Derivation of endothelial cells from human embryonic stem cells in fully defined medium enables identification of lysophosphatidic acid and platelet activating factor as regulators of eNOS localization

The limited availability of human vascular endothelial cells (ECs) hampers research into EC function whilst the lack of precisely defined culture conditions for this cell type presents problems for addressing basic questions surrounding EC physiology. We aimed to generate endothelial progenitors from human pluripotent stem cells to facilitate the study of human EC physiology, using a defined serum-free protocol. Human embryonic stem cells (hESC-ECs) differentiated under serum-free conditions generated CD34⁺KDR⁺ endothelial progenitor cells after 6 days that could be further expanded in the presence of vascular endothelial growth factor (VEGF). The resultant EC population expressed CD31 and TIE2/TEK, took up acetylated low-density lipoprotein (LDL) and up-regulated expression of ICAM-1, PAI-1 and ET-1 following treatment with TNFα. Immunofluorescence studies indicated that a key mediator of vascular tone, endothelial nitric oxide synthase (eNOS), was localised to a perinuclear compartment of hESC-ECs, in contrast with the pan-cellular distribution of this enzyme within human umbilical vein ECs (HUVECs). Further investigation revealed that that the serum-associated lipids, lysophosphatidic acid (LPA) and platelet activating factor (PAF), were the key molecules that affected eNOS localisation in hESC-ECs cultures. These studies illustrate the feasibility of EC generation from hESCs and the utility of these cells for investigating environmental cues that impact on EC phenotype. We have demonstrated a hitherto unrecognized role for LPA and PAF in the regulation of eNOS subcellular localization.     

EDHF is not K+ but may be due to spread of current from the endothelium in guinea pig arterioles

Endothelium-derived hyperpolarizing factor (EDHF)-attributed hyperpolarizations and relaxations were recorded simultaneously from submucosal arterioles of guinea pigs with the use of intracellular microelectrodes and a video-based system, respectively. Membrane currents were recorded from electrically short segments of arterioles under single-electrode voltage clamp. Substance P evoked an outward current with a current-voltage relationship that was well described by the Goldman-Hodgkin-Katz equation for a K+ current, consistent with the involvement of intermediate- and small-conductance Ca2+-activated K+ channels. 1-Ethyl-2-benzimidazolinone relaxed the arterioles and evoked hyperpolarizations that were blocked by charybdotoxin, but not by iberiotoxin. Application of K+ induced depolarization under conditions in which EDHF evoked hyperpolarization. The Ba2+-sensitive component of the K+-induced current was inwardly rectifying, in contrast to the outwardly rectifying current evoked by substance P. EDHF-attributed hyperpolarizations in dye-identified smooth muscle cells were indistinguishable from those recorded from dye-identified endothelial cells in the same arterioles. These results provide evidence that EDHF is not K+ but may involve electrotonic spread of hyperpolarization from the endothelial cells to the smooth muscle cells.     

Age-associated decrease in contraction-induced activation of downstream targets of Akt/mTor signaling in skeletal muscle

In this study, we investigated the effect of age on the association of eukaryotic initiation factor 4E (eIF4E) with eukaryotic initiation factor 4G (eIF4G), as well as the activity of its binding protein (4E-BP1) and the activity of glycogen synthase kinase-3 (GSK-3) after a single bout of rat hindlimb muscle contractile activity elicited by high-frequency electrical stimulation (HFES) of the sciatic nerve. Tibialis anterior (TA) and plantaris (Pla) muscles from adult (Y; 6 mo old) and aged (O; 30 mo old) Fischer 344 x Brown Norway rats were collected immediately or 6 h after HFES. eIF4E-eIF4G association was elevated at 6 h of recovery in TA (1.9 +/- 0.2-fold, P < 0.05) and immediately and 6 h after exercise in Pla (2.1 +/- 0.3- and 2.1 +/- 0.7-fold, P < 0.05) in Y rats. No significant increase was observed in O rats. An increase in 4E-BP1 phosphorylation was observed only 6 h after HFES in TA (5.0 +/- 2.0-fold, P < 0.05) in Y rats. Phosphorylation of GSK-3alpha was increased immediately and 6 h after contraction in TA (1.6 +/- 0.3- and 4.1 +/- 0.8-fold, P < 0.05) and Pla (1.7 +/- 0.2- and 2.1 +/- 0.4-fold, P < 0.05) in Y rats and remained unaffected in O rats. Phosphorylation of GSK-3beta was observed only immediately after HFES in TA (1.5 +/- 0.2-fold, P < 0.05) in Y rats. Overall, eIF4E-eIF4G association and phosphorylation of 4E-BP1 and GSK-3 are increased after HFES in adult, but not in aged, animals. These observations suggest that the anabolic response to muscle stimulation is attenuated with aging and may contribute to the limited capacity of hypertrophy in aged animals.     

Comparative study on the therapeutic potential of neurally differentiated stem cells in a mouse model of multiple sclerosis

Background

Transplantation of neural stem cells (NSCs) is a promising novel approach to the treatment of neuroinflammatory diseases such as multiple sclerosis (MS). NSCs can be derived from primary central nervous system (CNS) tissue or obtained by neural differentiation of embryonic stem (ES) cells, the latter having the advantage of readily providing an unlimited number of cells for therapeutic purposes. Using a mouse model of MS, we evaluated the therapeutic potential of NSCs derived from ES cells by two different neural differentiation protocols that utilized adherent culture conditions and compared their effect to primary NSCs derived from the subventricular zone (SVZ).

Methodology/Principal Findings

The proliferation and secretion of pro-inflammatory cytokines by antigen-stimulated splenocytes was reduced in the presence of SVZ-NSCs, while ES cell-derived NSCs exerted differential immunosuppressive effects. Surprisingly, intravenously injected NSCs displayed no significant therapeutic impact on clinical and pathological disease outcomes in mice with experimental autoimmune encephalomyelitis (EAE) induced by recombinant myelin oligodendrocyte glycoprotein, independent of the cell source. Studies tracking the biodistribution of transplanted ES cell-derived NSCs revealed that these cells were unable to traffic to the CNS or peripheral lymphoid tissues, consistent with the lack of cell surface homing molecules. Attenuation of peripheral immune responses could only be achieved through multiple high doses of NSCs administered intraperitoneally, which led to some neuroprotective effects within the CNS.

Conclusion/Significance

Systemic transplantation of these NSCs does not have a major influence on the clinical course of rMOG-induced EAE. Improving the efficiency at which NSCs home to inflammatory sites may enhance their therapeutic potential in this model of CNS autoimmunity.     

Fossil wood charcoal assemblages from Elands Bay Cave, South Africa: implications for Late Quaternary vegetation and climates in the winter-rainfall fynbos biome

Aim The aim of this paper is to analyse fossil charcoal deposits, largely identified to the species level and spanning a sequence from the late Holocene to < 40,000 BP , in order to reconstruct Late Quaternary vegetation and climatic patterns in the western (winter-rainfall) fynbos biome of South Africa. Location The charcoals were excavated from the Elands Bay Cave (32°19S, 18°20E) on the semiarid (200–250 mmyr^?1), winter-rainfall coastline of the western fynbos biome. Methods Patterns in the charcoal data set over time were sought by manual sorting of the charcoal×sample matrix, as well as by subjecting the data to multivariate analysis. Palaeoclimatic reconstruction was attempted by comparing the climatic controls on contemporary vegetation communities that resembled the fossil assemblages. Charcoal diversity was modelled using sample age and number of charcoal fragments as explanatory variables. Results The fossil assemblages ranged from xeric communities (similar to those presently occurring at the site) during the Holocene, to more mesic thicket and fynbos vegetation in the terminal Pleistocene, to Afromontane forest and riverine woodland communities after about 18,000 BP . Diversity of the charcoal samples increased monotonically with increasing sample age. Main conclusions The results suggest that, unlike the eastern fynbos biome, which is under fundamentally different climatic controls, soil moisture conditions in the western part of the biome were higher in the Last Glacial than during the Holocene. This scenario may help to explain the higher regional richness and associated diversification in the western than eastern part of the biome.