Adrenomedullin promotes cell-cell contact formation of podocytes through regulating Rho GTPases / 中华肾脏病杂志

Objective To investigate the regulatory effect of adrenomedullin (AM) on the cell-cell contact formation of podocytes and the possible mechanism.Methods Podocytes were treated with AM (10-7 mol/L),AM combined with a PKA inhibitor H89 (10-4 mol/L),and forskolin (10-5 mol/L) as positive control respectively for 12 hours.Immunofluorescent staining was applied to observe the distribution of cell adhesion molecules and actin-associated proteins.Western blotting assay was used to assess their protein levels.Rho GTPases activity was analyzed by GST-pull down assay and their protein levels were tested by Western blotting.Results AM induced the redistribution of adhesion molecules,actin-associated proteins as well as the F-actin at cell-cell contacts between podocytes.This effect was similar to that of forskolin and could be blocked by H89.The levels of those proteins did not change significantly (P ＞ 0.05).AM up-regulated the activities of RhoA,Rac1 and Cdc42 (P ＜ 0.05),which were partially blocked by H89.The protein levels of Rho GTPases showed no difference compared with the control (P ＞ 0.05).Conclusions AM may promote cell-cell contact formation of podocytes,probably through enhancing the activity of Rho GTPases and then resulting in the redistribution of adhesion molecules,actin-associated proteins and F-actin,which is partially mediated through cAMP-PKA signaling pathway.

Direct intercellular communications dominate the interaction between adipose-derived MSCs and myofibroblasts against cardiac fibrosis

The onset of cardiac fibrosis post myocardial infarction greatly impairs the function of heart. Recent advances of cell transplantation showed great benefits to restore myocardial function, among which the mesenchymal stem cells (MSCs) has gained much attention. However, the underlying cellular mechanisms of MSC therapy are still not fully understood. Although paracrine effects of MSCs on residual cardiomyocytes have been discussed, the amelioration of fibrosis was rarely studied as the hostile environment cannot support the survival of most cell populations and impairs the diffusion of soluble factors. Here in order to decipher the potential mechanism of MSC therapy for cardiac fibrosis, we investigated the interplay between MSCs and cardiac myofibroblasts (mFBs) using interactive co-culture method, with comparison to paracrine approaches, namely treatment by MSC conditioned medium and gap co-culture method. Various fibrotic features of mFBs were analyzed and the most prominent anti-fibrosis effects were always obtained using direct co-culture that allowed cell-to-cell contacts. Hepatocyte growth factor (HGF), a well-known anti-fibrosis factor, was demonstrated to be a major contributor for MSCs' anti-fibrosis function. Moreover, physical contacts and tube-like structures between MSCs and mFBs were observed by live cell imaging and TEM which demonstrate the direct cellular interactions.

Interleukin-18 enhances cytokine secretion by monocytes activated through direct contact with T lymphocytes / 第二军医大学学报

Objective: To study whether interleukin (IL)-18 is involved in the activation of monocytes through direct contact with T cells and the related intracellular mechanism. Methods: T cells and monocytes were isolated and purified from the peripheral blood of healthy donors by magnetic beads. Phytohemagglutinin (PHA) pre-stimulated T cells were fixed by 1% paraformaldehyde and were then co-cultured with monocytes at a T cell: monocyte ratio of 4:1. TNF-α and IL-18 levels in the supernatants were assayed by ELISA. Expression of IL-18 receptor α chain (IL-18Rα) on the surface of monocytes was analyzed by flow cytometry. Results: Monocytes activated by PHA-stimulated T cells produced significantly more TNF-α than by unstimulated T cells; non-cultured T cells or monocytes hardly produced any TNF-α. Upon direct cellular contact, PHA prestimulated T cells also up-regulated IL-18Rα expression on the surface of monocytes and induced IL-18 production in monocytes, which could be suppressed by nuclear factor (NF)-κB inhibitor (N-acetyl-L-cysteine, NAC) or phosphatidyl-inositol (PT) 3 kinase inhibitor (LY294002), but not by mitogen activated protein kinase (MAPK) inhibitor (SB203580). Neutralizing anti-IL-18 monoclonal antibody dose-dependently inhibited the production of TNF-α by monocyte-stimulated T cells. IL-18 failed to induce TNF-α production by cultured monocytes alone, while dose-dependently enhanced TNF-α production in monocyte-stimulated T cells, which could be inhibited by NAC or LY294002, but not by SB203580. Conclusion: By direct cellular contact T cells can stimulate monocytes to produce TNF-α and IL-18, up-regulate IL-18 receptor expression in monocytes, and activate intracellular NF-κB and PI3 kinase pathways. IL-18 can enhance T cell ability to stimulate TNF-α production by monocytes, which is dependent on the activation of NF-κB and PI3 kinase pathways.

Cell-cell contact induces mesenchymal stem cells differentiation to smooth muscle cells / 第三军医大学学报

Objective To establish a co-culture system between mesenchymal stem cells (MSCs) and smooth muscle cells (SMCs) in vitro, and study the influence of SMCs on the differentiation of bone marrow MSCs (BMSCs) into SMCs in the co-culture system. Methods Density gradient centrifugation was used to separate and culture BMSCs, and collagenase digestion was employed to separate and culture bladder smooth muscle cells(BSMCs). Then the 2 types of cells were identified respectively. Then the cells were divided into 4 groups, BMSCs culture group (positive control), BSMCs cells culture group (negative control), BMSCs and BSMCs co-culture groups with contact or non-contact. The expression of ?-smooth muscle actin (?-SMA) and calponin were detected by immunofluorescence staining. Western blot analysis was employed to determine protein expression of calponin. Results In contact co-culture group, the positive rate of CD90 abd CD44 were 99.78% and 60.27% respectively in BMSCs, while only 0.21% for CD45. Western blotting showed that BMSCs expressed little calponin, but those contacting with BSMCs had an increasingly stronger expression of calponin with the elapse of contact culture time, while there was no significant change in its expression in BMSCs with contact. Immunofluorescence staining indicated that part of BMSCs were calponin positive at the 8th day after culture, but no positive cell was observed in non-contact culture group and BMSCs culture group. Conclusion The contact between BSMCs and BMSCs can promote the differentiation of BMSCs into SMCs.