Giovanni E. Orlandini

Aponecrosis: Morphological and biochemical exploration of a syncretic process of cell death sharing apoptosis and necrosis†

A rat fibroblastic cell line (rat‐1/myc‐ER™) was treated with different concentration of Antimycin A, a metabolic poison that affects mitochondrial respiratory chain complex III. The modes of cell death were analyzed by time‐lapse videomicroscopy, in situ end‐labeling (ISEL) technique, and ultrastructural analysis. Intracellular ATP levels were also measured in order to detect whether the energetic stores were determinant for the type of cell death. It was found that while apoptosis was the prevalent cell death in the fibroblasts treated with low doses, 100 or 200 μM Antimycin A, a new type of cell demise that shared dynamic, molecular, and morphological features with both apoptosis and necrosis represents the most common cell death when the cells were exposed to high doses, 300 or 400 μM, of the hypoxic stimulus. This new type of cell death has been chimerically termed aponecrosis. The inhibition of caspase 3, an enzyme critical for the apoptotic DNA degradation, caused a clear shift from aponecrosis to necrosis in the cell culture, suggesting that this new type of cell death could account for an incomplete execution of the apoptotic program and the following degeneration in necrosis. After being treated with higher doses, i.e., 1000 μM Antimycin A, almost all of the cells died by true necrosis. The analysis of the cellular energetic stores showed that the levels of ATP were a primary determinant in directing toward active cell death (apoptosis), aponecrosis, or necrosis. We conclude that chemically induced hypoxia produces different types of cell death depending on the intensity of the insult and on the ATP availability of the cell, and that the classic apoptosis and necrosis may represent only two extremes of a continuum of intermediate forms of cell demise. J. Cell. Physiol. 182:41–49, 2000.

Paracrine effects of transplanted myoblasts and relaxin on post-infarction heart remodelling.

In the post‐infarcted heart, grafting of precursor cells may partially restore heart function but the improvement is modest and the mechanisms involved remain to be elucidated. Here, we explored this issue by transplanting C2C12 myoblasts, genetically engineered to express enhanced green fluorescent protein (eGFP) or eGFP and the cardiotropic hormone relaxin (RLX) through coronary venous route to swine with experimental chronic myocardial infarction. The rationale was to deliver constant, biologically effective levels of RLX at the site of cell engraftment. One month after engraftment, histological analysis showed that C2C12 myoblasts selectively settled in the ischaemic scar and were located around blood vessels showing an activated endothelium (ICAM‐1‐,VCAM‐positive). C2C12 myoblasts did not trans‐differentiate towards a cardiac phenotype, but did induce extracellular matrix remodelling by the secretion of matrix metalloproteases (MMP) and increase microvessel density through the expression of vascular endothelial growth factor (VEGF). Relaxin‐producing C2C12 myoblasts displayed greater efficacy to engraft the post‐ischaemic scar and to induce extracellular matrix re‐modelling and angiogenesis as compared with the control cells. By echocardio‐graphy, C2C12‐engrafted swine showed improved heart contractility compared with the ungrafted controls, especially those producing RLX. We suggest that the beneficial effects of myoblast grafting on cardiac function are primarily dependent on the paracrine effects of transplanted cells on extracellular matrix remodelling and vascularization. The combined treatment with myoblast transplantation and local RLX production may be helpful in preventing deleterious cardiac remodelling and may hold therapeutic possibility for post‐infarcted patients.

INSULIN-LIKE GROWTH FACTOR-I STIMULATES IN VITRO MIGRATION OF PREOSTEOCLASTS ACROSS BONE ENDOTHELIAL CELLS

Abstract.Little is known about the factors and the mechanisms involved in preosteoclast emigration from the vasculature. In this study, an in vitro model of bone endothelial lining was mimicked by culturing bone endothelial (BBE) cells at confluence on a 3-μm pore polycarbonate membranes. Preosteoclastic (FLG 29.1) cells were then added on top of the BBE cell monolayer and 10 nM insulin-like growth factor-1 (IGF-I) was added below the supporting membrane. Scanning and transmission electron microscopy were used to evaluate the chemotactic responses of preosteoclastic FLG 29.1 cells towards the IGF-I generated gradient. IGF-I potently stimulated chemotaxis in the FLG 29.1 cells, as shown by the migration of the preosteoclastic cells across the underlying BBE and through the intercellular junctions between adjacent endothelial cells. Subsequently, FLG 29.1 cells penetrated the pores of the supporting membrane and reached the lower face of the membrane. Thus, IGF-I, which is abundantly present in the bone tissue microenvironment, may play a paracrine role in the recruitment of the circulating preosteoclasts from the vascular compartment into the bone tissue. This in vitro model, which mimicks the in vivo phenomenon of preosteoclast extravasation, should prove useful in elucidating the molecular mechanisms that underlie this process.

Effects of ipriflavone on perialveolar bone formation.

Abstract. The effect of ipriflavone (IP), a synthetic isoflavonoid derivative, on in vivo bone formation was studied in rat perialveolar bone by surgically producing a hole in the mandibular bone. The holes were filled either with powdered IP or with compounds containing no osteoinductive properties such as biostite and Htr (hard tissue replacement). In control animals, the holes were left to heal spontaneously. The animals were killed 3, 28, and 40 days after surgery and a detailed morphological and morphometric study was performed on the perialveolar bone surrounding the wounds. Three days after surgery (inflammatory phase) the bone wounds were occupied by hemorragic and inflammatory cells in both the untreated and IP-treated bone defects. Twenty-eight days after surgery, bone formation was evident with new bone spiculae particularly concentrated in the area of the bone lesion closest to the adjacent periodontal ligament. Morphometric measurements of the areas occupied by new bone showed that the synthesis of perialveolar bone was significantly stimulated by IP. The repair of the bone defects by new bone formation progressed by day 40, but only in the presence of IP were the original holes almost completely repaired. Conversely, biostite and Htr did not influence promotion of new bone formation. In conclusion, the results of the present study are consistent with a role of IP in stimulating osteogenesis and suggest that this compound could represent a potential therapeutic tool to promote repair of injured perialveolar bone.

Scanning electron microscopic observations on the epithelium of the human prostatic urethra.

Summary: The human prostatic urethra has been investigated by means of scanning electron microscopy. On the posterior wall of the urethra, the seminal colliculus with the orifices of the ejaculatory ducts is clearly detectable. The upper portion of the prostatic urethra shows a typical transitional epithelium with large superficial cells of a ruffled appearance. In the lower portion of the organ (underneath the openings of the ejaculatory ducts), the apical pattern of the cells varies considerably. Four main aspects are recognizable: a) apices provided with microvilli, b) dome‐shaped apices with an almost smooth surface, c) large apices with labyrinthic microplicae and d) ciliated apices. Also, apices showing intermediate characteristics can be noted. The functional significance of the morphological patterns as well as the possibility of a transition among the various types of surface structures are discussed.

Scanning electron microscopic evaluation of the alterations induced by polluted air in the rabbit bronchial epithelium

With the aim of evaluating the influence of polluted air on the respiratory epithelium, ten New Zealand white rabbits, from a group of fifteen kept in the country, were transported to a site located in a metropolitan area. After 40 days, all the rabbits were killed, and the bronchial mucosa studied by scanning electron microscopy (SEM). In the animals exposed to polluted air, the ciliated cells, less numerous than in normal cases, show an evident decrease in the number and size of the cilia, exposing apical microvilli. It is therefore possible to hypothesize that a part of the non-ciliated cells is made up of cells that have lost their cilia. The number of non-ciliated elements and the amount of mucous secretion appear to have noticeably increased. The considerable response of the respiratory epithelium to inhaled agents appears to be confirmed, as is the irritant effect of polluted city air.

Ultrastructure of Human Male Urethra

The morphological features of human male urethra lining epithelium have been investigated by means of SEM at the various portions of the organ. The characteristics of the epithelial cells have been described and the significance of different morphological pattern is discussed.

Histochemical detection of sugar residues in chick embryo developing lingual glands with horseradish-peroxidase conjugated lectins

Tongue anlage were taken in chick embryos from the 7th to the 21st d of incubation and in 3 d old chicken. A battery of 7 different horseradish peroxidase-conjugated lectins (PNA, ConA, DBA, SBA, LTA, WGA, UEA I) was used to study the carbohydrate residues of glycoconjugates at the epithelial cells of the anterior and posterior lingual glands. Some sugar residues, detected at the surface of the epithelial cells in early developmental stages of glandular primordia, seemed to play a role in inducing and regulating the first differentiative steps of the glands. Differences in type, amount, time of appearance and cellular localization between the 2 groups of glands were detected. The group of the anterior lingual glands, adjacent to the entoglossal cartilage (paraentoglossal glands), showed some peculiar histochemical characteristics.

Vas Deferens and Seminal Vesicles Epithelium after Castration: a SEM Study

Vas deferens und Samenbläschen‐Epithel nach der Kastration: Eine Rasterelektronenmikroskopische Untersuchung

Histochemistry of mucosubstances in the gallbladder epithelium of the chick embryo

SummaryMucosubstances of epithelial cells of the chick embryo gallbladder were investigated by histochemical methods from days 12–21 of incubation (stages 38–46). On incubation day 14, only neutral mucins were detected; on day 15, neutral and sulfated mucosubstances were observed in the epithelial cells that invaginate the underlying mesenchyme. In the same sites, at day 16 of incubation, neutral, carboxylated and sulfated mucins were seen. From the 17th day of incubation until hatching, neutral, carboxylated and sulfated mucosubstances were present in the surface cells and in the cells lining the epithelial invaginations. During this period, the chemical characteristics of the secretory material are similar to those observed by Yamada and Hoshino (1972) in the fowl.