Targeted Biotherapy Key Laboratory of Ministry of Education

Wuhan, China

Targeted Biotherapy Key Laboratory of Ministry of Education

Wuhan, China
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Tang L.,Huazhong University of Science and Technology | Tang L.,Targeted Biotherapy Key Laboratory of Ministry of Education | Jian X.-R.,Huazhong University of Science and Technology | Jian X.-R.,Targeted Biotherapy Key Laboratory of Ministry of Education | And 11 more authors.
American Journal of Hematology | Year: 2013

Protein S (ProS) is a physiological inhibitor of coagulation with an important function in the down-regulation of thrombin generation. ProS deficiency is a major risk factor for venous thrombosis. This study enrolled 40 ProS-deficient probands to investigate the molecular basis of hereditary ProS deficiency in Chinese patients. A mutation analysis was performed by resequencing the PROS1 gene. Large deletions were identified by multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 20 different mutations, including 15 novel mutations, were identified in 21 of the 40 index probands. Small mutations were detected in 18 (45.0%) probands, and large deletions were found in 3 (7.5%) probands, leaving 19 (47.5%) patients without causative variants. To evaluate the functional consequences of 2 novel missense variants, ex vivo thrombin-generation assays, bioinformatics tools, and in vitro expression studies were employed. The p.Asn365Lys ProS variant was found to have moderately impaired secretion and reduced activated protein C cofactor activity. In contrast, the p.Pro410His mutant appeared to have severely impaired secretion but full anticoagulant activity. This study is the largest investigation of ProS deficiency in China and the first investigation of the influence of Type I ProS missense mutations on the global level of coagulation function. The p.K196E mutation, which is common in the neighboring Japanese population, was not found in our Chinese population, and null mutations were common in our Chinese population but not common in Japan. Further genetic analysis is warranted to understand the causes of ProS deficiency in patients without a genetic explanation. Am. J. Hematol. 88:899-905, 2013. © 2013 Wiley Periodicals, Inc.


Tang L.,Huazhong University of Science and Technology | Tang L.,Targeted Biotherapy Key Laboratory of Ministry of Education | Lu X.,Huazhong University of Science and Technology | Lu X.,Targeted Biotherapy Key Laboratory of Ministry of Education | And 12 more authors.
Journal of Thrombosis and Haemostasis | Year: 2012

Background: There are ethnic differences in the genetic risk factors for venous thrombosis (VT). The genetic causes of VT in the Chinese population are not fully understood. Objectives: To identify possible common abnormal factors that could contribute to thrombosis susceptibility. Methods/Results: We measured the levels of nine types of plasma coagulation factor, three types of anticoagulation factor and two types of fibrinolytic factor in 310 VT patients. Factor V activity was higher in 32 cases. Eleven of the 32 cases also had low protein C (PC) or protein S (PS) activities, indicating PC or PS deficiency. No other abnormalities were observed in the other 21 cases. All of the samples were sensitive to activated PC inactivation. Therefore, the abnormal factor involved may be FV inactivator or its cofactor rather than FV itself. Resequencing identified a common PROC c.574_576del variant in 10 of the 32 subjects. In a case-control study, this variant was detected in 68 of the 1003 patients and in 25 of the 1031 controls. It had an adjusted odds ratio of 2.71 (95% confidence interval [CI] 1.68-4.36). PC amidolytic activities of most variant carriers were similar to those of non-carriers, but the mean anticoagulant activity was only 72.7UdL-1. Expression studies in vitro showed that the anticoagulant activity of the mutant PC was 43.6% of that of the wild-type PC. Conclusions: We identified what is, so far, the most common genetic risk factor for VT in the Chinese population, with its prevalence being approximately 2.36%. © 2012 International Society on Thrombosis and Haemostasis.


Tang L.,Huazhong University of Science and Technology | Tang L.,Targeted Biotherapy Key Laboratory of Ministry of Education | Guo T.,Huazhong University of Science and Technology | Guo T.,Targeted Biotherapy Key Laboratory of Ministry of Education | And 14 more authors.
PLoS ONE | Year: 2012

Background: Protein C (PC) is one of the most important physiological inhibitors of coagulation proteases. Hereditary PC deficiency causes a predisposition to venous thrombosis (VT). The genetic characteristics of PC deficiency in the Chinese population remain unknown. Methods: Thirty-four unrelated probands diagnosed with hereditary PC deficiency were investigated. PC activity and antigen levels were measured. Mutation analysis was performed by sequencing the PROC gene. In silico analyses, including PolyPhen-2, SIFT, multiple sequence alignment, splicing prediction, and protein molecular modeling were performed to predict the consequences of each variant identified. One recurrent mutation and its relative risk for thrombosis in relatives were analyzed in 11 families. The recurrent mutation was subsequently detected in a case (VT patients)-control study, and the adjusted odds ratio (OR) for VT risk was calculated by logistic regression analysis. Results: A total of 18 different mutations, including 12 novel variants, were identified. One common mutation, PROC c.565C>T (rs146922325:C>T), was found in 17 of the 34 probands. The family study showed that first-degree relatives bearing this variant had an 8.8-fold (95%CI = 1.1-71.6) increased risk of venous thrombosis. The case-control (1003 vs. 1031) study identified this mutation in 5.88% patients and in 0.87% controls, respectively. The mutant allele conferred a high predisposition to venous thrombosis (adjusted OR = 7.34, 95%CI = 3.61-14.94). The plasma PC activity and antigen levels in heterozygotes were 51.73±6.92 U/dl and 75.17±4.84 U/dl, respectively. Conclusions: This is the first study on the genetic background of PC deficiency in the Chinese population. The PROC c.565C>T mutation is the most frequent cause of PC deficiency as well as a prevalent risk factor for VT in Chinese individuals. The inclusion of this variant in routine thrombophilic detection may improve the diagnosis and prevention of venous thrombosis. © 2012 Tang et al.


Ming Z.,Blood Research Institute | Ming Z.,Huazhong University of Science and Technology | Hu Y.,Huazhong University of Science and Technology | Hu Y.,Targeted Biotherapy Key Laboratory of Ministry of Education | And 6 more authors.
Blood | Year: 2011

Inhibition of platelet responsiveness is important to control pathologic thrombus formation. Platelet-endothelial cell adhesion molecule-1 (PECAM-1) and the Src family kinase Lyn inhibit platelet activation by the glycoprotein VI (GPVI) collagen receptor; however, it is not known whether PECAM-1 and Lyn function in the same or different inhibitory pathways. In these studies, we found that, relative to wild-type platelets, platelets derived from PECAM-1-deficient, Lyn-deficient, or PECAM-1/Lyn double-deficient mice were equally hyperresponsive to stimulation with a GPVI-specific agonist, indicating that PECAM-1 and Lyn participate in the same inhibitory pathway. Lyn was required for PECAM-1 tyrosine phosphorylation and subsequent binding of the Src homology 2 domain-containing phosphatase-2, SHP-2. These results support a model in which PECAM-1/SHP-2 complexes, formed in a Lyn-dependent manner, suppress GPVI signaling. © 2011 by The American Society of Hematology.


Huile G.,Fudan University | Huile G.,Key Laboratory of Smart Drug Delivery | Shuaiqi P.,Fudan University | Shuaiqi P.,Key Laboratory of Smart Drug Delivery | And 14 more authors.
Biomaterials | Year: 2011

Targeted drug delivery to selected brain cell types is a crucial step in enhancing therapeutic effects while limiting side effects in non-target cells. Here we report on the development and evaluation of a new cascade targeting delivery system that employs PEG-PCL nanoparticles modified with both an angiopep-2 peptide and a EGFP-EGF1 protein for precise targeting of brain neuroglial cells. Angiopep-2 penetrates the blood-brain barrier and EGFP-EGF1 binds neuroglial cells, providing the system with two stages of targeting. In vitro studies demonstrated that both bEnd.3 cells and neuroglial cells had a higher uptake of angiopep-2 and EGFP-EGF1 conjugated nanoparticles (AENP) as compared to unmodified nanoparticles. Ex vivo imaging showed that AENP had higher accumulation in the brain over unmodified nanoparticles and EGFP-EGF1 modified nanoparticles. Fluorescent in situ hybridization of brain slides demonstrated that AENP co-localized with neuroglial cells. Transmission electron microscopy further showed that AENP could target and enter neuroglial cells. This newly developed cascade targeting delivery system that precisely targets neuroglial cells has great potential in the diagnosis and treatment of neuroglial related diseases. Replacing EGFP-EGF1 and angiopep-2 with other ligands may extend the utility of the system to diagnose and treat organ diseases beyond brain. © 2011 Elsevier Ltd.


Ruan X.-L.,Huazhong University of Science and Technology | Ruan X.-L.,Targeted Biotherapy Key Laboratory of Ministry of Education | Li S.,Hubei University | Zeng X.-T.,Hubei University of Medicine | And 4 more authors.
Chinese Medical Journal | Year: 2013

Background Many studies indicated the human cytochrome P450 2D6 (CYP2D6) gene polymorphism was associated with acute leukemia (AL) susceptibility, however, the results were inconsistent. So we performed this meta-analysis to evaluate the relationship between CYP2D6*3 or CYP2D6*4 polymorphism and AL susceptibility. Methods We searched PubMed database up to February 20, 2013, and finally yielded 9 case-control studies including 1343 cases and 1843 controls which tested the association between CYP2D6*3 or *4 polymorphism and AL. After data extraction, we conducted a meta-analysis using the Comprehensive Meta Analysis software. Results Overall, no significant association between CYP2D6*3 or *4 polymorphism and AL risk was found in this meta-analysis (+ vs. -: OR=1.13, 95% CI=0.79-1.63; +/+ vs. -/-: OR=1.73, 95% CI=0.99-3.02; -/+ vs. -/-: OR=1.03, 95% CI=0.68-1.56; (-/+ and +/+) vs. -/-: OR=1.08, 95% CI=0.72-1.63; +/+ vs. (-/+ and -/-): OR=1.76, 95% CI=0.98-3.17). Similar results were also been found in stratified subgroup analysis. There was no publication bias. Conclusion CYP2D6*3 or *4 polymorphism might not be associated with AL susceptibility. However, the results need to be further confirmed by well-designed and high quality randomized controlled trials with larger sample sizes.


Mei H.,Huazhong University of Science and Technology | Mei H.,Targeted Biotherapy Key Laboratory of Ministry of Education | Shi W.,Huazhong University of Science and Technology | Shi W.,Targeted Biotherapy Key Laboratory of Ministry of Education | And 11 more authors.
Biomaterials | Year: 2010

In a strategy for anti-thrombotic therapy, we have expressed EGFP-EGF1 fusion protein, in which EGF1 can bind with tissue factor (TF). EGFP has previously been widely used as a fluorescent protein marker. EGFP-EGF1 protein was thiolated and conjugated to the malemide covering on the pegylated nanoparticles (NP) to form the EGFP-EGF1-NP. The EGFP-EGF1-NP was characterized in terms of morphology, size and zeta potential. In vitro cell viability experiment confirmed that the biodegradable EGFP-EGF1-NP was safe. To evaluate the delivering ability of EGFP-EGF1-NP, a fluorochrome dye, Dir, was incorporated into the nanoparticle, and the loading capacity and release property of the particle were examined. In vitro results showed that the binding ability of EGFP-EGF1-NP with TF-expressing cells was significantly stronger than that of non-conjugated NP. In vivo multispectral fluorescent imaging demonstrated that EGFP-EGF1-NP had high specificity and sensitivity in targeting thrombi. Our study demonstrated that EGFP-EGF1-NP is a promising TF-targeting drug delivery system for thrombolytic treatment. © 2010 Elsevier Ltd.


Shi W.,Huazhong University of Science and Technology | Shi W.,Targeted Biotherapy Key Laboratory of Ministry of Education | Mei H.,Huazhong University of Science and Technology | Mei H.,Targeted Biotherapy Key Laboratory of Ministry of Education | And 16 more authors.
Biomaterials | Year: 2013

In acute vascular events, the endothelium derived tissue factor (TF) is the trigger of the coagulation cascade. In this study, EGFP-EGF1 protein-conjugated PEG-PLGA nanoparticle was employed as a TF targeting vehicle, the NF-κB decoy oligonucleotides (ODNs) was incorporated into it and the resulting EGF1-EGFP-NP-ODNs were evaluated as a vector for therapy of cortex infarction. At 2 h after transfection of TF expressed rat brain capillary endothelial cell, EGF1-EGFP-NP-ODNs was more efficiently internalized and located in the cytoplasm than NP-ODNs. At 4 h and 6 h after administration, ODNs were present in the nuclei and obviously inhibited the TF expression. At 6 h after i.v. administration in vivo, most EGF1-EGFP-NP were accumulated in the embolism vessels, distributed in the damaged endothelial cells and lowered the TF expression. At 24 h after i.v. administration, MR imaging of cortex infarcts were predominantly dwindled. © 2013 Elsevier Ltd.


Chen C.,Huazhong University of Science and Technology | Chen C.,Targeted Biotherapy Key Laboratory of Ministry of Education | Mei H.,Huazhong University of Science and Technology | Mei H.,Targeted Biotherapy Key Laboratory of Ministry of Education | And 12 more authors.
PLoS ONE | Year: 2013

Injured endothelium is an important target for drug and/or gene therapy because brain microvascular endothelial cells (BMECs) play critical roles in various pathophysiological conditions. RNA-mediated gene silencing presents a new therapeutic approach for treating such diseases, but major challenge is to ensure minimal toxicity and target delivery of siRNA to injured BMECs. Injured BMECs overexpress tissue factor (TF), which the fusion protein EGFP-EGF1 could be targeted to. In this study, TNF alpha (TNF-α) was chosen as a stimulus for primary BMECs to produce injured endothelium in vitro. The EGFP-EGF1-PLGA nanoparticles (ENPs) with loaded TF-siRNA were used as a new carrier for targeted delivery to the injured BMECs. The nanoparticles then produced intracellular RNA interference against TF. We compared ENP-based transfections with NP-mediated transfections, and our studies show that the ENP-based transfections result in a more efficient downregulation of TF. Our findings also show that the TF siRNA-loaded ENPs had minimal toxicity, with almost 96% of the cells viable 24 h after transfection while Lipofectamine-based transfections resulted in only 75% of the cells. Therefore, ENP-based transfection could be used for efficient siRNA transfection to injured BMECs and for efficient RNA interference (RNAi). This transfection could serve as a potential treatment for diseases, such as stroke, atherosclerosis and cancer. © 2013 Chen et al.


Shi W.,Huazhong University of Science and Technology | Shi W.,Targeted Biotherapy Key Laboratory of Ministry of Education | Mei H.,Huazhong University of Science and Technology | Mei H.,Targeted Biotherapy Key Laboratory of Ministry of Education | And 17 more authors.
Biomaterials | Year: 2012

Tissue factor (TF) is a 47 kDa membrane-bound glycoprotein, which is present at high concentrations on damaged endothelium, atherosclerotic plaques or tumor vasculature, and is an important trigger of coagulation cascade. In this study, we have expressed and purified the TF targeting protein-EGFP-EGF1, which was thiolated and conjugated to the malemide of the PEG-PLGA nanoparticle to form a TF targeting nanomedical system: EGF1-EGFP-NP. The system was carefully characterized and the targeting efficiency was systematically evaluated. The EGF1-EGFP-NP could significantly facilitate specific uptake by TF overexpressed BCEC via EGF1/TF mediated endocytosis pathway. In addition, the pharmacokinetic study demonstrated that EGF1-EGFP-NP has the same blood circulation time as NP. Enhanced accumulation of EGF1-EGFP-NP in the cortex infarction region was also observed by real-time fluorescence image. Confocal microscopy and TEM further showed that EGF1-EGFP-NP combined with TF and further transfected through the damaged endothelium. Moreover, in vitro cell viability experiment and in vivo coagulation ability confirmed that the EGF1-EGFP-NP was safe. © 2012 Elsevier Ltd.

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