Recombinant Human ADAMTS13 Treatment Improves Myocardial Remodeling and Functionality After Pressure Overload Injury in Mice

Abstract

Background: A disintegrin-like metalloproteinase with thrombospondin motif type 1 member 13 (ADAMTS13), the von Willebrand factor-cleaving enzyme, decreases leukocyte and platelet recruitment and, thus, reduces thrombosis and inflammation. Recombinant human ADAMTS13 (rhADAMTS13) is a novel drug candidate for ischemia/reperfusion injury and has shown short-term benefits in mouse models of myocardial injury, but long-term outcome has not been investigated.

Methods and results: We evaluated the impact of rhADAMTS13 on cardiac remodeling, scarring, and contractile function, under chronic left ventricular pressure overload. The role of von Willebrand factor and the effect of rhADAMTS13 treatment were studied. This model of heart failure, based on ascending aortic constriction, produces a coronary inflammatory response and microvascular dysfunction, resulting in fibrotic remodeling and cardiac failure. Mice were treated with either rhADAMTS13 or vehicle and assessed for coronary vascular inflammation and ventricular function at several postsurgical time points, as well as for cardiac fibrosis after 4 weeks. Early upon induction of pressure overload under rhADAMTS13 treatment, we detected less endothelial-lumen-associated von Willebrand factor, fewer platelet aggregates, and decreased activated transforming growth factor-β1 levels than in vehicle-treated mice. We observed significant preservation of cardiac function and decrease in fibrotic remodeling as a result of rhADAMTS13 administration.

Conclusions: Herein, we show that rhADAMTS13 decreases coronary vascular dysfunction and improves cardiac remodeling after left ventricular pressure overload in mice. We propose that this effect may, at least in part, be the result of decreased von Willebrand factor-mediated recruitment of platelets, a major source of the activated profibrotic cytokine transforming growth factor-β1. Our study further supports the therapeutic potential of rhADAMTS13 for conditions characterized by inflammatory cardiac damage that results in fibrosis.

Keywords: ADAMTS13; cardiac remodeling; fibrosis; heart failure; von Willebrand factor.

Figure 1

Ascending aortic constriction (AAC) increases endothelial von Willebrand factor (VWF) secretion and inflammatory cell recruitment in coronary vessels. A, Immunohistochemical staining of VWF in the myocardium in mice that underwent AAC or sham surgery 3 days earlier. Each image depicts a representative myocardial left ventricular portion of 1 mouse. VWF staining was clearly stronger in myocardium exposed to afterload stress by AAC. The black arrows point to vessels that are filled with VWF‐rich material, indicating possible microvessel thrombosis. B, The myocardium of these mice was stained for platelets using immunofluorescent anti‐CD41 antibodies. Platelet aggregates in the tissue were quantified by an investigator blinded to treatment in 5 fields of view per mouse. Afterload‐stressed hearts (n=5) had significantly more myocardial platelet accumulation than control hearts (n=4). C, Representative image showing myeloperoxidase‐positive (MPO⁺) cells (green) and CD41⁺ platelets (red) accumulating along the myocardial vessel wall 3 days after AAC, showing the inflammatory response induced by the model. Myocardial neutrophil (Ly6G⁺, CD11b⁺, and CD45⁺ cells) infiltration was significantly increased in hearts that underwent AAC (n=4) compared with sham–operated hearts (n=3), as assessed by flow cytometric analysis of single‐cell suspensions prepared from digested heart tissue. Significance was assessed by Mann‐Whitney U tests. *P<0.05.

Figure 2

Infusion of recombinant human a disintegrin‐like metalloproteinase with thrombospondin motif type 1 member 13 (rhADAMTS13) has an immediate and dose‐dependent effect on platelet‐string formation in vivo. A, Mice deficient in ADAMTS13 (ADAMTS13^−/−) were prepared for intravital microscopy of the mesenteric venules. Rhodamine 6G was infused to visualize platelets. Representative images of ADAMTS13^−/− venules, forming visible platelet/von Willebrand factor (VWF) strings (left panel, strings indicated by red arrows). One minute after infusion of 3460 U/kg rhADAMTS13 in the same mouse, there were fewer platelet/VWF strings (middle panel); by 10 minutes, these were nearly entirely released from the vessel wall (right panel). B, Platelet‐strings observed per minute were quantified by a blinded investigator (T.W.) in ADAMTS13^−/− mice before (5 minutes) and after (10 minutes) treatment with indicated doses of rhADAMTS13. A highly significant effect on platelet/VWF string formation could only be observed after treatment with 3460 U/kg rhADAMTS13 (n=5). Treatment with 400 U/kg (n=4) had a smaller, but still significant, effect, whereas 40 U/kg (n=3) had no effect. C, Plasma ADAMTS13 activity was measured by fluorescence resonance energy transfer substrate (FRETS)‐VWF73 assay in mice that underwent ascending aortic constriction after treatment with either rhADAMTS13 (red) or vehicle (black), or in sham–operated mice (gray), 24 hours after injection and operation. A strong increase in plasma ADAMTS13 activity, even 24 hours after retro‐orbital injection of rhADAMTS13, demonstrated the enhanced and systemic effect of the treatment with a high dose of rhADAMTS13. No decrease in ADAMTS13 activity was observed in vehicle‐treated mice that underwent AAC compared with sham–operated mice. Both vehicle‐treated and sham–operated mice had similar activity to a pool of normal mouse plasma (NMP; blue). *P<0.05, ***P<0.001.

Figure 3

Recombinant human a disintegrin‐like metalloproteinase with thrombospondin motif type 1 member 13 (rhADAMTS13) treatment reduces platelet recruitment to the myocardium and plasma levels of active transforming growth factor (TGF)‐β1. Myocardial platelet aggregate accumulation (white arrows; A) and endothelial von Willebrand factor (VWF) adherence (VWF‐positive staining; B) 24 hours after surgery is significantly attenuated by high‐dose rhADAMTS13 treatment. Quantification was performed in 5 fields of view (FOVs) per mouse (sham, n=3; vehicle, n=4; rhADAMTS13, n=4) by an investigator (K.M.) blinded to treatment conditions. The number of platelet aggregates per field of view was averaged and, therefore, significance was assessed using a 1‐way ANOVA with Holm‐Sidak multiple‐comparisons test with multiplicity‐adjusted P values. VWF‐positive staining was summed and normalized to measured area and, therefore, expressed as a proportion of area. C, Plasma levels of total TGF‐β1 and the active form of TGF‐β1 were measured at day 1 after surgery by ELISA. Although total levels of TGF‐β1 were not different among the groups, the active form was significantly elevated in the ascending aortic constriction (AAC) vehicle‐treated group (n=4) compared with sham–operated (n=3) and unoperated control (n=5) groups. Active TGF‐β1 levels were lower than the detection limit in mice that underwent AAC and were given rhADAMTS13 (n=4). Mann‐Whitney U tests were performed to determine statistical significance. NS indicates not significant. *P<0.05, **P<0.01.

Figure 4

Recombinant human a disintegrin‐like metalloproteinase with thrombospondin motif type 1 member 13 (rhADAMTS13) treatment attenuates the left ventricular functional decline and hypertrophy resulting from chronic left ventricular (LV) pressure overload induced by ascending aortic constriction (AAC). A, Representative LV ejection fraction (LVEF) measurements in M‐mode echocardiography (sham, n=4; vehicle, n=5; rhADAMTS13, n=7). B, Mice that received rhADAMTS13 treatment for the first 7 days had significantly preserved left ventricular systolic function at 7 and 28 days. C, There was no significant difference in left ventricular diastolic diameters (LVDDs). D, Interventricular septum (IVS) hypertrophy was reduced in the rhADAMTS13 group compared with vehicle at 28 days. E, LV posterior wall (LVPW) hypertrophy was not different in the 2 groups that underwent AAC. d denotes diameter; D, denotes Diastole

Figure 5

Fibrotic and inflammatory changes of the myocardium resulting from left ventricular pressure overload are decreased in mice treated with recombinant human a disintegrin‐like metalloproteinase with thrombospondin motif type 1 member 13 (rhADAMTS13). A, Immunohistochemical staining for intercellular adhesion molecule‐1 (ICAM‐1), a marker for endothelial activation, demonstrated stronger staining in coronary vessel endothelium and endocardium of vehicle‐treated mice compared with rhADAMTS13‐treated mice 28 days after ascending aortic constriction (AAC), suggesting an anti‐inflammatory effect of rhADAMTS13. B, Collagen deposition in the heart 28 days after AAC was significantly lower in rhADAMTS13‐ than vehicle‐treated mice (sham, n=4; vehicle, n=5; rhADAMTS13, n=7).

Figure 6

Fibrosis induced by angiotensin II (AngII) infusion is attenuated with recombinant human a disintegrin‐like metalloproteinase with thrombospondin motif type 1 member 13 (rhADAMTS13) treatment or in von Willebrand factor–deficient (VWF^−/−) mice. A and B, Wild‐type mice were injected for 7 days after osmotic pump implantation with vehicle or rhADAMTS13. Control mice were implanted with osmotic pumps containing saline. A, Masson trichrome staining shows areas of interstitial collagen in hearts after 28 days of AngII infusion. The percentage of collagen‐positive area/total area was determined using color thresholding. B, Perivascular fibrosis was imaged and determined as a ratio of the collagen‐positive area/vessel interior area (saline, n=4; AngII vehicle, n=9; AngII rhADAMTS13, n=6). Perivascular (C) and interstitial (D) collagen were similarly analyzed in VWF^+/+ or VWF^−/− mice (VWF^+/+, n=6; AngII VWF^+/+, n=10; AngII VWF^−/−, n=8). Bar=100 μm. Mann‐Whitney U tests were used to determine statistical significance. NS indicates not significant. *P<0.05, **P<0.01.