Elizabeth Kelly

TNF-α and TGF-β synergistically stimulate elongation of human endothelial cells without transdifferentiation to smooth muscle cell phenotype

We earlier reported synergy between tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) for apoptosis in human umbilical vein endothelium (HUVEC). Here, we study morphological change by circularity measurement of HUVEC surviving this cytokine induced synergistic apoptosis. Contrasting with reports by others studying bovine endothelium, HUVEC did not change morphology in response to TGF-β1. TNF-α markedly elongated cells (p<0.001) and this further increased with combination of the two cytokines (p<0.001), while elongation was accompanied by increased actin stress fibres. Transdifferentiation of HUVEC to a smooth muscle cell phenotype as reported elsewhere was excluded in the current study.

The anti-apoptotic activity of albumin for endothelium is inhibited by advanced glycation end products restricting intramolecular movement

Human serum albumin (HSA) inhibits endothelial apoptosis in a highly specific manner. CNBr fragmentation greatly increases the effectiveness of this activity, suggesting that this type of protection is mediated by a partially cryptic albumin domain which is transiently exposed by intramolecular movement. Advanced glycation end-product (AGE) formation in HSA greatly reduces its intra-molecular movement. This study aimed to determine if this inhibits the anti-apoptotic activity of HSA, and if such inactivation could be reversed by CNBr fragmentation. HSA-AGE was prepared by incubating HSA with glucose, and assessed using the fructosamine assay, mass spectrometry, SDS-PAGE and fluorometry. Low levels of AGE in the HSA had little effect upon its anti-apoptotic activity, but when the levels of AGE were high and the intra-molecular movement was reduced, endothelial cell survival was also found to be reduced to levels equivalent to those in cultures without HSA or serum (p > 0.001). Survival was restored by the inclusion of native HSA, despite the presence of HSA with high levels of AGE. Also, CNBr fragmentation of otherwise inactive HSA-AGE restored the anti-apoptotic activity for endothelium. Apoptosis was confirmed by DNA gel electrophoresis, transmission electron microscopy and fluorescence-activated cell sorting analysis, and there was no evidence for direct toxicity in the HSA-AGE preparations. The results are consistent with the proposed role of intra-molecular movement in exposing the anti-apoptotic domain in HSA for endothelium. The levels of AGE formation required to inhibit the anti-apoptotic activity of HSA exceeded those reported for diabetes. Nonetheless, the data from this study seems to be the first example of reduced protein function due to AGE-restricted intra-molecular movement.

Arecoline increases basic fibroblast growth factor but reduces expression of IL-1, IL-6, G-CSF and GM-CSF in human umbilical vein endothelium

BACKGROUND Areca nut chewing is associated with oral submucous fibrosis (OSF). Raised vascular basic fibroblast growth factor may induce fibrosis. Arecoline is a muscarinic alkaloid in areca nut, which we earlier reported causes injury and necrosis of human endothelium. MATERIALS AND METHODS Human umbilical vein endothelial cells were exposed to arecoline with or without tumor necrosis factor-α, and separately to acetylcholine, muscarine, or nicotine. Protein levels of basic fibroblast growth factor, as well as the inflammatory cytokines: granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor, and Interleukins-6, 1-α and 1-β, were determined by enzyme-linked immunosorbent assay. mRNA levels were established by real-time reverse transcription polymerase chain reaction. RESULTS Basic fibroblast growth factor was released into the culture medium at arecoline levels causing necrosis (P < 0.05). This contrasted with an opposite effect of arecoline on levels of the inflammatory cytokines (P < 0.05). Tumor necrosis factor-α increased IL-6 and granulocyte-macrophage colony stimulated factor, but arecoline reduced this stimulated expression (P < 0.05). Arecoline had no effect on mRNA for basic fibroblast growth factor, although there was reduced mRNA for the separate inflammatory cytokines studied. The effect of acetylcholine, muscarine, and nicotine was minimal and dissimilar to that of arecoline. CONCLUSIONS Data raise the possibility that arecoline-induced, vascular basic fibroblast growth factor contributes to OSF, by combining increased growth factor expression with endothelial necrosis, and thus driving fibroblast proliferation.

Membrane and cytoplasmic marker exchange between malignant neoplastic cells and fibroblasts via intermittent contact: increased tumour cell diversity independent of genetic change

We previously demonstrated that human osteosarcoma cells (SAOS‐2) induce contact‐dependent apoptosis in endothelium, and expected similar apoptosis in human gingival fibroblasts (h‐GF) using SAOS‐2 alkaline phosphatase (AP) to identify cells. However, h‐GF apoptosis did not occur, despite reduction in AP‐negative h‐GF number (p < 0.01) and enhancement of this by h‐GF TNFα pretreatment (p < 0.01). We suggest that TNFα‐enhanced transfer of membrane AP from SAOS‐2 to h‐GF would explain these data. This idea was investigated using fluorescence prelabelled cells and confocal laser scanning microscopy. Co‐cultures of membrane‐labelled h‐GF (marker‐DiO) and SAOS‐2 (marker‐DiD) generated dual‐labelled cells, primarily at the expense of single labelled h‐GF (p < 0.001), suggesting predominant membrane transfer from SAOS‐2 to h‐GF. However, opposite directional transfer predominated when membrane labels were reversed; SAOS‐2 further expressed green fluorescent protein (GFP) in cytoplasm and nuclei, and h‐GF additionally bore nuclear label (Syto59) (p < 0.001). Cytoplasmic exchange was investigated using h‐GF prelabelled with cytoplasmic DDAO‐SE and nuclear Syto59, co‐cultured with SAOS‐2 expressing GFP in cytoplasm and nuclei, and predominant cytoplasmic marker transferred from h‐GF to SAOS‐2 (p < 0.05). Pretreating h‐GF with TNFα increased exchange of membrane markers (p < 0.04) but did not affect either cell surface area profile or circularity. Dual‐labelled cells had a morphological phenotype differing from SAOS‐2 and h‐GF (p < 0.001). Time‐lapse microscopy revealed extensive migration of SAOS‐2 and cell process contact with h‐GF, with the appearance of SAOS‐2 indulging in ‘cellular sipping’ from h‐GF. Similar exchange of membrane was seen between h‐GF and with other cell lines (melanoma MeIRMu, NM39, WMM175, MM200‐B12; osteosarcoma U20S; ovarian carcinoma cells PE01, PE04 and COLO316), while cytoplasmic sharing was also seen in all cell lines other than U20S. We suggest that in some neoplasms, cellular sipping may contribute to phenotypic change and the generation of diverse tumour cell populations independent of genetic change, raising the possibility of a role in tumour progression. Copyright

SAOS-2 osteosarcoma cells bind fibroblasts via ICAM-1 and this is increased by tumour necrosis factor-α.

We recently reported exchange of membrane and cytoplasmic markers between SAOS-2 osteosarcoma cells and human gingival fibroblasts (h-GF) without comparable exchange of nuclear markers, while similar h-GF exchange was seen for melanoma and ovarian carcinoma cells. This process of “cellular sipping” changes phenotype such that cells sharing markers of both SAOS-2 and h-GF have morphology intermediate to that of either cell population cultured alone, evidencing increased tumour cell diversity without genetic change. TNF-α increases cellular sipping between h-GF and SAOS-2, and we here study binding of SAOS-2 to TNF-α treated h-GF to determine if increased cellular sipping can be accounted for by cytokine stimulated SAOS-2 binding. More SAOS-2 bound h-GF pe-seeded wells than culture plastic alone (p<0.001), and this was increased by h-GF pre-treatment with TNF-α (p<0.001). TNF-α stimulated binding was dose dependent and maximal at 1.16nM (p<0.05) with no activity below 0.006 nM. SAOS-2 binding to h-GF was independent of serum, while the lipopolysaccharide antagonist Polymyxin B did not affect results, and TNF-α activity was lost on boiling. h-GF binding of SAOS-2 started to increase after 30min TNF-α stimulation and was maximal by 1.5hr pre-treatment (p<0.001). h-GF retained maximal binding up to 6hrs after TNF-α stimulation, but this was lost by 18hrs (p<0.001). FACS analysis demonstrated increased ICAM-1 consistent with the time course of SAOS-2 binding, while antibody against ICAM-1 inhibited SAOS-2 adhesion (p<0.04). Pre-treating SAOS-2 with TNF-α reduced h-GF binding to background levels (p<0.003), and this opposite effect to h-GF cytokine stimulation suggests that the history of cytokine exposure of malignant cells migrating across different microenvironments can influence subsequent interactions with fibroblasts. Since cytokine stimulated binding was comparable in magnitude to earlier reported TNF-α stimulated cellular sipping, we conclude that TNF-α stimulated cellular sipping likely reflects increased SAOS-2 binding as opposed to enhanced exchange mechanisms.

Inhibitory Effects of Angiotensin II Receptor Blockade on Human Tenon Fibroblast Migration and Reactive Oxygen Species Production in Cell Culture

Purpose We investigate the effect of angiotensin receptor blockade on the migration of human Tenon fibroblasts (HTF), using irbesartan, an angiotensin II receptor type 1 (AT1R) blocker (ARB) as a potential antifibrotic agent in glaucoma filtration surgery. Methods Confluent HTF cultures were scratched with a 1 mL pipette tip and treated with either irbesartan (10, 50, and 100 μg/mL) or angiotensin II (2 μg/mL). The extent of HTF migration up to 30 hours, and cell number and morphology at 72 hours was evaluated. To assess the effect on reactive oxygen species (ROS) level, HTF were treated with either irbesartan (10 μg/mL) or angiotensin II (2 μg/mL) for 24 hours after scratching, and then stained with dihydroethidium (DHE) before evaluation by confocal microscopy. Results Irbesartan inhibited HTF migration by 50% to 70% compared to controls (P < 0.05). Levels of ROS were almost completely attenuated by irbesartan (DHE fluorescence intensity of 5.68E-09) (P < 0.05). Irbesartan reduced cell numbers by 50% and induced morphologic changes with loss of pseudopods (P < 0.05). Conversely, angiotensin II increased cell numbers up to 4-fold while retaining cell viability. Conclusions Irbesartan inhibited HTF migration and ROS production. It also reduced cell numbers and altered HTF morphology. Angiotensin II increased cell number without altering morphology. This initial study warrants future investigations for further potential antifibrotic effects of this drug. Translational Relevance This in vitro study focused on investigations of irbesartans effects on HTF migration, ROS production, as well as HTF cell numbers and morphology. It suggests a potential therapeutic strategy worth further exploration with a view towards postoperative wound healing modulation in glaucoma filtration surgery.

A method for investigating the cellular response to cyclic tension or compression in three-dimensional culture

We have an interest in the cellular response to mechanical stimuli, and here describe an in-vitro method to examine the response of cells cultured in a three-dimensional matrix to mechanical compressive and tensile stress. Synthetic aliphatic polyester scaffolds coated with 45S5 bioactive glass were seeded with human dental follicular cells (HDFC), and attached to well inserts and magnetic endplates in six well palates. Scaffolds were subjected to either cyclic 10% tensile deformation, or 8% compression, at 1 Hz and 2 Hz respectively for 6, 24 or 48 h, by uniaxial motion of magnetically-coupled endplates. It was possible to isolate high quality mRNA from cells in these scaffolds, as demonstrated by high RNA integrity numbers scores, and ability to perform meaningful cRNA microarray analysis, in which 669 and 727 genes were consistently upregulated, and 662 and 518 genes down regulated at all times studied under tensile and compressive loading conditions respectively. MetaCore analysis revealed the most regulated gene ontogenies under both loading conditions to be for: cytoskeletal remodelling; cell adhesion-chemokines and adhesion; cytoskeleton remodelling-TGF WNT and cytoskeletal remodelling pathways. We believe the method here described will be of value for analysis of the cellular response to cyclic loading.