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(Hypertension. 2009;54:427.)
© 2009 American Heart Association, Inc.

Seventh International Workshop on Structure and Function of the Vascular System

Endothelial Cells Negatively Modulate Reactive Oxygen Species Generation in Vascular Smooth Muscle Cells

Role of Thioredoxin

Shaoping Xu; Ying He; Martina Vokurkova; Rhian M. Touyz

From the Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario, Canada.

Correspondence to Rhian M. Touyz, MD, PhD, Kidney Research Centre, University of Ottawa/Ottawa Hospital Research Institute, 451 Smyth Rd, Ottawa, ON, Canada KIH 8M5. E-mail rtouyz{at}uottawa.ca

In intact vessels, endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) act as an integrated system, possibly through reactive oxygen species (ROS). Using a coculture system we tested whether ECs modulate VSMC redox status by regulating activity of NAD(P)H oxidase and antioxidants. VSMC production of O₂^•–, H₂O₂, and NO was assessed using fluoroprobes and amplex-red. NAD(P)H oxidase subunit expression and oxidase activity were determined by Western blotting and chemiluminescence, respectively. Expression of thioredoxin, SOD, growth signaling pathways (PCNA, p21cip1, CDK4, ERK1/2, p38MAPK) was evaluated by immunoblotting. Thioredoxin activity was assessed by the insulin disulfide reduction assay. In cocultured conditions, VSMC ROS production was reduced by 50% without changes in NAD(P)H oxidase expression/activity versus monoculture (P<0.05). This was associated with decreased cell growth (P<0.05). Expression of Cu/Zn SOD and thioredoxin was increased in coculture versus monoculture VSMCs (P<0.01). Pretreatment of ECs with L-NAME (NOS inhibitor), NS-398 (Cox2 inhibitor), and HET0016 (20-HETE inhibitor) did not influence VSMC ROS formation, whereas CDNB, thioredoxin reductase inhibitor, abolished ROS modulating effects of ECs. These findings indicate that in a coculture system recapitulating intact vessels, ECs negatively regulate ROS production in VSMCs through thioredoxin upregulation. Functionally this is associated with growth inhibition. The modulatory actions of ECs are independent of NOS/NO, Cox2, and HETE and do not involve NAD(P)H oxidase. Our data identify novel mechanisms whereby ECs protect against VSMC oxidative stress, a process that may be important in maintaining vascular integrity.

Key Words: coculture • oxidative stress • vascular cells • antioxidants • signal transduction

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