ADAMTS13 Endopeptidase Protects against Vascular Endothelial Growth Factor Inhibitor-Induced Thrombotic Microangiopathy

Abstract

Thrombotic microangiopathy (TMA) is a life-threatening condition that affects some, but not all, recipients of vascular endothelial growth factor (VEGF) inhibitors given as part of chemotherapy. TMA is also a complication of preeclampsia, a disease characterized by excess production of the VEGF-scavenging soluble VEGF receptor 1 (soluble fms-like tyrosine kinase 1; sFlt-1). Risk factors for VEGF inhibitor-related TMA remain unknown. We hypothesized that deficiency of the VWF-cleaving ADAMTS13 endopeptidase contributes to the development of VEGF inhibitor-related TMA. ADAMTS13(-/-) mice overexpressing sFlt-1 presented all hallmarks of TMA, including thrombocytopenia, schistocytosis, anemia, and VWF-positive microthrombi in multiple organs. Similar to VEGF inhibitor-related TMA in humans, these mice exhibited severely impaired kidney function and hypertension. In contrast, wild-type mice overexpressing sFlt-1 developed modest hypertension but no other features of TMA. Recombinant ADAMTS13 therapy ameliorated all symptoms of TMA in ADAMTS13(-/-) mice overexpressing sFlt-1 and normalized BP in wild-type mice. ADAMTS13 activity may thus be a critical determinant for the development of TMA secondary to VEGF inhibition. Administration of recombinant ADAMTS13 may serve as a therapeutic approach to treat or prevent thrombotic complications of VEGF inhibition.

Keywords: VEGF; thrombosis; von Willebrand Factor.

Figure 1.

Lack of ADAMTS13 exacerbates the development of TMA under anti-VEGF treatment with sFlt-1. Blood parameters were measured before as well as 4, 7, and 10 days after Ad-sFlt-1 or Ad-null injection in WT and ADAMTS13^−/− mice. (A) Platelet counts and (B) presence of schistocytes in peripheral blood smears revealed thrombocytopenia and schistocytosis in the ADAMTS13^−/− Ad-sFlt-1 mice with a maximum on day 7. (C) Reticulocyte counts were strongly elevated by day 10 in the ADAMTS13^−/− Ad-sFlt-1 mice, but also increased in the WT Ad-sFlt-1 mice. (n=7–13 for A-C). (D) Representative blood smears appear normal in the WT Ad-sFlt-1 and ADAMTS13^−/− Ad-null mice, but show shistocytosis, polychromasia, and anisocytosis in the ADAMTS13^−/− Ad-sFlt-1 mice. Scale bars: 10 µm. (E) Hemoglobin was decreased in the ADAMTS13^−/− Ad-sFlt-1 mice at day 7, indicating hemolytic anemia (n=7–13). (F) VWF plasma levels were measured on day 7 after virus injection. Levels were compared with those of pooled untreated WT plasma, or pooled untreated ADAMTS13^−/− plasma, depending on the mouse genotype. Only WT mice injected with Ad-sFlt-1 showed an increase in plasma VWF levels (n=7–10). *P<0.05; **P<0.01; ***P<0.001; ****P<0.001.

Figure 2.

ADAMTS13^−/− mice show VWF-rich thrombi in multiple organs after sFlt-1 overexpression. Organs were collected from WT and ADAMTS13^−/− mice 7 days after injection of Ad-sFlt-1 or Ad-null and immunohistochemical staining for VWF (brown) was performed. Representative photographs of liver, kidney, lung, and heart sections are shown. Arrowheads indicate VWF-rich thrombi. Scale bar, 100 µm.

Figure 3.

ADAMTS13^−/− mice are prone to proteinuria, hypertension, and endotheliosis after Ad-sFlt-1 injection. (A) Proteinuria, determined as albumin-creatinine ratio, showed a dramatic increase in the ADAMTS13^−/− Ad-sFlt-1 mice by day 7. In WT Ad-sFlt-1 mice, albumin-creatinine ratios increased significantly on day 7, but overall stayed low (n=4–13). (B) Systolic BP was measured noninvasively by the tail-cuff method. Both WT Ad-sFlt-1 and ADAMTS13^−/− Ad-sFlt-1 mice had significant increases in their systolic BP. However, BP reached higher values in the ADAMTS13^−/− mice under Ad-sFlt-1 overexpression (n=5–20). (C) Upper panel: H&E micrographs of representative kidney glomeruli show normal size and open capillary loops (some of them filled with erythrocytes) in the WT Ad-sFlt-1 and the ADAMTS13^−/− Ad-null mice, but enlarged glomeruli and vascular loop occlusion in the ADAMTS13^−/− Ad-sFlt-1 group. Scale bar: 20 µm. Lower panel: electron microscopy images at ×5000 also show increased glomerular size and obstructed capillary loops in the ADAMTS13^−/− Ad-sFlt-1 mice, due to endothelial cell swelling. Asterisks indicate open capillary loops, which are visible either as white space or filled with light-gray erythrocytes. Scale bar: 4 µm. (D) Quantification of open capillary volume by ImageJ in H&E micrographs reveals a strong reduction of open capillary loops only in ADAMTS13^−/− Ad-sFlt-1 mice. (E) Glomerular area was increased in ADAMTS13^−/− mice after Ad-sFlt-1-injection (n=5–6 for D and E). *P<0.05; **P<0.01; ***P<0.001; ****P<0.001.

Figure 4.

Treatment with rhADAMTS13 rescues ADAMTS13^−/− mice from sFlt-1-induced TMA. ADAMTS13^−/− mice were injected with Ad-sFlt-1 virus and then received daily treatment with rhADAMTS13 (or PBS as vehicle control) starting at day 4 after virus injection. By day 7, rhADAMTS13-treated ADAMTS13^−/− Ad-sFlt-1 mice showed significant improvements compared with the vehicle-treated ADAMTS13^−/− Ad-sFlt-1 mice in (A) platelet counts, (B) schistocyte counts, (C) reticulocyte counts, and (D) hemoglobin values (n=6–9 for A–D). (E) Upon rhADAMTS13 treatment, VWF plasma levels increased significantly in ADAMTS13^−/− sFlt-1 mice compared with vehicle-treated mice (n=6–8). (F) Representative blood smears show schistocytes and an increased number of reticulocytes in the ADAMTS13^−/− Ad-sFlt-1 mice treated with PBS, but appear normal in the group that received treatment with rhADAMTS13. Scale bar, 10 µm. (G) Immunohistochemistry shows that VWF-rich thrombi (brown, arrowheads) are mostly lacking in mice treated with rhADAMTS13 at day 7 after injection of Ad-sFlt-1. Scale bar, 100 µm. Arrowheads indicate VWF-positive thrombi. *P<0.05; **P<0.01; ***P<0.001; ****P<0.001.

Figure 5.

Kidney damage is reduced by treatment with rhADAMTS13 in ADAMTS13^−/− Ad-sFlt-1 mice and BP is normalized in both WT and ADAMTS13^−/− mice overexpressing sFlt-1. (A) Representative H&E micrographs (upper panel) and electron microscope pictures (lower panel) at ×5000 show that treatment of ADAMTS13^−/− Ad-sFlt-1 mice with rhADATMS13 from day 4 to day 7 after Ad-sFlt-1 injection improved kidney histology with smaller glomeruli and less capillary occlusion, compared with ADAMTS13^−/− Ad-sFlt-1 mice that had received only PBS. Mice were sacrificed at day 7 after Ad-sFlt-1 injection. Scale bars: 20 and 4 µm, respectively. Asterisks indicate open capillary loops in the electron microscope pictures. (B) Quantification of open capillary volume and (C) glomerular area corroborated the improvement in kidney structure in the rhADAMTS13-treated ADAMTS13^−/− Ad-sFlt-1 mice compared with vehicle-treated mice (n=5–8 for B and C). (D) Proteinuria (n=4–9) and (E) BP (n=3–6) improved in ADAMTS13^−/− Ad-sFlt-1 mice upon rhADAMTS13 treatment. BP was also normalized in the rhADAMTS13-treated WT mice overexpressing sFlt-1. *P<0.05; **P<0.01.