Abstract-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 BMderived 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 ␣ as well as downregulated gene expressions of ER ␤. Under the physiological concentrations of estrogen (17␤-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 ␤-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 ␣, rather than ER ␤ 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␣. (Circ Res. 2007;101:598-606.)Key Words: estrogen Ⅲ endothelial progenitor cell Ⅲ estrogen receptor Ⅲ physiological postnatal vasculogenesis I n the female reproductive system, neovascularization is a recurring phenomemnon controlled by cyclic development of transient structure and cyclical repair of damaged tissue. 1 The ovarian sex steroid hormones, estrogen and progesterone, are primarily uterotropic and control the cyclical patterns of uterine cell proliferation and vascular growth that occur throughout the nonpregnant menstrual cycle. Given the synchronized nature of neovascularization in this cyclical mannter, it is assumed that angiogenic growth factor expression is induced by steroid hormones and regulates blood vessel formation in reproductive organogenesis. [2][3][4][5] Despite clinical evidence for the significant role of steroid hormones in endometrial neovascularization, further investigation using in vitro and in vivo experiments have yielded inconclusive results regarding pathophysiological mechanisms in angiogenesis. 6 -10 Moreover, estrogen has been sh...

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 STEM CELLS 2007;25: 2283-2290 Disclosure of potential conflicts of interest is found at the end of this article.

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 Ϫ/Ϫ mice, but not Delta-like (Dll)-1 Ϫ/Ϫ 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 Ϫ/Ϫ 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. Key Words: angiogenesis Ⅲ progenitor cells Ⅲ ischemia Ⅲ signal transduction G rowing evidence indicates that the perturbation of Notch signaling leads to dysfunctional behavior of the vascular system. 1 A human degenerative vascular disease, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), is related to mutations in the Notch3 receptor. Alagille syndrome, caused by mutation of the Jagged-1 (Jag-1) gene, is a pleiotropic developmental disease that is accompanied by features of congenital heart defects with cardiovascular anomalies. 2 Murine genetic studies that generate loss or gain of function of Notch receptors or ligands have exhibited abnormalities in blood vessel formation, such as impaired proliferation and migration of endothelial cells (ECs) 3 and arterial-venous identification. 4 -7 These findings indicate the involvement of Notch1, 7 Notch3, 8 and Notch4 7 receptors, as well as Deltalike ligand (Dll)-4 4,5 and Jag-1 9 ligands, in vascular formation. Recently, Notch ligand, especially Dll-4, has been focused on as an essential regulator for tumor angiogenesis and vascular development in terms of ligand signal control from tissue environment for EC bioactivity through Notch receptors. 10,11 Clinical Perspective p 165Although pioneered in the field of vascular biology, especially in terms of EC morphogenesis for blood vessel development and EC determination of arterial-venous specification, the role of Notch signal in stem cell-related postnatal vasculogenesis has not been investigated. Endothelial progenitor cells (EPCs) derived from bone marrow (BM) play an important role in the promotion of vascular and tissue repair in physiological and pathological conditions, such as coronary or periphe...

In order to establish the practical isolation and usage of skeletal muscle-derived stem cells (MDSCs), we determined reconstitution capacity of CD34(-)/CD45(-) (Sk-DN) cells as a candidate somatic stem cell source for transplantation. Sk-DN cells were enzymatically isolated from GFP transgenic mice (C57/BL6N) skeletal muscle and sorted using fluorescence activated cell sorting (FACS), and expanded by collagen gel-based cell culture with bFGF and EGF. The number of Sk-DN cells was small after sorting (2-8 x 10(4)); however, the number increased 10-20 fold (2-16 x 10(5)) after 6 days of expansion culture, and the cells maintained immature state and multipotency, expressing mRNAs for mesodermal and ectodermal cell lineages. Transplantation of expanded Sk-DN cells into the severe muscle damage model (C57/BL6N wild-type) resulted in the synchronized reconstitution of blood vessels, peripheral nerves and muscle fibers following significant recovery of total muscle mass (57%) and contractile function (55%), whereas the non-cell-transplanted control group showed around 20% recovery in both factors. These reconstitution capacities were supported by the intrinsic plasticity of Sk-DN cells that can differentiate into muscular (skeletal muscle), vascular (pericyte, endothelial cell and smooth muscle) and peripheral nerve (Schwann cells and perineurium) cell lineages that was revealed by transplantation to non-muscle tissue (beneath renal capsule) and fluorescence in situ hybridization (FISH) analysis.

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 × 10(7) 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.

BackgroundCellular 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 ResultsSkeletal 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 SignificanceSkeletal 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.

The mechanism underlying the development of osteopenia or osteoporosis in longstanding phenylketonuria (PKU) remains to be clarified. We investigated the details of bone metabolism in 21 female and 13 male classical PKU patients aged 20-35 years. Vitamin D (VD), parathyroid hormone (PTH), bone turnover markers, and daily nutrient intake were examined. The patients had lower daily energy and protein intake than did the age-matched controls (22 women, 14 men), but their respective fat, VD, and calcium intake did not differ. Serum 1,25-dihydroxy VD and 25-hydroxy VD levels in female and male patient groups were significantly higher and lower than those in respective control groups (females, P < 0.001; males, P < 0.05 and P < 0.01, respectively). Serum intact PTH levels were significantly higher in the female patient group (P < 0.05). Urinary calcium levels in the patient groups were significantly higher than those of the control subjects (females, P < 0.001; males, P < 0.05). Bone resorption markers were significantly higher in patients than in controls, although bone formation markers were not different. Patient serum levels of osteoprotegerin-inhibiting bone resorption were significantly lower (females, P < 0.001; males, P < 0.01). None of the bone parameters correlated significantly with serum phenylalanine or nutrient intake. PKU patients exhibited lower VD status and more rapid bone resorption despite normal calcium-VD intakes.

Abstract-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 ϩ /Sca-1 ϩ /Lineage Ϫ (KSL) subpopulations of bone marrow cells. The results of EPC colony-forming assays reveal that small (primitive) EPC colony formation by CD34 Ϫ KSLs and large (definitive) EPC colony formation by CD34 (dim) KSLs are more robust in lnk Ϫ/Ϫ mice. In hindlimb ischemia, perfusion recovery is augmented in lnk Ϫ/Ϫ 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 Ϫ/Ϫ 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. Key Words: endothelial progenitor cell Ⅲ lnk Ⅲ vascular regeneration S tem cell-related, postnatal neovascularization requires several activities of putative stem cells and their progeny, endothelial progenitor cells (EPCs), including the ability to self-renew in bone marrow (BM), commitment and differentiation into mature endothelial cells (ECs), mobilization from BM into the circulatory system, and recruitment to sites of neovascularization. 1,2 Many cytokines augment mobilization and/or recruitment of BM-derived EPCs, 3,4 including granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor; angiogenic growth factors such as vascular endothelial growth factor (VEGF) and stromal cellderived factor (SDF)-1; estrogen; and pharmaceutical drugs such as statins. However, these factors act not only on immature stem/progenitor cells but also on hematopoietic cells and mature ECs. Thus, the identification of a novel molecule that specifically regulates immature populations involved in EPC kinetics in BM is warranted.Differentiation of progenitor cells into hematopoietic and endothelial lineage cells has been intensively investigated. During development, hemangioblastic aggregates originate from the mesodermal yolk sac, migrate to the fetal liver, and finally establish themselves in the BM. The results of a number of gene-targeting studies contribute to our understanding of functional molecules such as Scl/Tal, 5 c-kit, CD34, Runx-1, 6 and Flk-1, 7 which regulate the developmental kinetics of hemangioblasts and are also expressed in the common precursors of hematopoietic cells and ECs. Postnatal hematopoietic stem cells (HSCs) and EPCs also share...