Self-assembly of differentiated dental pulp stem cells facilitates spheroid human dental organoid formation and prevascularization.

BACKGROUND: The self-assembly of solid organs from stem cells has the potential to greatly expand the applicability of regenerative medicine. Stem cells can self-organise into microsized organ units, partially modelling tissue function and regeneration. Dental pulp organoids have been used to recapitulate the processes of tooth development and related diseases. However, the lack of vasculature limits the utility of dental pulp organoids. AIM: To improve survival and aid in recovery after stem cell transplantation, we demonstrated the three-dimensional (3D) self-assembly of adult stem cell-human dental pulp stem cells (hDPSCs) and endothelial cells (ECs) into a novel type of spheroid-shaped dental pulp organoid in vitro under hypoxia and conditioned medium (CM). METHODS: During culture, primary hDPSCs were induced to differentiate into ECs by exposing them to a hypoxic environment and CM. The hypoxic pretreated hDPSCs were then mixed with ECs at specific ratios and conditioned in a 3D environment to produce prevascularized dental pulp organoids. The biological characteristics of the organoids were analysed, and the regulatory pathways associated with angiogenesis were studied. RESULTS: The combination of these two agents resulted in prevascularized human dental pulp organoids (Vorganoids) that more closely resembled dental pulp tissue in terms of morphology and function. Single-cell RNA sequencing of dental pulp tissue and RNA sequencing of Vorganoids were integrated to analyse key regulatory pathways associated with angiogenesis. The biomarkers forkhead box protein O1 and fibroblast growth factor 2 were identified to be involved in the regulation of Vorganoids. CONCLUSION: In this innovative study, we effectively established an in vitro model of Vorganoids and used it to elucidate new mechanisms of angiogenesis during regeneration, facilitating the development of clinical treatment strategies.

Endothelial HDAC1-ZEB2-NuRD Complex Drives Aortic Aneurysm and Dissection Through Regulation of Protein S-Sulfhydration.

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 (H2S) 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/H2S system and the findings were confirmed in transgenic mice. RESULTS: Higher plasma H2S 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 H2S donor GYY4137 or pharmacologically inhibiting HDAC1 activity with entinostat alleviated the progression of AAD. CONCLUSIONS: Decreased plasma H2S 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.

Dicer deficiency impairs proliferation but potentiates anti-tumoral effect of macrophages in glioblastoma.

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.

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.

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.

Pharmacological inhibition of SUMO-1 with ginkgolic acid alleviates cardiac fibrosis induced by myocardial infarction in mice.

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-ß1 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-ß1 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-ß1 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-ß1 pathway is crucial for GA-inhibited cardiac fibrosis.

Cucurbitacin B inhibits cell proliferation and induces apoptosis in human osteosarcoma cells via modulation of the JAK2/STAT3 and MAPK pathways.

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<0.05) and induced apoptosis, as assessed by MTT and Annexin V/propidium iodide staining, along with inhibiting cell migration. Gelatin zymography revealed supressed activities of matrix metalloproteinase (MMP-)2 and 9. Furthermore, cucurbitacin B effectively upregulated the apoptotic pathway and caused the effective inhibition of MAPK signalling and JAK2/STAT3 cascades. Multifold suppression of vascular endothelial growth factor by cucurbitacin B was also observed, indicating inhibition of angiogenesis. Thus, by downregulating major pathways-MAPK and JAK2/STAT3 and MMPs, cucurbitacin B has potent anti-proliferative and anti-metastatic effects that require further investigation with regards to cancer treatment.

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.

Neural Stem Cell-based Intraocular Administration of Pigment Epithelium-derived Factor Promotes Retinal Ganglion Cell Survival and Axon Regeneration after Optic Nerve Crush Injury in Rat: An Experimental Study.

BACKGROUND: Pigment epithelium-derived factor (PEDF) is regarded as a multifunctional protein possessing neurotrophic and neuroprotective properties. PEDF has a very short half-life, and it would require multiple injections to maintain a therapeutically relevant level without a delivery system. However, multiple injections are prone to cause local damage or infection. To overcome this, we chose a cell-based system that provided sustained delivery of PEDF and compared the effect of weekly injections of PEDF and neural stem cell (NSC)-based intraocular administration of PEDF on retinal ganglion cell (RGC) survival and axon regeneration after optic nerve injury. METHODS: Seventy-two rats were randomly assigned to 3 groups: group with injections of phosphate buffered saline (PBS) (n=24), group with weekly injections of PEDF (n=24), and group with NSC-based administration of PEDF (n=24). Western blot was used to analyze the PEDF protein level 2 weeks after injection. Retinal flat mounts and immunohistochemistry were employed to analyze RGC survival and axon regeneration 2 weeks and 4 weeks after injection. The data were analyzed with one-way ANOVA in SPSS (version 19.0). A P<0.05 was considered significant. RESULTS: The PEDF protein level in the group with NSC-based administration of PEDF increased compared with that in the groups with injections of PEDF and PBS (P<0.05). The PEDF-modified NSCs differentiated into GFAP-positive astrocytes andß-tubulin-III-positive neurons. NSC-based administration of PEDF effectively increased RGC survival and improved the axon regeneration of the optic nerve compared with weekly injections of PEDF. CONCLUSION: Subretinal space transplantation of PEDF-secreting NSCs sustained high concentrations of PEDF, differentiated into neurons and astrocytes, and significantly promoted RGC survival and axon regeneration after optic nerve injury.

Type III Transforming Growth Factor-ß Receptor Drives Cardiac Hypertrophy Through ß-Arrestin2-Dependent Activation of Calmodulin-Dependent Protein Kinase II.

The role of type III transforming growth factor-ß receptor (TßRIII) in the pathogenesis of heart diseases remains largely unclear. Here, we investigated the functional role and molecular mechanisms of TßRIII in the development of myocardial hypertrophy. Western blot and quantitative real time-polymerase chain reaction analyses revealed that the expression of TßRIII was significantly elevated in human cardiac hypertrophic samples. Consistently, TßRIII expression was substantially increased in transverse aortic constriction (TAC)- and isoproterenol-induced mouse cardiac hypertrophy in vivo and in isoproterenol-induced cardiomyocyte hypertrophy in vitro. Overexpression of TßRIII resulted in cardiomyocyte hypertrophy, whereas isoproterenol-induced cardiomyocyte hypertrophy was greatly attenuated by knockdown of TßRIII in vitro. Cardiac-specific transgenic expression of TßRIII independently led to cardiac hypertrophy in mice, which was further aggravated by isoproterenol and TAC treatment. Cardiac contractile function of the mice was not altered in TßRIII transgenic mice; however, TAC led to significantly decreased cardiac contractile function in TßRIII transgenic mice compared with control mice. Conversely, isoproterenol- and TAC-induced cardiac hypertrophy and TAC-induced cardiac contractile function impairment were partially reversed by suppression of TßRIII in vivo. Our data suggest that TßRIII mediates stress-induced cardiac hypertrophy through activation of Ca(2+)/calmodulin-dependent protein kinase II, which requires a physical interaction of ß-arrestin2 with both TßRIII and calmodulin-dependent protein kinase II. Our findings indicate that stress-induced increase in TßRIII expression results in cardiac hypertrophy through ß-arrestin2-dependent activation of calmodulin-dependent protein kinase II and that transforming growth factor-ß and ß-adrenergic receptor signaling are not involved in spontaneous cardiac hypertrophy in cardiac-specific transgenic expression of TßRIII mice. Our findings may provide a novel target for control of myocardial hypertrophy.

Tumor necrosis factor receptor-associated factor 5 (Traf5) acts as an essential negative regulator of hepatic steatosis.

BACKGROUND & AIMS: Obesity-related metabolic inflammation, insulin resistance (IR), and excessive fat accumulation are linked phenomena that promote the progression of nonalcoholic fatty liver disease (NAFLD). Previous research has indicated that CD40-TRAF5 signaling protects against obesity-related metabolic disorders; however, the precise roles and underlying mechanisms of TRAF5 in obesity-induced pathological processes have not been fully elucidated. METHODS: TRAF5 expression was evaluated in the livers of NAFLD patients, high-fat diet (HFD)-induced or genetically (ob/ob) induced obese mice, and in palmitate-treated hepatocytes. Gain- or loss-of-function approaches were used to investigate the specific roles and mechanisms of hepatic Traf5 under obesity-related pathological conditions. RESULTS: TRAF5 expression was decreased in the fatty livers of both NAFLD patients and obese mice, and in palmitate-treated hepatocytes in vitro. Traf5 overexpression significantly suppressed nonalcoholic steatohepatitis (NASH)-like phenotypes in mice after HFD treatment for 24weeks and inhibited the progression of NAFLD in ob/ob mice. Conversely, Traf5 deficiency resulted in the deterioration of metabolic disorders induced by HFD. Investigations of the underlying mechanisms revealed that Traf5 regulates hepatic steatosis by targeting Jnk signaling. Specifically, Jnk1 rather than Jnk2 is responsible for the function of Traf5 in metabolic disorders, as evidenced by the fact that Jnk1 ablation markedly ameliorates the detrimental effects of Traf5 deficiency on obesity, inflammation, IR, hepatic steatosis and fibrosis. CONCLUSIONS: Traf5 negatively regulates NAFLD/NASH and related metabolic dysfunctions by blocking Jnk1 activity, which represents a potential therapeutic target for obesity-related metabolic disorders. LAY SUMMARY: Lipid accumulation in the liver induces degradation of Traf5. Increasing Traf5 ameliorates nonalcoholic fatty liver by blocking Jnk1 activity.

Deletion of interleukin-6 alleviated interstitial fibrosis in streptozotocin-induced diabetic cardiomyopathy of mice through affecting TGFß1 and miR-29 pathways.

Interleukin 6 (IL-6) has been shown to be an important regulator of cardiac interstitial fibrosis. In this study, we explored the role of interleukin-6 in the development of diabetic cardiomyopathy and the underlying mechanisms. Cardiac function of IL-6 knockout mice was significantly improved and interstitial fibrosis was apparently alleviated in comparison with wildtype (WT) diabetic mice induced by streptozotocin (STZ). Treatment with IL-6 significantly promoted the proliferation and collagen production of cultured cardiac fibroblasts (CFs). High glucose treatment increased collagen production, which were mitigated in CFs from IL-6 KO mice. Moreover, IL-6 knockout alleviated the up-regulation of TGFß1 in diabetic hearts of mice and cultured CFs treated with high glucose or IL-6. Furthermore, the expression of miR-29 reduced upon IL-6 treatment, while increased in IL-6 KO hearts. Overexpression of miR-29 blocked the pro-fibrotic effects of IL-6 on cultured CFs. In summary, deletion of IL-6 is able to mitigate myocardial fibrosis and improve cardiac function of diabetic mice. The mechanism involves the regulation of IL-6 on TGFß1 and miR-29 pathway. This study indicates the therapeutic potential of IL-6 suppression on diabetic cardiomyopathy disease associated with fibrosis.

[Corrigendum] Activation of sonic hedgehog signaling enhances cell migration and invasion by induction of matrix metalloproteinase-2 and -9 via the phosphoinositide-3 kinase/AKT signaling pathway in glioblastoma.

Mol Med Rep 12: [Related article:] 6702­6710, 2015; DOI: 10.3892/mmr.2015.4229 After the publication of the article, it has been brought to the authors' attention by an interested reader that we had made an error regarding the presentation of certain data in the manuscript. The error relates to the presentation of Figs. 1 and 2 in the paper: The control panels for Fig. 1C [labelled 'cyclopamine (µM)'] and Figs. 2B and C [labelled 'rhSSH (µg/ml)'] were derived from the same image. The control U251 cells, featured in Fig. 1 and Figs. 2B and C, were treated without cyclopamine and rhSHH. Therefore, the U251 cells treated without cyclopamine and rhSHH were considered as a control group compared with U251 cells that were separately treated with cyclopamine or rhSHH, and these were photographed randomly. A new Fig. 2 is provided, which contains the correct data for the control panels for Figs. 2B and C.

Activation of sonic hedgehog signaling enhances cell migration and invasion by induction of matrix metalloproteinase-2 and -9 via the phosphoinositide-3 kinase/AKT signaling pathway in glioblastoma.

Aberrant hedgehog signaling contributes to the development of various malignancies, including glioblastoma (GBM). However, the potential mechanism of hedgehog signaling in GBM migration and invasion has remained to be elucidated. The present study showed that enhanced hedgehog signaling by recombinant human sonic hedgehog N­terminal peptide (rhSHH) promoted the adhesion, invasion and migration of GBM cells, accompanied by increases in mRNA and protein levels of matrix metalloproteinase­2 (MMP­2) and MMP­9. However, inhibition of hedgehog signaling with cyclopamine suppressed the adhesion, invasion and migration of GBM cells, accompanied by decreases in mRNA and protein levels of MMP­2 and ­9. Furthermore, it was found that MMP­2- and MMP­9-neutralizing antibodies or GAM6001 reversed the inductive effects of rhSHH on cell migration and invasion. In addition, enhanced hedgehog signaling by rhSHH increased AKT phosphorylation, whereas blockade of hedgehog signaling decreased AKT phosphorylations. Further experiments showed that LY294002, an inhibitor of phosphoinositide-3 kinase (PI3K), decreased rhSHH­induced upregulation of MMP­2 and ­9. Finally, the protein expression of glioblastoma-associated oncogene 1 was positively correlated with levels of phosphorylated AKT as well as protein expressions of MMP­2 and ­9 in GBM tissue samples. In conclusion, the present study indicated that the hedgehog pathway regulates GBM-cell migration and invasion by increasing MMP-2 and MMP-9 production via the PI3K/AKT pathway.

Hydrogen Sulfide Prevents Advanced Glycation End-Products Induced Activation of the Epithelial Sodium Channel.

Advanced glycation end-products (AGEs) are complex and heterogeneous compounds implicated in diabetes. Sodium reabsorption through the epithelial sodium channel (ENaC) at the distal nephron plays an important role in diabetic hypertension. Here, we report that H2S antagonizes AGEs-induced ENaC activation in A6 cells. ENaC open probability (P O ) in A6 cells was significantly increased by exogenous AGEs and that this AGEs-induced ENaC activity was abolished by NaHS (a donor of H2S) and TEMPOL. Incubating A6 cells with the catalase inhibitor 3-aminotriazole (3-AT) mimicked the effects of AGEs on ENaC activity, but did not induce any additive effect. We found that the expression levels of catalase were significantly reduced by AGEs and both AGEs and 3-AT facilitated ROS uptake in A6 cells, which were significantly inhibited by NaHS. The specific PTEN and PI3K inhibitors, BPV(pic) and LY294002, influence ENaC activity in AGEs-pretreated A6 cells. Moreover, after removal of AGEs from AGEs-pretreated A6 cells for 72 hours, ENaC P O remained at a high level, suggesting that an AGEs-related "metabolic memory" may be involved in sodium homeostasis. Our data, for the first time, show that H2S prevents AGEs-induced ENaC activation by targeting the ROS/PI3K/PTEN pathway.

ß-elemene induces caspase-dependent apoptosis in human glioma cells in vitro through the upregulation of Bax and Fas/ FasL and downregulation of Bcl-2.

BACKGROUND: ß-elemene, extracted from herb medicine Curcuma wenyujin has potent anti-tumor effects in various cancer cell lines. However, the activity of ß-elemene against glioma cells remains unclear. In the present study, we assessed effects of ß-elemene on human glioma cells and explored the underlying mechanism. MATERIALS AND METHODS: Human glioma U87 cells were used. Cell proliferation was determined with MTT assay and colony formation assay to detect the effect of ß-elemene at different doses and times. Fluorescence microscopy was used to observe cell apoptosis with Hoechst 33258 staining and change of glioma apoptosis and cell cycling were analyzed by flow cytometry. Real-time quantitative PCR and Western-blotting assay were performed to investigated the influence of ß-elemene on expression levels of Fas/FasL, caspase-3, Bcl-2 and Bax. The experiment was divided into two groups: the blank control group and ß-elemne treatment group. RESULTS: With increase in the concentration of ß-elemene, cytotoxic effects were enhanced in the glioma cell line and the concentration of inhibited cell viability (IC50) was 48.5 µg/mL for 24h. ß-elemene could induce cell cycle arrest in the G0/G1 phase. With Hoechst 33258 staining, apoptotic nuclear morphological changes were observed. Activation of caspase-3,-8 and -9 was increased and the pro-apoptotic factors Fas/FasL and Bax were upregulated, while the anti-apoptotic Bcl-2 was downregulated after treatment with ß-elemene at both mRNA and protein levels. Furthermore, proliferation and colony formation by U87 cells were inhibited by ß-elemene in a time and does- dependent manner. CONCLUSIONS: Our results indicate that ß-elemene inhibits growth and induces apoptosis of human glioma cells in vitro. The induction of apoptosis appears to be related with the upregulation of Fas/FasL and Bax, activation of caspase-3,-8 and -9 and downregulation of Bcl-2, which then trigger major apoptotic cascades.

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.

TRPP2 and TRPV4 form an EGF-activated calcium permeable channel at the apical membrane of renal collecting duct cells.

OBJECTIVE: Regulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear. METHODS AND RESULTS: We report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+)) or in which cilia are absent (cilia (-)). This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (-) compared to cilia (+) cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF) stimulated TRPP2\TRPV4 channel through the EGF receptor (EGFR) tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (-) but not cilia (+) cells. In addition EGF increased cell proliferation in cilia (-) cell that was dependent upon TRPP2\TRPV4 channel mediated increase in intracellular calcium. CONCLUSION: We conclude that in the absence of cilia, an EGF activated TRPP2\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis.