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Vascular endothelial growth factor (VEGF) triggers crucial signaling processes that regulate tumor angiogenesis and, therefore, represents an attractive target for the development of novel anticancer therapeutics. Several epidemiological studies have confirmed that abundant consumption of foods from plant origin is associated with reduced risk of developing cancers. In the Mediterranean basin, the consumption of extra virgin olive oil is an important constituent of the diet. Compared to other vegetable oils, the presence of several phenolic antioxidants in olive oil is believed to prevent the occurrence of a variety of pathological processes, such as cancer. While the strong antioxidant potential of these molecules is well characterized, their antiangiogenic activities remain unknown. The aim of this study is to investigate whether tyrosol (Tyr), hydroxytyrosol (HT), taxifolin (Tax), oleuropein (OL) and oleic acid (OA), five compounds contained in extra virgin olive oil, can affect in vitro angiogenesis. We found that HT, Tax and OA were the most potent angiogenesis inhibitors through their inhibitory effect on specific autophosphorylation sites of VEGFR-2 (Tyr951, Tyr1059, Tyr1175 and Tyr1214) leading to the inhibition of endothelial cell (EC) signaling. Inhibition of VEGFR-2 by these olive oil compounds significantly reduced VEGF-induced EC proliferation and migration as well as their morphogenic differentiation into capillary-like tubular structures in Matrigel. Our study demonstrates that HT, Tax and OA are novel and potent inhibitors of the VEGFR-2 signaling pathway. These findings emphasize the chemopreventive properties of olive oil and highlight the importance of nutrition in cancer prevention.

Although ischemia is the leading cause of acute renal failure in human, there is little information on the remodeling the kidney endothelium matrix during ischemic injury. In this study, we investigated the activity and expression of MMP-2 and MMP-9, in an isolated endothelial fraction following an acute in vivo reversible ischemia induced in rats by vascular clamping. Ischemia increased serum creatinine levels 1.4-fold, hallmark of acute renal failure. Isolation of the endothelial cell fraction was performed by affinity chromatography using an anti-PECAM-1 antibody. The isolated fraction was assessed by Western blotting analysis of endothelial cell markers. The positively selected fractions were enriched in the endothelial markers eNOS and PECAM-1 by 128-fold and 44-fold, respectively. Gelatin zymography showed that ischemia strongly stimulated proteolytic activity of proMMP-2 (1.8-fold), proMMP-9 (3-fold) and MMP-9 (4-fold) in the endothelial fractions. Western blot analysis indicated that TIMP-2 protein level increased by 3.2-fold in the endothelial fractions during ischemia. Surprisingly, TIMP-1 was absent from the endothelial preparations but was easily detected in the non-endothelial cells. Levels of the endocytic receptor LRP were increased by 2-fold during ischemia in the endothelial fractions. Occludin, a known in vivo MMP-9 substrate, was partly degraded in the endothelial fractions during ischemia, suggesting that the MMP-9 which was upregulated during ischemia was functional. These data suggest that ischemia in kidney could lead to the degradation of the vascular basement membrane and to increased permeability. This suggests new therapeutic approaches for ischemic pathologies by targeting MMP-9 and its regulators.

Protein l-isoaspartyl methyltransferase (PIMT) functions as a repair enzyme that acts upon damaged proteins bearing abnormal aspartyl residues. We previously reported that PIMT expression and activity are reduced by half in human epileptic hippocampus. Here we investigated PIMT regulation in astrocytic tumors, which are the most common human brain tumors. PIMT expression and enzyme activity were significantly decreased in all grades of human astrocytic tumors. More precisely, PIMT levels were significantly lower by 76% in pilocytic astrocytomas (grade I), 46% in astrocytomas (grade II), 69% in anaplastic astrocytomas (grade III), and a marked 80% in glioblastomas (grade IV) as compared to normal brains. RT-PCR analysis showed that levels of type I PIMT mRNA were up-regulated while those of type II PIMT mRNA were down-regulated in glioblastomas. Furthermore, the reduced PIMT levels correlated closely with a decrease in the number of neuron cells in astrocytic tumors as assessed by measuring the neuron-specific enolase level. Many proteins with abnormal aspartyl residues accumulated in brain tumors and some were specific to individual grades of astrocytic tumors. Similar results were obtained, either by measuring the reduction in PIMT activity and expression or by measuring the formation of abnormal proteins, in an orthotopic rat brain tumor model implanted with invasive CNS-1 glioma cells. The novelty of these findings was to provide the first evidence for a marked reduction of PIMT expression and activity during stage progression of astrocytic tumors in humans.

Background: The increased synthesis of matrix metalloproteinases (MMPs) by aortic smooth muscle cells (SMCs) is thought to be involved in the etiopathogenesis of abdominal aortic aneurysms (AAAs), but the functional regulation and the activation states of these MMPs remain unclear. In this study, we assessed the expression levels and the functional regulation of several MMPs in the pathogenesis of AAAs. Methods: Human healthy aorta and AAA specimens were homogenized, and the proteolytic activities of MMP-2 and MMP-9 and of the macrophage metalloelastase (MMP-12) were assessed with zymography. Protein expression of MMP-1, MMP-12, membrane-type 1 MMP (MT1-MMP), tissue inhibitor of MMP 1 (TIMP-1), TIMP-2, TIMP-3, α-actin, and β-actin was analyzed with electrophoresis on sodium dodecyl sulfate gels and immunoblotting. Results: MMP-1, MMP-9, and MMP-12 zymogen levels and proteolytic activities were increased in AAAs when compared with healthy aorta. A severe reduction in α-actin–positive vascular SMCs was observed in all the AAA specimens and was correlated with an increase in TIMP-3 but not TIMP-1 or TIMP-2 potential activities. Although pro–MMP-2 activity was decreased, the extent of activated MMP-2 remained unaffected in the AAAs. In accordance with this result, a highly activated MT1-MMP form was also observed in AAAs. Conclusion: These data suggest that chronic aortic wall inflammation is mediated by macrophage infiltration, which may account for the destruction of medial elastin, as reflected by SMC down regulation, through increased levels of active MMP-1 and MMP-12. Moreover, altered MT1-MMP proteolytic turnover and differential regulation of TIMP expression in AAAs suggest that tight regulatory mechanisms are involved in the molecular regulation of MMP activation processes in the pathogenesis of AAAs. (J Vasc Surg 2002;35:539-46.)

To date, pressure shock, heat shock, and chemical treatment with cytochalasin B have been the major methods used to induce triploid bivalves. In this study, triploid bivalves were induced by a new chemical treatment using 6-dimethylaminopurine (6-DMAP). The capacity of 6-DMAP to produce triploid eggs and larvae was investigated in the Pacific oyster, Crassostrea gigas , the giant sea scallop, Placopecten magellanicus , and the blue mussel, Mytilus edulis . The triploid yields from the 6-DMAP treatments were compared with those of cytochalasin treatments. The highest percentage of triploid production was 90% in the Pacific oysters when the fertilized eggs were treated for 20 min with 300 μM 6-DMAP at 15 min after fertilization and 95% in the giant scallops when treated for 15 min with 400 μM 6-DMAP at 70 min after fertilization. Increasing durations of 6-DMAP treatments improved the efficiency of triploid induction. However, long incubations with 6-DMAP, which overlapped the period of first mitotic cleavage, led to the development of abnormal larvae. These included low percentages of normal veliger larvae in the Pacific oysters and developmental arrest at the trochophore stage in the blue mussels. The percentage of abnormalities increased with increased treatment duration. Triploid larvae of Pacific oysters produced by 6-DMAP or cytochalasin treatments had equivalent growth rates and were similar to those of control diploid larvae. However, triploid larvae showed high mortalities following these two chemical treatments. Overall, the results clearly demonstrate that 6-DMAP was an efficient and practical inducer of triploidy in bivalve molluscs. Moreover, the described procedure is the most simple and reproducible method ever reported. In addition, 6-DMAP is safer to handle than cytochalasin which is classified as a carcinogen.