Pharmacological Regulation of In Situ Tissue Stem Cells Differentiation for Soft Tissue Calcification Treatment.

Calcification of soft tissues, such as heart valves and tendons, is a common clinical problem with limited therapeutics. Tissue specific stem/progenitor cells proliferate to repopulate injured tissues. But some of them become divergent to the direction of ossification in the local pathological microenvironment, thereby representing a cellular target for pharmacological approach. We observed that HIF-2alpha (encoded by EPAS1 inclined form) signaling is markedly activated within stem/progenitor cells recruited at calcified sites of diseased human tendons and heart valves. Proinflammatory microenvironment, rather than hypoxia, is correlated with HIF-2alpha activation and promoted osteochondrogenic differentiation of tendon stem/progenitor cells (TSPCs). Abnormal upregulation of HIF-2alpha served as a key switch to direct TSPCs differentiation into osteochondral-lineage rather than teno-lineage. Notably, Scleraxis (Scx), an essential tendon specific transcription factor, was suppressed on constitutive activation of HIF-2alpha and mediated the effect of HIF-2alpha on TSPCs fate decision. Moreover, pharmacological inhibition of HIF-2alpha with digoxin, which is a widely utilized drug, can efficiently inhibit calcification and enhance tenogenesis in vitro and in the Achilles's tendinopathy model. Taken together, these findings reveal the significant role of the tissue stem/progenitor cells fate decision and suggest that pharmacological regulation of HIF-2alpha function is a promising approach for soft tissue calcification treatment.

[Magnetic liposome mediated shRNA specifically suppresses the growth of non-small cell lung cancer in vitro and in vivo].

OBJECTIVE: To evaluate the inhibition of shRNA mediated by magnetic liposome in the growth of non-small cell lung cancer (NSCLC) under the interference of magnetic field in vitro and in vivo and explore the effects of magnetic field on the efficiency of magnetofection. METHODS: The plasmid of pGFPshIGF-1R was constructed for expressing GFP and shRNA against IGF-1R. CombiMAG as superparamagnetic iron oxide nanoparticles (SPIONs) and Lipofectamine2000 as cationic liposome comprised the magnetic liposome. pGFPshIGF-1R was transferred into A549 cells by magnetofection under a series of interaction durations and intensity of external magnetic fields. pGFPshIGF-1R was delivered into A549 cells in vitro and injected intravenously into the tumor-bearing mice every 48 h for four doses in vivo by way of lipofection or magnetofection. The magnetofection efficiency was analyzed by cytometry and the potency of IGF-1R knockdown by Western blot. At Week 3 after the 4th injection, the mice were sacrificed and the tumors removed and weighed. The tumor inhibition rate was calculated. RESULTS: The interaction durations and intensity of magnetic field could influence the magnetofection efficiency. In vitro, IGF-1R specific-shRNA transfected by lipofection inhibited IGF-1R protein by 56.1% ± 6.0% and by liposomal magnetofection by 85.1% ± 3.0%. In vivo, pGFPshIGF-1R delivered by both lipofection and magnetofection significantly inhibited the tumor growth by 41.3% (P < 0.01) and 65.2% (P < 0.01). CONCLUSIONS: Based on magnetic liposome as gene vectors, magnetofection may become a promising targeted therapy for lung cancer. And the transfection efficiency is influenced by magnetic field.

[One-stop hybrid cardiac surgery for adults with complex heart disease].

OBJECTIVE: To evaluate the efficacy and security of one-stop hybrid cardiac surgery for the treatment of adult patients with complex heart disease. METHODS: From November 2011 to March 2012, a total of 5 patients [4 male, mean age: (58.8 ± 14.7) years] underwent one-stop hybrid approach in the hybrid operating room. Two patients suffered from multi-coronary lesions, 2 patients were diagnosed with both valvular heart disease and coronary disease, and another 1 patient had valve disease and congenital heart disease (patent ductus arteriosus). Minimally invasive cardiac surgery (coronary artery bypass grafting for the left anterior descending or valvular surgery) and percutaneous intervention were performed in an enhanced operative unit. The efficacy and security of one-stop hybrid cardiac surgery were evaluated after the procedure. RESULTS: The one-stop hybrid procedure was successful in all patients. Left internal mammary artery grafts were unobstructed. A total of 6 non-left anterior descending coronary lesions were treated by percutaneous coronary intervention and 6 drug-eluting stents were implanted. There was no death, perioperative myocardial infarction, heart failure, prosthetic valve dysfunction, respiratory failure, stroke or repeat surgery during the procedure period. All patients remained free from angina, prosthetic valve dysfunction and patent ductus arteriosus recanalisation during the 3.2 months (rang 1 to 5 months) follow-up period. CONCLUSION: One-stop hybrid cardiac surgery provides a reasonable, feasible and safe alternative for treating adult patients with complex heart disease.

The first four cases of successful NeoChord procedure in mainland China.

BACKGROUND: Transapical off-pump NeoChord procedure is a novel minimally invasive surgical repair of degenerative mitral regurgitation (MR). Here, we report the first four cases of NeoChord procedure in patients with mitral valve prolapse in mainland China. METHODS: Four patients, aged 86, 84, 80 and 60 years, with severe MR due to posterior middle scallop prolapse (P2), underwent transapical off-pump artificial chordae implantation on April 9 and 10, 2019. The procedure was performed by left mini-thoracotomy under general anaesthesia and guided by 2D and 3D dimensional transoesophageal echocardiography (TEE). RESULTS: Mitral valve repair via NeoChord procedure was successfully performed with implantation of 3 artificial chordae in the first patient and 3, 2, and 3 artificial chordae in the following patients, respectively. Intraoperative TEE and pre-discharge transthoracic echocardiography (TTE) showed only mild to moderate MR of these four patients and no postoperative complications were noted. There were no changes of TTE finding between one-month follow-up and pre-discharge. CONCLUSION: The successful NeoChord procedures in four Chinese indicate that the valve repair using the NeoChord system for Chinese population is feasible.

[Biological behaviors and chemosensitivity of NSCLC A549 cells after IGF-IR gene silencing by targeting RNAi in vitro].

OBJECTIVE: To investigate the biological behaviors and chemosensitivity of non-small cell lung cancer (NSCLC) cell line A549 after IGF-IR gene silencing by RNA interference (RNAi) in vitro. METHODS: Two plasmids siRNA 1 and 2 expressing IGF-IR siRNA with human U6 promoter were constructed,and an unrelated siRNA was used as negative control. NSCLC A549 cells were transfected with sequence-specific siRNA or unrelated siRNA as control. Quantitative RT-PCR and Western blot were used to detect the expression of IGF-IR. NSCLC A549 cells were transfected with siRNA and treated with DDP. MTT assay and flow cytometry were used to assess the effects of IGF-IR silencing on tumor cell proliferation and chemosensitivity. RESULT: Transfection of NSCLC cells with siRNA resulted in reduction of IGF-IR mRNA expression by 78.9 % and protein production by 89.8%. The decrease in IGF-IR levels caused significant growth inhibition of A549 cells both at 48 h and at 72 h, and decrease of the IC50 of DDP at 24 h, 48 h and at 72 h. Flow cytometry showed that 77.5% of A549 cells retained in G0/G1 phase. CONCLUSION: The sequence specific suppression of IGF-IR gene expression by RNAi enhances sensitivity to DDP in NSCLC cell.

First-in-man implantation of the retrievable and repositionable VenusA-Plus valve.

BACKGROUND: No retrievable and repositionable second generation transcatheter aortic valve is available in China. Here, we report the first-in-man implantation of the retrievable and repositionable VenusA-Plus valve. METHODS: A 76-year-old patient with symptomatic severe aortic stenosis and high surgical risk (STS 13.8%) was recommended for transcatheter aortic valve replacement (TAVR) by heart valve team. Type 0 bicuspid aortic valve with asymmetric calcification was identified by dual source computed tomography, and the unfavorable anatomies increased the possibility of malposition and paravalvular leakage during TAVR. Therefore, we used the retrievable and repositionable VenusA-Plus valve for the patient. RESULTS: Transfemoral TAVR was performed under local anesthesia with sedation, and a 26mm VenusA-Plus valve was successfully implanted. No transvalvular pressure gradient and trace paravalvular leakage were found. CONCLUSION: The successful first-in-man implantation indicates the retrievable and repositionable VenusA-Plus valve is feasible in complicated TAVR cases such as bicuspid aortic valve.

[shRNA-mediated insulin-like growth factor I receptor gene silencing inhibits cell proliferation, induces cell apoptosis, and suppresses tumor growth in non-small cell lung cancer: in vitro and in vivo experiments].

OBJECTIVE: To study the effects of RNA interference (RNAi)-mediated insulin-like growth factor I receptor (IGF-IR) gene silencing on human lung cancer cells. METHODS: Plasmids expressing IGF-IR shRNA1 and IGF-IR shRNA2 were constructed. Human non-small cell lung cancer cells of the line A549 were cultured and transfected with sequence-specific shRNA. RT-PCR was used to monitor the IGF-IR mRNA expression. Western blotting was used to detect the expression of IGF-IR, bcl-2 and caspase-3, associated with apoptosis, and IGF-IR signaling pathways-associated proteins, total and phospho-ERK1/2 and Akt. MTT assay and flow cytometry were used to examine the cell activity and cell cycle. Twelve nude mice were injected subcutaneously with A549 cells, 20 days later the mice were randomly divided into 3 groups to be injected into the tumor with IGF-IR, PBS, or blank plasmid respectively 4 times with the interval of 5 days. Five days after the 4th injection the mice were killed and the tumors were taken out. TUNNEL assay was used to detect the apoptotic cell in the tumor. RESULTS: RT-PCR showed that the IGF-IR mRNA expression level of the A549 cells transfected with IGF-IR shRNA1 was only 24% +/- 4% that of the A549 cells transfected with blank plasmid (P < 0.05); however, the IGF-IR mRNA expression level of the A549 cells transfected with IGF-IR shRNA2 was 78% +/- 5% that of the A549 cells transfected with blank plasmid (P > 0.05). The IGF-IR protein expression level of the A549 cells of the IGF-IR shRNA1 group was only 10.2% +/- 2.8% that of the A549 cells of the blank plasmid group (P < 0.05). Western blotting showed that the protein expression levels of bcl-2 and caspase-3p20 of the A549 cells of the IGF-IR shRNA1 group were 46% +/- 6% and 156% +/- 8% those of the negative controls (both P < 0.05); however, the protein expression levels of bcl-2 and caspase-3p20 of the A549 cells of the IGF-IR shRNA2 group were not different from those of the negative control cells. The Akt kinase and ERK phosphorylation levels of the A549 cells of the IGF-IR shRNA1 group were 10% and 36% +/- 3% those of the negative control cells respectively (both P < 0.05). Since 48h after the transfection the active cell number of the IGF-IR shRNA1 group was 64% +/- 7% that of the negative group (P < 0.05), and this decrease effect lasted to 72 h after (67% +/- 6% that of the negative cells, P < 0.05). 48 h after the transfection the percentage of cells at G(0)/G(1) phase of the IGF-IR shRNA1 group was 77.5%, significantly higher than that of the negative control group, and the percentages of the cells at S and G(2)/M phases of the IGF-IR shRNA1 group were 15.7% and 7.3% respectively, both significantly lower than those of the negative control group (23.0% and 29.9% respectively). Since the second injection the tumor size of the mice of IGF-IR shRNA group was 40% - 50% that of the PBS group (P < 0.05), and the tumor size of the mice of the PBS group was 90% that of the control group. TUNNEL assay showed that the number of apoptotic cells in the tumors of the IGF-IR shRNA1 group mice was 118 +/- 8/high power, significantly higher than that of the control group (70 +/- 9, P < 0.05). CONCLUSION: RNAi technique effectively inhibits the expression of IGF-IR, thus decreasing the NSCLC cell proliferation inducing apoptosis and inhibiting the tumor growth.

Protective function of tocilizumab in human cardiac myocytes ischemia reperfusion injury.

OBJECTIVE: To investigate the protective function of tocilizumab in human cardiac myocytes ischemia-reperfusion injury. METHODS: The human cardiac myocytes were treated by tocilizumab with different concentrations(1.0 mg/mL, 3.0 mg/mL, 5.0 mg/mL) for 24 h, then cells were cultured in ischemia environment for 24 h and reperfusion environment for 1 h. The MTT and flow cytometry were used to detect the proliferation and apoptosis of human cardiac myocytes, respectively. The mRNA and protein expressions of Bcl-2 and Bax were measured by qRT-PCR and western blot, respectively. RESULTS: Compared to the negative group, pretreated by tocilizumab could significantly enhance the proliferation viability and suppress apoptosis of human cardiac myocytes after suffering ischemia reperfusion injury (P<0.05). The expression of Bcl-2 in tocilizumab treated group were higher than NC group (P<0.05), while the Bax expression were lower (P<0.05). CONCLUSIONS: Tocilizumab could significantly inhibit apoptosis and keep the proliferation viability of human cardiac myocytes after suffering ischemia reperfusion injury. Tocilizumab may obtain a widely application in the protection of ischemia reperfusion injury.

Anterolateral minithoracotomy versus median sternotomy for mitral valve disease: a meta-analysis.

OBJECTIVE: Mitral valve disease tends to be treated with anterolateral minithoracotomy (ALMT) rather than median sternotomy (MS), as ALMT uses progressively smaller incisions to promote better cosmetic outcomes. This meta-analysis quantifies the effects of ALMT on surgical parameters and post-operative outcomes compared with MS. METHODS: One randomized controlled study and four case-control studies, published in English from January 1996 to January 2013, were identified and evaluated. RESULTS: ALMT showed a significantly longer cardiopulmonary bypass time (P=0.001) and aortic cross-clamp time (P=0.05) compared with MS. However, the benefits of ALMT were evident as demonstrated by a shorter length of hospital stay (P<0.00001). According to operative complications, the onset of new arrhythmias following ALMT decreased significantly as compared with MS (P=0.05); however, the incidence of peri-operative mortality (P=0.62), re-operation for bleeding (P=0.37), neurologic events (P=0.77), myocardial infarction (P=0.84), gastrointestinal complications (P=0.89), and renal insufficiency (P=0.67) were similar to these of MS. Long-term follow-up data were also examined, and revealed equivalent survival and freedom from mitral valve events. CONCLUSIONS: Current clinical data suggest that ALMT is a safe and effective alternative to the conventional approach and is associated with better short-term outcomes and a trend towards longer survival.

Down-regulation of IGF-IR using small, interfering, hairpin RNA (siRNA) inhibits growth of human lung cancer cell line A549 in vitro and in nude mice.

Type I insulin-like growth factor receptor (IGF-IR), which is frequently overexpressed in a variety of human cancers including lung cancer, mediates cancer cell proliferation and tumor growth. In this study, we used a human U6 promoter-driven DNA-template approach to induce hairpin RNA (hpRNA)-triggered RNAi to silence IGF-IR gene expression in the human lung cancer cell line A549, and then evaluate its effects on apoptosis, apoptosis-related gene expression, and the growth of tumor cells in vitro and in nude mice. IGF-IR expression levels were found to markedly decrease in cells transfected with a plasmid expressing hairpin siRNA for IGF-IR (by more than 78.9%). Down-regulation of IGR-IR concomitantly accompanied reduction of bcl-2 as well as pERK and pAkt levels, activation of caspase-3, apoptosis and growth inhibition of A549 cells in vitro. Direct intratumoral injections of plasmid DNA expressing hpRNA for IGF-IR significantly regressed pre-established tumors in nude mice. Our results support the therapeutic potential of RNAi as a method for gene therapy in treating lung cancer.