Inhibition of YAP by lenvatinib in endothelial cells increases blood pressure through ferroptosis.

Lenvatinib, a multitarget tyrosine kinase inhibitor (TKI), increases the incidence of severe hypertension and thus the incidence of cardiovascular complications. Inhibition of ferroptosis, a newly recognized type of cell death, alleviates endothelial dysfunction. Here, we report that lenvatinib-induced hypertension is associated with ferroptosis of endothelial cells. RNA sequencing (RNA-seq) showed that lenvatinib led to ferroptosis of endothelial cells and that administration of mouse with ferrostatin-1 (Fer-1), a specific ferroptosis inhibitor, dramatically ameliorated lenvatinib-induced hypertension and reversed lenvatinib-induced impairment of endothelium-dependent relaxation (EDR). Furthermore, lenvatinib significantly reduced glutathione peroxidase 4 (GPX4) expressions in the mouse aorta and human umbilical vein endothelial cells (HUVECs) and increased lipid peroxidation, lactate dehydrogenase (LDH) release, and malondialdehyde (MDA) levels in HUVECs. Immunofluorescence and Western blotting showed that lenvatinib significantly reduced Yes-associated protein (YAP) nuclear translocation but not cytoplasmic YAP expression in HUVECs. The data, generated from both in vivo and in vitro, showed that lenvatinib reduced total YAP (t-YAP) expression and increased the phosphorylation of YAP at both Ser127 and Ser397, without affecting YAP mRNA levels in HUVECs. XMU-MP-1 mediated YAP activation or YAP overexpression effectively attenuated the lenvatinib-induced decrease in GPX4 expression and increases in LDH release and MDA levels. In addition, overexpression of YAP in HUVECs ameliorated lenvatinib-induced decrease in the mRNA and protein levels of spermidine/spermine N (1)-acetyltransferase-1 (SAT1), heme oxygenase-1 (HO-1), and ferritin heavy chain 1 (FTH1). Taken together, our data suggest that lenvatinib-induced inhibition of YAP led to ferroptosis of endothelial cells and subsequently resulted in vascular dysfunction and hypertension.

Aldosterone-stimulated endothelial epithelial sodium channel (EnNaC) plays a role in cold exposure-induced hypertension in rats.

Background: Previous studies have demonstrated that activated endothelial epithelial sodium channel (EnNaC) impairs vasodilatation, which contributes to salt-sensitive hypertension. Here, we investigate whether mesenteric artery (MA) EnNaC is involved in cold exposure-induced hypertension (CIH) and identify the underlying mechanisms in SD rats. Methods: One group of rats was housed at room temperature and served as control. Three groups of rats were kept in a 4°C cold incubator for 10 h/day; among which two groups were administrated with either benzamil (EnNaC blocker) or eplerenone (mineralocorticoid receptor antagonist, MR). Blood pressure (BP), vasodilatation, and endothelial function were measured with tail-cuff plethysmography, isometric myograph, and Total Nitric Oxide (NO) Assay kit, respectively. A cell-attached patch-clamp technique, in split-open MA, was used to determine the role of EnNaC in CIH rats. Furthermore, the plasma aldosterone levels were detected using an ELISA kit; and Western blot analysis was used to examine the relative expression levels of Sgk1 and Nedd4-2 proteins in the MA of SD rats. Results: We demonstrated that cold exposure increased BP, impaired vasodilatation, and caused endothelial dysfunction in rats. The activity of EnNaC significantly increased, concomitant with an increased level of plasma aldosterone and activation of Sgk1/Nedd4-2 signaling. Importantly, CIH was inhibited by either eplerenone or benzamil. It appeared that cold-induced decrease in NO production and impairment of endothelium-dependent relaxation (EDR) were significantly ameliorated by either eplerenone or benzamil in MA of CIH rats. Moreover, treatment of MAs with aldosterone resulted in an activation of EnNaC, a reduction of NO, and an impairment of EDR, which were significantly inhibited by either eplerenone or GSK650394 (Sgk1 inhibitor) or benzamil. Conclusion: Activation of EnNaC contributes to CIH; we suggest that pharmacological inhibition of the MR/Sgk1/Nedd4-2/EnNaC axis may be a potential therapeutic strategy for CIH.

Homocysteine Causes Endothelial Dysfunction via Inflammatory Factor-Mediated Activation of Epithelial Sodium Channel (ENaC).

BACKGROUND: Hyperhomocysteinemia (HHcy) causes cardiovascular diseases via regulating inflammatory responses. We investigated whether and how the epithelial sodium channel (ENaC), a recently identified ion channel in endothelial cells, plays a role in HHcy-induced endothelial dysfunction. METHODS: Cell-attached patch-clamp recording in acute split-open aortic endothelial cells, western blot, confocal imaging, and wire myograph combined with pharmacological approaches were used to determine whether HHcy-mediated inflammatory signaling leads to endothelial dysfunction via stimulating ENaC. RESULTS: The data showed that 4 weeks after L-methionine diet the levels of plasma Hcy were significantly increased and the ENaC was dramatically activated in mouse aortic endothelial cells. Administration of benzamil, a specific ENaC blocker, ameliorated L-methionine diet-induced impairment of endothelium-dependent relaxation (EDR) and reversed Hcy-induced increase in ENaC activity. Pharmacological inhibition of NADPH oxidase, reactive oxygen species (ROS), cyclooxygenase-2 (COX-2)/thromboxane B2 (TXB2), or serum/glucocorticoid regulated kinase 1 (SGK1) effectively attenuated both the Hcy-induced activation of endothelial ENaC and impairment of EDR. Our in vitro data showed that both NADPH oxidase inhibitor and an ROS scavenger reversed Hcy-induced increase in COX-2 expression in human umbilical vein endothelial cells (HUVECs). Moreover, Hcy-induced increase in expression levels of SGK-1, phosphorylated-SGK-1, and phosphorylated neural precursor cell-expressed developmentally downregulated protein 4-2 (p-Nedd4-2) in HUVECs were significantly blunted by a COX-2 inhibitor. CONCLUSION: We show that Hcy activates endothelial ENaC and subsequently impairs EDR of mouse aorta, via ROS/COX-2-dependent activation of SGK-1/Nedd4-2 signaling. Our study provides a rational that blockade of the endothelial ENaC could be potential method to prevent and/or to treat Hcy-induced cardiovascular disease.

NaHS or Lovastatin Attenuates Cyclosporine A-Induced Hypertension in Rats by Inhibiting Epithelial Sodium Channels.

The use of cyclosporine A (CsA) in transplant recipients is limited due to its side effects of causing severe hypertension. We have previously shown that CsA increases the activity of the epithelial sodium channel (ENaC) in cultured distal nephron cells. However, it remains unknown whether ENaC mediates CsA-induced hypertension and how we could prevent hypertension. Our data show that the open probability of ENaC in principal cells of split-open cortical collecting ducts was significantly increased after treatment of rats with CsA; the increase was attenuated by lovastatin. Moreover, CsA also elevated the levels of intracellular cholesterol (Cho), intracellular reactive oxygen species (ROS) via activation of NADPH oxidase p47phox, serum- and glucocorticoid-induced kinase isoform 1 (Sgk1), and phosphorylated neural precursor cell-expressed developmentally downregulated protein 4-2 (p-Nedd4-2) in the kidney cortex. Lovastatin also abolished CsA-induced elevation of α-, ß-, and γ-ENaC expressions. CsA elevated systolic blood pressure in rats; the elevation was completely reversed by lovastatin (an inhibitor of cholesterol synthesis), NaHS (a donor of H2S which ameliorated CsA-induced elevation of reactive oxygen species), or amiloride (a potent ENaC blocker). These results suggest that CsA elevates blood pressure by increasing ENaC activity via a signaling cascade associated with elevation of intracellular ROS, activation of Sgk1, and inactivation of Nedd4-2 in an intracellular cholesterol-dependent manner. Our data also show that NaHS ameliorates CsA-induced hypertension by inhibition of oxidative stress.

Cholesterol Stimulates the Transient Receptor Potential Melastatin 4 Channel in mpkCCDc14 Cells.

We have shown that cholesterol regulates the activity of ion channels in mouse cortical collecting duct (CCD) mpkCCDc14 cells and that the transient receptor potential melastatin 4 (TRPM4) channel is expressed in these cells. However, whether TRPM4 channel is regulated by cholesterol remains unclear. Here, we performed inside-out patch-clamp experiments and found that inhibition of cholesterol biosynthesis by lovastatin significantly decreased, whereas enrichment of cholesterol with exogenous cholesterol significantly increased, TRPM4 channel open probability (Po) by regulating its sensitivity to Ca2+ in mpkCCDc14 cells. In addition, inside-out patch-clamp data show that acute depletion of cholesterol in the membrane inner leaflet by methyl-ß-cyclodextrin (MßCD) significantly reduced TRPM4 Po, which was reversed by exogenous cholesterol. Moreover, immunofluorescence microscopy, Western blot, cell-surface biotinylation, and patch clamp analysis show that neither inhibition of intracellular cholesterol biosynthesis with lovastatin nor application of exogenous cholesterol had effect on TRPM4 channel protein abundance in the plasma membrane of mpkCCDc14 cells. Sucrose density gradient centrifugation studies demonstrate that TRPM4 was mainly located in cholesterol-rich lipid rafts. Lipid-protein overlay experiments show that TRPM4 directly interacted with several anionic phospholipids, including PI(4,5)P2. Depletion of PI(4,5)P2 with either wortmannin or PGE2 abrogated the stimulatory effects of exogenous cholesterol on TRPM4 activity, whereas exogenous PI(4,5)P2 (diC8-PI(4,5)P2, a water-soluble analog) increased the effects. These results suggest that cholesterol stimulates TRPM4 via a PI(4,5)P2-dependent mechanism.

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.

Stimulation of Epithelial Sodium Channels in Endothelial Cells by Bone Morphogenetic Protein-4 Contributes to Salt-Sensitive Hypertension in Rats.

Previous studies have shown that high salt induces artery stiffness by causing endothelial dysfunction via increased sodium influx. We used our unique split-open artery technique combined with protein biochemistry and in vitro measurement of vascular tone to test a hypothesis that bone morphogenetic protein 4 (BMP4) mediates high salt-induced loss of vascular relaxation by stimulating the epithelial sodium channel (ENaC) in endothelial cells. The data show that high salt intake increased BMP4 both in endothelial cells and in the serum and that exogenous BMP4 stimulated ENaC in endothelial cells. The data also show that the stimulation is mediated by p38 mitogen-activated protein kinases (p38 MAPK) and serum and glucocorticoid-regulated kinase 1 (Sgk1)/neural precursor cell expressed developmentally downregulated gene 4-2 (Nedd4-2) (Sgk1/Nedd4-2). Furthermore, BMP4 decreased mesenteric artery relaxation in a benzamil-sensitive manner. These results suggest that high salt intake stimulates endothelial cells to express and release BMP4 and that the released BMP4 reduces artery relaxation by stimulating ENaC in endothelial cells. Therefore, stimulation of ENaC in endothelial cells by BMP4 may serve as another pathway to participate in the complex mechanism of salt-sensitive (SS) hypertension.

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.

Transient Receptor Potential Melastatin 4 (TRPM4) Contributes to High Salt Diet-Mediated Early-Stage Endothelial Injury.

BACKGROUND/AIMS: The present study investigated whether the transient receptor potential melastatin 4 (TRPM4) channel plays a role in high salt diet (HSD)-induced endothelial injuries. METHODS: Western blotting and immunofluorescence were used to examine TRPM4 expression in the mesenteric endothelium of Dahl salt-sensitive (SS) rats fed a HSD. The MTT, TUNEL, and transwell assays were used to evaluate the cell viability, cell apoptosis, and cell migration, respectively, of human umbilical vein endothelial cells (HUVECs). Enzyme-linked immunosorbent assays were used to determine the concentrations of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion protein 1 (VCAM-1), and E-selectin. Carboxy-H2DCFDA, a membrane-permeable reactive oxygen species (ROS)-sensitive fluorescent probe, was used to detect intracellular ROS levels. RESULTS: TRPM4 was mainly expressed near the plasma membrane of mesenteric artery endothelial cells, and its expression level increased in SS hypertensive rats fed a HSD. Its protein expression was significantly upregulated upon treatment with exogenous hydrogen peroxide (H2O2) and aldosterone in cultured HUVECs. Cell viability decreased upon treatment with both agents in a concentration-dependent manner, which could be partially reversed by 9-phenanthrol, a specific TRPM4 inhibitor. Exogenous H2O2 induced apoptosis, enhanced cell migration, and increased the release of adhesion molecules, including ICAM-1, VCAM-1, and E-selectin, all of which were significantly attenuated upon treatment with 9-phenanthrol. Aldosterone and H2O2 induced the accumulation of intracellular ROS, which was significantly inhibited by 9-phenanthrol, suggesting that oxidative stress is one of the mechanisms underlying aldosterone-induced endothelial injury. CONCLUSIONS: Given the fact that oxidative stress and high levels of circulating aldosterone are present in hypertensive patients, we suggest that the upregulation of TRPM4 in the vascular endothelium may be involved in endothelial injuries caused by these stimuli.

Angiotensin-Converting Enzyme 3 (ACE3) Protects Against Pressure Overload-Induced Cardiac Hypertrophy.

BACKGROUND: Angiotensin-converting enzyme 3 (ACE3) is a recently defined homolog of ACE. However, the pathophysiological function of ACE3 is largely unknown. Here, we aim to explore the role of ACE3 in pathological cardiac hypertrophy. METHODS AND RESULTS: Neonatal rat cardiomyocytes (NRCMs) with gain and loss of function of ACE3 and mice with global knockout or cardiac-specific overexpression of ACE3 were used in this study. In cultured cardiomyocytes, ACE3 conferred protection against angiotensin II (Ang II)-induced hypertrophic growth. Cardiac hypertrophy in mice was induced by aortic banding (AB) and the extent of hypertrophy was analyzed through echocardiographic, pathological, and molecular analyses. Our data demonstrated that ACE3-deficient mice exhibited more pronounced cardiac hypertrophy and fibrosis and a strong decrease in cardiac contractile function, conversely, cardiac-specific ACE3-overexpressing mice displayed an attenuated hypertrophic phenotype, compared with control mice, respectively. Analyses of the underlying molecular mechanism revealed that ACE3-mediated protection against cardiac hypertrophy by suppressing the activation of mitogen-activated protein kinase kinase (MEK)-regulated extracellular signal-regulated protein kinase (ERK1/2) signaling, which was further evidenced by the observation that inhibition of the MEK-ERK1/2 signaling by U0126 rescued the exacerbated hypertrophic phenotype in ACE3-deficient mice. CONCLUSIONS: Our comprehensive analyses suggest that ACE3 inhibits pressure overload-induced cardiac hypertrophy by blocking the MEK-ERK1/2 signaling pathway.