Massimo Mariotti

OVER-EXPRESSION OF HEPATOCYTE GROWTH FACTOR IN HUMAN KAPOSI'S SARCOMA

Kaposis sarcoma is a highly vascularized multifocal tumor which frequently appears as a complication of HIV infection. It has been suggested that a disorder in the cytokine network may contribute to the development of the disease. We examined the expression of several cytokines in human sporadic Kaposis sarcoma specimens, as well as in spindle cells cultured from human lesions, and consistently found high levels of expression of hepatocyte growth factor (HGF). In addition, human lesion‐derived spindle cells synthesize and secrete biologically active hepatocyte growth factor and express the hepatocyte‐growth‐factor receptor (c‐MET). Moreover, elevated levels of transforming growth factor β1 (TGFβ1) mRNA were found in lesions of human sporadic Kaposis sarcoma and in lesion‐derived spindle cells which also over‐express urokinase. Since HGF, TGFβ1 and urokinase are all involved in capillary‐vessel organization, dysregulation of these interacting agents may play a role in the initiation and/or progression of Kaposis sarcoma, stimulating the growth of spindle cells and recruiting endothelial cells into the lesion.

HD-PTP inhibits endothelial migration through its interaction with Src

Endothelial migration, early step in angiogenesis, is tightly regulated by the coordinated action of tyrosine kinases and tyrosine phosphatases. HD-PTP contributes to endothelial motility, since endothelial cells silencing HD-PTP after transfection with iRNA acquire a scattered and spindle-shaped phenotype and migrate faster than controls. Since (i) the proto-oncogene Src contributes to the regulation of cell motility and (ii) HD-PTP has a potential binding site for Src, we investigated whether an interplay exists between these two proteins. We found that Src binds HD-PTP and this interaction is enhanced after exposure to basic fibroblast growth factor. While HD-PTP does not modulate the levels of Src phosphorylation both in vitro and in vivo, we found that Src phosphorylates HD-PTP on tyrosine residues. Here we show for the first time that (i) HD-PTP has a tyrosine phosphatase activity; (ii) HD-PTP phosphorylation by Src inhibits its enzymatic activity. Interestingly, pharmacological and genetic inhibition of Src abrogates the migratory phenotype of endothelial cells silencing HD-PTP. On these bases, and because we have previously demonstrated that HD-PTP binds and dephosphorylates focal adhesion kinase (FAK), another crucial regulator of cell migration, we hypothesize that HD-PTP participates to the regulation of endothelial motility through its interactions with Src and FAK.

Iterleukin 1 alpha is a marker of endothelial cellular senescent

BackgroundThe functional changes associated with endothelial senescence may be involved in human aging and age-related vascular disorders. Since the inflammatory cytokine interleukin (IL-)1 inhibits endothelial growth, we evaluated the expression of IL-1α, IL-1β and their antagonist, the IL-1 receptor antagonist (IL-1ra), in endothelial in vitro senescence and quiescence. We also examined the expression of IL-1α in human senescent and progeric fibroblasts.ResultsWe found that the overexpression of IL-1α specifically characterizes endothelial senescence. No modulation of this cytokine was observed in endothelial quiescence and in senescent or progeric human fibroblasts. The expression of IL-1β and IL-1ra was also assessed and found not to be affected by senescence.ConclusionOur results indicate that a dysfunction of the cytokine network associates with aging and point to a specific role of IL-1α in endothelial senescence.

Gravitational unloading induces an anti-angiogenic phenotype in human microvascular endothelial cells

Creating conditions similar to those occurring during exposure of cells to microgravity modulates endothelial functions. We have previously demonstrated that human macrovascular endothelial cells in simulated hypogravity proliferate faster than controls, partly because they downregulate interleukin 1α. On the contrary, murine microvascular endothelial cells are growth inhibited in simulated hypogravity, and this is due, at least in part, to the decrease of interleukin 6. Since endothelial cells are very heterogeneous and differences between various species have been reported, we exposed human microvascular cells to gravitational unloading and found that it retards cell growth without affecting cell migration. Interestingly, we detected the induction of Tissue Inhibitor of Metalloprotease‐2, which inhibits endothelial growth in vitro and angiogenesis in vivo. Together with the finding that hypogravity stimulates the synthesis of nitric oxide, involved also in neovascularization, our results underscore a modulation of the angiogenic properties of microvascular human endothelial cells. We also show that hypogravity inhibits proteasome activity, thus suggesting that post‐translational mechanisms are involved in the adaptations of these cells to hypogravity. These results underscore that hypogravity differently impacts on micro‐ and macro‐vascular human endothelial cells. In particular, these results may shed some light on the molecular mechanisms contributing to the impairment of angiogenesis observed in different models in space. Our data might also explain why bioengineered tissues to be used for regenerative medicine fail to neovascularize when assembled in simulated hypogravity. J. Cell. Biochem. 104: 129–135, 2008.

Inhibition of T24 human bladder carcinoma cell migration by RNA interference suppressing the expression of HD-PTP

Cell migration is fundamental for invasion and metastasis and is modulated by the reversible phosphorylation of tyrosine residues on target proteins. Here we report that the tyrosine phosphatase HD-PTP has a role in modulating the motility of T24 bladder carcinoma cells. Indeed, HD-PTP silencing by RNA interference (RNAi) markedly induced cell migration in a Src dependent fashion. We therefore investigated the interaction and the regulation of Src and HD-PTP. We found that, in Epidermal Growth Factor (EGF) stimulated cells, Src binds to and phosphorylates HD-PTP on tyrosine residues. On the contrary, HD-PTP does not modulate the levels of Src phosphorylation. Interestingly, HD-PTP also binds to FAK, another regulator of cell migration, and this interaction is inhibited after exposure to EGF. FAK phosphorylates HD-PTP and this event reduced the interactions between the two proteins. Interestingly, in cells silencing HD-PTP the phosphorylation of FAK is enhanced and this correlates with its localization in focal complexes both in the presence and in the absence of EGF. We hypothesize that in unstimulated T24 cells HD-PTP does not interact with Src, while it binds to FAK. Following stimulation with EGF, HD-PTP is tyrosine-phosphorylated and releases FAK which will ultimately contribute to the turn-over of focal adhesion and, therefore, to cell motility.

The dual role of endothelial differentiation-related factor-1 in the cytosol and nucleus: modulation by protein kinase A.

Endothelial differentiation-related factor (EDF)-1 is involved in the repression of endothelial cell differentiation and is the first studied calmodulin (CaM)-binding protein in endothelial cells. Here we report that (i) EDF-1 is in vitro and in vivo phosphorylated by protein kinase A (PKA); (ii) EDF-1/CaM interaction is modulated by the phosphorylation of EDF-1 by PKA; (iii) forskolin stimulates nuclear accumulation of EDF-1, and (iv) PKA phosphorylation enhances EDF-1 interaction with the TATA-binding protein. CaM modulates the activity of several enzymes, among which is nitric oxide synthase (NOS). EDF-1, but not phosphorylated EDF-1, inhibits the activity of NOS. Accordingly, we detected an increase in NOS activity in cells that express low amounts of EDF-1. Our results indicate that EDF-1 serves two main functions in endothelial cells: (i) it regulates CaM availability in the cytosol, and (ii) it acts in the nucleus as a transcriptional coactivator.

Nitric oxide mediates low magnesium inhibition of osteoblast-like cell proliferation

An adequate intake of magnesium (Mg) is important for bone cell activity and contributes to the prevention of osteoporosis. Because (a) Mg is mitogenic for osteoblasts and (b) reduction of osteoblast proliferation is detected in osteoporosis, we investigated the influence of different concentrations of extracellular Mg on osteoblast-like SaOS-2 cell behavior. We found that low Mg inhibited SaOS-2 cell proliferation by increasing the release of nitric oxide through the up-regulation of inducible nitric oxide synthase (iNOS). Indeed, both pharmacological inhibition with the iNOS inhibitor l-N(6)-(iminoethyl)-lysine-HCl and genetic silencing of iNOS by small interfering RNA restored the normal proliferation rate of the cells. Because a moderate induction of nitric oxide is sufficient to potentiate bone resorption and a relative deficiency in osteoblast proliferation can result in their inadequate activity, we conclude that maintaining Mg homeostasis is relevant to ensure osteoblast function and, therefore, to prevent osteoporosis.

Alendronate rescued osteoporotic phenotype in a model of glucocorticoid-induced osteoporosis in adult zebrafish scale.

Long‐term effects of glucocorticoid treatment in humans induce bone loss and increase the risk of fracture in the skeleton. The pathogenic mechanisms of glucocorticoid‐induced osteoporosis (GIOP) are still unclear. The GIOP and its effects have been reproduced in several animal models including Danio rerio (zebrafish) embryo. The treatment of adult fish with prednisolone (PN) has shown a dose‐dependent decrease of mineralized matrix in the scales. Large resorption lacunae are characterized by single TRAP‐positive cells which migrate to the margin of the scale merging into a multinucleated structures. The treatment with PN of cultured scales did not increase TRAP activity suggesting that the massive presence of osteoclasts in the resorption sites could be likely the result of a systemic recruitment of monocyte–macrophage precursors. We observed that treatment with PN induced a significant decrease of the alkaline phosphatase (ALP) activity in scale scleroblasts if compared with untreated controls. Then, we investigated the total mineral balance under prednisolone treatment using a time‐dependent double live staining. The untreated fish fully repaired the resorption lacuna induced by prednisolone, whereas treated fish failed. The presence of osteoclast resorption fingerprints on new matrix suggested that the osteoclast activity counterbalances the osteodepositive activity exerted by scleroblasts. The treatment with PN in association with alendronate (AL) has surprisingly resulted in a significant decrease of TRAP activity and increase of ALP compared to PN‐treated fish in biochemical and histological assays confirming the action of alendronate against GIOP in fish as well in humans.

The zebrafish scale as model to study the bone mineralization process.

Danio rerio (zebrafish) shows high similarity with humans in terms of bone architecture, bone cells, matrix proteins and molecular signalling. The fish body is covered by elasmoid scales which are part of the dermal skeleton. Since few data have been published about the function of the fish scale cells, we investigated the mineralization pattern of the scale and the role of the episquamal osteoblasts in the neodeposition of the bone tissue. First, we described a specific mineralization pattern and distribution of the bone forming cells in different areas of the scale. We observed along the external circuli that, during the scale growth, the marginal cells migrate and organize in a cord-like structure just before the mineralization process takes place generating a new circulus. These cells exhibit alkaline phosphatase activity, a well known mammalian osteoblastic differentiation marker. The internal circuli are also characterized by new matrix deposition. Thus, zebrafish scale represents a useful model for analyzing the osteoblast behaviour during bone formation and mineralization and it could be useful in physiological studies and pharmacological tests.

EDF-1 contributes to the regulation of nitric oxide release in VEGF-treated human endothelial cells.

Vascular endothelial growth factor (VEGF) induces nitric oxide (NO) release by triggering multiple intracellular signals, among others the calcium/calmodulin pathway and the activation of Akt, events which induce endothelial NO synthase (eNOS) activity. Because Endothelial Differentiation-related Factor (EDF)-1 is a calmodulin binding protein and plays a role in modulating endothelial functions, we evaluated whether EDF-1 is implicated in the regulation of eNOS activity in VEGF-treated human endothelial cells. While VEGF does not modulate the total amounts of EDF-1, it promotes the dissociation of calmodulin from EDF-1 which correlates with the increase of calmodulin bound to eNOS and the induction of NO release. To better characterize the contribution of EDF-1 to the regulation of VEGF-induced NO release, we stably silenced EDF-1 in endothelial cells. We here show that endothelial cells silencing EDF-1 produce more NO than controls and do not increase NO release in response to VEGF. The insensitivity to VEGF results from the incapability of cells silencing EDF-1 to phosphorylate eNOS Ser(1177), even though Akt is activated. Interestingly, okadaic acid, a pharmacologic inhibitor of the serine/threonine phosphatase PP2A, which preferentially dephosphorylates eNOS Ser(1177), restores NO release and eNOS Ser(1177) phosphorylation in cells silencing EDF-1. Our results suggest EDF-1 as a novel contributor to the complex regulation of eNOS activity in human endothelial cells.