Endothelial cells negatively modulate reactive oxygen species generation in vascular smooth muscle cells: role of thioredoxin.
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 O(2)(*-), H(2)O(2), 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 approximately 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.
Xu
Shaoping
S
Kidney Research Centre, University of Ottawa/Ottawa Hospital Research Institute, 451 Smyth Rd, Ottawa, ON, Canada KIH 8M5.
He
Ying
Y
Vokurkova
Martina
M
Touyz
Rhian M
RM
D000704:Analysis of Variance; D002450:Cell Communication; D002478:Cells, Cultured; D018920:Coculture Techniques; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism* / Q000502:physiology; D019255:NADPH Oxidases / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology*; D010101:Oxygen Consumption / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D013879:Thioredoxins / Q000378:metabolism*; D015854:Up-Regulation
D006893:Hydroxyeicosatetraenoic Acids; D017382:Reactive Oxygen Species; D009569:Nitric Oxide; D013879:Thioredoxins; D019255:NADPH Oxidases