Stable flow-induced expression of KLK10 inhibits endothelial inflammation and atherosclerosis

Abstract

Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), while regions exposed to stable flow (s-flow) are protected. The proatherogenic and atheroprotective effects of d-flow and s-flow are mediated in part by the global changes in endothelial cell gene expression, which regulates endothelial dysfunction, inflammation, and atherosclerosis. Previously, we identified Kallikrein-Related Peptidase 10 (Klk10, a secreted serine protease) as a flow-sensitive gene in mouse arterial endothelial cells, but its role in endothelial biology and atherosclerosis was unknown. Here, we show that KLK10 is upregulated under s-flow conditions and downregulated under d-flow conditions using in vivo& mouse models and in vitro studies with cultured endothelial cells (ECs). Single-cell RNA sequencing (scRNAseq) and scATAC sequencing (scATACseq) study using the partial carotid ligation mouse model showed flow-regulated Klk10 expression at the epigenomic and transcription levels. Functionally, KLK10 protected against d-flow-induced permeability dysfunction and inflammation in human artery ECs (HAECs), as determined by NFkB activation, expression of vascular cell adhesion molecule 1 (VCAM1) and intracellular adhesion molecule 1 (ICAM1), and monocyte adhesion. Further, treatment of mice in vivo with rKLK10 decreased arterial endothelial inflammation in d-flow regions. Additionally, rKLK10 injection or ultrasound-mediated transfection of Klk10-expressing plasmids inhibited atherosclerosis in Apoe-/- mice. Moreover, KLK10 expression was significantly reduced in human coronary arteries with advanced atherosclerotic plaques compared to those with less severe plaques. KLK10 is a flow-sensitive endothelial protein that serves as an anti-inflammatory, barrier-protective, and anti-atherogenic factor.

Sox13 is a novel flow-sensitive transcription factor that prevents inflammation by repressing chemokine expression in endothelial cells

“…For in vitro flow experiments, we exposed confluent cells to steady unidirectional flow (s-flow, 15 dyn/cm 2 ) or bidirectional oscillatory flow (d-flow, +5/-4 dyn/cm 2 at 1 Hz) to mimic in vivo flow profiles using the cone-and-plate viscometer for 24-or 48-h experiments (75). In brief, a cone controlled by a stepping motor is placed in a standard 10 cm tissue culture dish containing confluent HAEC monolayer.…”

“…Monocyte binding was determined using THP-1 monocytes (ATCC TIB-202, RRID:CVCL_0006) as we reported (75). In brief, THP-1 cells (1.5 × 10ˆ5 cells/mL) were labeled with a fluorescent dye 2-,7-bis(carboxyethyl)-5 (6)-carboxyfluorescein-AM (BCECF) (Thermo Fisher Scientific B1150; 1 mg/mL) in serum-free RPMI medium (Thermo Fisher Scientific 11875093) for 30 min at 37 • C. After exposure to flow or transfection treatments, BCECFloaded THP-1 cells were added directly to the ECs to the conditioned medium.…”

New Insights into Cerebral Vessel Disease Landscapes at Single-Cell Resolution: Pathogenetic and Therapeutic Perspectives

“…By reanalyzing these scRNA-seq and scATACseq datasets, the same group demonstrated a key role of kallikrein-related peptidase 10 (Klk10, a secreted serine protease) as a flow-regulated endothelial protein with positive anti-inflammatory, anti-atherogenic activities [ 38 ]. KLK10 expression is higher under stable flow but reduced under disturbed flow and protects against inflammatory signaling that counteracts the effect of disturbed flow.…”

“…KLK10 expression is higher under stable flow but reduced under disturbed flow and protects against inflammatory signaling that counteracts the effect of disturbed flow. Administration of KLK10 in mice reduces arterial endothelial inflammatory processes associated with disturbed flow [ 38 ].…”

Spatial control of robust transgene expression in mouse artery endothelium under ultrasound guidance

“…Kallikrein-10 (KLK10) is one of the atheroprotective genes that are downregulated by d-flow. 3 We determined the feasibility of targeted delivery of KLK10 gene with UGMAAV in the mouse partial carotid ligation (PCL) model. 4 Under the optimized condition (Supplementary Fig.…”