Pravastatin stimulates angiogenesis in a murine hindlimb ischemia model: a positron emission tomography imaging study with (64)Cu-NOTA-TRC105

Abstract

In this study, (64)Cu-NOTA-TRC105 (TRC105 is an anti-CD105 monoclonal antibody that binds to both human and murine CD105) positron emission tomography (PET) was used to assess the response to pravastatin treatment in a murine model of peripheral artery disease (PAD). Hindlimb ischemia was induced by ligation of the right femoral arteries in BALB/c mice under anesthesia, and the left hindlimb served as an internal control. Mice in the treatment group were given intraperitoneal pravastatin daily until the end of the study, whereas the animals in the control group were injected with 0.9% sodium chloride solution. Laser Doppler imaging showed that blood flow in the ischemic hindlimb plummeted to ~20% of the normal level after surgery, and gradually recovered to near normal level on day 10 in the treatment group and on day 20 in the control group. Angiogenesis was non-invasively monitored and quantified with (64)Cu-NOTA-TRC105 PET on postoperative days 3, 10, 17, and 24. Tracer uptake at 48 h post-injection in the ischemic hindlimb in the treatment group was significantly higher than that of the control group on day 10 (20.5 ± 1.9 %ID/g vs 11.4 ± 1.5 %ID/g), suggesting increased CD105 expression and higher level of angiogenesis upon pravastatin treatment, and gradually decreased to background levels in both groups (4.9 ± 0.8 %ID/g vs 3.4 ± 1.9 %ID/g on day 24). The in vivo PET data correlated well with ex vivo biodistribution studies performed on day 24. Increased CD105 expression on days 3 and 10 following ischemia was further confirmed by immunofluorescence staining. Taken together, our results indicated that (64)Cu-NOTA-TRC105 PET is a suitable and non-invasive method to monitor the angiogenesis and therapeutic response in PAD, which can also be utilized for non-invasive evaluation of other pro-angiogenic/anti-angiogenic drugs in other cardiovascular diseases and cancer.

Keywords: Angiogenesis; CD105 (endoglin); ischemia; peripheral artery disease (PAD); positron emission tomography (PET); pravastatin.

Figure 1

A: The change of blood perfusion in the ischemic hindlimb was documented by serial laser Doppler imaging. A steep decrease in blood flow was observed soon after surgical ligation of the femoral artery followed by a gradual recovery. Treatment: mice were injected with pravastatin daily. Control: mice were injected with 0.9% sodium chloride solution daily. B: Quantitative data based on laser Doppler imaging. Blood flow in the ischemic hindlimb is expressed as percentage of the blood flow in the control hindlimb (n = 3). Blood flow recovered to near-normal levels faster in pravastatin treated mice than in the control group. *: P < 0.05.

Figure 2

A: Representative coronal PET images at 4, 24, and 48 h post-injection of ⁶⁴Cu-NOTA-TRC105 on days 3, 10, 17, and 24 after surgical induction of hindlimb ischemia (white arrows) and daily pravastatin treatment. B: %ID/g values of ⁶⁴Cu-NOTA-TRC105 uptake in the ischemic hindlimb at 4, 24, and 48 h post-injection on days 3, 10, 17, and 24 after surgery, with (n = 4) or without (n = 3) daily pravastatin treatment. C: In the pravastatin treated mice, the differences between ⁶⁴Cu-NOTA-TRC105 uptake in the ischemic and non-ischemic hindlimb were statistically significant at all time points examined. n = 4. *: P < 0.05.

Figure 3

Biodistribution of ⁶⁴Cu-NOTA-TRC105 in major organs at 48 h post-injection on postoperative day 24 in pravastatin treated mice.

Figure 4

Immunofluorescence staining showed increased CD105 expression in the ischemic hindlimb on postoperative days 3 and 10 after daily pravastatin treatment, significantly higher than that on days 17 and 24, as well as the control non-ischemic hindlimb. Green: CD105; red: CD31; blue: DAPI. Scale bar: 100 μm.