Branched chain α-ketoacids aerobically activate HIF1α signaling in vascular cells.

Hypoxia-inducible factor 1α (HIF1α) is a master regulator of numerous biological processes under low oxygen tensions. Yet, the mechanisms and biological consequences of aerobic HIF1α activation by intrinsic factors, particularly in primary cells remain elusive. Here, we show that HIF1α signaling is activated in several human primary vascular cells under ambient oxygen tensions, and in vascular smooth muscle cells (VSMCs) of normal human lung tissue, which contributed to a relative resistance to further enhancement of glycolytic activity in hypoxia. Mechanistically, aerobic HIFα activation is mediated by paracrine secretion of three branched chain α-ketoacids (BCKAs), which suppress prolyl hydroxylase domain-containing protein 2 (PHD2) activity via direct inhibition and via lactate dehydrogenase A (LDHA)-mediated generation of L-2-hydroxyglutarate (L2HG). Metabolic dysfunction induced by BCKAs was observed in the lungs of rats with pulmonary arterial hypertension (PAH) and in pulmonary artery smooth muscle cells (PASMCs) from idiopathic PAH patients. BCKA supplementation stimulated glycolytic activity and promoted a phenotypic switch to the synthetic phenotype in PASMCs of normal and PAH subjects. In summary, we identify BCKAs as novel signaling metabolites that activate HIF1α signaling in normoxia and that the BCKA-HIF1α pathway modulates VSMC function and may be relevant to pulmonary vascular pathobiology.

SGLT2 inhibitor ertugliflozin decreases elevated intracellular sodium, and improves energetics and contractile function in diabetic cardiomyopathy.

BACKGROUND: Elevated myocardial intracellular sodium ([Na+]i) was shown to decrease mitochondrial calcium ([Ca2+]MITO) via mitochondrial sodium/calcium exchanger (NCXMITO), resulting in decreased mitochondrial ATP synthesis. The sodium-glucose co-transporter 2 inhibitor (SGLT2i) ertugliflozin (ERTU) improved energetic deficit and contractile dysfunction in a mouse model of high fat, high sucrose (HFHS) diet-induced diabetic cardiomyopathy (DCMP). As SGLT2is were shown to lower [Na+]i in isolated cardiomyocytes, we hypothesized that energetic improvement in DCMP is at least partially mediated by a decrease in abnormally elevated myocardial [Na+]i. METHODS: Forty-two eight-week-old male C57BL/6J mice were fed a control or HFHS diet for six months. In the last month, a subgroup of HFHS-fed mice was treated with ERTU. At the end of the study, left ventricular contractile function and energetics were measured simultaneously in isolated beating hearts by 31P NMR (Nuclear Magnetic Resonance) spectroscopy. A subset of untreated HFHS hearts was perfused with vehicle vs. CGP 37157, an NCXMITO inhibitor. Myocardial [Na+]i was measured by 23Na NMR spectroscopy. RESULTS: HFHS hearts showed diastolic dysfunction, decreased contractile reserve, and impaired energetics as reflected by decreased phosphocreatine (PCr) and PCr/ATP ratio. Myocardial [Na+]i was elevated > 2-fold in HFHS (vs. control diet). ERTU reversed the impairments in HFHS hearts to levels similar to or better than control diet and decreased myocardial [Na+]i to control levels. CGP 37157 normalized the PCr/ATP ratio in HFHS hearts. CONCLUSIONS: Elevated myocardial [Na+]i contributes to mitochondrial and contractile dysfunction in DCMP. Targeting myocardial [Na+]i and/or NCXMITO may be an effective strategy in DCMP and other forms of heart disease associated with elevated myocardial [Na+]i.

Biomimetic Nanosonosensitizers Combined with Noninvasive Ultrasound Actuation to Reverse Drug Resistance and Sonodynamic-Enhanced Chemotherapy against Orthotopic Glioblastoma.

Glioblastoma (GBM) is the most devastating brain tumor and highly resistant to conventional chemotherapy. Herein, we introduce biomimetic nanosonosensitizer systems (MDNPs) combined with noninvasive ultrasound (US) actuation for orthotopic GBM-targeted delivery and sonodynamic-enhanced chemotherapy. MDNPs were fabricated with biodegradable and pH-sensitive polyglutamic acid (PGA) and the chemotherapeutic agent and sonosensitizer doxorubicin (DOX), camouflaged with human GBM U87 cell membranes. MDNPs presented homologous targeting accumulation and in vivo long-term circulation ability. They effectively passed through the blood-brain barrier (BBB) under US assistance and reached the orthotopic GBM site. MDNPs exhibited controllable US-elicited sonodynamic effect by generation of reactive oxygen species (ROS). ROS not only induced cancer cell apoptosis but also downregulated drug-resistance-related factors to disrupt chemoresistance and increase sensitivity to chemotherapy. The in vivo study of orthotopic GBM treatments further proved that MDNPs exhibited US-augmented synergistic antitumor efficacy and strongly prolonged the survival rate of mice. The use of low-dose DOX and the safety of US enabled repeated treatment (4 times) without obvious cardiotoxicity. This effective and safe US-enhanced chemotherapy strategy with the advantages of noninvasive brain delivery and high drug sensitivity holds great promise for deep-seated and drug-resistant tumors.

L-2-Hydroxyglutarate Protects Against Cardiac Injury via Metabolic Remodeling.

BACKGROUND: L-2-hydroxyglutarate (L2HG) couples mitochondrial and cytoplasmic energy metabolism to support cellular redox homeostasis. Under oxygen-limiting conditions, mammalian cells generate L2HG to counteract the adverse effects of reductive stress induced by hypoxia. Very little is known, however, about whether and how L2HG provides tissue protection from redox stress during low-flow ischemia (LFI) and ischemia-reperfusion injury. We examined the cardioprotective effects of L2HG accumulation against LFI and ischemia-reperfusion injury and its underlying mechanism using genetic mouse models. METHODS AND RESULTS: L2HG accumulation was induced by homozygous (L2HGDH [L-2-hydroxyglutarate dehydrogenase]-/-) or heterozygous (L2HGDH+/-) deletion of the L2HGDH gene in mice. Hearts isolated from these mice and their wild-type littermates (L2HGDH+/+) were subjected to baseline perfusion and 90-minute LFI or 30-minute no-flow ischemia followed by 60- or 120-minute reperfusion. Using [13C]- and [31P]-NMR (nuclear magnetic resonance) spectroscopy, high-performance liquid chromatography, reverse transcription quantitative reverse transcription polymerase chain reaction, ELISA, triphenyltetrazolium staining, colorimetric/fluorometric spectroscopy, and echocardiography, we found that L2HGDH deletion induces L2HG accumulation at baseline and under stress conditions with significant functional consequences. In response to LFI or ischemia-reperfusion, L2HG accumulation shifts glucose flux from glycolysis towards the pentose phosphate pathway. These key metabolic changes were accompanied by enhanced cellular reducing potential, increased elimination of reactive oxygen species, attenuated oxidative injury and myocardial infarction, preserved cellular energy state, and improved cardiac function in both L2HGDH-/- and L2HGDH+/- hearts compared with L2HGDH+/+ hearts under ischemic stress conditions. CONCLUSION: L2HGDH deletion-induced L2HG accumulation protects against myocardial injury during LFI and ischemia-reperfusion through a metabolic shift of glucose flux from glycolysis towards the pentose phosphate pathway. L2HG offers a novel mechanism for eliminating reactive oxygen species from myocardial tissue, mitigating redox stress, reducing myocardial infarct size, and preserving high-energy phosphates and cardiac function. Targeting L2HG levels through L2HGDH activity may serve as a new therapeutic strategy for cardiovascular diseases related to oxidative injury.

Novel Small-Molecule Troponin Activator Increases Cardiac Contractile Function Without Negative Impact on Energetics.

BACKGROUND: Current heart failure therapies unload the failing heart without targeting the underlying problem of reduced cardiac contractility. Traditional inotropes (ie, calcitropes) stimulate contractility via energetically costly augmentation of calcium cycling and worsen patient survival. A new class of agents-myotropes-activates the sarcomere directly, independent of calcium. We hypothesize that a novel myotrope TA1 increases contractility without the deleterious myocardial energetic impact of a calcitrope dobutamine. METHODS: We determined the effect of TA1 in bovine cardiac myofibrils and human cardiac microtissues, ex vivo in mouse cardiac fibers and in vivo in anesthetized normal rats. Effects of increasing concentrations of TA1 or dobutamine on contractile function, phosphocreatine and ATP concentrations, and ATP production were assessed by 31P nuclear magnetic resonance spectroscopy on isolated perfused rat hearts. RESULTS: TA1 increased the rate of myosin ATPase activity in isolated bovine myofibrils and calcium sensitivity in intact mouse papillary fibers. Contractility increased dose dependently in human cardiac microtissues and in vivo in rats as assessed by echocardiography. In isolated rat hearts, TA1 and dobutamine similarly increased the rate-pressure product. Dobutamine increased both developed pressure and heart rate accompanied by decreased phosphocreatine-to-ATP ratio and decreased free energy of ATP hydrolysis (ΔG~ATP) and elevated left ventricular end diastolic pressure. In contrast, the TA1 increased developed pressure without any effect on heart rate, left ventricular end diastolic pressure, phosphocreatine/ATP ratio, or ΔG~ATP. CONCLUSIONS: Novel myotrope TA1 increased myocardial contractility by sensitizing the sarcomere to calcium without impairing diastolic function or depleting the cardiac energy reserve. Since energetic depletion negatively correlates with long-term survival, myotropes may represent a superior alternative to traditional inotropes in heart failure management.

Interferon-γ Impairs Human Coronary Artery Endothelial Glucose Metabolism by Tryptophan Catabolism and Activates Fatty Acid Oxidation.

BACKGROUND: Endothelial cells depend on glycolysis for much of their energy production. Impaired endothelial glycolysis has been associated with various vascular pathobiologies, including impaired angiogenesis and atherogenesis. IFN-γ (interferon-γ)-producing CD4+ and CD8+ T lymphocytes have been identified as the predominant pathological cell subsets in human atherosclerotic plaques. Although the immunologic consequences of these cells have been extensively evaluated, their IFN-γ-mediated metabolic effects on endothelial cells remain unknown. The purpose of this study was to determine the metabolic consequences of the T-lymphocyte cytokine, IFN-γ, on human coronary artery endothelial cells. METHODS: The metabolic effects of IFN-γ on primary human coronary artery endothelial cells were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux analyses and molecular mechanistic studies. Cellular phenotypic correlations were made by measuring altered endothelial intracellular cGMP content, wound-healing capacity, and adhesion molecule expression. RESULTS: IFN-γ exposure inhibited basal glycolysis of quiescent primary human coronary artery endothelial cells by 20% through the global transcriptional suppression of glycolytic enzymes resulting from decreased basal HIF1α (hypoxia-inducible factor 1α) nuclear availability in normoxia. The decrease in HIF1α activity was a consequence of IFN-γ-induced tryptophan catabolism resulting in ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF1ß sequestration by the kynurenine-activated AHR (aryl hydrocarbon receptor). In addition, IFN-γ resulted in a 23% depletion of intracellular nicotinamide adenine dinucleotide in human coronary artery endothelial cells. This altered glucose metabolism was met with concomitant activation of fatty acid oxidation, which augmented its contribution to intracellular ATP balance by >20%. These metabolic derangements were associated with adverse endothelial phenotypic changes, including decreased basal intracellular cGMP, impaired endothelial migration, and a switch to a proinflammatory state. CONCLUSIONS: IFN-γ impairs endothelial glucose metabolism by altered tryptophan catabolism destabilizing HIF1, depletes nicotinamide adenine dinucleotide, and results in a metabolic shift toward increased fatty acid oxidation. This work suggests a novel mechanistic basis for pathological T lymphocyte-endothelial interactions in atherosclerosis mediated by IFN-γ, linking endothelial glucose, tryptophan, and fatty acid metabolism with the nicotinamide adenine dinucleotide balance and ATP generation and their adverse endothelial functional consequences.

In situ poly I:C released from living cell drug nanocarriers for macrophage-mediated antitumor immunotherapy.

Immunotherapy is one of the most promising approaches to inhibit tumor growth and metastasis by activating host immune functions. However, the arising problems such as low immune response caused by complex tumor microenvironment and extremely systemic immune storm still limit the clinical applications of immunotherapy. Here, we construct Poly I: C-encapsulated poly (lactic-co-glycolic acid) nanoparticles (PLP NPs) with a slow release profile. A biomimetic system (MPLP), which loads PLP NPs on the surface of bone marrow-derived macrophage (BMDM) via the maleimide-thiol conjugation, is synthesized to effectively deliver PLP, control drug release and activate the tumor-specific immune response in situ. The results show that PLP NPs loading does not affect the activity and function of BMDM. Then, BMDM acts as a living cell drug vehicle and promotes the accumulation of PLP NPs in tumors, where Poly I: C is released from PLP NPs and reprograms BMDM into tumoricidal M1 macrophage. Furthermore, MPLP triggers potent antitumor immune responses in vivo and effectively inhibits local and metastatic tumors without causing adverse pathological immune reactions. This study offers an inspiration to facilitate clinical translation through the delivery of drugs by living immune cells for future anticancer therapy.

Click CAR-T cell engineering for robustly boosting cell immunotherapy in blood and subcutaneous xenograft tumor.

The adoptive transfer of chimeric antigen receptor-T (CAR-T) cells has shown remarkable clinical responses in hematologic malignancies. However, unsatisfactory curative results and side effects for tumor treatment are still unsolved problems. Herein we develop a click CAR-T cell engineering strategy via cell glycometabolic labeling for robustly boosting their antitumor effects and safety in vivo. Briefly, paired chemical groups (N3/BCN) are separately incorporated into CAR-T cell and tumor via nondestructive intrinsic glycometabolism of exogenous Ac4GalNAz and Ac4ManNBCN, serving as an artificial ligand-receptor. Functional groups anchored on cell surface strengthen the interaction of CAR-T cell and tumor via bioorthogonal click chemistry, further enhancing specific recognition, migration and selective antitumor effects of CAR-T cells. In vivo, click CAR-T cell completely removes lymphoma cells and minimizes off-target toxicity via selective and efficient bioorthogonal targeting in blood cancer. Surprisingly, compared to unlabeled cells, artificial bioorthogonal targeting significantly promotes the accumulation, deep penetration and homing of CAR-T cells into tumor tissues, ultimately improving its curative effect for solid tumor. Click CAR-T cell engineering robustly boosts selective recognition and antitumor capabilities of CAR T cells in vitro and in vivo, thereby holding a great potential for effective clinical cell immunotherapy with avoiding adverse events in patients.

An NIR-II-Emissive Photosensitizer for Hypoxia-Tolerant Photodynamic Theranostics.

As a common feature in a majority of malignant tumors, hypoxia has become the Achilles' heel of photodynamic therapy (PDT). The development of type-I photosensitizers that show hypoxia-tolerant PDT efficiency provides a straightforward way to address this issue. However, type-I PDT materials have rarely been discovered. Herein, a π-conjugated molecule with A-D-A configuration, COi6-4Cl, is reported. The H2 O-dispersible nanoparticle of COi6-4Cl can be activated by an 880 nm laser, and displays hypoxia-tolerant type I/II combined PDT capability, and more notably, a high NIR-II fluorescence with a quantum yield over 5%. Moreover, COi6-4Cl shows a negligible photothermal conversion effect. The non-radiative decay of COi6-4Cl is suppressed in the dispersed and aggregated state due to the restricted molecular vibrations and distinct intermolecular steric hindrance induced by its four bulky side chains. These features make COi6-4Cl a distinguished single-NIR-wavelength-activated phototheranostic material, which performs well in NIR-II fluorescence-guided PDT treatment and shows an enhanced in vivo anti-tumor efficiency over the clinically approved Chlorin e6, by the equal stresses on hypoxia-tolerant anti-tumor therapy and deep-penetration imaging. Therefore, the great potential of COi6-4Cl in precise PDT cancer therapy against hypoxia challenges is demonstrated.

Smart gold nanocages for mild heat-triggered drug release and breaking chemoresistance.

Chemotherapy is an important modality available for cancer treatment. However, the present chemotherapy is still far from being satisfactory mainly owing to the severe side effects of the chemotherapeutic agents and drug resistance of cancer cells. Thus, reversing drug resistance by constructing an ideal chemotherapeutic strategy with the least side effects and the best efficacy is greatly needed. Here, we designed a smart nanosystem of thermo-sensitive liposome coated gold nanocages with doxorubicin (DOX) loading (LAD) for near-infrared (NIR)-triggered drug release and chemo-photothermal combination therapy. The biocompatible liposomes coating facilitated the cellular uptake of LAD and meanwhile avoided drug leakage during the circulation. More importantly, LAD exhibited controllable photothermal conversion property and produced mild heat under NIR irradiation, which not only triggered DOX release and transferred DOX from lysosome to nucleus, but also elicited the mild heat cell killing effect to improve the curative efficiency. Further mechanism study revealed that mild heat could reverse drug resistance by down-regulation of the chemoresistance-related markers (e.g., HSF-1, p53, P-gp), and inhibited DOX export and increased drug sensitiveness, thereby prominently increased the anticancer efficiency. This versatile nanoplatform with enhanced curative efficacy and lower side effect is promising to apply in the field of drug controlled release and combination tumor therapy.

Increasing mitochondrial ATP synthesis with butyrate normalizes ADP and contractile function in metabolic heart disease.

Metabolic heart disease (MHD), which is strongly associated with heart failure with preserved ejection fraction, is characterized by reduced mitochondrial energy production and contractile performance. In this study, we tested the hypothesis that an acute increase in ATP synthesis, via short chain fatty acid (butyrate) perfusion, restores contractile function in MHD. Isolated hearts of mice with MHD due to consumption of a high fat high sucrose (HFHS) diet or on a control diet (CD) for 4 months were studied using 31 P NMR spectroscopy to measure high energy phosphates and ATP synthesis rates during increased work demand. At baseline, HFHS hearts had increased ADP and decreased free energy of ATP hydrolysis (ΔG~ATP ), although contractile function was similar between the two groups. At high work demand, the ATP synthesis rate in HFHS hearts was reduced by over 50%. Unlike CD hearts, HFHS hearts did not increase contractile function at high work demand, indicating a lack of contractile reserve. However, acutely supplementing HFHS hearts with 4mM butyrate normalized ATP synthesis, ADP, ΔG~ATP and contractile reserve. Thus, acute reversal of depressed mitochondrial ATP production improves contractile dysfunction in MHD. These findings suggest that energy starvation may be a reversible cause of myocardial dysfunction in MHD, and opens new therapeutic opportunities.

Tumor-targeted nanoplatform for in situ oxygenation-boosted immunogenic phototherapy of colorectal cancer.

Advanced colorectal cancer has a high mortality rate since conventional treatments have limited therapeutic effects and poor prognosis with high risks of metastasis and recurrence. Photodynamic therapy (PDT) is a promising treatment modality for the eradication of colorectal cancer, but its curative efficacy is severely affected by tumor hypoxia. Herein, we developed a core-shell gold nanocage coated with manganese dioxide and hyaluronic acid (AMH) for targeted delivery to colorectal tumors and oxygenation-boosted immunogenic phototherapy in situ. The AMH nanoparticles can generate abundant oxygen from mild acidic/H2O2 medium, which can further enhance the PDT efficacy of AMH itself under near infrared (NIR) irradiation. Meanwhile, AMH-based PDT induced immunogenic cell death (ICD) of tumor cells with damage-associated molecular patterns (DAMPs) release and facilitated the dendritic cells (DCs) maturation to further potentiate the systematic antitumor immunity against advanced tumors. In vivo experiment results exhibited that AMH nanoparticles not only had the ability of targeting tumor but also in situ produced sufficient oxygen to relieve the tumor hypoxia. Furthermore, AMH-mediated oxygen-boosted immunogenic PDT effectively inhibited the tumor growth and recurrence. Thus, this work provides a potent targeted delivery nanoplatform for enhanced immunogenic PDT against advanced cancers. STATEMENT OF SIGNIFICANCE: Local hypoxic tumor microenvironment not only greatly limits the photodynamic therapy (PDT) efficacy, but also has an association with tumor invasiveness and metastasis. This study provides an AMH nanoparticle for targeted delivery to colorectal tumors and oxygenation-boosted immunogenic PDT in situ. AMH nanoparticle exhibits a good tumor-targeted ability to in situ produce abundant oxygen to relieve the tumor hypoxia, and initiates the potent oxygen-boosted immunogenic PDT effect under NIR irradiation to effectively inhibit the growth and recurrence of colorectal tumor. This oxygen-boosted immunogenic PDT nanosystem can be a promising candidate for advanced tumor treatment.

Bioorthogonal Metabolic Labeling Utilizing Protein Biosynthesis for Dynamic Visualization of Nonenveloped Enterovirus 71 Infection.

Bioorthogonal metabolic labeling through the endogenous cellular metabolic pathways (e.g., phospholipid and sugar) is a promising approach for effectively labeling live viruses. However, it remains a big challenge to label nonenveloped viruses due to lack of host-derived envelopes. Herein, a novel bioorthogonal labeling strategy is developed utilizing protein synthesis pathway to label and trace nonenveloped viruses. The results show that l-azidohomoalanine (Aha), an azido derivative of methionine, is more effective than azido sugars to introduce azido motifs into viral capsid proteins by substituting methionine residues during viral protein biosynthesis and assembly. The azide-modified EV71 (N3-EV71) particles are then effectively labeled with dibenzocyclooctyl (DBCO)-functionalized fluorescence probes through an in situ bioorthogonal reaction with well-preserved viral infectivity. Dual-labeled imaging clearly clarifies that EV71 virions primarily bind to scavenger receptors and are internalized through clathrin-mediated endocytosis. The viral particles are then transported into early and late endosomes where viral RNA is released in a low-pH dependent manner at about 70 min postinfection. These results first reveal viral trafficking and uncoating mechanisms, which may shed light on the pathogenesis of EV71 infection and contribute to antiviral drug discovery.

Synergistic effect of all-trans-retinal and triptolide encapsulated in an inflammation-targeted nanoparticle on collagen-induced arthritis in mice.

Targeted delivery of nano-encapsulated anti-inflammatory agent represents a promising while challenging strategy in the treatment of rheumatoid arthritis (RA). Pro-inflammatory macrophages play a major role in the pathogenesis of RA. In this study, we investigated the effect of a macrophage-targeted pH-sensitive nanoparticle on collagen-induced arthritis (CIA) in mice. To target macrophage, all-trans-retinal was conjugated into dextran backbone through pH-sensitive hydrazone bond, then grafted with galactose (GDR). This nanoparticle was used for the encapsulation of triptolide (TPT), a potent anti-inflammatory compound isolated from Chinese herb. As expected, GDR nanoparticles preferentially accumulated in the inflammatory tissues. Treatment with GDR-TPT nanoparticles resulted in a marked decrease in the infiltration of CD3+ T cells and F4/80+ macrophages and reduction of the expression of TNF-α, IL-6 and IL-1ß in the inflamed lesions of CIA mice. Furthermore, Th1 and Th17 responses were also inhibited. Importantly, anti-arthritic effect of TPT was markedly enhanced while its toxic effect was attenuated by encapsulating with GDR. GDR by itself also had moderate effect in the inhibition of arthritis, due to its intrinsic anti-inflammatory property. Therefore, our results clearly show that GDR-TPT nanoparticle may represent a promising drug delivery system for the treatment of RA.

Interaction of α Carboxyl Terminus 1 Peptide With the Connexin 43 Carboxyl Terminus Preserves Left Ventricular Function After Ischemia-Reperfusion Injury.

Background α Carboxyl terminus 1 (αCT1) is a 25-amino acid therapeutic peptide incorporating the zonula occludens-1 (ZO-1)-binding domain of connexin 43 (Cx43) that is currently in phase 3 clinical testing on chronic wounds. In mice, we reported that αCT1 reduced arrhythmias after cardiac injury, accompanied by increases in protein kinase Cε phosphorylation of Cx43 at serine 368. Herein, we characterize detailed molecular mode of action of αCT1 in mitigating cardiac ischemia-reperfusion injury. Methods and Results To study αCT1-mediated increases in phosphorylation of Cx43 at serine 368, we undertook mass spectrometry of protein kinase Cε phosphorylation assay reactants. This indicated potential interaction between negatively charged residues in the αCT1 Asp-Asp-Leu-Glu-Iso sequence and lysines (Lys345, Lys346) in an α-helical sequence (helix 2) within the Cx43-CT. In silico modeling provided further support for this interaction, indicating that αCT1 may interact with both Cx43 and ZO-1. Using surface plasmon resonance, thermal shift, and phosphorylation assays, we characterized a series of αCT1 variants, identifying peptides that interacted with either ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 or Cx43-CT, but with limited or no ability to bind both molecules. Only peptides competent to interact with Cx43-CT, but not ZO-1-postsynaptic density-95/disks large/zonula occludens-1 2 alone, prompted increased pS368 phosphorylation. Moreover, in an ex vivo mouse model of ischemia-reperfusion injury, preischemic infusion only with those peptides competent to bind Cx43 preserved ventricular function after ischemia-reperfusion. Interestingly, a short 9-amino acid variant of αCT1 (αCT11) demonstrated potent cardioprotective effects when infused either before or after ischemic injury. Conclusions Interaction of αCT1 with the Cx43, but not ZO-1, is correlated with cardioprotection. Pharmacophores targeting Cx43-CT could provide a translational approach to preserving heart function after ischemic injury.

Energetic Dysfunction Is Mediated by Mitochondrial Reactive Oxygen Species and Precedes Structural Remodeling in Metabolic Heart Disease.

Aims: Metabolic syndrome is associated with metabolic heart disease (MHD) that is characterized by left ventricular (LV) hypertrophy, interstitial fibrosis, contractile dysfunction, and mitochondrial dysfunction. Overexpression of catalase in mitochondria (transgenic expression of catalase targeted to the mitochondria [mCAT]) prevents the structural and functional features of MHD caused by a high-fat, high-sucrose (HFHS) diet for ≥4 months. However, it is unclear whether the effect of mCAT is due to prevention of reactive oxygen species (ROS)-mediated cardiac remodeling, a direct effect on mitochondrial function, or both. To address this question, we measured myocardial function and energetics in mice, with or without mCAT, after 1 month of HFHS, before the development of cardiac structural remodeling. Results: HFHS diet for 1 month had no effect on body weight, heart weight, LV structure, myocyte size, or interstitial fibrosis. Isolated cardiac mitochondria from HFHS-fed mice produced 2.2- to 3.8-fold more H2O2, and 16%-29% less adenosine triphosphate (ATP). In isolated beating hearts from HFHS-fed mice, [phosphocreatine (PCr)] and the free energy available for ATP hydrolysis (ΔG∼ATP) were decreased, and they failed to increase with work demands. Overexpression of mCAT normalized ROS and ATP production in isolated mitochondria, and it corrected myocardial [PCr] and ΔG∼ATP in the beating heart. Innovation: This is the first demonstration that in MHD, mitochondrial ROS mediate energetic dysfunction that is sufficient to impair contractile function. Conclusion: ROS produced and acting in the mitochondria impair myocardial energetics, leading to slowed relaxation and decreased contractile reserve. These effects precede structural remodeling and are corrected by mCAT, indicating that ROS-mediated energetic impairment, per se, is sufficient to cause contractile dysfunction in MHD.

Bio-Orthogonal T Cell Targeting Strategy for Robustly Enhancing Cytotoxicity against Tumor Cells.

T cells can kill tumor cells by cell surface immunological recognition, but low affinity for tumor-associated antigens could lead to T cell off-target effects. Herein, a universal T cell targeting strategy based on bio-orthogonal chemistry and glycol-metabolic engineering is introduced to enhance recognition and cytotoxicity of T cells in tumor immunotherapy. Three kinds of bicycle [6.1.0] nonyne (BCN)-modified sugars are designed and synthesized, in which Ac4 ManN-BCN shows efficient incorporation into wide tumor cells with a BCN motif on surface glycans. Meanwhile, activated T cells are treated with Ac4 GalNAz to introduce azide (N3 ) on the cell surface, initiating specific tumor targeting through a bio-orthogonal click reaction between N3 and BCN. This artificial targeting strategy remarkably enhances recognition and migration of T cells to tumor cells, and increases the cytotoxicity 2 to 4 times for T cells against different kinds of tumor cells. Surprisingly, based on this strategy, the T cells even exhibit similar cytotoxicity with the chimeric antigen receptor T-cell against Raji cells in vitro at the effector: target cell ratios (E:T) of 1:1. Such a universal bio-orthogonal T cell-targeting strategy might further broaden applications of T cell therapy against tumors and provide a new strategy for T cell modification.

Dissecting complicated viral spreading of enterovirus 71 using in situ bioorthogonal fluorescent labeling.

Enterovirus 71 (EV71), the major pathogen of hand-foot-and-mouth disease (HFDM), can cause severe neurological and respiratory manifestations in young children. Viral spread route and tissue tropism are key factors contributing to different pathogenicity of EV71, however it remains a challenge to dynamically visualize EV71 infection in vivo. The present study applies an in situ bioorthogonal fluorescent labeling strategy to track clinically isolated EV71 strains with different pathogenicity in neonatal mice. The results show that the in situ labeling strategy effectively captures EV71 viruses through in vivo bioorthogonal reaction in multiple infected organs without interfering viral spread and tissue tropism. More importantly, the in situ labeling reveals different viral dynamics, dissemination, and tissue tropism of severe case EV71 (SC-EV71) and mild case EV71 (MC-EV71), consistent with their different pathogenicity in HFDM patients. Compared with MC-EV71, SC-EV71 not only enters the blood circulation and spreads out more quickly, but also shows more significant neuronal and respiratory tropism, which certainly contribute severe neurological complications and clinical manifestations in the patient. Hence, the in situ bioorthogonal fluorescent labeling is a plausible strategy to dissect complicated process of EV71 viral spread in the early stage of infection, thereby offering great opportunities to understand its pathogenesis and develop anti-viral drugs.

Bioinspired Hybrid Protein Oxygen Nanocarrier Amplified Photodynamic Therapy for Eliciting Anti-tumor Immunity and Abscopal Effect.

An ideal cancer therapeutic strategy is expected to possess potent ability to not only ablate primary tumors but also prevent distance metastasis and relapse. In this study, human serum albumin was hybridized with hemoglobin by intermolecular disulfide bonds to develop a hybrid protein oxygen nanocarrier with chlorine e6 encapsulated (C@HPOC) for oxygen self-sufficient photodynamic therapy (PDT). C@HPOC realized the tumor-targeted co-delivery of photosensitizer and oxygen, which remarkably relieved tumor hypoxia. C@HPOC was favorable for more efficient PDT and enhanced infiltration of CD8+ T cells in tumors. Moreover, oxygen-boosted PDT of C@HPOC induced immunogenic cell death, with the release of danger-associated molecular patterns to activate dendritic cells, T lymphocytes, and natural killer cells in vivo. Notably, C@HPOC-mediated immunogenic PDT could destroy primary tumors and effectively suppress distant tumors and lung metastasis in a metastatic triple-negative breast cancer model by evoking systemic anti-tumor immunity. This study provides a paradigm of oxygen-augmented immunogenic PDT for metastatic cancer treatment.

Oxygen-boosted immunogenic photodynamic therapy with gold nanocages@manganese dioxide to inhibit tumor growth and metastases.

Metastatic triple-negative breast cancer (mTNBC) is an aggressive disease among women worldwide, characterized by high mortality and poor prognosis despite systemic therapy with radiation and chemotherapies. Photodynamic therapy (PDT) is an important strategy to eliminate the primary tumor, however its therapeutic efficacy against metastases and recurrence is still limited. Here, we employed a template method to develop the core-shell gold nanocage@manganese dioxide (AuNC@MnO2, AM) nanoparticles as tumor microenvironment responsive oxygen producers and near-infrared (NIR)-triggered reactive oxygen species (ROS) generators for oxygen-boosted immunogenic PDT against mTNBC. In this platform, MnO2 shell degrades in acidic tumor microenvironment pH/H2O2 conditions and generates massive oxygen to boost PDT effect of AM nanoparticles under laser irradiation. Fluorescence (FL)/photoacoustic (PA)/magnetic resonance (MR) multimodal imaging confirms the effective accumulation of AM nanoparticles with sufficient oxygenation in tumor site to ameliorate local hypoxia. Moreover, the oxygen-boosted PDT effect of AM not only destroys primary tumor effectively but also elicits immunogenic cell death (ICD) with damage-associated molecular patterns (DAMPs) release, which subsequently induces DC maturation and effector cells activation, thereby robustly evoking systematic antitumor immune responses against mTNBC. Hence, this oxygen-boosted immunogenic PDT nanosystem offers a promising approach to ablate primary tumor and simultaneously prevent tumor metastases via immunogenic abscopal effects.