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Methods 8-week-age male ApoE −/− mice were fed with the atherogenic diet together with or without tested compounds (rosuvastatin calcium, α-LNA-LMWCS, LMWCS and α-LNA) for 16 weeks. When the animals were killed, blood plasma was isolated to test the level of TC, LDL-C, TNF-α, IL-6 and CRP by biochemistry analysis and ELISA method. The whole aorta and aortic root sections were also collected to study atherogenesis level and reveal the possible mechanism by histological examination, real-time PCR and Western blot analysis. Results The level of TC, LDL-C, TNF-α, IL-6 and CRP in plasma in H-LNA-LMWCS group were significantly lower than those of the control group (rosuvastatin calcium). Plaques in H-LNA-LMWCS group showed higher content of smooth muscle cells, lower content of lipid and macrophages, and lower mRNA levels of TNF-α, IL-6, CRP, MCP-1, VCAM-1 and ICAM-1 than those in the control group. In addition, α-LNA-LMWCS could reduce the nuclear translocation of NF-κB, inhibit expressions of p-ERK1/2, p-p38, MCP-1, VCAM-1 and ICAM-1 in mice aorta. Conclusion α-LNA-LMWCS exhibited anti-atherosclerosis effect through regulating the lipid metabolism and diminishing the synthesis of pro-inflammatory cytokines. The possible mechanism may be that α-LNA-LMWCS could influence MAPK/ NF-κB related signal pathway. General Significance The results may provide significant suggestions for the application of α-LNA-LMWCS in anti-atherosclerosis. [ABSTRACT FROM AUTHOR]

Background Diabetic retinopathy is a leading cause of blindness. The objective was to design a novel fusion protein, Tat PTD-Endostatin-RGD, to treat retinal neovascularization via eye drops instead of traditional intravitreal injection trepapeutical methods. Method The anti-angiogenesis ability was evaluated in vitro by chick embryo chorioallantoic membrane assay, wound healing assay and tube formation assay. Corneal barrier and blood-retina barrier were constructed in vitro to investigate the penetration ability of Tat PTD-Endostatin-RGD. Western blot was used to detect the integrin α v β 3 expression level in rat retina microvascular endothelial cells which was stimulated by S -nitroso- N -acetylpenicillamine. The binding affinity of Tat PTD-Endostatin-RGD to integrin α v β 3 was investigated by evaluating the penetration ability on blood-retina barriers treated with S -nitroso- N -acetylpenicillamine. The pharmacodynamics and efficacy analysis were further carried out in the oxygen-induced retinopathy model in vivo . In addition, the pharmacokinetic profile via eye drops was studied on a C57BL/6 mice model. Result Tat PTD-Endostatin-RGD showed high anti-angiogenesis activity and high ability to penetrate these two barriers in vitro . The Western blot results indicated S -nitroso- N -acetylpenicillamine upregulated the expression level of integrin α v β 3 in a dose-dependent manner. Tat PTD-Endostatin-RGD showed a high affinity to rat retina microvascular endothelial cells treated with S -nitroso- N -acetylpenicillamine. The results showed that Tat PTD-Endostatin-RGD could inhibit abnormal angiogenesis in retina via eye drops. Conclusion Tat PTD-Endostatin-RGD showed high penetration ability through ocular barriers, bound specifically to integrin α v β 3 and effectively inhibited the abnormal angiogenesis. General significance Tat PTD-Endostatin-RGD represents a potent novel drug applied via eye drops for fundus oculi neovascularization diseases. [ABSTRACT FROM AUTHOR]

Background Currently marketed chondroitin sulfate isolated from animal sources and structurally quite heterogeneous. Synthesis of structurally defined chondroitin sulfate is highly desired. The capsular polysaccharide from Escherichia coli strain K4 is similar to chondroitin, and its biosynthesis requires a chondroitin polymerase (KfoC). The essential step toward de novo enzymatic synthesis of chondroitin sulfate, synthesis of chondroitin, could be achieved by employing this enzyme. Methods Structurally defined acceptors and donor-sugars were prepared by chemoenzymatic approaches. In addition, surface plasmon resonance was employed to determine the binding affinities of individual substrates and donor–acceptor pairs for KfoC. Results KfoC has broad donor substrate specificity and acceptor promiscuity, making it an attractive tool enzyme for use in structurally-defined chimeric glycosaminoglycan oligosaccharide synthesis in vitro . In addition, the binding of donor substrate molecules regulated the affinity of KfoC for acceptors, then influenced the glycosyl transferase reaction catalyzed by this chondroitin polymerase. Conclusion and general significance These results assist in the development of enzymatic synthesis approaches toward chimeric glycosaminoglycan oligosaccharides and designing future strategies for directed evolution of KfoC in order to create mutants toward user-defined goals. [ABSTRACT FROM AUTHOR]

Background Endostatin, a specific inhibitor of endothelial cell proliferation and angiogenesis, has been proved to have effects on ocular neovascular diseases by intraocular injection. In order to increase its permeability to ocular barriers and make it effective on fundus oculi angiogenesis diseases via non-invasive administration (eye drops), endostatin was fused to Tat PTD via a genetic engineering method. Methods Most of the Tat PTD– endostatin was expressed as inclusion bodies in Escherichia coli , so pure and active Tat PTD–endostatin was prepared by a series of operations, including inclusion body denaturation, refolding and chromatography. The anti-angiogenesis activity of Tat PTD–endostatin was investigated by cell proliferation experiments and chick embryo chorioallantoic membrane assay. In addition, its translocating ability and concrete entry mechanism into cells were also investigated by fluorescence microscope and flow cytometry. The penetrating ability to ocular barriers was also studied by immunohistochemistry. A mouse choroidal neovascularization model was established to investigate the pharmacodynamics of Tat PTD–endostatin. Results The obtained Tat PTD–endostatin had excellent anti-angiogenesis activity and was superior to Es in cellular translocating. Macropinocytosis may be the dominant route of entry of Tat PTD–endostatin into cells. Tat PTD–endostatin could cross ocular barriers and arrive at the retina after eye-drop administration. In addition, it displayed inhibitory effects on choroidal neovascularization via eye drops. Conclusions Tat PTD–endostatin possessed excellent ocular penetrating ability and anti-angiogenesis effects. General significance Tat PTD is a promising ocular delivery tool, and Tat PTD–endostatin is a potential drug for curing fundus oculi angiogenesis diseases. [ABSTRACT FROM AUTHOR]