Effects of advanced glycation end-products (AGEs) on retinal pigment epithelial (RPE) cells lysosomal function:implications for age-related macular degeneration (AMD)

Purpose: RPE lysosomal dysfunction is a major contributor to AMD pathogenesis. Controlled activity of a major class of RPE proteinases, the cathepsins, is crucial in maintaining correct lysosomal function. Advanced glycation end-products (AGEs) accumulate in the Bruch’s membrane (BM) with age, impacting critical RPE functions and in turn, contributing to the development of AMD. The aim of this study was to assess the effect of AGEs on lysosomal function by analysing the expression, processing and activity of the cysteine proteinases cathepsins B, L and S, and the aspartic proteinase cathepsin D. Methods: ARPE-19 cells were cultured on AGE-containing BM mimics (matrigel) for 14 days and compared to untreated substrate. Expression levels and intracellular processing of cathepsins B, D, L and S, were assessed by qPCR and immunoblotting of cell lysates. Lysosomal activity was investigated using multiple activity assays specific to each of the analysed cathepsins. Statistical analysis was performed using the Student’s independent T-test. Results: AGE exposure produced a 36% decrease in cathepsin L activity when compared to non-treated controls (p=0.02, n= 3) although no significant changes were observed in protein expression/processing under these conditions. Both the pro and active forms of cathepsin S decreased by 40% (p=0.04) and 74% (p=0.004), respectively (n=3). In contrast, the active form of the cathepsin D increased by 125% (p=0.005, n= 4). However, no changes were observed in the activity levels of both cathepsins S and D. In addition, cathepsin B expression, processing and activity also remained unaltered following AGE exposure. Conclusions: AGEs accumulation in the extracellular matrix, a phenomenon associated with the natural aging process of the BM, attenuates the expression, intracellular processing and activity of specific lysosomal effectors. Altered enzymatic function may impair important lysosomal processes such as endocytosis, autophagy and phagocytosis of photoreceptor outer segments, each of which may influence the age-related dysfunction of the RPE and subsequently, AMD pathogenesis.

α-Tocopherol supplementation does not affect monocyte endothelial adhesion or C-reactive protein levels but reduces soluble vascular adhesion molecule-1 in the plasma of healthy subjects

Vascular monocyte retention in the subintima is pivotal to the development of cardiovascular disease and is facilitated by up-regulation of adhesion molecules on monocytes/endothelial cells during oxidative stress. Epidemiological studies have shown that cardiovascular disease risk is inversely proportional to plasma levels of the dietary micronutrients, vitamin C and vitamin E (α-tocopherol). We have tested the hypothesis that α-tocopherol supplementation may alter endothelial/monocyte function and interaction in subjects with normal ascorbate levels (> 50 μM), as ascorbate has been shown to regenerate tocopherol from its oxidised tocopheroxyl radical form in vitro. Healthy male subjects received α-tocopherol supplements (400 IU RRR-α-tocopherol /day for 6 weeks) in a placebo-controlled, double-blind intervention study. There were no significant differences in monocyte CD11b expression, monocyte adhesion to endothelial cells, plasma C-reactive protein or sICAM- 1 concentrations post-supplementation. There was no evidence for nuclear translocation of NF-κB in isolated resting monocytes, nor any effect of α-tocopherol supplementation. However, post-supplementation, sVCAM-1 levels were decreased in all subjects and sE-selectin levels were increased in the vitamin C-replete group only; a weak positive correlation was observed between sE-selectin and α-tocopherol concentration. In conclusion, α-tocopherol supplementation had little effect on cardiovascular disease risk factors in healthy subjects and the effects of tocopherol were not consistently affected by plasma vitamin C concentration. © W. S. Maney & Son Ltd.

A randomised controlled trial investigating the effect of lutein and antioxidant dietary supplementation on visual function in healthy eyes

Background & aims It has been suggested that retinal lutein may improve visual acuity for images that are illuminated by white light. Our aim was to determine the effect of a lutein and antioxidant dietary supplement on visual function. Methods A prospective, 9- and 18-month, double-masked randomised controlled trial. For the 9-month trial, 46 healthy participants were randomised (using a random number generator) to placebo (n=25) or active (n=21) groups. Twenty-nine of these subjects went on to complete 18 months of supplementation, 15 from the placebo group, and 14 from the active group. The active group supplemented daily with 6mg lutein combined with vitamins and minerals. Outcome measures were distance and near visual acuity, contrast sensitivity, and photostress recovery time. The study had 80% power at the 5% significance level for each outcome measure. Data were collected at baseline, 9, and 18 months. Results There were no statistically significant differences between groups for any of the outcome measures over 9 or 18 months. Conclusion There was no evidence of effect of 9 or 18 months of daily supplementation with a lutein-based nutritional supplement on visual function in this group of people with healthy eyes. ISRCTN78467674.

Structure and function in primary open angle glaucoma

The study utilised a Normal group, an Ocular Hypertensive (OHT) group and a Primary Open Angle Glaucoma (POAG) group to investigate two aspects. Firstly, the within- and between-visit variability for stereometric measurements of the optic nerve head (ONH) using the Heidelberg Retina Tomograph (HRT); retinal nerve fibre layer (RNFL) thickness using the HRT and using optical coherence tomography with the Optical Coherence Tomography Scanner (OCT); the visual field using white-on-white (W-W), short-wavelength (SWAP) and Frequency Doubling perimetry (FDT); and retinal haemodynamics using the Heidelberg Retinal Flowmeter (HRF). Secondly, the association demonstrated between some of the derived variables. The within- and between-visit variability for stereometric measurements of the entire ONH and the between-visit variability for sectoral measurements were similar for Normals and OHTs but greater for POAGs. The within-visit variability of the visual field pointwise parameters for SWAP were greater than for W-W and FDT particularly with increase in eccentricity and for the OHT group. The between-visit variability increased with increase in defect depth for the POAG group, across all types of perimetry. The MS was greater, the MD and PSD smaller and the examination duration shorter in FDT compared to W-W and SWAP across all groups. The within-visit variability was less than the between-visit variability for the OCT circumferential and sector RNFL thickness using the 1.5R, 2.0R and the fixed 1.73mm circular scan radii, across the three groups. The variability increased with decrease in the RNFL thickness, and was least for the 2.0R scan radius.

The effect of intraocular scattered light on the contrast sensitivity function

Intraocular light scatter is high in certain subject groups eg the elderly, due to increased optical media turbidity, which scatters and attenuates light travelling towards the retina. This causes reduced retinal contrast especially in the presence of glare light. Such subjects have depressed Contrast Sensitivity Functions (CSF). Currently available clinical tests do not effectively reflect this visual disability. Intraocular light scatter may be quantified by measuring the CSF with and without glare light and calculating Light Scatter Factors (LSF). To record the CSF on clinically available equipment (Nicolet CS2000), several psychophysical measurement techniques were investigated, and the 60 sec Method of Increasing Contrast was selected as the most appropriate. It was hypothesised that intraocular light scatter due to particles of different dimensions could be identified by glare sources at wide (30°) and narrow (3.5°) angles. CSFs andLSFs were determined for: (i) Subjects in young, intermediate and old age groups. (ii) Subjects during recovery from large amounts of induced corneal oedema. (iii) A clinical sample of contact lens (CL) wearers with a group of matched controls. The CSF was attenuated at all measured spatial frequencies with the intermediate and old group compared to the young group. High LSF values were found only in the old group (over 60 years). It was concluded that CSF attenuation in the intermediate group was due to reduced pupil size, media absorption and/or neural factors. In the old group, the additional factor was high intraocular light scatter levels of lenticular origin. The rate of reduction of the LSF for the 3.5° glare angle was steeper than that for the 30° angle, following induced corneal oedema. This supported the hypothesis, as it was anticipated that epithelial oedema would recover more rapidly than stromal oedema. CSFs and LSFs were markedly abnormal in the CL wearers. The analytical details and the value of these investigative techniques in contact lens research are discussed.

The investigation of the sensitivity gradient of the visual field, as a function of stimulus dynamic range, in the normal and abnormal eye

The study utilized the advanced technology provided by automated perimeters to investigate the hypothesis that patients with retinitis pigmentosa behave atypically over the dynamic range and to concurrently determine the influence of extraneous factors on the format of the normal perimetric sensitivity profile. The perimetric processing of some patients with retinitis pigmentosa was considered to be abnormal in either the temporal and/or the spatial domain. The standard size III stimulus saturated the central regions and was thus ineffective in detecting early depressions in sensitivity in these areas. When stimulus size was scaled in inverse proportion to the square root of ganglion cell receptive field density (M-scaled), isosensitive profiles did not result, although cortical representation was theoretically equivalent across the visual field. It was conjectured that this was due to variations in the ganglion cell characteristics with increasing peripheral angle, most notably spatial summation. It was concluded that the development of perimetric routines incorporating stimulus sizes adjusted in proportion to the coverage factor of retinal ganglion cells would enhance the diagnostic capacity of perimetry. Good general and local correspondence was found between perimetric sensitivity and the available retinal cell counts. Intraocular light scatter arising both from simulations and media opacities depressed perimetric sensitivity. Attenuation was greater centrally for the smaller LED stimuli, whereas the reverse was true for the larger projected stimuli. Prior perimetric experience and pupil size also demonstrated eccentricity-dependent effect on sensitivity. Practice improved perimetric sensitivity for projected stimuli at eccentricities greater than or equal to 30o; particularly in the superior region. Increase in pupil size for LED stimuli enhanced sensitivity at eccentricities greater than 10o. Conversely, microfluctuation in the accommodative response during perimetric examination and the correction of peripheral refractive error had no significant influence on perimetric sensitivity.

Improving the function of islet and beta-cell grafts

Background: Human islet transplantation would offer a less invasive and more physiological alternative than whole pancreas transplantation and insulin injections respectively for the treatment of diabetes mellitus if islet graft survival can be improved. Initial recipient post-transplant insulin independence declines to <10% after 5 years. Factors contributing to graft failure include enzymatic disruption of the islet microenvironment during isolation, diabetogenic effects of immunosuppressants and metabolic stress resulting from slow revascularisation. Aims: To investigate the effect of co-culture in both static (SC) and rotational culture (RC) of BRINBDII beta-cells (Dl1) and human umbilical vein endothelial cells (HUVEC) on Dl1 insulin secretion; and the effect of a thiazolidinedione (TZD) on DII function and HUVEC proliferation. To assess the effect of culture media, SC, RC and a TZD on human islet morphology, insulin secretion and VEGF production. To initiate in vivo protocol development for assessment of revascularisation of human islet grafts. Methods: D11 cells were cultured +/-TZD and co-cultured with HUVEC +/-TZD in SC and RC. Dl1 insulin secretion was induced by static incubation with low glucose (1.67mM), high glucose (l6.7mM: and high glucose with 10mM theophylline (G+T) and determined by ELISA. HUVEC were cultured +/-TZD in SC and RC and proliferation was assessed by ATP luminescence assay and VEGF ELISA. D II and HUVEC morphology was determined by immunocytochemistry. Human islets were cultured in SC and RC in various media +/-TZD. Insulin secretion was determined as above and VEGF production by fluorescence immunocytochemistry (FI) and ELISA. Revascularisation of islet grafts was assessed by vascular corrosion cast and FI. Results: Dll cultures showed significantly increased insulin secretion in response to 16.7mM and G+T over basal; this was enhanced by RC and further improved by adding 10mM TZD. Untreated Dll/HUVEC co-cultures displayed significantly increased insulin secretion in response to 16.7mM and G+T over basal, again enhanced by RC and improved with 10mM TZD. 10mM TZD significantly increased HUVEC proliferation over control. Human islets maintained in medium 199 (mI99) in SC and RC exhibited comparable maintenance of morphology and insulin secretory profiles compared to islets maintained in RPMI, endothelial growth media and dedicated islet medium Miami# I. All cultures showed significantly increased insulin secretion in response to 16.7mM and G+T over basal; this was enhanced by RC and in certain instances further improved by adding 25mM TZD. TZD increased VEGF production and release as determined by ELISA. Post-implant vascular corrosion casts of mouse kidneys analysed by x-ray micro tomography indicates a possible TZD enhancement of microvessel growth via VEGF upregulation. Conclusions: D II /HUVEC co-culture in SC or RC does not alter the morphology of either cell type and supports D 11 function. TZD improves 0 I I and D I I/HUVEC SC and RC co-culture insulin secretion while increasing HUVEC proliferation. Human islet RC supports islet functional viability and structural integrity compared to SC while the addition of TZD occasionally further improves secretagogue induced insulin secretion. Expensive, 'dedicated' islet media showed no advantage over ml99 in terms of maintaining islet morphology or function. TZD upregulates VEGF in islets as shown by ELISA and suggested by x-ray micro tomography analysis of vascular corrosion casts. Maintenance of islets in RC and treatment with TZD prior to transplant may improve the functional viability and revascularisation rate of islet grafts.

The role of heterodimerization between VEGFR-1 and VEGFR-2 in the regulation of endothelial cell homeostasis

VEGF-A activity is tightly regulated by ligand and receptor availability. Here we investigate the physiological function of heterodimers between VEGF receptor-1 (VEGFR-1; Flt-1) and VEGFR-2 (KDR; Flk-1) (VEGFR(1-2)) in endothelial cells with a synthetic ligand that binds specifically to VEGFR(1-2). The dimeric ligand comprises one VEGFR-2-specific monomer (VEGF-E) and a VEGFR-1-specific monomer (PlGF-1). Here we show that VEGFR(1-2) activation mediates VEGFR phosphorylation, endothelial cell migration, sustained in vitro tube formation and vasorelaxation via the nitric oxide pathway. VEGFR(1-2) activation does not mediate proliferation or elicit endothelial tissue factor production, confirming that these functions are controlled by VEGFR-2 homodimers. We further demonstrate that activation of VEGFR(1-2) inhibits VEGF-A-induced prostacyclin release, phosphorylation of ERK1/2 MAP kinase and mobilization of intracellular calcium from primary endothelial cells. These findings indicate that VEGFR-1 subunits modulate VEGF activity predominantly by forming heterodimer receptors with VEGFR-2 subunits and such heterodimers regulate endothelial cell homeostasis.

Rotational co-culture of clonal β-cells with endothelial cells:effect of PPAR-γ agonism in vitro on insulin and VEGF secretion

Aim: Delayed graft revascularization impedes the success of human islet transplantation. This study utilized rotational co-culture of insulin secreting ß-cells with human umbilical vein endothelial cells (HUVECs) and a peroxisome proliferator-activated receptor gamma (PPAR-?) agonist to promote insulin and vascular endothelial growth factor (VEGF) secretory function. Methods: Clonal BRIN-BD11 (D11) cells were maintained in static culture (SC) and rotational culture (RC) ± HUVEC and ± the TZD (thiazolidinedione) rosiglitazone (10 mmol/l) as a specific PPAR-? agonist. HUVECs were cultured in SC and RC ± D11 and ± TZD. D11 insulin secretion was induced by static incubation with low glucose (1.67 mmol/l), high glucose (16.7 mmol/l) and high glucose with 10 mmol/l theophylline (G+T) and assessed by enzyme-linked immunosorbent assay (ELISA). HUVEC proliferation was determined by ATP luminescence, whereas VEGF secretion was quantified by ELISA. Co-cultured cells were characterized by immunostaining for insulin and CD31. Results: D11 SC and RC showed enhanced insulin secretion in response to 16.7 mmol/l and G+T (p <0.01); without significant alteration by the TZD. Co-culture with HUVEC in SC and RC also increased D11 insulin secretion when challenged with 16.7 mmol/l and G+T (p <0.01), and this was slightly enhanced by the TZD. The presence of HUVEC increased D11 SC and RC insulin secretion in response to high glucose and G+T, respectively (p <0.01). Addition of the TZD increased SC and RC HUVEC ATP content (p <0.01) and VEGF production (p <0.01) in the presence and absence of D11 cells. Conclusions: Rotational co-culture of insulin secreting cells with endothelial cells, and exposure to a PPAR-? agonist may improve the prospects for graft revascularization and function after implantation. © 2011 Blackwell Publishing Ltd.

Celiac disease-specific TG2-targeted autoantibodies inhibit angiogenesis ex vivo and in vivo in mice by interfering with endothelial cell dynamics

A characteristic feature of celiac disease is the presence of circulating autoantibodies targeted against transglutaminase 2 (TG2), reputed to have a function in angiogenesis. In this study we investigated whether TG2-specific autoantibodies derived from celiac patients inhibit angiogenesis in both ex vivo and in vivo models and sought to clarify the mechanism behind this phenomenon. We used the ex vivo murine aorta-ring and the in vivo mouse matrigel-plug assays to address aforementioned issues. We found angiogenesis to be impaired as a result of celiac disease antibody supplementation in both systems. Our results also showed the dynamics of endothelial cells was affected in the presence of celiac antibodies. In the in vivo angiogenesis assays, the vessels formed were able to transport blood despite impairment of functionality after treatment with celiac autoantibodies, as revealed by positron emission tomography. We conclude that celiac autoantibodies inhibit angiogenesis ex vivo and in vivo and impair vascular functionality. Our data suggest that the anti-angiogenic mechanism of the celiac disease-specific autoantibodies involves extracellular TG2 and inhibited endothelial cell mobility. © 2013 Kalliokoski et al.

Celiac disease patient IgA antibodies induce endothelial adhesion and cell polarization defects via extracellular transglutaminase 2

We have recently found that celiac disease patient serum-derived autoantibodies targeted against transglutaminase 2 interfere with several steps of angiogenesis, including endothelial sprouting and migration, though the mechanism involved remained to be fully characterized. This study now investigated the processes underlying the antiangiogenic effects exerted by celiac disease patient antibodies on endothelial cells, with particular regard to the adhesion, migration, and polarization signaling pathway. We observed that celiac IgA reduced endothelial cell numbers by affecting adhesion without increasing apoptosis. Endothelial cells in the presence of celiac IgA showed weak attachment, a high susceptibility to detach from fibronectin, and a disorganized extracellular matrix due to a reduction of protein cross-links. Furthermore, celiac patient IgA led to secretion of active transglutaminase 2 from endothelial cells into the culture supernatants. Additionally, cell surface transglutaminase 2 mediated integrin clustering in the presence of celiac IgA was coupled to augmented expression of ß1-integrin. We also observed that celiac patient IgA-treated endothelial cells had migratory defects and a less polarized phenotype when compared to control groups, and this was associated with the RhoA signaling pathway. These biological effects mediated by celiac IgA on endothelial cells were partially influenced but not completely abolished by R281, an irreversible extracellular transglutaminase 2 enzymatic activity inhibitor. Taken together, our results imply that celiac patient IgA antibodies disturb the extracellular protein cross-linking function of transglutaminase 2, thus altering cell-extracellular matrix interactions and thereby affecting endothelial cell adhesion, polarization, and motility. © 2013 Springer Basel.

Retinal vessel analysis:flicker reproducibility, methodological standardisations and practical limitations

The Retinal Vessel Analyser (RVA) is a commercially available ophthalmoscopic instrument capable of acquiring vessel diameter fluctuations in real time and in high temporal resolution. Visual stimulation by means of flickering light is a unique exploration tool of neurovascular coupling in the human retina. Vessel reactivity as mediated by local vascular endothelial vasodilators and vasoconstrictors can be assessed non-invasively, in vivo. In brief, the work in this thesis • deals with interobserver and intraobserver reproducibility of the flicker responses in healthy volunteers • explains the superiority of individually analysed reactivity parameters over vendorgenerated output • links in static retinal measures with dynamic ones • highlights practical limitations in the use of the RVA that may undermine its clinical usefulness • provides recommendations for standardising measurements in terms of vessel location and vessel segment length and • presents three case reports of essential hypertensives in a -year follow-up. Strict standardisation of measurement procedures is a necessity when utilising the RVA system. Agreement between research groups on implemented protocols needs to be met, before it could be considered a clinically useful tool in detecting or predicting microvascular dysfunction.

Activation of vascular endothelial growth factor receptor-1 sustains angiogenesis and Bcl-2 expression via the phosphatidylinositol 3-kinase pathway in endothelial cells

Vascular insufficiency and retinal ischemia precede many proliferative retinopathies and stimulate secretion of various vasoactive growth factors, including vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF). It is unclear, however, how PlGF, which is elevated in proliferative diabetic retinopathy and is a VEGF homolog that binds only to VEGF receptor (VEGFR)-1, promotes pathological angiogenesis. When primary microvascular endothelial cells were grown on collagen gels, PlGF-containing ligands upregulated Bcl-2 expression and stimulated the formation of capillary-like tube networks that were retained for up to 14 days in culture. The inhibition of VEGFR-1 results in a dramatic decrease in the number of capillary connections, indicating that VEGFR-1 ligands promote branching angiogenesis. In contrast, VEGF-induced tube formations and Bcl-2 expression were significantly decreased at the end of this period. Flow cytometry analysis of annexin-V/propidium iodide-stained cells revealed that PlGF and PlGF/VEGF heterodimer inhibited apoptosis in serum-deprived endothelial cells. These two growth factors stimulated a survival signaling pathway phosphatidylinositol 3-kinase (PI3K), as identified by increased Akt phosphorylation and because blocking PI3K signalling by adenovirus-mediated overexpression of wild-type phosphatase and tensin homolog on chromosome 10 (PTEN) disrupted angiogenesis and decreased Bcl-2 expression by PlGF and PlGF/VEGF heterodimer, whereas a dominant-negative PTEN mutant enhanced endothelial sprout formation and Bcl-2 expression. Together, these findings indicate that PlGF-containing ligands contribute to pathological angiogenesis by prolonging cell survival signals and maintaining vascular networks.

Endothelial Nox4 NADPH oxidase enhances vasodilatation and reduces blood pressure in vivo

Objective- Increased reactive oxygen species (ROS) production is involved in the pathophysiology of endothelial dysfunction. NADPH oxidase-4 (Nox4) is a ROS-generating enzyme expressed in the endothelium, levels of which increase in pathological settings. Recent studies indicate that it generates predominantly hydrogen peroxide (H O ), but its role in vivo remains unclear. Methods and Results- We generated transgenic mice with endothelium-targeted Nox4 overexpression (Tg) to study the in vivo role of Nox4. Tg demonstrated significantly greater acetylcholine- or histamine-induced vasodilatation than wild-type littermates. This resulted from increased H O production and H O -induced hyperpolarization but not altered nitric oxide bioactivity. Tg had lower systemic blood pressure than wild-type littermates, which was normalized by antioxidants. Conclusion- Endothelial Nox4 exerts potentially beneficial effects on vasodilator function and blood pressure that are attributable to H O production. These effects contrast markedly with those reported for Nox1 and Nox2, which involve superoxide-mediated inactivation of nitric oxide. Our results suggest that therapeutic strategies to modulate ROS production in vascular disease may need to separately target individual Nox isoforms. © 2011 American Heart Association, Inc.

Upregulation of urotensin II receptor in preeclampsia causes in vitro placental release of soluble vascular endothelial growth factor receptor 1 in hypoxia

Preeclampsia is a hypertensive disorder of pregnancy caused by abnormal placental function, partly because of chronic hypoxia at the utero-placental junction. The increase in levels of soluble vascular endothelial growth factor receptor 1, an antiangiogenic agent known to inhibit placental vascularization, is an important cellular factor implicated in the onset of preeclampsia. We investigated the ligand urotensin II (U-II), a potent endogenous vasoconstrictor and proangiogenic agent, for which levels have been reported to increase in patients with preeclampsia. We hypothesized that an increased sensitivity to U-II in preeclampsia might be achieved by upregulation of placental U-II receptors. We further investigated the role of U-II receptor stimulation on soluble vascular endothelial growth factor receptor 1 release in placental explants from diseased and normal patients. Immunohistochemistry, real-time PCR, and Western blotting analysis revealed that U-II receptor expression was significantly upregulated in preeclampsia placentas compared with controls (P<0.01). Cellular models of syncytiotrophoblast and vascular endothelial cells subjected to hypoxic conditions revealed an increase in U-II receptor levels in the syncytiotrophoblast model. This induction is regulated by the transcriptional activator hypoxia-inducible factor 1a. U-II treatment is associated with increased secretion of soluble vascular endothelial growth factor receptor 1 only in preeclamptic placental explants under hypoxia but not in control conditions. Interestingly, normal placental explants did not respond to U-II stimulation.