Low-Dose Acetylsalicylic Acid and Mitochondria-Targeted Antioxidant Mitoquinone Attenuate Non-Alcoholic Steatohepatitis in Mice.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. NAFLD can evolve from simple fatty liver to non-alcoholic steatohepatitis (NASH), and ultimately, to cirrhosis. Inflammation and oxidative stress, promoted by mitochondrial dysfunction, play a crucial role in the onset and development of NASH. To date, no therapy has been approved for NAFLD and NASH. The aim of this study is to evaluate if the anti-inflammatory activity of acetylsalicylic acid (ASA) and the mitochondria-targeted antioxidant effect of mitoquinone could hinder the progression of non-alcoholic steatohepatitis. In mice, fatty liver was induced through the administration of a deficient in methionine and choline and rich in fat diet. Two experimental groups were treated orally with ASA or mitoquinone. Histopathologic evaluation of steatosis and inflammation was performed; the hepatic expression of genes associated with inflammation, oxidative stress, and fibrosis was evaluated; the protein expression of IL-10, cyclooxygenase 2, superoxide dismutase 1, and glutathione peroxidase 1 in the liver was analyzed; a quantitative analysis of 15-epi-lipoxin A4 in liver homogenates was performed. Mitoquinone and ASA significantly reduced liver steatosis and inflammation by decreasing the expression of TNFα, IL-6, Serpinb3, and cyclooxygenase 1 and 2 and restoring the anti-inflammatory IL-10. Treatment with mitoquinone and ASA increased the gene and protein expression of antioxidants, i.e., catalase, superoxide dismutase 1, and glutathione peroxidase 1, and decreased the expression of profibrogenic genes. ASA normalized the levels of 15-epi-Lipoxin A4. In mice fed with a deficient in methionine and choline and rich in fat diet, mitoquinone and ASA reduce steatosis and necroinflammation and may represent two effective novel strategies for the treatment of non-alcoholic steatohepatitis.

Contribution of Splenic Resistance Arteries to Splanchnic Blood Overflow in Cirrhosis.

BACKGROUND: In liver cirrhosis, a marked splanchnic vasodilation causes an increase in portal blood flow, contributing to the development of portal hypertension. AIM: To evaluate if, in experimental cirrhosis, a different vascular reactivity exists between splenic and mesenteric components of the splanchnic circulation. METHODS: Liver cirrhosis was induced in Sprague Dawley rats by common bile duct ligation. In sections of splenic and superior mesenteric arteries, cumulative dose-response curves were obtained. mRNA expression of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and prostaglandin I2 synthase (PTGIS) was evaluated. RESULTS: In cirrhotic rats, mesenteric but not splenic arteries showed a significant increase in endothelium-dependent relaxation to acetylcholine. In control and cirrhotic rats, COX inhibition alone did not significantly change the response of mesenteric arteries to acetylcholine; after inhibiting also NOS, the relaxation was completely abolished in control but only partially decreased in cirrhotic rats. After the inhibition of COX and NOS, the relaxation to acetylcholine was similarly decreased in splenic arteries from control and cirrhotic animals. The contraction induced by phenylephrine of both mesenteric and splenic arteries was decreased in cirrhotic rats. PTGIS mRNA expression did not differ in splenic and mesenteric arteries from control and cirrhotic rats; in cirrhotic rats, eNOS and iNOS mRNA expression was increased in mesenteric but not in splenic vascular bed. CONCLUSION: In cirrhotic rats, a decreased splenic arterial response to vasoconstrictors, rather than an increased response to vasodilators, contributes to splanchnic vasodilation, while in mesenteric arteries also an increased response to vasodilators secondary to, but not only, eNOS and iNOS overexpression, plays a role.

Mitochondria-targeted antioxidant mitoquinone attenuates liver inflammation and fibrosis in cirrhotic rats.

In liver cirrhosis, oxidative stress plays a major role in promoting liver inflammation and fibrosis. Mitochondria dysregulation is responsible for excessive reactive oxygen species production. Therefore, in an experimental model of cirrhosis, we investigated the effect of mitochondria-targeted antioxidant mitoquinone. Liver cirrhosis was induced in Spraque-Dawley rats by common bile duct ligation (CBDL). Mitoquinone (10 mg·kg-1·day-1, oral gavage) or vehicle was administered from 3rd to 28th day after CBDL, when animals were euthanized; liver oxidative stress, inflammation, fibrosis, mitophagy were evaluated; and in vivo and ex vivo hemodynamic studies were performed. In cirrhotic rats, mitoquinone prevented liver inflammation, hepatocyte necrosis, and fibrosis at histological examination; decreased circulating TNF-α, gene expression of transforming growth factor-ß1, collagen type 1a1, TNF-α, IL-6, IL-1ß, tissue inhibitor of metalloproteinase-1, matrix metalloproteinase (MMP)-2, and MMP-13; and reduced hepatic oxidative stress, as shown by reduced oxidative carbonylation of the proteins, by modulating antioxidants catalase, Mn superoxide dismutase, and Cu/Zn superoxide dismutase. Furthermore, mitoquinone attenuated apoptosis by reducing hepatic protein expression of cleaved caspase-3. A selective removal of dysfunctional mitochondria was improved by mitoquinone, as shown by the increase in Parkin translocation to mitochondria. Treatment with mitoquinone normalized the weight of the spleen; however, it increased portal blood flow and reduced splenic artery intrahepatic resistance, suggesting an effect on resistance index. Mitochondria-targeted antioxidant mitoquinone improves liver inflammation and fibrosis in cirrhotic rats by reducing hepatic oxidative stress, preventing apoptosis, and promoting removal of dysfunctional mitochondria. Therefore, it may represent a promising strategy for the prevention and treatment of liver cirrhosis.

Effects of levosimendan on isolated human internal mammary artery and saphenous vein: concurrent use with conventional vasodilators.

Graft spasm is a common problem in coronary artery bypass grafting (CABG). In this study, we aimed to investigate the interaction of levosimendan, a novel inodilator, with vasodilator agents that are clinically used for the treatment of graft spasm and with endogenous vasoconstrictors that are thought to play a role in graft vasospasm, in human internal mammary artery (IMA) and saphenous vein (SV). Isolated human IMA and SV segments derived from patients undergoing CABG were suspended in an organ bath. Responses to cumulative concentrations of noradrenaline (NA), serotonin (5-HT), papaverine, nitroglycerin (NG), and diltiazem were recorded before and after 10(-5) m levosimendan incubation (30 min). In addition, cumulative levosimendan responses were taken in vessels precontracted with NA or 5-HT. 10(-5) m levosimendan reduced NA Emax and sensitivity in IMA and SV, and 5-HT Emax responses in IMA. Moreover, levosimendan caused concentration-dependent relaxation in both grafts. Papaverine Emax or sensitivity was not altered by levosimendan neither in IMA nor in SV. Levosimendan diminished NG sensitivity in IMA and Emax responses in SV and decreased diltiazem Emax responses both in IMA and SV. Our results suggest that levosimendan may be used alone for prevention or treatment of graft spasm in IMA or in combination with papaverine in IMA and SV grafts. However, as concurrent administration with diltiazem or NG causes a reduction in relaxation in vitro, we suggest caution should be exercised when using levosimendan in combination with these agents.

Autophagy-regulating TP53INP2 mediates muscle wasting and is repressed in diabetes.

A precise balance between protein degradation and synthesis is essential to preserve skeletal muscle mass. Here, we found that TP53INP2, a homolog of the Drosophila melanogaster DOR protein that regulates autophagy in cellular models, has a direct impact on skeletal muscle mass in vivo. Using different transgenic mouse models, we demonstrated that muscle-specific overexpression of Tp53inp2 reduced muscle mass, while deletion of Tp53inp2 resulted in muscle hypertrophy. TP53INP2 activated basal autophagy in skeletal muscle and sustained p62-independent autophagic degradation of ubiquitinated proteins. Animals with muscle-specific overexpression of Tp53inp2 exhibited enhanced muscle wasting in streptozotocin-induced diabetes that was dependent on autophagy; however, TP53INP2 ablation mitigated experimental diabetes-associated muscle loss. The overexpression or absence of TP53INP2 did not affect muscle wasting in response to denervation, a condition in which autophagy is blocked, further indicating that TP53INP2 alters muscle mass by activating autophagy. Moreover, TP53INP2 expression was markedly repressed in muscle from patients with type 2 diabetes and in murine models of diabetes. Our results indicate that TP53INP2 negatively regulates skeletal muscle mass through activation of autophagy. Furthermore, we propose that TP53INP2 repression is part of an adaptive mechanism aimed at preserving muscle mass under conditions in which insulin action is deficient.

Methylglyoxal causes endothelial dysfunction: the role of endothelial nitric oxide synthase and AMP-activated protein kinase α.

BACKGROUND: Methylglyoxal is a major precursor in the formation of advanced glycation end products and is associated with the pathogenesis of diabetes-related vascular complications. The aim of this study was to evaluate whether methylglyoxal induces endothelial dysfunction and to determine the contributors involved in this process. METHODS: Rat thoracic aortic rings were treated for 24 h with 100 µM methylglyoxal by using an organ culture method. A cumulative dose-response curve to acetylcholine was obtained to determine endothelium-dependent relaxation. The protein levels of endothelial nitric oxide synthase (eNOS) and its phosphorylated form at the serine 1177 site [p-eNOS (Ser1177)], heat shock protein 90 (Hsp90), AMP-activated protein kinase α (AMPKα) and its phosphorylated form at the threonine 172 site [p-AMPKα (Thr172)] were evaluated. Superoxide production was determined by lucigenin-chemiluminescence. RESULTS: Treatment with 100 µM methylglyoxal for 24 h decreased acetylcholine-induced vascular relaxation. The levels of eNOS and p-eNOS (Ser1177) were reduced while no effect on Hsp90 was observed. Levels of p-AMPKα (Thr172) were significantly decreased without any change in total AMPKα protein levels. Superoxide level was not affected by methylglyoxal treatment. CONCLUSIONS: In rat aortic rings, methylglyoxal determines a reduction in endothelium-dependent relaxation. This effect seems to be mediated via a reduction in p-eNOS (Ser1177) and p-AMPKα (Thr172).

Prolonged AMP-activated protein kinase induction impairs vascular functions.

AMP-activated protein kinase (AMPK) is a regulator of cellular metabolism and is involved in the pathogenesis of several diseases, including type 2 diabetes and cardiovascular diseases. Data showing the effects of AMPK on vasculature are controversial. Therefore, the aim of this study was to determine the impact of prolonged AMPK activation on vascular functions. For this purpose we have examined the role of AMPK in endothelium-dependent and -independent relaxation and vascular contractions. For this, we incubated thoracic aortic rings, from rats, with AMPK activator 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR, 500 µmol/L or 2 mmol/L) in the presence or absence of AMPK inhibitor compound C (10 µmol/L). Next, cumulative dose-response curves to acetylcholine (ACh) (10(-9)-10(-4) mol/L), nitroglycerine (NG) (10(-9)-3 × 10(-5) mol/L), and noradrenaline (NA) (10(-9)-10(-4) mol/L) were obtained. Endothelial nitric oxide synthase (eNOS) protein expression was determined. Our results show that endothelium-dependent relaxation was inhibited after AICAR treatment, and that this effect was reversed by AMPK inhibition. Moreover, AICAR enhanced the contractile response to NA and caused a decrease in eNOS protein expression. In conclusion, prolonged AMPK induction causes endothelial impairment, possibly via increased degradation and (or) reduced expression of eNOS.

Heme oxygenase-2 deficiency contributes to diabetes-mediated increase in superoxide anion and renal dysfunction.

Heme oxygenase-1 (HO-1) and -2 play an important role in cytoprotection and are physiologic regulators of heme-dependent protein synthesis in renal tissues. The impact of HO-2 deletion comparing hyperglycemic HO-2 (+/+) mice and HO-2 knockout (-/-) mice was examined. Hyperglycemia was induced by streptozotocin (STZ) injection, and its effect on renal HO-1/HO-2 protein, HO activity, and creatinine levels were assessed. The effect of HO induction using systemic administration of the HO inducers heme or cobalt protoporphyrin and the effect of HO inhibition using systemic administration of the HO inhibitor tin mesoporphyrin also were assessed in STZ-treated mice. In STZ-treated HO-2 (-/-) mice, there was marked renal functional impairment as reflected by an increase in plasma creatinine, associated with acute tubular damage and microvascular pathology as compared with HO-2 (+/+). In these animals, HO activity was decreased with a concomitant increase in superoxide anion. Upregulation of HO-1 in HO-2 (-/-) mice by weekly administration of cobalt protoporphyrin prevented the increase in plasma creatinine levels and tubulointerstitial and microvascular pathology. Inhibition of HO activity by administration of tin mesoporphyrin accentuated superoxide production and increased creatinine levels in hyperglycemic HO-2 (-/-) mice. In conclusion, HO-2 deficiency enhanced STZ-induced renal dysfunction and morphologic injury and HO-1 upregulation in HO-2 (-/-) mouse rescue and prevented the morphologic damage. These observations indicate that HO activity is essential in preserving renal function and morphology in STZ-induced diabetic mice probably via mitigation of concomitant oxidative stress.

Chronic CO levels have [corrected] a beneficial effect on vascular relaxation in diabetes.

Heme oxygenase (HO) has been shown to provide cytoprotection to the vascular system in diabetes. Isolated femoral arteries from diabetic rats treated with cobalt protoporphyrin (CoPP) exhibited increased relaxation to acetylcholine (ACh), which was markedly decreased in control diabetic rats. In control rats treated with either CoPP or with CO releasing molecules-3 (CORM-3), but not in rats treated with biliverdin, we observed an increased dilatory response to ACh. The inhibition of guanylyl-cyclase (GC) with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) caused a contractile response to ACh in control rats and in biliverdin-treated rats, while in rats treated with CoPP and CORM-3, the ACh dilatory effect was only decreased. Moreover, the inhibition of HO with chromium mesoporphyrin did not change the response to ACh in rats treated with CoPP, suggesting that the improving effect of overproduction of CO on vascular reactivity is due to a decrease in iNOS and the beneficial effect on vascular function.

The role of endothelin receptor antagonism in collar-induced intimal thickening and vascular reactivity changes in rabbits.

Intimal thickening, due to smooth muscle cell migration and proliferation, is considered to be one of the major components of vascular proliferative disorders such as atherosclerosis and restenosis. One experimental model, resulting in intimal thickening in the rabbit, involves placing a silicon collar around the carotid artery, and is used in this study. Endothelin is known to act as a strong mitogen and to stimulate smooth muscle cell proliferation and migration. We investigated the contribution of endothelin to the development of collar-induced intimal thickening and the effects of TAK-044, (5 mg kg(-1) daily, s.c.), a non-selective ET(A)/ET(B) receptor antagonist, on intimal thickening and vascular reactivity changes in the collared rabbit carotid artery. Endothelin levels and the intimal cross-sectional area, as well as the ratio of intimal area to media (index), increased significantly in collared arteries as compared with those in sham-operated arteries. TAK-044 significantly inhibited intimal thickening and also decreased the index without affecting increased endothelin levels in collared arteries. Vascular reactivity changes in response to collaring produced predictable effects, such as decreased contractile responses to vasoconstrictor agents and increased sensitivity to serotonin (5-hydroxytryptamine, 5-HT). In terms of contractile responses in this model, TAK-044, in particular, did not affect collar-induced vascular reactivity changes. These results suggest that endothelin may be involved in the pathogenesis of collar-induced intimal thickening. As an endothelin receptor antagonist, TAK-044 may potentially be beneficial in the treatment of atherosclerosis.

D-4F induces heme oxygenase-1 and extracellular superoxide dismutase, decreases endothelial cell sloughing, and improves vascular reactivity in rat model of diabetes.

BACKGROUND: Apolipoprotein A1 mimetic peptide, synthesized from D-amino acid (D-4F), enhances the ability of HDL to protect LDL against oxidation in atherosclerotic animals. METHODS AND RESULTS: We investigated the mechanisms by which D-4F provides antioxidant effects in a diabetic model. Sprague-Dawley rats developed diabetes with administration of streptozotocin (STZ). We examined the effects of daily D-4F (100 microg/100 g of body weight, intraperitoneal injection) on superoxide (O2-), extracellular superoxide dismutase (EC-SOD), vascular heme oxygenase (HO-1 and HO-2) levels, and circulating endothelial cells in diabetic rats. In response to D-4F, both the quantity and activity of HO-1 were increased. O2- levels were elevated in diabetic rats (74.8+/-8x10(3) cpm/10 mg protein) compared with controls (38.1+/-8x10(3) cpm/10 mg protein; P<0.01). D-4F decreased O2- levels to 13.23+/-1x10(3) (P<0.05 compared with untreated diabetics). The average number of circulating endothelial cells was higher in diabetics (50+/-6 cells/mL) than in controls (5+/-1 cells/mL) and was significantly decreased in diabetics treated with D-4F (20+/-3 cells/mL; P<0.005). D-4F also decreased endothelial cell fragmentation in diabetic rats. The impaired relaxation typical of blood vessels in diabetic rats was prevented by administration of D-4F (85.0+/-2.0% relaxation). Western blot analysis showed decreased EC-SOD in the diabetic rats, whereas D-4F restored the EC-SOD level. CONCLUSIONS: We conclude that an increase in circulating endothelial cell sloughing, superoxide anion, and vasoconstriction in diabetic rats can be prevented by administration of D-4F, which is associated with an increase in 2 antioxidant proteins, HO-1 and EC-SOD.

Antioxidant mechanism of heme oxygenase-1 involves an increase in superoxide dismutase and catalase in experimental diabetes.

Increased heme oxygenase (HO)-1 activity attenuates endothelial cell apoptosis and decreases superoxide anion (O2-) formation in experimental diabetes by unknown mechanisms. We examined the effect of HO-1 protein and HO activity on extracellular SOD (EC-SOD), catalase, O2-, inducible nitric oxide synthase (iNOS), and endothelial nitric oxide synthase (eNOS) levels and vascular responses to ACh in control and diabetic rats. Vascular EC-SOD and plasma catalase activities were significantly reduced in diabetic compared with nondiabetic rats (P < 0.05). Upregulation of HO-1 expression by intermittent administration of cobalt protoporphyrin, an inducer of HO-1 protein and activity, resulted in a robust increase in EC-SOD but no significant change in Cu-Zn-SOD. Administration of tin mesoporphyrin, an inhibitor of HO-1 activity, decreased EC-SOD protein. Increased HO-1 activity in diabetic rats was associated with a decrease in iNOS but increases in eNOS and plasma catalase activity. On the other hand, aortic ring segments from diabetic rats exhibited a significant reduction in vascular relaxation to ACh, which was reversed with cobalt protoporphyrin treatment. These data demonstrate that an increase in HO-1 protein and activity, i.e., CO and bilirubin production, in diabetic rats brings about a robust increase in EC-SOD, catalase, and eNOS with a concomitant increase in endothelial relaxation and a decrease in O2-. These observations in experimental diabetes suggest that the vascular cytoprotective mechanism of HO-1 against oxidative stress requires an increase in EC-SOD and catalase.