Dual functions of angiopoietin-like protein 2 signaling in tumor progression and anti-tumor immunity.

Angiopoietin-like protein 2 (ANGPTL2) is a secreted glycoprotein homologous to angiopoietins. Previous studies suggest that tumor cell-derived ANGPTL2 has tumor-promoting function. Here, we conducted mechanistic analysis comparing ANGPTL2 function in cancer progression in a murine syngeneic model of melanoma and a mouse model of translocation renal cell carcinoma (tRCC). ANGPTL2 deficiency in tumor cells slowed tRCC progression, supporting a tumor-promoting role. However, systemic ablation of ANGPTL2 accelerated tRCC progression, supporting a tumor-suppressing role. The syngeneic model also demonstrated a tumor-suppressing role of ANGPTL2 in host tumor microenvironmental cells. Furthermore, the syngeneic model showed that PDGFRα+ fibroblasts in the tumor microenvironment express abundant ANGPTL2 and contribute to tumor suppression. Moreover, host ANGPTL2 facilitates CD8+ T-cell cross-priming and enhances anti-tumor immune responses. Importantly, ANGPTL2 activates dendritic cells through PIR-B-NOTCH signaling and enhances tumor vaccine efficacy. Our study provides strong evidence that ANGPTL2 can function in either tumor promotion or suppression, depending on what cell type it is expressed in.

Lung group 2 innate lymphoid cells differentially depend on local IL-7 for their distribution, activation, and maintenance in innate and adaptive immunity-mediated airway inflammation.

Interleukin-7 (IL-7) is a cytokine critical for the development and maintenance of group 2 innate lymphoid cells (ILC2s). ILC2s are resident in peripheral tissues such as the intestine and lung. However, whether IL-7 produced in the lung plays a role in the maintenance and function of lung ILC2s during airway inflammation remains unknown. IL-7 was expressed in bronchoalveolar epithelial cells and lymphatic endothelial cells (LECs). To investigate the role of local IL-7 in lung ILC2s, we generated two types of IL-7 conditional knockout (IL-7cKO) mice: Sftpc-Cre (SPC-Cre) IL-7cKO mice specific for bronchial epithelial cells and type 2 alveolar epithelial cells and Lyve1-Cre IL-7cKO mice specific for LECs. In steady state, ILC2s were located near airway epithelia, although lung ILC2s were unchanged in the two lines of IL-7cKO mice. In papain-induced airway inflammation dependent on innate immunity, lung ILC2s localized near bronchia via CCR4 expression, and eosinophil infiltration and type 2 cytokine production were reduced in SPC-Cre IL-7cKO mice. In contrast, in house dust mite (HDM)-induced airway inflammation dependent on adaptive immunity, lung ILC2s localized near lymphatic vessels via their CCR2 expression 2 weeks after the last challenge. Furthermore, lung ILC2s were decreased in Lyve1-Cre IL-7cKO mice in the HDM-induced inflammation because of decreased cell survival and proliferation. Finally, administration of anti-IL-7 antibody attenuated papain-induced inflammation by suppressing the activation of ILC2s. Thus, this study demonstrates that IL-7 produced by bronchoalveolar epithelial cells and LECs differentially controls the activation and maintenance of lung ILC2s, where they are localized in airway inflammation.

Plasma ANGPTL8 Levels and Risk for Secondary Cardiovascular Events in Japanese Patients With Stable Coronary Artery Disease Receiving Statin Therapy.

BACKGROUND: The ability to predict secondary cardiovascular events could improve health of patients undergoing statin treatment. Circulating ANGPTL8 (angiopoietin-like protein 8) levels, which positively correlate with proatherosclerotic lipid profiles, activate the pivotal proatherosclerotic factor ANGPTL3. Here, we assessed potential association between circulating ANGPTL8 levels and risk of secondary cardiovascular events in statin-treated patients. METHODS: We conducted a biomarker study with a case-cohort design, using samples from a 2018 randomized control trial known as randomized evaluation of high-dose (4 mg/day) or low-dose (1 mg/day) lipid-lowering therapy with pitavastatin in coronary artery disease (REAL-CAD [Randomized Evaluation of Aggressive or Moderate Lipid-Lowering Therapy With Pitavastatin in Coronary Artery Disease])." From that study's full analysis set (n=12 413), we selected 2250 patients with stable coronary artery disease (582 with the primary outcome, 1745 randomly chosen, and 77 overlapping subjects). A composite end point including cardiovascular-related death, nonfatal myocardial infarction, nonfatal ischemic stroke, or unstable angina requiring emergent admission was set as a primary end point. Circulating ANGPTL8 levels were measured at baseline and 6 months after randomization. RESULTS: Over a 6-month period, ANGPTL8 level changes significantly decreased in the high-dose pitavastatin group, which showed 19% risk reduction of secondary cardiovascular events compared with the low-dose group in the REAL-CAD [Randomized Evaluation of Aggressive or Moderate Lipid-Lowering Therapy With Pitavastatin in Coronary Artery Disease] study. In the highest quartiles, relative increases in ANGPTL8 levels were significantly associated with increased risk for secondary cardiovascular events, after adjustment for several cardiovascular disease risk factors and pitavastatin treatment (hazard ratio in Q4, 1.67 [95% CI, 1.17-2.39). Subgroup analyses showed relatively strong relationships between relative ANGPTL8 increases and secondary cardiovascular events in the high-dose pitavastatin group (hazard ratio in Q4, 2.07 [95% CI, 1.21-3.55]) and in the low ANGPTL8 group at baseline (166

Fasudil inhibits the expression of C/EBP homologous protein to protect against liver injury in acetaminophen-overdosed mice.

Acetaminophen (APAP) overdoses can cause severe liver injury. In this study, the protective effect of fasudil against APAP-induced liver injury was investigated. APAP (400 mg/kg) was administered to male C57BL/6J mice to induce liver injury, and fasudil (20 or 40 mg/kg) was injected 30 min before APAP administration. Fasudil markedly suppressed APAP-induced elevation in serum transaminase activity and hepatic necrosis and significantly reduced an increase in nitrotyrosine and DNA fragmentation. However, fasudil did not affect cytochrome P450 2E1 expression, N-acetyl-p-benzoquinone imine production or c-jun N-terminal kinase activation. In contrast, fasudil significantly inhibited an APAP-induced increase in expression of the transcription factor C/EBP homologous protein (CHOP) in the liver, accompanied by transcriptional suppression of ER stress-related molecules such as Ero1α, Atf4 and Grp78. These findings indicate that suppression of CHOP expression by fasudil exhibits a remarkable protective effect against APAP liver injury by regulating ER stress. We suggest that fasudil is a promising therapeutic candidate for treating APAP-induced liver injury.

ANGPTL2 expression in the intestinal stem cell niche controls epithelial regeneration and homeostasis.

The intestinal epithelium continually self-renews and can rapidly regenerate after damage. Dysregulation of intestinal epithelial homeostasis leads to severe inflammatory bowel disease. Additionally, aberrant signaling by the secreted protein angiopoietin-like protein 2 (ANGPTL2) causes chronic inflammation in a variety of diseases. However, little is known about the physiologic role of ANGPTL2 in normal tissue homeostasis and during wound repair following injury. Here, we assessed ANGPTL2 function in intestinal physiology and disease in vivo Although intestinal development proceeded normally in Angptl2-deficient mice, expression levels of the intestinal stem cell (ISC) marker gene Lgr5 decreased, which was associated with decreased transcriptional activity of ß-catenin in Angptl2-deficient mice. Epithelial regeneration after injury was significantly impaired in Angptl2-deficient relative to wild-type mice. ANGPTL2 was expressed and functioned within the mesenchymal compartment cells known as intestinal subepithelial myofibroblasts (ISEMFs). ANGPTL2 derived from ISEMFs maintained the intestinal stem cell niche by modulating levels of competing signaling between bone morphogenetic protein (BMP) and ß-catenin. These results support the importance of ANGPTL2 in the stem cell niche in regulating stemness and epithelial wound healing in the intestine.

Family with sequence similarity 13, member A modulates adipocyte insulin signaling and preserves systemic metabolic homeostasis.

Adipose tissue dysfunction is causally implicated in the impaired metabolic homeostasis associated with obesity; however, detailed mechanisms underlying dysregulated adipocyte functions in obesity remain to be elucidated. Here we searched for genes that provide a previously unknown mechanism in adipocyte metabolic functions and identified family with sequence similarity 13, member A (Fam13a) as a factor that modifies insulin signal cascade in adipocytes. Fam13a was highly expressed in adipose tissue, predominantly in mature adipocytes, and its expression was substantially reduced in adipose tissues of obese compared with lean mice. We revealed that Fam13a accentuated insulin signaling by recruiting protein phosphatase 2A with insulin receptor substrate 1 (IRS1), leading to protection of IRS1 from proteasomal degradation. We further demonstrated that genetic loss of Fam13a exacerbated obesity-related metabolic disorders, while targeted activation of Fam13a in adipocytes ameliorated it in association with altered adipose tissue insulin sensitivity in mice. Our data unveiled a previously unknown mechanism in the regulation of adipocyte insulin signaling by Fam13a and identified its significant role in systemic metabolic homeostasis, shedding light on Fam13a as a pharmacotherapeutic target to treat obesity-related metabolic disorders.

Circulating angiopoietin-like protein 2 levels and mortality risk in patients receiving maintenance hemodialysis: a prospective cohort study.

BACKGROUND: Patients undergoing hemodialysis treatment have a poor prognosis, as many develop premature aging. Systemic inflammatory conditions often underlie premature aging phenotypes in uremic patients. We investigated whether angiopoietin-like protein 2 (ANGPTL 2), a factor that accelerates the progression of aging-related and noninfectious inflammatory diseases, was associated with increased mortality risk in hemodialysis patients. METHODS: We conducted a multicenter prospective cohort study of 412 patients receiving maintenance hemodialysis and evaluated the relationship between circulating ANGPTL2 levels and the risk for all-cause mortality. Circulating ANGPTL2 levels were log-transformed to correct for skewed distribution and analyzed as a continuous variable. RESULTS: Of 412 patients, 395 were included for statistical analysis. Time-to-event data analysis showed high circulating ANGPTL2 levels were associated with an increased risk for all-cause mortality after adjustment for age, sex, hemodialysis vintage, nutritional status, metabolic parameters and circulating high-sensitivity C-reactive protein levels {hazard ratio [HR] 2.04 [95% confidence interval (CI) 1.10-3.77]}. High circulating ANGPTL2 levels were also strongly associated with an increased mortality risk, particularly in patients with a relatively benign prognostic profile [HR 3.06 (95% CI 1.86-5.03)]. Furthermore, the relationship between circulating ANGPTL2 levels and mortality risk was particularly strong in patients showing few aging-related phenotypes, such as younger patients [HR 7.99 (95% CI 3.55-18.01)], patients with a short hemodialysis vintage [HR 3.99 (95% CI 2.85-5.58)] and nondiabetic patients [HR 5.15 (95% CI 3.19-8.32)]. CONCLUSION: We conclude that circulating ANGPTL2 levels are positively associated with mortality risk in patients receiving maintenance hemodialysis and that ANGPTL2 could be a unique marker for the progression of premature aging and subsequent mortality risk in uremic patients, except those with significant aging-related phenotypes.

Age-dependent increase in angiopoietin-like protein 2 accelerates skeletal muscle loss in mice.

Skeletal muscle atrophy, or sarcopenia, is commonly observed in older individuals and in those with chronic disease and is associated with decreased quality of life. There is recent medical and broad concern that sarcopenia is rapidly increasing worldwide as populations age. At present, strength training is the only effective intervention for preventing sarcopenia development, but it is not known how this exercise regimen counteracts this condition. Here, we report that expression of the inflammatory mediator angiopoietin-like protein 2 (ANGPTL2) increases in skeletal muscle of aging mice. Moreover, in addition to exhibiting increased inflammation and accumulation of reactive oxygen species (ROS), denervated atrophic skeletal muscles in a mouse model of denervation-induced muscle atrophy had increased ANGPTL2 expression. Interestingly, mice with a skeletal myocyte-specific Angptl2 knockout had attenuated inflammation and ROS accumulation in denervated skeletal muscle, accompanied by increased satellite cell activity and inhibition of muscular atrophy compared with mice harboring wildtype Angptl2 Moreover, consistent with these phenotypes, wildtype mice undergoing exercise training displayed decreased ANGPTL2 expression in skeletal muscle. In conclusion, ANGPTL2 up-regulation in skeletal myocytes accelerates muscle atrophy, and exercise-induced attenuation of ANGPTL2 expression in those tissues may partially explain how exercise training prevents sarcopenia.

MicroRNA-204-5p: A novel candidate urinary biomarker of Xp11.2 translocation renal cell carcinoma.

Xp11.2 translocation renal cell carcinoma (Xp11 tRCC) is a rare sporadic pediatric kidney cancer caused by constitutively active TFE3 fusion proteins. Tumors in patients with Xp11 tRCC tend to recur and undergo frequent metastasis, in part due to lack of methods available to detect early-stage disease. Here we generated transgenic (Tg) mice overexpressing the human PRCC-TFE3 fusion gene in renal tubular epithelial cells, as an Xp11 tRCC mouse model. At 20 weeks of age, mice showed no histological abnormalities in kidney but by 40 weeks showed Xp11 tRCC development and related morphological and histological changes. MicroRNA (miR)-204-5p levels in urinary exosomes of 40-week-old Tg mice showing tRCC were significantly elevated compared with levels in control mice. MicroRNA-204-5p expression also significantly increased in primary renal cell carcinoma cell lines established both from Tg mouse tumors and from tumor tissue from 2 Xp11 tRCC patients. All of these lines secreted miR-204-5p-containing exosomes. Notably, we also observed increased miR-204-5p levels in urinary exosomes in 20-week-old renal PRCC-TFE3 Tg mice prior to tRCC development, and those levels were equivalent to those in 40-week-old Tg mice, suggesting that miR-204-5p increases follow expression of constitutively active TFE3 fusion proteins in renal tubular epithelial cells prior to overt tRCC development. Finally, we confirmed that miR-204-5p expression significantly increases in noncancerous human kidney cells after overexpression of a PRCC-TFE3 fusion gene. These findings suggest that miR-204-5p in urinary exosomes could be a useful biomarker for early diagnosis of patients with Xp11 tRCC.

Loss of Endogenous HMGB2 Promotes Cardiac Dysfunction and Pressure Overload-Induced Heart Failure in Mice.

BACKGROUND: The rapid increase in the number of heart failure (HF) patients in parallel with the increase in the number of older people is receiving attention worldwide. HF not only increases mortality but decreases quality of life, creating medical and social problems. Thus, it is necessary to define molecular mechanisms underlying HF development and progression. HMGB2 is a member of the high-mobility group superfamily characterized as nuclear proteins that bind DNA to stabilize nucleosomes and promote transcription. A recent in vitro study revealed that HMGB2 loss in cardiomyocytes causes hypertrophy and increases HF-associated gene expression. However, it's in vivo function in the heart has not been assessed. Methods and Results: Western blotting analysis revealed increased HMGB2 expression in heart tissues undergoing pressure overload by transverse aorta constriction (TAC) in mice. Hmgb2 homozygous knockout (Hmgb2-/-) mice showed cardiac dysfunction due to AKT inactivation and decreased sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a activity. Compared to wild-type mice, Hmgb2-/- mice had worsened cardiac dysfunction after TAC surgery, predisposing mice to HF development and progression. CONCLUSIONS: This study demonstrates that upregulation of cardiac HMGB2 is an adaptive response to cardiac stress, and that loss of this response could accelerate cardiac dysfunction, suggesting that HMGB2 plays a cardioprotective role.

Neuregulin-4 is an angiogenic factor that is critically involved in the maintenance of adipose tissue vasculature.

Adipose tissue (AT) contains well-developed vascular networks. Pathological AT expansion often accompany the reduction in AT blood vessels, which further exacerbates adipocyte dysfunction due to hypoxia; however, it remains unclear whether AT vascular rarefaction is simply secondary to adipocyte hypertrophy, or if there is an actively regulated pathway that mediates impaired AT angiogenesis in obesity. We searched for growth factors whose expression in AT is down-regulated in obesity; accordingly, we identified neuregulin-4 (Nrg4), a member of the EGF family of proteins. Nrg4 is highly and preferentially expressed in healthy adipocytes, while its expression was substantially reduced in obesity. Nrg4 activated endothelial angiogenic functions and angiogenesis both in vitro and in vivo. Genetic loss of Nrg4 caused reduction in brown and white AT blood vessels, and induced overweight even while consuming normal chow. Conditional knockout of Nrg4 in brown adipocytes caused blood vessel reduction in brown but not in white AT, and was sufficient to induce obese phenotype. Our data demonstrated that Nrg4 plays a critical role in maintaining AT vasculature and its metabolic functions. Considering the substantial reduction of Nrg4 in obesity, disruption of Nrg4-mediated angiogenesis could be an active mechanism for the obesity-associated vascular rarefaction in AT, and thus Nrg4 is an attracting pharmacotherapeutic target in the prevention and/or treatment of obesity-related metabolic disorders.

Circulating ANGPTL2 Levels Increase in Humans and Mice Exhibiting Cardiac Dysfunction.

BACKGROUND: Recently, it was reported that angiopoietin-like protein 2 (ANGPTL2) secreted from a pathologically stressed heart accelerates cardiac dysfunction in an autocrine/paracrine manner, and that suppression of ANGPTL2 production in the heart restored cardiac function and myocardial energy metabolism, thereby blocking heart failure (HF) development. Interestingly, circulating ANGPTL2 concentrations reportedly increase in HF patients, suggesting a possible endocrine effect on cardiac dysfunction. However, it remains unclear why circulating ANGPTL2 increases in those subjects and whether circulating ANGPTL2 alters cardiac function in an endocrine manner.Methods and Results:It was found that circulating ANGPTL2 levels are positively correlated with left atrial diameter and pulmonary capillary wedge pressure, and are inversely proportional to the percent of ejection fraction in patients with dilated cardiomyopathy. Furthermore, in mice, circulating ANGPTL2 concentrations increased as HF developed following transverse aorta constriction (TAC), and were inversely correlated with the percent of fractional shortening. Interestingly, although circulating ANGPTL2 concentrations significantly increased in transgenic mice overexpressing keratinocyte-derived ANGPTL2, no pathological cardiac remodeling was seen. Furthermore, it was observed that there was no difference in HF development between transgenic mice and controls following TAC surgery. CONCLUSIONS: Circulating ANGPTL2 levels increase in subjects experiencing cardiac dysfunction. However, circulating ANGPTL2 does not promote cardiac dysfunction in an endocrine manner, and increased levels of circulating ANGPTL2 seen during HF are a secondary effect of increased ANGPTL2 secretion from stressed hearts in HF pathologies.

Mice Deficient in Angiopoietin-like Protein 2 (Angptl2) Gene Show Increased Susceptibility to Bacterial Infection Due to Attenuated Macrophage Activity.

Macrophages play crucial roles in combatting infectious disease by promoting inflammation and phagocytosis. Angiopoietin-like protein 2 (ANGPTL2) is a secreted factor that induces tissue inflammation by attracting and activating macrophages to produce inflammatory cytokines in chronic inflammation-associated diseases such as obesity-associated metabolic syndrome, atherosclerosis, and rheumatoid arthritis. Here, we asked whether and how ANGPTL2 activates macrophages in the innate immune response. ANGPTL2 was predominantly expressed in proinflammatory mouse bone marrow-derived differentiated macrophages (GM-BMMs) following GM-CSF treatment relative to anti-inflammatory cells (M-BMMs) established by M-CSF treatment. Expression of the proinflammatory markers IL-1ß, IL-12p35, and IL-12p40 significantly decreased in GM-BMMs from Angptl2-deficient compared with wild-type (WT) mice, suggestive of attenuated proinflammatory activity. We also report that ANGPTL2 inflammatory signaling is transduced through integrin α5ß1 rather than through paired immunoglobulin-like receptor B. Interestingly, Angptl2-deficient mice were more susceptible to infection with Salmonella enterica serovar Typhimurium than were WT mice. Moreover, nitric oxide (NO) production by Angptl2-deficient GM-BMMs was significantly lower than in WT GM-BMMs. Collectively, our findings suggest that macrophage-derived ANGPTL2 promotes an innate immune response in those cells by enhancing proinflammatory activity and NO production required to fight infection.

ANGPTL2　- A New Causal Player in Accelerating Heart Disease Development in the Aging.

In parallel with the increase in the number of elderly people worldwide, the number of patients with heart disease is also rapidly increasing. Of the heart diseases, cardiovascular disease (CVD) and heart failure (HF) are strongly associated with adverse health outcomes that decrease productivity in later years. Recently, ANGPTL2, a secreted glycoprotein and member of the angiopoietin-like protein family, has received attention as a causal player in the development of CVD and HF. Prolonged ANGPTL2 autocrine/paracrine signaling in vascular tissue leads to chronic inflammation and pathologic tissue remodeling, accelerating CVD development. Excess ANGPTL2 autocrine/paracrine signaling induced in the pathologically stressed heart accelerates cardiac dysfunction by decreasing myocardial energy metabolism. Conversely, ANGPTL2 inactivation in vascular tissue and the heart delays development or progression of CVD and HF, respectively. Moreover, there is increased evidence for an association between elevated circulating ANGPTL2 levels and CVD and HF. Interestingly, ANGPTL2 expression is also associated with cellular senescence, which may promote premature aging and development of aging-associated diseases, including CVD and HF. Overall, ANGPTL2 autocrine/paracrine signaling is a new factor in accelerating heart disease development in the aging. Here, we focus on current topics relevant to ANGPTL2 function in heart disease.

Synthesis and Characterization of 8-Nitroguanosine 3',5'-Cyclic Monophosphorothioate Rp-Isomer as a Potent Inhibitor of Protein Kinase G1α.

Guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinases (PKG) are kinases regulating diverse physiological functions including vascular smooth muscle relaxation, neuronal synaptic plasticity, and platelet activities. Certain PKG inhibitors, such as Rp-diastereomers of derivatives of guanosine 3',5'-cyclic monophosphorothioate (Rp-cGMPS), have been designed and used to study PKG-regulated cell signaling. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is an endogenous cGMP derivative formed as a result of excess production of reactive oxygen species and nitric oxide. 8-Nitro-cGMP causes persistent activation of PKG1α through covalent attachment of cGMP moieties to cysteine residues of the enzyme (i.e., the process called protein S-guanylation). In this study, we synthesized a nitrated analogue of Rp-cGMPS, 8-nitroguanosine 3',5'-cyclic monophosphorothioate Rp-isomer (Rp-8-nitro-cGMPS), and investigated its effects on PKG1α activity. We synthesized Rp-8-nitro-cGMPS by reacting Rp-8-bromoguanosine 3',5'-cyclic monophosphorothioate (Rp-8-bromo-cGMPS) with sodium nitrite. Rp-8-Nitro-cGMPS reacted with the thiol compounds cysteine and glutathione to form Rp-8-thioalkoxy-cGMPS adducts to a similar extent as did 8-nitro-cGMP. As an important finding, a protein S-guanylation-like modification was clearly observed, by using Western blotting, in the reaction between recombinant PKG1α and Rp-8-nitro-cGMPS. Rp-8-Nitro-cGMPS inhibited PKG1α activity with an inhibitory constant of 22 µM in a competitive manner. An organ bath assay with mouse aorta demonstrated that Rp-8-nitro-cGMPS inhibited vascular relaxation induced by acetylcholine or 8-bromo-cGMP more than Rp-8-bromo-cGMPS did. These findings suggest that Rp-8-nitro-cGMPS inhibits PKG through induction of an S-guanylation-like modification by attaching the Rp-cGMPS moiety to the enzyme. Additional study is warranted to explore the potential application of Rp-8-nitro-cGMPS to biochemical and therapeutic research involving PKG1α activation.

Interstitial pneumonia induced by bleomycin treatment is exacerbated in Angptl2-deficient mice.

Angiopoietin-like protein 2 (ANGPTL2) is a chronic inflammatory mediator that, when deregulated, is associated with various pathologies. However, little is known about its activity in lung. To assess a possible lung function, we generated a rabbit monoclonal antibody that specifically recognizes mouse ANGPTL2 and then evaluated protein expression in mouse lung tissue. We observed abundant ANGPTL2 expression in both alveolar epithelial type I and type II cells and in resident alveolar macrophages under normal conditions. To assess ANGPTL2 function, we compared lung phenotypes in Angptl2 knockout (KO) and wild-type mice but observed no overt changes. We then generated a bleomycin-induced interstitial pneumonia model using wild-type and Angptl2 KO mice. Bleomycin-treated wild-type mice showed specifically upregulated ANGPTL2 expression in areas of severe fibrosing interstitial pneumonia, while Angptl2 KO mice developed more severe lung fibrosis than did comparably treated wild-type mice. Lung fibrosis seen following bone marrow transplant was comparable in wild-type or Angptl2 KO mice treated with bleomycin, suggesting that Angptl2 loss in myeloid cells does not underlie fibrotic phenotypes. We conclude that Angptl2 deficiency in lung epithelial cells and resident alveolar macrophages causes severe lung fibrosis seen following bleomycin treatment, suggesting that ANGPTL2 derived from these cell types plays a protective role against fibrosis in lung.

Angiopoietin-like protein 2 increases renal fibrosis by accelerating transforming growth factor-ß signaling in chronic kidney disease.

Renal fibrosis is a common pathological consequence of chronic kidney disease (CKD) with tissue fibrosis closely associated with chronic inflammation in numerous pathologies. However, molecular mechanisms underlying that association, particularly in the kidney, remain unclear. Here, we determine whether there is a molecular link between chronic inflammation and tissue fibrosis in CKD progression. Histological analysis of human kidneys indicated abundant expression of angiopoietin-like protein 2 (ANGPTL2) in renal tubule epithelial cells during progression of renal fibrosis. Numerous ANGPTL2-positive renal tubule epithelial cells colocalized with cells positive for transforming growth factor (TGF)-ß1, a critical mediator of tissue fibrosis. Analysis of M1 collecting duct cells in culture showed that TGF-ß1 increases ANGPTL2 expression by attenuating its repression through microRNA-221. Conversely, ANGPTL2 increased TGF-ß1 expression through α5ß1 integrin-mediated activation of extracellular signal-regulated kinase. Furthermore, ANGPTL2 deficiency in a mouse unilateral ureteral obstruction model significantly reduced renal fibrosis by decreasing TGF-ß1 signal amplification in kidney. Thus, ANGPTL2 and TGF-ß1 positively regulate each other as renal fibrosis progresses. Our study provides insight into molecular mechanisms underlying chronic inflammation and tissue fibrosis and identifies potential therapeutic targets for CKD treatment.

The Effect of Yokukansan, a Traditional Herbal Preparation Used for the Behavioral and Psychological Symptoms of Dementia, on the Drug-Metabolizing Enzyme Activities in Healthy Male Volunteers.

The concomitant use of herb and prescription medications is increasing globally. Herb-drug interactions are therefore a clinically important problem. Yokukansan (YKS), a Japanese traditional herbal medicine, is one of the most frequently used herbal medicines. It is effective for treating the behavioral and psychological symptoms of dementia. We investigated the potential effects of YKS on drug-metabolizing enzyme activities in humans. An open-label repeat-dose study was conducted in 26 healthy Japanese male volunteers (age: 22.7±2.3 years) with no history of smoking. An 8-h urine sample was collected after a 150-mg dose of caffeine and a 30-mg dose of dextromethorphan before and after the administration of YKS (2.5 g, twice a day for 1 week). The activities of cytochrome P450 (CYP) 1A2, CYP2D6, CYP3A, xanthine oxidase (XO) and N-acetyltransferase 2 (NAT2) were assessed based on the urinary metabolic indices of caffeine and dextromethorphan, and the urinary excretion ratio of 6ß-hydroxycortisol to cortisol. There were no statistically significant differences in the activities of the examined enzymes before or after the 7-d administration of YKS. Although further studies assessing the influence of YKS on the pharmacokinetics and pharmacodynamics of the substrates of the drug-metabolizing enzymes are needed to verify the present results, YKS is unlikely that a pharmacokinetic interaction will occur with concomitantly administered medications that are predominantly metabolized by the CYP1A2, CYP2D6, CYP3A, XO and NAT2.

IRAP deficiency attenuates diet-induced obesity in mice through increased energy expenditure.

UNLABELLED: Activation of the adipose renin-angiotensin system contributes to the development of obesity and metabolic syndrome. Insulin-regulated aminopeptidase (IRAP) has been identified a key regulator of GLUT4 transporter as well as angiotensin IV (AngIV) receptor (AT4R). Although AngII-AT1R axis appears as anorexigenic and as an effector of energy expenditure, the impact of AngIV-IRAP/AT4R axis on energy metabolism remains unknown. The aim was to determine the role of IRAP in energy metabolism in mice. METHODS AND RESULTS: In adipocyte culture, plasminogen activator inhibitor type 1 (PAI-1) expression levels were diminished in IRAP knockout (IRAP(-/-)) if compared with those of wild-type (C57Bl/6J, WT) mice. Mice were fed high-fat diet (32% fat) at age of 8 weeks. At the entry, body weight, body fat content, and parameters of saccharometabolism were similar between groups. However, IRAP(-/-) mice exhibited blunted body weight gain compared to that of WT mice, despite comparable food intake and physical activity. At 20weeks of age, IRAP(-/-) mice had 25% lower body weight than WT mice. Glucose and insulin tolerance tests revealed that the glucose disposal and the hypoglycemic effect of insulin were pronounced in IRAP(-/-) mice after a high fat diet. Indirect calorimetry demonstrated that whole-body oxygen consumption rates were significantly higher in IRAP(-/-) mice by 18% with mild hyperthermia. Analysis of brown adipose tissue (BAT) in IRAP(-/-) showed increased levels of uncoupling protein-1 (UCP-1) at basal level and adaptive thermogenesis was not impaired. CONCLUSIONS: IRAP deficiency may lead to suppression of PAI-1 expression in adipocytes and upregulation of UCP-1-mediated thermogenesis in BAT and increased energy expenditure to prevent the development of obesity, and these facts suggest a therapeutic potential of IRAP/AT4R blockade in diet-induced obesity.

The exacerbating roles of CCAAT/enhancer-binding protein homologous protein (CHOP) in the development of bleomycin-induced pulmonary fibrosis and the preventive effects of tauroursodeoxycholic acid (TUDCA) against pulmonary fibrosis in mice.

The purpose of this study was to evaluate the role of CCAAT/enhancer-binding protein homologous protein (CHOP), an important transcription factor that regulates the inflammatory reaction during the endoplasmic reticulum (ER) stress response, in the development of pulmonary fibrosis induced by bleomycin (BLM) in mice. An intratracheal injection of BLM transiently increased the expression of CHOP mRNA and protein in an early phase (days 1 and 3) in mice lungs. BLM-induced pulmonary fibrosis was significantly attenuated in Chop gene deficient (Chop KO) mice, compared with wild-type (WT) mice. Furthermore, the inflammatory reactions evaluated by protein concentration, the total number of leucocytes and neutrophils in the bronchoalveolar lavage fluid (BALF), the mRNA expression of interleukin 1b and caspase 11, and the apoptotic cell death were suppressed in Chop KO mice compared with those in WT mice. In addition, administration of tauroursodeoxycholic acid (TUDCA), a pharmacological agent that can inhibit CHOP expression, inhibited the BLM-induced pulmonary fibrosis and inflammation, and the increase in Chop mRNA expression in WT mice in a dose-dependent manner. These results suggest that the ER stress-induced transcription factor, CHOP, at least in part, plays an important role in the development of BLM-induced pulmonary fibrosis in mice, and that the inhibition of CHOP expression by a pharmacological agent, such as TUDCA, may be a promising strategy for the prevention of pulmonary fibrosis.