Nen Chung Chang

Antiarrhythmic effect of lithium in rats after myocardial infarction by activation of Nrf2/HO-1 signaling

Glycogen synthase kinase-3 (GSK-3) signaling has been shown to play a role in the regulation of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of antioxidant genes, including heme oxygenase-1 (HO-1). We assessed whether lithium, a GSK-3 inhibitor, attenuates cardiac sympathetic reinnervation after myocardial infarction, a status of high reactive oxygen species (ROS), by attenuating nerve growth factor (NGF) expression and whether Nrf2/HO-1 signaling is involved in the protection. Twenty-four hours after ligation of the left anterior descending artery, male Wistar rats were treated for 4 weeks. The postinfarction period was associated with increased oxidative-nitrosative stress, as measured by myocardial superoxide, nitrotyrosine, and dihydroethidium fluorescent staining. In concert, myocardial norepinephrine levels and immunohistochemical analysis of sympathetic nerve revealed a significant increase in innervation in vehicle-treated rats compared with sham-operated rats. Arrhythmic scores during programmed stimulation in the vehicle-treated rats were significantly higher than those in sham. This was paralleled by a significant upregulation of NGF protein and mRNA in the vehicle-treated rats, which was reduced after administration of LiCl. LiCl stimulated the nuclear translocation of Nrf2 and the transactivation of the Nrf2 target gene HO-1. Inhibition of phosphoinositide 3-kinase by wortmannin reduced the increase in Nrf2 nucleus translocation and HO-1 expression compared with lithium alone. In addition, the lithium-attenuated NGF levels were reversed in the presence of the Nrf2 inhibitor trigonelline, HO-1 inhibitor SnPP, and peroxynitrite generator SIN-1, indicating the role of Nrf2/HO-1/ROS. In conclusion, lithium protects against ventricular arrhythmias by attenuating NGF-induced sympathetic innervation via antioxidant activation of the Nrf2/HO-1 axis.

MK-0626, A Dipeptidyl Peptidase-4 Inhibitor, Improves Neovascularization by Increasing Both the Number of Circulating Endothelial Progenitor Cells and Endothelial Nitric Oxide Synthetase Expression

Current treatment modalities for critical limb ischemia (CLI) are of limited benefit; therefore, advances in therapeutic vasculogenesis may open an important new avenue for the treatment of CLI. This study examines the therapeutic potential of the DPP-4 inhibitor MK-0626 as a regulator of vasculogenesis in vivo. MK-0626 was administered daily to C57CL/B6 mice and eGFP-labeled bone marrow-transplanted ICR mice that had undergone hind limb ischemia surgery. Laser Doppler imaging and flow cytometry were used to evaluate the degree of neo-vasculogenesis and the number of circulating endothelial progenitor cells (EPCs), respectively. Cell surface markers of EPCs and the level of endothelial nitric oxide synthase (eNOS) were studied in the vessels. Mice that received MK-0626 had an elevated level of glucagon- like peptide-1 (GLP-1) and a decreased level of dipeptidyl peptidase-4 (DPP-4) in their plasma, in addition to an ischemia-induced increase in the level of stromal cell-derived factor-1 (SDF-1). In C57CL/B6 mice, blood flow in the ischemic limb was significantly improved by treatment with MK-0626. The number of circulating EPCs and both the synthesis and phosphorylation of eNOS were also increased in ischemic thigh muscle after MK-0626 treatment. In contrast, similar effects of MK-0626 were not observed in B6.129P2-Nos3(tm1Unc)/J mice (an eNOS knockout mouse). Additionally, MK-0626 treatment promoted the mobilization and homing of EPCs to ischemic tissue in eGFP transgenic mouse bone marrow-transplanted ICR mice. We conclude that both the number of circulating EPCs and neo-vasculogenesis are increased in response to DPP-4 inhibitor treatment and that this occurs via an eNOS-dependent mechanism. The results highlight the therapeutic vasculogenesis potential of the DPP-4 inhibitor MK-0626 using a hind limb ischemia mouse model.

Dapagliflozin, a selective SGLT2 Inhibitor, attenuated cardiac fibrosis by regulating the macrophage polarization via STAT3 signaling in infarcted rat hearts

ABSTRACT During myocardial infarction, infiltrated macrophages have pivotal roles in cardiac remodeling and delayed M1 toward M2 macrophage phenotype transition is considered one of the major factors for adverse ventricular remodeling. We investigated whether dapagliflozin, a sodium‐glucose cotransporter 2 (SGLT2) inhibitor, attenuates cardiac fibrosis via regulating macrophage phenotype by a reactive oxygen and nitrogen species (RONS)/STAT3‐dependent pathway in postinfarcted rats. Normoglycemic male Wistar rats were subjected to coronary ligation and then randomized to either saline, dapagliflozin (a specific SGLT2 inhibitor), phlorizin (a nonspecific SGLT1/2 inhibitor), dapagliflozin + S3I‐201 (a STAT3 inhibitor), or phlorizin + S3I‐201 for 4 weeks. There were similar infarct sizes among the infarcted groups at the acute and chronic stages of infarction. At day 3 after infarction, post‐infarction was associated with increased levels of superoxide and nitrotyrosine, which can be inhibited by administering either dapagliflozin or phlorizin. SGLT2 inhibitors significantly increased STAT3 activity, STAT3 nuclear translocation, myocardial IL‐10 levels and the percentage of M2 macrophage infiltration. At day 28 after infarction, SGLT2 inhibitors were associated with attenuated myofibroblast infiltration and cardiac fibrosis. Although phlorizin decreased myofibroblast infiltration, the effect of dapagliflozin on attenuated myofibroblast infiltration was significantly higher than phlorizin. The effects of SGLT2 inhibitors on cardiac fibrosis were nullified by adding S3I‐201. Furthermore, the effects of dapagliflozin on STAT3 activity and myocardial IL‐10 levels can be reversed by 3‐morpholinosydnonimine, a peroxynitrite generator. Taken together, these observations provide a novel mechanism of SGLT2 inhibitors‐mediated M2 polarization through a RONS‐dependent STAT3‐mediated pathway and selective SGLT2 inhibitors are more effective in attenuating myofibroblast infiltration during postinfarction remodeling. HIGHLIGHTSInfiltrated macrophages play a role for adverse postinfarction remodeling.It remained unclear whether dapagliflozin modulates M2 macrophage.Post‐infarction increased M1 macrophages, which were inhibited by dapagliflozin.The effects of dapagliflozin on M2 polarization were nullified by S3I‐201.Thus, dapagliflozin mediated M2 polarization through a STAT3‐related pathway.

Porphyromonas gingivalis GroEL induces osteoclastogenesis of periodontal ligament cells and enhances alveolar bone resorption in rats.

Porphyromonas gingivalis is a major periodontal pathogen that contains a variety of virulence factors. The antibody titer to P. gingivalis GroEL, a homologue of HSP60, is significantly higher in periodontitis patients than in healthy control subjects, suggesting that P. gingivalis GroEL is a potential stimulator of periodontal disease. However, the specific role of GroEL in periodontal disease remains unclear. Here, we investigated the effect of P. gingivalis GroEL on human periodontal ligament (PDL) cells in vitro, as well as its effect on alveolar bone resorption in rats in vivo. First, we found that stimulation of PDL cells with recombinant GroEL increased the secretion of the bone resorption-associated cytokines interleukin (IL)-6 and IL-8, potentially via NF-κB activation. Furthermore, GroEL could effectively stimulate PDL cell migration, possibly through activation of integrin α1 and α2 mRNA expression as well as cytoskeletal reorganization. Additionally, GroEL may be involved in osteoclastogenesis via receptor activator of nuclear factor κ-B ligand (RANKL) activation and alkaline phosphatase (ALP) mRNA inhibition in PDL cells. Finally, we inoculated GroEL into rat gingiva, and the results of microcomputed tomography (micro-CT) and histomorphometric assays indicated that the administration of GroEL significantly increased inflammation and bone loss. In conclusion, P. gingivalis GroEL may act as a potent virulence factor, contributing to osteoclastogenesis of PDL cells and resulting in periodontal disease with alveolar bone resorption.

The role of calpain-myosin 9-Rab7b pathway in mediating the expression of toll-like receptor 4 in platelets: A novel mechanism involved in α-granules trafficking

Toll-like receptors (TLRs) plays a critical role in innate immunity. In 2004, Aslam R. and Shiraki R. first determined that murine and human platelets express functional TLRs. Additionally, Andonegui G. demonstrated that platelets express TLR4, which contributes to thrombocytopenia. However, the underlying mechanisms of TLR4 expression by platelets have been rarely explored until now. The aim of this study was to identify the mechanism of TLR4 expression underlying thrombin treatment. The human washed platelets were used in this study. According to flowcytometry and western blot analysis, the surface levels of TLR4 were significantly enhanced in thrombin-activated human platelets and decreased by TMB-8, calpeptin, and U73122, but not Y27632 (a Rho-associated protein kinase ROCK inhibitor) indicating that thrombin-mediated TLR4 expression was modulated by PAR/PLC pathway, calcium and calpain. Co-immunoprecipitation (co-IP) assay demonstrated that the interaction between TLR4 and myosin-9 (a substrate of calpain) was regulated by calpain; cleavage of myosin-9 enhanced TLR4 expression in thrombin treated platelets. Transmission electron microscope data indicated that human platelets used α-granules to control TLR4 expression; the co-IP experiment suggested that myosin-9 did not coordinate with Rab7b to negatively regulate TLR4 trafficking in thrombin treated platelets. In summary, phospholipase Cγ-calpain-myosin 9-Rab7b axis was responsible for the mechanism underlying the regulation of TLR4 containing α-granules trafficking in thrombin-stimulated platelets, which was involved in coagulation.

Both GPER and membrane oestrogen receptor-α activation protect ventricular remodelling in 17β oestradiol-treated ovariectomized infarcted rats.

Clinical and experimental studies have established that gender is a factor in the development of ventricular hypertrophy. We investigated whether the attenuated hypertrophic effect of oestradiol was via activation of phosphatidylinositol 3‐kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS) through non‐genomic action. Twenty‐four hours after coronary ligation, female Wistar rats were randomized into control, subcutaneous oestradiol treatment or a G‐protein coupled oestrogen receptor (GPER) agonist, G‐1 and treated for 4 weeks starting from 2 weeks after bilateral ovariectomy. Ventricular hypertrophy assessed by cardiomyocyte size after infarction was similarly attenuated by oestradiol or G‐1 in infarcted rats. The phosphorylation of Akt and eNOS was significantly decreased in infarcted rats and restored by oestradiol and G‐1, implying the GPER pathway in this process. Oestradiol‐induced Akt phosphorylation was not abrogated by G‐15 (a GPER blocker). Akt activation was not inhibited by actinomycin D. When a membrane‐impermeable oestrogen‐albumin construct was applied, similar responses in terms of eNOS activation to those of oestradiol were achieved. Furthermore, PPT, an ERα receptor agonist, activated the phosphorylation of Akt and eNOS. Thus, membrane ERα receptor played a role in mediating the phosphorylation of Akt and eNOS. The specific PI3K inhibitor, LY290042, completely abolished Akt activation and eNOS phosphorylation in infarcted hearts treated with either oestradiol or oestradiol + G‐15. These data support the conclusions that oestradiol improves ventricular remodelling by both GPER‐ and membrane‐bound ERα‐dependent mechanisms that converge into the PI3K/Akt/eNOS pathway, unveiling a novel mechanism by which oestradiol regulates pathological cardiomyocyte growth after infarction.

Both PKA and Epac Pathways Mediate N-Acetylcysteine- Induced Connexin43 Preservation in Rats with Myocardial Infarction

Cardiac remodeling was shown to be associated with reduced gap junction expression after myocardial infarction. A reduction in gap junctional proteins between myocytes may trigger ventricular arrhythmia. Therefore, we investigated whether N-acetylcysteine exerted antiarrhythmic effect by preserving connexin43 expression in postinfarcted rats, focusing on cAMP downstream molecules such as protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). Male Wistar rats after ligating coronary artery were randomized to either vehicle, or N-acetylcysteine for 4 weeks starting 24 hours after operation. Infarct size was similar between two groups. Compared with vehicle, cAMP levels were increased by N-acetylcysteine treatment after infarction. Myocardial connexin43 expression was significantly decreased in vehicle-treated infarcted rats compared with sham operated rats. Attenuated connexin43 expression and function were blunted after administering N-acetylcysteine, assessed by immunofluorescent analysis, dye coupling, Western blotting, and real-time quantitative RT-PCR of connexin43. Arrhythmic scores during programmed stimulation in the N-acetylcysteine-treated rats were significantly lower than those treated with vehicle. In an ex vivo study, enhanced connexin43 levels afforded by N-acetylcysteine were partially blocked by either H-89 (a PKA inhibitor) or brefeldin A (an Epac-signaling inhibitor) and completely blocked when H-89 and brefeldin A were given in combination. Addition of either the PKA specific activator N6Bz or Epac specific activator 8-CPT did not have additional increased connexin43 levels compared with rats treated with lithium chloride alone. These findings suggest that N-acetylcysteine protects ventricular arrhythmias by attenuating reduced connexin43 expression and function via both PKA- and Epac-dependent pathways, which converge through the inactivation of glycogen synthase kinase-3β.

Sitagliptin attenuates sympathetic innervation via modulating reactive oxygen species and interstitial adenosine in infarcted rat hearts

We investigated whether sitagliptin, a dipeptidyl peptidase‐4 (DPP‐4) inhibitor, attenuates arrhythmias through inhibiting nerve growth factor (NGF) expression in post‐infarcted normoglycemic rats, focusing on adenosine and reactive oxygen species production. DPP‐4 bound adenosine deaminase has been shown to catalyse extracellular adenosine to inosine. DPP‐4 inhibitors increased adenosine levels by inhibiting the complex formation. Normoglycemic male Wistar rats were subjected to coronary ligation and then randomized to either saline or sitagliptin in in vivo and ex vivo studies. Post‐infarction was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle‐treated infarcted rats compared with sham. Compared with vehicle, infarcted rats treated with sitagliptin significantly increased interstitial adenosine levels and attenuated oxidative stress. Sympathetic hyperinnervation was blunted after administering sitagliptin, as assessed by immunofluorescent analysis and western blotting and real‐time quantitative RT‐PCR of NGF. Arrhythmic scores in the sitagliptin‐treated infarcted rats were significantly lower than those in vehicle. Ex vivo studies showed a similar effect of erythro‐9‐(2‐hydroxy‐3‐nonyl) adenine (an adenosine deaminase inhibitor) to sitagliptin on attenuated levels of superoxide and NGF. Furthermore, the beneficial effects of sitagliptin on superoxide anion production and NGF levels can be reversed by 8‐cyclopentyl‐1,3‐dipropulxanthine (adenosine A1 receptor antagonist) and exogenous hypoxanthine. Sitagliptin protects ventricular arrhythmias by attenuating sympathetic innervation via adenosine A1 receptor and xanthine oxidase‐dependent pathways, which converge through the attenuated formation of superoxide in the non‐diabetic infarcted rats.

Endothelin receptor blockade ameliorates renal injury by inhibition of RhoA/Rho-kinase signalling in deoxycorticosterone acetate-salt hypertensive rats.

Purpose of review: Excessive production of fibrosis is a feature of hypertension-induced renal injury. Activation of RhoA/Rho-kinase (ROCK) axis has been shown in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. We assessed whether selective endothelin receptor blockers can attenuate renal fibrosis by inhibiting RhoA/ROCK axis in DOCA-salt rats. Methods: At 4 weeks after the start of DOCA-salt treatment and uninephrectomization, male Wistar rats were randomized into three groups for 4 weeks: vehicle, ABT-627 (endothelin-A receptor inhibitor) and A192621 (endothelin-B receptor inhibitor). Results: DOCA-salt was characterized by increased blood pressure, decreased renal function, increased proteinuria, increased glomerulosclerosis and tubulointerstitial fibrosis with myofibroblast accumulation, increased renal endothelin-1 levels and RhoA activity along with increased expression of connective tissue growth factor at both mRNA and protein levels as compared with uninephrectomized control male Wistar rats. Treatment with a selective mineralocorticoid receptor antagonist, eplerenone, ameliorated proteinuria. Impaired renal function and histological changes were overcome by treatment with ABT-627, but not with A192621. The beneficial effects of bosentan, a nonspecific endothelin receptor blocker, on proteinuria, RhoA activity, and connective tissue growth factor levels were similar to ABT-627. Furthermore, in an isolated perfuse kidney, a RhoA inhibitor, C3 exoenzyme, and two ROCK inhibitors, fasudil and Y-27632, significantly attenuated connective tissue growth factor levels. Conclusions: These results indicate that DOCA-salt elevates renal endothelin-1 levels and RhoA activity via activation of mineralocorticoid receptor, resulting in renal fibrosis and proteinuria. Endothelin-A receptor blockade can attenuate DOCA-salt-induced renal fibrosis probably through the inhibition of RhoA/ROCK activity and connective tissue growth factor expression.

TNF-α-decreased thrombomodulin expression in monocytes is inhibited by propofol through regulation of tristetraprolin and human antigen R activities.

Thrombomodulin (TM) is expressed on the surface of monocytes and is a key regulator of actual immune capacity. Propofol is an anesthetic agent that exerts anti-inflammatory effects. The objective of this study was to determine whether propofol could modulate TM in TNF-&agr;-stimulated monocytes. THP-1 cells and male New Zealand rabbits were used in this study. The results showed that TNF-&agr; decreases the TM expression by mediating posttranscriptional modification, and this inhibition may be repressed by treatment with propofol. Immunofluorescence, immunoprecipitation, and pull-down assays were used to demonstrate that Rac1-dependent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, Cdc42, and p38 mitogen-activated protein kinase activation, as well as tristetraprolin (TTP) expression, all contributed to the downregulation of TM in TNF-&agr;-treated cells. Propofol reversed the effects of TNF-&agr; on TM downregulation. Propofol mediated the expression of intracellular TTP and the distribution of cytosolic human antigen R (HuR) and changed their interactions with the 3&vprime;-untranslated region of TM mRNA regulating by Cdc42 and Rac1. In addition, the animal study showed that propofol regulates TM, TTP, and HuR expression on monocytes in TNF-&agr;-treated rabbits. In conclusion, the inhibition of TM expression in TNF-&agr;-treated monocytes was mediated by the activation of NADPH oxidase and the expression of TTP. Propofol may inhibit the downregulation of TM by mediating NADPH oxidase and TTP inactivation and through the activation of HuR in vitro and in vivo. Utilizing TTP and HuR to control TM expression may be a promising approach for controlling systemic inflammation, and propofol may possess potential implications for the clinical immunity of monocytes after anesthesia or surgery.