Qinghua Hu

Calcium signaling and oxidant stress in the vasculature

Recent evidence suggests that oxidant stress plays a major role in several aspects of vascular biology. Oxygen free radicals are implicated as important factors in signaling mechanisms leading to vascular pathologies such as postischemic reperfusion injury and atherosclerosis. The role of intracellular Ca(2+) in these signaling events is an emerging area of vascular research that is providing insights into the mechanisms mediating these complex physiological processes. This review explores sources of free radicals in the vasculature, as well as effects of free radicals on Ca(2+) signaling in vascular endothelial and smooth muscle cells. In the endothelium, superoxides enhance and peroxides attenuate agonist-stimulated Ca(2+) responses, suggesting differential signaling mechanisms depending on radical species. In smooth muscle cells, both superoxides and peroxides disrupt the sarcoplasmic reticulum Ca(2+)-ATPase, leading to both short- and long-term effects on smooth muscle Ca(2+) handling. Because vascular Ca(2+) signaling is altered by oxidant stress in ischemia-related disease states, understanding these pathways may lead to new strategies for preventing or treating arterial disease.

[Ca2+] i Oscillation Frequency Regulates Agonist-stimulated NF-κB Transcriptional Activity

In nonexcitable cells, stimulation by high agonist concentrations typically produces a biphasic increase in cytosolic Ca2+ ([Ca2+] i ). This response is characterized by a transient initial increase because of intracellular Ca2+ release followed by a sustained elevation which varies in amplitude depending on the nature of the stimulus. In contrast, low-level stimulation often evokes oscillatory changes in [Ca2+] i . The specific information provided by repetitive [Ca2+] i spikes appears to be encoded in the frequency rather than in the amplitude of [Ca2+] i oscillations. The specific, membrane-permeable inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) receptor blocker Xestospongin C (XeC, 2–20 μm) was used to affect [Ca2+] i signaling in human aortic endothelial cells (HAEC) during an established response to low-level (1 μm) histamine stimulation. XeC produced a dose-dependent decrease in the frequency of [Ca2+] i oscillations during histamine stimulation without affecting oscillation amplitude. Histamine stimulated a 14-fold increase in NF-κB-chloramphenicol acetyltransferase reporter gene activity that was dose-dependently decreased by XeC. Thus, during low-level agonist stimulation, [Ca2+] i oscillation frequency regulates nuclear transcription in HAEC.

Hydrogen peroxide induces intracellular calcium oscillations in human aortic endothelial cells

BACKGROUND Because the vascular endothelium is exposed to oxidant stress resulting from ischemia/reperfusion and from the products of polymorphonuclear leukocytes or monocytes, studies were performed to examine the effect of hydrogen peroxide (1 micromol/L to 10 mmol/L) on endothelial Ca2+ signaling. METHODS AND RESULTS At low concentrations (1 to 10 micromol/L), hydrogen peroxide did not affect intracellular Ca2+ concentration in subconfluent, indo 1-loaded human aortic endothelial monolayers. At a concentration of 100 micromol/L hydrogen peroxide, intracellular free Ca2+ gradually increased from 125.3+/-6.8 to 286.3+/-19.9 nmol/L over 4.2+/-0.9 minutes before repetitive Ca2+ oscillations were observed, consisting of an initial large, transient spike of approximately 1 micromol/L followed by several spikes of decreasing amplitudes at a frequency of 0.7+/-0.1 min-1 over 12.0+/-1.1 minutes. After these oscillations, intracellular Ca2+ reached a plateau of 543.4+/-64.0 nmol/L, which was maintained above baseline levels for >5 minutes and then partially reversible on washout of hydrogen peroxide in most monolayers. Intracellular Ca2+ oscillations were typically observed when monolayers were exposed to 100 to 500 micromol/L hydrogen peroxide. Higher concentrations of hydrogen peroxide (1 and 10 mmol/L) increased intracellular Ca2+ but only rarely (2 of 6 monolayers at 1 mmol/L) or never (at 10 mmol/L) stimulated intracellular Ca2+ oscillations. Removal of Ca2+ from the buffer either before hydrogen peroxide stimulation or during an established response did not block intracellular Ca2+ oscillations in response to 100 micromol/L hydrogen peroxide, but prior depletion of an intracellular Ca2+ store with either caffeine, histamine, or thapsigargin abolished Ca2+ oscillations. CONCLUSIONS Hydrogen peroxide induces concentration-dependent intracellular Ca2+ oscillations in human endothelial cells, which results from release of an endoplasmic reticulum Ca2+ store. Because oxidant production appears to occur in the micromolar range in the postischemic/anoxic endothelium and is associated with impaired endothelium-dependent relaxation, the effects of micromolar concentrations of hydrogen peroxide on endothelial Ca2+ signaling described in the present study may be important in the pathogenesis of postischemic endothelial dysfunction.

NADPH Oxidase Activation Increases the Sensitivity of Intracellular Ca2+ Stores to Inositol 1,4,5-Trisphosphate in Human Endothelial Cells

Many stimuli that activate the vascular NADPH oxidase generate reactive oxygen species and increase intracellular Ca2+, but whether NADPH oxidase activation directly affects Ca2+ signaling is unknown. NADPH stimulated the production of superoxide anion and H2O2 in human aortic endothelial cells that was inhibited by the NADPH oxidase inhibitor diphenyleneiodonium and was significantly attenuated in cells transiently expressing a dominant negative allele of the small GTP-binding protein Rac1, which is required for oxidase activity. In permeabilized Mag-indo 1-loaded cells, NADPH and H2O2 each decreased the threshold concentration of inositol 1,4,5-trisphosphate (InsP3) required to release intracellularly stored Ca2+ and shifted the InsP3-Ca2+ release dose-response curve to the left. Concentrations of H2O2 as low as 3 μm increased the sensitivity of intracellular Ca2+ stores to InsP3 and decreased the InsP3 EC50 from 423.2 ± 54.9 to 276.9 ± 14.4 nm. The effect of NADPH on InsP3-stimulated Ca2+ release was blocked by catalase and by diphenyleneiodonium and was not observed in cells lacking functional Rac1 protein. Thus, NADPH oxidase-derived H2O2 increases the sensitivity of intracellular Ca2+ stores to InsP3 in human endothelial cells. Since Ca2+-dependent signaling pathways are critical to normal endothelial function, this effect may be of great importance in endothelial signal transduction.

Critical Role of NADPH Oxidase-derived Reactive Oxygen Species in Generating Ca2+ Oscillations in Human Aortic Endothelial Cells Stimulated by Histamine

There is increasing evidence that intracellular reactive oxygen species (ROS) play a role in cell signaling and that the NADPH oxidase is a major source of ROS in endothelial cells. At low concentrations, agonist stimulation of membrane receptors generates intracellular ROS and repetitive oscillations of intracellular Ca2+ concentration ([Ca2+] i ) in human endothelial cells. The present study was performed to examine whether ROS are important in the generation or maintenance of [Ca2+] i oscillations in human aortic endothelial cells (HAEC) stimulated by histamine. Histamine (1 μm) increased the fluorescence of 2′,7′-dihydrodichlorofluorescin diacetate in HAEC, an indicator of ROS production. This was partially inhibited by the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10 μm), by the farnesyltransferase inhibitor H-Ampamb-Phe-Met-OH (2 μm), and in HAEC transiently expressing Rac1N17, a dominant negative allele of the protein Rac1, which is essential for NADPH oxidase activity. In indo 1-loaded HAEC, 1 μm histamine triggered [Ca2+] i oscillations that were blocked by DPI or H-Ampamb-Phe-Met-OH. Histamine-stimulated [Ca2+] i oscillations were not observed in HAEC lacking functional Rac1 protein but were observed when transfected cells were simultaneously exposed to a low concentration of hydrogen peroxide (10 μm), which by itself did not alter either [Ca2+] i or levels of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3). Thus, histamine generates ROS in HAEC at least partially via NADPH oxidase activation. NADPH oxidase-derived ROS are critical to the generation of [Ca2+] i oscillations in HAEC during histamine stimulation, perhaps by increasing the sensitivity of the endoplasmic reticulum to Ins-1,4,5-P3.

Hypoxia/Reoxygenation Stimulates Intracellular Calcium Oscillations in Human Aortic Endothelial Cells

BackgroundWe have previously shown that hydrogen peroxide stimulates endothelial [Ca2+]i oscillations. This study was performed to determine whether posthypoxic reoxygenation stimulates [Ca2+]i oscillations in vascular endothelial cells. Methods and ResultsHypoxia (glucose-free 95% N2/5% CO2 bicarbonate buffer for 60 minutes) stimulated an increase in [Ca2+]i from 111.9±7.9 to 161.7±17.7 nmol/L (n=12, P <0.01) in indo 1–loaded human aortic endothelial cells. On reoxygenation (glucose-containing 95% air/5% CO2 bicarbonate buffer), 13 of 16 cells responded with repetitive [Ca2+]i oscillations with an average amplitude of 570.6±59.3 nmol/L, occurring at a mean interval of 0.28±0.04/min and persisting for ≥60 minutes. [Ca2+]i oscillations were still observed in 4 of 7 cells studied in Ca2+-free buffer but did not occur when the intracellular Ca2+ store was first depleted during hypoxia by either 1 &mgr;mol/L thapsigargin or by 10 mmol/L caffeine (n=6 for each). Reoxygenation-induced [Ca2+]i oscillations were abolished by 10 &mgr;mol/L diphenyleneiodonium, an inhibitor of NAD(P)H oxidase (n=7), and by polyethylene glycol (PEG)–catalase (5000 U/mL, n=4) but were not prevented by inhibitors of xanthine oxidase (n=5), cyclooxygenase (n=4), nitric oxide synthase (n=5), the mitochondrial electron transport chain (n=4), or by PEG–superoxide dismutase (n=5). ConclusionsPosthypoxic reoxygenation stimulates repetitive [Ca2+]i oscillations that are dependent on Ca2+ release from an intracellular pool and require extracellular Ca2+ to be maintained. These oscillations may be initiated by NAD(P)H oxidase–derived hydrogen peroxide and may play a role in signal transduction during ischemia/reperfusion in vivo.

Capillary leak syndrome in children with C4A-deficiency undergoing cardiac surgery with cardiopulmonary bypass: a double-blind, randomised controlled study

BACKGROUND Capillary leak syndrome is a life-threatening complication after cardiopulmonary bypass (CPB), with an incidence of about 4-37% in children worldwide. On the basis of previous results, we undertook a randomised controlled study to investigate the priming with plasma rich in the C4A isotype of complement component 4 on the incidence of capillary leak syndrome in children with C4A deficiency. METHODS In a hospital in Wuhan, China, we randomly assigned 116 neonates, infants, and children lacking complement component C4A to receive C4A-free or C4A-rich plasma priming (n=58 each, 20 mL/kg). The primary outcome was capillary leak syndrome, identified as an increased transvascular escape rate of Evans blue dye from plasma. Concentrations of activated complement components C4 and C3, inflammatory mediators interleukin 6, interleukin 8, tumour necrosis factor (TNF) alpha, plasma protein, and PaO2/F(I)O2 ratios (ratio of the partial arterial pressure of oxygen to the fractional concentration of oxygen in inspired air) were measured before and 4 h after CPB. Analysis was by intention to treat. FINDINGS Three (5%) patients given C4A-rich plasma priming had capillary leak syndrome compared with 56 (97%) given C4A-free plasma (p<0.0001). At 4 h after CPB, activated C4, interleukin 6, interleukin 8, and TNFalpha concentrations were higher, whereas PaO2/F(I)O2 ratios and plasma protein concentrations were significantly lower in the C4A-free group than changes in the C4A-rich group. Activated C3 rose equally in both groups. Activated C4 significantly correlated with interleukin 6, interleukin 8, and TNFalpha concentrations; PaO2/F(I)O2 ratios; and the escape rate of Evans blue dye at 4 h after CPB. Two patients in the C4A-free group died of respiratory and renal failure on day 3 after CPB. INTERPRETATION In paediatric patients with C4A deficiency, C4A-rich plasma priming reduces the incidence of CPB-related capillary leak syndrome by blocking the activated C4 increase and attenuating the systemic inflammatory response after CPB.

Opposite Effects of Pressurized Steady Versus Pulsatile Perfusion on Vascular Endothelial Cell Cytosolic pH Role of Tyrosine Kinase and Mitogen-Activated Protein Kinase Signaling

Endothelial cytosolic pH (pH(i)) modulates ion channel function, vascular tone, and cell proliferation. Steady shear induces rapid acidification in bicarbonate buffer. However, in vivo shear is typically pulsatile, potentially altering this response. We tested effects and mechanisms of pH(i) modulation by flow pulsatility, comparing pressurized steady versus pulse-flow responses in bovine aortic endothelial cells cultured within glass capillary tubes. Cells were loaded with the fluorescent pH(i) indicator carboxy seminaphthorhodafluor-1 and perfused with physiological pulsatile pressure and flow generated by a custom servo-control system. Raising mean pressure from 0 to 90 mm Hg at 0.5 mL/min steady flow in bicarbonate buffer induced sustained acidification (-0.33+/-0.09 pH units, P<0.01). A subsequent increase in steady flow resulted in further acidification. In contrast, if mean pressure and flow were unchanged but perfusion made pulsatile, pH(i) rose +0.3+/-0.03 (P<0. 0001) over 30 to 60 minutes. HCO(3)(-) removal and use of acid/base exchange inhibitors 5-(N-ethyl-N-isopropyl)amiloride or diisothiocyanato stilbene disulfonic acid identified both extracellular Na(+)-independent Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers as activated by static pressure, whereas pulsatility activated extracellular Na(+)-dependent Cl(-)-HCO(3)(-) and Na(+)-H(+) exchangers to raise pH(i). Pulse-perfusion alkalinization occurred with or without flow reversal and increased 1.6-fold in Ca(2+)-free buffer. Inhibition of c-Src tyrosine kinase (4-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo [3,4-d]pyrimidine; PP2) or MEK-1 (mitogen-activated protein kinase [MAP]/extracellular signal-regulated kinase [ERK]-1) (PD98059, blocking ERK1/2) blocked or reversed the pulsatile-flow pH(i) change to acidification. In contrast, PP2 had no effect on steady flow acidification, whereas MEK-1 inhibition converted it to alkalinization. Thus, pulsatile and steady flow trigger opposite effects on endothelial pH(i) by differential activation of acid/base exchangers linked to c-Src and MAP kinase phosphorylation, but not to Ca(2+). These data highlight specific signaling responses triggered by phasic shear profiles.