Hans H. Riese

Gold glyconanoparticles as new tools in antiadhesive therapy.

Gold glyconanoparticles (GNPs) have been prepared as new multivalent tools that mimic glycosphingolipids on the cell surface. GNPs are highly soluble under physiological conditions, stable against enzymatic degradation and nontoxic. Thereby GNPs open up a novel promising multivalent platform for biological applications. It has recently been demonstrated that specific tumor‐associated carbohydrate antigens (glycosphingolipids and glycoproteins) are involved in the initial step of tumor spreading. A mouse melanoma model was selected to test glyconanoparticles as possible inhibitors of experimental lung metastasis. A carbohydrate–carbohydrate interaction is proposed as the first recognition step for this process. Glyconanoparticles presenting lactose (lacto‐GNPs) have been used successfully to significantly reduce the progression of experimental metastasis. This result shows for the first time a clear biological effect of lacto‐GNPs, demonstrating the potential application of this glyconanotechnology in biological processes.

Inhibition of programmed cell death impairs in vitro vascular-like structure formation and reduces in vivo angiogenesis

Tissue remodeling during embryonic development and in the adult organism relies on a subtle balance between cell growth and apoptosis. As angiogenesis involves restructuring of preexisting endothelium, we examined the role of apoptosis in new vessel formation. We show that apoptosis occurs before capillary formation but not after vessels have assembled. Using the human umbilical vein endothelial cell (HUVEC) in vitro Matrigel angiogenesis model, we show that vascular‐like structure formation requires apoptotic cell death through activation of a caspasedependent mechanism and mitochondrial cytochrome c release. Vascular‐like structure formation was further blocked by caspase inhibitors such as z‐VAD or AcD‐EVD‐CHO, using HUVEC and human lung microvascular endothelial cells. Overexpression of anti‐apoptotic human Bcl‐2 or baculovirus p35 genes in HUVEC altered endothelial cell rearrangement during in vitro angiogenesis, causing impaired vessel‐like structure formation. Caspase inhibitors blocked VEGF‐ or bFGF‐induced HUVEC angiogenesis on 2‐ or 3‐D collagen gels, respectively, confirming that apoptosis was not the result of nonspecific cell death after seeding on the matrix. In an in vivo angiogenesis assay, caspase inhibitors blocked VEGF‐dependent vascular formation at the alignment step, as demonstrated histologically. This evidence indicates that endothelial cell apoptosis may be relevant for precise vascular tissue rearrangement in in vitro and in vivo angiogenesis.—Segura, I., Serrano, A., González de Buitrago, G., González, M. A., Abad, J. L., Clavería, C., Gómez, L., Bernad, A., Martínez‐A, C., Riese, H. H. Inhibition of programmed cell death impairs in vitro vascular‐like structure formation and reduces in vivo angiogenesis. FASEB J. 16, 833–841 (2002)

In vitro activation of macrophages by a novel proteoglycan isolated from corms of Crocus sativus L

Saffron corms contain a proteoglycan that is highly cytotoxic on human tumor cells. The present work was undertaken to study the possible immunomodulatory and anti-invasive properties of this compound. Non-cytotoxic concentrations of this glycoconjugate promoted significant macrophage activation, detected by the release of nitric oxide. A rapid activation of protein kinase C and NF-kappaB was obtained after proteoglycan treatment, which could explain the induction of nitric oxide synthase. Proteoglycan concentrations ranging from 10-1000 ng/ml specifically promoted apoptosis of macrophages, probably triggered by their activation. This molecule did not inhibit in vitro migration or invasion of human tumor cells. Altogether these results support a plausible immuno-modulating activity for this saffron Crocus compound.

Expression and purification of functional recombinant human pigment epithelium-derived factor (PEDF) secreted by the yeast Pichia pastoris

Pigment epithelium-derived factor (PEDF) combines neurotrophic, neuroprotective, anti-angiogenic, anti-tumor and neural stem cell self-renewal properties in a single molecule, making this protein a valuable potential therapeutic agent. We herein analyzed the expression of human recombinant full-length PEDF, and its N- and C-terminal regions (amino acids 1-243 and 195-418, respectively) in three mammalian cell lines (HEK-293T, COS-1, and 26HCMsv), and in the yeast Pichia pastoris. The highest production of recombinant PEDF was achieved in P. pastoris which secreted approximately 30 microg of full-length rPEDF, and 47 microg of C-terminal/ml of culture medium. Full-length rPEDF was purified by one-step Ni-chelating high-performance liquid chromatography, recovering almost 70% of secreted rPEDF with a purity of 98.6%. The C-terminal region of PEDF was isolated by low-pressure liquid chromatography, recovering around 4% of the recombinant molecule with a purity of 98%. The N-terminal region of PEDF was not secreted by any expression system assayed. The two isolated recombinant PEDF polypeptides inhibited in vitro endothelial cell migration, and full-length rPEDF also increased cerebellar granule cell survival, thus demonstrating their biological activity. These polypeptides can be used to investigate the therapeutic role of PEDF in cancer, neurodegenerative and ocular diseases, and stem cell-based therapies.

A Glycoconjugate Isolated from the Saffron Plant (Crocus sativus L.) is Cytolytic Against Tumoral Cells and Activates Macrophages In Vitro

Saffron corms contain a toxic glycoconjugate composed of 94.5% carbohydrate and 5.5% protein. We undertook to study the specificity and molecular events involved in cytotoxicity, as well as the possible immunomodulatory properties of this compound (Escribano et al 1999). Studies of intracellular calcium fluctuations, transmembrane potential and release of lactate dehydrogenase showed that this compound caused plasma membrane damage, allowing movements of both calcium and macromolecules, and leading to cell lysis. Microscopy of treated cells revealed cellular swelling and cytoplasmic collapse. This molecule was active in vitro against human tumoral cells derived from fibrosarcoma, cervical epithelioid carcinoma and breast carcinoma, the calculated IC50 values being 7, 9 and 22 μg/ml, respectively. Comparison of IC50 data for fibroblasts (60 μg/ml) and fibrosarcoma cells revealed that the glycoconjugate was nearly 8 times more cytotoxic for malignant cells than for their normal counterparts. A 100 μg/ml concentration of the compound produced 50% lysis of normal human erythrocytes, while 320 μg/ml were unable to induce 50% cell death of cultured human hair follicles. Non-cytotoxic concentrations promoted a significant macrophage activation, as detected by the release of nitric oxide, but did not stimulate either cAMP generation or mitogen-activated protein kinases. Activation of protein kinase C and transcriptional activator NF-KB was also observed. The activation of macrophages suggests a potential immuno-stimulating capacity of this compound.