Cardiovascular-specific PSEN1 deletion leads to abnormalities in calcium homeostasis.

Mutations of PSEN1 have been reported in dilated cardiomyopathy pedigrees. Understanding the effects and mechanisms of PSEN1 in cardiomyocytes might have important implications for treatment of heart diseases. Here, we showed that PSEN1 was downregulated in ischemia-induced failing hearts. Functionally, cardiovascular specific PSEN1 deletion led to spontaneous death of the mice due to cardiomyopathy. At the age of 11 months, the ratio of the heart weight/body weight was slightly lower in the Sm22a-PSEN1-KO mice compared with that of the WT mice. Echocardiography showed that the percentage of ejection fraction and fractional shortening was significantly reduced in the Sm22a-PSEN1-KO group compared with the percent of these measures in the WT group, indicating that PSEN1-KO resulted in heart failure. The abnormally regulated genes resulted from PSEN1-KO were detected to be enriched in muscle development and dilated cardiomyopathy. Among them, several genes encode Ca2+ ion channels, promoting us to investigate the effects of PSEN1 KO on regulation of Ca2+ in isolated adult cardiomyocytes. Consistently, in isolated adult cardiomyocytes, PSEN1-KO increased the concentration of cytosolic Ca2+ and reduced Ca2+ concentration inside the sarcoplasmic reticulum (SR) lumen at the resting stage. Additionally, SR Ca2+ was decreased in the failing hearts of WT mice, but with the lowest levels observed in the failing hearts of PSEN1 knockout mice. These results indicate that the process of Ca2+ release from SR into cytoplasm was affected by PSEN1 KO. Therefore, the abnormalities in Ca2+ homeostasis resulted from downregulation of PSEN1 in failing hearts might contribute to aging-related cardiomyopathy, which might had important implications for the treatment of aging-related heart diseases.

CAR exosomes derived from effector CAR-T cells have potent antitumour effects and low toxicity.

Genetically engineered T cells expressing a chimeric antigen receptor (CAR) are rapidly emerging a promising new treatment for haematological and non-haematological malignancies. CAR-T therapy can induce rapid and durable clinical responses but is associated with unique acute toxicities. Moreover, CAR-T cells are vulnerable to immunosuppressive mechanisms. Here, we report that CAR-T cells release extracellular vesicles, mostly in the form of exosomes that carry CAR on their surface. The CAR-containing exosomes express a high level of cytotoxic molecules and inhibit tumour growth. Compared with CAR-T cells, CAR exosomes do not express Programmed cell Death protein 1 (PD1), and their antitumour effect cannot be weakened by recombinant PD-L1 treatment. In a preclinical in vivo model of cytokine release syndrome, the administration of CAR exosomes is relatively safe compared with CAR-T therapy. This study supports the use of exosomes as biomimetic nanovesicles that may be useful in future therapeutic approaches against tumours.

Targeting RyR2 with a phosphorylation site-specific nanobody reverses dysfunction of failing cardiomyocytes in rats.

Chronic PKA phosphorylation of ryanodine receptor 2 (RyR2) has been shown to increase diastolic sarcoplasmic reticulum (SR) Ca2+ leakage and lead to cardiac dysfunction. We hypothesize that intracellular gene delivery of an RyR2-targeting phosphorylation site-specific nanobody could preserve the contractility of the failing myocardium. In the present study, we acquired RyR2-specific nanobodies from a phage display library that were variable domains of Camelidae heavy chain-only antibodies. One of the nanobodies, AR185, inhibited RyR2 phosphorylation in vitro and was chosen for further investigation. We investigated the potential of adeno-associated virus (AAV)9-mediated cardiac expression of AR185 to combat postischemic heart failure (HF). AAV gene delivery elevated the intracellular expression of the AR185 protein in a rat model of ischemic HF, and this treatment normalized the systolic and diastolic dysfunction of the failing myocardium in vivo by reversing myocardial Ca2+ handling. Furthermore, AR185 gene transfer to failing cardiomyocytes reduced the frequency of SR calcium leaks, thereby restoring the attenuated intracellular calcium transients and SR calcium load. Moreover, AR185 gene transfer inhibited the PKA-mediated phosphorylation of RyR2 in failing cardiomyocytes. Our results provide preclinical experimental evidence that the cardiac expression of RyR2 nanobodies with AAV9 vectors is a promising therapeutic strategy for HF.-Li, T., Shen, Y., Lin, F., Fu, W., Liu, S., Wang, C., Liang, J., Fan, X., Ye, X., Tang, Y., Ding, M., Yang, Y., Lei, C., Hu, S. Targeting RyR2 with a phosphorylation site-specific nanobody reverses dysfunction of failing cardiomyocytes in rats.

EGFR/Notch Antagonists Enhance the Response to Inhibitors of the PI3K-Akt Pathway by Decreasing Tumor-Initiating Cell Frequency.

PURPOSE: Both EGFR and PI3K-Akt signaling pathways have been used as therapeutically actionable targets, but resistance is frequently reported. In this report, we show that enrichment of the cancer stem cell (CSC) subsets and dysregulation of Notch signaling underlie the challenges to therapy and describe the development of bispecific antibodies targeting both HER and Notch signaling. EXPERIMENTAL DESIGN: We utilized cell-based models to study Notch signaling in drug-induced CSC expansion. Both cancer cell line models and patient-derived xenograft tumors were used to evaluate the antitumor effects of bispecific antibodies. Cell assays, flow cytometry, qPCR, and in vivo serial transplantation assays were employed to investigate the mechanisms of action and pharmacodynamic readouts. RESULTS: We found that EGFR/Notch targeting bispecific antibodies exhibited a notable antistem cell effect in both in vitro and in vivo assays. Bispecific antibodies delayed the occurrence of acquired resistance to EGFR inhibitors in triple-negative breast cancer cell line-based models and showed efficacy in patient-derived xenografts. Moreover, the EGFR/Notch bispecific antibody PTG12 in combination with GDC-0941 exerted a stronger antitumor effect than the combined therapy of PI3K inhibitor with EGFR inhibitors or tarextumab in a broad spectrum of epithelial tumors. Mechanistically, bispecific antibody treatment inhibits the stem cell-like subpopulation, reduces tumor-initiating cell frequency, and downregulates the mesenchymal gene expression. CONCLUSIONS: These findings suggest that the coblockade of EGFR and Notch signaling has the potential to increase the response to PI3K inhibition, and PTG12 may gain clinical efficacy when combined with PI3K blockage in cancer treatment.

Tumor Ablation Enhancement by Combining Radiofrequency Ablation and Irreversible Electroporation: An In Vitro 3D Tumor Study.

We hypothesized and demonstrated for the first time that significant tumor ablation enhancement can be achieved by combining radiofrequency ablation (RFA) and irreversible electroporation (IRE) using a 3D cervical cancer cell model. Three RFA (43, 50, and 60 °C for 2 min) and IRE protocols (350, 700, and 1050 V/cm) were used to study the combining effect in the 3D tumor cell model. The in vitro experiment showed that both RFA enhanced IRE and IRE enhanced RFA can lead to a significant increase in the size of the ablation zone compared to IRE and RFA alone. It was also noted that the sequence of applying ablation energy (RFA â†’ RE or IRE â†’ RFA) affected the efficacy of tumor ablation enhancement. The electrical conductivity of 3D tumor was found to be increased after preliminary RFA or IRE treatment. This increase in tumor conductivity may explain the enhancement of tumor ablation. Another explanation might be that there is repeat injury to the transitional zone of the first treatment by the second one. The promising results achieved in the study can provide us useful clues about the treatment of large tumors abutting large vessels or bile ducts.

Development of a statistical model for cervical cancer cell death with irreversible electroporation in vitro.

PURPOSE: The aim of this study was to develop a statistical model for cell death by irreversible electroporation (IRE) and to show that the statistic model is more accurate than the electric field threshold model in the literature using cervical cancer cells in vitro. METHODS: HeLa cell line was cultured and treated with different IRE protocols in order to obtain data for modeling the statistical relationship between the cell death and pulse-setting parameters. In total, 340 in vitro experiments were performed with a commercial IRE pulse system, including a pulse generator and an electric cuvette. Trypan blue staining technique was used to evaluate cell death after 4 hours of incubation following IRE treatment. Peleg-Fermi model was used in the study to build the statistical relationship using the cell viability data obtained from the in vitro experiments. A finite element model of IRE for the electric field distribution was also built. Comparison of ablation zones between the statistical model and electric threshold model (drawn from the finite element model) was used to show the accuracy of the proposed statistical model in the description of the ablation zone and its applicability in different pulse-setting parameters. RESULTS: The statistical models describing the relationships between HeLa cell death and pulse length and the number of pulses, respectively, were built. The values of the curve fitting parameters were obtained using the Peleg-Fermi model for the treatment of cervical cancer with IRE. The difference in the ablation zone between the statistical model and the electric threshold model was also illustrated to show the accuracy of the proposed statistical model in the representation of ablation zone in IRE. CONCLUSIONS: This study concluded that: (1) the proposed statistical model accurately described the ablation zone of IRE with cervical cancer cells, and was more accurate compared with the electric field model; (2) the proposed statistical model was able to estimate the value of electric field threshold for the computer simulation of IRE in the treatment of cervical cancer; and (3) the proposed statistical model was able to express the change in ablation zone with the change in pulse-setting parameters.

Cardiac adenovirus-associated viral Presenilin 1 gene delivery protects the left ventricular function of the heart via regulating RyR2 function in post-ischaemic heart failure.

Post-ischaemic heart failure is a major cause of death worldwide. Reperfusion of infarcted heart tissue after myocardial infarction has been an important medical intervention to improve outcomes. However, disturbances in Ca2+ and redox homeostasis at the cellular level caused by ischaemia/reperfusion remain major clinical challenges. In this study, we investigated the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of a Type-2 ryanodine receptor (RyR2) degradation-associated gene, Presenilin 1 (PSEN1), to combat post-ischaemic heart failure. Adeno-associated viral PSEN1 gene delivery elevated PSEN1 protein expression in a post-infarction rat heart failure model, and this administration normalised the contractile dysfunction of the failing myocardium in vivo and in vitro by reversing myocardial Ca2+ handling and function. Moreover, PSEN1 gene transfer to failing cardiomyocytes reduced sarcoplasmic reticulum (SR) Ca2+ leak, thereby restoring the diminished intracellular Ca2+ transients and SR Ca2+ load. Moreover, PSEN1 gene transfer reversed the phosphorylation of RyR2 in failing cardiomyocytes. However, selective autophagy inhibition did not prevent the PSEN1-induced blockade of RyR2 degradation, making the participation of autophagy in PSEN1-associated RyR2 degradation unlikely. Our results established a role of the cardiac expression of PSEN1 with AAV9 vectors as a promising therapeutic approach for post-ischaemic heart failure.

Conditionally targeted deletion of PSEN1 leads to diastolic heart dysfunction.

Recently, PSEN1 has been reported to have mutations in dilated cardiomyopathy pedigrees. However, the function and mechanism of PSEN1 in cardiomyopathy remains unresolved. Here, we established four types of genetically modified mice to determine the function of PSEN1 in cardiac development and pathology. PSEN1 null mutation resulted in perinatal death, retardation of heart growth, ventricular dilatation, septum defects, and valvular thickening. PSEN1 knockout in adults led to decreased muscle fibers, widened sarcomere Z lines and reduced lengths of sarcomeres in cardiomyocytes. Cardiovascular loss of function of PSEN1 induced by Sm22a-Cre or Myh6-Cre/ER/tamoxifen also resulted in severe ultrastructural abnormalities, such as relaxed gap junctions between neighboring cardiomyocytes. Functionally, cardiovascular deletion of PSEN1 caused spontaneous mortality from birth to adulthood and led to diastolic heart dysfunction, including decreased volume of the left ventricle at the end-systolic and end-diastolic stages. Additionally, in a myocardial ischemia model, deletion of PSEN1 in the cardiovascular system first protected mice by inducing adaptive hypertrophy but ultimately resulted in severe heart failure. Furthermore, a collection of genes was abnormally expressed in the hearts of cardiac-specific PSEN1 knockout mice. They were enriched in cell proliferation, calcium regulation, and so on. Taken together, dynamic regulation and abnormal function of PSEN1 underlie the pathogenesis of cardiovascular diseases due to ultrastructural abnormality of cardiomyocytes.

Antagonism of EGFR and Notch limits resistance to EGFR inhibitors and radiation by decreasing tumor-initiating cell frequency.

Epidermal growth factor receptor (EGFR) blockade and radiation are efficacious in the treatment of cancer, but resistance is commonly reported. Studies have suggested that dysregulation of Notch signaling and enrichment of the cancer stem cell population underlie these treatment challenges. Our data show that dual targeting of EGFR and Notch2/3 receptors with antibody CT16 not only inhibited signaling mediated by these receptors but also showed a strong anti-stem cell effect both in vitro and in vivo. Treatment with CT16 prevented acquired resistance to EGFR inhibitors and radiation in non-small cell lung cancer (NSCLC) cell line models and patient-derived xenograft tumors. CT16 also had a superior radiosensitizing impact compared with EGFR inhibitors. CT16 in combination with radiation had a larger antitumor effect than the combination of radiation with EGFR inhibitors or tarextumab. Mechanistically, CT16 treatment inhibits the stem cell-like subpopulation, which has a high mesenchymal gene expression and DNA repair activity, and reduces tumor-initiating cell frequency. This finding highlights the capacity of a combined blockade of EGFR and Notch signaling to augment the response to radiation and suggests that CT16 may achieve clinical efficacy when combined with radiation in NSCLC treatment.

TRIM14 regulates cell proliferation and invasion in osteosarcoma via promotion of the AKT signaling pathway.

Recent studies have shown that some members of the tripartite motif-containing protein (TRIM) family serve as important regulators of tumorigenesis. However, the biological role of TRIM14 in osteosarcoma remains to be established. In this study, we showed that TRIM14 is upregulated in human osteosarcoma specimens and cell lines, and correlated with osteosarcoma progression and shorter patient survival times. Functional studies demonstrated that overexpression of TRIM14 enhances osteosarcoma cell proliferation, clone formation, cell cycle procession, migration and invasion in vitro and promotes tumor growth in vivo, and conversely, its silencing has the opposite effects. Furthermore, TRIM14 overexpression induced activation of the AKT pathway. Inhibition of AKT expression reversed the TRIM14-mediated promotory effects on cell growth and mobility, in addition to TRIM14-induced epithelial-to-mesenchymal transition (EMT) and cyclin D1 upregulation. Our findings collectively suggest that TRIM14 functions as an oncogene by upregulating the AKT signaling pathway in osteosarcoma cells, supporting its potential utility as a therapeutic target for this disease.

Broad RTK-targeted therapy overcomes molecular heterogeneity-driven resistance to cetuximab via vectored immunoprophylaxis in colorectal cancer.

The human epidermal growth factor receptor (EGFR) targeting chimeric monoclonal antibody, cetuximab (Erbitux®), is a widely used drug in the treatment of metastatic colorectal cancer. However, the activation of the extensive crosstalk among the EGFR family receptors as well as other tyrosine kinase receptors (RTKs) impairs the efficacy of the drug by fueling acquired resistance. To identify the responsible potential activation pathway underlying cetuximab resistance and generate novel treatment strategies, cetuximab-resistant colorectal cancer cell lines were generated and validated and a functional RNAi screen targeting human RTKs was used to identify extensive receptor tyrosine kinase signaling networks established in resistant cancer cells. MET, Axl, and IGF-1R were identified as contributors to the acquired resistance to cetuximab. Targeting vectored immunoprophylaxis (VIPs) to different RTKs were generated and characterized. Different VIP approaches were evaluated in vivo with parental and cetuximab-resistance xenografts and the RTKs in resistant cancer xenografts were inhibited with VIPs via re-sensitization to cetuximab treatment. Combination of VIPs was more broadly efficacious, mechanistically, due to co-blocking the EGFR/Axl/MET signaling pathway, which was cross-activated in the resistant cell lines. Moreover, a VIP-based procedural treatment strategy not only eliminated the tumor but also afforded long-lasting protection from tumor recurrence and resistance. Overall, EGFR-related RTK pathway-network activation represents a novel mechanism underlying cetuximab resistance. A broad VIP combination strategy and VIP-based procedural treatment strategy may be a recommended addition to cetuximab-based targeted therapy. Our results establish a new principle to achieve combined RTK inhibition and reverse drug resistance using a VIP approach.

CoNi@SiO2 @TiO2 and CoNi@Air@TiO2 Microspheres with Strong Wideband Microwave Absorption.

The synthesis of CoNi@SiO2 @TiO2 core-shell and CoNi@Air@TiO2 yolk-shell microspheres is reported for the first time. Owing to the magnetic-dielectric synergistic effect, the obtained CoNi@SiO2 @TiO2 microspheres exhibit outstanding microwave absorption performance with a maximum reflection loss of -58.2 dB and wide bandwidth of 8.1 GHz (8.0-16.1 GHz, < -10 dB).

In situ visualizing T-Tubule/SR junction reveals the ultra-structures of calcium storage and release machinery.

In skeletal muscle, Ca(2+) release from sarcoplasmic reticulum (SR) following the action potential relies on the delicate architecture of the T-Tubule/SR junction. The T-Tubule/SR junction comprises two membrane systems: the integral proteins DHPR and RyR1 and the Ca(2+)-buffering apparatus within the SR lumen. The arrangement and interactions of the components have remained elusive due to technological limitations. Here, we determined whether electron tomography is effective fort the in situ determination of the relationships between RyR1 and DHPR, the SR membrane and other involved structures. First, we visually confirmed that RyR1 and DHPR are close neighbors that are mutually staggered with each other and directly interact with one of RyR1 subunits. Second, the Ca(2+) storage network within the SR lumen is directly correlated with RyR1. These results suggest that the excitation of the T-Tubule may induce Ca(2+) release through a direct interaction among DHPR, RyR1 and the Ca(2+) buffering apparatus. These results indicate that electron tomography has potential as an efficient method for the in situ characterization of the complex architecture and arrangement of two integral-integral-membrane proteins in the context of the surrounding phospholipid-bilayer and proteins.

In vitro cytotoxicity of gold nanorods in A549 cells.

Gold nanoparticles, which have unique physicochemical characteristics, are being used for an increasingly wide range of applications in biomedical research. In this study, gold nanorods (width of 25 nm, length of 52 nm) were found to be internalized by A549 cells and were primarily localized in the lysosomes and membranous vesicles. The integrity of the membranes of A549 cells exposed to gold nanorods for 4h was damaged, as indicated by laser scanning confocal microscopy (LSCM). Increased lactate dehydrogenase (LDH) leakage and decreased cell viability further indicated the concentration-dependent cytotoxicity of the gold nanorods to the A549 cells. Reactive oxygen species (ROS) production was induced in the A549 cells by the gold nanorods, and this effect was positively correlated with the concentration of the gold nanorods. The results of this study indicated that exposure to gold nanorods caused dose-dependent cytotoxicity in A549 cells and that oxidative stress may be the main factor causing cytotoxicity.

Distribution of graphene oxide and TiO2-graphene oxide composite in A549 cells.

Graphene and its derivatives are increasingly applied in nanoelectronics, biosensing, drug delivery, and biomedical applications. However, the information about its cytotoxicity remains limited. Herein, the distribution and cytotoxicity of graphene oxide (GO) and TiO2-graphene oxide composite (TiO2-GO composite) were evaluated in A549 cells. Cell viability and cell ultrastructure were measured. Our results indicated that GO could enter A549 cells and located in the cytoplasm and nucleus without causing any cell damage. TiO2 nanoparticles and GO would be separated after TiO2-GO composite entered A549 cells. TiO2-GO composite could induce cytotoxicity similar to TiO2 nanoparticles, which was probably attributed to oxidative stress. These results should be considered in the development of biological applications of GO and TiO2-GO composite.

Effects of fullerene C60 nanoparticles on A549 cells.

Fullerene C60 nanoparticles (C60 NPs) have been widely applied in many fields due to their excellent physical and chemical properties. As production and applications of C60 NPs expand, public concern about the potential risk to human health has also risen. The toxicity of C60 NPs was evaluated by the CCK-8 assay using the cultured human epithelial cell line A549. Cellular uptake of the C60 NPs was observed by TEM imaging. In our findings, C60 NPs could readily enter A549 cells and showed no significant toxicity. Exposure of cultured A549 cells to C60 NPs led to an increase of intracellular reactive oxygen species (ROS) while glutathione reductase activity was probably activated to generate more GSH to maintain a cellular oxidation-reduction equilibrium. The A549 cells responded to the ROS increases through the inauguration of autophagic responses, aimed at restoring cellular health and equilibrium.

Mesenchymal stem cells with modification of junctional adhesion molecule a induce hair formation.

The junctional adhesion molecule A (JAM-A) has been shown to serve a crucial role in the proliferation, differentiation, and tube-like formation of epithelial cells during angiogenesis. The role of JAM-A in hair follicle (HF) regeneration has not yet been reported. In this study, we used human JAM-A-modified human mesenchymal stem cells (MSCs) to repair HF abnormalities in BALB/c nu/nu mice. The JAM-A gene and JAM-A short hairpin RNA were transfected into cultured human MSCs to generate the JAM-A overexpression MSCs (JAM-A(ov) MSCs) and JAM-A knockdown MSCs (JAM-A(kd) MSCs), respectively. These cells were injected intradermally into the skin of nude mice during the first telogen phase of the HF that occurs 21 days postnatally. We found that JAM-A(ov) MSCs migrated into the HF sheath and remodeled HF structure effectively. The HF abnormalities such as HF curve and HF zigzag were remodeled, and hair formation was improved 7 days following injection in both the JAM-A(ov) MSC and MSC groups, compared with the JAM-A(kd) MSC group or negative control group. Furthermore, the JAM-A(ov) MSC group showed enhanced hair formation in contrast to the MSC group, and the number of curved and zigzagged HFs was reduced by 80% (p < .05). These results indicated that JAM-A(ov) MSCs improved hair formation in nude mice through HF structure remodeling.

Cytotoxicity of silica nanoparticles on HaCaT cells.

Despite the widespread use of silica nanoparticles (SiO2 NPs) in biological and medical fields, their adverse effects have not been clearly elucidated. In this study, spherical SiO2 NPs with a 50 nm diameter were used to study their interaction with HaCaT cells. SiO2 NPs were found to be readily internalized into HaCaT cells and localized in the cytoplasm, lysosomes and autophagosomes. Decreased cell viability and damaged cell membrane integrity showed the cytotoxicity of SiO2 NPs. Significant glutathione depletion and reactive oxygen species generation, which reduced the cellular antioxidant level, could be the major factor of cytotoxicity induced by SiO2 NPs.

Mitochondrial injury induced by nanosized titanium dioxide in A549 cells and rats.

The nanosized titanium dioxide (nano-TiO2) is an important nanoscale compound applied in many different fields because of its superior performance. Here, an anatase nano-TiO2 showed cytotoxicity in a dosage-dependent manner, which was in accordance with changes of A549 cell ultrastructure, A549 cell viability and intracellular ATP level. The lungs of rats treated with single intratracheal instillation of nano-TiO2 were injured, which was demonstrated by changes of alveolar epithelial cell ultrastructure, lung tissue pathology and lung tissue MDA level. The results of this study indicated that nano-TiO2 should be related to the generation of intracellular reactive oxygen species (ROS), which injured mitochondria and prevented the synthesis of ATP. The cells were approaching to apoptosis eventually. In macroscopic view, the lungs inevitably suffered.

Dexamethasone induces rapid promotion of norepinephrine­mediated vascular smooth muscle cell contraction.

The aim of the present study was to identify the rapid effect of dexamethasone (Dex) on norepinephrine (NE)­mediated contraction of vascular smooth muscle cells (VSMCs) and to establish the underlying mechanism(s). Rat VSMCs were preincubated with lipopolysaccharide to simulate acute septic shock. Myosin light chain (MLC20) phosphorylation of VSMCs was detected by western blot analysis to observe the effects of Dex on NE­mediated contraction. Activation of the RhoA/ RhoA kinase (ROCK), extracellular signal­regulated kinase (ERK) and p38 signaling pathways was detected by western blot analysis to explore the mechanism. It was identified that Dex rapidly promoted NE­induced phosphorylation of MLC20 in VSMCs and this effect may be non­genomic. The RhoA/ROCK, ERK and p38 pathways were demonstrated to be important for the rapid effect of Dex­induced promotion of NE­mediated contraction in VSMCs. The present results indicate that Dex may rapidly reverse the hyporeactivity of vasoconstriction to NE in vitro and this effect may be mediated by specific non­genomic mechanisms through increased activation of the RhoA/ROCK, ERK and p38 signaling pathways.