Evidence of protective effects of recombinant ADAMTS13 in a humanized model of sickle cell disease

Abstract

Sickle cell disease (SCD) is an inherited red blood cell disorder that occurs worldwide. Acute vaso-occlusive crisis is the main cause of hospitalization in patients with SCD. There is growing evidence that inflammatory vasculopathy plays a key role in both acute and chronic SCD-related clinical manifestations. In a humanized mouse model of SCD, we found an increase of von Willebrand factor activity and a reduction in the ratio of a disintegrin and metalloproteinase with thrombospondin type 1 motif, number 13 (ADAMTS13) to von Willebrand factor activity similar to that observed in the human counterpart. Recombinant ADAMTS13 was administered to humanized SCD mice before they were subjected to hypoxia/reoxygenation (H/R) stress as a model of vaso-occlusive crisis. In SCD mice, recombinant ADAMTS13 reduced H/R-induced hemolysis and systemic and local inflammation in lungs and kidneys. It also diminished H/R-induced worsening of inflammatory vasculopathy, reducing local nitric oxidase synthase expression. Collectively, our data provide for the firsttime evidence that pharmacological treatment with recombinant ADAMTS13 (TAK-755) diminished H/R-induced sickle cell-related organ damage. Thus, recombinant ADAMTS13 might be considered as a potential effective disease-modifying treatment option for sickle cell-related acute events.

Figure 1.

Recombinant ADAMTS13 reduced hypoxia/reoxygenation-induced hemolysis and modulated the systemic inflammatory response. (A) von Willebrand factor (VWF) activity/antigen concentration ratio in plasma of humanized healthy (AA) and sickle cell (SS) mice. VWF multimer gel analysis of SS and AA mouse plasma (see Online Supplementary Figure S2A for additional gels). (B) Plasma ADAMTS13 activity, VWF activity, ADAMTS13/VWF activity ratio in AA and SS mice (n=5-6 age-matched male mice; *P<0.05 vs. SS 21%). (C) Hemoglobin (Hb) in AA and SS mice under normoxia and exposed to hypoxia/reoxygenation (H/R) treated with either vehicle or recombinant ADAMTS13 (rADAMTS13); *P<0.05 compared to AA; ^P<0.05 compared to vehicle. (D) Left panel. Hemoglobin distribution width (HDW) in AA and SS mice under normoxia and exposed to H/R treated with either vehicle or rADAMTS13; *P<0.05 compared to AA; ^P<0.05 compared to vehicle. Right panel. Distribution histograms generated for red blood cell volume (RBC Volume) and cell hemoglobin concentration (RBC-HC) of red blood cells from humanized SS mice under normoxia and treated with either vehicle or rADAMTS13 and exposed to H/R. One experiment representative of six others with similar results is shown. The blue circle indicates the presence of a subpopulation of dense red cells. (E) Platelet (PLT) counts in humanized AA and SS mice under normoxia and treated with either vehicle or rADAMTS13 and exposed to H/R (n=6; age-matched female and male mice; ^P<0.05). (F) Peripheral neutrophils in AA and SS mice under normoxia or exposed to H/R (8% oxygen for 10 h followed by 3 h of reoxygenation) treated with either vehicle or rADAMTS13. Data are presented as box-and-whisker plots. *P<0.001 compared to vehicle-treated animals under normoxia. ^P<0.001 compared to vehicle-treated animals under hypoxia. P values were calculated using an unpaired one-tailed t-test with the Welch correction.

Figure 2.

In sickle cell disease mice, recombinant ADAMTS13 reduced hypoxia/reoxygenation-induced lung damage and inflammatory vasculopathy. (A) Representative hematoxylin and eosin-stained sections of lung tissue from healthy (AA) and sickle cell (SS) mice exposed to hypoxia (8% oxygen; 10 h), followed by reoxygenation (21% oxygen; 3 h) treated with vehicle or recombinant ADAMTS13 (rADAMTS13) (2940 U/kg), see also Table 1; black arrows indicate inflammatory cell infiltrate. (B) Leukocyte content (lower panel) and protein content (upper panel) in bronchoalveolar lavage (BAL) from AA and SS mice under normoxia or exposed to hypoxia (8% oxygen; 10 h), followed by reoxygenation (21% oxygen; 3 h) treated with either vehicle or rADAMTS13. Data are presented as mean ± standard error of mean (n=6, age-matched male and female animals). *P<0.001 compared to vehicle-treated animals under normoxia. ^P<0.001 compared to vehicle-treated animals under hypoxia. P values were calculated using an unpaired one-tailed t-test with the Welch correction. (C) Immunoblot analysis, using specific antibodies against phosphorylated (P-)NF-κB p65 and NF-κB p65 of lung from AA and SS mice under normoxia or exposed to hypoxia (8% oxygen; 10 h), followed by reoxygenation (21% oxygen; 3 h) treated with either vehicle or rADAMTS13. GAPDH was used as a protein loading control. One representative gel from three with similar results is shown. Densitometric analysis of immunoblots is shown in Online Supplementary Figure 4SA. (D) Immunoblot analysis, using specific antibodies against VCAM-1, ICAM-1, ET-1, TXAS and HO-1 of lung from AA and SS mice treated as in (C). GAPDH was used as a protein loading control. One representative gel from three with similar results is shown (n=3 age-matched male and female mice in each group). Densitometric analysis of immunoblots is shown in Online Supplementary Figure S4C. H/R: hypoxia/reoxygenation; VCAM-1: vascular endothelial cell adhesion molecule-1; ICAM-1: intracellular adhesion molecule-1; ET-1: endothelin-1; TXAS: thromboxane synthase; HO-1: heme oxygenase-1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Figure 3.

In sickle cell disease mice, recombinant ADAMTS13 diminished hypoxia/reoxygenation-induced kidney damage and vascular inflammatory activation. (A) Representative hematoxylin and eosin-stained sections of kidney tissue from healthy (AA) and sickle cell (SS) mice exposed to hypoxia (8% oxygen; 10 h), followed by reoxygenation (21% oxygen; 3 h) treated with either vehicle or recombinant ADAMTS13 (rADAMTS13) (2940 U/kg); see also Table 1; black arrows indicate inflammatory cell infiltrate. (B) Plasma creatinine (upper panel) and blood urea nitrogen (BUN) (lower panel) in AA and SS mice treated as in (A). Data are mean ± standard error of mean (n=6 age-matched male and female animals). *P<0.05 compared to AA, ^P<0.05 compared to vehicle-treated animals. (C) Immunoblot analysis, using specific antibodies against phosphorylated (P-)NF-κB p65 and NF-κB p65 of kidney from AA and SS mice under normoxia or exposed to hypoxia (8% oxygen; 10 h), followed by reoxygenation (21% oxygen; 3 h) treated with vehicle or rADAMTS13. GAPDH served as the protein loading control. One representative gel from three with similar results is shown (n=3 age-matched male and female in each group). Densitometric analysis of immunoblots is shown in Online Supplementary Figure 7SA. (D) Immunoblot analysis, using specific antibodies against VCAM-1, ET-1, TXAS and E-Selectin of kidney from AA and SS mice treated as in (A). GAPDH served as a protein loading control. One representative gel from three with similar results is shown (n=3 age-matched male and female in each group). Densitometric analysis of immunoblots is shown in Online Supplementary Figure 7SB. H/R: hypoxia/reoxygenation; VCAM-1: vascular endothelial cell adhesion molecule-1; ET-1: endothelin-1; TXAS: thromboxane synthase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Figure 4.

Recombinant ADAMTS13 reduces hypoxia/reoxygenation-induced kidney over-activation of endothelial nitric oxide synthase and protects against hypoxia/reoxygenation-induced worsening of inflammatory vasculopathy. (A) Left panel. Kidney soluble fraction from healthy (AA) and sickle cell (SS) mice treated under normoxia or exposed to hypoxia (8% oxygen; 10 h), followed by reoxygenation (21% oxygen; 3 h) treated with either vehicle or recombinant ADAMTS13 (rADAMTS13) (2940 U/kg). Samples were analyzed by 11% sodium dodecylsulfate polyacrylamide gel electrophoresis and subjected to OxyBlot. The carbonylated proteins (1 mg) were detected by treating with 2,4-dinitrophenylhydrazine (DNP) and blotted with anti-DNP antibody. Right panel. Band area was quantified by densitometry and expressed as percentage of that in AA mice in normoxia. The data are presented as means ± standard error of mean (SEM) (n=3 age-matched male and female mice in each group); ^P<0.05 compared to normoxia, *P<0.05 compared to AA mice; #P<0.05 compared to vehicle-treated animals by a one-tailed t-test with Welch correction. (B) Left panel. Immunoblot analysis, using specific antibodies against eNOS and iNOS in kidney from AA and SS mice under normoxia or exposed to hypoxia (8% oxygen; 10 h), followed by reoxygenation (21% oxygen; 3 h) treated with either vehicle or rADAMTS13 (2940 U/kg). GAPDH served as a protein loading control. One representative gel from three with similar results is shown. Right panel. Densitometric analysis of the immunoblot. Data are presented as means ± SEM (n=3 age-matched male and female mice in each group); ^P<0.05 compared to normoxia, *P<0.05 compared to AA mice; ^#P<0.05 compared to vehicle-treated animals by a one-tailed t-test with Welch correction. (C) Left panel. Immunoblot analysis, using specific antibodies against ET-1 and E-Selectin of isolated aorta from AA and SS mice under normoxia or exposed to hypoxia (8% oxygen; 10 h), followed by reoxygenation (21% oxygen; 3 h) treated with either vehicle or rADAMTS13 (2940 U/kg). Actin was used as a protein loading control. One representative gel from three with similar results is shown. Right panels. Densitometric analysis of immunoblots. Data are presented as means ± SEM (n=3 age-matched male and female in each group); ^P<0.05 compared to normoxia, *P<0.05 compared to AA; ^#P<0.05 compared to vehicle-treated animals, one-tailed t-test with Welch correction. H/R: hypoxia/reoxygenation; ET-1: endothelin-1; eNOS: endothelial nitric oxide synthase; iNOS: inducible nitric oxide synthase; DU: densitometric units.

Figure 5.

Schematic diagram of mechanisms involved in sickle cell-related microangiopathy and the role of recom-binant ADAMTS13 as a novel therapeutic option for acute vaso-occlusive crises. Vaso-occlusive crises in sickle cell disease are characterized by hypoxia/reoxygenation stress, promoting an amplified inflammatory response and severe hemolysis. Both factors contribute to a relative deficiency of ADAMTS13 associated with decreased susceptibility of von Willebrand factor (VWF) to ADAMTS13. This potentiates vascular endothelial activation and damage, characterized by increased expression of vascular pro-adhesion markers and abnormal local bioavailability of nitric oxide as part of severe sickle cell-related inflammatory vasculopathy and vascular dysfunction involved in acute organ damage. H/R: hypoxia/re-oxygenation; Hb: hemoglobin; ADAMTS13: a disintegrin and metalloproteinase with thrombospondin type 1 motif, number 13; VWF: von Willebrand factor; rADAMTS13: recombinant ADAMTS13; NO: nitric oxide; VCAM-1: vascular cell adhesion molecule-1.