Saverio Loddo

Prostaglandin E2 induces interleukin-8 gene transcription by activating C/EBP homologous protein in human T lymphocytes

The effect of prostaglandin E2 (PGE2) in regulating the synthesis of the pro-inflammatory chemokine inter-leukin-8 (IL-8) in T lymphocytes is not yet defined, even though it may reduce or enhance IL-8 synthesis in other cell types. Here, we demonstrate that, in human T cells, PGE2 induced IL-8 mRNA transcription through prostaglandin E2 receptors 1- and 4-dependent signal transduction pathways leading to the activation of the transcription factor C/EBP homologous protein (CHOP), never before implicated in IL-8 transcription. Several kinases, including protein kinase C, Src family tyrosine kinases, phosphatidylinositol 3-kinase, and p38 MAPK, were involved in PGE2-induced CHOP activation and IL-8 production. The transactivation of the IL-8 promoter by CHOP was NF-κB-independent. Our data suggest that PGE2 acts as a potent pro-inflammatory mediator by inducing IL-8 gene transcription in activated T cells through different signal transduction pathways leading to CHOP activation. These findings show the complexity with which PGE2 regulates IL-8 synthesis by inhibiting or enhancing its production depending on the cell types and environmental conditions.

Serum levels of interleukin 1β, interleukin 8 and tumour necrosis factor α as markers of gastric cancer

Abstract Despite the efforts made, a serum marker reliable for the screening and follow-up of patients with gastric cancer has not yet been identified. The aim of this preliminary study was to test the role of pro-inflammatory cytokines interleukin 1β, interleukin 8 and tumour necrosis factor α in patients with gastric cancer and in control groups. The statistical analysis of cytokines serum levels in the group with gastric cancer versus control groups has shown considerable differences (p<0.001) in their mean rates. The results indicate that the cytokines interleukin 1β, interleukin 8 and tumour necrosis factor α might perhaps act as diagnostic markers in patients with gastric cancer. Therefore, it is hypothesized that after more extended trials, their use in the screening and prognostic assessment of these patients could be a possibility.

Smoke exposure and circulating progenitor cells: Evidence for modulation of antioxidant enzymes and cell count

BACKGROUND Cigarette smoking is involved in vascular inflammation and impairment of circulating progenitor cells (CPCs), including endothelial progenitor cells (EPCs). The study aim was to evaluate the redox balance of these cells in relation to smoking exposure. METHODS Circulating cells from 36 healthy smokers and 26 controls were isolated and identified by flow cytometry. ROS generation, mRNA and protein cell expression, and enzymatic activity of MnSOD, catalase, and GPx-1 were evaluated. RESULTS Smokers showed higher levels of CRP and fibrinogen and lower levels of HDL-C. ROS and MnSOD were higher (p<0.001), while catalase and GPx-1 were lower (p<0.001) as was EPC number (p<0.001) in smokers. CPC and EPC correlated with HDL-C, CRP, ROS and enzyme expression and activity. CONCLUSIONS Our data suggest that smoking exposure involves antioxidant enzymes in CPCs and EPCs and that the inflammatory response in smokers plays an important role in impairing cells and their antioxidant functions.

Circulating progenitor cells are increased in newly diagnosed untreated hypertensive patients with arterial stiffening but normal carotid intima-media thickness

Circulating progenitor cells (CPCs), including endothelial progenitor cells (EPCs), have a key role in endothelium repair. Cellular NADPH oxidase (Nox) enzymes, including Nox-containing gp91phox, represent a source of reactive oxygen species (ROS); ROS trigger protective signals but may also have detrimental effects. Cellular defenses against ROS include the enzymes manganese superoxide dismutase (MnSOD), catalase (CAT) and glutathione peroxidase type-1 (GPx-1). We investigated the relationships of CPCs with cellular gp91phox, MnSOD, CAT, GPx-1 and ROS levels and with carotid intima-media thickness (cIMT) and stiffness in hypertensives without additional risk factors for cardiovascular disease. CPCs from 53 newly diagnosed, untreated hypertensives and from 29 controls were isolated and identified by flow cytometry. gp91phox, MnSOD, CAT, and GPx-1 mRNA and protein expression and ROS generation were evaluated in enriched samples of CD34+ cells; cIMT and stiffness were assessed. Hypertensives showed higher arterial stiffness (P<0.001) but no difference in cIMT with respect to controls. ROS generation was slightly increased (P=0.04), whereas gp91phox, MnSOD, CAT and GPx-1 were significantly higher (P<0.001) with respect to controls, as was CPC number (P<0.001), but EPCs were no different. CPC and EPC numbers correlated with gp91phox, ROS and fibrinogen (P<0.001); moreover, gp91phox, MnSOD, CAT and GPx-1 were correlated with CPC number. In early phases of arterial hypertension, before the development of wall thickening and even in the presence of arterial mechanical impairment, CPC number may be increased to maintain an adequate number of EPCs in peripheral blood.

Bisphosphonates Induce Apoptosis of Circulating Endothelial Cells in Multiple Myeloma Patients and in Subjects with Bisphosphonate-Induced Osteonecrosis of the Jaws

Bisphosphonates (BPs) are the current standard of care for bone lesions in patients with multiple myeloma (MM) but they are associated with a number of side effects such as osteonecrosis of the jaw. The exact mechanisms of osteonecrosis are not elucidated, and its physiopathology is based on several hypotheses such as a decrease in bone remodeling or an inhibitory effect on angiogenesis. The aim of our study was to investigate the mechanism involved in the pathogenesis of osteonecrosis. We examined the apoptosis of circulating endothelial progenitor cells in MM subjects before and after BP treatment and in osteonecrosis patients using a flow-cytometric analysis. Our data showed an increase in endothelial cell apoptosis in MM patients after BP administration and in osteonecrosis subjects. Our study seems in agreement with the hypothesis that BPs can inhibit angiogenesis interfering with endothelial cell proliferation and survival, leading to loss of blood vessels and avascular necrosis.

Chemical and Pathological Oxidative Influences on Band 3 Protein Anion-exchanger

Background/aims: The erythrocyte is a cell exposed to a high level of oxygen pressure and to oxidative chemical agents. This stress involves SH-groups oxidation, cell shrinkage by activation of K-Cl co-transport (KCC) and elevation of the band 3 tyrosine phosphorylation level. The aim of our study was to test whether oxidative stress could influence band 3-mediated anion transport in human red blood cells. Methods: To evaluate this hypothesis, normal and pathological (glucose 6 phosphate dehydrogenase (G6PDH) defficient) erythrocytes were treated with known sulphydryl-blocking or thiol-oxidizing agents, such as N-ethylmaleimide (NEM), azodicarboxylic acid bis[dimethylamide] (diamide), orthovanadate, Mg2+ and tested for sulphate (SO4-) uptake, K+ efflux, G6PDH activity and glutathione (GSH) concentration. Results: In normal red blood cells, the rate constants of SO4- uptake decreased by about 28 % when cells were incubated with NEM, diamide and orthovanadate. In G6PDH-deficient red blood cells, in which oxidative stress occurs naturally, the rate constant of sulphate uptake was decreased by about 40% that of normal red cells. Addition of oxidizing and phosphatase inhibitor agents to pathological erythrocytes further decreased anion transport. In contrast, G6PDH activity was increased under oxidative stress in normal as well as in pathological cells and was lower in the presence of exogenous Mg2+ in parallel to a significant increase in sulphate transport. In both cells, the oxidizing agents increased K+ efflux with depletion of GSH. Conclusion: The data are discussed in light of the possible opposite effects exerted by oxidative agents and Mg2+ on KCC and on the protein tyrosine kinase (PTK)-protein tyrosine phosphatase (PTP) equilibrium. The decreased sulphate uptake observed in the experimental and pathological conditions could be due to band 3 SH-groups oxidation or to oxidative stress-induced K-Cl symport-mediated cell shrinkage with concomitant band 3 tyrosine phosphorylation.

Effects of haemodialysis on circulating endothelial progenitor cell count

During haemodialysis (HD) the endothelium is the first organ to sense and to be impaired by mechanical and immunological stimuli. We hypothesized that a single HD session induces mobilization of endothelial progenitor cells (EPCs) and that cardiovascular risk factors may influence this process. We quantified EPCs at different maturational stages (CD34+, CD133+/VEGFR2+) in blood samples from 30 patients, during HD and on the interdialytic day, and in 10 healthy volunteers. Samples were drawn at the start of HD, 1, 2 and 3 h after, at the end of HD and at 24 h on the interdialytic day. Patients were divided into two groups based on a recent risk scoring system (SCORE project): low-risk (LR) and high-risk groups (HR). HD patients showed a significantly reduced basal number of EPCs with respect to healthy volunteers. In contrast, we observed increasing EPCs during HD whereas they diminished on the interdialytic day. The EPC number was directly correlated with HD time progression. The EPC number during HD was increased in the HR group with respect to the LR group. We had a direct correlation between risk score and number of EPCs. Cardiovascular risk factors influenced the mobilization of stem cells from the bone marrow. This feature could be the direct consequence of an augmented request of stem cells to respond to the most important endothelial impairment but could also show a defective capacity of EPCs to home in and repair the sites of vascular injury.

Vitamin D Status in Rheumatoid Arthritis: Inflammation, Arterial Stiffness and Circulating Progenitor Cell Number

Background and Aims Suboptimal vitamin D status was recently acknowledged as an independent predictor of cardiovascular diseases and all-cause mortality in several clinical settings, and its serum levels are commonly reduced in Rheumatoid Arthritis (RA). Patients affected by RA present accelerated atherosclerosis and increased cardiovascular morbidity and mortality with respect to the general population. In RA, it has been reported an impairment of the number and the activity of circulating proangiogenic haematopoietic cells (PHCs), including CD34+, that may play a role in endothelial homeostasis. The purpose of the study is to investigate the association between vitamin D levels and PHCs, inflammatory markers, and arterial stiffening in patients with RA. Methods and Results CD34+ cells were isolated from 27 RA patients and 41 controls. Vitamin D levels, C-reactive protein (CRP), fibrinogen, pulse wave velocity (PWV), and carotid intima-media thickness (cIMT) were also evaluated. CD34+ count and vitamin D levels were lower in RA patients as compared to controls, while fibrinogen, CRP, PWV and cIMT were higher in RA patients. CD34+ cell number appeared to be associated with vitamin D levels, and negatively correlated to fibrinogen and early atherosclerosis markers (PWV and cIMT); vitamin D levels appear also to be inversely associated to fibrinogen. Conclusions RA patients with moderate disease activity presented with low vitamin D levels, low CD34+ cell count, increased PWV and cIMT; we found that vitamin D deficiency is associated to CD34+ cell reduction in peripheral blood, and with fibrinogen levels. This suggests that vitamin D might contribute to endothelial homeostasis in patients with RA.

Role of osteopontin in breast cancer patients

Aim and background In breast cancer, as in almost all neoplastic diseases, the prognosis is strictly related to the invasive capacity, local and distant, that characterizes the growth of all tumors. Since the mechanisms that regulate replication of the neoplastic cells, with consequent capacity to metastasize, are not completely known, identification of new markers represents the gold standard of research in the stratification of patients with such a pathology. Osteopontin, a specific phosphoglycoprotein isolated from extracellular bone matrix and actively involved in mechanisms of bone reabsorption, appears to play a key role in osteoclastogenesis at the level of the skeleton in some pathologic situations. It has been found that patients with metastatic bone lesions from breast or prostate cancer present, with respect to subjects without repetitive bone lesions, elevated serum levels of the protein, indicating that osteopontin could play an important role in the development and progression of the neoplastic disease at the bone level. Methods and study design The authors studied 26 patients with breast cancer, evaluating as a marker also serum osteopontin levels. Results and conclusions The results, although obtained on a small number of patients, showed that osteopontin evaluation in breast cancer patients can be a particularly interesting method of research in staging of the disease as well as in the prognosis, thereby attributing a role of a biotumoral marker also in the follow-up of the therapy.

Circulating Progenitor Cells after Cold Pressor Test in Hypertensive and Uremic Patients

Endothelium was initially considered an inert lining of the blood vessels. Recently, it was suggested that damaged cells are continuously replaced by novel cells, hematopoietic stem cells (HSCs), which are directly mobilized by the bone marrow and then transformed into endothelial progenitor cells (EPCs). Initial triggers of vessel remodeling are physical forces such as blood pressure and fluid shear stress. We investigated whether or not a stress stimulus on vessels applied by a cold pressor test (CPT) would stimulate the mobilization of progenitor cells. Twenty-two healthy subjects, 20 patients with essential hypertension, and 18 with chronic kidney disease (CKD) underwent CPT by dipping their hands in icy water for 4 min. Immediately before and after 4 and 60 min, we quantified HSCs and EPCs identified by flow cytometry. We measured also adhesion soluble molecules (sICAM-1, sVCAM-1, and sE-selectin) as markers of endothelial activation. In healthy and hypertensive subjects, but not in CKD subjects, the number of HSCs was elevated as a direct response to CPT stress. Levels of EPCs and adhesion soluble molecules increased significantly, but to a different extent in every group. In CKD patients, the number of EPCs did not return to basal levels either after 60 min. Levels of adhesion soluble molecules directly correlated with the number of progenitor cells in hypertensive and healthy subjects. CPT caused an increase in adhesion soluble molecules. Discrepancies in the numbers of HSCs and EPCs in CKD patients could suggest a specific impairment in blood vessel remodeling correlated with recognized endothelial dysfunction.