Targeted inhibition of alphavbeta3 integrin with an RNA aptamer impairs endothelial cell growth and survival

Alphavbeta3 integrin is a crucial factor involved in a variety of physiological processes, such as cell growth and migration, tumor invasion and metastasis, angiogenesis, and wound healing. Alphavbeta3 integrin exerts its effect by regulating endothelial cell (EC) migration, proliferation, and survival. Inhibiting the function of alphavbeta3 integrin, therefore, represents a potential anti-cancer, anti-thrombotic, and anti-inflammatory strategy. In this study, we tested an RNA aptamer, Apt-alphavbeta3 that binds recombinant alphavbeta3 integrin, for its ability to bind endogenous alphavbeta3 integrin on the surface of cells in culture and to subsequently affect cellular response. Our data illustrate that Apt-alphavbeta3 binds alphavbeta3 integrin expressed on the surface of live HUVECs. This interaction significantly decreases both basal and PDGF-induced cell proliferation as well as inhibition of cell adhesion. Apt-alphavbeta3 can also reduce PDGF-stimulated tube formation and increase HUVEC apoptosis through inhibition of FAK phosphorylation pathway. Our results demonstrate that by binding to its target, Apt-alphavbeta3 can efficiently inhibit human EC proliferation and survival, resulting in reduced angiogenesis. It predicts that Apt-alphavbeta3 could become useful in both tumor imaging and the treatment of tumor growth, atherosclerosis, thrombosis, and inflammation.     

Agrobacterium rhizogenes transformation of the Phaseolus spp.: a tool for functional genomics

A fast, reproducible, and efficient transformation procedure employing Agrobacterium rhizogenes was developed for Phaseolus vulgaris L. wild accessions, landraces, and cultivars and for three other species belonging to the genus Phaseolus: P. coccineus, P. lunatus, and P. acutifolius. Induced hairy roots are robust and grow quickly. The transformation frequency is between 75 and 90% based on the 35-S promoter-driven green fluorescent protein and beta-glucuronidase expression reporter constructs. When inoculated with Rhizobium tropici, transgenic roots induce normal determinate nodules that fix nitrogen as efficiently as inoculated standard roots. The A. rhizogenes-induced hairy root transformation in the genus Phaseolus sets the foundation for functional genomics programs focused on root physiology, root metabolism, and root-microbe interactions.     

Culture conditions determine the balance between two different exopolysaccharides produced by Lactobacillus pentosus LPS26

Lactobacillus pentosus LPS26, isolated from a natural fermentation of green olives, produces a capsular polymer constituted of two exopolysaccharides (EPS): EPS A, a high-molecular-weight (high-Mw) polysaccharide (1.9x10(6) Da) composed of glucose and rhamnose (3:1), and EPS B, a low-Mw polysaccharide (3.3x10(4) Da) composed of glucose and mannose (3:1). Fermentation experiments in a chemically semidefined medium with different carbon sources (glucose, fructose, mannitol, and lactose) showed that all of them except fructose supported EPS A production rather than EPS B production. The influence of temperature and pH was further analyzed. As the temperature dropped, increased synthesis of both EPS was detected. The control of pH especially enhanced EPS B production. With regard to this, the maximum total EPS production (514 mg liter-1) was achieved at a suboptimal growth temperature (20 degrees C) and pH 6.0. Continuous cultures showed that EPS A, synthesized mainly at low dilution rates, is clearly dependent on the growth rate, whereas EPS B synthesis was hardly affected. EPS production was also detected in supplemented skimmed milk, but no increase on the viscosity of the fermented milk was recorded. This could be linked to the high proportion of the low-Mw polysaccharide produced in these conditions in contrast to that observed in culture media. Overall, the present study shows that culture conditions have a clear impact on the type and concentration of EPS produced by strain LPS26, and consequently, these conditions should be carefully selected for optimization and application studies. Finally, it should be noted that this is, to our knowledge, the first report on EPS production by L. pentosus.     

The Prolyl Hydroxylase PHD3 Identifies Proinflammatory Macrophages and Its Expression Is Regulated by Activin A

Modulation of macrophage polarization underlies the onset and resolution of inflammatory processes, with polarization-specific molecules being actively sought as potential diagnostic and therapeutic tools. Based on their cytokine profile upon exposure to pathogenic stimuli, human monocyte-derived macrophages generated in the presence of GM-CSF or M-CSF are considered as proinflammatory (M1) or anti-inflammatory (M2) macrophages, respectively. We report in this study that the prolyl hydroxylase PHD3-encoding EGLN3 gene is specifically expressed by in vitro-generated proinflammatory M1(GM-CSF) human macrophages at the mRNA and protein level. Immunohistochemical analysis revealed the expression of PHD3 in CD163(+) lung macrophages under basal homeostatic conditions, whereas PHD3(+) macrophages were abundantly found in tissues undergoing inflammatory responses (e.g., Crohn's disease and ulcerative colitis) and in tumors. In the case of melanoma, PHD3 expression marked a subset of tumor-associated macrophages that exhibit a weak (e.g., CD163) or absent (e.g., FOLR2) expression of typical M2-polarization markers. EGLN3 gene expression in proinflammatory M1(GM-CSF) macrophages was found to be activin A dependent and could be prevented in the presence of an anti-activin A-blocking Ab or inhibitors of activin receptor-like kinase receptors. Moreover, EGLN3 gene expression was upregulated in response to hypoxia only in M2(M-CSF) macrophages, and the hypoxia-mediated upregulation of EGLN3 expression was significantly impaired by activin A neutralization. These results indicate that EGLN3 gene expression in macrophages is dependent on activin A both under basal and hypoxic conditions and that the expression of the EGLN3-encoded PHD3 prolyl hydroxylase identifies proinflammatory macrophages in vivo and in vitro.