Tetsunori Seki

Mesenchymal lineage precursor cells induce vascular network formation in ischemic myocardium.

Mesenchymal lineage precursors can be reproducibly isolated from adult mammalian bone marrow and grown in culture. Immunoselection with monoclonal antibodies against STRO-1 and vascular-cell-adhesion molecule 1 (VCAM1/CD106) prior to expansion results in a 1,000-fold enrichment of mesenchymal precursors compared to standard isolation techniques. Intramyocardial injection of human STRO-1-selected precursors in an athymic rat model of acute myocardial infarction results in induction of vascular network formation and arteriogenesis coupled with global functional cardiac recovery.

Use of a differential subtraction method to identify genes that characterize the phenotype of cultured rheumatoid arthritis synoviocytes

OBJECTIVE To identify the genes that characterize the distinctive phenotype of cultured rheumatoid arthritis (RA) fibroblastoid synoviocytes. METHODS A representational difference method was used to subtract complementary DNA (cDNA) from cultured RA fibroblastoid synoviocytes with cDNA from noninflammatory osteoarthritis synoviocytes. The genes were identified by DNA sequencing, and their relative expression was determined by Northern blot analysis. RESULTS Twenty-four genes were identified, including novel genes such as a human homolog of mouse semaphorin E and one homologous to N-acetylglucosamine-6-sulfatase. Eleven of these genes were constitutively overexpressed in the rheumatoid synoviocyte line, including a chemokine, stromal cell-derived factor 1, and several genes capable of mediating synoviocyte-leukocyte interactions, including vascular cell adhesion molecule 1 and Mac-2 binding protein. Three genes (lumican, biglycan, and insulin-like growth factor binding protein 5) encoded extracellular matrix components, suggesting that distinct stromal-synoviocyte interactions may be mediated by this phenotype. Two interferon-inducible genes of unknown function were also found, emphasizing the presence of activation-like features in the phenotype. CONCLUSION A general method for the identification of differences in patterns of gene expression revealed that cultured RA fibroblastoid synoviocytes overexpress certain proinflammatory genes that are potentially relevant to lymphocyte and monocyte entry and interactions. The features of the genes identified in these mesenchymal cells suggest that they facilitate localization of immune reactions to the joint through leukocyte chemokinesis, cell-cell adhesion, and matrix specialization. The further characterization of these genes should help in resolving whether this phenotype is the consequence of modulation and imprinting by an inflammatory milieu or, more likely, whether it reflects the intrinsic lineage characteristics of intimal lining synoviocytes.

Catalytic Degradation of Vitamin D Up-regulated Protein 1 mRNA Enhances Cardiomyocyte Survival and Prevents Left Ventricular Remodeling after Myocardial Ischemia *

Vitamin D3 up-regulated protein 1 (VDUP1) is a key mediator of oxidative stress on various cellular processes via downstream effects on apoptosis signaling kinase 1 (ASK1) and p38 mitogen-activated protein kinase (MAPK). Here, we report that VDUP1 expression is significantly increased in rat hearts following acute myocardial ischemia, suggesting it may have important regulatory effects on cardiac physiological processes during periods of oxidative stress. Transfection of H9C2 cardiomyoblasts with a sequence-specific VDUP1 DNA enzyme to down-regulate VDUP1 mRNA expression significantly reduced apoptosis and enhanced cell survival under conditions of H2O2 stress, and these effects involved inhibition of ASK1 activity. Direct intracardiac injection of the DNA enzyme at the time of acute myocardial infarction reduced myocardial VDUP1 mRNA expression and resulted in prolonged reduction in cardiomyocyte apoptosis and ASK1 activity. Moreover, down-regulation of VDUP1 was accompanied by significant reduction in cardiac expression of pro-collagen type I α2 mRNA level, as well as marked reduction in myocardial scar formation. These features were accompanied by significant improvement in cardiac function. Together, these results suggest a direct role for VDUP1 in the adverse effects of ischemia and oxidative stress on cardiomyocyte survival, left ventricular collagen deposition, and cardiac function. Strategies to inhibit VDUP1 expression and/or function during acute ischemic events may be beneficial to cardiac functional recovery and prevention of left ventricular remodeling.

Down-regulation of Plasminogen Activator Inhibitor 1 Expression Promotes Myocardial Neovascularization by Bone Marrow Progenitors

Human adult bone marrow–derived endothelial progenitors, or angioblasts, induce neovascularization of infarcted myocardium via mechanisms involving both cell surface urokinase-type plasminogen activator, and interactions between β integrins and tissue vitronectin. Because each of these processes is regulated by plasminogen activator inhibitor (PAI)-1, we selectively down-regulated PAI-1 mRNA in the adult heart to examine the effects on postinfarct neovascularization and myocardial function. Sequence-specific catalytic DNA enzymes inhibited rat PAI-1 mRNA and protein expression in peri-infarct endothelium within 48 h of administration, and maintained down-regulation for at least 2 wk. PAI-1 inhibition enhanced vitronectin-dependent transendothelial migration of human bone marrow–derived CD34+ cells, and resulted in a striking augmentation of angioblast-dependent neovascularization. Development of large, thin-walled vessels at the peri-infarct region was accompanied by induction of proliferation and regeneration of endogenous cardiomyocytes and functional cardiac recovery. These results identify a causal relationship between elevated PAI-1 levels and poor outcome in patients with myocardial infarction through mechanisms that directly inhibit bone marrow–dependent neovascularization. Strategies that reduce myocardial PAI-1 expression appear capable of enhancing cardiac neovascularization, regeneration, and functional recovery after ischemic insult.

Therapeutic effects of human STRO‐3‐selected mesenchymal precursor cells and their soluble factors in experimental myocardial ischemia

Stromal precursor antigen (STRO)‐3 has previously been shown to identify a subset of adult human bone marrow (BM)‐derived mesenchymal lineage precursors, which may have cardioprotective potential. We sought to characterize STRO‐3‐immunoselected and culture‐expanded mesenchymal precursor cells (MPCs) with respect to their biology and therapeutic potential in myocardial ischemia. Immunoselection of STRO‐3+ MPCs enriched for fibroblastic colony forming units from unfractionated BM mononuclear cells (MNCs). Compared to mesenchymal stem cells conventionally isolated by plastic adherence, MPCs demonstrated increased proliferative capacity during culture expansion, expressed higher levels of early ‘stem cell’ markers and various pro‐angiogenic and cardioprotective cytokines, and exhibited greater trilineage developmental efficiency. Intramyocardial injection of MPCs into a rat model of myocardial infarction (MI) promoted left ventricular recovery and inhibited left ventricular dilatation. These beneficial effects were associated with cardioprotective and pro‐angiogenic effects at the tissue level, despite poor engraftment of cells. Treatment of MI rats with MPC‐conditioned medium (CM) preserved left ventricular function and dimensions, reduced myocyte apoptosis and fibrosis, and augmented neovascularization, involving both resident vascular cells and circulating endothelial progenitor cells (EPCs). Profiling of CM revealed various cardioprotective and pro‐angiogenic factors, which had biological activity in cultures of myocytes, tissue‐resident vascular cells and EPCs. Prospective immunoselection of STRO‐3+ MPCs from BM MNCs conferred advantage in maintaining a population of immature MPCs during ex vivo expansion. Transplantation of culture‐expanded MPCs into the post‐MI heart resulted in therapeutic benefit, attributable at least in part to paracrine mechanisms of action. Thus, MPCs represent a promising therapy for myocardial ischemia.

Expression of inhibitory receptor ILT3 on neoplastic B cells is associated with lymphoid tissue involvement in chronic lymphocytic leukemia.

T cell responses against leukemia‐associated antigens have been reported in chronic lymphocytic leukemia (CLL). However, the relentless accumulation of CLL B cells in some patients indicates that anti‐tumor immune responses are inefficient. Inhibitory receptors from the Ig‐like transcript (ILT) family, such as ILT3 and ILT4, are crucial to the tolerogenic activity of antigen presenting cells. In this study, we examined the expression of ILT3 on CD5+ B cells obtained from 47 patients with CLL. Using flow cytometry and RT‐PCR, we found that B CLL cells from 23 of 47 patients expressed ILT3 protein and mature ILT3 mRNA. ILT3 protein and mRNA were not found in normal B cells obtained from donors without CLL. Expression of ILT4 in normal and B CLL cells showed a pattern similar to ILT3. The frequency of ILT3 positive CLL B cells was higher in patients with lymphoid tissue involvement, suggesting that ILT3 may have prognostic value in CLL. Our findings indicate that expression of ILT3 and ILT4 on CLL B cells represents a phenotypic abnormality that may play a role in tolerization of tumor‐specific T cells.

Additive effects of endothelial progenitor cells combined with ACE inhibition and β-blockade on left ventricular function following acute myocardial infarction

Animal studies have demonstrated the efficacy of endothelial progenitor cells (EPCs) in preventing left ventricular (LV) remodelling following myocardial infarction (MI). Preliminary human studies are underway, yet no studies have demonstrated efficacy in combination with standard medical therapy, i.e. angiotensin-converting enzyme (ACE) inhibitors and β-blockers. Nude rats underwent left anterior descending coronary artery ligation to induce MI. Animals were randomised to receive no treatment (MI, n=5), quinapril 200 mg/L + metoprolol 2 g/L (ACE/BB, n=5), two million EPCs intravenously (EPC, n=5)or both (ACE/BB + EPC [n=5]), then sacrificed after two weeks treatment. ACE/BB resulted in a 75% reduction in fibrosis in the region remote from the MI (p<0.05), but EPC therapy had little effect here. Conversely, EPC therapy induced neovascularisation at the peri-infarct rim, thereby preventing peri-infarct apoptosis by 81% (p<0.05). Acting via different but complementary mechanisms, the combination of ACE/BB + EPCs resulted in a greater overall improvement in LV function on echocardiography than either therapy alone. Clinical trials using stem cell therapy in conjunction with standard medical treatment are warranted.

CCR3- and CXCR4-mediated interactions regulate migration of CD34+ human bone marrow progenitors to ischemic myocardium and subsequent tissue repair

OBJECTIVE Hematopoietic progenitor cells are able to induce neovascularization of ischemic myocardium, inhibit apoptosis, and prevent heart failure. They express functional CC chemokine-binding receptor 3 (CCR3) and CXC chemokine-binding receptor 4 (CXCR4); however, the role of those receptors in migration of progenitor cells into the ischemic myocardium is unknown. METHODS Myocardial infarction was surgically induced in athymic nude rats, and human bone marrow-derived CD34+ cells or saline was injected into the tail vein. Cell chemotaxis was studied in vitro using chemotaxis chambers with or without concomitant stimulation with eotaxin or stromal cell-derived factor-1. Cell migration into ischemic myocardium was evaluated by immunohistochemistry. CCR3 and CXCR4 antibodies or local injections of stromal cell-derived factor-1 were used to investigate the role of chemokine expression in the migration capacity of the injected cells. Morphologic analysis included evaluation of apoptosis and capillary density in the ischemic myocardium. RESULTS Ischemic rat myocardium demonstrated induced messenger RNA expression for the CCR3-binding chemokines eotaxin, RANTES (regulated on activation, normal T expressed and secreted), and monocyte chemotactic protein-3, but not the CXCR4-binding chemokine stromal cell-derived factor-1. Migration of human angioblasts to ischemic rat myocardium was inhibited by a blocking anti-CCR3 monoclonal antibody, but not by a blocking anti-CXCR4 monoclonal antibody, which instead inhibited migration to bone marrow. Finally, intramyocardial injection of stromal cell-derived factor-1 redirected migration of human angioblasts to ischemic rat hearts, resulting in augmented neovascularization, enhanced cardiomyocyte survival, and functional cardiac recovery. CONCLUSIONS CCR3-dependent chemokine interactions regulate endogenous migration of CD34+ progenitors from bone marrow to ischemic but not to normal myocardium. Manipulating CXCR4-dependent interactions could enhance the efficacy of cell therapy after myocardial infarction.

A DNA enzyme against plasminogen activator inhibitor- type 1 (PAI-1) limits neointima formation after angioplasty in an obese diabetic rodent model.

We investigated whether targeted cleavage of PAI-1 mRNA might prevent post-angioplasty neointima formation in diabetic JCR:LA-cp/cp rats with naturally elevated PAI-1 levels. Catalytic DNA enzymes targeting rat PAI-1 mRNA (PAI-1 DNA enzyme, n = 12) or a random sequence as control (scrambled DNA enzyme, n = 12) were infused at the site of arterial damage. Control animals demonstrated prominent PAI-1 protein expression in the arterial endothelium at 48 hours, and robust neointimal proliferation by two weeks, with 60 ± 10% mean occlusion of the artery lumen. The neointimal lesion consisted of dense fibrin deposition and numerous proliferating smooth muscle cells, as determined by dual α-smooth muscle actin/Ki67 expression. Treatment with PAI-1 DNA enzyme resulted in marked early (48 hour) reduction of endothelial PAI-1 protein expression, which persisted for the next two weeks as well as a two fold reduction of expression of PAI-1 mRNA by RT-PCR at the same time point, (P < 0.05). By two weeks, PAI-1 DNA enzyme treated animals demonstrated significantly reduced levels of fibrin deposition and 5-fold lower levels of proliferating smooth muscle cells at the site of arterial injury compared to controls (P < 0.01), and a 2-fold lower neointima/media ratio (0.67 ± 0.11 vs 1.39 ± 0.12) (P < 0.05). Treatment with a catalytic PAI-1 DNA enzyme successfully prevents neointimal proliferation after balloon injury in diabetic animals.

Downregulation of the CXC chemokine receptor 4/stromal cell–derived factor 1 pathway enhances myocardial neovascularization, cardiomyocyte survival, and functional recovery after myocardial infarction

OBJECTIVES Although adequate numbers of hematopoietic progenitor cells reside in the human bone marrow, the extent of endogenous neovascularization after myocardial infarction remains insufficient. The aim of this study was to identify the role of the CXC chemokine receptor 4/stromal cell-derived factor 1 axis in the mobilization and homing of hematopoietic progenitor cells in the ischemic heart. METHODS Human bone marrow-derived hematopoietic progenitor cells or saline were injected systemically into athymic nude rats 48 hours after myocardial infarction. Myocardial and bone marrow expression of stromal cell-derived factor 1 and chemotaxis of hematopoietic progenitor cells were measured in vitro in the presence or absence of stromal cell-derived factor 1. The role of the CXC chemokine receptor 4/stromal cell-derived factor 1 axis was investigated by means of antibody blockade or systemic administration of granulocyte colony-stimulating factor. Morphologic analysis included measurement of the infarct area, capillary density, and apoptosis, whereas left ventricular function was measured by means of echocardiographic analysis. RESULTS Expression of postinfarct stromal cell-derived factor 1 was increased by 67% in the bone marrow and decreased by 43% in myocardium. Disruption of bone marrow stromal cell-derived factor 1/CXC chemokine receptor 4 interactions by antibody blockade resulted in a redirection of human hematopoietic progenitor cells from the bone marrow to the ischemic heart and augmented neovascularization and cardiomyocyte survival. Similarly, systemic administration of granulocyte colony-stimulating factor to block CXC chemokine receptor 4/stromal cell-derived factor 1 interaction resulted in increased mobilization and homing of hematopoietic progenitor cells to the ischemic heart, which translated to augmented myocardial neovascularization, prevention of apoptosis, and improved cardiac function. CONCLUSIONS Bone marrow stromal cell-derived factor 1 upregulation after myocardial ischemia prevents mobilization of endogenous hematopoietic progenitor cells. We provide evidence that disruption of stromal cell-derived factor 1/CXC chemokine receptor 4 interactions allows redirection of hematopoietic progenitor cells to ischemic myocardium and enhances recovery of left ventricular function.