Igor Malyshev

Nitric oxide donor induces HSP70 accumulation in the heart and in cultured cells

As our group has shown, the NO‐synthase inhibitor L‐NNA decreased 2–3 times heat shock‐induced synthesis of the heat shock protein HSP70 (FEBS Lett. 370 (1995) 159–162). It was suggested that NO is involved in such induction. In the present study, it was found that (1) injection of the NO donor dinitrosyl iron complex (DNIC) into rats results in accumulation of HSP70 in the heart; (2) heat shock is accompanied by increased generation of NO (EPR assay) and HSP70 accumulation in cultured cells; (3) DNIC induces HSP70 accumulation in cultured cells not exposed to heat shock.

Current Concept and Update of the Macrophage Plasticity Concept: Intracellular Mechanisms of Reprogramming and M3 Macrophage “Switch” Phenotype

Macrophages play a key role in immunity. In this review, we consider the traditional notion of macrophage plasticity, data that do not fit into existing concepts, and a hypothesis for existence of a new switch macrophage phenotype. Depending on the microenvironment, macrophages can reprogram their phenotype toward the proinflammatory M1 phenotype or toward the anti-inflammatory M2 phenotype. Macrophage reprogramming involves well-coordinated changes in activities of signalling and posttranslational mechanisms. Macrophage reprogramming is provided by JNK-, PI3K/Akt-, Notch-, JAK/STAT-, TGF-β-, TLR/NF-κB-, and hypoxia-dependent pathways. Posttranscriptional regulation is based on micro-mRNA. We have hypothesized that, in addition to the M1 and M2 phenotypes, an M3 switch phenotype exists. This switch phenotype responds to proinflammatory stimuli with reprogramming towards the anti-inflammatory M2 phenotype or, contrarily, it responds to anti-inflammatory stimuli with reprogramming towards the proinflammatory M1 phenotype. We have found signs of such a switch phenotype in lung diseases. Understanding the mechanisms of macrophage reprogramming will assist in the selection of new therapeutic targets for correction of impaired immunity.

Dinitrosyl iron complexes with thiol-containing ligands and S-nitroso-D,L-penicillamine as inductors of heat shock protein synthesis in H35 hepatoma cells

The concentration‐dependent effect of various nitric oxide donors on synthesis of different heat shock proteins was evaluated in Reuber H35 hepatoma cells and their heat shock protein‐inducing ability was compared with the effect of a heat shock. A 6 h incubation of H35 cells with the dimeric (diamagnetic) form of dinitrosyl iron complex with glutathione or N‐acetyl‐L‐cysteine activated synthesis of various heat shock proteins, heat shock protein 28, 32, 60, 70, 90 and 100. Synthesis of these proteins was evaluated by [35S]methionine and [35S]cysteine labelling with subsequent separation of proteins by polyacrylamide gel electrophoresis. The dinitrosyl iron complex with glutathione appeared to be the most efficient inductor of heat shock protein synthesis and initiated the synthesis of heat shock protein 28 even more efficiently than a 30 min heating of cells. In the same experiments, S‐nitroso‐D,L‐penicillamine exerted a considerably lesser effect on the synthesis of heat shock proteins. It was suggested that the active moiety of dinitrosyl iron complexes as inductors of heat shock protein synthesis is represented by their Fe+(NO+)2 groups which move to thiol groups of the proteins participating in the initiation of heat shock protein synthesis.

Adaptation to intermittent hypoxia restricts nitric oxide overproduction and prevents beta-amyloid toxicity in rat brain.

This study tested the hypothesis that adaptation to intermittent hypoxia (AIH) can prevent overproduction of nitric oxide (NO) in brain and neurodegeneration induced by beta-amyloid (Aβ) toxicity. Rats were injected with a Aβ protein fragment (25-35) into the nucleus basalis magnocellularis. AIH (simulated altitude of 4000 m, 14 days, 4h daily) was produced prior to the Aβ injection. A passive, shock-avoidance, conditioned response test was used to evaluate memory function. Degenerating neurons were visualized in stained cortical sections. NO production was evaluated in brain tissue by the content of nitrite and nitrate. Expression of nNOS, iNOS, and eNOS was measured in the cortex and the hippocampus using Western blot analysis. 3-Nitrotyrosine formation, a marker of protein nitration, was quantified by slot blot analysis. Aβ injection impaired memory of rats; AIH significantly alleviated this disorder. Histological examination confirmed the protective effect of AIH. Degenerating neurons, which were numerous in the cortex of Aβ-injected, unadapted rats, were essentially absent in the brain of hypoxia-adapted rats. Injections of Aβ resulted in significant increases in NOx and in expression of all NOS isoforms in brain; AIH blunted these increases. NO overproduction was associated with increased amounts of 3-nitrotyrosine in the cortex and hippocampus. AIH alone did not significantly influence tissue 3-nitrotyrosine, but significantly restricted its increase after the Aβ injection. Therefore, AIH affords significant protection against experimental Alzheimers disease, and this protection correlates with restricted NO overproduction.

Macrophages Reprogrammed In Vitro Towards the M1 Phenotype and Activated with LPS Extend Lifespan of Mice with Ehrlich Ascites Carcinoma

Background The majority of tumors trigger macrophage reprogramming from an anti-tumor M1 phenotype towards a pro-tumor M2 phenotype. The M2 phenotype promotes tumor growth. We hypothesized that increasing the number of M1 macrophages in a tumor would limit carcinogenesis and extend the lifespan of the tumor host. The aim of this study was to verify this hypothesis in Ehrlich ascites carcinoma (EAC). The objectives were to evaluate effects of 1) EAC on a macrophage phenotype and NO-producing macrophage activity in vivo; 2) ascitic fluid from mice with EAC on a macrophage phenotype and NO-producing macrophage activity in vitro; and 3) in vitro reprogrammed M1 macrophages on lifespan of mice with EAC. Material/Methods The study was conducted using C57BL/6J mice. Results Concentration of nitrite, a stable NO metabolite and an index of NO production, was measured spectrophotometrically. Shifts of macrophage phenotype were assessed by changes in NO production as well as by amounts of CD80, a marker of M1 phenotype, and CD206, a marker of M2 phenotype. The CD markers were measured by flow cytometry. Macrophages were reprogrammed towards the M1 phenotype using two reprogramming factors: 0% FBS and 20 ng/ml IFN-γ. The study results showed that 1) EAC inhibited the macrophage NO production in vivo and reprogrammed macrophages towards the M2 phenotype; 2) ascitic fluid of mice with EAC inhibited the macrophage NO production in vitro and reprogrammed macrophages towards the M2 phenotype; and 3) injection of in vitro reprogrammed M1 macrophages into mice with EAC significantly increased the lifespan of mice. Conclusions These findings suggest that promising biotechnologies for restriction of tumor growth could be developed based on the in vitro macrophage reprogramming.

The Role of HSP70 in the Protection of: (A) The Brain in Alzheimer’s Disease and (B) The Heart in Cardiac Surgery

The accumulation of aggregated, misfolded proteins and the appearance of neurotoxic aggregates of Aβ and tau proteins play a key role in the development of Alzheimer’s disease. HSP70 can inhibit neurodegeneration associated with Alzheimer’s disease because this protein can: (i) aid in the degradation of intracellular and extracellular Aβ aggregates; (ii) restrict tau protein hyperphosphorylation and facilitate the degradation of dysfunctional tau proteins; (iii) limit NO overproduction; and (iv) regulate apoptosis. It is also likely that HSP70 may delay the development of Alzheimer’s disease by limiting insulin receptor desensitization. HSP70 can limit ischemia myocardial injury by: (i) maintaining protein homeostasis in cells; (ii) stabilizing lysosomal membranes; (iii) inhibiting the excessive activation of ADP-ribose polymerase; and (iv) blocking ischemia-induced apoptosis. During the excessive systemic inflammatory response syndrome (SIRS) that occurs in heart surgery, extracellular HSP70 initiates inflammatory effects through the stimulation of immune cell receptors. In contrast, intracellular HSP70, exerts anti-inflammatory effects on the inflammatory balance of SIRS by inhibiting proinflammatory signaling in immune cells.

The Functions of HSP70 in Normal Cells

In normal cells, the HSP70 ATPase cycle performs several fundamental functions: (1) together with co-chaperones, HSP70 forms a protein folding mechanism and provides protein transportation into organelles; (2) assisted by HSP40, HSP70 recognizes irreversibly damaged proteins and, assisted by CHIP, Bag-1 and HSJ1 ubiquitinates these proteins, thereby targeting them for degradation via proteasomes; and (3) together with the co-chaperones HSP90, HSP40, Hip, Hop and Bag-1, HSP70 recognizes normal proteins containing the marker sequence KFPRQ and sends these proteins for degradation in lysosomes. Thus, the HSP70 ATPase cycle forms a protein quality control system or the FOlding Refolding Degradation machinery (FORD) and, depending on the state of the protein, sends the protein either for re-folding or for degradation. Because of the FORD machinery, a cell maintains protein homeostasis. The HSP70 ATPase cycle also controls the activity of key signalling proteins by maintaining these proteins in an inactive or active state by regulating their levels and by intracellular transport.

Protective Effects of Adaptation to Hypoxia in Experimental Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by formation of amyloid plaques, intracellular neurofibrillary tangles, and cell death in the brain, resulting in progressive loss of memory and cognitive ability. Efficacy of drugs currently used for prevention and treatment of AD is limited by the fact that each drug influences only a single step of the pathogenesis in AD, and the drugs affect both damaged and normal cells. This is why major attention is now paid to nonpharmacological means that may enhance the adaptive capacity and mobilize the self-defense systems of the body. This chapter focuses on protective effects of adaptation to intermittent hypobaric hypoxia on the memory, brain neurons, and cerebral blood vessels in rats with experimental AD induced by intracerebral injections of beta-amyloid (Aβ) and mechanisms of these protective effects. Special attention is paid to intermittent hypobaric hypoxia’s ability to limit early stages in AD pathogenesis, such as nitrosative and oxidative stress in brain tissue. Presented data show that adaptation to hypoxia may be a promising approach to prevention and treatment of AD.

C57BL/6N Mice Are More Resistant to Ehrlich Ascites Tumors Than C57BL/6J Mice: The Role of Macrophage Nitric Oxide

Background Effectiveness of the immune defense formed by the genotype often determines the predisposition to cancer. Nitric oxide (NO) produced by macrophages is an important element in this defense. Material/Methods We hypothesized that genetic characteristics of NO generation systems can predetermine the vulnerability to tumor development. The study was conducted on mice of 2 genetic substrains – C57BL/6J and C57BL/6N – with Ehrlich ascites carcinoma (EAC). NO production in the tumor was changed using ITU, an iNOS inhibitor; c-PTIO, a NO scavenger; and SNP, a NO donor. Macrophage NO production was estimated by nitrite concentration in the culture medium. iNOS content was measured by Western blot analysis. Macrophage phenotype was determined by changes in NO production, iNOS level, and CD markers of the phenotype. Results The lifespan of C57BL/6N mice (n=10) with EAC was 25% longer (p<0.01) than in C57BL/6J mice (n=10). Decreased NO production 23% reduced the survival duration of C57BL/6N mice (p<0.05), which were more resistant to tumors. Elevated NO production 26% increased the survival duration of C57BL/6J mice (p<0.05), which were more susceptible to EAC. Both the NO production and the iNOS level were 1.5 times higher in C57BL/6N than in C57BL/6J mice (p<0.01). CD markers confirmed that C57BL/6N macrophages had the M1 and C57BL/6J macrophages had the M2 phenotype. Conclusions The vulnerability to the tumor development can be predetermined by genetic characteristics of the NO generation system in macrophages. The important role of NO in anti-EAC immunity should be taken into account in elaboration of new antitumor therapies.

M3 Macrophages Stop Division of Tumor Cells In Vitro and Extend Survival of Mice with Ehrlich Ascites Carcinoma

Background M1 macrophages target tumor cells. However, many tumors produce anti-inflammatory cytokines, which reprogram the anti-tumor M1 macrophages into the pro-tumor M2 macrophages. We have hypothesized that the problem of pro-tumor macrophage reprogramming could be solved by using a special M3 switch phenotype. The M3 macrophages, in contrast to the M1 macrophages, should respond to anti-inflammatory cytokines by increasing production of pro-inflammatory cytokines to retain its anti-tumor properties. Objectives of the study were to form an M3 switch phenotype in vitro and to evaluate the effect of M3 macrophages on growth of Ehrlich ascites carcinoma (EAC) in vitro and in vivo. Material/Methods Tumor growth was initiated by an intraperitoneal injection of EAC cells into C57BL/6J mice. Results 1) The M3 switch phenotype can be programed by activation of M1-reprogramming pathways with simultaneous inhibition of the M2 phenotype transcription factors, STAT3, STAT6, and/or SMAD3. 2) M3 macrophages exerted an anti-tumor effect both in vitro and in vivo, which was superior to anti-tumor effects of cisplatin or M1 macrophages. 3) The anti-tumor effect of M3 macrophages was due to their anti-proliferative effect. Conclusions Development of new biotechnologies for restriction of tumor growth using in vitro reprogrammed M3 macrophages is very promising.