Moderate Increase of Indoxyl Sulfate Promotes Monocyte Transition into Profibrotic Macrophages.

OBJECTIVE: The uremic toxin Indoxyl-3-sulphate (IS), a ligand of Aryl hydrocarbon Receptor (AhR), raises in blood during early renal dysfunction as a consequence of tubular damage, which may be present even when eGFR is normal or only moderately reduced, and promotes cardiovascular damage and monocyte-macrophage activation. We previously found that patients with abdominal aortic aneurysms (AAAs) have higher CD14+CD16+ monocyte frequency and prevalence of moderate chronic kidney disease (CKD) than age-matched control subjects. Here we aimed to evaluate the IS levels in plasma from AAA patients and to investigate in vitro the effects of IS concentrations corresponding to mild-to-moderate CKD on monocyte polarization and macrophage differentiation. METHODS: Free IS plasma levels, monocyte subsets and laboratory parameters were evaluated on blood from AAA patients and eGFR-matched controls. THP-1 monocytes, treated with IS 1, 10, 20 µM were evaluated for CD163 expression, AhR signaling and then induced to differentiate into macrophages by PMA. Their phenotype was evaluated both at the stage of semi-differentiated and fully differentiated macrophages. AAA and control sera were similarly used to treat THP-1 monocytes and the resulting macrophage phenotype was analyzed. RESULTS: IS plasma concentration correlated positively with CD14+CD16+ monocytes and was increased in AAA patients. In THP-1 cells, IS promoted CD163 expression and transition to macrophages with hallmarks of classical (IL-6, CCL2, COX2) and alternative phenotype (IL-10, PPARγ, TGF-ß, TIMP-1), via AhR/Nrf2 activation. Analogously, AAA sera induced differentiation of macrophages with enhanced IL-6, MCP1, TGF-ß, PPARγ and TIMP-1 expression. CONCLUSION: IS skews monocyte differentiation toward low-inflammatory, profibrotic macrophages and may contribute to sustain chronic inflammation and maladaptive vascular remodeling.

Effects of Pleiotrophin on endothelial and inflammatory cells: Pro-angiogenic and anti-inflammatory properties and potential role for vascular bio-prosthesis endothelialization.

PURPOSE: One of the limitations emerged with both synthetic and degradable vascular grafts is the lack of endothelialization after implantation that is known to be the main reason leading to unfavourable outcomes. It emerges the need to find new strategies to promote a rapid endothelialization of the scaffold. Pleiotrophin is a growth/differentiation cytokine for various cell type. We here evaluated the effect of Pleiotrophin on endothelial cells (EC), monocytes and macrophages that have been shown as key cells promoting neovascularization. MATERIAL/METHODS: EA.hy926 endothelial cells, THP-1 monocytes and PMA-differentiated macrophages were treated with Pleiotrophin (10 and 100ng/ml). VEGF, Flk-1, Nrp-1, COX-2, ICAM-1 and TGFß expression were detected by Western Blot, IL-10, MCP-1 and TNFα levels by ELISA. Chemotaxis was performed in Boyden chambers. Wound healing was performed by scratch wound assay. RESULTS: Pleiotrophin induces in EC the expression of VEGF and its receptors Flk-1 and Nrp-1 and improves the migratory capacity. In THP-1 monocytes, Pleiotrophin induces the expression of VEGF and its receptor Nrp-1 and decreases the levels of COX-2 and TNFα. In PMA-differentiated macrophages COX-2 expression was significantly reduced by Pleiotrophin, while IL-10 and TGFß were increased. CONCLUSIONS: Pleiotrophin acts as an angiogenesis 'driver' by promoting the creation of a pro-angiogenic environment, a migratory behaviour in EC and a pro-regenerative alternative phenotype in macrophages. Our results suggest that Pleiotrophin might be considered for vascular prosthesis engineering.

Simultaneous ultrasound-assisted water extraction and ß-cyclodextrin encapsulation of polyphenols from Mangifera indica stem bark in counteracting TNFα-induced endothelial dysfunction.

This study proposes an alternative technique to prevent heat degradation induced by classic procedures of bioactive compound extraction, comparing classical maceration/decoction in hot water of polyphenols from Mango (Mangifera indica L.) (MI) with ultrasound-assisted extraction (UAE) in a water solution of ß-cyclodextrin (ß-CD) at room temperature and testing their biological activity on TNFα-induced endothelial dysfunction. Both extracts counteracted TNFα effects on EAhy926 cells, down-modulating interleukin-6, interleukin-8, cyclooxygenase-2 and intracellular adhesion molecule-1, while increasing endothelial nitric oxide synthase levels. ß-CD extract showed higher efficacy in improving endothelial function. These effects were abolished after pre-treatment with the oestrogen receptor inhibitor ICI1182,780. Moreover, the ß-CD extract induced Akt activation and completely abolished the TNFα-induced p38MAPK phosphorylation. UAE and ß-CD encapsulation provide an efficient extraction protocol that increases polyphenol bioavailability. Polyphenols from MI play a protective role on endothelial cells and may be further considered as oestrogen-like molecules with vascular protective properties.

Telomere shortening and increased oxidative stress are restricted to venous tissue in patients with varicose veins: A merely local disease?

Shortened telomere length (TL) and oxidative stress have been described in several vascular disorders at both the tissue and circulating level. However, to our knowledge, there are no reports about TL associated with varicose vein (VV) disease. This paper aimed to evaluate, at the tissue and circulating level, TL and oxidative stress in VV disease, compared to the corresponding counterparts from abdominal aortic aneurysm (AAA) patients and control healthy subjects. TL was measured using quantitative fluorescence in situ hybridization (Q-FISH). Oxidative stress was evaluated by measuring the malondialdehyde (MDA) concentration by thiobarbituric acid reactive substance/s (TBARS) assay. At the vascular tissue level, VV patients had shortened TL and a high MDA concentration, similarly to AAA patients. Conversely, blood lymphocytes and epidermal cells from VV patients had a TL similar to healthy controls and significantly longer than the same cells from AAA patients. Moreover, the MDA concentration in plasma from VV patients was significantly lower than from the AAA group. Linear regression analysis showed a statistically significant inverse correlation between the blood lymphocyte TL and plasma MDA level. Our results suggest that, unlike AAA, telomere attrition in VV tissue is not a systemic phenomenon but it may be attributable to tissue microenvironment conditions and possibly to high local oxidative stress.

Estrogen receptor activation protects against TNF-α-induced endothelial dysfunction.

Lack of estrogen is a cause of cardiovascular disease in men and postmenopausal women. We examined the effects of estrogen receptor (ERs) activation/inactivation on endothelial cells subjected to tumor necrosis factor (TNF) α, which is involved in vascular disease pathogenesis. Endothelial nitric oxide synthase (eNOS) and matrix metalloproteinases (MMP) 9 expression, as well as protein kinase B (PKB) activation were evaluated as markers of endothelial dysfunction. The TNF-α induces eNOS and MMP-9 expression and PKB activation. The ER activation by apigenin, a nonsteroidal compound with estrogen-like activity mediated through ER binding-dependent pathways, counteracts these effects. These effects are reversed by classic (ER-α and ER-ß) and nonclassic (G-protein-coupled receptor) ER inhibitors (ICI182 780 and pertussis toxin, respectively). Our data suggest that ER activation counteracts endothelial dysfunction induced by TNF-α. The use of ER activators, such as apigenin, may represent a strategy to prevent vascular disease associated with endothelial dysfunction, while avoiding the feminizing effects of estrogens.

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.

Circulating fibrocytes prepare the lung for cancer metastasis by recruiting Ly-6C+ monocytes via CCL2.

Fibrocytes are circulating, hematopoietic cells that express CD45 and Col1a1. They contribute to wound healing and several fibrosing disorders by mechanisms that are poorly understood. In this report, we demonstrate that fibrocytes predispose the lung to B16-F10 metastasis by recruiting Ly-6C(+) monocytes. To do so, we isolated fibrocytes expressing CD45, CD11b, CD13, and Col1a1 from the lungs of wild type (WT) and Ccr5(-/-) mice. WT but not Ccr5(-/-) fibrocytes increased the number of metastatic foci when injected into Ccr5(-/-) mice (73 ± 2 versus 32 ± 5; p < 0.001). This process was MMP9 dependent. Injection of WT enhanced GFP(+) fibrocytes also increased the number of Gr-1(Int), CD11b(+), and enhanced GFP(-) monocytes. Like premetastatic-niche monocytes, these recruited cells expressed Ly-6C, CD117, and CD45. The transfer of these cells into Ccr5(-/-) mice enhanced metastasis (90 ± 8 foci) compared with B cells (27 ± 2), immature dendritic cells (31 ± 6), or alveolar macrophages (28 ± 3; p < 0.05). WT and Ccl2(-/-) fibrocytes also stimulated Ccl2 expression in the lung by 2.07 ± 0.05- and 2.78 ± 0.36-fold compared with Ccr5(-/-) fibrocytes (1.0 ± 0.06; p < 0.05). Furthermore, WT fibrocytes did not increase Ly-6C(+) monocytes in Ccr2(-/-) mice and did not promote metastasis in either Ccr2(-/-) or Ccl2(-/-) mice. These data support our hypothesis that fibrocytes contribute to premetastatic conditioning by recruiting Ly-6C(+) monocytes in a chemokine-dependent process. This work links metastatic risk to conditions that mobilize fibrocytes, such as inflammation and wound repair.

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 CD14(dim)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(+), CD14(dim)CD16(+) monocytes and monocyte CD143 expression in AAA patients, and their relationship with Ddimer, eGFR and other inflammatory parameters. Blood from 74 AAA patients and 30 healthy controls was analyzed to determine the frequency of CD14(+)CD16(+), CD14(dim)CD16(+) monocytes and the monocyte CD143 expression by means of flow-cytometry. AAA patients had expanded CD16+ subsets (CD14(+)CD16(+): 7.66 ± 0.31% vs 5.42 ± 0.27%; CD14(dim)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. CD14(dim)CD16(+) cells were associated to uric acid, surface CD143, and reduced count of total leukocytes and neutrophils. Within AAA patients, the two CD16(+) subsets and the monocyte CD143 expression display different relationships with D-dimer, parameters of renal function and circulating biochemical and inflammatory biomarkers.

Apigenin inhibits the TNFα-induced expression of eNOS and MMP-9 via modulating Akt signalling through oestrogen receptor engagement.

Apigenin is a naturally occurring plant flavone with strong anti-oxidant and anti-inflammatory activity. While the anticancer properties of Apigenin have been extensively studied, little is known about its effects on endothelial dysfunction. We investigated the effects of Apigenin in EAhy926 endothelial cells exposed to TNFα by evaluating the expression of eNOS and MMP-9, two key molecules in endothelial dysfunction. MMP-9 activity was measured by gel zymography. Western blot analysis was performed to analyze eNOS expression and signal transduction. Treatment with Apigenin (50 µM) counteracted the TNFα-induced expression of eNOS and MMP-9 and the TNFα- triggered activation of Akt, p38MAPK and JNK signalling suggesting that multiple signalling pathways are involved in mediating the protective effects of Apigenin on endothelial function. To better understand the molecular mechanisms underlying the protective effects of Apigenin, we used a pharmacological approach with specific inhibitors. The use of an Akt inhibitor mimicked the inhibitory effects of Apigenin on eNOS and MMP-9 expression, suggesting that eNOS and MMP-9 induction by TNFα depends on Akt activation. The TNFα-induced expression of MMP-9 was also affected by the JNK inhibitor SP600125. No effect on eNOS and MMP-9 expression was observed in the presence of the p38MAPK inhibitor SB203580 or the ERK 1/2 inhibitor PD98059. Pretreatment with 'classic' (ERα and ERß) or 'non classic' (GPR30) oestrogen receptor (ER) inhibitors (ICI182,780 and PTX, respectively) counteracted the ability of Apigenin to decrease the TNFα-triggered activation of the Akt pathway. Consistently, the use of both ER inhibitors reversed the inhibitory effects of Apigenin on the TNFα-induced expression of eNOS and, to a lesser extent, MMP-9. We can conclude that Apigenin exerts its inhibitory effect on the TNFα-induced expression of eNOS and MMP-9 through the Akt signalling inhibition generated by ER activation. Oestrogen signalling has been implicated in protection from cardiovascular disease. Therefore, having regard to its ability to bind to ERs, Apigenin may be considered an oestrogen-like molecule to potentially be used against the onset and progression of vascular diseases associated with endothelial dysfunction.

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.

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 µg(TE) L(-1) and 15.80 ± 0.62 µg(DPPH) µL(extract) (-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).

Soluble form of the endothelial adhesion molecule CD146 binds preferentially CD16+ monocytes.

The cell adhesion molecule CD146 is normally located at the endothelial cell-to-cell junction and colocalizes with actin cytoskeleton. The soluble form of CD146 (sCD146) has been identified in the endothelial cell supernatant and in normal human plasma, and is increased in pathologic conditions with altered endothelial function. Soluble CD146 binding to monocytes promotes their transendothelial migration, which represents a central step in the development of atherosclerotic plaque. Since peripheral blood monocytes are characterized by a phenotypic and functional heterogeneity, with different transendothelial migration capacity, we hypothesized that monocyte subsets differently bind sCD146. Based on surface CD14 and CD16 expression monocytes were distinguished by flow cytometry (FACS) into three subsets: CD14++/CD16-, CD14++/CD16+ and CD14+/CD16+. CD16+ monocytes have been found to possess higher transendothelial migration ability. FACS analysis on blood monocytes from 30 healthy subjects revealed that higher percentages of CD14++/CD16+ (median, first and third quartile: 2.26, 1.62-3.87) and of CD14+/CD16+ (2.59, 1.28-4.80) were positive for CD146 (both p < 0.01), in comparison to CD14++/CD16- (0.66, 0.47-1.01). Moreover, in vitro treatment of ficoll separated monocytes with recombinant CD146 showed that both CD16+ subsets increased their percentage of CD146-positive events compared to CD16- monocytes (p < 0.01). Soluble CD146 levels were evaluated by ELISA in plasma samples of subjects from our study group and showed a correlation with percentage of CD146-positive CD14+/CD16+ monocyte subset. In this work we have demonstrated that monocyte subsets behave differently with regard to their sCD146 binding activity; because binding of CD146 influences transendothelial migration of monocytes, modulation of monocyte-CD146 interaction may represent a potential target to limit atherosclerotic plaque development.

Direct relationship between cell density and FDG uptake in asymptomatic aortic aneurysm close to surgical threshold: an in vivo and in vitro study.

PURPOSE: Conflicting results have been reported about the clinical value of fluorodeoxyglucose (FDG) imaging in predicting the risk of rupture of abdominal aortic aneurysm (AAA). The present study tests the hypothesis that FDG uptake is low in asymptomatic noninflammatory AAA due to the low cell density in aneurysmal walls. METHODS: Positron emission tomography (PET)/CT imaging was performed in 12 consecutive candidates for AAA surgical repair and in 12 age- and sex-matched controls. At intervention, aneurysmal walls were cut into three sequential blocks. Block A was frozen to cut three 5-µm slices for incubation with 2-3 MBq of FDG for 5 min. Block C was first incubated with the same tracer solution for the same time and subsequently frozen to cut three 5-µm slices. Autoradiographic images were coregistered with immunohistochemical pictures of cell density, type and DNA synthesis as assessed on block B. RESULTS: No visible uptake in abdominal aorta occurred in any patient or control subject. Immunohistochemistry documented a severe loss of wall structure, with low numbers of cells. Tracer retention directly correlated with overall cell density and with prevalence of cells synthesizing DNA. The metabolic nature of FDG uptake was confirmed by the selective effect of preliminary freezing that decreased tracer content by 90% in regions with high cell density and only by 34% in cold acellular areas. CONCLUSION: The loss of tissue structure and the marked decrease in cell density account for the low prevalence of positive findings at FDG PET imaging, at least in asymptomatic patients bearing AAAs whose diameter is close to surgical indication.

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.

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

BACKGROUND: We 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. METHODS: We 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). RESULTS: We 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. CONCLUSIONS: HU 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.

The carboxyl terminal trimer of procollagen I induces pro-metastatic changes and vascularization in breast cancer cells xenografts.

BACKGROUND: The COOH terminal peptide of Pro-collagen type I (PICP, also called C3) is chemotactic for endothelial melanoma and breast cancer cells. PICP induces the expression of Metalloproteinases-2 and -9, of Vascular endothelial growth factor and of the chemokine CXCL-12 receptor CXCR4 in MDA MB231 breast carcinoma cells in vitro. METHODS: We used a model of xenografts in BalbC/nude mice obtaining tumors by implanting in contro-lateral subcutaneous positions MDA MB231 cells added or not with purified PICP and studied the earlier phases of tumor development, up to 48 days from implant, by histology, immunostain and in situ hybridization. RESULTS: Addition of PICP promotes rapid vascularization of the tumors while does not affect mitotic and apoptotic indexes and overall tumor growth. PICP-treated, relative to control tumors, show up-modulation of Vascular endothelial factor, Metalloproteinase-9 and CXCR4, all tumor prognostic genes; they also show down-modulation of the endogenous Metalloproteinase inhibitor, reversion-inducing-cysteine-rich protein with kazal motifs, and a different pattern of modulation of Tissue Inhibitor of Metalloproteinase-2. These changes occur in absence of detectable expression of CXCL-12, up to 38 days, in control and treated tumors. CONCLUSION: PICP has an early promoting effect in the acquisition by the tumors of prometastatic phenotype. PICP may be play a relevant role in the productive interactions between stroma and tumor cells by predisposing the tumor cells to respond to the proliferation stimuli ensuing the activation of signaling by engagement of CXCR4 by cytokines and by fostering their extravasion, due to the induction of increased vascular development.

Prevalence and long-term course of macro-aspartate aminotransferase in children.

OBJECTIVES: To investigate the prevalence, association with clinical conditions, and long-term course of macro-aspartate aminotransferase (macro-AST). STUDY DESIGN: Forty-four children with an isolated elevation of serum AST were screened for macro-AST with electrophoresis and % polyethylene glycol (PEG) precipitable activity (PPA). RESULTS: All children were healthy, except they had elevated AST values. Seventeen children (38.6%) were macro-AST-positive. They had higher AST values than the 27 children who were macro-AST-negative (P = .001). Values <67.1% PPA and >82.2% PPA were associated with a very low probability of being macro-AST-positive and macro-AST-negative, respectively. Thirty-eight children underwent clinical and laboratory follow-up (mean, 4.7 +/- 3.8; range, 1-16 years). All remained symptom-free. AST levels decreased significantly only in children who were macro-AST-negative (P = .006). Macroenzyme persisted in 6 of the 9 children who were macro-AST-positive after 6.0 +/- 4.1 years. CONCLUSIONS: Macro-AST was present in more than one-third of children with an isolated increase of AST levels. The lack of pathological correlates in a long period argues for the benign nature of this phenomenon in childhood. We suggest that our %PPA thresholds can be used as a screening test and that electrophoresis be reserved for confirming positive screen test results and cases in which %PPA levels are of intermediate discriminant accuracy.

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.

Malignancy after kidney transplantation: results of 400 patients from a single center.

BACKGROUND: Post-transplant malignancies (PTM) occur in a percentage as high as 50% in patients followed 20 yr and have become a main cause of mortality and are expected to be the first cause of death within the next 20 yr in kidney transplant recipients. PATIENTS AND METHODS: We analyzed the PTM incidence in our kidney transplant recipients, and its main risk factors. The records of 400 patients (min follow up = one yr) have been retrospectively reviewed and categorized into three groups: patients without any tumor, with a non-melanoma skin cancer and with a solid or hematologic cancer. A cancer-free multivariate survival study was performed stratified by age, sex, immunosuppressive therapy, time on dialysis, body mass index (BMI), smoke, diabetes and nephropathy. RESULTS: Thirty patients developed PTM: 12 non-melanoma skin cancer, three lymphomas and 15 solid malignancies (seven genitourinary, three lung, two breast, two gastrointestinal and one sarcoma). The mean age at diagnosis was 55 yr, with a mean time from transplant of 27 months. We observed six deaths and two graft losses. Non-melanoma skin cancer-free survival and the solid/hematologic cancer-free survival was 99.5% and 98.5% at one yr, and 95.2% and 94.6% at five yr, respectively. At univariate analysis, age and induction therapy were significant risk factors for both types of PTM, while only recipient age significantly increased the risk of all PTM, and anti CD25 significantly reduced the risk of non-melanoma skin cancer at the multivariate study. CONCLUSIONS: These data confirm the role of age and induction strategies in modulating the risk of neoplasia. To look for which strategies might reduce the PTM risk, including a personalized therapy to minimize the effects of chronic immunosuppressant, will be a crucial goal.