Effects And Mechanism Of Bone Marrow Mononuclear Cells Transplantation And Tanshinone IIA On Diabetes Accompanied By Ischeamic Heart Disease

BackgroundDiabetes is intimately correlated with cardiovascular disease. ATPⅢguidline indicated that diabetes has the same cardiac risk with coronary heart disease. In china, diabetic popularity of patients hospitalized for coronary heart disease is about 52.9%. Diabetes is associated with 2–4 times increased risk of CHD mortality compared with patients without diabetes. More than 65% of deaths in patients with diabetes are from cardiovascular causes. Current therapies for diabetes accompanied by ischeamic heart disease such as percutaneous coronary intervention (PCI),coronary artery bypass graft (CABG) and medical therapy can normalize coronary perfusion. However, many patients develop left ventricular (LV) remodeling and progressive heart failure after AMI. The restoration of cardiac function in such situations remains a major challenge.Stem cells may contribute to heart muscle repair in patients with AMI. In recent years, a variety of clinical trials have explored the hypothesis that BMMNC transplantation may enhance the recovery of LV function after AMI. The aim of the first part of the present study was to evaluate the safety and efficacy of bone marrow mononuclear cells (BMMNC) transplantation in patients with diabetes accompanied by ST-segment elevation myocardial infarction (STEMI).A variety of medicines have been used in the treatment of diabetes accompanied by ischemic heart disease including aspirin, clopidogrel, ACEI, statins and so on. TanshinoneⅡA (TSN), one of the most abundant components of tanshinones, exhibits a variety of cardiovascular activities including vasorelaxation and protection against ischemia-reperfusion (I/R) injury and anti-arrhythmic effects. However, the pretreatment effects and mechanisms of TSN on experimental acute myocardial infarction in diabetic rats are not well understood. Akt is known to regulate many survival pathways of the cardiac cells. Alongside other pathways, this regulates cardiac contractility and also cardiac apoptosis. Interestingly, the Akt activation has been reported to preserve cardiac function and prevent injury after transient cardiac ischemia. Therefore, the aims of the second part of the present study were 1) to determine whether TSN protects diabetic rats from I/R injury; 2) to identify whether the underlying mechanisms are associated with the phosphatidylinositol 3-kinase (PI3K)/ Akt- dependent pathway.AIM1. To evaluate the safety profile and efficacy of bone marrow mononuclear cells (BMMNC) transplantation for patients with diabetes accompanied by ST-segment elevation myocardial infarction (STEMI) at up to 4 years follow-up.2. After the establishment of the rat model of diabetes with cardiac ischemic/ reperfusion injury, effects of TSN on infarct size, left ventricular function and cardiomyocyte apoptosis were examined.3. To identify whether the underlying mechanisms of TSN are associated with the phosphatidylinositol 3-kinase (PI3K)/ Akt- dependent pathway.Methods1. Safety and efficacy of bone marrow mononuclear cells (BMMNC) transplantation in patients with diabetes accompanied by ST-segment elevation myocardial infarction (STEMI)1.1 STEMI patients with the culprit lesion in the left anterior descending artery proximal to the 2 diagonal branches were enrolled within 12 hours of the onset of symptoms. All of the patients gave written, informed consent. 86 patients were randomized into BMMNC (n=41) and control group (n=45). The number of diabetic patients is 10 in BMMNC group and 12 in control group.1.2 All of the patients received medication in accordance with current guidelines for the management of patients with diabetes accompanied by ST-elevation myocardial infarction. A detailed history was recorded. Blood samples, ECG, UCG, SPECT and coronary angiography data were collected.1.3 Bone marrow (40 ml) aspiration was conducted 7 days after successful PCI under local anesthesia. Density gradient centrifugation was used to isolate BMMNC. BMMNC was resuspended in 10 ml heparinized saline. Cell number was 5±1.2×108 , cell viability was 96±3.2% and CD34+ cell fraction was 1.8±0.6%.1.4 Cell suspension (10ml) containing around 5×108 BMMNC was injected into the infarct-related coronary artery. The control group did not undergo bone marrow aspiration and was injected with the same volume of heparinized saline as the BMMNC groups.1.5 Blood samples, ECG, UCG, SPECT and coronary angiography data were collected 1 month, 3 month, 1 year after cell transplantation. Coronary angiography was performed 6 month, 4 years after cell transplantation. Cumulative major adverse cardiac events (MACE), including cardiac death, non-fatal myocardial infarction and target lesion revascularization, were also recorded.2.Effects and mechanism of tanshinone IIA (TSN) on ischemia/reperfusion injury in diabetic rats2.1 Diabetes mellitus (DM) was induced in adult male Sprague-Dawley (SD) rats, weight 180 to 220 g by intraperitoneal injections (i.p.) of STZ (50 mg/kg). Repeated random blood glucose≥16.7mmol/L confirmed the establishment of diabetic rats.2.2 Seven weeks after STZ was given, TSN (5mg/kg) was administered by i.p. injection for seven days. Wortmannin (a specific PI3K inhibitor, 16μg/kg) was injected via the tail vein 10 min before TSN injection. Sham group and I/R group received the same volume of 0.9% saline by i.p. injection for seven days.2.3 Eight weeks after STZ was given, rats were anesthetized with 3% isoflurane, I/R was produced by left anterior descending artery (LAD) ligation.2.4 Cardiac function was continuously determined by invasive hemodynamic evaluation methods before STZ injection, 7w after STZ injection, before and after the I/R injury (±LV dp/dt max).2.5 Echocardiography was conducted at 24 h after infarction. Sedated rats (3% isoflurane) were studied on an echocardiography system (Sequoia Acuson, Siemens; 15-MHz linear transducer) before STZ injection, 7w after STZ injection, before and after the I/R injury (ESV, EDV, LVEF).2.6 Evans blue and TTC staining evaluated the myocardial infarct size.2.7 TUNEL staining and caspase activity assay assessed the apoptotic index of cardiomyocytes.2.8 Western-Blot analysis evaluated Akt, p-Akt (Ser 473), NF-κB expression.2.9 The activity of MPO was measured spectrophotometrically at 460nm. The concentrations of TNF-αand IL-6 were measured by enzyme-linked immunosorbent assay (ELISA).Results1. Safety and efficacy of bone marrow mononuclear cells (BMMNC) transplantation for patients with diabetes accompanied by ST-segment elevation myocardial infarction (STEMI).1.1 BMMNC transplantation is safe for patients with diabetes accompanied by ST-segment elevation myocardial infarction (STEMI). MACE: One case (2.2%) of in-stent restenosis and one death in the control group was confirmed. One patient (2.4%) had transient acute heart failure in the BMMNC group.1.2 Echocardiography showed that LVEF improved both in the BMMNC and the control group. LVEF measured at 6 months (0.484±0.5 vs 0.457±0.6, p = 0.001), 1 year (0.482±0.7 vs 0.446±0.6, p<0.001) and 4 years (0.505±0.8 vs 0.464±0.8, p<0.001) follow up was increased significantly in the BMMNC group as compared to the control group. WMSI was also decreased in both the BMMNC and the control group. Comparison between the two groups at 6 months (1.43±0.01 vs 1.48±0.02, p = 0.04), 1 year (1.35±0.02 vs 1.41±0.02, p = 0.02) and 4 years (1.23±0.01 vs 1.37±0.03, p<0.001) after cell or standard therapy demonstrated significant differences in WMSI according to the regional wall motion analysis.1.3 There was a statistically significant increase in LVEF when comparing the BMMNC group to the control group (mean change 3.5 (Cl 95%, 1.4 to 5.5), p = 0.001). Also, the reduction in ESV was significantly greater in the BMMNC group as compared to the control group (mean change -7.6 (Cl 95%, -11.9 to -3.3), p<0.001). The change in WMSI was also significantly higher in BMMNC group than in control group (mean change -0.16(Cl 95%,-0.24 to -0.08), p<0.001).1.4 SPECT evaluation indicated that infarct size decreased in both the BMMNC and the control groups, although comparison of infarct size between the two groups showed no statistical difference at 4 years post-transplant (0.263±0.007 in BMMNC group vs 0.281±0.008 in control group, p = 0.10). Changes in infarct size between day 7 and 4 yr also showed no significant difference between the two groups (-9.5±0.9 in BMMNC group vs -7.1±0.9 in control group, p = 0.06).1.5 Diabetic patients with STEMI who underwent BMMNC transplantation had a significant improvement in LVEF measured at 4 years'follow up (47.9±0.6 vs 40.3±0.7, p<0.001). No significant improvement in LVEF was observed in diabetic patients with STEMI who did not underwent cell transplantation (43.1±0.8 vs 40.1±1.0, p=0.06).1.6 Changes in LVEF between baseline (Day 7) and 4 years were greater in non-diabetic patients as compared with diabetic patients (BMMNC: (9.7±1.0) vs (7.6±0.9), p = 0.258; Control (6.7±0.8) vs (3.0±0.7), p = 0.014).2.Effects and mechanism of tanshinone IIA (TSN) on ischemia/reperfusion injury in diabetic rats2.1 TSN Preserves Left Ventricular Function after I/R Injury Hemodynamic measurements showed that TSN significantly enhanced + LV dp / dt max compared with the I/R group (4378.4±365.0 vs 3989.9±415.8 mmHg / s, p<0.05). The - LV dp / dt max was also significantly increased in the TSN group compared with the I/R group and the wortmannin group (4205.4±329.6 vs 3722.0±541.9 vs 3830.9±382.0 mmHg / s, respectively; p<0.05).Echocardiography showed that compared with I/R group, TSN group had significantly smaller decreases in LVEF (0.774±0.058 vs 0.716±0.054, p<0.05). LVEF was also significantly higher in TSN group compared with the wortmannin group (0.774±0.058 vs 0.713±0.045, p<0.05). TSN significantly inhibited the increase of LVESV compared with the I/R group (0.307±0.079 vs 0.397±0.071 ml, p<0.05) and the wortmannin group (0.307±0.079 vs 0.391±0.060 ml, p<0.05).2.2 TSN Decreases Infarct Size after I/R Injury in Rats with DiabetesEvans Blue and TTC staining showed that pretreatment with TSN decreased infarct size at 3 h (0.252±0.038 vs 0.327±0.027, p<0.05) after I/R injury compared with the I/R group. This effect was abolished by wortmannin administration (0.312±0.040 in wortmannin group vs 0.252±0.038 in TSN group, p<0.05).2.3 TSN decreases apoptotic index after I/R Injury in Diabetic RatsQuantitative analyses demonstrated that the number of TUNEL-positive cardiomyocytes was significantly less in the TSN group than in the I/R group (0.114±0.026 vs 0.191±0.023, p<0.05) and the wortmannin group (0.114±0.026 vs 0.154±0.024, p<0.05).Caspase-3 activity indicated that TSN significantly decreased caspase-3 enzymatic activity compared with the I/R group (60.0±17.2 vs 114.7±13.0, p<0.05) and the wortmannin group (60.0±17.2 vs 108.9±13.4, p<0.05).2.4 Western blot analysis revealed that TSN treatment was associated with a significant increase in phosphorylation of Akt in cardiac tissue that was exposed to I/R injury (p<0.05). The effect of TSN on P-Akt was inhibited by wortmannin administration (p<0.05).2.5 The levels of phospho- NF-κB p65 protein were elevated in the I/R group which indicated NF-κB activation during the procedure of I/R injury. TSN administration also associated with reduced expression of phospho-NF-κB p65 protein and this effect was abolished by wortmannin administration.2.6 I/R resulted in a noticeable increase in IL-6 (p<0.05) and TNF-α(p<0.05) compared with the sham group. TSN reduced the levels of IL-6 (p<0.05) and TNF-α(p < 0.05) compared with the I/R group. However, wortmannin administration significantly increased IL-6 (p<0.05) and TNF-α(p<0.05) production compared with the TSN group.2.7 The activity of MPO was significantly elevated after I/R injury when compared to the sham group (30.3±4.7 vs 6.4±0.5 U/100mg, p<0.05). Treatment with TSN reduced MPO activity compared with the I/R group (23.7±3.4 vs 30.3±4.7 U/100mg, p<0.05). Pretreatment with wortmannin abolished the effects of TSN (29.0±4.2 vs 23.7±3.4 U/100mg, p<0.05).Conclusion1. BMMNC transplantation is safe and feasible for diabetic patients accompanied by STEMI who have undergone PCI, and can lead to long-term improvement in myocardial function. Patients without diabetes may benifit more from BMMNC transplantation.2. TSN pretreatment reduces infarct size and improves cardiac dysfunction after I/R injury in diabetic rats. This was accompanied with decreased cardiac apoptosis and inflammation. The possible mechanism responsible for the effects of TSN is associated with the phosphatidylinositol 3-kinase (PI3K)/ Akt- dependent pathway.