Abstract 15299: Diabetes Causes Bone Marrow Endothelial Barrier Dysfunction through Activation of Redox-Sensitive Rho Gtpases and Associated Protein Kinases
Background: Diabetes causes endothelial dysfunction in bone marrow (BM). Here, we investigate the mechanisms behind BM endotheliopathy in streptozotocin-induced type-1 diabetic (T1D) mice
Methods: Using primary BM endothelial cells (BMECs) from T1D and age-matched non diabetic mice, we screened transcriptional targets and verified expressional and functional changes by qRT-PCR, western blot and in vitro assays. Moreover, vascular permeability was studied in vivo by assessing the biodistribution of fluorescent-labeled dextrans.
Results: Illumina gene arrays indicate expressional changes in signaling pathways controlling cell cycle, migration, adhesion and oxidative stress. In particular, RhoA, which modulates endothelial permeability, was upregulated at mRNA levels in T1D BMECs (P<0.05). Likewise, Rho activity was increased (pull-down assay) along with the RhoA targets Rho–associated protein kinases (ROCK1/ROCK2) (P<0.05), these effects being prevented by the ROS-scavenger N-acetyl-cysteine (NAC, P<0.01). Increased ROS were confirmed in T1D BMECs using the Mitotracker dye. We next investigated if RhoA activation results in Akt downregulation and impairment of Akt-related functions. T1D BMECs showed reduced Akt activity (P<0.001) as well as impaired migration, network formation and angiocrine factor releasing capacities (P<0.01), all being rescued by ROCK inhibitor Y-27632 or transfection with constitutively active Akt (myrAkt). Likewise, Y-27632, myrAkt or NAC contrasted the increased permeability of T1D BMEC monolayers to dextrans and mononuclear cells. Redox-dependent kinases modulate vascular permeability through VE-cadherin phosphorylation at adherens junctions. Phosphorylation of tyrosine kinase Pyk2 was increased in T1D BMECs, resulting in VE-cadherin phosphorylation at Y731. This was prevented by both NAC and Src inhibitors, suggesting a role of Src kinase in adherens junction disruption. Finally, vascular permeability to dextrans was increased in BM of T1D mice (P<0.01 vs controls) and restored by insulin replacement (P<0.01).
Conclusions: We newly show that ROS-dependent activation of RhoA/ROCK and Src kinase signaling pathways contribute to altered endothelial barrier function in diabetic BM.
- © 2012 by American Heart Association, Inc.