Methods to prepare pure, bioactive recombinant human vascular endothelial growth inhibitor (rhVEGI), a potent inhibitor of angiogenesis potentially applicable in antiangiogenic cancer therapy, are in urgent demand for preclinical investigation as well as future clinical trials of the protein. Here, we report expression and purification of rhVEGI-192, a recombinant VEGI isoform, comparatively using host strains BL21 (DE3) pLysS and Origami B (DE3) with IPTG-induction and autoinduction techniques. Our study identified that a combined use of Origami B (DE3) strain and autoinduction expression system gave rise to a high yield of purified rhVEGI-192 at 105.38 mg/L culture by immobilized-metal affinity chromatography on Ni-NTA column. The antiangiogenic activity was effectively restored after the insoluble fractions being dissolved in 8M urea and subsequently subjected to a gradient-dialysis refolding process. Functional tests demonstrated that the purified rhVEGI-192 potently inhibited endothelial growth, induced endothelial apoptosis and suppressed neovascularization in chicken chorioallantoic membrane, indicating that the developed method allows preparation of rhVEGI-192 with high yield, solubility, and bioactivity. Most importantly, our study also demonstrates that VEGI-192 is capable of forming polymeric structure, which is possibly required for its antiangiogenic activity.

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Expanded polytetrafluoroethylene (ePTFE) polymers do not support endothelialization because of nonconductive characteristics towards cellular attachment. Inner surface modification of the grafts can improve endothelialization and increase the long-term patency rate of the ePTFE vascular grafts. Here we reported a method of inner-surface modification of ePTFE vascular graft with extracellular matrix (ECM) and CD34 monoclonal antibodies (CD34 mAb) to stimulate the adhesion and proliferation of circulating endothelial progenitor cells on ePTFE graft to enhance graft endothelialization. The inner surface of ECM-coated ePTFE grafts were linked with CD34 mAb in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide (EDC/NHS) solution and the physicochemical properties, surface morphology, biocompatibility, and hemocompatibility of the grafts were studied. The hydrophilicity of CD34 mAb-coated graft inner surface was significantly improved. Fourier transform infrared spectroscopy analysis confirmed ECM and CD34 mAb cross-linking in the ePTFE vascular grafts with our method. Scanning electron microscopy analysis showed protein layer covering uniformly on the inner surface of the modified grafts. The cell-counting kit-8 (CCK-8) assay confirmed that the modified graft has no obvious cytotoxicity. The modified graft showed a low hemolytic rate (0.9%) in the direct contact hemolysis test, suggesting the modification improved hemocompatibility of biopolymers. The modification also decreased adhesion of platelets, while significantly increased the adhesion of endothelial cells on the grafts. We conclude that our method enables ePTFE polymers modification with ECM and CD34 mAb, facilitates endothelialization, and inhibits platelet adhesion on the grafts, thus may increase the long-term patency rate of the prosthetic bypass grafts. Keywords ePTFE vascular grafts Á Anti-CD34 antibody Á Extracellular matrix Á EDC/NHS solution Á Endothelial cells Lei Chen and Haipeng He are co-first authors.

PurposeThe purpose of this study is to identify a prospective association between CA125 and tumorigenic ovarian cancer cells, using the new method of orthotopic transplantation (1).MethodAfter making the surgical ovarian cancer specimen into cell suspension, we separated the tumorigenic cells from the nontumorigenic cancer cells based on cell surface marker (cancer antigen CA125 and lineage markers) expression. We developed a SCID mice model in which the CA125+/ lineage- and CA125-/ lineage- cells were injected into ovarian parenchyma by use of a microinjector. As a measure of effectiveness of tumor-forming, tumor weight, abdominal distension, ascites volume and activity, subcutaneous fat were determined or observed. Immunohistochemistry was done to determine tumor cell markers.ResultsWe found that the cells of CA125+/ lineage- were able to form new tumors; whereas, an equal quantity of CA125-/lineage- cells failed to form any tumors. The new generated tumor contained additional CA125-/lineage- tumorigenic cells as well as the phenotypically diverse population of nontumorigenic cells. Quantities were judged to be significantly different P < 0.0001.ConclusionCA125+/ lineage- cells, which may be ovarian cancer stem cells, were the source for tumor recurrence. The strategies designed to target this cell population may lead to more effective therapies.

WHAT THIS PAPER ADDS Plaque calcification and stent oversizing have been confirmed as two key factors for in stent restenosis in atherosclerotic peripheral artery occlusive disease. An effective plaque scoring system and specific stent oversizing standard have been well established in the coronary artery. However, there are still no similar assessment methods for the femoropopliteal artery. In this study, automatic computer software was used to analyse the pre-operative computed tomography angiography (CTA) images of patients receiving femoropopliteal artery stenting for atherosclerotic occlusive disease. Plaque calcification and stent oversizing were assessed by several quantitative parameters. Stent oversizing and plaque calcification were linked with adverse clinical outcomes. The results suggest potential cut off values of plaque calcification and stent oversizing, which may be useful for clinical intervention, and also imply the feasibility of quantitatively assessing plaques in the femoropopliteal artery on pre-operative CTA. Objective: Plaque calcification and stent oversizing are two key factors contributing to in stent restenosis (ISR) following femoropopliteal stent angioplasty. This study aimed to explore a pre-operative quantitative assessment method of plaque calcification and rational parameters of stent oversizing in the femoropopliteal artery. Methods: A total of 115 patients with atherosclerotic femoropopliteal arterial occlusive disease treated from January 2013 to January 2016 were included retrospectively. Computed tomography angiography (CTA) imaging was performed to analyse calcified plaque parameters (calcified plaque volume [CV], standard CV [SCV], burden of calcified plaque) and stent oversizing parameters at different vessel segments (distal oversizing, maximum oversizing, plaque oversizing). Optimal cut offs for the six parameters were determined by the maximum Youden's index. The relationship between calcified plaque, stent oversizing, and clinical outcomes were assessed by correlation analysis and multivariable Cox regression models. Results: The one year primary patency rate was 77.4%; the rates of ISR, major amputation, target lesion revascularisation, and mortality were 40.9%, 8.7%, 17.4%, and 12.2%, respectively. For all six parameters, patients with values greater than the cut offs had a significantly higher incidence of ISR than those with values below the cut offs. ISR was positively correlated with all six calcification and oversizing parameters. Amputation and mortality were positively correlated with calcification parameters. Multivariable Cox regression analysis demonstrated that all six parameters were independent risk factors for ISR. All calcification parameters were identified as independent risk factors for amputation, while only CV and SCV were independent risk factors for mortality. Conclusion: Calcified plaque in the femoropopliteal artery can be quantitatively analysed on pre-operative CTA images. High calcified plaque burden and excessive stent oversizing were ass...