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Background: Aortic aneurysm and aortic dissection (AAD) are life-threatening vascular diseases, with endothelium being the primary target for AAD treatment. Protein S-sulfhydration is a newly discovered posttranslational modification whose role in AAD has not yet been defined. This study aims to investigate whether protein S-sulfhydration in the endothelium regulates AAD and its underlying mechanism. Methods: Protein S-sulfhydration in endothelial cells (ECs) during AAD was detected and hub genes regulating homeostasis of the endothelium were identified. Clinical data of patients with AAD and healthy controls were collected, and the level of the cystathionine γ lyase (CSE)/hydrogen sulfide (H 2 S) system in plasma and aortic tissue were determined. Mice with EC-specific CSE deletion or overexpression were generated, and the progression of AAD was determined. Unbiased proteomics and coimmunoprecipitation combined with mass spectrometry analysis were conducted to determine the upstream regulators of the CSE/H 2 S system and the findings were confirmed in transgenic mice. Results: Higher plasma H 2 S levels were associated with a lower risk of AAD, after adjustment for common risk factors. CSE was reduced in the endothelium of AAD mouse and aorta of patients with AAD. Protein S-sulfhydration was reduced in the endothelium during AAD and protein disulfide isomerase (PDI) was the main target. S-sulfhydration of PDI at Cys343 and Cys400 enhanced PDI activity and mitigated endoplasmic reticulum stress. EC-specific CSE deletion was exacerbated, and EC-specific overexpression of CSE alleviated the progression of AAD through regulating the S-sulfhydration of PDI. ZEB2 (zinc finger E-box binding homeobox 2) recruited the HDAC1-NuRD complex (histone deacetylase 1–nucleosome remodeling and deacetylase) to repress the transcription of CTH , the gene encoding CSE, and inhibited PDI S-sulfhydration. EC-specific HDAC1 deletion increased PDI S-sulfhydration and alleviated AAD. Increasing PDI S-sulfhydration with the H 2 S donor GYY4137 or pharmacologically inhibiting HDAC1 activity with entinostat alleviated the progression of AAD. Conclusions: Decreased plasma H 2 S levels are associated with an increased risk of aortic dissection. The endothelial ZEB2-HDAC1-NuRD complex transcriptionally represses CTH , impairs PDI S-sulfhydration, and drives AAD. The regulation of this pathway effectively prevents AAD progression.

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.

Pathological cardiac hypertrophy is a process characterized by significant disturbance of protein turnover. Cullin-associated and Neddylation-dissociated 1 (CAND1) acts as a coordinator to modulate substrate protein degradation by promoting the formation of specific cullin-based ubiquitin ligase 3 complex in response to substrate accumulation, which thereby facilitate the maintaining of normal protein homeostasis. Accumulation of calcineurin is critical in the pathogenesis of cardiac hypertrophy and heart failure. However, whether CAND1 titrates the degradation of hypertrophy related protein eg. calcineurin and regulates cardiac hypertrophy remains unknown. Therefore, we aim to explore the role of CAND1 in cardiac hypertrophy and heart failure and the underlying molecular mechanism. Here, we found that the protein level of CAND1 was increased in cardiac tissues from heart failure (HF) patients and TAC mice, whereas the mRNA level did not change. CAND1-KO+ /- aggravated TAC-induced cardiac hypertrophic phenotypes; in contrast, CAND1-Tg attenuated the maladaptive cardiac remodeling. At the molecular level, CAND1 overexpression downregulated, whereas CAND1-KO+ /- or knockdown upregulated calcineurin expression at both in vivo and in vitro conditions. Mechanistically, CAND1 overexpression favored the assembly of Cul1/atrogin1/calcineurin complex and rendered the ubiquitination and degradation of calcineurin. Notably, CAND1 deficiency-induced hypertrophic phenotypes were partially rescued by knockdown of calcineurin, and application of exogenous CAND1 prevented TAC-induced cardiac hypertrophy. Taken together, our findings demonstrate that CAND1 exerts a protective effect against cardiac hypertrophy and heart failure partially by inducing the degradation of calcineurin.

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.

Glioblastoma is a lethal primary brain tumor with abundant immune-suppressive glioblastoma-associated macrophage (GAM) infiltration. Skewing immune suppressive GAMs towards an immune-activating phenotype represents a promising immunotherapeutic strategy against glioblastoma. Herein, we reported that genetic deletion of miRNA-processing enzyme Dicer in macrophages inhibited the growth of GL261 murine glioblastoma xenografts and prolonged survival of tumor-bearing mice. Single cell RNA sequencing (scRNA-seq) of the tumor-infiltrating immune cells revealed that Dicer deletion in macrophages reduced the proportion of cell-cycling GAM cluster and reprogramed the remaining GAMs towards a proinflammatory activation state (enhanced phagocytotic and IFN-producing signature). Dicer-deficient GAMs showed reduced level of cyclin-dependent kinases (CDK1 and CDK2) and increased expression of CDK inhibitor p27 Kip1, thus manifesting impaired proliferation. Dicer knockout enhanced phagocytotic activity of GAMs to eliminate GL261 tumor cells. Increased proinflammatory GAM clusters in macrophage Dicer-deficient mice actively interacted with tumor-infiltrating T cells and NK cells through TNF paracrine signaling to create a pro-inflammatory immune microenvironment for tumor cell elimination. Our work identifies the role of Dicer deletion in macrophages in generating an immune-activating microenvironment, which could be further developed as a potential immunotherapeutic strategy against glioblastoma.

In the past, it was believed that the expression of the epithelial sodium channel (ENaC) was restricted to epithelial tissues, such as the distal nephron, airway, sweat glands, and colon, where it is critical for sodium homeostasis. Over the past two decades, this paradigm has shifted due to the finding that ENaC is also expressed in various nonepithelial tissues, notably in vascular endothelial cells. In this review, the recent findings of the expression, regulation, and function of the endothelial ENaC (EnNaC) are discussed. The expression of EnNaC subunits is reported in a variety of endothelial cell lines and vasculatures, but this is controversial across different species and vessels and is not a universal finding in all vascular beds. The expression density of EnNaC is very faint compared to ENaC in the epithelium. To date, little is known about the regulatory mechanism of EnNaC. Through it can be regulated by aldosterone, the detailed downstream signaling remains elusive. EnNaC responds to increased extracellular sodium with the feedforward activation mechanism, which is quite different from the Na+ self-inhibition mechanism of ENaC. Functionally, EnNaC was shown to be a determinant of cellular mechanics and vascular tone as it can sense shear stress, and its activation or insertion into plasma membrane causes endothelial stiffness and reduced nitric oxide production. However, in some blood vessels, EnNaC is essential for maintaining the integrity of endothelial barrier function. In this context, we discuss the possible reasons for the distinct role of EnNaC in vasculatures.

BackgroundHyperhomocysteinemia (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.MethodsCell-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.ResultsThe 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.ConclusionWe 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.

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.

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 phosphatidylinositol‑4,5‑bisphosphate (PIP2), an ENaC activator. Confocal microscopy data show that both α‑ENaC and PIP2 were localized in microvilli via a Cho-dependent mechanism. Deletion of membrane Cho reduced the levels of γ‑ENaC 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.

Pathological cardiac hypertrophy is a process characterized by significant disturbance of protein turnover. Cullin-associated and Neddylation-dissociated 1 (CAND1) acts as a coordinator to modulate substrate protein degradation by promoting the formation of specific cullin-based ubiquitin ligase 3 complex in response to the accumulation of specific proteins, which thereby maintains the normal protein homeostasis. However, whether CAND1 titrates the degradation of hypertrophic related proteins and manipulates cardiac hypertrophy remains unknown. In this study, we found that the protein level of CAND1 was increased in the heart tissues from heart failure (HF) patients and TAC mice. CAND1-KO+/- aggravated TAC-induced cardiac hypertrophic phenotypes; in contrast, CAND1-Tg attenuated the maladaptive cardiac remodeling. At the protein level, CAND1 overexpression downregulated, whereas CAND1-KO+/- or knockdown upregulated the expression of calcineurin, a critical pro-hypertrophic protein at both in vivo and in vitro conditions. Mechanistically, CAND1 overexpression favored, whilst CAND1 knockdown blocked the assembly of Cul1/atrogin1/calcineurin complex, an E3 that renders the ubiquitination and degradation of calcineurin. It turned out that calcineurin ubiquitination was suppressed by CAND1 knockdown, but enhanced by CAND1 overexpression. In addition, overexpression of truncated calcineurin that cannot be recognized by atrogin1 abrogated the antihypertrophic effects of CAND1. Notably, CAND1 deficiency-induced hypertrophic phenotypes were partially rescued by knockdown of calcineurin, and application of exogenous CAND1 prevented TAC-induced cardiac hypertrophy and heart failure. In conclusion, CAND1 exerts a protective effect against cardiac hypertrophy and heart failure partially by inducing the degradation of calcineurin. CAND1 represents a novel, promising therapeutic target for cardiac hypertrophy and heart failure. Keywords: CAND1; heart failure; calcineurin; ubiquitination; cullin1

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-β-cyclodextrin (MβCD) 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.

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.

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.

We previously showed that increased epithelial sodium channel (ENaC) activity in endothelial cells induced by oxidized low-density lipoprotein (ox-LDL) contributes to vasculature dysfunction. Here, we investigated whether ENaC participates in the pathological process of atherosclerosis using LDL receptor-deficient (LDLr−/−) mice. Male C57BL/6 and LDLr−/− mice were fed a normal diet (ND) or high fat diet (HFD) for 10 weeks. Our data show that treatment of LDLr−/− mice with a specific ENaC blocker, benzamil, significantly decreased atherosclerotic lesion formation and expression of matrix metalloproteinase 2 (MMP2) and metalloproteinase 9 (MMP9) in aortic arteries. Furthermore, benzamil ameliorated HFD-induced impairment of aortic endothelium-dependent dilation by reducing expression of proinflammatory cytokines, including TNF-α, IL-1β, and IL-6 and production of adhesion molecules including VCAM-1 and ICAM-1 in both C57BL/6 and LDLr−/− mice fed with HFD. In addition, HFD significantly increased ENaC activity and the levels of serum lipids, including ox-LDL. Our in vitro data further demonstrated that exogenous ox-LDL significantly increased the production of TNF-α, IL-1β, IL-6, VCAM-1 and ICAM-1. This ox-LDL-induced increase in inflammatory cytokines and adhesion molecules was reversed by γ-ENaC silencing or by treatment with the cyclooxygenase-2 (COX-2) antagonist celecoxib. Benzamil inhibited HFD-induced increase in COX-2 expression in aortic tissue in both C57BL/6 and LDLr−/− mice, and γ-ENaC gene silencing attenuated ox-LDL-induced COX-2 expression in HUVECs. These data together suggest that HFD-induced activation of ENaC stimulates inflammatory signaling, thereby contributes to HFD-induced endothelial dysfunction and atherosclerotic lesion formation. Thus, targeting endothelial ENaC may be a promising strategy to halt atherogenesis.

Background/Aims: The epithelial sodium channel (ENaC) in cortical collecting duct (CCD) principal cells plays a critical role in regulating systemic blood pressure. We have previously shown that cholesterol (Cho) in the apical cell membrane regulates ENaC; however, the underlying mechanism remains unclear. Methods: Patch-clamp technique and confocal microscopy were used to evaluate ENaC activity and density. Results: Here we show that extraction of membrane Cho with methyl-β-cyclodextrin (MβCD) significantly reduced amiloride-sensitive current and ENaC single-channel activity. The effects were reproduced by inhibition of Cho synthesis in the cells with lovastatin. We have previously shown that phosphatidylinositol-4,5-bisphosphate (PIP2), an ENaC activator, is predominantly located in the microvilli, a specialized apical membrane domain. Here, our confocal microscopy data show that α-ENaC was co-localized with PIP2 in the microvilli and that Cho was also co-localized with PIP2 in the microvilli. Either extraction of Cho with MβCD or inhibition of Cho synthesis with lovastatin consistently reduced the levels of Cho, PIP2, and ENaC in the microvilli. Conclusions: Since PIP2 can directly stimulate ENaC and also affect ENaC trafficking, these data suggest that depletion of Cho reduces ENaC apical density and activity at least in part by decreasing PIP2 in the microvilli.

Background and purpose The epithelial sodium channel (ENaC) is expressed in endothelial cells and acts as a negative modulator of vasodilatation. Oxidized LDL (ox-LDL) is a key pathological factor in endothelial dysfunction. In the present study we examined the role of ENaC in ox-LDL-induced endothelial dysfunction and its associated signal transduction pathway. Experimental approach Patch clamp techniques combined with pharmacological approaches were used to examine ENaC activity in the endothelial cells of a split-open mouse thoracic aorta. Western blot analysis was used to determine ENaC expression in the aorta. The aorta relaxation was measured using a wire myograph assay. Key results Ox-LDL, but not LDL, significantly increased ENaC activity in the endothelial cells attached to split-open thoracic aortas, and the increase was inhibited by a lectin-like ox-LDL receptor-1 (LOX-1) antagonist (κ-carrageenan), an NADPH oxidase inhibitor (apocynin), and a scavenger of ROS (TEMPOL). Sodium nitroprusside, an NO donor, diminished the ox-LDL-mediated activation of ENaC, and this effect was abolished by inhibiting soluble guanylate cyclase (sGC) and PKG. Ox-LDL reduced the endothelium-dependent vasodilatation of the aorta pectoralis induced by ACh, and this reduction was partially restored by blocking ENaC. Conclusion and implications Ox-LDL stimulates ENaC in endothelial cells through LOX-1 receptor-mediated activation of NADPH oxidase and accumulation of intracellular ROS. Since the stimulation of ENaC can be reversed by elevating NO, we suggest that both inhibition of ENaC and an elevation of NO may protect the endothelium from ox-LDL-induced dysfunction. Linked articles This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

Sulfur-containing gaseous signal molecules including hydrogen sulphide and sulfur dioxide were previously recognized as toxic gases. However, extensive studies have revealed that they can be generated in the cardiovascular system via a sulfur-containing amino acid metabolic pathway, and have an important role in cardiovascular physiology and pathophysiology. Ion channels are pore-forming membrane proteins present in the membrane of all biological cells; their functions include the establishment of a resting membrane potential and the control of action potentials and other electrical signals by conducting ions across the cell membrane. Evidence has now accumulated suggesting that the sulfur-containing gaseous signal molecules are important regulators of ion channels and transporters. The aims of this review are (1) to discuss the recent experimental evidences in the cardiovascular system regarding the regulatory effects of sulfur-containing gaseous signal molecules on a variety of ion channels, including ATP-sensitive potassium, calcium-activated potassium, voltage-gated potassium, L- and T-type calcium, transient receptor potential and chloride and sodium channels, and (2) to understand how the gaseous signal molecules affect ion channels and cardiovascular diseases. Linked articles This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

Background and purpose Our recent studies show that the reduced activity of epithelial sodium channels (ENaC) in endothelial cells accounts for the adaptation of vasculature to salt in Sprague-Dawley rats. The present study examines a hypothesis that enhanced ENaC activity mediates the loss of vasorelaxation in Dahl salt-sensitive (SS) rats. Experimental approach We used the cell-attached patch-clamp technique to record ENaC activity in split-open mesenteric arteries. Western blot and immunofluorescence staining were used to evaluate the levels of aldosterone, ENaC, eNOS and NO. Blood pressure was measured with the tail-cuff method and the artery relaxation was measured with the wire myograph assay. Key results High-salt (HS) diet significantly increased plasma aldosterone and ENaC activity in the endothelial cells of Dahl SS rats. The endothelium-dependent artery relaxation was blunted by HS challenge in these rats. Amiloride, a potent blocker of ENaC, increased both phosphorylated eNOS and NO and therefore prevented the HS-induced loss of vasorelaxation. As, in SS rats, endogenous aldosterone was already elevated by HS challenge, exogenous aldosterone did not further elevate ENaC activity in the rats fed with HS. Eplerenone, a mineralocorticoid receptor antagonist, attenuated the effects of HS on both ENaC activity and artery relaxation. Conclusions and implications These data suggest that HS diet blunts artery relaxation and causes hypertension via a pathway associated with aldosterone-dependent activation of ENaC in endothelial cells. This pathway provides one of the mechanisms by which HS causes hypertension in Dahl SS rats. Linked articles This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.

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.

Background and purpose We have shown that cholesterol is synthesized in the principal cells of renal cortical collecting ducts (CCD) and stimulates the epithelial sodium channels (ENaC). Here we have determined whether lovastatin, a cholesterol synthesis inhibitor, can antagonize the hypertension induced by activated ENaC, following deletion of the cholesterol transporter (ATP-binding cassette transporter A1; ABCA1). Experimental approach We selectively deleted ABCA1 in the principal cells of mouse CCD and used the cell-attached patch-clamp technique to record ENaC activity. Western blot and immunofluorescence staining were used to evaluate protein expression levels. Systolic BP was measured with the tail-cuff method. Key results Specific deletion of ABCA1 elevated BP and ENaC single-channel activity in the principal cells of CCD in mice. These effects were antagonized by lovastatin. ABCA1 deletion elevated intracellular cholesterol levels, which was accompanied by elevated ROS, increased expression of serum/glucocorticoid regulated kinase 1 (Sgk1), phosphorylated neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) and furin, along with shorten the primary cilium, and reduced ATP levels in urine. Conclusions and implications These data suggest that specific deletion of ABCA1 in principal cells increases BP by stimulating ENaC channels via a cholesterol-dependent pathway which induces several secondary responses associated with oxidative stress, activated Sgk1/Nedd4-2, increased furin expression, and reduced cilium-mediated release of ATP. As ABCA1 can be blocked by cyclosporine A, these results suggest further investigation of the possible use of statins to treat CsA-induced hypertension.

Background/aims Atherosclerosis is a chronic inflammatory cardiovascular disease. Macrophages are major components of atherosclerotic plaques and play a key role in the development of atherosclerosis by secreting a variety of pro-inflammatory factors. Our previous studies have confirmed that upconversion nanoparticles encapsulating chlorin e6 (UCNPs-Ce6) mediated photodynamic therapy (PDT) can promote cholesterol efflux and induce apoptosis in THP-1 macrophages. In this study, we investigated whether reactive oxygen species (ROS) generated by UCNPs-Ce6-mediated PDT can induce autophagy to inhibit the expression of pro-inflammatory factor in M1 peritoneal macrophages. Methods Peritoneal macrophages were collected from C57/BL6 mice injected with 3% thioglycollate broth medium and induced by lipopolysaccharides and interferon-γ. Intracellular ROS production was assessed by 2'-7'-dichloroflorescein diacetate and flow cytometry. Autophagy was assayed by western blot, transmission electron microscopy and immunofluorescence. Pro-inflammatory cytokines were detected by enzyme-linked immunosorbent assay and western blot. Results Model M1 peritoneal macrophages were established after 24 h induction. Protein expression levels of LC3 II and Beclin1, and degradation of p62 increased and peaked at 2 h in the PDT group. Meanwhile, levels of inflammatory cytokines iNOS, IL-12, and TNF-α markedly decreased after PDT. The increase in autophagy levels and decrease in pro-inflammatory cytokines were significantly inhibited by 3-methyladenine. Furthermore, ROS generated by UCNPs- Ce6 mediated PDT activated autophagy. The expression of autophagy related-protein and inflammatory cytokines iNOS, IL-12, and TNF-α were inhibited by the ROS inhibitor N-acetyl cysteine. Conclusion ROS generated by UCNPs-Ce6-mediated PDT activated autophagy and inhibited the expression of pro-inflammatory factors of M1 peritoneal macrophage via the PI3K/AKT/mTOR signaling pathway.

Previous studies indicate that the epithelial sodium channel (ENaC) in the kidney is upregulated in diabetes mellitus. Here, we show that ENaC single-channel activity in distal nephron cells was significantly increased by palmitate, a free fatty acid which is elevated in diabetes mellitus. We also show that palmitate increased intracellular Ca2+ and that after chelating intracellular Ca2+ with BAPTA-AM, palmitate failed to affect ENaC activity. Treatment of the cells with 2-aminoethoxydiphenyl borate (2-APB, an inhibitor of IP3 receptors) abolished the elevation of both intracellular Ca2+ and ENaC activity. Treatment of the cells with apocynin (an NADPH oxidase inhibitor), dithiothreitol/NaHS (reducing agents), or LY294002 (a phosphoinositide 3-kinase (PI3K) inhibitor) prevented palmitate-induced ENaC activity, whereas thimerosal (an oxidizing agent) mimicked the effects of palmitate on ENaC activity. However, these treatments did not alter the levels of intracellular Ca2+, indicating that elevation of reactive oxygen species (ROS) and activation of PI3K are downstream of the signaling cascade. Since we have shown that ROS stimulate ENaC by activating PI3K, these data together suggest that palmitate first elevates intracellular Ca2+, then activates an NADPH oxidase to elevate intracellular ROS and PI3K activity, and finally increases ENaC activity via the activated PI3K.

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.

Background and purpose Protein modification by small ubiquitin-like modifier (SUMO) plays a critical role in the pathogenesis of heart diseases. The present study was designed to determine whether ginkgolic acid (GA) as a SUMO-1 inhibitor exerts an inhibitory effect on cardiac fibrosis induced by myocardial infarction (MI). Experimental approach GA was delivered by osmotic pumps in MI mice. Masson staining, electron microscopy (EM) and echocardiography were used to assess cardiac fibrosis, ultrastructure and function. Expression of SUMO-1, PML, TGF-β1 and Pin1 was measured with Western blot or Real-time PCR. Collagen content, cell viability and myofibroblast transformation were measured in neonatal mouse cardiac fibroblasts (NMCFs). Promyelocytic leukemia (PML) protein was over-expressed by plasmid transfection. Key results GA improved cardiac fibrosis and dysfunction, and decreased SUMO-1 expression in MI mice. GA (>20 μM) inhibited NMCF viability in a dose-dependent manner. Nontoxic GA (10 μM) restrained angiotensin II (Ang II)-induced myofibroblast transformation and collagen production. GA also inhibited expression of TGF-β1 mRNA and protein in vitro and in vivo. GA suppressed PML SUMOylation and PML nuclear body (PML-NB) organization, and disrupted expression and recruitment of Pin1 (a positive regulator of TGF-β1 mRNA), whereas over-expression of PML reversed that. Conclusions and implications Inhibition of SUMO-1 by GA alleviated MI-induced heart dysfunction and fibrosis, and the SUMOylated PML/Pin1/TGF-β1 pathway is crucial for GA-inhibited cardiac fibrosis.

Inactivation of hedgehog-interacting protein (HHIP) and overexpression of Gli1 play vital roles in the development of diverse human cancers. The aim of this study is to examine the association of HHIP and Gli1 with the clinicopathologic features and prognosis of patients with glioblastoma (GBM). The expression of HHIP and Gli1 in 103 patients with GBM and 32 control patients was investigated by immunohistochemistry. Statistical analysis was utilized to evaluate the association of HHIP as well as Gli1 with clinicopathological characteristics and prognosis of patients. HHIP and Gli1 were dysregulated in GBM. Spearman's rank analysis showed that HHIP and Gli1 had an inverse correlation (r = -0.386, P = 0.000). Expression of HHIP was significantly correlated with age (P = 0.000), gender (P = 0.003), seizure (P = 0.013), resection degree (P = 0.033), adjuvant treatment (P = 0.030), and O(6)-methylguanine-DNA methyltransferase (MGMT) methylation (P = 0.021), while Gli1 expression was significantly correlated with age (P = 0.002), gender (P = 0.033), Karnofsky performance status (KPS) score (P = 0.028), resection degree (P = 0.000), adjuvant treatment (P = 0.014), and MGMT methylation (P = 0.030). Kaplan-Meier method showed that patients with low Gli1 expression had longer overall survival (OS) than those with high Gli1 expression (P = 0.000) and the OS of the patients with HHIP-positive GBM was significantly longer than that of the patients with HHIP-negative GBM (P = 0.000). Univariate and multivariate analyses confirmed that HHIP expression and Gli1 expression were independent prognostic factors. Our data suggested that expression of HHIP could be considered as significant prognostic marker for patients with GBM.

The primary aim of the present study was to examine the effects of microRNA‑21 (miR‑21) on the proliferation and differentiation of rat primary neural stem cells (NSCs) in vitro. miR‑21 was overexpressed in NSCs by transfection with a miR‑21 mimic. The effects of miR‑21 overexpression on NSC proliferation were revealed by Cell Counting kit 8 and 5‑ethynyl‑2'‑deoxyuridine incorporation assay, and miR‑21 overexpression was revealed to increase NSC proliferation. miR‑21 overexpression was confirmed using reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). mRNA and protein expression levels of key molecules (β‑catenin, cyclin D1, p21 and miR‑21) in the Wnt/β‑catenin signaling pathway were studied by RT‑qPCR and western blot analysis. RT‑qPCR and western blot analyses revealed that miR‑21 overexpression increased β‑catenin and cyclin D1 expression, and decreased p21 expression. These results suggested that miR‑21‑induced increase in proliferation was mediated by activation of the Wnt/β‑catenin signaling pathway, since overexpression of miR‑21 increased β‑catenin and cyclin D1 expression and reduced p21 expression. Furthermore, inhibition of the Wnt/β‑catenin pathway with FH535 attenuated the influence of miR‑21 overexpression on NSC proliferation, indicating that the factors activated by miR‑21 overexpression were inhibited by FH535 treatment. Furthermore, overexpression of miR‑21 enhanced the differentiation of NSCs into neurons and inhibited their differentiation into astrocytes. The present study indicated that in primary rat NSCs, overexpression of miR‑21 may promote proliferation and differentiation into neurons via the Wnt/β‑catenin signaling pathway in vitro.

Interleukin 17 (IL-17) plays an important role in the pathogenesis of cardiac interstitial fibrosis. In this study, we explored the role of interleukin-17 in the development of diabetic cardiomyopathy and the underlying mechanisms. The level of IL-17 increased in both the serum and cardiac tissue of diabetic mice. Knockout of IL-17 improved cardiac function of diabetic mice induced by streptozotocin (STZ), and significantly alleviated interstitial fibrosis as manifested by reduced collagen mRNA expression and collagen deposition evaluated by Masson's staining. High glucose treatment induced collagen production were abolished in cultured IL-17 knockout cardiac fibroblasts (CFs). The levels of long noncoding RNA-AK081284 were increased in the CFs treated with high glucose or IL-17. Knockout of IL-17 abrogated high glucose induced upregulation of AK081284. Overexpression of AK081284 in cultured CFs promoted the production of collagen and TGFβ1. Both high glucose and IL-17 induced collagen and TGFβ1 production were mitigated by the application of the siRNA for AK081284. In summary, deletion of IL-17 is able to mitigate myocardial fibrosis and improve cardiac function of diabetic mice. The IL-17/AK081284/TGFβ1 signaling pathway mediates high glucose induced collagen production. This study indicates the therapeutic potential of IL-17 inhibition on diabetic cardiomyopathy disease associated with fibrosis.

Osteosarcoma (OS) is the most commonly diagnosed tumor of the bones in children and young adults. Even with conventional therapies the 5-year survival rate is ~65% in patients with OS. Considering the side effects and aggressiveness of malignant bone tumors, research is focussing on multi-targeted strategies in treatment. Cucurbitacin B, a triterpenoid compound has been demonstrated to induce apoptosis in various cancer cell types. The Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) signalling cascades and mitogen activated protein kinases (MAPK) signalling cascades are critical regulators of tumorigenesis. The present study assessed the influence of cucurbitacin B on the viability and expression of MAPKs and proteins of the JAK2/STAT3 cascades in human OS cells (U-2 OS). Cucurbitacin B (20-100 µM) significantly reduced cell viability (P

BACKGROUND AND PURPOSE: The epithelial sodium channel (ENaC) is expressed in endothelial cells and acts as a negative modulator of vasodilatation. Oxidized LDL (ox‐LDL) is a key pathological factor in endothelial dysfunction. In the present study we examined the role of ENaC in ox‐LDL‐induced endothelial dysfunction and its associated signal transduction pathway. EXPERIMENTAL APPROACH: Patch clamp techniques combined with pharmacological approaches were used to examine ENaC activity in the endothelial cells of a split‐open mouse thoracic aorta. Western blot analysis was used to determine ENaC expression in the aorta. The aorta relaxation was measured using a wire myograph assay. KEY RESULTS: Ox‐LDL, but not LDL, significantly increased ENaC activity in the endothelial cells attached to split‐open thoracic aortas, and the increase was inhibited by a lectin‐like ox‐LDL receptor‐1 (LOX‐1) antagonist (κ‐carrageenan), an NADPH oxidase inhibitor (apocynin), and a scavenger of ROS (TEMPOL). Sodium nitroprusside, an NO donor, diminished the ox‐LDL‐mediated activation of ENaC, and this effect was abolished by inhibiting soluble guanylate cyclase (sGC) and PKG. Ox‐LDL reduced the endothelium‐dependent vasodilatation of the aorta pectoralis induced by ACh, and this reduction was partially restored by blocking ENaC. CONCLUSION AND IMPLICATIONS: Ox‐LDL stimulates ENaC in endothelial cells through LOX‐1 receptor‐mediated activation of NADPH oxidase and accumulation of intracellular ROS. Since the stimulation of ENaC can be reversed by elevating NO, we suggest that both inhibition of ENaC and an elevation of NO may protect the endothelium from ox‐LDL‐induced dysfunction. LINKED ARTICLES: This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc

Anoctamin1 (ANO1) encodes a Ca(2+)-activated chloride (Cl(-)) channel (CaCC) in variety tissues of many species. Whether ANO1 expresses and functions as a CaCC in cardiomyocytes remain unknown. The objective of this study is to characterize the molecular and functional expression of ANO1 in cardiac myocytes and the role of ANO1-encoded CaCCs in ischemia-induced arrhythmias in the heart. Quantitative real-time RT-PCR, immunofluorescence staining assays, and immunohistochemistry identified the molecular expression, location, and distribution of ANO1 in mouse ventricular myocytes (mVMs). Patch-clamp recordings combined with pharmacological analyses found that ANO1 was responsible for a Ca(2+)-activated Cl(-) current (I(Cl.Ca)) in cardiomyocytes. Myocardial ischemia led to a significant increase in the current density of I(Cl.Ca), which was inhibited by a specific ANO1 inhibitor, T16A(inh)-A01, and an antibody targeting at the pore area of ANO1. Moreover, cardiomyocytes isolated from mice with ischemia-induced arrhythmias had an accelerated early phase 1 repolarization of action potentials (APs) and a deeper "spike and dome" compared to control cardiomyocytes from non-ischemia mice. Application of the antibody targeting at ANO1 pore prevented the ischemia-induced early phase 1 repolarization acceleration and caused a much shallower "spike and dome". We conclude that ANO1 encodes CaCC and plays a significant role in the phase 1 repolarization of APs in mVMs. The ischemia-induced increase in ANO1 expression may be responsible for the increased density of I(Cl.Ca) in the ischemic heart and may contribute, at least in part, to ischemia-induced arrhythmias.

Both senile plaques and intracellular neurofibrillary tangles are important pathological characteristics in Alzheimer's disease. However, the relationship between Aβ deposition and tau hyperphosphorylation is unknown. In this study, the increased levels of full-length amyloid precursor protein (APP), APP C-terminal fragment (β-CTF) and BACE1 were found in streptozotocin-induced tau hyperphosphorylation models by quantitative polymerase chain reaction, Western blotting and immunohistochemistry methods. In the previous studies, few strategies focusing on inhibiting β-secretase (BACE1) in a tau hyperphosphorylation model were utilized. Here, BACE1 RNAi was used to treat the streptozotocin-induced tau hyperphosphorylation animal models. BACE1 RNAi treatment improved the behavioural ability of animal models and reduced the amount of Aβ1-40 and Aβ1-42, accompanied by decreasing the levels of BACE1 and β-CTF. Our results demonstrated that neurological defects and neurotoxic fragments, including Aβ and β-CTF, were eliminated by BACE1 RNAi in the tau hyperphosphorylated model, implying the efficiency and safety of BACE1RNAi treatment against Alzheimer's disease.

Vascular endothelial cells (ECs) line the luminal surface of blood vessels, which are exposed constantly to mechanical stimuli, such as fluid shear stress, cyclic strain, and blood pressure. In recent years, more and more evidence indicates that ECs sense these mechanical stimuli and subsequently convert mechanical stimuli into intracellular signals. The properties of ECs that sense the mechanical stimuli are defined as mechanosensors. There are a variety of mechanosensors that have been identified in ECs. These mechanosensors play an important role in regulating the function of the endothelium and vascular function, including blood pressure. This review focuses on the mechanosensors that have been identified in ECs and on the roles that mechanosensors play in the regulation of endothelium function, and in the regulation of vascular function.

MicroRNAs are ~22 nt long small noncoding RNA molecules that silence post-transcription gene expression. It has proven that microRNAs are widely expressed in eukaryotes and play an important role in the regulation of cell differentiation and development, growth metabolism, and many other cell activities. Induced pluripotent stem cells (iPS) are a type of pluripotent stem cells reprogrammed from somatic cells and exhibit the essential characteristics of embryonic stem cells. iPS technology has been widely applied in the biological and medical fields, and the key of it is reprogramming of somatic epigenetic state. Therefore, it is of important theoretical and practical significance to study the mechanisms of somatic reprogramming for establishment of an improved iPS technology. The methods of transfection of defined exogenetic stem factors, such as Oct4, Sox2, Klf4, and c-Myc into somatic cells through viral vectors have been continuously improving, but the genome integration and reactivation of the oncogenic gene increase the tumorigenicity of induced cells. The integration-free ways, such as adenovirus, plasmid, recombinant proteins, and L-myc replacement used in iPS technology significantly reduce the risk of cancer. However, the inducing mechanisms are still unclear. Recent studies showed that microRNA affect the process of somatic cell reprogramming, especially embryonic stem cell regulating (ESCC) family of microRNAs (miR302/367, miR200, miR-34, and miR290/295) enhances the reprogramming of embryonic fibroblasts to iPS. This article reviews the recent progresses of roles of microRNA in iPS.

Summary Aims To examine a novel strategy to enhance the survival of grafted neural stem cells (NSCs) in stroke model. Methods Using a cell counting kit-8 (CCK-8) and TUNEL assay to test the protective effects of p53 inhibitor, pifithrin-α (PFT-α), on oxygen glucose deprivation (OGD) in NSCs. We compared the effects of vehicle + NSCs and FFT-α + NSCs on the efficacy of transplantation in stroke rat model using behavioral analysis, immunohistochemistry, etc. Results Pifithrin-α increased viability and decreased apoptosis in NSCs after OGD in vitro. By in vivo studies, we showed that the best recovery of neurological function in the stroke rats and the maximum survival of grafted NSCs were found in the PFT-α + NSCs group. Twelve hours after cell transplantation, p53 was localized to the nuclei of grafted NSCs in the vehicle + NSCs group but was primarily localized to the cytoplasm in the PFT-α + NSCs group. The p53-upregulated modulator of apoptosis (PUMA) was highly expressed among the grafted cells in the vehicle + NSCs group compared with that in the PFT-α + NSCs group. Conclusion Our results indicate that PFT-α enhances the survival of grafted NSCs through the inhibition of p53 translocation into the nucleus.

Pathological cardiac hypertrophy is a key risk factor for heart failure. We found that the protein expression levels of the ZNF307 (zinc finger protein 307) were significantly increased in heart samples from both human patients with dilated cardiomyopathy and mice subjected to aortic banding. Therefore, we aimed to elucidate the role of ZNF307 in the development of cardiac hypertrophy and to explore the signal transduction events that mediate the effect of ZNF307 on cardiac hypertrophy, using cardiac-specific ZNF307 transgenic (ZNF307-TG) mice and ZNF307 global knockout (ZNF307-KO) mice. The results showed that the deletion of ZNF307 potentiated aortic banding–induced pathological cardiac hypertrophy, fibrosis, and cardiac dysfunction; however, the aortic banding–induced cardiac hypertrophic phenotype was dramatically diminished by ZNF307 overexpression in mouse heart. Mechanistically, the antihypertrophic effects mediated by ZNF307 in response to pathological stimuli were associated with the direct inactivation of NF-κB (nuclear factor-κB) signaling and blockade of the nuclear translocation of NF-κB subunit p65. Furthermore, the overexpression of a degradation-resistant mutant of IκBα (IκBα S32A/S36A ) reversed the exacerbation of cardiac hypertrophy, fibrosis, and dysfunction shown in aortic banding–treated ZNF307-KO mice. In conclusion, our findings demonstrate that ZNF307 ameliorates pressure overload–induced cardiac hypertrophy by inhibiting the activity of NF-κB–signaling pathway.

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 mpkCCDc14cells, a mouse CCD principal cell line, to determine whether NSCCarepresents 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 mpkCCDc14cells. 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 mpkCCDc14cells. Unlike those previously reported in other cell types, the TRPM4 in mpkCCDc14cells was unable to be activated by hydrogen peroxide (H2O2). Conversely, after treatment with H2O2, TRPM4 density in the apical membrane of mpkCCDc14cells was significantly decreased. The channel in intact cell-attached patches was activated by ionomycin (a Ca2+ionophore), but not by ATP (a purinergic P2receptor agonist). These data suggest that the NSCCacurrent 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.