Mixed Hematopoietic Chimerism for Sickle Cell Disease Prevents Intravascular Hemolysis and Corrects Biomarkers of Endothelial Function.
Authors: Wu Catherine J. , Krishnamurti Lakshmanan , Gladwin Mark T. , Biernacki Melinda , Rogers Shelby A. , Wang Xunde , Zahrieh David , Antin Joseph H. , Ritz Jerome 
Published: April 2020

Recent studies have highlighted the impact of chronic intravascular hemolysis on endothelial function and end-organ injury, such as pulmonary hypertension, in patients with sickle cell disease (SCD). Preservation of organ function is a critical criterion of the success of therapy. Nonmyeloablative allogeneic stem cell transplantation is relatively non-toxic and frequently results in mixed hematopoietic chimerism. However, it is not known if mixed chimerism reduces end-organ toxicity. To assess the effects of mixed chimerism on reduction in hemolysis and endothelial function, we measured multiple plasma parameters of intravascular hemolysis (free hemoglobin concentration, LDH, haptoglobin) and vascular function (sVCAM, NO consumption, arginine/ornithine ratio), recently identified to be associated with pulmonary hypertension and endothelial dysfunction, in 7 SCD patients who developed 25–90% donor engraftment. For each of the 7 patients, we identified normalization in the degree of intravascular hemolysis and endothelial function following transplant. In the analysis of 5 of the 7 patients from whom paired pre and post samples were available, LDH was found to decrease from a median of 376 U/L (range 255–850 U/L) to 162 U/L (range 122–260) (p=0.06, Wilcoxan signed rank test). Similarly, free Hb concentration was observed to decrease from a median of 29.6 μM (range 5.5 to 75.9) to 1.6 μM (range 0.71–8.22) (p=0.06). Haptoglobin rose from a median value of 5.0 mg/dL (range 5–20), to 33 mg/dL (range 7–47) (p=0.06). Parameters of vascular function were also analyzed in a similar fashion. NO bioavailability and sVCAM levels decreased from a median of 7.63 μM (range 1.9–49.7) to 1.22 μM (range 0.6–8.6) (p=0.06), and from a median of 952.1 ng/ml (range 498.2 to 1067) to 543.1 ng/ml (range 193.9 to 627.2) (p= 0.06), respectively. Average arginine/ornithine ratio was inverted prior to transplant to a level of 0.95 (range 0.10–1.4), and normalized following transplantation to a ratio of 2.2 (range 0.14–2.3) at levels that approached significance (p= 0.19). After d180, Pt 1 experienced graft rejection, and all hemolysis parameters again approached pre-transplant levels, demonstrating that reduction in intravascular hemolysis was directly attributable to the presence of circulating donor erythrocytes. Studies of RBC chimerism more accurately assessed the functional impact of donor hematopoiesis. RBC chimerism was serially measured by quantifying donor-derived beta-globin RNA in PBMC. Surprisingly, this revealed a ~2-fold higher level of donor erythroid precursor expression (70–100%) compared to the level of total donor cell engraftment, attributable to the improved survival of engrafted cells. This near-to full replacement of peripheral erythrocytes with donor derived RBCs accounts for the improvements in hemolysis. Our data reveal that levels of donor engraftment as low as 25% can ameliorate the degree of hemolysis and can potentially protect against ongoing chronic end-organ damage. Taken together, our data mechanistically supports mixed chimerism as a suitable endpoint of stem cell transplantation for SCD.