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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., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

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., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

This work was supported by grants 08/0744 and 11/01048 and by RETICS Program, RD08/0075 (RIER) of the Instituto de Salud Carlos III (Spain) that are partially financed by the European Regional Development Fund of the European Union, and by grants from the Xunta de Galicia and by DFG KFO 250, TP 03 to TW., Alonso-Perez, E., Suarez-Gestal, M., Calaza, M., Ordi-Ros, J., Balada, E., Bijl, M., Papasteriades, C., Carreira, P., Skopouli, F.N., Witte, T., Endreffy, E., Marchini, M., Migliaresi, S., Sebastiani, G.D., Santos, M.J., Suarez, A., Blanco, F.J., Barizzone, N., Pullmann, R., Ruzickova, S., Lauwerys, B.R., Gomez-Reino, J.J., Gonzalez, A., Liz, M., Schmidt, R.E., Kappou-Rigatou, I., Scorza, R., Kovacs, A., Balada, E., Kallenberg, C.G., Vinagre, F., Dostal, C., Pullmann Jr., R., Mavromati, M., D'Alfonso, S., Gutierrez, C., Rego, I.

This work was supported by Fondo de Investigacion Sanitaria of the Instituto de Salud Carlos III(Spain) grant 08/0744 and by RETICS Program RD08/0075 that are partially financed by the European Regional Development Fund of the European Union, and by grants from the Xunta de Galicia and BMBF KN Rheuma (C2.12 to TW)., Alonso-Perez, E., Suarez-Gestal, M., Calaza, M., Sebastiani, G.D., Pullmann, R., Papasteriades, C., Kovacs, A., Skopouli, F.N., Bijl, M., Suarez, A., Marchini, M., Migliaresi, S., Carreira, P., Ordi-Ros, J., Witte, T., Ruzickova, S., Santos, M.J., Barizzone, N., Blanco, F.J., Lauwerys, B.R., Gomez-Reino, J.J., Gonzalez, A.

Introduction: Systemic Lupus Erythematosus (SLE) shows a spectrum of clinical manifestations that complicate its diagnosis, treatment and research. This variability is likely related with environmental exposures and genetic factors among which known SLE susceptibility loci are prime candidates. The first published analyses seem to indicate that this is the case for some of them, but results are still inconclusive and we aimed to further explore this question. Methods: European SLE patients, 1444, recruited at 17 centres from 10 countries were analyzed. Genotypes for 26 SLE associated SNPs were compared between patients with and without each of 11 clinical features: ten of the American College of Rheumatology (ACR) classification criteria (except ANAs) and age of disease onset. These analyses were adjusted for centre of recruitment, top ancestry informative markers, gender and time of follow-up. Overlap of samples with previous studies was excluded for assessing replication. Results: There were three new associations: the SNPs in XKR6 and in FAM167A-BLK were associated with lupus nephritis (OR = 0.76 and 1.30, P-corr = 0.007 and 0.03, respectively) and the SNP of MECP2, which is in chromosome X, with earlier age of disease onset in men. The previously reported association of STAT4 with early age of disease onset was replicated. Some other results were suggestive of the presence of additional associations. Together, the association signals provided support to some previous findings and to the characterization of lupus nephritis, autoantibodies and age of disease onset as the clinical features more associated with SLE loci. Conclusion: Some of the SLE loci shape the disease phenotype in addition to increase susceptibility to SLE. This influence is more prominent for some clinical features than for others. However, results are only partially consistent between studies and subphenotype specific GWAS are needed to unravel their genetic component.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease with a very varied spectrum of clinical manifestations that could be partly determined by genetic factors. We aimed to determine the relationship between prevalence of 11 clinical features and age of disease onset with European population genetic substructure. Data from 1413 patients of European ancestry recruited in nine countries was tested for association with genotypes of top ancestry informative markers. This analysis was done with logistic regression between phenotypes and genotypes or principal components extracted from them. We used a genetic additive model and adjusted for gender and disease duration. Three clinical features showed association with ancestry informative markers: autoantibody production defined as immunologic disorder (P = 6.8×10 -4), oral ulcers (P = 6.9×10 -4) and photosensitivity (P = 0.002). Immunologic disorder was associated with genotypes more common in Southern European ancestries, whereas the opposite trend was observed for photosensitivity. Oral ulcers were specifically more common in patients of Spanish and Portuguese self-reported ancestry. These results should be taken into account in future research and suggest new hypotheses and possible underlying mechanisms to be investigated. A first hypothesis linking photosensitivity with variation in skin pigmentation is suggested. © 2011 Alonso-Perez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Systemic Lupus Erythematosus (SLE) is an autoimmune disease with a very varied spectrum of clinical manifestations that could be partly determined by genetic factors. We aimed to determine the relationship between prevalence of 11 clinical features and age of disease onset with European population genetic substructure. Data from 1413 patients of European ancestry recruited in nine countries was tested for association with genotypes of top ancestry informative markers. This analysis was done with logistic regression between phenotypes and genotypes or principal components extracted from them. We used a genetic additive model and adjusted for gender and disease duration. Three clinical features showed association with ancestry informative markers: autoantibody production defined as immunologic disorder (P = 6.8x10(-4)), oral ulcers (P = 6.9x10(-4)) and photosensitivity (P = 0.002). Immunologic disorder was associated with genotypes more common in Southern European ancestries, whereas the opposite trend was observed for photosensitivity. Oral ulcers were specifically more common in patients of Spanish and Portuguese self-reported ancestry. These results should be taken into account in future research and suggest new hypotheses and possible underlying mechanisms to be investigated. A first hypothesis linking photosensitivity with variation in skin pigmentation is suggested.

Raman micro-spectroscopy is a qualitative and quantitative non-destructive analytical technique in which a monochromatic radiation emitted from a laser source is focused on a sample through microscope optics, and the light inelastically scattered by the sample is thus collected and analyzed by means of an optical spectrograph. The bands observed in Raman spectra correspond to molecular vibrations of the biochemical species constituting the sample, with each species contributing with a specific molecular “spectral fingerprint”. Exploiting this analytical capability, “chemical images” showing the distribution of different biochemical species are produced by collecting spectra from each point of the sample: an approach called “Raman mapping”. With diagnostic purposes, Raman mapping is being increasingly exploited to characterize pathological tissues. 30-mμ-thick cryosections of samples from aortic valve leaflets surgically excised from patients affected by calcific aortic valve stenosis (CAVS) were mounted on calcium-fluoride-slides for Raman-analysis. Nearly adjacent 8-mμ-thick cryosections were mounted on histological slides for histochemical analysis, including von Kossa silver staining. The localization of prominent hydroxyapatite quantities closely correlated with von Kossa staining at level of calcific nodules and single neighbouring dead cells and cell debris. In addition, Raman imaging revealed great amounts of phspholipids and cholesterol around the calcific nodules, where a distinct cell degeneneration occurs, as previuosly ultrastructurally described (Ortolani et al., Histol. Histopathol. 22, 261-272, 2007), which includes increasing in cell membranous component, its subsequent dissolution, and release/clustering of lipid-containing material acting as major hydroxyapatite nucleator. Such a lipid distribution in Raman-maps was superimposable to specific reactivity for von Kossa and Cuprolinic blue staining on histological sections. Interestingly, Raman-maps also showed further co-localization of carotenoids with lipid moiety and in particular cholesterol. In conclusion, more detailed chemical characterization was provided of the calcific process underlying CAVS by implementing histochemical and ultrastructural procedures with Raman micro-spectroscopy. In addition, considering that Raman spectra could be obtained in-vivo upon shining the laser light on the valvular tissues via an optical fiber, the intense and characteristic signal of carotenoids might be used as an optical marker for precocious diagnosis of valve calcification., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011