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The subdermal model: new insights in aortic valve calcification
No full textAlthough "in vivo" subdermal model is very usefully to predict tissue propensity to calcification, the inherent calcification mechanism is unclear and the reliability of this "in vivo" simulation is still debated. Samples were obtained from (i) native aortic valve leaflets excised from pig (AVLs), (ii) AVLs undergone pre-implantation treatment alone, including fixation in 0.6% glutaraldehyde under nitrogen atmosphere, (pre-TAVLs), and (iii) AVLs implanted into rat subcutis for 2 days, 2 weeks, and 6 weeks (SI-AVLs). TUNEL-reactivity was found for pre-T-AVLs and all SI-AVLs, and ultrastructural apoptosisrelated features were observed. Immunoreactivity was found for Annexin-V, with diffuse intracytoplasmic distribution, for AVLs, pre-T-AVLs and 2d-SI-AVLs, and pericellular localization, for 2w- and 6w-SI-AVLs. Ultrastructural colocalization was evident between Annexin-V (immunogold labeling) and endogenous acidic lipids clustering at cell surfaces (modified pre-embedding reactions with cuprolinic blue) and acting as apatite nucleators (additional post-embedding von Kossa silver staining). These data indicate that hypoxic/anoxic conditions characterizing the pre-T-step prime uncomplete, apoptotic cell death and a distinct cascade of cell reaction/degradation steps leading to initial apatite precipitation. New parameters are now available for more proper comparison between the experimental calcification occuring in the subdermal model and physiological or pathological calcific processes
Lipid and calcium-binding protein involvement in aortic valve calcification as revealed by ultrastructural-histochemical and immunohistochemical data.
No full textThe subdermal model: apoptosis, protein expression and associated tissue responses in aortic valve calcification
No full textSelective silver precipitation and malachite green uptake reveal calcium-binding sites and phospholipid involvement on calcified aortic valve thin sections
No full textPrevious study on porcine aortic valves implantated in rat subcutis showed that glutaraldehyde-Cuprolinic blue reactions (GA-CB), at low pH, induce tissue unmasking from calcium and subsequent visualization of cellsand matrix-vesicles-like bodies outlined by peculiar, CB-reactive layers; because of anionic nature and differential chemical/enzymatic extra activity, these structures were assumed to be composed by acidic phospholipids (Ortolani et al. Connect Tiss Res 2002; 43:44-55; Histochem J 34:41-50). In the present investigation, pre-embedding GA-CB reactions followed by post-embedding von Kossa silver staining (GA-CB-S) showed major metal precipitation just occurring on the pericellular CB-reactive layers, and minor one at three additional sites: (i) nuclear heterocromatin; (ii) juxtacellular, filamentous material; and (iii) collagen fibrils. Moreover, glutaraldehyde-malachite green (GA-MG) pre-embedding reactions, at lowered pH, followed by osmium post-fixation gave rise to the appearance of pericellular osmium-MG-reactive layers, which were comparable to the silver-CB-reactive ones. These data show that a unique process of cell degeneration occurs in this calcification model, in which acidic phospholipids accumulate at cell surface replacing cell membranes and acting as major apatite nucleator. In addition, the overall data are consistent with the concept that common steps would exist for the various pathways in normal and pathological calcification
Calcification in human stenotic aortic valves: involvement of acidic lipids and annexin-V
No full textAcidic lipid clustering at the surface of cells and matrix vesicles and associated calcium-binding protein Annexin-V (Anx-V) have been found to act as major apatite nucleators in aortic valves subjected to experimental calcification (Ortolani F et al: Connect Tissue Res 43: 44-55, 2002 - Histochem J 34: 41-50, 2002 - Histol Histopathol 18: 1131-40, 2003). Here, possibility was investigated that these degenerative processes also occur in pathological valve calcification. After explantation, human calcified valves affected by aortic stenosis were subjected to reaction with 0.05% Cuprolinic Blue + 2.5% glutaraldehyde + 0.05M MgCl2 in phosphate solutions, pH 4,8 (GA-CB). Semithin sections of GA-CB-reacted samples underwent von-Kossa-silver-staining and re-embedded to achieve reacted thin sections (GA-CB-S). Histological sections underwent von-Kossa-silver-staining (S). Cryosections underwent immunohistochemical reactions for Anx-V. LR-White-thin sections underwent immunogold reactions for Anx-V. Ployclonal AB anti-Anx-V R88 was used (courtesy of Klaus von der Mark). As for experimental valve calcification, it was observed clustering of acid lipids around cell debris, matrix vesicles and elastic fibers, colocalization between GA-CB and GA-CB-S reactivity, and immunogold labelling for Anx-V which was closely associated with the accumulating lipidic material. These results suggest common mechanisms to be shared by this pathological calcification and experimental one, including initial hypoxia-induced up-regulation of Anx-V (Denko N et al: Clin Cancer Res 6: 480-7, 2000) and subsequent translocation due to protein avidity for the exudating lipids
Experimental calcification induction in aortic valve interstitial cells using a novel in vitro model
No full textAnnexin-V and acidic lipids in calcification of human stenotic aortic valves as revealed by histochemical reactions and immuno-gold labelling
No full textIntroduction. In spite of the concept that physiological, pathological and experimental calcification processes are very similar to each other, accumulating data indicate specific differences to exist. A distinct process of cell degeneration/mineralization occurs in aortic valves subjected to calcification subdermal models. This was found to be characterized by acidic lipid clustering at the surface of cells and matrix-vesicle-like bodies, that acts as major apatite nucleators (Ortolani F et al: Connect Tissue Res 43:44-55, 2002; Ortolani F et al: Histochem J 34: 41-50, 2002; Ortolani F et al: Histol Histopathol 18: 1131-40, 2003) in association with calcium-binding protein Annexin-V (Anx-V). Here the possibility was investigated that these degenerative processes also occur in pathological valve calcification. Material and Methods. After surgical replacement with valve prostheses in patients affected by aortic valve stenosis, samples were excised from the explanted, calcified valves and subjected to pre-embedding reaction with 0.05% Cuprolinic Blue in phosphate solutions containing 2.5% glutaraldehyde plus 0.05M MgCl2, pH 4,8 (GA-CB). Semithin sections of GA-CB-reacted samples underwent von-Kossa-silver-staining and re-embedded to achieve reacted ultrathin sections (GA-CB-S). Histological sections underwent von-Kossa-silver-staining (S). Cryosections underwent reactions for alkaline phosphatase activity (AP) and immunohistochemical reactions for Anx-V. LR-White-thin sections underwent immunogold reactions for Anx-V. Polyclonal AB anti-Anx-V R88 was used (courtesy of Klaus von der Mark). Results. Light microscopy showed colocalized positivity between GA-CB and S reactions for several cell groups, whereas non-colocalized AP-reactivity appeared for several others. At the ultrastructural level, degenerative features were observed which were closely superimposable to those in the subdermal model. Clustering of acid lipids was observed around cell debris, matrix-vesicle-like bodies and elastic fibers as well as colocalization between GA-CB and GA-CB-S reactivity. Immunogold labelling showed Anx-V to be closely associated with the accumulating lipidic material. Conclusions. In spite of the heterogenous patterns observed and undependently from eziopathogenesis, these results suggest that common mechanisms are shared by the different forms of this pathological calcification and experimental one, including initial hypoxia-induced up-regulation (Denko N et al: Clin Cancer Res 6: 480-7, 2000) of Anx-V and subsquent protein translocation due to its avidity for the exudating lipids resulting from concurrent cell phospholipidosis
Ab interno trabeculectomy: Ultrastructural evidence and early tissue response in a human eye
No full textDeletion of alpha 7 integrin gene results in structural heart modifications
No full textWithin the integrin family, the alpha7beta1 integrin is the major transmembrane laminin-receptor of skeletal, cardiac and smooth muscle cells. Due to interactions with the actin cytoskeleton, this integrin is essential for structural integrity and mechanical properties of muscle cells, but it is also involved in signal transduction pathways triggered by laminin. Knock out mice have been obtained by generation of null allele of the alpha 7 gene (itga7) in the germline of mice by homologous recombination in embryonic stem cells (1). Absence of integrin alpha 7 chain expression in the skeletal muscle has been reported to correlate with histological changes as for muscular dystrophy and damage of the myotendinous junctions (1,2). Previously, we reported the localization of alpha 7 integrin chain in the heart at the surface of cardiomyocytes with predominance at the myo-tendinous junction between papillary muscles and "chordae tendinae". In the present investigation we examined myo-cardium of 5 K.O. mice at the ultrastructural level. A series of distinct alterations was found to affect myocardium in all animals in different heart areas but at varying degrees. Ultrastructural analysis revealed the presence of cross corrugation at the myocardium fiber surface, following the sarcomeric patterns, being each valley bottom connected with a sarcomeric Z line; often sub-sarcolemmal material connecting longitudinally subsequent Z lines was also detectable. In apical cardiomyocytes of papillary muscle the presence of prominent increase of plasmalemma infolding involved in myo-tendinous junction was observed, as well as abnormal thickening of basal laminae and accumulation of intracytoplasmic material. These dsata suggest that integrin alpha 7 deficiency causes severe alterations in the myocariocyte cytoskeleton associated with a disturbance in cell-extracellular matrix interactions. Moreover, the lateral modifications exhibited by the cardiomyocytes suggest that minor cohesiveness between cells and extracellular matrix should result in less reduction of the heart chambre volume during systole. On the other hand, the increase in the interface between papillary muscles and "chordae tendinae" should represent a compensatory adaptation for the absence of these major adhesive molecules. REFERENCES. (1) U. Mayer et al., Nat Genet, 17: 318-323, 1997. (2) N. Miosge et al., Lab Invest, 79: 1591-1599, 1999
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