Inactivation of Malic Enzyme 1 in Endothelial Cells Alleviates Pulmonary Hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a progressive cardiopulmonary disease with a high mortality rate. Although growing evidence has revealed the importance of dysregulated energetic metabolism in the pathogenesis of PH, the underlying cellular and molecular mechanisms are not fully understood. In this study, we focused on ME1 (malic enzyme 1), a key enzyme linking glycolysis to the tricarboxylic acid cycle. We aimed to determine the role and mechanistic action of ME1 in PH. METHODS: Global and endothelial-specific ME1 knockout mice were used to investigate the role of ME1 in hypoxia- and SU5416/hypoxia (SuHx)-induced PH. Small hairpin RNA and ME1 enzymatic inhibitor (ME1*) were used to study the mechanism of ME1 in pulmonary artery endothelial cells. Downstream key metabolic pathways and mediators of ME1 were identified by metabolomics analysis in vivo and ME1-mediated energetic alterations were examined by Seahorse metabolic analysis in vitro. The pharmacological effect of ME1* on PH treatment was evaluated in PH animal models induced by SuHx. RESULTS: We found that ME1 protein level and enzymatic activity were highly elevated in lung tissues of patients and mice with PH, primarily in vascular endothelial cells. Global knockout of ME1 protected mice from developing hypoxia- or SuHx-induced PH. Endothelial-specific ME1 deletion similarly attenuated pulmonary vascular remodeling and PH development in mice, suggesting a critical role of endothelial ME1 in PH. Mechanistic studies revealed that ME1 inhibition promoted downstream adenosine production and activated A2AR-mediated adenosine signaling, which leads to an increase in nitric oxide generation and a decrease in proinflammatory molecule expression in endothelial cells. ME1 inhibition activated adenosine production in an ATP-dependent manner through regulating malate-aspartate NADH (nicotinamide adenine dinucleotide plus hydrogen) shuttle and thereby balancing oxidative phosphorylation and glycolysis. Pharmacological inactivation of ME1 attenuated the progression of PH in both preventive and therapeutic settings by promoting adenosine production in vivo. CONCLUSIONS: Our findings indicate that ME1 upregulation in endothelial cells plays a causative role in PH development by negatively regulating adenosine production and subsequently dysregulating endothelial functions. Our findings also suggest that ME1 may represent as a novel pharmacological target for upregulating protective adenosine signaling in PH therapy.

Ahf-Caltide, a Novel Polypeptide Derived from Calpastatin, Protects against Oxidative Stress Injury by Stabilizing the Expression of CaV1.2 Calcium Channel.

Reperfusion after ischemia would cause massive myocardial injury, which leads to oxidative stress (OS). Calcium homeostasis imbalance plays an essential role in myocardial OS injury. CaV1.2 calcium channel mediates calcium influx into cardiomyocytes, and its activity is modulated by a region of calpastatin (CAST) domain L, CSL54-64. In this study, the effect of Ahf-caltide, derived from CSL54-64, on myocardial OS injury was investigated. Ahf-caltide decreased the levels of LDH, MDA and ROS and increased heart rate, coronary flow, cell survival and SOD activity during OS. In addition, Ahf-caltide permeated into H9c2 cells and increased CaV1.2, CaVß2 and CAST levels by inhibiting protein degradation. At different Ca2+ concentrations (25 nM, 10 µM, 1 mM), the binding of CSL to the IQ motif in the C terminus of the CaV1.2 channel was increased in a H2O2 concentration-dependent manner. CSL54-64 was predicted to be responsible for the binding of CSL to CaV1.2. In conclusion, Ahf-caltide exerted a cardioprotective effect on myocardial OS injury by stabilizing CaV1.2 protein expression. Our study, for the first time, proposed that restoring calcium homeostasis by targeting the CaV1.2 calcium channel and its regulating factor CAST could be a novel treatment for myocardial OS injury.

Advances in the Study of Bioactive Nanoparticles for the Treatment of HCC and Its Postoperative Residual Cancer.

Primary hepatocellular carcinoma (HCC, hepatocellular carcinoma) is the third leading cause of tumor death in the world and the second leading cause in China. The high recurrence rate at 5 years after surgery also seriously affects the long-term survival of HCC patients. For reasons such as poor liver function, large tumors, or vascular invasion, only relatively limited palliative treatment is available. Therefore, effective diagnostic and therapeutic strategies are needed to improve the complex microenvironment and block the mechanism of tumor development in order to treat the tumor and prevent recurrence. A variety of bioactive nanoparticles have been shown to have therapeutic effects on hepatocellular carcinoma and have the advantages of improving drug solubility, reducing drug side effects, preventing degradation in the blood, increasing drug exposure time, and reducing drug resistance. The development of bioactive nanoparticles is expected to complete the current clinical therapeutic approach. In this review, we discuss the therapeutic advances of different nanoparticles for hepatocellular carcinoma and discuss their potential for postoperative applications with respect to possible mechanisms of hepatocellular carcinoma recurrence. We further discuss the limitations regarding the application of NPs and the safety of NPs.

Adenovirus-mediated Overexpression of FcγRIIB Attenuates Pulmonary Inflammation and Fibrosis.

Progressive fibrosing interstitial lung diseases (PF-ILDs) result in high mortality and lack effective therapies. The pathogenesis of PF-ILDs involves macrophages driving inflammation and irreversible fibrosis. Fc-γ receptors (FcγRs) regulate macrophages and inflammation, but their roles in PF-ILDs remain unclear. We characterized the expression of FcγRs and found upregulated FcγRIIB in human and mouse lungs after exposure to silica. FcγRIIB deficiency aggravated lung dysfunction, inflammation, and fibrosis in silica-exposed mice. Using single-cell transcriptomics and in vitro experiments, FcγRIIB was found in alveolar macrophages, where it regulated the expression of fibrosis-related genes Spp1 and Ctss. In mice with macrophage-specific overexpression of FcγRIIB and in mice treated with adenovirus by intratracheal instillation to upregulate FcγRIIB, silica-induced functional and histological changes were ameliorated. Our data from three genetic models and a therapeutic model suggest that FcγRIIB plays a protective role that can be enhanced by adenoviral overexpression, representing a potential therapeutic strategy for PF-ILDs.

FcεRI deficiency alleviates silica-induced pulmonary inflammation and fibrosis.

Silicosis is one of the most important occupational diseases worldwide, caused by inhalation of silica particles or free crystalline silicon dioxide. As a disease with high mortality, it has no effective treatment and new therapeutic targets are urgently needed. Recent studies have identified FCER1A, encoding α-subunit of the immunoglobulin E (IgE) receptor FcεRI, as a candidate gene involved in the biological pathways leading to respiratory symptoms. FcεRI is known to be important in allergic asthma, but its role in silicosis remains unclear. In this study, serum IgE concentrations and FcεRI expression were assessed in pneumoconiosis patients and silica-exposed mice. The role of FcεRI was explored in a silica-induced mouse model using wild-type and FcεRI-deficient mice. The results showed that serum IgE concentrations were significantly elevated in both pneumoconiosis patients and mice exposed to silica compared with controls. The mRNA and protein expression of FcεRI were also significantly increased in the lung tissue of patients and silica-exposed mice. FcεRI deficiency significantly attenuated the changes in lung function caused by silica exposure. Silica-induced elevations of IL-1ß, IL-6, and TNF-α were significantly attenuated in the lung tissue and bronchoalveolar lavage fluid (BALF) of FcεRI-deficient mice compared with wild-type controls. Additionally, FcεRI-deficient mice showed a significantly lower score of pulmonary fibrosis than wild-type mice following exposure to silica, with significantly lower hydroxyproline content and expression of fibrotic genes Col1a1 and Fn1. Immunofluorescent staining suggested FcεRI mainly on mast cells. Mast cell degranulation took place after silica exposure, as shown by increased serum histamine levels and ß-hexosaminidase activity, which were significantly reduced in FcεRI-deficient mice compared with wild-type controls. Together, these data showed that FcεRI deficiency had a significant protective effect against silica-induced pulmonary inflammation and fibrosis. Our findings provide new insights into the pathophysiological mechanisms of silica-induced pulmonary fibrosis and a potential target for the treatment of silicosis.

Gefitinib and fostamatinib target EGFR and SYK to attenuate silicosis: a multi-omics study with drug exploration.

Silicosis is the most prevalent and fatal occupational disease with no effective therapeutics, and currently used drugs cannot reverse the disease progress. Worse still, there are still challenges to be addressed to fully decipher the intricated pathogenesis. Thus, specifying the essential mechanisms and targets in silicosis progression then exploring anti-silicosis pharmacuticals are desperately needed. In this work, multi-omics atlas was constructed to depict the pivotal abnormalities of silicosis and develop targeted agents. By utilizing an unbiased and time-resolved analysis of the transcriptome, proteome and phosphoproteome of a silicosis mouse model, we have verified the significant differences in transcript, protein, kinase activity and signaling pathway level during silicosis progression, in which the importance of essential biological processes such as macrophage activation, chemotaxis, immune cell recruitment and chronic inflammation were emphasized. Notably, the phosphorylation of EGFR (p-EGFR) and SYK (p-SYK) were identified as potential therapeutic targets in the progression of silicosis. To inhibit and validate these targets, we tested fostamatinib (targeting SYK) and Gefitinib (targeting EGFR), and both drugs effectively ameliorated pulmonary dysfunction and inhibited the progression of inflammation and fibrosis. Overall, our drug discovery with multi-omics approach provides novel and viable therapeutic strategies for the treatment of silicosis.

BPA disrupts the cardioprotection by 17ß-oestradiol against ischemia/reperfusion injury in isolated guinea pig hearts.

Bisphenol A (BPA) is an environmental oestrogen or xenoestrogen (XEs). XEs represent a health risk due to their potential for endocrine disruption and ability to mimic estrogenic activity. The effects of BPA on isolated hearts under normal and ischemia/reperfusion (I/R) conditions were investigated for the first time, with a focus on the effects of BPA and 17ß-oestradiol (E2) co-administration on I/R injury. Our results indicated that BPA at 10-7 M and 10-5 M did not significantly affect heart rate (HR), coronary flow (CF), lactate dehydrogenase (LDH) or creatine kinase (CK) release in normal or I/R isolated hearts within the 90 min. However, E2 exerted a protective effect against I/R injury, whereas, BPA inhibited the cardio-protective effects of E2 on HR, CF, and LDH and CK release. Furthermore, BPA in combination with E2 aggravated I/R injury by increasing infarct size and causing a more severe ultrastructural disruption as compared to treatment with E2 alone. Based on our results, we conclude that BPA inhibits the cardio-protective effects of E2 on I/R-injured hearts, despite not significantly affecting normal or I/R isolated hearts.

Gut Epithelial Vitamin D Receptor Regulates Microbiota-Dependent Mucosal Inflammation by Suppressing Intestinal Epithelial Cell Apoptosis.

Recent studies show that colonic vitamin D receptor (VDR) signaling protects the mucosal epithelial barrier and suppresses colonic inflammation, but the underlying molecular mechanism remains to be fully understood. To investigate the implication of colonic VDR downregulation seen in patients with inflammatory bowel disease, we assessed the effect of gut epithelial VDR deletion on colonic inflammatory responses in an experimental colitis model. In a 2,4,6-trinitrobenzenesulfonic acid-induced colitis model, mice carrying VDR deletion in gut epithelial cells [VDRflox/flox (VDRf/f);Villin-Cre or VDRΔIEC] or in colonic epithelial cells (VDRf/f;CDX2-Cre or VDRΔCEC) developed more severe clinical colitis than VDRf/f control mice, characterized by more robust T-helper (TH)1 and TH17 responses, with greater increases in mucosal interferon (IFN)-γ+, interleukin (IL)-17+, and IFN-γ+IL-17+ T cells. Accompanying the severe mucosal inflammation was more profound colonic epithelial cell apoptosis in the mutant mice. Treatment with caspase inhibitor Q-VD-OPh dramatically reduced colitis severity and attenuated TH1 and TH17 responses in VDRΔCEC mice. The blockade of cell apoptosis also prevented the increase in mucosal CD11b+CD103+ dendritic cells (DCs), known to be critical for TH17-cell activation. Moreover, depletion of gut commensal bacteria with antibiotics eliminated the robust TH1 and TH17 responses and CD11b+CD103+ DC induction. Taken together, these observations demonstrate that gut epithelial VDR deletion aggravates epithelial cell apoptosis, resulting in increases in mucosal barrier permeability. Consequently, invading luminal bacteria activate CD11b+CD103+ DCs, which promote mucosal TH1 and TH17 responses. Therefore, gut epithelial VDR signaling controls mucosal inflammation by suppressing epithelial cell apoptosis.

Nonylphenol affects myocardial contractility and L-type Ca(2+) channel currents in a non-monotonic manner via G protein-coupled receptor 30.

Nonylphenol (NP) is one of the widely spread xenoestrogens (XEs) and is used in the production of industrial and consumer surfactants. In the present study, we examined the acute effects of NP on myocardial contractility to determine its rapid-response cardiac effects in the isolated heart. We also investigated the mechanism of action of NP by determining its effects on the L-type Ca(2+) channel (LTCC) currents (ICa-L) in ventricular myocytes. Lower concentrations (10(-12)-10(-10)M) of NP increased cardiac contractility while higher concentrations (10(-8)-10(-6)M) exhibited opposite effects. These apparently opposing effects suggest that NP has biphasic concentration-dependent rapid effects on cardiac contractility. These non-monotonic changes in contractility correlated with the effects of NP on ICa-L, indicating that ion channels, as rapidly responding membrane proteins, play a very important role in mediating the rapid-response effects of XEs. Further studies revealed that the G protein coupled receptor 30 (GPR30) mediates the effects of NP on LTCC at lower but not higher concentrations, implying that the differential involvement of GPR30 might be responsible for the non-monotonic effects of NP.

Low-Mg(2+) treatment increases sensitivity of voltage-gated Na(+) channels to Ca(2+)/calmodulin-mediated modulation in cultured hippocampal neurons.

Culture of hippocampal neurons in low-Mg(2+) medium (low-Mg(2+) neurons) results in induction of continuous seizure activity. However, the underlying mechanism of the contribution of low Mg(2+) to hyperexcitability of neurons has not been clarified. Our data, obtained using the patch-clamp technique, show that voltage-gated Na(+) channel (VGSC) activity, which is associated with a persistent, noninactivating Na(+) current (INa,P), was modulated by calmodulin (CaM) in a concentration-dependent manner in normal and low-Mg(2+) neurons, but the channel activity was more sensitive to Ca(2+)/CaM regulation in low-Mg(2+) than normal neurons. The increased sensitivity of VGSCs in low-Mg(2+) neurons was partially retained when CaM12 and CaM34, CaM mutants with disabled binding sites in the N or C lobe, were used but was diminished when CaM1234, a CaM mutant in which all four Ca(2+) sites are disabled, was used, indicating that functional Ca(2+)-binding sites from either lobe of CaM are required for modulation of VGSCs in low-Mg(2+) neurons. Furthermore, the number of neurons exhibiting colocalization of CaM with the VGSC subtypes NaV1.1, NaV1.2, and NaV1.3 was significantly higher in low- Mg(2+) than normal neurons, as shown by immunofluorescence. Our main finding is that low-Mg(2+) treatment increases sensitivity of VGSCs to Ca(2+)/CaM-mediated regulation. Our data reveal that CaM, as a core regulating factor, connects the functional roles of the three main intracellular ions, Na(+), Ca(2+), and Mg(2+), by modulating VGSCs and provides a possible explanation for the seizure discharge observed in low-Mg(2+) neurons.

A new method for maturity-dependent fractionation of neutrophil progenitors applicable for the study of myelodysplastic syndromes.

We applied our new method, maturity-dependent fractionation of bone marrow-derived neutrophil progenitors, to a study of gene expression profiles during granulopoiesis in myelodysplastic syndromes. CD34+ cells with low density [F1], CD11b-/CD16- [F2], CD11b+/CD16- [F3] and CD11b+/CD16low [F4] with intermediate density, CD11b+/CD16int [F5] and CD11b+/CD16high [F6] with high density were isolated from six patients. Although AML1 and C/EBP-ϵ mRNA peaked at F1 and F4, respectively, in healthy individuals, C/EBP-ϵ was maximized at F2/F3 in all patients, two of whom showed simultaneous peaks of AML1 at F2. Thus, this fractionation is useful to detect mistimed induction of granulopoiesis-regulating genes in myelodysplastic syndromes.

Nonylphenol disrupts the cardio-protective effects of 17ß-estradiol on ischemia/reperfusion injury in isolated hearts of guinea pig.

Nonylphenol (NP), a widely distributed, toxic, endocrine-disrupting chemical, has estrogenic properties. However, its cardiac effects remain unclear. In this study, the effects of NP on isolated guinea pig hearts were studied in three separate experiments. First, hearts were perfused with 10⁻7 M NP or 10⁻5 M NP to determine whether NP was toxic to isolated healthy hearts. Next, hearts were subjected to 50 min of ischemia and 60 min of reperfusion (I50R60) with 10⁻7 M NP or 10⁻5 M NP to determine whether NP could aggravate ischemia/reperfusion (I/R) injury. Finally, the interaction of the cardio-protective agent 17ß-estradiol (E2) with NP was studied using 10⁻7 M E2, 10⁻7 M E2 plus 10⁻7 M NP, and 10⁻7 M E2 plus 10⁻5 M NP. Heart rate (HR) and coronary flow (CF) were significantly decreased and the leakage of lactate dehydrogenase (LDH) in effluent was increased in the 10⁻5 M NP group. However, there were no obvious changes in HR, CF, the leakage of LDH or creatine kinase (CK), or the activity of superoxide dismutase in either of the NP treatments in the I50R60 model. Treatment with 10⁻7 M E2 attenuated I/R injury by increasing HR, decreasing the leakage of LDH and CK, and decreasing infarct size. However, these effects were reversed by both concentrations of NP. These data demonstrate that NP had direct toxic effects on normal hearts and NP might disrupt the cardio-protective effects of E2 on I/R injury.

Nonylphenol, an environmental estrogen, affects voltage-gated K⁺ currents and L-type Ca²âº currents in a non-monotonic manner in GH3 pituitary cells.

We have investigated the characteristics of voltage-gated K(+) channels and L-type Ca(2+) channels in GH3 rat pituitary cells and the effects of the xenoestrogen (XEs) nonylphenol (NP) on these ion channel currents. Our results have shown that the lower concentrations (10(-15)-10(-14)M) of NP decreased the amplitudes of voltage-gated K(+) currents (IKv) and activated L-type Ca(2+) currents (ICa-L) by reducing half-activation membrane potentials of activation kinetics curves. However, the higher concentrations (10(-10)-10(-9)M) of NP increased the amplitudes of IKv and inhibited ICa-L by reducing the peak values of ICa-L. Thus, NP affects IKv and ICa-L in an opposite and non-monotonic manner.

Maturity-dependent fractionation of neutrophil progenitors: a new method to examine in vivo expression profiles of differentiation-regulating genes.

To investigate differentiation-dependent gene expression during granulopoiesis, we established a new method to isolate six sequential differentiation stages of neutrophil progenitors from bone marrow. Neutrophil progenitors were divided into three populations by density centrifugation, followed by depletion of other lineages, and further separated by fluorescence-activated cell sorting based on the expressions of CD34, CD11b, and CD16: CD34(+) fraction from a low-density population (F1), CD11b(-)/CD16(-) (F2), CD11b(+)/CD16(-) (F3), and CD11b(+)/CD16(low) (F4) fractions with intermediate density, and CD11b(+)/CD16(int) (F5) and CD11b(+)/CD16(high) (F6) fractions from a high-density population. To examine whether this fractionation was applicable to the study of in vivo gene expression profiles during granulopoiesis, we analyzed messenger RNA levels of AML-1 and CCAAT/enhancer binding protein (EBP)-ε and two target genes of C/EBP-ε, granulocyte-macrophage colony-stimulating factor receptor common ß subunit and lactoferrin, in the six fractions and peripheral blood-derived neutrophils (F7). Expression of AML-1 and C/EBP-ε peaked at F1 and F4, respectively, followed by a gradual decrease. Although granulocyte-macrophage colony-stimulating factor receptor common ß subunit messenger RNA levels remained low from F1 through F6 and elevated at F7, lactoferrin messenger RNA showed a drastic increase at F3 and dropped at F5. The difference in the expression profiles of the two C/EBP-ε target genes suggests the involvement of regulators other than C/EBP-ε in the induction of the two genes. The new fractionation method is able to provide new information on maturation-dependent gene expression during granulopoiesis.

Transcripts expressed using a bicistronic vector pIREShyg2 are sensitized to nonsense-mediated mRNA decay.

BACKGROUND: pIREShyg2 has been widely used as a bicistronic expression vector. However, it is not known if the vector would affect the expression of cloned genes via nonsense-mediated mRNA decay (NMD), an mRNA surveillance system that degrades mRNA with a premature termination codon (PTC). In mammalian cells, the induction of NMD requires either a long 3'UTR or the presence of an exon-junction complex downstream of a PTC. The efficiency of NMD is greater when a PTC generates longer 3'UTR. pIREShyg2 provides the first cistron gene with a long 3'UTR consisting of a downstream intervening sequence (IVS), an internal ribosomal entry site (IRES) and the second cistron. Therefore, we hypothesized that the first cistron genes in pIREShyg2 are sensitized to NMD, which affects their expression levels. To examine this hypothesis, cDNAs encoding human granulocyte-macrophage colony-stimulating factor receptor beta chain (betac) and its splice variant (betac79), in which the retention of a 79-base intron caused a frameshift generating 18 PTCs, were cloned into pIREShyg2 and stably expressed in a murine cell line, Ba/F3. RESULTS: Compared with wild-type betac, the mRNA levels of betac79 were less than one tenth and decayed faster. Both translation inhibition and Upf1 knockdown led to significantly greater up-regulation of betac79 than wild-type betac. However, the use of a monocistronic pMT21 vector abolished the up-regulatory effects of translation inhibition and Upf1 knockdown on both wild-type betac and betac79, suggesting that the NMD is attributable to a structural determinant in pIREShyg2. The elimination of the intron and the proximal 3' 17 PTCs did not alter the greater effects of translation inhibition on betac79, suggesting that the first PTC, which determines 3'UTR length, was sufficient to enhance NMD efficiency. Thus, transcripts of PTC-harboring genes with longer 3'UTR are more efficiently degraded by the vector-dependent NMD than those of wild-type genes with relatively shorter 3'UTR, resulting in minimized expression of truncated mutants. CONCLUSIONS: We conclude that pIREShyg2, which sensitizes its bicistronic transcripts to NMD, may be useful for studying NMD but should be avoided when maximum expressions of PTC-harboring genes are required.

Terminally differentiated neutrophils predominantly express Survivin-2 alpha, a dominant-negative isoform of survivin.

Survivin, which is a member of the inhibitor of apoptosis protein family, was found originally in immature cells and cancer cells but not in non-neoplastic adult tissues. The subsequent identification of four other alternative splice variants that possess distinct functions and localizations suggested the diverse roles of survivin isoforms. An unspecified isoform of survivin was found recently to be induced in terminally differentiated neutrophils by cytokines that prolong the neutrophil lifespan, such as GM-CSF and G-CSF, suggesting the importance of survivin in blocking apoptosis in neutrophils. To examine the mechanism by which survivin inhibits neutrophil apoptosis, we attempted to induce survivin by GM-CSF/G-CSF in an HL60 cell line that was differentiated into neutrophils by all-trans retinoic acid and DMSO and freshly isolated human neutrophils. The antiapoptotic isoform "Survivin," which was decreased during differentiation, was re-induced by GM-CSF in neutrophil-like, differentiated HL60. In contrast, in normal neutrophils, survivin mRNA was observed to increase spontaneously after 24 h incubation, and no additional elevation was induced by GM-CSF/G-CSF, which exerted their antiapoptotic effects on the neutrophils in 6 h, despite the lack of survivin induction. PCR and Western blotting detected Survivin-2 alpha, a dominant-negative of antiapoptotic Survivin, with no other isoforms in the freshly isolated or incubated neutrophils. Our study revealed that the expressed isoforms and the response to GM-CSF were different between the HL60-derived and normal neutrophils, which predominantly expressed Survivin-2 alpha, not likely involved in apoptosis inhibition by GM-CSF/G-CSF.