The expression of MMP‐1 and MMP‐9 is up‐regulated by smooth muscle cells after their cross‐talk with macrophages in high glucose conditions

Patients with diabetes mellitus have an increased risk of myocardial infarction and coronary artery disease‐related death, exhibiting highly vulnerable plaques. Many studies have highlighted the major role of macrophages (MAC) and smooth muscle cells (SMC) and the essential part of metalloproteases (MMPs) in atherosclerotic plaque vulnerability. We hypothesize that in diabetes, the interplay between MAC and SMC in high glucose conditions may modify the expression of MMPs involved in plaque vulnerability. The SMC‐MAC cross‐talk was achieved using trans‐well chambers, where human SMC were grown at the bottom and human MAC in the upper chamber in normal (NG) or high (HG) glucose concentration. After cross‐talk, the conditioned media and cells were isolated and investigated for the expression of MMPs, MCP‐1 and signalling molecules. We found that upon cross‐talk with MAC in HG, SMC exhibit: (i) augmented expression of MMP‐1 and MMP‐9; (ii) significant increase in the enzymatic activity of MMP‐9; (iii) higher levels of soluble MCP‐1 chemokine which is functionally active and involved in MMPs up‐regulation; (iv) activated PKCα signalling pathway which, together with NF‐kB are responsible for MMP‐1 and MMP‐9 up‐regulation, and (v) impaired function of collagen assembly. Taken together, our data indicate that MCP‐1 released by cell cross‐talk in diabetic conditions binds to CCR2 and triggers MMP‐1 and MMP‐9 over‐expression and activity, features that could explain the high vulnerability of atherosclerotic plaque found at diabetic patients.     

Molecular markers for X‐ray‐insensitive differentiated cells in the Inner and outer regions of the mesenchymal space in planarian Dugesia japonica

Planarian's strong regenerative ability is dependent on stem cells (called neoblasts) that are X‐ray‐sensitive and proliferative stem cells. In addition to neoblasts, another type of X‐ray‐sensitive cells was newly identified by recent research. Thus, planarian's X‐ray‐sensitive cells can be divided into at least two populations, Type 1 and Type 2, the latter corresponding to planarian's classically defined “neoblasts”. Here, we show that Type 1 cells were distributed in the outer region (OR) immediately underneath the muscle layer at all axial levels from head to tail, while the Type 2 cells were distributed in a more internal region (IR) of the mesenchymal space at the axial levels from neck to tail. To elucidate the biological significance of these two regions, we searched for genes expressed in differentiated cells that were locate close to these X‐ray‐sensitive cell populations in the mesenchymal space, and identified six genes mainly expressed in the OR or IR, named OR1, OR2, OR3, IR1, IR2 and IR3. The predicted amino acid sequences of these genes suggested that differentiated cells expressing OR1, OR3, IR1, or IR2 provide Type 1 and Type 2 cells with specific extracellular matrix (ECM) environments.     

Extracellular matrix proteins secreted from both the endometrium and the embryo are required for attachment: A study using a co‐culture model of rat blastocysts and Ishikawa cells

Integrins are expressed in a highly regulated manner at the maternal‐fetal interface during implantation. However, the significance of extracellular matrix (ECM) ligands during the integrin‐mediated embryo attachment to the endometrium is not fully understood. Thus, the distribution of fibronectin in the rat uterus and blastocyst was studied at the time of implantation. Fibronectin was absent in the uterine luminal epithelial cells but was intensely expressed in the trophoblast cells and the inner cell mass suggesting that fibronectin secreted from the blastocyst may be a possible bridging ligand for the integrins expressed at the maternal‐fetal interface. An Arg‐Gly‐Asp (RGD) peptide was used to block the RGD recognition sites on integrins, and the effect on rat blastocyst attachment to Ishikawa cells was examined. There was a significant reduction in blastocyst attachment when either the blastocysts or the Ishikawa cells were pre‐incubated with the RGD‐blocking peptide. Thus, successful attachment of the embryo to the endometrium requires the interaction of integrins on both the endometrium and the blastocyst with the RGD sequence of ECM ligands, such as fibronectin. Pre‐treatment of both blastocysts and Ishikawa cells with the RGD peptide also inhibited blastocyst attachment, but not completely, suggesting that ECM bridging ligands that do not contain the RGD sequence are also involved in embryo attachment. J. Morphol. 2013. © 2012 Wiley Periodicals, Inc.     

Chitosan‐triggered immunity to Fusarium in chickpea is associated with changes in the plant extracellular matrix architecture,stomatal closure and remodeling of the plant metabolome and proteome

Pathogen‐/microbe‐associated molecular patterns (PAMPs/MAMPs) initiate complex defense responses by reorganizing the biomolecular dynamics of the host cellular machinery. The extracellular matrix (ECM) acts as a physical scaffold that prevents recognition and entry of phytopathogens, while guard cells perceive and integrate signals metabolically. Although chitosan is a known MAMP implicated in plant defense, the precise mechanism of chitosan‐triggered immunity (CTI) remains unknown. Here, we show how chitosan imparts immunity against fungal disease. Morpho‐histological examination revealed stomatal closure accompanied by reductions in stomatal conductance and transpiration rate as early responses in chitosan‐treated seedlings upon vascular fusariosis. Electron microscopy and Raman spectroscopy showed ECM fortification leading to oligosaccharide signaling, as documented by increased galactose, pectin and associated secondary metabolites. Multiomics approach using quantitative ECM proteomics and metabolomics identified 325 chitosan‐triggered immune‐responsive proteins (CTIRPs), notably novel ECM structural proteins, LYM2 and receptor‐like kinases, and 65 chitosan‐triggered immune‐responsive metabolites (CTIRMs), including sugars, sugar alcohols, fatty alcohols, organic and amino acids. Identified proteins and metabolites are linked to reactive oxygen species (ROS) production, stomatal movement, root nodule development and root architecture coupled with oligosaccharide signaling that leads to Fusarium resistance. The cumulative data demonstrate that ROS, NO and eATP govern CTI, in addition to induction of PR proteins, CAZymes and PAL activities, besides accumulation of phenolic compounds downstream of CTI. The immune‐related correlation network identified functional hubs in the CTI pathway. Altogether, these shifts led to the discovery of chitosan‐responsive networks that cause significant ECM and guard cell remodeling, and translate ECM cues into cell fate decisions during fusariosis.     

Biogenesis,characterization, and the effect of vicenin‐gold nanoparticles on glucose utilization in 3T3‐L1 adipocytes: A bioinformatic approach to illuminate its interaction with PTP 1B and AMPK

This study reported the synthesis of Vicenin‐2 gold nanoparticles (VN‐AuNPs) and evaluated their effect on the glucose utilization efficiency of 3T3‐L1 adipocytes. The VN‐AuNPs were characterized by microscopic, DLS and spectral analysis. The bio‐reducing efficiency of Vicenin‐2 (VN) was computed and confirmed by HPLC analysis. The stability of VN‐AuNPs in various physiological media was explored. The cytotoxicity and glucose uptake assays were performed in 3T3‐L1 adipocytes. The docking of VN with PTP1B and AMPK was also performed. The color change and UV absorption at 537 nm preliminarily confirmed the VN reduced gold nanoparticles. The VN‐AuNPs appeared as spherical particles (57 nm) and face centered cubic crystals under TEM and XRD analysis, respectively. Its zeta potential was found to be ?6.53 mV. The FT‐IR spectra of VN and its AuNPs confirmed its stability. The computed reducing potential of VN was similar to the extent of VN utilized during the synthesis of VN‐AuNPs. The VN‐AuNPs showed a remarkable stability in different physiological media. At 100 µM concentration, VN‐AuNPs displayed 78.21% cell viability. A concentration dependent increase in glucose uptake was noted in 3T3‐L1 adipocytes when incubated with VN‐AuNPs. The docking data revealed a strong interaction of VN with the binding pockets of PTP1B and AMPK. This demonstrates that the fabricated VN‐AuNPs might enhance the intracellular VN availability mediated cellular glucose utilization and this would serve as a novel nanodrug for the management of diabetes. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1096–1106, 2015     

β cell membrane remodelling and procoagulant events occur in inflammation‐driven insulin impairment: a GLP‐1 receptor dependent and independent control

Inflammation and hyperglycaemia are associated with a prothrombotic state. Cell‐derived microparticles (MPs) are the conveyors of active procoagulant tissue factor (TF) and circulate at high concentration in diabetic patients. Liraglutide, a glucagon‐like peptide (GLP)‐1 analogue, is known to promote insulin secretion and β‐cell preservation. In this in vitro study, we examined the link between insulin impairment, procoagulant activity and plasma membrane remodelling, under inflammatory conditions. Rin‐m5f β‐cell function, TF activity mediated by MPs and their modulation by 1 μM liraglutide were examined in a cell cross‐talk model. Methyl‐β‐cyclodextrine (MCD), a cholesterol depletor, was used to evaluate the involvement of raft on TF activity, MP shedding and insulin secretion as well as Soluble N‐éthylmaleimide‐sensitive‐factor Attachment protein Receptor (SNARE)‐dependent exocytosis. Cytokines induced a two‐fold increase in TF activity at MP surface that was counteracted by liraglutide. Microparticles prompted TF activity on the target cells and a two‐fold decrease in insulin secretion via protein kinase A (PKA) and p38 signalling, that was also abolished by liraglutide. Large lipid raft clusters were formed in response to cytokines and liraglutide or MCD‐treated cells showed similar patterns. Cells pre‐treated by saturating concentration of the GLP‐1r antagonist exendin (9‐39), showed a partial abolishment of the liraglutide‐driven insulin secretion and liraglutide‐decreased TF activity. Measurement of caspase 3 cleavage and MP shedding confirmed the contribution of GLP‐1r‐dependent and ‐independent pathways. Our results confirm an integrative β‐cell response to GLP‐1 that targets receptor‐mediated signalling and membrane remodelling pointing at the coupling of insulin secretion and inflammation‐driven procoagulant events.     

Expression of class A scavenger receptor is enhanced by high glucose in vitro and under diabetic conditions in vivo: one mechanism for an increased rate of atherosclerosis in diabetes

In the early stage of atherosclerosis, macrophages take up chemically modified low density lipoproteins (LDL) through the scavenger receptors, leading to foam cell formation in atherosclerotic lesions. To get insight into a role of the scavenger receptors in diabetes-enhanced atherosclerotic complications, the effects on class A scavenger receptor (SR-A) of high glucose exposure in vitro as well as the diabetic conditions in vivo were determined in the present study. The in vitro experiments demonstrated that high glucose exposure to human monocyte-derived macrophages led to an increased SR-A expression with a concomitant increase in the endocytic uptake of acetylated LDL and oxidized LDL. The endocytic process was significantly suppressed by an anti-SR-A neutralizing antibody. Stability analyses revealed a significant increased stability of SR-A at a mRNA level but not a protein level, indicating that high glucose-induced up-regulation of SR-A is due largely to increased stability of SR-A mRNA. High glucose-enhanced SR-A expression was prevented by protein kinase C and NAD(P)H oxidase inhibitors as well as antioxidants. High glucose-enhanced production of intracellular peroxides was visualized in these cells, which was attenuated by an antioxidant. The in vivo experiments demonstrated that peritoneal macrophages from streptozotocin-induced diabetic mice increased SR-A expression when compared with those from nondiabetic mice. Endocytic degradation of acetylated LDL and oxidized LDL were also increased with these macrophages but not with the corresponding macrophages from diabetic SR-A knock-out mice. These in vitro and in vivo results probably suggest that reactive oxygen species generated from a protein kinase C-dependent NAD(P)H oxidase pathway plays a role in the high glucose-induced up-regulation of SR-A, leading to the increased endocytic degradation of modified LDL for foam cell formation. This could be one mechanism for an increased rate of atherosclerosis in patients with diabetes.     

Glutamate‐induced changes in the pattern of hippocampal dendrite outgrowth: A role for calcium‐dependent pathways and the microtubule cytoskeleton

Glutamate regulation of a variety of aspects of dendrite development may be involved in neuronal plasticity and neuropathology. In this study, we examine the calcium‐dependent pathways and alterations in the microtubule (MT) cytoskeleton that may mediate glutamate‐induced changes in the pattern of dendrite outgrowth. We used Fura‐2 AM and inhibitors of the calcium‐dependent proteins, calmodulin and calpain, to identify the role of specific calcium‐dependent pathways in glutamate‐regulated dendrite outgrowth. Additionally, we used a quantitative fluorescence technique to correlate changes in MT levels with glutamate‐induced changes in dendrite outgrowth. We show that the intracellular calcium concentration ([Ca²⁺ ]_i) changes in a biphasic manner over a 12‐h period in the presence of glutamate. A transient increase in [Ca²⁺ ]_i over the first hour of glutamate exposure correlated with a calmodulin‐associated increase in the rate of dendrite outgrowth, whereas a sustained increase in [Ca²⁺ ]_i was correlated with calpain‐associated dendrite retraction. Quantitative fluorescence measurements showed no net change in the level of MTs during calmodulin‐associated increases in dendrite outgrowth, but showed a significant decline in the level of MTs during calpain‐associated dendrite retraction. These findings provide insights into the intracellular mechanisms involved in activity‐dependent regulation of dendrite morphology during development and after pathology. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 159–172, 2000     

HIV‐1 promonocytic and lymphoid cell lines: an in vitro model of in vivo mitochondrial and apoptotic lesion

To characterize mitochondrial/apoptotic parameters in chronically human immunodeficiency virus (HIV‐1)‐infected promonocytic and lymphoid cells which could be further used as therapeutic targets to test pro‐mitochondrial or anti‐apoptotic strategies as in vitro cell platforms to deal with HIV‐infection. Mitochondrial/apoptotic parameters of U1 promonocytic and ACH2 lymphoid cell lines were compared to those of their uninfected U937 and CEM counterparts. Mitochondrial DNA (mtDNA) was quantified by rt‐PCR while mitochondrial complex IV (CIV) function was measured by spectrophotometry. Mitochondrial‐nuclear encoded subunits II–IV of cytochrome‐c‐oxidase (COXII‐COXIV), respectively, as well as mitochondrial apoptotic events [voltage‐dependent‐anion‐channel‐1(VDAC‐1)‐content and caspase‐9 levels] were quantified by western blot, with mitochondrial mass being assessed by spectrophotometry (citrate synthase) and flow cytometry (mitotracker green assay). Mitochondrial membrane potential (JC1‐assay) and advanced apoptotic/necrotic events (AnexinV/propidium iodide) were measured by flow cytometry. Significant mtDNA depletion spanning 57.67% (P < 0.01) was found in the U1 promonocytic cells further reflected by a significant 77.43% decrease of mitochondrial CIV activity (P < 0.01). These changes were not significant for the ACH2 lymphoid cell line. COXII and COXIV subunits as well as VDAC‐1 and caspase‐9 content were sharply decreased in both chronic HIV‐1‐infected promonocytic and lymphoid cell lines (<0.005 in most cases). In addition, U1 and ACH2 cells showed a trend (moderate in case of ACH2), albeit not significant, to lower levels of depolarized mitochondrial membranes. The present in vitro lymphoid and especially promonocytic HIV model show marked mitochondrial lesion but apoptotic resistance phenotype that has been only partially demonstrated in patients. This model may provide a platform for the characterization of HIV‐chronicity, to test novel therapeutic options or to study HIV reservoirs.     

Laying the groundwork for growth: Cell–cell and cell–ECM interactions in cardiovascular development

Cardiac development is reliant upon the spatial and temporal regulation of both genetic and chemical signals. Central to the communication of these signals are direct interactions between cells and their surrounding environment. The extracellular matrix (ECM) plays an integral role in cell communication and tissue growth throughout development by providing both structural support and chemical signaling factors. The present review discusses elements of cell–cell and cell–ECM interactions involved in cardiogenesis, and how disruption of these interactions can result in numerous heart defects. Examining the relationships between cells and their immediate environment has implications for novel and existing therapeutic strategies to combating congenital disorders. Birth Defects Research (Part C) 90:1–7, 2010. © 2010 Wiley‐Liss, Inc.     

Ram spermatozoa cocultured with epithelial cell monolayers: An in vitro model for the study of capacitation and the acrosome reaction

The effects of different epithelial cells, namely, hamster oviduct, sheep oviduct, and pig kidney epithelial cells (IBRS-2), on the viability, percentage of progressive motility (PPM), and acrosome reactions of ejaculated ram spermatozoa were investigated. Sperm aliquots were cultured on cells, cell-conditioned medium 199, or control medium 199. The PPM of unattached spermatozoa was estimated after 0, 3, 6, 9, 12, and 24 hr of incubation at 37°C under 5% CO₂ in air. Viability and the occurrence of true acrosome reactions were assessed using a triple-stain technique. Spermatozoa started to attach within 1 hr of coculture with the hamster or sheep oviductal epithelial cell (OEC) monolayers, and these spermatozoa showed vigorous tail motion. No spermatozoa were found to attach to the IBRS-2 monolayer. The PPM of unattached spermatozoa cocultured with the various types of epithelial cell monolayers for 12 hr was significantly higher than that of spermatozoa incubated in conditioned media or medium 199 alone (54% in hamster OEC vs. 40% in conditioned; 68% in sheep OEC vs. 38% in conditioned; 36% in control medium). On the other hand, after 24 hr of incubation, there were no differences in the PPM of spermatozoa cocultured with epithelial cells or incubated in conditioned media. The percentages of cells undergoing a true acrosome reaction reached maximum values (P < 0.05) in spermatozoa incubated for 9 hr in the presence of hamster OEC (22.5%) or for 12 hr on sheep OEC (20.5%) monolayers. IBRS-2, a commercial nonreproductive cell type, had a positive influence on both PPM and sperm viability but no effect on the occurrence of the acrosome reaction. Interactions leading to the acrosome reaction were thus observed only when spermatozoa were cocultured with OEC monolayers. The values of PPM in unattached sperm cells seen after 12 hr of coculture with OEC or IBRS-2 were still at a high level (52–67%) for in vitro fertilization. The coculture with OECs provides an “in vitro” model to study the capacitation processes in a situation that may resemble that occurring in vivo. Moreover, the coculture with hamster OECs may provide a convenient and standardized in vitro system to study mechanisms underlying capacitation and the acrosome reaction. © 1993 Wiley-Liss, Inc.     

The ecology and evolution of temperature‐dependent reaction norms for sex determination in reptiles: a mechanistic conceptual model

Sex‐determining mechanisms are broadly categorised as being based on either genetic or environmental factors. Vertebrate sex determination exhibits remarkable diversity but displays distinct phylogenetic patterns. While all eutherian mammals possess XY male heterogamety and female heterogamety (ZW) is ubiquitous in birds, poikilothermic vertebrates (fish, amphibians and reptiles) exhibit multiple genetic sex‐determination (GSD) systems as well as environmental sex determination (ESD). Temperature is the factor controlling ESD in reptiles and temperature‐dependent sex determination (TSD) in reptiles has become a focal point in the study of this phenomenon. Current patterns of climate change may cause detrimental skews in the population sex ratios of reptiles exhibiting TSD. Understanding the patterns of variation, both within and among populations and linking such patterns with the selection processes they are associated with, is the central challenge of research aimed at predicting the capacity of populations to adapt to novel conditions. Here we present a conceptual model that innovates by defining an individual reaction norm for sex determination as a range of incubation temperatures. By deconstructing individual reaction norms for TSD and revealing their underlying interacting elements, we offer a conceptual solution that explains how variation among individual reaction norms can be inferred from the pattern of population reaction norms. The model also links environmental variation with the different patterns of TSD and describes the processes from which they may arise. Specific climate scenarios are singled out as eco‐evolutionary traps that may lead to demographic extinction or a transition to either male or female heterogametic GSD. We describe how the conceptual principles can be applied to interpret TSD data and to explain the adaptive capacity of TSD to climate change as well as its limits and the potential applications for conservation and management programs.     

An engineered mouse embryonic stem cell model with survivin as a molecular marker and EGFP as the reporter for high throughput screening of embryotoxic chemicals in vitro

Embryonic stem cell test (EST) is the only generally accepted in vitro method for assessing embryotoxicity without animal sacrifice. However, the implementation and application of EST for regulatory embryotoxicity screening are impeded by its technical complexity, long testing period, and limited endpoint data. In this study, a high throughput embryotoxicity screening based on mouse embryonic stem cells (mESCs) expressing enhanced green fluorescent protein (EGFP) driven by a human survivin promoter and a human cytomegalovirus promoter, respectively, was developed. These EGFP expressing mESCs were cultured in three-dimensional (3D) fibrous scaffolds in microbioreactors on a multiwell plate with EGFP fluorescence signals as cell responses to chemicals monitored noninvasively in a high throughput manner. Nine chemicals with known developmental toxicity were used to validate the survivin-based embryotoxicity assay, which showed that strongly embryotoxic compounds such as 5-fluorouracil, retinoic acid, and methotrexate downregulated survivin expression by more than 50% in 3 days, while weakly embryotoxic compounds such as boric acid, methoxyacetic acid, and tetracyclin showed modest downregulation effect and nonembryotoxic saccharin, penicillin G, and acrylamide had negligible downregulation effect on survivin expression, confirming that survivin can be used as a molecular endpoint for high throughput screening of embryotoxicants. The potential developmental toxicity of three Chinese herbal medicines were also evaluated using this assay, demonstrating its application in in vitro developmental toxicity test for drug safety assessment.     

Restriction of transpiration rate under high vapour pressure deficit and non‐limiting water conditions is important for terminal drought tolerance in cowpea

Drought stress is a major constraint on cowpea productivity, since the crop is grown under warm conditions on sandy soils having low water‐holding capacity. For enhanced performance of crops facing terminal drought stress, like cowpea, water‐saving strategies are crucial. In this work, the growth and transpiration rate (TR) of 40 cowpea genotypes with contrasting response to terminal drought were measured under well‐watered conditions across different vapour pressure deficits (VPD) to investigate whether tolerant and sensitive genotypes differ in their control of leaf water loss. A method is presented to indirectly assess TR through canopy temperature (CT) and the index of canopy conductance (Ig). Overall, plants developed larger leaf area under low than under high VPD, and there was a consistent trend of lower plant biomass in tolerant genotypes. Substantial differences were recorded among genotypes in TR response to VPD, with tolerant genotypes having significantly lower TR than sensitive ones, especially at times with the highest VPD. Genotypes differed in TR response to increasing VPD, with some tolerant genotypes exhibiting a clear VPD breakpoint at about 2.25 kPa, above which there was very little increase in TR. In contrast, sensitive genotypes presented a linear increase in TR as VPD increased, and the same pattern was found in some tolerant lines, but with a smaller slope. CT, estimated with thermal imagery, correlated well with TR and Ig and could therefore be used as proxy for TR. These results indicate that control of water loss discriminated between tolerant and sensitive genotypes and may, therefore, be a reliable indicator of terminal drought stress tolerance. The water‐saving characteristics of some genotypes are hypothesised to leave more soil water for pod filling, which is crucial for terminal drought adaptation.