Molecular analysis of endothelial progenitor cell (EPC) subtypes reveals two distinct cell populations with different identities

BACKGROUND: The term endothelial progenitor cells (EPCs) is currently used to refer to cell populations which are quite dissimilar in terms of biological properties. This study provides a detailed molecular fingerprint for two EPC subtypes: early EPCs (eEPCs) and outgrowth endothelial cells (OECs). METHODS: Human blood-derived eEPCs and OECs were characterised by using genome-wide transcriptional profiling, 2D protein electrophoresis, and electron microscopy. Comparative analysis at the transcript and protein level included monocytes and mature endothelial cells as reference cell types. RESULTS: Our data show that eEPCs and OECs have strikingly different gene expression signatures. Many highly expressed transcripts in eEPCs are haematopoietic specific (RUNX1, WAS, LYN) with links to immunity and inflammation (TLRs, CD14, HLAs), whereas many transcripts involved in vascular development and angiogenesis-related signalling pathways (Tie2, eNOS, Ephrins) are highly expressed in OECs. Comparative analysis with monocytes and mature endothelial cells clusters eEPCs with monocytes, while OECs segment with endothelial cells. Similarly, proteomic analysis revealed that 90% of spots identified by 2-D gel analysis are common between OECs and endothelial cells while eEPCs share 77% with monocytes. In line with the expression pattern of caveolins and cadherins identified by microarray analysis, ultrastructural evaluation highlighted the presence of caveolae and adherens junctions only in OECs. CONCLUSIONS: This study provides evidence that eEPCs are haematopoietic cells with a molecular phenotype linked to monocytes; whereas OECs exhibit commitment to the endothelial lineage. These findings indicate that OECs might be an attractive cell candidate for inducing therapeutic angiogenesis, while eEPC should be used with caution because of their monocytic nature.

Abnormal endothelial tight junctions in active lesions and normal-appearing white matter in multiple sclerosis

Blood-brain barrier (BBB) breakdown, demonstrable in vivo by enhanced MRI is characteristic of new and expanding inflammatory lesions in relapsing remitting and chronic progressive multiple sclerosis (MS). Subtle leakage may also occur in primary progressive MS. However, the anatomical route(s) of BBB leakage have not been demonstrated. We investigated the possible involvement of interendothelial tight junctions (TJ) by examining the expression of TJ proteins (occludin and ZO-1 ) in blood vessels in active MS lesions from 8 cases of MS and in normal-appearing white (NAWM) matter from 6 cases. Blood vessels (10-50 per frozen section) were scanned using confocal laser scanning microscopy to acquire datasets for analysis. TJ abnormalities manifested as beading, interruption, absence or diffuse cytoplasmic localization of fluorescence, or separation of junctions (putative opening) were frequent (affecting 40% of vessels) in oil red-O-positive active plaques but less frequent in NAWM (15%), and in normal (

Oxaliplatin induces hyperexcitability at motor and autonomic neuromuscular junctions through effects on voltage-gated sodium channels

Oxaliplatin, an effective cytotoxic treatment in combination with 5-fluorouracil for colorectal cancer, is associated with sensory, motor and autonomic neurotoxicity. Motor symptoms include hyperexcitability while autonomic effects include urinary retention, but the cause of these side-effects is unknown. We examined the effects on motor nerve function in the mouse hemidiaphragm and on the autonomic system in the vas deferens. In the mouse diaphragm, oxaliplatin (0.5 mM) induced multiple endplate potentials (EPPs) following a single stimulus, and was associated with an increase in spontaneous miniature EPP frequency. In the vas deferens, spontaneous excitatory junction potential frequency was increased after 30 min exposure to oxaliplatin; no changes in resting Ca(2+) concentration in nerve terminal varicosities were observed, and recovery after stimuli trains was unaffected.In both tissues, an oxaliplatin-induced increase in spontaneous activity was prevented by the voltage-gated Na(+) channel blocker tetrodotoxin (TTX). Carbamazepine (0.3 mM) also prevented multiple EPPs and the increase in spontaneous activity in both tissues. In diaphragm, beta-pompilidotoxin (100 microM), which slows Na(+) channel inactivation, induced multiple EPPs similar to oxaliplatin's effect. By contrast, blockers of K(+) channels (4-aminopyridine and apamin) did not replicate oxaliplatin-induced hyperexcitability in the diaphragm. The prevention of hyperexcitability by TTX blockade implies that oxaliplatin acts on nerve conduction rather than by effecting repolarisation. The similarity between beta-pompilidotoxin and oxaliplatin suggests that alteration of voltage-gated Na(+) channel kinetics is likely to underlie the acute neurotoxic actions of oxaliplatin.

Outgrowth Endothelial Cells: Characterization and Their Potential for Reversing Ischemic Retinopathy

Purpose. Endothelial progenitor cells (EPCs) have potential for promoting vascular repair and revascularization of ischemic retina. However, the highly heterogeneous nature of these cells causes confusion when assessing their biological functions. The purpose of this study was to provide a comprehensive comparison between the two main EPC subtypes, early EPCs (eEPCs) and outgrowth endothelial cells (OECs), and to establish the potential of OECs as a novel cell therapy for ischemic retinopathy.

Methods. Two types of human blood-derived EPCs were isolated and compared using immunophenotyping and multiple in vitro functional assays to assess interaction with retinal capillary endothelial cells and angiogenic activity. OECs were delivered intravitreally in a mouse model of ischemic retinopathy, and flat mounted retinas were examined using confocal microscopy.

Results. These data indicate that eEPCs are hematopoietic cells with minimal proliferative capacity that lack tube-forming capacity. By contrast, OECs are committed to an endothelial lineage and have significant proliferative and de novo tubulogenic potential. Furthermore, only OECs are able to closely interact with endothelial cells through adherens and tight junctions and to integrate into retinal vascular networks in vitro. The authors subsequently chose OECs to test a novel cell therapy approach for ischemic retinopathy. Using a murine model of retinal ischemia, they demonstrated that OECs directly incorporate into the resident vasculature, significantly decreasing avascular areas, concomitantly increasing normovascular areas, and preventing pathologic preretinal neovascularization.

Conclusions. As a distinct EPC population, OECs have potential as therapeutic cells to vascularize the ischemic retina.

Plasmonic junctions with cucurbit[5]uril 'glue': fabrication of precise sub-nm junctions in gold nanoparticle assemblies

Aggregation of gold nanoparticles with rigid cucurbit[5]uril molecules generates fixed inter-particle separations of 0.91 nm. These nanoparticle assemblies possess discrete plasmonic modes which elucidate nanoscale growth and serve as molecular-recognition based SERS substrates.

Precise Subnanometer Plasmonic Junctions for SERS within Gold Nanoparticle Assemblies Using Cucurbit[n]uril "Glue"

Cucurbit[n]urils (CB[n]) are macrocyclic host molecules with subnanometer dimensions capable of binding to gold surfaces. Aggregation of gold nanoparticles with CB[n] produces a repeatable, fixed, and rigid interparticle separation of 0.9 nm, and thus such assemblies possess distinct and exquisitely sensitive plasmonics. Understanding the plasmonic evolution is key to their use as powerful SERS substrates. Furthermore, this unique spatial control permits fast nanoscale probing of the plasmonics of the aggregates "glued" together by CBs within different kinetic regimes using simultaneous extinction and SERS measurements. The kinetic rates determine the topology of the aggregates including the constituent structural motifs and allow the identification of discrete plasmon modes which are attributed to disordered chains of increasing lengths by theoretical simulations. The CBs directly report the near-field strength of the nanojunctions they create via their own SERS, allowing calibration of the enhancement. Owing to the unique barrel-shaped geometry of CB[n] and their ability to bind "guest" molecules, the aggregates afford a new type of in situ self-calibrated and reliable SERS substrate where molecules can be selectively trapped by the CB[n] and exposed to the nanojunction plasmonic field. Using this concept, a powerful molecular-recognition-based SERS assay is demonstrated by selective cucurbit[n]uril host-guest complexation.

OPTIMIZATION OF THE SLOW-MODE PLASMON POLARITON IN LIGHT-EMITTING TUNNEL-JUNCTIONS

Light emitted from metal/oxide/metal tunnel junctions can originate from the slow-mode surface plasmon polariton supported in the oxide interface region. The effective radiative decay of this mode is constrained by competition with heavy intrinsic damping and by the need to scatter from very small scale surface roughness; the latter requirement arises from the mode's low phase velocity and the usual momentum conservation condition in the scattering process. Computational analysis of conventional devices shows that the desirable goals of decreased intrinsic damping and increased phase velocity are influenced, in order of priority, by the thickness and dielectric function of the oxide layer, the type of metal chosen for each conducting electrode, and temperature. Realizable devices supporting an optimized slow-mode plasmon polariton are suggested. Essentially these consist of thin metal electrodes separated by a dielectric layer which acts as a very thin (a few nm) electron tunneling barrier but a relatively thick (several 10's of nm) optically lossless region. (C) 1995 American Institute of Physics.

DEPENDENCE OF LIGHT OUTPUT FROM NOMINALLY SMOOTH AL-OX-AU TUNNEL-JUNCTIONS ON ELECTRODE MORPHOLOGY

The light output from nominally smooth Al-Ox-Au tunnel junctions is observed to be substantially independent of the deposition rate of the Au film electrode. Films deposited quickly (2 nm s-1) and those deposited slowly (0.16 nm s-1) have similar spectral dependences and intensities. (This is in contrast to roughened films where those deposited quickly give out less light, especially towards the blue end of the spectrum.) The behaviour can be interpreted in terms of the ratio l(ph)/l(em) where l(ph) and l(em) are the mean free paths of surface plasmons between external photon emissions and internal electromagnetic absorptions respectively. Once l(ph)/l(em) exceeds 100, as it does on smooth films, grain size has little further effect on the spectral shape of the light output. In fast-deposited films there are two compensating effects on the output intensity: grain boundary scattering decreases it and greater surface roughness increases it.

CHARACTERIZATION OF THE AU SURFACE-STRUCTURE OF AL-L-AU LIGHT-EMITTING TUNNEL-JUNCTIONS

Visible light is emitted from the Au-air interface of Al-I-Au thin-film tunnel junctions (deposited over a thin layer of CaF2 on glass) as a result of the decay of surface plasmon polaritons (SPPs). We show the surface topography of such a Au film and relate its large-scale features to the outcoupling of fast SPP's to photons. The absence of short-scale roughness features is explained by thier disappearance through surface diffusion. To confirm this a controlled sequence of 5-nm, 20-ms scanning tunneling microscope (STM) W tip crashes has been used to produce indentations 3 nm deep with a lateral dimension of 5-7 nm on a Au crystal in air at room temperature. Four sequences of indentations were drawn in the form of a square box. Right from the start, feature decay is observed and over a period of 2 h a succession of images shows that the structure disappears into the background as a result of surface diffusion. The surface diffusion constant is estimated to be 10(-18) cm2 s-1. The lack of light output via slow mode SPPs is an inevitable consequence of surface annealing.