A mitochondrial contribution to anti-inflammatory shear stress signaling in vascular endothelial cells.

Atherosclerosis, the major cause of myocardial infarction and stroke, results from converging inflammatory, metabolic, and biomechanical factors. Arterial lesions form at sites of low and disturbed blood flow but are suppressed by high laminar shear stress (LSS) mainly via transcriptional induction of the anti-inflammatory transcription factor, Kruppel-like factor 2 (Klf2). We therefore performed a whole genome CRISPR-Cas9 screen to identify genes required for LSS induction of Klf2. Subsequent mechanistic investigation revealed that LSS induces Klf2 via activation of both a MEKK2/3-MEK5-ERK5 kinase module and mitochondrial metabolism. Mitochondrial calcium and ROS signaling regulate assembly of a mitophagy- and p62-dependent scaffolding complex that amplifies MEKK-MEK5-ERK5 signaling. Blocking the mitochondrial pathway in vivo reduces expression of KLF2-dependent genes such as eNOS and inhibits vascular remodeling. Failure to activate the mitochondrial pathway limits Klf2 expression in regions of disturbed flow. This work thus defines a connection between metabolism and vascular inflammation that provides a new framework for understanding and developing treatments for vascular disease.

Monitoring Aggregate Clearance and Formation in Cell-Based Assays.

Understanding the fundamental mechanism underlying the accumulation and clearance of misfolded proteins can lead to insights into the synthetic and degradative pathways that maintain the homeostasis of proteins in all cells. Given the interconnection between protein homeostasis and cell health, as well as the complexity of aggregate formation and the degradation pathways with which it is intertwined, the design of the tools that are used to examine protein aggregation and accumulation can have a profound impact on the interpretation of results. We rely on two previously published stable cell lines that use conditional expression and the ligand-receptor tag known as HaloTag, to temporally distinguish distinct pools of aggregates, and use a combination of biochemical- and imaging-based methods to measure aggregation of a canonical aggregation-prone protein. We measure aggregate load biochemically using Filter Trap Analysis, which combines a filter trap retardation assay and immunoblotting to measure detergent soluble and insoluble protein levels, and visually, using confocal microscopy to monitor simultaneously aggregate formation and growth events in the background of aggregate clearance. As a secondary screen to more simplistic screen based approaches, this method permits further insight into how aggregate load is affected.