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.
Received November 25, 2007; accepted April 18, 2008.
Growing 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 Notch47 receptors, as well as Delta-like ligand (Dll)-44,5 and Jag-19 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 165
Although 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 peripheral vascular diseases.12–14 BM-derived EPCs are committed and differentiated from hemangioblastic stem cells,15–17 a common stem cell for EPCs and hematopoietic stem cells (HSCs), into endothelial lineage and mobilized from BM into circulating blood, then recruited into sites of ischemia and interaction with tissue-specific cells to regenerate blood vessels in organs. Because vasculogenesis is essential for adult neovascularization,12–14 and given the angiogenesis mechanism, the Notch ligand/receptor systems could play a key role in the functional kinetics of BM-derived EPCs.
In BM, Notch ligands, especially Jag-1 and Dll-1, are expressed mainly by osteoblasts, stromal cells, ECs, and hematopoietic stem/progenitor cells.3,18–20 These cells consist of microenvironmental niches for HSC self-renewal and commitment for hematopoietic maintenance, which has been of great interest recently.21–24 The interaction between osteoblasts that express Notch ligands and HSCs that express Notch receptors is considered to be one of the key molecular mechanisms underlying the regulation of HSC function in the BM niche.
Considering the common origin and localization of HSCs and EPCs,15–17 we were interested in controlling EPC maintenance and kinetics by modulating Notch signals in BM niches. EPC proliferation, commitment from hemangioblast, differentiation as an endothelial phenotype, and mobilization into circulation for vascular maintenance could be regulated by certain pathways triggered by specific Notch ligand–mediated signals in BM environments. The purpose of the present study was to investigate the role of specific Notch ligands, Jag-1 and Dll-1, on EPC biology in BM through a loss-of-function study using conditional knockout mice and a gain-of-function study using a coculture system with a gene-modified stromal cell line.
Animals and Stromal Cell Line
Conditional Jag-1−/− mice (loxP/loxP, mxCre) or conditional Dll-1−/− mice (loxP/loxP, mxCre) were generated as reported previously.20,25 For gene targeting, polyinosinic:polycytidylic acid ( poly I:C; 200 μg/200 μL) was administered intravenously 4 times over a period of 12 days (once every 3 days). For the gain-of-function study, 3T3 stromal cells in which Jag-1, Dll-1, or empty vector was transduced retrovirally were cultured in DMEM with 10% fetal bovine serum.
Evaluation of EPC Bioactivity: EPC Colony Assay, Migration Assay, Proliferation Assay, Apoptosis, and Gene or Protein Assay
After BM c-kit+/Sca-1+/lineage (Lin)− cells (KSLs) and peripheral blood (PB)–mononuclear cells were isolated, we performed an EPC colony-forming assay, recently established in our laboratory. To investigate the different functions of EPCs under various conditions, a migration assay, proliferation assay, apoptosis assay, and expression analysis of both gene and protein were performed.
Evaluation of EPC Kinetics in the Hindlimb Ischemia Model
A hindlimb ischemia model was generated to evaluate in vivo EPC functions, such as capacity for blood vessel regeneration, mobilization from BM, incorporation into sites of neovascularization, and survival of endogenous cells. A more detailed and expanded description of the materials and methods used is provided in the online-only Data Supplement.
All data are presented as mean±SEM. The results were analyzed statistically with the use of the software package Statview 5.0 (Abacus Concepts Inc, Berkeley, Calif). A paired t test was performed to compare the bromodeoxyuridine (BrdU) incorporation rate of EPCs before and after hindlimb ischemia. Scheffé’s test was performed for multiple comparisons after ANOVA between each group. A P value <0.05 was considered to denote statistical significance.
The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.
Loss of Jag-1–Mediated Notch Signal Impairs EPC Commitment and Mobilization From BM
To prove the significance of Notch ligand for EPC biology, we analyzed EPC kinetics in conditional Jag-1 or Dll-1 null mice, which were generated by cre/loxP systems and induced in a timely manner by administration of poly I:C in postnatal stages. In this system, reverse-transcription polymerase chain reaction revealed that expression of Jag-1 or Dll-1 was decreased drastically in BM stromal cells (Figure 1A) and KSLs (online-only Data Supplement Figure Ib) in Jag-1 null or Dll-1 null mice, respectively, compared with wild-type mice. In contrast, no significant differences between Jag-1 null, Dll-1 null, and wild-type mice were found with regard to expression levels of Notch receptors in BM stromal cells (online-only Data Supplement Figure Ia) or KSLs (online-only Data Supplement Figure Ic). The frequency of KSLs in BM-Lin− cells was similar in Jag-1 null, Dll-1 null, and WT mice (Figure 1B). Flow cytometry analysis of BM mononuclear cell samples demonstrated that the frequency of Flk-1 (VEGFR2 [vascular endothelial growth factor receptor-2])+/CD31+ or Flt-1 (VEGFR1)+/CD31+ cells in Sca-1+/Lin− cells, the EPC-enriched cells, decreased significantly in Jag-1−/− mice compared with Dll-1−/− mice and wild-type mice (Figure 1C). To determine whether the impaired EPC commitment was accompanied by a defect in vasculogenic capacity in Jag-1−/− mice, BM KSLs from Jag-1−/−, Dll-1−/−, or wild-type mice were allowed to form a cluster of EPC colonies with spindle-like morphology. KSLs from Jag-1−/− mice indeed formed fewer EPC colonies than wild-type KSLs, although the absence of Dll-1 in KSLs did not lead to significant defects in vasculogenic capacity (Figure 1D). The effect of Notch signaling on the vasculogenic capacity of EPCs was also evaluated by experimental inhibition of Notch signals in BM-KSLs with γ-secretase II, which blocks the cleavage steps of the intracellular domain of Notch receptors, revealing that the inhibition of Notch signals resulted in a significant decrease in EPC colony-formation activity (online-only Data Supplement Figure II).
To evaluate the kinetics of EPCs mobilized from BM, PB mononuclear cells were isolated and analyzed by both EPC culture assay and EPC colony-forming assay. Importantly, the EPC culture assay indicated that the number of attached EPCs that represented uptake of acetylated LDL and expression of the endothelial markers isolectin B4, Flk-1 (VEGFR2), and/or endothelial nitric oxide synthase was significantly less in Jag-1−/− mice than in either Dll-1−/− or wild-type mice (online-only Data Supplement Figures IIIa, IIIb, and IIIc). The EPC colony-forming assay also demonstrated the significantly impaired vasculogenic capacity of PB mononuclear cells in Jag-1−/− mice compared with Dll-1−/− or wild-type mice (online-only Data Supplement Figure IIId). These findings suggest that Notch signaling, especially the Jag-1–mediated signal, is crucial for EPC commitment and mobilization in BM.
Loss of Jag-1–Mediated Notch Signal in BM Impairs EPC Bioactivities In Vitro
To test the effect of switching off Notch signals on ischemia-induced EPC proliferation, we examined the frequency of BrdU+ cells in Sca-1+/Lin− cells obtained from Jag-1−/−, Dll-1−/−, or wild-type mice before and 4 days after hindlimb ischemia. Ischemia partially induced BrdU incorporation in wild-type EPCs (preischemia 1.71±0.45%, postischemia 3.4±0.55%, P<0.01) and Dll-1 null EPCs (preischemia 1.65±0.52%, postischemia 3.51±0.43%, P<0.01); however, such an ischemia-induced effect was not observed in Jag-1 null EPCs (preischemia 1.52±0.32%, postischemia 1.72±0.65%, P=NS; Figure 1E). These data support the hypothesis that Jag-1–mediated signals are critical for the proliferation of EPC-enriched cells in response to ischemia.
To clarify the effect of Notch signals on the survival potential of EPCs, we performed an in vitro terminal dUTP nick end-labeling (TUNEL) assay using BM Sca-1+/Lin− cells obtained from Jag-1−/−, Dll-1−/−, or wild-type mice 4 days after hindlimb ischemia. As shown in Figure 1F, the frequency of apoptotic cells in EPC-enriched cells was significantly greater in Jag-1−/− mice than in Dll-1−/− or wild-type mice.
To further investigate the modulation of BM EPC biology by Notch signals, we performed an in vitro invasiveness assay, a modified Boyden chamber invasiveness analysis. The assay exhibited marked impairment of the stromal cell–derived factor-1–induced invasiveness in Jag-1 null EPCs but not Dll-1 null EPCs (Figure 1G), which implies that Jag-1–mediated Notch signals play an important role in the motility of EPCs in the BM microenvironment. Thus, Jag-1–mediated Notch signals are crucial for various EPC functions such as proliferation, antiapoptosis, and invasiveness.
Loss of Jag-1–Mediated Notch Signals in BM Attenuates EPC Contribution for Vasculogenesis In Vivo
To test the involvement of Notch signals in vascular regeneration, we induced hindlimb ischemia by ligating the femoral arterial structure in conditional Jag-1−/−, Dll-1−/−, or wild-type mice. The recovery of hindlimb perfusion was delayed significantly by inactivation of Jag-1–mediated but not Dll-1–mediated Notch signals (Figures 2A and 2B), which suggests a specific role for Jag-1–mediated Notch signals in ischemic neovascularization.
To evaluate the possible role and contribution of Notch signals for EPC function in ischemic recovery in vivo, we next transplanted the EPC-enriched cells (Sca-1+/Lin− cells) obtained from BM of Jag-1−/−, Dll-1−/−, or wild-type mice into nude mice with hindlimb ischemia. Transplantation of the BM-EPCs from wild-type or Dll-1−/− mice significantly improved hindlimb perfusion compared with PBS injection. In contrast, BM-EPCs from Jag-1−/− mice failed to augment hindlimb perfusion (Figure 3A). Histological assessment of capillary density also revealed enhanced neovascularization after transplantation of EPCs from wild-type or Dll-1−/− mice but not Jag-1−/− mice (Figure 3B). These findings strongly indicate the essential role of Jag-1–mediated Notch signaling in BM-EPCs with regard to their vasculogenic potential in ischemic diseases.
Gain of Jag-1–Mediated Notch Signal From Stromal Cells Stimulates BM-EPC Commitment and Differentiation
The present data from loss-of-function studies indicate that Notch systems could regulate the kinetics of BM-EPCs for vasculogenesis. To further confirm the critical role of Notch signals from BM microenvironments for EPC bioactivity, we established an insert culture system by coculturing BM Lin− cells together with 3T3 stromal cells overexpressing Notch ligand, Jag-1, or Dll-1 (Figure 4A), in which activation of each Notch signal was confirmed by reverse-transcription polymerase chain reaction analysis (Figure 4B and 4C) and luciferase assay (data not shown). This analysis revealed that expression of Notch receptor 1, 2, 3, and 4 was similar after coculture with either of the Notch ligand–expressing stromal cells (online-only Data Supplement Figure IVb).
To assess the effect of Notch ligand signaling on EPC differentiation, the percentage of BM-Lin− cells positive for CD31 and Flk-1 (VEGFR 2), which are typical EPC markers, was determined by flow cytometric analysis. Importantly, the population of CD31+/Flk-1+ cells was remarkably increased in BM Lin− cells stimulated by Jag-1–mediated signals but not Dll-1–mediated signals (Figure 4D). The signal intensity of CD31 and Flk-1 in BM Lin− cells also increased after stimulation with Jag-1–mediated signals but not Dll-1–mediated signals (online-only Data Supplement Figure Va). Moreover, the cellular mRNA level of EPC markers such as CD31, Flk-1, or vascular endothelial cadherin was elevated in the Jag-1 group compared with the Dll-1 and empty-vector groups. In contrast, the cellular mRNA level of vascular endothelial growth factor was similar in all groups (Figure 4E). To obtain more concrete evidence for enhancement of EPC differentiation by Notch signals, we performed in vitro EPC culture assay using BM-Lin− cells cocultured with stromal cells expressing various Notch ligands. Fluorescent microscopic examination revealed that the number of cells demonstrating both acetylated LDL uptake and isolectin B4 binding was significantly greater in the Jag-1 group than in the empty-vector group, whereas in the Dll-1 group, the number was comparable to that in the control group (online-only Data Supplement Figure Vb). EPC colony-forming assay also clearly disclosed that specific induction of Jag-1–mediated signals but not Dll-1–mediated signals contributed significantly to enhancement of the vasculogenic activity of BM-KSLs, which are considered to be the putative origin of EPCs in mice (Figure 4F). TUNEL staining further indicated that Jag-1–mediated, not Dll-1–mediated, signals significantly inhibited the apoptosis of the cultured EPCs (Figure 4G). Importantly, Jag-1–derived signals enabled the BM-Lin− cells to form a tubelike structure just 4 days after coculture. In contrast, Dll-1–derived signals, as well as empty-vector–derived signals, did not affect the morphological features of the EPC-enriched cells (online-only Data Supplement Figure IVa). These data indicate that Jag-1–mediated Notch signal augments the commitment and differentiation of BM stem/progenitor cells toward endothelial lineage.
Gain of Jag-1–Mediated Notch Signal Promotes Vasculogenic Property of BM-EPCs
To explore the effects of gain of function from Notch signals on the therapeutic potential of EPCs, we serially examined perfusion recovery after hindlimb ischemia and transplantation of BM-Lin− cells in which Notch signals were stimulated by coculturing with 3T3 stromal cells. Laser Doppler perfusion imaging revealed that recovery of blood flow in the ischemic hindlimb was significantly enhanced by transplantation of EPC-enriched cells stimulated by Jag-1–mediated but not Dll-1–mediated signals compared with infusion of PBS or empty-vector–transduced EPCs (Figure 5A). The favorable effect of stimulating Jag-1–mediated signal was also confirmed by histological assessment of capillary density (Figure 5B). Thus, augmentation of Jag-1–mediated signal may specifically enhance the therapeutic potential of the BM EPC-enriched fraction for ischemic neovascularization.
Homing of EPCs to sites of ischemia is an essential step for neovascularization. Therefore, we examined the effect of specific Notch ligand stimulation on the incorporation of putative EPCs into blood vessels of ischemic tissues. BM-Lin− cells obtained from GFP (green fluorescent protein) transgenic mice, cocultured with stromal cells overexpressing the distinct Notch ligand, were infused intravenously into nude mice with hindlimb ischemia. Histochemical staining for CD31, a typical marker of endothelial cells, revealed significantly abundant incorporation of GFP+/CD31+ cells into ischemic tissue in the Jag-1 group but not the Dll-1 group compared with the empty-vector and PBS groups (Figure 5C).
Finally, we examined the therapeutic potency of Jag-1– or Dll-1–stimulated EPCs in Jag-1−/− mice with hindlimb ischemia, which is a model of Alagille syndrome and represents severe impairment of ischemic neovascularization. Recovery of hindlimb perfusion was augmented significantly in both the Dll-1– and empty-vector–stimulated EPC groups compared with the PBS group. Notably, perfusion recovery in Jag-1−/− mice was further enhanced after transplantation of Jag-1–stimulated EPCs compared with infusion of Dll-1– or empty-vector–stimulated EPCs (Figure 6A and 6B). These data provide critical evidence that augmentation of specific Jag-1–mediated signaling, not Dll-1–mediated signaling, from stromal cells enhances the vasculogenic potential of BM-EPCs for ischemic recovery.
The novel finding in the present study is that the specific Jag-1–mediated Notch signal promotes adult neovascularization by regulating functional kinetics of stem/progenitor cells in the BM microenvironment. We demonstrated that the Jag-1–induced signal evokes EPC commitment and differentiation in an in vitro gain-of-function study, as well as in an in vivo loss-of-function study, which eventually resulted in improvement of ischemia-induced neovascularization. In contrast, the Dll-1–induced Notch signal appeared to be dispensable for both commitment of EPCs in BM and recovery of blood flow from organ ischemia, at least in the postnatal stage, although Limbourg et al26 recently showed that inadequate Dll-1–induced Notch signal from the embryonic to adult stages appeared to affect arteriogenesis.
In the loss of Jag-1 ligand function, but not Dll-1, we observed (1) fewer BM cells expressing endothelium-specific genes; (2) lower EPC colony-forming ability in BM; (3) less proliferative activity, invasive capacity, and survival bioactivity of the EPC-enriched fraction in BM; (4) impaired neovascularization in ischemic tissue; and (5) impaired potential of therapeutic vasculogenesis after EPC transplantation. A surprising finding in Jag-1 knockout mice was the drastic decrease in functional EPCs (ie, an 80% decrease of Flk-1+/CD31+/Sca-1+/Lin− cells in BM and >50% reduction in total EPC colony-forming capacity compared with wild-type or Dll-1 knockout mice). The fact that the loss of Jag-1 function resulted in a lower number of EPCs and impaired EPC biological function for vasculogenesis indicates the essential regulatory role of Jag-1 for EPC commitment from stem cells and EPC differentiation to acquire vasculogenic properties in BM.
Several reports have proposed that Notch signaling is actively involved in HSC maintenance/growth in osteoblastic niches in various experimental animal models. A study23 using transgenic mice constitutively expressing active parathyroid hormone receptor under the control of collagen type IV promoter reported an increase in trabecular bone mass associated with overexpression of a Notch ligand, Jag-1, in osteoblasts. The authors of that report argued that the increase in BM HSCs is a direct consequence of the increased osteoblastic niche area and overexpression of Jag-1 in the niche cells.23 Taken together with the present data in the loss-of-function studies, signal transmission between stromal cells expressing a Notch ligand, Jag-1, and EPCs expressing Notch receptors is considered to be the most essential molecular mechanism underlying the differential regulation of EPCs in the stromal niche in BM.
In the present analysis of Jag-1−/−, Dll-1−/−, and WT mice, we did not observe a significant difference in the number of KSLs (Figure 1B) or their hematopoietic colony-forming capacity (data not shown) among the 3 groups as reported previously.19 However, the development of EPCs from the stem cell pool evaluated by the in vitro EPC colony-forming assay was significantly impaired only in Jag-1−/− mice. These facts suggest that the Jag-1–mediated Notch signal may exist in the marrow structure for specific regulation of EPC kinetics in response to demands of neovascularization, such as ischemic conditions.
To confirm the mechanism of regulating EPC commitment and differentiation in the BM stroma, we used a study of gain of Notch ligand function. An insert coculture system of BM-Lin− cells together with 3T3 stromal cells overexpressing Notch ligand (Jag-1 or Dll-1) demonstrated that precisely controlled gain of Jag-1 function in vitro promoted (1) endothelium-specific gene expression, (2) activity of EPC colony formation, (3) antiapoptosis bioactivity, (4) activity of both vascular endothelial growth factor–dependent proliferation and migration (online-only Data Supplement Figure Va and Vb), and (5) the potential of therapeutic vasculogenesis of hindlimb ischemia in BM-EPCs. These findings suggest that Jag-1 strongly drives the immature BM population to commit and differentiate into endothelial lineage, whereas Dll-1 is not involved at all, although downstream signals, such as Hes-1 and Hes-5, are equally stimulated.
The finding that EPCs preconditioned by specific Jag-1–dependent signaling were able to rescue the impaired vasculogenic potential in both athymic nude and Jag-1 null mice may open a novel gate for enhancing the potential of therapeutic neovascularization. Key mechanisms underlying this favorable phenomenon may be upregulation of EPC functions, including proliferation, differentiation, and migration, by exogenous Jag-1 signal, because the impaired EPC bioactivity in the Jag-1–deficient KSLs could be rescued by Jag-1–mediated signals (Figure 4; online-only Data Supplement Figures VI and VII). Another possible mechanism may relate to the rescue signals for prevention of programmed cell death, because the present study indicates the antiapoptotic effect of preconditioning by Jag-1 signal on EPCs (Figure 4G). The antiapoptotic effect of Jag-1 signals was also confirmed in ischemic tissue after transplantation of the distinct Notch ligand–stimulated EPCs (online-only Data Supplement Figure VIII). These combined effects of Jag-1 signaling on EPCs may contribute to augmentation of the vasculogenic potential.
The predominant view of Notch signaling is that any Notch ligand is capable of inducing consequential structural changes of Notch receptors for their cleavage and initiating the proteolytic cascade that ultimately leads to generation of a Notch intracellular domain. Very recently, several reports have propounded the concept that each Notch ligand might independently communicate with the receptor for a separate signaling cascade even in the same cell for hematopoiesis or ear regeneration.25,27 Ligand-specific signaling for vascular development in postnatal stages, however, has never been demonstrated. We demonstrated for the first time the specific role of Jag-1 in stimulating postnatal vasculogenesis, which was not observed in Dll-1–dependent signaling. As indicated by the recently discovered concept, elucidation of distinct Notch ligand/receptor communication would be fundamental to illustrating the governed and elaborated mechanisms of stem cell biology in BM environments, as well as the vascular biology involved in postnatal neovascularization for vascular repair.
We would like to thank Rie Ito and Michiru Kobori for providing technical assistance and Sachie Ota and Yumiko Masukawa for secretarial work. We appreciate assistance from the Research Center for Regenerative Medicine and from members of the animal facility in Tokai University School of Medicine, as well as technical advice and support from the Teaching and Research Support Center. We also thank all our colleagues for their helpful advice and encouragement.
Source of Funding
This work was supported by the Academic Frontier Promotion Program of the Ministry of Education, Culture, Sports, Science, and Technology in Japan.
Iso T, Hamamori Y, Kedes L. Notch signaling in vascular development. Arterioscler Thromb Vasc Biol. 2003; 23: 543–553.
Gale NW, Dominguez MG, Noguera I, Pan L, Hughes V, Valenzuela DM, Murphy AJ, Adams NC, Lin HC, Holash J, Thurston G, Yancopoulos GD. Haploinsufficiency of delta-like 4 ligand results in embryonic lethality due to major defects in arterial and vascular development. Proc Natl Acad Sci U S A. 2004; 101: 15949–15954.
Krebs LT, Shutter JR, Tanigaki K, Honjo T, Stark KL, Gridley T. Haploinsufficient lethality and formation of arteriovenous malformations in Notch pathway mutants. Genes Dev. 2004; 18: 2469–2473.
Krebs LT, Xue Y, Norton CR, Shutter JR, Maguire M, Sundberg JP, Gallahan D, Closson V, Kitajewski J, Callahan R, Smith GH, Stark KL, Gridley T. Notch signaling is essential for vascular morphogenesis in mice. Genes Dev. 2000; 14: 1343–1352.
Domenga V, Fardoux P, Lacombe P, Monet M, Maciazek J, Krebs LT, Klonjkowski B, Berrou E, Mericskay M, Li Z, Tournier-Lasserve E, Gridley T, Joutel A. Notch3 is required for arterial identity and maturation of vascular smooth muscle cells. Genes Dev. 2004; 18: 2730–2735.
Xue Y, Gao X, Lindsell CE, Norton CR, Chang B, Hicks C, Gendron-Maguire M, Rand EB, Weinmaster G, Gridley T. Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum Mol Genet. 1999; 8: 723–730.
Ridgway J, Zhang G, Wu Y, Stawicki S, Liang WC, Chanthery Y, Kowalski J, Watts RJ, Callahan C, Kasman I, Singh M, Chien M, Tan C, Hongo JA, de Sauvage F, Plowman G, Yan M. Inhibition of Dll4 signalling inhibits tumour growth by deregulating angiogenesis. Nature. 2006; 444: 1083–1087.
Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, Witzenbichler B, Schatteman G, Isner JM. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275: 964–967.
Drake CJ, Fleming PA. Vasculogenesis in the day 6.5 to 9.5 mouse embryo. Blood. 2000; 95: 1671–1679.
Sato TN, Qin Y, Kozak CA, Audus KL. Tie-1 and tie-2 define another class of putative receptor tyrosine kinase genes expressed in early embryonic vascular system. Proc Natl Acad Sci U S A. 1993; 90: 9355–9358.
Mancini SJ, Mantei N, Dumortier A, Suter U, MacDonald HR, Radtke F. Jagged1-dependent Notch signaling is dispensable for hematopoietic stem cell self-renewal and differentiation. Blood. 2005; 105: 2340–2342.
Brooker R, Hozumi K, Lewis J. Notch ligands with contrasting functions: Jagged1 and Delta1 in the mouse inner ear. Development. 2006; 133: 1277–1286.
Limbourg A, Ploom M, Elligsen D, Sorensen I, Ziegelhoeffer T, Gossler A, Drexler H, Limbourg FP. Notch ligand Delta-like 1 is essential for postnatal arteriogenesis. Circ Res. 2007; 100: 363–371.
Although accumulating evidence has indicated that therapy with endothelial progenitor cells (EPCs) could be a promising modality for vascular regeneration, the problems of quantity and quality control need to be resolved to achieve translational application in humans. Pathological conditions such as aging, diabetes mellitus, and hypercholesterolemia lead to a decrease in circulating EPCs and impairment of their proliferative and migratory function. These limitations may be solved by the integration of both in vitro expansion and quality control of EPCs by genetic modification, such as transducing vascular endothelial growth factor, glycogen synthase kinase-1β, human telomerase reverse transcriptase expression, or adjunctive cytokines that promote EPC mobilization. The promise of our therapeutic strategy is that governed Notch signaling in culture can produce the preferred quality and quantity of EPCs needed to enhance vasculogenic potential. The manipulation of Jag-1 ligand–mediated signals in culture before transplantation would allow EPCs to increase in number and augment their vasculogenic potential in patients with ischemic diseases.
The online-only Data Supplement, consisting of Methods and figures, is available with this article at http://circ.ahajournals.org/cgi/content/full/ CIRCULATIONAHA.107.754978/DC1.