Adriano Spreafico

Biochemical investigation of the effects of human platelet releasates on human articular chondrocytes

The aim of the present study was to demonstrate the mitogenic and differentiating properties of platelet‐rich plasma releasates (PRPr) on human chondrocytes in mono‐ and three‐dimensional cultures. In order to assess if PRPr supplementation could maintain the chondrocyte phenotype or at least inhibit the cell de‐differentiation even after several days in culture, we performed a proteomic study on several cell cultures independently grown, for different periods of time, in culture medium with FCS, human serum (HS), and releasates obtained from PRP and platelet‐poor plasma (PPP). We found that PRP treatment actually induced in chondrocytes the expression of proteins (some of which novel) involved in differentiation. J. Cell. Biochem. 108: 1153–1165, 2009.

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.

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)

Hierarchical affinity maturation of a phage library derived antibody for the selective removal of cytomegalovirus from plasma

Recombinant antibody fragments can be produced in large quantities using bacterial expression systems and could potentially be useful for the generation of biofilters for the selective removal of viral particles from fluids. A human single chain-Fv antibody library, derived from synthetic repertoires of germ line VH-gene segments rearranged in vitro and paired to a single light chain (Nissim et al., 1994, EMBO J., 13, 692-698), has recently been used to isolate hundreds of different binding specificities by panning with antigen. Antibodies from this library typically have affinities in the 10(6)-10(7) M-1 range. Occasionally, better binders are isolated but at other times the affinities recovered are poor. In the latter situation binding cannot be detected with soluble antibodies, but only by high-avidity display of multiple copies of antibodies on phage. By panning with human cytomegalovirus (HCMV)-coated immunotubes, we have isolated a number of antibody clones from this library that bound to the antigen only if displayed on the filamentous phage, but not in soluble form. One of these clones was selected for an affinity maturation procedure, achieved by combinatorial mutagenesis of the complementarity determining region 3 (CDR3) of the antibody light chain, followed by selection of the resulting library for HCMV binding. By this means, we were able to isolate a number of binders, some of which exhibited specific HCMV binding in soluble form. The clone that gave the strongest ELISA signal was expressed in bacteria, purified in solution, characterised using a novel capture methodology with surface plasmon resonance detection on a BIAcore instrument and used for the production of an immunofilter for the removal of HCMV form human serum. The filter removed more than 99% of applied HCMV in 10 min circulation time, while the amount of HCMV retained non-specifically in a cartridge derivatised with a non-specific antibody was less than 10% under similar conditions.

Evaluation of antioxiodant drugs for the treatment of ochronotic alkaptonuria in an in vitro human cell model

Alkaptonuria (AKU) is a rare autosomal recessive disease, associated with deficiency of homogentisate 1,2‐dioxygenase activity in the liver. This leads to an accumulation of homogentisic acid (HGA) and its oxidized derivatives in polymerized form in connective tissues especially in joints. Currently, AKU lacks an appropriate therapy. Hence, we propose a new treatment for AKU using the antioxidant N‐acetylcysteine (NAC) administered in combinations with ascorbic acid (ASC) since it has been proven that NAC counteracts the side‐effects of ASC. We established an in vitro cell model using human articular primary chondrocytes challenged with an excess of HGA (0.33 mM). We used this experimental model to undertake pre‐clinical testing of potential antioxidative therapies for AKU, evaluating apoptosis, viability, proliferation, and metabolism of chondrocytes exposed to HGA and treated with NAC and ASC administered alone or in combination addition of both. NAC decreased apoptosis induced in chondrocytes by HGA, increased chondrocyte growth reduced by HGA, and partially restored proteoglycan release inhibited by HGA. A significantly improvement in efficacy was found with combined addition of the two antioxidants in comparison with NAC and ASC alone. Our novel in vitro AKU model allowed us to demonstrate the efficacy of the co‐administration of NAC and ASC to counteract the negative effects of HGA for the treatment of ochronotic arthropathy. J. Cell. Physiol. 225: 84–91, 2010.

Semen characteristics and inflammatory mediators in infertile men with different clinical diagnoses.

This study was aimed at investigating whether semen characteristics in different clinical diagnoses of infertility are associated with PMN elastase, IL-6, IL-8, IL-1beta and TNFalpha levels detected in seminal plasma. Sixty-eight patients were divided into groups according to their clinical diagnosis: idiopathic infertility (group I), varicocele with infections (group II), varicocele (group III), infections (group IV), controls (group V). Physical examination and scrotal Eco-color Doppler was used to detect the varicocele. Patients with positive bacteriological semen analysis were considered as having an infection of the male reproductive tract. Samples were examined by light microscopy and transmission electron microscopy (TEM). TEM data were quantified with a mathematical formula furnishing a fertility index and the percentage of sperm apoptosis, immaturity and necrosis. PMN elastase/alpha1-PI complex levels were determined by ELISA and IL-6, IL-8, IL-1beta, TNFalpha by Bio-Plex Cytokine assay. Sperm concentration (I-II: p < 0.005; III-IV: p < 0.0001), motility (I-IV: p < 0.0001) and the fertility index (I: p < 0.005; II-IV: p < 0.0001) were significantly lower in the groups vs. controls, whereas sperm pathologies, except for apoptosis, were significantly higher in group I and apoptosis and necrosis were higher in group III. An increase in immaturity (p < 0.005) with a decrease in necrosis (p < 0.005) were observed in group III vs. group IV. Significantly higher levels of inflammatory mediators were detected in groups III and IV vs. controls. Despite a broad relationship among different inflammatory mediators, no correlation was found among them and the semen parameters, including indices from TEM analysis. In conclusion, patients with idiopathic infertility showed altered semen quality and normal levels of inflammatory mediators. Genitourinary infection and varicocele induced an inflammatory effect which could play a detrimental role in spermatogenesis, revealed by a decrease in sperm motility and the fertility index, concomitant with an increase in immaturity mainly in varicocele and necrosis in infection.

Homogentisate 1,2 dioxygenase is expressed in human osteoarticular cells: Implications in alkaptonuria†

Alkaptonuria (AKU) results from defective homogentisate1,2‐dioxygenase (HGD), causing degenerative arthropathy. The deposition of ochronotic pigment in joints is so far attributed to homogentisic acid produced by the liver, circulating in the blood and accumulating locally. Human normal and AKU osteoarticular cells were tested for HGD gene expression by RT‐PCR, mono‐ and 2D‐Western blotting. HGD gene expression was revealed in chondrocytes, synoviocytes, osteoblasts. Furthermore, HGD expression was confirmed by Western blotting, that also revealed the presence of five enzymatic molecular species. Our findings indicate that AKU osteoarticular cells produce the ochronotic pigment in loco and this may strongly contribute to induction of ochronotic arthropathy. J. Cell. Physiol. 227: 3254–3257, 2012.

Biochemical and proteomic characterization of alkaptonuric chondrocytes

Alkaptonuria (AKU) is a rare genetic disease associated with the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products which leads to the deposition of melanin‐like pigments (ochronosis) in connective tissues. Although numerous case reports have described ochronosis in joints, little is known on the molecular mechanisms leading to such a phenomenon. For this reason, we characterized biochemically chondrocytes isolated from the ochronotic cartilage of AKU patients. Based on the macroscopic appearance of the ochronotic cartilage, two sub‐populations were identified: cells coming from the black portion of the cartilage were referred to as “black” AKU chondrocytes, while those coming from the white portion were referred to as “white” AKU chondrocytes. Notably, both AKU chondrocytic types were characterized by increased apoptosis, NO release, and levels of pro‐inflammatory cytokines. Transmission electron microscopy also revealed that intracellular ochronotic pigment deposition was common to both “white” and “black” AKU cells. We then undertook a proteomic and redox‐proteomic analysis of AKU chondrocytes which revealed profound alterations in the levels of proteins involved in cell defence, protein folding, and cell organization. An increased post‐translational oxidation of proteins, which also involved high molecular weight protein aggregates, was found to be particularly relevant in “black” AKU chondrocytes. J. Cell. Physiol. 227: 3333–3343, 2012.

Osteogenic growth peptide effects on primary human osteoblast cultures: Potential relevance for the treatment of glucocorticoid‐induced osteoporosis

The osteogenic growth peptide (OGP) is a naturally occurring tetradecapeptide that has attracted considerable clinical interest as a bone anabolic agent and hematopoietic stimulator. In vivo studies on animals have demonstrated that the synthetic peptide OGP (10–14), reproducing the OGP C‐terminal active portion [H‐Tyr‐Gly‐Phe‐Gly‐Gly‐OH] increases bone formation, trabecular bone density and fracture healing. In vitro studies performed on cellular systems based on osteoblastic‐like cell lines or mouse stromal cells, have demonstrated that OGP (10–14) increases osteoblast proliferation, alkaline phosphatase (ALKP) activity and matrix synthesis and mineralization. In view of a potential application of OGP (10–14) in clinical therapy, we have tested different concentrations of OGP (10–14) on primary human osteoblast (hOB) cultures. We have observed significant increases of hOB proliferation (+35%), ALKP activity (+60%), osteocalcin secretion (+50%), and mineralized nodules formation (+49%). Our experimental model based on mature hOBs was used to investigate if OGP (10–14) could prevent the effects on bone loss induced by sustained glucocorticoid (GC) treatments. A strong decrease in bone formation has been attributed to the effects of GCs on osteoblastogenesis and osteocyte apoptosis, while an increase in bone resorption was due to a transient osteoblastic stimulation, mediated by the OPG/RANKL/RANK system, of osteoclasts recruitment and activation. Moreover, GCs act on hOBs decreasing the release of osteoprotegerin (OPG) a regulator of the RANKL/RANK interaction. Here, we provide evidences that OGP (10–14) inhibits hOB apoptosis induced by an excess of dexamethasone (−48% of apoptotic cells). Furthermore, we show that OGP (10–14) can increase OPG secretion (+20%) and can restore the altered expression of OPG induced by GCs to physiological levels. Our results support the employment of OGP (10–14) in clinical trials addressed to the treatment of different bone remodeling alterations including the GC‐induced osteoporosis. J. Cell. Biochem. 98: 1007–1020, 2006.

Evaluation of anti-oxidant treatments in an in vitro model of alkaptonuric ochronosis

OBJECTIVES Alkaptonuria (AKU) is a rare genetic disease associated with deficient homogentisate 1,2-dioxygenase activity in the liver. This leads to the accumulation of homogentisic acid (HGA) and its oxidized/polymerized products in connective tissues, which in turn become characterized by the presence of melanin-like pigments (ochronosis). Since at present, further studies are necessary to support the use of drugs for the treatment of AKU, we investigated the effects of various anti-oxidants in counteracting melanin-like pigmentation and oxidative stress related to HGA and its metabolites. METHODS We set up an in vitro model using human serum treated with 0.33 mM HGA and tested the anti-oxidants ascorbic acid, N-acetylcysteine, phytic acid (PHY), taurine (TAU), ferulic acid (FER) and lipoic acid (LIP) for their ability to prevent or delay the production of melanin-like pigments, as well as to reduce oxidative post-translational modifications of proteins. Monitoring of intrinsic fluorescence of HGA-induced melanin-like pigments was used to evaluate the efficacy of compounds. RESULTS Our model allowed us to prove efficacy especially for PHY, TAU, LIP and FER in counteracting the production of HGA-induced melanin-like pigments and protein oxidation induced by HGA and its metabolites. CONCLUSIONS Our model allows the opening of new anti-oxidant therapeutic strategies to treat alkaptonuric ochronosis.