Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to the development of pulmonary arterial hypertension

Aims: Pulmonary arterial hypertension [1] is a proliferative disorder associated with enhanced proliferation and suppressed apoptosis of pulmonary artery smooth muscle cells (PASMCs). Reactive oxygen species (ROS) is implicated in the development of PAH and regulates the vascular tone and functions. However, which cellular signaling mechanisms are triggered by ROS in PAH is still unknown. Hence, here we wished to characterize the signaling mechanisms triggered by ROS. Methods and Results: By Western blots, we showed that increased intracellular ROS caused inhibition of the glycolytic pyruvate kinase M2 (PKM2) activity through promoting the phosphorylation of PKM2. Monocrotaline (MCT)-induced rats developed severe PAH and right ventricular hypertrophy, with a significant increase in the P-PKM2 and decrease in pyruvate kinase activity which could be attenuated with the treatments of PKM2 activators, FBP and l-serine. The antioxidant NAC, apocynin and MnTBAP had the similar protective effects in the development of PAH. In vitro assays confirmed that inhibition of PKM2 activity could modulate the flux of glycolytic intermediates in support of cell proliferation through the increased pentose phosphate pathway (PPP). Increased ROS and decreased PKM2 activity also promoted the Cav1.2 expression and intracellular calcium. Conclusion: Our data provide new evidence that PKM2 makes a critical regulatory contribution to the PAHs for the first time. Decreased pyruvate kinase M2 activity confers additional advantages to rat PASMCs by allowing them to sustain anti-oxidant responses and thereby support cell survival in PAH. It may become a novel treatment strategy in PAH by using of PKM2 activators.

The signal transduction pathways involved in the respiratory burst of murine peritoneal macrophages

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT

25-hydroxycholesterol, 7β-hydroxycholesterol and 7-ketocholesterol upregulate interleukin-8 expression independently of Toll-like receptor 1, 2, 4 or 6 signalling in human macrophages

Recent studies have shown that Toll-like receptor (TLR)- signalling contributes significantly to the inflammatory events of atherosclerosis. As products of cholesterol oxidation (oxysterols) accumulate within atherosclerotic plaque and have been proposed to contribute to inflammatory signalling in the diseased artery, we investigated the potential of 7-ketocholesterol (7-KC), 7β-hydroxycholesterol (7β-HC) and 25-hydroxycholesterol (25-HC) to stimulate inflammatory signalling via the lipid-recognising TLRs 1, 2, 4 and 6. Each oxysterol stimulated secretion of the inflammatory chemokine interleukin-8 (IL-8), but not I?B degradation or tumour necrosis factor- release from monocytic THP-1 cells. Transfection of TLR-deficient HEK-293 cells with TLRs 1, 2, 4 or 6 did not increase sensitivity to the tested oxysterols. Moreover, blockade of TLR2 or TLR4 with specific inhibitors did not reduce 25-hydroxycholesterol (25-HC) induced IL-8 release from THP-1 cells. We conclude that although the oxysterols examined in this study may contribute to increased expression of certain inflammatory genes, this occurs by mechanisms independent of TLR signalling.

Gene delivery of the elastase inhibitor elafin protects macrophages from neutrophil elastase-mediated impairment of apoptotic cell recognition

The resolution of inflammation is dependent on recognition and phagocytic removal of apoptotic cells by macrophages. Receptors for apoptotic cells are sensitive to degradation by human neutrophil elastase (HNE). We show in the present study that HNE cleaves macrophage cell surface CD14 and in so doing, reduces phagocytic recognition of apoptotic lymphocytic cells (Mutu 1). Using an improved method of adenovirus-mediated transfection of macrophages with the HNE inbibitor elafin, we demonstrate that elafin overexpression prevents CD14 cleavage and restores apoptotic cell recognition by macrophages. This approach of genetic modification of macrophages could be used to restore apoptotic cell recognition in inflammatory conditions. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

CD14-dependent clearance of apoptotic cells by human macrophages:the role of phosphatidyiserine

Apoptotic-cell clearance is dependent on several macrophage surface molecules, including CD14. Phosphatidylserine (PS) becomes externalised during apoptosis and participates in the clearance process through its ability to bind to a novel receptor, PS-R. CD14 has the proven ability to bind phospholipids and may function as an alternative receptor for the externallsed PS of apoptotic cells. Here we demonstrate that CD14 does not function preferentially as a PS receptor in apoptotic-cell clearance. Compared with phosphatidylcholine and phosphatidylethanolamine, PS was the least active phospholipid binding to human monocyte-derived macrophages and showed no specificity for soluble or membrane-anchored CD14. Significantly, PS-containing liposomes a e to inhibit CD14-dependent uptake of apoptotic cells by macrophages. PS exposure was, however, found to be insufficient for either CD14-dependent or CD14-independent apoptotic-cell uptake by phagocytes. The additional features that enable apoptotic-cell clearance are derived from mechanisms that can be divorced temporally from those responsible for the morphological features of apoptosis.

Transglutaminase 2 interacts with syndecan-4 and CD44 at the surface of human macrophages to promote removal of apoptotic cells

Tissue transglutaminase (TG2) is a multifunctional protein cross-linking enzyme that has been implicated in apoptotic cell clearance but is also important in many other cell functions including cell adhesion, migration and monocyte to macrophage differentiation. Cell surface-associated TG2 regulates cell adhesion and migration, via its association with receptors such as syndecan-4 and β1 and β3 integrins. Whilst defective apoptotic cell clearance has been described in TG2-deficient mice, the precise role of TG2 in apoptotic cell clearance remains ill-defined. Our work addresses the role of macrophage extracellular TG2 in apoptotic cell corpse clearance. Here we reveal TG2 expression and activity (cytosolic and cell surface) in human macrophages and demonstrate that inhibitors of protein crosslinking activity reduce macrophage clearance of dying cells. We show also that cell-impermeable TG2 inhibitors significantly inhibit the ability of macrophages to migrate and clear apoptotic cells through reduced macrophage recruitment to, and binding of, apoptotic cells. Association studies reveal TG2-syndecan-4 interaction through heparan sulphate side chains, and knockdown of syndecan-4 reduces cell surface TG2 activity and apoptotic cell clearance. Furthermore, inhibition of TG2 activity reduces crosslinking of CD44, reported to augment AC clearance. Thus our data define a role for TG2 activity at the surface of human macrophages in multiple stages of AC clearance and we propose that TG2, in association with heparan sulphates, may exert its effect on AC clearance via a mechanism involving the crosslinking of CD44.

Ideal Criteria for both Ideal Criteria for both X2-dy2 = M1 And X2-dy2 = M2 to have Primitive Solutions for any Integers M1, M2 Prime to D > 0

This article provides necessary and sufficient conditions for both of the Diophantine equations X^2 − DY^2 = m1 and x^2 − Dy^2 = m2 to have primitive solutions when m1 , m2 ∈ Z, and D ∈ N is not a perfect square. This is given in terms of the ideal theory of the underlying real quadratic order Z[√D].

Optical trapping with superfocused high-M2 laser diode beam

Many applications of high-power laser diodes demand tight focusing. This is often not possible due to the multimode nature of semiconductor laser radiation possessing beam propagation parameter M2 values in double-digits. We propose a method of 'interference' superfocusing of high-M2 diode laser beams with a technique developed for the generation of Bessel beams based on the employment of an axicon fabricated on the tip of a 100 μm diameter optical fiber with highprecision direct laser writing. Using axicons with apex angle 140º and rounded tip area as small as 10 μm diameter, we demonstrate 2-4 μm diameter focused laser 'needle' beams with approximately 20 μm propagation length generated from multimode diode laser with beam propagation parameter M2=18 and emission wavelength of 960 nm. This is a few-fold reduction compared to the minimal focal spot size of 11 μm that could be achieved if focused by an 'ideal' lens of unity numerical aperture. The same technique using a 160º axicon allowed us to demonstrate few-μm-wide laser 'needle' beams with nearly 100 μm propagation length with which to demonstrate optical trapping of 5-6 μm rat blood red cells in a water-heparin solution. Our results indicate the good potential of superfocused diode laser beams for applications relating to optical trapping and manipulation of microscopic objects including living biological objects with aspirations towards subsequent novel lab-on-chip configurations.

Superfocusing of high-M2 semiconductor laser beams:experimental demonstration

The focusing of multimode laser diode beams is probably the most significant problem that hinders the expansion of the high-power semiconductor lasers in many spatially-demanding applications. Generally, the 'quality' of laser beams is characterized by so-called 'beam propagation parameter' M2, which is defined as the ratio of the divergence of the laser beam to that of a diffraction-limited counterpart. Therefore, M2 determines the ratio of the beam focal-spot size to that of the 'ideal' Gaussian beam focused by the same optical system. Typically, M2 takes the value of 20-50 for high-power broad-stripe laser diodes thus making the focal-spot 1-2 orders of magnitude larger than the diffraction limit. The idea of 'superfocusing' for high-M2 beams relies on a technique developed for the generation of Bessel beams from laser diodes using a cone-shaped lens (axicon). With traditional focusing of multimode radiation, different curvatures of the wavefronts of the various constituent modes lead to a shift of their focal points along the optical axis that in turn implies larger focal-spot sizes with correspondingly increased values of M2. In contrast, the generation of a Bessel-type beam with an axicon relies on 'self-interference' of each mode thus eliminating the underlying reason for an increase in the focal-spot size. For an experimental demonstration of the proposed technique, we used a fiber-coupled laser diode with M2 below 20 and an emission wavelength in ~1μm range. Utilization of the axicons with apex angle of 140deg, made by direct laser writing on a fiber tip, enabled the demonstration of an order of magnitude decrease of the focal-spot size compared to that achievable using an 'ideal' lens of unity numerical aperture. © 2014 SPIE.

On strongly regular graphs with m2 = qm3 and m3 = qm2

2010 Mathematics Subject Classification: 05C50.

Climate variability in the Aegean Sea (marine record M2, Athos basin) during the past 1500 years

We provide new evidence on sea surface temperature (SST) variations and paleoceanographic/paleoenvironmental changes over the past 1500 years for the north Aegean Sea (NE Mediterranean). The reconstructions are based on multiproxy analyses, obtained from the high resolution (decadal to multi-decadal) marine record M2 retrieved from the Athos basin. Reconstructed SSTs show an increase from ca. 850 to 950 AD and from ca. 1100 to 1300 AD. A cooling phase of almost 1.5 °C is observed from ca. 1600 AD to 1700 AD. This seems to have been the starting point of a continuous SST warming trend until the end of the reconstructed period, interrupted by two prominent cooling events at 1832 ± 15 AD and 1995 ± 1 AD. Application of an adaptive Kernel smoothing suggests that the current warming in the reconstructed SSTs of the north Aegean might be unprecedented in the context of the past 1500 years. Internal variability in atmospheric/oceanic circulations systems as well as external forcing as solar radiation and volcanic activity could have affected temperature variations in the north Aegean Sea over the past 1500 years. The marked temperature drop of approximately ~2 °C at 1832 ± 15 yr AD could be related to the 1809 ?D 'unknown' and the 1815 AD Tambora volcanic eruptions. Paleoenvironmental proxy-indices of the M2 record show enhanced riverine/continental inputs in the northern Aegean after ca. 1450 AD. The paleoclimatic evidence derived from the M2 record is combined with a socio-environmental study of the history of the north Aegean region. We show that the cultivation of temperature-sensitive crops, i.e. walnut, vine and olive, co-occurred with stable and warmer temperatures, while its end coincided with a significant episode of cooler temperatures. Periods of agricultural growth in Macedonia coincide with periods of warmer and more stable SSTs, but further exploration is required in order to identify the causal links behind the observed phenomena. The Black Death likely caused major changes in agricultural activity in the north Aegean region, as reflected in the pollen data from land sites of Macedonia and the M2 proxy-reconstructions. Finally, we conclude that the early modern peaks in mountain vegetation in the Rhodope and Macedonia highlands, visible also in the M2 record, were very likely climate-driven.

Identification and expression modulation of a C-type lectin domain family 4 homologue that is highly expressed in monocytes/macrophages in rainbow trout (Oncorhynchus mykiss)

Peer reviewed

Identification and expression modulation of a C-type lectin domain family 4 homologue that is highly expressed in monocytes/macrophages in rainbow trout (Oncorhynchus mykiss)

Peer reviewed

Identification et caractérisation de gènes impliqués dans la virulence de Salmonella typhi suite à une analyse globale par biopuces de l'infection de macrophages humains en culture

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Overexpression of notch1 ectodomain in macrophages induces vascular defects and promotes tumor progression

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.