Francesca Ronca

Anti-inflammatory activity of chondroitin sulfate

The pharmacokinetics of chondroitin sulfate (CS, Condrosulf, IBSA, Lugano, Switzerland) were investigated in rats and in healthy volunteers using CS tritiated at the reducing end and CS labeled with 131I or 99mTc respectively. A rapid absorption of orally administered CS is observed in rats and in humans when the drug is dissolved in water. Lower and delayed absorption is observed when CS is administered in gastroresistant capsules. The absolute bio-availability is 15 and 12% for rats and humans respectively. The CS shows a tropism for cartilagineous tissues in rats and for knee tissues in humans as demonstrated by scintigraphic analysis with 99mTc-CS. Monomers, oligo and polysaccharides produced by enzymatic hydrolysis of CS appear in the blood and tissues together with native CS. The effects of partially depolymerized (m.m. 3 to 15 kD) and desulfated fractions on human leukocytes were investigated. CS and its fractions inhibit the directional chemotaxis induced by zymosan-activated serum, are able to decrease the phagocytosis and the release of lysozyme induced by zymosan and to protect the plasma membrane from oxygen reactive species. In rats the oral administration of CS significantly decreases granuloma formation due to sponge implants and cell migration and lysosomal enzyme release in carrageenan pleurisy. Compared with nonsteroidal anti-inflammatory drugs (indomethacin, ibuprofen), CS appears to be more effective on cellular events of inflammation than on edema formation. It is noteworthy that CS is devoid of dangerous effects on the stomach, platelets and kidneys. In synovial fluid of patients requiring joint aspiration, treated orally for 10 days with CS (800 mg/day) the hyaluronate concentration and the intrinsic viscosity significantly increased, while collagenolytic activity, phospholipase A2 and N-acetylglucosaminidase (NAG) decreased. These results give an insight into the mechanism of the anti-inflammatory and chondroprotective actions demonstrated by this drug in a number of clinical trials in patients with osteoarthritis.Osteoarthritis is primarily characterized by areas of destruction of articular cartilage and by synovitis. Articular damage and synovitis are secondary to local increase of pro-inflammatory cytokines (interleukin-1beta and tumor necrosis factor-alpha), enzymes with proteolytic activity (matrix metalloproteinases), and enzymes with pro-inflammatory activity (cyclooxygenase-2 and nitric oxide synthase-2). Enhanced expression of these proteins in chondrocytes and in synovial membrane appears associated to the activation and nuclear translocation of nuclear factor-kappaB (NF-kappaB). Chondroitin sulfate (CS) prevents joint space narrowing and reduces joint swelling and effusion. To produce these effects, CS elicits an anti-inflammatory effect at the chondral and synovial levels. CS and its disaccharides reduce NF-kappaB nuclear translocation, probably by diminishing extracellular signal-regulated kinase1/2, p38mitogen-activated protein kinase and c-Jun N-terminal kinase activation. This review discusses the evidence supporting that CS pleiotropic effects in chondrocytes and synoviocytes are primarily due to a common mechanism, e.g., the inhibition of NF-kappaB nuclear translocation.

Modulation of sarcoplasmic reticulum function: a new strategy in cardioprotection?

This article reviews the experimental evidence suggesting that cytosolic Ca(2+) overload plays a major role in the development of myocardial injury during ischemia-reperfusion and that Ca(2+) release from the sarcoplasmic reticulum (SR) is of crucial importance in the early phase of ischemia. It is suggested that interventions able to deplete the SR Ca(2+) pool and/or to reduce the rate of SR Ca(2+) release should be cardioprotective. This thesis is supported by the review of experimental studies in which modulators of the SR Ca(2+)-ATPase or SR Ca(2+) release channel (ryanodine receptor) have been used. In addition, the role of the SR in ischemic preconditioning and in some instances of toxic myocardial injury (particularly, anthraquinone-induced injury) is discussed.

A3 adenosine receptor stimulation modulates sarcoplasmic reticulum Ca2+ release in rat heart

OBJECTIVE Stimulation of A3 adenosine receptors has been shown to protect cardiac myocytes from ischemic injury, but the mechanism of this action is unknown. We evaluated the effect of adenosine agonists and antagonists on the sarcoplasmic reticulum (SR) Ca(2+) channels. METHODS Isolated rat hearts were perfused with control buffer or different adenosine agonists and antagonists. Hearts were then homogenized and used to determine SR Ca(2+)-induced Ca(2+) release, assayed by quick filtration technique after loading with 45Ca(2+), and the binding of [3H]ryanodine, a specific ligand of the SR Ca(2+) release channel. In parallel experiments, hearts were challenged with 30 min of global ischemia and 120 min of reperfusion, and the extent of tissue necrosis was evaluated by triphenyltetrazolium chloride staining. RESULTS Perfusion with the A1>A3 agonist R-PIA and the A3>A1 agonist IB-MECA was associated with reduced [3H]ryanodine binding, due to reduced B(max) (by about 20%), whereas K(d) and Ca(2+)-dependence of the binding reaction were unaffected. These actions were abolished by the A3 antagonist MRS 1191, while they were not affected by A1 and A2 antagonists. The rate constant of SR Ca(2+) release decreased by 25-30% in hearts perfused with R-PIA or IB-MECA. Tissue necrosis was significantly reduced in the presence of R-PIA or IB-MECA. Protection was removed by MRS 1191, and it was not affected by A1 and A2 antagonists. Hearts were also protected by administration of dantrolene, a ryanodine receptor antagonist. In the presence of dantrolene, no further protection was provided by IB-MECA. CONCLUSION A3 adenosine receptor stimulation modulates the SR Ca(2+) channel. This action might account for the protective effect of adenosine.

Pilot in vivo investigation of cerium oxide nanoparticles as a novel anti-obesity pharmaceutical formulation

UNLABELLED Obesity is a worldwide pathological condition that strongly impairs human health, and, to date, no effective therapy against excessive fat accumulation has been found yet. Since overweight correlates with an increased oxidative stress, our aim is to investigate the antioxidant effects of cerium oxide nanoparticles (nanoceria) as a potential pharmaceutical approach for the treatment of obesity. Nanoceria were tested both in vitro and in vivo; they were proven to interfere with the adipogenic pathway by reducing the mRNA transcription of genes involved in adipogenesis, and by hindering the triglycerides accumulation in 3T3-L1 pre-adipocytes. Nanoceria, intraperitonally injected in Wistar rats, did not show appreciable toxic effects, but instead efficiently contributed in reducing the weight gain and in lowering the plasma levels of insulin, leptin, glucose and triglycerides. FROM THE CLINICAL EDITOR Obesity is now a significant problem worldwide. To date, obesity surgery remains the best treatment for weight reduction. Much research has been conducted to discover an effective pharmacological treatment against obesity. In this article, the authors continued their previous work in studying the anti-adipogenic properties of cerium oxide nanoparticles. The antioxidant effects of nanoceria were studied in in vitro and in vivo experiments. It was shown in animal model that nanoceria could reduce body weight effectively. These promising results may provide a novel treatment in the clinical setting in the future.

Active Targeting of Sorafenib: Preparation, Characterization, and In Vitro Testing of Drug-Loaded Magnetic Solid Lipid Nanoparticles.

Sorafenib is an anticancer drug approved by the Food and Drug Administration for the treatment of hepatocellular and advanced renal carcinoma. The clinical application of sorafenib is promising, yet limited by its severe toxic side effects. The aim of this study is to develop sorafenib-loaded magnetic nanovectors able to enhance the drug delivery to the disease site with the help of a remote magnetic field, thus enabling cancer treatment while limiting negative effects on healthy tissues. Sorafenib and superparamagnetic iron oxide nanoparticles are encapsulated in solid lipid nanoparticles by a hot homogenization technique using cetyl palmitate as lipid matrix. The obtained nanoparticles (Sor-Mag-SLNs) have a sorafenib loading efficiency of about 90% and are found to be very stable in an aqueous environment. Plain Mag-SLNs exhibit good cytocompatibility, whereas an antiproliferative effect against tumor cells (human hepatocarcinoma HepG2) is observed for drug-loaded Sor-Mag-SLNs. The obtained results show that it is possible to prepare stable Sor-Mag-SLNs able to inhibit cancer cell proliferation through the sorafenib cytotoxic action, and to enhance/localize this effect in a desired area thanks to a magnetically driven accumulation of the drug. Moreover, the relaxivity properties observed in water suspensions hold promise for Sor-Mag-SLN tracking through clinical magnetic resonance imaging.

Evidence of a species-differentiated regulatory domain within the N-terminal region of skeletal muscle AMP deaminase

Rabbit skeletal muscle AMP deaminase was submitted to limited proteolysis by trypsin that converts the native 80 kDa enzyme subunit to a stable product of approx. 70 kDa, which, in contrast to the native enzyme, is not sensitive to regulation by ATP at pH 6.5. Tryptic peptide mapping indicates that proteolysis is confined to the N-terminal region of the molecule, identifying in this region of AMP deaminase a non-catalytic, 95 residue regulatory domain that stabilises the binding of ATP to a distant site in the molecule. Protein sequence analysis reveals a marked degree of divergence between rat and rabbit skeletal muscle AMP deaminases in the regions containing residues 7-12 and 51-52, giving molecular basis to the hypothesis of the existence of isoenzymes of AMP deaminase in the mature skeletal muscle of the mammals.

Vascular Endothelial Growth Factor Polymorphisms in Mantle Cell Lymphoma

In this study, we determined the allele and genotype frequencies of vascular endothelial growth factor (VEGF) G+405C, C–460T, C+936T and C–2578A single nucleotide polymorphisms (SNPs) in 32 patients affected by mantle cell lymphoma (MCL) and 58 healthy controls. Real-time PCR combined with melting curve analysis was used for the determination of SNP alleles. A significant difference in the allele frequency of VEGFC–460T and C+936T SNPs in MCL and healthy cases was not observed. On the contrary, VEGF G+405C and C–2578A SNP allele distribution was significantly lower in the patient group than among normal controls (p = 0.014, p = 0.001). This observation suggests that further investigation is warranted, both in vitro and in a larger series of patients, to further examine the role of VEGF polymorphisms in the pathogenesis of MCL. In addition, the use of quantitative real-time PCR combined with a melting curve analysis method in the detection of the 4 VEGF SNPs may have the potential to replace older and more time-consuming PCR-RFLP methods and bears further investigation.

Ca2+ channel remodeling in perfused heart: effects of mechanical work and interventions affecting Ca2+ cycling on sarcolemmal and sarcoplasmic reticulum Ca2+ channels

We investigated whether changes in cardiac work or in Ca2+ fluxes may affect the expression of sarcolemmal or sarcoplasmic reticulum Ca2+ channels (DHPRs and RyRs, respectively). Isolated rat hearts were perfused at low Ca2+ concentration (0.8 mM instead of 1.5 mM), at low preload (5 cm instead of 20 cm), in the presence of 100 nM nifedipine or with a cardioplegic solution. After 60 min, hypocalcemic perfusion produced significant reduction in [3H]‐PN 200–110 and [3H]‐ryanodine binding, due to ≈30% reduction in Bmax (P<0.01), with unchanged Kd. Such modifications were reversible. Similar results were obtained in the nifedipine and cardioplegia groups. Low preload perfusion produced similar contractile effects as hypocalcemic perfusion, but it had no effect on radioligand binding. After hypocalcemic perfusion, DHPR and RyR gene expression, evaluated by RT‐PCR, were not modified. Chelerythrine (protein kinase C inhibitor) and lavendustin C (Ca2+/calmodulin‐dependent protein kinase II inhibitor), but not H‐89 (protein kinase A inhibitor), abolished the effects of hypocalcemic perfusion on [3H]‐PN 200–110 and [3H]‐ryanodine binding. We conclude that reduced Ca2+ entry and/or intracellular Ca2+ cycling determines DHPR and RyR remodeling through posttranslational protein modifications. Both protein kinase C and Ca2+/calmodulin‐dependent protein kinase II appear to play a role in this phenomenon.

Structure-function relationships in mammalian histidine-proline-rich glycoprotein

Histidine-proline-rich glycoprotein (HPRG), or histidine-rich glycoprotein (HRG), is a serum protein that is synthesized in the liver and is actively internalised by different cells, including skeletal muscle. The multidomain arrangement of HPRG comprises two modules at the N-terminus that are homologous to cystatin but void of cysteine proteinase inhibitor function, and a second half consisting of a histidine-proline-rich region (HPRR) located between two proline-rich regions (PRR1 and PRR2), and a C-terminus domain. HPRG has been reported to bind various ligands and to modulate angiogenesis via the histidine residues of the HPRR. However, the secondary structure prediction of the HPRR reveals that more than 98% is disordered and the structural basis of the hypothesized functions remains unclear. Comparison of the PRR1 of several mammalian species indicates the presence of a conserved binding site that might coordinate the Zn(2+) ion with an amino acid arrangement compatible with the cysteine-containing site that has been identified experimentally for rabbit HPRG. This observation provides a structural basis to the function of HPRG as an intracellular zinc chaperone which has been suggested by the involvement of the protein in the maintenance of the quaternary structure of skeletal muscle AMP deaminase (AMPD). During Anthropoidea evolution, a change of the primary structure of the PRR1 Zn(2+) binding site took place, giving rise to the sequence M-S-C-S/L-S/R-C that resembles the MxCxxC motif characteristic of metal transporters and metallochaperones.

Poly(vinyl alcohol)/gelatin Hydrogels Cultured with HepG2 Cells as a 3D Model of Hepatocellular Carcinoma: A Morphological Study

It has been demonstrated that three-dimensional (3D) cell culture models represent fundamental tools for the comprehension of cellular phenomena both for normal and cancerous tissues. Indeed, the microenvironment affects the cellular behavior as well as the response to drugs. In this study, we performed a morphological analysis on a hepatocarcinoma cell line, HepG2, grown for 24 days inside a bioartificial hydrogel composed of poly(vinyl alcohol) (PVA) and gelatin (G) to model a hepatocellular carcinoma (HCC) in 3D. Morphological features of PVA/G hydrogels were investigated, resulting to mimic the trabecular structure of liver parenchyma. A histologic analysis comparing the 3D models with HepG2 cell monolayers and tumor specimens was performed. In the 3D setting, HepG2 cells were viable and formed large cellular aggregates showing different morphotypes with zonal distribution. Furthermore, β-actin and α5β1 integrin revealed a morphotype-related expression; in particular, the frontline cells were characterized by a strong immunopositivity on a side border of their membrane, thus suggesting the formation of lamellipodia-like structures apt for migration. Based on these results, we propose PVA/G hydrogels as valuable substrates to develop a long term 3D HCC model that can be used to investigate important aspects of tumor biology related to migration phenomena.