Abnormal shear stress induces ferroptosis in endothelial cells via KLF6 downregulation
Stable laminar flow maintains vascular tone regulation, while abnormal blood flow, such as disturbed flow or extreme shear stress, causes endothelial dysfunction, but the underlying mechanism is yet to be explored. We used a microfluidic device to deform flat microchannel into tunnel-like macrochannel. The cross-sectional area of this macrochannel varies with the flow rate and the thickness of the deformable layer, creating three levels of shear stress: low (0.99 dynes/cm², LSS), medium (4.78 dynes/cm², MSS), and high (24 dynes/cm², HSS). Comparing different shear stress exposure to endothelial cells for 24 h, prominent ferroptosis features emerged under either LSS or HSS compared to MSS. These features included increased C11 BODIPY-labeled lipid peroxidation and 4-hydroxynonenal accumulation, CoQ10 depletion, reduced SLC7A11 protein expression, and diminished cell death with Ferrostatin-1 treatment. RNA-seq analysis (LSS/MSS) showed that LSS significantly downregulated transcription of cholesterol homeostasis and unfolded protein response (UPR). Compared to MSS, Western blot results showed that both LSS and HSS reduced the expression of two key enzymes (MVD and IDI1) in the mevalonate pathway, as well as the expression of two main UPR signaling regulators (PERK and BiP). Based on the binding prediction between transcription factors and gene promoters from differentially expressed genes identified through RNA-seq, we found KLF6 to be a key transcription factor. It regulates the PERK-mediated UPR and the mevalonate pathway, which are associated with SLC7A11 expression and CoQ10 synthesis, respectively. The overexpression of KLF6 restored SLC7A11 and CoQ10 levels under both LSS and HSS, significantly reducing foam cell formation, monocyte adhesion, and lipid peroxidation. Our findings reveal KLF6 as a crucial regulator of atherosclerosis induced by abnormal shear stress. Overall design: Total RNA was extracted from the shear stress treated endothelial cells using RNAsimple Total RNA Kit (DP419,TIANGEN BIOTECH) according to the manufacturer's protocol. RNA quantification and purity were determined using Nanodrop One (Thermo Fisher Scientific). Qualified RNA samples were sent to BGI-Shenzhen (Shenzhen, China) for strand-specific library construction using the NEBNext Ultra II RNA Library Prep Kit (NEB #E7770), followed by sequencing on an Illumina NovaSeq 6000 platform with 150-bp paired-end reads. Raw data processing and downstream analyses were conducted on BGI's Dr. TOM platform (https://biosys.bgi.com).