Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

Peroxisome proliferator-activated receptor gamma ligands improve the antitumor efficacy of thrombospondin peptide ABT510

An expanding capillary network is critical for several pathologic conditions. In cancer, the decrease of antiangiogenic thrombospondin-1 (TSP1) often enables an angiogenic switch, which can be reversed with exogenous TSP1 or its peptide derivative ABT510. TSP1 acts by inducing endothelial cell apoptosis via signaling cascade initiated at CD36, a TSP1 antiangiogenic receptor. Here, we show that the ligands of nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma), 15-deoxy-delta(12,14)-prostaglandin J2, troglitazone, and rosiglitazone increased PPARgamma and CD36 expression in endothelial cells and improved the efficacy of TSP1 and ABT510 in a CD36-dependent manner. The ABT510 and PPARgamma ligands cooperatively blocked angiogenic endothelial functions in vitro and neovascularization in vivo. In tumor xenografts, 15-deoxy-delta(12,14)-prostaglandin J2 and troglitazone synergistically improved antiangiogenic and antitumor effects of ABT510. Our data provide one mechanism for the in vivo angioinhibitory effect of PPARgamma ligands and show fine-tuning of the antiangiogenic efficacy via targeted up-regulation of the endothelial receptor.     

Contrasting effects of VEGF gene disruption in embryonic stem cell-derived versus oncogene-induced tumors

Previous gene targeting studies have implicated an indispensable role of vascular endothelial growth factor (VEGF) in tumor angiogenesis, particularly in tumors of embryonal or endocrine origin. In contrast, we report here that transformation of VEGF-deficient adult fibroblasts (MDF528) with ras or neu oncogenes gives rise to highly tumorigenic and angiogenic fibrosarcomas. These aggressive VEGF-null tumors (528ras, 528neu) originated from VEGF(-/-) embryonic stem cells, which themselves were tumorigenically deficient. We also report that VEGF production by tumor stroma has a modest role in oncogene-driven tumor angiogenesis. Both ras and neu oncogenes down-regulated at least two endogenous inhibitors of angiogenesis [pigment epithelium derived factor (PEDF) and thrombospondin 1 (TSP-1)]. This is functionally important as administration of an antiangiogenic TSP-1 peptide (ABT-526) markedly inhibited growth of VEGF(-/-) tumors, with some ingress of pericytes. These results provide the first definitive genetic demonstration of the dispensability of tumor cell-derived VEGF in certain cases of 'adult' tumor angiogenesis, and thus highlight the importance of considering VEGF-independent as well as VEGF-dependent pathways when attempting to block this process pharmacologically.     

Structure-directed discovery of potent non-peptidic inhibitors of human urokinase that access a novel binding subsite

BACKGROUND: Human urokinase-type plasminogen activator has been implicated in the regulation and control of basement membrane and interstitial protein degradation. Because of its role in tissue remodeling, urokinase is a central player in the disease progression of cancer, making it an attractive target for design of an anticancer clinical agent: Few urokinase inhibitors have been described, which suggests that discovery of such a compound is in the early stages. Towards integrating structural data into this process, a new human urokinase crystal form amenable to structure-based drug design has been used to discover potent urokinase inhibitors. RESULTS: On the basis of crystallographic data, 2-naphthamidine was chosen as the lead scaffold for structure-directed optimization. This co-crystal structure shows the compound binding at the primary specificity pocket of the trypsin-like protease and at a novel binding subsite that is accessible from the 8-position of 2-napthamidine. This novel subsite was characterized and used to design two compounds with very different 8-substituents that inhibit urokinase with K(i) values of 30-40 nM. CONCLUSIONS: Utilization of a novel subsite yielded two potent urokinase inhibitors even though this site has not been widely used in inhibitor optimization with other trypsin-like proteases, such as those reported for thrombin or factor Xa. The extensive binding pockets present at the substrate-binding groove of these other proteins are blocked by unique insertion loops in urokinase, thus necessitating the utilization of additional binding subsites. Successful implementation of this strategy and characterization of the novel site provides a significant step towards the discovery of an anticancer clinical agent.     

Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.