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Background/Aims: Acetylcholine (ACh) is known to modulate the cardiac redox environment and thereby suppress reactive oxygen species (ROS) generation during oxidative stress. However, there is little information about its regulation on ROS clearance. Here we investigate the beneficial effects of ACh on superoxide dismutase (SOD) as key ROS-detoxifying enzyme system in cultured rat cardiomyoblasts. Methods: H9c2 cells were subjected to hypoxia/reoxygenation (H/R) to mimic oxidative stress. Western blot was used to detect the expression of SOD and related signaling molecules. Specific protein knockdown was performed with siRNA transfection. Results: ACh treatment on the beginning of reoxygenation decreased ROS and apoptosis. ACh increased ATP synthesis and mitochondrial DNA. Furthermore, ACh significantly reversed H/R-induced reduction in protein expressions and activities of SOD. ACh stimulated peroxisome proliferator activated receptor γ co-activator 1α (PGC-1α) and decreased forkhead box subfamily O3a (FoxO3a) phosphorylation. Atropine (muscarinic receptor antagonist) abolished the cytoprotection afforded by ACh. PGC-1α siRNA blocked ACh-induced invigorating effects on SOD2, whereas it did not alter SOD1 and FoxO3a phosphorylation. FoxOSa siRNA drastically decreased the expressions of SOD2 and PGC-1α, while it did not affect SOD1. Conclusion: ACh activates SOD2 within mitochondria through FoxO3a/PGC-1α pathway and up-regulates SOD1 in the cytoplasm, thus protecting against oxidative injury induced by H/R. Our findings provide new insights into mechanisms underlying the cardioprotection of ACh on ROS detoxifying. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: The present study was designed to investigate the relaxant effect of isopropyl 3-(3, 4-dihydroxyphenyl)-2-hydroxypropanoate (IDHP), a new metabolite from Salvia miltiorrhiza, on rat mesenteric artery. Isolated mesenteric arterial rings were mounted in organ baths and the isometric tension changes were measured continuously by a sensitive myograph system. The results showed that IDHP at concentrations greater than 0.1 nM produced a concentration-dependent relaxation of artery contracted by norepinephrine with pEC50 of 7.41±0.08. Removal of the endothelium did not affect this relaxation, suggesting that IDHP exerted a direct effect on vascular smooth muscle cells. Meanwhile, the vasorelaxant effect of IDHP was unaffected by pre-treatment with ATP-sensitive K+ channel inhibitor glibenclamide, delayed rectifier K+ channel inhibitor 4-aminopyridine, inwardly rectifying K+ channel inhibitor barium chloride and β-adrenoceptor antagonist propranolol. However, the non-specific K+ channel inhibitor tetraethylammonium (TEA, 3 mM) produced a rightward shift of 1.8 fold on the concentration–response curve of IDHP. Moreover, IDHP shifted the concentration–response curve of CaCl2 as well as two receptor-mediated constrictors, phenylephrine and 5-hydroxytryptamine, to the right in a non-parallel manner. In the absence of extracellular Ca2+, IDHP depressed the contractions induced by norepinephrine and CaCl2, and the maximal inhibitions were 48.3±18.9% and 58.4±10.9%, respectively. These results suggest that IDHP exerts a vasorelaxant effect by inhibiting both Ca2+ release from intracellular stores and Ca2+ influx through voltage-dependent calcium channels, and receptor-operated calcium channels in vascular smooth muscle cells. In addition, activation of vascular TEA-sensitive K+ channels may be partially involved in the relaxant effect of IDHP. [Copyright &y& Elsevier]

Abstract: Heart failure is correlated with attenuation of parasympathetic nervous function and enhanced sympathetic activity. Carvedilol, a third-generation β-blocker, may improve the prognosis of heart failure better than selective β1-blockers. Not all of its effects, however, can be explained by direct actions on the sympathetic nervous system. This study was therefore performed to investigate the possible alterations of muscarinic cholinergic (M)2 receptors and cholinesterase-positive nerves in different regions of the adriamycin-induced failing rat heart, and the potential effects of carvedilol on these M2 receptors and cholinesterase-positive nerves. Karnovsky–Roots histochemical staining combined with point counting methods, and immunochemical streptavidin–biotin complex staining and image analysis were used to test the distribution of cholinesterase-positive nerves and the expression of M2 receptors, respectively. Our results show that the cholinesterase-positive nerve system was downregulated in the adriamycin-induced failing heart group, while the density of M2 receptors was increased in the carvedilol 3- and 10-mg/kg body weight groups, especially in the endocardial tissues of the left-ventricular free wall. It is concluded that upregulation of M2 receptors may be one of the potential mechanisms by which carvedilol exert its action on heart failure. [Copyright &y& Elsevier]

Abstract: In this study, protective effects of adenosine and acetylcholine-induced postconditioning were investigated on the contractile function of the ischemic isolated rat ventricular myocytes. A video-based edge-detection system was used to monitor single ventricular myocytes contraction. Adenosine and acetylcholine were administrated for 6 min before ischemia as preconditioning, or 15 min after ischemia as postconditioning. Adenosine and acetylcholine receptor antagonists and mitoKATP inhibitor were used to analyze pathways underlying the effects on postconditioning. Results: (1) The peak shortening of ischemic heart cells was improved by both adenosine and acetylcholine during preconditioning (84.72±5.34% and 68.61±8.10% vs. control: 8.43±5.35% of the pre-ischemia value), as well as postconditioning (76.47±7.87% and 57.48±6.97% vs. control: 8.43±5.35% of the pre-ischemia value) and the effects of preconditioning and postconditioning were comparable. More datum in the normal text. (2) Observed effects of adenosine and acetylcholine postconditioning were missing in the presence of adenosine A1 receptor and muscarinic M2 receptor antagonists, respectively. (3) Adenosine and acetylcholine-induced postconditioning was also blocked by mitoKATP antagonist. These results suggest that both adenosine and acetylcholine protect the contractile function of ischemic heart cells to a similar extent during preconditioning and postconditioning. The postconditioning of adenosine and acetylcholine is relative to the adenosine A1 and muscarinic M2 receptors, respectively. MitoKATP is implicated in the postconditioning of both acetylcholine and adenosine. [Copyright &y& Elsevier]

This study was designed to examine the in vivo and in vitro effects of captopril, an angiotensin-converting enzyme inhibitor, on nicotine-induced endothelial dysfunction in rats. Endothelial dysfunction was induced by exposing isolated rat mesenteric arteries to nicotine (0.01, 0.1, or 1 mM) for 24 hr using an organ culture system, or by treating rats with nicotine (2 mg/kg/day, intraperitoneally) for 4 weeks. The protective effects of captopril were tested by exposing isolated mesenteric arteries to captopril (0.01, 0.03, or 0.1 mM) + nicotine (0.1 mM) for 24 hr, or by treating rats with captopril (3 mg/kg/day, intravenously) + nicotine (2 mg/kg/day, intraperitoneally) for 4 weeks. Exposure of the isolated mesenteric arteries to nicotine induced a significant concentration -dependent inhibition of endothelium-dependent relaxation. Co-culture of segments of mesenteric artery with captopril (0.03 or 0.1 mM) attenuated the nicotine-induced impairment of vasorelaxation in a dose-dependent manner. Administration of nicotine to rats for 4 weeks significantly impaired endothelium-dependent relaxation compared with control rats. This impairment was accompanied by a reduction in nitrite/nitrate, nitric oxide (NO) synthase (NOS), and superoxide dismutase (SOD) activities in the serum and aorta. Chronic captopril treatment not only improved the impairment of endothelium-dependent relaxation, but also prevented the reduction of nitrite/nitrate contents and of NOS and SOD activities in the serum and aorta. However, there were no significant differences in serum angiotensin-converting enzyme activity among the three groups. These results indicate that captopril can be used to attenuate nicotine-induced endothelial dysfunction, an effect that may be related not only to antioxidation, but also to enhancing NO production by preventing the decrease in NOS. [ABSTRACT FROM AUTHOR]

Abstract: It has been demonstrated that the number and differentiating potential of bone marrow mesenchymal stem cells (MSCs) decrease with age. Therefore, the search for alternative sources of MSCs is of significant value. In the present study, MSCs were isolated from umbilical cord blood (UCB) by combining gradient density centrifugation with plastic adherence. Cultured cells were treated with ascorbate acid-2-phosphate, dexamethasone, β-glycerophosphate dexamethasone, insulin, 1-methyl-3-isobutylxamthine, indomethacin, β-mercaptoethanol, butylated hydroxyanisole, FGF-4 and HGF. Differentiating characterization of UCB-derived MSCs were detected by cytochemistry, immunocytochemistry, radioimmunoassay, RT-PCR and urea assay. The results showed UCB-derived MSCs could differentiate into osteoblasts, adipocytes and neuron-like cells. When MSCs were cultured with FGF-4 and HGF, approximately 63.6% of cells became small, round and epithelioid on day 28 by morphology. Compared with the control, levels of AFP in the supernatant liquid increased significantly from day 12 and were higher on day 28 (P <0.01). Albumin increased significantly from day 16 (P <0.01). Urea was first detected on day 20 (P <0.01), and continued to increase on day 28 (P <0.01). Cells first expressed CK-18 on day 16 through immunocytochemistry analysis. RT-PCR analysis showed that differentiated cells could express a number of hepatocyte-specific genes in a time-dependent manner. Glycogen storage was first seen on day 24. Our results suggest that UCB-derived MSCs can differentiate not only into osteoblasts, adipocytes and neuron-like cells, but also into hepatocytes. Human UCB-derived MSCs are a new source of cell types for cell transplantation and therapy. [Copyright &y& Elsevier]

Mitochondria participate in essential cellular functions, including energy production, metabolism, redox homeostasis regulation, intracellular Ca2+ handling, apoptosis, and cell fate determination. Disruption of mitochondrial homeostasis under pathological conditions results in mitochondrial reactive oxygen species (ROS) generation and energy insufficiency, which further disturb mitochondrial and cellular homeostasis in a deleterious loop. Mitochondrial redox status has therefore become a potential target for therapy against cardiovascular diseases. In this review, we highlight recent progress in determining the roles of mitochondrial processes in regulating mitochondrial redox status, including mitochondrial dynamics (fusion–fission pathways), mitochondrial cristae remodeling, mitophagy, biogenesis, and mitochondrion–organelle interactions (endoplasmic reticulum–mitochondrion interactions, nucleus–mitochondrion communication, and lipid droplet–mitochondrion interactions). The strategies that activate vagal system include direct vagal activation (electrical vagal stimulation and administration of vagal neurotransmitter acetylcholine) and pharmacological modulation (choline and cholinesterase inhibitors). The vagal system plays an important role in maintaining mitochondrial homeostasis and suppressing mitochondrial oxidative stress by promoting mitochondrial biogenesis and mitophagy, moderating mitochondrial fusion and fission, strengthening mitochondrial cristae stabilization, regulating mitochondrion–organelle interactions, and inhibiting mitochondrial Ca2+ overload. Therefore, enhancement of vagal activity can maintain mitochondrial homeostasis and represents a promising therapeutic strategy for cardiovascular diseases. [Display omitted] • Vagal system regulates mitochondrial homeostasis in cardiovascular diseases. • Protective effects of vagal system on mitochondrial quality control and redox status. • Roles of vagal system in regulation of multiple mitochondrion-organelle interactions. [ABSTRACT FROM AUTHOR]

Mitochondrial dysfunction is associated with obesity-induced cardiac remodelling. Recent research suggests that the cristae are the true bioenergetic components of cells. Acetylcholine (ACh), the major neurotransmitter of the vagus nerve, exerts cardio-protective effects against ischaemia. This study investigated the role of cristae remodelling in palmitate (PA)-induced neonatal rat cardiomyocyte hypertrophy and explored the beneficial effects of ACh. We found loose, fragmented and even lysed cristae in PA-treated neonatal cardiomyocytes along with declines in mitochondrial network and complex expression and overproduction of mitochondrial reactive oxygen species (ROS); these changes ultimately resulted in increased myocardial size. Overexpression of mitofilin by adenoviral infection partly improved cristae shape, mitochondrial network, and ATP content and attenuated cell hypertrophy. Interestingly, siRNA-mediated AMP-activated protein kinase (AMPK) silencing increased the number of cristae with a balloon-like morphology without disturbing mitofilin expression. Furthermore, AMPK knockdown abolished the effects of mitofilin overexpression on cristae remodelling and inhibited the interaction of mitofilin with sorting and assembly machinery 50 (Sam50) and coiled-coil helix coiled-coil helix domain-containing protein 3 (CHCHD3), two core components of the mitochondrial contact site and cristae organizing system (MICOS) complex. Intriguingly, ACh upregulated mitofilin expression and AMPK phosphorylation via the muscarinic ACh receptor (MAChR). Moreover, ACh enhanced protein-protein interactions between mitofilin and other components of the MICOS complex, thereby preventing PA-induced mitochondrial dysfunction and cardiomyocyte hypertrophy; however, these effects were abolished by AMPK silencing. Taken together, our data suggest that ACh improves cristae remodelling to defend against PA-induced myocardial hypertrophy, presumably by increasing mitofilin expression and activating AMPK to form the MICOS complex through MAChR. These results suggest new and promising therapeutic approaches targeting mitochondria to prevent lipotoxic cardiomyopathy. • Loose and lysed cristae occurred in PA-induced cardiomyocytes along with mitochondrial damage. • Overexpression of mitofilin improved cristae shape in PA-induced cardiomyocytes. • SiRNA AMPK inhibited mitofilin to form MICOS in cristae remodelling. • ACh increased mitofilin expression and activated AMPK to form MICOS via MAChR. [ABSTRACT FROM AUTHOR]

Insulin resistance and autonomic imbalance are important pathological processes in metabolic syndrome–induced cardiac remodeling. Recent studies determined that disruption of mitochondrial cristae shape is associated with myocardial ischemia; however, the change in cristae shape in metabolic syndrome–induced cardiac remodeling remains unclear. This study determined the effect of pyridostigmine (PYR), which reversibly inhibits cholinesterase to improve autonomic imbalance, on high-fat diet (HFD)–induced cardiac insulin resistance and explored the potential effect on the shape of mitochondrial cristae. Feeding of a HFD for 22 weeks led to an irregular and even lysed cristae structure in cardiac mitochondria, which contributed to decreased mitochondrial content and ATP production and increased oxygen species production, ultimately impairing insulin signaling and lipid metabolism. Interestingly, PYR enhanced vagal activity by increasing acetylcholine production and exerted mito-protective effects by activating the LKB1/AMPK/ACC signal pathway. Specifically, PYR upregulated OPA1 and Mfn1/2 expression, promoted the formation of the mitofilin/CHCHD3/Sam50 complex, and decreased p-Drp1 and Fis1 expression, resulting in tight and parallel cristae and increasing cardiac mitochondrial complex subunit expression and ATP generation as well as decreasing release of cytochrome C from mitochondria and oxidative damage. Furthermore, PYR improved glucose and insulin tolerance and insulin-stimulated Akt phosphorylation, decreased lipid toxicity, and ultimately ameliorated HFD-induced cardiac remodeling and dysfunction. In conclusion, PYR prevented cardiac and insulin insensitivity and remodeling by stimulating vagal activity to regulate mitochondrial cristae shape and function in HFD-induced metabolic syndrome in mice. These results provide novel insights for the development of a therapeutic strategy for obesity-induced cardiac dysfunction that targets mitochondrial cristae. fx1 • Loose and lysed cristae were observed in cardiac mitochondria of obesity mice. • Pyridostigmine improved cardiac vagal activity and mitochondrial morphology. • Pyridostigmine improved cristae form by regulating mitochondria-shaping proteins. • Pyridostigmine alleviated cardiac insulin insensitivity and cardiac remodeling. [ABSTRACT FROM AUTHOR]

Abstract Aims Obesity is associated with increased cardiovascular morbidity and mortality. It is accompanied by augmented O-linked β- N -acetylglucosamine (O-GlcNAc) modification of proteins via increasing hexosamine biosynthetic pathway (HBP) flux. However, the changes and regulation of the O-GlcNAc levels induced by obesity are unclear. Main methods High fat diet (HFD) model was induced obesity in mice with or without the cholinergic drug pyridostigmine (PYR, 3 mg/kg/d) for 22 weeks and in vitro human umbilical vein endothelial cells (HUVECs) was treated with high glucose (HG, 30 mM) with or without acetylcholine (ACh). Key findings PYR significantly reduced body weight, blood glucose, and O-GlcNAcylation levels and attenuated vascular endothelial cells detachment in HFD-fed mice. HG addition induced endoplasmic reticulum (ER) stress and increased O-GlcNAcylation levels and apoptosis in HUVECs in a time-dependent manner. Additionally, HG decreased levels of phosphorylated AMP-activated protein kinase (AMPK). Interestingly, ACh significantly blocked damage to HUVECs induced by HG. Furthermore, the effects of ACh on HG-induced ER stress, O-GlcNAcylation, and apoptosis were prevented by treating HUVECs with 4-diphenylacetoxy- N -methylpiperidine methiodide (4-DAMP, a selective M3 AChR antagonist) or compound C (Comp C, an AMPK inhibitor). Treatment with 5-aminoimidazole-4-carboxamide ribose (AICAR, an AMPK activator), 4-phenyl butyric acid (4-PBA, an ER stress inhibitor), and 6-diazo-5-oxonorleucine (DON, a GFAT antagonist) reproduced a similar effect with ACh. Significance Activation of cholinergic signaling ameliorated endothelium damage, reduced levels of ER stress, O-GlcNAcylation, and apoptosis in mice and HUVECs under obese conditions, which may function through M3 AChR-AMPK signaling. [ABSTRACT FROM AUTHOR]

Aims Cardiac hypertrophy is characterized by a shift in metabolic substrate utilization, but the molecular events underlying the metabolic remodelling remain poorly understood. We explored metabolic remodelling and mitochondrial dysfunction in cardiac hypertrophy and investigated the cardioprotective effects of choline. Methods and results The experiments were conducted using a model of ventricular hypertrophy by partially banding the abdominal aorta of Sprague Dawley rats. Cardiomyocyte size and cardiac fibrosis were significantly increased in hypertrophic hearts. In vitro cardiomyocyte hypertrophy was induced by exposing neonatal rat cardiomyocytes to angiotensin II (Ang II) (10−6 M, 24 h). Choline attenuated the mito-nuclear protein imbalance and activated the mitochondrial-unfolded protein response (UPRmt) in the heart, thereby preserving the ultrastructure and function of mitochondria in the context of cardiac hypertrophy. Moreover, choline inhibited myocardial metabolic dysfunction by promoting the expression of proteins involved in ketone body and fatty acid metabolism in response to pressure overload, accompanied by the activation of sirtuin 3/AMP-activated protein kinase (SIRT3-AMPK) signalling. In vitro analyses demonstrated that SIRT3 siRNA diminished choline-mediated activation of ketone body metabolism and UPRmt, as well as inhibition of hypertrophic signals. Intriguingly, serum from choline-treated abdominal aorta banding models (where β-hydroxybutyrate was increased) attenuated Ang II-induced myocyte hypertrophy, which indicates that β-hydroxybutyrate is important for the cardioprotective effects of choline. Conclusion Choline attenuated cardiac dysfunction by modulating the expression of proteins involved in ketone body and fatty acid metabolism, and induction of UPRmt; this was likely mediated by activation of the SIRT3-AMPK pathway. Taken together, these results identify SIRT3-AMPK as a key cardiac transcriptional regulator that helps orchestrate an adaptive metabolic response to cardiac stress. Choline treatment may represent a new therapeutic strategy for optimizing myocardial metabolism in the context of hypertrophy and heart failure. [ABSTRACT FROM AUTHOR]

Obesity, a major contributor to the development of cardiovascular diseases, is associated with an autonomic imbalance characterized by sympathetic hyperactivity and diminished vagal activity. Vagal activation plays important roles in weight loss and improvement of cardiac function. Pyridostigmine is a reversible acetylcholinesterase inhibitor, but whether it ameliorates cardiac lipid accumulation and cardiac remodeling in rats fed a high-fat diet has not been determined. This study investigated the effects of pyridostigmine on high-fat diet-induced cardiac dysfunction and explored the potential mechanisms. Rats were fed a normal or high-fat diet and treated with pyridostigmine. Vagal discharge was evaluated using the BL-420S system, and cardiac function by echocardiograms. Lipid deposition and cardiac remodeling were determined histologically. Lipid utility was assessed by qPCR. A high-fat diet led to a significant reduction in vagal discharge and lipid utility and a marked increase in lipid accumulation, cardiac remodeling, and cardiac dysfunction. Pyridostigmine improved vagal activity and lipid metabolism disorder and cardiac remodeling, accompanied by an improvement of cardiac function in high-fat diet-fed rats. An increase in the browning of white adipose tissue in pyridostigmine-treated rats was also observed and linked to the expression of UCP-1 and CIDEA. Additionally, pyridostigmine facilitated activation of brown adipose tissue via activation of the SIRT-1/AMPK/PGC-1α pathway. In conclusion, a high-fat diet resulted in cardiac lipid accumulation, cardiac remodeling, and a significant decrease in vagal discharge. Pyridostigmine ameliorated cardiomyopathy, an effect related to reduced cardiac lipid accumulation, and facilitated the browning of white adipose tissue while activating brown adipose tissue. [ABSTRACT FROM AUTHOR]

It is well-accepted that inflammation plays an important role in the development of cardiac remodelling and that therapeutic approaches targeting inflammation can inhibit cardiac remodelling. Although a large amount of evidence indicates that activation of α7 nicotinic acetylcholine receptor (α7n AChR) causes an anti-inflammatory effect, the role of α7n AChR in cardiac remodelling and the underlying mechanism have not been established. To investigate the effect of the specific α7n AChR agonist, PNU282987, on cardiac remodelling induced by isoproterenol ( ISO 60 mg/kg per day) in mice, the cardiomyocyte cross-sectional area ( CSA) and collagen volume fraction were evaluated by hematoxylin and eosin ( HE) and Masson staining, respectively. Cardiac function and ventricular wall thickness were measured by echocardiography. The protein expressions of collagen I, matrix metalloproteinase 9 ( MMP-9), transforming growth factor β1 ( TGF-β1), and Smad3 were analyzed by Western blot. ISO-induced cardiac hypertrophy, characterized by an increase in the heart weight/body weight ratio, CSA and ventricular wall thickness. Moreover, cardiac fibrosis indices, such as collagen volume fraction, MMP-9 and collagen I protein expression, were also increased by ISO. PNU282987 not only attenuated cardiac hypertrophy but also decreased the cardiac fibrosis induced by ISO. Furthermore, PNU282987 suppressed TGF-β1 protein expression and the phosphorylation of Smad3 induced by ISO. In conclusion, PNU282987 ameliorated the cardiac remodelling induced by ISO, which may be related to the TGF-β1/Smad3 pathway. These data imply that the α7n AChR may represent a novel therapeutic target for cardiac remodelling in many cardiovascular diseases. [ABSTRACT FROM AUTHOR]

The endoplasmic reticulum (ER) forms discrete junctions with the plasma membrane (PM) that play a critical role in the regulation of Ca 2 + signaling during cellular bioenergetics, apoptosis and autophagy. We have previously confirmed that acetylcholine can inhibit ER stress and apoptosis after inflammatory injury. However, limited research has focused on the effects of acetylcholine on ER-PM junctions. In this work, we evaluated the structure and function of the supramolecular sodium-calcium exchanger 1 (NCX1)-transient receptor potential canonical 3 (TRPC3)-inositol 1,4,5-trisphosphate receptor 1 (IP3R1) complex, which is involved in regulating Ca 2 + homeostasis during inflammatory injury. The width of the ER-PM junctions of human umbilical vein endothelial cells (HUVECs) was measured in nanometres using transmission electron microscopy and a fluorescent probe for Ca 2 + . Protein-protein interactions were assessed by immunoprecipitation. Ca 2 + concentration was measured using a confocal microscope. An siRNA assay was employed to silence specific proteins. Our results demonstrated that the peripheral ER was translocated to PM junction sites when induced by tumour necrosis factor-alpha (TNF-α) and that NCX1-TRPC3-IP3R1 complexes formed at these sites. After down-regulating the protein expression of NCX1 or IP3R1, we found that the NCX1-mediated inflow of Ca 2 + and the release of intracellular Ca 2 + stores were reduced in TNF-α-treated cells. We also observed that acetylcholine attenuated the formation of NCX1-TRPC3-IP3R1 complexes and maintained calcium homeostasis in cells treated with TNF-α. Interestingly, the positive effects of acetylcholine were abolished by the selective M3AChR antagonist darifenacin and by AMPK siRNAs. These results indicate that acetylcholine protects endothelial cells from TNF-alpha-induced injury, [Ca 2 + ] cyt overload and ER-PM interactions, which depend on the muscarinic 3 receptor/AMPK pathway, and that acetylcholine may be a new inhibitor for suppressing [Ca 2 + ] cyt overload. [ABSTRACT FROM AUTHOR]

Periodontitis is a severe inflammatory response, leading to characteristic periodontal soft tissue destruction and alveolar bone resorption. Baicalin possesses potent anti-inflammatory activity; however, it is still unclear whether baicalin regulates toll-like receptor (TLR) 2/4 expression and downstream signaling during the process of periodontitis. In this study, the cervical area of the maxillary second molars of rats was ligated and inoculated with Porphyromonas gingivalis ( P. gingivalis ) for 4 weeks to induce periodontitis. Some rats with periodontitis were treated intragastrically with baicalin (50, 100 or 200 mg/kg/day) or vehicle for 4 weeks. Compared with the sham group, the levels of TLR2, TLR4 and MyD88 expression and the p38 MAPK and NF-κB activation were up-regulated in the experimental periodontitis group (EPG), accompanied by marked alveolar bone loss and severe inflammation. Treatment with 100 or 200 mg/kg/day baicalin dramatically reduced the alveolar bone loss, the levels of HMGB1, TNF-α, IL-1β, and MPO expression, and the numbers of inflammatory infiltrates in the gingival tissues. Importantly, treatment with 100 or 200 mg/kg/day baicalin mitigated the periodontitis-up-regulated TLR2, TLR4 and MyD88 expression, and the p38 MAPK and NF-κB activation. Hence, the blockage of the TLR2 and TLR4/MyD88/p38 MAPK/NF-κB signaling by baicalin may contribute to its anti-inflammatory effects in rat model of periodontitis. In conclusion, these novel findings indicate that baicalin inhibits the TLR2 and TLR4 expression and the downstream signaling and mitigates inflammatory responses and the alveolar bone loss in rat experimental periodontitis. Therefore, baicalin may be a potential therapeutic agent for treatment of periodontitis. [ABSTRACT FROM AUTHOR]

Acetylcholine (ACh) protected against cardiac injury via promoting autophagy and mitochondrial biogenesis, however, the involvement of mitophagy in ACh-elicited cardioprotection remains unknown. In the present study, H9c2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) and ACh treatment during reoxygenation. Mitophagy markers PTEN-induced kinase 1 (PINK1) and Parkin translocation were examined using western blot and confocal fluorescence microscopy. Mitochondrial membrane potential and reactive oxygen species (ROS) were detected with fluorescence staining. We found that H/R-treated cells exhibited reduced levels of PINK1 and Parkin in mitochondria, accompanied with decreased autophagy flux (reduced LC3-II/LC3-I and increased p62). Conversely, ACh increased PINK1 and Parkin translocation to mitochondria and enhanced autophagy proteins. Confocal imaging of Parkin and MitoTracker Green-labeled mitochondria further confirmed ACh-induced mitochondrial translocation of Parkin, which was reversed by M2 receptor antagonist methoctramine and M2 receptor siRNA, suggesting ACh could induce mitophagy by M2 receptor after H/R. Mitophagy inhibitor 3-methaladenine abolished ACh-induced mitoprotection, manifesting as aggravated mitochondrial morphology disruption, ATP and membrane potential depletion, increased ROS overproduction, and apoptosis. Furthermore, PINK1/Parkin siRNA attenuated the protective effects of ACh against ATP loss and oxidative stress due to mitochondrial-dependent injury. Taken together, ACh promoted mitochondrial translocation of PINK1/Parkin to stimulate cytoprotective mitophagy via M2 receptor, which may provide beneficial targets in the preservation of cardiac homeostasis against H/R injury. J. Cell. Physiol. 231: 1171-1181, 2016. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background and Purpose: The activation of M3 cholinoceptors (M3 receptors) by choline reduces cardiovascular risk, but it is unclear whether these receptors can regulate ischaemia/reperfusion (I/R)-induced vascular injury. Thus, the primary goal of the present study was to explore the effects of choline on the function of mesenteric arteries following I/R, with a major focus on Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) regulation.Experimental Approach: Rats were given choline (10 mg · kg(-1), i.v.) and then the superior mesenteric artery was occluded for 60 min (ischaemia), followed by 90 min of reperfusion. The M3 receptor antagonist, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), was injected (0.12 μg · kg(-1), i.v.) 5 min prior to choline treatment. Vascular function was examined in rings of mesenteric arteries isolated after the reperfusion procedure. Vascular superoxide anion production, CaMKII and the levels of Ca(2+)-cycling proteins were also assessed.Key Results: Choline treatment attenuated I/R-induced vascular dysfunction, blocked elevations in the levels of reactive oxygen species (ROS) and decreased the up-regulated expression of oxidised CaMKII and phosphorylated CaMKII. In addition, choline reversed the abnormal expression of Ca(2+)-cycling proteins, including Na(+)Ca(2+) exchanger, inositol 1,4,5-trisphosphate receptor, sarcoplasmic reticulum Ca(2+)-ATPase and phospholamban. All of these cholinergic effects of choline were abolished by 4-DAMP.Conclusions and Implications: Our data suggest that inhibition of the ROS-mediated CaMKII pathway and modulation of Ca(2+)-cycling proteins may be novel mechanisms underlying choline-induced vascular protection. These results represent a significant addition to the understanding of the pharmacological roles of M3 receptors in the vasculature, providing a new therapeutic strategy for I/R-induced vascular injury. [ABSTRACT FROM AUTHOR]

Background: Excessive activation of matrix metalloproteinase 9 (MMP-9) has been found in several inflammatory diseases. Previous studies have shown that acetylcholine (ACh) reduced the levels of pro-inflammatory cytokines and decreased tissue damage. Therefore, this study was designed to explore the potential effects and mechanisms of ACh on MMP-9 production and cell migration in response to lipopolysaccharide (LPS) stimulation in RAW264.7 cells. Methods: MMP-9 expression and activity were induced by LPS in RAW264.7 cells, and examined by real-time PCR, western blotting and gelatin zymography, respectively. ELISA was used to determine the changes in MMP-9 secretion among the groups. Macrophage migration was evaluated using transwell migration assay. Knockdown of α7 nicotinic acetylcholine receptor (α7 nAChR) expression was performed using siRNA transfection. Results: Pre-treatment with ACh inhibited LPS-induced MMP-9 production and macrophage migration in RAW264.7 cells. These effects were abolished by the α7 nAChR antagonist methyllycaconitine (MLA) and α7 nAChR siRNA. The α7 nAChR agonist PNU282987 was found to have an effect similar to that of ACh. Moreover, ACh enhanced the expression of JAK2 and STAT3, and the JAK2 inhibitor AG490 and the STAT3 inhibitor static restored the effect of ACh. Meanwhile, ACh decreased the phosphorylation and nuclear translocation of NF-κB, and this effect was abrogated in the presence of MLA. In addition, the JAK2 and STAT3 inhibitor abolished the inhibitory effects of ACh on phosphorylation of NF-κB. Conclusions: Activation of α7 nAChR by ACh inhibited LPS-induced MMP-9 production and macrophage migration through the JAK2/STAT3 signaling pathway. These results provide novel insights into the anti-inflammatory effects and mechanisms of ACh. [ABSTRACT FROM AUTHOR]

Previous findings have shown that acetylcholine (ACh) decreased hypoxia-induced tumor necrosis factor alpha (TNF α) production, thus protected against cardiomyocyte injury. However, whether and how ACh affects TNF α-induced endoplasmic reticulum (ER) stress and cell apoptosis remain poorly defined. This study was aimed at determining the effect of ACh in H9c2 cells after TNF α stimulation. Presence of ER stress was verified using the ER stress protein markers glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP). Cell apoptosis was shown by caspase-3 activation and terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling. Exogenously administered ACh significantly decreased these TNF α-induced changes. Moreover, when the cells were exposed to nonspecific muscarinic receptor (M AChR) inhibitor atropine, methoctramine (M2 AChR inhibitor) or the epidermal growth factor receptor (EGFR) inhibitor AG1478, the cardioprotection elicited by ACh was diminished. Furthermore, the above effects were also blocked by M2 AChR or EGFR siRNA, indicating that EGFR transactivation by M2 AChR may be the major pathway responsible for the benefits of ACh. In addition, LY294002, a phosphatidylinositol-3-kinase (PI3K) inhibitor, displayed the similar trends as AG1478, suggesting that PI3K/Akt signaling may be the downstream of EGFR in ACh-elicited anti-apoptotic property. Together, these data indicate that EGFR-PI3K/Akt signaling is involved in M2 AChR-mediated ER apoptotic pathway suppression and the subsequent survival of H9c2 cardiomyocytes. We have identified a novel pathway underlying the cardioprotection afforded by ACh. J. Cell. Physiol. 230: 767-774, 2015. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Objectives: This study sought to explore molecular mechanisms underlying the adrenergic-induced QT prolongation associated with KCNQ1 mutations. Background: The most frequent type of congenital long QT syndrome is LQT1, which is caused by mutations in the gene (KCNQ1) that encodes the alpha subunit of the slow component of delayed rectifier K+ current (I Ks) channel. We identified 11 patients from 4 unrelated families that are heterozygous for KCNQ1-G269S. Most patients remained asymptomatic, and their resting corrected QT intervals ranged from normal to borderline but were prolonged significantly during exercise. Methods: Wild-type (WT) KCNQ1 and/or KCNQ1-G269S (G269S) were expressed in mammalian cells with KCNE1. I Ks-like currents were measured in control conditions or after isoproterenol or protein kinase A (PKA) stimulation using the patch-clamp technique. Additionally, experiments that incorporated the phosphomimetic KCNQ1 substitution, S27D, in WT or KCNQ1-G269S were also performed. Results: The coexpression of WT-KCNQ1 with varying amounts of G269S decreased I Ks, shifted the current-voltage I-V relation of I Ks to more positive potentials, and accelerated the I Ks deactivation rates in a concentration-dependent manner. In addition, the coexpression of G269S and WT blunted the activation of I Ks in response to isoproterenol or PKA stimulation. Lastly, a phosphomimetic substitution in G269S did not show an increased I Ks. Conclusions: G269S modestly affected I Ks in control conditions, but it almost completely blunted I Ks responsiveness in conditions that simulate or mimic PKA phosphorylation of KCNQ1. This insensitivity to PKA stimulation may explain why patients with G269S mutation showed an excessive prolongation of QT intervals on exercise. [ABSTRACT FROM AUTHOR]

Aerobic interval training ( AIT) exerts beneficial effects on cardiovascular disease. However, its cardioprotective mechanisms are not fully understood. The aim of the present study was to evaluate AIT-mediated anti-oxidation by focusing on anti-oxidase and mitochondrial biogenesis in rats after myocardial infarction ( MI)., Sprague-Dawley rats were divided into three groups: (i) a sham-operated control ( CON); (ii) an MI group; and (iii) an MI + AIT group. Myocardial microstructure and function, markers of oxidative stress, mitochondrial anti-oxidase, Phase II enzymes and mitochondrial biogenesis were assessed. In addition, levels of nuclear factor-erythroid 2-related factor (Nrf2) and phosphorylated (p-) AMP-activated protein kinase ( AMPK) were determined. The anti-oxidative gene sirtuin 3 ( SIRT3) and the prosurvival phosphatidylinositol-3 kinase ( PI3-K)-protein kinase B (Akt) signalling cascade were also evaluated., Compared with CON, there was noticeable microstructure injury, cardiac dysfunction and oxidative damage in rats after MI. In addition, decreased mitochondrial anti-oxidase content, Phase II enzyme (except heme oxygenase-1) expression and mitochondrial biogenesis were observed in the post- MI rats as well as reduced protein levels of the regulators Nrf2 and p- AMPK and suppression of SIRT3 levels and PI3-K/Akt signalling. These detrimental modifications were considerably ameliorated by AIT, as evidenced by increases in anti-oxidase, mitochondrial biogenesis, Nrf2 and AMPK phosphorylation, as well as SIRT3 upregulation and PI3-K/Akt signalling activation. Moreover, PI3-K inhibitor- LY294002 (20 mg/kg) treatment partly attenuated AIT-elicited increases in Nrf2 levels and AMPK phosphorylation., Based on these results, we conclude that AIT effectively alleviates MI-induced oxidative injury, which may be closely correlated with activation of the anti-oxidase system and mitochondrial biosynthesis. Increased SIRT3 expression and activation of PI3-K/Akt signalling may play key roles in AIT-mediated anti-oxidation. These results open up new avenues for exercise intervention therapies for MI patients. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis ( RA) is a systemic autoimmune disease mediated by T cells. Proinflammatory cytokines plays a critical role in the pathogenesis of RA. The aim of the present study was to investigate the effects of synthetic peptides ( HP- R1, HP- R2 and HP- R3), derived from the sequence of 65 kDa mycobacterial heat shock protein ( HSP), on the proliferation of and cytokine secretion by peripheral blood mononuclear cells ( PBMC) from RA patients., The PBMC were obtained from RA patients and collected by Ficoll- Hypaque density centrifugation. Peripheral blood mononuclear cells were treated with one of the three synthetic peptides for 4 h, after which time proliferation and cytokine production were determined. The effects of the three peptides on the proliferation of PBMC were analysed by the colorimetric cell proliferation ( CCK-8) assay. Cytokine production was measured in culture supernatants using specific ELISAs., None of the three peptides had any significant effect on the proliferation of PBMC from healthy controls. However, the proliferation of PBMC from RA patients was inhibited by all three peptides. The production of tumour necrosis factor-α from RA patients was significantly inhibited by all three peptides. The secretion of interferon-γ was significantly suppressed by HP- R1 and HP- R2. Unlike the other two peptides, HP- R2 increased the secretion of interleukin ( IL)-4. None of the peptides had any significant effect on the production of IL-10., The results of the present study suggest that the synthetic peptides derived from HSP65 exhibit antiproliferative and anti-inflammatory activity, and support the potential use of synthetic peptides as therapeutic drugs in RA patients. [ABSTRACT FROM AUTHOR]

Background: Acetylcholine (ACh), a neurotransmitter of vagal nerve, offers tolerance to ischemia/reperfusion injury. Given the regulation of autophagy in cardioprotection, this study was to examine the role of autophagy in ACh-elicited protection against hypoxia/reoxygenation (HR) injury. Methods: H9c2 cells were subjected to HR injury. Autophagy was determined by transmission electron microscopy, MDC staining and western blot. MTT kit, LDH and CK release, ATP content and TUNEL assay were used to evaluate cardiomyocytes injury. Atg7 and AMPK knockdown was performed with siRNA transfection. Results: Following 4, 8, 12 and 16 h reoxygenation, autophagosomes were decreased along with reduced cell viability. ACh during 4 h reoxygenation facilitated autophagy as evidence by increased autophagosomes and MDC labeling autophagic vacuoles. H9c2 cells treated with ACh also underwent a biochemical changes by increased ratio of LC3-II/LC3-I and autophagy flux (decreased p62), while muscarinic receptor antagonist atropine suppressed these effects. Induction of autophagy was correlated with enhanced cell survival and decreased apoptosis. Autophagy inhibition with chloroquine and Atg7 siRNA significantly attenuated ACh-induced cytoprotection. ACh-elicited autophagy activation could be related to increased AMPK phosphorylation and decreased mTOR phosphorylation. AMPK siRNA exhibited an elevation in mTOR phosphorylation and reduced the ratio of LC3-II/LC3-I. Importantly, AMPK knockdown desensitized H9c2 cells to ACh-mediated protection. Conclusions: These data provided first evidence that ACh-induced autophagy elicited cytoprotective effects through muscarinic receptor activated-AMPK-mTOR pathway, and suggested a novel mechanism of ACh-induced tolerance against HR injury. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background and Purpose The glycoprotein IIb/ IIIa receptor is the final common pathway of platelet aggregation, regardless of the agonist, and thus represents an ideal therapeutic target for blocking coronary thrombosis. In this study, the anti-platelet and antithrombotic actions of Z4A5, a new glycoprotein IIb/ IIIa receptor inhibitor, were evaluated in a canine model of acute unstable angina. Experimental Approach Z4A5 was given i.v. as a bolus followed by 60 min of continuous infusion at doses of 30 μg·kg−1 + 1 μg·kg−1·min−1, 30 μg·kg−1 + 5 μg·kg−1·min−1 or 300 μg·kg−1 + 5 μg·kg−1·min−1. Its antithrombotic effect was evaluated in a model of coronary thrombosis, the injured, stenosed left circumflex coronary artery, in which platelet-dependent cyclic flow reductions (CFRs) were induced by vascular compression and constriction to simulate clinical acute unstable angina. Platelet aggregation and coagulation parameters were determined in platelet-rich plasma and platelet poor plasma respectively. Key Results The Z4A5 infusion induced a dose-dependent reduction in CFR frequency, which returned to baseline levels after the termination of the infusion at low doses. At medium dose that inhibited most part of platelet aggregation, it increased tongue bleeding time marginally with no dramatic changes in haemodynamic and coagulation parameters. Furthermore, the inhibition of ADP-induced platelet aggregation and prolonged bleeding time observed during Z4A5 infusion reverted to baseline levels after the termination of the infusion. Conclusions and Implications Z4A5 is an effective antithrombotic agent for coronary artery thrombosis with a rapid-on and rapid-off pharmacological profile, and could be used as an alternative treatment of coronary artery ischaemic syndromes. [ABSTRACT FROM AUTHOR]

Mitochondrial biogenesis disorders appear to play an essential role in cardiac dysfunction. Acetylcholine as a potential pharmacologic agent exerts cardioprotective effects. However, its direct action on mitochondria biogenesis in acute cardiac damage due to ischemia/reperfusion remains unclear. The present study determined the involvement of mitochondrial biogenesis and function in the cardiopotection of acetylcholine in H9c2 cells subjected to hypoxia/reoxygenation (H/R). Our findings demonstrated that acetylcholine treatment on the beginning of reoxygenation improved cell viability in a concentration-dependent way. Consequently, acetylcholine inhibited the mitochondrial morphological abnormalities and caused a significant increase in mitochondrial density, mass, and mitochondrial DNA (mtDNA) copy number. Accordingly, acetylcholine enhanced ATP synthesis, membrane potentials, and activities of mitochondrial complexes in contrast to H/R alone. Furthermore, acetylcholine stimulated the transcriptional activation and protein expression of peroxisome proliferator-activated receptor co-activator 1 alpha (PGC-1α, the central factor for mitochondrial biogenesis) and its downstream targets including nuclear respiration factors and mitochondrial transcription factor A. In addition, acetylcholine activated phosphorylation of AMP-activated protein kinase (AMPK), which was located upstream of PGC-1α. Atropine (muscarinic receptor antagonist) abolished the favorable effects of acetylcholine on mitochondria. Knockdown of PGC-1α or AMPK by siRNA blocked acetylcholine-induced stimulating effects on mtDNA copy number and against cell injury. In conclusion, we suggested, acetylcholine as a mitochondrial nutrient, protected against the deficient mitochondrial biogenesis and function induced by H/R injury in a cellular model through muscarinic receptor-mediated, AMPK/PGC-1α-associated regulatory program, which may be of significance in elucidating a novel mechanism underlying acetylcholine-induced cardioprotection. J. Cell. Physiol. 228: 1238-1248, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Myocardial ischemia/reperfusion (I/R) induces inflammatory response that may lead to remote vascular injury. Vagal nerve elicits the cholinergic anti-inflammatory pathway by activating α7 nicotinic acetylcholine receptors (α7nAChR). Nevertheless, the role of vagal nerve-mediated anti-inflammatory pathway in the vasculature has not been studied previously. Therefore, we aimed to clarify the potential role of vagal stimulation (VNS) in regulating remote vascular injury after myocardial I/R. Adult male Sprague-Dawley rats were subjected to VNS starting 15 min prior to ischemia until the end of reperfusion. VNS not only reduced infarct size and improved cardiac function, but also ameliorated myocardial I/R-induced dysfunctional vasoconstriction and vasodilatation and degradation of endothelial structure in mesenteric arteries. VNS decreased serum and vascular levels of tumor necrosis factor-α and IL-1β. Interestingly, in vivo microdialysis studies demonstrated that VNS increased ACh concentration in the mesenteric circulation. Furthermore, VNS up-regulated expressions of muscarinic ACh receptors-3 (MAChR) and α7nAChR in mesenteric arteries. Preserved endothelial relaxations by VNS were inhibited by atropine or methyllycaconitine, indicating that functional protection was associated with M and α7nAChR activation. Finally, VNS increased STAT3 phosphorylation and inhibited NF-κB activation in mesenteric arteries, and these effects were abolished by α7nAChR shRNA treatment, indicating VNS-mediated anti-inflammatory effect mainly involved α7nAChR. These results demonstrated for the first time that VNS protected against remote vascular dysfunction, through the cholinergic anti-inflammatory pathway which is dependent on α7nAChR. Our findings represent a significant addition to the understanding of vagal nerve-mediated pathways and the potential roles they play in regulating the vasculature. [ABSTRACT FROM AUTHOR]

1. The role of celecoxib in cardiovascular events remains contentious. The aim of the present study was to investigate the effects of celecoxib in acute myocardial ischaemia (AMI) in rabbits in comparison with those of another non-steroidal anti-inflammatory drug, namely aspirin. 2. Male New Zealand white rabbits were divided into four groups: (i) a sham-operated group; (ii) an AMI group, in which the left anterior descending coronary arteries were occluded for 60 min; (iii) the celecoxib + AMI group, pretreated with 3 mg/kg celecoxib, twice a day, for 3 days before AMI induction; and (iv) the aspirin + AMI group, pretreated with 12.5 mg/kg aspirin, twice a day, for 3 days before AMI induction. Haemodynamic parameters were monitored using a multichannel physiological recorder. Serum levels of creatine kinase (CK), malondialdehyde (MDA), cyclo-oxygenase-2 (COX-2), tumour necrosis factor (TNF)-a, total nitrate/nitrite (NOx), nitric oxide synthase (NOS) and myocardial infarct size were determined. Changes in isometric tension of isolated coronary rings were recorded by a myograph system. 3. Compared with the sham group, the AMI group had lower blood pressure, higher left ventricular (LV) end-diastolic pressure, depressed maximum dP/dt of LV pressure, a larger infarct size and higher CK and MDA levels. Celecoxib, but not aspirin, pretreatment significantly ameliorated these effects of AMI. Celecoxib reversed AMI-induced increases in COX-2 levels to a similar extent as aspirin. Pretreatment with celecoxib resulted in a significant reduction in TNF-a levels and an increase in NOx and NOS levels compared with the AMI group. The dysfunctional vasoconstriction and vasodilation of coronary arteries were ameliorated by celecoxib administration. 4. In conclusion, the experimental evidence suggests that celecoxib exerts its protective effects in a COX-independent manner. [ABSTRACT FROM AUTHOR]

In most previous studies, ischaemia-reperfusion (I/R)-induced vascular injury referred to injury in the tissue or blood vessel that was directly subjected to I/R. However, less attention has been focused on remote vascular injury that might be caused by cardiac I/R. In the present study, we aimed to assess whether cardiac I/R could affect vasoconstriction and vasodilatation in mesenteric arteries from Sprague-Dawley rats. Left anterior descending coronary arteries from adult male Sprague-Dawley rats were occluded (60 min) and then reperfused (120 min). Changes in haemodynamic parameters indicated that this procedure caused evident cardiac dysfunction. In mesenteric arteries isolated from the animals, cardiac I/R significantly increased the maximal contractions in response to KCl, 5-hydroxytryptamine, phenylephrine and U46619 and decreased the maximal relaxation in response to acetylcholine, but not to sodium nitroprusside, compared with sham-operated animals. The nitric oxide synthase inhibitor l-NAME abolished differences of contractile responses to phenylephrine between sham-operated and I/R rats. The antioxidant N-acetyl- l-cysteine reversed the impairment of acetylcholine-stimulated vasodilatation induced by regional cardiac I/R. However, l-NAME caused a similar degree of inhibition of acetylcholine-stimulated relaxation in mesenteric arteries from sham-operated and I/R rats. Electron microscopy revealed that mesenteric arterial endothelial structure was degraded in the I/R group and that N-acetyl- l-cysteine treatment prevented this structural damage. In conclusion, regional cardiac I/R caused by transient occlusion and reperfusion of the left anterior descending coronary artery results in peripheral vascular endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Abstract: Urokinase (uPA) is used widely for thrombosis therapy in the clinic. However, ways to minimize its adverse effect of hemorrhage are still being studied. As a new technique for the local delivery of genes and drugs, ultrasound (US) contrast agents, microbubbles (MBs), have been mentioned. The purpose of this study is to explore a more efficacious and safer thrombolytic method by preparing three groups of self-made microbubble-loading uPA (uPA-MBs) (1 uPA-MBs, 5 uPA-MBs and 10 uPA-MBs) using freeze-drying methods and measuring their thrombolysis when combined in vitro with low-frequency US. The results showed the mean concentration, mean diameter, pH value and encapsulation efficiency of uPA of the three groups of uPA-MBs were approximately 2.08–2.82 × 108/mL, ∼3.13 μm, 6.89–6.99 and from (78.08% ± 0.57%) to (57.23% ± 0.94%), respectively. Under US exposure, the loaded uPA demonstrated bioactivity by agarose fibrin plate and in vitro thrombolysis of the three uPA-MBs also showed higher effects than in the group of those who received uPA-MBs alone, the control group or the US group. In conclusion, the physiochemical properties of these self-made uPA-MBs are suitable for intravenous administration but 1 uPA-MB and 5 uPA-MBs are better than 10 uPA-MBs. uPA-MBs combined with US can decrease the in vitro dosage of uPA for thrombolysis. [Copyright &y& Elsevier]

Abstract: Introduction: The antithrombotic effect of the glycopreotein IIb/IIIa (GP IIb/IIIa) receptor antagonist Z4A5, exert alone or combination with heparin, and/or aspirin, was examined in a rabbit arteriovenous shunt thrombosis model. Materials and Methods: Thrombosis was induced by the insertion of a silk thread (thrombogenic substrate) into an extracorporeal shunt. Before and after drug administration (0, 5, and 15min), ex vivo adenosine diphosphate (ADP)-induced platelet aggregation and coagulation parameters (prothrombin time (PT) and activated partial thromboplastin time (APTT)) were determined in platelet-rich plasma (PRP) and platelet poor-plasma (PPP), respectively. Results: Our data demonstrated that, compared to the control, Z4A5 decreased the thrombus weight (31-65%) in a dose-dependent manner and inhibited ADP-induced platelet aggregation (47-98%) 5min after Z4A5 administration (25–100mg/kg). However, PT and APTT remained stable, even at the highest dose (100mg/kg). Heparin (100U/kg) and aspirin (15mg/kg) also significantly reduced thrombus mass, but this effect was accompanied by an increase of APTT by heparin. Furthermore, the combination of heparin (100U/kg) and a low dose of Z4A5 (25mg/kg) failed to produce an additional benefit beyond that provided by heparin or Z4A5 alone, whereas Z4A5 (25mg/kg) plus aspirin (15mg/kg) potentiated the antithrombotic effects of both compounds without further increasing the values of coagulation. Conclusions: Our results indicate that Z4A5 is an effective antithrombotic agent with no significant effects on values of coagulation. Furthermore, Z4A5 can potentiate these antithrombotic effects when prescribed with aspirin. [Copyright &y& Elsevier]

1. Acute myocardial infarction (AMI) often activates the sympathetic system and inhibits the vagal system. Long-term vagal nerve stimulation (VNS) exerts several beneficial effects on the ischaemic heart, including an anti-inflammatory effect. The aim of the present study was to investigate whether short-term VNS during AMI could inhibit tumour necrosis factor (TNF)-α expression and the effect of TNF receptor (TNFR), key components in inflammatory responses to AMI, in a rodent model. 2. Adult male Sprague-Dawley rats were divided into four groups, namely a control (C), VNS (S), AMI (M) and an AMI group subjected to prior VNS (MS). In the S and MS groups, the right vagus nerve was stimulated electrically for 4 h; in the M and MS groups, AMI was induced by occlusion of the left anterior descending coronary artery. Haemodynamic data were monitored continuously using a multichannel physiological recorder. Lactate dehydrogenase (LDH) leakage, creatine kinase (CK) leakage and infarct size were determined. The expression of TNF-α and its receptors were analysed by reverse transcription-polymerase chain reaction, western blotting and ELISA. 3. Compared with the control group, rats in the M group had low blood pressure, high left ventricular (LV) end-diastolic pressure, a depressed maximum dP/dt of LV pressure, higher LDH and CK leakage, a larger infarct size, increased TNF-α levels and an increased TNFR1/TNFR2 ratio. However, these presumably harmful effects of AMI were all significantly ameliorated by VNS during AMI (MS group). 4. In conclusion, VNS can rectify ischaemia-induced cardiac dysfunction partly via inhibition of a TNF-α-mediated signalling pathway. [ABSTRACT FROM AUTHOR]

Recent findings have reported that up-regulation of tumor necrosis factor-alpha (TNF-α) induced by myocardial hypoxia aggravates cardiomyocyte injury. Acetylcholine (ACh), the principle vagal neurotransmitter, protects cardiomyocytes against hypoxia by inhibiting apoptosis. However, it is still unclear whether ACh regulates TNF-α production in cardiomyocytes after hypoxia. The concentration of extracellular TNF-α was increased in a time-dependent manner during hypoxia. Furthermore, ACh treatment also inhibited hypoxia-induced TNF-α mRNA and protein expression, caspase-3 activation, cell death and the production of reactive oxygen species (ROS) in cardiomyocytes. ACh treatment prevented the hypoxia-induced increase in p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) phosphorylation. Co-treatment with atropine, a non-selective muscarinic acetylcholine receptor antagonist, or methoctramine, a selective type-2 muscarinic acetylcholine (M) receptor antagonist, abrogated the effects of ACh treatment in hypoxic cardiomyocytes. Co-treatment with hexamethonium, a non-selective nicotinic receptor antagonist, and methyllycaconitine, a selective alpha7-nicotinic acetylcholine receptor antagonist, had no effect on ACh-treated hypoxic cardiomyocytes. In conclusion, these results demonstrate that ACh activates the M receptor, leading to regulation of MAPKs phosphorylation and, subsequently, down-regulation of TNF-α production. We have identified a novel pathway by which ACh mediates cardioprotection against hypoxic injury in cardiomyocytes. J. Cell. Physiol. 226: 1052-1059, 2011. © 2010 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

1. Endothelial dysfunction plays a critical role in the development and progression or pathogenesis of hypertension. Amlodipine, a calcium channel blocker, is an effective antihypertensive agent. We investigated the effects of amlodipine on endothelial dysfunction in mesenteric arteries from spontaneously hypertensive rats (SHR). 2. Eight-week-old SHR were treated with amlodipine (10 mg/kg per day) for 8 weeks. Control SHR and Wistar-Kyoto (WKY) rats were treated with saline. Systolic blood pressure (SBP) was measured by the tail-cuff method. Isometric tension changes of isolated mesenteric arterial rings were recorded continuously by a myograph system. Serum contents of malondialdehyde (MDA) and total nitrate/nitrite (NO) were determined. Vascular superoxide anion production was analysed with dihydroethidium (DHE) fluorescence. 3. The contractile responses to KCl and phenylephrine were greater in untreated SHR than in WKY. Acetylcholine (ACh)-induced relaxation was significantly impaired in untreated SHR. Amlodipine treatment reduced the contractions and improved relaxation to ACh. In WKY, relaxation to ACh was inhibited by N-nitro--arginine methyl ester (-NAME) and not changed by ascorbic acid. In untreated SHR, the response to ACh was unaffected by -NAME, whereas it was improved by ascorbic acid. Amlodipine restored the inhibitory effect of -NAME on ACh-induced relaxation, but ascorbic acid no longer exerted its facilitating effect. Amlodipine prevented the rise in SBP and ameliorated abnormalities in serum MDA and NO in untreated SHR. DHE assay showed an increased intravascular superoxide generation in untreated SHR, which was abrogated by amlodipine. 4. Treatment of SHR with amlodipine resulted in amelioration of endothelial dysfunction by anti-oxidant activity and improvement in NO availability. [ABSTRACT FROM AUTHOR]

1. Cardiac acetylcholine receptors are involved in the negative inotropic effect of the vagus and the protection of the stimulated vagal nerve against myocardial ischaemic injury. Acetylcholine receptors consist of five types of muscarinic acetylcholine receptors (M AChR) and several nicotinic acetylcholine receptors (nAChR). Notably, ischaemic heart disease is accompanied by substantial withdrawal of vagal activity. However, it is not entirely clear what the changes of M AChR and α7-nAChR expression are after cardiac ischaemia/reperfusion (I/R) injury. 2. Cardiac functions were continuously recorded in Langendorff mode during 30 min of ischaemia and 60 min of reperfusion. Lactate dehydrogenase (LDH) leakage was measured. M AChRs and α7-nAChR expression were measured by reverse transcription polymerase chain reaction and western blot. 3. In hearts exposed to I/R injury, left ventricular development pressure, heart rate and ±d P/d t decreased significantly compared with the controls. LDH leakage increased with respect to the controls during reperfusion. 4. In normal hearts, expression of M AChR in the left ventricle were lower than in atria and the right ventricle, whereas expression of α7-nAChR was dramatically higher in the left ventricle and right ventricle than the atria. After reperfusion, the mRNA and protein expression of M AChR increased notably in the left and right ventricle, and α7-nAChR was enhanced significantly in the left ventricle. M AChR mRNA expression reduced notably after ischaemia and recovered to the control level after reperfusion in the atria, but the protein level did not change. 5. In conclusion, the increase in M AChR and α7-nAChR after reperfusion might be the compensatory response to myocardial I/R injury, providing new information for treatment of myocardial I/R injury. [ABSTRACT FROM AUTHOR]

1. Obesity is a significant challenge in terms of public health and preventive medicine. Inhibition of pre-adipocyte proliferation is believed to be important in the proposed anti-obesity mechanism. The aim of the present study was to examine the interplay between Cl− channels and their possible involvement in the proliferation of undifferentiated human pre-adipocytes. 2. Pre-adipocytes were isolated from human abdominal subcutaneous adipose tissue. Membrane ion currents were recorded using the whole-cell patch-clamp technique. Expression of the Cl− channel ClC-3 gene and protein was determined by reverse transcription–polymerase chain reaction (RT-PCR) and western blot, respectively. Cell proliferation was evaluated using the [3H]-thymidine incorporation assay. 3. Electrophysiological recordings revealed a volume-sensitive Cl− current ( ICl.vol) expressed in pre-adipocytes that was activated under hyposmotic conditions (external osmolarity decreased by 80%) and inhibited by the Cl− channel blocker tamoxifen. 4. Expression of the ClC-3 channel gene and protein was confirmed by RT-PCR and western blot analysis. Blocking ICl.vol with tamoxifen supressed the proliferation of pre-adipocytes in a concentration-dependent manner. 5. Collectively, the results of the present study indicate that the volume-sensitive Cl− channel participates in regulation of the proliferation of human subcutaneous pre-adipocytes. [ABSTRACT FROM AUTHOR]

Ion channels participate in regulation of cell proliferation. However, though preadipocyte (the progenitor of fat cell) is a type of highly proliferating cells, ion channel expression and their role in proliferation is not understood in human preadipocytes. The present study was designed to characterize ion channels using whole-cell patch clamp technique, RT-PCR, and Western blotting. It was found that a 4-aminopyridine- (4-AP) sensitive transient outward K+ current (Ito) was present in a small population of (32.0%) cells, and an outward “noisy” big conductance Ca2+-activated K+ current (IKCa) was present in most (92.7%) preadipocytes. The noisy current was inhibited by the big conductance IKCa channel blocker paxilline (1 µM), and enhanced by the Ca2+ ionophore A23187 (5 µM) and the big conductance IKCa channel activator NS1619 (10 µM). RT-PCR and Western blot revealed the molecular identities (i.e., KCa1.1 and Kv4.2) of the functional ionic currents IKCa and Ito. Blockade of IKCa or Ito with paxilline or 4-AP reduced preadipocyte proliferation, and similar results were obtained with specific siRNAs targeting to KCa1.1 and Kv4.2. Flow cytometric analysis showed ion channel blockade or knockdown of KCa1.1 or Kv4.2 with specific siRNA increased the cell number of G0/G1 phase. The present study demonstrates for the first time that two types of functional ion channel currents, Ito and big conductance IKCa, are present in human preadipocytes and that these two types of ion channels participate in regulating proliferation of human preadipocytes. J. Cell. Physiol. 218: 427–435, 2009. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Cardiac remodeling is characterized by overactivity of the renin-angiotensin system (RAS) and withdrawal of vagal activity. We hypothesized that improving vagal activity could attenuate cardiac fibrosis induced by angiotensin II (Ang II) in vivo and in vitro. Rats were subjected to abdominal aorta constriction (AAC) with or without pyridostigmine (PYR) (31 mg/kg/d). After 8 weeks, PYR significantly decreased Ang II level, AT1 protein expression, and collagen deposition in cardiac tissue and improved heart rate variability, baroreflex sensitivity and cardiac function, which were abolished by atropine. In vitro, treatment of cardiac fibroblasts (CFs) with Ang II (10−7 M) increased cell proliferation, migration, transformation, and secretory properties, which were significantly diminished by acetylcholine (ACh, 10−6 M). Subsequently, Ang II significantly increased collagen type I expression as well as metalloproteinase (MMP)-2 expression and activity. Transforming growth factor (TGF)-β1 expression and Smad3 phosphorylation presented a similar trend. Notably, the knockdown of the acetylcholine M2 receptor by siRNA could abolish ACh anti-fibrotic action. These data implicated cholinesterase inhibitor can increase vagal activity and reduce local Ang II level, and ACh inhibit Ang II pro-fibrotic effects. Our findings suggested that the parasympathetic nervous system can serve as a promising target for cardiac remodeling treatment. [ABSTRACT FROM AUTHOR]

Background: Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease mediated by T cells. The aim of the present study was to investigate the therapeutic efficacy of synthetic peptides (HP-R1, HP-R2 and HP-R3), derived from the sequence of 65-kD mycobacterial heat shock protein (HSP), in the treatment of RA using adjuvant-induced arthritis (AA) animal model.Methods: AA was induced by a single intradermal injection Freund's complete adjuvant in male Lewis rats. At the first clinical sign of disease, rats were administered nasally by micropipette of peptides or phosphate buffer saline (PBS). Disease progression was monitored by measurement of body weight, arthritis score and paw swelling. The changes of histopathology were assessed by hematoxylin eosin staining. The serum levels of tumor necrosis factor (TNF) - alpha and interleukin (IL)-4 were measured by enzyme-linked immunosorbent assay (ELISA).Results: The peptides efficiently inhibited the footpad swelling and arthritic symptoms in AA rats. The synthetic peptides displayed significantly less inflammatory cellular infiltration and synovium hyperplasia than model controls. This effect was associated with a suppression of pro-inflammatory cytokine TNF-alpha production and an increase of anti-inflammatory cytokine IL-4 production after peptides treatment.Conclusions: These results suggest that the synthetic peptides derived from HSP65 induce highly effective protection against AA, which is mediated in part by down-regulation of inflammatory cytokines, and support the view that the synthetic peptides is a potential therapy for RA that may help to diminish both joint inflammation and destruction. [ABSTRACT FROM AUTHOR]

Recent studies have suggested that mitochondria may play important roles in the Ca2+ homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca2+ flux can regulate the generation of Ca2+ waves (CaWs) and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca2+ (Cai2+) was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR) Ca2+ release and CaWs were induced in the presence of high (4 mM) external Ca2+ (Cao2+). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) reversibly raised basal Cai2+ levels even after depletion of SR Ca2+ in the absence of Cao2+ , suggesting Ca2+ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψm) and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψm) or Ru360 (a mitochondrial Ca2+ uniporter inhibitor), but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca2+ uniporter activator kaempferol. Our results suggest that mitochondrial Ca2+ release and uptake exquisitely control the local Ca2+ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis. [ABSTRACT FROM AUTHOR]

1. The physiological activities of the mammalian heart are regulated by the autonomic nervous system. An imbalanced autonomic nervous system with increased sympathetic tone and reduced vagal tone has been implicated in cardiovascular diseases. 2. Experimental and clinical reports have demonstrated that vagal nerve activation is able to improve outcomes for multiple cardiovascular diseases, such as ischaemic heart disease, heart failure, arrhythmia and hypertension. 3. In this paper, we mainly focus on the potential cardioprotective mechanisms of vagal nerve activation. Based on the knowledge gained from our experiments and other published reports, vagal activation results in cardioprotection is not only associated with heart rate, anti-adrenergic effect but also related to anti-inflammatory activity, regulation of cellular redox states and regulation of mitochondrial targets. 4. In conclusion, vagal nerve activation may be a promising new therapeutic approach for the treatment of cardiovascular diseases. [ABSTRACT FROM AUTHOR]