Thrombin promotes arteriogenesis and hemodynamic recovery in a rabbit hindlimb ischemia model

Abstract

Compared with angiogenesis, arteriogenesis is a distinct process based on the remodeling and maturation of pre-existing arterioles into large conductance arteries. Therapeutic angiogenesis has been proposed as a potential treatment for ischemic atherosclerotic diseases. Since a variety of angiogenic factors have been tested with inconsistent so far clinical results, the challenge remains in identifying the factor(s) that will stimulate functional neovascularization. Thrombin has been reported to play a pivotal role in the initiation of angiogenesis by regulating and organizing a network of angiogenic mediators. Also, it was recently demonstrated that thrombin is a potent anti-apoptotic factor for endothelial cells, providing evidence on a potential role of thrombin in vascular protection and maintenance of vessel integrity. Based on these observations, we hypothesized that thrombin may promote the development of mature functional blood vessels. Seventy-four (n=74) rabbits underwent bilateral femoral artery surgical excision. On the 20th postsurgical day increasing doses of VEGF or bFGF or thrombin were injected in one ischemic limb per rabbit and an equal volume of normal saline to the contralateral control limbs. Quantification of newly developed collateral vessels (diameter >500 mum) was performed by transauricular intra-arterial subtraction angiography. Computerized quantitative analysis of collateral vessels in angiography images was based on the concept of multiscale structural tensor. Perfusion analysis of an in vivo dynamic computed tomography study was performed to investigate hemodynamic recovery of the distal ischemic limbs. Tissue perfusion analysis was performed with the semiquantitative slope methodology, which focuses on the first-pass arterial phase. A single administration of thrombin exhibited a dose-dependent increase of arteriogenic outcome. Thrombin at 5000 IU induced a 30.2 +/- 7.4% (P < 0.05) increase of total collateral area and length. Both VEGF and bFGF were without any significant effect at the concentrations used. Functional estimation of limb perfusion showed a statistically significant increase of blood flow recovery only for thrombin. The semiquantitative slope method perfusion score differed significantly in the 5000 IU thrombin treated limbs (5.7 +/- 0.3 vs 5.0 +/- 0.3 in control ischemic limbs; P < .05), and was not significantly inferior from the score of normal nonoperated limbs (6.5 +/- 0.3) suggesting a trend towards hemodynamic recovery of distal limb perfusion. In a rabbit hindlimb ischemia model, thrombin promoted the formation of large collateral vessels and improved the perfusion of distal ischemic tissue. These results provide new insights in understanding the involvement of thrombin in vascular formation and point to a novel role of thrombin in arteriogenesis.