Hesperidin Attenuates Oxidative Stress, Inflammation, Apoptosis, and Cardiac Dysfunction in Sodium Fluoride-Induced Cardiotoxicity in Rats.

Excessive fluoride intake has been reported to cause toxicities to brain, thyroid, kidney, liver and testis tissues. Hesperidin (HSP) is an antioxidant that possesses anti-allergenic, anti-carcinogenic, anti-oxidant and anti-inflammatory activities. Presently, the studies focusing on the toxic effects of sodium fluoride (NaF) on heart tissue at biochemical and molecular level are limited. This study was designed to evaluate the ameliorative effects of HSP on toxicity of NaF on the heart of rats in vivo by observing the alterations in oxidative injury markers (MDA, SOD, CAT, GPX and GSH), pro-inflammatory markers (NF-κB, IL-1ß, TNF-α), expressions of apoptotic genes (caspase-3, -6, -9, Bax, Bcl-2, p53, cytochrome c), levels of autophagic markers (Beclin 1, LC3A, LC3B), expression levels of PI3K/Akt/mTOR and cardiac markers. HSP treatment attenuated the NaF-induced heart tissue injury by increasing activities of SOD, CAT and GPx and levels of GSH, and suppressing lipid peroxidation. In addition, HSP reversed the changes in expression of apoptotic (caspase-3, -6, -9, Bax, Bcl-2, p53, cytochrome c), levels of autophagic and inflammatory parameters (Beclin 1, LC3A, LC3B, NF-κB, IL-1ß, TNF-α), in the NaF-induced cardiotoxicity. HSP also modulated the gene expression levels of PI3K/Akt/mTOR signaling pathway and levels of cardiac markers (LDH, CK-MB). Overall, these findings reveal that HSP treatment can be used for the treatment of NaF-induced cardiotoxicity.

PTX3/TWIST1 Feedback Loop Modulates Lipopolysaccharide-Induced Inflammation via PI3K/Akt Signaling Pathway.

Chronic inflammation of nasal mucosal tissue is an obvious feature of allergic rhinitis. Pentraxin 3 (PTX3) is a member of the pentraxin family and plays important roles in inflammation. We aimed to investigate the roles and mechanisms of PTX3 in inflammatory factors and MUC5AC production in human nasal epithelia cells. Loss- and gain-of-function experiments were performed. We found that the silencing of PTX3 dramatically blocked the expression of interleukin (IL)-6, IL-8, IL-1ß, and MUC5AC induced by lipopolysaccharide (LPS). Gain-of-function of PTX3 displayed the opposite results. Interestingly, the ablation of PTX3 blocked activation of the PI3K/Akt signaling pathway, whereas the administration of an agonist of PI3K, 740Y-P, partially reversed the inhibitory functions of PTX3 silencing on inflammation and MUC5AC production. Moreover, PTX3 was a positive regulator of TWIST1, which is one of the transcription factors of PTX3. We noticed that TWIST1 downregulation reduced the expression of PTX3. Furthermore, chromatin immunoprecipitation assay and dual-luciferase reporter assay demonstrated that TWIST1 could bind to the promoter of PTX3. Importantly, the depletion of TWIST1 attenuated the LPS-mediated expression and secretion of inflammatory cytokines, whereas these effects were partially abolished upon PTX3 overexpression. Taken together, our findings revealed that the PTX3/TWIST1 feedback loop modulates LPS-induced inflammation and MUC5AC production via the PI3K/Akt signaling pathway.

MFAP4 deletion attenuates the progression of angiotensin II-induced atrial fibrosis and atrial fibrillation.

AIMS: Microfibrillar-associated protein 4 (MFAP4) is associated with atrial fibrillation (AF). Nevertheless, the specific role and underlying mechanism of MFAP4 in atrial fibrosis, the hallmark of AF, remain undefined. This study aims to elucidate the role of MFAP4 in the regulation of atrial fibrosis and to explore the underlying mechanism. METHODS AND RESULTS: This study used MFAP4 knockout (MFAP4-KO) mice and their wild-type (WT) littermates to investigate the effect of angiotensin II (Ang II) (2000 ng/kg/min for 3 weeks) on atrial fibrosis and susceptibility to AF in terms of morphology, histology, electrophysiology, and molecular biology. MFAP4 deletion in mice did not alter cardiac structure and function at baseline. After treatment with Ang II, the MFAP4-KO mice showed a decreased left atrial enlargement and fibrosis, slowed atrial conduction, and reduced susceptibility to AF compared with the WT mice. Regarding the mechanism, we found that MFAP4 deletion markedly inhibited activated focal adhesion kinase (FAK)-mediated PI3K-AKT signalling and MEK1/2-ERK1/2 signalling after Ang II treatment. CONCLUSIONS: Overall, our study showed that loss of MFAP4 attenuates Ang II-mediated left atrial fibrosis and dilation and decreases susceptibility to AF by decreasing the phosphorylation of FAK and inhibiting the activation of the PI3K-AKT and MEK1/2-ERK1/2 signalling pathways. These findings further indicate that targeting MFAP4 may be a potential upstream therapeutic option for atrial fibrosis and AF.

Exosomal miR-132-3p from mesenchymal stem cells alleviated LPS-induced acute lung injury by repressing TRAF6.

Exosomes isolated from mesenchymal stem cells (MSC) had shown beneficial effect on acute lung injury (ALI). However, the effective components in MSC-derived exosomes need further investigation. ALI mice model was established by lipopolysaccharide (LPS) injection. In vitro inflammatory model was established by LPS stimulation of MLE-12 cells. The cell proliferation was evaluated by EdU assay. TUNEL and Annexin V/PI were applied to evaluate the apoptosis of tissue and cell respectively. HE staining was performed to evaluate the lung injury. Transmission electronic microscope was used to observe isolated exosomes. Level of cytokines, MDA, KGF were determined by ELISA kit. Direct interaction of miR-132-3p and TRAF6 were verified by dual luciferase assay. The level of mRNA or proteins were determined by qRT-PCR or western blots respectively. TRAF6 was upregulated while miR-132-3p was downregulated in LPS-stimulated ALI model. MiR-132-3p negatively regulated TRAF6 by direct binding. MiR-132-3p potentiated proliferation and suppressed apoptosis of LPS-induced MLE-12 cells at least partly dependent on targeting TRAF6. Treatment of exosome alleviated the LPS-induced ALI in mice and LPS-induced inflammatory response in MLE-12 cells. Moreover, overexpression of miR-132-3p promoted the protective effect of exosomes in LPS-induced MLE-12 cells injury and LPS-induced ALI. Mechanically, it was suggested that miR-132-3p inactivated PI3K/Akt signalling via targeting TRAF6. In the present study, our results indicated that miR-132-3p mediated protective effect of MSC-derived exosomes on LPS-induced ALI. Exosomal miR-132-3p ameliorated LPS-induced ALI via targeting TRAF6 and inactivating PI3K/Akt signalling.

Hypertension induces compensatory left ventricular hypertrophy by a mechanism involving gap junction lateralization and overexpression of CD36, PKC and MMP-2.

Hypertension-induced left ventricular hypertrophy evolves initially as an adaptive response meant to minimize ventricular wall stress. The mechanisms involved in the preservation of the cardiac function during the "compensatory" phase of the left ventricular hypertrophy are still unclear. Therefore, we aimed at uncovering fine changes that aid the heart to cope with the increased stress in hypertension. Male golden Syrian hamsters were given NG-nitro-L-arginine methyl ester (L-NAME) for 16 weeks, and they became hypertensive (HT), developing left ventricular hypertrophy with no impaired contractility or fibrosis. As compared to age-matched control hamsters, the hypertrophied left ventricles in L-NAME-induced HT hamsters exhibited the following structural and molecular changes: (i) accumulation of lipid droplets (LDs) within cardiomyocytes and relocation of gap junctions to the lateral membrane of cardiomyocytes or close to mitochondria (revealed by electron microscopy); (ii) overexpression of the cluster of differentiation 36 (CD36) fatty acid transporter, protein kinase C (PKC), and matrix metalloproteinase-2 (MMP-2), enhanced activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway, and unchanged expression of the connexin 43 (Cx43) and N-cadherin junctional proteins (assessed by Western blot); (iii) increased protein carbonyl content, assessed with a 2,4-Dinitrophenylhydrazine (DNPH)-based spectrophotometric assay, indicative of an enhanced reactive oxygen species (ROS) production; and (iv) augmented MMP-2 activity (determined by gelatin zymography). These changes may participate in an orchestrated adaptive hypertrophic growth response that helps to maintain cardiac performance, in HT hamsters. Together, these findings could provide support for designing future strategies meant to prevent the transition from compensatory left ventricular hypertrophy to decompensated heart failure.

Infection risk with PI3K-AKT-mTOR pathway inhibitors and immune checkpoint inhibitors in patients with advanced solid tumours in phase I clinical trials.

BACKGROUND: Patients undergoing chemotherapy are known to be at risk for infection from myelosuppression by cytotoxic agents (CTAs) or immunosuppressive effects from mTOR inhibitors. The infection risk of newly developed anticancer agents has not been fully evaluated. It remains unknown how T-cell activation induced by immune checkpoint inhibitors (ICIs) relates to infection. METHODS: We retrospectively examined infection risk in patients with cancer treated with investigational agents in a phase I study. The investigational agents were classified into four groups: CTA, phosphatidylinositol 3 kinase/Akt/mammalian target of rapamycin inhibitor (PAM), molecular targeted agent (MTA) and ICI. All infection-related adverse events (AEs) during treatment were recorded. We compared the CTA, PAM and ICI with MTA, because MTA are already considered low risk and were used in the largest number of patients. RESULTS: A total of 641 patients were enrolled: 35 CTAs (5.5%), 61 PAMs (9.5%), 445 MTAs (69.4%) and 100 ICIs (15.6%). Among all patients, 132 (20.6%) experienced infection-related AEs and 46 (7.2%) developed 50 ≥grade 3 infection-related AEs. In any infection-related AEs, the ORs compared with MTAs were 2.19 (95% CI 1.03 to 4.66) for CTAs, 3.55 (95% CI 2.02 to 6.24) for PAMs and 1.05 (95% CI 0.60 to 1.85) for ICIs, respectively. In time to the first infection-related AE analysis, the risks for any infection-related AE from CTAs and PAMs were higher than those from MTAs (HR 1.84 (95% CI 0.82 to 4.11); p=0.05 and 3.96 (95% CI 2.18 to 7.22); p<0.001). The risk from ICIs was not significantly different from that of MTAs (HR 0.71 (95% CI 0.46 to 1.10); p=0.19). CONCLUSION: Our results validate that PAMs and CTAs carry a higher infection risk in patients with advanced solid tumours compared with MTAs. We suggest that the infection risk of ICIs is a similar infection risk to MTAs.

Infección pulmonar polimicrobiana asociada al uso de idelalisib / Polymicrobial lung infection associated with idelalisib administration

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Pulmonary-derived phosphoinositide 3-kinase gamma (PI3Kγ) contributes to ventilator-induced lung injury and edema.

BACKGROUND: Ventilator-induced lung injury (VILI) occurs in part by increased vascular permeability and impaired alveolar fluid clearance. Phosphoinositide 3-kinase gamma (PI3Kγ) is activated by mechanical stress, induces nitric oxide (NO) production, and participates in cyclic adenosine monophosphate (cAMP) hydrolysis, each of which contributes to alveolar edema. We hypothesized that lungs lacking PI3Kγ or treated with PI3Kγ inhibitors would be protected from ventilation-induced alveolar edema and lung injury. METHODS: Using an isolated and perfused lung model, wild-type (WT) and PI3Kγ-knockout (KO) mice underwent negative-pressure cycled ventilation at either -25 cmH2O and 0 cmH2O positive end-expiratory pressure (PEEP) (HIGH STRESS) or -10 cmH2O and -3 cmH2O PEEP (LOW STRESS). RESULTS: Compared with WT, PI3Kγ-knockout mice lungs were partially protected from VILI-induced derangement of respiratory mechanics (lung elastance) and edema formation [bronchoalveolar lavage (BAL) protein concentration, wet/dry ratio, and lung histology]. In PI3Kγ-knockout mice, VILI induced significantly less phosphorylation of protein kinase B (Akt), endothelial nitric oxide synthase (eNOS), production of nitrate and nitrotyrosine, as well as hydrolysis of cAMP, compared with wild-type animals. PI3Kγ wild-type lungs treated with AS605240, an inhibitor of PI3Kγ kinase activity, in combination with enoximone, an inhibitor of phosphodiesterase-3 (PDE3)-induced cAMP hydrolysis, were protected from VILI at levels comparable to knockout lungs. CONCLUSIONS: Phosphoinositide 3-kinase gamma in resident lung cells mediates part of the alveolar edema induced by high-stress ventilation. This injury is mediated via altered Akt, eNOS, NO, and/or cAMP signaling. Anti-PI3Kγ therapy aimed at resident lung cells represents a potential pharmacologic target to mitigate VILI.

ErbB4 activation inhibits MPP+-induced cell death in PC12-ErbB4 cells: involvement of PI3K and Erk signaling.

The neuroprotective effects of neuregulin (NRG), a polypeptide growth factor, on 1-methyl-4-phenylpyridinium ion (MPP+)-induced cell death and oxidative stress in PC12-ErbB4 cells were investigated. Treatment of PC12-ErbB4 cells with MPP+ induced cell death that was markedly attenuated by NRG. The PI3K/PKB/Akt and Ras/MapK signaling pathways probably mediate the survival effect of NRG. NRG induces prolonged activation of PKB/Akt and Erk. Moreover, inhibition of the PI3K and MEK activities prevented the NRG-induced survival effect. Overexpression of constitutively active PI3K or H-Ras (12V) inhibited MPP+-mediated cell death. In addition, MPP+- mediated reactive oxygen species (ROS) elevation was also inhibited by NRG. The effect of NRG on ROS levels was blocked by PI3K and MEK inhibitors, indicating that both signaling pathways can regulate the toxic ROS levels induced by MPP+. Taken together, these results indicate that in PC12-ErbB4 cells, the NRG-induced neuroprotective effect from MPP+ treatment, requires PI3K/PKB/Akt and Ras/MapK signaling networks.

Effect of vectors on human endothelial cell signal transduction: implications for cardiovascular gene therapy.

OBJECTIVE: Endothelium is an important target for gene therapy. We have investigated the effect of viral and nonviral vectors on the phenotype and function of endothelial cells (ECs) and developed methods to block any activation caused by these vectors. METHODS AND RESULTS: Transduction of ECs with viral vectors, including adenovirus, lentiviruses, and Moloney murine leukemia virus, can induce a pro-inflammatory phenotype. This activation was reduced when nonviral vectors were used. We demonstrate that after transduction there is upregulation of dsRNA-triggered antiviral and PI3K/Akt signaling pathway. Blockade of the NFkappaB, PI3-K, or PKR signaling pathways all operated to inhibit partially virally induced activation, and inhibition of both PKR and PI3-K pathways totally blocked EC activation. Furthermore, inhibition of IFN-alpha/beta in addition to PI3-K was effective at preventing EC activation. CONCLUSIONS: Viral vectors, although efficient at transducing ECs, result in their activation. Blockade of the signaling pathways involved in viral activation may be used to prevent such activation.

Calphostin C as a rapid and strong inducer of apoptosis in human coronary artery smooth muscle cells.

Vascular smooth muscle cells (VSMCs) play a major role in the development of atherosclerotic and restenotic lesions. The apoptotic process has been implicated in the development of this pathology. In this study, we characterized the induction of apoptosis by calphostin C (CC), a protein kinase C (PKC) inhibitor, in primary human coronary artery smooth muscle cells in the presence and absence of insulin-like growth factor-I (IGF-I). Additionally, we investigated the signal transduction pathways important for IGF-I mediated protection. Calphostin C induced apoptosis, as measured by terminal deoxy-UTP nick-end labeling (TUNEL), in a time- and dose-dependent manner, approaching 20% within 6 h of 50 nM calphostin C treatment. The amount of apoptosis increased to 44.58+/-8.08%, 47.54+/-1.66% and 78.1+/-11.9% after 8, 10 and 12 h of treatment, respectively (p<0.01 vs. control). IGF-I offered significant protection (p<0.05) at 8 and 10 h of treatment (60.6% and 52.5% protection, respectively). DNA ELISA confirmed the apoptotic effect of calphostin C and the protective effect of IGF-I. After 6 h of calphostin C treatment, DNA ELISA revealed 11.20+/-1.53 fold greater apoptosis as compared to baseline values. IGF-I treatment offered a level of protection of 46.6% as measured by DNA ELISA (p=0.06). Apoptosis was further qualitatively confirmed by time-lapse video microscopy and scanning electron microscopy. Interestingly, inhibitors of phosphatidylinositol-3-kinase (PI-3-K), p38 and extracellular regulated kinase (ERK) activation significantly (p<0.05 vs. calphostin C only treatment) increased apoptosis when used in conjunction with calphostin C. Inhibitors of phospatidylinositol-3-kinase and ERK activation reversed IGF-I protection. However, the p38 inhibitor SB203580 failed to reverse IGF-I protection. This study characterized an apoptotic system for human coronary artery smooth muscle cells offering a rapid and strong induction of programmed cell death (PCD) that remains responsive to the survival effects of IGF-I. Studies utilizing this system may prove useful in understanding the apoptotic response of VSMCs in the arterial wall.

Synergistic activation of MAP kinase (ERK1/2) by erythropoietin and stem cell factor is essential for expanded erythropoiesis.

Stem cell factor (SCF) and erythropoietin (EPO) work synergistically to support erythropoiesis, but the mechanism for this synergism is unknown. By using purified human erythroid colony-forming cells (ECFC), we have found that SCF and EPO synergistically activate MAP kinase (MAPK, ERK1/2), which correlates with the cell growth and thus may be responsible for the synergistic effects. Treatment of the cells with PD98059 and wortmannin, inhibitors of MEK and PI-3 kinase, respectively, inhibited the synergistic activation of MAPK and also the cell growth, further supporting this conclusion. Wortmannin only inhibits MAPK activation induced by EPO but not that by SCF, suggesting that SCF and EPO may activate MAPK through different pathways, which would facilitate synergy. Furthermore, EPO, but not SCF, led to activation of STAT5, whereas SCF and wortmannin had no effect on the EPO-induced STAT5 activation, suggesting that STAT5 is not involved in the synergistic action of SCF and EPO. Together, the data suggest that synergistic activation of MAPK by SCF and EPO is essential for expanded erythropoiesis.