Endothelin-1 activation of JAK2 in vascular smooth muscle cells involves NAD(P)H oxidase-derived reactive oxygen species.

Endothelin-1 (ET-1) and JAK2 are both implicated in diabetic complications. Therefore, we investigated whether ET-1 differentially activates JAK2 under conditions of normal (5 mM) and high (25 mM) glucose. We tested the hypothesis that reactive oxygen species mediate the activation of JAK2 in response to ET-1. In rat aortic vascular smooth muscle cells (VSMC), ET-1 (10 (- 7) M, 5 min) stimulated the activation of JAK2, which was further enhanced under high glucose conditions. Allopurinol (xanthine oxidase inhibitor, 1 microM) and l-NAME (nitric oxide synthase inhibitor, 1 mM) had no effect on ET-1-induced JAK2 activation, while apocynin (NAD(P)H oxidase inhibitor 100 microM) resulted in a significant inhibition of ET-1-induced JAK2 and MAPK activation. Overexpression of SOD did not inhibit ET-1-induced activation of JAK2, but catalase (50 units/mL) treatment resulted in complete inhibition. In vivo administration of apocynin (1.5 mM) resulted in a significant decrease ( 50%), while the ETA receptor antagonist ABT-627 completely inhibited phosphorylation of JAK2 in aortae from STZ-induced diabetic rats. Additionally, DHE staining of aortic sections was significantly reduced in diabetic rats treated with ABT-627. These data suggest that in VSMC, ET-1 via the ETA receptor, utilizes NAD(P)H oxidase to activate JAK2.

Hyperglycemia and reactive oxygen species mediate apoptosis in aortic endothelial cells through Janus kinase 2.

The generation of reactive oxygen species (ROS) has been implicated in the perturbation of endothelial function and cell death. However, the specific signaling pathways which mediate and modifying this response have not been fully elucidated. Therefore, in this study we tested the hypothesis that activation of JAK2 is involved in the aortic endothelial cell (EC) response to ROS. When ECs were exposed to HG (25 mM) for 6 h or ROS (i.e., H(2)O(2) (100 microM)) for 1 h and returned to normal medium we found a decrease in cell density and morphologic signs of apoptosis. Furthermore, incubation of ECs with HG and H(2)O(2) also resulted in the tyrosine phosphorylation of JAK2. In addition, pretreatment of ECs with AG-490, an inhibitor of JAK2, prevented nuclear fragmentation, whereas inhibitors of Jun kinase (SP 600125), MAP kinase (PD 98059), Src kinase (PP2) or PI-3 kinase (wortmannin) were without effect. Finally, immunoblot analysis of caspase-3 and PARP cleavage confirmed a role for activation of JAK2 in both HG- or ROS-induced apoptosis, based on inhibition by either AG-490 or adenoviral transfection with a dominant-negative JAK2 mutant. In conclusion the activation of JAK2 plays a pivotal role in oxidant stress-induced commitment of ECs to apoptosis, based on studies with HG and H(2)O(2).