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Enhanced p62 accumulation occurs in endothelial cells lining in human CCM lesions To examine the clinical relevance of our findings in cellular and animal models of CCM disease, we analyzed p62 expression in human CCM lesions.
Hy926 cells (Appendix Fig S5A) induced p62 accumulation, as well as increased levels of LC3-I and LC3-II (Appendix Fig S5B).
The autophagy defects observed in krit1-KO cells could be attributed to alterations of the mTOR-ULK1 pathway (Fig and ).
The loss of krit1 in both endothelial cells (Appendix Fig S1D) and MEFs (Appendix Fig S1E) promoted increased levels of p62 in both the soluble and insoluble fractions, which is consistent with previous observations made under defective autophagy and high protein aggregation conditions (Waguri Komatsu.To complete the analysis of all three CCM genes, we investigated the effect of CCM2 down-regulation in human endothelial cells on autophagy.Direct selective inhibition of mTOR, through the allosteric inhibitor rapamycin or the small molecule ATP-competitive inhibitor Torin1, induces autophagy in many cell types (Kundu, ).Defective autophagy underlies major phenotypic signatures of CCM disease To further clarify whether defective autophagy is involved in the pathogenesis of CCM, we investigated the relationship between autophagy and endothelial-to-mesenchymal sonicwall vpn client ubuntu 12.04 transition (EndMt a pathological signature that contributes to CCM progression (Maddaluno et al, ).Therefore, we examined the role of autophagy in CCM3-depleted endothelial cells derived from Ccm3 fl/fl mice (Bravi et al, ).As expected, treatment with the antioxidant N-acetylcysteine (NAC) decreased p62 levels, but the disruption.Notably, in one of the eight tissue samples that displayed marked positive p62 staining in CCM lesions, typical normal vessels surrounding the lesion were also present and stained negative for p62, resulting in an internal negative control (Fig).We used krit1-KO lung endothelial cells derived from.This result might be related to the dual suppressive role played by city hunter episode 2 english sub mTOR, which inhibits autophagy not only at the initiation stage via suppression of the ULK1 complex but also at the degradation stage via inhibition of lysosomal function (Zhou et al,b).Importantly, when autophagy-mediated degradation is inhibited, p62 appears to be partially detergent insoluble (Klionsky et al, therefore, the lysates were divided between Triton X-100 (TX-100)-soluble and TX-100-insoluble fractions and subsequently analyzed for their protein content.Indeed, p62 accumulates in several autophagy-deficient mouse tissues (Zatloukal et al, ; Martinet et al, ) and represents a reliable marker for tissues with reduced autophagic activity (Waguri Komatsu, ).Importantly, the induction of autophagy was more robust in Torin1-treated cells, as evidenced by the greater inhibition of the mTOR pathway by Torin1 (Appendix Fig S2B).Moreover, immunofluorescence staining showed a punctate pattern of p62 and the accumulation of aggresomes (Appendix Fig S5C).Overall, these data suggest that krit1 loss inhibits autophagy through the up-regulation of the mTOR pathway and that the restoration of autophagy by mTOR inhibitors could significantly mitigate the metabolic disorders resulting from krit1 loss-of-function.
Moreover, similar results were obtained using the protein synthesis inhibitor cycloheximide (CHX) (Appendix Fig S1B further supporting the notion that the inhibition of autophagy-dependent protein turnover upon krit1 loss contributes to p62 accumulation.
As shown in Fig, krit1 deficiency was associated with defective autophagy, displaying increased levels of p62 and total LC3.

Program after activation resides in computer memory, without performing any activities and not interrupting other programs.As in patients with CCM (Labauge et al, an inducible and endothelial-specific CCM3-KO mouse model ( CCM3- ecko) presented venous malformations at the periphery of the retinal vascular plexus.The silencing of krit1 suppressed autophagy in both the human cerebral microvascular endothelial cell line hbmec (Fig) and the human umbilical vein cell line.Immunoblot analysis revealed marked up-regulation of mTOR signaling in krit1-KO endothelial cells, as evidenced by the increased phosphorylation of both mTOR and its downstream targets p70S6k and 4E-BP1 (Fig).Moreover, ATG7 silencing in huvecs slowed the formation of capillary-like structures (Fig) but significantly increased the migratory capacity of these cells (Appendix Fig S4B).Both Torin1 and rapamycin treatments inhibited the EndMt switch by lowering the expression of mesenchymal markers (Fig) and by increasing the levels of key endothelial markers such as CD31 (also known as Pecam-1) and vascular endothelial cadherin (VE-cadherin) (Fig).Because mutations in any of the three CCM genes lead to the onset of similar pathological signatures, the three CCM proteins likely share a common mechanism of action.The use of rapamycin and mTOR kinase inhibitors significantly re-established autophagy (Fig).As shown in Appendix Fig S2A, ambra1 phosphorylation at Serine 52 is more abundant upon krit1 deletion compared to wt endothelial cells.As shown in Fig, we observed greater colocalization between p62 and aggresomes in endothelial krit1-KO cells, as well as extremely high fluorescence intensity of aggresome-like inclusion bodies.Similar to krit1 down-regulation, we observed autophagy inhibition upon CCM3 ablation, which could be re-activated by treatment with mTOR inhibitors (Fig).
Krit1 fl/fl mice treated with Tat-Cre recombinase (Maddaluno et al, ).
One of the most useful methods for measuring autophagy is based on the mRFP-GFP-LC3 tandem construct assay (Mizushima et al, ).

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