Research

Expression of A Novel Large Non-coding RNA Is Associated with Neoplasia, Proliferation, and Differentiation

Recent genomic and transcriptomic studies have revealed the existence of a substantial number of large non-coding RNAs (ncRNAs). Only a very few large ncRNAs have been characterized, but with little knowledge of their functional roles. In the present study, we discovered a novel very large ncRNA. A ~360 bp RNA hit was identified by differential display analysis of CD31-enriched tumor microvascular endothelial cells from a syngeneic murine colon carcinoma. The full-length gene is a 7 Kb mRNA-like transcript but lacks extensive or conserved open reading frames, as for a large ncRNA transcript. Bioinformatics analysis suggested that the full-length gene was evolutionally highly conserved among vertebrates, inferring functional significance. More importantly, Northern blot analysis and in situ hybridization indicated that expression of the full-length transcript was highly up-regulated in carcinogen (DEN) induced primary hepatomas and represents a novel marker for hepatomas as well as other neoplasms so far analyzed. We designate this ncRNA Hepatomin. In addition, we have found that Hepatomin transcription is induced in liver undergoing regeneration, skin undergoing wound healing, hair follicles in growth, and cultured cells synchronized at the mitosis stage. These results suggest that Hepatomin plays roles in proliferation and differentiation. We conclude that Hepatomin is a member of the new class of large ncRNAs, and a new marker for hepatomas. This appears to provide unique murine models for analysis of the role of this ncRNA in neoplasia, proliferation, and differentiation as an apparent regulator of changed gene expression associated with these processes.

The Tumor Microvasculature. Novel Redirected Molecular Expression, Architecture, and Properties Imparted by the Local Modified Endothelial Surface Proteome.

Neoplastic cells as they seek nutrient, oxygen, and freedom to invade and metastasize impart remarkable changes upon their new angiogenic microvasculature. This is observed as the discard of normal vascular development and architecture results in a bizarre functionally incompetent maze of capillary-like vessels lined by endothelium with strikingly divergent gene transcription and aberrant cell surface proteome display. An example of aberrations is expression of molecules and molecular complexes that not only may broaden knowledge of vascular molecular biology of these complex angiogenic and post-angiogenic vascular mazes, but also some that may provide novel targets for therapeutic intervention. Search yielded an endothelial receptor complex that appears quite unique for the tumor microvascular environment. VEGF Receptor-2 and its co-receptor Neuropilin-1 can be found elsewhere within the vascular tree; but the association of an oligosaccharide (chondroitin C sulfate) to create a ternary complex which is unique to the tumor microvasculature. This complex represents a novel target for therapeutic strategies, which we have exploited by developing a tumor vascular specific thrombogen recognizing this ternary complex selectively for tumor neoangiogenic vessels. This soluble hybrid protein, HBDt.TFt incorporates the modified exon 7 encoded heparin-binding domain of the VEGF gene at its N-terminus with the extracellular domain of tissue factor at the C-terminus. It is capable of selectively docking on the surface of tumor endothelium at this trimolecular complex and initiating the coagulation protease cascade. Consequently, the tumor microvasculature is thrombosed resulting in infarctive local tumor eradication. Utilizing intravital microscopy, we have optimized the dose and rate of infusion of HBDt.TFt and monitored microcirculation within the tumor as well as thrombus formation. We have also demonstrated a central participation of platelets in tumor specific microvascular thrombosis by imaging fluorescein-labled platelets.

Regulation of Tissue Factor-Mediated Initiation of the Coagulation (Thrombogenic) Cascade by the Cell Surface Chaperon Grp78.

Biopanning with phage displayed peptidyl libraries has identified peptide probes which bind selectively to the surface of atherosclerotic plaque endothelium. The highest affinity peptide, EKO130, binds the 78 kDa glucose regulated protein (Grp78). Grp78 participates in numerous pathological processes including the regulation of the coagulation cascade; but the mechanism of Grp78 regulation of coagulation is unknown. To characterize this function of Grp78 we analyzed the effect of Grp78 on TF-mediated procoagulant activity on mouse brain endothelial cells (bEND.3) and macrophage-like (RAW) cells, which are relevant in mediation of atherothrombosis. We show that Grp78 is present on the endothelium and monocyte/macrophage-like cells in atherosclerotic lesions. Inhibition of Grp78 resulted in increased procoagulant activity. We also demonstrate that Grp78 negatively regulates procoagulant activity by interacting directly with TF extracellular domain on the cell surface. Thus, our results indicate that Grp78 negatively regulates TF functional activity via direct binding to and functional inhibition of TF. Identification of the mechanism by which Grp78 regulates TF function may advance insight into the pathobiology of atherosclerosis and associated arterial thrombosis.

We welcome collaborative studies and investigators who wish to gain further experience as post-doctoral fellows or research associates.

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