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An in vitro human 3D co-culture model to study endothelial-astrocyte interactionsPeddagangannagari, Sreekanth Reddy January 2012 (has links)
At the gliovascular interface, reciprocal inductive influences between brain microvascular endothelial cells (BMVEC) and astrocytes occur. Most of the knowledge in this area of research is derived from in vitro eo-culture models in which astrocytes are cultured on a stiff, two-dimensional (2D) surface. Three-dimensional (3D) culture models closely mimic the in lJZVO cellular architecture and they bridge the gap between 2D culture models and animal models. Hence, an in vitro 3D eo-culture model was developed and characterised, to study the interactions between BMVEC and astrocytes. In this model, human astrocytes (HA) were seeded inside a collagen type--I gel while human immortalised cerebral microvascular endothelial cells (hCMEC/D3) were cultured on the gel surface. Both cell types were of human origin to improve the translatability of findings to humans in vivo. Additional important features of the model are the culture of endothelial cells on a soft matrix, and the simulation of the geometric relationship that exists in vivo i.e., the interaction of astrocytes with BMVEC from their ab luminal side. To determine the effect of the 3D environment on the HA, the proliferation rate and expression of four molecules namely, glial fibrillary acidic protein (GF AP), aquaporin-4 (AQP4), endothelin-l (Et-l) and endothelin receptor type-B (EDNRB), were compared between 3D and 2D cultured HA. The decreased expression of AQP4 and EDNRB and the much-decreased proliferation rate of 3D HA suggested their reduced reactivity and a similarity to their in lJiVO counterparts. However, 3D HA did not differ from the 2D HA in their ability to release soluble factors that induce barrier properties on BMVEC, as observed by similar levels of three expression markers of barrier phenotype on hCMEC/D3 cells namely, zonula occludens-l, claudin-5, and P-glycoprotein and similar paracellular permeability coefficients to fluorescent-dextrans (70 kDa).
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Cellular and molecular basis of the mouse oesophagus epithelial developmentYu, Wei-Yuan January 2005 (has links)
No description available.
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The effects of garlic upon endothelial function, vascular inflammation, oxidative stress and insulin resistance in patients with type 2 diabetes at high cardiovascular risk : a double blind randomised placebo controlled trialAtkin, Marc January 2011 (has links)
Background and aims: Endothelial dysfunction, vascular inflammation and oxidative stress have been integrally linked to the pathogenesis of both type 2 diabetes and cardiovascular disease. Aged Garlic Extract (AGE), a potent antioxidant, has been shown in previous studies to attenuate these novel risk factors in a non-diabetic population. Aims: This study tested the hypothesis that AGE may improve endothelial function , oxidative stress, vascular inflammation and insulin resistance in high risk cardiovascular subjects with type 2 diabetes (defined as >30% Cardiovascular risk over 10 yrs). Methods: A double blind, placebo controlled cross-over study was performed in 26 type 2 diabetic patients who received 1200mg of AGE or placebo daily for 4 weeks with a 4 week washout period. Plasma HsCRP was measured as a marker of inflammation. TAOS,GSH/GSSG and LHP were measured as markers of oxidative stress/anti-oxidant defence. Insulin resistance was measured using the HOMA-IR method. Endothelial function was measured using change in the reflective index (RI) post salbutamol using digital photoplethysmography and urinary albumin/creatinine ratio was measured as a biochemical surrogate. Measurements were taken at baseline and after intervention with AGE or placebo. Results: Of the 26 patients studied (Male 17, Female 9), mean age was 61 ± 8 yrs, HbA1c 7.2 ± 1.1%, BP 130/75 ± 15.9/9.8 mmHg, total cholesterol 4.2 ± 0.81 mmol/l, triglyceride 2.11 ± 1.51 mmol/l, HDL-cholesterol 1.04 ± 0.29 mmol/l. The majority of patients were being treated with metformin (59%), aspirin (50%) and statin (96%) therapy. 36% were treated with an ACEI. There were no changes in these therapies throughout the study. Treatment with AGE had no significant effect upon the above metabolic parameters including insulin resistance. Systolic blood pressure pre AGE 130 ± 15mmHg vs post AGE 130± 14mmHg. Total cholesterol pre AGE 4.2 ± 0.9 mmol/l vs post AGE 4.2 ± 0.8 mmol/l. Triglycerides pre AGE 1.4 IQ range 0.7 mmol/l vs post AGE 1.4 IQ range 0.8 mmol/l. HDL cholesterol pre AGE 1.0 ± 0.3 mmol/l vs post AGE 1.0 ± 0.3mmol/l. In addition, no statistically significant difference was found in plasma HsCRP (pre AGE: median 2.0mg/l, IQ range 0.8-2.7 vs post AGE: median 1.83mg/l, IQ range 1.1-3.2, p = 0.89) or urinary albumin/creatinine ratio (pre AGE: 0.55, IQ range 0.4-1.65 vs post AGE: 0.6, IQ range 0.47-1.5, p = 0.43) endothelial function (change in RI pre AGE: 6.5%, IQ range 2.75-11 vs post AGE: 6.5%, IQ range 2.75-13, p = 0.95) with AGE or placebo. Conclusion: In this group of type 2 diabetic patients at high cardiovascular risk, 4 weeks treatment with AGE did not significantly improve endothelial function, vascular inflammation, oxidative stress or insulin resistance.
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The relationship between in-vitro endothelial permeability and molecules of the intercellular junctionBudworth, Rachel Ann January 2003 (has links)
Changes in endothelial permeability are known to contribute to many pathologies, including inflammation seen in skin irritation. The regulation of permeability has been linked to inter-endothelial junctions, specifically the tight and adherens junctions (TJ and AJ). The functional state of the junction is thought to be associated with numerous factors including cytoskeletal changes and the interactions between junctional components. How these factors interact and respond to inflammatory stimuli is still not fully understood. This project examined two human endothelial cell-lines for use in in-vitro permeability studies, ECV304 and HMEC-1. Changes in permeability along with arrangement of the F-actin cytoskeleton and the adherens junction molecule VE-cadherin were studied in response to a variety of compounds. Macromolecular permeability was assessed by measuring the leakage of fluorescently labelled dextrans of varying molecular weights. The F-actin and VE-cadherin were visualised using immunocytochemical techniques. TEM was undertaken to examine the ultrastructure of the junctions The ECV304 cells, whilst showing an increased permeability to a variety to vaso-active mediators, did not express some of the pertinent junctional molecules of the endothelium and thus are not recommended for use. The basal permeability of the HMEC-l cell-line was shown to act in predictable fashion, giving comparable permeability coefficients to other endothelial cells. The increase in permeability following exposure to A23187, CAPB, EGTA and PMA was shown to correlate to an altered expression of VE-cadherin and F-actin. These observations were furthered using histamine, where a quantifiable change in the levels of continuous and stitch VE-cadherin staining was demonstrated. The permeability response to histamine occurred much later than the VE-cadherin and F-actin changes. This could be due to methodology and/or the cells lack of TJs. The apparent lack of mature AJs was approached by exposing the cultures to cAMP-raising media. This significantly reduced the basal permeability and increased the expression of AJ components, apart from β-catenin, at the cell-cell contacts. Indeed α-catenin was redistributed from the triton-soluble fraction of the cells to the triton-insoluble fraction, which is proposed to contain the junctional components. These results demonstrate additional information on the role that the adherens junction molecules play in endothelial permeability and characterise the HMEC-1 cell-line for further use in this field.
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Platelet and endothelial cell interactions in vitro / Kathryn Moira Wilson.Wilson, Kathryn Moira January 1994 (has links)
Bibliography: leaves 300-326. / 326 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Evaluates an in vitro experimental system which was designed to assess functions of platelets and cultured endothelial cells when they were incubated either independently or in combination. / Thesis (Ph.D.)--University of Adelaide, Dept. of Clinical and Experimental Pharmacology, 1995
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Cell fate specification and polarisation in mouse preimplantation epitheliaDoughton, Gail Louise January 2014 (has links)
Understanding the establishment of polarity and the cell fate specification of epithelial cells is important for developmental biology, regenerative medicine and the study of cancer. In this thesis, models of pre-implantation epithelial development are used to investigate the relationship between these two processes. The trophoblast is an extraembryonic epithelial tissue which contributes to the placenta. Addition of BMP4 to mouse and human embryonic stem (mES) cells grown in culture has been suggested to induce differentiation of cells to the trophoblast lineage. The use of this differentiation method was investigated as a possible model of trophoblast polarisation and cell fate specification. Unfortunately, with the protocol and reagents available this model did not appear to physiologically recapitulate trophoblast development and was not reliable. The primitive endoderm is an epithelium which arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. When mES cells are grown in suspension they form aggregates of differentiating cells known as embryoid bodies. The outermost cell layer of an embryoid body is an epithelial cell type comparable to the primitive endoderm. Embryoid bodies were used here to study the polarisation and cell fate specification of the primitive endoderm. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. The acquisition of epithelial polarity occurred prior to the maximal expression of cell fate markers. Fgfr/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier which normally blocks free diffusion across the epithelial cell layer occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erkmediated polarisation. This data shows that the formation of a polarised primitive endoderm layer in embryoid bodies requires the Fgfr/Erk signalling pathway.
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Characterisation of human PETA-3 : a member of the transmembrane 4 superfamily / by Paul Martin Sincock.Sincock, Paul Martin January 1998 (has links)
Copy of author's previously published article in pocket on back end-paper. / Includes bibliography (leaves 135-185). / 185, [94] leaves, [32] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to characterise the expression of PETA-3 (Platelet Endothelial Tetraspan Antigen-3), CD9, CD63 and ?gb?s1 integrins in normal human tissue ; to determine the subcellular localisation in endothilial cells and platelets ; to investigate protein-protein interactions involving PETA-3 ; and to examine the effects of anti-PETA-3 monoclonial antibodies on platelet and endothilial cell function. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1999
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