Impact of implanted bone marrow progenitor cell composition on limb salvage after cell implantation in patients with critical limb ischemia.

OBJECTIVE: The aim of this study is to identify which factors influence limb salvage after bone marrow mononuclear cell implantation (BMI) in patients with chronic critical limb ischemia (CLI). METHODS: Thirteen no-option CLI patients treated with BMI were enrolled in the present study. Limb ischemia was assessed using the ankle-brachial index (ABI), transcutaneous oxygen tension (TcO(2)), and rest pain score. The cell populations among the implanted cells were determined by May-Giemsa staining and flow cytometry. RESULTS: Major lower extremity amputations after BMI were performed in seven patients. Before implantation, there were no significant differences between the amputation group (n=7) and the salvage group (n=6) in clinical characteristics, the ABI, the TcO(2) level, or the rest pain score. After implantation, there were no differences between the groups in the serum levels of angiogenic or inflammatory cytokines. The number of implanted BM cells was the same in the two groups, but the cells implanted in the limb salvage group were composed of significantly higher numbers of hematopoietic progenitors (erythroblasts and myeloblasts) and lymphocytes (p<0.05, respectively). The number of CD34-positive cells was somewhat greater in the salvage group than in the amputation group (p=0.09) and was positively associated with the number of erythroblasts (r(2)=0.29, p=0.06) and the number of myeloblasts (r(2)=0.59, p<0.01). CONCLUSIONS: The cellular composition of the BM cells injected may affect limb salvage after the implantation in patients with severe CLI. The favorable effects of BMI appear to reflect the impact of the progenitor cell doses.

Serum hepcidin-20 is elevated during the acute phase of myocardial infarction.

Hepcidin, a key iron-regulator secreted from the liver, consists of 25 amino acids (hepcidin-25), blocks iron release from macrophages via internalization and degradation of cellular iron exporter ferroportin, and restrains the use of iron in organs. Hepcidin mRNA and protein are also expressed in the human heart. A short form of hepcidin that lacks 5 amino-acid residues in the N-terminus (hepcidin-20) has been found in human serum, although its physiological role is unknown. Here, we successfully measured the serum levels of hepcidin-25 and hepcidin-20 in 12 patients with acute myocardial infarction (AMI) using surface-enhanced laser desorption ionization time of flight mass spectrometry. Among the selected 10 patients, whose blood samples were taken within 4 hours after a heart attack, all the patients showed elevated serum levels of hepcidin-20 [between 31.7 and 285.1 arbitrary unit (AU); normal level < 9.3 AU], while 8 patients showed high levels of hepcidin-25 (9.3-271.4; normal < 25.5 AU). The hepcidin-20 level was decreased to nearly the normal level on day 7 (range of 2.9 to 12.5 AU) in the 12 patients, whereas the hepcidin-25 level remained high on day 7 in 8 patients. Furthermore, the elevated levels of hepcidin-25 and hepcidin-20 were not correlated with the serum levels of markers for inflammation, interleukin-6 and C-reactive protein, in the patients with AMI. In conclusion, the serum hepcidin-20 is transiently elevated in response to acute cardiac ischemia. Measurement of serum hepcidin-20, rather than hepcidin-25, is helpful for diagnosis of acute myocardial infarction.

High-dose granulocyte-colony stimulating factor promotes neointimal hyperplasia in the early phase and inhibits neointimal hyperplasia in the late phase after vascular injury.

BACKGROUND: Granulocyte-colony stimulating factor (G-CSF) affects injured arteries through early endothelialization. Some reports, however, have cautioned that the restenosis rate may increase after G-CSF injection. In the present study, high-dose G-CSF was administered to mice with vascular injury to clarify its effect. METHODS AND RESULTS: Mice were received daily subcutaneous injections of saline or a high dose (300 microg/kg) of G-CSF for 5 days after vascular injury. In the FACS analysis, CD34-/Sca-1-positive progenitor cells were more abundant in the G-CSF group (p<0.05). Neointimal hyperplasia was more evident in the G-CSF group at 1 week (p<0.05), whereas at 4 weeks it was more evident in the control group (p<0.01). TUNEL-positive cells in the arterial wall were more numerous in the G-CSF group at day 1 (p<0.01). CD34-positive cells were observed in the G-CSF group at 1 week. Re-endothelialization appeared earlier in the G-CSF group (at 4 weeks; p<0.01). An increased number of 1A4-positive smooth muscle cells were found in bone marrow cell culture treated with G-CSF. CONCLUSION: High-dose G-CSF induced neointimal proliferation through excessive inflammation and bone marrow cell mobilization in the early phase. In the late phase, however, it induced early re-endothelialization and thereby inhibited neointimal hyperplasia.

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.

Bone marrow cells contribute to neointimal formation after stent implantation in swine.

BACKGROUND: Recent studies suggest that neointimal cells in atherosclerotic lesions are partly derived from bone marrow (BM) cells. However, studies with large animal models have not yet clarified how BM cells contribute to neointimal formation in restenotic lesions. We examined the expression of CD34, a hematopoietic stem cell marker, in the neointima after coronary stent implantation in porcine. METHODS: Pigs underwent balloon injury in the coronary arteries followed by stent implantation. The arteries were harvested at 3, 7, and 28 days after the stenting. The samples were used for immunohistochemistry for CD34, smooth muscle embryo (SMemb), alpha-smooth muscle cell actin (alpha-SMA), macrophage, c-kit, and AC133 antibodies. In morphometric analysis, each layer of vascular wall was calculated in the sections. RESULTS: At 3 days, the expressions of CD34 and SMemb were minimal, and many macrophages were seen around the stent struts. At 7 days, co-expression of CD34 and SMemb was observed around the struts, and 11.5% of the neointimal cells were stained by CD34. In addition, c-kit positive cells and AC133 positive cells are detected in neointimal area. At 28 days, the neointima had thickened and expressed alpha-SMA rather than SMemb, and a few CD34-positive cells were detected. In morphometric analysis, luminal area/total vascular area was significantly smaller and intimal area/total vascular area was significantly bigger in 7 and 28 days than in the day of implantation. CONCLUSION: BM cells of possibly hematopoietic origin partially contributed to neointimal formation after coronary stent implantation in a large animal model.

Nocturnal oxygen therapy prevents progress of congestive heart failure with central sleep apnea.

BACKGROUND: Sleep disordered breathing has been reported to be associated with congestive heart failure (CHF). Nocturnal oxygen has been shown to abolish apnea. The aim of this study is to examine whether nocturnal oxygen reduces sympathetic nerve activity, and prevents progress of CHF. METHODS: 93 patients with left ventricular ejection fractions < 60%, were examined with overnight saturation monitoring for an oxygen desaturation index. Subjects with oxygen desaturation of 4% > or = 4/h were examined with polysomnography. Apnea-hypopnea index (AHI) was calculated as the total number of episodes of apnea and hypopnea per hour of sleep. We started nocturnal oxygen for the patients with AHI > or = 20. Urinary and plasma catecholamines concentrations, serum brain natriuretic peptide, human atrial natriuretic peptide, and endothelial nitric oxide synthase levels were measured before and after starting oxygen. RESULTS: Compared among the three groups, CHF with central sleep apnea (CHF-CSA) group had significantly higher 24-h urinary adrenaline (CHF-CSA: 4.411+/-2.940 micromol/day, CHF with obstructive sleep apnea (CHF-OSA): 2.686+/-1.084 micromol/day, CHF without apnea (CHF-N): 3.178+/-1.778 micromol/day, P<0.05). Oxygen therapy significantly decreased AHI and 4 serum BNP levels (from 91.75+/-80.35 pg/ml to 52.75+/-45.70 pg/ml, mean change=33.85 pg/ml, P=0.0208). Serum eNOS levels were lower in CHF-CSA group and CHF-OSA group than in CHF-N group (CHF-CSA: 15.89+/-10.75 pg/ml, CHF-OSA: 7.46+/-3.91 pg/ml, CHF-N: 27.33+/-14.83 pg/ml, P<0.05). CONCLUSIONS: Nocturnal oxygen may prevent progress of CHF with central sleep apnea.

Inhibitory effect of candesartan cilexetil on left ventricular remodeling after myocardial infarction.

Although angiotensin-converting enzyme inhibitors (ACEIs) have been shown to reduce left ventricular remodeling after acute myocardial infarction (AMI), the effects of angiotensin receptor blockers have yet to be established. This study was conducted to examine the effects of candesartan on left ventricular remodeling after AMI. Consecutive AMI patients were assigned to a candesartan group or ACEI group after successful coronary intervention. The patients in the candesartan group (n = 77, mean age, 62.8 +/- 1.3) received candesartan and the patients in the ACEI group (n = 80, mean age, 63.3 +/- 1.2) received lisinopril, enalapril, or trandolapril. Four mg was the most frequent dose in the candesartan group at 6 months. Lisinopril, enalapril, and trandolapril were administered to 52%, 27%, and 21% of the patients in the ACEI group, respectively. No significant differences in the incidences of cardiac death, nonfatal MI, or hospitalization for heart failure (P = NS) were found between the groups. The candesartan group exhibited a somewhat higher percent increase in left ventricular ejection fraction and significantly lower percent increases in left ventricular end-diastolic volume index and left ventricular end-systolic volume index compared to the ACEI group (P < 0.05, P < 0.05, respectively). Candesartan is more effective than ACEI in preventing left ventricular remodeling after AMI.

Tumor-necrosis-factor-alpha-gene-deficient mice have improved cardiac function through reduction of intercellular adhesion molecule-1 in myocardial infarction.

BACKGROUND: Tumor necrosis factor (TNF)-alpha is linked to the pathogenesis of cardiovascular diseases, but how it affects myocardial infarction (MI), so the present study examined the effects of TNF-alpha and the involvement of intercellular adhesion molecule (ICAM)-1 on MI. METHODS AND RESULTS: Left coronary arteries of C57BL/6 wild type (WT) and TNF-alpha knockout (KO) mice were ligated and the mice were killed 1, 3, and 7 days later. Fractional shortening on echocardiography of the KO mice was significantly higher than that of the WT mice from day 1 to 7 (p<0.01). The ICAM-1 mRNA in the infarcted area of the KO mice was significantly lower than that of the WT from day 1 (p<0.01) to 7. In immunohistochemistry, the expression of ICAM-1 was weaker in the KO than in the WT mice. The number of neutrophils in the KO mice peaked at day 1, but even this peak level failed to reach the levels in the infarcted (p<0.01) and peri-infarcted areas (p<0.05) in the WT. The number of macrophages in the KO mice peaked at day 7, but this peak level failed to reach the levels in the infarcted (p<0.01) and peri-infarcted areas (p<0.05) in the WT. CONCLUSION: In a permanent occlusion model of MI TNF-alpha decreased cardiac function and ameliorated myocardial remodeling through the induction of ICAM-1.

Insulin resistance in nondiabetic patients with acute myocardial infarction.

BACKGROUND: Recent studies have shown that insulin resistance (IR) is an independent predictor of early restenosis after coronary stenting. The aim of this study was to examine the effects of IR and its linkage to late loss with bare metal stenting in nondiabetic patients with acute myocardial infarction (AMI). MATERIALS AND METHODS: We enrolled 61 nondiabetic patients with AMI who have undergone coronary stenting. Quantitative analyses of coronary angiographic data before and after the procedure and at 4 months were performed. Fasting plasma glucose (FPG) and insulin were measured every week until the subjects' hospital discharge. Stress hormones, endothelial nitric oxide synthase, tumor necrosis factor alpha, interleukin-6, leptin, and adiponectin were measured on admission and at 4 months after coronary stenting. RESULTS: Simple linear regression analyses showed a relationship between FPG and insulin [IR group: r=0.297, P=.0428; no insulin resistance (NIR) group: r=0.539, P=.0466] and that late loss was associated with the homeostasis model assessment of IR (HOMA-IR) at 4 months (r=0.435, P=.03). At multiple regression analyses, HOMA-IR on admission in the IR group significantly correlated with thyroid-stimulating hormone, glucagon, and cortisol. The HOMA-IR at 4 months correlated with leptin. CONCLUSIONS: Nondiabetic patients with AMI can be classified into two groups: the IR group and the NIR group. The IR consisted of the transient IR, which correlated with stress hormones, and the continuous IR, which correlated with leptin and contributed to restenosis after coronary stenting.

A randomized comparison of pioglitazone to inhibit restenosis after coronary stenting in patients with type 2 diabetes.

OBJECTIVE: Recent studies have demonstrated that the treatment with thiazolidinediones reduces in-stent restenosis. The aim of this study was to elucidate the mechanism of the efficacy of pioglitazone for preventing in-stent restenosis in type 2 diabetic patients. RESEARCH DESIGN AND METHODS: We conducted a prospective, randomized trial involving 54 type 2 diabetic patients referred for coronary stenting who were randomly assigned to either the control or the pioglitazone group. Quantitative coronary angiography was performed at study entry and at 6 months follow-up. Endothelial nitric oxide synthase (eNOS), tumor necrosis factor alpha, interleukin-6, leptin, and adiponectin were measured at study entry and at 6 months follow-up. RESULTS: A total of 28 patients were randomly assigned to the control group, and 26 patients were assigned to the pioglitazone group. There were no significant differences in glycemic control levels or in lipid levels in the two groups at baseline or at follow-up. Insulin, homeostasis model assessment of insulin resistance, eNOS, and leptin at follow-up were significantly reduced in the pioglitazone group compared with the control group. The late luminal loss and in-stent restenosis were significantly less in the pioglitazone group than in the control group. Leptin independently correlated with late luminal loss at multiple regression analysis. CONCLUSIONS: The treatment with pioglitazone in type 2 diabetic patients significantly reduced leptin. This decreased leptin improved insulin resistance and endothelial function with the reduction of insulin. The improved endothelial function affected the reduction of in-stent restenosis.