Thrombopoietin mimetic stimulates bone marrow vascular and stromal niches to mitigate acute radiation syndrome

Abstract

Background: Acute radiation syndrome (ARS) manifests after exposure to high doses of radiation in the instances of radiologic accidents or incidents. Facilitating the regeneration of the bone marrow (BM), namely the hematopoietic stem and progenitor cells (HSPCs), is a key in mitigating ARS and multi-organ failure. JNJ-26366821, a PEGylated thrombopoietin mimetic (TPOm) peptide, has been shown as an effective medical countermeasure (MCM) to treat hematopoietic-ARS (H-ARS) in mice. However, the activity of TPOm on regulating BM vascular and stromal niches to support HSPC regeneration has not yet been elucidated.

Methods: C57BL/6J mice (9-14 weeks old) received sublethal or lethal total body irradiation (TBI), a model for H-ARS, by ¹³⁷Cs or X-rays. At 24 hours post-irradiation, mice were subcutaneously injected with a single dose of TPOm (0.3 mg/kg or 1.0 mg/kg) or PBS (vehicle). At homeostasis and on days 4, 7, 10, 14, 18, and 21 post-TBI with and without TPOm treatment, BM was harvested for histology, BM flow cytometry of HSPCs, endothelial (EC) and mesenchymal stromal cells (MSC), and whole-mount confocal microscopy. For survival, irradiated mice were monitored and weighed for 30 days. Lastly, BM triple negative cells (TNC; CD45^-, TER-119^-, CD31^-) were sorted for single-cell RNA-sequencing to examine transcriptomics after TBI with or without TPOm treatment.

Results: At homeostasis, TPOm expanded the number of circulating platelets and HSPCs, ECs, and MSCs in the BM. Following sublethal TBI, TPOm improved BM architecture and promoted recovery of HSPCs, ECs, and MSCs. Furthermore, TPOm elevated VEGF-C levels in normal and irradiated mice. Following lethal irradiation, mice improved body weight recovery and 30-day survival when treated with TPOm after ¹³⁷Cs and X-ray exposure. Additionally, TPOm reduced vascular dilation and permeability. Finally, single-cell RNA-seq analysis indicated that TPOm increased the expression of collagens in MSCs to enhance their interaction with other progenitors in BM and upregulated the regeneration pathway in MSCs.

Conclusions: TPOm interacts with BM vascular and stromal niches to locally support hematopoietic reconstitution and systemically improve survival in mice after TBI. Therefore, this work warrants the development of TPOm as a potent radiation MCM for the treatment of ARS.

Keywords: bone marrow; endothelial cells; hematopoietic acute radiation syndrome; mesenchymal stromal cells; thrombopoietin mimetic; total body irradiation.

Figure 1. TPOm expands megakaryocytes, hematopoietic stem and progenitor, endothelial, and stromal cells in murine bone marrow at homeostasis.

(a–d)Complete blood count of peripheral blood for (a) platelets, (b)white blood cells, (c) neutrophils, and (d) lymphocytes. (e)Representative H&E images of sternal bone marrow from naïve and mice treated with TPOm on day 6 after injection. Yellow arrow, megakaryocytes. Scale bar is 100 μm. (f) Count of megakaryocytes in the sternal bone marrow of naive and TPOm-treated mice at the indicated day post-injection (n=3/group). (g)The number of MPC, MkP, LSK, ST-, and LT-HSC per femur of naive and TPOm-treated mice (n=3–4/group) over time. (h) The number of EC, EPC, AEC, SEC, and MSC per femur of naive and TPOm-treated mice (n=3–4/group) over time. Data are expressed as mean ± SEM. *p < 0.05 vs. naïve assessed by one-way ANOVA with post hoc Dunnett test for multiple comparisons.

Figure 2. TPOm promotes recovery of hematopoietic stem and progenitor, endothelial, and stromal cells in murine bone marrow following 7.0 Gy sublethal total body irradiation.

(a) Representative H&E images of sternal bone marrow from vehicle and TPOm-treated mice on days 2, 4, 7, and 14 after irradiation. Yellow arrow, megakaryocytes; light blue arrow, adipocytes; dark blue box, hemorrhaging. Scale bar is 100 μm. (b) The number of megakaryocytes in the sternal bone marrow of naïve, vehicle, and TPOm-treated mice over time (n=3/group). (c) The number of adipocytes using MarrowQuant through QuPath in the sternal bone marrow of naïve, vehicle, and TPOm-treated mice over time (n=3/group). (d) Live cell count of femoral bone marrow of naïve, vehicle, and TPOm-treated mice over time (n=4/group). (e)The number of MPC, MkP, LSK, ST-, and LT-HSC per femur of naïve, vehicle, and TPOm-treated mice (n=4–29/group) over time. (f) The number of EC, EPC, AEC, SEC, and MSC per femur of naïve, vehicle, and TPOm-treated mice (n=4–29/group) over time. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vehicle vs. TPOm-treated assessed by unpaired Student’s t-test with post hoc Holm-Sidak method for multiple comparisons. Outliers were determined using ROUT with a Q = 0.2%.

Figure 3. TPOm significantly increases survival of mice after lethal total body irradiation.

Kaplan-Meier survival curve of vehicle and TPOm treated male mice for 30 days after (a) 8.8 Gy ¹³⁷Cs, (c) 6.7 Gy X-ray, and (e) 7.2 Gy X-ray TBI. The percentage of body weight change over 30 days after (b) 8.8 Gy ¹³⁷Cs, (d) 6.7 Gy X-ray, and (f) 7.2 Gy X-ray TBI. For survival, the Log-rank (Mantel-Cox) test was used for curve comparison. For the percent weight change data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, vehicle vs. TPOm-treated by unpaired Student’s t-test with post hoc Holm-Sidak method for multiple comparisons.

Figure 4. TPOm systemically reduces bone marrow vascular dilatation and vascular leakage and promotes production of VEGF-A and VEGF-C in irradiated mice.

(a) Representative immunofluorescent images of femurs stained with DAPI (blue), VEGFR3 (green), and CD31/CD144 (red) on days 4 and 10 after irradiation in vehicle and TPOm-treated mice. Non-irradiated mice were represented as naïve for reference. Scale bar is 10 μm. (b) VEGFR3⁺ vessel area in the bone marrow on days 4 and 10 after irradiation, quantitated by using Volocity software (n=3/group). (c) Representative immunofluorescent images of sternum stained with DAPI (blue), Sca-1 (green), and TIE2 (red) on days 2 and 4 after irradiation in vehicle and TPOm- treated mice from 2 independent experiments. Non-irradiated mice were represented as naïve for reference. (d) Quantification of Sca-1⁺ cells per 100× field in sternal bone marrow quantitated by using Volocity software (n=3/group). (e) IVIS images of 7.2 Gy (X-rays) TBI mice imaged 5 days after irradiation with AngioSense750 EX i.v. injection performed on day 3 after irradiation. (f) Quantification of total radiant efficiency (n=3/group). (g,h) ELISA of VEGF-A in (g) serum and in (h) BM after 7.0 Gy (¹³⁷Cs) TBI. (i,j) ELISA of VEGF-C in (i) serum and in (j) BM after 7.0 Gy (¹³⁷Cs) TBI. Data are expressed as mean ± SEM. *p < 0.05, **p < 0.01 vehicle vs. TPOm-treated assessed by unpaired Student’s t-test with post hoc Holm-Sidak method for multiple comparisons.

Figure 5. TPOm increases subpopulations of hematopoietic progenitors and Mki67 expression in sorted bone marrow TNCs from mice after sublethal total body irradiation.

Single-cell RNA-seq analysis of sorted CD45⁻, TER-119⁻, CD31⁻ (TNC) cells derived from BM of mice 10 days after 6 Gy (X-ray) TBI. (a) Heatmap of highly expressed genes used to identify different cell clusters. (b) Overall UMAP clustering of TNC cells, (c) individual UMAP clusters following each group: naïve (Control), TPOm alone, irradiated (IR), and irradiated plus TPOm-treated (IR_TPOm). (d) Each of the cluster’s distribution by percentage iterated by treatment condition. (e) Expression distribution of Mki67 on the overall UMAP of TNC cells. (f) Violin plots of Mki67 expression iterated by identified clusters per treatment group. (g) Cell cycle analysis of each identified cluster per treatment group.

Figure 6. TPOm stimulates the interaction of MSCs with other cell clusters by upregulating the regeneration pathway and collagens expression.

(a) A chord diagram of cell-to-cell communications between MSCs and other identified clusters of the TNC in the BM. (b) MSCs (sender) and other identified clusters (receivers) interaction by ligand and receptor. (c) Relative contributions of each ligand-receptor interaction. (d) Violin plots of the expression levels of different collagens expressed by MSCs iterated in each treatment group. (e) Volcano plot of differentially expressed genes of TPOm vs. Naïve groups and (f) Gene Ontology pathways significantly overrepresented among up- and down-regulated genes (g) Volcano plot of differentially expressed genes of irradiated (IR) vs. TPOm-irradiated (IR_TPOm) groups and (h) Gene Ontology pathways significantly overrepresented among down-regulated genes. Differential expression was performed with MAST model adjusting for Sex, nCount_RNA, percent.mt, S.Score.

Figure 7

Summary of TPOm’s effect on BM vascular and stromal niches for HSPC regeneration after irradiation to mitigate H-ARS.