Insulin Resistance and Pellino-1 Mediated Decrease in the Activities of Vasodilator Signaling Contributes to Sunitinib-Induced Hypertension.

Antiangiogenic tyrosine kinases inhibitors induce hypertension, which may increase the incidents of cardiovascular complications and limit their use. However, the mechanisms by which usage of TKIs results in hypertension have not been fully understood. Here, we report the potential mechanisms of how sunitinib, a widely used TKI, induces hypertension. Male SD rats were randomly divided into control group and sunitinib-administrated group. We show that sunitinib administration for seven days caused a significant increase in artery blood pressure, along with glycerolipid metabolism abnormalities including decreased food intake and low body weight, hypoglycemia, hyperinsulinemia. Sunitinib administration also resulted in a significant increase in the levels of insulin autoantibody (IAA), cyclic adenosine monophosphate and free fatty acid in serum; whereas, sunitinib administration had no effects on serum glucagon levels. Sunitinib led to the decreased insulin sensitivity as determined by insulin tolerance test (ITT) and glucose tolerance test (GTT), reflecting insulin resistance occurred in sunitinib-treated rats. The results obtained from wire myograph assay in the mesenteric arteries show that endothelium-dependent relaxation, but not endothelium-independent relaxation, was impaired by sunitinib. Furthermore, western blot analysis revealed that the expressions levels of phosphorylated IRS-1, Pellino-1, AKT and eNOS were significantly attenuated by sunitinib in rat mesenteric artery tissues and in the sunitinib-treated primary cultured mesenteric artery endothelial cells. The levels of serum and endothelium-derived nitric oxide were also significantly decreased by sunitinib. Moreover, sunitinib-induced decrease in the expression levels of phosphorylated AKT and eNOS was further reduced by knocking down of Pellino-1 in MAECs. Our results suggest that sunitinib causes vascular dysfunction and hypertension, which are associated with insulin resistance- and Pellino-1-mediated inhibition of AKT/eNOS/NO signaling. Our results may provide a rational for preventing and/or treating sunitinib-induced endothelial dysfunction and hypertension.

Long non-coding RNAs in recurrent ovarian cancer: Theranostic perspectives.

Nearly 70% of ovarian cancer (OC) patients experience recurrence within the first 2 years after initial treatment. Emerging evidence indicates that long non-coding RNAs (lncRNAs) play a pivotal role in the pathogenesis of OC progression, resistance to therapy and recurrent OC (ROC). Transcriptome profiling studies have reported differential expression patterns of lncRNAs in OC which are related to increased cell invasion, metastasis and drug resistance. In this review, we highlighted the roles of lncRNAs in OC progression and outlined the potential molecular mechanisms by which lncRNAs impact on ROC. Recent advances using lncRNAs as potential biomarkers for screening, detection, prediction, response to therapy and as therapeutic targets are discussed.

The vascular endothelial growth factor trap aflibercept induces vascular dysfunction and hypertension via attenuation of eNOS/NO signaling in mice.

Aflibercept, as a soluble decoy vascular endothelial growth factor receptor, Which has been used as a first-line monotherapy for cancers. Aflibercept often causes cardiovascular toxicities including hypertension, but the mechanisms underlying aflibercept-induced hypertension remain unknown. In this study we investigated the effect of short-term and long-term administration of aflibercept on blood pressure (BP), vascular function, NO bioavailability, oxidative stress and endothelin 1 (ET-1) in mice and cultured endothelial cells. We showed that injection of a single-dose of aflibercept (18.2, 36.4 mg/kg, iv) rapidly and dose-dependently elevated BP in mice. Aflibercept treatment markedly impaired endothelial-dependent relaxation (EDR) and resulted in NADPH oxidases 1 (NOX1)- and NADPH oxidases 4 (NOX4)-mediated generation of ROS, decreased the activation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) concurrently with a reduction in nitric oxide (NO) production and elevation of ET-1 levels in mouse aortas; these effects were greatly attenuated by supplementation of L-arginine (L-arg, 0.5 or 1.0 g/kg, bid, ig) before aflibercept injection. Similar results were observed in L-arg-pretreated cultured endothelial cells, showing markedly decreased ROS accumulation and AKT/eNOS/NO signaling impairment induced by aflibercept. In order to assess the effects of long-term aflibercept on hypertension and to evaluate the beneficial effects of L-arg supplementation, we administered these two drugs to WT mice for up to 14 days (at an interval of two days). Long-term administration of aflibercept resulted in a sustained increase in BP and a severely impaired EDR, which are associated with NOX1/NOX4-mediated production of ROS, increase in ET-1, inhibition of AKT/eNOS/NO signaling and a decreased expression of cationic amino acid transporter (CAT-1). The effects caused by long-term administration were greatly attenuated by L-arg supplementation in a dose-dependent manner. We conclude that aflibercept leads to vascular dysfunction and hypertension by inhibiting CAT-1/AKT/eNOS/NO signaling, increasing ET-1, and activating NOX1/NOX4-mediated oxidative stress, which can be suppressed by supplementation of L-arg. Therefore, L-arg could be a potential therapeutic agent for aflibercept-induced hypertension.

Intracellular cholesterol stimulates ENaC by interacting with phosphatidylinositol­4,5­bisphosphate and mediates cyclosporine A-induced hypertension.

We have previously shown that blockade of ATP-binding cassette transporter A1 (ABCA1) with cyclosporine A (CsA) stimulates the epithelial sodium channel (ENaC) in cultured distal nephron cells. Here we show that CsA elevated systolic blood pressure in both wild-type and apolipoprotein E (ApoE) knockout (KO) mice to a similar level. The elevated systolic blood pressure was completely reversed by inhibition of cholesterol (Cho) synthesis with lovastatin. Inside-out patch-clamp data show that intracellular Cho stimulated ENaC in cultured distal nephron cells by interacting with phosphatidylinositol­4,5­bisphosphate (PIP2), an ENaC activator. Confocal microscopy data show that both α­ENaC and PIP2 were localized in microvilli via a Cho-dependent mechanism. Deletion of membrane Cho reduced the levels of γ­ENaC in the apical membrane. Reduced ABCA1 expression and elevated intracellular Cho were observed in old mice, compared to young mice. In parallel, cell-attached patch-clamp data from the split-open cortical collecting ducts (CCD) show that ENaC activity was significantly increased in old mice. These data suggest that elevation of intracellular Cho due to blockade of ABCA1 stimulates ENaC, which may contribute to CsA-induced hypertension. This study also implies that reduced ABCA1 expression may mediate age-related hypertension by increasing ENaC activity via elevation of intracellular Cho.

TRUSS Exacerbates NAFLD Development by Promoting IκBα Degradation in Mice.

There is no effective treatment method for nonalcoholic fatty liver disease (NAFLD), the most common liver disease. The exact mechanism underlying the pathogenesis of NAFLD remains to be elucidated. Here, we report that tumor necrosis factor receptor-associated ubiquitous scaffolding and signaling protein (TRUSS) acts as a positive regulator of NAFLD and in a variety of metabolic disorders. TRUSS expression was increased in the human liver specimens with NAFLD or nonalcoholic steatohepatitis, and in the livers of high-fat diet (HFD)-induced and genetically obese mice. Conditional knockout of TRUSS in hepatocytes significantly ameliorated hepatic steatosis, insulin resistance, glucose intolerance, and inflammatory responses in mice after HFD challenge or in spontaneous obese mice with normal chow feeding. All of these HFD-induced pathological phenotypes were exacerbated in mice overexpressing TRUSS in hepatocytes. We show that TRUSS physically interacts with the inhibitor of nuclear factor κB α (IκBα) and promotes the ubiquitination and degradation of IκBα, which leads to aberrant activation of nuclear factor κB (NF-κB). Overexpressing IκBαS32A/S36A , a phosphorylation-resistant mutant of IκBα, in the hepatocyte-specific TRUSS overexpressing mice almost abolished HFD-induced NAFLD and metabolic disorders. Conclusion: Hepatocyte TRUSS promotes pathological stimuli-induced NAFLD and metabolic disorders, through activation of NF-κB by promoting ubiquitination and degradation of IκBα. Our findings may provide a strategy for the prevention and treatment of NAFLD by targeting TRUSS.

Trefoil factor 3 mediation of oncogenicity and chemoresistance in hepatocellular carcinoma is AKT-BCL-2 dependent.

The efficacious treatment of hepatocellular carcinoma (HCC) remains a challenge, partially being attributed to intrinsic chemoresistance. Previous reports have observed increased TFF3 expression in HCC. Herein, we investigated the functional role of TFF3 in progression of HCC, and in both intrinsic and acquired chemoresistance. TFF3 expression was observed to be upregulated in HCC and associated with poor clinicopathological features and worse patient survival outcome. Functionally, forced expression of TFF3 in HCC cell lines increased cell proliferation, cell survival, anchorage-independent and 3D matrigel growth, cell invasion and migration, and in vivo tumor growth. In contrast, depleted expression of TFF3 decreased the oncogenicity of HCC cells as indicated by the above parameters. Furthermore, forced expression of TFF3 decreased doxorubicin sensitivity of HCC cells, which was attributed to increased doxorubicin efflux and cancer stem cell-like behavior of Hep3B cells. In contrast, depletion of TFF3 increased doxorubicin sensitivity and decreased cancer stem cell-like behavior of Hep3B cells. Correspondingly, TFF3 expression was markedly increased in Hep3B cells with acquired doxorubicin resistance, while the depletion of TFF3 resulted in re-sensitization of the Hep3B cells to doxorubicin. The increased doxorubicin efflux and enhanced cancer stem cell-like behavior of the doxorubicin-resistant Hep3B cells was observed to be dependent on TFF3 expression. In addition, we determined that TFF3-stimulated oncogenicity and chemoresistance in HCC cells was mediated by AKT-dependent expression of BCL-2. Hence, therapeutic inhibition of TFF3 should be considered to hinder HCC progression and overcome intrinsic and acquired chemoresistance in HCC.

Hydrogen peroxide suppresses TRPM4 trafficking to the apical membrane in mouse cortical collecting duct principal cells.

A Ca2+-activated nonselective cation channel (NSCCa) is found in principal cells of the mouse cortical collecting duct (CCD). However, the molecular identity of this channel remains unclear. We used mpkCCDc14 cells, a mouse CCD principal cell line, to determine whether NSCCa represents the transient receptor potential (TRP) channel, the melastatin subfamily 4 (TRPM4). A Ca2+-sensitive single-channel current was observed in inside-out patches excised from the apical membrane of mpkCCDc14 cells. Like TRPM4 channels found in other cell types, this channel has an equal permeability for Na+ and K+ and has a linear current-voltage relationship with a slope conductance of ~23 pS. The channel was inhibited by a specific TRPM4 inhibitor, 9-phenanthrol. Moreover, the frequency of observing this channel was dramatically decreased in TRPM4 knockdown mpkCCDc14 cells. Unlike those previously reported in other cell types, the TRPM4 in mpkCCDc14 cells was unable to be activated by hydrogen peroxide (H2O2). Conversely, after treatment with H2O2, TRPM4 density in the apical membrane of mpkCCDc14 cells was significantly decreased. The channel in intact cell-attached patches was activated by ionomycin (a Ca2+ ionophore), but not by ATP (a purinergic P2 receptor agonist). These data suggest that the NSCCa current previously described in CCD principal cells is actually carried through TRPM4 channels. However, the physiological role of this channel in the CCD remains to be further determined.

The Polarized Effect of Intracellular Calcium on the Renal Epithelial Sodium Channel Occurs as a Result of Subcellular Calcium Signaling Domains Maintained by Mitochondria.

The renal epithelial sodium channel (ENaC) provides regulated sodium transport in the distal nephron. The effects of intracellular calcium ([Ca(2+)]i) on this channel are only beginning to be elucidated. It appears from previous studies that the [Ca(2+)]i increases downstream of ATP administration may have a polarized effect on ENaC, where apical application of ATP and the subsequent [Ca(2+)]i increase have an inhibitory effect on the channel, whereas basolateral ATP and [Ca(2+)]i have a stimulatory effect. We asked whether this polarized effect of ATP is, in fact, reflective of a polarized effect of increased [Ca(2+)]i on ENaC and what underlying mechanism is responsible. We began by performing patch clamp experiments in which ENaC activity was measured during apical or basolateral application of ionomycin to increase [Ca(2+)]i near the apical or basolateral membrane, respectively. We found that ENaC does indeed respond to increased [Ca(2+)]i in a polarized fashion, with apical increases being inhibitory and basolateral increases stimulating channel activity. In other epithelial cell types, mitochondria sequester [Ca(2+)]i, creating [Ca(2+)]i signaling microdomains within the cell that are dependent on mitochondrial localization. We found that mitochondria localize in bands just beneath the apical and basolateral membranes in two different cortical collecting duct principal cell lines and in cortical collecting duct principal cells in mouse kidney tissue. We found that inhibiting mitochondrial [Ca(2+)]i uptake destroyed the polarized response of ENaC to [Ca(2+)]i. Overall, our data suggest that ENaC is regulated by [Ca(2+)]i in a polarized fashion and that this polarization is maintained by mitochondrial [Ca(2+)]i sequestration.

Regulation of ASIC1 by Ca2+/calmodulin-dependent protein kinase II in human glioblastoma multiforme.

Recent studies have implicated the acid-sensing ion channel 1 (ASIC1), a proton-gated cation channel that belongs to the epithelial sodium channel (ENaC)/Degenerin family, plays an important role in glioma cell migration. Among the ASIC subunits, only ASIC1a has been found be calcium permeable. However, it has not been determined whether Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates ASIC1 in glioblastoma multiforme (GBM). Herein, we report that ASIC1 and CaMKII assemble to form a functional complex at the plasma membrane of GBM cells. We found that migration ability was significantly attenuated in GBM cells that were pre-treated with autocamtide-2-related inhibitory peptide (AIP), a CaMKII-specific inhibitor, or psalmotoxin 1 (PcTX-1), a selective ASIC1 blocker. Furthermore, the inhibitory effect of AIP or PcTX-1 on migration was diminished when ASIC1 was knocked down in GBM cells; when ASIC1 knockdown GBM cells were concurrently treated with these two inhibitors, cell migration was slightly but significantly decreased. Using whole-cell patch-clamp recordings, we detected an amiloride-sensitive current in GBM cells, and this current was significantly inhibited by both PcTX-1 and AIP. Moreover, the magnitude of this current was dramatically decreased when ASIC1 was knocked down in GBM cells. The addition of AIP failed to further decrease the amplitude of this current. Taken together, these data suggest that ASIC1 and CaMKII form a functional complex in GBM cells. Furthermore, it can be concluded that CaMKII regulates the activity of ASIC1, which is associated with the ability of GBM cells to migrate.