Effect of Stromal Vascular Fractions on Angiogenesis of Injected Diced Cartilage.

OBJECTIVES: This study explored the effect of adipose-derived stromal vascular fractions (SVFs) on angiogenesis in injected autologous diced cartilage. METHODS: Stromal vascular fractions were extracted by enzymatic digestion. Cartilage grafts were harvested from 1 side of the auricular cartilage of New Zealand rabbit and then diced to a size of 1.0 mm3. The grafts were divided into 2 groups. The control group was diced cartilage mixed with culture medium, and the experimental group was diced cartilage mixed with SVFs. The 2 groups of composite grafts were subcutaneously implanted on both sides of the back of each rabbit. After 4, 12 and 24 weeks, the tissue structure, number of blood vessels, and angiogenic factors in the grafts were observed. RESULTS: The SVFs conformed to the current standard of the biological evaluation. Under an inverted microscope, the number of layers of chondrocytes in the experimental group was higher than that in the control group at 4 weeks. A small number of inflammatory cells and blood vessels were observed around the cartilage grafts. At 12 and 24 weeks, the volume of tissue was increased gradually by general observation. And a large number of chondrocytes were observed microscopically, whereas the number of inflammatory cells decreased. And meanwhile additional new blood vessels were observed. Immunohistochemical analysis of CD31 showed that the number of capillaries in the control group was significantly lower than that in the experimental group at 4, 12 and 24 weeks. Further, the expression of Hypoxia inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) mRNA and protein were measured by RT-PCR and Western bloting, respectively. The results showed that the mRNA expression of VEGF and HIF-1α in the experimental group was increased. The mRNA level remained higher than that of the control group at 24 weeks (P < 0.05). And the relative expression levels of VEGF and HIF-1α protein in the experimental group were higher than those in the control group at 4, 12 and 24 weeks (P < 0.05). CONCLUSION: Autologous diced cartilage mixed with adipose-derived SVFs can promote angiogenesis when transplanted by injection. Further research showed that SVFs could increase the expression levels of VEGF and HIF-1α in the grafts, which may be part of the mechanism that SVFs promoted the angiogenesis of diced cartilage.

Different strategies for preparation of non-tagged rV270 protein and its efficacy against Yersinia pestis challenge.

OBJECTIVE: LcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study. METHODS: A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co(2+) affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography. RESULTS: Removal of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 106 CFU of Y. pestis virulent strain 141. CONCLUSION: The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.

The HMGB1 signaling pathway activates the inflammatory response in Schwann cells.

Schwann cells are not only myelinating cells, but also function as immune cells and express numerous innate pattern recognition receptors, including the Toll-like receptors. Injury to peripheral nerves activates an inflammatory response in Schwann cells. However, it is unclear whether specific endogenous damage-associated molecular pattern molecules are involved in the inflammatory response following nerve injury. In the present study, we demonstrate that a key damage-associated molecular pattern molecule, high mobility group box 1 (HMGB1), is upregulated following rat sciatic nerve axotomy, and we show colocalization of the protein with Schw-ann cells. HMGB1 alone could not enhance expression of Toll-like receptors or the receptor for advanced glycation end products (RAGE), but was able to facilitate migration of Schwann cells. When Schwann cells were treated with HMGB1 together with lipopolysaccharide, the expression levels of Toll-like receptors and RAGE, as well as inflammatory cytokines were upregulated. Our novel findings demonstrate that the HMGB1 pathway activates the inflammatory response in Schwann cells following peripheral nerve injury.