[CD137-CD137L signaling promotes angiogenesis in atherosclerosis plaque of mice through activating nuclear factor of activated T cells c1].

Objective: To explore whether CD137-CD137L signaling can promote angiogenesis in atherosclerosis plaque via activating nuclear factor of activated T cells c1 (NFATc1). Methods: Apolipoprotein E knock out mice were divided into the following groups: control group (n=5), CD137 activated group(n=5)and CD137 inhibited group (n=5). Immunohistochemistry was performed to detect the expression of CD31 in aortic plaque. Endothelial cells (bEnd.3) were purchased from ATCC and divided into the following groups: control group, IgG isotype control group, CD137 activated group and CD137 inhibited group. Western blot was used to determine total protein and nucleoprotein expression of NFATc1. The expression level of CD137 protein on the surface of endothelial cells was detected by flow cytometry(FCM) and CD137 protein of lysate of endothelial cells was detected by enzyme-linked immunosorbent assay (ELISA). Transwell assay was used to observe the migration ability of endothelial cells.Matrigel tube formation ability of endothelial cells were tested in the following groups: control group, CD137 activated group, silent NFATc1 + CD137 activated group, CD137 inhibited group, and over expressed NFATc1+ CD137 inhibited group. Results: (1) In vivo, the expression level of CD31 was significantly higher in the aortic plaque of CD137 activated group than in control group(1 191±187 vs. 115±30, P<0.05), while which was significantly downregulated in CD137 inhibited group(450±92, P<0.05). (2) The level of nucleoprotein(3.07±0.03 vs. 1.00±0.00, P<0.05) and total protein(2.18±0.30 vs. 1.00±0.00, P<0.05) of NFATc1 were significantly higher in CD137 activated group than in IgG isotype control group. The level of nucleoprotein(0.82±0.04) and total protein(0.84 ± 0.09) of NFATc1 were significantly lower in CD137 inhibited group than in CD137 activated group(both P<0.05). (3) FCM results showed that the fluorescence intensity of CD137 on the cell membrane was significantly higher in endothelial cells stimulated by TNF-α than in normal endothelial cells(5 163±329 vs. 1 660±162, P<0.05). (4) ELISA examination showed that the level of CD137 protein was significantly higher in endothelial cells stimulated by TNF-α than in normal endothelial cells ((573.4±23.7)pg/mg vs.(69.5±16.7)pg/mg, P<0.05). (5) Migration cell number was remarkably higher in CD137 activated group than in IgG isotype control group(1.19±0.13 vs. 1.00±0.00, P<0.05) and significantly lower in CD137 inhibited group(0.82±0.06)than in control group (P<0.05). (6) Values of the formation of the tube length ((5.76±0.18)mm vs. (4.21±0.11)mm, P<0.05) and branch number (29.38±1.28 vs. 21.13±0.96, P<0.05) were both significantly higher in CD137 activated group than in the control group. The formation of the tube length ((1.90±0.11)mm) and branch number(8.91±0.72)were significantly lower in silent NFATc1 + CD137 activated group than in the CD137 activated group (both P<0.05). The formation of the tube length((1.28±0.34)mm) and branch number(5.07±0.35)were also significantly decreased in the CD137 inhibited group compared with the CD137 activated group (both P<0.05). Compared with the CD137 inhibited group, the formation of the tube length((4.82±0.09)mm) and branch number(24.44±1.05) in the over expressed NFATc1+ CD137 inhibited group was increased (both P<0.05). Conclusion: CD137 can promote the angiogenesis in atherosclerosis plaque by activating NFATc1.

[In vivo and in vitro effects of CD137 stimulation on vascular calcification in high fat diet fed ApoE-/- mice].

Objective: To investigate the effect and related mechanism of CD137 stimulation on aortic atherosclerotic plaque calcification in high fat diet fed ApoE-/- mice and on calcification of vascular smooth muscle cells (VSMCs). Methods: (1) ApoE-/- mice fed with high fat diet were randomly divided into 3 groups: CD137 activated group (treated by 200 µg CD137 agonist i. p. once per week for 6 weeks, n=5); CD137 inhibited group (anti-CD137 group: 200 µg anti-CD137 antibody + 200 µg CD137 agonist, i. p., once per week for 6 weeks, n=5) and control group (n=5). Von kossa staining was used to observe the calcification of the aortic plaque and VSMCs. Immunohistochemistry was used to observe the expression of BMP-2 and Runx2 which are known mediators of osteogenic differentiation. (2) The mouse aortic VSMCs were obtained by Patch-attaching method. The calcium content was measured by Methylthymol Blue complexone method. The mRNA expressions of bone morphogenetic protein 2 (BMP-2) and Runx2 were measured by real-time fluorescent quantitative PCR (RT-PCR). The protein levels of BMP-2, Runx2 of the VSMCs were determined by Western blot. Results: (1) In vivo, the plaque calcified area in ApoE-/- mice was significantly larger in CD137-agonist group than that in control group ((1.75±0.33)×104 µm2 vs. (0.23±0.07)×104 µm2,P<0.01), and this effect was significantly reduced by cotreatment with CD137-antagonist ((0.83±0.30)×104 µm2 vs. (1.75 ±0.33)×104 µm2,P<0.05). The levels of BMP-2 and Runx2 were all significantly upregulated in CD137-agonist group than in control group (both P<0.01), again, this effect was blocked by cotreatment with CD137-antagonist (P<0.05). (2) Consistent with the in vivo results, VSMCs calcification was also more serious in CD137-agonist group than in control group, which could be significantly attenuated by cotreatment with CD137-antagonist. In VSMCs, calcium content level in CD137-agonist group was higher than in control group ((0.001 3±0.000 2) mmol/mg protein vs. (0.000 7±0.000 1) mmol/mg protein, P<0.01), which could be significantly reduced by co-treatment with CD137-antagonist ((0.000 9±0.000 2) mmol/mg protein vs. (0.001 3±0.000 2) mmol/mg protein, P<0.01). The mRNA and protein levels of BMP-2 and Runx2 were significantly upregulated in CD137-agonist group compared with the control group (P<0.05), which could be significantly down-regulated by cotreatment with CD-137 antagonist (P<0.05). Conclusion: CD137 activation can promote vascular calcification in high fat diet fed ApoE-/- mice both in vivo and in vitro.

[Artesunate attenuate chronic graft-versus-host disease by regulating Th17/Treg balance].

Objective: To investigate the effects of artesunate treatment on chronic graft-versus-host disease (cGVHD). Methods: Recipient BALB/c mice received 8 × 10(6) bone marrow cells with 8×10(6) spleen cells from B10D2 mice. Artesunate solubilized in acetone was injected intraperitoneally every day at the dose of 1 mg/kg at Day 28 after BMT. The clinical scores, survival and histopathological damage were analyzed. The frequency of Th17 and Tregs in PB and spleens from the mice were evaluated by flow cytometry. In addition, CD4(+) T cells from the spleens of mice were cultured in vitro, then stimulated with artesunate, the frequency of Th17 and Tregs in these splenocytes were evaluated by flow cytometry. Results: Artesunate administration diminished clinical and histopathological damage, and improved the survival of cGVHD mice[(46.57±7.83)% vs (55.71±6.99)%, χ(2)=5.457, P=0.020]; Artesunate contributed to Tregs development [(4.45±0.04)% vs (8.40±0.23)%, t=15.679, P<0.001; (6.62±0.24)% vs (10.48±0.48)%, t=6.587, P=0.003] while decreased Th17 cells [(1.51±0.18)% vs (0.58±0.19)%, t=7.233, P<0.001; (1.48±0.38)% vs (0.71±0.18)%, t=3.653, P=0.011] expressions in both PB and spleens, and decreased the Th17/Treg ratio (0.34±0.05 vs 0.09±0.03, t=7.621, P=0.002; 0.19±0.03 vs 0.06±0.02, t=6.993, P=0.002). Moreover, artesunate suppressed the Th17 cells expressions [(0.82±0.37) % vs (3.39±1.22) %, t=4.044, P=0.007] and contributed to Tregs development [(34.63±1.29) % vs (14.28±1.69) %, t=19.119, P<0.001], and also decreased the Th17/Treg ratio (0.24±0.09 vs 0.02±0.01, t=4.780, P=0.003) in vitro. Conclusions: Artesunate suppressed the Th17 cells expressions and contributed to Tregs development, which provided new sights into the development of a novel drug for cGVHD, e.g., artemisinin.

[Changes of WT1 mRNA expression level in patients with myelodysplastic syndromes after hypomethylating agents and its prognostic significance].

Objective: To monitor the WT1 mRNA level and its dynamic changes in patients with myelodysplastic syndromes (MDS) after hypomethylating agents (HMA) , as well as to assess the significance of WT1 mRNA levels and its dynamic changes in evaluating the efficacy of HMA and distinguishing the disease status of heterogeneous patients with stable disease (SD) . Methods: Bone marrow or peripheral blood samples of 56 patients with MDS who underwent hypomethylating agents (≥4 cycles) from November 2009 to March 2018 were tested by real-time quantitative polymerase chain reaction (PCR) to detect the expression of WT1 mRNA, and to observe the correlation between the dynamic changes of WT1 mRNA expression and clinical efficacy and prognosis of patients. Results: WT1 mRNA expression levels of MDS patients decreased significantly after 3 cycles of hypomethylating agent treatment. Besides, the WT1 mRNA expression levels of patients increased significantly after diseases progression. According to the dynamic changes of WT1 mRNA expression levels during SD, 45 cases could be further divided into increased group and non-increased group. In those SD patients with increased WT1 mRNA expression level, the ratio of suffering disease progression or transformation to AML was 95.65% (22/23) , whereas the ratio turned to be 9.09% (2/22) for the non-increased group (χ(2)=33.852, P<0.001) . Compared with those SD patients reporting no increase in WT1 mRNA expression level, the overall survival[17 (95%CI 11-23) months vs not reached, P<0.001] and progression-free survival [13 (95%CI 8-18) months vs not reached, P<0.001] of those SD patients reporting increase in WT1 mRNA expression level were significantly shorter. Conclusion: WT1 mRNA expression level is a useful indicator to assess the efficacy of hypomethylating agents in MDS patients. Especially in patients with SD, detection of the changes in WT1 mRNA expression level is able to predict disease progression and help to make clinical decision.

M(1)-like muscarinic acetylcholine receptors regulate fast-spiking interneuron excitability in rat dentate gyrus.

Cholinergic transmission through muscarinic acetylcholine receptors (mAChRs) plays a key role in cortical oscillations. Although fast-spiking (FS), parvalbumin-expressing basket cells (BCs) are proposed to be the cellular substrates of gamma oscillations, previous studies reported that FS nonpyramidal cells in neocortical areas are unresponsive to cholinergic modulation. Dentate gyrus (DG) is an independent gamma oscillator in the hippocampal formation. However, in contrast to other cortical regions, the direct impact of mAChR activation on FS BC excitability in this area has not been investigated. Here, we show that bath-applied muscarine or carbachol, two mAChR agonists, depolarize DG BCs in the acute brain slices, leading to action potential firing in the theta-gamma bands in the presence of blockers of ionotropic glutamate and gamma-aminobutyric acid type A receptors at physiological temperatures. The depolarizing action persists in the presence of tetrodotoxin, a voltage-gated Na(+) channel blocker. In voltage-clamp recordings, muscarine markedly reduces background K(+) currents. These effects are mimicked by oxotremorine methiodide, an mAChR-specific agonist, and largely reversed by atropine, a non-selective mAChR antagonist, or pirenzepine, an M(1) receptor antagonist, but not by gallamine, an M(2/4) receptor antagonist. Interestingly, in contrast to M(1)-receptor-mediated depolarization, M(2) receptor activation by the specific agonist arecaidine but-2-ynyl ester tosylate down-regulates GABA release at BC axons-the effect is occluded by gallamine, an M(2) receptor antagonist. Overall, muscarinic activation results in a net increase in phasic inhibitory output to the target cells. Thus, cholinergic activation through M(1)-like receptor enhances BC activity and promotes the generation of nested theta and gamma rhythms, thereby enhancing hippocampal function and associated performance.

Mesenchymal stem cell as salvage treatment for refractory chronic GVHD.

Refractory chronic GVHD (cGVHD) is an important complication after allogeneic hematopoietic SCT and is prognostic of poor outcome. MSCs are involved in tissue repair and modulating immune responses in vitro and in vivo. From April 2005 to October 2008, 19 patients with refractory cGVHD were treated with MSCs derived from the BM of volunteers. The median dose of MSCs was 0.6 × 10(6) cells per kg body weight. Fourteen of 19 patients (73.7%) responded well to MSCs, achieving a CR (n=4) or a PR (n=10). The immunosuppressive agent could be tapered to less than 50% of the starting dose in 5 of 14 surviving patients, and five patients could discontinue immunosuppressive agents. The median duration between MSC administration and immunosuppressive therapy discontinuation was 324 days (range, 200-550 days). No patients experienced adverse events during or immediately after MSC infusion. The 2-year survival rate was 77.7% in this study. Clinical improvement was accompanied by the increasing ratio of CD5+CD19+/CD5-CD19+ B cells and CD8+CD28-/CD8+CD28+ T cells. In conclusion, transfusion of MSCs expanded in vitro, irrespective of the donor, might be a safe and effective salvage therapy for patients with steroid-resistant, cGVHD.