After escaping from the hyperacute rejection (HAR), the xenograft has to be faced the challenge of acute vascular, acute cellular and even chronic rejection. Endothelial cells have been confirmed as a kind of antigen processing cell (APC) in allo-rejection. The porcine aortic endothelial cell (PAEC) expressed SLA-II and B7 which are the characteristics of professional APC. PAEC also has plenty of alpha-Gal residues, whether the antigen play any role in the post-HAR is still unknown. Human and porcine peripheral blood lymphocyte (PBLC) were isolated and divided into two parts, one for the effectors and the another were incubated with mitomycin C (MMC) as stimulators. The two kinds of PBLC were mixed-cultured within five days. Cultured PAEC from NJZ Pig was incubated with MMC and divided into two: One digested with alpha-galactosidase. The two kinds of PAEC were taken as stimulators to mixed-culture with human PBLC for five days. All the proliferation was detected with 3H-TdR intermingled in the system. The results showed that allo-MLR was ber than xeno-MLR in the cases. The proliferation was much ber when PAEC was used as the stimulator than that of porcine PBLC. However, the response was remarkably decreased after the digestion of alpha-Gal with alpha-galactosidase. The conclusion was that the low response of porcine-to-human MLR in vitro might be related to the predominant indirect pathway of antigen recognition in this system. While PAEC was used as the stimulator the proliferation in MLR was ber which might be concerned that PAEC itself was an APC as well as xeno-antigen sources, thus the direct pathway was predominant and worked more efficiently. The alpha-Gal might induce T cell proliferation through the linkage with the biological big molecules working as a complete antigen. The other post-HAR antigen might also exist in PAEC such as SLA-II, etc.
ObjectiveTo review the research progress on the role and mechanism of matrix stiffness in regulating endothelial cell sprouting. MethodsThe related literature at home and abroad in recent years was extensively reviewed, and the behaviors of matrix stiffness related endothelial cell sprouting in different cell cultivation conditions were analyzed, and the specific molecular mechanism of matrix stiffness regulating related signal pathways in endothelial cell sprouting was elaborated. Results In two-dimensional cell cultivation condition, increase of matrix stiffness stimulates endothelial cell sprouting within a certain range. However, in three-dimensional cell cultivation condition, the detailed function of matrix stiffness in regulating endothelial cell sprouting and angiogenesis are still unclear. At present, the research of the related molecular mechanism mainly focuses on YAP/TAZ, and roles of its upstream and downstream signal molecules. Matrix stiffness can regulate endothelial cell sprouting by activating or inhibiting signal pathways to participate in vascularization. ConclusionMatrix stiffness plays a vital role in regulating endothelial cell sprouting, but its specific role and molecular mechanism in different environments remain ambiguous and need further study.
ObjectiveTo evaluate the effects of icariin on autophagy induced by low-concentration of glucocorticoid and exosome production in bone microvascular endothelial cells (BMECs).MethodsBMECs were isolated from femoral heads resected in total hip arthroplasty and then intervened with hydrocortisone of low concentration (0, 0.03, 0.06, 0.10 mg/mL), which were set as groups A, B, C, and D, respectively. On the basis of hydrocortisone intervention, 5×10?5 mol/L of icariin was added to each group (set as groups A1, B1, C1 and D1, respectively). Western blot was used to detect the expressions of microtubule-associated protein 1 light chain 3B (LC3B) and dead bone slice 1 (p62) after 24 hours. Exosomes were extracted from BMECs treated with icariin (intervention group) and without icariin (non-intervention group), and the diameter and concentration of exosomes were evaluated by nanoparticle tracking analysis technique. The total protein content of exosomes was detected by BCA method, and the expressions of proteins carried by exosomes including CD9, CD81, transforming growth factor β1 (TGF-β1), and vascular endothelial growth factor A (VEGFA) were assessed by Western blot. The BMECs were further divided into three groups: BMECs in the experimental group and the control group were co-cultured with exosomes secreted by BMECs treated with or without icariin, respectively; the blank control group was BMECs without exosome intervention. The three groups were treated with hydrocortisone and Western blot was used to detect the expressions of LC3B and p62. The scratching assay was used to detect cell migration ability; angiogenic ability of BMECs was also assessed.ResultsWith the increase of hydrocortisone concentration, the protein expression of LC3B-Ⅱ increased gradually, and the protein expression of p62 decreased gradually (P<0.01). Compared with group with same concentration of hydrocortisone, the protein expression of LC3B-Ⅱ decreased and the protein expression of p62 increased after the administration of icariin (P<0.01). The concentration of exosomes in the intervention group was significantly higher than that in the non-intervention group (t=?10.191, P=0.001); and there was no significant difference in exosome diameter and total protein content between the two groups (P>0.05). CD9 and CD81 proteins were highly expressed in the non-intervention group and the intervention group, and the relative expression ratios of VEGFA/CD9 and TGF-β1/CD9 proteins in the intervention group were significantly higher than those in the non-intervention group (P<0.01). After co-culture of exosomes, the protein expression of p62 increased in blank control group, control group, and experimental group, while the protein expression of LC3B-Ⅱ decreased. There were significant differences among groups (P<0.05). When treated with hydrocortisone for 12 and 24 hours, the scratch closure rate of the control group and experimental group was significantly higher than that of the blank control group (P<0.05), and the scratch closure rate of the experimental group was significantly higher than that of the control group (P<0.05). When treated with hydrocortisone for 4 and 8 hours, the number of lumens, number of sprouting vessels, and length of tubule branches in the experimental group and the control group were significantly greater than those in the blank control group (P<0.05); the length of tubule branches and the number of lumens in the experimental group were significantly greater than those in the control group (P<0.05).ConclusionIcariin and BMECs-derived exosomes can improve the autophagy of BMECs induced by low concentration of glucocorticoid.
Objective To observe the proliferation and migration of endothelial cells after 30% total burn surface area (TBSA) of deep partial thickness scald, and the effect of basic fibroblast growth factor (bFGF) on angiogenesis during wound healing.Methods A total of 133 male Wistar ratswere divided randomly into normal control (n=7), injured control group (n=42), bFGF group (n=42) andanti-c-fos group (n=42). The apoptosis expression of fibroblasts was determinedwith in situ hybridization and the changes of proliferation cell nuclear antigen(PCNA), focal adhesion rinase(FAK), c-fos and extracellular signalregulated kinase(ERK) proteins expression were detected with immunohistochemistry staining technique after 3 hours, 6 hours, 1 day, 3 days, 7 days, 14 days and 21 days of scald.Results In injured control group and bFGF group, theproliferation rate of the vascular endothelial had evident changes 7 days and14 days after scald； the expression of FAK was increased 14 days after scald. ERK proteins expression was different between injury control group and bFGF group at initial stage after scald. Stimulation of ERKs by bFGF led to up-regulation of c-fos and b expression of FAK. Conclusion Exogenous bFGF extended the influence on wound healing process by ERK signaling pathway, affecting migration cascade of vascular endothelial cell. The oncogene proteins play an important role on accelerating angiogenesis duringwound healing.
Objective
To investigate the role and relative mechanism of stromal cell derived factorl (SDF-1) secreted by nucleus pulposus cells (NPCs) on the proliferation of vascular endothelial cells (VECs).
Methods
The NPCs were isolated from the degenerated disc specimens after discectomy. NPCs at passage 1 were transfected with lentivirus-mediated SDF-1 over-expression; transfected and untransfected NPCs at passage 2 were cultured in the three-dimensional alvetex? scaffold, then they were co-cultured with HMEC-1 cells. The morphology of NPCs was observed by scanning electron microscope (SEM), and the apoptosis of HMEC-1 cells was detected by Annexin V/propidiumiodide staining after 72 hours co-culutre. The proliferation of HMEC-1 cells was detected by cell counting kit 8 at 12, 24, 48, and 72 hours in transfected group and untransfected group, respectively. ELISA was used to measure the vascular endothelial growth factor (VEGF) expression level. The virus transfection efficiency and relative Akt pathway were determined by Western blot.
Results
The NPCs maintained cell phenotype and secreted much extracellular matrix in three-dimensional-culture by SEM observation. In the co-culutre system, after NPCs were transfected with SDF-1 over-expression lentivirus, the proliferation of HMEC-1 cells was significantly increased, while the apoptosis was decreased obviously. The ELISA results demonstrated that the amount of VEGF was remarkably increased in the culture medium. Furthermore, SDF-1 promoted the up-regulation of phosphorylate Akt expression; after inhibition of Akt expression by GSK690693, the proliferation rate of VECs decreased significantly.
Conclusion
Over-expression of SDF-1 by NPCs is beneficial for VECs proliferation, which is involved in SDF-1-Akt signalling pathway.
Objective
To investigate the effects of vascular endothelial growth factor C (VEGF-C) gene modified lymph nodes on promoting proliferation of lymphatic endothelial cells in the surrounding tissues.
Methods
Thirty-six Sprague Dawley rats, weighing 200.1-271.5 g, were randomly divided into 2 groups (n=18). After the in situ axillary lymph nodes transplantation models were established in both groups, 1.5 × 108 PFU Ad-VEGF-C-Flag and Ad-Flag were injected into the transplanted lymph nodes in experimental group and control group, respectively. At 3 days after injection, the axillary lymph nodes were harvested to observe the expression of Flag; at 1, 2, and 4 weeks after injection, the axillary lymph nodes and the surrounding tissues were harvested to observe the expression of Prxo-1 protein and to calculate the fluorescence density; at 2 and 4 weeks after injection, the absorbance (A) value of treated blood at 620 nm was calculated to observe lymphatic back-flow function improvement; the rats without treatment served as normal control group, and the rats with in situ axillary lymph nodes transplantation model served as blank control group.
Results
At 3 days after injection, the expression of Flag could be detected in experimental group and control group. The fluorescence density of Prox-1 protein in experimental group increased at 1, 2, and 4 weeks, and it was significantly higher than that in control group (P lt; 0.05). The A values of normal control group and blank control group were 0.539 ± 0.020 and 0.151 ± 0.007, respectively. The A values of experimental group and control group were 0.170 ± 0.011 and 0.168 ± 0.010 at 2 weeks, and 0.212 ± 0.016 and 0.197 ± 0.006 at 4 weeks, which were significantly lower than those of normal control group (P lt; 0.05), but no significant difference was found when compared with blank control group, and between the experimental group and control group (P gt; 0.05).
Conclusion
The VEGF-C gene modified lymph nodes can stimulate the proliferation of lymphatic endothelial cells in the surrounding tissues. However, it has no improved effect on lymphatic back-flow function in the affected limb.
ObjectiveTo explore a new strategy for constructing three-dimensional dermoid tissue in vitro by using cell sheets technology.MethodsRabbit bone marrow mesenchymal stem cells (rBMSCs) were isolated from bone marrow of New Zealand white rabbits and cultured by whole bone marrow adherent method. Human dermal fibroblasts (HDFs) were cultured and passaged in vitro. The 2nd generation rBMSCs and the 3rd generation HDFs were cultured in a culture dish for 2 weeks with cell sheets conditioned medium respectively to obtain a monolayer cell sheets. Human umbilical vein endothelial cells (HUVECs) were inoculated on rBMSCs sheet to construct pre-vascularized cell sheet. During the culture period, the morphological changes of the cell sheet were observed under an inverted phase contrast microscope. At 1, 3, 7, and 14 days, HE staining and CD31 immunofluorescence staining were performed to observe the cell distribution and microvascular network formation. The rBMSCs sheet was used as control. The pre-vascularized cell sheet (experimental group) and rBMSCs sheet (control group) cultured for 7 days were placed in the middle of two HDFs sheets, respectively, to prepare three-dimensional dermoid tissues. After 24 hours of culture, CD31 immunofluorescence staining and collagen type Ⅰ and collagen type Ⅲ immunohistochemical stainings were performed to evaluate cell distribution and collagen expression.ResultsHDFs and rBMSCs sheets were successfully prepared after 2 weeks of cell culture. After inoculation of HUVECs on rBMSCs sheet for 3 days, HUVECs could be seen to rearrange on rBMSCs sheet and forming vacuoles. The reticular structure was visible at 7 days and more obvious at 14 days. The formation of vacuoles between the cell sheets was observed by HE staining, and the vacuoles became more and more obvious, the thickness of the membranes increased significantly with time. CD31 immunofluorescence staining showed the microvascular lumen formation. However, only the thickness of rBMSCs sheet increasing was observed, with no changes in cell morphology or cavitation structure. The three-dimensional dermoid tissue observation showed that the endothelial cells in the experimental group were positive expressions, and the rBMSCs, HDFs, and HUVECs cells were arranged neatly. The endothelial cells were negative expressions and randomly arranged in the control group. The collagen type Ⅰ and collagen type Ⅲ were positive expression in the experimental group and the control group. But compared with control group, experimental group presented a " honeycomb” network connection, where the matrix was distributed regularly, and cells were arranged tightly. The difference in the expression of collagen type Ⅰ and collagen type Ⅲ between the experimental group and the control group was not significant (P>0.05).ConclusionThree-dimensional dermoid tissue is successfully constructed by using cell sheet technology. The cell matrix distribution of the pre-vascularized cell sheet constructed by HUVECs and rBMSCs sheet is relatively regular, which has the potential to form tissue engineered dermis.
OBJECTIVE: To prepare the compound biodegradable matrices, polyglycolic acid (PGA), polylactic acid (PLA) mesh and poly-beta-hydroxybutyrate(PHB) which precoated with collagen, and to observe the growth and differentiation of bovine vascular endothelial cells on these scaffolds. METHODS: By enzymatic digestion methods, bovine vascular endothelial cell (VEC) were isolated from calf thoracic aorta, then cultured and purified. PGA, PLA, PHB meshes were dipped into cross-linked type I collagen solution, dried under vacuum frozen condition. VEC were seeded into these scaffolds. The growth of VEC on scaffolds was analyzed by MTT method. RESULTS: The collagen, PGA/collagen, PLA/collagen scaffolds were elasticity and tenacity. VEC grew better on collagen, PGA/collagen, and PLA/collagen membranes than on the PHB/collagen one. CONCLUSION: The PGA/collagen scaffold has elasticity, plasticity and tenacity. VEC grow best on it. It is an ideal scaffold for tissue engineered vessel reconstruction for it integrating both advantages of biomaterials and degradable materials.
Objective To examine the effect of zinc finger protein A20 on regeneration of small-for-sized liver allograft, graft rejection and recipient rat survival time. Methods Small-for-sized liver transplantation with 30% partial liver allograft was performed by using a b-rejection combination rat model of DA (RT1a) to Lewis (RT1l) rats. The rats were grouped into rAdEasy-A20 treatment group （A20 group）, the control empty Ad vector rAdEasy treatment group (rAdEasy group) and PS control treatment group (PS group). Ex vivo gene transfer in donor liver graft was performed through portal vein infusion. Animals were assessed for survival days, expression of A20 in liver graft, liver graft regeneration, hepatocyte apoptosis, graft rejection, NF-κB activation and ICAM-1 mRNA expression in liver graft sinusoidal endothelial cells (LSECs), number of liver graft infiltrating mononuclear cells (LIMCs) and the subproportion of NK/NKT cells, and serum IFN-γ level. Results Survival day of A20 group rats was prominently longer than that of PS group rats and rAdEasy group rats (P=0.001 8), whereas survival day of rAdEasy group rats was remarkably shorter than that of PS group rats (P=0.001 8). Regeneration of the small-for-sized liver allograft was markedly augmented by A20, BrdU labelling index of hepatocyte on postoperative day 4 was significantly increased in the A20 group compared with the PS group and rAdEasy group (P＜0.01). Hepatocyte apoptosis on postoperative day 4 was significantly inhibited by A20 (P＜0.01). On postoperative day 4, histologic examination revealed a mild rejection in the A20 group but a more severe rejection in the PS and rAdEasy groups. NF-κB activity and ICAM-1 mRNA expression in LSECs on postoperative day 1 were notably suppressed by A20 overexpression. Flow cytometry analysis showed a marked downregulation of LIMCs number by A20, including more prominent decrease in the subproportion of NK/NKT cells on postoperative day 1 and 4, respectively (P＜0.05). Serum IFN-γ level on postoperative day 4 was also significantly suppressed by A20 overexpression (P＜0.05). Conclusion These data suggest that A20 could effectively promote small-for-sized liver allograft regeneration, suppresses rejection and prolong survival days of recipient rats. These effects of A20 could be related to an inhibition of LSECs activation, suppression of infiltration of LIMCs and the subpopulations such as NK cells and NKT cells into liver graft, and inhibition of hepatocyte apoptosis.
Objective To investigate the effect of Nodal protein on retinal neovascularization under hypoxia. MethodsIn vivo animal experiment: 48 healthy C57BL/6J mice were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+dimethyl sulfoxide (DMSO) group and OIR+SB431542 group, with 12 mice in each group. Retinal neovascularization was observed in mice at 17 days of age by retina flat mount. Counts exceeded the number of vascular endothelial nuclei in the retinal inner boundary membrane (ILM) by hematoxylin eosin staining. In vivo cell experiment: human retinal microvascular endothelial cells (hRMEC) were divided into normal group, hypoxia group, hypoxia+DMSO group and hypoxia +SB431542 group. The cell proliferation was detected by thiazolyl blue colorimetry (MTT). The effect of SB431542 on hRMEC lumen formation was detected by Matrigel three-dimensional in vitro molding method. Cell migration in hRMEC was detected by cell scratch assay. The Seahorse XFe96 Cell Energy Metabolism analyzer measured extracellular acidification rate (ECAR) of intracellular glycolysis, glycolysis reserve, and glycolysis capacity. One-way analysis of variance was used to compare groups. ResultsIn vivo animal experiment: compared with normal group, the neovascularization increased in OIR group (t=41.621, P＜0.001). Compared with OIR group, the number of vascular endothelial nuclei breaking through ILM in OIR+SB431542 group was significantly reduced, and the difference was statistically significant (F=36.183, P＜0.001). MTT test results showed that compared with normal group and hypoxia+SB431542 group, the cell proliferation of hypoxia group and hypoxia+DMSO group was significantly increased, and the difference was statistically significant (F=39.316, P＜0.01). The cell proliferation of hypoxia+SB431542 group was significantly lower than that of hypoxia+DMSO group, and the difference was statistically significant (t=26.182, P＜0.001). The number of intact lumen formation and migration cells in normal group, hypoxia group, hypoxia+DMSO group and hypoxia+SB431542 group were statistically significant (F=34.513, 41.862; P＜0.001, ＜0.01). Compared with the hypoxia+DMSO group, the number of intact lumen formation and migrating cells in the hypoxia+SB431542 group decreased significantly, and the differences were statistically significant (t=44.723, 31.178; P＜0.001, ＜0.01). The results of cell energy metabolism showed that compared with the hypoxia +DMSO group, the ECAR of intracellular glycolysis and glycolysis reserve in the hypoxia +SB431542 group was decreased, and the ECAR of glycolysis capacity was increased, with statistical significance (t=26.175, 33.623, 37.276; P＜0.05). ConclusionSB431542 can inhibit the proliferation, migration and the ability to form lumens, reduce the level of glycolysis of hRMECs cells induced by hypoxia.
