A. N. Murashev

Exogenous mammalian extracellular HSP70 reduces endotoxin manifestations at the cellular and organism levels

In this study, we checked whether HSP70 preparations of different origins are able to protect model animals (rats) from endotoxic shock and modify the response of myeloid cells to lipopolysaccharide (LPS) challenge. It was shown that HSP70 preparations can effectively protect organisms from endotoxic shock by strongly decreasing mortality and restoring both homeostasis and various hemodynamic characteristics. At the cellular level, HSP70 preparations significantly inhibit LPS‐induced reactive oxygen species production in various myeloid cells and decrease NO expression in macrophages, which is enhanced after LPS priming. In parallel, HSP70 preconditioning partially normalizes neutrophil apoptosis, which is disturbed as a result of LPS stimulation. These results suggest that the antiseptic actions of HSP70 preparations are probably realized at the level of receptor membrane complexes of myeloid cells, which represent the major target of LPS action. Taken together, our findings show that extracellular mammalian HSP70 may play an important role in innate immunity modulation and stimulation of endogenous protective mechanisms, both at the cellular and organism levels, which make this protein a promising base for the development of efficient antiseptic drugs.

Recombinant human Hsp70 protects against lipoteichoic acid-induced inflammation manifestations at the cellular and organismal levels

It has been previously reported that pretreatment with exogenous heat shock protein 70 (Hsp70) is able to protect cells and animals from the deleterious effects of bacterial lipopolysaccharide (LPS) produced by Gram-negative bacteria. However, the effects of Hsp70 pretreatment on lipoteichoic acid (LTA) challenge resulted from Gram-positive bacteria infection have not been fully elucidated. In this study, we demonstrated that preconditioning with human recombinant Hsp70 ameliorates various manifestations of systematic inflammation, including reactive oxygen species, TNFα, and CD11b/CD18 adhesion receptor expression induction observed in different myeloid cells after LTA addition. Therefore, exogenous Hsp70 may provide a mechanism for controlling excessive inflammatory responses after macrophage activation. Furthermore, in a rat model of LTA-induced sepsis, we demonstrated that prophylactic administration of exogenous human Hsp70 significantly exacerbated numerous homeostatic and hemodynamic disturbances induced by LTA challenge and partially normalized the coagulation system and multiple biochemical blood parameters, including albumin and bilirubin concentrations, which were severely disturbed after LTA injections. Importantly, prophylactic intravenous injection of Hsp70 before LTA challenge significantly reduced mortality rates. Thus, exogenous mammalian Hsp70 may serve as a powerful cellular defense agent against the deleterious effects of bacterial pathogens, such as LTA and LPS. Taken together, our findings reveal novel functions of this protein and establish exogenous Hsp70 as a promising pharmacological agent for the prophylactic treatment of various types of sepsis.

COG1410, a Novel Apolipoprotein-E Mimetic, Improves Functional and Morphological Recovery in a Rat Model of Focal Brain Ischemia

Apolipoprotein E (apoE) is the primary apolipoprotein synthesized in the brain in response to injury with known neuroprotective effects exerted through antioxidant, antiinflammatory, antiexcitotoxic, and neurotrophic mechanisms. We have previously demonstrated that COG1410, an apoE mimetic peptide, exerts neuroprotective and antiinflammatory effects in a murine model of traumatic brain injury (TBI). As in TBI, ischemia‐reperfusion injury is a component of acute stroke, which displays a pharmacogenetic association with the APOE4 gene. Using an intraluminal middle cerebral occlusion (MCAO) model in rats, we found that a single intravenous injection of COG1410 at 120 min post‐MCAO significantly improved vestibulomotor function, decreased poststroke locomotor asymmetry, and decreased infarct volume of the ipsilateral hemisphere. These results support further exploration of a novel apoE‐mimetic peptide, COG1410, as a therapeutic treatment for stroke.

Anti-inflammatory cytokine interleukin-10 increases resistance to brain ischemia through modulation of ischemia-induced intracellular Ca2+ response

It is suggested that anti-inflammatory cytokine interleukin-10 (IL-10) mediates the delayed protective effects through activation of Jak-Stat3, PI3K-Akt and NF-κB signaling pathways. However, our previous experiments have demonstrated that IL-10 is capable to exert the rapid neuroprotective action through modulation of hypoxia-induced intracellular Ca(2+) ([Ca(2+)]i) response. The first purpose of the present study was to evaluate the neuroprotective effects of IL-10 using three models of the ischemic insults in rats: permanent middle cerebral artery occlusion, ischemia in acute hippocampal slices in vitro and ischemia in cultured hippocampal cells in vitro. The second purpose of the study was to elucidate a role of [Ca(2+)]i changes in the mechanisms underlying IL-10 elicited protection of neurons and astrocytes from ischemia-induced death in cultures of primary hippocampal cells. The data presented here shown that anti-inflammatory cytokine IL-10 is capable to induce a resistance of the brain cells to ischemia-evoked damages in in vivo and in vitro models of the ischemic insults in rats. This protective effect in cultured hippocampal cells is developed rapidly after application of IL-10 and strongly associated with the IL-10 elicited elimination of [Ca(2+)]i response to ischemia. Thus, our results provide the evidence that anti-inflammatory cytokine IL-10, in addition to an activation of the canonical signaling pathways, is capable to exert the rapid neuroprotective effects through transcription-independent modulation of ischemia-induced intracellular Ca(2+) responses in the brain cells.

Role of Reactive Oxygen Species in the Sensitivity of Rat Hypertrophied Myocardium to Ischemia

The relationship between hydroxyl radical (OH·) generation in the zone of ischemia/reperfusion and the size of infarction formed was investigated in 18-22-week-old anaesthetized male SHRSP and Wistar rats using a myocardial microdialysis technique. The marker of OH· generation, 2,3-dihydroxybenzoic acid (2,3-DHBA), was analyzed in dialyzates by high performance liquid chromatography with electrochemical detection. Myocardial ischemia was induced by ligation of the descending branch of the left main coronary artery for 30 min. The mean value of basal 2,3-DHBA level in the dialyzate samples from SHRSP (243 ± 21 pg for 30 min) was significantly higher than that from Wistar rats (91 ± 4 pg for 30 min, p < 0.0002); it positively correlated with left ventricular hypertrophy (r = 0.806; p < 0.05). During reperfusion total 2,3-DHBA output was 1.8-fold higher in SHRSP than in Wistar rats (659 ± 60 pg versus 364 ± 66 pg for 60 min, respectively, p < 0.0002). At the same time, 2,3-DHBA increase above the basal level was the same in Wistar and SHRSP rats (181 ± 25 and 172 ± 36 pg for 60 min, respectively). The infarct size in SHRSP (45.4 ± 4.3%) was significantly higher (p < 0.05) than in Wistar rats (32.8 ± 3.3%). There was a significant positive correlation between basal level of 2,3-DHBA and total reperfusion 2,3-DHBA content in SHRSP (r = 0.752; p < 0.05). Thus, data obtained clearly indicate that the hypertrophied myocardium of SHRSP was less tolerant to ischemia/reperfusion than that of Wistar rats due to chronically increased OH· production and enhanced total OH· output during reperfusion. Greater myocardial damage in SHRSP than in Wistar rats following the equal increase in OH· production above the basal level suggests the existence of deficit of the antioxidant defense in the hypertrophied myocardium.

Influence of sino-aortic barodenervation on the cardiovascular effects of imidazoline-like drugs.

Earlier findings have shown that hypotensive effects of centrally acting drugs, such as clonidine, are enhanced in animals after denervation of arterial baroreceptors. The purpose of this study was to investigate the dynamic of changes in arterial pressure, heart rate and hypotensive effects of clonidine, rilmenidine and moxonidine in Wistar rats after sino-aortic denervation (SAD) using radio-telemetry. SAD was followed by significant elevation of arterial pressure lability (the standard deviation of the mean arterial pressure), while the baseline mean arterial pressure (MAP) and heart rate in barodenervated rats (12 days after SAD) was similar to intact rats. The hypotension produced by clonidine, rilmenidine and moxonidine was much greater in SAD rats than in intact rats. The study suggests that baroreflex mechanisms are not only important for maintaining levels of blood pressure in the very short term, but also for buffering the effects of centrally acting antihypertensive drugs.

Autonomic Dysfunction Determines Stress‐Induced Cardiovascular and Immune Complications in Mice

Background Clinical studies suggest that acute inflammation in patients with elevated heart rate (HR) increases morbidity and mortality. The SJL/J (SJL) inbred mouse strain is a unique genetic model that has higher HR and systemic and vascular inflammation compared with C3HeB/FeJ (C3HeB) mice. The goal of this study was to investigate the role of stress on cardiac and vascular complications between 2 strains. Methods and Results Radiotelemetry was used for continuous recordings of HR and blood pressure in mice. Hemodynamic differences between mouse strains were very small without stress; however, tail-cuff training generated mild stress and significantly increased HR (≈2-fold) in SJL compared with C3HeB mice. Circulating proinflammatory monocytes (CD11b+Ly6CHi) significantly increased in SJL mice but not in C3HeB mice after stress. Presence of Ly6C+ cells in injured carotids was elevated only in SJL mice after stress; however, a transfer of bone marrow cells from SJL/C3HeB to C3HeB/SJL chimeras had no effect on HR or vascular inflammation following stress. Arterial inflammation (VCAM-1+) was greater in SJL inbred mice or SJL recipient chimeras, even without stress or injury. HR variability was reduced in SJL mice compared with C3HeB mice. Conclusions We found that impaired parasympathetic activity is central for stress-induced elevation of HR and systemic and vascular inflammation; however, immune cells from stress-susceptible mice had no effect on HR or vascular inflammation in stress-protected mice.

Bone marrow-derived multipotent stromal cells promote myocardial fibrosis and reverse remodeling of the left ventricle.

Cell therapy is increasingly recognized as a beneficial practice in various cardiac conditions, but its fundamentals remain largely unclear. The fates of transplanted multipotent stromal cells in postinfarction cardiac microenvironments are particularly understudied. To address this issue, labeled multipotent stromal cells were infused into rat myocardium at day 30 after myocardial infarction, against the background of postinfarction cardiosclerosis. Therapeutic effects of the transplantation were assessed by an exercise tolerance test. Histological examination at 14 or 30 days after the transplantation was conducted by means of immunostaining and quantitative image analysis. An improvement in the functional status of the cardiovascular system was observed after both the autologous and the allogeneic transplantations. Location of the label-positive cells within the heart was restricted to the affected part of myocardium. The transplanted cells could give rise to fibroblasts or myofibroblasts but not to cardiac myocytes or blood vessel cells. Both types of transplantation positively influenced scarring processes, and no expansion of fibrosis to border myocardium was observed. Left ventricular wall thickening associated with reduced dilatation index was promoted by transplantation of the autologous cells. According to the results, multipotent stromal cell transplantation prevents adverse remodeling and stimulates left ventricular reverse remodeling.

Soluble fas antigen in serum of cancer patients

Blood concentration of soluble Fas antigen is higher in patients with benign and malignant tumors in comparison with healthy subjects, which probably suggests its involvement into tumorigenesis.

Reparation of the Myocardium after Transplantation of Mononuclear Bone Marrow Cells

Safety and efficiency of intracoronary transventricular transplantation of autologous mononuclear bone marrow cells in rats with postinfarction cardiosclerosis were studied. The cells migrated to the damaged area and were detected only in the cicatricial tissue; they have fibroblast-like phenotype and some of them were stained with Fapα (marker of reactive fibroblasts). More active proliferation of non-muscular cells and formation and maturation of collagen fibers in the cicatricial tissue were observed after transplantation of mononuclear cells. This led to thickening of the cicatricial wall, but the size of the scar and index of dilatation of the left ventricle remained unchanged. The number and volume density of newly formed blood vessels in the damaged area increased after transplantation, but no labeled cells were seen in the vascular walls. It can be hypothesized that stimulation of neoangiogenesis is mediated by paracrine mechanisms, which also explains improvement of global contractility of the left ventricle (increased contractility index in functional tests). Thus, transplantation of mononuclear bone marrow cells leads to thickening and strengthening of the cicatricial wall, stimulates angiogenesis, and improves global myocardial contractility. However, no morphological signs of reverse remodeling of the left-ventricular myocardium were revealed.