Lu Wenbin, Tang Yong, Zhang Ziwei, Zhang Xiaofeng, Yao Yuyu, Fu Cong, Wang Xin, Ma Genshan
Department of Cardiology, Zhongda Hospital Affiliated with Southeast University China ; Department of Cardiology, The Second Hospital Affiliated with Southeast University China.
Department of Cardiology, Zhongda Hospital Affiliated with Southeast University China.
Am J Transl Res. 2015 Mar 15;7(3):587-97. eCollection 2015.
Ischemia related inflammation is the most critical factor for the survival of transplanted mesenchymal stem cells (MSCs), and strategies for controlling excessive inflammation after acute myocardial infarction (AMI) are essential and necessary for cell transplantation therapy. Our present study tested the effect of decreased Ly6C(high) monocytes on mouse MSCs transplantation after AMI.
BALB/c AMI mice were treated systemically with a CCR2 antagonist (RS 504393, 2 mg/kg, subcutaneously) or normal saline (control group). Next, 10(5) EdU-labeled MSCs were administered by intramyocardial injection to the mice in each group. TUNEL kits were used to identify the apoptotic cardiomyocytes in the infarct. The slides of the infarct border zone were stained with wheat germ agglutinin to measure the vessel density, and anti-myosin heavy chain eFluor 660 was used to measure the cardiac myosin-positive area. A transwell chamber was used to examine the interactions between Ly6C(high) monocytes and MSCs. The inflammatory cytokines expressed by Ly6C(high) monocytes and the SDF-1 expressed by MSCs were detected using ELISA kits. MSC viability was further examined by MTT and mitochondrial membrane potential assays by flow cytometry using JC-1 kits.
We first observed the increased survival of transplanted MSCs (11.2 ± 3.4/mm(2) vs. 3.5 ± 1.6/mm(2), p < 0.001), and the decreased apoptosis of cardiomyocytes (11.20% ± 3.55% vs. 20.51% ± 8.17%, p < 0.001) in the infarcts at 3 days in the CCR2 antagonist group. An increased number of capillaries and small arterioles (139.6 ± 21.7/mm(2) vs. 95.4 ± 17.6/mm(2), p < 0.001) and an increased cardiac myosin-positive area (17.9% ± 6.6% vs. 11.8% ± 3.5%, p < 0.001) were also observed in the infarct zone at 21 days post MSC infusion in the CCR2 antagonist group. In addition, a significantly increased LvEF% (50.17 ± 10.06 vs. 45.44 ± 9.45, p < 0.001) was detected at the same time compared to the control mice. We further demonstrated that both the mitochondrial membrane potential of the MSCs (0.45 ± 0.11 vs. 3.4 ± 0.3, p < 0.001) and stromal cell-derived factor-1 (SDF-1) secreted by the MSCs significantly decreased (80.77 ± 39.02 pg/ml vs. 435.5 ± 77.41 pg/ml, p < 0.001) when co-cultured with Ly6C(high) monocytes. This is possibly mediated by the over-expressed cytokines secreted by the Ly6C(high) monocytes compared to the Ly6C(low) monocytes, including IL-1 (139.45 ± 30.44 vs. 80.05 ± 19.33, p < 0.001), IL-6 (187.82 ± 40.43 vs. 135.5 ± 22.09, p < 0.001), TNF-α (121.77 ± 31.65 vs. 75.3 ± 22.14, p < 0.001) and IFN-γ (142.46 ± 27.55 vs. 88.25 ± 19.91, p < 0.001).
缺血相关炎症是移植间充质干细胞（MSCs）存活的最关键因素，控制急性心肌梗死（AMI）后过度炎症的策略对于细胞移植治疗至关重要且必不可少。我们目前的研究测试了减少Ly6C（高）单核细胞对AMI后小鼠MSCs移植的影响。
将BALB/c AMI小鼠全身给予CCR2拮抗剂（RS 504393，2mg/kg，皮下注射）或生理盐水（对照组）。接下来，通过心肌内注射将10（5）个EdU标记的MSCs给予每组小鼠。使用TUNEL试剂盒鉴定梗死区凋亡心肌细胞。梗死边缘区的切片用小麦胚芽凝集素染色以测量血管密度，并用抗肌球蛋白重链eFluor 660测量心肌肌球蛋白阳性面积。使用Transwell小室检测Ly6C（高）单核细胞与MSCs之间的相互作用。使用ELISA试剂盒检测Ly6C（高）单核细胞表达的炎性细胞因子和MSCs表达的SDF-1。通过MTT进一步检测MSCs活力，并使用JC-1试剂盒通过流式细胞术检测线粒体膜电位。
我们首先观察到CCR2拮抗剂组在第3天时梗死区移植MSCs的存活率增加（11.2±3.4/mm（2）对3.5±1.6/mm（2），p<0.001），心肌细胞凋亡减少（11.20%±3.55%对20.51%±8.17%，p<0.001）。在输注MSCs后第21天，CCR2拮抗剂组梗死区还观察到毛细血管和小动脉数量增加（139.6±21.7/mm（2）对95.4±17.6/mm（2），p<0.001）以及心肌肌球蛋白阳性面积增加（17.9%±6.6%对11.8%±3.5%，p<0.001）。此外，与对照小鼠相比，同时检测到左室射血分数（LvEF%）显著增加（50.17±10.06对45.44±9.45，p<0.001）。我们进一步证明，当与Ly6C（高）单核细胞共培养时，MSCs的线粒体膜电位（0.45±0.11对3.4±0.3，p<0.001）和MSCs分泌的基质细胞衍生因子-1（SDF-1）均显著降低（80.77±39.02 pg/ml对435.5±77.41 pg/ml，p<0.001）。这可能是由Ly6C（高）单核细胞与Ly6C（低）单核细胞相比分泌的过表达细胞因子介导的，包括IL-1（139.45±30.44对80.05±19.33，p<0.001）、IL-6（187.82±40.43对135.5±22.09，p<0.001）、TNF-α（121.77±31.65对75.3±22.14，p<0.001）和IFN-γ（142.46±27.55对88.25±19.91，p<0.001）。
