Masamichi Eguchi

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.

Endothelial progenitor cells for postnatal vasculogenesis

Bone marrow-derived endothelial progenitor cells (EPCs) are present in the systemic circulation, are augmented in response to certain cytokines and/or tissue ischemia, and are home to – as well as incorporate into – sites of neovascularization. On the basis of these aspects, EPCs have attractive potential therapeutic applications for cardiovascular ischemic diseases as a novel cell-based strategy, mainly via a vasculogenesis mechanism. This review provides an update of the biology of EPCs, as well as highlighting the potential use of these cells for therapeutic regeneration.

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.

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.

Lesion-targeted thrombopoietin potentiates vasculogenesis by enhancing motility and enlivenment of transplanted endothelial progenitor cells via activation of Akt/mTOR/p70S6kinase signaling pathway

Thrombopoietin (TPO), a physiological regulator of megakaryocyte and platelet development, is a multifunctional positive regulator in early hematopoiesis by hematopoietic stem cells. In this study, we investigated the effect of TPO on endothelial progenitor cells (EPCs) for therapeutic vasculogenesis in vitro and in vivo, and the intracellular signaling mechanism exerting the activity of EPCs. 7-day culture-expanded EPCs derived from human peripheral blood mononuclear cells were applied to each assay. Flow cytometry demonstrated the expression of c-Mpl, the receptor of TPO, in cultured EPCs. In vitro experiments revealed enhanced migration and survival of cultured EPCs by TPO. In vivo, TPO was intramuscularly administered into the foci of ischemic hindlimbs in athymic nude mice, immediately followed by intravenous injection of cultured EPCs, to assess the booster effect of TPO on vascular regeneration. At day 4 post-transplantation, transplanted EPCs were 1.7-fold higher in TPO-treated animals compared to control. At day 28, blood perfusion was recovered in the TPO-treated group, accompanied by an increase in microvascular density. The signaling transduction pathway underlying TPO-mediated activities of cultured EPCs was assessed by Western blotting. TPO induced sequential phosphorylations of Akt to p70S6kinase through mTOR. Inhibition of the PI3-kinase/Akt/mTOR/p70S6kinase signaling pathway negated the biological functions of cultured EPCs, either migration (by LY294002 for PI3-kinase and Rapamycin for mTOR) or survival and tubulogenesis (by Rapamycin). These findings provide evidence that TPO possesses booster potential for therapeutic vasculogenesis, by activating the PI3-kinase/Akt/mTOR/p70S6kinase pathway crucial to the biological activities of EPCs.

Jagged-1 Signaling in the Bone Marrow Microenvironment Promotes Endothelial Progenitor Cell Expansion and Commitment of CD133+ Human Cord Blood Cells for Postnatal Vasculogenesis.

Notch signaling is involved in cell fate decisions during murine vascular development and hematopoiesis in the microenvironment of bone marrow. To investigate the close relationship between hematopoietic stem cells and human endothelial progenitor cells (EPCs) in the bone marrow niche, we examined the effects of Notch signals [Jagged-1 and Delta-like ligand (Dll)-1] on the proliferation and differentiation of human CD133+ cell-derived EPCs. We established stromal systems using HESS-5 murine bone marrow cells transfected with human Jagged-1 (hJagged-1) or human Dll-1 (hDll-1). CD133+ cord blood cells were co-cultured with the stromal cells for 7 days, and then their proliferation, differentiation, and EPC colony formation was evaluated. We found that hJagged-1 induced the proliferation and differentiation of CD133+ cord blood EPCs. In contrast, hDll-1 had little effect. CD133+ cells stimulated by hJagged-1 differentiated into CD31+/KDR+ cells, expressed vascular endothelial growth factor-A, and showed enhanced EPC colony formation compared with CD133+ cells stimulated by hDll-1. To evaluate the angiogenic properties of hJagged-1- and hDll-1-stimulated EPCs in vivo, we transplanted these cells into the ischemic hindlimbs of nude mice. Transplantation of EPCs stimulated by hJagged-1, but not hDll-1, increased regional blood flow and capillary density in ischemic hindlimb muscles. This is the first study to show that human Notch signaling influences EPC proliferation and differentiation in the bone marrow microenvironment. Human Jagged-1 induced the proliferation and differentiation of CD133+ cord blood progenitors compared with hDll-1. Thus, hJagged-1 signaling in the bone marrow niche may be used to expand EPCs for therapeutic angiogenesis.