Daniela Palmieri

Resveratrol counteracts systemic and local inflammation involved in early abdominal aortic aneurysm development

BACKGROUND Monocyte activation, macrophage infiltration, vascular oxidative stress and matrix proteolysis are inflammatory key steps contributing to abdominal aortic aneurysm (AAA) development. A phenotypical and functional heterogeneity is recognizable in monocytes by the differential expression of surface molecules: CD62L- subset corresponds to activated monocytes, while CD143/ACE surface expression increases during their differentiation into macrophages. In this work, Resveratrol, which is an antioxidant polyphenol with vasoprotective properties, has been evaluated for its potential to limit aneurysm development and monocyte-dependent inflammatory response in a model of elastase-induced AAA. METHODS Male Sprague-Dawley rats received Resveratrol (10 mg/kg/die) (Rsv group, n=15) or vehicle (ethanol) alone (Et-OH group, n=15) continuously from 7 d before until 14 d after the AAA induction with elastase; five littermates were used as untreated control group (Ctr group, n=5). At the end of treatment, CD143 and CD62L monocyte expression was analyzed by flow cytometry, serum antioxidant capacity was evaluated using the TRAP method and circulating TNFα, and MMP-9 were measured with ELISA and gel zymography, respectively. Aortas were subjected to histology and immunohistochemistry for morphological analysis, macrophage infiltration, and MMP-9, TNFα, and VEGF expression. RESULTS Resveratrol counteracted the CD62L-monocyte subset expansion, CD143 monocyte expression, and circulating levels of MMP-9 activity and TNFα associated to AAA induction. Similarly, treatment with Resveratrol significantly attenuated AAA expansion, vessel wall macrophage infiltration and MMP-9, VEGF, and TNFα expression, compared with AAA from Et-OH group. CONCLUSIONS Resveratrol limited the monocyte-dependent inflammatory response, macrophage differentiation and aortic lumen enlargement in elastase-induced AAA. These data suggest that Resveratrol might be tested in selected patients with small AAA to modulate the early systemic and local inflammatory response associated to AAA progression.

Effects of polyphenol extract from olive pomace on anoxia-induced endothelial dysfunction.

Anoxia modulates the expression of molecules associated with endothelial dysfunction and vascular diseases. Polyphenols have potent antioxidant properties due to their ability to modulate genes involved in oxidative tissue damage. In this study, we investigated the effect of polyphenol extract from olive pomace (PEOP) and its main constituents, Tyrosol and Oleuropein, on endothelial cells subjected to anoxia by evaluating the expression of molecules critical for endothelial function, proliferation and migration, and the signaling pathway involved. EAhy926 human endothelial cells were exposed to anoxic stress in the presence or absence of PEOP. Anoxia increased the nitric oxide (NO) level and the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNFα). These effects were prevented by PEOP treatment in a dose-dependent manner. Moreover, PEOP prevented the proliferation and migration associated with anoxia in EAhy926 cells, down-regulated the levels of matrix metalloproteinase (MMP)-2, MMP-9 and membrane type-1 MMP (MT1-MMP) and increased tissue MMP inhibitor-1 (TIMP-1) expression. Purified Oleuropein or Tyrosol restored to a basal level the anoxia-induced expression of MMP-9 and partially of MMP-2. The expression of TNFα was reduced by both polyphenols in a dose-dependent manner, but more efficiently by Tyrosol. Conversely, Oleuropein and Tyrosol had no significant effects on iNOS, COX-2 and TIMP-1 expression when used at the concentration found in PEOP. PEOP induced a time-dependent phosphorylation of p38 MAPK and ERK1/2 and inhibited anoxia-induced NF-κB activation. PEOP treatment restores the endothelial functions that are impaired by anoxia by regulating the expression of genes involved in proteolysis, angiogenesis and inflammation more efficiently than the single purified components. Therefore, the combined use of polyphenols, as in PEOP, could represent a powerful tool for the treatment and chemoprevention of endothelial dysfunction-associated vascular diseases.

Trimer Carboxyl Propeptide of Collagen I Produced by Mature Osteoblasts Is Chemotactic for Endothelial Cells

During the second phase of osteogenesis in vitro, rat osteoblasts secrete inducer(s) of chemotaxis and chemoinvasion of endothelial and tumor cells. We report here the characterization and purification from mature osteoblast conditioned medium of the agent chemotactic for endothelial cells. The chemoactive conditioned medium specifically induces directional migration of endothelial cells, not affecting the expression and activation of gelatinases, cell proliferation, and scattering. Directional migration induced in endothelial cells by conditioned medium from osteoblasts is inhibited by pertussis toxin, by blocking antibodies to integrins α1, β1, and β3, and by antibodies to metalloproteinase 2 and 9. The biologically active purified protein has two sequences, coincident with the amino-terminal amino acids, respectively, of the α1 and of the α2 carboxyl propeptides of type I collagen, as physiologically produced by procollagen C proteinase. Antibodies to type I collagen and to the carboxyl terminus of α1or α2 chains inhibit chemotaxis. The chemoattractant is the propeptide trimer carboxyl-terminal to type I collagen, and its activity is lost upon reduction. These data illustrate a previously unknown function for the carboxyl-terminal trimer, possibly relevant in promoting endothelial cell migration and vascularization of tissues producing collagen type I.

Role of MT1-MMP in the osteogenic differentiation.

Metalloproteinase MT1-MMP is induced and Pro-MMP-2 up modulated early in rat preosteoblasts (ROB) set to differentiate. We here show that the induction of MMPs, accompanied by activation of Pro-MMP-2, occurs by 6 h of adhesion on endogenous extracellular matrix (ECM), Fibronectin (FN) and Collagen type I (CI). These events do not occur after adhesion on Collagen III (CIII), Vitronectin (VN) or BSA. Within the first hour on inducing substrata or plastic, FAK is unchanged and ERK(1,2), is activated, but this activation is not sufficient for MT1-MMP induction. The function of p38 MAPK and PTKs is not required for the induction by substrata of MMPs. Six hours after plating preosteoblasts on MMP-inducing substrata, complexes of beta1 integrin with MT1-MMP are formed, that contain integrin dimers specifically engaged by the substratum, alpha4 and alpha5 chains for cells plated on FN, and alpha2 chain for cells plated on CI and ECM. Induction of MT1-MMP and its expression during osteogenesis pleiotropically regulate alkaline phosphatase (AP) expression. During differentiation, variant clones derived from preosteoblasts and MMPs-over-expressing osteoblasts show high MT1-MMP level associated with high AP level both persisting in time, while inhibition of MMPs is accompanied by inhibition of AP. Up or down modulation of AP, transcriptionally or by inhibition of the enzyme activity, has no effect on level or timing of expression of MT1-MMP and Pro-MMP-2. The persistence in expression of MT1-MMP during differentiation, and the associated persistence in expression of AP, as well as their inhibition, both impair the formation of nodules and mineral deposition. A transient pattern of expression of MT1-MMP is required for the establishment of nodules, and MT1-MMP decrease is permissive for nodule mineralization. The expression of AP is required for nodule formation and its level modulates the mineralization. MT1-MMP has multiple functions and is implicated in multiple steps of the differentiation process, acting to regulate homeostasis of the osteogenic differentiation.

TNFα induces the expression of genes associated with endothelial dysfunction through p38MAPK-mediated down-regulation of miR-149.

MicroRNAs have been proposed as novel regulators of vascular inflammation and dysfunction. This study aimed to evaluate the role of miR-149 in regulating the expression of key molecules associated with TNFα-induced endothelial activation. miR-149 was selected by in silico analysis and microRNA target prediction. Endothelial dysfunction was induced by TNFα treatment in Eahy926 endothelial cells and HUVEC. miR-149 level was evaluated by quantitative real time-polymerase chain reaction (RT-qPCR). Metalloproteinase-9 (MMP-9) was measured by zymography, Inducible Nitric Oxide Synthase (iNOS) by immunoblotting, Interleukin-6 (IL-6) and Interleukin-8 (IL-8) by ELISA. miR-149 regulatory effect was evaluated by gain-of-function technique upon miR-149 mimics transfection. TNFα down-modulated miR-149 level in Eahy926 and HUVEC. This effect was significantly abolished in Eahy926 by treatment with p38MAPK inhibitor. miR-149 mimic transfection counteracted the TNFα-induced expression of MMP-9, iNOS and IL-6. No effect was detected on IL-8 expression. Our results suggest that miR-149 represents an important new regulator of endothelial function through negative regulation of molecules associated with TNFα-induced endothelial dysfunction.

CD16 + monocyte subsets are increased in large abdominal aortic aneurysms and are differentially related with circulating and cell-associated biochemical and inflammatory biomarkers

Proinflammatory components are present in abdominal aortic aneurysm (AAA). Circulating monocytes display heterogeneity, and three subsets have been identified, based on the differential expression for CD14 and CD16 receptors: CD14+CD16-, classical, CD14+CD16+, intermediate and CD14dim CD16+, non-classical monocytes. Increased proinflammatory CD16+ monocytes with high expression of CD143 are present in CKD patients. D-dimer is increased in AAA patients, and might contribute to the pro-inflammatory response associated to circulating monocytes. We aimed to investigate the frequency of CD14+CD16+, CD14dim CD16+ monocytes and monocyte CD143 expression in AAA patients, and their relationship with D-dimer, eGFR and other inflammatory parameters. Blood from 74 AAA patients and 30 healthy controls was analyzed to determine the frequency of CD14+, CD16+, CD14dim CD16+ monocytes and the monocyte CD143 expression by means of flow-cytometry. AAA patients had expanded CD16+ SUPsets (CD14+CD16+: 7.66 ± 0.31% vs 5.42 ± 0.27%; CD14dim CD16+: 7.43 ± 0.48% vs 5.54 ± 0.38%, AAA vs controls, mean ± SE, both p<0.05). CD14+ CD16+ cells were associated to D-dimer and age, and to reduced eGFR. CD14dim CD16+ cells were associated to uric acid, surface CD143, and reduced count of total leukocytes and neutrophils. Within AAA patients, the two CD16+ supsets and the monocyte CD143 expression display different relationships with D-dimer, parameters of renal function and circulating biochemical and inflammatory biomarkers.

Antioxidant activity and biological evaluation of olive pomace extract.

The antiradical power of the methanol extracts of olive pomace (Taggiasca cultivar) achieved by high-pressure–high-temperature reactor were investigated using ABTS•+ and DPPH• assays. The highest antioxidant activity was quantified at 90 min of contact time and 180°C of extraction temperature (64.19 ± 0.16 µgTE L−1 and 15.80 ± 0.62 µgDPPH µLextract −1). The extract with high-antioxidant power resulted to be effective to counteract key aspects of cellular oxidation sensitive mechanisms and inflammation associated to vascular diseases. A linear correlation (p < 0.05) between total polyphenol contents and antioxidant capacity was given by the ABTS•+ method (R 2 = 0.9184) and DPPH assay (R 2 = 0.7062).

Hind limb unloading of mice modulates gene expression at the protein and mRNA level in mesenchymal bone cells

BackgroundWe investigated the extent, modalities and reversibility of changes at cellular level in the expression of genes and proteins occurring upon Hind limb unloading (HU) in the tibiae of young C57BL/6J male mice. We focused on the effects of HU in chondrogenic, osteogenic, and marrow mesenchymal cells.MethodsWe analyzed for expression of genes and proteins at two time points after HU (7 and 14 days), and at 14 days after recovery from HU. Levels of mRNAs were tested by in situ hybridization. Protein levels were tested by immunohistochemistry. We studied genes involved in osteogenesis (alkaline phosphatase (AP), osteocalcin (OC), bonesialoprotein (BSP), membrane type1 matrix metalloproteinase (MT1-MMP)), in extracellular matrix (ECM) formation (procollagenases (BMP1), procollagenase enhancer proteins (PCOLCE)) and remodeling (metalloproteinase-9 (MMP9), RECK), and in bone homeostasis (Stro-1, CXCL12, CXCR4, CD146).ResultsWe report the following patterns and timing of changes in gene expression induced by HU: 1) transient or stable down modulations of differentiation-associated genes (AP, OC), genes of matrix formation, maturation and remodelling, (BMP1, PCOLCEs MMP9) in osteogenic, chondrogenic and bone marrow cells; 2) up modulation of MT1-MMP in these same cells, and uncoupling of its expression from that of AP; 3) transient down modulation of the osteoblast specific expression of BSP; 4) for genes involved in bone homeostasis, up modulation in bone marrow cells at distal epiphysis for CXCR4, down modulation of CXCL12, and transient increases in osteoblasts and marrow cells for Stro1. 14 days after limb reloading expression returned to control levels for most genes and proteins in most cell types, except AP in all cells, and CXCL12, only in bone marrow.ConclusionsHU induces the coordinated modulation of gene expression in different mesenchymal cell types and microenvironments of tibia. HU also induces specific patterns of expression for homeostasis related genes and modulation of mRNAs and proteins for ECM deposition, maturation and remodeling which may be key factors for bone maintenance.

Fibroblasts from FAD-linked presenilin 1 mutations display a normal unfolded protein response but overproduce Aβ42 in response to tunicamycin

Many patients affected by early onset familial Alzheimers disease (FAD), carry mutations in the presenilin 1 (PS1) gene. Since it has been suggested that FAD-linked PS1 mutations impair the unfolded protein response (UPR) due to endoplasmic reticulum (ER) stress, we analyzed the UPR and amyloid beta-protein processing in fibroblasts bearing various PS1 mutations. Neither in normal conditions nor after induction of ER stress with DTT or tunicamycin were the mRNA levels of UPR-responsive genes (BiP and PDI) significantly different in control and FAD fibroblasts. DTT, which blocked APP transport to the Golgi, caused a 30% decrease of secreted Abeta42 in wild type and PS1 mutant fibroblasts. In contrast, tunicamycin, which allowed exit of APP from the ER, increased secreted Abeta42 only in PS1 mutant fibroblasts. Our findings suggest that, although the UPR is active in fibroblasts from FAD patients, mutant PS1 may selectively increase Abeta42 secretion when N-glycosylation is impaired.

Pro-collagen I COOH-terminal Trimer Induces Directional Migration and Metalloproteinases in Breast Cancer Cells

Breast and prostatic carcinomas, melanoma, and endothelial cell lines are chemoattracted by medium conditioned by mature osteoblasts. The chemoattractant for endothelial cells was identified with C3, carboxyl-terminal trimer of pro-collagen type I. We report that C3 induces directional migration and proliferation, the expression of tissue inhibitor of metalloproteinases-2, pro-metalloproteinase-2 and –9, and their activation in MDA MB231 cells, without changing the expression of tissue inhibitor of metalloproteinases-1 and of metalloproteinase-14. Antiserum against metalloproteinase-2 or -9 or -14, tissue inhibitor of metalloproteinases-1, or GM6001 inhibits the C3-induced migration. Urokinase and its receptor are detected and unchanged upon exposure to C3. The antibody against urokinase or addition of plasminogen activator inhibitor inhibits migration. Blocking antibodies to integrins α2, α6, β1, and β3 inhibit chemotaxis and do not change urokinase and urokinase receptor expression. Blockage of α2, β1, and β3 integrins affect differently the induction by C3 of pro-metalloproteinase-2 and -9 and of tissue inhibitor of metalloproteinases-2. Chemotaxis to C3 is also inhibited by genistein, by pertussis toxin, which also inhibits C3-induced pro-metalloproteinase -2 and -9, but not urokinase expression. Wortmannin partially inhibits C3-induced cell migration. Other, but not all, breast carcinoma lines tested responded to C3 with migration and pro-metalloproteinase-2 induction. Presently C3 is the only agent known to induce migration specifically of both endothelial and breast carcinoma cells. The mitogenic and motogenic role of C3in vitro might prefigure a role in in vivo carcinogenesis and in the establishment of metastasis.