Enzymatic Formation of an Injectable Hydrogel from a Glycopeptide as a Biomimetic Scaffold for Vascularization

The construction of functional vascular networks in regenerative tissues is a crucial technology in tissue engineering to ensure the sufficient supply of nutrients. Although natural hydrogels are highly prevalent in fabricating three-dimensional scaffolds to induce neovascular growth, their widespread applicability was limited by the potential risk of immunogenicity or pathogen transmission. Therefore, developing hydrogels with good biocompatibility and cell affinity is highly desirable for fabricating alternative matrices for tissue regeneration applications. Herein, we report the generation of a new kind of hydrogel from supramolecular assembling of a synthetic glycopeptide to mimic the glycosylated microenvironment of extracellular matrix. In the presence of a tyrosine phosphate group, this molecule can undergo supramolecular self-assembling and gelation triggered by alkaline phosphatase under physiological conditions. Following supramolecular self-assembling, the glycopeptide gelator tended to form nanofilament structures displaying a high density of glucose moieties on their surface for endothelial cell adhesion and proliferation. On further incorporation with deferoxamine (DFO), the self-assembled hydrogel can serve as a reservoir for sustainably releasing DFO and inducing endothelial cell capillary morphogenesis in vitro. After subcutaneous injection in mice, the glycopeptide hydrogel encapsulating DFO can work as an effective matrix to trigger the generation of new blood capillaries in vivo

<i>ErmF</i> and <i>ereD</i> Are Responsible for Erythromycin Resistance in <i>Riemerella anatipestifer</i>

<div><p>To investigate the genetic basis of erythromycin resistance in <i>Riemerella anatipestifer</i>, the MIC to erythromycin of 79 <i>R</i>. <i>anatipestifer</i> isolates from China and one typed strain, ATCC11845, were evaluated. The results showed that 43 of 80 (53.8%) of the tested <i>R</i>. <i>anatipestifer</i> strains showed resistance to erythromycin, and 30 of 43 erythromycin-resistant <i>R</i>. <i>anatipestifer</i> strains carried <i>ermF</i> or <i>ermFU</i> with an MIC in the range of 32–2048 μg/ml, while the other 13 strains carrying the <i>ereD</i> gene exhibited an MIC of 4–16 μg/ml. Of 30 <i>ermF</i> + <i>R</i>. <i>anatipestifer</i> strains, 27 (90.0%) carried the <i>ermFU</i> gene which may have been derived from the CTnDOT-like element, while three other strains carried <i>ermF</i> from transposon Tn4351. Moreover, sequence analysis revealed that <i>ermF</i>, <i>ermFU</i>, and <i>ereD</i> were located within the multiresistance region of the <i>R</i>. <i>anatipestifer</i> genome.</p></div

Distribution of <i>R</i>. <i>anatipestifer</i> strains with different MICs for erythromycin.

<p>Distribution of <i>R</i>. <i>anatipestifer</i> strains with different MICs for erythromycin.</p

The glycosylation of PTPRT by GnT-V promotes galectin-3 binding, resulting in dimerized PTPRT with increased cell-surface retention.

<p>(A) Cell-surface retention of galectin-3 was examined after cell-surface biotinylation. The stable transfectants were re-cultured for 3 and 6 hours before lysis and streptavidin bounded agarose precipitation. After that galectin-3 was detected by immunoblot using anti-galectin-3 antibody. The graph (right panel) is the quantification of the band intensity. The relative amount of galectin-3 at 3 or 6 hour time point was normalized to that at 0 h in both Mock-7721 and GnT-V-7721. The data represent the mean ± SEM of three independent analyses. (B) More colocalization of galectin-3 with PTPRT was visualized at the cell surface in GnT-V overexpressing cells. Confocal microscopy was taken to detect the localization of galectin-3 and PTPRT in Mock-7721, GnT-V-7721, Mock-HT29, GnT-V-HT29. Mouse anti-PTPRT and rabbit anti-galectin-3 were used as primary antibodies. Cy3-conjugated donkey anti-rabbit IgG and FITC-conjugated goat anti-mouse IgG were secondary antibodies. Cell nuclei were visualized by DAPI staining. Merged panels show the overlapped channels. (C) Increased association of galectin-3 with PTPRT is observed in GnT-V overexpressing cells. Mock-7721 and GnT-V-7721 cells were cross-linked with 3 mM BS³ at 4°C for 2 hours before immunoprcipitation (IP). IP was performed using anti-PTPRT antibody followed by immunoblot with anti-PTPRT and anti-galectin-3 antibodies. (D) Mock-HT29 and GnT-V-HT29 cells were treated as described in Fig. 4C, except for that anti-galectin-3 was used for IP.</p

Increased β1,6 GlcNAc branching N-glycans on PTPRT prolongs PTPRT cell-surface retention time and promotes PTPRT's dimerization.

<p>(A) Confocal microscopy was performed to observe the distribution of PTPRT in Mock-7721 and GnT-V-7721 cells. The mouse anti-human PTPRT and FITC-conjugated goat anti-mouse IgG were used. The bottom panel represents the magnified images of indicated area in the top panel. (B) Cell-surface retention of PTPRT was examined after cell-surface protein was biotinylated. The stable transfectants were re-cultured for 3 and 6 hours before they were homogenized and pulled down by streptavidin bounded agarose precipitation. After the precipitation, PTPRT was detected by immunoblot using anti-PTPRT antibody. The bottom chart is the quantification of band intensity in the upper panel. The band intensity of PTPRT at 3 or 6 hour time point was normalized to that at 0 hour in both Mock-7721 and GnT-V-7721. The data represent the mean ± SEM of three independent analyses. (C) Mock-7721, GnT-V-7721, Mock-HT29 and GnT-V-HT29 cells were grown to confluence in 6-well dishes and cross-linked with 3 mM BS³ at 4°C for 2 hours, followed by immunoblot for PTPRT. The graph (right panel) is the band intensity analysis of dimer band intensity normalized to its total monomer. The data represent the mean ± SEM of three independent analyses.</p

Activation of STAT3 promotes cell migration.

<p>(A) Mock-7721cells were treated with IL-6. STAT3 was activated when Mock-7721 cells were treated with 30 µg/ml of IL-6 for 24 hours. The levels of pY705 STAT3 and STAT3 were detected using immunoblot with indicated antibodies.(B) Mock-7721 cells were tested in the transwell assay with or without IL-6 at a concentration of 30 µg/ml for 24 hours. Migrated cells were visualized by staining with crystal violet. The graph (right panel) is the fold changes of migrated cells from the IL-6 treated to the untreated cells. The data represent the mean ± SEM of three independent analyses. (C) Mock-7721 and GnT-V-7721 cells were transfected with synthetic double-stranded control (-) or STAT3 siRNA (+). Two days posttransfection, immunoblot was performed to check the efficiency of knockdown of STAT3. (D) Mock-7721 and GnT-V-7721 cells were transfected with synthetic double-stranded control (−) or STAT3 siRNA (+). Two days posttransfection, transwell assay, described in procedure, was performed to observe cell migration. The graph (right panel) is the fold changes of migrated cells. The data represent the mean ± SEM of three independent analyses. (E) Mock and GnT-V-7721 cells were transfected with synthetic double-stranded control (−) or PTPRT siRNA (+). Two days posttransfection, immunoblot was performed to check the efficiency of knockdown of PTPRT and the level of pY705 STAT3. (F) Mock and GnT-V-7721 cells were transfected with synthetic double-stranded control (−) or PTPRT siRNA (+). Two days posttransfection, transwell assay, described in procedure, was performed to observe cell migration. The graph (right panel) is the fold changes of migrated cells. The data represent the mean ± SEM of three independent analyses.</p

Overexpression of GnT-V causes aberrant N-glycosylation in the transfected cells.

<p>(A) The level of GnT-V protein was detected in different cell lines by immunoblot. (B) The GnT-V-7721 or GnT-V-HT29 stable cells were identified by detecting the level of GnT-V protein using immunoblot. (C) GnT-V-7721 and Mock-7721 cells were seeded on coverslips, followed by fixation and staining with biotinylated L-PHA and FITC-conjugated avidin, and then visualized under fluorescence microscopy. (D) Flow cytometry was performed with biotinylated L-PHA in GnT-V-7721 and GnT-V-HT29 stable cells, as well as Mock cells. FITC-conjugated avidin D staining was used as negative control.</p

GnT-V overexpression attenuates phosphatase activity of PTPRT, resulting in activation of STAT3.

<p>(A) The protein levels of phosphorylated STAT3 at Y705 and total STAT3 were detected using anti-pY705 STAT3 and anti-STAT3 in Mock-7721 and GnT-V-7721, Mock-HT29 and GnT-V-HT29. (B) Cytoplasmic and nuclear fractions were prepared and separated by immunoblot and probed with indicated antibodies. Histone H1 and β-tublin were served as nuclear and cytoplasmic markers, respectively. (C) Subcellular localization of STAT3 in stable transfectants was detected using confocal microscopy. Mock-7721, GnT-V-7721 cells were fixed, permeabilized, and incubated with anti-pY705 STAT3 and Cy3-conjugated secondary antibody. DAPI was used to counter-stain the nuclei. Merged pictures show the overlap of red and blue channels. Zoom, indicated by the white lines, are magnified images of upper panel. (D)Tyrosine phosphatase activity assay was performed in Mock-7721 and GnT-V-7721 (left panel), Mock-HT29 and GnT-V-HT29 (right panel) cells. PTPRT was immunoprecipitated, and phosphatase activity was measured after 15 minutes using tyrosine-phosphorylated peptide as substrate.</p

PTPRT is identified as a target of GnT-V.

<p>(A) 4 types of RPTPs were evaluated in Mock-7721 and GnT-V-7721 using immunofluorescent staining. Cells were placed on coverslips overnight and stained with anti-PTPRM, anti-PTPRT, anti-PTPRK, or anti-PTPRU antibody, respectively. Then, FITC-labeled anti-mouse IgG or Cy3-labeled anti-rabbit IgG was used based on the specificity of the primary antibody. Intensity of fluorescent was visualized under confocal microscopy. (B) Lectin precipitation was performed with L-PHA bounded agarose, followed by immunoblot with anti-PTPRT antibody in GnT-V-7721 and Mock-7721. (C) PTPRT was immunoprecipitated in GnT-V-7721 and Mock-7721, and then subjected to the immunoblot with biotinylated L-PHA, biotinylated DSL and anti-PTPRT antibody, respectively.</p

Clustal W alignment of the upstream sequences from the <i>ermF</i> / <i>ermFU</i> genes from CTnDOT, Tn4351, and different <i>R</i>. <i>anatipestifer</i> strains.

<p>The start codon ATG of <i>ermFU</i>/<i>ermF</i> gene was underlined.</p