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

Abstract

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...

Development of Serum-Free Quality and Quantity Control Culture of Colony-Forming Endothelial Progenitor Cell for Vasculogenesis

“…In EPC-CFA of cultured cells in all Combs, as previously indicated in mouse or human EPC-CFA [32][33][34][35][36][37], two types of EPC colonies were morphologically detected: PEPC-CFU, formed of small, round cells, and DEPC-CFU, formed of large, spindle-like cells (supplemental online Fig. 1A).…”

“…Considering the necessity of creating a defined assay, a novel adhesive clonogenic assay for the quantitative and qualitative analysis of EPCs based on differentiation hierarchy has been developed recently [32][33][34][35][36][37]. The new EPC colony-forming assay (EPC-CFA) enabled the distinction and the definition of two different types of EPC-colony-forming units (EPC-CFUs), that is, primitive and definitive EPC-CFUs, composed of small and large cells, respectively.…”

“…To investigate the vasculogenic potential of pre-or post-QQc cells, we quantified adhesive EPC colonies by EPC-CFA using semi-solid culture medium (MethoCult SF BIT ; StemCell Technologies) with pro-angiogenic growth factors in 35-mm Primaria dishes (BD Biosciences), as described in supplemental online Methods 2 [32][33][34][35]37]. Eighteen days after initiation of the culture, the number of adherent colonies per dish was measured using a gridded scoring dish (StemCell Technologies) under light The top three rows are counted EPC-CFU numbers per 500 QQc cells in each period.…”

A small interfering RNA targeting Lnk accelerates bone fracture healing with early neovascularization

“…27,28 The number of EPC colonies was assessed 16 days after seeding of Lnk siRNA and control siRNA-transfected KSL cells (1 Â 10 3 cells/well) in a six-well plate with methyl cellulose-containing medium M3236 (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 20 ng/ml SCF (Kirin, Tokyo, Japan), 50 ng/ml VEGF (R&D Systems, Minneapolis, MN), 20 ng/ml interleukin-3 (Kirin) and 50 ng/ml basic fibroblast growth factor (Wako, Osaka, Japan). Two different types of attaching cell colonies made of small EPCs and large EPCs were counted separately.…”

Protective Effect of Catalpol on Myocardium in Rats with Isoprenaline-Induced Myocardial Infarcts via Angiogenesis through Endothelial Progenitor Cells and Notch1 Signaling Pathway

“…EPCs were found to decline dramatically in mice that had been knocked out for Jagged1, the ligand of Notch1 receptor [6]. Therefore, Jagged1 is believed to have an important role in determining the number of EPCs and their mobilization into blood for restoration of injured blood vessels [7]. Clinical data have shown that prescriptions that nourish kidney and activate blood are effective in treatment of acute myocardial infarction [8].…”