Yasutoshi Omori

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.

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.

Neutrophil and erythroblast regulate the effects of bone marrow cell implantation

Sir, Efficacy of autologous bone-marrow cell implantation as therapeutic angiogenesis has been reported in patients with severe peripheral artery disease [1]. In addition to containing CD34-positive cells, the main drivers of vasculogenesis and probably the main contributors to this therapy, sorted bone marrow-mononuclear cells (BM-MNC), also contain an abundance of CD34-negative cells. No studies have yet elucidated which types of CD34-negative cells influence the clinical appearance in BM-MNC implantation. We investigated the correlations of morphologically classified cell types of sorted BM-MNCs with changes in the ankle brachial index (ABI) and transcutaneous oxygen pressure (TcO 2 ) as clinical parameters.

Cardioprotective effect of G-CSF administration after coronary reperfusion in swine AMI model

[Purpose] Recent studies have suggested that granulocyte colony-stimulating factor (G-CSF) may accelerate angiogenesis or cardio-myogenesis. No previous studies, however, have used large animal models to investigete how clinical doses of G-CSF affect cardiac function after acute myocardial infarction (AMI). [Methods] Diagonal branch of the left anterior descending coronary artery of domestic swine was balloon-occluded for 1-hour and then reperfused. The G-CSF group received a subcutaneous injection of G-CSF at a dose of 5.0/µg/kg/day for 6 days after MI. Left ventriculography was performed 4 weeks after Ml. The number of vessels in the infarcted area were calculated using sections stained by anti-α-smooth muscle actin (SMA) and anti-von Willebrand factor (vWF). Reverse transcription polymerase chain reactions for collagen I, collagen III, and transforming growth factor (TGF)-β were also examined. [Results] The G-CSF group showed a significantly higher ejection fraction and lower end-diastolic volume in left ventriculography. The numbers of α-SMA- and vWF-positive vessels in the G-CSF group were significantly larger. The expression of collagen III mRNA was significantly lower in the G-CSF group in the infarct and border areas. The expression of TGF-β mRNA was significantly lower in the G-CSF group in the border area. [Conclusions] The administration of clinical doses of G-CSF improved cardiac function after reperfusion in AMI. G-CSF confers its effects by accelerating angio-genesis and modifying the wound-healing process.

Improvement of severe ulcer of buerger’s disease by bone-marrow mononuclear cell transplantation: a case report

[Case Report] A 47-year-old man with Buerger’s disease was admitted with complaints of progressive ulcer of left planta pedis and resting pain of the left leg. The ulcer was not healed under medical therapy nor lumbar sympathetic ganglion block, and angioplasty or bypass surgery was not applicable. All his toes showed cyanotic and left third and fifth toes were gangrene, as well as ulcer in left planta pedis. Angiography showed arterial occlusion in the crus and formation of corkscrew like changes. Although ankle-brachial index was kept in almost normal range, transcutaneous oxygen pressure (TcO2) was decreased. Leg pain was not relieved with medication and he could not walk for pain due to his ulcer. We performed bone-marrow mononuclear cell implantation to his inferior limbs to achieve therapeutic angiogenesis. After aspirating the bone marrow (600 ml) from the ileum, the bone-marrow mononuclear cells were sorted. The separated cells were implanted into the ischemic legs by intramuscular injection. Reduction of leg pain was observed as early as 3 days, and completely disappeared at rest after 4 weeks. Improvement of TcO2 was observed at 1 week, and the ulcer of planta pedis was almost healed 4 weeks after the therapy. [Conclusion] Bone-marrow mononuclear cell implantation is very effective in patients with Buerger’s disease, even they have large severe ulcer.

Importance of Neutrophil and Erythroblast for the efficacy of Bone-marrow Cell Implantation in Peripheral Artery Disease

[Background] Efficacy of autologous bone marrow-mononuclear cell (BM-MNC) implantation as therapeutic angiogenesis has been reported in patients with severe peripheral artery disease. In addition to containing CD34 positive-cells, sorted BM-MNCs contain an abundance of CD34-negative cells. No studies have yet elucidated which types of CD34-negative cells influence the clinical appearance in BM-MNC implantation. We investigated the correlations of morphologically classified cell types of sorted BM-MNCs with changes in the ankle brachial index (ABI) and transcutaneous oxygen pressure (TcO2). [Material and Methods] Seven patients with severe peripheral arterial disease who were not candidates for angioplasty or surgical operation underwent BM-MNC implantation. The sorted BM-MNCs using a cell separator were classified on the basis of May-Giemsa staining, and CD34-positive cells were counted. ABI and TcO2 were performed before and after BM-MNC implantation. [Results] Mean ABI (p<0.05) and mean TcO2 (p<0.005) from baseline to 4 weeks after the implantation were significantly increased. The numbers of total injected cells and CD34-positive cells were not correlated with Δ TcO2 from before to 4 weeks. Among the cell types analyzed, ΔTcO2 showed significant negative correlations with the percentage of mature neutrophils (p<0.01) and significant positive correlations with the percentage of erythroblasts (p<0.05). [Conclusions] Neutrophils could be an inverse regulator and erythroblasts could be a positive regulator in clinical BM-MNC implantation.

Bone marrow derived cells contribute to arterial remodeling

Exuberant smooth muscle cells hyperplasia is the major cause of postangioplasty restenosis. We suggested that circulating smooth muscle progenitor cells might contribute to lesion formation after vascular injury. We extensively investigated the cellular constituents during neointimal formation after mechanical injury. A large wire was inserted into the mouse femoral artery. At 2 hours, the injured artery remained dilated with a thin media containing very few cells. One week after the injury, CD45 positive hematopoietic cells accumulated at the luminal side. Those CD 45 positive cells gradually disappeared, whereas neointimal was formed with alpha smooth muscle actin positive cells. Bone marrow cells and peripheral mono-nuclear cells differentiated into alpha smooth muscle cells in the presence of PDGF and basic FGF. These results suggest that early accumulation of hematopoietic cells may play a role in the pathogenesis of smooth muscle cells hyperplasia under certain circumstances.

TNF-α mobilizes bone marrow derived cells to vascular wall, resulting in neointima formation through its inflammatory effects

Background: Recent studies suggest that bone marrow cells contribute to neointimal formation after vascular injury. However, the relationship between the inflammatory reactions and bone marrow cell invasion has not been clarified. Method and Results: We insert a large wire (0.38 mm in a diameter) into the femoral arteries of 6–8 week-old male balb/c (WT) and TNF-α Knockout (KO) mice. In imunohistochmistry using CD34 at 1 week, positive cells possibly containing bone marrow derived cells, were hardly observed in KO, but some were observed in WT in neointima. At 4 weeks, CD45 positive cells were rarely seen, and α-smooth muscle actin positive cells were main component of thickened neointima in both groups. In morphometric analysis at 4 weeks after the injury, developed neointimal area was smaller in KO. Furthermore, re-endothelialization appeared earlier in KO than WT. Conclusion: TNF-α is involved in neointimal formation after vascular injury, possible through its inflammatory effects to induce bone marrow cells.