THE ROLE OF LIPOPROTEIN(a)/APOLIPOPROTEIN(a) IN ENDOTHELIAL DYSFUNCTION: MECHANISTIC STUDIES IN VASCULAR ENDOTHELIUM

Multiple lines of evidence suggest that elevated plasma lipoprotein(a) (Lp(a)) concentrations are a significant risk factor for the development of a number of vascular diseases including coronary heart disease and stroke. Lp(a) consists of a low-density lipoprotein (LDL)-like moiety and an unique glycoprotein, apolipoprotein(a) (apo(a)), that is covalently attached to the apolipoproteinB-100 (apoB-100) component of LDL by a single disulfide bond. Many studies have suggested a role for Lp(a) in the process of endothelial dysfunction. Indeed, Lp(a) has been shown to increase both the expression of adhesion molecules on endothelial cells (EC), as well as monocyte and leukocyte chemotactic activity in these cells. We have previously demonstrated that Lp(a), through its apo(a) moiety, increases actomyosin-driven EC contraction which, as a consequence, increases EC permeability. In this thesis, we have demonstrated a role for the strong lysine-binding site in the kringle IV type 10 domain of apo(a) in increasing EC permeability, which occurs through a Rho/Rho kinase-dependent pathway. We have further validated these findings using mouse mesenteric arteries in a pressure myograph system. We also have dissected another major signaling pathway initiated by apo(a) that involves in a disruption of adherens junctions in EC. In this pathway, apo(a)/Lp(a) activates the PI3K/Akt/GSK3β-dependent pathway to facilitate nuclear translocation of beta-catenin. In the nucleus beta-catenin induced the expression of cyclooxygenase-2 (COX-2) and the secretion of prostaglandin E2 (PGE2) from the EC. Finally, we have presented data to suggest a novel inflammatory role for apo(a) in which it induces the activation of nuclear factor-kappaB through promotion of the dissociation of IkappaB from the inactive cytoplasmic complex; this allows the nuclear translocation of NFkappaB with attendant effects on the transcription of pro-inflammatory genes. Taken together, our findings may facilitate the development of new drug targets for mitigating the harmful effects of Lp(a) on vascular EC which corresponds to an early step in the process of atherogenesis.

Tight junctional abnormality in multiple sclerosis white matter affects all calibres of vessel and is associated with blood-brain barrier leakage and active demyelination

Blood-brain barrier (BBB) hyperpermeability in multiple sclerosis (MS) is associated with lesion pathogenesis and has been linked to pathology in microvascular tight junctions (TJs). This study quantifies the uneven distribution of TJ pathology and its association with BBB leakage. Frozen sections from plaque and normal-appearing white matter (NAWM) in 14 cases were studied together with white matter from six neurological and five normal controls. Using single and double immunofluorescence and confocal microscopy, the TJ-associated protein zonula occludens-1 (ZO-1) was examined across lesion types and tissue categories, and in relation to fibrinogen leakage. Confocal image data sets were analysed for 2198 MS and 1062 control vessels. Significant differences in the incidence of TJ abnormalities were detected between the different lesion types in MS and between MS and control white matter. These were frequent in oil-red O (ORO)+ active plaques, affecting 42% of vessel segments, but less frequent in ORO- inactive plaques (23%), NAWM (13%), and normal (3.7%) and neurological controls (8%). A similar pattern was found irrespective of the vessel size, supporting a causal role for diffusible inflammatory mediators. In both NAWM and inactive lesions, dual labelling showed that vessels with the most TJ abnormality also showed most fibrinogen leakage. This was even more pronounced in active lesions, where 41% of vessels in the highest grade for TJ alteration showed severe leakage. It is concluded that disruption of TJs in MS, affecting both paracellular and transcellular paths, contributes to BBB leakage. TJ abnormality and BBB leakage in inactive lesions suggests either failure of TJ repair or a continuing pathological process. In NAWM, it suggests either pre-lesional change or secondary damage. Clinically inapparent TJ pathology has prognostic implications and should be considered when planning disease-modifying therapy

Independent particle descriptions of tunneling using the many-body quantum transport approach

Currents across thin insulators are commonly taken as single electrons moving across classically forbidden regions; this independent particle picture is well-known to describe most tunneling phenomena. Examining quantum transport from a different perspective, i.e., by explicit treatment of electron-electron interactions, we evaluate different single particle approximations with specific application to tunneling in metal-molecule-metal junctions. We find maximizing the overlap of a Slater determinant composed of single-particle states to the many-body current-carrying state is more important than energy minimization for defining single-particle approximations in a system with open boundary conditions. Thus the most suitable single particle effective potential is not one commonly in use by electronic structure methods, such as the Hartree-Fock or Kohn-Sham approximations.

Current-induced forces in atomic-scale conductors

We present a self-consistent tight-binding formalism to calculate the forces on individual atoms due to the flow of electrical current in atomic-scale conductors. Simultaneously with the forces, the method yields the local current density and the local potential in the presence of current flow, allowing a direct comparison between these quantities. The method is applicable to structures of arbitrary atomic geometry and can be used to model current-induced mechanical effects in realistic nanoscale junctions and wires. The formalism is implemented within a simple Is tight-binding model and is applied to two model structures; atomic chains and a nanoscale wire containing a vacancy.

Fast Response Exhaust Gas Temperature Measurement in IC Engines

The harsh environment presented by engines, particularly in the exhaust systems, often necessitates the use of robust and therefore low bandwidth temperature sensors. Consequently, high frequencies are attenuated in the output. One technique for addressing this problem involves measuring the gas temperature using two sensors with different time-constants and mathematically reconstructing the true gas temperature from the resulting signals. Such a technique has been applied in gas turbine, rocket motor and combustion research. A new reconstruction technique based on difference equations has been developed and its effectiveness proven theoretically. The algorithms have been successfully tested and proven on experimental data from a rig that produces cyclic temperature variations. These tests highlighted that the separation of the thermocouple junctions must be very small to ensure that both sensors are subjected to the same gas temperatures. Exhaust gas temperatures were recorded by an array of thermocouples during transient operation of a high performance two-stroke engine. The results show that the increase in bandwidth arising from the dual sensor technique allowed accurate measurement of exhaust gas temperature with relatively robust thermocouples. Finally, an array of very fine thermocouples (12.5 - 50 microns) was used to measure the in-cycle temperature variation in the exhaust.

The role of advanced glycation end products in retinal microvascular leukostasis

PURPOSE: A critical event in the pathogenesis of diabetic retinopathy is the inappropriate adherence of leukocytes to the retinal capillaries. Advanced glycation end-products (AGEs) are known to play a role in chronic inflammatory processes, and the authors postulated that these adducts may play a role in promoting pathogenic increases in proinflammatory pathways within the retinal microvasculature. METHODS: Retinal microvascular endothelial cells (RMECs) were treated with glycoaldehyde-modified albumin (AGE-Alb) or unmodified albumin (Alb). NFkappaB DNA binding was measured by electromobility shift assay (EMSA) and quantified with an ELISA: In addition, the effect of AGEs on leukocyte adhesion to endothelial cell monolayers was investigated. Further studies were performed in an attempt to confirm that this was AGE-induced adhesion by co-incubation of AGE-treated cells with soluble receptor for AGE (sRAGE). Parallel in vivo studies of nondiabetic mice assessed the effect of intraperitoneal delivery of AGE-Alb on ICAM-1 mRNA expression, NFkappaB DNA-binding activity, leukostasis, and blood-retinal barrier breakdown. RESULTS: Treatment with AGE-Alb significantly enhanced the DNA-binding activity of NFkappaB (P = 0.0045) in retinal endothelial cells (RMECs) and increased the adhesion of leukocytes to RMEC monolayers (P = 0.04). The latter was significantly reduced by co-incubation with sRAGE (P <0.01). Mice infused with AGE-Alb demonstrated a 1.8-fold increase in ICAM-1 mRNA when compared with control animals (P <0.001, n = 20) as early as 48 hours, and this response remained for 7 days of treatment. Quantification of retinal NFkappaB demonstrated a threefold increase with AGE-Alb infusion in comparison to control levels (AGE Alb versus Alb, 0.23 vs. 0.076, P <0.001, n = 10 mice). AGE-Alb treatment of mice also caused a significant increase in leukostasis in the retina (AGE-Alb versus Alb, 6.89 vs. 2.53, n = 12, P <0.05) and a statistically significant increase in breakdown of the blood-retinal barrier (AGE Alb versus Alb, 8.2 vs. 1.6 n = 10, P <0.001). CONCLUSIONS: AGEs caused upregulation of NFkappaB in the retinal microvascular endothelium and an AGE-specific increase in leukocyte adhesion in vitro was also observed. In addition, increased leukocyte adherence in vivo was demonstrated that was accompanied by blood-retinal barrier dysfunction. These findings add further evidence to the thinking that AGEs may play an important role in the pathogenesis of diabetic retinopathy.

Cardiovascular disease and hypertension are strong risk factors for choroidal neovascularization.

Purpose: To investigate the association of cardiovascular risk factors and inflammatory markers with neovascular age-related macular degeneration (AMD). Design: Cross-sectional case-control study. Participants: Of the 410 of the =65-year-old community sample invited to attend, 205 participated (50% response rate). Of the 215 clinic attendees who were invited to participate, 212 agreed to take part (98% response rate). A diagnosis of neovascular AMD in at least one eye was made in 193 clinic attendees and 2 of the community sample. Methods: Clinic and community participants underwent a detailed ophthalmic examination with fundus imaging, were interviewed for assessment of putative risk factors, and provided a blood sample. Analysis included levels of serum lipids, intercellular adhesion molecule 1 (ICAM), vascular cellular adhesion molecule (VCAM), and C-reactive protein (CRP). All participants were classified by fundus image grading on the basis of the eye with more severe AMD features. Main Outcome Measure: Neovascular AMD. Results: There were 195 participants with choroidal neovascularization in at least one eye, 97 nonneovascular AMD participants, and 115 controls (no drusen or pigmentary irregularities in either eye). In confounder-adjusted logistic regression, a history of cardiovascular disease was strongly associated with neovascular AMD (odds ratio [OR], 7.53; 95% confidence interval [CI], 2.78-20.41). Cigarette smoking (OR, 3.71; 95% CI, 1.25-11.06), being in the highest quartile of body mass index (OR, 3.82; 95% CI, 1.22-12.01), stage 2 hypertension (OR, 3.21; 95% CI, 1.14-8.98), and being in the highest quartile of serum cholesterol (OR, 4.66; 95% CI, 1.35-16.13) were positively associated with neovascular AMD. There was no association between AMD status and serum CRP, ICAM, or VCAM. Conclusions: Our results suggest that cardiovascular disease plays an etiological role in the development of choroidal neovascularization in a proportion of older adults and highlight the importance of control of blood pressure and cholesterol, avoidance of smoking, and maintenance of a normal body weight. © 2008 American Academy of Ophthalmology.

The effect of treatment of cystic fibrosis pulmonary exacerbations on airways and systemic inflammation

Background: Chronic infection in cystic fibrosis (CF) and airway inflammation leads to progressive lung injury Neutrophils are considered to be responsible for the onset and promotion of the inflammatory response within the CF lung. The relationship between infection and inflammation is complex but circulating inflammatory markers may not truly reflect the local inflammatory response in the lung. The aims of this study were to investigate the change of inflammatory biomarkers and cells within sputum and blood before and after intravenous antibiotics for a pulmonary exacerbation of CF Methods: Assays included neutrophil elastase (NE) and complex, interleukin-8 (IL-8) and soluble intercellular adhesion molecule-1 (sICAM-1), fas ligand (FAS-L), and TNFr-1. Analysis of sputum cell differential and absolute cell counts and immunocytochemistry (CD11b and CD95) on sputum and isolated blood neutrophils were carried out. Results: There were no significant differences in absolute or differential sputum cell counts or sputum sol measurements following antibiotics. There was a significant increase in the percentage of blood neutrophils with minimal CD11b staining, 28 (4.1) mean percentage (SEM) versus41 (2.9) and a decrease in the percentage showing maximal staining 30 (0.5) versus 15 (2.5). There was a significant increase in the percentage of blood neutrophils without CD95 staining, 43 (5.4) mean percentage versus 52 (5.1). Conclusion: These data suggest a modifiable systemic response to IV antibiotics but a local sustained inflammatory response in the lung.

Getting to the root of neuronal signalling in plant-parasitic nematodes using RNA interference

A variety of genes expressed in preparasitic second-stage juveniles (J2) of plant-parasitic nematodes appear to be vulnerable to RNA interference (RNAi) in vitro by coupling double-stranded (ds)RNA soaking with the artificial stimulation of pharyngeal pumping. Also, there is mounting evidence that the in planta generation of nematode-specific double-stranded RNAs (dsRNAs) has real utility in the control of these pests. Although neuronally-expressed genes in Caenorhabditis elegans are commonly refractory to RNAi, we have discovered that neuronally-expressed genes in plant-parasitic nematodes are highly susceptible to RNAi and that silencing can be induced by simple soaking procedures without the need for pharyngeal stimulation. Since most front-line anthelmintics that are used for the control of nematode parasites of animals and humans act to disrupt neuromuscular coordination, we argue that intercellular signalling processes associated with neurons have much appeal as targets for transgenic plant-based control strategies for plant-parasitic nematodes. FMRFamide-like peptides (FLPs) are a large family of neuropeptides which are intimately associated with neuromuscular regulation, and our studies on flp gene function in plant-parasitic nematodes have revealed that their expression is central to coordinated locomotory activities. We propose that the high level of conservation in nervous systems across nematodes coupled with the RNAi-susceptibility of neuronally-expressed genes in plant-parasitic nematodes provides a valuable research tool which could be used to interrogate neuronal signalling processes in nematodes.

Compact ridged-waveguide bandpass filters and diplexers

A ridged-waveguide diplexer incorporating integrated compact ridged-waveguide filters and a ridged-waveguide T-junction is presented. The compact size of the filter is achieved by integrating of bandpass periodic filter, and a low-pass structure is used in order to suppress spurious responses. Simulation using the mode-matching method has been carried out for ridge-waveguide filters and T junctions and the results are presented. The measured results are in good agreement with the simulated results. (C) 2004 Wiley Periodicals, Inc.

Design of asymmetrical RF and microwave bandpass filters by computer optimization

This paper presents an optimization-based approach to the design of asymmetrical filter structures having the maximum number of return- or insertion-loss ripples in the passband such as those based upon Chebyshev function prototypes. The proposed approach. has the following advantages over the general purpose optimization techniques adopted previously such as: less frequency sampling is required, optimization is carried out with respect to the Chebyshev (or minimax) criterion, the problem of local minima does not arise, and optimization is usually only required for the passband. When implemented around an accurate circuit simulation, the method can be used to include all the effects of discontinuities, junctions, fringing, etc. to reduce the amount of tuning required in the final filter. The design of asymmetrical ridged-waveguide bandpass filters is considered as an example. Measurements on a fabricated filter confirm the accuracy of the design procedure.

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.

Interstitial Cells in the Urinary Bladder-Localization and Function

Aims: This review summarizes the currently available literature on the localization and proposed functions of a novel group of cells in the urinary bladder known as interstitial cells or interstitial cells of Cajal (ICC).

Methods: On-line searches of "Pubmed" for bladder, c-Kit, ICC, interstitial cell and myofibroblast were performed to identify relevant studies for the review.

Results: The literature contains substantial data that several sub-populations of ICC are present in the wall of the mammalian urinary bladder. These are located in the lamina propria and within the detrusor with distinctive cell shapes and morphological arrangements. Bladder ICC are identified with transmission electron microscopy or by immunohistochemical labeling using antibodies to the Kit receptor which is an established ICC marker. Lamina propria-ICC form a loose network connected via Cx43 gap junctions and are associated with mucosal nerves. Detrusor ICC track the smooth muscle bundles and make frequent contacts with intramural nerves. Both groups of ICC exhibit spontaneous electrical and Ca²⁺-signalling and also respond to application of neurotransmitter substances including ATP and carbachol. There is emerging evidence that the expression of ICC is upregulated in pathophysiological conditions including the overactive bladder.

Conclusions: There is now a convincing body of evidence that specialized ICC are present in the urinary bladder making important associations with other cells that make up the bladder wall and possessing physiological properties consistent with a role of bladder activity modulation. Neurourol. Urodynam. 29: 82–87, 2010. © 2009 Wiley-Liss, Inc.

Cytopathogenesis of Sendai virus in well-differentiated primary pediatric bronchial epithelial cells

Sendai virus (SeV) is a murine respiratory virus of considerable interest as a gene therapy or vaccine vector, as it is considered nonpathogenic in humans. However, little is known about its interaction with the human respiratory tract. To address this, we developed a model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs). These physiologically authentic cultures are comprised of polarized pseudostratified multilayered epithelium containing ciliated, goblet, and basal cells and intact tight junctions. To facilitate our studies, we rescued a replication-competent recombinant SeV expressing enhanced green fluorescent protein (rSeV/eGFP). rSeV/eGFP infected WD-PBECs efficiently and progressively and was restricted to ciliated and nonciliated cells, not goblet cells, on the apical surface. Considerable cytopathology was evident in the rSeV/eGFP-infected cultures postinfection. This manifested itself by ciliostasis, cell sloughing, apoptosis, and extensive degeneration of WD-PBEC cultures. Syncytia were also evident, along with significant basolateral secretion of proinflammatory chemokines, including IP-10, RANTES, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), interleukin 6 (IL-6), and IL-8. Such deleterious responses are difficult to reconcile with a lack of pathogenesis in humans and suggest that caution may be required in exploiting replication-competent SeV as a vaccine vector. Alternatively, such robust responses might constitute appropriate normal host responses to viral infection and be a prerequisite for the induction of efficient immune responses.

Inflammation markers are associated with Cardiovascular diseases risk in adolescents The Young Hearts Project 2000

Purose: The traditional approach for identifying subjects at risk from cardiovascular diseases (CVD) is to determine the extent of clustering of biological risk factors adjusted for lifestyle. Recently, markers of endothelial dysfunction and low grade inflammation, including high sensitivity C-reactive protein (hsCRP), soluble intercellular adhesion molecules (sICAM), and soluble vascular adhesion molecules (sVCAM), have been included in the detection for high risk individuals. However, the relationship of these novel biomarkers with CVD risk in adolescents remains unclear. The purpose of this study, therefore, was to establish the association of hsCRP, sICAM, and sVCAM with CVD risk in an adolescent population.
Methods: Data from the Young Hearts 2000 cross-sectional cohort study, carried out in 1999-2001, were used. From a total of 2,017 male and female participants, 95 obese subjects were identified and matched according to age, sex, and cigarette smoking, with 95 overweight and 95 normal-weight adolescents. Clustered CVD risk was computed using a sum of Z-scores of biological risk factors. The relationship was described using multiple linear regression analyses.
Results: hsCRP, sICAM, and sVCAM showed significant associations with CVD risk. hsCRP and sICAM had a positive relation with CVD risk, whereas sVCAM showed an inverse relationship. In this study, lifestyle factors showed no relation with CVD risk.
Conclusion: The results fit the hypothesized role of low grade inflammation and endothelial dysfunction in CVD risk in asymptomatic adolescents. The inverse relationship of VCAM, however, is hard to explain and indicates the complex mechanisms underlying CVD. Further research is needed to draw firm conclusions on the biomarkers used.