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Apelin+ Endothelial Niche Cells Control Hematopoiesis and Mediate Vascular Regeneration after Myeloablative Injury

Summary Radiotherapy and chemotherapy disrupt bone vasculature, but the underlying causes and mechanisms enabling vessel regeneration after bone marrow (BM) transplantation remain poorly understood. Here, we show that loss of hematopoietic cells per se, in response to irradiation and other treatments, triggers vessel dilation, permeability, and endothelial cell (EC) proliferation. We further identify a small subpopulation of Apelin-expressing (Apln+) ECs, representing 0.003% of BM cells, that is critical for physiological homeostasis and transplant-induced BM regeneration. Genetic ablation of Apln+ ECs or Apln-CreER-mediated deletion of Kitl and Vegfr2 disrupt hematopoietic stem cell (HSC) maintenance and contributions to regeneration. Consistently, the fraction of Apln+ ECs increases substantially after irradiation and promotes normalization of the bone vasculature in response to VEGF-A, which is provided by transplanted hematopoietic stem and progenitor cells (HSPCs). Together, these findings reveal critical functional roles for HSPCs in maintaining vascular integrity and for Apln+ ECs in hematopoiesis, suggesting potential targets for improving BM transplantation.

, Summary of genetic mouse models used in this study (A-B) DAPI labelled (A) and Emcn-immunostained (B) sections from control or lethally irradiated bone (9 Gy; 7 days post irradiation). The overview images are stitched from several individual z-stacked images.
(C) Representative images showing extravasation of intravenously injected Texas red-coupled Dextran (red) in control bone and 7 days post lethal irradiation (9 Gy). The overview images are stitched from several individual z-stacked images.
(D) Confocal overview and high-magnification images of diaphyseal vessels at 3 hours, 1 day and 4 days after lethal irradiation (9 Gy). Green: Emcn. The overview images are stitched from several individual z-stacked images.
(G) Flow cytometry validating Emcn expression in GFP+ cells from Cdh5-mTnG mice. Representative histogram of Emcn expression in GFP-or GFP+ cells and quantification of normalized mean fluorescence intensity (MFI      Figure S4. Characterization of Apln+ ECs and vascular defects in DTA in bone, related to Figure 4 (A) Overview and high magnification confocal images of Emcn+ (red) capillaries and GFP+ (green) arteries in Bmx-CreERT2 R26-mTmG mice at 7 days after lethal irradiation (9 Gy). The overview images are stitched from several individual z-stacked images. (B) Representative images of arterial vessels in Bmx-CreER R26-mTmG mice 7 days after irradiation. GFP (green), Caveolin-1 (red) and Bcam (blue). (C) Overview and high magnification confocal images of Emcn+ (red) capillaries and GFP+ (green) arteries in Bmx-CreERT2 R26-mTmG mice at 3 weeks after lethal irradiation and BM transplantation.   peripheral blood (PB) components (Ctrl=8; Vegfr2 =8). Error bars, mean± s.e.m. P values, two-tailed unpaired Student's t-Test. WBC, white blood cells; RBC, red blood cells; GRA, granulocyte. (K) Flow cytometric analysis of hematopoietic cell subpopulations in Apln (T/y) and control bone marrow and peripheral blood (PB). Animals in this experiment were not treated with tamoxifen to evaluate the effect of the loss of Apln expression in Apln-CreERT knock-in males (equivalent to full knockout mice) (n=5 per group). Error bars, mean± s.e.m. P values, two-tailed unpaired Student's t-Test. CLP, common lymphoid progenitors; CMP, common myeloid progenitors; GMP, granulocyte-monocyte progenitors; MEP, Megakaryocyte-erythroid progenitors; RBC, red blood cells; WBC, white blood cells; PLT, platelets; Lym, lymphocytes. (L) Flow cytometric analysis of hematopoietic reconstitution after loss of Apln expression in Apln-CreERT T/y knockout (n=6) and control males (n=7) without tamoxifen (TMX) administration. Vav1-Cre R26-mTmG hematopoietic cells were used as donors. Error bars, mean± s.e.m. P values, two-tailed unpaired Student's t-Test.   Figure S6. Role of Apln+ ECs but not Esm1+ ECs in hematopoiesis and transplantation, related to Figure 6.
(A) FACS analysis of DTA peripheral blood (PB) components 3 days after irradiation and transplantation (Ctrl=9, DTA =9). Error bars, mean± s.e.m. P values, two-tailed unpaired Student's t-Test. (B) Cytokine levels in peripheral blood plasma of DTA mice 3 days after irradiation and transplantation. Cytokine level lower than detection limit was excluded from quantification. n=2-8 in each group. Error bars, mean± s.e.m. P values, two-tailed unpaired Student's t-Test. P values higher than 0.1 are not indicated. (C) Survival (in days) of DTA mutants (n=5) relative to irradiated (9 Gy) and transplanted control (Apln-CreER+/y, n=11) and DTA mice (n=14). (D) Genetic lineage tracing of GFP+ cells in Apln-mTmG long bone at 8 days after a single 4-OHT administration with or without irradiation at day 2. Confocal overview images and magnification of diaphysis. The overview images are stitched from several individual z-stacked images. Quantification of GFP+ area relative to Emcn+ area in diaphysis (n=3 in each group). Error bars, mean± s.e.m. P values, two-tailed unpaired Student's t-Test. (E) Genetic lineage tracing of GFP+ cells in Apln-mTmG long bone at 7 weeks after a single 4-OHT administration with or without irradiation and transplantation at day 2. Confocal overview images and magnification of diaphysis. The overview images are stitched from several individual z-stacked images. Quantification of GFP+ area relative to Emcn+ area in diaphysis (Ctrl=3; 9 Gy=5