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名古屋大学博士学位論文 学位の種類 : 博士(医学)(論文) 学位授与年月日:平成5年9月14日 水谷大裕氏の博士論文として提出された

Ameliorating cardiac microvascular injury is the most effective means to mitigate diabetes-induced cardiovascular complications. Inositol-requiring 1α (IRE1α), a sensor of endoplasmic reticulum stress, is activated by Toll like receptors (TLRs), and then promotes cardiac microvascular injury. Peli1 is a master regulator of TLRs and activates IRE1α. This study aims to investigate whether Peli1 in endothelial cells promotes diabetes-induced cardiac microvascular injury through activating IRE1α. Here we found that Peli1 was markedly up-regulated in cardiac endothelial cells of both diabetic mice and in AGEs-treated cardiac microvascular endothelial cells (CMECs). Peli1 deficiency in endothelial cells significantly alleviated diabetes-induced cardiac microvascular permeability, promoted microvascular regeneration, and suppressed apoptosis, accompanied by the attenuation of adverse cardiac remodeling. Furthermore, Peli1 deletion in CMECs ameliorated AGEs-induced damages in vitro. We identified heat shock protein 90 (Hsp90) as a potential binding partner for Peli1, and the Ring domain of Peli1 directly bound with Hsp90 to enhance IRE1α phosphorylation. Our study suggests that blocking Peli1 in endothelial cells may protect against diabetes-induced cardiac microvascular injury by restraining ER stress.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Endothelial injury and dysfunction precede the development of cardiovascular diseases. The endothelium may be regarded as the first line of defence against inflammation / obesity-induced vascular injury, therefore gaining more information on the mechanisms of injury and response to injury, as well as modulating endothelial function may be key in the prevention of cardiovascular diseases. Endothelial cells differ in structure and function, therefore endothelial heterogeneity may be relevant when investigating endothelial function and dysfunction. Understanding endothelial heterogeneity in response to pathophysiological stimuli may be of significance in the prevention of cardiovascular diseases. Oleanolic acid (OA), a plant-derived triterpenoid, has been shown to possess endothelio-protective properties; however, its role in reversing endothelial injury is poorly understood. This study investigated endothelial heterogeneity between aortic endothelial cells (AECs) and cardiac microvascular endothelial cells (CMECs) at baseline and in response to an inflammatory insult via the cytokine, tumour necrosis factor-alpha (TNF-α). An in vitro model of endothelial injury was developed by treating AECs and CMECs with 20 ng/ml TNF-α for 24 hours. Endothelial heterogeneity was investigated by comparing intracellular nitric oxide (NO) and reactive oxygen species (ROS) production, protein expression and phosphorylation, and large-scale protein expression and regulation in AECs and CMECs. The experimental techniques included flow cytometry, western blots and proteomic analyses. An ex vivo model of endothelial injury was included to investigate vascular function in aortic rings from lean and high fat diet (HFD) rats. The role of OA in reversing TNF-α-induced injury and modulating vascular function in the ex vivo model was investigated. Although baseline NO-levels were similar between AECs and CMECs, heterogeneity was observed with regards to the NO biosynthetic pathway in terms of increased eNOS expression in CMECs. Baseline ROS levels were heterogeneous between AECs and CMECs, interestingly CMECs possessed higher anti-oxidant capacity. An in vitro model of TNF-α-induced injury was confirmed by decreased NO-levels, increased ROS-levels and necrosis, up-regulation of apoptotic proteins and activation of inflammatory pathways in AECs and CMECs. Here, heterogeneity between AECs and CMECs was also observed: endothelial activation was mediated through different proteins in AECs (CD9 molecule, galectin) and CMECs (ICAM-1 and IL-36α). Apoptosis was mediated by caspase 3 in AECs and Bid in CMECs. AECs appeared to advance to a dysfunctional state shown by up-regulation of endothelin-converting enzyme and angiotensin II-converting enzyme, while CMECs maintained an activated state. OA reversed TNF-α-induced injury through restoring NO-production, decreasing ROS-production in both AECs and CMECs, and inhibiting necrosis in AECs. In the ex vivo model of injury, aortic rings from 16-week HFD rats showed a pro-contractile response to phenylephrine-induced contraction, a response that was reversed by OA. In conclusion, we demonstrated novel findings with regards to endothelial heterogeneity between AECs and CMECs under baseline and TNF-α-treated conditions. Although reduced NO-bioavailability may be the hallmark of endothelial dysfunction, signalling pathways mediating endothelial injury may differ between cell types as was shown in this study. We demonstrated that OA possess protective properties in AECs and CMECS, an observation which was translated to the ex vivo model. / AFRIKAANSE OPSOMMING: Endoteelbesering en –disfunksie gaan die ontwikkeling van kardiovaskulêre siektes vooraf. Die endoteel word as die eerste linie van verdediging teen inflammasie / vetsug-geïnduseerde vaskulêre skade beskou; dus is die ontginning van nuwe inligting betreffende die meganismes van en respons tot besering, asook die modulering van endoteelfunksie essensieël in die voorkoming van kardiovaskulêre siektes. Endoteelselle verskil t.o.v. struktuur en funksie, en dus is endoteel-heterogeniteit relevant tydens die ondersoek van endoteelfunksie en –disfunksie. ‘n Beter begrip van endoteel-heterogeniteit in die respons tot patofisiologiese stimuli kan betekenisvol tot die voorkoming van kardiovaskulêre siektes bydra. Oleanoliese suur (OA), ‘n triterpenoïed afkomstig van plante is voorheen bewys om endoteelbeskermende eienskappe te besit; die rol van OA in die omkering van endoteelbesering is egter minder bekend. Hierdie studie het endoteel-heterogeniteit tussen aorta endoteelselle (AECs) en hart mikrovaskulêre endoteeelselle (CMECs) by basislyn en in respons tot ‘n inflammatoriese besering via die sitokien, tumor nekrose faktor-alfa (TNF-α), ondersoek. ‘n In vitro model van endoteelbesering is ontwikkel deur AECs en CMECs met 20 ng/ml TNF-α vir 24 uur te behandel. Endoteel-heterogeniteit was ondersoek deur intrasellulêre stikstofoksied (NO) en reaktiewe suurstofspesies (ROS) produksie, proteïenuitdrukking en fosforilering, en grootskaalse proteïenuitdrukking en regulering in AECs en CMECs te vergelyk. Die eksperimentele tegnieke het ingesluit: vloeisitometrie, western blots en proteomika. ‘n Ex vivo model van endoteelbesering was ook ingesluit deur die vaskulêre funksie in aortaringe van normale en hoë vet dieet-gevoerde (HFD) rotte te meet. Die rol van OA in die omkering van TNF-α-geïnduseerde besering en modulering van vaskulêre funksie was in hierdie model is ondersoek. Alhoewel basislyn NO-vlakke vergelykbaar was in AECs en CMECs, is heterogeniteit wel aangetoon m.b.t. die NO biosintese pad met verhoogde eNOS uitdrukking in die CMECs. Basislyn ROS-vlakke was verskillend in AECs en CMECs en die CMECs het hoër anti-oksidant kapasiteit getoon. ‘n In vitro model van TNF-α-geïnduseerde besering is bevestig met die waarneming van verlaagde NO-vlakke, verhoogde ROS-vlakke en nekrose, opregulering van apoptotiese proteïene en aktivering van inflammatoriese paaie in AECs en CMECs. Hier was heterogeniteit ook opmerkbaar: endoteelaktivering was deur verskillende proteïene in AECs (CD9 molekule, galektien) en CMECs (ICAM-1, IL-36α) bemiddel. Apotose was deur kaspase 3 in AECs en Bid in CMECs bemiddel. Dit het geblyk dat AECs tot ‘n staat van endoteeldisfunksie gevorder het met die opregulering van endotelien-omsettingsensiem en angiotensien II-omsettingsensiem, terwyl CMECs eerder ‘n geaktiveerde staat gehandhaaf het. OA het TNF-α-geïnduseerde besering omgekeer deur NO-produksie te herstel, ROS-produksie te onderdruk in beide AECs en CMECs, en nekrose te inhibeer in AECs. In die ex vivo model van besering, het aortaringe van 16-week HFD rotte ‘n pro-kontraktiele respons tot fenielefrien-geïnduseerde kontraksie getoon, wat deur OA omgekeer is. Ten slotte, nuwe bevindinge is waargeneem m.b.t. endoteel-heterogeniteit tussen AECs en CMECs onder basislyn en TNF-α-behandelde omstandighede. Alhoewel verlaagde NO-biobeskikbaarheid die waarmerk van endoteeldisfunksie is, het hierdie studie getoon dat seintransduksiepaaie wat endoteelbesering medieer verskillend is tussen seltipes. Die studie het verder ook gedemonstreer dat OA beskermende eienskappe toon in AECs en CMECs, ‘n waarneming wat ook in die ex vivo model aangetoon kon word.