Qian Lingbo Protein Kinase C–Dependent Increase in Reactive Oxygen Species Production in Vascular Tissues of Diabetes: Role of Vascular NAD(P)H Oxidase 1

ONOO - NO O 2 -. NOS Short of L-arginine or H 4 B Oxidation of H 4 B Pathological circumstances Damage Effects of ROS on Vascular Cells Background 2

Major ROS in diabetic processes 3

EDVDAtherosclerosis Hypertension Hyperglycemia ROS PKC ? etc. Note: PKC-  is more important in diabetic vascular damages DiabetesDiabetic vascular complication 4

diacylglycerol (DAG)-protein kinase C (PKC) pathway Diabetic state NAD(P)H oxidase ROS Vascular damage PKC-dependent activation of NAD(P)H oxidase may be an essential mechanism for increased ROS in diabetic vessels 5

6 Activation of PKC in Diabetic Vascular Tissues

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aortic smooth muscle cells aortic endothelial cells PKC-Dependent Activation of NAD(P)H Oxidase Induced by High Glucose 8

Effect of STZ treatment on NAD(P)H oxidase activity (A) and on the NAD(P)H oxidase subunit mRNA gp91phox (B) NAD(P)H oxidase protein subunits in human diabetes Internal mammary arteries (IMA) and saphenous veins (HSV) 9

molecular mechanism High glucose Rac-1 PKC inhibitors NAD(P)H oxidase Ros 10

 saturated nonesterified fatty acids (NEFA) can stimulate de novo DAG synthesis and PKC activity in cultured aortic EC and smooth muscle cells 11

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Upregulation of NAD(P)H Oxidase Components in Diabetic Vascular Tissues and Kidney 13

NOX4 mRNA expression in kidney of control rats and streptozotocin induced diabetic rats p22phox mRNA expression in kidney of control rats and streptozotocin induced diabetic rats 14

15 A 1 B 1 A 2 B 2 Immunostaining analysis of NOX4 and p22phox expression (brown particles) in kidney of control rats (A 1, A 2 ) and diabetic rats (B 1, B 2 ) NOX4p22phox

44 Effects of interventive insulin treatment on NOX4, p22phox and 8-OHdG expression levels in renal tissue from diabetic rats 16

Effect of PKC inhibitor on high glucose induced increases in NOX4 (A) and p22phox (B) mRNA expression in cultured mesangial cells 17

Antioxidative Agents Targeting the Mechanism of NAD(P)H Oxidase 18

A. Effect of CGP41251 on spin clearance rates in streptozotocin induced diabetic rats B. Effect of apocynin on spin clearance rates in streptozotocin induced diabetic rats 19

40 Effect of the inhibition of PKC in vivo on vascular superoxide production (red particles) in rat aorta as detected with hydroethidine 20

PKC inhibitor decreased expression of PKC-  2 in high glucose incubated HUVECs and improved EDVD in diabetic aorta 21

 Statins, angiotensin II-converting enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB) reduce ROS productions In diabetic vessels by inhibiting the activity of NAD(P)H oxidase 22

23 ？ High glucose level PKC activation ARB, ACEI PKC inhibitors NAD(P)H oxidase activation Angiotensin II ROS Statin ？ Possible antioxidative agents for diabetic vascular complications

Conclusion  PKC-NAD(P)H oxidase-ROS pathway  a new target of antioxidative therapy for preventing diabetic micro- or macro-vascular complications 24