-degrading activity, we hypothesized that cPLA2α may play a direct role in priming and advancing the peculiar lipid-release-associated AVIC pro-calcific degeneration we previously described. Using in vitro pro-calcific models as above, possible involvement of cPLA2α in both metastatic and severe dystrophic-like AVIC calcification was ascertained in the present study. Whether enzyme expression may be influenced by Pi concentration was also assayed. The obtained results indicate that (i) cPLA2α is over-expressed by calcifying AVICs, (ii) its expression rate is positively related to Pi concentration, and (iii) increases in enzyme amounts are time-dependent and consistent with the advancement of cell calcification. To some extent, anti-calcific effects were also found subsequent to enzyme inhibition.

cPLA2α Expression in Control and Pro-Calcific AVIC Cultures
Western blotting quantification revealed cPLA2α content in 3.0-Pi-LPS-CM cultures underwent a significant time-dependent increase between days 3 and 6 (p < 0.025), followed by a remarkable drop at day 9, although values remained significantly higher than those in controls (Figure 1a,c). For 2.0-Pi-LPS-CM cultures (Figure 1b,c), enzyme content underwent significant time-dependent increases up to day 21 (p < 0.0125), with values significantly higher than those in controls starting from day 15. A remarkable drop in enzyme amounts occurred at day 28, with values resulting nearly identical to those in controls. In paralleled 2.0-Pi-LPS-CM cultures, qPCR-estimated cPLA2α mRNA amounts underwent significant time-dependent increases up to day 15 (p < 0.0125), with values significantly higher than those in controls starting from day 9. A moderate decrease at day 21 followed, with values remaining nearly unchanged up to day 28 ( Figure 1d). For 3.0-Pi-LPS-CM cultures, qPCR analyses revealed cPLA2α mRNA content to be significantly increased compared to controls at days 6 and 9, with values showing no significant differences to each other over time (p > 0.025) (not shown). Compared to Western blotting estimations, a similar trend resulted for percentages of cPLA2α-immunopositive AVICs. Namely, for 3.0-Pi-LPS-CM cultures, immunopositive cells underwent a significant time-dependent increase between days 3 and 6 (p < 0.025), followed by a remarkable drop at day 9, with values remaining significantly higher than those in controls ( Figure 2a). For 2.0-Pi-LPS-CM cultures, immunopositive AVICs underwent significant time-dependent increases up to day 21 (p < 0.0125), with values being significantly higher than those in controls starting from day 15. A remarkable drop occurred at day 28, with percentages of cPLA2α-positive AVICs reaching values close to those in controls (Figure 2b).

Discussion
In the present study, cPLA2α involvement was ascertained in AVIC calcification using pre-validated in vitro pro-calcific models, indicating the enzyme to be over-expressed by calcifying AVICs, with time-dependent enzyme increases being closely associated with progression of the inherent cell calcific degeneration. Quantitative estimations revealed cPLA2α and its mRNA to be constitutively expressed at low rates by control AVICs, undergoing significant time-dependent increases in the pro-calcific cultures exclusively. In the latter, cPLA2α over-expression can be conceivably ascribed to simultaneous cell exposition to high Pi, LPS, and CM-derived pro-inflammatory mediators. Consistently, LPS and pro-inflammatory stimuli are widely reported to act as potent cPLA2α up-regulators [5][6][7][8], whereas Pi is known to contribute to enzyme activation [2]. Further evidence that Pi could directly activate gene expression in vitro [31,32] raises the intriguing question whether direct Pi-mediated activation of cPLA2α mRNA synthesis can also take place in cultured AVICs. Although this query needs more in-depth investigation, the fact that remarkable increases in cPLA2α mRNA amounts were restricted to the pro-calcific AVIC cultures suggests that an additional pro-transcriptional effect of high Pi on the cPLA2α gene cannot be excluded. Taking also into account that higher levels of enzyme content were reached in metastatic calcification-like AVIC cultures even within shorter incubation times compared to severe dystrophic calcification-like ones, the cPLA2α expression rate appears to be positively related to Pi concentration. Evidence that high-phosphate-mediated inflammation can cause vascular cell calcification [33,34] leads to the assumption that cPLA2α over-expression by calcifying AVICs may be due to a possible pro-inflammatory effect of high phosphate, boosting that exerted by the employed pro-inflammatory stimuli. Occurrence of cPLA2α over-expression in the employed pro-calcific cultures was also consistent with failure of enzyme cleavage by caspase-3 [35,36], supporting our previous finding that apoptosis is not involved in the induced cell calcification processes [28]. Despite cPLA2α over-expression being associated with cell osteoblastic differentiation in vitro [37,38], it is worth noting that, in the present study as well as our previous ones on AVIC calcification in vitro [26][27][28], cells exhibiting osteoblast-like features were never encountered at the ultrastructural level, with the sole cell fate consisting in the described process of lipid-release-associated pro-calcific cell death. This intracellular release of pro-calcific lipid material we previously reported to occur during AVIC mineralization actually might depend on massive cell membrane dissolution subsequent to cPLA2α over-expression, since time-dependent increases in enzyme content were found to be closely associated with progression of cell mineralization. The pro-calcific role played by cPLA2α might also be exerted via the downstream activation of alkaline phosphatase, an additional enzyme contributing to mineralization progression [39]. Consistently in analogous AVIC cultures [27,28], maximum alkaline phosphatase activity was observed at incubation times longer than those corresponding to the maximal cPLA2α amounts found in the present study. This assumption is also consistent with previous reports showing cPLA2α-dependent activation of alkaline phosphatase in vitro [40,41]. Activity of downstream enzymes involved in the arachidonic acid metabolism was reported to be associated with the occurrence of calcific aortic valve stenosis [42,43], strongly suggesting that cPLA2α may actually be involved in calcific valve disease priming also in vivo. On the other hand, decreases in cPLA2α content observed at the longest incubation times may be due to pro-calcific cell degeneration, with associated enzyme breakdown. Far more substantial decreases in mRNA and enzyme amounts resulted for the pro-calcific AVIC cultures supplemented with the anti-inflammatory agent dexamethasone [44], as previously reported for other culture types [45,46]. Even taking into account the non-specific inhibitory effect of dexamethasone, the drastic lowering in mineralization as observed ultrastructurally for the pro-calcific AVIC cultures strongly suggests that AVIC calcification advances hand-in-hand with the found time-dependent cPLA2α over-expression. The identification of sporadic mineralizing cells in 3.0-Pi-LPS-CM-Dex cultures and, to a much lesser extent, 2.0-Pi-LPS-CM-Dex cultures suggests that cPLA2α can be regarded as a co-factor in AVIC calcification, high Pi being a pro-calcific factor per se, as previously reported [27,28]. High Pi is capable of activating sodium/phosphate cotransporter PiT-1 [47,48], alkaline phosphatase [49,50], and oxidative responses [51,52], also triggering cell calcification in absence of cPLA2α over-expression, although at much lower degrees. This leads to speculation that sole inflammation-lowering therapies might not be effective enough for hyperphosphatemic patients.
In conclusion, the obtained results indicate that (i) cPLA2α contributes to in vitro AVIC calcification, with its over-expression occurring in parallel with AVIC calcific progression and its inhibition resulting in drastic mineralization lowering, and (ii) cPLA2α expression rate is positively related to Pi concentration, with higher enzyme levels being reached in metastatic calcification-like cultures within shorter incubation times compared to severe dystrophic calcification-like ones.

qPCR Quantification of cPLA2α mRNA
Triplicates of control cultures, 3.0-Pi-LPS-CM cultures, and 2.0-Pi-LPS-CM cultures were prepared (n = 3). After medium removal, AVICs were washed twice with cold PBS, scraped away, and treated with RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions for total mRNA extraction. Then, 500 ng of total mRNA was reverse transcribed to cDNA using random exaprimers and SuperScript III reverse transcriptase (Life Technologies, Carlsbad, CA, USA). Real-time PCR was performed using PowerUp Sybr Green Master Mix (Life Technologies, Carlsbad, CA, USA) on the QuantStudio3 Real Time PCR System (Applied BioSystems, Foster City, CA, USA). To calculate mRNA levels the ∆∆CT method via the SDS software (Applied Biosystems, Foster City, CA, USA) was used. β-actin mRNA levels were used as endogenous control. All oligonucleotides were purchased from Sigma-Aldrich.

cPLA2α Inhibition in Pro-Calcific AVIC Cultures
AVICs were cultured in (i) pro-calcific medium as for 3.0-Pi-LPS-CM cultures supplemented with 100 nM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA) for 6 days (3.0-Pi-LPS-CM-Dex cultures) and (ii) pro-calcific medium for 2.0-Pi-LPS-CM cultures supplemented with 100 nM dexamethasone for 21 days (2.0-Pi-LPS-CM-Dex cultures). Treatment with dexamethasone was carried out for 6 and 21 days, since maximal cPLA2α amounts were estimated at these incubation times for 3.0-Pi-LPS-CM cultures and 2.0-Pi-LPS-CM cultures, respectively. To ascertain whether actual cPLA2α inhibition occurred, parallel 6-day-long 3.0-Pi-LPS-CM cultures and 21-day-long 2.0-Pi-LPS-CM cultures, including their respective control cultures, were prepared. According to the manufacturer's instructions, dexamethasone was dissolved in absolute ethanol and additionally diluted in DMEM. After dexamethasone supplementation, the final ethanol concentration in the pro-calcific media was equal to 0.0002%. However, to ascertain whether or not toxic effects occurred on AVICs, 6-and 21-day-long control cultures supplemented with 100 nM dexamethasone were also prepared (control-Dex cultures). All AVIC treatments were performed in triplicate (n = 3). For each treatment, AVIC cultures were subjected to Western blotting and qPCR, as above, as well as transmission electron microscopy.

Transmission Electron Microscopy
Cells were fixed with a 25 mM sodium acetate/acetic acid buffer containing 2.5% glutaraldehyde, 0.05% cuprolinic blue (Electron Microscopy Sciences, Hatfield, PA, USA), and 0.05 M magnesium chloride overnight at room temperature, keeping Petri dishes on a rocking platform. Cells were then post-fixed with 2% osmium tetraoxide (Agar Scientific, Stansted, Essex, UK), dehydrated with graded ethanol solutions, and embedded into Epon 812 resin. Ultrathin sections were collected onto formvar-coated 2 × 1 mm slot copper grids and contrasted with uranyl acetate and lead citrate. Observations and photographic recording were made using a Philips CM12 STEM transmission electron microscope.

Statistical Analysis
Continuous variables were summarized as mean ± standard error. Data obtained from qPCR, Western blotting, and immunocytochemical estimations were tested for normal distribution using the Shapiro-Wilk test. Variation of data between groups (controls and pro-calcific cultures) throughout incubation times was explored using the analysis of variance (ANOVA) for repeated measures. Paired comparisons between groups (controls and pro-calcific cultures) for each incubation time were performed using the Student's t-test or Mann-Whitney test, as appropriate. Comparisons between incubation times within each group (controls and pro-calcific cultures) were performed using the paired Student's t-test or Wilcoxon signed rank test. Bonferroni correction for multiple comparisons was applied.

Conclusions
The present results strongly suggest that cPLA2α over-expression by calcifying AVICs is crucial in triggering and sustaining their dramatic lipid-release-associated pro-calcific degeneration in vitro. Hence, cPLA2α could be seen as an attractive target in conceiving novel therapeutic strategies aimed to prevent or mitigate actual calcific aortic valve disorders.