A Study On The Clinical Effect And Mechanism Of Extracorporeal Cardiac Shock Waves Therapy In Coronary Heart Disease

Background:The current management of coronary artery disease (CAD) has three major therapeutic options including medical treatment, percutaneous coronary intervention (PCI), and coronary artery bypass grafting (CABG). However, these approaches are invasive and often inadequate in the treatment of advanced CAD and are associated with serious cardiovascular risks and complications. Prognosis for patients diagnosed with end stage CAD without indication of PCI or CABG surgery is poor.Angiogenesis by gene or cell therapy may be effective but it require invasive procedures such as open-chest surgery or catheter intervention and is still at a preclinical stage. Thus, it is imperative that an effective and noninvasive therapy for ischemic cardiomyopathy be developed. The extracorporeal cardiac shock waves therapy (CSWT) just appears in this background, and in vitro and animal experiments has already demonstrated that the low energy shock wave can promote injured tissues to produce nitric oxide (NO), up-regulate the expression of the receptor of vascular endothelial growth factor (VEGF), so as to promote the new capillary growth in the ischemic tissues and accelerate the collateral circulation establishment, improve myocardial ischemia, increase heart ejection fraction, reduce left ventricular remodeling and lower mortality. But the mechanism of improving ischemic heart microenvironment, promoting angiogenesis inside ischemic tissue is unclear. Therefore, it is very important to further study on the mechanism of CSWT promoting angiogenesis in vitro, find the action targets.PartⅠ:The clinical efficacy and safety of CSWT in CADPurpose:To explore the effectiveness, safety and the mechanism of CSWT treatment in CAD, summarize the CSWT methodology and technical details, and seek the population suitable to CSWT treatment.Methods:A total of 26 patients with history 1-16 years of old myocardial infarction, stable and unstable angina pectoris admitted to the Cardiology Department from August 2008 to December 2010 were enrolled in this study. All patients were performed the multi-slice spiral CT coronary angiography and demonstrated mild and severe arterial stenosis, regardless of whether they were operated with PCI or CABG and received standard medical treatment and the CSWT procedure was explained to each patien. The reasons for hospitalization included repeated episodes of chest tightness, shortness of breath, and fatigue after PCI/CABG. Exclusion criteria were absence of the inclusion criteria, acute myocardial infarction (AMI) with 1 month, uncontrolled heart failure, and severe chronic obstructive pulmonary disease. Prior to initiation of CSWT, all patients received Dobutamine stress echocardiography (DSE) and 99mTc-MIBI myocardial perfusion imaging (MPI) at rest and stress state to identify areas of ischemic myocardium. Under the guidance of echocardiography, we applied shock wave in R-wave-triggered manner with low energy (0.09mJ/mm2) at 200 shoots/spot for 9 spots (-1-0-+1combination). During the procedure, ECG, blood pressure, breathing, and blood oxygen saturation were concurrently monitored and vital signs and symptoms including palpitations, chest pain, breathing difficulty, and dizziness was closely inquired. The efficacy of CSWT was assessed using the Canada Cardiovascular Society (CCS) angina scale, NYHA class, SAQ scale (angina), 6-min walk test (6MWT) and nitroglycerin dose. Myocardial perfusion, regional myocardium function and wall motion were evaluated by MPI, peak systolic strain rate (PSSR) and M-mode measure at rest and Dob stress state. The left ventricular ejection fraction (LVEF), left ventricular diastolic diameter (LVDd) was measured by Simpson method. The myocardial enzymes were respectively tested before and after the third time and the ninth times CSWT.Results:All of the 26 patients were successfully completed CSWT treatment, without dyspnoea, heart failure, faint, embolism, palpitations, bleeding or shock these complications. A total of 40 viable ischemic myocardial segments were identified by stress MPI and DSE.The 26 patients after treatment significantly improved symptoms [6MWT (360.69±116.79) m vs (434.15±86.29) m, NYHA (1.85±0.21) vs (1.23±0.08), SAQ (67.58±13.03) vs (77.54±10.84), all P<0.01, CCS (1.85±0.15) vs (1.19±0.08), P<0.001]; The Nitroglycerin dosage significantly reduced (1.00±0.27 vs 0.50±0.19, P<0.01). The rest and stress MPI significantly increased [rest (1.04±0.19) vs (2.16±0.16), P<0.001; stress (0.80±0.16 vs 1.40±0.16), P<0.01]; The rest PSSR had no significant changes (1.04±0.43vs 1.21±0.62, P>0.05) while the stress PSSR significantly enhanced (1.35±0.66 vs 2.02±1.00, P<0.001). The LVDd and LVEF had no significant changes [LVDd (53.81±7.34) mm vs (53.27±6.68) mm; LVEF (53.69±12.70)% vs (55.27±10.87)%, P>0.05], Myocardial enzymes had no significant changes before and after the treatment (P>0.05).Conclusions:①The CSWT is a noninvasive, safe and effective non-invasive intervention in the management of patients with CAD, and at the early stage it already can relieve the angina pectoris symptom, improve myocardial perfusion and the life quality.②The CSWT possibly plays therapy in CAD through promoting the myocardial angiogenesis in ischemia area.③The CSWT therapeutic population seems to be expanded to those without end-stage CAD.④DSE combined with MPI is a preferable method to locate the viable ischemic myocardial segments and guarantees the accuracy and effect of CSWT.⑤The scope and energy of CSWT should be implemented individually based on the patient's specific situation.PartⅡ:The study on CWST inducing EPCs proliferation and stimulating ischemic myocardial angiogenesisPurpose:To investigate the changes of EPCs proliferation and related factors in peripheral blood after the CSWT treatment.Methods:A total of 26 CAD patients undergoing extracorporeal cardiac shock wave therapy were selected.Mononuclear cells obtained from peripheral blood were cultured in EGM-2-MV medium. Cell morphology and the number of colonies formed were observed and analyzed. After 7 days of culture, adherent cells were analyzed and counted. Whether EPCs differentiated or not was identified by laser confocal microscopy; the number of circulating EPCs were studied by flow cytometry; the plasma level of VEGF, IL-8, SDF-1, and MMP-9 was determined by enzyme linked immunosorbent assay.Results:The cultured EPCs and EPC-CFU number in vitro were significantly increase after therapy [EPCs (18.85±4.30) cell/high power field vs (30.12±6.77) cell/high power field(5.08±1.79) cell/high power field vs (12.27±2.75) cell/high power field, all P<0.001],Circulating EPCs number were significantly increased [(0.015±0.003)% vs (0.021±0.005)%, P<0.001],VEGF, IL-8 level were significantly increased[VEGF (120.26±19.85) pg/ml vs (155.19±24.67)pg/ml, IL-8 (21.81±5.94) pg/ml vs (149.70±44.11)pg/ml, all P<0.01], whereas SDF-1 and MMP-9 had no significant changes[SDF-1(2750.87±636.74)pg/ml vs (2700.47±415.19)pg/ml,MMP-9 (19.66±3.96)ng/ml vs (18.55±3.78)ng/ml, all P>0.05], compared with the group before treatment.Conclusions:The CSWT appears to promote the expression of VEGF and IL-8 proteins, also stimulates the EPCs proliferation, significantly increases the EPCs and improves its function in peripheral blood, whereas the CSWT likes not influence so obviously on the expression of SDF-1,MMP-9.PartⅢ:The study on Human umbilical vein endothelial cells (HUVECs) proliferation and the best energy in CSWTPurpose:To explore the different intensity of ultrasonic shock energy how to influence the human umbilical vein endothelial cells (HUVECs) proliferation, differentiation and the related cytokines, and ascertain the best energy of CSWT.Methods:The HUVECs cell lines were performed different levels of energy (0,0.03, 0.09,0.18,0.24mJ/mm2) shock wave treatment in vitro, Utilizing CCK8 colorimetric method to detect HUVECs proliferation, real time PCR, Western blotting and Flow Cytometry were utilized to detect the changes in mRNA and protein of VEGF,IL-8,ICAM-1 before and after CSWT treatment.Results:The 0.09mJ/mm2 shock energy significantly promoted the HUVECs proliferation (P<0.05). The results from real time PCR revealed that 0.09mJ/mm2 treatment markedly increased the expression of VEGF,IL-8,ICAM-1(P<0.001, respectively)compared with the non-treated control,the expression of ICAM-10.03 mJ/mm2 group were increased compared with the control group (P<0.01) While the expression of VEGF, IL-8 showed no significant changes(P>0.05, respectively),0.18mJ/mm2 treatment markedly increased the expression of VEGF (P<0.001) compared with the non-treated control While the expression of ICAM-1, IL-8 showed no significant changes(P>0.05, respectively),0.24mJ/mm2 treatment no significant increased the expression of VEGF, IL-8, ICAM-1 (P>0.05, respectively) compared with the non-treated control. Western blot analysis and Flow Cytometry showed that the expression of VEGF, IL-8, ICAM-1 in 0.09mJ/mm2 group were marked increased than the control group group(P<0.05, respectively); the expression of VEGF in 0.03mJ/mm2 group was obviously higher than the control group (P<0.05) While the expression of ICAM-1, IL-8 was not obviously higher than the control group P>0.05, respectively); the expression of VEGF in 0.18,0.24mJ/mm2 group was obviously higher than the control group (P<0.05) While the expression of ICAM-1, IL-8 was not obviously higher than the control group(P>0.05, respectively).Conclusions:①The different extracorporeal shock wave energy promote the HUVECs proliferations in different degree, the effect of 0.09mJ/mm2 energy is the most evident.②The 0.09mJ/mm2 energy increase the expression of IL-8,ICAM-1 mRNA and protein most significantly.③The 0.09mJ/mm2 energy increased the expression of VEGFmRNA and protein most remarkably.④The 0.03-0.24 mJ/mm2 energies also have some effects in facilitating the secretion of VEGF, IL-8 and ICAM-1.In this study, the CAD patients and HUVECs cell culture in vitro were administrated with CSWT. Our findings suggest that the CSWT is a safe, simple treatment and it can improve the curative effect and those objective indicators about myocardial perfusion. May be this effective mechanism is that the appropriate shock wave promotes some cytokines secretion, expression, promotes the EPCs and HUVECs proliferation, and thus facilitates the angiogenesis in myocardial ischemia area.