| Background:Coronary artery disease (CAD) is the leading cause of death and hospital admissions worldwide. Although many treatment as percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG) etc. for acute coronary disease has been proved to improve mortality and morbidity, the prognosis for patients with ischemic heart failure without indications for PCI or CABG still remains poor. A high prevalence of hibernated myocardium in patients with ischemic heart failure and limited revascularization options in this population lead to unfavorable prognosis. Despite possible symptomatic relief and cardiac function improvement, surgical treatments limited in heart failure patients due to a high incidence of major adverse events. So it is crucial to develop alternative therapeutic strategies for ischemic heart failure. Extracorporeal cardiac shock wave therapy (CSWT) has been proved as a new, noninvasive, angiogenesis-based option in patients with refractory angina pectoris. Despite that exact mechanism of shock wave-induced angiogenesis is unknown, it is thought to be mediated through increased production of vascular endothelial growth factor and endothelial nitric oxide synthase (eNOS) activation. Thus, we hypothesized CSWT may help in improving prognosis of patients with ischemic heart failure. In this study, we explored clinical effect and possible mechanisms of CSWT in ischemic heart failure patients. Objective:To evaluate the feasibility, security and efficiency of CSWT in patients with ischemic heart failure. And to investigate the effect of CSWT on cell proliferation, cell cycle, the expression of VEGF and its receptor in cultured Human umbilical veins endothelial cells (HUVECs)Methods:Part I:Clinical trialFifty patients with ischemic heart failure and left ventricular ejection fraction (LVEF)<50%were randomized to either CSWT group (200shots/spot at0.09mJ/mm2for9spots,9times within3month/series, n=25) or control group (exactly same procedures without shock wave energy, n=25). Dual isotope simultaneous acquisition single-photon emission computed tomography(SPECT) with99mTc-sestamibi/18F-fluorodeoxy glucose (99m Tc-M IBI/18F-FDG) and Dobutamine stress echocardiograPhy (DSE) were performed before randomization and1month after CSWT to locate and evaluate viable region. Follow-ups were performed at0,3and6months after the therapy. Canadian Cardiovascular Society (CCS) class sores, New York Heart Association Classification of Heart Failure (NYHA), Seattle Angina Questionnaire(SAQ),6-min walk test(6MWT), Left ventricular ejection fraction (LVEF), left ventricular end-diastolic dimension (LVEDD), the dosage of nitroglycerin and the blood plasma of Brain natriuretic peptide (BNP),VEGF were compared between2groups at each time point during the6months follow-up. Study design was approved by the hospital Ethical Committee and consent forms were obtained from all patients.Part II:Basic studyUsing in vitro HUVEC method, the HUVECs cell lines were performed using different levels of energy (0：onchol group,1:0.035mJ/mm2,2:0.065mJ/mm2,3:0.10mJ/mm2,4:0.155mJ/mm2) shock wave treatment in vitro. MTT method and flow cytometer were used to observe cell proliferation and cell cycle. Real-time PCR were used to determine the mRNA level of VEGF and its receptors (VEGFR1/Flt-1, VEGFR2/KDR). VEGF in supernatant of cultured HUVECs were detected using ELISA kit. The expression of VEGFRl/Flt-1, VEGFR2/KDR were determined by flow cytometer. The number of cells which expressed Ki-67were redetected after inhibited VEGF, VEGFR1/Flt-1and VEGFR2/KDR using ZM323881(2uM)〠anti-human VEGF (10ul/ml). anti-human VEGFR1/Flt-1(10ul/ml) to validated the relationship between the promotion of endothelial cell proliferation by CSWT and VEGF/VEGFR.Results:Part I:Clinical trial1. All patients completed the study without procedural complications or adverse effect including4patients implanted permanent heart pacemaker and3patients with atrial fibrillation.2. Combining9m Tc-M IBI/18F-FDG-DISA SPECT with DSE, we located65viable myocardium segments in CSWT group, and62in control group.3.At3months follow-up(l month after CSWT), summed perfusion score (19.40±5.2vs.22.10±2.10P=0.006), metabolism score (21.10±5.28vs.23.80±3.08P=0.031),base peak systolic strain rate (PSSR)(-1.09±0.71vs.-0.62±0.36P=0.007) and strain PSSR (-1.36±0.23vs.-0.97±0.40P<0.001) were improved in CSWT group compared to baseline. When compared between control and CSWT group, the parameters above were better in patients in CSWT group (P<0.05).4. At3and6months follow-up, patients in CSWT group experienced continuous improvement in symptoms:NYHA (2.36±0.50vs.1.46±0.21vs.1.67±0.52, for baseline,3month and6month respectively, P=0.008), CCS (2.56±0.07vs.1.25±0.12vs.1.10±0.33, P=0.001), and Dosage of nitroglycerin use (3.77±0.55/week vs.2.18±0.34/week vs.2.51±0.43/week P=0.006). Seattle Angina Questionnaire (59.01±9.43vs.65.0±10.09vs.66.94±11.22, P=0.031), Six-minute walk test (m)(286.17±34.22vs.306.04±33.56vs.304.78±45.25, P=0.027), LVEF (%)(45.02±6.37vs.49.30±7.06vs.48.70±10.53, P=0.022) and LVEDD(mm)(63.10±11.36vs.60.13±7.70vs.58.10±4.01,P=0.033)were increased in CSWT group. Continuous increasing of VEGF(pg/ml)(127.61±31.69vs.147.29±34.37vs.159.56±55.36, P=0.022) and decreasing of BNP(pg/ml)(1702.25±122.75vs.1492.33±389.55vs.1334.78±227.91, P=0.001) were also observed in CSWT group. However, no significant changes were found in the control group (P>0.05).5. CSWT was independent predict factor for improved cardiac function, quality of life and echocardiography parameters after adjusting for known factors that may affect outcome.Part II:Basic study1.MTT test shows the OD value of group3and4were larger than the control group(P all<0.05).2.The proportions of the cells that expressed Ki-67were higher in all groups received CSWT than in control group(P<0.05), especially in group3and4(P<0.01).3. Cells in phase G0/G1reduced while cells in phase S increased were observed in all groups received CSWT compared to control group (P<0.05or0.01)4.When compared with the control group, the expression of VEGF and VEGFR2/KDR mRNA increased in almost all intervented groups (P<0.05)5.The level of VEGF in supernatant of cultured HUVECs in all groups received CSWT were higher than that of control group(P<0.05).6.The cells expressed VEGFRl/Flt-1were more in group1,3and4than in the control group(P all<0.05), while cells expressed VEGFR2/KDR were more in group2,3and4than in the control group(P all<0.05).7.Among cells received CSWT after inhibiting VEGF or VEGFR1/Flt-1or VEGFR2/KDR, the proportions of the Ki-67expressing cells were lower compared to those receiving CSWT without any inhibitors.Conclusion:1. CSWT improved the perfusion and metabolism of viable myocardium and then ameliorated the symptom, cardiac function, quality of life and exercise tolerance in ischemic heart failure patients, and could be considered as a new, non-invasive, safe and efficient therapy for these patients. 2. Combining9m Tc-M IBI/18F-FDG-DISA SPECT with DSE to locate/evaluate the viable myocardium segments can guarantee the accuracy and effect of CSWT.3. CSWT could up-regulate the level of VEGF in patients with ischemic heart failure which chould be one of the mechanism of CSWT.4. The most appropriate candidates for CSWT might include patient with ischaemic heart failure, patients with permanent heart pacemaker or atrial fibrillation may also benefit from CSWT.5. CSWT could increase the proportion of HUVECs in the period of proliferation, so as to promote cell proliferation, especially with the energy of0.10mJ/mm2and0.155mJ/mm2.6. CSWT could enhance gene expression of VEGF and VEGFR2/KDR, especially with the energy of0.10mJ/mm2.7. CSWT can improve the protein expression of VEGF and it’s receptor (VEGFRl/Flt-1and VEGFR2/KDR), especially with the energy of0.10mJ/mm2.8. After inhibiting VEGF or VEGFRl/Flt-1or VEGFR2/KDR, the effect of CSWT in endothelial cell proliferation were weakend, thus VEGF and its receptor may play an important role in the mechanism of angiogenesis of CSWT. |
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