Carlo Storelli

A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Their use in investigating some properties of D-glucose and choline transport systems.

We have worked out a simplification of the procedure described by Schmitz et al. (Biochim. Biophys. Acta (1973) 323, 98--112) for the preparation of brush border membranes from small intestine. The procedure ultimately adopted is simple, rapid, does not necessarily require scraping and can be started from fresh or frozen material. It can be scaled up easily, allowing a quick production of large amounts of brush border membrane vesicles. These vesicles prove to be excellently suited for transport studies, as suggested by our measurements of D-glucose transport. Using these vesicles, the mode of choline transport across the brush border membrane was also investigated. Choline transport was found to occur by a saturable component with a Km of 83 +/- 4 micrometer (at 20 degrees C) and by a non-saturable component. It is independent of the presence of Na+ and appears to be non-electrogenic.

Olive Oil and Red Wine Antioxidant Polyphenols Inhibit Endothelial Activation. Antiatherogenic Properties of Mediterranean Diet Phytochemicals

Objective—Epidemiology suggests that Mediterranean diets are associated with reduced risk of cardiovascular disease. Because monocyte adhesion to the endothelium is crucial in early atherogenesis, we evaluated whether typical olive oil and red wine polyphenols affect endothelial–leukocyte adhesion molecule expression and monocyte adhesion. Methods and Results—Phytochemicals in olive oil and red wine, including oleuropein, hydroxytyrosol, tyrosol, elenolic acid, and resveratrol, with or without antioxidant activity, were incubated with human umbilical vein endothelial cells for 30 minutes, followed by co-incubation with bacterial lipopolysaccharide or cytokines to trigger adhesion molecule expression. At nutritionally relevant concentrations, only oleuropein, hydroxytyrosol, and resveratrol, possessing a marked antioxidant activity, reduced monocytoid cell adhesion to stimulated endothelium, as well as vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein by Northern analysis and cell surface enzyme immunoassay. Reporter gene assays with deletional VCAM-1 promoter constructs indicated the relevance of nuclear factor-&kgr;B, activator protein-1, and possibly GATA binding sites in mediating VCAM-1 transcriptional inhibition. The involvement of nuclear factor-&kgr;B and activator protein-1 was finally demonstrated at electrophoretic mobility shift assays. Conclusions—Olive oil and red wine antioxidant polyphenols at nutritionally relevant concentrations transcriptionally inhibit endothelial adhesion molecule expression, thus partially explaining atheroprotection from Mediterranean diets.

Oleic Acid Inhibits Endothelial Activation: A Direct Vascular Antiatherogenic Mechanism of a Nutritional Component in the Mediterranean Diet

Because oleic acid is implicated in the antiatherogenic effects attributed to the Mediterranean diet, we investigated whether this fatty acid can modulate endothelial activation, ie, the concerted expression of gene products involved in leukocyte recruitment and early atherogenesis. We incubated sodium oleate with human umbilical vein endothelial cells for 0 to 72 hours, followed by coincubation of oleate with human recombinant tumor necrosis factor, interleukin (IL)-1alpha, IL-1beta, IL-4, Escherichia coli lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate for a further 6 to 24 hours. The endothelial expression of vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and intercellular adhesion molecule-1 was monitored by cell surface enzyme immunoassays or flow cytometry, and steady-state levels of VCAM-1 mRNA were assessed by Northern blot analysis. At 10 to 100 micromol/L for >24 hours, oleate inhibited the expression of all adhesion molecules tested. After a 72-hour incubation with oleate and a further 16-hour incubation with oleate plus 1 microg/mL LPS, VCAM-1 expression was reduced by >40% compared with control. Adhesion of monocytoid U937 cells to LPS-treated endothelial cells was reduced concomitantly. Oleate also produced a quantitatively similar reduction of VCAM-1 mRNA levels on Northern blot analysis and inhibited nuclear factor-kappaB activation on electrophoretic mobility shift assays. Incubation of endothelial cells with oleate for 72 hours decreased the relative proportions of saturated (palmitic and stearic) acids in total cell lipids and increased the proportions of oleate in total cell lipids without significantly changing the relative proportions of polyunsaturated fatty acids. Although less potent than polyunsaturated fatty acids in inhibiting endothelial activation, oleic acid may contribute to the prevention of atherogenesis through selective displacement of saturated fatty acids in cell membrane phospholipids and a consequent modulation of gene expression for molecules involved in monocyte recruitment.

Mediterranean diet polyphenols reduce inflammatory angiogenesis through MMP-9 and COX-2 inhibition in human vascular endothelial cells: a potentially protective mechanism in atherosclerotic vascular disease and cancer.

Diets with high content of antioxidant polyphenols are associated with low prevalence of cardiovascular diseases and cancer. Inflammatory angiogenesis is a key pathogenic process both in cancer and atherosclerosis, and is tightly regulated by the proinflammatory enzyme cyclooxygenase (COX)-2 and the matrix degrading enzymes matrix metalloproteinases (MMPs). We studied the effects of antioxidant polyphenols from virgin olive oil (oleuropein and hydroxytyrosol) and red wine (resveratrol and quercetin) on endothelial cell angiogenic response in vitro, and explored underlying mechanisms. Cultured endothelial cells were pre-incubated with 0.1-50 μmol/L polyphenols before stimulation with phorbol myristate acetate (PMA). All tested polyphenols reduced endothelial cell tube formation on matrigel and migration in wound healing assays. The reduced angiogenesis was associated with the inhibition of PMA-induced COX-2 protein expression and prostanoid production, as well as MMP-9 protein release and gelatinolytic activity. These effects were accompanied by a significant reduction in the stimulated intracellular reactive oxygen species levels and in the activation of the redox-sensitive transcription factor nuclear factor (NF)-κB. Our findings reveal that olive oil and red wine polyphenols reduce inflammatory angiogenesis in cultured endothelial cells, through MMP-9 and COX-2 inhibition, supporting a potential protective role for dietary polyphenols in atherosclerotic vascular disease and cancer.

D-glucose transport in decapod crustacean hepatopancreas.

Physiological mechanisms of gastrointestinal absorption of organic solutes among crustaceans remain severely underinvestigated, in spite of the considerable relevance of characterizing the routes of nutrient absorption for both nutritional purposes and formulation of balanced diets in aquaculture. Several lines of evidence attribute a primary absorptive role to the digestive gland (hepatopancreas) and a secondary role to the midgut (intestine). Among absorbed organic solutes, the importance of D-glucose in crustacean metabolism is paramount. Its plasma levels are finely tuned by hormones (crustacean hyperglycemic hormone, insulin-like peptides and insulin-like growth factors) and the function of certain organs (i.e. brain and muscle) largely depends on a balanced D-glucose supply. In the last few decades, D-glucose absorptive processes of the gastrointestinal tract of crustaceans have been described and transport mechanisms investigated, but not fully disclosed. We briefly review our present knowledge of D-glucose transport processes in the crustacean hepatopancreas. A discussion of previous results from experiments with hepatopancreatic epithelial brush-border membrane vesicles is presented. In addition, recent advances in our understandings of hepatopancreatic D-glucose transport are shown, as obtained (1) after isolation of purified R-, F-, B- and E-cell suspensions from the whole organ by centrifugal elutriation, and (2) by protein expression in hepatopancreatic mRNA-injected Xenopus laevis oocytes. In a perspective, the applicability of these novel methods to the study of hepatopancreatic absorptive function will certainly improve our knowledge of this structurally complex organ.

Molecular and functional characterisation of the zebrafish (Danio rerio) PEPT1-type peptide transporter1

We report the molecular and functional characterisation of a novel peptide transporter from zebrafish, orthologue to mammalian and avian PEPT1. Zebrafish PEPT1 is a low‐affinity/high‐capacity system. However, in contrast to higher vertebrate counterparts in which maximal transport activity is independent of extracellular pH, zebrafish PEPT1 maximal transport rates unexpectedly increase at alkaline extracellular pH. Zebrafish pept1 is highly expressed in the proximal intestine since day 4 post‐fertilisation, thus preceding functional maturation of the gut, first feeding and complete yolk resorption. Zebrafish PEPT1 might help to understand the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, zebrafish pept1 can be a useful marker for screening mutations that affect gut regionalisation, differentiation and morphogenesis.

Angiotensin II activates extracellular signal regulated kinases via protein kinase C and epidermal growth factor receptor in breast cancer cells.

Angiotensin II (Ang II) induces, through AT1, intracellular Ca2+ increase in both normal and cancerous breast cells in primary culture (Greco et al., 2002 Cell Calcium 2:1–10). We here show that Ang II stimulated, in a dose‐dependent manner, the 24 h‐proliferation of breast cancer cells in primary culture, induced translocation of protein kinase C (PKC)‐α, ‐β1/2, and δ (but not ‐ε, ‐η, ‐θ, ‐ζ, and ‐ι), and phosphorylated extracellular‐regulated kinases 1 and 2 (ERK1/2). The proliferative effects of Ang II were blocked by the AT1 antagonist, losartan. Also epidermal growth factor (EGF) had mitogenic effects on serum‐starved breast cancer cells since induced cell proliferation after 24 h and phosphorylation of ERK1/2. The Ang II‐induced proliferation of breast cancer cells was reduced by (a) Gö6976, an inhibitor of conventional PKC‐α and ‐β1, (b) AG1478, an inhibitor of the tyrosine kinase of the EGF receptor (EGFR), and (c) downregulation of 1,2‐diacylglycerol‐sensitive PKCs achieved by phorbol 12‐myristate 13‐acetate (PMA). A complete inhibition of the Ang II‐induced cell proliferation was achieved using the inhibitor of the mitogen activated protein kinase kinase (MAPKK or MEK), PD098059, or using Gö6976 together with AG1478. These results indicate that in human primary cultured breast cancer cells AT1 regulates mitogenic signaling pathways by two simultaneous mechanisms, one involving conventional PKCs and the other EGFR transactivation. J. Cell. Physiol. 196: 370–377, 2003.

Statins inhibit cyclooxygenase-2 and matrix metalloproteinase-9 in human endothelial cells: anti-angiogenic actions possibly contributing to plaque stability

AIMS Cyclooxygenase (COX)-2 expression is increased in inflammation and angiogenesis and also in atherosclerotic plaques, where it co-localizes with metalloproteinases (MMPs) involved in the fibrous cap weakening. Insight into the regulation of COX-2 and MMP-9 expression suggests the involvement of a Rho-dependent pathway. Because statins interfere with Rho activation, we investigated the statin effect on COX-2 and MMP expressions in the human endothelium. METHODS AND RESULTS Simvastatin and atorvastatin were incubated with endothelial cells for 12 h before stimulation with phorbol myristate acetate or tumour necrosis factor-alpha, for times suitable to assess the endothelial tube differentiation on Matrigel and COX-2 and MMPs activities, proteins, and mRNA expressions. At 0.1-10 micromol/L, both statins reduced COX-2 expression and activity, without affecting COX-1. The statin effect was reversed by mevalonate and geranylgeranyl-pyrophosphate and mimicked by the Rho inhibitor C3 transferase, indicating the involvement of Rho in the signal transduction pathway leading to COX-2 expression. In parallel, statins, as well as COX-2 inhibitors, reduced the MMP-9 stimulated release and the endothelial tubular differentiation. CONCLUSION In the human vascular endothelium, statins reduce COX-2 and MMP-9 expression and activity. Through this mechanism, statins exert an anti-angiogenic effect possibly contributing to the cholesterol-lowering-unrelated protective effects of statins against plaque inflammatory angiogenesis and rupture.

Reconstitution of a sucrase‐mediated sugar transport system in lipid membranes

Digestive diand oligosaccharidases are bound to, or are building blocks of, the brush border membrane of the enterocytes. This has been shown consistently by a variety of approaches, including the essentially complete recovery of some disaccharidase activities in isolated brush border membranes [l] and the immunochemical localisation by electron microscopy [2] . (For review, see [3] ). In 1970 Crane et al. [4,5] have investigated the intestinal uptake of glucose from combinations of free glucose and disaccharides. The presence of sucrose, maltose, lactose or trehalose in the medium produces an additional glucose uptake, even if the medium already contains enough free glucose to saturate completely the transport system(s) for the free monosaccharide. The extra glucose uptake from the disaccharides must be attributed to additional system(s) not accessible to free glucose. One notable characteristic of this (these) newly detected system(s) is that of not being Na-dependent. (The transport of free glucose across the brush border membrane of the small intestine is, of course, highly Na-dependent: for reviews see [6,7] ). We have now made lipid bilayers in the presence of the small intestine sucrase-isom~tase complex and observed a specific increase of the permeability of the bilayers for the sugars arising from sucrose.

Resveratrol inhibits the epidermal growth factor-induced epithelial mesenchymal transition in MCF-7 cells

Carcinoma progression is associated with the loss of epithelial features, and the acquisition of a mesenchymal phenotype by tumour cells. Herein we show that exposure of MCF-7 cells to epidermal growth factor (EGF) resulted in morphological alterations characteristic of epithelial-to-mesenchymal transition (EMT). EGF treatment resulted in increased motility along with an up-regulation of transcription factors Slug, Zeb1, Zeb2, and mesenchymal markers Vimentin and N-cadherin. Treatment of MCF-7 cells with a combined stimulation of EGF and resveratrol, a naturally occurring stilbene with antitumor properties, failed to alter cell morphology, motility and overexpression of EMT markers induced by EGF. Using specific chemical inhibitors, we demonstrated that EGF-induced EMT is mediated by extracellular signal-regulated kinase 1/2 (ERK 1/2) signalling pathway and that resveratrol is able to repress EGF-induced ERK activation. In summary, these data provide new evidence of the inhibitory effect of resveratrol on EGF-induced EMT cell transformation.