Characterization of a distinct population of circulating human non-adherent endothelial forming cells and their recruitment via intercellular adhesion molecule-3.

Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133(+) population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from 'early' endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis.

Over-expression of sphingosine kinase-1 enhances a progenitor phenotype in human endothelial cells.

OBJECTIVES: The use of endothelial progenitor cells in vascular therapies has been limited due to their low numbers present in the bone marrow and peripheral blood. The aim of this study was to investigate the effect of sphingosine kinase on the de-differentiation of mature human endothelial cells toward a progenitor phenotype. METHODS: The lipid enzyme sphingosine kinase-1 was lentivirally over-expressed in human umbilical vein endothelial cells and cells were analyzed for progenitor phenotype and function. RESULTS: Sphingosine kinase-1 mRNA expression was induced approximately 150-fold with a resultant 20-fold increase in sphingosine kinase-1 enzymatic activity. The mRNA expression of the progenitor cell markers CD34, CD133, and CD117 and transcription factor NANOG increased, while the endothelial cell markers analyzed were largely unchanged. The protein level of mature endothelial cell surface markers CD31, CD144, and von Willebrand factor significantly decreased compared to controls. In addition, functional assays provided further evidence for a de-differentiated phenotype with increased viability, reduced uptake of acetylated low-density lipoprotein and decreased tube formation in Matrigel in the cells over-expressing sphingosine kinase-1. CONCLUSIONS: These findings suggest that over-expression of sphingosine kinase-1 in human endothelial cells promotes, in part, their de-differentiation to a progenitor cell phenotype, and is thus a potential tool for the generation of a large population of vascular progenitor cells for therapeutic use.

Characterization of the zebrafish (Danio rerio) mineralocorticoid receptor.

Comparison between evolutionarily distant receptors can provide critical insights into both structure and function. Sequence comparison between the mineralocorticoid receptors (MR) of the zebrafish (zMR) and human (hMR) reveals a high degree of sequence conservation in the major functional domains. We isolated a zMR cDNA to contrast the transcriptional response to a range of ligands and to establish whether a teleost MR exhibits the amino/carboxyl-terminal interaction (N/C-interaction) previously reported for the hMR. Aldosterone, deoxycorticosterone (DOC) and cortisol induced zMR transcriptional activity with similar efficacy to that observed with the hMR. The hMR antagonist, spironolactone, acted as an agonist with the zMR. The zMR exhibited an N/C-interaction in response to aldosterone but, in contrast to the hMR, cortisol and DOC predominantly stimulated the interaction in the zMR. Conservation of the N/C-interaction between evolutionarily distant MR provides evidence of functional significance.

Structural and functional characterization of the interdomain interaction in the mineralocorticoid receptor.

The mineralocorticoid receptor (MR) plays a central role in electrolyte homeostasis and in cardiovascular disease. We have previously reported a ligand-dependent N/C-interaction in the MR. In the present study we sought to fully characterize the MR N/C-interaction. By using a range of natural and synthetic MR ligands in a mammalian two-hybrid assay we demonstrate that in contrast to aldosterone, which strongly induces the interaction, the physiological ligands deoxycorticosterone and cortisol weakly promote the interaction but predominantly inhibit the aldosterone-mediated N/C-interaction. Similarly, progesterone and dexamethasone antagonize the interaction. In contrast, the synthetic agonist 9alpha-fludrocortisol robustly induces the interaction. The ability of the N/C interaction to discriminate between MR agonists suggests a subtle conformational difference in the ligand-binding domain induced by these agonists. We also demonstrate that the N/C interaction is not cell specific, consistent with the evidence from a glutathione-S-transferase pull-down assay, of a direct protein-protein interaction between the N- and C-terminal domains of the MR. Examination of a panel of deletions in the N terminus suggests that several regions may be critical to the N/C-interaction. These studies have identified functional differences between physiological MR ligands, which suggest that the ligand-specific dependence of the N/C-interaction may contribute to the differential activation of the MR that has been reported in vivo.

Structure-function relationships in the mineralocorticoid receptor.

The signature action of aldosterone in the regulation of electrolyte and fluid balance is well established. However, the role of aldosterone as an important contributor to morbidity and mortality in heart failure has gained a heightened interest in recent years, but the mechanisms of this action are not well understood. Aldosterone is the principal physiological ligand for the mineralocorticoid receptor (MR), a ligand-activated transcription factor, that also binds to the physiological glucocorticoid, cortisol. Both classes of hormones bind with similar affinity to the MR, but the molecular basis of selective and tissue-specific effects of MR ligands is not yet fully documented. The structural and functional determinants of MR function are described and their significance is discussed.

The peroxisome proliferator-activated receptor beta/delta agonist, GW501516, regulates the expression of genes involved in lipid catabolism and energy uncoupling in skeletal muscle cells.

Lipid homeostasis is controlled by the peroxisome proliferator-activated receptors (PPARalpha, -beta/delta, and -gamma) that function as fatty acid-dependent DNA-binding proteins that regulate lipid metabolism. In vitro and in vivo genetic and pharmacological studies have demonstrated PPARalpha regulates lipid catabolism. In contrast, PPARgamma regulates the conflicting process of lipid storage. However, relatively little is known about PPARbeta/delta in the context of target tissues, target genes, lipid homeostasis, and functional overlap with PPARalpha and -gamma. PPARbeta/delta, a very low-density lipoprotein sensor, is abundantly expressed in skeletal muscle, a major mass peripheral tissue that accounts for approximately 40% of total body weight. Skeletal muscle is a metabolically active tissue, and a primary site of glucose metabolism, fatty acid oxidation, and cholesterol efflux. Consequently, it has a significant role in insulin sensitivity, the blood-lipid profile, and lipid homeostasis. Surprisingly, the role of PPARbeta/delta in skeletal muscle has not been investigated. We utilize selective PPARalpha, -beta/delta, -gamma, and liver X receptor agonists in skeletal muscle cells to understand the functional role of PPARbeta/delta, and the complementary and/or contrasting roles of PPARs in this major mass peripheral tissue. Activation of PPARbeta/delta by GW501516 in skeletal muscle cells induces the expression of genes involved in preferential lipid utilization, beta-oxidation, cholesterol efflux, and energy uncoupling. Furthermore, we show that treatment of muscle cells with GW501516 increases apolipoprotein-A1 specific efflux of intracellular cholesterol, thus identifying this tissue as an important target of PPARbeta/delta agonists. Interestingly, fenofibrate induces genes involved in fructose uptake, and glycogen formation. In contrast, rosiglitazone-mediated activation of PPARgamma induces gene expression associated with glucose uptake, fatty acid synthesis, and lipid storage. Furthermore, we show that the PPAR-dependent reporter in the muscle carnitine palmitoyl-transferase-1 promoter is directly regulated by PPARbeta/delta, and not PPARalpha in skeletal muscle cells in a PPARgamma coactivator-1-dependent manner. This study demonstrates that PPARs have distinct roles in skeletal muscle cells with respect to the regulation of lipid, carbohydrate, and energy homeostasis. Moreover, we surmise that PPARbeta/delta agonists would increase fatty acid catabolism, cholesterol efflux, and energy expenditure in muscle, and speculate selective activators of PPARbeta/delta may have therapeutic utility in the treatment of hyperlipidemia, atherosclerosis, and obesity.