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TUMOUR NECROSIS FACTOR (TNF-Α) ENHANCES MONOCYTES TRANS-ENDOTHELIAL MIGRATION (TEM) THROUGH GAP JUNCTION (GJ); A ROLE FOR PROTEIN KINASE C (PKC) BUT NOT MAPK
  1. Ahmed H Qasem,
  2. Matt Warner,
  3. Christopher H Fry,
  4. Rita I Jabr
  1. Biochemistry and Physiology, University of Surrey, Surrey, United Kingdom

Abstract

Atherosclerosis is a chronic inflammatory disease characterised by accumulation of monocytic cells and lipids within the sub-endothelial space by direct MO-to-EC contact through GJ. Both cell types express connexin (Cx)43 isoforms that permit formation of GJ. This is enhanced by adhesion molecules in the presence of pro-inflammatory stimuli such as TNF-α.TNF-α suggested to have a role on Cx43 expression through MAPK mainly over other intercellular pathways, however, so far the mechanism is still unclear which our hypothesis is to investigate it. Experiments were carried out in the absence and presence of 25ng/ml TNF-α.Human umbilical vein endothelial cells (HUVECs) monolayer functional integrity was assessed by measuring trans-endothelial electrical resistance (TEER). TEM assay used as a model for the transmigration of MOs to the sub-endothelial space. Upper and lower contents were collected, marked with CD14+ and measured by flow cytometry. MAPk inhibitors, Chelerythrine Chloride(ChC) and SB 203580. TEER measurements shown a reduction in the resistance in the presence of TNF-α compared to the control respectively (13.58±2.82 Ω.cm2, 43.94±3.32 Ω.cm2; n=6), however, in the presence of PKC inhibitor; ChC, the resistant was the same as control (48.64±3.62 Ω.cm2, 45.76±4.83 Ω.cm2; n=6). In TEM,TNF-α enhanced the migration of MOs (66.3±2.1%; n=3) compared to the control (9.9±4.5%; n=3; P<0.001). Such an effect was prevented in monolayer treated with ChC (13.70±2.45%; n=3; P<0.001). Also, this fraction was attenuated by GAP26 and GAP27 and was comparable to the fraction of HUVECs Cx43-siRNA (8.33±4.66%; n=3). These results show that GJs is formed between ECs and MOs. TNF-α increased TEM, reduced the TEER and had no effects in HUVECs with Cx43-siRNA.The effect of TNF-α is mediated through PKC as it is effect attenuated by PKC inhibitor; ChC.

  • CARDIAC PROCEDURES AND THERAPY

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