Detection and characterization of circulating endothelial progenitor cells in normal rat blood.

INTRODUCTION: There are currently few widely accepted noninvasive detection methods for drug-induced vascular damage. Circulating endothelial progenitor cell (EPC) enumeration in humans has recently gained attention as a potential biomarker of vascular injury/endothelial damage/dysfunction. The rat is commonly used in preclinical drug development toxicity testing and lacks consensus noninvasive methodologies for immunophenotypic identification of EPCs. Identification of immunophenotypic markers of EPCs in the rat would enable transfer of technologies used in human for potential development of biomarkers for vascular injury the rat. Therefore, the aim of this work was to develop methods to consistently identify a discreet population of EPCs from rat peripheral blood. METHODS: EPCs were identified phenotypically from rat blood using cell culture, immunolabeling, fluorescence microscopy, and flow cytometry. EPCs isolated using immunolabeling coupled with magnetic separation and flow cytometric cell sorting were characterized genotypically using mRNA analysis. RESULTS: A modified colony forming unit (CFU)-Hill assay confirmed existence of immature EPCs in peripheral blood. Extended in vitro culture resulted in a morphology and immunophenotype consistent with mature endothelial cells as noted by positive staining for CD31, von Willebrand factor, rat endothelial cell antigen, and negative staining for smooth muscle cell alpha-actin. The majority of the cells identified as LDL+/CD11b/c(-) did not stain positively for either vWF or CD31. EPC populations isolated using magnetic separation and cell sorting were consistently positive for PECAM1, EDN1, FLK1, VWF, ITGAD, CCR1, IP30, and MMP2 mRNA expression. Cells identified as EPCs express cell-surface and gene expression markers consistent with endothelial cells and endothelial progenitor cell populations. DISCUSSION: Vascular trauma induces transient mobilization of EPCs in humans and their enumeration and characterization have been proposed as a surrogate biomarker for assessment of vascular injury. Potential exists for using rat circulating EPCs as a surrogate sampling population for biomarker development in drug-related injury in preclinical toxicity studies. A prerequisite to biomarker development is the ability to consistently identify a discreet population of EPCs from peripheral rat blood. This work describes novel methods for isolation and validation of phenotypically and genotypically consistent populations of rat EPCs from peripheral blood. These methods are well suited for potential future use in validation of enumeration and/or biomarker development methods in the rat.

Role of p38 in regulation of hematopoiesis: effect of p38 inhibition on cytokine production and transcription factor activity in human bone marrow stromal cells.

This study was designed to evaluate effects of specific p38 MAP kinase inhibition on gene and protein expression of essential hematopoietic cytokines in primary human bone marrow stromal cells (HBMSC) and to identify downstream transcription factors (TF) regulated by the p38 MAP kinase signalling pathway. In vitro effects of p38 inhibitors (p38i) on cytokine regulation were compared to inhibitors of other major signalling pathways including PI3 kinase, JNK, MEK-1, NF-kappaB or protein kinase C (PKC). HBMSC were pre-treated with p38i (SB-203580) for 1 h and then stimulated with 200 ng/ml lipopolysaccharide (LPS). Supernatants and RNA were collected 6 h post LPS treatment for quantitative protein and mRNA analyses by ELISA and real-time RT-PCR, respectively, for interleukin-6 (IL-6), interleukin-11 (IL-11), granulocyte-monocyte colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and Activin A. Effects of the inhibitors of PI3 kinase (LY294002), JNK (synthetic inhibitory peptide), MEK-1 (PD90859), NF-kappaB (pyrrolidinedithiocarbamate (PDTC)) and protein kinase C (calphostin C) on HBMSC expression hematopoietic cytokines were evaluated and compared. SB-203580 caused dose-dependent decreases in cytokine protein expression and decreased IL-6 and IL-11 mRNA expression. Of the pathway inhibitors examined, only NF-kappaB elicited similar effects on cytokine protein and mRNA expression. p38-regulated transcription factor activity was assessed using a DNA/Protein array. Several TFs linked to cytokine regulation were modulated by SB-203580, with 10 of 21 p38-regulated TFs identified have not been previously linked to downstream p38 signalling. These observations in cultured HBMSC have illustrated the involvement of cytokine proteins, mRNA and TF activities and may improve the current understanding of the in vivo p38i suppression of erythropoiesis. In addition, these results suggest that IL-6, IL-11, GM-CSF, G-CSF and Activin A are similarly regulated by p38 and NF-kappaB and that the MEK1, JNK and PKC pathways appear to play a more limited role in modulating cytokine expression in HBMSC.

Confounding effects of platelets on flow cytometric analysis and cell-sorting experiments using blood-derived cells.

BACKGROUND: Flow cytometric analysis and cell-sorting of peripheral blood leukocytes is commonplace; however, platelet contamination is typically ignored during immunophenotypic analysis and sorting of blood-derived cells. METHODS: Red blood cells, platelets, T & B lymphocytes, monocytes, and granulocytes were sorted from rat blood preparations. Presort enrichment was performed by differential centrifugation for all cell types. Additionally, leukocyte samples were prepared by ammonium chloride lysis of red blood cells. RESULTS: Unless proper precautions were taken, significant numbers of platelets were sorted along with (nonplatelet) cells of interest. The amount of platelet contamination varied greatly from experiment to experiment with the highest level of leukocyte-platelet association observed in the neutrophil/granulocyte population in samples prepared using ammonium chloride-based red blood cell-lysing solution. CONCLUSIONS: Addition of an immunophenotypic marker for platelet identification is a simple, yet prudent, measure to help evaluate the impact of platelets on immunophenotypic staining when performing flow cytometric analysis or sorting of blood-derived cells and should become a routine practice. Platelet presence in postsort fractions can be due to free platelets as well as target cell-associated platelets and both sources of contamination must be addressed.

Effects of 6-hydroxydopamine on mitochondrial function and glutathione status in SH-SY5Y human neuroblastoma cells.

SH-SY5Y human neuroblastoma cells were incubated with 6-hydroxydopamine (6-OHDA) for 4 and 24 h to examine the mechanism of cell death and to determine the time-dependent effects of 6-OHDA on cellular glutathione status. After 4 h, 6-OHDA significantly depleted cellular ATP and GSH concentrations with only slight increases in cell death. GSH:GSSG ratios and mitochondrial membrane potential (Deltapsim) were significantly decreased during 4 h incubations with 6-OHDA. High concentrations of 6-OHDA (100 microM) induced oxidative stress and mitochondrial dysfunction in SH-SY5Y cells within 4 h leading to cell death. In 24 h incubations, 25 and 50 microM 6-OHDA significantly decreased ATP concentrations; however, significant increases in cell death were only observed with 50 microM 6-OHDA. 6-OHDA induced a concentration-dependent increase in GSH and total glutathione concentrations after 24 h. After exposure to 50 microM 6-OHDA, GSH concentrations were increased up to 12-fold after 24 h with no change in the GSH:GSSG ratio. Gene analysis suggests that the increase in GSH concentration was due to increased expression of the GSH synthesis genes glutamate cysteine ligase modifier and catalytic subunits. Our results suggest that 6-OHDA induces oxidative stress in SH-SY5Y cells resulting in an adaptive increase in cellular GSH concentrations.

In vitro expansion of human cord blood CD36+ erythroid progenitors: temporal changes in gene and protein expression.

OBJECTIVE: Erythropoiesis involves proliferation and differentiation of committed erythroid progenitors to mature red blood cells. The objective of this study was to characterize growth characteristics of human CD36+ erythroid progenitors and to profile temporal expression of lineage-specific transcription factors, structural proteins, and growth factor receptors involved in erythropoiesis. MATERIALS AND METHODS: Erythropoietin-induced differentiation of human cord blood CD36+ erythroid progenitors was profiled for GATA-1, GATA-2, NFE2, EKLF, SCL, PU.1, Id1, Evi-1, c-myb, Hox2.2, c-kit, EpoR, glycophorin A (GPA), CD71, beta- and gamma-globin, and protein 4.2 gene and/or protein expression and DNA content analysis on days 4, 7, and 15 of culture. RESULTS: Real-time RT-PCR analysis revealed upregulation of GATA-1, Id1, glycophorin A, and protein 4.2 mRNA expression on day 7 when compared to day 4 and decreased expression on day 15. EKLF, GATA-2, Hox2.2, c-myb, Evi-1, c-kit, and PU.1 mRNA expression decreased on days 7 and 15. NFE2, CD71, SCL, and EPO-R mRNA expression remained similar on days 4 and 7 but decreased on day 15. Expression of globin genes beta- and gamma-globin increased on both day 7 and day 15 compared to day 4. Values from flow cytometric quantitation of glycophorin A, transferrin receptor (CD71), and hemoglobin A proteins correlated with gene expression results. DNA analysis demonstrated that most cells lacked DNA content by day 15, a finding consistent with enucleation and terminal erythroid differentiation. CONCLUSION: These data indicate that in vitro liquid cultures of committed CD36+ erythroid progenitor cells retain, in part, many features of erythropoiesis at the cellular and molecular level and may provide a useful model for assessment of disease-related or drug-induced erythropoietic abnormalities.

Etomoxir-induced oxidative stress in HepG2 cells detected by differential gene expression is confirmed biochemically.

Although they are known to be effective antidiabetic agents, little is published about the toxic effects of carnitine palmitoyltransferase-1 (CPT-1) inhibitors, such as etomoxir (ET). These compounds inhibit mitochondrial fatty acid beta-oxidation by irreversibly binding to CPT-1 and preventing entry of long chain fatty acids into the mitochondrial matrix. Treatment of HepG2 cells with 1 mM etomoxir for 6 h caused significant modulations in the expression of several redox-related and cell cycle mRNAs as measured by microarray analysis. Upregulated mRNAs included heme oxygenase 1 (HO1), 8-oxoguanine DNA glycosylase 1 (OGG1), glutathione reductase (GSR), cyclin-dependent kinase inhibitor 1A (CDKN1 [p21(waf1)]) and Mn+ superoxide dismutase precursor (SOD2); while cytochrome P450 1A1 (CYP1A1) and heat shock 70kD protein 1 (HSPA1A) were downregulated. Real time quantitative PCR (RT-PCR) confirmed the significant changes in 4 of 4 mRNAs assayed (CYP1A1, HO1, GSR, CDKN1), and identified 3 additional mRNA changes; 2 redox-related genes, gamma-glutamate-cysteine ligase modifier subunit (GCLM) and thioredoxin reductase (TXNRD1) and 1 DNA replication gene, topoisomerase IIalpha (TOP2A). Temporal changes in selected mRNA levels were examined by RT-PCR over 11 time points from 15 min to 24 h postdosing. CYP1A1 exhibited a 38-fold decrease by 4 h, which rebounded to a 39-fold increase by 20 h. GCLM and TXNRD1 exhibited 13- and 9-fold increases, respectively at 24 h. Etomoxir-induced oxidative stress and impaired mitochondrial energy metabolism were confirmed by a significant decrease in reduced glutathione (GSH), reduced/oxidized glutathione ratio (GSH/GSSG), mitochondrial membrane potential (MMP), and ATP levels, and by concurrent increase in oxidized glutathione (GSSG) and superoxide generation. This is the first report of oxidative stress caused by etomoxir.