Oligosaccharides of hyaluronan (o-HA; 3-10 disaccharides) are pro-angiogenic and are believed to act through two receptors, C044 and RHAMM (receptor for hyluronanmediated motility). Utilizing human liver micro-vessel-derived endothelial cells (HLMVEC)and bovine aortic endothelial cells (BAEC), the roles of C044 and RHAMM in o-HA induced signalling were examined. In vitro assays were employed for proliferation, migration and tube formati~m by endothelial cells, prior to and following C044 and RHAMM knock-down, using small interference RNA (siRNA) technology. Epidermal growth factor (EGF), which has stimulatory effect on endothelial cells, but does not act through C044 or RHAMM, was used to assess specificity of the results. All data were analysed using SSP statistical package and p values S 0.05 were considered significant. The results are summarised as follows: • Following knock-down of CD44 and RIIAMM, using siRNA technology, in HLMVEC and BAEC, the result of RT-PCR and Western blotting revealed a highly significant decrease in their gene and protein expression in both cell types. • A significant increase in cell proliferation, as quantified by cell number, was obtained in the presence of o-HA and EGF, compared with untreated controls. • Knock-down of RIIAMM, significantly inhibited BAEC (-30%) and Hr_.MVEC (-25%), whereas in neither cell type knock-down of C044 had no effect on cell proliferation in either cell type. RHAMM and CD44 knock-down almost completely inhibited o-HA-induced cell proliferation, compared with controls and negative control (NC) siRNA-treated cells. In contrast, an angiogenic growth factor, epidermal growth factor (EGF) which operates independently of CD44 or RHAMM was used as a control. After knocking-down C044 and RHAMM in BAEC or HLMVEC, EGF induced proliferation remained unaffected compared with, controls • In wound healing migration assay, treatment with o-HA significantly stimulated migration of BAEC compared with controls. Following knock-down of CD44 and RHAMM, o-HA-induced wound healing was markedly decreased compared with controls. • In another assay for migration that utilizes Boyden chamber, again o-HA and EGF significantly promoted migration of HLMVEC. Following knock-down of RHAMM and CD44, o-HA, unlike EGF, induced significantly lower (-5-fold) migration compared with controls. • In Matrigel assay for tube (capillary net-work) formation, o-HA and EGF significantly enhanced tube formation by BAEC and HLMVEC. Knock-down of CD44 even in the absence of o-HA induced a significant increase in tube formation compared with non-transfected cells. In o-HA stimulated BAEC and HLMVEC tube formation significantly decreased in comparison to controls, but the effect of EGF remained the same in knock-down and controls. • To ascertain possible signal transduction mechanisms, it was shown that CD44 and RHAMM acted through phosphor-ERK expression in o-HA treated cells, compared with control cells. In both BAEC and HLMVEC. a significant increase was observed in phospho-ERK1I2, compared with the controls. After knocking down CD44 or RHAMM there was a significant decrease in phospho-ERK1I2 expression, compared with untreated controls, in both BAEC and HLMVEC. Collectively, these results suggest that o-HA induced signalling pathways are independently mediated by RHAMM and CD44 receptors in micro- and macro-vascular EC, through a mechanism that remains to be fully elucidated
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:516733 |
| Date | January 2009 |
| Creators | Al-Baradie, Raid Saleem |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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