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SDF-1 dynamically mediates megakaryocyte niche occupancy and thrombopoiesis at steady state and following radiation injury

Lisa M. Niswander, Katherine H. Fegan, Paul D. Kingsley, Kathleen E. McGrath and James Palis

Data supplements

Article Figures & Data

Figures

  • Figure 1

    Vascular elevation of SDF-1 by IV administration acutely promotes the association of MKs with vasculature and thrombopoiesis. (A) MKP, MK, and platelet kinetics 24 hours after 400 ng IV SDF-1 (blue) or vehicle control (gray). SDF-1-treated mice have an acute increase in circulating platelets with no change in MK or MKP number in the marrow. MKP (colony assay) and MK (imaging flow cytometry) numbers are normalized to per femur values. Mean absolute numbers for vehicle controls: 16 905 MKP/femur, 45 939 MK/femur, 430 × 103 platelets/μL. (B) MFI of thiozole orange (TO) in platelets identified as CD41+Ter119- by imaging flow cytometry 24 hours after IV SDF-1 or vehicle. Platelets from SDF-1–treated mice have increased TO staining. (C) The proportion of exhausted MKs was identified by imaging flow cytometry and presented as percent of vehicle control for each experiment. Mean proportion of exhausted MKs for vehicle controls: 0.14. (D) Representative images of femoral marrow immunohistochemistry (IHC) for Gp1Bβ (MKs, red) and MECA32 (vascular endothelium, green) 24 hours after IV treatment with vehicle (left panel) or SDF-1 (right panel). White arrows indicate examples of MKs physically associated with MECA32+ vessels. Some MKs express the pan-endothelial antigen recognized by MECA32.57 Images were processed as described in the “Immunohistochemistry” section. (E) Quantification of Gp1Bβ+ MKs physically associated with MECA32+ vessels by manual counting of IHC 1 hour and 24 hours after IV SDF-1 (blue) or vehicle (gray). IV SDF-1 acutely increases MK association with the vasculature. (F) MFI/A of surface CXCR4 on primary MKs by imaging flow cytometry of flushed marrow samples prepared 1 hour and 24 hours after IV SDF-1 (blue) or vehicle (gray). (G) MFI/A of surface CXCR4 on in vitro–derived MKs 1 hour after SDF-1 treatment (blue) by imaging flow cytometry. SDF-1 treatment increases MK surface CXCR4 both in vivo (F) and in vitro (G). Error bars represent standard error of the mean of ≥3 independent experiments (n = 6-18 total mice per group). Statistical analyses comparing SDF-1 to vehicle controls were performed using a 2-tailed Student’s t test. Bar represents 100 μm (D). *P < .04; **P < .005.

  • Figure 2

    Native SDF-1 enhances MKs in the vascular niche and increases circulating platelets. (A) MKP, MK, and platelet kinetics 24 hours after 1.7 mg (5 μmol) intraperitoneal Diprotin A (red) or vehicle control (gray). Stabilization of SDF-1 with Diprotin A acutely increases circulating platelets with no change in MK or MKP number in the marrow. MKP (colony assay) and MK (imaging flow cytometry) numbers are normalized to per femur values and all compartments expressed as percent of vehicle control. Mean absolute numbers for vehicle controls: 11 593 MKP/femur, 49 856 MK/femur, 436 × 103 platelets/μL. (B) MFI/A of surface CXCR4 on primary MKs by imaging flow cytometry of flushed marrow samples prepared 1 hour after treatment with Diprotin A (red) or vehicle control (gray). (C) Quantification of Gp1Bβ+ MKs physically associated with MECA32+ vessels by IHC 1 hour and 24 hours following treatment with Diprotin A (red) or vehicle (gray). Diprotin A-mediated stabilization of SDF-1 acutely increases MK association with the vasculature. (D) Representative images of femoral marrow IHC for Gp1Bβ (MKs, red) and MECA32 (vascular endothelium, green) 24 hours after administration of vehicle (left panel) or Diprotin A (right panel). White arrows indicate examples of MKs physically associated with MECA32+ vessels. Images were processed as described in the “Immunohistochemistry” section. Error bars represent standard error of the mean of ≥3 independent experiments (n = 7-8 total mice per group). Statistical analyses comparing Diprotin A to vehicle controls were performed using a 2-tailed Student’s t test. Bar represents 100 μm (D). *P < .02; **P < .003.

  • Figure 3

    Temporal and spatial changes in marrow SDF-1 following sublethal radiation. (A) Representative images of radioactive in situ hybridization with SDF-1 antisense probe (red pseudocolor indicates SDF-1 transcripts) on femoral marrow sections from (left to right panels): unirradiated (0 Gy), and days 1, 2, and 3 post-4 Gy TBI. Dotted lines delineate the endosteal region. Images were acquired and processed as described in the “RNA in situ hybridization.” (B) Expression of SDF-1 transcripts by qPCR in flushed marrow cells from unirradiated mice (black) and days 1, 2, and 3 post-4 Gy TBI (green). SDF-1 transcripts increase in the marrow following TBI. (C) Ratio of SDF-1 transcript area in the endosteal region (between 0-100 μm from the endosteal surface within the diaphysis) compared to an immediately adjacent region of the same size (between 100-200 μm from the endosteal surface) for each biologic replicate of unirradiated mice (black), and days 1, 2, and 3 post-4 Gy TBI (green). An endosteal gradient of SDF-1 transcript is apparent at day 2, but is lost at day 3. Error bars represent standard error of the mean of ≥3 independent experiments (n = 3-7 total mice per group). Statistical analyses either comparing irradiated samples to unirradiated control (B) or indicated samples (C) were performed using a 2-tailed Student’s t test. Bar represents 100 μm (A). *P < .02; **P < .007.

  • Figure 4

    Altered niche occupancy of radioresistant MKs following sublethal radiation. (A) MK lineage injury kinetics following 4 Gy TBI. Circulating platelets (black) and marrow MKs (red) remain at steady-state levels for 3 days (one-way analysis of variance, P = .45), while upstream MKPs (blue) are rapidly lost. MKP (colony assay) and MK (imaging flow cytometry) numbers are normalized to per femur values and all compartments expressed as percent of unirradiated control. Mean absolute numbers for 0 Gy controls: 13 550 MKP/femur, 56 022 MK/femur, 484 × 103 platelets/μL. (B) MFI/A of surface CXCR4 on primary MKs by imaging flow cytometry of flushed marrow samples prepared from unirradiated controls (black) or days 1 to 3 post-4 Gy TBI (green). MKs from irradiated mice display increased surface CXCR4 expression. (C) Representative images of femoral IHC for MECA32 (vasculature, green) with hindlimbs isolated from unirradiated mice (top panel) or day 2 post-4 Gy TBI (bottom panel). The marrow vasculature dilates after TBI. (D) Quantification of vascular area within MECA32+ marrow vessels by IHC. Vascular dilation occurs by day 1 after injury and remains constant from days 1 to 4 post-4 Gy TBI. (E) Quantification of Gp1Bβ+ MKs physically associated with MECA32+ vessels by femoral IHC prepared from unirradiated control mice (black) or days 1 to 4 post-4 Gy TBI (green). MK association with vasculature changes dynamically following radiation, increasing between days 0 and 1 and between days 2 and 3, but decreasing between days 3 and 4. (F) Quantification of Gp1Bβ+ MKs in the endosteal niche (within 100 μm of the endosteal surface within diaphysis) by IHC in unirradiated control mice (black) and days 1 to 4 post-4 Gy TBI (green). MKs increase in the endosteal niche between days 1 and 2, and decrease between days 2 and 3. Image processing and analysis is described in the “Immunohistochemistry” section. The vascular niche and endosteal niche measurements were not made in a mutually exclusive manner and there may be overlap in occupancy between these niches. Error bars represent standard error of the mean of ≥3 independent experiments (n = 3-12 total mice per group). Statistical analyses either comparing irradiated samples to unirradiated control (B and D) or indicated samples (E and F) were performed using a 2-tailed Student’s t test. Bar represents 100 μm (C). *P < .04; **P < .001.

  • Figure 5

    Changes in SDF-1 regulate MK spatial location and platelet production following 4 Gy TBI. (A) Analysis of MK lineage kinetics (right panel) and Gp1Bβ+ MKs physically associated with MECA32+ vasculature (left panel) 24 hours after treatment with Diprotin A (red) or vehicle (gray) at day 2 post-4 Gy TBI. Diprotin A-mediated stabilization of SDF-1 at day 2 following TBI decreases platelets and MKs in the vascular niche. MKP (colony assay) and MK (imaging flow cytometry) numbers are normalized to per femur values and all compartments are expressed as percent of unirradiated control. Mean absolute numbers for 0 Gy controls: 12 420 MKP/femur, 53 946 MK/femur, 470 × 103 platelets/μL. (B) Analysis of MK lineage kinetics (right panel) and Gp1Bβ+ MKs physically associated with MECA32+ vasculature (left panel) 24 hours after treatment with Diprotin A (red) or vehicle (gray) at day 3 post-4 Gy TBI. Diprotin A-mediated stabilization of SDF-1 at day 3 increases platelets and MKs in the vascular niche. MKP (colony assay) and MK (imaging flow cytometry) numbers are normalized to per femur values and all compartments expressed as percent of unirradiated control. Mean absolute numbers for 0 Gy controls: 13 272 MKP/femur, 52 414 MK/femur, 491 × 103 platelets/μL. (C) Model depicting the regulation of MK niche occupancy in the marrow by SDF-1 at steady state (top panel) and days 2 (middle panel) and 3 (bottom panel) after 4 Gy TBI. At a steady state, Diprotin A-mediated stabilization of SDF-1 increases MKs in the vascular niche. At day 2 following TBI, when our studies reveal a gradient of SDF-1 toward the endosteum, stabilization of SDF-1 decreases both MK association with vasculature and the number of circulating platelets. In contrast, at day 3 post-TBI, when the endosteal SDF-1 gradient is lost, stabilization of SDF-1 resembles the steady-state condition with increased MKs in the vascular niche. (D) Analysis of MK lineage kinetics (right panel) and Gp1Bβ+ MKs physically associated with MECA32+ vasculature (left panel) 24 hours after IV SDF-1 administration (blue) or vehicle (gray) at day 2 post-4 Gy TBI. At this time point, elevation of vascular SDF-1 with IV administration counteracts the endogenous endosteal SDF-1 gradient and leads to enhanced MK-vasculature association and increased circulating platelets. Mean absolute numbers for 0 Gy controls: 10 996 MKP/femur, 52 615 MK/femur, 459 × 103 platelets/μL. Error bars represent standard error of the mean of ≥3 independent experiments (n = 5-9 total mice per group). Statistical analyses comparing treated mice to vehicle controls were performed using a 2-tailed Student’s t test. *P ≤ .04; **P < .006.

  • Figure 6

    Acute administration of IV SDF-1 improves radiation-induced thrombocytopenia and is an additive with earlier TPO administration. (A) MK lineage kinetics at day 5 post-4 Gy TBI in mice treated with IV SDF-1 at day 4 (blue), 0.3 μg IP TPO (red) at 2 hours, both 2-hour TPO and day 4 SDF-1 (purple), or vehicle control (gray). IV SDF-1 increases platelet count following radiation injury. TPO administration at 2 hours post-TBI increases MK and MKP numbers, and IV SDF-1 at day 4 following initial TPO treatment results in an additive improvement in circulating platelets. MKP (colony assay) and MK (imaging flow cytometry) numbers are normalized to per femur values and all compartments expressed as a percentage of unirradiated control. Mean absolute numbers for 0 Gy controls: 10 939 MKP/femur, 50 595 MK/femur, 473 × 103 platelets/μL. (B) Quantification of Gp1Bβ+ MKs in the vascular niche (physically associated with MECA32+ vessels) by femoral IHC prepared on day 5 post-4 Gy from irradiated vehicle control mice (gray), or mice treated with IV SDF-1 at day 4 (blue), 0.3 μg IP TPO (red) at 2 hours, or both 2-hour TPO and day 4 SDF-1 (purple). Percentage of MKs associated with vasculature (IHC) are normalized to per femur values (imaging flow cytometry). Mean 20 104 MK/femur associated with vasculature for 0 Gy controls. Sequential TPO and SDF-1 administration increases MKs in the vascular niche above either treatment alone. (C) Representative images of femoral marrow IHC for Gp1Bβ (MKs, red) and MECA32 (vascular endothelium, green) at day 5 post-4 Gy with hindlimbs isolated from vehicle controls (left panel), mice receiving IV SDF-1 at day 4 (middle panel), and mice receiving 2-hour TPO followed by day 4 SDF-1 (right panel) as described above. White arrows indicate examples of MKs physically associated with MECA32+ vessels. Images were acquired and processed as described in the “Immunohistochemistry” section. Error bars represent standard error of the mean of ≥3 independent experiments (n = 3-11 total mice per group). Statistical analyses comparing indicated platelet samples in panel A were performed by one-way analysis of variance with Tukey’s multiple comparisons test, and all other indicated comparisons were performed by a 2-tailed Student’s t test. Bar represents 100 μm (C). *P < .05; **P < .006.