The Function And Molecular Mechanisms Of Hairy/enhancer Of Split Homologue-1 Suppresses Vascular Endothelial Growth Factor-induced Angiogenesis Via Downregulation Of Osteopontin Expression In The Development And Progression Of Atherosclerosis

Backgroundand purpose:Atherosclerosis is the principal contributing factor to the pathology of cardiovascular disease.The clinical complications of atherosclerosis are mainly caused by thrombus formation,which results from rupture of a vulnerable atherosclerotic plaque.Importantly,angiogenesis is a key feature of atherosclerosis.The formation of microvessels(angiogenesis)in an atherosclerotic plaque contributes to the development of vulnerable plaques,thereby increasing the risk of rupture Although the regulation of angiogenesis in atherosclerotic plaques is still under investigation,suppression of angiogenesis is considered a promising therapeutic strategy for the treatment of atherosclerosis.Hairy/enhancer of split homologue-1(Hes-1)is a transcriptional repressor of members of the basic helix-loop-helix family of transcription factors.It has been shown that Hes-1 plays a critical role in the regulation of various physiological processes,including cellular differentiation,cycling,apoptosis,and self-renewal.Hes-1-regulated transcription is essential to cardiovascular development and its function has been linked to smooth muscle differentiation,angiogenic processes,arterial-venous cell fate determination,and vascular morphogenesis.In addition,increasing evidence has shown that Hes-1 is involved in the regulation of endothelial cell(EC)proliferation,differentiation,and apoptosis,which are fundamental processes necessary for angiogenesis.Curry et al.demonstrated that Hes-1 expression is regulated in confluent ECs by the JNK signaling pathway,which induces EC growth arrest.However,it remains unknown whether Hes-1 is involved in the progression and vulnerability of atherosclerotic plaques by affecting angiogenesis.Osteopontin(OPN)is an acidic glycoprotein that is a member of the small integrin-binding N-linked glycoprotein family that was recently found to play both proinflammatory and proatherogenic roles in the pathogenesis of atherosclerotic plaques induced by various atherosclerotic risk factors Moreover,initial studies have demonstrated that OPN not only promotes arteriosclerosis but is also closely associated with angiogenesis.Wang et al.revealed that OPN directly stimulates angiogenesis via the avβ3/PI3-K/AKT/eNOS/NO signaling pathway and Junaid et al.found that OPN improves survival and differentiation of ECs during angiogenesis by binding to the integrin αvβ3 and triggering downstream signaling.These results indicate that OPN is a promising therapeutic target for angiogenesis in atherosclerosis.Although the mechanism underlying OPN regulation during angiogenesis remains unclear,Matsue et al.showed that Hes-1 overexpression suppressed OPN transcription in osteoblastic cells,suggesting that Hes-1 is a potential regulator ofOPN.In atherosclerotic plaques,vascular endothelial growth factor(VEGF)produced by inflammatory cells and/or ECs has been implicated in the initiation of intraplaque angiogenesis and promotion of plaque formation,which eventually results in intraplaque hemorrhage.Hes-1-monitored transcription has been shown to be involved the VEGF signaling pathway.Thomas et al.showed that Hes-1 was a signaling hub in early retinal progenitor cells that integrates downstream VEGF signaling,although the role of Hes-1 in VEGF-induced angiogenesis remains unclear.In addition to Hes-1,increasing studies have found that VEGF induces OPN expression in ECs.However,whether Hes-1 plays a role in VEGF-induced angiogenesis in atherosclerotic plaque formation via OPN regulation has not yet been elucidated.Methods1.Tissuemicroarrayanalysis found that Hes-1 and OPNdifferentially expressed between atherosclerotic plaquesnormal arterial intima tissues;2.Immunohistochemistry,immunofluorescence and real-time PCR analysis were used to determined expression of Hes-1 and OPN in four atherosclerotic plaques and four normal arterial intima tissues;3.Underlying mechanism of atherosclerosis was detected by identification of targets of VEGF and their associations by real-time PCR analysis and western blotting;4.Transfected with siRNA targeting Hes-1 or recombinant plasmid over-expressing Hes-1(pcDNA-Hes-1)on regulation of OPN induced by VEGFwere used to investigate whether VEGF-stimulated up-regulation of OPN could be affected by Hes-1 throughwestern blot.5.Tube formation test was used to delineate the role of VEGF regulated Hes-1 on angiogenesis and whether Hes-1 attenuates angiogenesis by suppressing OPN.ResultThe results of this study demonstrated that Hes-1 was significantly downregulated,while OPN was upregulated in atherosclerotic plaques,and that both Hes-1 and OPN were expressed in ECs of neovessels in atherosclerotic plaques.Moreover,Hes-1 knockdown in human umbilical vein endothelial cells(HUVECs)augmented VEGF-induced upregulation of OPN and promoted angiogenesis,but Hes-1 overexpression suppressed VEGF-induced elevation of OPN and inhibited angiogenesis,suggesting that decreased Hes-1 expression may promote VEGF-angiogenesis,likely by upregulating OPN in atherosclerotic plaques and restoring Hes-1 expression to stabilize vulnerable plaques by suppressing intraplaque angiogenesis.These findings indicate that Hes-1 and OPN are novel modulators of the progression and vulnerability of atherosclerotic plaques,and that dysregulation of these factors may contribute to the pathogenesis of vulnerable atherosclerotic plaques.ConclusionThis is the first study to reveal that Hes-1 in ECs plays an important role in angiogenesis by OPN regulation,and that Hes-1 downregulation and OPN upregulation may contribute to the progression and vulnerability of atherosclerotic plaques.This knowledge may shed new light on the therapy of vulnerable plaque formation in patients with atherosclerosis.