Vinsamlegast notið þetta auðkenni þegar þið vitnið til verksins eða tengið í það: http://hdl.handle.net/1946/16269
Healthy, normally functioning endothelium is critical for maintenance of vascular homeostasis. Endothelial dysfunction, on the other hand, is a key early step in numerous vascular diseases. A vascular surface that normally is thromboresistant, antiinflammatory, vasodilatory and antiproliferative can turn into a surface that is thrombogenic, proinflammatory, vasoconstrictive and stimulatory of smooth muscle cell proliferation. In certain disease states such as atherosclerosis, hypertension and diabetes such changes may involve a chronically perturbed vascular behaviour critical for disease progression. The signaling that mediates the response to environmental changes has in recent years attracted attention. The transduction of these signals is critical for an appropriate cellular response while derailment of the signaling can be a key feature of a pathologic response or disease. One of the crucial responses of the vascular endothelium to a variety of signals is the production of NO from arginine through the activation of eNOS.
The main goal of this work was to enhance our understanding of the biological and physiological properties of the healthy vascular endothelium with regards to the activity of eNOS. Specifically, we sought to elucidate the signaling pathway involved in mediating endothelial stimulation by the G-protein linked agonists thrombin and histamine with emphasis on the role of AMPK in maintaining normal NO-production.
We demonstrated the presence of a PI3K-Akt independent pathway activating eNOS in HUVEC after treatment of cells with histamine or thrombin. We showed that both agonists inhibited Akt phosphorylation by activation of PKCδ and that the phosphorylation of eNOS at Ser1177 by these agonists was unaffected by the PI3K-inhibitor wortmannin. After having ruled out several kinases known to phosphorylate eNOS we hypothesized a role for AMPK based on preliminary results showing AMPK phosphorylation by histamine and thrombin.
We were able to demonstrate a role for AMPK in this previously unrecognised pathway, making AMPK an important link in mediating thrombin and histamine induced activation of eNOS independent of PI3K-Akt. AMPK was phosphorylated by these agonists and activation was demonstrated by phosphorylation of ACC a known target of AMPK. eNOS phosphorylation and activation after histamine or thrombin was inhibited by H89, an inhibitor of AMPK and PKA. Also, AICAR and CCCP, both known to activate AMPK, caused phosphorylation of eNOS. The Ca+2-chelator BAPTA inhibited the phosphorylation of AMPK, eNOS and NO-formation, demonstrating the Ca+2-dependency of this pathway. With LKB1 being the only kinase known to activate AMPK in mammals at this time, we suggested an LKB1-AMPK dependent pathway causing eNOS activation after treatment with histamine or thrombin, possibly explained by activation of energy-consuming pathways by the elevation of intracellular Ca+2.
We discovered that this pathway was only activated under culture conditions that allowed a sharp but brief fall in cellular ATP after thrombin or histamine stimulation. Under culture conditions that prevented or did not allow such a fall in the energy level of the endothelial cells AMPK was still activated but exclusively via a different upstream pathway and downstream this AMPK-activation played no role in the activation of eNOS. Also, we demonstrated, using siRNA, that the AMPK-eNOS pathway is dependent on the activation of AMPK through LKB1. Downregulation of the α2-isoform of AMPK but not α1 resulted in shrinkage of the cells and loss of contact between cells suggesting that the α2-isoform of AMPK is critical for endothelial integrity. The inhibiting effects of the SOD-mimetic Tempol on these morphologic changes seen in AMPKα2-downregulated cells and the increased expression of the ER stress marker GRP78 caused by this downregulation also suggested a role for AMPKα2 in oxidant defences.
We confirm a pathway dependent on CaMKK causing activation of eNOS under culture conditions where intracellular ATP is unchanged without the involvement of (LKB1-)AMPK, as also described by others. However, we also demonstrate a previously unknown pathway, activated by a rise in intracellular AMP/ATP ratio, causing LKB1-AMPK-dependent phosphorylation and activation of eNOS by thrombin or histamine. This pathway is activated under culture conditions where the intracellular AMP/ATP ratio is elevated by these agonists. Our fundamental discovery that environmental conditions dictate which pathway is activated is interesting but even more so the key role played by the ATP-level, a fall in the cellular energy level being a well known consequence of pathologic conditions such as ischemia, hypoxia and infection. Although thrombin can activate eNOS through an AMPK-independent pathway the activation of the LKB1-AMPK pathway greatly enhances the NO-production and may therefore be of major importance for endothelial function and endothelial health.