Myeloid lineage progenitors give rise to vascular endothelium

Abstract

Despite an important role in vascular development and repair, the origin of endothelial progenitors remains unknown. Accumulating evidence indicates that cells derived from the hematopoietic system participate in angiogenesis. However, the identity and functional role of these cells remain controversial. Here we show that vascular endothelial cells can differentiate from common myeloid progenitors and granulocyte/macrophage progenitors. Endothelial cells derived from transplanted bone marrow-derived myeloid lineage progenitors expressed CD31, von Willebrand factor, and Tie2 but did not express the hematopoietic markers CD45 and F4/80 or the pericyte markers desmin and smooth muscle actin. Lineage tracing analysis in combination with a Tie2-driven Cre/lox reporter system revealed that, in contrast to bone marrow-derived hepatocytes, bone marrow-derived endothelial cells are not the products of cell fusion. The establishment of both hematopoietic and endothelial cell chimerism after parabiosis demonstrates that circulating cells can give rise to vascular endothelium in the absence of acute radiation injury. Our findings indicate that endothelial cells are an intrinsic component of myeloid lineage differentiation and underscore the close functional relationship between the hematopoietic and vascular systems.

Fig. 1.

Bone marrow-derived progenitor cells have endothelial cell potential. Based on the expression of hematopoietic cell markers, sorted populations of bone marrow progenitors were injected into lethally irradiated recipient mice at a dose equivalent to their frequency in 1 × 10⁶ bone marrow cells. (a) Fractionation of bone marrow cells based on the expression of the B cell marker B220. (b) Portal vein branches were assayed for donor-derived endothelial cells after transplanting either marker-positive (filled bars) or marker-negative (open bars) fractions of bone marrow. The percentage of vessel cross-sections containing EGFP or ROSA26 donor-derived endothelial cells is indicated. Both Sca-1-negative and lineage-positive (Lin⁺) subpopulations of bone marrow gave rise to endothelial cells, indicating the existence of endothelial progenitors distinct from the HSC phenotype. ∗, below level of detection of 0.05%. Error bars indicate SEM. (c) Isolation of HSCs [c-kit⁺Sca-1⁺Lin⁻ (KSL)], CMPs, and GMPs. Lineage-negative cells (Left) were sorted for c-kit and Sca-1 (Center). Sca-1⁻, c-kit⁺ cells were further fractionated into CMP and GMP populations based on CD34 and FcγR expression (Right). ∗∗, KSL lineage marker mixture differs from CMP/GMP lineage markers as indicated in Methods. (d) Transplantation schema for detecting donor-derived endothelial cells after transplantation of CMPs and GMPs.

Fig. 2.

Myeloid lineage progenitor cells give rise to vascular endothelial cells. After transplantation of either CMPs or GMPs, recipient livers were analyzed for the presence of donor-derived endothelial cells. (a) X-Gal detection (blue) of a ROSA26-marked GMP-derived endothelial cell expressing VWF (red). (Inset) Higher magnification. (b) X-Gal detection of a CMP-derived endothelial cell. (c) An EGFP⁺ (green) CMP-derived VWF⁺ (red)-expressing endothelial cell. (Inset) Higher magnification. (d) Deconvoluted images of a CMP-derived β-gal⁺ (green), VWF⁺ (red), DAPI⁺ (blue) endothelial cell (arrow in Left) that is negative for CD45 (magenta) expression (Center). A single host-derived CD45⁺ (magenta), VWF⁻ blood cell is present next to the vessel wall (arrowhead). (Right) Merged image. (e) Z-stack images of a β-gal⁺ (green), VWF⁺ (red), DAPI⁺ (blue) endothelial cell taken at 0.5-μm intervals demonstrating colocalization of β-gal and VWF expression in a single cell. (f) A host-derived F4/80⁺, CD45⁺ cell (magenta, arrowhead) in the liver parenchyma of a GMP transplant recipient. (Inset) A donor-derived F4/80⁺, CD45⁺ hematopoietic cell (green). The same cell is indicated with an arrow (magenta). (g) Frequency of portal vessels containing donor-derived endothelial cells after transplantation of either CMPs or GMPs. (Scale bars: a and c, 20 μm; b, d, and f, 5 μm.) L, vessel lumen.

Fig. 3.

Donor-derived endothelial cells are not products of cell fusion. (a–e) Donor hematopoietic cells fuse with host hepatocytes. (b) X-Gal detection of fusion-derived hepatocytes (blue) after transplantation of EGFP, Tie2-Cre HSCs into a R26R, Fah recipient [portal vein (PV)]. (Inset) Higher magnification. (c–e) Fusion-derived hepatocytes are both EGFP⁺ (green channel in c) and β-gal⁺ (red channel in d). (e) Merged image of c and d. (f–i) Donor-derived endothelial cells do not arise through cell fusion. (g) A donor-derived, EGFP⁺ (green) endothelial cell (arrowhead) expresses the endothelial cell marker CD31 (magenta). (h) The same cell does not express the β-gal marker (red). (i) A Tie2 (red)-expressing EGFP⁺ (green) endothelial cell showing activation of the Tie2 promoter. (j–m) The Cre recombinase experimental approach in the reverse direction was performed by transplanting EGFP, R26R HSCs into Tie2-Cre recipients. (k and l) An endothelial cell (arrowhead) that expresses both the donor marker (green) and CD31 (magenta) (k) and is negative for β-gal expression (not red) (l). (m) Tie2 expression (red) in a donor-marked endothelial cell (green). (n–r) To evaluate cell fusion without the requirement of Cre-mediated recombination, EGFP⁺ HSCs were transplanted into ROSA26 mice. Recipient livers were analyzed for the presence of fusion-derived EGFP⁺ (green), β-gal⁺ (red) endothelial cells and hepatocytes. (o) EGFP⁺ (green) donor-derived endothelial cells were negative for β-gal staining (box), whereas host endothelial cells were β-gal-positive (red, arrow). (p) High magnification of the EGFP⁺, β-gal⁻ endothelial cell shown in o. (q and r) An EGFP⁺ donor-derived hepatocyte (q) that coexpresses β-gal (r), indicating that the hepatocyte is fusion-derived. (Scale bars: b, 20 μm; c–e, g–i, k–m, and p–r, 5 μm; o, 10 μm.) L, vessel lumen.

Fig. 4.

Donor-derived endothelial cells contribute to the vascular endothelium after parabiosis. Mice were separated and analyzed for donor-derived cells in the blood and in blood vessels. (a) Flow cytometric analysis of the peripheral blood of a wild-type mouse after parabiosis with a transgenic, EGFP-expressing mouse reveals the presence of donor-derived blood cells. (b) The percentage of B cells (B220), T cells (CD3), and myeloid cells (Mac-1/Gr-1) of host (open bars) and donor (filled bars) origin; n = 5, error bars indicate SEM. (c) The frequency of donor-derived endothelial cells in the liver vessels of individual parabiotic recipients.