Adipose-derived regenerative cells exert beneficial effects on systemic responses following myocardial ischemia/reperfusion

Background: Acute coronary syndrome leads to systemic responses, including activation of the sympathetic nervous system, inflammation of atherosclerotic lesions, changes in metabolism and gene expressions of remote organs such as the spleen, bone marrow, and liver. Clinical trials and experimental studies have demonstrated that therapy with adipose-derived regenerative cells (ADRCs) attenuates myocardial ischemia/reperfusion (I/R) injury. The aim of this study is to investigate the role of ADRCs in regulating systemic reactions following I/R.Methods: Isolated ADRCs were obtained from green fluorescent protein transgenic male mice. Flow cytometry revealed that freshly isolated ADRCs expressed stem cell markers CD90 and Sca-1, and mesenchymal lineage marker. These cells exhibited multilineage differentiation into adipogenic, osteogenic, and chondrogenic lineages. Wild-type mice were subjected to 30 min of left ascending coronary ischemia and 24 h reperfusion. Freshly isolated ADRCs (105 cells) or vehicle (VEH), were administered intravenously through the tail at the time of reperfusion.Results: Compared to VEH, administration of ADRCs significantly reduced circulating troponin levels 24 h after I/R. Using quantitative real-time polymerase chain reaction analysis, the present study confirms that I/R-induced increase of factor X mRNA expression in the liver and was significantly inhibited by ADRCs compared to VEH. Administration of ADRCs significantly reduced the I/R-induced increase in serum levels of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-18 seen in mice receiving VEH.Conclusions: These results suggest that administration of ADRCs could have an important role in reducing myocardial injury and regulating the hepatic gene expression profile following I/R

Altered Interaction Between Plasminogen Activator Inhibitor Type 1 Activity and Sympathetic Nerve Activity With Aging

金沢大学大学院医学系研究

Rho-kinase activation in leukocytes plays a pivotal role in myocardial ischemia/reperfusion injury.

The Rho/Rho-kinase pathway plays an important role in many cardiovascular diseases such as hypertension, atherosclerosis, heart failure, and myocardial infarction. Although previous studies have shown that Rho-kinase inhibitors reduce ischemia/reperfusion (I/R) injury and cytokine production, the role of Rho-kinase in leukocytes during I/R injury is not well understood. Mice were subjected to 30-min ischemia and reperfusion. Rho-kinase activity was significantly greater in leukocytes subjected to myocardial I/R compared to the sham-operated mice. Administration of fasudil, a Rho-kinase inhibitor, significantly reduced the I/R-induced expression of the proinflammatory cytokines interleukin (IL)-6, C-C motif chemoattractant ligand 2 (CCL2), and tumor necrosis factor (TNF)-α, in leukocytes, compared with saline as the vehicle. Furthermore, fasudil decreased I/R-induced myocardial infarction/area at risk (IA) and I/R-induced leukocyte infiltration in the myocardium. Interestingly, IA in fasudil-administered mice with leukocyte depletion was similar to that in fasudil-administered mice. I/R also resulted in remarkable increases in the mRNA expression levels of the proinflammatory cytokines TNF-α, IL-6, and CCL2 in the heart. Inhibition of Rho-kinase activation in leukocytes has an important role in fasudil-induced cardioprotective effects. Hence, inhibition of Rho-kinase may be an additional therapeutic intervention for the treatment of acute coronary syndrome

Fasudil reduces myocardial infarct size and apoptosis after I/R through leukocyte infiltration.

<p>Representative examples of myocardial infarction stained with Evans blue (EB) and triphenyl tetrazolium chloride (TTC) 24 h after reperfusion (A). Infarct area is marked with yellow lines. Risk area is marked with white line. The area at risk (AAR) was expressed as a percentage of the left ventricle (LV) (B). Myocardial infarct size expressed as a percentage of AAR (C). Representative photographs of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in heart tissues obtained from mice that were subjected to 30-min ischemia and 24-h reperfusion and treated with vehicle (V) or fasudil (F), neutrophil depletion, or neutrophil depletion + fasudil. Fasudil reduces the I/R-induced increase in the number of TUNEL-positive myocytes (D). Quantitative measurement of the percentage of apoptotic myocytes (E). Data represent means from at least 5 mice each. ** p<0.01, compared with I/R+V; ## p<0.01, compared with the sham-operated group.</p

Fasudil reduces the I/R-induced inflammatory status in hearts partially through its action on leukocytes.

<p>I/R significantly induced expression levels of IL-6 mRNA (A), CCL2 mRNA (B), TNF-α mRNA (C), and IL-10 (D) in the heart. Data represent means from at least 4 mice each. * p<0.05, ** p<0.01. Fasudil and neutrophil depletion influence the expression of various cytokines 24 h after I/R in the heart. Expression levels of IL-6 mRNA (E), CCL2 mRNA (F), TNF-α mRNA (G), and IL-10 (H) in the heart. Data represent means from at least 5 mice each. * p<0.05, ** p<0.01.</p

Fasudil reduces neutrophil infiltration after I/R.

<p>Immunohistochemical analysis of neutrophils stained with anti-Gr-1 are shown (A). Myeloperoxidase (MPO) activity at 6 h after ischemia/reperfusion (I/R) (B). Relative rate of accumulation of leukocytes and neutrophils during I/R (C). Representative FACS data, in which anti-CD45 or the isotype control was used to stain cells isolated from the sham, I/R, or I/R+F groups 12 h after I/R (D). Quantitative measurements of CD45+ cell number in the sham, I/R plus vehicle (I/R+V), and I/R plus fasudil (I/R+F) groups (E). Representative FACS data for CD45+ cells purified using immunobeads and stained with anti-Gr-1+ (F). Summary data of FACS analysis. The results of quantitative analysis of the number of Gr-1+ cells among CD45+ cells are shown (G). Data represent means from at least 4 mice each. * p<0.05, ** p<0.01. Fasudil attenuates chemotaxis and cytokine production of leukocytes in vitro. Chemotaxis and production of IL-6 were significantly reduced in the presence of fasudil (10μM) (H and I).</p

Altered firing pattern of single-unit muscle sympathetic nerve activity during handgrip exercise in chronic heart failure

Sympathetic activation in chronic heart failure (CHF) is greatly augmented at rest but the response to exercise remains controversial. We previously demonstrated that single-unit muscle sympathetic nerve activity (MSNA) provides a more detailed description of the sympathetic response to physiological stress than multi-unit nerve recordings. The purpose of this study was to determine whether the reflex response and discharge properties of single-unit MSNA are altered during handgrip exercise (HG, 30% of maximum voluntary contraction for 3 min) in CHF patients (New York Heart Association functional class II or III, n= 16) compared with age-matched healthy control subjects (n= 13). At rest, both single-unit and multi-unit indices of sympathetic outflow were augmented in CHF compared with controls (P < 0.05). However, the percentage of cardiac intervals that contained one, two, three or four single-unit spikes were not different between the groups. Compared to the control group, HG elicited a larger increase in multi-unit total MSNA (Δ1002 ± 50 compared with Δ636 ± 76 units min−1, P < 0.05) and single-unit MSNA spike incidence (Δ27 ± 5 compared with Δ8 ± 2 spikes (100 heart beats)−1), P < 0.01) in the CHF patients. More importantly, the percentage of cardiac intervals that contained two or three single-unit spikes was increased (P < 0.05) during exercise in the CHF group only (Δ8 ± 2% and Δ5 ± 1% for two and three spikes, respectively). These results suggest that the larger multi-unit total MSNA response observed during HG in CHF is brought about in part by an increase in the probability of multiple firing of single-unit sympathetic neurones