Effects Of Low Frequency Pulsed Magnetic Fields On Cardiac Microvascular Endothelial Cells, Cardiac Myocytes And Bone Marrow-derived Endothelial Progenitor Cells

Background and objectivePulsed electromagnetic field (PEMF) has been demonstrated to be a safe and effective non-invasive physical treatment of treating bone fractures. Some study showed that the application of electromagnetic fields (EMFs) improved the vascularization of human umbilical vein endothelial cells and bovine aortic endothelial cells. The effects of low frequency pulsed magnetic fields (LF-PMFs) varies when the cells are exposed to different frequency, time and field intensity. This is known as the"window"effects. Cardiac microvascular endothelial cells (CMECs) and cardiac myocytes (CMs) are the main components in heart tissue. Impaired function of both of them are closely related to the occurrence and development of heart disease, while the original cells of the endothelial cells that endothelial progenitor cells (EPCs) are important cells in the procedure of angiogenesis and cardiac tissue engineering. In this study, we investigated the impact of LF-PMFs on cell viability and 3D tube-like structure formation of CMECs, EPCs, CMs and CMEC-CM coculture system with different intensity and a fixed frequency and exposure time. Changes caused by LF-PMFs on proliferation, apoptosis, migration and tube formation of CMECs and EPCs which represent important relevant events of angiogenesis were examined, too. We also observed cytoskeleton and ultrastructures changes of CMECs in response to LF-PMFs.MethodsPart I: Effects of LF-PMFs on CMECs, CMs and CMEC-CM coculture system1. CMECs and CMs were isolated by enzyme digestion and differential adhesion method. CMECs were characterized by finding Weible-Palade (WP) body under transmission electron microscopy.2. The cells were randomly divided into 4 groups (control, 1.0mT, 1.4mT and 1.8mT). CMECs and CMs were exposed to 15Hz square wave LF-PMFs for 2h per day with field intensity of 1.0mT, 1.4mT and 1.8mT, respectively. After 5d, effects of LF-PMFs on CMECs proliferation was detected with cells growth curve, MTT assay and flow cytometry (FCM). Cellular apoptosis was assessed by flow cytometry. Ultrastructures of CMECs were observed under transmission electron microscopy. Cells scratch assay and transwell migration assay were used to evaluate CMECs migration. Changes of cytoskeleton were detected by immunofluorescent staining and CMECs angiogenic potential was measured by tube formation assay and 3D- culture assay.3. CMEC-CM coculture system: effects of LF-PMFs on CMECs proliferation was detected with MTT assay in both CMEC-CM coculture system and CMECs culture alone.4. CMEC-CM coculture system 3D tube-like structures formation assay and hematoxylin-eosin (HE) staining. 4 experimental groups were established: (1) CMs alone; (2) CMECs alone; (3) CMEC-CM coculture system (in a 1:1 CMEC-CM cell ratio); (4) CMEC-CM coculture system (in a 1:1 CMEC-CM cell ratio) were exposed to 1.4mT LF-PMFs.PartⅡ: Effects of LF-PMFs on EPCs1. EPCs were isolated from cell suspension of bone marrow by density gradient centrifugation. EPCs were characterized by Dil-ac-LDL and FITC-UEA-1 double staining.2. The cells were randomly divided into 4 groups (control, 1.0mT, 1.4mT and 1.8mT). After 5 days, we determined the effects of LF-PMFs on EPCs proliferation by MTT assay. Cell cycle and apoptosis were analysed by flow cytometry. EPCs migration ability were examined by cell scratch assay and EPCs angiogenic potential were assessed by tube formation assay and 3D- culture assay.Results1. CMECs were cultured and identified in vitro successfully.2. 1.4mT magnetic field accelerated CMECs proliferation. The peak of cells growth curve was higher and moved forward, and the percentage of cells at S phase increased significantly compared with the control group〔(13.10±0.15)% vs (3.90±0.18)%, P<0.01〕. No obvious changes were detected on cellular apoptosis rate. After exposed to 1.4mT magnetic fields, CMECs ultrastructures changed and showed more viable and powerful. Their nuclear nucleolus became bigger and clearer than the control group. The number of mitochondria increased and the endoplasmic reticulum was rich etc. Magnetic fields facilitated migration and tube formation of CMECs significantly. The effects were correlated with the intensity of magnetic fields (1.4mT>1.8mT>1.0mT). The cell migratory percentage in 1.4mT group was 86.1% while in the control group was only 45.3%. Immunofluorescent staining showed that cytoskeleton components reorganized after exposed to LF-PMFs (2 hr /day) for 5 days. When CMECs were cultured on collagen mixture and in 3D cultured, they were able to spontaneously reorganize in tube-like structures. After persistently cultured for 5d, they grew to cord-like structures which were similar to vascular trees in vivo. The numbers of tube-like and cord-like structures of CMECs exposed to magnetic fields were much more than the control group.3. The viability of CMs was enhanced when exposed to 1.4mT and 1.8mT LF-PMFs (2 hr /day) for 5 days (0.229±0.021 vs 0.196±0.012 ,0.252±0.012 vs 0.196±0.012,P<0.05). No obvious changes were detected on cellular apoptosis rate in CMs.4. MTT assays of CMEC-CM coculture system and 3D tube-like structures formation assay confirmed that both coculture with CMs and exposure to LF-PMFs increased proliferation and angiogenic capacity of CMECs significantly, and showed a more obvious increasing in double effects.5. EPCs were cultured and identified in vitro successfully. 6. After 5 days of intervention, 1.0mT and 1.4mT magnetic fields accelerated EPCs proliferation. The percentage of cells at the (S+ G2) phase was increased significantly. 1.8mT magnetic field also accelerated EPCs proliferation. However, it didn't change the distribution of cell cycle. FCM results suggested that magnetic field induced EPCs apoptosis. EPCs had poor migration ability in vitro and didn't show difference after exposure to magnetic field. All three magnetic fields elevated EPCs angiogenic potential, while 1.0mT and 1.4mT are better than 1.8mT.ConclusionsDifferent intensity of magnetic fields provides different biological effects.In this study, 1.4mT LF-PMFs significantly accelerated proliferation, migration and tube formation of CMECs. LF-PMFs promoted reorganization of cytoskeletal components of CMECs. No cytotoxicity was induced by LF-PMFs with low field intensity (1.0mT, 1.4mT, 1.8mT) and short exposure time (2 hr/d, 5 days) in both CMECs and CMs. Proliferation and vascular capacity of CMECs increased significantly both in the coculture system with CMs and when exposured to LF-PMFs. LF-PMFs had double effects on EPCs which exhibited as accelerating EPCs proliferation and tube formation while inducing their apoptosis.These findings suggest a potential role of LF-PMFs in the angiogenesis in ischemic myocardium and in the cardiac tissue engineering.