Bone marrow–derived CMPs and GMPs represent highly functional proangiogenic cells: implications for ischemic cardiovascular disease

Abstract

Clinical studies using bone marrowderived proangiogenic cells (PACs) have demonstrated modest improvements of function and/or perfusion of ischemic myocardium or skeletal muscle. Because the identities of these PACs and their functional ability to promote neovascularization remain poorly understood, it is possible that a subset of robust PACs exists but is obscured by the heteroge- IntroductionTherapeutic angiogenesis has emerged as an innovative strategy for the treatment of patients with cardiovascular ischemic disease states, such as myocardial ischemia or peripheral artery disease. Several clinical trials using autologous, bone marrow (BM)-derived mononuclear cells have demonstrated only modest improvements of function and/or perfusion of ischemic myocardium or limb in patients. [1][2][3][4][5][6] However, the identities of these BM-derived mononuclear cells and their functional ability to promote neovascularization remain poorly understood.Accumulating evidence has indicated that proangiogenic cells (PACs) may originate from the BM and are capable of being recruited to sites of ischemic injury, where they contribute to neovascularization and tissue repair through paracrine/autocrine mechanisms or by direct incorporation into the vessel wall. [7][8][9][10] Indeed, PAC function and/or number have been found to be markedly impaired in patients with coronary or peripheral artery disease. 11-17 Herein, we provide evidence that a hierarchy exists among mouse hematopoietic stem cell (HSC) progenitors in their ability to differentiate into PACs and promote neovascularization. Complementary functional studies revealed that common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) preferentially differentiate into PACs as opposed to megakaryocyte-erythrocyte progenitors (MEPs), HSCs, and common lymphoid progenitors. Moreover, only PACs derived from BM-derived CMPs and GMPs were able to enhance neovascularization in vivo. Taken together, these findings reveal a novel and unexpected hierarchy among BM-derived progenitors in their ability to promote neovascularization. Methods Isolation and in vitro culture of PACsProgenitor cells were isolated from C57BL/6 mice (n ϭ 8-10) BM by using multicolor FACSAria (BD Biosciences) as previously described. 18,19 Briefly, approximately 2 to 5 ϫ 10 5 BM-derived cells were prospectively isolated and purified as follows: HSCs (Lin Ϫ Sca1 ϩ c-kit ϩ ), CMPs (Lin Ϫ Sca1 Ϫ ckit ϩ CD34 ϩ Fc␥RII/II lo ), GMPs (Lin Ϫ Sca1 Ϫ c-kit ϩ CD34 ϩ Fc␥RII/III hi ), and MEPs (Lin Ϫ Sca1 Ϫ c-kit ϩ CD34 Ϫ Fc␥RII/III lo ; supplemental Figure 1A, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). Subsequently, cells were incubated in endothelial growth media-2 (EGM-2; Lonza) for 8 to 10 days and subjected to flow cytometry to detect expression of vascular endothelial growth factor receptor-2 (VEGFR2), CD31, CD133, CD34, CD45, Tie2, von Willebrand factor, endothelial nitric oxide synthase, and VE-cadherin (eBioscience). Cells were a...

TGF-β1 signaling and Krüppel-like factor 10 regulate bone marrow–derived proangiogenic cell differentiation, function, and neovascularization

“…preferentially differentiated into PACs and exhibited high angiogenic activity in vivo. 10 Signals emanating from TGF-␤ and IFN-␥ are known to antagonize normal cell growth and differentiation events and have been implicated in regulating different aspects of angiogenesis. [20][21][22] To assess the effect of TGF-␤1 signaling on PAC differentiation, BM progenitorderived PACs were cultured for 7 days in the presence or absence of recombinant TGF-␤1; modulation of the PAC marker VEGFR2 was then examined.…”

“…In addition, VEGFR2 expression was also induced by ϳ 2-fold in KLF10-overexpressing HSCs ( Figure 2E), which possess lower angiogenic properties than CMPs and GMPs. 10 To assess whether the defect in PAC differentiation from TGF-␤1 ϩ/Ϫ GMPs could be rescued by exogenous KLF10, GFP-EV (Ctrl), or KLF10 (GFP-RV-KLF10) was transduced in TGF-␤1 ϩ / ϩ or TGF-␤1 ϩ/Ϫ GMPs, and VEGFR2 expression was measured by FACS. As shown in Figure 2F, KLF10-overexpressing cells were capable of rescuing VEGFR2 expression in TGF-␤1 ϩ/Ϫ GMPs to levels even higher than that achieved in TGF-␤1 ϩ / ϩ GMPs transduced with EV Ctrl.…”

“…We have demonstrated that among hematopoietic progenitor stem cells, the common myeloid progenitors (CMPs) and granulocytemacrophage progenitors (GMPs) constitute a population of BMderived cells that preferentially differentiate into PACs and possess robust angiogenic activity under ischemic conditions in vivo. 10 However, the signaling pathways and downstream factors that mediate these proangiogenic functions remain poorly understood.…”

“…We have demonstrated that among hematopoietic progenitor stem cells, the common myeloid progenitors (CMPs) and granulocytemacrophage progenitors (GMPs) constitute a population of BMderived cells that preferentially differentiate into PACs and possess robust angiogenic activity under ischemic conditions in vivo. 10 However, the signaling pathways and downstream factors that mediate these proangiogenic functions remain poorly understood.The pleiotropic TGF-␤1 plays an important role in cell growth, differentiation, and activation in a number cell types. 11 TGF-␤1 has been shown to contribute to various aspects of neovascularization, including cell adhesion, migration, and homing.…”

Endothelial progenitor cells in hematologic malignancies

“…Basically, two types of proangiogenic hematopoietic cells have been identified, one with a more mature phenotype and corresponding to mature Tie2 + monocytic cells and an immature progenitor cell population, corresponding to proangiogenic hematopoietic progenitor cells or CFU-Hill (17). The studies carried out in these last years on proangiogenic hematopoietic cells have led to the conclusion that these cells derive from the differentiation of a subset of hematopoietic progenitor cells, characterized by positivity for CD34, CD133 and VEGF-R2, mobilized from bone marrow by angiogenic growth factors and contributing in vivo to an angiogenetic response only through an indirect effect based on paracrine mechanisms (18)(19)(20)(21).…”