Abstract 17601: The Dual RhoGEF Kalirin Reduces Atherosclerosis Through Endothelial Cell-Specific Mechanisms
Associated with human atherosclerosis (athero) by genetic studies, KALRN encodes a ~270-kDa protein encompassing 2 GTP exchange factors (GEFs). Kalirin is expressed in endothelial cells (ECs), smooth muscle cells (SMCs) and macrophages, and up-regulates in atherosclerotic arteries. To test the hypothesis that Kalirin is atherogenic, we compared Kalrn-/+/Apoe-/- and Kalrn+/+/Apoe-/- mice fed a Western diet for 14 wks. Kalrn-/+/Apoe-/- mice developed 2.0±0.5- and 2.0±0.7-fold more aortic and innominate artery athero, respectively (p<0.05), despite equivalent blood pressures and cholesterol levels. To determine cell-specific anti-atherogenic roles for Kalirin, we orthotopically transplanted into Apoe-/- mice carotid arteries from congenic WT, Kalrn-/+ and SMC-specific Kalrn-/+ (SM22-Cre+/Kalrnflox/+) mice. Post-op 7 wk, carotid athero was 33% less in Kalrn-/+ than in WT grafts, and 50% less in SMC-Kalrn-/+ than in Kalrn-/+ grafts (p<0.05, n=7/group). Thus, while SMC Kalirin appeared atherogenic, EC Kalirin appeared to be atheroprotective. Compared with WT ECs, Kalrn-/+ ECs demonstrated 35±9% less FBS-induced Rac1 activity, assessed as PAK1 autophosphorylation (p<0.05). Congruently, in response to fluid shear stress (12 dyn/cm2), Kalirin siRNA-transfected HUVECs compared with controls showed 80±30% less PAK1 autophosphorylation (p<0.05), but equivalent VEGFR2 autophosphorylation. The highly selective PAK inhibitor IPA-3 not only abolished VEGF-induced PAK1 autophosphorylation but also reduced (by 90±10%, p<0.05) phosphorylation of eNOS on Ser633 (an activating site); however, IPA-3 did not affect VEGF-induced Akt phosphorylation. In response to shearing and VEGF, respectively, Kalirin-silenced HUVECs and HAECs showed less autophosphorylation of PAK1 (20±15% and 40±10% of control) and less phosphorylation of eNOS-Ser633 (40±10% and 25±10% of control) (p<0.05 for all). Concordantly, relative to WT, Kalrn-/+ ECs showed 50±10% less carbachol-induced NO production (assessed by DAF-2 fluorescence, p<0.05) and 40% less migration by monolayer scratch-wound assay (p<0.05). We conclude that Kalirin is atheroprotective through its Rac1 GEF activity, which promotes PAK1-mediated phosphorylation/activation of eNOS in ECs.
- © 2013 by American Heart Association, Inc.