Spatially Resolved Identification of Transglutaminase Substrates by Proteomics in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by the invariably progressive deposition of fibrotic tissue in the lungs and overall poor prognosis. TG2 (transglutaminase 2) is an enzyme that crosslinks glutamine and lysine residues and is involved in IPF pathogenesis. Despite the accumulating evidence implicating TG2 as a critical enzyme, the causative function and direct target of TG2 relating to this pathogenesis remain unelucidated. Here, we clarified the distributions of TG2 protein/activity and conducted quantitative proteomics analyses of possible substrates crosslinked by TG2 on unfixed lung sections in a mouse pulmonary fibrosis model. We identified 126 possible substrates as markedly TG2-dependently increased in fibrotic lung. Gene ontology analysis revealed that these identified proteins were mostly enriched in the lipid metabolic process, immune system process, and protein transport. In addition, these proteins were enriched in 21 pathways, including phagosome, lipid metabolism, several immune responses, and protein processing in endoplasmic reticulum. Furthermore, the network analyses screened out the six clusters and top 20 hub proteins with higher scores, which are related to endoplasmic reticulum stress and peroxisome proliferator-activated receptor signals. Several enriched pathways and categories were identified, some of which were the same terms based on transcription analysis in IPF. Our results provide novel pathological molecular networks driven by protein crosslinking via TG2, which can lead to the development of new therapeutic targets for IPF.

Abaloparatide Exerts Bone Anabolic Effects with Less Stimulation of Bone Resorption-Related Factors: A Comparison with Teriparatide.

Abaloparatide (ABL) is a novel synthetic peptide analog of parathyroid hormone-related protein. In previous reports, intermittent ABL administration showed robust bone mineral density (BMD) increase and reduced the incidence of fractures in patients with osteoporosis, while its calcemic effect was reduced, as compared with teriparatide (TPTD), a parathyroid hormone N-terminal fragment. The present study aimed to elucidate the effects of ABL on bone anabolism and bone turnover as compared with TPTD. In ovariectomized (OVX) rats, ABL increased the bone strength and BMD of lumbar spine by intermittent administration similar to TPTD. Both ABL and TPTD increased the bone formation marker serum P1NP with little effect on the bone resorption maker urine DPD/Cr, suggesting anabolic effects on bone. In human osteoblastic cells, both peptides increased the expression of bone resorption-related factors such as RANKL/OPG and M-CSF, and the effects of ABL were significantly attenuated as compared with those of TPTD under transient 6-h treatment, although no significant differences were found under continuous treatment. In contrast, ABL and TPTD similarly promoted the expression of bone formation-related factors, IGF-1 and osteocalcin. In addition, there were no significant differences in the effects on WNT signaling inhibitors such as sclerostin and dickkopf-related protein 1 (DKK1) between the two peptides. These results demonstrate that ABL exerts bone anabolic effects in OVX rats. It is also indicated that ABL stimulates the expression of RANKL/OPG and M-CSF less than TPTD, while showing similar effects on bone formation-related factors and WNT signaling inhibitors in vitro. The profile of ABL indicates that it would be a suitable bone anabolic agent for osteoporosis.

Chymase activities and survival in endotoxin-induced human chymase transgenic mice.

We examined the effects of overexpressed human chymase on survival and activity in lipopolysaccharide (LPS)-treated mice. Human chymase transgenic (Tg) and wild-type C57BL/6 (WT) mice were treated with LPS (0.03, 0.1 and 0.3 mg/day; intraperitoneal) for 2 weeks. Treatment with 0.03 mg LPS did not affect survival in either WT or Tg mice. WT mice were not affected by 0.1 mg/day of LPS, whereas 25% of Tg mice died. Survival of mice treated with 0.3 mg/day of LPS was 87.5% and 0% in WT and Tg, respectively. LPS-induced increases in chymase activity in the heart and skin were significantly greater in Tg than WT mice. These data suggest a possible contribution of human chymase activation to LPS-induced mortality.

Chymase inhibition prevents fibronectin and myofibrillar loss and improves cardiomyocyte function and LV torsion angle in dogs with isolated mitral regurgitation.

BACKGROUND: The left ventricular (LV) dilatation of isolated mitral regurgitation (MR) is associated with an increase in chymase and a decrease in interstitial collagen and extracellular matrix. In addition to profibrotic effects, chymase has significant antifibrotic actions because it activates matrix metalloproteinases and kallikrein and degrades fibronectin. Thus, we hypothesize that chymase inhibitor (CI) will attenuate extracellular matrix loss and LV remodeling in MR. METHODS AND RESULTS: We studied dogs with 4 months of untreated MR (MR; n=9) or MR treated with CI (MR+CI; n=8). Cine MRI demonstrated a >40% increase in LV end-diastolic volume in both groups, consistent with a failure of CI to improve a 25% decrease in interstitial collagen in MR. However, LV cardiomyocyte fractional shortening was decreased in MR versus normal dogs (3.71±0.24% versus 4.81±0.31%; P<0.05) and normalized in MR+CI dogs (4.85±0.44%). MRI with tissue tagging demonstrated an increase in LV torsion angle in MR+CI versus MR dogs. CI normalized the significant decrease in fibronectin and FAK phosphorylation and prevented cardiomyocyte myofibrillar degeneration in MR dogs. In addition, total titin and its stiffer isoform were increased in the LV epicardium and paralleled the changes in fibronectin and FAK phosphorylation in MR+CI dogs. CONCLUSIONS: These results suggest that chymase disrupts cell surface-fibronectin connections and FAK phosphorylation that can adversely affect cardiomyocyte myofibrillar structure and function. The greater effect of CI on epicardial versus endocardial titin and noncollagen cell surface proteins may be responsible for the increase in torsion angle in chronic MR.

Blood glucose level and survival in streptozotocin-treated human chymase transgenic mice.

A growing body of evidence suggests the potential role of chymase in organ injury in diabetes. We investigated blood glucose levels and survival in transgenic mice carrying the human chymase gene (Tg). Intraperitoneal injections of streptozotocin (STZ) (200, 100, 75 and 50 mg/kg in total, i.p.) were given to uninephrectomized Tg mice and wild-type C57BL/6 (BL) mice. Before STZ injection, the Tg mice had significantly lower body weights and slightly higher systolic blood pressure as compared with the BL mice. STZ-treated Tg mice showed significantly higher postprandial blood glucose levels as compared with the STZ-treated BL mice. The survival prevalence of STZ-treated Tg mice was zero, whereas BL mice showed a value of 40% until 42 days. STZ (100, 75 or 50 mg/kg, i.p.)-treated Tg mice also showed a similar pattern as compared with the STZ-treated BL mice. These data suggest that human chymase contributes to blood glucose levels and mortality during the progression of diabetes.

Frequent administration of abaloparatide shows greater gains in bone anabolic window and bone mineral density in mice: A comparison with teriparatide.

Abaloparatide (ABL) is a novel 34-amino acid peptide analog of parathyroid hormone-related protein. In clinical studies, although ABL showed a greater bone mineral density (BMD) increase than teriparatide (TPTD, human parathyroid hormone 1-34), the responses of ABL to bone formation and resorption markers were weaker, making it difficult to understand the relationship between the bone anabolic window (increase in bone formation versus resorption) and bone mass. In the present study, the effects of ABL and TPTD were compared in mice. Given that the rate of bone turnover is higher in rodents than in humans, the comparison was made with several administration regimens providing equivalent daily dosages: once daily (QD, 30 µg/kg every 24 h), twice daily (BID, 15 µg/kg every 12 h), or three times a day (TID, 10 µg/kg every 8 h). Frequent administration of ABL showed higher BMD with enhancement of trabecular and cortical bone mass and structures than that of TPTD, consistent with the clinical results seen with once daily administration. ABL increased bone formation marker levels more than TPTD with more frequent regimens, while bone resorption marker levels were not different between ABL and TPTD in all regimens. Analysis of bone histomorphometry and gene expression also suggested that ABL increased bone formation more than TPTD, while the effect on bone resorption was almost comparable between ABL and TPTD. The bone anabolic windows calculated from bone turnover markers indicated that ABL enhanced the anabolic windows more than TPTD, leading to a robust increase in BMD. The mechanism by which ABL showed a better balance of bone turnover was suggested to be partly due to the enhanced remodeling-based bone formation involved in Ephb4. Taken together, our findings would help elucidate the mechanism by which ABL shows excellent BMD gain and reduction of fractures in patients with osteoporosis.

Activity of xanthine oxidase in plasma correlates with indices of insulin resistance and liver dysfunction in patients with type 2 diabetes mellitus and metabolic syndrome: A pilot exploratory study.

AIMS/INTRODUCTION: There is controversy as to whether hyperuricemia is an independent risk factor for cardiometabolic diseases. The serum level of uric acid is affected by a wide variety of factors involved in its production and excretion. In contrast, evidence has accumulated that locally- and systemically-activated xanthine oxidase (XO), a rate-limiting enzyme for production of uric acid, is linked to metabolic derangement in humans and rodents. We therefore explored the clinical implication of plasma XO activity in patients with type 2 diabetes mellitus and metabolic syndrome (MetS). MATERIALS AND METHODS: We enrolled 60 patients with type 2 diabetes mellitus and MetS. MetS was defined according to the 2005 International Diabetes Federation guidelines. Plasma XO activity was measured by highly-sensitive fluorometric assay measuring the conversion of pterin to isoxanthopterin, and explored associations between the value of plasma XO activity and metabolic parameters. RESULTS: The value of plasma XO activity was correlated with indices of insulin resistance and the level of circulating liver transaminases. In contrast, the level of serum uric acid was not correlated with indices of insulin resistance. The value of plasma XO activity was not correlated with the serum uric acid level. CONCLUSIONS: Plasma XO activity correlates with indices of insulin resistance and liver dysfunction in Japanese patients with type 2 diabetes mellitus and MetS. Through assessing the plasma XO activity, patients showing normal levels of serum uric acid with higher activity of XO can be screened, thereby possibly providing a clue to uncovering metabolic risks in type 2 diabetes mellitus and MetS patients.

Chymase mediates injury and mitochondrial damage in cardiomyocytes during acute ischemia/reperfusion in the dog.

Cardiac ischemia and reperfusion (I/R) injury occurs because the acute increase in oxidative/inflammatory stress during reperfusion culminates in the death of cardiomyocytes. Currently, there is no drug utilized clinically that attenuates I/R injury in patients. Previous studies have demonstrated degranulation of mast cell contents into the interstitium after I/R. Using a dog model of I/R, we tested the role of chymase, a mast cell protease, in cardiomyocyte injury using a specific oral chymase inhibitor (CI). 15 adult mongrel dogs had left anterior descending artery occlusion for 60 min and reperfusion for 100 minutes. 9 dogs received vehicle and 6 were pretreated with a specific CI. In vivo cardiac microdialysis demonstrated a 3-fold increase in interstitial fluid chymase activity in I/R region that was significantly decreased by CI. CI pretreatment significantly attenuated loss of laminin, focal adhesion complex disruption, and release of troponin I into the circulation. Microarray analysis identified an I/R induced 17-fold increase in nuclear receptor subfamily 4A1 (NR4A1) and significantly decreased by CI. NR4A1 normally resides in the nucleus but can induce cell death on migration to the cytoplasm. I/R caused significant increase in NR4A1 protein expression and cytoplasmic translocation, and mitochondrial degradation, which were decreased by CI. Immunohistochemistry also revealed a high concentration of chymase within cardiomyocytes after I/R. In vitro, chymase added to culture HL-1 cardiomyocytes entered the cytoplasm and nucleus in a dynamin-dependent fashion, and promoted cytoplasmic translocation of NR4A1 protein. shRNA knockdown of NR4A1 on pre-treatment of HL-1 cells with CI significantly decreased chymase-induced cell death and mitochondrial damage. These results suggest that the beneficial effects of an orally active CI during I/R are mediated in the cardiac interstitium as well as within the cardiomyocyte due to a heretofore-unrecognized chymase entry into cardiomyocytes.

Mast cell chymase limits the cardiac efficacy of Ang I-converting enzyme inhibitor therapy in rodents.

Ang I-converting enzyme (ACE) inhibitors are widely believed to suppress the deleterious cardiac effects of Ang II by inhibiting locally generated Ang II. However, the recent demonstration that chymase, an Ang II-forming enzyme stored in mast cell granules, is present in the heart has added uncertainty to this view. As discussed here, using microdialysis probes tethered to the heart of conscious mice, we have shown that chronic ACE inhibitor treatment did not suppress Ang II levels in the LV interstitial fluid (ISF) despite marked inhibition of ACE. However, chronic ACE inhibition caused a marked bradykinin/B2 receptor-mediated increase in LV ISF chymase activity that was not observed in mast cell-deficient KitW/KitW-v mice. In chronic ACE inhibitor-treated mast cell-sufficient littermates, chymase inhibition decreased LV ISF Ang II levels substantially, indicating the importance of mast cell chymase in regulating cardiac Ang II levels. Chymase-dependent processing of other regulatory peptides also promotes inflammation and tissue remodeling. We found that combined chymase and ACE inhibition, relative to ACE inhibition alone, improved LV function, decreased adverse cardiac remodeling, and improved survival after myocardial infarction in hamsters. These results suggest that chymase inhibitors could be a useful addition to ACE inhibitor therapy in the treatment of heart failure.

Contribution of chymase-dependent angiotensin II formation to the progression of tubulointerstitial fibrosis in obstructed kidneys in hamsters.

Recent studies indicate a role of chymase in the regulation of angiotensin II (AngII) formation in cardiovascular and renal tissues. We investigated a possible contribution of chymase to AngII formation and to renal fibrosis in unilateral ureteral obstruction (UUO). Eight-week-old Syrian hamsters were subjected to UUO and treated with vehicle, the specific chymase inhibitor (CI) 4-[1-(4-methyl-benzo[b]thiophen-3-ylmethyl)-1H-benzimidazol-2-ylsulfanyl]-butyric acid (50 mg/kg, twice a day, p.o.), or the selective AT(1)-receptor blocker olmesartan (10 mg/kg per day, p.o.) for 14 days. UUO-induced renal interstitial fibrosis was associated with increases in renal mRNA levels of alpha-smooth muscle actin (SMA), type I collagen, and transforming growth factor (TGF)-beta. The UUO hamsters showed markedly higher AngII contents and increased AT(1)-receptor mRNA level in the obstructed kidney than sham-operated ones. In contrast, angiotensin-converting enzyme (ACE) protein expression was significantly lower in UUO hamsters. In UUO hamsters, treatment with CI or olmesartan significantly decreased AngII levels in renal tissue and mRNA levels of alpha-SMA, type I collagen, and TGF-beta and ameliorated tubulointerstitial injury. On the other hand, neither CI nor olmesartan changed systolic blood pressure, renal ACE, and AT(1)-receptor protein levels. These data suggest that chymase-dependent intrarenal AngII formation contributes to the pathogenesis of interstitial fibrosis in obstructed kidneys of hamsters.

Enhanced intrarenal oxidative stress and angiotensinogen in IgA nephropathy patients.

This study was performed to determine whether immunoreactivity of intrarenal hemeoxygenase-1 and angiotensinogen are increased in IgA nephropathy (IgAN) patients. Hemeoxygenase-1 and angiotensinogen immunoreactivity were determined by immunohistochemistry robot system in renal specimens from 39 patients with IgAN. Normal portions of surgically resected kidney served as controls. IgAN patients showed moderate proteinuria (1.1+/-0.2 g/day); however, the control group did not show any proteinuria. Immunoreactivity of intrarenal hemeoxygenase-1 and angiotensinogen in IgAN were significantly increased compared to normal kidneys (2.42+/-0.42 vs 1.00+/-0.26 for hemeoxygenase-1 and 4.05+/-0.40 vs 1.00+/-0.21 for angiotensinogen, arbitrary unit). Even though these IgAN patients did not show massive renal damage, hemeoxygenase-1 and angiotensinogen immunoreactivity were increased in these patients at this time point. These data suggest that activated intrarenal reactive oxygen species-angiotensinogen axis plays some roles in development of IgAN at the early stage and will provide supportive foundation of effectiveness of the renin-angiotensin system blockade in IgAN.

Kidney-specific enhancement of ANG II stimulates endogenous intrarenal angiotensinogen in gene-targeted mice.

This study was performed in transgenic mice to test the hypothesis that the selective intrarenal overproduction of ANG II increases intrarenal mouse (m) angiotensinogen (AGT) expression. We used the following three groups: 1) single transgenic mice (group A, n = 14) expressing human (h) AGT only in the kidney, 2) double-transgenic mice (group D, n = 13) expressing human renin systemically in addition to hAGT only in the kidney, and 3) wild-type (group W, n = 12) mice. Exogenous hAGT protein is inactive in group A because endogenous mouse renin cannot cleave hAGT to ANG I because of a high species specificity. All mice were monitored from 12 to 18 wk of age. Systolic blood pressure progressively increased from 116 +/- 5 mmHg (12 wk) to 140 +/- 7 (18 wk) in group D. This increase was not observed in groups A or W. Intrarenal hAGT levels were similar in groups A and D; however, hAGT was not detectable in kidneys of group W. Kidney ANG II levels were increased in group D (216 +/- 43 fmol/g) compared with groups A (117 +/- 16) and W (118 +/- 17). However, plasma ANG II concentrations were similar among the three groups. Endogenous renal mAGT mRNA was increased significantly in group D (1.46 +/- 0.19, ratio) compared with groups A (0.97 +/- 0.12) and W (1.00 +/- 0.08). Endogenous renal mAGT protein was also significantly increased in group D compared with groups A and W. Interstitial collagen-positive area, interstitial macrophage/monocyte infiltration, and afferent arteriolar wall thickness were increased significantly in group D compared with groups A and W. These data indicate for the first time that the selective stimulation of intrarenal production of ANG II from hAGT augments endogenous intrarenal mAGT mRNA and protein expression.

Generation and characterization of new monoclonal antibodies against human chymase.

We have succeeded in producing monoclonal antibodies directed against a wide variety of epitopes of human chymase by using two different immunogens: a recombinant human chymase-heparin mixture, and chymase alone. Hybridomas were screened by ELISA, and 7 clones were selected based on antibody titers. Epitopes were localized by Western blotting with a C-terminal-deletion series of chymase-GST fusion proteins, and it was possible to use the antibodies for Western blotting and immunohistochemistry. Dot-blot analysis for species specificity revealed that the MAbs bound canine chymase as well as human chymase, and that two of them also bound rodent chymases. These results indicate that the antibodies can be used for various immunological analyses in further investigations of chymase.