The effect of circulating iron on barrier integrity of primary human endothelial cells.

Iron is hypothesized to be one of the contributors to cardiovascular disease and its levels in the circulation may correlate with cardiovascular risk. The aim of this study is to investigate the mechanisms that underlie the effects of iron on the barrier function of primary human endothelium. We used Human Umbilical Vein Endothelial Cells (HUVEC) to investigate the effects of Fe3+ using electric cell-substrate impedance sensing, microscopy, western blot and immunofluorescence microscopy. Exposure to Fe3+ caused EC elongation and upregulation of stress-induced proteins. Analysis of barrier function showed a dose-dependent drop in endothelial integrity, which was accompanied by Reactive Oxygen Species (ROS) production and could partly be prevented by ROS scavengers. Inhibition of contractility by the ROCK inhibitor Y27632, showed even more effective rescue of barrier integrity. Using western blot, we detected an increase in expression of the small GTPase RhoB, an inducer of EC contraction, and a small decrease in VE-cadherin, suggestive for an iron-induced stress response. Co-stimulation by TNFα and iron, used to investigate the role of low-grade inflammation, revealed an additive, negative effect on barrier integrity, concomitant with an upregulation of pro-inflammatory markers ICAM-1 and RhoB. Iron induces a response in HUVEC that leads to endothelial activation and a pro-inflammatory state measured by loss of barrier integrity which can be reversed by ROS scavengers, combined with inhibition of contractility. These data suggest that ROS-mediated damage of the vascular endothelium could contribute to the increased cardiovascular risk which is associated with elevated levels of circulating iron.

Evening aspirin intake results in higher levels of platelet inhibition and a reduction in reticulated platelets - a window of opportunity for patients with cardiovascular disease?

Cardiovascular events occur most frequently in the early morning. Similarly, the release of reticulated platelets (RP) by megakaryocytes has a peak in the late night and early morning. Which aspirin regimen most effectively inhibits platelets during these critical hours is unknown. Hence, the primary objective of this trial was to assess platelet function and RP levels at 8.00 AM, in stable cardiovascular (CVD) patients, during three different aspirin regimens. In this open-label randomized cross-over study subjects were allocated to three sequential aspirin regimens: once-daily (OD) 80 mg morning; OD-evening, and twice-daily (BID) 40 mg. Platelet function was measured at 8.00 AM & 8.00 PM by serum Thromboxane B2 (sTxB2) levels, the Platelet Function Analyzer (PFA)-200® Closure Time (CT), Aspirin Reaction Units (ARU, VerifyNow®), and RP levels. In total, 22 patients were included. At 8.00 AM, sTxB2 levels were the lowest after OD-evening in comparison with OD-morning (p = <0.01), but not in comparison with BID. Furthermore, RP levels were similar at 8.00 AM, but statistically significantly reduced at 8.00 PM after OD-evening (p = .01) and BID (p = .02) in comparison with OD-morning. OD-evening aspirin intake results in higher levels of platelet inhibition during early morning hours and results in a reduction of RP levels in the evening. These findings may, if confirmed by larger studies, be relevant to large groups of patients taking aspirin to reduce cardiovascular risk.