Pharmacological targeting of the thrombomodulin-activated protein C pathway mitigates radiation toxicity

Abstract

Tissue damage induced by ionizing radiation in the hematopoietic and gastrointestinal systems is the major cause of lethality in radiological emergency scenarios and underlies some deleterious side effects in patients undergoing radiation therapy. The identification of target-specific interventions that confer radiomitigating activity is an unmet challenge. Here we identify the thrombomodulin (Thbd)-activated protein C (aPC) pathway as a new mechanism for the mitigation of total body irradiation (TBI)-induced mortality. Although the effects of the endogenous Thbd-aPC pathway were largely confined to the local microenvironment of Thbd-expressing cells, systemic administration of soluble Thbd or aPC could reproduce and augment the radioprotective effect of the endogenous Thbd-aPC pathway. Therapeutic administration of recombinant, soluble Thbd or aPC to lethally irradiated wild-type mice resulted in an accelerated recovery of hematopoietic progenitor activity in bone marrow and a mitigation of lethal TBI. Starting infusion of aPC as late as 24 h after exposure to radiation was sufficient to mitigate radiation-induced mortality in these mice. These findings suggest that pharmacologic augmentation of the activity of the Thbd-aPC pathway by recombinant Thbd or aPC might offer a rational approach to the mitigation of tissue injury and lethality caused by ionizing radiation.

Figure 1. Elevated expression of Thbd selects for primitive hematopoietic cells upon irradiation in vivo

(a) EGFP chimerism in PB of animal 9 after exposure to 3 Gy TBI every week for 3 consecutive weeks versus controls (same experiment, non-selected). (b) Graphical representation of the provirus integration into chromosome 2 of animal 9 (BM) as determined by LM-PCR followed by sequencing of the dominant integron product (see Supplementary Table 1 for a list of all integration sites in animal 9). (c) Transcriptional level of expression of the genes surrounding 5′ and 3′ side of integrated provirus of animal 9 (BM cells). Determination by quantitative real-time RT-PCR compared to a pool of control samples (BM cells from C57BL/6CD45.1 mice, and animals 11 and 18, which were transplanted with transduced HSPCs but not irradiated). (d) Expression of Thbd in the BM of animal 9 compared to control animals (C57BL/6), determined by Western blot analysis. (e) Experimental setup: over-expression of Thbd in HSPCs by retroviral transduction, followed by transplantation and subsequent selection by irradiation. Recipient animals were pre-conditioned by irradiation with 11.75 Gy to accept the graft. Animals consistently presented with a graft chimerism (CD45.2+ cells in PB) of higher than 90% 3 weeks post transplantation. (f) Representative data depicting the level of EGFP expression/selection among CD45.2+ cells in PB of individual animals with and without irradiation in Thbd-transduced compared to control-transduced cells transplanted. (g) Quantification of the radioselection of Thbd-transduced hematopoietic cells in PB in vivo post-irradiation relative to control (GFP only)–transduced hematopoietic cells; n = 3 independent experiments with at least 3 recipients per single experiment. * p = < 0.05.

Figure 2. Solulin and recombinant aPC confer mitigation of radiation toxicity after TBI

(a,b) 30 d survival of mice injected subcutaneously (n = 8 per group) 30 min after exposure to (a) 8.5 Gy or (b) 9.5 Gy TBI, p<0.05 for both Solulin at 3 mg kg⁻¹ and control. (c) Experimental setup for experiments depicted in (d). (d) 30 d survival of C57BL/6 animals injected with recombinant murine aPC (0.35 to 0.4 mg kg⁻¹) or vehicle (PBS) i.v. 30 min after single-dose TBI; n = 8 for 8 Gy vehicle, n = 33 for 9 Gy aPC, n = 23 for 9 Gy vehicle, n = 8 for 10 Gy vehicle, n = 20 for 10 Gy multiple aPC (30 min, 1 h and 2 h post-irradiation, 9 Gy vehicle vs. 9 Gy aPC, p < 0.0001; 10 Gy vehicle vs. 10 Gy multiple aPC, p<0.0001). (e) Experimental setup: mitigation of TBI toxicity when aPC is given no earlier than 24 hours post-TBI. (f) 30 d survival of C57BL/6 animals injected with vehicle or murine aPC at a dose of 5 μg/mouse through a single i.v. injection at 24 and 48 hours post TBI (9.5 Gy).

Figure 3. Mechanisms of action of mitigation by soluble Thbd and aPC

(a) Experimental design. (b) Total number of leukocytes per femur in control animals and animals treated with aPC 10 d post irradiation. (c) Frequency of Lin⁻, c-Kit⁺ cells in BM 10 d post irradiation. (d) Frequency of CFCs in BM cells 10 d post irradiation. (e) Experimental design for data presented in s(f). (f) Frequency of CFCs in BM cells 10 days post irradiation. (g) 30 d survival of mice (n = 15 per group) irradiated with an LD50 receiving either 5 μg/mouse of the hyper-anticoagulant E149A mutant form of aPC or vehicle control through a single tail vein injection 30 min after TBI, p < 0.001 for E149A-aPC as well as for aPC versus saline and 5A-aPC. (h) 30 d survival of mice given a LD50/30 TBI and treated with the histone blocking antibody BWA3 (i.p. 30 mg kg⁻¹) 0 h (black lines) or 16 h (green lines) post irradiation, compared to control injections of IgG Ab, n ≥ 12 per experimental group. (i) 30 d survival of mice given a LD50/30 TBI and treated with an anti-factor XIa antibody (14E11) at 0 h post irradiation (i.p. 30 mg kg⁻¹) compared to control Ab treated animals, n ≥ 16 per experimental group.

Figure 4. A role of endogenous Thbd in radiation protection

(a) Expression of Thbd relative to β-actin in hematopoietic cells. As an additional control, RNA from BM cells from an floxed Thbd allele crossed to an Mx-Cre animals treated with pIC to delete the allele in BM cells (601 BM, deletion of up to 80% confirmed by PCR) was used. (b) Expression of Thbd relative to β-actin in BM stroma cells containing endothelial cells (CD45⁻, Ter119⁻ cells) and in non-endothelial stroma cells (CD45⁻,Ter119⁻, CD31⁻ cells). (c) Femoral bone marrow of Thbd^Pro/lacZ mice stained in situ with a substrate for β-galactosidase. Blue staining indicates expression of the LacZ reporter gene controlled by the endogenous Thbd-promoter. (left) Staining occurs in the endothelium of blood vessel on the outer surface of the bone or penetrating the bone (black arrowhead), and in some vessels within the bone marrow mass. Intense staining is seen in a loose meshwork of cells located between the entire inner surface of the bone and the central bone marrow (white arrow; BM: bone marrow partially exposed by removal of the lacZ-positive layer). No staining was noted in parallel-processed WT controls. (middle) LacZ-positive clusters of cells are predominantly associated with the periphery of the marrow extruded from the bone cavity. (right) lacZ-positive endothelial cells seen in small blood vessels supplying the outer bone surface. The expression of lacZ correlated well with the data obtained by real-time RT-PCR in (a,b). White bar represent 1 mm. (d) 30 d survival of Thbd^+/+ and Thbd^Pro/lacZ mice (n = 8 per group) subjected to TBI with doses ranging between 7-11 Gy, p = 0.0001. (e) 20 d survival of Thbd^Pro/Pro animals irradiated with an LD50/30 TBI, n ≥ 11 per group, p = 0.09. (f) 30 d survival of animals treated with an LD50/30 TBI, n = 10 for APC^HI, n = 15 for Thbd^Pro/Pro/APC^HI and WT control, p < 0.05 for Thbd^Pro/Pro/APC^HI over WT. (g) Experimental setup: competitive transplantation/radioselection experiments with BM cells from Thbd^Pro/Pro mice. (h) Donor chimerism in PB of animals competitively transplanted according to (g) 3 weeks post–3 Gy irradiation of recipients compared to controls, n ≥ 4 recipients per group, * = p < 0.05. (i) Experimental setup: transplantation/radioselection experiments with Thbd^Pro/Pro and WT mice reconstituted with WT BM cells. (j) 30 d survival of WT and Thbd^Pro/Pro recipients treated with a second dosage of 7.75 Gy, n = 20 for WT and n = 9 for Thbd^Pro/Pro recipients, p < 0.05 for Thbd^Pro/Pro recipients compared to WT recipients.