Effect of membrane stiffness and cytoskeletal element density on mechanical stimuli within cells: an analysis of the consequences of ageing in cells

A finite element model of a single cell was created and used to investigate the effects of ageing on biophysical stimuli generated within a cell. Major cellular components were incorporated in the model: the membrane, cytoplasm, nucleus, microtubules, actin filaments, intermediate filaments, nuclear lamina, and chromatin. The model used multiple sets of tensegrity structures. Viscoelastic properties were assigned to the continuum components. To corroborate the model, a simulation of Atomic Force Microscopy (AFM) indentation was performed and results showed a force/indentation simulation with the range of experimental results.

Ageing was simulated by both increasing membrane stiffness (thereby modelling membrane peroxidation with age) and decreasing density of cytoskeletal elements (thereby modelling reduced actin density with age). Comparing normal and aged cells under indentation predicts that aged cells have a lower membrane area subjected to high strain compared to young cells, but the difference, surprisingly, is very small and would not be measurable experimentally. Ageing is predicted to have more significant effect on strain deep in the nucleus. These results show that computation of biophysical stimuli within cells are achievable with single-cell computational models whose force/displacement behaviour is within experimentally observed ranges. the models suggest only small, though possibly physiologically-significant, differences in internal biophysical stimuli between normal and aged cells.

Glycation does not alter LDL-induced secretion of tissue plasminogen activator and plasminogen activator inhibitor-1 from human aortic endothelial cells

Diabetes may induce both quantitative and qualitative changes in lipoproteins, especially low-density lipoprotein (LDL). Effects of LDL glycation on endothelial cell secretion of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) have not been fully elucidated. Human aortic endothelial cell (HAEC) tPA and PAI-1 production were determined after incubation with LDL (50 to 500 microg/mL protein, 24 h) from three sources: (1) nondiabetic LDL (N-LDL) modified in vitro to form six preparations: native, nonmodified (N); glycated (G); minimally oxidized (MO); minimally oxidized and glycated (MOG); heavily oxidized (HO); and heavily oxidized and glycated (HOG); (2) in vivo glycated and relatively nonglycated LDL subfractions from type 1 diabetic patients; (3) LDL from type 1 diabetic patients and matched controls, which was subfractionated using density gradient ultracentrifugation. In experiments using LDL modified in vitro, the rate of tPA release by HAECs incubated with N-LDL (83 +/- 4 ng/mg cell protein/24 h) did not differ significantly from those incubated with G-LDL (73 +/- 7), MO-LDL (74 +/- 13), or MOG-LDL (66 +/- 15) and was not influenced by LDL concentration. The rate of PAI-1 release was similar in HAECs incubated with N-LDL (5.7 +/- 0.6 mug/mg cell protein/24 h), G-LDL (5.7 +/- 0.7), MO-LDL (5.5 +/- 0.8), or MOG-LDL (5.7 +/- 0.9) and was not influenced by LDL concentration. In contrast, tPA release was significantly decreased in cells incubated with LDL (10 microg/mL) modified extensively by oxidation, and averaged 45.2 +/- 5.0 and 43.7 +/- 9.9 ng/mg/24 h for HO-LDL and HOG-LDL, respectively, and was further decreased with increasing concentrations of the heavily oxidized LDL preparations. PAI-1 release was not significantly decreased relative to N-LDL in cells incubated with low concentrations (5 to 50 microg/mL) of HO-LDL and HOG-LDL, but was decreased to 3.2 +/- 0.5 and 3.1 +/- 0.7 microg/mg/24 h for HO-LDL and HOG-LDL at 200 microg/mL, respectively. Results using in vivo glycated versus nonglycated LDL showed that tPA and PAI-1 release did not differ between subfractions. Release of tPA averaged 5.11 +/- 0.6 and 5.12 +/- 0.7 ng/mg/24 h, whereas release of PAI-1 averaged 666 +/- 27 ng/mg/24 h and 705 +/- 30 ng/mg/24 h for nonglycated and glycated LDL subfractions, respectively. Using LDL of different density subclasses, tPA and PAI-1 release in response to LDL from diabetic patients compared with control subjects did not differ when HAECs were incubated with LDLs of increasing density isolated from each subject pair. We conclude that oxidation of LDL, but not glycation, may contribute to the altered fibrinolysis observed in diabetes.

Activation of MAPK by modified low-density lipoproteins in vascular smooth muscle cells

A high concentration of circulating low-density lipoproteins (LDL) is a major risk factor for atherosclerosis. Native LDL and LDL modified by glycation and/or oxidation are increased in diabetic individuals. LDL directly stimulate vascular smooth muscle cell (VSMC) proliferation; however, the mechanisms remain undefined. The extracellular signal-regulated kinase (ERK) pathway mediates changes in cell function and growth. Therefore, we examined the cellular effects of native and modified LDL on ERK phosphorylation in VSMC. Addition of native, mildly modified (oxidized, glycated, glycoxidized) and highly modified (highly oxidized, highly glycoxidized) LDL at 25 microg/ml to rat VSMC for 5 min induced a fivefold increase in ERK phosphorylation. To elucidate the signal transduction pathway by which LDL phosphorylate ERK, we examined the roles of the Ca(2+)/calmodulin pathway, protein kinase C (PKC), src kinase, and mitogen-activated protein kinase kinase (MEK). Treatment of VSMC with the intracellular Ca(2+) chelator EGTA-AM (50 micromol/l) significantly increased ERK phosphorylation induced by native and mildly modified LDL, whereas chelation of extracellular Ca(2+) by EGTA (3 mmol/l) significantly reduced LDL-induced ERK phosphorylation. The calmodulin inhibitor N-(6-aminohexyl)-1-naphthalenesulfonamide (40 micromol/l) significantly decreased ERK phosphorylation induced by all types of LDL. Downregulation of PKC with phorbol myristate acetate (5 micromol/l) markedly reduced LDL-induced ERK phosphorylation. Pretreatment of VSMC with a cell-permeable MEK inhibitor (PD-98059, 40 micromol/l) significantly decreased ERK phosphorylation in response to native and modified LDL. These findings indicate that native and mildly and highly modified LDL utilize similar signaling pathways to phosphorylate ERK and implicate a role for Ca(2+)/calmodulin, PKC, and MEK. These results suggest a potential link between modified LDL, vascular function, and the development of atherosclerosis in diabetes.

Aminoguanidine and the effects of modified LDL on cultured retinal capillary cells

Compared with normal low density lipoprotein (N-LDL), LDL minimally modified in vitro by glycation, minimal oxidation, or glycoxidation (G-, MO-, GO-LDL) decreases survival of cultured retinal capillary endothelial cells and pericytes. Similar modifications occurring in vivo in diabetes may contribute to retinopathy. The goal of this study was to determine whether low concentrations of aminoguanidine might prevent cytotoxic modification of LDL and/or protect retinal capillary cells from previously modified LDL.

Native and modified LDL activate extracellular signal-regulated kinases in mesangial cells

Glycation and/or oxidation of LDL may promote diabetic nephropathy. The mitogen-activated protein kinase (MAPK) cascade, which includes extracellular signal-regulated protein kinases (ERKs), modulates cell function. Therefore, we examined the effects of LDL on ERK phosphorylation in cultured rat mesangial cells. In cells exposed to 100 microg/ml native LDL or LDL modified by glycation, and/or mild or marked (copper-mediated) oxidation, ERK activation peaked at 5 min. Five minutes of exposure to 10-100 microg/ml native or modified LDL produced a concentration-dependent (up to sevenfold) increase in ERK activity. Also, 10 microg/ml native LDL and mildly modified LDL (glycated and/or mildly oxidized) produced significantly greater ERK activation than that induced by copper-oxidized LDL +/- glycation (P <0.05). Pretreatment of cells with Src kinase and MAPK kinase inhibitors blocked ERK activation by 50-80% (P <0.05). Native and mildly modified LDL, which are recognized by the native LDL receptor, induced a transient spike of intracellular calcium. Copper-oxidized (+/- glycation) LDL, recognized by the scavenger receptor, induced a sustained rise in intracellular calcium. The intracellular calcium chelator (EGTA/AM) further increased ERK activation by native and mildly modified LDL (P <0.05). These findings demonstrate that native and modified LDL activate ERKs 1 and 2, an early mitogenic signal, in mesangial cells and provide evidence for a potential link between modified LDL and the development of glomerular injury in diabetes.

Toxicity of mildly modified low-density lipoproteins to cultured retinal capillary endothelial cells and pericytes

To investigate the role of modified low-density lipoproteins (LDL) in the pathogenesis of diabetic retinopathy, we studied the cytotoxicity of normal and mildly modified human LDL to bovine retinal capillary endothelial cells and pericytes in vitro. Pooled LDL was incubated (in phosphate-buffered saline-EDTA, 3 days, 37 degrees C) under 1) nitrogen with additional chelating agents and 2) air, to prepare normal and minimally oxidized LDL, respectively. Similar conditions, but with the addition of 50 mM D-glucose, were used to prepare glycated and glycoxidized LDL. None of the LDL preparations was recognized by the macrophage scavenger receptor, confirming limited modification. Retinal capillary endothelial cells and pericytes were grown to confluence and then exposed for 2 or 3 days to serum-free medium (1% albumin) supplemented with normal or modified LDL (100 mg/l) or to serum-free medium alone. Cytotoxicity was assessed by cell counting (live and total cells) and by cell protein determination. Compared with normal LDL, modified LDL were cytotoxic to both cell types at both time points, causing highly significant decreases in live and total cell counts (P <0.001) (analysis of variance). Reductions in cell protein also were significant for pericytes at day 3 (P = 0.016) and of borderline significance for endothelial cells at day 2 (P = 0.05) and day 3 (P = 0.063). Cytotoxicity increased as follows: normal <glycated <or = minimally oxidized <glycoxidized LDL. We conclude that, in diabetes, mild modification of LDL resulting from separate or combined processes of glycation and oxidation may contribute to chronic retinal capillary injury and thus to the development of diabetic retinopathy.

Novel 5-aryloxypyrimidine SEN1576 as a candidate for the treatment of Alzheimer's disease

Prefibrillar assembly of amyloid-ß (Aß) is a major event underlying the development of neuropathology and dementia in Alzheimer's disease (AD). This study determined the neuroprotective properties of an orally bioavailable Aß synaptotoxicity inhibitor, SEN1576. Binding of SEN1576 to monomeric Aß 1–42 was measured using surface plasmon resonance. Thioflavin-T and MTT assays determined the ability of SEN1576 to block Aß 1–42-induced aggregation and reduction in cell viability, respectively. In vivo long-term potentiation (LTP) determined effects on synaptic toxicity induced by intracerebroventricular (i.c.v.) injection of cell-derived Aß oligomers. An operant behavioural schedule measured effects of oral administration following i.c.v. injection of Aß oligomers in normal rats. SEN1576 bound to monomeric Aß 1–42, protected neuronal cells exposed to Aß 1–42, reduced deficits in in vivo LTP and behaviour. SEN1576 exhibits the necessary features of a drug candidate for further development as a disease modifying treatment for the early stages of AD-like dementia.

Preparation and characterization of new anodes based on Ti mesh for direct methanol fuel cells

A novel anode structure based on Ti mesh for the direct methanol fuel cell (DMFC) has been prepared by thermal deposition of ~5 µm PtRuO2 catalyst layer on ~50 µm Ti mesh. The preparation procedures and the main characteristics of the anode were studied by half-cell testing, scanning electron microscopy analysis, energy-dispersive X-ray measurement, and single-cell testing. The optimum calcination temperature is 450°C, calcination time is 90- 120 min, PtRuO2 catalyst loading is 5.0 mg cm-2, Pt precursor concentration range of solution is 0.14- 0.4 M, and solution aging time is 1 day. The performances of the anodes prepared using the solution kept within 20 days showed no significant difference. When it was used in DMFC feed with low-concentration methanol solution at 90°C, this new anode shows better performance than that of the conventional anode, because its thin hydrophilic structure is a benefit to the transport of methanol and carbon dioxide. However, due to its opening structure, when higher concentration methanol was employed, the performance of the cell with new anode became worse. © 2006 The Electrochemical Society. All rights reserved.

Selective loss of vascular smooth muscle cells in the retinal microcirculation of diabetic dogs

This study was undertaken to further characterise the fine structural changes occurring in the retinal circulation in early diabetes. The eyes of eight alloxan/streptozotocin and three spontaneously diabetic dogs were examined by trypsin digest and electron microscopy after durations of diabetes of between 1 and 7 years. Basement membrane (BM) thickening in the retinal capillaries was the only obvious fine structural change identified during the first 3 years of diabetes and was established within 1 year of induction. Widespread pericyte loss was noted after 4 years of diabetes and was paralleled by loss of smooth muscle (SM) cells, in the retinal arterioles. SM cell loss was most obvious in the smaller arterioles of the central retina. No microaneurysms were noted in the experimental diabetic dogs with up to 5 years' duration of diabetes but were widespread in a spontaneously diabetic animal at 7 years. This study has shown that SM cell loss, a hitherto unrecognised feature of diabetic microangiopathy, accompanies pericyte loss in the retinal circulation of diabetic dogs.

Effects of D- and L-glucose and mannitol on retinal capillary cells:Inhibition by nanomolar aminoguanidine

Hyperglycemia may contribute directly to pericyte loss and capillary leakage in early diabetic retinopathy. To elucidate relative contributions of glycation, glycoxidation, sugar autoxidation, osmotic stress and metabolic effects in glucose-mediated capillary damage, we tested the effects of D-glucose, L-glucose, mannitol and the potentially protective effects of aminoguanidine on cultured bovine retinal capillary pericytes and endothelial cells. Media (containing 5 mM D-glucose) were supplemented to increase the concentration of each sugar by 5, 10, or 20 mM. Subconfluent pericytes and endothelial cells were exposed to the supplemented media in the presence or absence of aminoguanidine (1 nM-100 µM) for three days. Cell counts, viability and protein were determined. For both cell types, all three sugars produced concentration-dependent decreases in cell counts and protein content (p

Grape-seed procyanidins modulate cellular membrane potential and nutrient-induced GLP-1 secretion in STC-1 cells

Grape-seed procyanidins (GSPE) modulate glucose homeostasis and it was suggested that GSPE may achieve this by enhancing the secretion of incretin hormones such as glucagon-like peptide-1 (GLP-1). Therefore, the aim of the present study is to examine in detail the effects of GSPE on intestinal endocrine cells (STC-1). GSPE was found to modulate plasma membrane potential in enteroendocrine cells, inducing depolarization at low concentrations (0.05 mg/L) and hyperpolarization at high concentrations (50 mg/L), and surprisingly this was also accompanied by suppressed GLP-1 secretion. Furthermore, how GSPE affects STC-1 cells under nutrient-stimulated conditions (i.e. glucose, linoleic acid and L-proline) was also explored, and we found that the higher GSPE concentration was effective in limiting membrane depolarization and reducing GLP-1 secretion. Next, it was also examined whether GSPE affected mitochondrial membrane potential, finding that this too is altered by GSPE, however this does not appear to explain the observed effects on plasma membrane potential and GLP-1 secretion. In conclusion, our results show that grape-seed procyanidins modulate cellular membrane potential and nutrient-induced enteroendocrine hormone secretion in STC-1 cells.

Accurate and Efficient Modelling to Calculate the Voltage Dependence of Liquid Crystal Based Reflectarray Cells

Two models that can predict the voltage-dependent scattering from liquid crystal (LC)-based reflectarray cells are presented. The validity of both numerical techniques is demonstrated using measured results in the frequency range 94-110 GHz. The most rigorous approach models, for each voltage, the inhomogeneous and anisotropic permittivity of the LC as a stratified media in the direction of the biasing field. This accounts for the different tilt angles of the LC molecules inside the cell calculated from the solution of the elastic problem. The other model is based on an effective homogeneous permittivity tensor that corresponds to the average tilt angle along the longitudinal direction for each biasing voltage. In this model, convergence problems associated with the longitudinal inhomogeneity are avoided, and the computation efficiency is improved. Both models provide a correspondence between the reflection coefficient (losses and phase-shift) of the LC-based reflectarray cell and the value of biasing voltage, which can be used to design beam scanning reflectarrays. The accuracy and the efficiency of both models are also analyzed and discussed.

Endothelial progenitor cells in diabetic retinopathy

Diabetic retinopathy (DR) is a leading cause of visual impairment worldwide. Patients with DR may irreversibly lose sight as a result of the development of diabetic macular edema (DME) and/or proliferative diabetic retinopathy (PDR); retinal blood vessel dysfunction and degeneration plays an essential role in their pathogenesis. Although new treatments have been recently introduced for DME, including intravitreal vascular endothelial growth factor inhibitors (anti-VEGFs) and steroids, a high proportion of patients (~40-50%) do not respond to these therapies. Furthermore, for people with PDR, laser photocoagulation remains a mainstay therapy despite this being an inherently destructive procedure. Endothelial progenitor cells (EPCs) are a low-frequency population of circulating cells known to be recruited to sites of vessel damage and tissue ischemia where they promote vascular healing and re-perfusion. A growing body of evidence suggests that the number and function of EPCs are altered in patients with varying degrees of diabetes duration, metabolic control, and in the presence or absence of DR. Although there are no clear-cut outcomes from these clinical studies, there is mounting evidence that some EPC sub-types may be involved in the pathogenesis of DR and may also serve as biomarkers for disease progression and stratification. Moreover, some EPC sub-types have considerable potential as therapeutic modalities for DME and PDR in the context of cell therapy. This study presents basic clinical concepts of DR and combines this with a general insight on EPCs and their relation to future directions in understanding and treating this important diabetic complication.

Clinical grade allogeneic human mesenchymal stem cells restore alveolar fluid clearance in human lungs rejected for transplantation

The lack of suitable donors for all solid-organ transplant programs is exacerbated in lung transplantation by the low utilization of potential donor lungs, due primarily to donor lung injury and dysfunction, including pulmonary edema. The current studies were designed to determine if intravenous clinical-grade human mesenchymal stem (stromal) cells (hMSCs) would be effective in restoring alveolar fluid clearance (AFC) in the human ex vivo lung perfusion model, using lungs that had been deemed unsuitable for transplantation and had been subjected to prolonged ischemic time. The human lungs were perfused with 5% albumin in a balanced electrolyte solution and oxygenated with continuous positive airway pressure. Baseline AFC was measured in the control lobe and if AFC was impaired (defined as

Efficient Drug Delivery and Induction of Apoptosis in Colorectal Tumors Using a Death Receptor 5-Targeted Nanomedicine

Death Receptor 5 (DR5) is a pro-apoptotic cell-surface receptor that is a potential therapeutic target in cancer. Despite the potency of DR5-targeting agents in preclinical models, the translation of these effects into the clinic remains disappointing. Herein, we report an alternative approach to exploiting DR5 tumor expression using antibody-targeted, chemotherapy-loaded nanoparticles. We describe the development of an optimized polymer-based nanotherapeutic incorporating both a functionalized polyethylene glycol (PEG) layer and targeting antibodies to limit premature phagocytic clearance whilst enabling targeting of DR5-expressing tumor cells. Using the HCT116 colorectal cancer model, we show that following binding to DR5, the nanoparticles activate caspase 8, enhancing the anti-tumor activity of the camptothecin payload both in vitro and in vivo. Importantly, the combination of nanoparticle-induced DR5 clustering with camptothecin delivery overcomes resistance to DR5-induced apoptosis caused by loss of BAX or overexpression of anti-apoptotic FLIP. This novel approach may improve the clinical activity of DR5-targeted therapeutics while increasing tumor-specific delivery of systemically toxic chemotherapeutics.Molecular Therapy (2014); doi:10.1038/mt.2014.137.