Bone marrow transplant protects mice from sickle cell-mediated large artery remodeling

Introduction: Sickle cell disease (SCD) is a genetic blood disorder associated with progressive arterial damage and heightened risk of stroke, particularly hemorrhagic strokes in early adulthood. In previous studies, we have shown pathological expansive remodeling in the common carotid arteries of mice with SCD with evidence of breaks in the elastic lamina and collagen degradation in the arterial wall [1]. Bone marrow transplant (BMT), or hematopoietic stem cell transplant, is the only curative therapy currently for SCD. Even with the recent approvals of CRISPR/cas-9 for therapy, transplants after ablative therapy will still be necessary. This study aims to determine the optimal timing for the costly and risky transplant therapies and the benefits of protecting the vasculature at its earliest phases. The effectiveness of BMT in halting or reversing SCD-mediated arteriopathy remains unclear. Human clinical indications have shown that a fraction of people with SCD who have undergone curative BMT may continue to be at risk for strokes and cerebrovascular events post-transplant, even after the sickle cell condition has been corrected. Due to the variability of endpoint analyses, we aim to use magnetic resonance angiography (MRA) with a label-free method that enables longitudinal scanning to monitor carotid anomalies in Townes mice. We hypothesize that BMT at an earlier age will mitigate the initiation and accumulation of sickle cell-mediated expansive arterial remodeling.

Materials and

Methods: This study employed two distinct conditioning regimens, radioablation (X-ray irradiation), and chemotherapy (using busulfan), in a humanized murine model of SCD that consists of 3 genotypes: homozygous for β-globin S mutation (SS), littermate heterozygous (AS), and littermate normal homozygous (AA). Label-free magnetic resonance angiography (MRA) was performed to longitudinally monitor common carotid artery luminal areas in living mice pre- and post-BMT over time (Fig. 1A). Mimics Materialise software was used for segmentation and 3D reconstruction of the common carotid arteries. Cross-sectional luminal areas were measured at a resolution of 0.15 mm along the whole length of the left common carotid artery (Fig. 1B). AS and SS Townes mice underwent BMT at 2 months or 4 months of age using AA bone marrow cells, time points we deemed early or late, respectively (Fig. 1C). Endpoint histological analysis of the common carotid arteries and plasma inflammatory cytokine analysis was performed at sacrifice. Both males and females were used in this longitudinal study. All experiments were approved by Georgia Tech’s IACUC.

Results, Conclusions, and Discussions: Myeloablative chemotherapy demonstrated significantly higher survivability in SS (95% survival, n=20) and AS mice (100% survival, n=19) compared to X-ray irradiation by which all SS mice died within 1 week while AS mice had 60-75% survival (n=8 SS, n=9 AS; p< 0.05). Therefore, chemotherapy was chosen as the primary method of conditioning for the rest of the study. Results show that SS mice exhibited significant arterial outward expansion by 3 months and thinning of the medial layer characteristics of a weakened artery wall, heightening the risk of hemorrhagic stroke (n=8 SS, 0.40±0.03 mm2; n=10 AA, 0.32±0.01 mm2; n=11 AS, 0.32±0.02 mm2, p< 0.05). BMT at an early age (2 months) effectively halted this expansion, maintaining smaller luminal areas in SS mice at 3 months post-BMT which was 5 months of age (SS early-BMT: 0.34±0.02 mm2 versus SS-no BMT: 0.42±0.02 mm2, p< 0.01). However, curative BMT in mature adult SS mice (4 months) did not reverse arteriopathy, indicating the importance of early intervention. There was no statistically significant difference in luminal mean area between SS late-BMT (0.39±0.01 mm2, n=8) and SS control groups (0.42±0.02 mm2, n=11) at 5 months (Fig. 1D). Expansive arterial remodeling persisted till 7 months in both non-transplanted and late-transplanted SS mice. In addition, inflammatory cytokines IL-6 and TNF-a concentrations were elevated in plasma of non-transplanted SS mice, and significantly decreased at 5 months following early-BMT in SS mice.
Our study demonstrates that early intervention with BMT effectively halts the progression of arterial expansion, a hallmark of SCD-associated vasculopathy. Our comparison of early (2 months) and late (4 months) BMT in the murine model corresponds to a critical developmental stage in humans, akin to early adolescence and early 20s, respectively. This work emphasizes the significance of MRA as a non-invasive method of assessing arteriopathy progression in SS mice as well as identifying abnormalities such as stenosis and tortuosity in some SS carotid arteries. This study also demonstrates that the timing of BMT is crucial in mitigating sickle cell-induced large artery remodeling. Understanding these age-related dynamics is vital for informing clinical interventions and improving outcomes for individuals with SCD.

Acknowledgements (Optional): Funding sources:
National Institutes of Health grant R01HL158159,
Intramural funding from the National Institute of Biomedical Imaging and Bioengineering

References:
[1] Song, H., Keegan, P. M., ... & Platt, M. O. (2020). ATVB, 40(5), 1220-1230.