Circadian Rhythm Regulates VIP-induced Schlemm’s Canal Expansion Via Bmal1-Atg14-VPAC2 Pathway

There exists an internal clock within the human body,which is defined as “circadian rhythm” since it synchronizes physiological activities to daily light-dark cycle.The circadian rhythm manifests various outputs such as sleep-wake cycle,blood pressure and intraocular pressure(IOP),under the regulation of which tissues and organs collaborate to meet dynamics needs across the day.The circadian rhythm therefore prepares one to anticipate environmental changes rather than just reaction.It is well acknowledged that circadian rhythm is involved with not only the onset but also the progression of numerous diseases that are either metabolic,cardiovascular,neoplastic or neurological.Moreover,it is now appreciated that circadian rhythm is also relevant to clinical outcomes.Thus,to improve circadian rhythm is to take initiative for one’s well-being.Primary open-angle glaucoma(POAG)is one of the primary causes that lead to irreversible blindness.The elevation of measured IOP is consistently involved with POAG onset,diagnosis and treatment.IOP is generated and maintained through aqueous humor inflow and outflow,with Schlemm’s canal playing a key role in the latter.The expansion or collapse of Schlemm’s canal is therefore closely related with IOP fluctuation.Our research group previously demonstrated that vasoactive intestinal polypeptide(VIP)expands the lumen of Schlemm’s canal and reduces IOP via receptor VPAC2.These findings preliminarily indicated the potential for VIP as anti-glaucomatous drugs.In the pre-experiments concerning disrupted circadian rhythm,however,the hypotensive efficacy of VIP was dampened.To further illustrate the underlying mechanism,relative experiments in the dissertation were carried out with three primary objectives.The first,to explore the potential for Schlemm’s canal as a target under circadian regulation.The second,to acquire more knowledge of Schlemm’s canal since it is a rather special type of endothelial lumen.The third,to improve competency of VIP as ocular hypotensive drugs in multiple settings.The dissertation would be illustrated in two parts.Part Ⅰ: An animal model of disrupted circadian rhythm was established and confirmed.The potential for Schlemm’s canal as a target under circadian regulation was also explored.The power of VIP to expand Schlemm’s canal and reduce IOP was then examined under the intended setting.Part Ⅱ: To confirm that decreased VPAC2 expression at Schlemm’s canal endothelial cells was the key to dampened VIP efficacy.The underpinning mechanism by which Bmal1(the key circadian gene)regulates VPAC2 via Atg14 was further explored in vitro.Additionally,melatonin was introduced adjuvant to VIP under disrupted circadian rhythm to achieve effective ocularhypotensive effect.Part Ⅰ: Dampened efficacy of VIP to expand Schlemm’s canal under disrupted circadian rhythmObjective: To explore the potential for Schlemm’s canal as a target under circadian regulation,and to examine whether VIP-induced Schlemm’s canal expansion is affected under disrupted circadian rhythm.Methods: Disrupted circadian rhythm was established in a rat model by keeping rats under constant darkness,of which the reliability was examined by voluntary wheel-running activities and serum DLMO.IOP under different rhythm was recorded after VIP administration,of which Schlemm’s canal was visualized by HE-staining.Bmal1 expression was IHC-stained at Schlemm’s canal endothelium under different rhythm.Results: Constant darkness caused circadian disruption of rat voluntary wheel-running activity and serum DLMO.Disruption of circadian rhythm reduced Bmal1 expression of Schlemm’s canal endothelium,which was more significant at objective night than day when VIP-induced Schlemm’s canal expansion was also diminished.Conclusions: Constant darkness leads to disruption of circadian rhythm,under which Schlemm’s canal is a potential target.The VIP-induced Schlemm’s canal expansion and IOP reduction are diminished under disrupted circadian rhythm.Part Ⅱ: VIP-induced Schlemm’s canal expansion is regulated by Bmal1-Atg14-VPAC2 pathwayObjective: To investigate Bmal1-regulated VPAC2 expression at Schlemm’s canal endothelium as well as the underlying mechanism,and turn to “VIP and melatonin” as an alternative to promote VIP-induced Schlemm’s canal expansion under disrupted circadian rhythm.Methods: VPAC2 expression at Schlemm’s canal endothelium was detected by IHCstaining under different rhythm.In HUVEC model,Bmal1 was interfered or overexpressed prior to WB of VPAC2 protein.By means Of CHX chase assay,GFP-LC3 probe and WB,the autophagic flux was detected when Bmal1 was down-regulated.After transduction of flag-VPAC2 plasmids into 293 T cells,the colocalization of VPAC2 with early/recycling/late endosomes and lysosome was quantified under confocal microscopy.The role of Atg14 as an intermediate between VPAC2 and Bmal1 was demonstrated by Co-IP,WB and RT-q PCR.Under disrupted circadian rhythm,melatonin was orally-administered adjuvant to VIP,of which IOP and Schlemm’s canal expansion were measured.The capability of melatonin to promote Bmal1 and membrane VPAC2 expression was examined by IHC-staining or WB.Results: Under disrupted circadian rhythm,VPAC2 expression decreased at Schlemm’s canal endothelium.Bmal1 could regulate VPAC2 expression in vitro via increased lysosomal degradation of membrane VPAC2.Atg14 could be transcriptionally-regulated by Bmal1 and showed cytoplasmic interaction with VPAC2.Adjuvant therapy of “VIP and melatonin” significantly expanded Schlemm’s canal,reduced IOP compared with VIP alone.Melatonin increased Bmal1 and membrane VPAC2 expression of endothelium in vivo or in vitro.Conclusions: VPAC2 expression at Schlemm’s canal endothelium is regulated by circadian rhythm via Bmal1-Atg14-VPAC2 pathway.Melatonin “rescued” VIP-induced Schlemm’s canal expansion and hypotensive effect under disrupted circadian rhythm possibly via increased Bmal1 and membrane VPAC2 expression.