Trimetazidine Preconditioning Potentiates the Effect of Mesenchymal Stem Cells Secretome on the Preservation of Rat Pancreatic Islet Survival and Function In Vitro.

Islet transplantation offers improved glycemic control in individuals with type 1 diabetes mellitus. However, in vitro islet culture is associated with islet apoptosis and eventually will lose their functionality prior to transplantation. In this study, we examined the effects of mesenchymal stem cells (MSCs) secretome preconditioned with diazoxide (DZ) and trimetazidine (TMZ) on rat islet cells during pre-transplant culture. With and without preconditioned hAD-MSCs' concentrated conditioned media (CCM) were added to the culture medium containing rat islets every 12 h for 24 and 48 h, after testing for selected cytokine concentrations (interleukin (IL)-4, IL-6, IL-13). Insulin content, glucose-stimulated insulin secretion, islet cell apoptosis, and mRNA expression of pro-apoptotic (BAX, BAK-1, and PUMA) and anti-apoptotic factors (BCL-2, BCL-xL, and XIAP) in rat islets were assessed after 24 and 48 h of culture. The protein level of IL-6 and IL-4 was significantly higher in TMZ-MSC-CM compared to MSC-non-CM. In rat isolated islets, normalized secreted insulin in the presence of 16.7 mM glucose was significantly higher in treated islet groups compared to control islets at both 24 and 48 h cultivation. Also, the percentage of apoptotic islet cells TMZ-MSC-CCM-treated islets was significantly lower compared to MSC-CM and MSC-CCM-treated islets in both 24 and 48 h cultivation. Consistent with the number of apoptotic cells, after 24 h culture, the expression of BCL-2 and BCL-xL genes in the control islets was lower than all treatment islet groups and in 48 h was lower than only TMZ-MSC-CM-treated islets. Also, the expression of the XIAP gene in control islets was significantly lower compared to the TMZ-MSC-CCM-treated islets at both at 24 and 48 h. In addition, mRNA level of the BAX gene in TMZ-MSC-CCM-treated islets was significantly lower compared to other groups at 48 h. Our findings revealed that TMZ proved to be more effective than DZ and could enhance the potential of hAD-MSCs-CM to improve the function and viability of islets prior to transplantation.

Fatty acid oxidation fuels agonist-induced platelet activation and thrombus formation: Targeting ß-oxidation of fatty acids as an effective anti-platelet strategy.

Platelet mitochondria possess remarkable plasticity for oxidation of energy substrates, where metabolic dependency on glucose or fatty acids is higher than glutamine. Since platelets metabolize nearly the entire pool of glucose to lactate rather than fluxing through mitochondrial tricarboxylic acid cycle, we posit that majority of mitochondrial ATP, which is essential for platelet granule secretion and thrombus formation, is sourced from oxidation of fatty acids. We performed a comprehensive analysis of bioenergetics and function of stimulated platelets in the presence of etomoxir, trimetazidine and oxfenicine, three pharmacologically distinct inhibitors of ß-oxidation. Each of them significantly impaired oxidative phosphorylation in unstimulated as well as thrombin-stimulated platelets leading to a small but consistent drop in ATP level in activated cells due to a lack of compensation from glycolytic ATP. Trimetazidine and oxfenicine attenuated platelet aggregation, P-selectin externalization and integrin αIIb ß3 activation. Both etomoxir and trimetazidine impeded agonist-induced dense granule release and platelet thrombus formation on collagen under arterial shear. The effect of inhibitors on platelet aggregation and dense granule release was dose- and incubation time- dependent with significant inhibition at higher doses and prolonged incubation times. Neither of the inhibitors could protect mice from collagen-epinephrine-induced pulmonary embolism or prolong mouse tail bleeding times. However, mice pre-administered with etomoxir, trimetazidine and oxfenicine were protected from ferric chloride-induced mesenteric thrombosis. In conclusion, ß-oxidation of fatty acids sustains ATP level in stimulated platelets and is therefore essential for energy-intensive agonist-induced platelet responses. Thus, fatty acid oxidation may constitute an attractive therapeutic target for novel antiplatelet agents.

Trimetazidine Reduces Cardiac Fibrosis in Rats by Inhibiting NOX2-Mediated Endothelial-to-Mesenchymal Transition.

Purpose: Endothelial-to-mesenchymal transition (EndMT) is an important mechanism underlying cardiac fibrosis. The anti-ischemic drug trimetazidine (TMZ) is reportedly useful in ventricular remodeling and associated with NADPH oxidase (NOX) 2. This study aimed to investigate the possible effect of TMZ on cardiac fibrosis exerted via the inhibition of NOX2-mediated EndMT. Methods: A cardiac fibrosis model was established in Sprague-Dawley rats through a subcutaneous injection of isoproterenol (ISO, 5 mg/kg/d). Echocardiographic parameters, myocardial fibrosis, NOX2 expression and EndMT were assessed. An in vitro model of EndMT was developed using human umbilical vein endothelial cells (HUVECs) via treatment with transforming growth factor-ß (TGF-ß) at 10 ng/mL for 24 h. HUVECs were administrated with TMZ or TMZ and lentivirus, the expression of EndMT and related proteins was observed by wound healing assay, immunoblotting, and immunofluorescence. Results: Rats injected with ISO exhibited severe interstitial cardiac fibrosis and perivascular fibrosis, decreased left ventricular ejection fraction, and increased NOX activity. TMZ treatment mitigated cardiac fibrosis, ameliorated left ventricular dysfunction, and reduced NOX activity. In addition, TMZ effectively inhibited EndMT in ISO-treated rat hearts and TGF-ß-treated HUVECs, as manifested by increased CD31 expression, decreased α-SMA expression, and suppressed cell migration. Compared with the control group, the expression of NOX2, nuclear factor-κB (NF-κB), and Snail was increased in vivo and in vitro but decreased with TMZ treatment. Furthermore, the overexpression of NOX2 by lentivirus abolished the protective effects of TMZ on TGF-ß-induced EndMT. Conclusion: TMZ may ameliorate EndMT and ISO-induced cardiac fibrosis through the NOX2/NF-κB/Snail pathway. The findings of the study may provide new insights into the potential role of TMZ in the pathophysiology of cardiac fibrosis.

Synthesis and in†Vivo Evaluation of Triphenylphosphonium Conjugated Trimetazidine with Enhanced Cardioprotection and Ability to Restore Mitochondrial Function.

Trimetazidine exhibits great therapeutic potential in cardiovascular diseases and mitochondria-mediated cardioprotection by trimetazidine has been widely reported. In this study, to enhance its cardioprotection, the triphenylphosphonium-based modification of trimetazidine was conducted to deliver it specifically to mitochondria. Fifteen triphenylphosphonium (TPP) conjugated trimetazidine analogs were designed and synthesized. Their protective effects were evaluated in vivo using a tert-butyl hydroperoxide (t-BHP) induced zebrafish injury model. Structure-activity relationship correlations revealed the best way to couple the TPP moiety to trimetazidine, and led to a new conjugate (18a) with enhanced therapeutic properties. Compared to trimetazidine, 18a effectively protects against heart injury in the zebrafish model at a much lower concentration. Further study in t-BHP treated zebrafish and H9c2 cells demonstrated that 18a protects against cardiomyocyte death and damage by inhibiting excessive production of ROS, maintaining mitochondrial morphology, and preventing mitochondrial dysfunction. Consequently, 18a can be regarded as a potential therapeutic agent for cardioprotection.

Trimetazidine and exercise offer analogous improvements to the skeletal muscle insulin resistance of mice through Nrf2 signaling.

INTRODUCTION: Insulin resistance (IR) plays a key role in the pathogenesis and clinical course of patients with multiple metabolic diseases and diabetes. This study aimed to explore the effect of trimetazidine (TMZ) on skeletal muscle IR in mice fed a high-fat diet (HFD) and explore the possible underlying mechanism. RESEARCH DESIGN AND METHODS: In vivo, a HFD mouse IR model was adopted and TMZ and exercise were used to intervene. Postintervention the following were determined: blood levels of glucose and insulin, homeostasis model assessment of IR index, expression of skeletal muscle insulin signaling-related proteins phosphorylated insulin receptor substrate 1 (p-IRS1/IRS1) and phosphorylated protein kinase B (p-AKT/AKT), nuclear factor erythroid 2 related factor 2 (Nrf2) signaling pathway, and oxidative stress. In vitro, a palmitate-treated C2C12 myotube IR model was constructed. Cellular glucose uptake, p-IRS1/IRS1, and p-AKT/AKT were determined, and reactive oxygen species (ROS) production was analyzed based on treatments with specific small interfering RNA of Nrf2 with or without TMZ. Western blot was used to obtain the protein expression level and ROS production by functional analysis kits. RESULTS: In vivo, TMZ and exercise decreased the blood glucose and insulin levels and homeostasis model assessment of IR index, increased skeletal muscle insulin signaling-related protein ratios of p-IRS1/IRS1 and p-AKT/AKT, and both interventions activated Nrf2 signaling and reduced oxidative stress production in HFD mice. In vitro, TMZ reduced the oxidative stress reaction, increased the ratios of p-AKT/AKT and p-IRS1/IRS1, and attenuated the insulin stimulation of PA-induced glucose uptake. However, in the absence of Nrf2, TMZ failed to resist the effects of IR. CONCLUSIONS: This study showed that TMZ, like exercise, brought about marked improvements to HFD-induced skeletal muscle IR through TMZ, a common pathway with exercise in the form of Nrf2, regulating oxidative stress. We provide new evidence to support the use of TMZ for diabetes treatment.

Trimetazidine inhibits liver fibrosis and hepatic stellate cell proliferation and blocks transforming growth factor-ß (TGFß)/Smad signaling in vitro and in vivo.

Trimetazidine (TMZ) has been used extensively to treat coronary artery disease and to reduce fibrosis. Liver fibrosis is a reversible process. However, the impacts of TMZ on liver fibrosis triggered by CCl4 and on hepatic stellate cells in liver fibrosis remain to be elaborated. In the current study, the liver fibrosis models were constructed by using CCl4-induced mice and TGF-ß-induced hepatic stellate cells. The involvement of TMZ in liver fibrosis was subsequently investigated. In the CCl4-induced hepatic fibrosis mouse model, it was shown that the expression levels of alanine aminotransferase and aspartate aminotransferase were reduced after TMZ treatment; the expression levels of the extracellular matrix proteins colla1 and α-SMA were down-regulated; furthermore, the expression levels of TGFß/Smad signaling proteins were inhibited. In TGF-ß-induced hepatic stellate cells, compared to the TGF-ß-induced group, cell proliferation and migration were inhibited after TMZ treatment; meanwhile, extracellular matrix protein and TGFß/Smad signaling protein expression levels followed the same trend as in the hepatic fibrosis model. In conclusion, TMZ could block the TGFß/Smad signaling in liver fibrosis model, with inhibiting liver fibrosis and hepatic stellate cell proliferation. This may broaden the application sphere of TMZ in liver fibrosis therapy.

Trimetazidine mitigates high glucose-induced retinal endothelial dysfunction by inhibiting PI3K/Akt/mTOR pathway-mediated autophagy.

Trimetazidine (TMZ), as a metabolic regulator, has been widely testified to exhibit positive therapeutic effects on various disease models, but its role in diabetic retinopathy has not been reported. Therefore, this study was designed with the purpose of exploring the effects of TMZ on high-glucose (HG)-induced retinal endothelial dysfunction and its underlying mechanism. To establish DR model in vitro, 30 mM glucose was applied to induce human retinal endothelial cells (HRECs). Cell proliferation, invasion, and migration were examined by means of Cell Counting Kit-8, transwell, and wound healing assays, respectively. The tubule formation experiment was used to test the tubulogenesis ability and fluorescein isothiocyanate (FITC)-albumin was utilized to measure the permeability of monolayer HRECs. In addition, immunofluorescence and Western blot were employed to detect protein expression. Compared with the HG-induced group, TMZ concentration dependently inhibited the proliferation, migration, and angiogenesis of HG-induced HRECs, decreased the permeability of monolayer HRECs, and increased the protein expression levels of Claudin-5 and VE-cadherin. In addition, TMZ intervention increased the expression of p-PI3K, p-AKT, and p-mTOR but decreased the expression of LC3I, LC3II, and Beclin 1, which were then partially reversed by P13 K inhibitor (LY294002). Moreover, the autophagy agonist rapamycin (RAPA) was also testified to reverse the inhibitory effects of TMZ on the proliferation, migration, and angiogenesis of HG-induced HRECs. In summary, TMZ inhibited excessive autophagy by activating PI3K/Akt/mTOR pathway, thereby improving retinal endothelial dysfunction induced by HG.

Trimetazidine enhances myocardial angiogenesis in pressure overload-induced cardiac hypertrophy mice through directly activating Akt and promoting the binding of HSF1 to VEGF-A promoter.

Latest clinical research shows that trimetazidine therapy during the perioperative period relieves endothelial dysfunction in patients with unstable angina induced by percutaneous coronary intervention. In this study we investigated the effects of TMZ on myocardial angiogenesis in pressure overload-induced cardiac hypertrophy mice. Cardiac hypertrophy was induced in mice by transverse aortic constriction (TAC) surgery. TAC mice were administered trimetazidine (2.8 mg/100 µL, i.g.) for 28 consecutive days. We showed that trimetazidine administration significantly increased blood vessel density in the left ventricular myocardium and abrogated cardiac dysfunction in TAC mice. Co-administration of a specific HSF1 inhibitor KRIBB11 (1.25 mg/100 µL, i.h.) abrogated the angiogenesis-promoting effects of trimetazidine in TAC mice. Using luciferase reporter and electrophoretic mobility shift assays we demonstrated that the transcription factor HSF1 bound to the promoter region of VEGF-A, and the transcriptional activity of HSF1 was enhanced upon trimetazidine treatment. In molecular docking analysis we found that trimetazidine directly bound to Akt via a hydrogen bond with Asp292 and a pi-pi bond with Trp80. In norepinephrine-treated HUVECs, we showed that trimetazidine significantly increased the phosphorylation of Akt and the synergistic nuclear translocation of Akt and HSF1, as well as the binding of Akt and HSF1 in the nucleus. These results suggest that trimetazidine enhances myocardial angiogenesis through a direct interaction with Akt and promotion of nuclear translocation of HSF1, and that trimetazidine may be used for the treatment of myocardial angiogenic disorders in hypertensive patients.

Feasibility and application of trimetazidine in 18F-FDG PET myocardial metabolic imaging of diabetic mellitus patients with severe coronary artery disease: A prospective, self-controlled study.

BACKGROUND: 18F-FDG PET myocardial metabolic imaging (MMI) is sometimes uninterpretable due to background activity from uncontrolled glucose homeostasis in diabetic mellitus (DM) patients. Trimetazidine is an oral medication that promotes the transformation of myocardial energy supply from free fatty acids to glucose. We aimed to investigate the feasibility and application of trimetazidine in 18F-FDG PET MMI of DM patients. METHODS: With DM patients exhibiting severe coronary artery disease (CAD) symptoms serving as self-controls, the effects of trimetazidine on PET MMI image quality, myocardial viability assessment, quantitative analytical parameters, and 18F-FDG uptake of different myocardial segments were elucidated. RESULTS: The image quality of 18F-FDG MMI was graded visually as good, moderate, and uninterpretable. After trimetazidine, grades of good, moderate, and uninterpretable were observed in 14 (60.9%), 8 (34.8%), and 1 (4.3%) patients, respectively, and in 4 (17.4%), 15 (65.2%), 4 (17.4%) patients without trimetazidine. The myocardial SUV and myocardial to blood pool SUV ratio (M/B ratio) were significantly higher after trimetazidine administration than those before (3.11 ± 1.07 vs 2.32 ± 1.00, 2.67 ± 1.41 vs 1.81 ± 0.75, P all < 0.01). 6 (3, 7) viable myocardium segments were detected with a mismatch score of 10 (6, 17) after trimetazidine, significantly higher than those before trimetazidine [5 (2, 7) and 8 (2, 17), P < 0.05]. Meanwhile, the 18F-FDG uptake in myocardial segments with decreased and normal perfusion showed different ranges of increase (by 15.30%-57.77%). CONCLUSION: Trimetazidine is feasible and effective in DM patients with severe CAD before 18F-FDG PET MMI, which can significantly improve the image quality and increase the number of viable myocardium segments detected. TRIAL REGISTRY: The study was registered in the Chinese Clinical Trial Registry (ChiCTR2000038559).

Lomerizine, trimetazidine and bis-(4-fluorophenyl)-methylpiperazine in human urine after oral administration of lomerizine dihydrochloride: analysis by liquid chromatography-high resolution-tandem mass spectrometry.

In sports drugs testing, the differentiation between the abuse of the prohibited substance trimetazidine and that of the permitted drug lomerizine is required because trimetazidine is one of the metabolites of lomerizine. Therefore, it is important to identify a lomerizine-specific metabolite in urine that allows making the distinction. In this study, a simple dilute-and-shoot method employing liquid chromatography-high resolution-tandem mass spectrometry for the quantification of trimetazidine, lomerizine and the specific metabolite bis-(4-fluorophenyl)-methylpiperazine (M6) in urine was developed. An oral dose of 15 mg was administered to 10 male volunteers, after which urine samples collected during the following 276 hours were analyzed using the developed method, allowing for examination of the target analytes' excretion profile. The limit of detection of all target analytes was <0.02 ng/mL. In all volunteers, the metabolite M6 was detected up to 276 hours after administration. After more than 12 hours, all volunteers were found to have higher concentrations of the metabolite M6 than of trimetazidine. The concentrations of trimetazidine, lomerizine, M6, and the M6/trimetazidine ratio in the final sample collected after 276 hours were 0.2-0.9 ng/mL, <0.05-0.1 ng/mL, 14.1-38.3 ng/mL, and 28.8-122.9, respectively. The urinary excretion of trimetazidine, unchanged lomerizine, and the metabolite M6 within the first 276 hours was 0.64%, 0.006%, and 6.1%, respectively. Consequently, the absence of the metabolite M6 in doping control urine samples corroborates the conclusion that lomerizine is unlikely to be the source of trimetazidine. The results confirm that the M6 metabolite is the longest-lasting urinary metabolite of lomerizine currently known.

Analytical detection of trimetazidine produced by metabolic conversion of lomerizine in doping control analysis.

The identification of trimetazidine in urine samples might result from administration of the permitted drug lomerizine. Laboratories are therefore urged to carefully investigate suspicious cases where trimetazidine is detected. Differentiation of abuse of the banned substance trimetazidine from use of the permitted drug lomerizine would be supported by analysis of the intact drug lomerizine and/or specific metabolites. Copyright © 2015 John Wiley & Sons, Ltd.

Ultrasonic microdialysis coupled with capillary electrophoresis electrochemiluminescence study the interaction between trimetazidine dihydrochloride and human serum albumin.

The paper describes a homemade ultrasonic microdialysis device coupled with capillary electrophoresis electrochemiluminescence (CE-ECL) for studying the interaction between human serum albumin (HSA) and trimetazidine dihydrochloride (TMZ). The time required for equilibrium by ultrasonic microdialysis was 45min, which was far less than that by traditional dialysis (240min). It took 80min to achieve the required combination equilibrium by normal incubation and only 20min by ultrasonic. Compared with traditional dialysis, the use of ultrasonic microdialysis simplified experimental procedures, shortened experimental time and saved consumption of sample. A simple, sensitive and selective determination of TMZ was developed using CE-ECL and the parameters that affected ECL intensity were optimized. Under the optimized conditions, the linear range of TMZ was from 0.075 to 80µmol/L (r(2)=0.9974). The detection limit was 26nmol/L with RSD of 2.8%. The number of binding sites and binding constant were 1.54 and 15.17L/mol, respectively.

Last performance with VIAGRA: post-mortem identification of sildenafil and its metabolites in biological specimens including hair sample.

A 43-year-old man was found dead in a hotel room during a sexual relation with a colleague.He was treated both for cardiovascular disease and for erectile dysfunction with VIAGRA. A pillbox was found in the room with several tablets of verapamil (Isoptine), trimetazidine (Vastarel), yohimbine and bromazepam (Lexomil). A box of VIAGRA 25mg was found in his raincoat and two tablets were missing. His wife declared during the investigation that he was also treated by trinitrine. Autopsy revealed severe coronary artery sclerosis as well as signs of previous myocardial infarctions. Blood, urine, bile, gastric content and hair and representative tissues for histology were collected for toxicological analysis. Sildenafil and yohimbine were screened with liquid chromatography/mass spectrometry (LC/MS) and trinitrine with headspace injection (HS)/GC/MS. Verapamil and trimetazidine were identified and quantified with LC/diode array detection (DAD). Sildenafil was identified in blood, urine, bile and gastric content at 105, 246, 1206 and 754ng/ml, respectively. Hair concentration was 177pg/mg. The desmethyl metabolite was quantified in urine at 143ng/ml. Blood concentrations of verapamil and trimetazidine were measured at 659 and 2133ng/ml, respectively and were above therapeutic ranges. Trinitrine and yohimbine were not identified. These results confirm the absorption of sildenafil, verapamil and trimetazidine before the death and hair analysis indicates the chronic use of sildenafil. To the author's knowledge, this is the first report of a fatal sildenafil-verapamil association, probably by hypotension and cardiac dysrhythmia.

[(3)H]-trimetazidine mitochondrial binding sites: regulation by cations, effect of trimetazidine derivatives and other agents and interaction with an endogenous substance.

Trimetazidine, an antiischaemic drug, has been shown to restore impaired mitochondrial functions. Specific binding sites for [(3)H]-trimetazidine have been previously detected in liver mitochondria. In the present study we confirm this observation and provide additional evidence for the involvement of these sites in the pharmacological effects of the drug. Inhibition experiments using a series of trimetazidine derivatives revealed the presence of three classes of binding sites. An N-benzyl substituted analogue of trimetazidine exhibited a very high affinity (K(i)=7 nM) for one of these classes of sites. Compounds from different pharmacological classes were evaluated for their ability to inhibit [(3)H]-trimetazidine binding. Among the drugs tested pentazocine, ifenprodil, opipramol, perphenazine, haloperidol, and to a lower extent prenylamine, carbetapentane and dextromethorphan competed with high affinity, suggesting a similarity of high affinity [(3)H]-trimetazidine sites with sigma receptors. [(3)H]-Trimetazidine binding was modulated by pH. Neutral trimetazidine had about 10 fold higher affinity than protonated trimetazidine for its mitochondrial binding sites. Various cations also affected [(3)H]-trimetazidine binding. Ca(2+) was the most potent inhibitor and totally suppressed the binding of [(3)H]-trimetazidine to the sites of medium affinity. An endogenous cytosolic ligand was able to displace [(3)H]-trimetazidine from its binding sites. Its activity was not affected by boiling for 15 min, suggesting a non-protein compound. These data suggest that mitochondrial [(3)H]-trimetazidine binding sites could have a physiological relevance and be involved in the antiischaemic effects of the drug.

Evidence for the existence of [3H]-trimetazidine binding sites involved in the regulation of the mitochondrial permeability transition pore.

1. Trimetazidine is an anti-ischaemic drug effective in different experimental models but its mechanism of action is not fully understood. Data indicate that mitochondria could be the main target of this drug. The aim of this work was to investigate the binding of [3H]-trimetazidine on a purified preparation of rat liver mitochondria. 2. [3H]-trimetazidine binds to two populations of mitochondrial binding sites with Kd values of 0.96 and 84 microM. The total concentration of binding sites is 113 pmol mg(-1) protein. Trimetazidine binding sites are differently distributed. The high-affinity ones are located on the outer membranes and represent only a small part (4%) of total binding sites, whereas the low-affinity ones are located on the inner membranes and are more abundant (96%) with a Bmax=108 pmol mg(-1) protein. 3. Drug displacement studies with pharmacological markers for different mitochondrial targets showed that [3H]-trimetazidine binding sites are different from previously described mitochondrial sites. 4. The possible involvement of [3H]-trimetazidine binding sites in the regulation of the mitochondrial permeability transition pore (MTP), a voltage-dependent channel sensitive to cyclosporin A, was investigated with mitochondrial swelling experiments. Trimetazidine inhibited the mitochondrial swelling induced by Ca2+ plus tert-butylhydroperoxide (t-BH). This effect was concentration-dependent with an IC50 value of 200 microM. 5. Assuming that trimetazidine effectiveness may be related to its structure as an amphiphilic cation, we compared it with other compounds exhibiting the same chemical characteristic both for their ability to inhibit MTP opening and to displace [3H]-trimetazidine bound to mitochondria. Selected compounds were drugs known to interact with various biological membranes. 6. A strong correlation between swelling inhibition potency and low-affinity [3H]-trimetazidine binding sites was observed: r=0.907 (n=24; P<0.001). 7. These data suggest that mitochondrial sites labelled with [3H]-trimetazidine may be involved in the MTP inhibiton.