Inhibition of osteopontin reduce the cardiac myofibrosis in dilated cardiomyopathy via focal adhesion kinase mediated signaling pathway.

BACKGROUND: Osteopontin (OPN) is a pleiotropic cytokine, which has been shown to a close relationship with cardiac fibrosis. Overexpression of OPN in cardiomyocytes induces dilated cardiomyopathy (DCM). This research is to study whether inhibition of OPN could reduce myocardial remodelling in DCM, and if this process is focal adhesion kinase (FAK) dependent, which is recently found an important signal molecule in fibrosis. METHOD: Eight-week-old cTnTR141W transgenic mouse of DCM were injected with OPN-shRNA in left ventricular free wall, which could inhibit the OPN expression. Six weeks later, echocardiographic examinations were performed to test left ventricle function and heart tissues were harvested to test the quality of FAK by western blot and severity of fibrosis by masson staining. Human cardiac fibroblast was administrated with OPN, and FAK inhibition by PP2 was treated 2 h before OPN was given. Expression of α-SMA and collagen-I were tested by western blot and real-time PCR assay. RESULTS: OPN-shRNA group has a relatively high ejection fraction (EF), fractional shortening (FS), LV free wall thickness and a less sever cardiac fibrosis. In vitro, OPN could increase collagen-I and α-SMA expression, and this process can be inhibited by FAK inhibitor. CONCLUSION: Inhibition of OPN could reduce the LV remodeling and dysfunction in DCM mice, which may attribute to the suppression of collagen-I secretion in fibroblast through a FAK/Akt dependent pathway.

Pharmacological Inhibition of Focal Adhesion Kinase Attenuates Cardiac Fibrosis in Mice Cardiac Fibroblast and Post-Myocardial-Infarction Models.

BACKGROUND: To investigate the role of focal adhesion kinase (FAK)-mediated signaling in hypoxia-induced cardiac fibroblasts (CFs) differentiation and cardiac fibrosis post-myocardial infarction (MI) on a mice model. METHODS: CFs of neonatal C57BL/6 mice were treated under normoxic, hypoxic, or hypoxic+PP2 (known as a Src kinase family inhibitor) conditions. Gene expressions of FAK, alpha-smooth muscle actin (α-SMA) and collagen type I alpha 1 (Col1α1), or α-SMA and vimentin levels were performed by RT-PCR and immunofluorescence staining, respectively. Thirty mice were surgically treated into Sham (n=7) and MI (n=23) groups; and FAK inhibitor PF-562271 was given to six survivor MI mice (as PF group, from 15 survivors). Heart function and collagenous tissues were examined by echocardiography, as well as by Masson's trichrome and Sirius red staining, respectively. Type I collagen, FAK protein, mTOR, ERK1/2, AKT, P70S6K and phospho-FAK levels were also analyzed. RESULTS: FAK inhibition with PP2 significantly decreased CFs differentiation and collagen synthesis under hypoxia treatment. In vivo, PF-562271 treatment resulted in fibrosis attenuation; however, deteriorated heart function of MI mice could not be significantly improved. PF-562271 may affect phospho-mTOR (p<0.05), phospho-ERK1/2 (p<0.01), phospho-AKT (p<0.001) and phospho-P70S6K (p<0.05) to exert its benefits. FAK can be activated either under hypoxia in CFs or MI in a mouse model to promote fibrosis. However, pharmacological inhibition of FAK can attenuate fibrosis response. CONCLUSION: This study provides novel evidence that FAK inhibition may become a promising pharmaceutical strategy to attenuate fibrosis post-MI.

Co-application of ricin A chain and a recombinant adenovirus expressing ricin B chain as a novel approach for cancer therapy.

AIM: To develop a novel ricin-based approach for the safe and effective therapy of cancer. METHODS: The ricin A chain (RTA) was expressed in Escherichia coli in the form of a 6XHis-tagged fusion protein and purified with Ni(2+)-NTA affinity resin. A replication-deficient ricin B chain (RTB)-expression adenovirus green fluorescence protein (AdGFP-RTB) was constructed. RTA and AdGFP-RTB were tested for cytotoxicity either individually or in combination in human cell lines HEK293, HeLa, SMMC7721, and HL7702. Cell viability was determined with trypan blue staining or MTT assay. RESULTS: The expression and release of RTB, as well as the entry of RTA into AdGFP-RTB-infected cells were confirmed. When RTA and AdGFP-RTB was used individually, neither was toxic to the cells. When they were applied together, significant cell death was observed in all of the cell lines tested. The cell-killing effect correlated with the amount of RTA protein used, with cell mortality at about 60% at 4.8 mu g RTA in combination with AdGFP-RTB at 100 pfu/cell. No major cell killing was seen when RTA was used in combination with a control adenovirus AdGFP. The treatment of healthy HeLa cells with the virusfree supernatant from AdGFP-RTB/RTA-treated HeLa cells resulted in cell death, suggesting the formation of RTA/RTB complex, and a potential by-stander effect. CONCLUSION: The new approach was successful in vitro. Further modifications of the adenovirus vector, as well as an in vivo study are needed to confirm its potential in cancer therapy.

[Antitumor effect of recombinant T7 phage vaccine expressing xenogenic vascular endothelial growth factor on Lewis lung cancer in mice].

BACKGROUND & OBJECTIVE: Angiogenesis plays an important role in growth and metastasis of tumors. Vascular endothelial growth factor (VEGF) is considered as a fundamental regulator for angiogenesis. This study was to construct a recombinant T7 phage vaccine expressing xenogenic VEGF on the capsid, and test its inhibitory effect on Lewis lung cancer cells in mice. METHODS: VEGF gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR) from human lung cancer tissues, and inserted into phage using T7 Select10-3b kit to construct T7 Select10-3b_VEGF vaccine. The titer of prepared phage reached 1x10(13) pfu/ml. C57BL/6J mice were randomly divided into 3 groups: T7 Select10-3b_VEGF vaccine group (T7-VEGF), T7 phage (T7) group, normal saline (NS) group (10 mice/group). Each mouse was injected with Freundos adjuvant mixed with 1x10(12) pfu/200 microl T7 Select10-3b_VEGF, or T7, or normal saline once a week for 4 weeks. Lewis lung carcinoma model (LL/2) was established in C57BL/6J mice after 4-week immunization. Tumor growth and mouse's physical status were observed during immunization. Tumor weight and serum level of specific anti-VEGF antibody were measured by enzyme-linked immunosorbent assay (ELISA). Microvessel density (MVD) of tumors was detected by immunohistochemistry 14 days after the inoculation of tumor cells. RESULTS: Tumor weight of T7-VEGF vaccine group,T7 group, and NS group were (0.543+/-0.259)g, (0.982+/-0.359)g, (1.169+/-0.460)g, respectively. Tumor weight of T7-VEGF vaccine group was significantly lower than that of NS group (P<0.01). Serum anti-VEGF antibody level in T7-VEGF vaccine group was 1:1,000. MVD was significantly lower in T7-VEGF vaccine group than in NS group (8.5+/-0.8 vs 18.5+/-1.6, P<0.05). MVD in T7 group was 16.4+/-1.3. CONCLUSION: Recombinant T7 phage vaccine expressing xenogenic VEGF can break immunologic tolerance against self-VEGF and inhibit the growth of Lewis lung cancer cells.

[An anti-tumor research of recombinant phage vaccine expressed xenogenic EGFR].

A recombinant phage vaccine expressing EGFR on it's capsid was constructed and used to study the anti-tumor effect. The T7 phage display system was applied to display seven xenogenic (human, chicken) epidermal growth factor receptor extracellular domain fragments. The EGFR fragment was expressed as fused protein with 10B capsid on the surface of T7 phage. The T7-EGFR phage vaccines were injected into C57BL/6J mice, and then Lewis lung cancer cells were inoculated after 4 weeks immunization. The tumor tissue was excised and weighed after 10 days to evaluate the anti-tumor effect of each experimental group. The EGFR expression of the phage vaccine was verified by western-blot analysis. The A431 cells with high expressed EGFR was used to detect the anti-EGFR antibody by flow cytometry analysis. The results showed that the A431 cell can react with the serum obtained from the mice after three-week immunization. The experimental results confirmed that special EGFR antibody could be induced by the T7-EGFR phage vaccine. The T7-EGFR phage vaccine can elicit endogenous special EGFR antibody in mice and is capable of suppressing the tumor proliferation and retarding the growth of Lewis lung cancer. This research can be used to develop an anti-tumor vaccine for the target-therapy of EGFR(+) tumor.