Yo Iwami

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: past, state of the art, and future

Recent evidences suggest that endothelial progenitor cells (EPCs) derived from bone marrow (BM) contribute to de novo vessel formation in adults occurring as physiological and pathological responses. Emerging preclinical trials have shown that EPCs home to sites of neovascularization after ischemic events in limb and myocardium. On the basis of these aspects, EPCs are expected to develop as a key strategy of therapeutic applications for the ischemic organs. Such clinical requirements of EPCs will tentatively accelerate the translational research aiming at the devices to acquire the optimized quality and quantity of EPCs. In this review, we attempt to discuss about biological features of EPCs and speculate on the clinical potential of EPCs for therapeutic neovascularization.

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.