Deciphering Ectopic Calcification: Contribution of the Rare, Inherited Disorder Pseudoxanthoma Elasticum

Soft connective tissue calcification is still an intriguing problem due to the high number of genes, proteins, and enzymes involved in the process. Numerous epidemiological and experimental studies of the ectopic calcification associated with metabolic, inflammatory, and degenerative disorders have been performed. Moreover, in the last decade, great efforts have been made in studying the genetic disorders leading to soft connective tissue calcification, trying to understand the imbalance between pro and anti-calcifying factors in the different disorders, and why calcification occurs only in certain body regions (which often differ between the various genetic defects). The rare, inherited disorder pseudoxanthoma elasticum (PXE), which is caused by mutations in the ABCC6 gene, is an interesting model because the gene responsible is mainly expressed in the liver, whereas calcification affects peripheral soft connective tissues. It has been suggested that liver deficiency of the protein encoded by ABCC6 directly induces peripheral calcification, although, in contrast, several studies both in humans and in transgenic mice indicate that peripheral mesenchymal cells might be permanently involved in PXE calcification. In this review, the author suggests that early in development PXE cells may undergo epigenetic changes and acquire a permanent pro-calcific signature. However, given the complexity of the calcification process and the metabolic inter-exchanges among the different calcific genetic disorders, a bioinformatic approach analysing data ranging from genes to functional proteins and clinical features may complete the puzzle and provide new therapeutic perspectives in PXE, as well as in other calcific disorders

Connective tissue and diseases: from morphology to proteomics towards the development of new therapeutic appproach

Connective tissue consists of cells separated by the extracellular matrix, whose composition and amount vary according to age, to functional requirements, and to the presence of pathologic conditions. Within this non-random macromolecular assembly, collagens, elastin, proteoglycans and structural glycoproteins are mutually interdependent and modifications of one component, by extrinsic (environmental) and/or intrinsic (systemic, genetic, age-related) factors, may have consequences on the tissue as a whole. Since decades, different microscopical techniques have been applied mainly for diagnostic purposes and for detailed descriptions of changes occurring in cells and in matrix components. More recently, in order to dissect the molecular complexity of the matrix network, to analyse the interactions between cells and matrix and to look for modulators of cell phenotype, histomorphologic investigations have been implemented with proteomic studies that allow to identify possible diagnostic markers, and to better understand patho-mechanisms enabling the design of novel therapeutic strategies. Therefore, the progressively expanding, although incomplete, knowledge on connective tissue biology, sheds new light on the pathogenesis of diseases affecting single molecules (i.e. collagenopathies, mucopolysaccharidoses, elastinopathies) and discloses the importance of matrix components as fundamental regulators of cell phenotype, in relation, for instance, to the aging process and/or to cancer development and progression. Few examples will be presented demonstrating the promises of proteomics as a technique leading to the discovery of new therapies and possibly to the development of individualized treatments for a better patient care

The effect on rat thymocytes of the simultaneous in vivo exposure to 50-Hz electric and magnetic field and to continuous light

Thymus plays an important role in the immune system and can be modulated by numerous environmental factors, including electromagnetic fields (EMF). The present study has been undertaken with the aim to investigate the role of long-term exposure to extremely low frequency electric and magnetic fields (ELF-EMF) on thymocytes of rats housed in a regular dark/light cycle or under continuous light. Male Sprague-Dawley rats, 2 months old, were exposed or sham exposed for 8 months to 50-Hz sinusoidal EMF at two levels of field strength (1 kV/m, 5 µT and 5 kV/m, 100 µT, respectively). Thymus from adult animals exhibits signs of gradual atrophy mainly due to collagen deposition and fat substitution. This physiological involution may be accelerated by continuous light exposure that induces a massive death of thymocytes. The concurrent exposure to continuous light and to ELF-EMF did not change significantly the rate of mitoses compared to sham-exposed rats, whereas the amount of cell death was significantly increased, also in comparison with animals exposed to EMF in a 12-h dark-light cycle. In conclusion, long-term exposure to ELF-EMF, in animals housed under continuous light, may reinforce the alterations due to a photic stress, suggesting that, in vivo, stress and ELF-EMF exposure can act in synergy determining a more rapid involution of the thymus and might be responsible for an increased susceptibility to the potentially hazardous effects of ELF-EMF. 91 Quaglino et al.: Rat thymus, Photic Stress, and EMF exposure TheScientificWorldJOURNAL (2004) 4(S2), 91-99 A progressively expanded literature has been devoted in the last decades to the beneficial or noxious effects of electromagnetic fields (EMF) to live organisms KEYWORDS In previous in vitro experiments, it has been observed that exposure to extremely low frequency (ELF) EMF is not possibly affecting the functional characteristics of human lymphocytes Furthermore, thymocytes can be severely affected by other environmental factors such as light We performed in vivo experiments in order to (1) add further information on the role of a prolonged exposure to continuous light on rat thymocytes and (2) evaluate the possibility that a photic stress may act in synergy with a long-term exposure to ELF-EMF. MATERIALS AND METHODS Animals and Exposure System Two-month-old, male Sprague-Dawley rats (Charles River, Calco, Italy) were housed in the CESI (Milan, Italy) animal care exposure facilities and were fed ad libitum in a dark-light cycle (12:12 h) or under continuous light. Animals were further divided into three groups that were sham exposed or exposed to 50-Hz sinusoidal EMF at two levels of field strength (1 kV/m, 5 µT and 5 kV/m, 100 µT), respectively. Exposure to EMF was performed for 8 months, 5 days/week (Monday through Friday), 8 h/day (9 a.m. to 5 p.m.). Rats were individually housed in polycarbonate cages placed on electrically grounded metal nets in three identical exposure units. The electric field was generated by parallel electrodes with a 40-cm air gap. The magnetic field was generated by five pairs of vertically arranged rectangular coils (width 1.9 m, height 0.95 m). The external size of the system was 1.9 × 1.9 m and the distance between two adjacent pairs of coils was 0.6 m. Magnetic coils were fixed to wooden frames and the absence of vibrations was confirmed by acceleration measurements. Frames with coils were mechanically separated from the one holding the animal cages. The coil geometry was optimized in order to reduce the joule-heating losses at a level not affecting the microenvironment temperature. Sham-exposed animals were held in a similar nonenergized system. In the sham unit, after energizing the system, the measured magnetic field corresponded to the background level (0.04 µT). At least six animals from each experimental condition were sacrificed by decapitation between 9 a.m. and 12.30 p.m., then thymus was removed and processed for electron microscopy. No abnormalities were noted from the gross and anatomo-pathological points of view. Morphology At sacrifice, the left part of the thymus from each animal was cut into 1-mm 3 pieces, fixed in 2.5% glutaraldehyde in phosphate buffered saline (PBS) pH 7.4 for 24 h at room temperature, postfixed in 1% osmium tetroxide in the same buffer for 2 h at room temperature, dehydrated in alcohol, and embedded in epoxy resin. Sections, 1 µm thick, were stained with 1% toluidine blue and observed in a Zeiss Axiophot light microscope. Selected areas were further analyzed on ultrathin sections with a Jeol 1200 EX electron microscope. 92 Quaglino et al.: Rat thymus, Photic Stress, and EMF exposure TheScientificWorldJOURNAL (2004) 4(S2), 91-99 Morphometry Semi-thin sections of at least five specimens containing lymphoid tissue from each animal were carefully observed by light microscopy. The resin embedded material shows an excellent resolution allowing the evaluation of details that cannot be appreciated by classical histology. Only specimens comprehensive of both cortex and medulla were considered. In order to homogeneously compare the trend of data obtained using different criteria of evaluation, i.e., frequency and appearance, scores from 0-5 have been attributed, at 40× magnification, by two different investigators to the following parameters: cell-cell adhesion, mitoses, and cell death. All data have been referred to 0.1 mm 2 of tissue. Mitoses and apoptoses were counted and scored on a semi-quantitative scale from 0-5 (0, absent; 1, from 1-5; 2, from 6-10; 3, from 11-15; 4, from 16-20; 5, more than 20 on random fields). Necrosis and clustered cell death were graded semi-quantitatively on a scale from 0-5 (0, absent; 1, very scarce but present; 2; scarce; 3; moderately represented; 4, relatively abundant; 5, exuberant). Cell-cell adhesion was evaluated according to the space between thymocytes and their shape; score 0 was given when thymocytes were round and spread in an abundant necrotic or fibrotic material; score 1 was attributed to round thymocytes only rarely in close apposition and separated by areas devoid of cells; score 2 was assigned when at least 70% of thymocytes were round and dissociated, while the others were polygonal and maintained contact among themselves; score 3 was ascribed when at least 50% of thymocytes were round and dissociated, while the others were polygonal and maintained contact among themselves; score 4 was given when the majority of thymocytes presented a polygonal shape with limited disconnection; score 5 was attributed when almost all thymocytes were polygonal, in close apposition one to the other. Kruskal-Wallis and Dunn nonparametric statistical analyses were performed in order to assess the significance of our data. RESULTS Continuous Light Exposure In 10-month-old animals, thymus exhibited the morphology typical of adult animals: the organ was surrounded by a collagen capsule penetrating into the parenchyma and exhibiting few fibroblasts and small blood vessels. Areas of lymphoid tissue were frequently replaced with lipid degeneration. The separation between cortex and medulla was not always well defined and cells appeared quite separated one from the others Morphometric analyses revealed that, compared to animals housed in a DL, those caged under CL did not exhibit significant changes in the amount of mitoses The more pronounced cell death 95 Quaglino et al.: Rat thymus, Photic Stress, and EMF exposure TheScientificWorldJOURNAL (2004) 4(S2), 91-9

Fibroblast involvement in soft connective tissue calcification

Soft connective tissue calcification is not a passive process, but the consequence of metabolic changes of local mesenchymal cells that, depending on both genetic and environmental factors, alter the balance between pro- and anti-calcifying pathways. While the role of smooth muscle cells and pericytes in ectopic calcifications has been widely investigated, the involvement of fibroblasts is still elusive. Fibroblasts isolated from the dermis of pseudoxanthoma elasticum (PXE) patients and of patients exhibiting PXE-like clinical and histopathological findings offer an attractive model to investigate the mechanisms leading to the precipitation of mineral deposits within elastic fibers and to explore the influence of the genetic background and of the extracellular environment on fibroblast-associated calcifications, thus improving the knowledge on the role of mesenchymal cells on pathologic mineralization