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The effects of high glucose on connexin 43 in human endothelial cells
Author(s) -
Willmott T.,
Leach L.
Publication year - 2002
Publication title -
journal of anatomy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.932
H-Index - 118
eISSN - 1469-7580
pISSN - 0021-8782
DOI - 10.1046/j.1469-7580.2002.00047_25.x
Subject(s) - gap junction , connexin , cytosol , paracellular transport , biology , microbiology and biotechnology , angiogenesis , cell , endothelial stem cell , intracellular , homeostasis , cell culture , biochemistry , cancer research , genetics , membrane , in vitro , permeability (electromagnetism) , enzyme
Connexins (cx) are a family of gap junctional proteins found in most mammalian cells with cx43 being one of the residents in endothelial cells. Gap junctional communications are thought to play a major role in maintaining tissue homeostasis and in angiogenesis. The latter functions are impaired in diabetes mellitus but the specific role of connexins in this pathology is currently not known. The aim of this study was to identify any changes in surface expression of cx43 in primary endothelial cells isolated from the human umbilical cord (HUVEC) and exposed to different duration of glycaemic insult. Cells were grown in euglycaemic (5 m m D glucose) and hyperglycaemic (25 m m d ‐glucose) conditions with l ‐glucose as the osmotic control for 2, 7 and 14 d. Confocal microscopy using identical laser settings was employed to look at both pattern of localisation and fluorescence intensity. At d 2, under euglycaemic conditions cx43 was localised throughout the cytosol (punctate labelling) but predominantly at perinuclear regions. Cx43 was also seen at cell‐cell margins as a diffuse ‘zigzag’ pattern. By 7 d this latter staining at cell‐cell borders resembled a ‘beads on a string’ formation, indicative of discrete gap junctions. By 14 d the cells were forming tubes with punctate labelling throughout the cytosol and beads on a string localisation in the paracellular clefts. Cells cultured in 25 m m d ‐glucose showed a different pattern of expression of cx43; at 2 d cx43 was present as discrete puncta at cell‐cell borders and throughout the cytosol and perinuclear region. Both fluorescent intensity and frequency of puncta appear diminished. By 7 d cell‐cell borders are immuno‐negative to cx43. By 14 d HUVEC cells had formed tubes. There was intense immunoreactivity to cx43 in the cytosol but not at paracellular clefts. This change in expression pattern is not an osmotic effect as the cells cultured in 25 m m l ‐glucose reflected a similar pattern of expression as cells grown in euglycaemic conditions. In conclusion this study is a first indication that hyperglycaemia alters targetting of cx43 to cell‐cell borders and formation of connexons. Funded by Smith & Nephew & The University of Nottingham