P. V. Avdonin

Reactive oxygen species in pathogenesis of atherosclerosis.

The volume of publications on the role of reactive oxygen species (ROS) in biological processes has been increasing exponentially over the last decades. ROS in large amounts clearly have detrimental effects on cell physiology, whereas low concentrations of ROS are permanently produced in cells and play a role as signaling molecules. An imbalance in ROS production and defense mechanisms can lead to pathological vascular remodeling, atherosclerosis being among them. The aim of this review is to examine different sources of ROS from the point of view of their participation in pathogenesis of atherosclerosis and related cardiovascular risk. Among the possible sources of ROS discussed here are mitochondria, NADPH-oxidases, xanthine oxidase, peroxidases, NO-synthases, cytochrome P450, cyclooxygenases, lipoxygenases, and hemoglobin of red blood cells. A great challenge for future research is to establish interrelations, feedback and feed-forward regulation mechanisms of various sources of ROS in development of atherosclerosis and other vascular pathologies.

Upregulation of vasopressin V1A receptor mRNA and protein in vascular smooth muscle cells following cyclosporin A treatment

The major side effects of the immunosuppressive drug cyclosporin A (CsA) are hypertension and nephrotoxicity. It is likely that both are caused by local vasoconstriction. We have shown previously that 20 h treatment of rat vascular smooth muscle cells (VSMC) with therapeutically relevant CsA concentrations increased the cellular response to [Arg8]vasopressin (AVP) by increasing about 2 fold the number of vasopressin receptors. Displacement experiments using a specific antagonist of the vasopressin V1A receptor (V1AR) showed that the vasopressin binding sites present in VSMC were exclusively receptors of the V1A subtype. Receptor internalization studies revealed that CsA (10−6 M) did not significantly alter AVP receptor trafficking.  V1AR mRNA was increased by CsA, as measured by quantitative polymerase chain reaction. Time‐course studies indicated that the increase in mRNA preceded cell surface expression of the receptor, as measured by hormone binding. A direct effect of CsA on the V1AR promoter was investigated using VSMC transfected with a V1AR promoter‐luciferase reporter construct. Surprisingly, CsA did not increase, but rather slightly reduced V1AR promoter activity. This effect was independent of the cyclophilin‐calcineurin pathway. Measurement of V1AR mRNA decay in the presence of the transcription inhibitor actinomycin D revealed that CsA increased the half‐life of V1AR mRNA about 2 fold. In conclusion, CsA increased the response of VSMC to AVP by upregulating V1AR expression through stabilization of its mRNA. This could be a key mechanism in enhanced vascular responsiveness induced by CsA, causing both hypertension and, via renal vasoconstriction, reduced glomerular filtration.

Calcium ions as a factor of cell—cell cooperation in hepatocyte cultures

Ultradian protein synthesis rhythm was used as a marker of cell cooperation in synchronous dense and non‐synchronous sparse hepatocyte cultures. Phenylephrine (2 μM, 2 min), an α 1‐adrenoreceptor agonist, which exerts [Ca2+]cytelevation from intracellular stores, affected protein synthesis rhythm in sparse cultures, i.e. initiated cooperative activity of the cells. The same effect was produced by 2,5‐di(tertiary‐butyl)‐1,4‐benzohydroquinone (10 μM, 2 min), which increases [Ca2+]cytby a non‐receptor pathway. Pretreatment of dense cultures with the intracellular calcium chelator, 1,2‐bis (2‐aminophenoxy) ethane‐N,N,N′,N′‐ tetraacetic acid (acetoxymethyl) ester (BAPTA‐AM) at 10–20 μM for, 30–60 min resulted in loss of the rhythm of protein synthesis, i.e. loss of cooperative activity between the cells. The medium conditioned by control dense cultures initiated rhythm in sparse cultures, whereas the conditioned medium of cultures pretreated with BAPTA‐AM did not. [Ca2+]cytincrease is known to occur with monosialoganglioside GM1 treatment. By ELISA estimation, the GM1 content in 3 h conditioned medium was similar in control dense cultures to that in cultures pretreated with BAPTA‐AM. Bearing in mind data on the Ca2+‐dependence of vesicle formation and shedding, the conditioned medium was separated by 150,000 g centrifugation to supernatant containing monomers and micelles, and a pellet containing vesicular form of gangliosides. Only the latter initiated cooperative activity of the cells of sparse cultures. These cultures were also synchronized by GM1‐containing liposomes at lower concentrations than added free GM1, 0.0003 and 0.06 μM respectively. Thus, GM1 and calcium are both involved in cell—cell synchronization. Activation of gangliosides, including GM1 and elevation of [Ca2+]cyt, is known to lead to changes of protein kinase activity and protein phosphorylation resulting in modulation of oscillations in protein metabolism.

Inversion of the response to serotonin in rats with traumatic shock

Normally serotonin reduced blood pressure. It was shown that in rats with traumatic shock its hypotensive effect was transformed into hypertensive one. In vitro serotonin exhibited a slight vasoconstrictor effect on isolated rat aorta, while 24 h after injury, the strength of aortic contractions in response to serotonin increased 2.2 times. Desensitization of glucocorticoid receptors caused by injection of high doses of dexamethasone (3 mg/kg) to rats for 5 days led to similar changes in serotonin effect. We hypothesized that inversion of the response to serotonin in shock was caused by increased activity and/or expression of vasoconstrictor serotonin receptors in blood vessels.

Changes in Concentration of Calcium Ions and the Rhythm of Protein Synthesis in the Culture of Hepatocytes

We studied the effects of the chelating agents of extra- and intracellular calcium ions, EGTA and BAPTA-AM, and of the inhibitor of Ca2+release from the reticulum, TMB-8, in the kinetics of protein synthesis in hepatocyte cultures. We also studied dense cultures capable of self-synchronization of protein synthesis oscillations and diluted cultures, in which synchronization is induced by phenylephrine or gangliosides (standard preparation of total gangliosides from the bovine brain). Preincubation of the diluted or dense cultures in the presence of 2 mM EGTA for 1–2 h with subsequent protein assay in a medium with EGTA did not affect the kinetics of protein synthesis: no rhythm was found in the diluted cultures, while it was preserved in the dense cultures. When the diluted cultures preincubated in the presence of EGTA were placed in a medium with EGTA and 2 μM phenylephrine for 2 min, the rhythm was visualized. The treatment of diluted cultures with 100 μM TMB-8 for 5 or 10 min with subsequent washing and incubation in a medium with 3 μM gangliosides led to visualization of the protein synthesis rhythm, i.e., to the synchronization of oscillations, while no rhythm was found in the standard cultures. Preincubation of the diluted cultures in a medium with 10, 15, or 20 μM BAPTA-AM for 1 h did not affect the kinetics of protein synthesis. When, after such preincubation, the diluted cultures were placed in the medium with gangliosides, the rhythm was visualized. In the dense cultures, normally capable of self-synchronization, no rhythm of protein synthesis was found after their treatment with 10–20 μM BAPTA-AM for 1 h. The transfer of such cultures in the medium with gangliosides led to visualization of the rhythm. Thus, calcium affects the kinetics of protein synthesis: after the rise of Ca2+in the cytoplasm was blocked, the rhythm of protein synthesis was not visualized due, supposedly, to disturbed mechanisms of medium conditioning. However, exogenous gangliosides in the dense or diluted cultures preincubated in the presence of BAPTA-AM ore TMB-8 allowed the rhythm visualization, i.e., synchronization may not depend on changes in the intracellular calcium concentration.

Dual proapoptotic and pronecrotic effect of hydrogen peroxide on human umbilical vein endothelial cells

Human umbilical vein endothelial cells were exposed in culture to hydrogen peroxide (H2O2), keeping them close to physiological conditions (high cell density, high serum content, H2O2 concentration not over 500 µM). Cell viability was assessed by flow cytometry using simultaneous staining with the fluorescent dye PO-PRO-1 to detect early apoptotic cells and DRAQ7 to detect late apoptotic and necrotic cells. The data obtained suggest that the primary mechanism of the cytotoxic response to H2O2 is apoptosis. The critical concentration of H2O2 causing death in a dense monolayer is 250 µM. Lower H2O2 concentrations (up to 200 µM) cause death of individual cells. The population of endothelial cell retains viability and response to calcium activating agonists does not change compared to control cells.

Plasma membrane depolarization and activation of receptors for endogenous vasoconstrictors as possible mechanisms of potentiation of vasoconstrictive response to serotonin in traumatic shock in rats

The goal of this work was to study possible mechanisms underlying the potentiation of vasopressor response to serotonin observed in traumatic shock. Experiments with isolated aorta and mesenteric artery of the rat showed that vasoconstriction is caused by the activation of 5HT2A receptors. Agonists of 5HT1B, 5HT1D, 5HT2B, and 5HT4 receptors induced vasodilation. Agonists of 5HT1A receptors had a dual effect determined by interaction with α1-adrenergic receptors and 5HT1A receptors. Plasma membrane depolarization with 15 mM KCl increased the vasoconstrictive force in response to serotonin. This effect was determined by the ability of KCl to activate voltage-gated calcium channels, as a result of which the intracellular calcium stores are replenished. Inhibition of the response to serotonin by ketanserin, a 5HT2A receptor blocker, did not depend on the presence of 15 mM KCl. Constriction in response to serotonin was potentiated after its addition to vessels preconstricted with noradrenaline or endothelin-1. The constriction response partially retained in the presence of 2 × 10−7 M ketanserin, which completely suppressed the serotonin-induced constriction of dilated vessels both at normal membrane potential and after plasma membrane depolarization. It can be assumed that noradrenalin and endothelin-1 alter the characteristics of 5HT2A receptors and possibly 5HT1A receptors as a result of their heterodimerization with the receptors for these vasoconstrictive hormones or receptor-receptor interaction at the level of signaling systems. Along with the potentiating effect of KCl, this mechanism may underlie the enhancement of vasopressor response to serotonin in shock.

[Activation of "silent" vasoconstrictive 5-HT1A receptors as a possible mechanism of synergism in angiotensin II and serotonin effect on vascular tone].

It has been shown that the agonist of 5HT1A-receptors 8-OH-DPAT induces contraction of aortic rings in the presence of angiotensin II. This effect is not associated with activation of α1-adrenoceptors by 8-OH-DPAT as it is reproduced in the presence of prazosin which completely suppresses the nonspecific vasoconstrictive effect of 8-OH-DPAT via α1-adrenoceptors on the aorta incubated without angiotensin II. Synergism in the action of angiotensin II and 8-OH-DPAT is completely preserved after partial desensitization of the receptors of angiotensin II. It has been found that 8-OH-DPAT increases the free cytoplasmic calcium concentration in cultured smooth muscle cells from the rat aorta. The data obtained support the hypothesis about the existence of “silent” vasoconstrictive 5HT1A-receptors. It has been suggested that activation of these receptors underlies synergism in vasoconstrictive action of serotonin and angiotensin II.

Control of thrombotic thrombocytopenic purpura by sirolimus in a child with juvenile myelomonocytic leukemia and somatic N‐RAS mutation

We describe an infant who developed juvenile myelomonocytic leukemia (JMML) at the age of 6 months. Myeloproliferation was effectively controlled by low‐dose cytosine arabinoside and 13‐cis retinoic acid therapy. Two years after therapy for JMML was stopped, at the age of 5 years, the patient developed autoimmune thrombotic thrombocytopenic purpura (TTP). TTP was transiently controlled by plasma exchange, prednisolone, rituximab, and cyclophosphamide, but relapsed within a short time. Long‐term control of TTP was established by sirolimus. Somatic N‐RAS G38A→Gly13Asp substitution was restricted to hematopoietic cells. The somatic N‐RAS mutation may link myeloproliferation and autoimmunity. Pediatr Blood Cancer 2014; 61:1871–1873.

Arachidonic acid activates release of calcium ions from reticulum via ryanodine receptor channels in C2C12 skeletal myotubes

Arachidonic acid causes an increase in free cytoplasmic calcium concentration ([Ca2+]i) in differentiated skeletal multinucleated myotubes C2C12 and does not induce calcium response in C2C12 myoblasts. The same reaction of myotubes to arachidonic acid is observed in Ca2+-free medium. This indicates that arachidonic acid induces release of calcium ions from intracellular stores. The blocker of ryanodine receptor channels of sarcoplasmic reticulum dantrolene (20 μM) inhibits this effect by 68.7 ± 6.3% (p < 0.001). The inhibitor of two-pore calcium channels of endolysosomal vesicles trans-NED19 (10 μM) decreases the response to arachidonic acid by 35.8 ± 5.4% (p < 0.05). The phospholipase C inhibitor U73122 (10 μM) has no effect. These data indicate the involvement of ryanodine receptor calcium channels of sarcoplasmic reticulum in [Ca2+]i elevation in skeletal myotubes caused by arachidonic acid and possible participation of two-pore calcium channels from endolysosomal vesicles in this process.