A Nrf2-OSGIN1&2-HSP70 axis mediates cigarette smoke-induced endothelial detachment: implications for plaque erosion.

AIMS: Endothelial erosion of plaques is responsible for ∼30% of acute coronary syndromes (ACS). Smoking is a risk factor for plaque erosion, which most frequently occurs on the upstream surface of plaques where the endothelium experiences elevated shear stress. We sought to recreate these conditions in vitro to identify potential pathological mechanisms that might be of relevance to plaque erosion. METHODS AND RESULTS: Culturing human coronary artery endothelial cells (HCAECs) under elevated flow (shear stress of 7.5 Pa) and chronically exposing them to cigarette smoke extract (CSE) and tumour necrosis factor-alpha (TNFα) recapitulated a defect in HCAEC adhesion, which corresponded with augmented Nrf2-regulated gene expression. Pharmacological activation or adenoviral overexpression of Nrf2 triggered endothelial detachment, identifying Nrf2 as a mediator of endothelial detachment. Growth/Differentiation Factor-15 (GDF15) expression was elevated in this model, with protein expression elevated in the plasma of patients experiencing plaque erosion compared with plaque rupture. The expression of two Nrf2-regulated genes, OSGIN1 and OSGIN2, was increased by CSE and TNFα under elevated flow and was also elevated in the aortas of mice exposed to cigarette smoke in vivo. Knockdown of OSGIN1&2 inhibited Nrf2-induced cell detachment. Overexpression of OSGIN1&2 induced endothelial detachment and resulted in cell cycle arrest, induction of senescence, loss of focal adhesions and actin stress fibres, and disturbed proteostasis mediated in part by HSP70, restoration of which reduced HCAEC detachment. In ACS patients who smoked, blood concentrations of HSP70 were elevated in plaque erosion compared with plaque rupture. CONCLUSION: We identified a novel Nrf2-OSGIN1&2-HSP70 axis that regulates endothelial adhesion, elevated GDF15 and HSP70 as biomarkers for plaque erosion in patients who smoke, and two therapeutic targets that offer the potential for reducing the risk of plaque erosion.

Acetylcholine Inhibits Monomeric C-Reactive Protein Induced Inflammation, Endothelial Cell Adhesion, and Platelet Aggregation; A Potential Therapeutic?

Objectives: In this study, we examined the possibility of using targeted antibodies and the potential of small molecular therapeutics (acetylcholine, nicotine and tacrine) to block the pro-inflammatory and adhesion-related properties of monomeric C-reactive protein (mCRP). Methods: We used three established models (platelet aggregation assay, endothelial leucocyte binding assay and monocyte inflammation via ELISA and Western blotting) to assess the potential of these therapeutics. Results: The results of this study showed that monocyte induced inflammation (raised tumor necrosis factor-alpha-TNF-α) induced by mCRP was significantly blocked in the presence of acetylcholine and nicotine, whilst tacrine and targeted antibodies (clones 8C10 and 3H12) had less of or no significant effects. Western blotting confirmed the ability of acetylcholine to inhibit mCRP-induced cell signaling phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), p38 and nuclear factor-kappa B (NF-κB). There was no evidence of direct binding between small molecules and mCRP. mCRP also induced endothelial cell-monocyte adhesion in a dose dependent fashion, however, both acetylcholine and nicotine as well as targeting antibodies notably inhibited adhesion. Finally, we investigated their effects on mCRP-induced platelet aggregation. All three small molecules significantly attenuated platelet aggregation as did the antibody 8C10, although 3H12 had a weaker effect. Discussion: Acetylcholine and to a lesser extent nicotine show potential for therapeutic inhibition of mCRP-induced inflammation and cell and platelet adhesion. These results highlight the potential of targeted antibodies and small molecule therapeutics to inhibit the binding of mCRP by prevention of membrane interaction and subsequent activation of cellular cascade systems, which produce the pro-inflammatory effects associated with mCRP.

The effect of vascular origin, oxygen, and tumour necrosis factor alpha on trophoblast invasion of maternal arteries in vitro.

Extravillous trophoblasts (EVTs) invade and remodel uterine spiral arteries. Regulatory factors may include inherent vessel susceptibility, local oxygen levels and tumour necrosis factor alpha (TNFalpha). We have used an in vitro model to investigate interstitial and endovascular invasion of myometrial spiral arteries from pregnant and non-pregnant uteri and also omental arteries. To model endovascular invasion, fluorescent-labelled EVTs were perfused into the lumen of these dissected vessels. For interstitial invasion, labelled EVTs were layered on top. Cultures were either maintained in 17% or 3% oxygen, or cultured with TNFalpha. The invasion of arteries from pregnant women occurred via both routes at 17% oxygen, with endovascular invasion more efficient than interstitial. In omental arteries and spiral arteries from non-pregnant women, endovascular invasion was limited. Endovascular and interstitial invasion were lower in all arteries at 3% oxygen. Typically, endovascular events were clustered, with an associated disruption in the adjacent endothelium and smooth muscle. A role for TNFalpha in limiting invasion was also supported. In conclusion, priming of uterine arteries may be necessary prior to EVT invasion. Oxygen is a sensitive regulator within this physiological model and increased invasion at higher pO2 may explain the homing of EVT to maternal arteries rather than veins. Adequate vascular transformation may therefore rely on a balance between vascular receptivity, oxygen partial pressure, and exposure to inflammatory mediators.