Elevated Angiopoietin-2 Level in Patients With Continuous-Flow Left Ventricular Assist Devices Leads to Altered Angiogenesis and Is Associated With Higher Nonsurgical Bleeding

Abstract

Background: Nonsurgical bleeding is the most common adverse event in patients with continuous-flow left ventricular assist devices (LVADs) and is caused by arteriovenous malformations. We hypothesized that deregulation of an angiogenic factor, angiopoietin-2 (Ang-2), in patients with LVADs leads to increased angiogenesis and higher nonsurgical bleeding.

Methods: Ang-2 and thrombin levels were measured by ELISA and Western blotting, respectively, in blood samples from 101 patients with heart failure, LVAD, or orthotopic heart transplantation. Ang-2 expression in endothelial biopsy was quantified by immunofluorescence. Angiogenesis was determined by in vitro tube formation from serum from each patient with or without Ang-2-blocking antibody. Ang-2 gene expression was measured by reverse transcription-polymerase chain reaction in endothelial cells incubated with plasma from each patient with or without the thrombin receptor blocker vorapaxar.

Results: Compared with patients with heart failure or those with orthotopic heart transplantation, serum levels and endothelial expression of Ang-2 were higher in LVAD patients (P=0.001 and P<0.001, respectively). This corresponded to an increased angiogenic potential of serum from patients with LVADs (P<0.001), which was normalized with Ang-2 blockade. Furthermore, plasma from LVAD patients contained higher amounts of thrombin (P=0.003), which was associated with activation of the contact coagulation system. Plasma from LVAD patients induced more Ang-2 gene expression in endothelial cells (P<0.001), which was reduced with thrombin receptor blockade (P=0.013). LVAD patients with Ang-2 levels above the mean (12.32 ng/mL) had more nonsurgical bleeding events compared with patients with Ang-2 levels below the mean (P=0.003).

Conclusions: Our findings indicate that thrombin-induced Ang-2 expression in LVAD patients leads to increased angiogenesis in vitro and may be associated with higher nonsurgical bleeding events. Ang-2 therefore may contribute to arteriovenous malformation formation and subsequent bleeding in LVAD patients.

Keywords: LVAD; angiogenesis; angiopoietin-2; cardiac transplant; nonsurgical bleeding; thrombin.

Figure 1. Altered blood levels of angiogenic proteins in patients with LVADs

Blood levels of Ang-1, Ang-2, VEGF, and Tie-2 were measured by ELISA and patients with HF, LVAD, or OHT (n=17, 38, and 14 repsectively). (a) Ang-2 was significantly higher in LVAD patients compared with HF and OHT. (b) Soluble Tie-2 (sTie-2) was significantly higher in LVAD patients compared with OHT and increased non-significantly from patients with HF. (c) Ang-1 trended lower in LVAD patients compared with HF. (d) Plasma VEGF remained elevated in patients with LVADs compared with HF but trended lower in patients with OHT. * p<0.05, † p<0.01, ‡ p=0.055

Figure 2. Endothelial Angiopoietin-2 expression is increased in patients with LVADs

Endothelial biopsy was performed in patients with HF, LVAD, or OHT (n=10, 13, and 12 respectively) and endothelial cells were isolated by centrifugation. Cells were plated onto microscope slides and stained with fluorescent-labeled antibodies. Protein content was measured by quantitative immunofluorescence. Endothelial expression of Angiopoietin-2 (red) was significantly higher in patients with LVADs compared with HF or OHT. * p<0.05, † p<0.01

Figure 3. Elevated Angiopoietin-2 in serum from patients with LVADs induces angiogenesis in human endothelium

(a) Human umbilical vein endothelial cells were assayed on Matrigel and incubated overnight with serum from patients with HF, LVAD, or OHT (n=17, 35, and 14 respectively) in the presence or absence of an Ang-2 blocking antibody. Tubule formation was then quantified visually by an investigator blinded to sample identity. Tubule formation was significantly greater in cultures treated with serum from patients with LVADs compared with controls. Co-treatment with Ang-2 blocking antibody significantly reduced angiogenic growth in cells treated with serum from patients with LVADs but had no significant effect in cells treated with serum from HF or OHT patients. † p<0.01 (b) Among LVAD patients, tubule formation correlated strongly with serum Ang-2 concentration. R²=0.645, p<0.001

Figure 3. Elevated Angiopoietin-2 in serum from patients with LVADs induces angiogenesis in human endothelium

(a) Human umbilical vein endothelial cells were assayed on Matrigel and incubated overnight with serum from patients with HF, LVAD, or OHT (n=17, 35, and 14 respectively) in the presence or absence of an Ang-2 blocking antibody. Tubule formation was then quantified visually by an investigator blinded to sample identity. Tubule formation was significantly greater in cultures treated with serum from patients with LVADs compared with controls. Co-treatment with Ang-2 blocking antibody significantly reduced angiogenic growth in cells treated with serum from patients with LVADs but had no significant effect in cells treated with serum from HF or OHT patients. † p<0.01 (b) Among LVAD patients, tubule formation correlated strongly with serum Ang-2 concentration. R²=0.645, p<0.001

Figure 4. Elevated plasma levels of thrombin in patients with LVADs

Thrombin was measured by Western Blot in plasma from patients with HF, LVAD, or OHT (n=14, 13, and 15 respectively). (a–b) Thrombin was significantly higher in plasma from patients with LVADs compared with HF or OHT while prothrombin was unchanged. To investigate one possible source of this elevated thrombin, we measured Factor XIIa and XIa by Western Blot in plasma from these patients. (c–f) Both Factor XIIa and XIa were significantly higher in LVAD patients. Taken together, our findings suggest that activation of the contact coagulation system contributes to increased production of thrombin in LVAD patients. * p<0.05, † p<0.01, ‡ p=0.060

Figure 5. Elevated plasma levels of thrombin in patients with LVADs induce endothelial overexpression of Ang-2

Plasma samples from patients with HF, LVAD, or OHT (n=14, 13, and 15 respectively) were anticoagulated with fondaparinux to prevent artefactual thrombin generation ex vivo. Cultured HUVECs were starved overnight and then incubated with plasma from each patient in the presence or absence of Vorapaxar, a thrombin receptor blocker. Ang-2 gene expression was measured by RT-PCR. Plasma from patients with LVADs induced significantly higher expression of Ang-2 compared with HF or OHT. This effect was normalized with thrombin receptor blockade. * p<0.05, † p<0.01