Pulsatile flow regulates monocyte adhesion to oxidized lipid-induced endothelial cells.

Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC), a component of minimally modified low density lipoprotein, induces monocyte adhesion to endothelial cells. It is not known whether the upstroke slopes of pulsatile flow, defined as shear stress slew rates (tau(r)/tauT)), can regulate monocyte binding to ox-PAPC-treated bovine aortic endothelial cells (BAECs). At 60 cycles per minute, ox-PAPC-treated BAECs were exposed to 3 conditions representing known vascular conditions: (1) high shear stress slew rates (tau(r)/tau(T)=293 dyne. cm(-2). s(-1)), with time-averaged shear stress=50 dyne/cm(2); (2) low shear stress slew rate (tau(r)/tau(t)=71 dyne. cm(-2). s(-1)), with identical time-averaged shear stress; and (3) reversing oscillating flow (0+/-2.6 mm Hg). Reverse transcription-polymerase chain reaction and quantification were performed for monocyte chemoattractant protein-1 (MCP-1) mRNA expression. High tau(r)/tau(t) reduced monocyte binding to ox-PAPC-treated BAECs by 64+/-3.2% compared with static conditions, and low tau(r)/tau(t) reduced monocyte binding by 31+/-3.4%, whereas oscillating flow increased monocyte binding by 22+/-1.7% (P<0.005). High partial tau(r)/tau(t) downregulated MCP-1 expression by 33+/-8%, and low partial tau(r)/tau(t) downregulated MCP-1 expression by 15+/-4%, but oscillating flow upregulated MCP-1 by 13+/-5%. These results suggest that shear stress slew rates regulate monocyte binding by modulating the expression of a potent monocyte chemoattractant.

A complex flow pattern of low shear stress and flow reversal promotes monocyte binding to endothelial cells.

Predilection sites for atherosclerosis within the vasculature are characterized by low shear stress and flow reversal. In this study, endothelial cells were exposed to a complex flow pattern that was characterized by particle velocity determination. Bovine aortic endothelial cells exposed to low shear stress and flow reversal demonstrated higher levels of monocyte binding compared to endothelial cells exposed to one-directional flow. In addition, endothelial cells exposed to low shear stress and flow reversal responded to inflammatory stimuli with substantial increases in monocyte binding, similar to that seen in cells exposed to one-directional flow. These findings suggest a mechanism by which areas of low shear stress and flow reversal are predisposed to the development of atherosclerotic lesions.

Regulation of the mitochondrial permeability transition by matrix Ca(2+) and voltage during anoxia/reoxygenation.

We studied the interplay between matrix Ca(2+) concentration ([Ca(2+)]) and mitochondrial membrane potential (Deltapsi) in regulation of the mitochondrial permeability transition (MPT) during anoxia and reoxygenation. Without Ca(2+) loading, anoxia caused near-synchronous Deltapsi dissipation, mitochondrial Ca(2+) efflux, and matrix volume shrinkage when a critically low PO(2) was reached, which was rapidly reversible upon reoxygenation. These changes were related to electron transport inhibition, not MPT. Cyclosporin A-sensitive MPT did occur when extramitochondrial [Ca(2+)] was increased to promote significant Ca(2+) uptake during anoxia, depending on the Ca(2+) load size and ability to maintain Deltapsi. However, when [Ca(2+)] was increased after complete Deltapsi dissipation, MPT did not occur until reoxygenation, at which time reactivation of electron transport led to partial Deltapsi regeneration. In the setting of elevated extramitochondrial Ca(2+), this enhanced matrix Ca(2+) uptake while promoting MPT because of less than full recovery of Deltapsi. The interplay between Deltapsi and matrix [Ca(2+)] in accelerating or inhibiting MPT during anoxia/reoxygenation has implications for preventing reoxygenation injury associated with MPT.

High-density lipoprotein increases intracellular calcium levels by releasing calcium from internal stores in human endothelial cells.

Elevated levels of high-density lipoproteins (HDL) appear to delay or prevent the development of atherosclerosis. The intracellular signaling mechanisms activated by HDL in vascular cells are currently under active investigation. In this study the effects of HDL on endothelial intracellular Ca levels (EC Ca(i)) are investigated. We show that HDL, like low density lipoproteins (LDL), increases EC Ca(i) in a dose-dependent fashion by releasing Ca from internal stores. Neither apolipoprotein A-I (apo A-I) nor apolipoprotein A-II (apo A-II) was responsible for the increase in EC Ca(i). HDL appeared to release Ca from the same internal stores as did LDL, since preincubation of EC with LDL prevented subsequent responses to HDL but not to the vasodilator ATP. In addition, preincubation of EC with pertussis toxin, an inhibitor of specific G proteins, as well as U73122, an inhibitor of phospholipase C, prevented a rise in EC Ca(i) in response to HDL. These findings suggest that HDL, like LDL, can modulate EC Ca(i) and that this occurs via a pertussis toxin-sensitive G protein-mediated pathway which involves phospholipase C.

Induction of monocyte binding to endothelial cells by MM-LDL: role of lipoxygenase metabolites.

Treatment of human aortic endothelial cells (EC) with minimally oxidized LDL (or minimally modified LDL, MM-LDL) produces a specific pattern of endothelial cell activation distinct from that produced by LPS, tumor necrosis factor-alpha, and interleukin-1, but similar to other agents that elevate cAMP. The current studies focus on the signal transduction pathways by which MM-LDL activates EC to bind monocytes. We now demonstrate that, in addition to an elevation of cAMP, lipoxygenase products are necessary for the MM-LDL response. Treatment of EC with inhibitors of the lipoxygenase pathway, 5,8,11, 14-eicosatetraynoic acid (ETYA) or cinnamyl-3, 4-dihydroxy-alpha-cyanocinnamate (CDC), blocked monocyte binding in MM-LDL-treated EC (MM-LDL=118+/-13%; MM-LDL+ETYA=33+/-4%; MM-LDL+CDC=23+/-4% increase in monocyte binding) without reducing cAMP levels. To further investigate the role of the lipoxygenase pathway, cellular phospholipids were labeled with arachidonic acid. Treatment of cells for 4 hours with 50 to 100 microg/mL MM-LDL, but not native LDL, caused a 60% increase in arachidonate release into the medium and increased the intracellular formation of 12(S)-HETE (approximately 100% increase). There was little 15(S)-HETE present, and no increase in its levels was observed. We demonstrated that 12(S)-HETE reversed the inhibitory effect of CDC. We also observed a 70% increase in the formation of 11,12-epoxyeicosatrienoic acid (11, 12-EET) in cells treated with MM-LDL. To determine the mechanism of arachidonate release induced by MM-LDL, we examined the effects of MM-LDL on intracellular calcium levels. Treatment of EC with both native LDL and MM-LDL caused a rapid release of intracellular calcium from internal stores. However, several pieces of evidence suggest that calcium release alone does not explain the increased arachidonate release in MM-LDL-treated cells. The present studies suggest that products of 12-lipoxygenase play an important role in MM-LDL action on the induction of monocyte binding to EC.

Ca2+-independent myosin II phosphorylation and contraction in chicken embryo fibroblasts.

1. Non-muscle contraction is widely believed to be mediated through Ca2+-stimulated myosin II regulatory light chain (LC20) phosphorylation, similar to the contractile regulation of smooth muscle. However, this hypothesis lacks conclusive experimental support. 2. By modulating chicken embryo fibroblast cytosolic Ca2+ concentration ([Ca2+]i), we investigated the putative role of [Ca2+]i in fetal bovine serum (FBS)-stimulated LC20 phosphorylation and force development in these cells. 3. Eliminating the FBS-stimulated rise in [Ca2+]i with the Ca2+ chelator BAPTA only partially inhibited FBS-stimulated LC20 phosphorylation and did not significantly alter the magnitude of FBS-stimulated isometric contraction. 4. Ionomycin (1 microM) produced a larger but shorter lasting rise in [Ca2+]i relative to FBS. However, ionomycin only stimulated a small and transient increase in LC20 phosphorylation and did not cause contraction. 5. We conclude that fibroblasts differ from smooth muscle in that LC20 phosphorylation and contraction are predominantly regulated independently of [Ca2+]i.

Endothelium-dependent vasodilators do not cause propagated intercellular Ca2+ waves in vascular endothelial monolayers.

Local application of a number of vasoactive agents affects vasomotor tone not only downstream to the point of application but also upstream. The mechanism(s) of upstream propagation is unknown. In endothelial cell monolayers, mechanical stimulation of one cell leads to intercellular propagation of increases in endothelial cell (EC) [Ca2+]i. In this study, we tested whether increases in EC [Ca2+]i induced by the local application of the endothelium-dependent vasodilators ATP, bradykinin and acetylcholine could spread across the monolayer. We demonstrate that unlike the response seen to a mechanical stimulus, there was no significant propagation of increases in EC [Ca2+]i levels in response to localized application of these agents. These findings suggest that upstream vasodilation in response to endothelium-dependent vasodilators is not mediated by propagation of EC [Ca2+]i waves and suggest that other electrical or chemical signals are responsible.

Oxidation of LDL to a biologically active form by derivatives of nitric oxide and nitrite in the absence of superoxide. Dependence on pH and oxygen.

A key factor in atherogenesis is oxidation of LDL in the subendothelial space. In the normal vessel wall or in the thickened intima of diseased vessels, this space is rich in nitric oxide (NO.) released from endothelial cells, smooth muscle cells, and macrophages. To determine whether NO. has a role in LDL oxidation, we exposed human LDL to NO. under aerobic and anaerobic conditions and at acidic and neutral pH. Spectrophotometric detection of beta-carotene in the LDL was used as a marker for LDL oxidation. Depletion of beta-carotene was observed in LDL treated with NO. under aerobic conditions but not under anaerobic conditions. In contrast, treatment of LDL with sodium nitrite did not require oxygen for beta-carotene depletion, although depletion was increased when O2 was present. Furthermore, low pH greatly accelerated LDL oxidation by either NO. gas or by nitrite (NO2-). Depletion of beta-carotene corresponded with formation of conjugated dienes, increased susceptibility to further oxidation, and aggregation of apolipoprotein B-100, but did not increase electrophoretic mobility of LDL. Also, nitrite-oxidized LDL demonstrated biological properties similar to minimally oxidized LDL, including stimulation of monocyte adhesion and inhibition of lipopolysaccharide-induced neutrophil binding to endothelium. These results indicate that NO. under certain circumstances may contribute to oxidative modification of LDL and may have a role in atherogenesis.