Advanced glycation end products and diabetic retinopathy

Diabetic retinopathy (DR) has a complex pathogenesis which is impacted by a raft of systemic abnormalities and tissue-specific alterations occurring in response to the diabetes milieu. Many pathogenic processes play key roles in retinal damage in diabetic patients. One such pathway is the formation and accumulation of advanced glycation endproducts (AGEs) and advanced lipoxidation end products (ALEs) which are relevant modifications with roles in the initiation and progression of pathology. In this review, AGE/ALE formation in the diabetic retina is discussed alongside their impact on retinal cell function. In addition, various inhibitors of the AGE-RAGE system and their therapeutic utility for DR will also be evaluated.

Towards optimal software engineering: learning from agile practice

In essence, optimal software engineering means creating the right product, through the right process, to the overall satisfaction of everyone involved. Adopting the agile approach to software development appears to have helped many companies make substantial progress towards that goal. The purpose of this paper is to clarify that contribution from comparative survey information gathered in 2010 and 2012. The surveys were undertaken in software development companies across Northern Ireland. The paper describes the design of the surveys and discusses optimality in relation to the results obtained. Both surveys aimed to achieve comprehensive coverage of a single region rather than rely on a voluntary sample. The main outcome from the work is a collection of insights into the nature and advantages of agile development, suggesting how further progress towards optimality might be achieved.

The Maturation of Agile Software Development Principles and Practice: Observations on Successive Industrial Studies in 2010 and 2012

The agile model of software development has been mainstream for several years, and is now in a phase where its principles and practices are maturing. The purpose of this paper is to describe the results of an industry survey aimed at understanding how maturation is progressing. The survey was taken across 40 software development companies in Northern Ireland at the beginning of 2012. The paper describes the design of the survey and examines maturity by comparing the results obtained in 2012 with those from a study of agile adoption in the same region in 2010. Both surveys aimed to achieve comprehensive coverage of a single area rather than rely on a voluntary sample. The main outcome from the work is a collection of ‘insights’ into the nature and practice of agile development, the main two of which are reported in this paper.

Advanced glycation endproduct modified basement membrane attenuates endothelin-1 induced [Ca2+]i signalling and contraction in retinal microvascular pericytes

PURPOSE: To assess the effects of advanced glycation endproduct (AGE) modification of vascular basement membrane (BM) on endothelin-1 (Et-1) induced intracellular [Ca2+] ([Ca2+]i) homeostasis and contraction in retinal microvascular pericytes (RMP). METHODS: RMPs were isolated from bovine retinal capillaries and propagated on AGE modified BM extract (AGE-BM) or non-modified native BM. Cytosolic Ca2+ was estimated using fura-2 microfluorimetry and cellular contraction determined by measurement of planimetric cell surface area. ETA receptor mRNA and protein expression was assessed by real time RT-PCR and western blotting, respectively. RESULTS: Exogenous endothelin-1 (Et-1) evoked rises in [Ca2+]i and contraction in RMPs were found to be mediated entirely through ETA receptor (ETAR) activation. Both peak and plateau phases of the Et-1 induced [Ca2+]i response and contraction were impaired in RMPs propagated on AGE modified BM. ETAR mRNA expression remained unchanged in RMPs exposed to native or AGE-BM, but protein expression for ETAR (66 kDa) was lower in the AGE exposed cells. CONCLUSIONS: These results suggest that substrate derived AGE crosslinks can influence RMP physiology by mechanisms which include disruption of ETA receptor signalling. AGE modification of vascular BMs may contribute to the retinal hemodynamic abnormalities observed during diabetes.

Substrates modified by advanced glycation end-products cause dysfunction and death in retinal pericytes by reducing survival signals mediated by platelet-derived growth factor

AIMS/HYPOTHESIS: Premature death of retinal pericytes is a pathophysiological hallmark of diabetic retinopathy. Among the mechanisms proposed for pericyte death is exposure to AGE, which accumulate during diabetes. The current study used an in vitro model, whereby retinal pericytes were exposed to AGE-modified substrate and the mechanisms underlying pericyte death explored. METHODS: Pericytes were isolated from bovine retinal capillaries and propagated on AGE-modified basement membrane (BM) extract or non-modified native BM. The extent of AGE modification was analysed. Proliferative responses of retinal pericytes propagated on AGE-modified BM were investigated using a 5-bromo-2-deoxy-uridine-based assay. The effect of extrinsically added platelet-derived growth factor (PDGF) isoforms on these proliferative responses was also analysed alongside mRNA expression of the PDGF receptors. Apoptotic death of retinal pericytes grown on AGE-modified BM was investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling labelling, mitochondrial membrane depolarisation and by morphological assessment. We also measured both the ability of PDGF to reverse Akt dephosphorylation that was mediated by AGE-modified BM, and increased pericyte apoptosis. RESULTS: Retinal pericytes exposed to AGE-modified BM showed reduced proliferative responses in comparison to controls (p

Increased expression of bronchial epithelial transient receptor potential vanilloid 1 channels in severe asthma

BACKGROUND: The airway epithelium is exposed to a range of physical and chemical irritants in the environment that are known to trigger asthma. Transient receptor potential (TRP) cation channels play a central role in sensory responses to noxious physical and chemical stimuli. Recent genetic evidence suggests an involvement of transient receptor potential vanilloid 1 (TRPV1), one member of the vanilloid subfamily of TRP channels, in the pathophysiology of asthma. The functional expression of TRPV1 on airway epithelium has yet to be elucidated.

OBJECTIVE: In this study we examined the molecular, functional, and immunohistochemical expression of TRPV1 in asthmatic and healthy airways.

METHODS: Bronchial biopsy specimens and bronchial brushings were obtained from healthy volunteers (n = 18), patients with mild-to-moderate asthma (n = 24), and patients with refractory asthma (n = 22). Cultured primary bronchial epithelial cells from patients with mild asthma (n = 4), nonasthmatic coughers (n = 4), and healthy subjects (n = 4) were studied to investigate the functional role of TRPV1.

RESULTS: Quantitative immunohistochemistry revealed significantly more TRPV1 expression in asthmatic patients compared with healthy subjects, with the greatest expression in patients with refractory asthma (P = .001). PCR and Western blotting analysis confirmed gene and protein expression of TRPV1 in cultured primary bronchial epithelial cells. Patch-clamp electrophysiology directly confirmed functional TRPV1 expression in all 3 groups. In functional assays the TRPV1 agonist capsaicin induced dose-dependent IL-8 release, which could be blocked by the antagonist capsazepine. Reduction of external pH from 7.4 to 6.4 activated a capsazepine-sensitive outwardly rectifying membrane current.

CONCLUSIONS: Functional TRPV1 channels are present in the human airway epithelium and overexpressed in the airways of patients with refractory asthma. These channels might represent a novel therapeutic target for the treatment of uncontrolled asthma.

AXL Is a Key Regulator of Inherent and Chemotherapy-Induced Invasion and Predicts a Poor Clinical Outcome in Early-Stage Colon Cancer

Purpose: Despite the use of 5-fluorouracil (5-FU)–based adjuvant treatments, a large proportion of patients with high-risk stage II/III colorectal cancer will relapse. Thus, novel therapeutic strategies are needed for early-stage colorectal cancer. Residual micrometastatic disease from the primary tumor is a major cause of patient relapse.

Experimental Design: To model colorectal cancer tumor cell invasion/metastasis, we have generated invasive (KRASMT/KRASWT/+chr3/p53-null) colorectal cancer cell subpopulations. Receptor tyrosine kinase (RTK) screens were used to identify novel proteins that underpin the migratory/invasive phenotype. Migration/invasion was assessed using the XCELLigence system. Tumors from patients with early-stage colorectal cancer (N = 336) were examined for AXL expression.

Results: Invasive colorectal cancer cell subpopulations showed a transition from an epithelial-to-mesenchymal like phenotype with significant increases in migration, invasion, colony-forming ability, and an attenuation of EGF receptor (EGFR)/HER2 autocrine signaling. RTK arrays showed significant increases in AXL levels in all invasive sublines. Importantly, 5-FU treatment resulted in significantly increased migration and invasion, and targeting AXL using pharmacologic inhibition or RNA interference (RNAi) approaches suppressed basal and 5-FU–induced migration and invasion. Significantly, high AXL mRNA and protein expression were found to be associated with poor overall survival in early-stage colorectal cancer tissues.

Conclusions: We have identified AXL as a poor prognostic marker and important mediator of cell migration/invasiveness in colorectal cancer. These findings provide support for the further investigation of AXL as a novel prognostic biomarker and therapeutic target in colorectal cancer, in particular in the adjuvant disease in which EGFR/VEGF–targeted therapies have failed.

Acridine orange leukocyte fluorography in mice

Simultaneous non-invasive visualization of blood vessels and nerves in patients can be obtained in the eye. The retinal vasculature is a target of many retinopathies. Inflammation, readily manifest by leukocyte adhesion to the endothelial lining, is a key pathophysiological mechanism of many retinopathies, making it a valuable and ubiquitous target for disease research. Leukocyte fluorography has been extensively used in the past twenty years; however, fluorescent markers, visualization techniques, and recording methods have differed between studies. The lack of detailed protocol papers regarding leukocyte fluorography, coupled with lack of uniformity between studies, has led to a paucity of standards for leukocyte transit (velocity, adherence, extravasation) in the retina. Here, we give a detailed description of a convenient method using acridine orange (AO) and a commercially available scanning laser ophthalmoscope (SLO, HRA-OCT Spectralis) to view leukocyte behavior in the mouse retina. Normal mice are compared to mice with acute and chronic inflammation. This method can be readily adopted in many research labs.

The Role of K+ and Cl- Channels in the Regulation of Retinal Arteriolar Tone and Blood Flow

Purpose: This study tested the role of K(+)- and Cl(-)-channels in retinal arteriolar smooth muscle in the regulation of retinal blood flow.

Methods: Studies were carried out in adult Male Hooded Lister rats. Selectivity of ion channel blockers was established using electrophysiological recordings from smooth muscle in isolated arterioles under voltage clamp conditions. Leukocyte velocity and retinal arteriolar diameters were measured in anesthetised animals using leukocyte fluorography and fluorescein angiography imaging with a confocal scanning laser ophthalmoscope. These values were used to estimate volumetric flow, which was compared between control conditions and following intravitreal injections of ion channel blockers, either alone or in combination with the vasoconstrictor potent Endothelin 1 (Et1).

Results: Voltage activated K(+)-current (IKv) was inhibited by correolide, large conductance (BK) Ca(2+)-activated K(+)-current (IKCa) by Penitrem A, and Ca(2+)-activated Cl(-)-current (IClCa) by disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). Intravitreal injections (10µl) of DIDS (estimated intraocular concentration 10mM) increased flow by 22%, whereas the BK-blockers Penitrem A (1µM) and iberiotoxin (4µM), and the IKv-inhibitor correolide (40µM) all decreased resting flow by approximately 10%. Et1 (104nM) reduced flow by almost 65%. This effect was completely reversed by DIDS but was unaffected by Penitrem A, iberiotoxin or correolide.

Conclusions: These results suggest that Cl(-)-channels in retinal arteriolar smooth muscle limit resting blood flow and play an obligatory role in Et1 responses. K(+)-channel activity promotes basal flow but exerts little modifying effect on the Et1 response. Cl(-)-channels may be appropriate molecular targets in retinal pathologies characterised by increased Et1 activity and reduced blood flow.

Automated detection and measurement of isolated retinal arterioles by a combination of edge enhancement and cost analysis

Pressure myography studies have played a crucial role in our understanding of vascular physiology and pathophysiology. Such studies depend upon the reliable measurement of changes in the diameter of isolated vessel segments over time. Although several software packages are available to carry out such measurements on small arteries and veins, no such software exists to study smaller vessels (<50 µm in diameter). We provide here a new, freely available open-source algorithm, MyoTracker, to measure and track changes in the diameter of small isolated retinal arterioles. The program has been developed as an ImageJ plug-in and uses a combination of cost analysis and edge enhancement to detect the vessel walls. In tests performed on a dataset of 102 images, automatic measurements were found to be comparable to those of manual ones. The program was also able to track both fast and slow constrictions and dilations during intraluminal pressure changes and following application of several drugs. Variability in automated measurements during analysis of videos and processing times were also investigated and are reported. MyoTracker is a new software to assist during pressure myography experiments on small isolated retinal arterioles. It provides fast and accurate measurements with low levels of noise and works with both individual images and videos. Although the program was developed to work with small arterioles, it is also capable of tracking the walls of other types of microvessels, including venules and capillaries. It also works well with larger arteries, and therefore may provide an alternative to other packages developed for larger vessels when its features are considered advantageous.