Non-alcoholic steatohepatitis (NASH) has pathological characteristics similar to those of alcoholic hepatitis, despite the absence of a drinking history. The greatest threat associated with NASH is its progression to cirrhosis and hepatocellular carcinoma. The pathophysiology of NASH is not fully understood to date. In this study, we investigated the pathophysiology of NASH from the perspective of glycolysis and the Warburg effect, with a particular focus on microRNA regulation in liver-specific macrophages, also known as Kupffer cells. We established NASH rat and mouse models and evaluated various parameters including the liver-to-body weight ratio, blood indexes, and histopathology. A quantitative phosphoproteomic analysis of the NASH rat model livers revealed the activation of glycolysis. Western blotting and immunohistochemistry results indicated that the expression of pyruvate kinase muscle 2 (PKM2), a rate-limiting enzyme of glycolysis, was upregulated in the liver tissues of both NASH models. Moreover, increases in PKM2 and p-PKM2 were observed in the early phase of NASH. These observations were partially induced by the downregulation of microRNA122-5p (miR-122-5p) and occurred particularly in the Kupffer cells. Our results suggest that the activation of glycolysis in Kupffer cells during NASH was partially induced by the upregulation of PKM2 via miR-122-5p suppression.

To clarify the role of chymase in hypertension, we evaluated the effect of a chymase inhibitor, TY-51469, on vascular dysfunction and survival in stroke-prone spontaneously hypertensive rats (SHR-SP). SHR-SP were treated with TY-51469 (1 mg/kg per day) or placebo from 4 to 12 weeks old or until death. Wistar-Kyoto rats were used as a normal group. SBP was significantly higher in both the placebo and TY-51469 groups than in the normal group, but there was no significant difference between the two treatment groups. Plasma renin, angiotensin-converting enzyme activity and angiotensin II levels were not different between the placebo and TY-51469 groups. In contrast, vascular chymase-like activity was significantly higher in the placebo than in the normal group, but it was reduced by TY-51469. Acetylcholine-induced vascular relaxation was significantly higher in the TY-51469 group than in the placebo group. There was significant augmentation of the number of monocytes/macrophages and matrix metalloproteinase-9 activity in aortic tissue from the placebo group compared with the normal group, and these changes were attenuated by TY-51469. There were also significant increases in mRNA levels of monocyte chemoattractant protein-1 and tumor necrosis factor-α in the placebo group that were attenuated by TY-51469. Cumulative survival was significantly prolonged in the TY-51469 group compared with the placebo group. Chymase might play an important role in vascular dysfunction via augmentation both of matrix metalloproteinase-9 activity and monocyte/macrophage accumulation in SHR-SP, and its inhibition may be useful for preventing vascular remodeling and prolonging survival.

Chymase present in mast cells can directly form matrix metalloproteinase (MMP)-9 from proMMP-9. Chymase-activated MMP-9 has been reportedly closely related to the pathogenesis of various diseases, and inflammation-related diseases in particular. Upregulated chymase and MMP-9 have been observed in tissues from patients and animal models of aortic aneurysm, inflammatory gastrointestinal and hepatic diseases, acute pancreatic failure, atopic dermatitis and rheumatoid arthritis. Chymase at these regions is only derived from mast cells, while MMP-9 is derived from macrophages and neutrophils in addition to mast cells. Chymase inhibitors attenuate MMP-9 formation from pro-MMP-9, and ameliorate the development and progression of these disorders, along with reduction in inflammatory cell numbers. MMP-9 activated by chymase might also be involved in angiogenesis in the tumor environment. Development of angiogenesis around several cancers is closely related to the expression of chymase and MMP-9, and postoperative survival curves have revealed that patients with a higher number of chymase positive cells have lower survival rates. In this review, we wanted to clarify the role of chymase-activated MMP-9, which might become an important therapeutic target for various inflammatory disorders.

Oxidative stress plays an important role in the pathophysiology of acute kidney injury (AKI). Previously, we reported that vanin-1, which is involved in oxidative stress, is associated with renal tubular injury. This study was aimed to determine whether urinary vanin-1 is a biomarker for the early diagnosis of AKI in two experimental models: in vivo and in vitro. In a rat model of AKI, ischemic AKI was induced in uninephrectomized rats by clamping the left renal artery for 45 min and then reperfusing the kidney. On Day 1 after renal ischemia/reperfusion (I/R), serum creatinine (SCr) in I/R rats was higher than in sham-operated rats, but this did not reach significance. Urinary N-acetyl-β-D-glucosaminidase (NAG) exhibited a significant increase but decreased on Day 2 in I/R rats. In contrast, urinary vanin-1 significantly increased on Day 1 and remained at a significant high level on Day 2 in I/R rats. Renal vanin-1 protein decreased on Days 1 and 3. In line with these findings, immunofluorescence staining demonstrated that vanin-1 was attenuated in the renal proximal tubules of I/R rats. Our in vitro results confirmed that the supernatant from HK-2 cells under hypoxia/reoxygenation included significantly higher levels of vanin-1 as well as KIM-1 and NGAL. In conclusion, our results suggest that urinary vanin-1 might be a potential novel biomarker of AKI induced by I/R.

In the present study, we investigated whether Akt is involved in insulin-like growth factor-I (IGF-I)-stimulated activity of alkaline phosphatase, a marker of mature osteoblast phenotype, in osteoblast-like MC3T3-E1 cells. IGF-I induced the phosphorylation of Akt in these cells. Akt inhibitor significantly suppressed the IGF-I-stimulated alkaline phosphatase activity. The phosphorylation of Akt induced by IGF-I was reduced by the Akt inhibitor. LY294002 and wortmannin, inhibitors of phosphatidylinositol 3-kinase, significantly suppressed the IGF-I-induced alkaline phosphatase activity. The phosphorylation of Akt induced by IGF-I was markedly reduced by LY294002 and wortmannin. These results strongly suggest that phosphatidylinositol 3-kinase/Akt plays a role in the IGF-I-stimulated alkaline phosphatase activity in osteoblasts.

Background: Deep vein thrombosis (DVT) is the primary cause of pulmonary embolism and the third most life‐threatening cardiovascular disease in North America. Post‐DVT anticoagulants, such as warfarin, heparin, and direct oral anticoagulants, reduce the incidence of subsequent venous thrombi. However, all currently used anticoagulants affect bleeding time at various degrees, and there is therefore a need for improved therapeutic regimens in DVT. It has recently been shown that mast cells play a crucial role in a DVT murine model. The underlying mechanism involved in the prothrombotic properties of mast cells, however, has yet to be identified. Methods and Results: C57BL/6 mice and mouse mast cell protease‐4 (mMCP‐4) genetically depleted mice (mMCP‐4 knockout) were used in 2 mouse models of DVT, partial ligation (stenosis) and ferric chloride–endothelial injury model of the inferior vena cava. Thrombus formation and impact of genetically repressed or pharmacologically (specific inhibitor TY‐51469) inhibited mMCP‐4 were evaluated by morphometric measurements of thrombi immunochemistry (mouse and human DVT), color Doppler ultrasound, bleeding times, and enzymatic activity assays ex vivo . Recombinant chymases, mMCP‐4 (mouse) and CMA‐1 (human), were used to characterize the interaction with murine and human plasmin, respectively, by mass spectrometry and enzymatic activity assays. Inhibiting mast cell–generated mMCP‐4, genetically or pharmacologically, resolves and prevents venous thrombus formation in both DVT models. Inferior vena cava blood flow obstruction was observed in the stenosis model after 6 hours of ligation, in control‐ but not in TY‐51469–treated mice. In addition, chymase inhibition had no impact on bleeding times of healthy or DVT mice. Furthermore, endogenous chymase limits plasmin activity in thrombi ex vivo. Recombinant mouse or human chymase degrades/inactivates purified plasmin in vitro. Finally, mast cell–containing immunoreactive chymase was identified in human DVT. Conclusions: This study identified a major role for mMCP‐4, a granule‐localized protease of chymase type, in DVT formation. These findings support a novel pharmacological strategy to resolve or prevent DVT without affecting the coagulation cascade through the inhibition of chymase activity.