Levosimendan Relaxes Thoracic Aortic Smooth Muscle in Mice by Inhibiting PKC and Activating Inwardly Rectifying Potassium Channels.

ABSTRACT: Studies have examined the therapeutic effect of levosimendan on cardiovascular diseases such as heart failure, perioperative cardiac surgery, and septic shock, but the specific mechanism in mice remains largely unknown. This study aimed to investigate the relaxation mechanism of levosimendan in the thoracic aorta smooth muscle of mice. Levosimendan-induced relaxation of isolated thoracic aortic rings that were precontracted with norepinephrine or KCl was recorded in an endothelium-independent manner. Vasodilatation by levosimendan was not associated with the production of the endothelial relaxation factors nitric oxide and prostaglandins. The voltage-dependent K + channel (K V ) blocker (4-aminopyridine) and selective K Ca blocker (tetraethylammonium) had no effect on thoracic aortas treated with levosimendan, indicating that K V and K Ca channels may not be involved in the levosimendan-induced relaxation mechanism. Although the inwardly rectifying K + channel (K ir ) blocker (barium chloride) and the K ATP channel blocker (glibenclamide) significantly inhibited levosimendan-induced vasodilation in the isolated thoracic aorta, barium chloride had a much stronger inhibitory effect on levosimendan-induced vasodilation than glibenclamide, suggesting that levosimendan-induced vasodilation may be mediated by K ir channels. The vasodilation effect and expression of K ir 2.1 induced by levosimendan were further enhanced by the PKC inhibitor staurosporine. Extracellular calcium influx was inhibited by levosimendan without affecting intracellular Ca 2+ levels in the isolated thoracic aorta. These results suggest that K ir channels play a more important role than K ATP channels in regulating vascular tone in larger arteries and that the activity of the K ir channel is enhanced by the PKC pathway.

TXNIP knockout improves cardiac function after myocardial infarction by promoting angiogenesis and reducing cardiomyocyte apoptosis.

Background: Myocardial infarction (MI) is a common cause of death. Thioredoxin-interacting protein (TXNIP) expression increases after MI, and it exerts a negative regulatory effect on cardiac function after MI. Our study aimed to investigate the specific regulatory mechanism of TXNIP on angiogenesis and cardiomyocyte apoptosis after MI. Methods: The TXNIP gene knock-in (TXNIP-KI) and knock-out (TXNIP-KO) mice were generated, respectively. Eight-week-old male TXNIP-KO, TXNIP-KI, and wild type (WT) mice were subjected to MI by permanent ligation of the left anterior descending artery. Cardiomyocyte apoptosis was detected by TUNEL assay on the 4th post-surgery day. The expressions of TXNIP, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), phosphorylated protein kinase B (p-AKT), p-AMP-activated protein kinase (p-AMPK), cleaved caspase-3, and caspase-3 were detected by Western blot. Quantitative real-time PCR was performed to detect the expression of TXNIP, HIF-1α, VEGF, prolyl hydroxylase (PHD) 1, and factor inhibiting HIF (FIH). In addition, the superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in each group were also measured. On day 7 after MI, the hearts of sacrificed animals were analyzed by immunohistochemistry to assess CD31 expression and determine the density of angiogenesis. One month after treatment, the cardiac functional and structural changes were determined by echocardiography and the level of myocardial fibrosis was observed by Masson staining. Results: Compared with WT mice, TXNIP-KO mice had a significantly improved cardiac functional recovery after MI, and the proportion of myocardial fibrosis area was dramatically reduced, cardiomyocyte apoptosis was decreased, and angiogenesis was significantly increased; TXNIP-KI mice reversed in these changes. The expression of HIF-1α, p-AKT, and p-AMPK increased after MI in TXNIP-KO mice, and the mRNA expression of PHD 1 and FIH decreased. TXNIP-KI mice reversed in these changes. Conclusions: After MI, TXNIP down-regulated the level of HIF-1α and VEGF, reduced the number of angiogenesis, increased cardiomyocyte apoptosis, and ultimately led to a poor prognosis of ischemic myocardium. TXNIP was a protein with negative effects after MI and was expected to be a target for the prevention and treatment of MI.

Delayed metformin treatment improves functional recovery following traumatic brain injury via central AMPK-dependent brain tissue repair.

Accumulating evidence suggests that chronic metformin posttreatment offers potent neuroreparative effects against acute brain injury. However, in previous studies, metformin was not initially administered beyond 24 h postinjury, and the effects of delayed metformin treatment in traumatic brain injury (TBI) and other types of acute brain injury and the related mechanisms are unclear. To test this, male C57BL/6 mice received once daily metformin treatment (20, 50 or 100 mg/kg/d, i.p.) at day 1-14, day 1-2, day 1-10, day 3-10, day 5-12 or day 5-28 after cryogenic TBI (cTBI). The results showed that 100 mg/kg/d metformin administered at day 1-14 postinjury significantly promoted motor functional recovery in the beam walking and gait tests and reduced the infarct volume. Metformin (100 mg/kg/d) administered at day 1-10 or day 3-10 but not day 1-2 or day 5-12 after cTBI significantly improved motor functional outcomes at day 7 and 14, and reduced the infarct volume at day 14. Interestingly, the therapeutic time window was further expanded when the duration of metformin treatment starting at day 5 postinjury was extended to 2 weeks. Furthermore, compared with cTBI, the administration of metformin at day 3-10 or day 5-28 after cTBI significantly elevated the expression of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and growth associated protein 43 (an axonal regeneration marker) and the number of vascular branch points and decreased the area of glial scar and the number of amoeboid microglia in the peri-infarct area at day 14 or 28 postinjury. The above beneficial effects of metformin were blocked by the intracerebroventricular injection of the AMPK inhibitor compound C (40 µg/mouse/d). Our data provide the first evidence that metformin has a wide therapeutic time window for at least 5 days after cTBI, during which it can improve functional recovery by promoting tissue repair and inhibiting glial scar formation and microglial activation in a central AMPK-dependent manner.

[Adenovirus-mediated overexpression of thioredoxin interaction protein inhibits INS-1 islet ß cell proliferation].

Diabetes can cause a significant increase in the expression of thioredoxin (Trx)-interacting protein (TXNIP), which binds to Trx and inhibits its activity. The present study was aimed to investigate the effect of TXNIP on proliferation of rat INS-1 islet ß cells and the underlying mechanism. TXNIP overexpressing adenovirus vectors (Ad-TXNIP-GFP and Ad-TXNIPc247s-GFP) were constructed and used to infect INS-1 cells. Ad-TXNIPc247s-GFP vector carries a mutant C247S TXNIP gene, and its expression product (TXNIPc247s) cannot attach and inhibit Trx activity. The expression of TXNIP was detected by real-time PCR and Western blot. EdU and Ki67 methods were used to detect cell proliferation. Protein phosphorylation levels of ERK and AKT were detected by Western blot. The results showed that both TXNIP and TXNIPc247s protein overexpressions inhibited the proliferation of INS-1 cells, and the former's inhibitory effect was greater. Moreover, both of the two kinds of overexpressions inhibited the phosphorylation of ERK and AKT. These results suggest that TXNIP overexpression may inhibit the proliferation of INS-1 cells through Trx-dependent and non-Trx-dependent pathways, and the mechanism involves the inhibition of ERK and AKT phosphorylation.

[Role of autophagy in TXNIP overexpression-induced apoptosis of INS-1 islet cells].

Thioredoxin (Trx) interacting protein (TXNIP) is a Trx-binding protein that inhibits the antioxidative function of Trx and is highly expressed in the serum and tissue samples from diabetes patients. This study was to explore whether TXNIP overexpression could cause INS-1 cell autophagy under normal glucose and lipid concentrations, and to analyze the role of autophagy in the apoptosis of INS-1 cells. The INS-1 cells cultured under normal conditions were divided into three groups: normal control, empty adenovirus vector (Ad-eGFP) and TXNIP overexpression (Ad-TXNIP-eGFP) groups. Forty-eight hours after transfection, the expression levels of TXNIP mRNA and protein were measured. Western blot was used to examine the protein expression levels of Beclin-1 and P62, as well as LC3-II/LC3-I ratio, which are associated with autophagy. IF/ICC was used to measure the autophagosome. In addition, the cleaved caspase-3/caspase-3 ratio, the apoptosis marker, was also measured, and the apoptotic rates were detected by flow cytometry (FCM). The results showed that the TXNIP mRNA and protein levels were significantly up-regulated in Ad-TXNIP-eGFP group, suggesting that TXNIP overexpression model was successfully established. In Ad-TXNIP-eGFP group, the protein levels of Beclin-1 and LC3-II/LC3-I ratio were increased, while the protein expression of P62 was decreased, compared with those in Ad-eGFP group. Red fluorescent intensity, representing autophagy level, was higher in Ad-TXNIP-eGFP group than that in Ad-eGFP group. These results suggested that TXNIP overexpression can significantly promote INS-1 cell autophagy. Meanwhile, cleaved caspase 3/caspase 3 ratio and the number of apoptotic cells were significantly increased in Ad-TXNIP-eGFP group. The inhibitor of autophagy, 3-MA, reduced TXNIP overexpression-induced apoptosis in INS-1 cells. Taken together, our data demonstrate that autophagy appears to be an important pathway in TXNIP overexpression-induced apoptosis in INS-1 cells.

[Effects of Indomethacin on Proliferation and Senescence of Leukemia Cells].

OBJECTIVE: To investigate the effects of indomethacin on proliferation and senescence of leukemia cells and to analyze whether non-steroidal anti-inflammatory drugs have any adjunctively therapeutic effects on leukemia. METHODS: Three kinds of leukemia cell lines (U266, K562 and U937) were treated with either indomethacin (final concentration 30 µmol/L) or solvent DMSO (control) for 7 days. Cell viability was determined by trypan blue, cell apoptosis and cell cycle were determined by flow cytometry, mRNA expression of hyperplastic suppression gene P21 and P27 was determined by RT-PCR, and cell senescence rate was assayed at day 0, 4, 7 after treatment. RESULTS: After treatment with indomethacin, the cell viability in U266 and U937 groups decreased significantly, while it was not changed in K562 group. Cell cycle in U266 and U937 groups was blocked at G(2)/M phase, but the blooking effect was not found in K562 cells. The cell apoptotic rate was enhanced in 3 treated groups (P < 0.01). The mRNA expression of P21 and P27 was significantly increased after indomethacin treatment in U266 and K562 cell lines except for the P27 mRNA expression of U937. The expression levels of P21 and P27 mRNA in U266 and K562 increased obviously. The cell senescence rate in K562 and U937 group also increased. CONCLUSION: The indomethacin possesses inhibitory effects on leukemia cells. However, its mechanisms are varied from different types of leukemia cells. It suggests that non-steroidal anti-inflammatory drugs can play a role in adjuvant therapy for leukemia, while its application would be adjusted according to different types of leukemia.

[Effect of adenovirus-mediated TXNIP overexpression on apoptosis and injury of H9C2 cardiomyocytes].

Adenovirus transfection technique was used in the current study to show if thioredoxin-interacting protein (TXNIP) overexpression can induce cell apoptosis and injury in H9C2 cardiomyocytes cultured in normal glucose condition. And the mechanisms were then investigated. Briefly, H9C2 cardiomyocytes in logarithmic growth phase were randomly divided into three groups: normal cultured group, empty adenovirus vector group (Ad-eGFP) and TXNIP overexpression group (Ad-TXNIP-eGFP). All cells were cultured in DMEM containing normal concentration of glucose (5 mmol/L) and lipid. 72 h after adenovirus transfection, cells and culture mediums were collected for further assay. The results showed that Ad-eGFP and Ad-TXNIP-eGFP adenovirus transfected H9C2 cells successfully, and the transfection efficiency reached the peak at 72 h. Compared with Ad-eGFP group, Ad-TXNIP-eGFP transfection significantly increased TXNIP mRNA (P < 0.05) and protein expression level (P < 0.01). TXNIP overexpression induced remarkable cell apoptosis and injury as evidenced by increased caspase-3 activity (P < 0.05), apoptotic rate (P < 0.01) and LDH activity (P < 0.01). To further analysis the mechanisms of TXNIP-induced cell apoptosis, we also determined Trx activity, Trx related free radical injury and p38 kinase activation, which are involved in free radical induced apoptosis. The results showed that, compared with those in Ad-eGFP group, Trx activity was significantly decreased (P < 0.01), while malondialdehyde (MDA), 3-nitrotyrosine contents and p38 kinase activity were significantly increased (P < 0.01) in TXNIP overexpression group. These results suggest that TXNIP overexpression alone can induce severe apoptosis and injury in H9C2 cardiomyocytes even they are cultured in normal glucose and lipid concentration conditions. The mechanism involved is that overexpressed TXNIP can bind and inhibit Trx, impairs its antioxidative and antiapoptotic function, and then increases free radical induced injury and p38 kinase dependent apoptosis.

INO-4885 [5,10,15,20-tetra[N-(benzyl-4'-carboxylate)-2-pyridinium]-21H,23H-porphine iron(III) chloride], a peroxynitrite decomposition catalyst, protects the heart against reperfusion injury in mice.

Oxidative/nitrative stress caused by peroxynitrite, the reaction product of superoxide (O2(.-)) and nitric oxide (NO), is the primary cause of myocardial ischemia/reperfusion injury. The present study determined whether INO-4885 [5,10,15,20-tetra[N-(benzyl-4'-carboxylate)-2-pyridinium]-21H,23H-porphine iron(III) chloride], a new peroxynitrite decomposition catalyst, may provide cellular protection and protect heart from myocardial ischemia/reperfusion injury. Adult male mice were subjected to 30 min of ischemia and 3 or 24 h of reperfusion. Mice were randomized to receive vehicle, INO-4885 without catalytic moiety, or INO-4885 (3-300 microg/kg i.p.) 10 min before reperfusion. Infarct size, apoptosis, nitrotyrosine content, NO/O2(.-) production, and inducible nitric-oxide synthase (iNOS)/NADPH oxidase expression were determined. INO-4885 treatment reduced ischemia/reperfusion-induced protein nitration and caspase 3 activation in a dose-dependent fashion in the range of 3 to 100 microg/kg. However, doses exceeding 100 microg/kg produced nonspecific effects and attenuated its protective ability. At the optimal dose (30 microg/kg), INO-4885 significantly reduced infarct size (p < 0.01), decreased apoptosis (p < 0.01), and reduced tissue nitrotyrosine content (p < 0.01). As expected, INO-4885 had no effect on ischemia/reperfusion-induced iNOS expression and NO overproduction. To our surprise, this compound significantly reduced superoxide production and partially blocked NADPH oxidase overexpression in the ischemic/reperfused cardiac tissue. Additional experiments demonstrated that INO-4885 provided better cardioprotection than N-(3-(aminomethyl)benzyl)acetamidine (1400W, a selective iNOS inhibitor), apocynin (an NADPH oxidase inhibitor), or Tiron (a cell-permeable superoxide scavenger). Taken together, our data demonstrated that INO-4885 is a cardioprotective molecule that attenuates myocardial reperfusion injury by facilitating peroxynitrite decomposition and inhibiting NADPH oxidase-derived O2(.-) production.