Maurizio Orlandini

Embryonic expression pattern of the murine figf gene, a growth factor belonging to platelet-derived growth factor/vascular endothelial growth factor family.

Morphogenesis, growth and differentiation of tissues and organs require cell interactions mediated by signal molecules, their receptors and transcriptional control systems. c-fos-induced growth factor (figf) is a new secreted member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family with mitogenic activity on fibroblasts. Here we studied figf expression during murine embryonic development. figf expression was detected with a dynamic pattern in several body structures and organs such as limb buds, acoustic ganglion, teeth, heart, anterior pituitary as well as lung and kidney mesenchyme, liver, derma, and periosteum of the vertebral column.

Vascular Endothelial Growth Factor-D Activates VEGFR-3 Expressed in Osteoblasts Inducing Their Differentiation

Vascular endothelial growth factor (VEGF)-D is a member of the VEGF family of angiogenic growth factors that recognizes and activates the vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 on blood and/or lymphatic vessels. We show that in the long bones of newborn mice, VEGF-D and VEGFR-3 are expressed in the osteoblasts of the growing plate. The treatment of primary human osteoblasts with recombinant VEGF-D induces the expression of osteocalcin and the formation of mineralized nodules in a dose-dependent manner. A monoclonal neutralizing antibody, anti-VEGF-D, or silencing of VEGFR-3 by lentiviral-mediated expression of VEGFR-3 small hairpin RNA affects VEGF-D-dependent osteocalcin expression and nodule formation. Moreover, in primary human osteoblasts, VEGF-D expression is under the control of VEGF, and inhibition of VEGF-D/VEGFR-3 signaling, by monoclonal antibodies or VEGFR-3 silencing, affects VEGF-dependent osteoblast differentiation. These experiments establish that VEGF-D/VEGFR-3 signaling plays a critical role in osteoblast maturation and suggest that VEGF-D is a downstream effector of VEGF in osteogenesis.

Endothelial Cell Adhesion to the Extracellular Matrix Induces c-Src–Dependent VEGFR-3 Phosphorylation Without the Activation of the Receptor Intrinsic Kinase Activity

Rationale: Integrins cooperate with growth factor receptors to promote downstream signaling for cell proliferation and migration. However, the mechanism of receptor activation is still unknown. Objective: To analyze the mechanism of phosphorylation of the vascular endothelial growth factor receptor (VEGFR)-3 by cell adhesion. Methods and Results: We show that VEGFR-3 phosphorylation, induced by cell attachment to the extracellular matrix, is independent from the intrinsic kinase activity of the receptor, as evidenced from phosphorylation cell adhesion experiments with a mutant kinase dead receptor or in the presence of the specific kinase inhibitor MAZ 51. Cell adhesion experiments in the presence of the c-Src inhibitor PP2 or in fibroblast triple knockout for c-Src, Yes, and Fyn (SYF) demonstrate that VEGFR-3 phosphorylation, induced by extracellular matrix, is mediated by c-Src. Kinase assays in vitro with recombinant c-Src show that VEGFR-3 is a direct c-Src target and mass spectrometry analysis identified the sites phosphorylated by c-Src as tyrosine 830, 833, 853, 1063, 1333, and 1337, demonstrating that integrin-mediated receptor phosphorylation induces a phosphorylation pattern that is distinct from that induced by growth factors. Furthermore, pull-down assays show that integrin-mediated VEGFR-3 phosphorylation activates the recruitment to the receptor of the adaptor proteins CRKI/II and SHC inducing activation of JNK. Conclusions: These data suggest that cell adhesion to extracellular matrix induces a downstream signaling using the tyrosine kinase receptor VEGFR-3 as scaffold.

Antiproliferative and proapoptotic activities of new pyrazolo[3,4-d]pyrimidine derivative Src kinase inhibitors in human osteosarcoma cells

Osteosarcoma is the most frequent primitive malignant tumor of the skeletal system, char acterized by an extremely aggressive clinical course that still lacks an effective treatment. Src kinase seems to be involved in the osteosarcoma malignant phenotype. We show that the treatment of human osteosarcoma cell lines with a new pyrazolo[3,4‐d]pyrimidine derivative Src inhibitor, namely SI‐83, impaired cell viability, with a half‐maximal inhibitory concentration of 12 μ.M in nonstarved cells and a kinetic different from that known for the Src inhibitor PP2. Analysis by terminal deoxynucleotidyl transferase‐mediated nick end labeling, Hoechst, and flow cytometric assay showed that SI‐83 induced apoptosis in SaOS‐2 cells. Moreover, SI‐83, by inhibiting Src phosphorylation, decreased in vivo osteosarcoma tumor mass in a mouse model. Finally, SI‐83 showed selectivity for osteosarcoma, since it had a far lower effect in primary human osteoblasts. These results show that human osteosarcoma had Src‐ dependent proliferation and that modulation of Src activity may be a therapeutic target of this new com pound with low toxicity for nonneoplastic cells—Spreafico, A., Schenone, S., Serchi, T., Orlandini, M., Angelucci, A., Magrini, D., Bernardini, G., Collodel, G., Di Stefano, A., Tintori, C., Bologna, M., Manetti, F., Botta, M., Santucci, A. Antiproliferative and proapo‐ ptotic activities of new pyrazolo[3,4‐d]pyrimidine deriv ative Src kinase inhibitors in human osteosarcoma cells. FASEBJ. 22, 1560–1571 (2008)

β-Catenin Inversely Regulates Vascular Endothelial Growth Factor-D mRNA Stability

The angiogenic and lymphangiogenic vascular endothelial growth factor (VEGF)-D is the only member of the VEGF family that is not induced by hypoxia or by serum factors, but its induction is mediated by direct cell-cell contact. Here we show that VEGF-D mRNA is down-modulated either by β-catenin mobilization from the cell membrane, by activation of the Wnt signaling pathway, or by transfection with the β-catenin stable mutant. Down-modulation of β-catenin by means of RNA interference showed an increase of VEGF-D mRNA steady state in fibroblasts. The β-catenin-dependent decrease of VEGF-D mRNA is indirect and mainly due to reduced VEGF-D mRNA stability, as demonstrated by experiments of mRNA decay in the presence of transcription or translation inhibitors. By transient transfection of chimeric constructs carrying fusion of VEGF-D sequences under the control of the cytomegalovirus early promoter, we demonstrated that β-catenin negative regulation is on the VEGF-D mRNA 3′-untranslated region. We mapped the VEGF-D mRNA-destabilizing element to a sequence, conserved between mouse and human VEGF-D, which contains an AU-rich element of group I. These results unveiled a new regulatory pathway for VEGF-D, which explains, at least in part, VEGF-D regulation in tumor progression.

Growth factor stimulation induces cell survival by c-Jun. ATF2-dependent activation of Bcl-XL.

Growth factor stimulation induces c-Jun-dependent survival of primary endothelial cells. However, the mechanism of c-Jun anti-apoptotic activity has not been identified. We here demonstrate that in response to growth factor treatment, primary human endothelial cells as well as mouse fibroblasts respond with an increased expression of c-Jun that forms a complex with ATF2. This complex activates the expression of the anti-apoptotic protein Bcl-XL. By site-directed mutagenesis experiments, we identified two AP-1-binding sites located within the proximal promoter of the Bcl-X gene. Site-directed mutagenesis demonstrated that these AP-1 sites are required for the transcriptional activation of the promoter. Chromatin immunoprecipitation experiments show that in response to growth factor treatment, the heterodimer c-Jun·ATF2 binds to these functional AP-1 sites. Silencing of either c-Jun or ATF2 demonstrated that both nuclear factors are required for the activation of the proximal Bcl-X promoter. Taken together, our experiments provide evidence that growth factor-independent signaling pathways converge in the formation of an active c-Jun·AFT2 dimer, which induces the expression of the anti-apoptotic factor Bcl-XL that mediates a pro-survival response.

Amyloidosis, Inflammation, and Oxidative Stress in the Heart of an Alkaptonuric Patient

Background. Alkaptonuria, a rare autosomal recessive metabolic disorder caused by deficiency in homogentisate 1,2-dioxygenase activity, leads to accumulation of oxidised homogentisic acid in cartilage and collagenous structures present in all organs and tissues, especially joints and heart, causing a pigmentation called ochronosis. A secondary amyloidosis is associated with AKU. Here we report a study of an aortic valve from an AKU patient. Results. Congo Red birefringence, Th-T fluorescence, and biochemical assays demonstrated the presence of SAA-amyloid deposits in AKU stenotic aortic valve. Light and electron microscopy assessed the colocalization of ochronotic pigment and SAA-amyloid, the presence of calcified areas in the valve. Immunofluorescence detected lipid peroxidation of the tissue and lymphocyte/macrophage infiltration causing inflammation. High SAA plasma levels and proinflammatory cytokines levels comparable to those from rheumatoid arthritis patients were found in AKU patient. Conclusions. SAA-amyloidosis was present in the aortic valve from an AKU patient and colocalized with ochronotic pigment as well as with tissue calcification, lipid oxidation, macrophages infiltration, cell death, and tissue degeneration. A local HGD expression in human cardiac tissue has also been ascertained suggesting a consequent local production of ochronotic pigment in AKU heart.

FOSL1 Controls the Assembly of Endothelial Cells into Capillary Tubes by Direct Repression of αv and β3 Integrin Transcription

ABSTRACT To form three-dimensional capillary tubes, endothelial cells must establish contacts with the extracellular matrix that provides signals for their proliferation, migration, and differentiation. The transcription factor Fosl1 plays a key role in the vasculogenic and angiogenic processes as Fosl1 knockout embryos die with vascular defects in extraembryonic tissues. Here, we show that Fosl1−/− embryonic stem cells differentiate into endothelial cells but fail to correctly assemble into primitive capillaries and to form tube-like structures. FOSL1 silencing affects in vitro angiogenesis, increases cell adhesion, and decreases cell mobility of primary human endothelial cells (HUVEC). We further show that FOSL1 is a repressor of αv and β3 integrin expression and that the down-modulation of αvβ3 rescues the angiogenic phenotype in FOSL1-silenced HUVEC, while the ectopic expression of αvβ3 alone reproduces the phenotypic alterations induced by FOSL1 knockdown. FOSL1 represses the transcription of both αv and β3 integrin genes by binding together with JunD to their proximal promoter via the transcription factor SP1. These data suggest that FOSL1-dependent negative regulation of αvβ3 expression on endothelial cells is required for endothelial assembly into vessel structures.

Chondroptosis in Alkaptonuric Cartilage

Alkaptonuria (AKU) is a rare genetic disease that affects the entire joint. Current standard of treatment is palliative and little is known about AKU physiopathology. Chondroptosis, a peculiar type of cell death in cartilage, has been so far reported to occur in osteoarthritis, a rheumatic disease that shares some features with AKU. In the present work, we wanted to assess if chondroptosis might also occur in AKU. Electron microscopy was used to detect the morphological changes of chondrocytes in damaged cartilage distinguishing apoptosis from its variant termed chondroptosis. We adopted histological observation together with Scanning Electron Microscopy and Transmission Electron Microscopy to evaluate morphological cell changes in AKU chondrocytes. Lipid peroxidation in AKU cartilage was detected by fluorescence microscopy. Using the above‐mentioned techniques, we performed a morphological analysis and assessed that AKU chondrocytes undergo phenotypic changes and lipid oxidation, resulting in a progressive loss of articular cartilage structure and function, showing typical features of chondroptosis. To the best of our knowledge, AKU is the second chronic pathology, following osteoarthritis, where chondroptosis has been documented. Our results indicate that Golgi complex plays an important role in the apoptotic process of AKU chondrocytes and suggest a contribution of chondroptosis in AKU pathogenesis. These findings also confirm a similarity between osteoarthritis and AKU. J. Cell. Physiol. 230: 1148–1157, 2015.

Role of the AP-1 transcription factor FOSL1 in endothelial cells adhesion and migration

Vasculogenesis and angiogenesis, the fundamental processes by which new blood vessels are formed, involve the proliferation, migration, and remodeling of endothelial cells. Dynamic adhesion of endothelial cells to extracellular matrix plays a fundamental role in all these events. Key regulators of endothelial cells adhesion and migration are the αvβ3 and uPA-uPAR complexes. The αvβ3 integrin heterodimer is the receptor for extracellular matrix components such as vitronectin and is overexpressed on the cell surface of angiogenic endothelial cells, but not quiescent cells lining normal vessels. The uPA-uPAR complex contributes to extracellular matrix remodeling by mediating proteolytic activity at the leading edge of migrating cells. We recently reported that the FOSL1 transcription factor of the AP-1 family plays a pivotal role in the regulation of the level of the αvβ3 and uPA-uPAR complexes on the surface of endothelial cells. In this commentary, we review the current knowledge of αv and β3 transcriptional regulation in endothelial cells and discuss the role of FOSL1 in angiogenesis.