The Oxidative Stress-Induced miR-200c Is Upregulated in Psoriasis and Correlates with Disease Severity and Determinants of Cardiovascular Risk.

Psoriasis is a chronic inflammatory skin disease associated with reactive oxygen species (ROS) increase and a higher risk of cardiovascular (CV) events. We previously showed that the miR-200 family (miR-200s) is induced by ROS, miR-200c being the most upregulated member responsible for apoptosis, senescence, ROS increase, and nitric oxide decrease, finally causing endothelial dysfunction. Moreover, circulating miR-200c increases in familial hypercholesterolemic children and in plaques and plasma of atherosclerotic patients, two pathologies associated with increased ROS. Given miR-200s' role in endothelial dysfunction, ROS, and inflammation, we hypothesized that miR-200s were modulated in lesional skin (LS) and plasma of psoriatic patients (Pso) and that their levels correlated with some CV risk determinants at a subclinical level. All Pso had severe psoriasis, i.e., Psoriasis Area and Severity Index (PASI) > 10, and one of the following: at least two systemic psoriasis treatments, age at onset < 40 years, and disease duration > 10 years. RNA was extracted from plasma (Pso, N = 29; Ctrl, N = 29) and from nonlesional skin (NLS) and LS of 6 Pso and 6 healthy subject skin (HS) biopsies. miR-200 levels were assayed by quantitative RT-PCR. We found that all miR-200s were increased in LS vs. NLS and miR-200c was the most expressed and upregulated in LS vs. HS. In addition, circulating miR-200c and miR-200a were upregulated in Pso vs. Ctrl. Further, miR-200c positively correlated with PASI, disease duration, left ventricular (LV) mass, LV relative wall thickness (RWT), and E/e', a marker of diastolic dysfunction. Multiple regression analysis indicates a direct association between miR-200c and both RWT and LV mass. Circulating miR-200a correlated positively only with LV mass and arterial pressure augmentation index, a measure of stiffness, although the correlations were nearly significant (P = 0.06). In conclusion, miR-200c is upregulated in LS and plasma of Pso, suggesting its role in ROS increase and inflammation associated with CV risk in psoriasis.

UVC inhibits HIF-1alpha protein translation by a DNA damage- and topoisomerase I-independent pathway.

Hypoxia inducible factor 1 (HIF-1) is a key player in cancer progression and an attractive target for cancer therapy. Several small molecule inhibitors of HIF-1alpha also induce a DNA damage response. However, whether or not DNA damage is required for or associated with the inhibition of HIF-1alpha protein accumulation is poorly understood. In this report we investigated the effects of distinct DNA damaging conditions on the hypoxic induction of HIF-1alpha protein in cancer cell lines. We demonstrate that in addition to topotecan (TPT), a known inhibitor of HIF-1alpha, UVC, but not other DNA damaging agents (cisplatin, ionizing radiation and doxorubicin), inhibited HIF-1alpha protein accumulation in a dose-dependent, p53-independent fashion. Low doses UVC decreased HIF-1alpha translation without affecting global protein synthesis. Inhibition of HIF-1alpha by UVC required ongoing RNA transcription, but not DNA replication. Moreover, a functional ATR was required for the activation of DNA damage-dependent responses by both UVC and TPT, but was dispensable for the inhibition of HIF-1alpha protein. Notably, unlike TPT, inhibition of HIF-1alpha protein by UVC did not require topoisomerase I, suggesting a similar yet distinct mode of action. Our data reveal that UVC is a novel signal associated with inhibition of HIF-1alpha protein accumulation, and they uncouple the DNA damage-dependent signaling pathway exerted by UVC and TPT from HIF-1alpha inhibition.

The slide rule: a new method for the assessment of acid-base equilibrium disorders.

AIM: Maps and nomograms are routinely used to evaluate acid-base equilibrium (ABE), but often require previous skilled practice and time to be used in the clinical setting; moreover, some definite alterations may be missed. The aim of this study was to evaluate the new slide rule (patented by Authors) for the rapid, precise and complete assessment and diagnosis of altered blood gas analysis (ABG) parameters and compare it to traditional methods. METHODS: Once pH, bicarbonate and PaCO(2) values are known by arterial blood gas analysis (ABG), the slide rule can calculate, show and instantly diagnose the related alteration, including possible mixed partial compensated ones. In this regard, 330 patients coming from 6 (4 national and 2 foreign) clinics were studied; each patient underwent evaluation of ABG alterations using traditional methods and the slide rule immediately thereafter. RESULTS: The results of consecutive evaluations on involved patients made by specialists in all clinics were in agreement; nonetheless, the slide rule was far more user friendly, rapid and complete in the ABE alterations' diagnostic range, in comparison with traditional methods. CONCLUSION: All involved specialists confirmed that the new slide rule was able to rapidly diagnose ABE alterations, including mixed or partially compensated ones that may be missed by traditional methods.

Gene therapy in peripheral artery disease.

In the last decade, studies of the biological mechanisms underlying angiogenesis, i.e. the development of a new vasculature from pre-existing blood vessels, have suggested a new approach to peripheral obstructive artery disease based on the treatment of ischemic tissues with angiogenic growth factors. As demonstrated by experimental studies in animal models, a therapeutic effect can be reached as the newly formed vascular network, functioning as a biologic by-pass, restores a normal blood supply to the ischemic territories. New techniques of gene therapy proved effective in reaching sustained concentrations of angiogenic factors in the target tissues. This review concerns the pre-clinical background and the results of the early clinical trials of angiogenic gene therapy, which have shown the safety and feasibility of this new approach.

A potential role for imaging technology in anticancer efficacy evaluations.

The introduction of imaging methods suitable for rodents offers opportunities for new anticancer efficacy models. Traditional models do not provide the level of sensitivity afforded by these precise and quantitative techniques. Bioluminescent endpoints, now feasible because of sensitive charge-coupled device cameras, can be non-invasively detected in live animals. Currently, the most common luminescence endpoint is firefly luciferase, which, in the presence of O(2) and ATP, catalyses the cleavage of the substrate luciferin and results in the emission of a photon of light. In vivo implantation of tumour cells transfected with the luciferase gene allows sequential monitoring of tumour growth within the viscera by measuring these photon signals. Furthermore, tumour cell lines containing the luciferase gene transcribed from an inducible promoter offer opportunities to study molecular-target modulation without the need for ex vivo evaluations of serial tumour samples. In conjunction with this, transgenic mice bearing a luciferase reporter mechanism can be used to monitor the tumour microenvironment as well as to signal when transforming events occur. This technology has the potential to reshape the efficacy evaluations and drug-testing algorithms of the future.

Picolinic acid- or desferrioxamine-inducible autocrine activation of macrophages engineered to produce IFNgamma: an approach for gene therapy.

Macrophage (Mphi)-based vectors are highly mobile cellular shuttles designed to deliver therapeutic genes within the tissues. We engineered a mouse Mphi cell line to express the murine interferon-gamma (IFNgamma) under the control of an inducible promoter containing the hypoxia-responsive element, which can be triggered by hypoxia and other stimuli. We show that this Mphi vector can be induced to produce IFNgamma under normoxic conditions by stimulation with picolinic acid (PA), a catabolite of tryptophan, or desferrioxamine (DFX), an iron-chelating drug. The Mphi vector responds to IFNgamma with the induction of IRF-1 and of other IFNgamma-inducible genes, the expression of Ia antigens and induction of phagocytic activity. Inducible nitric oxygen synthase gene expression, nitric oxide production, as well as TNFalpha secretion were enhanced by PA or DFX as the sole stimuli. None of the above responses could be triggered individually by PA or DFX in control, normal Mphi, indicating that the Mphi vector overcame the need for costimulatory molecules derived from the immune system for its full activation. Furthermore, we demonstrate that extracellular iron can downregulate such response, thereby identifying an additional tool for the fine tuning of the Mphi vector response to stimulation.

Adenovirus-mediated VEGF(165) gene transfer enhances wound healing by promoting angiogenesis in CD1 diabetic mice.

It has been previously shown that vascular endothelial growth factor (VEGF) plays a central role in promoting angiogenesis during wound repair and that healing-impaired diabetic mice show decreased VEGF expression levels. In order to investigate the potential benefits of gene therapy with growth factors on wound repair, a replication-deficient recombinant adenovirus vector carrying the human VEGF(165) gene (AdCMV.VEGF(165)) was topically applied on excisional wounds of streptozotocin-induced diabetic mice. Treatment with AdCMV.VEGF(165) significantly accelerated wound closure when compared with AdCMV.LacZ-treated, as well as saline-treated control mice, by promoting angiogenesis at the site of injury. Our findings suggest that AdCMV.VEGF(165) may be regarded as a therapeutic tool for the treatment of diabetic ulcers.

In vitro permeation screening of a new formulation of thiocolchicoside containing various enhancers.

Thiocolchicoside, a muscle relaxant agent with anti-inflammatory and analgesic actions, also is used topically for the treatment of muscular spasms and for rheumatologic, orthopedic, and traumatologic disorders. In this study, thiocolchicoside was formulated to use as foam to avoid contact with the afflicted area during the spreading phase. To enhance drug penetration, various enhancers were added to the base formulation. The tested enhancers were ethoxyethylendiglycol (Transcutol), highly purified phosphatidylcholine (Lipoid S20), capsaicin, propylene glycol dipelargonate (DPPG), and glycolysed ethoxylated glycerides (Labrafil M1944 CS). The transdermal absorption of the tested formulations containing enhancers, in comparison with base formulation, was evaluated in vitro through rat skin using standard Franz diffusion cells. Base formulation was found to have a higher permeation profile than the simple aqueous and hydroalcoholic solutions of the drug, meaning that the base formulation by itself enhances the drug permeation. Among the tested formulations, only the formulation containing DPPG/ethanol was found to be statistically different, showing an enhancement factor of 3.58. In the same experimental session, Muscoril ointment, the commercially available pharmaceutical product containing the same thiocolchicoside concentration (0.25%), also was tested. The formulation containing DPPG/ethanol showed a 4.86 times increase of permeability constant in comparison with Muscoril ointment. The formulation containing DPPG/ethanol as an enhancer could be a good candidate for a new topical foam, considering its good characteristics of permeability and compliance.

Engineering of macrophages to produce IFN-gamma in response to hypoxia.

Activation of murine macrophages (Mphi) requires the collaboration of signals derived from the immune system and the environment. In this study, we engineered a murine Mphi cell line to become activated in response to an environmental signal, hypoxia, as the sole stimulus. Hypoxia is a condition of low oxygen tension, occurring in several pathological tissues, which acts in synergy with IFN-gamma to induce full Mphi activation. We transfected the ANA-1 murine Mphi cell line with a construct containing the IFN-gamma gene controlled by a synthetic promoter inducible by hypoxia (HRE3x-Tk), and we characterized the cellular and molecular biology of the engineered Mphi under normoxia or hypoxia. Engineered Mphi in normoxia expressed basal levels of IFN-gamma mRNA and protein that were strongly augmented by shifting the cells to hypoxia. Furthermore, they responded to the synthesized IFN-gamma with induction of IFN-responsive factor-1 and 2'-5'-oligoadenylate synthase expression. Under normoxic conditions, the engineered Mphi had a significant constitutive level of Ia Ags and Fc receptors. Hypoxia induced further augmentation of Ia and Fc expression. Finally, hypoxia induced inducible NO synthase expression, and subsequent reoxygenation led to the production of NO. In conclusion, the engineered Mphi, which produce IFN-gamma in an inducible manner, express new biochemical and functional properties in response to low oxygen environment as the sole stimulus, thereby circumventing the need for costimulation by other immune system-derived signals.

[Perspectives of gene therapy in ischemic syndromes of the lower limbs]. / Prospettive di terapia genica delle sindromi ischemiche degli arti inferiori.

In recent years, new knowledge has been provided from the study of the biological mechanisms underlying the angiogenic process, i.e. the development of a new vasculature from preexisting blood vessels. These advances suggested a possible new approach for the therapy of peripheral obstructive arterial disease, based on the treatment of ischemic tissues with angiogenic growth factors. As demonstrated by experimental studies in animal models, a therapeutic effect can be reached as the newly formed vascular network, functioning as a biologic by-pass, restores a normal blood supply to the ischemic territories. New techniques of gene therapy proved effective in reaching sustained concentrations of angiogenic factors in the target tissues. This paper will briefly describe the methodological background, the results of the early clinical applications of angiogenic gene therapy and the open questions that need to be addressed before this new approach can be proposed as an effective option instead of existing medical and surgical therapies.

Shear stress downregulation of platelet-derived growth factor receptor-beta and matrix metalloprotease-2 is associated with inhibition of smooth muscle cell invasion and migration.

BACKGROUND: After endovascular injury, smooth muscle cells (SMCs) may be exposed to hemodynamic shear stress (SS), and these forces modulate neointima accumulation. The effect of SS on SMC migration and invasion is unknown, and it was examined in the present study. METHODS AND RESULTS: Bovine aortic SMCs were exposed to laminar SS of 12 dyne/cm(2) for 3 (SS3) or 15 (SS15) hours; control (C3 and C15) SMCs were kept under static conditions. Platelet-derived growth factor (PDGF)-BB-directed SMC migration and invasion were evaluated by a modified Boyden chamber assay with filters coated with either gelatin or reconstituted basement membrane proteins (Matrigel), respectively. SS15 inhibited both SMC migration and invasion (P<0.0001). There was no significant difference between SS3 and C3 cells. Media conditioned with SS15 cells exhibited a reduction in matrix metalloprotease-2 (MMP-2) by zymography and Western analysis. Northern blot analysis revealed no effect of SS15 on MMP-2 mRNA. In contrast, SS15 decreased MMP-2 activator and membrane-type MMP (MT-MMP or MMP-14) mRNA and protein. Furthermore, SS15 decreased PDGF receptor-beta (PDGF-Rbeta) mRNA and protein (P<0.05), and the SS-dependent decrease in PDGF-BB-directed cell migration was rescued by overexpressing PDGF-Rbeta. CONCLUSIONS: SS inhibits SMC migration and invasion via diminished PDGF-Rbeta expression. This effect of SS is associated with decreased MMP-2 secretion and MT-MMP downregulation.

Wild-type p53 gene transfer inhibits invasion and reduces matrix metalloproteinase-2 levels in p53-mutated human melanoma cells.

The tumor suppressor gene p53 has inhibitory effects on cell growth and angiogenesis and induces apoptosis when overexpressed in melanoma and in a variety of tumor cells by adenovirus-mediated gene transfer. The invasive ability of tumor cells, facilitating local infiltration and metastasis, is related to matrix metalloproteinase levels. In melanoma, matrix metalloproteinase-2 and matrix metalloproteinase-9 have a prominent role in this process. The aim of this study was to evaluate whether wild-type p53 overexpression, obtained by a recombinant adenovirus vector (AdCMV.p53), affects cell invasiveness through modulation of matrix metalloproteinase-2 and matrix metalloproteinase-9. Two human melanoma cell lines were used in this study: the SK-MEL-110, carrying a mutated p53 gene, and the SK-MEL-147, carrying the wild-type p53 gene. SK-MEL-110 cells infected with AdCMV.p53 exhibited decreased invasion capability from day 1 after infection, compared with cells not infected or infected with the control vector AdCMV.Null. This reduced invasiveness was associated with decreased matrix metalloproteinase-2 levels in conditioned media whereas no changes were detected in matrix metalloproteinase-9 secreted levels. No modulation in matrix metalloproteinase-2 mRNA levels was detectable, however, after wild-type p53 gene transfer. Furthermore, protein expression of secreted tissue inhibitor of metalloproteinase-2 was not altered by AdCMV.p53 treatment. In contrast, in SK-MEL-147 cells, AdCMV.p53 did not affect cell invasiveness and levels of secreted matrix metalloproteinase-2. Gene transfer of wild-type p53 inhibited proliferation of both cell lines, showing that also SK-MEL-147 cells respond to wild-type p53 overexpression. This novel mechanism of action of wild-type p53 gene transfer may contribute to its antitumor effect by downregulating cell invasion and matrix metalloproteinase-2 secreted levels in mutated p53 human melanoma cell lines.

Lipopolysaccharide-induced lung injury in mice. I. Concomitant evaluation of inflammatory cells and haemorrhagic lung damage.

Intratracheal instillation of lipopolysaccharide (LPS) induces an inflammatory response characterized by infiltration of polymorphonuclear neutrophils (PMNs) into the extracellular matrix and by the release of mediators that play a fundamental role in lung damage. In the present study, we developed a mouse model which allows correlation of the inflammatory response and haemorrhagic tissue injury in the same animal. In particular, the different steps of the inflammatory response and tissue damage were evaluated by the analysis of three parameters: myeloperoxidase (MPO) activity in the parenchyma, reflecting PMNs accumulation into the lung, inflammatory cells count in the bronchoalveolar lavage fluid (BALF), reflecting their extravasation, and total haemoglobin estimation in BALF, a marker of haemorrhagic tissue damage consequent to PMNs degranulation. In our experimental conditions, intra-tracheal administration of 10 microg/mouse of LPS evoked an increase of MPO activity in the lung at 4 h (131%) and 6 h (147%) from endotoxin challenge. A significant increase of PMNs in the BALF was noticed at these times with a plateau between the 12nd and 24th h. PMN accumulation produced a time-dependent haemorrhagic lung damage until 24 h after LPS injection (4 h: +38%; 6 h: +23%; 12 h: +44%; 24 h: +129% increase of haemoglobin concentration in the BALF vs. control). Lung injury was also assessed histopathologically. Twenty-four hours after the challenge, diffuse alveolar haemorrhage, as well as PMN recruitment in the interstitium and alveolus were observed in the LPS group. This model was pharmacologically characterized by pretreatment of LPS-treated mice with antiinflammatory drugs acting on different steps of the <>. We demonstrated that: a) betamethasone (1, 3, 10, 30 mg/kg p.o.) reduced in a dose-dependent manner the MPO activity, the number of inflammatory cells and, at the same time, lung injury; b) pentoxifylline, a TNFalpha production inhibitor (200 mg/kg i.p.), inhibited PMN extravasation and lung haemorrhage but it was not able to reduce MPO activity in the lung; c) L-680,833, an anti-elastase compound (30 mg/kg po), decreased significantly only the haemorrhagic lung damage; d) indomethacin, a non steroidal antiinflammatory drug (5 mg/kg p.o.), did not show any effect on any of the parameters considered. In conclusion, our in vivo mouse model is a practical alternative to animal models of ARDS (Adult Respiratory Distress Syndrome) recently described and it permits to dissect and to characterize the different steps of PMNs infiltration and, at the same time, the damage caused by their activation.

A dominant negative RAS-specific guanine nucleotide exchange factor reverses neoplastic phenotype in K-ras transformed mouse fibroblasts.

Ras proteins are small GTPases playing a pivotal role in cell proliferation and differentiation. Their activation state depends on the competing action of GTPase Activating Proteins (GAP) and Guanine nucleotide Exchange Factors (GEF). A tryptophan residue (Trp1056 in CDC25Mm-GEF), conserved in all ras-specific GEFs identified so far has been previously shown to be essential for GEF activity. Its substitution with glutamic acid results in a catalytically inactive mutant, which is able to efficiently displace wild-type GEF from p21ras and to originate a stable ras/GEF binary complex due to the reduced affinity of the nucleotide-free ras/GEF complex for the incoming nucleotide. We show here that this 'ras-sequestering property' can be utilized to attenuate ras signal transduction pathways in mouse fibroblasts transformed by oncogenic ras. In fact overexpression of the dominant negative GEFW1056E in stable transfected cells strongly reduces intracellular ras-GTP levels in k-ras transformed fibroblasts. Accordingly, the transfected fibroblasts revert to wild-type phenotype on the basis of morphology, cell cycle and anchorage independent growth. The reversion of the transformed phenotype is accompanied by DNA endoreduplication. The possible use of dominant negative ras-specific GEFs as a tool to down-regulate tumor growth is discussed.

Quality control of spirometry in the elderly. The SA.R.A. study. SAlute Respiration nell'Anziano = Respiratory Health in the Elderly.

We evaluated the outcome of the spirometry quality control program of the SA.R.A. multicenter project, the aim of which is the multidimensional assessment of asthma and COPD in the elderly (>/= 65 yr). The factors determining this quality were also evaluated. The program was based on standardized procedures (ATS recommendations), performed by specifically trained and certified personnel; a fully-computerized spirometer with customized software was used for spirometry. A reference center made monthly controls. Overall, 638 cases and 984 controls were examined. Spirometric measurements were obtained in 607 cases and 912 controls; 508 and 747 tests with at least three acceptable curves were obtained in cases and in controls, respectively (NS). The percentage of reproducible tests ranged between 95.8% for FEV(1) in controls and 87.6% for FVC in cases. The average reproducibility for FEV(1) was 61.6 ml in cases and 58.3 ml in controls (NS). Cognitive impairment, shorter 6-min walk distance, and lower educational level were found to be independent risk factors for a poorer acceptability rate (logistic regression analysis). Male sex and age were risk factors for a poorer reproducibility of FEV(1). Reproducibility tended to improve with time (p < 0.001). Although spirometry becomes increasingly difficult in aging patients, a rigorous quality control program can ensure that reliable data are obtained in the majority of patients.

The tryptophan catabolite picolinic acid selectively induces the chemokines macrophage inflammatory protein-1 alpha and -1 beta in macrophages.

We previously found that the tryptophan catabolite picolinic acid (PA) is a costimulus for the activation of macrophage effector functions. In this study, we have investigated the ability of PA to modulate the expression of chemokines in macrophages. We demonstrate that PA is a potent activator of the inflammatory chemokines MIP (macrophage inflammatory protein)-1 alpha and MIP-1 beta (MIPs) mRNA expression in mouse macrophages in a dose- and time-dependent fashion and through a de novo protein synthesis-dependent process. The induction by PA occurred within 3 h of treatment and reached a peak in 12 h. The stimulatory effects of PA were selective for MIPs because other chemokines, including monocyte chemoattractant protein-1, RANTES, IFN-gamma-inducible protein-10, MIP-2, and macrophage-derived chemokine, were not induced under the same experimental conditions and were not an epiphenomenon of macrophage activation because IFN-gamma did not affect MIPs expression. Induction of both MIP-1 alpha and MIP-1 beta by PA was associated with transcriptional activation and mRNA stabilization, suggesting a dual molecular mechanism of control. Iron chelation could be involved in MIPs induction by PA because iron sulfate inhibited the process and the iron-chelating agent, desferrioxamine, induced MIPs expression. We propose the existence of a new pathway leading to inflammation initiated by tryptophan catabolism that can communicate with the immune system through the production of PA, followed by secretion of chemokines by macrophages. These results establish the importance of PA as an activator of macrophage proinflammatory functions, providing the first evidence that this molecule can be biologically active without the need for a costimulatory agent.

Acidosis inhibits endothelial cell apoptosis and function and induces basic fibroblast growth factor and vascular endothelial growth factor expression.

Endothelial cells are exposed to an acidotic environment in a variety of pathological and physiological conditions. However, the effect of acidosis on endothelial cell function is still largely unknown, and it was evaluated in the present study. Bovine aortic endothelial cells (BAECs) were grown in bicarbonate buffer equilibrated either with 20% CO(2) (pH 7.0, acidosis) or 5% CO(2) (pH 7.4, control). Acidosis inhibited BAEC proliferation in 10% FCS, whereas by day 7 in serum-free medium, cell number was 3-fold higher in acidotic cells than in control cells. Serum deprivation enhanced BAEC apoptosis, and apoptotic cell death was markedly inhibited by acidosis. Additionally, acidosis inhibited FCS-stimulated migration in a modified Boyden chamber assay and FCS-stimulated differentiation into capillary-like structures on reconstituted basement membrane proteins. Conditioned media from BAECs cultured for 48 hours either at pH 7.0 or pH 7.4 enhanced BAEC proliferation and migration at pH 7.4, and both effects were more marked with conditioned medium from BAECs grown in acidotic than in control conditions. Acidosis enhanced vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) mRNA expression as well as bFGF secretion, and a blocking bFGF antibody inhibited enhanced BAEC migration in response to conditioned medium from acidotic cells. These results show that acidosis protects endothelial cells from apoptosis and inhibits their proangiogenic behavior despite enhanced VEGF and bFGF mRNA expression and bFGF secretion.