Relation between dysfunction of nitric oxide synthase and angiogenesis

活性氧（ROS）在缺血再灌注和炎症中引起组织损伤。然而，最近有报道称- ros调节细胞功能，如增殖。有趣的是，当一氧化氮合酶（NOS）的辅助因子四氢生物蝶呤（BH4）减少时，大脑中的一氧化氮合酶（NOS）释放ROS而不是一氧化氮（NO）。本研究的目的是确定NOS内皮异构体是否也随着BH4的降低而产生ROS， NOS功能障碍是否影响血管生成。钙离子载体加入内皮细胞（ECs）释放0_2，用荧光素类似物MCLA测定。用BH4合成抑制剂- 2,4-二氨基-6-羟基嘧啶（DAHP）进一步刺激钙离子载体诱导的0_2释放。此外，NOS抑制剂能明显抑制钙离子载体诱导的DAHP处理细胞的O_2释放。这些结果表明，内皮型NOS也产生ROS， BH4含量降低。接下来，我们研究了ROS中的一种H_20_2对体外血管生成的影响。低浓度H_20刺激血管生成。ets -1是调控尿激酶纤溶酶原激活物和基质金属蛋白酶1表达的转录因子ets基因家族成员。有趣的是，H_20_2增加了ECs中ets-l mRNA的表达。h_2_2刺激的血管生成被ets-1反义寡核苷酸完全阻断。这些结果表明，低浓度H_2O_2刺激血管生成，H_2O_2诱导的血管生成可能由转录因子ets- 1介导。在本研究中，我们无法确定NOS中的ROS是否影响血管生成，因为DAHP本身具有抑制血管生成的作用。未来的研究将需要寻找更多选择性的BH4合成抑制剂。

Reactive oxygen species (ROS) have been known to induce tissue injury in ischemialreperfusion and inflammation. Recently, however, it has beenTeported that-ROS regulates cel-lular-function such as proliferation. Interestingly, nitric oxide synthase (NOS) from brain releases ROS instead of nitric oxide (NO) when tetrahydrobiopterin (BH4), a cofactor of NOS, is decreased. The purpose of this study was to determine whether endothelial isoform of NOS also produces ROS with decreasing BH4, and the dysfunction of NOS affects angiogenesis. Addition of calcium ionophore to endothelial cells (ECs) released 0_2 which was measured by using MCLA, a Cypridina luciferin analogue. The calcium ionophore-induced 0_2 release was further stimulated by the treatment with 2,4-diamino-6-hydroxyprimidine (DAHP), an inhibitor of BH4 synthesis. Moreover, he calcium ionophore-induced O_2 release in the DAHP treated cells was strongly inhibited by NOS inhibitor. These findings suggest that endothelial isoform of NOS also produces ROS with decreasing BH4 content. We next examined the effect of H_20_2, one of the ROS, on in vitro angiogenesis. The low concentrations of H_20 stimulated angiogenesis. Ets-1 is a member of the ets gene family of transcription factors, which regulates the expression of urokinase plasminogen activator and matrix metalloprotease-1. Interestingly, H_20_2, increased the ets-l mRNA in ECs. The H_2_2-stimulated angiogenesis was completely blocked by an ets-1 antisense oligonucleotide. These results indicate that low concentrations of H_2O_2 stimulate angiogenesis, and the H_20_2-induced angiogenesis is likely to be mediated by the transcription factor ets-l. In the present study, we were not able to determine whether ROS from NOS affect angiogenesis, since DAHP itself has an inhibiting effect of angiogenesis. Future studies will be needed to find more selective inhibitor for BH4 synthesis.