Jia Wei Liu

Characterization of endothelial-like cells derived from human mesenchymal stem cells.

Summary.  Background:  Blood‐derived endothelial progenitor cells (EPC) have been used to treat ischemic disease. However, the number of EPC that can be obtained from adult blood is limited.

Gene conversion limits divergence of mammalian TLR1 and TLR6

BackgroundToll-like receptors (TLR) recognize pathogen-associated molecular patterns and are important mediators of the innate immune system. TLR1 and TLR6 are paralogs and located in tandem on the same chromosome in mammals. They form heterodimers with TLR2 and bind lipopeptide components of gram-positive and gram-negative bacterial cell walls. To identify conserved stretches in TLR1 and TLR6, that may be important for their function, we compared their protein sequences in nine mammalian species(Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, Rattus norvegicus; Erinaceus europaeus, Bos Taurus, Sus scrofa and Canis familiaris).ResultsThe N-terminal sequences of the orthologous proteins showed greater similarity than corresponding paralog sequences. However, we identified a region of 300 amino acids towards the C-terminus of TLR1 and TLR6, where paralogs had a greater degree of sequence identity than orthologs. Preservation of DNA sequence identity of paralogs in this region was observed in all nine mammalian species investigated, and is due to independent gene conversion events. The regions having undergone gene conversion in each species are almost identical and encode the leucine-rich repeat motifs 16 to 19, the C-terminal cap motif, the transmembrane domain and most of the intracellular Toll/interleukin-1 receptor (TIR) domain.ConclusionOur results show that, for a specific conserved region, divergence of TLR1 and TLR6 is limited by gene conversion, most likely because of the need for co-evolution with multiple intracellular and extracellular binding partners. Thus, gene conversion provides a mechanism for limiting the divergence of functional regions of protein paralogs, while allowing other domains to evolve diversified functions.

CD146 Short Isoform Increases the Proangiogenic Potential of Endothelial Progenitor Cells In Vitro and In Vivo

Rationale: CD146, a transmembrane immunoglobulin mainly expressed at the intercellular junction of endothelial cells, is involved in cell-cell cohesion, paracellular permeability, monocyte transmigration and angiogenesis. CD146 exists as 2 isoforms, short (sh) and long (lg), but which isoform is involved remains undefined. Objective: The recently described role of CD146 in angiogenesis prompted us to investigate which isoform was involved in this process in human late endothelial progenitors (EPCs), with the objective of increasing their proangiogenic potential. Methods and Results: Immunofluorescence experiments showed that, in subconfluent EPCs, shCD146 was localized in the nucleus and at the migrating edges of the membrane, whereas lgCD146 was intracellular. In confluent cells, shCD146 was redistributed at the apical membrane and lgCD146 was directed toward the junction. In contrast to lgCD146, shCD146 was overexpressed in EPCs as compared to mature endothelial cells and upregulated by vascular endothelial growth factor and SDF-1 (stromal cell–derived factor 1). Study of the properties of both isoforms in vitro provided evidence that shCD146 was involved in EPC adhesion to activated endothelium, migration, and proliferation, with a paracrine secretion of interleukin-8 or angiopoietin 2, whereas lgCD146 was implicated in stabilization of capillary-like structures in Matrigel and transendothelial permeability. In an animal model of hindlimb ischemia, transplantation of shCD146-modified EPCs selectively promoted both EPC engraftment and blood flow. Conclusions: Altogether, these findings establish that CD146 isoforms display distinct functions in vessels regeneration. Selective improvement of therapeutic angiogenesis by shCD146 overexpression suggests a potential interest of shCD146-transduced EPCs for the treatment of peripheral ischemic disease.

Promoter Dependence of Transgene Expression by Lentivirus‐Transduced Human Blood–Derived Endothelial Progenitor Cells

Peripheral blood– derived endothelial progenitor cells (EPCs) have considerable potential for the autologous therapy of vascular lesions or ischemic tissues. By introducing stable genetic modifications into these cells, this potential might be further enhanced. We investigated to what extent transgene expression can be controlled by using different transgene promoters. This was investigated in early‐ or late‐outgrowth human EPCs obtained by culturing blood mononuclear cells for 1 or 4 weeks on type 1 collagen in medium containing endothelial growth supplements. A large fraction of these cells were stably transduced using lentiviral vectors for expression of the enhanced green fluorescent protein (EGFP). Transgene expression in vitro or in vivo after injection into nude mice was highest when under the control of the cytomegalovirus (CMV) promoter, intermediate with the EF1α promoter, and lowest with the phosphoglycerate kinase promoter. When blood mononuclear cells were cultured for 1 week in the absence of endothelial growth supplements, CMV promoter– driven expression of EGFP was two orders of magnitude lower than in similarly transduced EPCs. Our results show that lentiviral vectors are useful tools for the stable introduction of exogenous genes into EPCs and for their expression at desired levels using the appropriate gene promoter.

The Anti-Estrogen Hydroxytamoxifen Is a Potent Antagonist in a Novel Yeast System

Abstract The budding yeast Saccharomyces cerevisiae has been used extensively as a biological ‘test tube’ to study the regulation of the human estrogen receptor (ER) α. However, anti-estrogens, which are of great importance as therapeutic agents and research tools, fail to antagonize the activation by estrogen in yeast. Here, we have surveyed the antagonistic potential of five different anti-estrogens of diverse chemical nature. While they all act as agonists for wild-type ERα we have established a novel yeast assay system for anti-estrogens, in which at least the commonly used anti-estrogen hydroxytamoxifen is a potent antagonist.

Characterization of human late outgrowth endothelial progenitor-derived cells under various flow conditions.

Background: Endothelial progenitor-derived cells (EPC) are a cell therapy tool in peripheral arterial disease and for re-endothelialization of bypasses and stents. Objective: To assess EPC behavior under flow conditions normally found in vivo. Results: EPC were isolated from human cord blood, cultured on compliant tubes and exposed in an in vitro flow system mimicking hemodynamic environments normally found in medium and large arteries. EPC exposed for 24 h to unidirectional (0.3 ± 0.1 or 6 ± 3 dynes/cm2) shear stress oriented along flow direction, while those exposed to bidirectional shear stress (0.3 ± 3 dynes/cm2) or static conditions had random orientation. Under bidirectional flow, tissue factor (TF) activity and mRNA expression were significantly increased (2.5- and 7.0-fold) compared to static conditions. Under low shear unidirectional flow TF mRNA increased 4.9 ± 0.5-fold. Similar flow-induced increases were observed for TF in mature umbilical vein-derived endothelial cells. Expression of tissue-type plasminogen activator (t-PA), urokinase (u-PA) and monocyte chemotactic protein 1 (MCP1) were reduced by 40–60% in late outgrowth endothelial progenitor-derived cells (LO-EPC) exposed to any flow environment, while MCP1, but not t-PA or u-PA, was decreased in HUVEC. Conclusions: Flow, in particular bidirectional, modifies the hemostatic balance in LO-EPC with increased TF and decreased plasminogen activator expression.

Increasing lentiviral vector titer using inhibitors of protein kinase R.

Lentiviral Production Looks Up Stable gene transfer methods increasingly involve lentiviral vectors because of the vectors large cloning capacity, stable integration into the host genome, and easy...

Generation of Human Inflammation-Resistant Endothelial Progenitor Cells by A20 Gene Transfer

Inflammatory activation of the vascular endothelium is a major contributory factor to ischemic cardiovascular disease. Endothelial progenitor cells (EPCs) are being investigated for the treatment of ischemic disease or to coat vein grafts for bypass surgery. As an inflammatory environment might reduce their therapeutic efficacy, we sought to generate EPCs that are less sensitive to inflammatory activation. EPCs were obtained from human umbilical cord blood and transduced with a lentiviral vector for stable expression of A20, an anti-inflammatory protein. Nontransduced and green-fluorescent-protein-transduced cells were used as controls. Expression of A20 by EPCs did not modify cell morphology or expression of a panel of 20 proteins known to contribute to angiogenesis. Also, A20 had no effect on the capacity of EPCs to form tube-like structures in Matrigel™. A20 expression reduced EPC activation by tumor necrosis factor-α and interleukin-1β as determined from changes in vascular cell adhesion molecule 1 and E-selectin expression and decreased monocyte transmigration through a monolayer of EPCs. In conclusion, EPCs can be genetically modified to overexpress A20 in a stable fashion. These cells become less sensitive to inflammatory stimuli. This may be of interest in cell-based therapeutic approaches for clinical settings where inflammation is an important pathogenic factor.