In vitro cultured progenitors and precursors of cardiac cell lineages from human normal and post-ischemic hearts.

The demonstration of the presence of dividing primitive cells in damaged hearts has sparked increased interest about myocardium regenerative processes. We examined the rate and the differentiation of in vitro cultured resident cardiac primitive cells obtained from pathological and normal human hearts in order to evaluate the activation of progenitors and precursors of cardiac cell lineages in post-ischemic human hearts. The precursors and progenitors of cardiomyocyte, smooth muscle and endothelial lineage were identified by immunocytochemistry and the expression of characteristic markers was studied by western blot and RT-PCR. The amount of proteins characteristic for cardiac cells (alpha-SA and MHC, VEGFR-2 and FVIII, SMA for the precursors of cardiomyocytes, endothelial and smooth muscle cells, respectively) inclines toward an increase in both alpha-SA and MHC. The increased levels of FVIII and VEGFR2 are statistically significant, suggesting an important re-activation of neoangiogenesis. At the same time, the augmented expression of mRNA for Nkx 2.5, the trascriptional factor for cardiomyocyte differentiation, confirms the persistence of differentiative processes in terminally injured hearts. Our study would appear to confirm the activation of human heart regeneration potential in pathological conditions and the ability of its primitive cells to maintain their proliferative capability in vitro. The cardiac cell isolation method we used could be useful in the future for studying modifications to the microenvironment that positively influence cardiac primitive cell differentiation or inhibit, or retard, the pathological remodeling and functional degradation of the heart.

Effect of human granulocyte macrophage-colony stimulating factor on differentiation and apoptosis of the human osteosarcoma cell line SaOS-2.

We investigated the effects of human granulocyte macrophage-colony stimulating factor (GM-CSF) on the relation between differentiation and apoptosis in SaOS-2 cells, an osteoblast-like cell line. To determine the relationship between these cellular processes, SaOS-2 cells were treated in vitro for 1, 7 and 14 days with 200 ng/mL GM-CSF and compared with untreated cells. Five nM insulin-like growth factor (IGF-I) and 30 nM okadaic acid were used as negative and positive controls of apoptosis, respectively. Effects on cell differentiation were determined by ECM (extracellular matrix) mineralization, morphology of some typical mature osteoblast differentiation markers, such as osteopontin and sialoprotein II (BSP-II), and production of bone ECM components such as collagen I. The results showed that treatment with GM-CSF caused cell differentiation accompanied by increased production of osteopontin and BSP-II, together with increased ECM deposition and mineralization. Flow cytometric analysis of annexin V and propidium iodide incorporation showed that GM-CSF up-regulated apoptotic cell death of SaOS-2 cells after 14 days of culture in contrast to okadaic acid, which stimulated SaOS-2 apoptosis only during the early period of culture. Endonucleolytic cleavage of genomic DNA, detected by "Aúladdering analysis"Aù, confirmed these data. The results suggest that GM-CSF induces osteoblastic differentiation and long-term apoptotic cell death of the SaOS-2 human osteosarcoma cell line, which in turn suggests a possible in vivo physiological role for GM-CSF on human osteoblast cells.

Granulocyte macrophage colony stimulating factor (GM-CSF) biological actions on human dermal fibroblasts.

Fibroblasts are involved in all pathologies characterized by increased ExtraCellularMatrix synthesis, from wound healing to fibrosis. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) is a cytokine isolated as an hemopoietic growth factor but recently indicated as a differentiative agent on endothelial cells. In this work we demonstrated the expression of the receptor for GM-CSF (GM-CSFR) on human normal skin fibroblasts from healthy subjects (NFPC) and on a human normal fibroblast cell line (NHDF) and we try to investigate the biological effects of this cytokine. Human normal fibroblasts were cultured with different doses of GM-CSF to study the effects of this factor on GM-CSFR expression, on cell proliferation and adhesion structures. In addition we studied the production of some Extra-Cellular Matrix (ECM) components such as Fibronectin, Tenascin and Collagen I. The growth rate of fibroblasts from healthy donors (NFPC) is not augmented by GM-CSF stimulation in spite of increased expression of the GM-CSFR. On the contrary, the proliferation of normal human dermal fibroblasts (NHDF) cell line seems more influenced by high concentration of GM-CSF in the culture medium. The adhesion structures and the ECM components appear variously influenced by GM-CSF treatment as compared to fibroblasts cultured in basal condition, but newly only NHDF cells are really induced to increase their synthesis activity. We suggest that the in vitro treatment with GM-CSF can shift human normal fibroblasts towards a more differentiated state, due or accompanied by an increased expression of GM-CSFR and that such "differentiation" is an important event induced by such cytokine.

Extra cellular matrix features in human meninges.

We collected human fetal and adult normal meninges to relate the age of the tissue with the presence of collagenous and non-collagenous components of Extra Cellular Matrix (ECM). Immunohistochemistry led us to observe some differences in the amount and in the distribution of these proteins between the two sets of specimens. In particular, laminin and tenascin seem to be expressed more intensely in fetal meninges when compared to adult ones. In order to investigate whether the morphofunctional characteristics of fetal meninges may be represented in pathological conditions we also studied meningeal specimens from human meningiomas. Our attention was particularly focused on the expression of those non-collagenous proteins involved in nervous cell migration and neuronal morphogenesis as laminin and tenascin, which were present in lesser amount in normal adult specimens. Microscopical evidences led us to hipothesize that these proteins which are synthesized in a good amount during the fetal development of meninges can be newly produced in tumors. On the contrary, the role of tenascin and laminin in adult meninges is probably only interesting for their biophysical characteristics.

Morpho-functional aspects of human placental vessels.

This study reports some morphological characteristics of human placenta that play an important physiological role in normal pregnancies. The structures were the spiral arteries, the extracellular matrix and the MHC-antigens bearing cells. We studied these important tissue compartments in 80 placentas from normal and abnormal pregnancies (affected by in utero growth-retardation), where we could hypothesize an altered pattern of the same structures. Placental specimens were evaluated using histochemistry and immunohistochemical methods, like echo-doppler and echography. Our data show uterine and spiral arteries as markedly changing during pregnancy in normal women, but surely involved in some different morphological alterations in the pathological groups. The extracellular matrix compartment is strictly related with the vascular one, both under a physiological point of view (artery resistance and sometimes blood flow inversion) and under a non-cellular component pattern. Normal specimens are rich in sialomucins, solfomucins and glycoproteins. Collagen bundles are mainly of fetal type III, by also adult type I is present at the physiological end of pregnancy. In placentas with blood supply alteration, morphological studies suggest an early aging of the extracellular matrix (with prevalence of adult type I collagen in perivascular muffs). These changes could make more difficult all the exchanges from the blood to the tissues. The immunocompetent cell population is normally well represented in placental tissue: these cells are dentritic-shaped and lie in the perivascular spaces and in the placental and villous stroma. In the altered placentas examined, we were able to identify HLA-DR+ cells that exhibit Langerhans cell markers and are strongly suggestive of an alteration of the normal immunitary relationship between fetal antigens and mother T-cell populations.

Neuropeptides and 5-HT immunoreactivity in the gastric nerves of the dogfish (Scyliorhinus stellaris).

The gastric autonomic innervation of the dogfish was examined for regulatory peptides and serotonin by immunochemical techniques. Bouin's-fixed, paraffin-embedded or benzoquinone-fixed frozen sections were used for light microscopical immunocytochemistry and glutaraldehyde-fixed resin-embedded sections for electron microscopical immunocytochemistry. Bombesin-, somatostatin-, gastrin/cholecystokinin-, substance P-, peptide histidine isoleucine-, vasoactive intestinal peptide- and serotonin-immunoreactive nerves were found in all layers of the stomach wall. Bombesin and vasoactive intestinal peptide-containing nerves were identified at ultrastructural level. Radioimmunoassay of acetic acid extracts of tissue confirmed the presence of immunoreactivity for bombesin, somatostatin, substance P, peptide histidine isoleucine and vasoactive intestinal peptide. Reverse phase high performance liquid chromatography indicated that the peptides identified were broadly similar to their mammalian counterparts.