Increasing HDL levels by inhibiting cholesteryl ester transfer protein activity in rabbits with hindlimb ischemia is associated with increased angiogenesis

Abstract

Background

High density lipoprotein (HDL) infusions increase new blood vessel formation (angiogenesis) in rodents with ischemic injury. This study asks if increasing HDL levels by inhibiting cholesteryl ester transfer protein (CETP) activity increases angiogenesis in New Zealand White (NZW) rabbits with hindlimb ischemia.

Methods and results

NZW rabbits were maintained for 6 weeks on chow or chow supplemented with 0.07% or 0.14% (wt/wt) of the CETP inhibitor, des-fluoro-anacetrapib. The left femoral artery was ligated after 2 weeks of des-fluoro-anacetrapib treatment. The animals were sacrificed 4 weeks after femoral artery ligation. Treatment with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib reduced CETP activity by 63 ± 12% and 81 ± 8.6%, increased plasma apoA-I levels by 1.3 ± 0.1- and 1.4 ± 0.1-fold, and increased plasma HDL-cholesterol levels by 1.4 ± 0.1- and 1.7 ± 0.2-fold, respectively. Treatment with 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib increased the number of collateral arteries by 60 ± 16% and 84 ± 27%, and arteriole wall area in the ischemic hindlimbs by 84 ± 16% and 94 ± 13%, respectively. Capillary density in the ischemic hindlimb adductor muscle increased from 1.1 ± 0.2 (control) to 2.1 ± 0.3 and 2.2 ± 0.4 in the 0.07% and 0.14% (wt/wt) des-fluoro-anacetrapib-treated animals, respectively. Incubation of HDLs from des-fluoro-anacetrapib-treated animals with human coronary artery endothelial cells at apoA-I concentrations comparable with their plasma levels increased tubule network formation. These effects were abolished by knockdown of scavenger receptor-B1 (SR-B1) and PDZK1, and pharmacological inhibition of PI3K/Akt.

Conclusion

Increasing HDL levels by inhibiting CETP activity is associated with increased collateral blood vessel formation in NZW rabbits with hindlimb ischemia in an SR-B1- and PI3K/Akt-dependent manner.

Introduction

Plasma high density lipoproteins (HDLs) have several well-documented cardioprotective functions, including an ability to reduce ischemia–reperfusion injury. Increasing HDL levels in rodents with intravenous infusions of HDLs isolated from human plasma, or reconstituted HDLs containing apolipoprotein (apo) A-I complexed with egg yolk phosphatidylcholine, (A-I)rHDLs, reduce cardiac ischemia reperfusion injury by preserving mitochondrial integrity, by decreasing tumour necrosis factor-α levels and by increasing prostaglandin release [1], [2]. HDL infusions also inhibit neutrophil recruitment and reduce cardiomyocyte apoptosis in areas of ischemic damage in mice in a nitric oxide-dependent manner, possibly by a mechanism that involves the delivery of sphingosine-1-phosphate from the HDLs to the areas of damage [3], [4].

Increasing plasma HDL levels with twice weekly infusions of (A-I)rHDLs has also been shown to enhance angiogenesis and improve blood flow by increasing capillary density and the recruitment of endothelial progenitor cells to areas of damage in a mouse model of hindlimb ischemia [5]. These events were dependent on nitric oxide and activation of the phosphatidyl inositol 3-kinase (PI3K)/Akt signalling pathway [5].

While the beneficial effects of intravenous HDL and rHDL infusions on angiogenesis under ischemic conditions are well established, it is not known if ischemic damage is also attenuated when HDL levels are increased by inhibiting the activity of cholesteryl ester transfer protein (CETP). Although the use of CETP inhibitors to increase plasma HDL levels has been questioned following the recent failure of two human clinical cardiovascular outcome trials with the CETP inhibitors torcetrapib and dalcetrapib, evidence has emerged that the failure of torcetrapib in the ILLUMINATE trial may have been due to off-target effects unrelated to CETP inhibition [6], and that the failure of dalcetrapib in the dal-OUTCOMES trial may have been a consequence of the study being conducted in people with dysfunctional HDL [7]. In spite of these adverse outcomes, the ability of CETP inhibitors to increase HDL levels and reduce cardiovascular risk is still actively under investigation in two large clinical outcome trials using anacetrapib and evacetrapib, which do not have the off-target effects of torcetrapib, to inhibit activity of CETP. These trials are being conducted in people who are either at increased risk of developing coronary heart disease, or in people with established coronary heart disease [8], [9].

In the present study new blood vessel formation was assessed in New Zealand White (NZW) rabbits with hindlimb ischemia in which plasma HDL levels were increased by treatment with the CETP inhibitor, des-fluoro-anacetrapib. This inhibitor is structurally analogous to anacetrapib but contains 10 fluorine molecules, compared with 9 fluorine molecules for anacetrapib. The results establish that the des-fluoro-anacetrapib-mediated increase in plasma HDL levels in NZW rabbits is accompanied by enhanced collateral blood vessel formation and capillary density in ischemic hindlimbs and that these effects are mediated by a mechanism that is dependent on scavenger receptor Class B Type 1 (SR-B1), the adaptor PDZ domain-containing protein 1 (PDZ1) and activation of the PI3K/Akt signal transduction pathway.