Svetlana A. Gavrilova

Chronic hypoxia leads to a glycolytic phenotype and suppressed HIF-2 signaling in PC12 cells.

BACKGROUND Along with other regulators of cell metabolism, hypoxia-inducible factors HIF-1 and HIF-2 differentially regulate cell adaptation to hypoxia. Switches in HIF-1/HIF-2 signaling in chronic hypoxia have not been fully investigated. METHODS Proliferation, viability, apoptosis, neuronal and bioenergetic markers, mitochondrial function, respiration, glycolysis, HIF signalling, responses to O2 and glucose deprivation (OGD) were examined using tumor PC12 and SH-SY5Y cells continuously grown at 3% O2. RESULTS Hypoxic PC12 cells (H-cells) exhibit reduced proliferation and histone H4 acetylation, NGF-independent differentiation, activation of AMPK, inhibition of Akt, altered mitochondria and response to NGF. Cellular cytochrome c is increased with no effect on apoptosis. Reduction in respiration has minor effect on cellular ATP which is maintained through activated uptake (GLUT1) and utilization (HK2, PFK2) of glucose. H-cells exhibit resistance to OGD linked to increased glycogen stores. HIF-2alpha protein is decreased without changes in mRNA. Unlike HIF-1alpha, HIF-2alpha is not stabilized pharmacologically or by O2 deprivation. Capacity for HIF-2alpha stabilization is partly restored when H-cells are cultured at normoxia. In low-respiring SH-SY5Y cells cultured under the same conditions HIF-2alpha stabilization and energy budget are not affected. CONCLUSIONS In chronically hypoxic PC12 cells glycolytic energy budget, increased energy preservation and low susceptibility to OGD are observed. HIF-2alpha no longer orchestrates adaptive responses to anoxia. GENERAL SIGNIFICANCE Demonstrated switch in HIF-1/HIF-2 signaling upon chronic hypoxia can facilitate cell survival in energy crisis, by regulating balance between energy saving and decrease in proliferation, on one hand and active cell growth and tumor expansion, on the other.

SEMAX INFLUENCE ON RAT CARDIOMYOCITE APOPTOTIC CELL DEATH IN IRREVERSIBLE ISCHEMIA AND ISCHEMIA-REPERFUSION

5 groups of male rats were investigated: control group; irreversible ischemia with drug and without; the animals with reperfusion after 2.5 hours of ischemia with drug and without it. Samples for light microscopy (haematoxylin-eosin and TUNEL-analysis) of the risk area (anterior wall of left ventricle) and conditionally intact myocardium (lateral wall and interventricular septum) were taken 72 hours after intervention. Semax were injected intraperitoneally at a dose of 150 mg / kg after 15 minutes and after 2 hours 15 minutes after ligation of coronary artery. The necrosis was formed in the anterior wall area both under irreversible ischemia and ischemia-reperfusion. Substantial morphological changes also developed in areas of relatively intact myocardium. These signs are accompanied by a significant increase of apoptotic index. Semax had no effects on the morphology of myocardium, but significantly reduced apoptosis of cardiomyocytes.

Expression of cyclooxygenases and trophic and growth factors in epiretinal membranes at late stages of proliferative vitreoretinopathy

Dear Editor, Proliferative vitreoretinopathy (PVR) is one of the main causes of failure after rhegmatogenous retinal detachment (RRD) surgery. Inflammation is a key factor in the development of PVR. This pathology is characterized by proliferation of retinal pigment epithelial (RPE) cells, macrophages, glial cells, fibroblasts, and vitreous cells on the retinal surface, which leads to avascular membranes formation. Growth factors and inflammatory mediators stimulate cell proliferation. Cyclooxygenases (COX) catalyze the production of prostaglandins from arachidonic acid; this is one of the mechanisms of initiation and maintenance of the inflammatory response. The first and the second types of cyclooxygenase are normally present in the retina. COX-2 expression has been found in human choroidal neovascular membranes [1], in vascularized epiretinal membranes in diabetic retinopathy, and in idiopathic epiretinal membranes [2]. COX-1 was not detected in membranes. Brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF) regulate the viability of retinal cells and their normal functioning. However, retinal detachment and its ischemia are factors that promote the proliferation of glial and RPE cells [3, 4] which actively take part in the development of membranes during PVR. The purpose of this research was to study the level of expression of pro-inflammatory COX-1 and COX-2, VEGF, BDNF, and NGF in epiretinal membranes from patients with severe PVR. Eleven patients suffering from RRD complicated by PVR at late stages were included in the study (Table 1). Patients with other ocular and systemic pathologies were excluded. The localization of retinal breaks was peripheral in all patients. All vitreoretinal surgeries were performed with standard 25-gauge instruments by a single surgeon. During the surgery, the epiretinal membrane of all the eyes was made more visible with triamcinolone acetonide (Kenalog; KRKA, Slovenia). Membranes were immediately placed in liquid nitrogen, after which they were transferred to 500 ml TRI reagent (SigmaAldrich, St. Louis, MO, USA) for a period of 12 h. The standard method of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used. We used specific primers for β-actin (forward: 5’AGGCCAACCGCGAGAAGATGAC-3’; reverse: 5’TCGGCCGTGGTGGTGAAGC-3’), COX-1 (forward: 5’GCTCCAACCTTATCCCCAGTCCC-3’; reverse: 5’CATCAACACAGGCGCCTCTTCTAC-3’), COX-2 (forward: 5’-CCTGATGATTGCCCGACTCCC-3’; reverse: 5’ATACATCATCAGACCAGGCACCAGAC-3’), VEGF (forward: 5’-GGAGGAGGGGGAGGAGGAAGAAGA-3’; reverse: 5’-AGCCCCCGCATCGCATCAG-3’), NGF (forward: * Marina Tikhonovich marina.tikhonovich@gmail.com

Long-term normalization of blood pressure in SHR and 1-kidney 1-clip rats by synthetic precursor of stable PAF analogue without systemic effects in normotensive rats

This study characterized the actions of the newly synthesized PAF precursor 1-hexadecyl-2-alkylcarbamoyl-glycerol (HAG) on blood pressure (BP) in male spontaneously hypertensive rats (SHR), SHR-stroke prone (SHRSP) and Wistar rats with 1-kidney 1-clip (1K1C) renovascular hypertension used as experimental models of human primary and secondary hypertension. Systolic blood pressure (SBP) in the tail artery and mean arterial pressure (MAP) in the abdominal aorta were measured by tail plethysmography and invasive pressure transducer, respectively. Intravenous treatment with 1mg/kg HAG in SHR resulted in a rapid decline of MAP from 151±4 to 127±4mmHg in 50min (p<0.001) that was maintained for 24h after injection (128±5mmHg, p<0.01). We also observed a profound hypotensive effect of HAG in SHRSP but not in normotensive Wistar rats. In 1K1C rats, the magnitude of the BP decline evoked by HAG was correlated with MAP measured before drug administration (R=0.74, p<0.005). In 1K1C rats with SBP>140mmHg, 5mg/kg/48h HAG, given orally for 14 days, decreased SBP by 20-30mmHg without an increase in the death rate and other adverse effects. Thus, our results show that intravenous and oral administration of HAG led to a long-lasting reduction of BP in experimental models of primary and secondary hypertension. In contrast to PAF and its derivatives, the hypotensive action of HAG was preserved for 24h after a single administration, was absent in normotensive animals, and was not accompanied by visible side-effects, at least during 2 weeks of treatment.

Inducible NO Synthase mRNA Expression and Infiltration of Rats Myocardium with Inflammatory Cells in 2-4 Hours after Modeled Permanent Ischemia or Ischemia/Reperfusion

Expression of inducible NO-synthase mRNA and myocardial infiltration with neutrophils were studied in rats with modeled permanent ischemia and ischemia/reperfusion models. Expression of inducible NO synthase mRNA in the ischemic region increased significantly in 3, 3.5, and 4 h in modeled ischemia/reperfusion and in 3.5 and 4 h in permanent ischemia. Myocardial infiltration with neutrophils was significantly higher than in intact controls throughout the experiment without significant intergroup differences. In non-ischemic myocardium, enhanced expression of inducible NO synthase mRNA and moderate neutrophilic-lymphocytic myocardial infiltration were also observed in 3.5, and 4 h after ischemia.

Molecular Bases of Brain Preconditioning

Preconditioning of the brain induces tolerance to the damaging effects of ischemia and prevents cell death in ischemic penumbra. The development of this phenomenon is mediated by mitochondrial adenosine triphosphate-sensitive potassium (KATP+) channels and nitric oxide signaling (NO). The aim of this study was to investigate the dynamics of molecular changes in mitochondria after ischemic preconditioning (IP) and the effect of pharmacological preconditioning (PhP) with the KATP+-channels opener diazoxide on NO levels after ischemic stroke in rats. Immunofluorescence-histochemistry and laser-confocal microscopy were applied to evaluate the cortical expression of electron transport chain enzymes, mitochondrial KATP+-channels, neuronal and inducible NO-synthases, as well as the dynamics of nitrosylation and nitration of proteins in rats during the early and delayed phases of IP. NO cerebral content was studied with electron paramagnetic resonance (EPR) spectroscopy using spin trapping. We found that 24 h after IP in rats, there is a two-fold decrease in expression of mitochondrial KATP+-channels (p = 0.012) in nervous tissue, a comparable increase in expression of cytochrome c oxidase (p = 0.008), and a decrease in intensity of protein S-nitrosylation and nitration (p = 0.0004 and p = 0.001, respectively). PhP led to a 56% reduction of free NO concentration 72 h after ischemic stroke simulation (p = 0.002). We attribute this result to the restructuring of tissue energy metabolism, namely the provision of increased catalytic sites to mitochondria and the increased elimination of NO, which prevents a decrease in cell sensitivity to oxygen during subsequent periods of severe ischemia.

Changes in Sympathetic Innervation of the Heart in Rats with Experimental Myocardial Infarction. Effect of Semax

The effect of peptide Semax on remodeling of cardiac sympathetic innervation was examined in rats with experimental myocardial infarction. In 28 days after ischemia/reperfusion injury, Semax diminished the growth of sympathetic innervation of ventricular septum, although it produced no effect on the density of β1 and β2 adrenoceptors.

Changes in Sympathetic Innervation of Rat Caudal Artery in Experimental Myocardial Infarction. Effect of Semax Peptide.

Activation of the sympathetic nervous system aggravates the course of myocardial infarction. Semax peptide moderated the degree of this activation and prevented the increase in the density of sympathetic endings in rat caudal artery in 28 days after ischemia or ischemia/reperfusion. The peptide reduced the density of α-adrenoreceptors in the caudal artery of rats with myocardial infarction. Semax produced no effect on β-adrenoreceptors in both experimental models. The experiments on isolated segments of the caudal artery revealed reduced vascular responsiveness to electrical stimulation and norepinephrine infusion in rats treated with Semax after ischemia/reperfusion injury.