Background Mesenchymal stem cells (MSCs) can improve limb perfusion and increase vessel density in a murine model of hindlimb ischemia. But low engraftment rate of those cells limited their therapeutic effect. Endothelial cells (ECs) play an important role in neovascularization. And MSCs can differentiate into ECs in vitro. The aim of this study is to investigate if EC differentiation of MSCs in vitro before transplantation is effective in improving therapeutic outcomes in the treatment of ischemic disease in a murine ischemia animal model. Methods MSCs were isolated from the bone marrow of EGFP-transgenic mice by density gradient centrifugation. The identity of the MSCs was determined by their cluster of differentiation (CD) marker profile by flow cytometry. Inducing medium containing a few cytokines was applied to induce the MSCs to differentiate into ECs. Endothelial differentiation was quantitatively evaluated using flow cytometry, quantitative real-time PCR (qRT-PCR), immunofluorescence, Matrigel tube formation assay, and Dil-labeled acetylated low-density lipoprotein uptake assay. Mouse hindlimb ischemia model was made by excision of the femoral artery. Uninduced EGFP+ MSCs, induced EGFP+ MSCs, and PBS were intramuscularly injected into the gastrocnemius following ischemia no later than 24 h after operation. Restoration of blood flow and muscle function was evaluated by laser Doppler perfusion imaging. Immunofluorescence was conducted to evaluate the engraftment of transplanted MSCs. Histological analysis was performed to evaluate blood vessel formation. Results Induced EGFP+ MSCs expressed endothelial markers and exhibited tube formation capacity. Mice in the induced EGFP+ MSCs group had a better blood perfusion recovery, enhanced vessel densities, higher engraftment, and improved function of the ischemic limb than those in the uninduced EGFP+ MSCs or PBS groups. Conclusions This study reveals that after short-term pre-treatment in the EC-inducing medium, induced MSCs acquire stronger vessel formation capability and enhanced angiogenic therapeutic effect in the murine hindlimb ischemia model.

Objective: The aim was to identify the change in gene expression between mesenchymal stem cells (MSCs) and induced endothelial cells (ECs) and to investigate the potential mechanism of endothelial differentiation based on ribonucleic acid sequencing (RNA-Seq) analysis. Methods: MSCs were isolated from bone marrow and exposed to inducing medium. The dynamic transcription profiles of MSCs were identified and ECs were induced through RNA-seq. Differentially expressed genes (DEGs) were identified. Enrichment of functions and signalling pathways analysis were performed based on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Quantitative real time polymerase chain reaction (qRT-PCR) was used to validate the genes selected from RNA-Seq. Results: In total, 2769 DEGs were identified, of which 1117 genes were upregulated and 1652 genes were downregulated. GO and KEGG pathway analyses identified significantly enriched pathways in DEGs, including extracellular matrix organisation, blood vessel morphogenesis, angiogenesis, extracellular matrix binding, growth factor binding and glycosaminoglycan binding extracellular matrixereceptor interaction pathway, cytokineereceptor interaction pathway and transforming growth factor (TGF)-beta signalling pathway. All genes found to be associated with the TGF-beta pathway were significantly downregulated. Eleven novel genes were also identified that most likely are involved in endothelial differentiation and were upregulated with more than 10 fold change, which were further validated by qRT-PCR. Conclusion: The GO and KEGG analysis revealed that extracellular matrix, cytokines and the TGF-beta pathway play an important role in the process of endothelial differentiation. Furthermore, 11 genes were found that may be involved in the differentiation of MSCs into ECs and contribute to current understanding of the differentiation mechanism.

目的 采用Meta分析评价骨髓间充质干细胞(BMSCs)治疗下肢缺血性疾病的疗效.方法 对PubMed、EMbase及Cochrane Library数据库进行检索,收集与BMSCs治疗下肢缺血性疾病相关的临床对照研究,按照制定的纳入和排除标准进行文献筛选、提取资料.以RevMan 5.3软件对纳入文献进行Meta分析,比较BMSCs治疗组与对照组相关指标,包括截肢率、无截肢生存率、踝肱指数、溃疡愈合率、疼痛评分以及无痛行走距离.结果 最终纳入5篇文献.Meta分析结果显示,与对照组比较,BMSCs组患者踝肱指数[均数差(MD) =0.15,95 ％CI(0.12,0.18),P＜0.000 01]、疼痛评分[MD=-1.38,95％CI(-1.65,-1.11),P＜0.000 01]、无痛行走距离[MD=202.20,95％CI(154.30,250.10),P＜0.000 01]及溃疡愈合率[相对危险度(RR)=1.42,95％CI(0.82,2.46),P=0.021]均明显改善;但两组患者截肢率RR=0.52,95％CI(0.24,1.10),P=0.09]、无截肢生存率[RR=1.09,95％CI(0.98,1.21),P=0.12]差异均无统计学意义.结论 BMSCs治疗下肢缺血性疾病虽不能显著降低截肢率和提高无截肢生存率,但可以改善患者的临床症状.

Objective To evaluate the efficacy and safety of super-selective renal arterial embolization (SRAE) for treatment of huge renal angiomyolipoma (RAML).Methods Data of 16 patients with huge RAML treated with SRAE were retrospectively analyzed.The clinical symptoms,tumor size,serum creatinine and complications were compared before and after SRAE.Results A total of 26 SRAE treatments were performed on 16 patients with huge RAML.The technical success rate of SRAE was 100％ (26/26).Seven cases (7/16,43.75 ％) received one SRAE treatment,whereas 8 (8/16,50.00％) required two SRAE treatments.Only one case (1/16,6.25％) received three SRAE treatments.The mean follow-up period was (16.60± 15.60) months.The maximum diameter of the tumor reduced significantly after SRAE at final follow-up than before embolization ([9.00±2.80]cm vs [12.60±2.40]cm,t=12.41,P＜0.01).The symptoms of flank pain and hematuria gradually relieved after SRAE.And there was no statistical difference of mean serum creatinine before and after SRAE ([76.00±14.90]μmol/L] vs [79.10±12.80]μmol/L,t=0.89,P=0.39).Fourteen cases (14/ 16,87.50％) experienced post-embolization syndrome including varying degrees of fever,local pain or nausea on 1-3 days after embolization.No serious complications occurred.Conclusion SRAE is an effective method for stopping bleeding of ruptured huge RAML,as well as relieving symptoms and reserving nephron.

目的 探讨无血清内皮细胞生长培养基套装(EGM-2 BulletKit)对骨髓间充质干细胞(MSC)向血管内皮细胞分化的诱导作用.方法 通过贴壁培养获取MSC,用流式细胞仪检测MSC表面标志物表达.将获得的MSC随机分为对照组与诱导组.对照组用无血清EGM-2基础培养基进行培养,诱导组选用无血清EGM-2基础培养基+BulletKit进行诱导.于诱导第2、6、10、14天用流式细胞仪、细胞免疫荧光法检测内皮细胞表面分子CD31表达.采用DiI标记的乙酰化低密度脂蛋白吞噬实验检测MSC诱导分化后的吞噬功能,用基底膜基质胶成管实验检测细胞体外成管功能.结果 贴壁培养获得的MSC呈长梭形,流式细胞仪检测结果显示MSC标志物表达阳性的比例均大于95％,而内皮细胞标志物无表达.诱导组诱导第2天CD31开始表达,随诱导时间增加,CD31表达量逐渐升高,诱导第14天CD31表达量最高,CD31表达阳性的内皮细胞比例为55.8％;对照组无CD31表达.诱导第14天,诱导组能够摄取脂质,而对照组无摄取现象.诱导组细胞能够形成典型的管状结构;对照组细胞未形成典型的线状、管状结构.结论 无血清EGM-2Bullet Kit可诱导MSC分化为血管内皮细胞,且具有内皮细胞的特性、功能.

To evaluate the efficacy of prophylactic selective arterial embolization (SAE) of angiomyolipomas (AMLs) and to find out predictive factors of significant shrinkage of AMLs after SAE.

The purpose of this study was to evaluate the efficacy and safety of transarterial embolization (TAE) for iatrogenic renal arterial pseudoaneurysm and arteriovenous fistula at our center. Our retrospective analysis included 27 patients who received TAE for iatrogenic renal arterial pseudoaneurysm and arteriovenous fistula between January 2006 and January 2016. Data on demographics, type of minimally invasive renal procedures, clinical manifestation, imaging features, embolization procedure, and perioperative details were collected. The technical and clinical success rates were analyzed. Furthermore, the changes in serum creatinine and eGFR before and after embolization were recorded and compared by t test. The median time between iatrogenic renal injury and TAE was 3 days (range, 0-110 days), with most patients (24/27, 88.9%) receiving TAE within 14 days. Only 1 patient was diagnosed with renal artery pseudoaneurysm 110 days after laproscopic partial nephrectomy. The technical and clinical success rates were 100% and 96.3%, respectively, with 1 patient requiring a second embolotherapy at the third postoperative day. No other patient required additional endovascular or surgical intervention due to recurrent hemorrhage. The mean serum creatinine before TAE was 92.8 +/- 25.3 mu mol/L and after TAE, 96.1 +/- 27.7 mu mol/L (P=.095). The eGFR of pre-and postembolization was 75.2 +/- 26.5 mL/min/1.73m(2) and 72.5 +/- 26.2mL/min/1.73m(2) (P=.16). No severe complications were observed during follow-up. This retrospective review demonstrated that TAE for the treatment of iatrogenic renal artery pseudoaneurysm and/or arteriovenous fistula was safe and associated with high technical and clinical success rate.

Objective To evaluate the efficacy and safety of super-selective renal artery embolization in treatment of iatrogenie renal pseudoaneurysm and arteriovenous fistula.Methods Twenty-nine patients with iatrogenic renal pseudoaneurysm and arteriovenous fistula underwent renal angiography after ineffective conservative treatment.After identifying the location and characteristic of lesions by DSA,super-selective renal artery embolization was performed.Symptoms and signs,renal function changes before and after embolization and complications were recorded to evaluate the therapeutic effect.Results The technical success rate of super-selective renal artery embolization was 100％ (29/29),and clinical success rate was 96.55 ％ (28/29).The serum creatinine level before and after embolotherapy was (93.26 ± 28.79) mmol/L and (91.51 ± 27.68) mmol/L respectively,and there were no significant differences (t=1.28,P=0.22).No serious complications such as nephrapostasis or renal failure occurred in the study.Conclusion Super-selective renal artery embolization has technically and clinically high success rate with limited effect on renal function.It is a safe and effective therapy method in patients with iatrogenic renal pseudoaneurysm and arteriovenous fistula.

Objective: Endothelial cells (ECs) play an important role in neovascularisation, but are too limited in number for adequate therapeutic applications. Mesenchymal stem cells (MSCs) have the potential to differentiate into endothelial lineage cells, which makes them attractive candidates for therapeutic angiogenesis. The aim of this study was to investigate efficient differentiation of MSCs into ECs by inducing medium in vitro. Methods: MSCs were isolated from bone marrow by density gradient centrifugation. The characterisation of the MSCs was determined by their cluster of differentiation (CD) marker profile. Inducing medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin like growth factor (IGF), epidermal growth factor (EGF), ascorbic acid, and heparin was applied to differentiate the MSCs into ECs. Endothelial differentiation was quantitatively evaluated using flow cytometry. Real time quantitative PCR (qRT-PCR) was used to analyse mRNA expression of endothelial markers. Tube formation assay was further performed to examine the functional status of the differentiated MSCs. Results: Flow cytometry analysis demonstrated that CD31(+) and CD34(+) cells increased steadily from 12% at 3 days, to 40% at 7 days, and to 60% at 14 days. Immunofluorescence staining further confirmed the expression of CD31 and CD34. qRT-PCR showed that expression of von Willebrand factor (vWF), vascular endothelial cadherin (VE-cadherin) and vascular endothelial growth factor receptor-2 (VEGFR-2) were significantly higher in the induced MSCs group compared with the uninduced MSCs group. The functional behavior of the differentiated cells was tested by tube formation assay in vitro on matrigel. Induced MSCs were capable of developing capillary networks, and progressive formation of vessel like structures was associated with increased EC population. Conclusions: These results provide a method to efficiently promote differentiation of MSCs into ECs in vitro for potential application in the treatment of peripheral arterial disease. (C) 2017 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.

Objective To evaluate the long-term efficacy and safety of selective arterial embolization (SAE) in the treatment of renal angiomyolipomas (AMLs). Methods This was a retrospective review of medical records and imaging findings from patients with renal AMLs who attended our clinic and received SAE between January 2007 and January 2014. Only patents with complete medical records, preoperative computed tomography scans using typical imaging and follow-up data were included. Results A total of 79 patients were enrolled in the study. Technical and clinical success rates were 100% and 91% ( n = 72), respectively. Only two patients experienced major complications. Post-embolization syndrome (i.e. fever, abdominal pain, nausea or vomiting) was reported in 68 (86%) patients, but all symptoms were mild and resolved with conservative measures. Mean radiological and clinical follow-up periods were 16.8 and 35.9 months, respectively. In 75 (95%) patients, tumours decreased in size; mean ± SD tumour size significantly decreased from 8.4 ± 3.5 cm pre-embolization to 6.7 ± 3.0 cm post-embolization . Conclusions This study provides long-term evidence that SAE is a safe and effective method in the treatment of patients with renal AMLs.