Abstract
The intracellular scaffold KRIT1/CCM1 is an established regulator of vascular barrier function. Loss of KRIT1 leads to decreased microvessel barrier function and to the development of the vascular disorder Cerebral Cavernous Malformation (CCM). However, how loss of KRIT1 causes the subsequent deficit in barrier function remains undefined. Previous studies have shown that loss of KRIT1 increases the production of reactive oxygen species (ROS) and exacerbates vascular permeability triggered by several inflammatory stimuli, but not TNF−α. We now show that endothelial ROS production directly contributes to the loss of barrier function in KRIT1 deficient animals and cells, as targeted antioxidant enzymes reversed the increase in permeability in KRIT1 heterozygous mice as shown by intravital microscopy. Rescue of the redox state restored responsiveness to TNF-α in KRIT1 deficient arterioles, but not venules. In vitro, KRIT1 depletion increased endothelial ROS production via NADPH oxidase signaling, up-regulated Nox4 expression, and promoted NF-κB dependent promoter activity. Recombinant yeast avenanthramide I, an antioxidant and inhibitor of NF-κB signaling, rescued barrier function in KRIT1 deficient cells. However, KRIT1 depletion blunted ROS production in response to TNF-α. Together, our data indicate that ROS signaling is critical for the loss of barrier function following genetic deletion of KRIT1.
Highlights
The intracellular scaffold KRIT1/CCM1 is an established regulator of vascular barrier function
As it had been previously reported that loss of KRIT1 expression can increase reactive oxygen species (ROS) production[12], we tested whether scavenging ROS from the endothelium could reverse the increased permeability in KRIT1 deficient mice
Because vascular abnormalities make intravital permeability measurements in KRIT1 null animals extremely challenging, we elected to treat Krit1+/− mice with a novel antioxidant agent designed to target its delivery and effect to the endothelium. This agent is comprised of two antioxidant enzymes (AOE): superoxide dismutase (SOD), which converts the superoxide radical (O2·−) into hydrogen peroxide (H2O2), and catalase, which converts H2O2 into oxygen and water, each conjugated with an antibody that targets platelet-endothelial cell adhesion molecule-1 (PECAM-1)
Summary
The intracellular scaffold KRIT1/CCM1 is an established regulator of vascular barrier function. Loss of KRIT1 leads to decreased microvessel barrier function and to the development of the vascular disorder Cerebral Cavernous Malformation (CCM). Previous studies have shown that loss of KRIT1 increases the production of reactive oxygen species (ROS) and exacerbates vascular permeability triggered by several inflammatory stimuli, but not TNF−α. We show that endothelial ROS production directly contributes to the loss of barrier function in KRIT1 deficient animals and cells, as targeted antioxidant enzymes reversed the increase in permeability in KRIT1 heterozygous mice as shown by intravital microscopy. Our finding that KRIT1-depleted cells are insensitive to TNF-α —yet respond to other stimuli, suggests that reduced KRIT1 expression does not merely reduce barrier function in vivo but actively modifies signaling responses to inflammatory mediators
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