PI3KCIIα-Dependent Autophagy Program Protects From Endothelial Dysfunction and Atherosclerosis in Response to Low Shear Stress in Mice.

BACKGROUND: The ability to respond to mechanical forces is a basic requirement for maintaining endothelial cell (ECs) homeostasis, which is continuously subjected to low shear stress (LSS) and high shear stress (HSS). In arteries, LSS and HSS have a differential impact on EC autophagy processes. However, it is still unclear whether LSS and HSS differently tune unique autophagic machinery or trigger specific autophagic responses in ECs. METHODS: Using fluid flow system to generate forces on EC and multiscale imaging analyses on ApoE-/- mice whole arteries, we studied the cellular and molecular mechanism involved in autophagic response to LSS or HSS on the endothelium. RESULTS: We found that LSS and HSS trigger autophagy activation by mobilizing specific autophagic signaling modules. Indeed, LSS-induced autophagy in endothelium was independent of the class III PI3K (phosphoinositide 3-kinase) VPS34 (vacuolar sorting protein 34) but controlled by the α isoform of class II PI3K (phosphoinositide 3-kinase class II α [PI3KCIIα]). Accordingly, reduced PI3KCIIα expression in ApoE-/- mice (ApoE-/-PI3KCIIα+/-) led to EC dysfunctions associated with increased plaque deposition in the LSS regions. Mechanistically, we revealed that PI3KCIIα inhibits mTORC1 (mammalian target of rapamycin complex 1) activation and that rapamycin treatment in ApoE-/-PI3KCIIα+/- mice specifically rescue autophagy in arterial LSS regions. Finally, we demonstrated that absence of PI3KCIIα led to decreased endothelial primary cilium biogenesis in response to LSS and that ablation of primary cilium mimics PI3KCIIα-decreased expression in EC dysfunction, suggesting that this organelle could be the mechanosensor linking PI3KCIIα and EC homeostasis. CONCLUSIONS: Our data reveal that mechanical forces variability within the arterial system determines EC autophagic response and supports a central role of PI3KCIIα/mTORC1 axis to prevent EC dysfunction in LSS regions.

A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene of Kingella kingae and Application to 201 Pediatric Clinical Specimens.

Kingella kingae is a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting the groEL gene and the RTX locus of K. kingae However, recent studies showed that real-time PCR (RT-PCR) assays targeting the Kingella sp. RTX locus that are currently available for the diagnosis of K. kingae infection lack specificity because they could not distinguish between K. kingae and the recently described Kingella negevensis species. Furthermore, in silico analysis of the groEL gene from a large collection of 45 K. kingae strains showed that primers and probes from K. kingaegroEL-based RT-PCR assays display a few mismatches with K. kingae groEL variations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative to groEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, a K. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of the K. kingae mdh gene from 20 distinct sequence types of K. kingae This novel K. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnose K. kingae infections and carriage in 104 clinical specimens from children between 7 months and 7 years old.