The Angiogenesis Effects And Mechanism Of The Applied Low Voltage Steady Direct Current Electrical Fields On An Acute Myocardial Infarction Rabbit Model

1. Background and purposeCoronary artery disease (CAD) has become one of the leading causes of morbidity and mortality world wide. How to find effective methods to treat CAD is still the main topic for the clinical cardiology. Although therapies such as percataneous coronary intervention (PCI), coronary artery bypass graft (CABG), or intensive medicine pharmacotherapy are available, some patients still could not restore normal myocardium infusion. Approximately 20%~70% of CAD patients could not have PCI or CABG treatment or the complete revascularization treatment they had accepted were incomplete, thus further increasing the mortality and worsening the clinical prognosis of CAD. Therapeutic angiogenesis is a method which can promote the angiogenensis in the ischemia myocardium and then restore the coronary perfusion. This method has become the alternative treatment for the patients with serious ischemia heart disease.Electric potential difference is generated around an injured tissue. The electric current generated at the wound area and the surrounding epithelial area is a steady direct current (DC). Injured and ischemic tissues such as myocardium are also polarized electrically, these endogenous EFs (EEFs) produce biological effects on promoting the wound healing by facilitating cell division, cell proliferation, cell directional migration toward the wound, and by remodeling and stimulating the release of various cytokines and growth factors such as vascular endothelial growth factor (VEGF). When EEFs is insufficient to stimulate the wound healing, application of DC to the injured and the non-injured regions could greatly benefit this process. Application of 150 ~ 400 mV/mm DC EFs could trigger the angiogenesis, the maturation of the neovessel and the forming of functional collateral circulation. Ischemia of the tissue is the original stimulating factor of angiogenesis and VEGF is the specific key growth factor for the angiogenesis. The expression of VEGF was regulated by the phosphatidylinositol 3-kinase (PI3K)/ Protein kinase B (Akt) pathway through hypoxia-inducible factor-1α(HIF-1α). Signal transduction via the PI3K/Akt pathway plays a pivotal role in regulating cell proliferation, survival, migration, development, differentiation, and apoptosis. Phosphatase and tensin homologue deleted from chromosome 10 (PTEN) can negatively regulate the PI3K/Akt pathway and they are crucial for the electrotaxis-regulated wound healing of whole tissue.In this study, we evaluated the safety and efficacy of low-voltage DC electrical stimulation of angiogenesis in rabbits with myocardial infarction. We also observed and monitored its effect on the cardiac function after the angiogenesis was completed. The possible mechanism of the applied low voltage DC EFs to the angiogenesis in the marginal zone of the infarction myocardium was also investigated.2. MethodsPartⅠ. The safety of the applied steady DC EFs on the myocardiumThirty Japanese white rabbit were randomly divided into treatment group and control group. Myocardial infarction (MI) was induced by ligation of the left circumflex (LCX) of coronary artery. Two platinum electrodes were placed directly on the epicardium on either side of LCX artery. The stimulated electrodes were made by 99% Platinum. The length, width and the thickness of the electrodes were 10.0mm, 1.0mm and 0.5mm respectively. Low-voltage DC stimulation (4.0 V/cm, 30 min/day) was performed in the treatment group immediately after surgery until four weeks postoperation. Cardio-electrophysiological, respiratory, hematological, blood biochemical, histopathological and immunohistochemical parameters at the marginal zone of myocardial infarct were compared between the treatment and the control groups.PartⅡ. Angiogenesis effect of the DC EFs on the marginal zone of myocardial infarct in rabbitNinety-five Japanese white rabbit were randomly divided into sham operative group (SH group), myocardial infarction group (MI group) and electric fields (EFs) group. The EFs group was then divided into 2.0 V/cm EFs group (2EFs) and 4.0 V/cm EFs group (4EFs) according to the different EFs intensity. There were 5 rabbits in each group and the electrodes were implanted in all rabbits, but only the EFs group received electrical stimulation. Low-voltage DC stimulation (2.0V/cm or 4.0 V/cm, 30 min/day respectively) was performed in the EFs group immediately after surgery until four weeks postoperation. Samples were collected at 1, 3, 5, 7 days and 2, 4 weeks post-operation in each group except the SH group. Hemodynamics parameters of left ventricular and femoral artery pressure were detected with multiplying channel physiograph. The weights of the left and right ventricular were measured after the rabbit were sacrificed. The infarct size of the myocardium was detected with Masson’s staining. The capillary density were counted using VIII immunohistochemisty staining. The expression of monocyte chemoattractant protein-1 (MCP-1) were detected using RT-PCR while the expression of matrix metalloproteinases -2 (MMP-2) were detected using RT-PCR and Western-blot.PartⅢ. The mechanism of the angiogenesis effect of DC EFs after myocardial infarctionBased on the results of part II, another eighteen rabbits were included in order to observe the effect of rosiglitazone. The expression of VEGF in the myocardium was detected using immunohistochemisty, RT-PCR and Western-blot methods. HIF-1αwas detected with immunohistochemisty and RT-PCR methods. The expressions of PTEN before and after the rosiglitazone interfere were detected with RT-PCR and Western-blot methods. The capillary density after rosiglitazone interfere was calculated with VIII immunohistochemisty staining.3. Results(1) The acute MI was induced by ligation of the LCX artery after the sternotomy. The purpose of using this sternotomy approach was to simplifiy the surgical procedure because no tracheal incision or cannula was needed using this method. The infection rate or hemorrhage incidence from the surgery was decreased and there were no pleura or vessel injury.(2) The size of the electrode tip used in our study was increased about 2/3 than the one used in the early study and there had a sulcule on the tip to help fixing the electrode. The electrodes were then fixed up on the back of the neck running through a subcutaneous tunnel. The displacement rate of the electrodes was dramatically reduced which has benefit the safety of the rabbits.(3) Application of low voltage steady DC EFs of 4.0V/cm, 30min/d was safe for the rabbits in terms of their cardio-electrophysiological, respiratory, hematological, blood biochemical, histopathological and the general physical conditions in the entire 4 weeks post-operation compared with the control group. (4) Low-voltage DC electrical stimulation with electrodes directly placed on the epicardium could induce and/or promote angiogenesis in the post-MI rabbit heart. The capillary density in EFs group was significantly higher than that of MI group, especially in the 4EFs group. The highest capillary densities of the EFs groups were observed in the EFs 5 days post-operation. The capillary density decreased gradually from then on and stabilized on 4th week post-operation at the end,. At the mean time, myocardial infarction areas of the EFs groups decreased and left ventricular function of the EFs groups were also improved significant.(5) The expression of the MCP-1 protein in the myocardium tissue in the MI group and the EFs group was increased significantly compared to those of the SH group, while there were no difference between the EFs group and the MI group. The expression of the MMP-2 was reduced in the EFs group compared to that of the MI group on the 4th week post-operation.(6) The expressions of HIF-1αand VEGF in the ischemic myocardium were higher than those of the SH group. Following the intervention of the EFs, expressions of the HIF-1αand VEGF in the ischemic myocardium increased within a certain time period.(7) The expression of PTEN in the normal myocardium was high and it decreased after the MI was induced. The expressions of PTEN in EFs group in the early 3 days were rather lower than those of the MI group. But the expressions of PTEN in EFs group increased quickly from then on and approached to a high level after four weeks of surgery.(8) The expression of PTEN increased after the treatment of rosiglitazone, meanwhile the expressions of the HIF-1αand VEGF decreased. All these changes lead to the reduction of the capillary density in the marginal zone of the infarcted myocardium. These results indicated that the low voltage steady DC EFs may have potentially improved the angiogenesis in the marginal zone of the infarcted myocardium through the PTEN / PI3K / Akt / HIF-1α/ VEGF pathway.4. Conclusion(1) Compared to the control group, application of 4.0V/cm, 30min/d low voltage steady DC EFs was safe for the rabbits in terms of their cardio-electrophysiological, respiratory, hematological, blood biochemical, histopathological and the general physical conditions in the entire 4 weeks post-operation. (2) The expression of MMP-2 was significantly decreased in the EFs group compared to that of the MI group after four weeks surgery, while the infarction area was reduced. The left ventricular myocardial hypertrophy index was also decreased and the left ventricular function improved significantly, especially in the 4EFs group.(3) The capillary density of the EFs group in the marginal zone of the infarcted myocardium was higher than that of the MI group, especially in the 4EFs group. However, the capillary density was not increased further despite the extension of the electrical stimulation application time.(4) The expression of PTEN of the EFs group was lower than that of the MI group at the beginning and the expressions of VEGF and HIF-1αwere increased at the same time. The expression of PTEN of the EFs group increased rapidly along with the EFs intervention. The rosiglitazone treatment could promote the expression of PTEN and inhibit the expressions of VEGF and HIF-1α, thus leading to the reduction of the capillary density. These results suggested that the low voltage steady DC EFs could promote the angiogenesis in the marginal zone of the infarcted myocardium through PTEN/PI3K/Akt/HIF-1α/VEGF pathway.