Histomorphometric, biochemical and ultrastructural changes in the aorta of salt-loaded stroke-prone spontaneously hypertensive rats fed a Japanese-style diet.

BACKGROUND AND AIM: It is demonstrated that dietary habits play a role in cardiovascular diseases. In stroke-prone spontaneously hypertensive rats (SHRsp), concomitant salt loading and a Japanese-style diet greatly accelerate hypertension and the appearance of cerebrovascular lesions by directly damaging arterial vessels. A number of studies have characterised medium and small vessel lesions in SHRsp, but little attention has been paid to the changes in the wall structure of large arteries induced by exposure to a salt-enriched diet. The aim of this study was to investigate the effects of a Japanese-style diet and salt loading on the thoracic aorta. METHODS AND RESULTS: Two-month-old SHRsp were kept on a Japanese-style diet with 1% sodium chloride solution replacing tap water. Two months later, they were sacrificed and compared with age-matched or two-month-old control SHRsp kept on a standard diet and tap water in terms of the histomorphometry, ultrastructure and biochemical composition of the thoracic aorta. The vessel was consistently thicker in the four-month-old SHRsp (+20%, p < 0.05 vs two-month-old rats) regardless of diet. The salt-loaded SHRsp showed a significant reduction in elastic fibre density (-20%, p < 0.05 vs two-month-old rats) and an increase in the other matrix components (%), whereas the four-month-old controls showed preserved elastic fibres and a significant increase in the other matrix components (+65%, p < 0.05 vs two-month-old rats). There was a considerable increase in the amounts of 4-OH-proline (+147%), 5-OH-lysine (+174%) and desmosines (+360%) in the four-month-old controls vs their two-month-old counterparts (p < 0.01), but not in the salt-loaded animals. Ultrastructural analysis revealed clear damage and accelerated aging in the thoracic aorta of the salt-loaded SHRsp. CONCLUSIONS: Salt loading and a Japanese-style diet destabilize thoracic aorta architecture in SHRsp after two months of treatment.

Characterization of pseudoxanthoma elasticum-like lesions in the skin of patients with beta-thalassemia.

BACKGROUND: Pseudoxanthoma elasticum (PXE), an inherited disorder of unknown pathogenesis, is characterized by elastic fiber mineralization, collagen fibril alterations, and accumulation of thread material in the extracellular space. PXE-like clinical lesions have been described in patients with beta-thalassemia. OBJECTIVE AND METHODS: Dermal lesions in these two genetic disorders were compared by light and electron microscopy and by immunocytochemistry. RESULTS: In both disorders, elastic fiber polymorphism, fragmentation, and mineralization were structurally identical. Elastic fiber mineralization in beta-thalassemia was associated with vitronectin, bone sialoprotein, and alkaline phosphatase, similar to what was observed in inherited PXE. Furthermore, abnormalities of collagen fibrils and filament aggregates were identical in both disorders. In both inherited and beta-thalassemia-associated PXE, unrelated gene defects seem to induce cell metabolic abnormalities that lead to identical clinical and structural phenotypes. CONCLUSION: Data indicate that patients with beta-thalassemia may undergo important alterations of connective tissues, a better understanding of which may help in preventing clinical complications.

Antimicrobial properties and morphological characteristics of two Photorhabdus luminescens strains.

The biological properties of two Photorhabdus luminescens isolates (MU1 and MU2) of environmental source and the activity of antimicrobial agar diffusible agents (AADA) produced by the same are reported. With regard to cultural features, two variant forms for P. luminescens MU1 and three for P. luminescens MU2 (including an intermediate phase I-like form) have been found. These three forms differ in biological and biochemical properties: beta-lactamase, urease, bioluminescence and antimicrobial agar diffusible substance production associated with the phase I form, were less evident in the intermediate phase I-like MU2 and were absent in phase II form. Antimicrobial activity was present in both strains, with the production of a large amount of a diffusible compound with a wide spectrum of action against bacteria of other genera; a reduced activity against correlated species was also observed. Examination by electron microscopy of MU1 and MU2 purified broth cultures revealed the presence of particles belonging to the class of the phage tail-like bacteriocins, described in recent studies as responsible for antibacterial activity against correlated bacteria, a result never confirmed "in vitro". A plasmid of 21 Mdal was observed in all the form variants of P. luminescens MU2, suggesting that plasmids are not involved in the transition from primary to secondary phase; no plasmid was detected in P. luminescens MU1.

Inhibition of myogenesis by transforming growth factor beta is density-dependent and related to the translocation of transcription factor MEF2 to the cytoplasm.

Transforming growth factor beta (TGF-beta) was found to inhibit differentiation of myogenic cells only when they were grown to high density. Inhibition also occurred when myogenic cells were cocultured with other types of mesenchymal cells but not when they were cocultured with epithelial cells. It is therefore possible that some density-dependent signaling mediates the intracellular response to TGF-beta. Within 30 min of treatment, TGF-beta induced translocation of MEF2, but not MyoD, myogenin, or p21, to the cytoplasm of myogenic cells grown to high density. Translocation was reversible on withdrawal of TGF-beta. By using immune electron microscopy and Western blot analysis on subcellular fractions, MEF2 was shown to be tightly associated with cytoskeleton membrane components. To test whether MEF2 export from the nucleus was causally related to the inhibitory action of TGF-beta, we transfected C2C12 myoblasts with MEF2C containing the nuclear localization signal of simian virus 40 large T antigen (nlsSV40). Myogenic cells expressing the chimerical MEF2C/nlsSV40, but not wild-type MEF2C, retained this transcription factor in the nucleus and were resistant to the inhibitory action of TGF-beta. We propose a mechanism in which the inhibition of myogenesis by TGF-beta is mediated through MEF2 localization to the cytoplasm, thus preventing it from participating in an active transcriptional complex.

Study of elastic fiber organization by scanning force microscopy.

Elastic fibers of beef ligamentum nuchae were observed by atomic force microscopy and data compared with those obtained by conventional and freeze-fracture electron microscopy. Fresh isolated elastin fibers as well as thin sections of ligament fragments, which were fixed and embedded either in relaxed or in stretched conditions, were analysed. The results confirm that, at least in beef ligamentum nuchae, elastic fibers consist of beaded filaments which can be oriented by stretching in the direction of the force applied. Moreover, atomic force microscopy revealed that these beaded filaments are laterally connected by periodical bridges which become more pronounced upon stretching. The data clearly show that elastin molecules are organized in a rather ordered array, at least at the super-molecular level, and a depiction of the elastin organization in beef ligamentum nuchae is attempted.

Elastic fiber during development and aging.

Elastin molecules aggregate in the extracellular space where they are crosslinked by stable desmosine bridges. The resulting polymer is structurally organized as branched fibers and lamellae, which, in skin, are wider (a few microns) in the deep dermis and become progressively thinner (fraction of a micron) towards the papillary dermis. Several general and local factors seem to regulate elastin gene expression, deposition and degradation. In skin, the volume density of the elastin network increases from birth up to maturity, when it accounts for about 3-4% of the tissue. However, its amount and distribution depend on dermis areas, which are different among subjects and change with age. Several matrix molecules (glycosaminoglycans, decorin, biglycan, osteopontin) have been found to be associated with elastin into the normal fiber, and several others have been recognized within pathologic elastic fiber (osteonectin, vitronectin, alkaline phosphatase in PXE). With age, and in some pathologic conditions, skin elastin may undergo irreversible structural and compositional changes, which seem to progress from localized deposition of osmiophilic materials to the substitution of the great majority of the amorphous elastin with interwoven filaments negative for elastin specific antibodies.

Proteoglycan alterations in skin fibroblast cultures from patients affected with pseudoxanthoma elasticum.

Proteoglycans (PGs) were investigated in fibroblast cultures from both apparently normal and involved areas of skin from two patients affected with Pseudoxanthoma elasticum (PXE) and compared to control normal cells. Biochemical analysis showed that cells from the PXE-affected patients produced a PG population with stronger polyanion properties, as well as a markedly increased amount of high hydrodynamic-size PGs. Moreover, PGs from PXE-affected cells showed abnormal hydrophobic interaction properties when examined under associative conditions and included heparan sulphate (HS)-containing populations with anomalous electrophoretic mobility. These phenomena were particularly evident in the case of PGs secreted into the growth medium. In agreement with these findings immunohistochemical study showed alterations affecting decorin and biglycan, as well as a different content and distribution of HS-PGs in PXE-affected cells. The same biochemical and morphological alterations were confirmed for both patients on different cell cultures and were present in cells from both apparently normal and affected skin areas, being more pronounced in the latter. Our results indicate that PXE-affected fibroblasts in culture exhibit an abnormal PG metabolism, which could affect the normal assembly of extracellular matrix.

Osteopontin is a constitutive component of normal elastic fibers in human skin and aorta.

Osteopontin is an acidic matrix protein, mainly expressed in mineralized tissues, kidney and atherosclerotic vessels; its biological role is still largely undefined. In the present study, immunocytochemical approaches showed that osteopontin is localized within normal elastic fibers of human skin and aorta. Antibodies raised against human bone osteopontin (LF7) or against human osteopontin synthetic peptide (amino acids 1-10, LF19) recognized epitopes associated with the amorphous material within the elastic fibers. Elastic fiber-associated microfibrils were always negative. The positivity for osteopontin of the elastic fibers was independent of age and could be observed in fetal skin and aorta as well as in the same of children, young adults and old subjects. The altered elastic fibers in the skin of old individuals were only fairly positive for osteopontin. The presence of osteopontin within the elastic fibers suggests that it may play a role against the observed tendency of elastic fibers to favor mineral precipitation. A role of osteopontin in modulating crystal nucleation and growth in mineralizing tissues and, more generally, in conditions in which mineral precipitation should be controlled is also possible.

Ultrastructure of elastin.

Almost all structural studies on elastin have been done in higher vertebrates, in which it is organized as an extracellular network of branched fibres which vary from fractions f microns to several microns in diameter. By conventional electron microscopy, elastin appears amorphous. By both freeze-fracture and negative staining on cryosections, it can be resolved as beaded filaments 5 nm in diameter forming a 3D meshwork that, upon stretching, becomes oriented in the direction of the force applied. This filamentous aggregation of elastin molecules is confirmed in vitro by the observation that its soluble precursor, tropoelastin, shows a strong tendency to associate into short 5 nm-thick filaments that, with time, become longer and aggregate into bundles of various dimensions. If chemically fixed and embedded, these aggregates appear amorphous and identical to natural elastin fibres. The tendency of tropoelastin to aggregate into 4-5 nm-thick beaded filaments, which then associate into 12 nm-thick filaments forming a 3D network, has been observed by atomic force microscopy for recombinant human tropoelastin. Therefore, the amorphous structure of elastin seems to be a technical artefact. Apart from elastin-associated microfibrils, which are always present at the periphery of growing elastic fibres and probably have a role more complex than being a scaffold for tropoelastin aggregation in vivo, the elastic fibres seem to be composed of several matrix constituents, which are different in different organs and change with age and in pathological conditions. This is demonstrated by immunocytochemical studies on ultrathin sections.

Ultrastructural analysis of skin and aorta from a patient with Menkes disease.

Ultrastructural studies of the skin and aorta of a patient with Menkes disease, an X-linked recessive disorder of copper metabolism, are described. Dermal thickness was normal, while dermal collagen fibrils exhibited a heterogeneous size range, with a mean diameter smaller than normal. Long-spacing collagen was often observed near fibroblasts, the plasma membranes of which were decorated by aggregates of interwoven filaments. Dermal elastin fibers were scarce and consisted of thin strands of amorphous elastin associated with numerous microfibrils. In the aorta, the amount of collagen was normal, although the fibrils displayed a broader range of diameters than normal, with a slightly smaller mean. Elastin fibers showed considerable disruption, appearing fragmented and wider than normal, and displaying irregular contours. The inclusion of cationic dyes during tissue fixation gave rise to numerous electron-dense precipitates within the elastin fibers, suggesting the presence there of glycosaminoglycans or proteoglycans, among which unsulfated and sulfated chondroitins were demonstrated by immunoelectron microscopy to be prominent. Heparan sulfate, observed to be a constituent of normal elastin fibers, was much reduced in amount. Elastin was also found associated with glycosaminoglycans in the soluble matrix of the aortic wall.

Immunochemical identification of abnormal constituents in the dermis of pseudoxanthoma elasticum patients.

Pseudoxanthoma elasticum (PXE) is a connective tissue inherited disease characterized by dermal alterations and mineralization of the elastin fibres. To investigate its pathogenesis, which is still unknown, antibodies against the principal connective tissue components were assayed on ultrathin sections of dermis from 7 PXE subjects and 5 age matched controls. Both control and PXE elastin fibres were positive for heparan, dermatan and chondroitin 0-sulphates, decorin and biglycan. In PXE, elastin fibres were also highly positive for chondroitin 6-sulphate, vitronectin, fibronectin and serum amyloid antigen. Vitronectin and fibronectin were mostly concentrated in the areas of dense mineralization within the elastin fibres. The abnormal microfilament aggregates, often seen in PXE dermis, were positive for all the above mentioned molecular species as well as for collagen types I and III and fibrillin; on the contrary, they were always negative for elastin. The results suggest that PXE is a complex disorder, in which the whole extracellular matrix is deeply disturbed. Therefore, without excluding an elastin gene defect, the data seem rather to suggest that PXE is a disorder of the mechanisms controlling the production of matrix constituents and that elastin mineralization is caused by molecules abnormally produced and entrapped within the fibre during elastin fibrogenesis.

A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1).

A new method for the cytofluorimetric analysis of mitochondrial membrane potential in intact cells has been developed by using the lipophilic cationic probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1), whose monomer emits at 527 nm after excitation at 490 nm. Depending on the membrane potential, JC-1 is able of forming J-aggregates that are associated with a large shift in emission (590 nm). The color of the dye changes reversibly from green to greenish orange as the mitochondrial membrane becomes more polarized. In two human cell lines (K562 and U937), we have studied by flow cytometry the changes in membrane potential provoked by the K+ ionophor valinomycin, a drug known to affect mitochondrial membrane potential, while the K+/H+ ionophor nigericin, known to affect intracellular pH but not mitochondrial membrane potential, was used as control. The incubation with valinomycin for 10 min. at 37 degrees C in a low K+ medium provoked a marked and dose-dependent reduction in JC-1 greenish orange fluorescence, while nigericin had no effect.

A clinical, ultrastructural and immunochemical study of Dupuytren's disease.

Aponeurotic tissue from seven normal subjects and from apparently unaffected branches, nodules and cords of 16 Dupuytren's patients were compared. Control tissue was characterized by polymorphous cells, showing cytoplasmic microfilament bundles, numerous pinocytic vesicles, basement membrane-like structures, and a thick coat of interwoven filaments, and by type I- and III-positive heterogeneous collagen fibrils, fibronectin, vitronectin, decorin and proteoglycans. The clinically normal branches consisted of fibroblast-like cells, small type III-highly positive collagen fibrils, fibronectin and proteoglycans. Nodules and fibrotic cords contained fibroblast-like cells, type I and III collagen, fibronectin and proteoglycans. Myofibroblast-like cells in only five out of 16 patients were present. There was no relation between clinical stage and structural alterations; the whole aponeurosis always seemed to be involved; cord retraction would seem to depend on the interactions among fibroblast-like cells and matrix components and among matrix macromolecules themselves.

Cell-matrix interactions in cultured dermal fibroblasts from patients with an inherited connective-tissue disorder.

Indirect-immunofluorescence studies were performed on cultured dermal fibroblasts from patients with Pseudoxanthoma Elasticum (PXE), an inherited connective-tissue disorder the pathogenesis of which is still unknown. Apparent abnormalities of cytoskeletal structures were revealed by using phalloidin and specific antibodies to alfa-smooth muscle actin and to vimentin. Altered expression of integrin receptors for different extracellular matrix components seems to be present in the pathological cells as preliminary data suggest by using antibodies against alfa subunits of integrins. This study was designed to test the presence of abnormal cell-matrix interactions responsible for the clinical features and involved in the pathogenesis of the PXE disease.

Cell-matrix interactions in cultured dermal fibroblasts from patients with an inherited connective-tissue disorder.

Indirect-immunofluorescence studies were performed on cultured dermal fibroblasts from patients with Pseudoxanthoma Elasticum (PXE), an inherited connective-tissue disorder the pathogenesis of which is still unknown. Apparent abnormalities of cytoskeletal structures were revealed by using phalloidin and specific antibodies to alfa-smooth muscle actin and to vimentin. Altered expression of intergrin receptors for different extracellular matrix components seems to be present in the pathological cells as preliminary data suggest by using antibodies against alfa subunits of integrins. This study was designed to test the presence of abnormal cell-matrix interactions responsible for the clinical features and involved in the pathogenesis of the PXE disease.

Aging of the human synovium: an in vivo and ex vivo morphological study.

Age-associated changes of the human synovium have been investigated by microarthroscopy, optical and electron microscopy, immunohistochemistry, and cytochemistry. The knee joints of nineteen 15- to 56-year-old subjects, classified as normal by inspection, were carefully examined by microarthroscopy; small synovial tissue biopsy specimens from both the suprapatellar pouch and the medial tibiofemoral gutter were taken. Microarthroscopy showed that the villi were more numerous and the vascular network and cell distribution and profiles less regular in aged individuals. These data were confirmed by scanning electron microscopy, which also showed large areas of the synovial surface devoid of cells and collagen bundles in contact with the joint cavity in aged subjects. Light and transmission electron microscopy confirmed these data and allowed evaluation of the number, distribution, shape, and internal organization of cells as well as the distribution of vessels and the organization of the extracellular matrix in the full thickness of the synovium (down to 2 mm). Particular attention was paid to synovial lining cells, among which three main phenotypes could be recognized: synthetic type (present at all ages and hypertrophied in aged subjects), macrophagelike (increasing with age), and fibroblastlike. Collagen increased with age. Further studies are needed for comprehensive understanding of age-associated changes in the human synovium.

Liver iron overload and desferrioxamine treatment of porphyria cutanea tarda.

The aim of this paper is to evaluate invasive and non-invasive indices of iron store and compare the effectiveness of different ferrodepletive protocols in 150 patients with porphyria cutanea tarda (PCT). Iron removal was performed either by intensive phlebotomy (22 cases) or slow subcutaneous and high intravenous doses of desferrioxamine (18 and 5 cases, respectively), and several laboratory parameters were studied; among these, oligo-elements and urinary porphyrins (detected by HPLC) were taken into account before and after the treatments. Serum iron, transferrin saturation, ferritin (RIA) and nuclear magnetic resonance results were compared with invasive findings in order to detect the metal deposition in liver tissue (atomic absorption concentration, optic or electron-microscopic detection). Liver iron overload was observed in 95% of cases. Full normalization of the disease took place by all the treatments, even if it required slightly more time in the phlebotomy group. We may conclude that ferrodepletive treatments are highly effective in PCT and, considering the fact that siderosis and liver damage always accompany the disease, these treatments are proposed as first choice in such cases.

Immunocytochemical localization of proteoglycans within normal elastin fibers.

Appreciable amounts of glycosaminoglycans have been found by immunocytochemistry within mature elastin fibers of human dermis. On thin sections, elastin fibers showed antigenic sites for monoclonal antibodies recognizing the unsaturated units remaining after digestion of hyaluronic acid with Streptomyces hyaluronidase and after digestion of dermatan and chondroitin sulfates with chondroitinase ABC. Moreover, sectioned elastin fibers were positive towards antibodies raised against synthetic peptides corresponding to amino acid sequences near the N-terminus of the protein core of small matrix proteoglycans PGI and PGII, respectively (Fisher et al., J. Biol. Chem. 262, 9702-9708 (1987)). This is the first demonstration that highly hydrophylic molecules are strictly associated with normally cross-linked elastin. The presence of highly hydrated molecules within the elastin polymer could greatly influence its physiological properties and behavior in pathology.

Localization of human placenta lysyl oxidase on human placenta, skin and aorta by immunoelectronmicroscopy.

Polyclonal antibodies to human placenta lysyl oxidase (Kuivaniemi et al., 1984) were used to localize the enzyme at ultrastructural level in human placenta, skin and aorta, by using the indirect immunogold method. The antibodies were tested on thin sections of tissues fixed and embedded in various experimental conditions. With all methods employed, the immunoreaction was always positive on collagen fibers in all tissues examined, independently of the age of the subjects. In placenta, the reaction was also slightly positive on matrix microfilaments and cells. In dermis, fibroblasts and elastin were scarcely positive in a normal 5 day-old child, in a child with skin hyperelasticity, and in two babies with osteogenesis imperfecta type II; whereas they were negative in two 16 and 40 year-old normal subjects. In aorta, the immunoreaction was always positive on collagen, scarcely positive on cells and on elastin of a 24 week-old fetus, of a normal child, and of two babies who died of complications associated with O.I. type II; on the contrary, the reaction was negative on cells and elastin fibers of a 16 week-old fetus, and of a normal 19 year-old girl. When present on elastin, gold particles were localized mostly inside the fibers. Contrary to what was observed by Kagan and coworkers on bovine aorta by using antibodies against aortic lysyl oxidase (Kagan et al., 1986), no specific localization of gold particles could be observed on or adjacent to the elastin/associated microfibrils. The results indicate that antibodies raised against placenta lysyl oxidase recognize collagen-associated as well as elastin-associated epitopes.(ABSTRACT TRUNCATED AT 250 WORDS)

Dermal alterations in patients with Wilson's disease treated with D-penicillamine.

Wilson's disease is characterized by accumulation of copper and D-penicillamine favors its elimination. However, penicillamine binds to precursors of intermolecular crosslinks both in collagen and elastin, and could lead to alterations of these two fibrous proteins. In the present report skin biopsies from patients with Wilson's disease, treated with 900 mg/day of D-penicillamine, for 5, 9, 58 and 60 months, were studied by electron microscopy and compared with findings obtained from skin biopsies of age-matched normal subjects. Clinically, the elasticity and consistency of the skin of Wilson's patients was not modified by D-penicillamine treatment. The ultrastructural organization of collagen fibrils appeared normal in the adults treated with D-penicillamine for 5-9 months. In a 15-year-old girl, treated for 48 months, a high number of collagen fibrils were swollen and unreeved. Elastin fibers were altered in all patients. The alterations were mostly pronounced in the reticular dermis, were proportional to the time of treatment, and consisted of polymorphous aggregates of elastin connected to apparently normal elastin fibers. A stereological analysis, on EM pictures from the patient treated for 60 months, and from an age-matched control, showed a significant decrease in the percentage of collagen and of the mean area occupied by each collagen bundle in the reticular dermis of the patient compared to control; on the contrary, the number of elastin fibers per unit area increased significantly, and the mean area of each elastin fiber decreased. The volume density of elastin was similar to control. The results indicate that prolonged administration of penicillamine to humans induces alterations in the deposition of dermal collagen and elastin.