Tomono Takahashi

Bone Marrow Origin of Endothelial Progenitor Cells Responsible for Postnatal Vasculogenesis in Physiological and Pathological Neovascularization

Circulating endothelial progenitor cells (EPCs) have been isolated in peripheral blood of adult species. To determine the origin and role of EPCs contributing to postnatal vasculogenesis, transgenic mice constitutively expressing beta-galactosidase under the transcriptional regulation of an endothelial cell-specific promoter (Flk-1/LZ or Tie-2/LZ) were used as transplant donors. Localization of EPCs, indicated by flk-1 or tie-2/lacZ fusion transcripts, were identified in corpus luteal and endometrial neovasculature after inductive ovulation. Mouse syngeneic colon cancer cells (MCA38) were implanted subcutaneously into Flk-1/LZ/BMT (bone marrow transplantation) and Tie-2/LZ/BMT mice; tumor samples harvested at 1 week disclosed abundant flk-1/lacZ and tie-2/lacZ fusion transcripts, and sections stained with X-gal demonstrated that the neovasculature of the developing tumor frequently comprised Flk-1- or Tie-2-expressing EPCs. Cutaneous wounds examined at 4 days and 7 days after skin removal by punch biopsy disclosed EPCs incorporated into foci of neovascularization at high frequency. One week after the onset of hindlimb ischemia, lacZ-positive EPCs were identified incorporated into capillaries among skeletal myocytes. After permanent ligation of the left anterior descending coronary artery, histological samples from sites of myocardial infarction demonstrated incorporation of EPCs into foci of neovascularization at the border of the infarct. These findings indicate that postnatal neovascularization does not rely exclusively on sprouting from preexisting blood vessels (angiogenesis); instead, EPCs circulate from bone marrow to incorporate into and thus contribute to postnatal physiological and pathological neovascularization, which is consistent with postnatal vasculogenesis.

Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization

Endothelial progenitor cells (EPCs) have been isolated from circulating mononuclear cells in human peripheral blood and shown to be incorporated into foci of neovascularization, consistent with postnatal vasculogenesis. We determined whether endogenous stimuli (tissue ischemia) and exogenous cytokine therapy (granulocyte macrophage-colony stimulating factor, GM-CSF) mobilize EPCs and thereby contribute to neovascularization of ischemic tissues. The development of regional ischemia in both mice and rabbits increased the frequency of circulating EPCs. In mice, the effect of ischemia-induced EPC mobilization was demonstrated by enhanced ocular neovascularization after cornea micropocket surgery in mice with hindlimb ischemia compared with that in non-ischemic control mice. In rabbits with hindlimb ischemia, circulating EPCs were further augmented after pretreatment with GM-CSF, with a corresponding improvement in hindlimb neovascularization. There was direct evidence that EPCs that contributed to enhanced corneal neovascularization were specifically mobilized from the bone marrow in response to ischemia and GM-CSF in mice transplanted with bone marrow from transgenic donors expressing β-galactosidase transcriptionally regulated by the endothelial cell-specific Tie-2 promoter. These findings indicate that circulating EPCs are mobilized endogenously in response to tissue ischemia or exogenously by cytokine therapy and thereby augment neovascularization of ischemic tissues.

VEGF contributes to postnatal neovascularization by mobilizing bone marrow‐derived endothelial progenitor cells

Vascular endothelial growth factor (VEGF) has been shown to promote neovascularization in animal models and, more recently, in human subjects. This feature has been assumed to result exclusively from its direct effects on fully differentiated endothelial cells, i.e. angiogenesis. Given its regulatory role in both angiogenesis and vasculogenesis during fetal development, we investigated the hypothesis that VEGF may modulate endothelial progenitor cell (EPC) kinetics for postnatal neovascularization. Indeed, we observed an increase in circulating EPCs following VEGF administration in vivo. VEGF‐induced mobilization of bone marrow‐derived EPCs resulted in increased differentiated EPCs in vitro and augmented corneal neovascularization in vivo. These findings thus establish a novel role for VEGF in postnatal neovascularization which complements its known impact on angiogenesis.

Tie2 Receptor Ligands, Angiopoietin-1 and Angiopoietin-2, Modulate VEGF-Induced Postnatal Neovascularization

Angiopoietin-1 (Ang1) has been recently identified as the major physiological ligand for the tyrosine kinase receptor Tie2 and assigned responsibility for recruiting and sustaining periendothelial support cells. Angiopoietin-2 (Ang2) was found to disrupt blood vessel formation in the developing embryo by antagonizing the effects of Ang1 and Tie2 and was thus considered to represent a natural Ang1/Tie2 inhibitor. In vivo effects of either angiopoietin on postnatal neovascularization, however, have not been previously described. Accordingly, we used the cornea micropocket assay of neovascularization to investigate the impact of angiopoietins on neovascularization in vivo. Neither Ang1 nor Ang2 alone promoted neovascularization. Pellets containing vascular endothelial growth factor (VEGF) alone induced corneal neovascularity extending from the limbus across the cornea. Addition of Ang 1 to VEGF (Ang1+VEGF) produced an increase in macroscopically evident perfusion of the corneal neovasculature without affecting macroscopic measurements of length (0.58+/-0.03 mm) or circumferential neovascularity (136+/-10 degrees). In contrast, pellets containing Ang2+VEGF promoted significantly longer (0.67+/-0.05 mm) and more circumferential (160+/-15degrees) neovascularity than VEGF alone or Ang1+VEGF (P<0.05). Excess soluble Tie2 receptor (sTie2-Fc) precluded modulation of VEGF-induced neovascularization by both Ang2 and Ang1. Fluorescent microscopic findings demonstrated enhanced capillary density (fluorescence intensity, 2.55+/-0.23 e+9 versus 1.23+/-0.17 e+9, P<0.01) and increased luminal diameter of the basal limbus artery (39.0+/-2.8 versus 27.9+/-1.3 microm, P<0.01) for Ang1+VEGF compared with VEGF alone. In contrast to Ang1+VEGF, Ang2+VEGF produced longer vessels and, at the tip of the developing capillaries, frequent isolated sprouting cells. In the case of Ang2+VEGF, however, luminal diameter of the basal limbus artery was not increased (26.7+/-1.9 versus 27.9+/-1.3, P=NS). These findings constitute what is to our knowledge the first direct demonstration of postnatal bioactivity associated with either angiopoietin. In particular, these results indicate that angiopoietins may potentiate the effects of other angiogenic cytokines. Moreover, these findings provide in vivo evidence that Ang1 promotes vascular network maturation, whereas Ang2 works to initiate neovascularization.

Vascular Endothelial Growth Factor165 Gene Transfer Augments Circulating Endothelial Progenitor Cells in Human Subjects

Preclinical studies in animal models and early results of clinical trials in patients suggest that intramuscular injection of naked plasmid DNA encoding vascular endothelial growth factor (VEGF) can promote neovascularization of ischemic tissues. Such neovascularization has been attributed exclusively to sprout formation of endothelial cells derived from preexisting vessels. We investigated the hypothesis that VEGF gene transfer may also augment the population of circulating endothelial progenitor cells (EPCs). In patients with critical limb ischemia receiving VEGF gene transfer, gene expression was documented by a transient increase in plasma levels of VEGF. A culture assay documented a significant increase in EPCs (219%, P<0.001), whereas patients who received an empty vector had no change in circulating EPCs, as was the case for volunteers who received saline injections (VEGF versus empty vector, P<0.001; VEGF versus saline, P<0.005). Fluorescence-activated cell sorter analysis disclosed an overall increase of up to 30-fold in endothelial lineage markers KDR (VEGF receptor-2), VE-cadherin, CD34, alpha(v)beta(3), and E-selectin after VEGF gene transfer. Constitutive overexpression of VEGF in patients with limb ischemia augments the population of circulating EPCs. These findings support the notion that neovascularization of human ischemic tissues after angiogenic growth factor therapy is not limited to angiogenesis but involves circulating endothelial precursors that may home to ischemic foci and differentiate in situ through a process of vasculogenesis.

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;.

Vaskulärer endothelialer Wachstumsfaktor (VEGF): Therapeutische Angiogenese und Vaskulogenese in der Behandlung kardiovaskulärer Erkrankungen

The formation of new blood vessel is essential for a variety of physiological processes like embryogenesis and the female reproduction as well as pathological processes like tumor growth, wound healing and neovascularization of ischemic tissue. Vasculogenesis and angiogenesis are the mechanisms responsible for the development of the blood vessels. While angiogenesis refers to the formation of capillaries from pre-existing vessels in the embryo and adult organism, vasculogenesis, the development of new blood vessels from in situ differentiating endothelial cells, has been previously considered restricted to embryogenesis. Recent investigations, however, show the existence of endothelial progenitor cells (EPCs) in the peripheral blood of the adult and their participation in ongoing neovascularization. Molecular and cell-biological experiments suggest that different cytokines and growth factors have a stimulatory effect on these bone-marrow derived EPCs. Results with GM-CSF (granulocyte macrophage-colony stimulating factor) and VEGF (vascular endothelial growth factor) open a new insight into the clinical use of cytokines and in particular the use of growth factors in gene therapy. The administration via protein or plasmid-DNA for neovascularization seems to enhance both pathways, angiogenesis and vasculogenesis.ZusammenfassungDie Bildung neuer Blutgefäße ist bei einer Vielzahl von physiologischen Vorgängen, wie zum Beispiel der Embryogenese und dem weiblichen Reproduktionszyklus, sowie bei pathologischen Prozessen, wie zum Beispiel dem Tumorwachstum, der Wundheilung und der Neovaskularisation ischämischer Gewebe, von Bedeutung. Vaskulogenese und Angiogenese sind die Mechanismen, die für die Neubildung von Gefäßen verantwortlich sind. Während die Angiogenese die Neubildung von Gefäßen aus vorbestehenden Kapillaren im Embryo und im erwachsenen Organismus beschreibt, schien die Vaskulogenese, die Gefäßneubildung aus in situ differenzierenden Endothelvorläuferzellen, bislang auf die Embryogenese beschränkt zu sein. Neueste Ergebnisse zeigen jedoch, daß aus dem Knochenmark stammende endotheliale Vorläuferzellen in der erwachsenen Spezies im peripheren Blut nachweisbar und an der Neovaskularisation beteiligt sind. Molekular- und zellbiologische Untersuchungen deuten daraufhin, daß verschiedene Zytokine und Wachstumsfaktoren stimulierend auf die Mobilisierung dieser endothelialen Vorläuferzellen aus dem Knochenmark wirken.Die Ergebnisse mit GM-CSF (granulocyte macrophage-colony stimulating factor) und VEGF (vascular endothelial growth factor) eröffnen eine neue Betrachtungsweise für die klinische Verwendung von Zytokinen und vor allem für den Einsatz gentherapeutisch anwendbarer Wachstumsfaktoren. Exogen als Protein zugeführte oder als Plasmid-DNS kodierte Wachstumsfaktoren können nicht nur auf dem Wege der Angiogenese, sondern auch unter Mobilisierung von Endothelvorläuferzellen an der Neovaskularisation beteiligt sein.AbstractThe formation of new blood vessel is essential for a variety of physiological processes like embryogenesis and the female reproduction as well as pathological processes like tumor growth, wound healing and neovascularization of ischemic tissue. Vasculogenesis and angiogenesis are the mechanisms responsible for the development of the blood vessels. While angiogenesis refers to the formation of capillaries from pre-existing vessels in the embryo and adult organism, vasculogenesis, the development of new blood vessels from in situ differentiating endothelial cells, has been previously considered restricted to embryogenesis. Recent investigations, however, show the existence of endothelial progenitor cells (EPCs) in the peripheral blood of the adult and their participation in ongoing neovascularization. Molecular and cell-biological experiments suggest that different cytokines and growth factors have a stimulatory effect on these bone-marrow derived EPCs.Results with GM-CSF (granulocyte macrophage-colony stimulating factor) and VEGF (vascular endothelial growth factor) open a new insight into the clinical use of cytokines and in particular the use of growth factors in gene therapy. The administration via protein or plasmid-DNA for neovascularization seems to enhance both pathways, angiogenesis and vasculogenesis.