Takehiro Yamaguchi

Repeated remote ischemic conditioning attenuates left ventricular remodeling via exosome-mediated intercellular communication on chronic heart failure after myocardial infarction.

BACKGROUND Remote ischemic conditioning (RIC) by repeated treatment of transient limb ischemia is a clinically applicable method for protecting the heart against injury at the time of reperfusion. In this study, we investigated the effects of repeated RIC on cardiac dysfunction after myocardial infarction (MI). METHODS AND RESULTS At 4weeks after MI, rats were separated into the untreated (UT) group or the RIC-treated group. RIC treatment was performed by 5cycles of 5min of bilateral hindlimb ischemia and 5min of reperfusion once a day for 4weeks. Despite comparable MI size, left ventricular (LV) ejection fraction (LVEF) was significantly improved in the RIC group compared with the UT group. Furthermore, the LVEF in the RIC group was improved, although not significantly, after treatment. RIC treatment also prevented the deterioration of LV diastolic function. MI-induced LV interstitial fibrosis in the boundary region and oxidant stress were significantly attenuated by RIC treatment. MicroRNA-29a (miR-29a), a key regulator of tissue fibrosis, was highly expressed in the exosomes and the marginal area of the RIC group. Even in the differentiated C2C12-derived exosomes, miR-29a expression was significantly increased under hypoxic condition. As well as miR-29a, insulin-like growth factor 1 receptor (IGF-1R) was highly expressed both in the exosomes and remote non-infarcted myocardium of the RIC group. IGF-1R expression was also increased in the C2C12-derived exosomes under hypoxic conditions. CONCLUSIONS Repeated RIC reduces adverse LV remodeling and oxidative stress by MI. Exosome-mediated intercellular communication may contribute to the beneficial effect of RIC treatment.

Lipid synthesis is promoted by hypoxic adipocyte-derived exosomes in 3T3-L1 cells.

Hypoxia occurs within adipose tissues as a result of adipocyte hypertrophy and is associated with adipocyte dysfunction in obesity. Here, we examined whether hypoxia affects the characteristics of adipocyte-derived exosomes. Exosomes are nanovesicles secreted from most cell types as an information carrier between donor and recipient cells, containing a variety of proteins as well as genetic materials. Cultured differentiated 3T3-L1 adipocytes were exposed to hypoxic conditions and the protein content of the exosomes produced from these cells was compared by quantitative proteomic analysis. A total of 231 proteins were identified in the adipocyte-derived exosomes. Some of these proteins showed altered expression levels under hypoxic conditions. These results were confirmed by immunoblot analysis. Especially, hypoxic adipocyte-released exosomes were enriched in enzymes related to de novo lipogenesis such as acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and fatty acid synthase (FASN). The total amount of proteins secreted from exosomes increased by 3-4-fold under hypoxic conditions. Moreover, hypoxia-derived exosomes promoted lipid accumulation in recipient 3T3-L1 adipocytes, compared with those produced under normoxic conditions. FASN levels were increased in undifferentiated 3T3-L1 cells treated with FASN-containing hypoxic adipocytes-derived exosomes. This is a study to characterize the proteomic profiles of adipocyte-derived exosomes. Exosomal proteins derived from hypoxic adipocytes may affect lipogenic activity in neighboring preadipocytes and adipocytes.

Acute effect of percutaneous transvenous mitral commissurotomy on ventilatory and hemodynamic responses to exercise. Pathophysiological basis for early symptomatic improvement.

BackgroundImprovement of exertional dyspnea occurs immediately after percutaneous transvenous mitral commissurotomy (PTMC), but the pathophysiological basis for this early symptomatic improvement has not been elucidated. Methods and ResultsExercise hemodynamic measurement and exercise ventilatory measurement with arterial blood gas analysis were performed in 21 patients aged 50.4+9.5 years (mean±SD) with symptomatic mitral stenosis before and a few days after PTMC. Exercise ventilatory measurement were also performed in 14 normal control subjects aged 48.9±4.9 years. After PTMC, mitral valve area increased (from 1.0±03 to 1.7±03 cm2, p<.001), mean mitral gradient (from 12.2±5.2 to 5.2±2.2 mm Hg, p<.001), and mean left atrial pressure (from 18.7±6.1 to 12.1±4.0 mm Hg, p<.001) decreased. All patients experienced significant symptomatic improvement soon after PTMC. Comparison of hemodynamic parameters at the same ergometer work rate showed a significant decrease in pulmonary artery systolic pressure (from 77±_18 to 67±+14 mm Hg, p<.001) and diastolic pressure (from 36±10 to 28±7 mm Hg, p<.001) and a significant increase in cardiac output (from 6.4±1.4 to 8.1 ± 1.9 L/min, p<.001). Despite the improvement in exercise hemodynamics and symptoms, exercise capacity determined by peak oxygen uptake (from 18.0+2.9 to 18.6+3.1 mL- kg-1 min-1) and anaerobic threshold (from 11.7±2.4 to 12.0±2.4 mL* kg-1 min-1) remained unchanged. Excessive exercise ventilation, as assessed by the slope of the regression line between expired minute ventilation and carbon dioxide output, decreased significantly from 37.2±6.7 to 33.9±5.8 (P<.001), but remained significantly higher than that in the normal subjects (27.9±3.6, p<.01). The ratio of total dead space to tidal volume and total dead space per breath during exercise decreased significantly after PTMC (P<.05). The change in excessive exercise ventilation after PTMC was correlated with the change in dead space to tidal volume ratio (r=.59) ConclusionsSignificant relief of exertional dyspnea immediately after PTMC is not accompanied by an improvement in exercise capacity. A decrease in excessive exercise ventilation due to a decrease in physiological dead space resulting from hemodynamic improvement partly contributes to the early relief of symptoms after PTMC. However, lung compliance, which was not measured in the present study, may have changed after PTMC. This change may also contribute to the symptomatic improvement.

Macrophage-derived exosomes induce inflammatory factors in endothelial cells under hypertensive conditions

Hypertension is one of the most important cardiovascular risk factors and results in macrophage infiltration of blood vessels. However, how macrophages coordinate inflammatory responses with endothelial cells (ECs) remains unclear. In this study, we investigated whether exosomes upregulate the expression of inflammatory factors in ECs under hypertensive conditions. Hypertension was induced in rats by continuous infusion of angiotensin II (Ang II). Exosomes were purified from rat serum by density gradient and ultracentrifugation and used to stimulate human coronary artery ECs (HCAECs). Moreover, the interactions between HCAECs and exosomes from human THP-1-derived macrophages were analyzed. Administration of Ang II enhanced the expression of CD68, a macrophage marker, in rat hearts, suggesting enhanced infiltration of macrophages. In addition, the expression of intracellular adhesion molecule-1 (ICAM1) and plasminogen activator inhibitor-1 (PAI-1), a proinflammatory factor, was increased in hypertensive rat hearts compared with control rats. CD68 protein expression and an increase in the expression of some exosome markers were detected in exosomes from hypertensive rat serum. Moreover, the exosomes upregulated the expression levels of ICAM1 and PAI-1 in HCAECs. The level of miR-17, a negative regulator of ICAM1 expression, was markedly decreased in exosomes from hypertensive rat serum compared with exosomes from control rats. Interestingly, Ang II-stimulated THP-1-derived exosomes also enhanced the expression of ICAM1 and PAI-1 and contained reduced levels of miR-17 compared with exosomes from unstimulated cells. These results suggest that inflammation of ECs under hypertensive conditions is caused, at least in part, by macrophage-derived exosomes.

Mycobacterial DNA-binding protein 1 is critical for long term survival of Mycobacterium smegmatis and simultaneously coordinates cellular functions

Bacteria can proliferate perpetually without ageing, but they also face conditions where they must persist. Mycobacteria can survive for a long period. This state appears during mycobacterial diseases such as tuberculosis and leprosy, which are chronic and develop after long-term persistent infections. However, the fundamental mechanisms of the long-term living of mycobacteria are unknown. Every Mycobacterium species expresses Mycobacterial DNA-binding protein 1 (MDP1), a histone-like nucleoid associated protein. Mycobacterium smegmatis is a saprophytic fast grower and used as a model of mycobacterial persistence, since it shares the characteristics of the long-term survival observed in pathogenic mycobacteria. Here we show that MDP1-deficient M. smegmatis dies more rapidly than the parental strain after entering stationary phase. Proteomic analyses revealed 21 upregulated proteins with more than 3-fold in MDP1-deficient strain, including DnaA, a replication initiator, NDH, a NADH dehydrogenase that catalyzes downhill electron transfer, Fas1, a critical fatty acid synthase, and antioxidants such as AhpC and KatG. Biochemical analyses showed elevated levels of DNA and ATP syntheses, a decreased NADH/NAD+ ratio, and a loss of resistance to oxidative stress in the MDP1-knockout strain. This study suggests the importance of MDP1-dependent simultaneous control of the cellular functions in the long-term survival of mycobacteria.

Percutaneous Carbon Dioxide Treatment using a Gas Mist Generator Enhances the Collateral Blood Flow in the Ischemic Hindlimb

AIM Highly concentrated carbon dioxide (CO2) is thought to be useful for ischemic diseases. We investigated whether treatment with a few micrometers of CO2 molecules atomized via two fluidnozzles (CO2 mist) exerts an angiogenic effect in a mouse ischemic hindlimb model. METHODS Mice with unilateral hindlimb ischemia were divided into untreated (UT), 100% CO2 gas alone-treated (CG), mixed air (O2; 20%, N2; 80%) mist-treated (AM) and 100% CO2 mist-treated (CM) groups. The lower body of the mice was encased in a polyethylene bag filled with each gaseous agent using a gas mist generator for 10 minutes daily. RESULTS According to a laser Doppler analysis, the ischemic hindlimb blood flow was persistently higher after the seventh day of induction of ischemia in the CM group than in the UT group. The capillary density was also greater in the CM group on day 28 compared with that observed in the UT group. In addition, the parameters in the AM and CG groups were similar to those obtained in the UT group. The observed effects were abolished by the administration of an inhibitor of nitric oxide synthase (NOS). The vascular endothelial growth factor mRNA expression and protein levels and the phosphorylated endothelial NOS level were increased in the CM group compared with that observed in the UT group. A proteomic analysis using liquid chromatography-tandem mass spectrometry identified novel protein candidates regulated by CO2 mist. CONCLUSION Percutaneous CO2 mist therapy may be useful for treating ischemia-induced angiogenesis.

C-terminal intrinsically disordered region-dependent organization of the mycobacterial genome by a histone-like protein

The architecture of the genome influences the functions of DNA from bacteria to eukaryotes. Intrinsically disordered regions (IDR) of eukaryotic histones have pivotal roles in various processes of gene expression. IDR is rare in bacteria, but interestingly, mycobacteria produce a unique histone-like protein, MDP1 that contains a long C-terminal IDR. Here we analyzed the role of IDR in MDP1 function. By employing Mycobacterium smegmatis that inducibly expresses MDP1 or its IDR-deficient mutant, we observed that MDP1 induces IDR-dependent DNA compaction. MDP1-IDR is also responsible for the induction of growth arrest and tolerance to isoniazid, a front line tuberculosis drug that kills growing but not growth-retardated mycobacteria. We demonstrated that MDP1-deficiency and conditional knock out of MDP1 cause spreading of the M. smegmatis genome in the stationary phase. This study thus demonstrates for the first time a C-terminal region-dependent organization of the genome architecture by MDP1, implying the significance of IDR in the function of bacterial histone-like protein.

High-density lipoprotein suppresses tumor necrosis factor alpha production by mycobacteria-infected human macrophages

Immune responses to parasitic pathogens are affected by the host physiological condition. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) are transporters of lipids between the liver and peripheral tissues, and modulate pro-inflammatory immune responses. Pathogenic mycobacteria are parasitic intracellular bacteria that can survive within macrophages for a long period. Macrophage function is thus key for host defense against mycobacteria. These basic facts suggest possible effects of HDL and LDL on mycobacterial diseases, which have not been elucidated so far. In this study, we found that HDL and not LDL enhanced mycobacterial infections in human macrophages. Nevertheless, we observed that HDL remarkably suppressed production of tumor necrosis factor alpha (TNF-α) upon mycobacterial infections. TNF-α is a critical host-protective cytokine against mycobacterial diseases. We proved that toll-like receptor (TLR)-2 is responsible for TNF-α production by human macrophages infected with mycobacteria. Subsequent analysis showed that HDL downregulates TLR2 expression and suppresses its intracellular signaling pathways. This report demonstrates for the first time the substantial action of HDL in mycobacterial infections to human macrophages.

RIVAROXABAN ATTENUATES CARDIAC REMODELING DUE TO INTERMITTENT HYPOXIA BY SUPPRESSING THE SYNERGISTIC EFFECTS OF PAR-1 AND PAR-2

Background: The cumulative frequency of atrial fibrillation (AF) is high in patients with sleep apnea. We have reported that intermittent hypoxia (IH: repeated cycles of 1.5 minute of 5% oxygen followed by 5 minutes of 21% oxygen) relevant to sleep apnea increases oxidative stress causing vascular

Capillary Degeneration and Right Ventricular Remodeling Due to Hypoxic Stress with Sugen5416

BACKGROUND Sugen5416 (semaxinib) is an inhibitor of the vascular endothelial growth factor (VEGF) receptor. A rat model of Pulmonary Arterial Hypertension (PAH), created with Sugen5416 and chronic hypoxia, is known to have similar histological findings to those of PAH patients. OBJECTIVE To evaluate the pathophysiological mechanisms of cardiac remodeling due to hypoxic stress with Sugen5416 in vivo. METHODS Male Sprague-Dawley rats were exposed to hypoxia (10 ± 1% O2) for 2 weeks after a single injection of Sugen5416 (SU-hypoxia group) or the vehicle (V-hypoxia group). RESULTS Hypoxia elevated right ventricular (RV) systolic pressure and caused RV remodeling on Day 14. By electron microscopy, metamorphosis of capillaries with endothelial cell occlusive degeneration was observed in the RV myocardium of the SU-hypoxia group from Day 3. After reoxygenation, progressive RV remodeling with extensive degeneration of cardiomyocytes was observed in the SUhypoxia group, associated with a significant increase of oxidative stress and TUNEL-positive cells in both RV and left ventricular myocardium on Day 84. The expression of VEGF mRNA in the RV myocardium was significantly suppressed in the SU-hypoxia group on Day 3, whereas delayed activation of VEGF/extracellular signal-regulated kinase (ERK) signaling pathway on Day 14 were observed. CONCLUSION Capillary degeneration and activation of VEGF/ERK signaling pathway might be crucial to accelerate the cardiac remodeling due to hypoxic stress with Sugen5416.