br Materials and methods br

Materials and methods
Results
Discussions Our results indicated that autophagy was suppressed in GECs under HAGG treatment, through Akt/mTOR-dependent pathways. Previous reports provided clues for the linkages between ICs and endothelial cell autophagy. Fujii and colleagues reported that self-aggregated antibodies could be internalized by endothelial cells via Glucose Uptake Fluorometric Assay Kit polymerization induced by fibronectin-integrin interaction [29]. In relation, autophagy requires membrane remodelling and actin rearrangement participated in early events of autophagy [37]. Besides, activated mTOR pathway has been detected in renal endothelial cells in patients with anti-phospholipid syndrome (APS) nephropathy, which often occurs secondary to lupus and characterized by presence of circulating anti-phospholipid antibodies. After incubation with anti-phospholipid antibodies isolated from APS patients, cultured human microvascular endothelial cells exhibited increased S6RP and Akt phosphorylations, also suggesting the activated mTOR pathways [38]. In transplant vasculopathy model, anti-HLA antibodies ligation to HLA class I molecules expressed in human aortic endothelial cells, activated mTOR pathways, demonstrated by increased phosphorylations of S6K and Akt [39]. Activation of Akt/mTOR pathway was also illustrated in GECs in NZBW/F1 female lupus mice, as over-expressed phosphorylated AKT and mTOR were detected and visualized in renal endothelial cells by double-immunofluorescence staining [40]. These results implied activated mTOR pathways in endothelial cells in IC-mediated diseases. However, the authors have not reported the autophagy status. Together with the changed expressions of autophagic markers LC3 and p62, our results showed the suppressed autophagy in GECs under HAGG treatment, by activating Akt/mTOR pathways. For the effects of TNF-alpha on autophagy, Lee and colleagues demonstrated that TNF-alpha activated mTOR signaling, through inactivation of TSC1 by activating IKK-beta activity in breast cancers [41]. p62 was also reported to be upregulated after TNF-alpha treatment. Kojima and colleagues reported that p62 was upregulated in rat optic nerve after intravitreal injection of TNF-alpha [42]. In another study, Opperman et al observed accumulated p62 and increased LC3 II/I ratio in rat cardiomyoblasts under TNF-alpha stimulation [43]. Here, our results showed that TNF-alpha upregulated expressions of p-mTOR and p62, together with slightly increased LC3 II/I ratio in GECs. These results suggested that the clearance step in autophagy may be inhibited. Moreover, Mostowy et al reported not only significantly increased p62 and LC3-II levels in HeLa cells treated with TNF-alpha, but also the increased p62 mRNA expression under TNF-alpha stimulation [44]. Their results implied that, besides the inhibited clearance step in autophagy, TNF-alpha may regulate p62 at transcriptional level and then the autophagy process. We have also investigated the interaction between ICs and TNF-alpha on endothelial cell functions. HAGG and TNF-alpha co-incubation led to further reduced ratio of LC3 conversion and increased expression of p-mTOR and p-Akt, implying aggravated suppression of autophagy in GECs. HAGG and TNF-alpha co-incubation also inhibited cell viabilities. Actually, there are evidences implying the interactive effects of ICs and TNF-alpha on endothelial cell functions. In TNF−/− mice, ICs-induced inflammation and microvascular dysfunctions were attenuated [45]. TNF-alpha increased the expressions of FcγRII and FcγRIII on aortic endothelial cells, which may facilitate circulating ICs localization on ECs [46]. Further investigations on Fc receptor expressions is needed for GECs. In this study, we observed decreased cell viabilities under HAGG and TNF-alpha treatment separately. As both HAGG and TNF-alpha suppressed autophagy in GECs, we investigated whether the decreased viabilities were resulted from suppressed autophagy. We treated GECs with autophagic activator rapamycin and inhibitor 3MA and measured the corresponding cell viabilities. However, these autophagic regulators decreased cell viabilities significantly. This may be partly because that these two drugs have multiple effects on cell functions other than autophagy. Rapamycin, an inhibitor of mTOR, also exhibits immunosuppressive and anti-proliferation properties [47]. 3MA is a non-specific inhibitor for phosphatidylinositol 3-kinase, and has dual roles in the autophagy process [48]. More specific techniques to interfere autophagy are needed to investigate the relationship between autophagy and cell viability.