Distinct mobilization of circulating CD271+ mesenchymal progenitors from hematopoietic progenitors during aging and after myocardial infarction.

The specific cell surface markers on mesenchymal stem/progenitor cells (MSCs) have been poorly defined in vivo, but in one recent study, an MSC subpopulation was directly isolated from a CD271-positive fraction of human bone marrow cells. The aim of this study was to identify circulating CD271(+) MSCs in human peripheral blood and investigate whether the cells are mobilized after acute myocardial infarction (MI). A flow cytometric analysis identified CD45(low/-)CD34(+)CD271(+) cells in adult human peripheral blood. The numbers of circulating CD45(low/-)CD34(+)CD133(+) cells (hematopoietic linage progenitors) were significantly lower in elderly subjects without coronary artery disease than in healthy young subjects, whereas the numbers of CD45(low/-)CD34(+)CD271(+) cells were comparable between elderly subjects and younger subjects. The CD45(low/-)CD34(+)CD271(+) and CD133(+) cell counts were both higher in patients with acute MI than in patients with stable coronary artery disease. In our investigation of the time course changes after acute MI, the CD45(low/-)CD34(+)CD133(+) cell counts gradually increased up to day 7. Over the same period, the CD45(low/-)CD34(+)CD271(+) cell counts peaked at day 3 and then declined up to day 7. Importantly, the CD271(+) cell counts at day 3 were positively correlated with the peak concentrations of creatine kinase after acute MI. Results of the present study suggest that the CD271(+) MSCs are mobilized differently from the CD133(+) hematopoietic progenitors and may play a specific role in the tissue repair process during age-related changes and after acute myocardial infarction.

Effects of erythropoietin on angiogenesis after myocardial infarction in porcine.

Erythropoietin (EPO) has recently been shown to confer cardioprotective effects via angiogenesis and antiapoptosis. The administration of EPO after myocardial infarction (MI) reduces infarct size and improves cardiac function in small animals. The purpose of this study is to investigate the protective effects of EPO in porcine MI. Each animal in the EPO group received four injections of recombinant human EPO (rhEPO; 6000 U per injection) at 2-day intervals, starting after coronary occlusion. Animals in the control group received saline. Left ventriculography was performed just after coronary occlusion and at 28 days. Time-course changes in serum levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and fibroblast growth factor (FGF) were measured. The number of vessels was calculated, and the mRNA expressions of VEGF and insulin-like growth factor (IGF) were examined. Left ventricular function was similar between the groups. The numbers of cells positive for anti-α-smooth muscle actin, von Willebrand factor, and c-kit were significantly higher in the EPO group than in the controls (P < 0.05). The EPO group exhibited significantly higher HGF and FGF concentrations (P < 0.05) and higher expression of VEGF and IGF mRNA (P < 0.05) compared with the controls. In conclusion, EPO accelerates angiogenesis via the upregulation of systemic levels such as HGF and FGF, and the local expression of VEGF and IGF, in porcine MI.

Coronary vein infusion of multipotent stromal cells from bone marrow preserves cardiac function in swine ischemic cardiomyopathy via enhanced neovascularization.

Few reports have examined the effects of adult bone marrow multipotent stromal cells (MSCs) on large animals, and no useful method has been established for MSC implantation. In this study, we investigate the effects of MSC infusion from the coronary vein in a swine model of chronic myocardial infarction (MI). MI was induced in domestic swine by placing beads in the left coronary artery. Bone marrow cells were aspirated and then cultured to isolate the MSCs. At 4 weeks after MI, MSCs labeled with dye (n=8) or vehicle (n=5) were infused retrogradely from the anterior interventricular vein without any complications. Left ventriculography (LVG) was performed just before and at 4 weeks after cell infusion. The ejection fraction (EF) assessed by LVG significantly decreased from baseline up to a follow-up at 4 weeks in the control group (P<0.05), whereas the cardiac function was preserved in the MSC group. The difference in the EF between baseline and follow-up was significantly greater in the MSC group than in the control group (P<0.05). The MSC administration significantly promoted neovascularization in the border areas compared with the controls (P<0.0005), though it had no affect on cardiac fibrosis. A few MSCs expressed von Willebrand factor in a differentiation assay, but none of them expressed troponin T. In quantitative gene expression analysis, basic fibroblast growth factor and vascular endothelial growth factor (VEGF) levels were significantly higher in the MSC-treated hearts than in the controls (P<0.05, respectively). Immunohistochemical staining revealed VEGF production in the engrafted MSCs. In vitro experiment demonstrated that MSCs significantly stimulated endothelial capillary network formation compared with the VEGF protein (P<0.0001). MSC infusion via the coronary vein prevented the progression of cardiac dysfunction in chronic MI. This favorable effect appeared to derive not from cell differentiation, but from enhanced neovascularization by angiogenic factors secreted from the MSCs.

Cardioprotective effect of endogenous pituitary adenylate cyclase-activating polypeptide on Doxorubicin-induced cardiomyopathy in mice.

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) is known as a cytoprotective polypeptide. PACAP and its receptors are expressed in the heart, but it is unclear whether PACAP exerts its protective effect on the myocardium in vivo. The aim of the present study was to investigate whether endogenous PACAP has a cardioprotective effect on Doxorubicin (Dox)-induced cardiomyopathy. METHODS AND RESULTS: Dox was intraperitoneally injected to induce cardiomyopathy in wild type (WT) and PACAP knockout (ie, PACAP+/- and PACAP-/-) mice. The survival rates up to 15 days of PACAP+/- mice and PACAP-/- mice were significantly less than that of WT mice. Cardiac function, measured by echocardiography, was significantly lower in PACAP+/- mice than in WT mice at day 10. Morphological examination of sections of myocardium showed degenerative change and fibrosis in PACAP+/- mice at day 10. Serum reactive oxygen metabolites (a marker of oxidative stress), the number of 8-hydroxy-deoxyguanosine-positive nuclei and TdT-mediated dUTP nick end-labeling (TUNEL) positive nuclei in the myocardium were higher in PACAP+/- mice than WT mice. However, continuous subcutaneous administration of PACAP38 was able to prevent the myocardial damage typically caused by Dox injection in PACAP+/-. CONCLUSIONS: These results suggest that endogenous PACAP might attenuate Dox-induced myocardial damage and that its mechanism of action is likely to be associated with the reduction of oxidative stress and mediated via anti-apoptotic effects.

Upper gastrointestinal bleeding in patients receiving dual antiplatelet therapy after coronary stenting.

OBJECTIVE: We investigated the risk of upper gastrointestinal (UGI) bleeding and the protective effect of concomitant anti-secretory drugs during dual antiplatelet therapy administered following implantation of drug-eluting stents (DES) for coronary heart disease. Because proton pump inhibitors (PPIs) are reported to decrease the platelet inhibitory effects of clopidogrel, we also assessed cardiovascular outcomes in patients taking thienopyridine derivatives with or without anti-secretory drug. METHODS: We retrospectively analyzed 243 patients, who underwent DES implantation between January 2006 and December 2007 and were receiving dual anti-platelet therapy post-surgery. The main outcome measurement was the presence of UGI bleeding. Cardiovascular outcomes were assessed by follow-up coronary angiography (CAG) findings. Data were collected from medical records. RESULTS: Eight cases of UGI bleeding were observed during the follow-up period, none of whom were taking anti-secretory drugs. Among the 243 cases, 108 cases were taking anti-secretory drugs: a PPI (67 cases), and an H2 receptor antagonist (41 cases). No UGI bleeding was observed among patients who were taking concomitant anti-secretory drugs. The 1- and 2-year cumulative incidences of UGI bleeding among patients who were not taking anti-secretory drugs were 4.5% and 9.2%, respectively. When CAG findings were compared between patients not taking any anti-secretory drug, taking PPI, or taking H2RA, significantly more stenotic lesions of the coronary artery were observed in the PPI-treatment group. CONCLUSION: Concomitant use of an anti-secretory agent was associated with a reduced risk of UGI bleeding. Use of PPI may be associated with an attenuation of the effect of dual antiplatelet therapy.

Hydroxyzine, a first generation H(1)-receptor antagonist, inhibits human ether-a-go-go-related gene (HERG) current and causes syncope in a patient with the HERG mutation.

QT prolongation, a risk factor for arrhythmias, can result from genetic variants in one (or more) of the genes governing cardiac repolarization as well as intake of drugs known to affect a cardiac K(+) channel encoded by human ether-a-go-go-related gene (HERG). In this paper, we will report a case of drug-induced long QT syndrome associated with an H(1)-receptor antagonist, hydroxyzine, in which a mutation was identified in the HERG gene. After taking 75 mg of hydroxyzine for several days, a 34-year-old female began to experience repetitive syncope. The deleterious effect of hydroxyzine was suspected because QTc interval shortened from 630 to 464 ms after cessation of the drug. Later on, the patient was found to harbor an A614V-HERG mutation. By using the patch-clamp technique in the heterologous expression system, we examined the functional outcome of the A614V mutation and confirmed a dominant-negative effect on HERG expression. Hydroxyzine concentration-dependently inhibited both wild-type (WT) and WT/A614V-HERG K(+) currents. Half-maximum block concentrations of WT and WT/A614V-HERG K(+) currents were 0.62 and 0.52 microM, respectively. Thus, accidental combination of genetic mutation and intake of hydroxyzine appeared to have led to a severe phenotype, probably, syncope due to torsade de pointes.

G-CSF after myocardial infarction accelerates angiogenesis and reduces fibrosis in swine.

OBJECTIVE: Recent studies have suggested that granulocyte colony-stimulating factor (G-CSF) may improve cardiac function after acute myocardial infarction (AMI) by accelerating angiogenesis or cardiomyogenesis, but negative results and side effect of G-CSF have also been reported. However, no previous studies have used large animal models of ischemia/reperfusion to investigate the effect and side effect of G-CSF after AMI. METHODS: The diagonal branch of the left anterior descending coronary artery of swine was balloon-occluded for 1 h and then reperfused. The animals of the G-CSF group were injected with G-CSF subcutaneously (5.0 microg/kg/day) for 6 days after MI and then sacrificed after 4 weeks. The control group received the same volume of saline. RESULTS: There were no differences between the groups in the rate of thrombotic obstruction or progression of stenosis lesion in coronary angiography. The ejection fraction and end-diastolic volume in the G-CSF group were not significantly improved over the control values. The fibrotic area was significantly smaller in the G-CSF group than in the controls (P<0.05), and the numbers of vessels counted in anti-von Willebrand factor and anti-alpha-smooth muscle actin-stained sections were significantly larger (P<0.005 and P<0.05, respectively). The expression of collagen III mRNA was significantly lower in the G-CSF group than in the control in the infarct (P<0.0005) and border areas (P<0.005), and TGF-beta mRNA was significantly lower in the G-CSF group in the border area (P<0.05). CONCLUSIONS: G-CSF could modify the healing process after AMI by accelerating angiogenesis in a swine ischemia/reperfusion model. At the dose administered, however, G-CSF did not seem to improve the global cardiac function.

Eplerenone suppresses neointimal formation after coronary stent implantation in swine.

BACKGROUND: Enhanced extracellular matrix accumulation rather than cell proliferation contributes to later stages of in-stent restenosis. Aldosterone itself has been shown to increase cardiovascular fibrosis, therefore, we studied the suppressive effects of eplerenone, a new aldosterone receptor antagonist, on neointimal hyperplasia after coronary stent implantation in swine. METHODS: Palmatz-Shatz stents were implanted in the left anterior descending artery of 36 pigs. One hundred milligrams of Eplerenone was orally administered from 1 week before, to 4 weeks after stent implantation in Group E (n=18), and vehicle was given to Group C (n=18). Pigs were sacrificed 1 or 4 weeks after stent implantation. The number of infiltrating macrophages was calculated at 1 week. Morphometrical analysis was performed to measure the area of each layer, and %area of fibrosis and mRNA for collagen I, III and TGF-beta was analyzed by RT-PCR at 4 weeks. RESULTS: The number of infiltrating macrophages was less in Group E than in Group C (p<0.01). The overall size of coronary arteries at 4 weeks was similar in both groups. However, the luminal area was larger in Group E than in Group C (p<0.05), and the intimal area was smaller in Group E than in Group C (p<0.05). The %area of fibrosis was significantly less in Group E than in Group C at 4 weeks (p<0.01). In Group E, the expression of mRNA for collagen I, III and TGF-beta was significantly reduced. CONCLUSION: Orally administered eplerenone attenuated collagen accumulation within the neointima, thereby inhibiting neointimal hyperplasia after stent implantation.

Mechanisms of restenosis after coronary intervention: difference between plain old balloon angioplasty and stenting.

BACKGROUND: Restenosis after coronary intervention remains an unsolved and important clinical problem. We histologically examined the mechanism of restenosis after both balloon injury and stenting. METHODS: Coronary arteries of swine were subjected to balloon injury and stenting. Next, just after stenting or at 7, 14, or 28 days, the animals were sacrificed for the evaluation by morphometric analysis, histological observation, and immunostaining. RESULTS: The neointimal area peaked at 14 days in the balloon injury group (BG) and increased linearly up to 28 days in the stent group (SG). At 28 days, the total vascular area in the BG was reduced to 78% of the control values. In the SG, the total vascular area remained enlarged. According to the phenotypic analysis, the vascular smooth muscle cells (VSMCs) in the neointimal area at 28 days were the contractile type in the BG and the synthetic type in the SG. Proliferating cell nuclear antigen (PCNA) and macrophage-positive cells were not observed in neointima in the BG at 28 days, whereas they were observed around the stent struts in the SG. In addition, numerous inflammatory cells, such as neutrophils and eosinophils, were also present in the SG. CONCLUSIONS: Restenosis after balloon injury consisted of arterial remodeling and neointimal hyperplasia, whereas that after stenting consisted mostly of neointimal hyperplasia. The neointimal area in the SG lasted longer than that in the BG. Continuous inflammation may be an important factor in the restenosis of stenting.

Comparison of the morphological changes of restenosis after the implantation of various types of stents in a swine model.

BACKGROUND: Stent design causes the differences of restenosis rate, but the morphological differences after the various types of stent implantation have not been clarified. DESIGN: Seven types of stents were implanted in pig coronary arteries to clarify how the mechanism of restenosis differs with coil stents and tube stents. METHODS: The left anterior descending coronary arteries (LADs) of pigs were injured using coronary angioplasty balloons (diameter: 3.0 mm; length: 20 mm; balloon/artery ratio: 1 : 2). Fourteen days after the injury, four types of coil stents (Cordis, Wiktor, GR-I, and GR-II) and three types of tube stents (Palmaz-Schatz, gfx, and Multilink) were implanted, and the LADs were extracted 28 days after the implantation. RESULTS: The proliferated neointima was eccentric in the coil stents and concentric in the tube stents. Although there was no significant difference in the area of neointima, the area of the lumen was significantly larger in the tube stents than in the coil stents ( < 0.01) because of the larger area of stent. Cells positive for anti-proliferating cell nuclear antigen antibody were mainly observed around the stent struts, and most of these cells were also positive for either anti-macrophage or anti-smooth muscle actin antibodies. CONCLUSIONS: Compared to the coil stents, the tube stents induce less negative remodelling including stent recoil, resulting in a wider luminal area. In order to prevent restenosis, it is crucial to implant a stent that will cause less negative remodelling.

Clinical characteristics and follow-up in patients with microvascular angina.

Arteriosclerosis of the small arteries is one of the main causes of microvascular angina, and although some reports have shown favorable prognoses, there is progressive reduction in left ventricular function. The present study evaluated the prognosis of microvascular angina in 86 Japanese patients (51 women, 35 men; average age, 59+/-9 years) who had ischemic ST segment depression, normal coronary angiograms and small artery sclerosis confirmed by endomyocardial biopsies. The mean follow-up period was 7.2+/-3.4 years. Questionnaires regarding their symptoms, cardiac medication, and new events were sent to all patients. Eighty-five patients (98.9%) were still alive at the end of the follow-up period. Chest pain remained in 35.3%; the degree of pain was unchanged in 18.8%, and had lessened in 11.8%. None of the patients died of cardiac events or suffered from a myocardial infarction. At the end of the follow-up period, calcium antagonist was used in 63.5% of patients. Seventeen patients (20.0%) were free of antianginal medication. The prognosis of microvascular angina diagnosed by strict criteria was favorable.