S. Zecchi 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.

Characterization and function of the receptor for IGF-I in human preosteoclastic cells

Using a coculture system, we have recently demonstrated that insulin-like growth factor I (IGF-I) is a mediator of preosteoclastic cell migration toward bone-derived endothelial cells. To better characterize the mechanisms of IGF-I action on preosteoclastic cells we evaluated the expression of type I IGFs receptor in the human leukemic cell line, FLG 29.1, which differentiates toward the osteoclastic phenotype following phorbol ester (TPA) treatment. Scatchard analysis of 125I-labeled IGF-I to FLG 29.1 cells revealed the presence of a single high affinity binding site in both untreated and TPA-treated cells with a similar Kd value (0.3 +/- 0.2 nmol/L and 0.4 +/- 0.1 nmol/L, respectively). In untreated cells, IGF-I binding capacity (1.43 +/- 0.41 fmol/10(6) cells) was significantly (p < 0.05) lower than in TPA-treated cells (2.62 +/- 0.87 fmol/10(6) cells). Competition analyses and crosslinking studies revealed the presence of type I IGF receptor both in untreated and TPA-treated cells. Northern analysis demonstrated that mRNA for IGF-I receptor was expressed by both untreated and TPA-treated FLG 29.1 cells. In addition, FLG 29.1 cells released in the conditioned medium IGFBP-2 and IGFBP-4, whose expression was increased by TPA treatment as demonstrated by ligand and immunoblot analyses. The previous observations of chemotactic action of IGF-I on FLG 29.1 cells was confirmed by ultrastructural observations. Indeed, these cells revealed a marked migratory activity in response to nanomolar concentrations of IGF-I. In addition, the IGF-I receptor alpha IR-3 antiserum inhibited the IGF-I-induced FLG 29.1 cells migratory activity. These findings clearly show that type IIGF receptor is expressed by osteoclast precursors and that IGF-I induces migration of these through the binding to type I IGF receptors. Binding proteins expressed by osteoclast precursors may play an autocrine role in modulating the IGF-I bioeffects.

Prevention of corneal keratocyte apoptosis after argon fluoride excimer laser irradiation with the free radical scavenger ubiquinone Q10.

Purpose To assess in vitro the potential of the free radical scavenger ubiquinone Q10 in preventing keratocyte apoptosis after argon fluoride (ArF) excimer laser irradiation. Methods Cultured rabbit keratocytes were irradiated at very low single-pulse laser fluences. The cumulative effects generated by three total fluence doses between 12 and 45 mJ/cm2, representative of single-pulse subablative doses during photorefractive keratectomy (PRK) in humans, were evaluated. We employed the following parameters to compare pretreated (10 μM ubiquinone Q10) and untreated samples: 1) number and morphology of living cells by Trypan blue test and ultramicroscopy, respectively; 2) level of free-radical formation assessed by malonaldehyde quantitation; 3) cellular energy level evaluated by ATP assay. Results Excimer laser irradiation kills cultured keratocytes by inducing apoptosis. The effect increases with the cumulative fluence dose. In the samples pretreated with ubiquinone Q10 there were significantly fewer cumulative apoptotic events than in the untreated ones. Quantitative analysis of malonaldehyde cellular levels suggested this protective action of ubiquinone Q10 was connected with its ability to scavenge laser-generated free radicals. ATP assay also confirmed that it raised cellular energy levels. Conclusions The treatment of corneal keratocytes with relatively low concentrations of ubiquinone Q10 can prevent apoptosis after ArF excimer laser irradiation. If these findings are confirmed on human keratocytes this treatment could be usefully exploited in the PRK surgical procedure. That might lead to a reduction in the occurrence of haze and curvature regression triggered by programmed cell death.

Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cells.

We have previously shown that sphingosine 1‐phosphate (S1P) can induce intracellular Ca2+ mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca2+‐independent mechanisms of cell contraction have been the focus of numerous studies on Ca2+ sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho‐dependent kinase mediate most of Ca2+‐independent contractile responses. In the present study, we examined the potential role of Rho/Rho‐kinase cascade activation in S1P‐induced C2C12 cell contraction. First, we showed that depletion of Ca2+, by pre‐treatment with BAPTA, did not affect S1P‐induced myoblastic contractility, whereas it abolished S1P‐induced Ca2+ transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P‐stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist‐stimulation in cells depleted of Ca2+ and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y‐27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKCα and PKCδ, respectively, failed to inhibit the agonist‐induced myoblastic contraction. Single particle tracking of G‐actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca2+‐independent/Rho‐Rho kinase‐dependent pathways may exert an important role in S1P‐induced myoblastic cell contraction. J. Cell. Physiol. 198: 1–11, 2004© 2003 Wiley‐Liss, Inc.

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.

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.

Morphological study of fetal nasopharyngeal epithelium in man.

In 30 human fetuses between 8 and 13 weeks of intrauterine life the lateral wall of the nasopharynx was examined by light microscopy and transmission and scanning electron microscopy. In the subjects between 8 and 9 weeks in utero the mucosa displays still an immature appearance, being mono- or bistratified and lacking the characteristic structures of the respiratory epithelium. Nevertheless, signs of differentiation are to be noticed, with the presence of two distinct cellular types that, in the later periods, will give rise to ciliated cells and microvillus-provided cells. An almost complete differentiation will be reached at 12-13 weeks in utero, even if goblet cells are still lacking in the examined zone during the considered period. Nonrespiratory types of epithelium, such as transitional or squamous, were never found in the studied subjects.