Abstract
Purpose:
Previous studies from our group have shown that calcium/calmodulin-dependent protein kinase II (CaMKII) plays a critical role in VEGF-induced retinal angiogenic signalling. In the present study, we have extended this work to examine the wider contribution of CAMKII signalling to growth factor (GF)-induced retinal angiogenesis in vitro and the specific involvement of the γ and δ isoforms of CAMKII in this process.
Methods:
Human retinal microvascular endothelial cells (hRMECs) were cultured and stimulated over a 24h period with a range of GFs (vascular endothelial growth factor [VEGF], fibroblast growth factor [FGF], insulin-like growth factor [IGF], hepatocyte growth factor [HGF], placental growth factor [PlGF] and platelet derived growth factor [PDGF]) at 50ng/ml. Total and phosphorylated protein levels of CaMKII were detected using western blotting. The effects of CaMKII inhibition using 10μM KN93 (CaMKII inhibitor) and its inactive analogue KN92 on GF-induced sprouting angiogenesis in vitro were evaluated. Small interference RNA (siRNA; 50nM) mediated knockdown of CaMKIIγ and δ isoforms was used to investigate the relevance of these isoforms in GF-induced retinal endothelial cell migration, tube formation and sprouting angiogenesis.
Results:
Exposing hRMECS to VEGF, FGF, HGF, IGF and PlGF triggered a time-dependent increase in total and phospho-CAMKII protein levels. In contrast, PDGF had no effect on CAMKII phosphorylation or total CAMKII levels. All GFs with the exception of PlGF and PDGF stimulated sprout formation compared with controls. VEGF, FGF, HGF and IGF also stimulated hRMEC migration and tube formation. KN93 reduced GF-induced sprout formation to control levels, whereas KN92 (inactive analogue) had no effect. siRNA knockdown of CaMKIIδ isoform significantly reduced GF-induced sprouting angiogenesis, migration and tube formation to control levels, whereas siRNA targeting of CaMKIIγ had only a partial effect.
Conclusions:
These results suggest that both CaMKIIδ and γ isoforms are involved in mediating GF-induced angiogenic activity. CaMKII is thus an important regulator of GF-induced retinal angiogenesis and treatments targeting the γ and δ isoforms of this protein have the potential to reduce abnormal angiogenesis in ocular diseases.
Keywords: 499 diabetic retinopathy •
498 diabetes •
543 growth factors/growth factor receptors