Title:
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The role of hyaluronan in angiogenesis through RHAMM and CD44 receptors in endothelial cells
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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
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