Erika Rimondi

Systemic Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Delivery Shows Antiatherosclerotic Activity in Apolipoprotein E-Null Diabetic Mice

Background— Although in vitro studies have suggested that tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) might be involved in vascular biology, its potential role in the pathogenesis and/or treatment of atherosclerosis has not been investigated. Methods and Results— Both recombinant human TRAIL and an adeno-associated virus vector expressing human TRAIL were used to deliver TRAIL in apolipoprotein E (apoE)–null mice in which diabetes mellitus was induced by destruction of islet cells with streptozotocin. Diabetes in apoE-null mice was associated with a significant increase in atherosclerotic plaque area and complexity in the aorta as assessed by a marked increase in interstitial collagen, cellular proliferation, and macrophage infiltration and a focal loss of endothelial coverage. Repeated intraperitoneal injections of recombinant human TRAIL and a single intravenous injection of adeno-associated virus–human TRAIL significantly attenuated the development of atherosclerotic plaques in apoE-null animals. TRAIL also markedly affected the cellular composition of plaque lesions by inducing apoptosis of infiltrating macrophages and increasing the vascular smooth muscle cell content. Moreover, TRAIL promoted the in vitro migration of cultured human aortic vascular smooth muscle cells but not of monocytes or macrophages. Conversely, TRAIL selectively induced apoptosis of human cultured macrophages but not of vascular smooth muscle cells. Conclusions— Overall, data from the present study indicate that atherosclerosis in diabetic apoE-null mice is ameliorated by systemic TRAIL administration and that adeno-associated virus–mediated TRAIL gene delivery might represent an innovative method for the therapy of diabetic vascular diseases.

Tumour necrosis factor-related apoptosis-inducing ligand sequentially activates pro-survival and pro-apoptotic pathways in SK-N-MC neuronal cells.

The SK‐N‐MC neuroblastoma cell line, which expresses surface tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) receptors TRAIL‐R2 and TRAIL‐R4, was used as a model system to examine the effect of TRAIL on key intracellular pathways involved in the control of neuronal cell survival and apoptosis. TRAIL induced distinct short‐term (1–60 min) and long‐term (3–24 h) effects on the protein kinase B (PKB)/Akt (Akt), extracellular signal‐regulated kinase (ERK), cAMP response element‐binding protein (CREB), nuclear factor kappa B (NF‐κB) and caspase pathways. TRAIL rapidly (from 20 min) induced the phosphorylation of Akt and ERK, but not of c‐Jun NH2‐terminal kinase (JNK). Moreover, TRAIL increased CREB phosphorylation and phospho‐CREB DNA binding activity in a phosphatidylinositol 3‐kinase (PI 3K)/Akt‐dependent manner. At later time points (from 3 to 6 h onwards) TRAIL induced a progressive degradation of inhibitor of κB (IκB)β and IκBε, but not IκBα, coupled to the nuclear translocation of NF‐κB and an increase in its DNA binding activity. In the same time frame, TRAIL started to activate caspase‐8 and caspase‐3, and to induce apoptosis. Remarkably, caspase‐dependent cleavage of NF‐κB family members as well as of Akt and CREB proteins, but not of ERK, became prominent at 24 h, a time point coincident with the peak of caspase‐dependent apoptosis.

GM-CSF exhibits anti-inflammatory activity on endothelial cells derived from chronic venous disease patients.

Twenty patients affected by chronic venous disease (CVD) in tertiary venous network and/or saphenous vein were analyzed before surgical ablation by echo-color-doppler for the hemodynamic parameters reflux time (RT) and resistance index (RI), a negative and a positive prognostic factor, respectively. RT and RI were next correlated with relevant in vitro parameters of venous endothelial cells (VEC) obtained from surgical specimens, such as cell migration in response to serum gradient, proliferation index, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression, as well as cytokines release. Of interest, ICAM-1 expression in patient-derived VEC cultures correlated positively with RT and negatively with RI. Moreover, RT showed a positive correlation with the baseline osteoprotegerin (OPG) expression by VEC and an inverse correlation with VEC proliferation index. On the other hand, RI correlated positively with TNF-related apoptosis inducing ligand (TRAIL) expression. Among the cytokines released by VEC, GM-CSF showed a positive correlation with VEC proliferation and TRAIL expression and a negative correlation with OPG, ICAM-1 and VCAM-1 expression. Since in vitro recombinant GM-CSF induced VEC proliferation and counteracted the induction of ICAM-1, VCAM-1 and OPG upon exposure to TNF-α, our data suggest an anti-inflammatory activity of GM-CSF on venous endothelial cells.

Perifosine selectively induces cell cycle block and modulates retinoblastoma and E2F1 protein levels in p53 mutated leukemic cell lines.

SummaryThe effect of the single-chain alkylphospholipid perifosine was analyzed in p53wild-type (SKW6.4, OCI and MOLM), p53mutated (BJAB, MAVER) and p53null (HL-60) leukemic cell lines. Perifosine promoted cytotoxicity with a combination of apoptosis induction in all cell lines and cell cycle block at the G2M checkpoint, which was selectively observed in p53mutated BJAB and MAVER cell lines. At the molecular level, perifosine induced hypophosphorylation of retinoblastoma protein and the degradation of E2F1 protein in p53mutated but not in p53wild-type cells. These data indicate that perifosine potentially represents an innovative therapeutic approach for p53mutated hematological malignancies.

Levels of TNF-Related Apoptosis-Inducing Ligand (TRAIL) Show a Long-term Stability in the Breast Milk of Mothers of Preterm Infants

Background: The immune modulator TNF-related apoptosis-inducing ligand (TRAIL) has been found at extremely high levels in human milk of women with normal gestation at day 5 after delivery. Objective: To investigate the presence and the levels of soluble TRAIL in human milk of women with preterm delivery at different time points post-partum (32, 34, and 36 weeks from conception). Methods: The levels of soluble TRAIL were analyzed by ELISA in the breast milk of a group of 25 women with preterm delivery at different gestational ages. Results: Soluble TRAIL was present at high levels in human milk since early post-conceptional ages (32 weeks). No significant differences in TRAIL levels were noticed with respect to different gestational ages, or with respect to time of collection when comparing, in a selected group of patients, samples obtained between 15 and 26 days with those obtained 27 and 40 days after birth. Conclusion: Due to the key immunoregulatory role of human soluble TRAIL, the presence of high levels of TRAIL in the milk of women with preterm delivery and its maintenance at high levels up to 72 days after birth support the importance of breastfeeding the preterm newborn.

Ibrutinib synergizes with MDM-2 inhibitors in promoting cytotoxicity in B chronic lymphocytic leukemia.

Objective The aim of this study was to investigate the anti-leukemic activity of the Bruton tyrosine kinase inhibitor Ibrutinib in combination with the small molecule MDM-2 inhibitor Nutlin-3 in preclinical models. Methods The potential efficacy of the Ibrutinib/Nutlin-3 combination was evaluated in vitro in a panel of B leukemic cell lines (EHEB, JVM-2, JVM-3, MEC-1, MEC-2) and in primary B-chronic lymphocytic leukemia (B-CLL) patient samples, by assessing cell viability, cell cycle profile, apoptosis and intracellular pathway modulations. Validation of the combination therapy was assessed in a B leukemic xenograft mouse model. Results Ibrutinib exhibited variable anti-leukemic activity in vitro and the combination with Nutlin-3 synergistically enhanced the induction of apoptosis independently from the p53 status. Indeed, the Ibrutinib/Nutlin-3 combination was effective in promoting cytotoxicity also in primary B-CLL samples carrying 17p13 deletion and/or TP53 mutations, already in therapy with Ibrutinib. Molecular analyses performed on both B-leukemic cell lines as well as on primary B-CLL samples, while confirming the switch-off of the MAPK and PI3K pro-survival pathways by Ibrutinib, indicated that the synergism of action with Nutlin-3 was independent by p53 pathway and was accompanied by the activation of the DNA damage cascade signaling through the phosphorylation of the histone protein H2A.X. This observation was confirmed also in the JVM-2 B leukemic xenograft mouse model. Conclusions Taken together, our data emphasize that the Ibrutinib/Nutlin-3 combination merits to be further evaluated as a therapeutic option for B-CLL.

Kinetic Profiles of Inflammatory Mediators in the Conjunctival Sac Fluid of Patients upon Photorefractive Keratectomy

Photorefractive keratectomy (PRK) represents a therapeutic option to remodel corneal stroma and to compensate refractive errors, which involves inflammatory and/or regenerative processes. In this context, the modulation of cytokines/chemokines in the conjunctival sac fluid and their role in the maintenance of the corneal microenvironment during the healing process upon refractive procedures has not been deeply investigated. In this study, serial samples of conjunctival sac fluid of patients (n = 25) undergoing PRK were harvested before and at different time points after surgery. The levels of 29 cytokines/chemokines/growth factors involved in inflammatory/immune processes were measured with a multiplex array system. The results have firstly highlighted the different pattern of cytokine expression between the microenvironment at the anterior surface of the eye and the systemic circulation. More importantly, the kinetic of modulation of cytokines/chemokines at the conjunctival level following PRK revealed that while the majority of cytokines/chemokines showed a significant decrease, MCP-1 emerged in light of its pronounced and significant increase soon after PRK and during the follow-up. This methodological approach has highlighted the role of MCP-1 in the healing process following PRK and has shown a potential for the identification of expression/modulation of soluble factors for biomarker profiling in ocular surface diseases.