Description: The anti-inflammatory function of tanshinone IIA (TIIA), an active natural compound from Chinese herbal medicine Danshen, has been well recognized, and therefore TIIA has been widely used to treat various inflammatory conditions associated with cardiac and lung diseases. Mucin 1 (Muc1) plays important anti-inflammatory roles in resolution of acute lung inflammation. In this study, we investigated the effects of TIIA on LPS-induced acute lung inflammation, as well as its relationship to Muc1 expression in mouse lung and MUC1 in human alveolar epithelial cells. TIIA pretreatment significantly inhibited LPS-induced pulmonary inflammation in both Muc1 wild-type ( Muc1 +/+ ) and knockout ( Muc1 –/– ) mice, as manifested by reduced neutrophil infiltration and reduced TNF-α and keratinocyte chemoattractant levels in bronchoalveolar lavage fluid. The inhibitory effects of TIIA on airway inflammation were associated with reduced expression of Muc1 in Muc1 +/+ mouse lung. Moreover, pretreatment with TIIA significantly inhibited LPS-induced MUC1 expression and TNF-α release in A549 alveolar epithelial cells. TNF-α upregulated MUC1 mRNA and protein expression in A549 cells, which was inhibited by pretreatment with TIIA. The LPS-induced MUC1 expression was blocked when A549 cells were transfected with siRNA targeting for TNF-α receptor 1. Furthermore, TIIA inhibited LPS-induced nuclear translocation of NF-B and upregulation of Toll-like receptor 4 in A549 cells. Taken together, these results demonstrate that TIIA suppressed LPS-induced acute lung inflammation regardless of the presence of Muc1, and TIIA inhibited LPS- and TNF-α-induced MUC1/Muc1 expression in airway epithelial cells, suggesting that MUC1/Muc1 does not account for the mechanisms of the anti-inflammatory effects of TIIA in the airway.

Description: We hypothesized that potentiating the bioavailability of endothelial epoxyeicosatrienoic acids (EETs) via deletion of the gene for soluble epoxide hydrolase (sEH), or downregulation of sEH expression, enhances flow/shear stress-induced dilator responses (FID) of arterioles. With the use of male (M) and female (F) wild-type (WT) and sEH-knockout (KO) mice, isolated gracilis muscle arterioles were cannulated and pressurized at 80 mmHg. Basal tone and increases in diameter of arterioles as a function of perfusate flow (5, 10, 15, 20, and 25 μl/min) were recorded. The magnitude of FID was significantly smaller and associated with a greater arteriolar tone in M-WT than F-WT mice, revealing a sex difference in FID. This sex difference was abolished by deletion of the sEH gene, as evidenced by an enhanced FID in M-KO mice to a level comparable with those observed in F-KO and F-WT mice. These three groups of mice coincidentally exhibited an increased endothelial sensitivity to shear stress (smaller WSS 50 ) and were hypotensive. Endothelial EETs participated in the mediation of enhanced FID in M-KO, F-KO, and F-WT mice, without effects on FID of M-WT mice. Protein expression of sEH was downregulated by approximately fourfold in vessels of F-WT compared with M-WT mice, paralleled with greater vascular EET levels that were statistically comparable with those observed in both male and female sEH-KO mice. In conclusion, sex-different regulation of sEH accounts for sex differences in flow-mediated dilation of microvessels in gonadally intact mice.

Description: Phenotypic divergence in diet-induced obesity (DIO) and hepatic steatosis has been reported in two independently generated lines of L-Fabp –/– mice [New Jersey (NJ) L-Fabp –/– vs. Washington University (WU) L-Fabp –/– mice]. We performed side-by-side studies to examine differences between the lines and investigate the role of genetic background, intestinal microbiota, sex, and diet in the divergent phenotypes. Fasting-induced steatosis was attenuated in both L-Fabp –/– lines compared with C57BL/6J controls, with restoration of hepatic triglyceride levels following adenoviral L-Fabp rescue. Both lines were protected against DIO after high-saturated-fat diet feeding. Hepatic steatosis was attenuated in WU but not NJ L-Fabp –/– mice, although this difference between the lines disappeared upon antibiotic treatment and cohousing. In contrast, there was phenotypic divergence in L-Fabp –/– mice fed a high cocoa butter fat diet, with WU L-Fabp –/– mice, but not NJ L-Fabp –/– mice, showing protection against both DIO and hepatic steatosis, with some sex-dependent (female 〉 male) differences. Dense mapping revealed no evidence of unintended targeting, duplications, or deletions surrounding the Fabp1 locus in either line and only minor differences in mRNA expression of genes located near the targeted allele. However, a C57BL/6 substrain screen showed that the NJ L-Fabp –/– line contains ~40% C57BL/6N genomic DNA, despite reports that these mice were backcrossed six generations. Overall, these findings suggest that some of the phenotypic divergence between the two L-Fabp –/– lines may reflect unanticipated differences in genetic background, underscoring the importance of genetic background in phenotypic characterization.

Description: MAD2B, an anaphase-promoting complex/cyclosome (APC/C) inhibitor and a small subunit of DNA polymerase , is indispensible for mitotic checkpoint control and DNA repair. Previously, we established that MAD2B is expressed in glomerular and tubulointerstitial compartments and participates in high glucose-induced podocyte injury. However, its role in other renal diseases remains elusive. In the present study, we aim to illustrate the potential role of MAD2B in the pathogenesis of renal fibrosis. By immunofluorescence and Western blotting, we found MAD2B expression is obviously increased in tubulointerstitial fibrosis (TIF) patients and unilateral ureteral obstruction (UUO) mice. It is widely accepted that resident fibroblasts are the major source of collagen-producing myofibroblasts during TIF. Therefore, we evaluated the level of MAD2B in fibroblasts (NRK-49F) exposed to transforming growth factor (TGF)-β1 by immunoblotting and revealed that MAD2B is upregulated in a time-dependent manner. Intriguingly, SnoN, a transcriptional repressor of the TGF-β1/Smad signaling pathway, is decreased in TGF-β1-treated fibroblasts as well as the kidney cortex from TIF patients and UUO mice. Either in vitro or in vivo, local genetic depletion of MAD2B by lentiviral transfection could preserve SnoN abundance and suppress Smad3 phosphorylation, which finally dampens fibroblast activation, ECM accumulation, and alleviates the severity of TIF. However, the ubiquitin ligase APC/C is not involved in the MAD2B-mediated SnoN decline, although this process is ubiquitination dependent. In conclusion, our observation proposes that besides cell cycle management, MAD2B has a profibrotic role during fibroblast activation and TIF by suppressing SnoN expression. Targeting the MAD2B-SnoN pathway is a promising intervention for TIF.

Description: Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid via CYP/epoxygenases, which are catabolized by soluble epoxide hydrolase (sEH) and known to possess cardioprotective properties. To date, the role of sEH in the modulation of pressure-induced myogenic response/constriction in coronary arteries, an important regulatory mechanism in the coronary circulation, and the issue as to whether the disruption of the sEH gene affects the myogenic response sex differentially have never been addressed. To this end, experiments were conducted on male (M) and female (F) wild-type (WT) and sEH-knockout (KO) mice. Pressure-diameter relationships were assessed in isolated and cannulated coronary arteries. All vessels constricted in response to increases in intraluminal pressure from 60 to 120 mmHg. Myogenic vasoconstriction was significantly attenuated, expressed as an upward shift in the pressure-diameter curve of vessels, associated with higher cardiac EETs in M-KO, F-WT, and F-KO mice compared with M-WT controls. Blockade of EETs via exposure of vessels to 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) prevented the attenuated myogenic constriction in sEH-KO mice. In the presence of 14,15-EEZE, pressure-diameter curves of females presented an upward shift from those of males, exhibiting a sex-different phenotype. Additional administration of N -nitro- l -arginine methyl ester eliminated the sex difference in myogenic responses, leading to four overlapped pressure-diameter curves. Cardiac sEH was downregulated in F-WT compared with M-WT mice, whereas expression of endothelial nitric oxide synthase and CYP4A (20-HETE synthase) was comparable among all groups. In summary, in combination with NO, the increased EET bioavailability as a function of genetic deletion and/or downregulation of sEH accounts for the female-favorable attenuation of pressure-induced vasoconstriction.

Description: The mechanisms by which prostanoids contribute to the maintenance of whole body water homeostasis are complex and not fully understood. The present study demonstrates that an EP3-dependent feedback mechanism contributes to the regulation of water homeostasis under high-salt conditions. Rats on a normal diet and tap water were placed in metabolic cages and given either sulprostone (20 μg·kg –1 ·day –1 ) or vehicle for 3 days to activate EP3 receptors in the thick ascending limb (TAL). Treatment was continued for another 3 days in rats given either 1% NaCl in the drinking water or tap water. Sulprostone decreased expression of cyclooxygenase 2 (COX-2) expression by ~75% in TAL tubules from rats given 1% NaCl concomitant with a ~60% inhibition of COX-2-dependent PGE 2 levels in the kidney. Urine volume increased after ingestion of 1% NaCl but was reduced ~40% by sulprostone. In contrast, the highly selective EP3 receptor antagonist L-798106 (100 μg·kg –1 ·day –1 ), which increased COX-2 expression and renal PGE 2 production, increased urine volume in rats given 1% NaCl. Sulprostone increased expression of aquaporin-2 (AQP2) in the inner medullary collecting duct plasma membrane in association with an increase in phosphorylation at Ser269 and decrease in Ser261 phosphorylation; antagonism of EP3 with L-798106 reduced AQP2 expression. Thus, although acute activation of EP3 by PGE 2 in the TAL and collecting duct inhibits the Na-K-2Cl cotransporter and AQP2 activity, respectively, chronic activation of EP3 in vivo limits the extent of COX-2-derived PGE 2 synthesis, thereby mitigating the inhibitory effects of PGE 2 on these transporters and decreasing urine volume.

Description: Lung infections are major causes of acute lung injury (ALI), with limited effective treatment available. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is an essential adaptor regulating Toll-like receptors (TLRs). We recently identified Cullin-5 (Cul-5) as a prominent component in the regulation of TRAF6 polyubiquitination, but its physiological significance in ALI has not been explored. In this study, we investigated the potential role of Cul-5 in regulating ALI using mice receiving intratracheal instillation of LPS. We observed that Cul-5-deficient mice displayed reduced lung injury compared with wild-type mice as evidenced by histological analysis, alveolar neutrophil infiltration, and lung liquid accumulation. In addition, inflammatory cytokine expression in bronchoalveolar lavage fluid and lung tissue was also markedly reduced in LPS-treated Cul-5-deficient mice. Interestingly, intratracheal adoptive transfer of Cul-5 +/– but not Cul-5 +/+ macrophages attenuated neutrophil recruitment, alveolar inflammation, and loss of barrier function in LPS-challenged wild-type mice. Finally, we demonstrated that Cul-5 neddylation following LPS exposure induced Cul-5 and TRAF6 interaction and, thereby, TFAR6 polyubiquitination, leading to NF-B activation and generation of proinflammatory cytokines. Our data show that neural precursor cell expressed developmentally downregulated protein 8 (Nedd8) modification of Cul-5 is required for its interaction with TRAF6 and activation of the TLR4-TRAF6 signaling pathway in LPS-induced ALI, a feature that may be explored for therapeutic intervention.

Description: We have previously reported that epoxyeicosatrienoic acid (EET) has multiple beneficial effects on vascular function; in addition to its antiapoptotic action, it increases insulin sensitivity and inhibits inflammation. To uncover the signaling mechanisms by which EET reduces cardiomyopathy, we hypothesized that EET infusion might ameliorate obesity-induced cardiomyopathy by improving heme oxygenase (HO)-1, Wnt1, thermogenic gene levels, and mitochondrial integrity in cardiac tissues and improved pericardial fat phenotype. EET reduced levels of fasting blood glucose and proinflammatory adipokines, including nephroblastoma overexpressed (NOV) signaling, while increasing echocardiographic fractional shortening and O 2 consumption. Of interest, we also noted a marked improvement in mitochondrial integrity, thermogenic genes, and Wnt 1 and HO-1 signaling mechanisms. Knockout of peroxisome proliferator-activated receptor- coactivator-1α (PGC-1α) in EET-treated mice resulted in a reversal of these beneficial effects including a decrease in myocardial Wnt1 and HO-1 expression and an increase in NOV. To further elucidate the effects of EET on pericardial adipose tissues, we observed EET treatment increases in adiponectin, PGC-1α, phospho-AMP-activated protein kinase, insulin receptor phosphorylation, and thermogenic genes, resulting in a "browning" pericardial adipose phenotype under high-fat diets. Collectively, these experiments demonstrate that an EET agonist increased Wnt1 and HO-1 signaling while decreasing NOV pathways and the progression of cardiomyopathy. Furthermore, this report presents a portal into potential therapeutic approaches for the treatment of heart failure and metabolic syndrome. NEW & NOTEWORTHY The mechanism by which EET acts on obesity-induced cardiomyopathy is unknown. Here, we describe a previously unrecognized function of EET infusion that inhibits nephroblastoma overexpressed (NOV) levels and activates Wnt1, hence identifying NOV inhibition and enhanced Wnt1 expression as novel pharmacological targets for the prevention and treatment of cardiomyopathy and heart failure. Listen to this article's corresponding podcast at http://ajpheart.physiology.org/content/early/2017/05/31/ajpheart.00093.2017 .

Description: In mice, myocardial hypertrophic preconditioning (HP), which is produced by the removal of short-term transverse aortic constriction (TAC), was recently reported to render the heart resistant to hypertrophic responses induced by subsequent reconstriction (Re-TAC). However, there is no efficient noninvasive method for ensuring that the repeated aortic manipulations were successfully performed. We previously demonstrated that ultrasound biomicroscopy (UBM) is a noninvasive and effective approach for predicting TAC success. Here, we investigated the value of UBM for serial predictions of load conditions in establishing a murine HP model. C57BL/6J mice were subjected to a sham operation, TAC, or Re-TAC, and the peak flow velocity at the aortic banding site (PVb) was measured by UBM. Left ventricular end-systolic pressure (LVESP) was examined by micromanometric catheterization. The PVb was positively associated with LVESP ( R 2 = 0.8204, P 〈 0.001, for TAC at 3 days and R 2 = 0.7746, P 〈 0.001, for Re-TAC at 4 wk). PVb and LVESP values were markedly elevated after aortic banding, became attenuated to the sham-operated level after debanding, and increased after aortic rebanding. The cardiac hypertrophic responses were examined by UBM, histology, RT-PCR, and Western blot analysis. Four weeks after the last operation, with PVb ≥ 3.5 m/s as an indicator of successful aortic constriction, Re-TAC mice showed less cardiac hypertrophy, fetal gene expression, and ERK1/2 activation than TAC mice. Therefore, we successfully established a UBM protocol for the serial assessment of aortic flow and the prediction of LVESP during repeated aortic manipulations in mice, which might be useful for noninvasive evaluations of the murine HP model. NEW & NOTEWORTHY We successfully developed an ultrasound biomicroscopy protocol for the serial assessment of aortic bandings and the relevant left ventricular pressure in a murine model of cardiac hypertrophic preconditioning. The protocol may be of great importance in the successful establishment of the hypertrophic preconditioning model for further mechanistic and pharmacological studies.

Description: Adiponectin (APN), the most abundant adipocyte-secreted adipokine, regulates energy homeostasis and exerts well-characterized insulin-sensitizing properties. The peripheral or central effects of APN regulating bone metabolism are beginning to be explored but are still not clearly understood. In the present study, we found that APN-knockout (APN-KO) mice fed a normal diet exhibited decreased trabecular structure and mineralization and increased bone marrow adiposity compared with wild-type (WT) mice. APN intracerebroventricular infusions decreased uncoupling protein 1 (UCP1) expression in brown adipose tissue, epinephrine and norepinephrine serum levels, and osteoclast numbers, whereas osteoblast osteogenic marker expression and trabecular bone mass increased in APN-KO and WT mice. In addition, centrally administered APN increased hypothalamic tryptophan hydroxylase 2 (TPH2), cocaine- and amphetamine-regulated transcript (CART), and 5-hydroxytryptamine (serotonin) receptor 2C (Htr2C) expressions but decreased hypothalamic cannabinoid receptor-1 expression. Treatment of immortalized mouse neurons with APN demonstrated that APN-mediated effects on TPH2, CART, and Htr2C expression levels were abolished by downregulating adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif (APPL)-1 expression. Pharmacological increase in sympathetic activity stimulated adipogenic differentiation of bone marrow stromal cells (BMSC) and reversed APN-induced expression of the lysine-specific demethylases involved in regulating their commitment to the osteoblastic lineage. In conclusion, we found that APN regulates bone metabolism via central and peripheral mechanisms to decrease sympathetic tone, inhibit osteoclastic differentiation, and promote osteoblastic commitment of BMSC.