Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenation.

Ischemic stroke is associated with a surge in reactive oxygen species generation during reperfusion. The narrow therapeutic window for the delivery of intravenous thrombolysis and endovascular thrombectomy limits therapeutic options for patients. Thus, understanding the mechanisms regulating neurovascular redox defenses are key for improved clinical translation. Our previous studies in a rodent model of ischemic stroke established that activation of Nrf2 defense enzymes by pretreatment with sulforaphane (SFN) affords protection against neurovascular and neurological deficits. We here further investigate SFN mediated protection in mouse brain microvascular endothelial cells (bEnd.3) adapted long-term (5 days) to hyperoxic (18 kPa) and normoxic (5 kPa) O2 levels. Using an O2-sensitive phosphorescent nanoparticle probe, we measured an intracellular O2 level of 3.4 ± 0.1 kPa in bEnd 3 cells cultured under 5 kPa O2. Induction of HO-1 and GCLM by SFN (2.5 µM) was significantly attenuated in cells adapted to 5 kPa O2, despite nuclear accumulation of Nrf2. To simulate ischemic stroke, bEnd.3 cells were adapted to 18 or 5 kPa O2 and subjected to hypoxia (1 kPa O2, 1 h) and reoxygenation. In cells adapted to 18 kPa O2, reoxygenation induced free radical generation was abrogated by PEG-SOD and significantly attenuated by pretreatment with SFN (2.5 µM). Silencing Nrf2 transcription abrogated HO-1 and NQO1 induction and led to a significant increase in reoxygenation induced free radical generation. Notably, reoxygenation induced oxidative stress, assayed using the luminescence probe L-012 and fluorescence probes MitoSOX™ Red and FeRhoNox™-1, was diminished in cells cultured under 5 kPa O2, indicating an altered redox phenotype in brain microvascular cells adapted to physiological normoxia. As redox and other intracellular signaling pathways are critically affected by O2, the development of antioxidant therapies targeting the Keap1-Nrf2 defense pathway in treatment of ischemia-reperfusion injury in stroke, coronary and renal disease will require in vitro studies conducted under well-defined O2 levels.

The anti-ageing hormone klotho induces Nrf2-mediated antioxidant defences in human aortic smooth muscle cells.

Vascular ageing in conditions such as atherosclerosis, diabetes and chronic kidney disease, is associated with the activation of the renin angiotensin system (RAS) and diminished expression of antioxidant defences mediated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2). The anti-ageing hormone klotho promotes longevity and protects against cardiovascular and renal diseases. Klotho has been shown to activate Nrf2 and attenuate oxidative damage in neuronal cells, however, the mechanisms by which it protects against vascular smooth muscle cell VSMC dysfunction elicited by Angiotensin II (AngII) remain to be elucidated. AngII contributes to vascular ageing and atherogenesis by enhancing VSMC oxidative stress, senescence and apoptosis. This study demonstrates that soluble klotho (1 nM, 24 hrs) significantly induces expression of Nrf2 and the antioxidant enzymes haeme oxygenase (HO-1) and peroxiredoxin-1 (Prx-1) and enhances glutathione levels in human aortic smooth muscle cells (HASMC). Silencing of Nrf2 attenuated the induction of HO-1 and Prx-1 expression by soluble klotho. Furthermore, soluble klotho protected against AngII-mediated HASMC apoptosis and senescence via activation of Nrf2. Thus, our findings highlight a novel Nrf2-mediated mechanism underlying the protective actions of soluble klotho in HAMSC. Targeting klotho may thus represent a therapeutic strategy against VSMC dysfunction and cardiovascular ageing.

Phosphatidylinositol-3,4,5-trisphosphate stimulates Ca(2+) elevation and Akt phosphorylation to constitute a major mechanism of thromboxane A2 formation in human platelets.

Phosphatidylinositol trisphosphate (PIP3) has been implicated in many platelet functions however many of the mechanisms need clarification. We have used cell permeable analogues of PIP3,1-O-(1,2-di-palmitoyl-sn-glyero-3-O-phosphoryl)-D-myo-inositol-3,4,5-trisphosphate (DiC16-PIP3) or 1-O-(1,2-di-octanoyl-sn-glyero-3-O-phosphoryl)-D-myo-inositol-3,4,5-trisphosphate (DiC8-PIP3) to study their effects on activation on washed human platelets. Addition of either DiC8- or DiC16-PIP3 to human platelets induced aggregation in the presence of extracellular Ca(2+). This was reduced by the presence of indomethacin, the phospholipase C inhibitor U73122 and apyrase. DiC8-PIP3 induced the phosphorylation of Akt-Ser(473) which was reduced by the Akt inhibitor IV, wortmannin and EGTA (suggesting a dependence on Ca(2+) entry). In Fura2 loaded platelets DiC8-PIP3 was effective at increasing intracellular Ca(2+) in a distinct and transient manner that was reduced in the presence of indomethacin, U73122 and 2-aminoethyl diphenylborinate (2APB). Ca(2+) elevation was reduced by the non-SOCE inhibitor LOE908 and also by the SOCE inhibitor BTP2. DiC8-PIP3 induced the release of Ca(2+) from stores which was not affected by the proton dissipating agent bafilomycin A1 and was more potent than the two-pore channel agonist DiC8-PI[3,5]P2 suggesting release from an endoplasmic reticulum type store. DiC8-PIP3 weakly induced the tyrosine phosphorylation of Syk but not of PLCγ2. Finally like thrombin DiC8-PIP3 induced the formation of thromboxane B2 that was inhibited by the Akt inhibitor IV. These studies suggest that PIP3 via Ca(2+) elevation and Akt phosphorylation forms a central role in thromboxane A2 formation and the amplification of platelet activation.