Citations

Mechanisms that mediate migration, homing and integration of blood outgrowth endothelial cells (BOEC) to sites of vascular injury are incompletely understood, although matrix metalloproteinases (MMPs) have been implicated in this process. Farnesoid X receptor (FXR) signaling pathway has recently been recognized to regulate vascular wall signaling. The aim of this study was to characterize the expression of FXR in human BOEC and examine signaling pathways relevant to BOEC migration. FXR mRNA and protein levels were detected by reverse‐transcriptase PCR and Western blot. Microarray analysis using a focused human endothelial cell biology array (Superarray) suggested that natural (Chenodeoxycholic acid (CDCA), 0.1–100μM) and synthetic (6‐ethylchenodeoxycholic acid (6‐ECDCA) 0.01–10μM) FXR ligands significantly increased MMP9 transcription which was confirmed by real time PCR and gelatin zymography. SiRNA based silencing of FXR prevented CDCA induced increases in MMP9 mRNA, while retroviral over‐expression of FXR in BOEC enhanced MMP9 mRNA levels. CDCA induced expression of Small Heterodimeric Partner (SHP), a downstream target of FXR. Retroviral over‐expression of SHP increased, whereas siRNA silencing of SHP inhibited CDCA induced MMP9 mRNA levels. Migration analysis using Boyden assay demonstrated that CDCA stimulated migration of BOEC which was blocked by transfection with FXR or MMP9 siRNA (p<0.05; n=5). These studies identify a novel signaling pathway by which a key protein important in cell homing and migration, MMP9, is regulated through a previously unrecognized FXR‐SHP pathway in BOEC. Funded by NIH Grant # DK59615

Epoxyeicosatrienoic acids (EETs) potently and stereospecifically activate the cardiac ATP‐sensitive K + (Kir6.2) channels by reducing channel sensitivity to ATP. In this study, we determined the effects of EETs on the binding of ATP to Kir6.2 by using 2,4,6‐trinitrophenyl (TNP)‐ATP, which increases fluorescence emission >8‐fold with binding to protein, and by using GST fusion protein containing Kir6.2. TNP‐ATP binds to the GST fusion protein containing the Kir6.2C‐terminus but not to that with the Kir6.2N‐terminus. Binding of TNP‐ATP to GST‐Kir6.2C (K D 5.0 μ M) was unchanged by 5 μ M 11,12‐EET (K D 4.4 μ M) but B max was reduced by half, suggesting non‐competitive inhibition of ATP binding. The effect of 11,12‐EET was dose‐dependent and 8,9‐ and 14,15‐EETs were equipotent inhibitors of TNP‐ATP binding to GST‐Kir6.2C. Arachidonic acid and 11,12‐DHET were not effective inhibitors, whereas the methyl ester of 11,12‐EET was as effective as 11,12‐EET in inhibiting TNP‐ATP binding to GST‐Kir6.2C. R195A mutation in GST‐Kir6.2C abolished the EET effects on TNP‐ATP binding, while the K185A mutation, which reduced ATP binding to Kir6.2, had no effect. These findings are consistent with previous patch clamp results that showed the epoxide group in EET and R195 in Kir6.2 are critical for K ATP channel activation by EET. We conclude that EETs bind to the C‐terminus of the K ATP channel and allosterically alter the ATP binding site, reducing the channel sensitivity to ATP.

Background and Aims During liver fibrosis, liver sinusoidal endothelial cells (LSECs) release angiocrine signals to recruit inflammatory cells into the liver. p300, a master regulator of gene transcription, is associated with pathological inflammatory response. Therefore, we examined how endothelial p300 regulates angiocrine signaling and inflammation related to portal hypertension and fibrogenesis. Approach and Results CCl4 or partial inferior vena cava ligation (pIVCL) was used to induce liver injury. Mice with LSEC‐specific p300 deletion (p300 LSECΔ/Δ) or C‐C motif chemokine ligand 2 (Ccl2) deficiency, nuclear factor kappa B (NFκB)–p50 knockout mice, and bromodomain containing 4 (BRD4) inhibitors in wild‐type mice were used to investigate mechanisms of inflammation regulation. Leukocytes were analyzed by mass cytometry by time‐of‐flight. Epigenetic histone marks were modified by CRISPR endonuclease‐deficient CRISPR‐associated 9‐fused with the Krüppel associated box domain (CRISPR‐dCas9‐KRAB)–mediated epigenome editing. Portal pressure and liver fibrosis were reduced in p300 LSECΔ/Δ mice compared to p300 fl/fl mice following liver injury. Accumulation of macrophages was also reduced in p300 LSECΔ/Δ mouse livers. Ccl2 was the most up‐regulated chemokine in injured LSECs, but its increase was abrogated in p300 LSECΔ/Δ mice. While the macrophage accumulation was increased in NFκB‐p50 knockout mice with enhanced NFκB activity, it was reduced in mice with LSEC‐specific Ccl2 deficiency and mice treated with specific BRD4 inhibitors. In vitro, epigenome editing of CCL2 enhancer and promoter regions by CRISPR‐dCas9‐KRAB technology repressed TNFα‐induced CCL2 transcription through H3K9 trimethylation. In contrast, TNFα activated CCL2 transcription by promoting p300 interaction with NFκB and BRD4, leading to histone H3 lysine 27 acetylation at CCL2 enhancer and promoter regions. Conclusions In summary, endothelial p300 interaction with NFκB and BRD4 increases CCL2 expression, leading to macrophage accumulation, portal hypertension, and liver fibrosis. Inhibition of p300 and its binding partners might serve as therapy in the treatment of liver diseases.

AbstractWe previously demonstrated that cancer‐associated fibroblasts (CAFs) promoted the proliferation of gallbladder cancer (GBC) cells, but the mechanism is not clear. Neuropilin‐1 (NRP‐1) plays an important role in various malignancies as transmembrane glycoprotein. Our goal was to reveal the relationship between CAFs and NRP‐1 and their potential functions in GBC. In this study, we found NRP‐1 was overexpressed in GBC tissue, associated with poor survival and was up‐regulated by CAFs. The cytokine array cluster analysis revealed IL‐8 secreted by CAFs facilitated the up‐regulation of NRP‐1 in tumour cells. NRP‐1 knockdown suppressed tumour growth in vivo. Gene expression microarray analysis showed 581 differentially regulated genes under NRP‐1 knockdown conditions. Ingenuity pathway analysis demonstrated that NRP‐1 knockdown may inhibit tumour progression by affecting cell proliferation. We then confirmed that NRP‐1 knockdown in NOZ and GBC‐SD cells significantly inhibited cell proliferation. Additionally, the IL‐8 mediated MDM2 and CCNA2 expression were affected by NRP‐1 knockdown. Our findings suggested that NRP‐1 was up‐regulated by CAF‐secreted IL‐8, which subsequently promoted GBC cell proliferation, and these molecules may serve as useful prognostic biomarkers and therapeutic targets for GBC.

OBJECTIVES: Severe alcoholic hepatitis (sAH) confers substantial mortality, but the disease course is difficult to predict. As iron parameters are attractive outcome predictors in other liver diseases, we tested their prognostic ability in sAH. METHODS: Serum ferritin, transferrin, iron, transferrin saturation, nontransferrin-bound iron, soluble transferrin receptor, and hepcidin were measured in 828 patients with sAH recruited prospectively through the STOPAH trial. The cohort was randomly divided into exploratory (n = 200) and validation sets (n = 628). RESULTS: Patients with sAH had diminished serum transferrin but increased transferrin saturation. Among iron parameters, baseline transferrin was the best predictor of 28-day (area under the receiver operated characteristic 0.72 [95% confidence interval 0.67–0.78]) and 90-day survival (area under the receiver operated characteristic 0.65 [0.61–0.70]). Transferrin's predictive ability was comparable with the composite scores, namely model of end-stage liver disease, Glasgow alcoholic hepatitis score, and discriminant function, and was independently associated with survival in multivariable analysis. These results were confirmed in a validation cohort. Transferrin did not correlate with markers of liver synthesis nor with non–transferrin-bound iron or soluble transferrin receptor (as markers of excess unbound iron and functional iron deficiency, respectively). DISCUSSION: In patients with sAH, serum transferrin predicts mortality with a performance comparable with commonly used composite scoring systems. Hence, this routinely available parameter might be a useful marker alone or as a component of prognostic models.

In carbon tetrachloride-induced liver cirrhosis, diminution of hepatic endothelial nitric oxide synthase (eNOS) activity may contribute to impaired hepatic vasodilation and portal hypertension. The mechanisms responsible for these events remain unknown; however, a role for the NOS-associated proteins caveolin and calmodulin has been postulated. The purpose of this study is to characterize the expression and cellular localization of the NOS inhibitory protein caveolin-1 in normal rat liver and to then examine the role of caveolin in conjunction with calmodulin in regulation of NOS activity in cholestatic portal hypertension. In normal liver, caveolin protein is expressed preferentially in nonparenchymal cells compared with hepatocytes as assessed by Western blot analysis of isolated cell preparations. Additionally, within the nonparenchymal cell populations, caveolin expression is detected within both liver endothelial cells and hepatic stellate cells. Next, studies were performed 4 wk after bile duct ligation (BDL), a model of portal hypertension characterized by prominent cholestasis, as evidenced by a significant increase in serum cholesterol in BDL animals. After BDL, caveolin protein levels from detergent-soluble liver lysates are significantly increased as assessed by Western blot analysis. Immunoperoxidase staining demonstrates that this increase is most prominent within sinusoids and venules. Additionally, caveolin-1 upregulation is associated with a significant reduction in NOS catalytic activity in BDL liver lysates, an event that is corrected with provision of excess calmodulin, a protein that competitively binds eNOS from caveolin. We conclude that, in cholestatic portal hypertension, caveolin may negatively regulate NOS activity in a manner that is reversible by excess calmodulin.

Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) contributes to hepatic vascular homeostasis. The aim of this study was to examine whether delivery of an adenoviral vector encoding eNOS gene to liver affects vasomotor function in vivo and the mechanism of NO production in vitro. Rats were administered adenoviruses encoding β-galactosidase (AdCMVLacZ) or eNOS (AdCMVeNOS) via tail vein injection and studied 1 wk later. In animals transduced with AdCMVLacZ, β-galactosidase activity was increased in the liver, most prominently in hepatocytes. In AdCMVeNOS-transduced animals, eNOS protein levels and catalytic activity were significantly increased. Overexpression of eNOS diminished baseline perfusion pressure and constriction in response to the α1-agonist methoxamine in the perfused liver. Transduction of cultured hepatocytes with AdCMVeNOS resulted in the targeting of recombinant eNOS to a perinuclear distribution and binding with the NOS-activating protein heat shock protein 90. These events were associated with increased ionomycin-stimulated NO release. In summary, this is the first study to demonstrate successful delivery of the recombinant eNOS gene to liver in vivo and in vitro with ensuing NO production.

The scaffold protein synectin plays a critical role in the trafficking and regulation of membrane receptor pathways. As platelet-derived growth factor receptor (PDGFR) is essential for hepatic stellate cell (HSC) activation and liver fibrosis, we sought to determine the role of synectin on the PDGFR pathway and development of liver fibrosis. Mice with deletion of synectin from HSC were found to be protected from liver fibrosis. mRNA sequencing revealed that knockdown of synectin in HSC demonstrated reductions in the fibrosis pathway of genes, including PDGFR-β. Chromatin IP assay of the PDGFR-β promoter upon synectin knockdown revealed a pattern of histone marks associated with decreased transcription, dependent on p300 histone acetyltransferase. Synectin knockdown was found to downregulate PDGFR-α protein levels, as well, but through an alternative mechanism: protection from autophagic degradation. Site-directed mutagenesis revealed that ubiquitination of specific PDGFR-α lysine residues was responsible for its autophagic degradation. Furthermore, functional studies showed decreased PDGF-dependent migration and proliferation of HSC after synectin knockdown. Finally, human cirrhotic livers demonstrated increased synectin protein levels. This work provides insight into differential transcriptional and posttranslational mechanisms of synectin regulation of PDGFRs, which are critical to fibrogenesis.

Fibrogenesis encompasses the deposition of matrix proteins, such as collagen I, by hepatic stellate cells (HSCs) that culminates in cirrhosis. Fibrogenic signals drive transcription of procollagen I, which enters the endoplasmic reticulum (ER), is trafficked through the secretory pathway, and released to generate extracellular matrix. Alternatively, disruption of procollagen I ER export could activate the unfolded protein response (UPR) and drive HSC apoptosis. Using a small interfering RNA screen, we identified Transport and Golgi organization 1 (TANGO1) as a potential participant in collagen I secretion. We investigated the role of TANGO1 in procollagen I secretion in HSCs and liver fibrogenesis. Depletion of TANGO1 in HSCs blocked collagen I secretion without affecting other matrix proteins. Disruption of secretion led to procollagen I retention within the ER, induction of the UPR, and HSC apoptosis. In wild‐type (WT) HSCs, both TANGO1 and the UPR were induced by transforming growth factor β (TGFβ). As the UPR up‐regulates proteins involved in secretion, we studied whether TANGO1 was a target of the UPR. We found that UPR signaling is responsible for up‐regulating TANGO1 in response to TGFβ, and this mechanism is mediated by the transcription factor X‐box binding protein 1 (XBP1). In vivo , murine and human cirrhotic tissue displayed increased TANGO1 messenger RNA levels. Finally, TANGO1 +/– mice displayed less hepatic fibrosis compared to WT mice in two separate murine models: CCl 4 and bile duct ligation. Conclusion : Loss of TANGO1 leads to procollagen I retention in the ER, which promotes UPR‐mediated HSC apoptosis. TANGO1 regulation during HSC activation occurs through a UPR‐dependent mechanism that requires the transcription factor, XBP1. Finally, TANGO1 is critical for fibrogenesis through mediating HSC homeostasis. The work reveals a unique role for TANGO1 and the UPR in facilitating collagen I secretion and fibrogenesis. (H epatology 2017;65:983‐998).