Chiara Armani

Expression, pharmacology, and functional role of somatostatin receptor subtypes 1 and 2 in human macrophages

Somatostatin (SRIF)‐14 is recognized as an important mediator between the nervous and the immune system, although the functional role of its receptors (sst1–sst5) is poorly understood in humans. In our study, we demonstrate that human macrophages, differentiated from PBMC‐derived monocytes, express sst1 and sst2 mRNAs. sst1 and sst2 are mostly localized at the cell surface and display active binding sites. In particular, sst1/sst2 activation results in a weak internalization of sst1, and the sst2 internalization appears more efficient. At the functional level, the activation of SRIF receptors by the multiligand analogs SOM230 and KE108, but not by SRIF‐14 or cortistatin‐14, reduces macrophage viability. Their effects are mimicked by the selective activation of sst1 and sst2 using CH‐275 and SMS 201‐995/L‐779,976, respectively. Further, sst1‐ and sst2‐mediated effects are reversed by the sst1 antagonist SRA‐880 or the sst2 antagonist CYN 154806, respectively. CH‐275, SMS 201‐995, and L‐779,976, but not SRIF‐14, decrease mRNA expression and secretion of the MCP‐1. In addition, SRIF‐14, CH‐275, SMS 201‐995, and L‐779,976 decrease IL‐8 secretion, and they do not affect IL‐8 mRNA expression. In contrast, SRIF‐14 and sst1/sst2 agonists do not affect the secretion of matrix metalloproteinase‐9. Collectively, our results suggest that the SRIF system, through sst1 and sst2, exerts mainly an immunosuppressive effect in human macrophages and may, therefore, represent a therapeutic window that can be exploited for the development of new strategies in pharmacological therapy of inflammation.

Inflammatory markers and cardiac function in acute coronary syndrome: difference in ST-segment elevation myocardial infarction (STEMI) and in non-STEMI models.

PURPOSE No studies have been addressed to the differences in inflammation kinetics between ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI). PATIENTS AND METHODS Forty consecutive patients with acute coronary syndrome (ACS) (n=23 STEMI, age=61.7+/-10.3 years; n=17 NSTEMI, age=65.6+/-11.3 years) were enrolled within 12h after symptoms. All patients received therapy according to the current Guidelines. Blood samples were collected at admission (t0), on days 7 (t1) and 30 (t2) to evaluate CD40 ligand (CD40L), transforming growth factor (TGF)-beta, interleukin (IL)-6, tumor necrosis factor (TNF)-alpha and its receptors TNFRI and TNFRII, high sensitivity C-reactive protein (hs-CRP), serum amyloid A (SAA) and white blood cells (WBC). Echocardiographic parameters were also evaluated. RESULTS STEMI patients, at admission, had significantly higher median values of hs-CRP (p<0.001), WBC (p<0.01), ferritin (p<0.0005) and IL-6 (p<0.05) than NSTEMI. On the contrary, NSTEMI patients had lower median levels of every inflammatory marker except for CD40L (p<0.05) that was significantly higher. Moreover, three out of four deceased patients presented levels of CD40L higher than the median. At admission, STEMI showed a reduced ejection fraction (EF, p<0.01) and increased wall motion score index (WMSI, p<0.001) and end-diastolic volume (EDV, p<0.05) vs NSTEMI. An inverse correlation between admission values of inflammatory markers (SAA and WBC) and cardiac function was observed (p<0.05). Moreover, the necrosis marker troponin I was positively correlated with both WMSI (p<0.05) and hs-CRP (p<0.05). Regarding the inflammation kinetics, a difference was observed in the two groups only for WBC (p<0.05) and SAA (p<0.05). SAA showed higher values in STEMI at t0 and t1. In both groups, TGF-beta had an increase at t1 and t2 with respect to admission, while IL-6 had a decreasing trend. The total incidence of major adverse clinical events (MACE) was 22.5% at t2, with a mortality rate of 10%. CONCLUSION These observations suggest a differential inflammatory pattern in STEMI and NSTEMI patients. The absence of significant correlations between inflammatory indexes and myocardial infarction in NSTEMI supports the hypothesis that a different pattern of inflammation occurs in these patients. CD40L may have an important role as a marker for risk stratification in patients with ACS.

Fibrin acts as biomimetic niche inducing both differentiation and stem cell marker expression of early human endothelial progenitor cells

Objectives:  Transplantation of endothelial progenitor cells (EPCs) is a promising approach for revascularization of tissue. We have used a natural and biocompatible biopolymer, fibrin, to induce cell population growth, differentiation and functional activity of EPCs.

Role of NF-κB and PPAR-γ in lung inflammation induced by monocyte-derived microparticles

Microparticles (MP) are phospholipid vesicles shed by cells upon activation or apoptosis. Monocyte-derived MP upregulate the synthesis of proinflammatory mediators by lung epithelial cells; the molecular bases of such activity are unknown. Peroxisome proliferator-activated receptors (PPAR) have been demonstrated to be involved in the modulation of nuclear factor (NF)-&kgr;B transcriptional activity and inflammation. We investigated whether the upregulation of the synthesis of proinflammatory cytokines by human lung epithelial cells induced by monocyte/macrophage-derived MP involves NF-&kgr;B activation and is modulated by PPAR-&ggr;. MP were generated by stimulation of human monocytes/macrophages with the calcium ionophore, A23187. MP were incubated with human lung epithelial cells. NF-&kgr;B translocation was assessed by electrophoretic mobility shift assay. Interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1 synthesis was assessed by ELISA and RT-PCR. Stimulation of A549 alveolar cells with monocyte/macrophage-derived MP caused an increase in NF-&kgr;B activation and IL-8 and MCP-1 synthesis that was inhibited by pre-incubation with the PPAR-&ggr; agonists, rosiglitazone and 15-deoxy-&Dgr;12,14-prostaglandin-J2. Parallel experiments with normal human bronchial epithelial cells largely confirmed the results. The effects of PPAR-&ggr; agonists were reversed by the specific antagonist, GW9662. Upregulation of the synthesis of proinflammatory mediators by human lung epithelial cells induced by monocyte/macrophage-derived MP is mediated by NF-&kgr;B activation through a PPAR-&ggr; dependent pathway.

Procoagulant, Tissue Factor-Bearing Microparticles in Bronchoalveolar Lavage of Interstitial Lung Disease Patients: An Observational Study

Coagulation factor Xa appears involved in the pathogenesis of pulmonary fibrosis. Through its interaction with protease activated receptor-1, this protease signals myofibroblast differentiation in lung fibroblasts. Although fibrogenic stimuli induce factor X synthesis by alveolar cells, the mechanisms of local posttranslational factor X activation are not fully understood. Cell-derived microparticles are submicron vesicles involved in different physiological processes, including blood coagulation; they potentially activate factor X due to the exposure on their outer membrane of both phosphatidylserine and tissue factor. We postulated a role for procoagulant microparticles in the pathogenesis of interstitial lung diseases. Nineteen patients with interstitial lung diseases and 11 controls were studied. All subjects underwent bronchoalveolar lavage; interstitial lung disease patients also underwent pulmonary function tests and high resolution CT scan. Microparticles were enumerated in the bronchoalveolar lavage fluid with a solid-phase assay based on thrombin generation. Microparticles were also tested for tissue factor activity. In vitro shedding of microparticles upon incubation with H2O2 was assessed in the human alveolar cell line, A549 and in normal bronchial epithelial cells. Tissue factor synthesis was quantitated by real-time PCR. Total microparticle number and microparticle-associated tissue factor activity were increased in interstitial lung disease patients compared to controls (84±8 vs. 39±3 nM phosphatidylserine; 293±37 vs. 105±21 arbitrary units of tissue factor activity; mean±SEM; p<.05 for both comparisons). Microparticle-bound tissue factor activity was inversely correlated with lung function as assessed by both diffusion capacity and forced vital capacity (r2 = .27 and .31, respectively; p<.05 for both correlations). Exposure of lung epithelial cells to H2O2 caused an increase in microparticle-bound tissue factor without affecting tissue factor mRNA. Procoagulant microparticles are increased in interstitial lung diseases and correlate with functional impairment. These structures might contribute to the activation of factor X and to the factor Xa-mediated fibrotic response in lung injury.

Different growth conditions for peripheral blood endothelial progenitors

PURPOSE To compare different growth conditions for endothelial progenitor cells (EPCs) from peripheral blood mononuclear cells (PBMNCs). METHODS AND MATERIALS PBMNCs of healthy volunteers were cultured on fibronectin as follows: M199 with VEGF, bFGF, IGF-I; the same medium with bovine retina-derived extract (RDE); freshly isolated or depleted of adherent cells PBMNCs in HUVEC conditioned medium; DiI-stained PBMNCs with HUVECs (1:4 ratio) in Ml99 with RDE. PBMNCs were analysed by FACS using mAbs for endothelial markers. EPCs migration was determined using a modified Boyden chamber assay and VEGF as chemoattractant. EPCs were seeded alone or with HUVECs on Matrigel to assess in vitro angiogenesis. RESULTS With growth factors, numerous cell clusters appeared within 1 week. Spindle-shaped and attached cells sprouted, differentiating in endothelial cell (EC)-like cells within 2 weeks and forming cobblestone-like monolayers within 3 weeks. With RDE, numerous large cell clusters appeared within 1 week, but the number of cells with an EC morphology decreased during culture. FACS confirmed the endothelial phenotype and attached cells were able to migrate in response to VEGF. When nonadherent cells were cultured in HUVEC conditioned medium, they proliferated readily and EPCs were induced while freshly isolated cells neither proliferated nor induced EPCs. FACS analysis of the cocultures showed the presence of double-labeled PBMNCs expressing endothelial antigens. Capillary-like structures were observed on Matrigel only from cocultures and PBMNCs were able to incorporate in these networks. CONCLUSIONS PBMNCs are able to differentiate in EPCs when stimulated with appropriate culture conditions (growth factors, HUVEC conditioned medium, HUVECs).

The mechanisms of nadroparin-mediated inhibition of proliferation of two human lung cancer cell lines.

Clinical data suggest that heparin treatment improves survival of lung cancer patients, but the mechanisms involved are not fully understood. We investigated whether low molecular weight heparin nadroparin, directly affects lung cancer cell population growth in conventionally cultured cell lines.

Human peripheral blood endothelial progenitor cells synthesize and express functionally active tissue factor

INTRODUCTION Endothelial progenitor cells are circulating cells able to home to sites of vascular damage and to contribute to the revascularization of ischemic areas. We evaluated whether endothelial progenitor cells synthesize tissue factor, a procoagulant protein also involved in angiogenesis. MATERIALS AND METHODS Endothelial progenitor cells were obtained from the peripheral blood mononuclear fraction of normal donors and cultured in endothelial medium supplemented with specific growth factors. The procoagulant activity expressed by cells disrupted by freeze-thaw cycles was assessed by a one stage clotting assay. Tissue factor mRNA expression was evaluated by RT-PCR. RESULTS Endothelial progenitor cells do not express procoagulant activity in baseline conditions. However, lipopolysaccharide induces the expression of procoagulant activity. The effect is dose-dependent and reaches statistical significance at 100 ng/mL lipopolysaccharide. Inhibition with an anti-tissue factor antibody and amplification of cDNA with primers based on the tissue factor sequence confirm the identity of this activity with tissue factor. The kinetics of tissue factor expression by endothelial progenitor cells is identical to that of human umbilical vein endothelial cells showing maximal activity within 4 hours, and then decreasing; in contrast, tissue factor expression by mononuclear cells lasts for longer times. Both 5,6-dichloro-beta D-ribofuranosyl-benzimidazole and cycloheximide prevented the expression of procoagulant activity. Stimulation of endothelial progenitor cells with tumor necrosis factor-alpha did not elicit any detectable procoagulant activity. CONCLUSIONS Endothelial progenitor cells can be stimulated by lipopolysaccharide to synthesize tissue factor. This protein might be involved in thrombotic phenomena and might contribute to endothelial progenitor cells related neovascularization.

HAS-1 genetic polymorphism in sporadic abdominal aortic aneurysm

The hyaluronan synthase 1 (HAS-1) gene encodes a plasma membrane protein that synthesizes hyaluronan (HA), an extracellular matrix molecule. Accumulating evidence emphasizes the relevance of HA metabolism in an increasing number of processes of clinical interest, including abdominal aortic aneurysm (AAA). The existence of aberrant splicing variants of the HAS-1 gene could partly explain the altered extracellular matrix architecture and influence various biological functions, resulting in progressive arterial wall failure in the development of AAA. In the present study, we assessed the hypothesis that HAS-1 genetic 833A/G polymorphism could be associated with the risk of AAA by performing a case-control association study, involving AAA patients and healthy matched donors.

Endothelial progenitor cells induction by short-term stimulation with phytohaemagglutinin of mononuclear cells

BACKGROUND The identification of circulating endothelial progenitors cells (EPCs) in the adult has forced to reconsider how new blood vessels grow in physiological and pathological conditions. Neovascularization during adult life has long been attributed to angiogenesis only. However, recent studies have revealed that peripheral blood EPCs may be recruited and incorporated into sites of active neovascularization. PURPOSE To verify that EPCs are induced from peripheral blood mononuclear cells (PBMCs) and bone marrow derived mononuclear cells (BMMCs) upon short-term stimulation with phytohaemoagglutinin (PHA), a potent T-cell mitogen. METHODS PBMCs and BMMCs were isolated from healthy donors. Freshly isolated or depleted of adherent cells (one day and three days of adherence) mononuclear cells (MCs) were cultured in RPMI, 10% FBS, containing PHA (10 microl/10(6) cells) for 24 h. After stimulation with PHA, clusters of adherent cells were further propagated in M199 containing L-glutammine, Hepes, 20% FBS, heparin, antibiotics and bovine retina extract for 1 and 2 weeks. PBMCs and BMMCs cultured without PHA stimulation served as controls. FACS of EPCs was performed on attached cells after 7 and 14 days of culture. RESULTS AND CONCLUSION After stimulation of MCs with PHA for 24 h, many cells clusters were observed and around these clusters some adherent EC-like cells were observed. These cells were ovoid but a very little of these were elongated in morphology, however their number and size gradually increased during culture. However a longer time was needed for obtaining EPCs from MCs harvested after adherence. Thus this indicates that short-term signals provided by PHA must be sufficient for MCs to express the ligands necessary for the induction of EPCs but signals from monocytes/macrophages are important for a more rapid differentiation.