Mika Wada

Estrogen-Mediated Endothelial Progenitor Cell Biology and Kinetics For Physiological Postnatal Vasculogenesis

Estrogen has been demonstrated to promote therapeutic reendothelialization after vascular injury by bone marrow (BM)–derived endothelial progenitor cell (EPC) mobilization and phenotypic modulation. We investigated the primary hypothesis that estrogen regulates physiological postnatal vasculogenesis by modulating bioactivity of BM-derived EPCs through the estrogen receptor (ER), in cyclic hormonally regulated endometrial neovascularization. Cultured human EPCs from peripheral blood mononuclear cells (PB-MNCs) disclosed consistent gene expression of ER &agr; as well as downregulated gene expressions of ER &bgr;. Under the physiological concentrations of estrogen (17&bgr;-estradiol, E2), proliferation and migration were stimulated, whereas apoptosis was inhibited on day 7 cultured EPCs. These estrogen-induced activities were blocked by the receptor antagonist, ICI182,780 (ICI). In BM transplanted (BMT) mice with ovariectomy (OVX) from transgenic mice overexpressing &bgr;-galactosidase (lacZ) regulated by an endothelial specific Tie-2 promoter (Tie-2/lacZ/BM), the uterus demonstrated a significant increase in BM-derived EPCs (lacZ expressing cells) incorporated into neovasculatures detected by CD31 immunohistochemistry after E2 administration. The BM-derived EPCs that were incorporated into the uterus dominantly expressed ER &agr;, rather than ER &bgr; in BMT mice from BM of transgenic mice overexpressing EGFP regulated by Tie-2 promoter with OVX (Tie-2/EGFP/BMT/OVX) by ERs fluorescence immunohistochemistry. An in vitro assay for colony forming activity as well as flow cytometry for CD133, CD34, KDR, and VE-cadherin, using human PB-MNCs at 5 stages of the female menstrual-cycle (early-proliferative, pre-ovulatory, post-ovulatory, mid-luteal, late-luteal), revealed cycle-specific regulation of EPC kinetics. These findings demonstrate that physiological postnatal vasculogenesis involves cyclic, E2-regulated bioactivity of BM-derived EPCs, predominantly through the ER&agr;.

Specific Jagged-1 Signal From Bone Marrow Microenvironment Is Required for Endothelial Progenitor Cell Development for Neovascularization

Background— Despite accumulating evidence that proves the pivotal role of endothelial progenitor cells (EPCs) in ischemic neovascularization, the key signaling cascade that regulates functional EPC kinetics remains unclear. Methods and Results— In this report, we show that inactivation of specific Jagged-1 (Jag-1)–mediated Notch signals leads to inhibition of postnatal vasculogenesis in hindlimb ischemia via impairment of proliferation, survival, differentiation, and mobilization of bone marrow–derived EPCs. Bone marrow–derived EPCs obtained from Jag-1−/− mice, but not Delta-like (Dll)-1−/− mice, demonstrated less therapeutic potential for ischemic neovascularization than EPCs from the wild type. In contrast, a gain-of-function study using 3T3 stromal cells overexpressing Notch ligand revealed that Jag-1–mediated Notch signals promoted EPC commitment, which resulted in enhanced neovascularization. The impaired neovascularization in Jag-1−/− mice was profoundly rescued by transplantation of Jag-1–stimulated EPCs. Conclusions— These data indicate that specific Jag-1–derived Notch signals from the bone marrow microenvironment are critical for EPC–mediated vasculogenesis, thus providing an important clue for modulation of strategies for therapeutic neovascularization.

Clonal multipotency of skeletal muscle-derived stem cells between mesodermal and ectodermal lineage.

The differentiation potential of skeletal muscle‐derived stem cells (MDSCs) after in vitro culture and in vivo transplantation has been extensively studied. However, the clonal multipotency of MDSCs has yet to be fully determined. Here, we show that single skeletal muscle‐derived CD34−/CD45− (skeletal muscle‐derived double negative [Sk‐DN]) cells exhibit clonal multipotency that can give rise to myogenic, vasculogenic, and neural cell lineages after in vivo single cell‐derived single sphere implantation and in vitro clonal single cell culture. Muscles from green fluorescent protein (GFP) transgenic mice were enzymatically dissociated and sorted based on CD34 and CD45. Sk‐DN cells were clone‐sorted into a 96‐well plate and were cultured in collagen‐based medium with basic fibroblast growth factor and epidermal growth factor for 14 days. Individual colony‐forming units (CFUs) were then transplanted directly into severely damaged muscle together with 1 × 105 competitive carrier Sk‐DN cells obtained from wild‐type mice muscle expanded for 5 days under the same culture conditions using 35‐mm culture dishes. Four weeks after transplantation, implanted GFP+ cells demonstrated differentiation into endothelial, vascular smooth muscle, skeletal muscle, and neural cell (Schwann cell) lineages. This multipotency was also confirmed by expression of mRNA markers for myogenic (MyoD, myf5), neural (Musashi‐1, Nestin, neural cell adhesion molecule‐1, peripheral myelin protein‐22, Nucleostemin), and vascular (α‐smooth muscle actin, smoothelin, vascular endothelial‐cadherin, tyrosine kinase‐endothelial) stem cells by clonal (single‐cell derived) single‐sphere reverse transcription‐polymerase chain reaction. Approximately 70% of clonal CFUs exhibited expression of all three cell lineages. These findings support the notion that Sk‐DN cells are a useful tool for damaged muscle‐related tissue reconstitution by synchronized vasculogenesis, myogenesis, and neurogenesis.

Pivotal Role of Lnk Adaptor Protein in Endothelial Progenitor Cell Biology for Vascular Regeneration

Despite the fact that endothelial progenitor cells (EPCs) are important for postnatal neovascularization, their origins, differentiation, and modulators are not clear. Here, we demonstrate that Lnk, a negative regulator of hematopoietic stem cell proliferation, controls endothelial commitment of c-kit+/Sca-1+/Lineage− (KSL) subpopulations of bone marrow cells. The results of EPC colony–forming assays reveal that small (primitive) EPC colony formation by CD34− KSLs and large (definitive) EPC colony formation by CD34(dim) KSLs are more robust in lnk−/− mice. In hindlimb ischemia, perfusion recovery is augmented in lnk−/− mice through enhanced proliferation and mobilization of EPCs via c-Kit/stem cell factor. We found that Lnk-deficient EPCs are more potent actors than resident cells in hindlimb perfusion recovery and ischemic neovascularization, mainly via the activity of bone marrow-EPCs. Similarly, lnk−/− mice show augmented retinal neovascularization and astrocyte network maturation without an increase in indicators of pathogenic angiogenesis in an in vivo model of retinopathy. Taken together, our results provide strong evidence that Lnk regulates bone marrow-EPC kinetics in vascular regeneration. Selective targeting of Lnk may be a safe and effective strategy to augment therapeutic neovascularization by EPC transplantation.

Cardiomyocyte Formation by Skeletal Muscle-Derived Multi-Myogenic Stem Cells after Transplantation into Infarcted Myocardium

BACKGROUND Cellular cardiomyoplasty for myocardial infarction has been developed using various cell types. However, complete differentiation and/or trans-differentiation into cardiomyocytes have never occurred in these transplant studies, whereas functional contributions were reported. METHODS AND RESULTS Skeletal muscle interstitium-derived CD34(+)/CD45(-) (Sk-34) cells were purified from green fluorescent protein transgenic mice by flowcytometory. Cardiac differentiation of Sk-34 cells was examined by in vitro clonal culture and co-culture with embryonic cardiomyocytes, and in vivo transplantation into a nude rat myocardial infarction (MI) model (left ventricle). Lower relative expression of cardiomyogenic transcription factors, such as GATA-4, Nkx2-5, Isl-1, Mef2 and Hand2, was seen in clonal cell culture. However, vigorous expression of these factors was seen on co-culture with embryonic cardiomyocytes, together with formation of gap-junctions and synchronous contraction following sphere-like colony formation. At 4 weeks after transplantation of freshly isolated Sk-34 cells, donor cells exhibited typical cardiomyocyte structure with formation of gap-junctions, as well as intercalated discs and desmosomes, between donor and recipient and/or donor and donor cells. Fluorescence in situ hybridization (FISH) analysis detecting the rat and mouse genomic DNA and immunoelectron microscopy using anti-GFP revealed donor-derived cells. Transplanted Sk-34 cells were incorporated into infarcted portions of recipient muscles and contributed to cardiac reconstitution. Significant improvement in left ventricular function, as evaluated by transthoracic echocardiography and micro-tip conductance catheter, was also observed. CONCLUSIONS AND SIGNIFICANCE Skeletal muscle-derived multipotent Sk-34 cells that can give rise to skeletal and smooth muscle cells as reported previously, also give rise to cardiac muscle cells as multi-myogenic stem cells, and thus are a potential source for practical cellular cardiomyoplasty.

Establishment of a Functionally Active Collagen-Binding Vascular Endothelial Growth Factor Fusion Protein In Situ

Objective—Tissue regeneration requires both growth factor and extracellular matrix such as collagen, serving as a scaffold for cell growth. We established FNCBD-VEGF121, consisting of the fibronectin collagen-binding domain (FNCBD) and vascular endothelial growth factor (VEGF) 121, and investigated its properties. Methods and Results—FNCBD-VEGF121 specifically bound to gelatin and type I, II, III, IV, and V collagen. This collagen-bound FNCBD-VEGF121 captured soluble VEGF receptor 2 (VEGFR-2)/Fc chimeric protein. Cell growth-promoting activity of FNCBD-VEGF121 was almost identical to that of VEGF121. The VEGF fusion protein significantly enhanced the expression of VEGFR-2 (71.6±0.8%) on endothelial progenitor cells (EPCs) derived from umbilical cord blood. Expectably, the collagen-bound VEGF fusion protein not only promoted the growth of endothelial cells (ECs) but also induced the expression of VEGFR-2 (63.7±0.8%) on non-adherent cells expanded from bone marrow CD34+ cells. Moreover, the VEGF fusion protein enhanced sprout formation of ECs in a matrigel model. In vivo experiments revealed that FNCBD-VEGF121 had local effects but not systemic effect on EPC mobilization. Conclusions—These results suggest that FNCBD-VEGF121 stably maintains an optimally high and local concentration of VEGF with collagen matrix and stimulates both ECs and EPCs in situ, supplying a vascular regeneration niche.

The effects of flap ischemia on normal and diabetic progenitor cell function.

Background: Endothelial progenitor cells play an important role in neovascularization of ischemic flaps, a process that is significantly impaired in diabetes. This is the first investigation into the effects of flap ischemia on circulating and bone marrow–derived endothelial progenitor cells. Potential mechanisms for impaired vasculogenesis in diabetes are also investigated. Methods: Circulating and bone marrow–derived endothelial progenitor cells were isolated from wild-type (n = 24) and diabetic mice (n = 24) with ischemic flaps (days 0, 1, 3, and 7). The number and vasculogenic function of primitive and definitive endothelial progenitor cells were determined by fluorescence-activated cell sorting analysis, culture assay, and vasculogenic colony-forming assay. Results: Ischemia mobilized endothelial progenitor cells (25 ± 0.5 cells per high-power field at day 7 versus 9.0 ± 0.6 cells per high-power field, p < 0.01) and enhanced the vasculogenic potential of circulating primitive endothelial progenitor cells (23 ± 3.2 at day 3 versus 14 ± 0.8, p < 0.01) relative to baseline. In the bone marrow, endothelial progenitor cell number and vasculogenic potential peaked at day 3 (2.1 ± 0.3 × 105 cells versus 1.3 ± 0.1 × 105 cells, p < 0.05; 36 ± 1.9 versus 27 ± 1.6, p < 0.05, respectively). In diabetes, circulating endothelial progenitor cell mobilization (5.8 ± 0.4 cells per high-power field versus 9.0 ± 0.6 cells per high-power field, p < 0.01) and vasculogenic potential (36 ± 1.7 versus 43 ± 2.6, p < 0.05) were impaired relative to the wild-type animals. Bone marrow–derived endothelial progenitor cell number was normal in diabetic animals, but the vasculogenic potential of these cells was significantly impaired (5.7 ± 0.8 day 1 versus 13.4 ± 2.5, p < 0.05). Conclusions: Flap ischemia induces phenotypic changes in bone marrow–derived endothelial progenitor cells that subsequently traffic through the circulation. The vasculogenic potential of endothelial progenitor cells at various stages of differentiation is impaired in diabetes and thus may account for impaired ischemia-induced vasculogenesis observed clinically.

Role of phospholipase C-L2, a novel phospholipase C-like protein that lacks lipase activity, in B-cell receptor signaling.

ABSTRACT Phospholipase C (PLC) plays important roles in phosphoinositide turnover by regulating the calcium-protein kinase C signaling pathway. PLC-L2 is a novel PLC-like protein which lacks PLC activity, although it is very homologous with PLC δ. PLC-L2 is expressed in hematopoietic cells, but its physiological roles and intracellular functions in the immune system have not yet been clarified. To elucidate the physiological function of PLC-L2, we generated mice which had a genetic PLC-L2 deficiency. PLC-L2-deficient mice grew with no apparent abnormalities. However, mature B cells from PLC-L2-deficient mice were hyperproliferative in response to B-cell receptor (BCR) cross-linking, although B2 cell development appeared to be normal. Molecular biological analysis revealed that calcium influx and NFATc accumulation in nuclei were increased in PLC-L2-deficient B cells. Extracellular signal-regulated kinase activity was also enhanced in PLC-L2-deficient B cells. These mice had a stronger T-cell-independent antigen response. These results indicate that PLC-L2 is a novel negative regulator of BCR signaling and immune responses.

Reconstitution of human haematopoiesis in non-obese diabetic/severe combined immunodeficient mice by clonal cells expanded from single CD34+CD38- cells expressing Flk2/Flt3.

Summary. In the present study, we examined the expression of Flk2/Flt3, a tyrosine kinase receptor, on human cord blood CD34+ haematopoietic progenitor/stem cells. In flow cytometric analysis, Flk2/Flt3 was expressed on 80% of CD34+ cells and their immature subpopulations, CD34+CD33– and CD34+CD38– cells. Methycellulose clonal culture of sorted CD34+Flk2/Flt3+ and CD34+Flk2/Flt3– cells showed that most of myelocytic progenitors expressed Flk2/Flt3, but erythroid and haematopoietic multipotential progenitors were shared by both fractions. When 1 × 104 lineage marker‐negative (Lin–)CD34+Flk2/Flt3– cells were transplanted into non‐obese diabetic/severe combined immunodeficient (NOD/SCID) mice, none of the recipients possessed human CD45+ cells in bone marrow 11–12 weeks after the transplantation. In contrast, all recipients transplanted with 1 × 104 Lin–CD34+Flk2/Flt3+ cells showed successful engraftment. Furthermore, clonal cells expanded from single Lin–CD34+CD38–Flk2/Flt3+ cells in the culture with Flk2/Flt3 ligand, stem cell factor, thrombopoietin, and a complex of interleukin 6/soluble interleukin 6 receptor were individually transplanted into NOD/SCID mice. At 20 to 21 weeks after the transplantation, three out of 10 clones harvested at d 7 of culture, and three out of six clones at d 14 could reconstitute human haematopoiesis in recipient marrow. These results demonstrated that Flk2/Flt3 was expressed on a wide variety of human haematopoietic cells including long‐term‐repopulating haematopoietic stem cells.

Autologous G-CSF-Mobilized Peripheral Blood CD34 + Cell Therapy for Diabetic Patients With Chronic Nonhealing Ulcer

Recently, animal studies have demonstrated the efficacy of endothelial progenitor cell (EPC) therapy for diabetic wound healing. Based on these preclinical studies, we performed a prospective clinical trial phase I/IIa study of autologous G-CSF-mobilized peripheral blood (PB) CD34+ cell transplantation for nonhealing diabetic foot patients. Diabetic patients with nonhealing foot ulcers were treated with 2 × 107 cells of G-CSF-mobilized PB CD34+ cells as EPC-enriched population. Safety and efficacy (wound closure and vascular perfusion) were evaluated 12 weeks posttherapy and further followed for complete wound closure and recurrence. A total of five patients were enrolled. Although minor amputation and recurrence were seen in three out of five patients, no death, other serious adverse events, or major amputation was seen following transplantation. Complete wound closure was observed at an average of 18 weeks with increased vascular perfusion in all patients. The outcomes of this prospective clinical study indicate the safety and feasibility of CD34+ cell therapy in patients with diabetic nonhealing wounds.