Changhai Lei

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.

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

An anti-EGFR/Notch antibody limits acquired resistance to EGFR inhibitors and radiation by reducing tumor-initiating cell frequency. Two targets for a bigger punch The epidermal growth factor receptor (EGFR) is a common target of therapeutics for a variety of different tumors, but tumors eventually become resistant to these drugs. One route to resistance is an increase in the number of cancer stem cells driven by the Notch pathway. To overcome this type of resistance, Hu et al. created a customized antibody called CT16, which can recognize both EGFR and Notch, simultaneously inhibiting both pathways. The authors tested this antibody in mouse models of non–small cell lung cancer, showing that it inhibits cancer stem cells and is more effective than antibodies against the individual pathways alone and in combination, particularly in the presence of radiation treatment. 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.

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.

Targeting EGFR/HER2 heterodimerization with a novel anti-HER2 domain II/III antibody

HighlightsA novel anti‐HER2 antibody was developed.There was a structural conformation change in HER2 in complex with 7C3.The antibody could block HER2/EGFR heterodimerization and signaling.It reveals a unique potential to complement current anti‐HER2 treatment strategy. Abstract HER2, a ligand‐free tyrosine kinase receptor of the HER family, is frequently overexpressed in breast cancer. The anti‐HER2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer. Despite the effectiveness of trastuzumab, its efficacy remains variable and often modest. Thus, there is an urgent need to improve ErbB2‐targeting therapy. Here, we describe a novel anti‐HER2 antibody, 7C3, which was developed using hybridoma technique. Structural analysis confirms that the epitope of this antibody is in domain II/III of HER2. Moreover, a structural conformation change was observed in HER2 in complex with 7C3. Interestingly, this novel anti‐HER2 antibody exhibits efficacy in blocking HER2/EGFR heterodimerization and signaling. The results highlight the different function role of HER2 domains and the unique potential of 7C3 to inhibit the HER2/EGFR heterodimer, which may complement current anti‐HER2 treatments.

Large-Scale Clinical Data Management and Analysis System Based on Cloud Computing

With the exponential increase of Electronic Health Record systems in China, large-scale clinical data management and analysis have become big challenges. This paper depicts a novel system named Clinical Data Managing and Analyzing System, which uses hybrid XML database, and HBase/Hadoop infrastructure to handle big amount of heart disease clinical data analysis online. Using standardized format of Clinical Document Architecture, the system now has integrated more than 50,000 valvular heart disease clinical documents and provided efficient distributed data mining tools as well as data managing tools for doctor users from multi heart clinical centers in six different 3A hospitals of China.

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.

Dynamic expression of early responsible genes to acute left-ventricular ischemia in a time-dependent pattern

Acute myocardial infarction remains one of the leading causes of death and disability worldwide. The mechanisms underlying myocardial infarction involve a complex of signaling molecules, such as tumor necrosis factor α (TNFα), interleukin-6 (IL6), C-Myc, atria natriuretic peptide (ANP), superoxide dismutase 1 (SOD1), and so on. The aim of this study is to understand the time-dependent expressional pattern of these early responsible genes following acute myocardial ischemia established by left anterior descending (LAD) coronary artery ligation. After LAD ligation, a collection of genes was detected using real-time polymerase chain reaction (PCR). The expression of inflammation-related genes, such as TNFα and IL6, was immediately upregulated at 2 h, reached to the highest point at 12 h, and then decreased to nearly basis level at 24 h after ligation, suggesting inflammation appeared and disappeared rapidly after acute ischemia. C-Myc, an important transcription factor, was significantly upregulated at 2 h, and thereafter persisted at high level to 24 h. The secretary peptide, ANP, was consistently upregulated from 2 to 24 h, reached to 40-folds at 24 h. The calcium-regulated gene, FK506-binding protein 12.6, was not significantly altered after ischemia. SOD1 was not altered at the first 4 h, and began to downregulate at 12 and 24 h. These results indicate that several genes were dynamically and transiently regulated after acute myocardial infarction (AMI) in a time-dependent pattern, suggesting that there is an immediate molecular response to acute myocardial ischemia, which might provide us a new insight to understand molecular mechanisms of AMI.

Targeting RyR2 with a phosphorylation site-specific nanobody Reverses Dysfunction of Failing Cardiomyocytes in Rat

Chronic PKA phosphorylation of RyR2 has been shown to increased diastolic SR Ca2+ leak and lead to cardiac dysfunction. Since the change of phosphorylation level of RyR2 is a biomarker of failing heart, we attempted to verify the hypothesis that intracellular gene delivery of a RyR2 targeting phosphorylation site-specific nanobody could preserve contractility of failing myocardium. In present study, we acquired the RyR2-specific nanobodies from a phage display library which are variable domains of camellidae heavy chain-only antibodies (VHH). One of the monoclonal nanobodies, AR185, inhibiting RyR2 phosphorylation in an in vitro assay was then chosen for further investigation. We investigated the potential of adeno-associated virus (AAV)-9-mediated cardiac expression of AR185 against post-ischemic heart failure. Adeno-associated virus gene delivery elevated the intracellular expression AR185 protein in the ischemic heart failure model of rats, and this treatment normalized the systolic and diastolic dysfunction of the failing myocardium in vivo and in vitro by reversing myocardial Ca2+ handling. Furthermore, AR185 gene transfer to failing cardiomyocytes reduced the frequency of sarcoplasmic reticulum (SR) calcium leak, thereby restoring the attenuated intracellular calcium transients and SR calcium load. Moreover, AR185 gene transfer inhibited PKA phosphorylation of RyR2 in failing cardiomyocytes. Our results provided strong pre-clinical experimental evidence of the cardiac expression of RyR2 nanobody with AAV9 vectors as a promising therapeutic strategy for ischemic heart failure.

Development and Optimization of Therapeutic Analogues of Anti-TNFα Antibody Infliximab

Previously, we have reported the crystal structures of Fab fragment of Infliximab in complex with TNFα. The structurally identified epitope on TNFα revealed the mechanism of TNFα inhibition by partially overlapping with the TNFα-receptor interface and the possibility to optimize the binding affinity. In this study, we launched a screen of a phage display library to isolate novel anti-TNFα antibodies based on the infliximab epitope. To develop novel anti-TNFα antibodies, structural analysis, the phage display antibody isolation, step by step antibody optimization, CDR residues random mutagenesis, and binding affinity characterization were performed. One of the novel antibodies generated on the backbone of infliximab, Inf3D6, has the superior binding affinity to TNFα, thus, demonstrating the potential for structure guided optimization for improvement of existing antibody-based therapeutics.

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

Abstract 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.