Inhibition of transforming growth factor-β restores endothelial thromboresistance in vein grafts

Background: Thrombosis is a major cause of the early failure of vein grafts (VGs) implanted during peripheral and coronary arterial bypass surgeries. Endothelial expression of thrombomodulin (TM), a key constituent of the protein C anticoagulant pathway, is markedly suppressed in VGs after implantation and contributes to local thrombus formation. While stretch-induced paracrine release of transforming growth factor-beta (TGF-beta) is known to negatively regulate TM expression in heart tissue, its role in regulating TM expression in VGs remains unknown. Methods: Changes in relative rnRNA expression of major TGF-beta isoforms were measured by quantitative polymerase chain reaction (qPCR) in cultured human saphenous vein smooth muscle cells (HSVSMCs) subjected to cyclic stretch. To determine the effects of paracrine release of TGF-beta on endothelial TM mRNA expression, human saphenous vein endothelial cells (HSVECs) were co-cultured with stretched HSVSMCs in the presence of 1D11, a pan-neutralizing TGF-beta antibody, or 13C4, an isotype-control antibody. Groups of rabbits were then administered 1D11 or 13C4 and underwent interpositional grafting of jugular vein segments into the carotid circulation. The effect of TGF-beta inhibition on TM gene expression was measured by qPCR; protein C activating capacity and local thrombus formation were measured by in situ chromogenic substrate assays; and VG remodeling was assessed by digital morphometry. Results: Cyclic stretch induced TGF-beta(1) expression in HSVSMCs by 1.9 +/- 0.2-fold (P < .001) without significant change in the expressions of TGF-beta(2) and TGF-beta(3). Paracrine release of TGF-beta(1) by stretched HSVSMCs inhibited TM expression in stationary HSVECs placed in co-culture by 57 +/- 12% (P = .03), an effect that was abolished in the presence of 1D11. Similarly, TGF-beta(1) was the predominant isoform induced in rabbit VGs 7 days after implantation (3.5 +/- 0.4-fold induction; P < .001). TGF-beta(1) protein expression localized predominantly to the developing neointima and coincided with marked suppression of endothelial TM expression (16% +/- 2% of vein controls; P < .03), a reduction in situ activated protein C (APC)-generating capacity (53% +/- 9% of vein controls; P = .001) and increased local thrombus formation (3.7 +/- 0.8-fold increase over vein controls; P < .01). External stenting of VGs to limit vessel distension significantly reduced TGF-beta(1) induction and TM downregulation. Systemic administration of 1D11 also effectively prevented TM downregulation, preserved APC-generating capacity, and reduced local thrombus in rabbit VGs without observable effect on neointima formation and other morphometric parameters 6 weeks after implantation. Conclusion: TM downregulation in VGs is mediated by paracrine release of TGF-beta(1) caused by pressure-induced vessel stretch. Systemic administration of an anti-TGF-beta antibody effectively prevented TM downregulation and preserved local thromboresistance without negative effect on VG remodeling. (J Vasc Surg 2011;54:1117-23.) Clinical Relevance: Vein grafts (VGs) are commonly used conduits for coronary and peripheral arterial bypass surgeries. Thrombosis is a major cause of early VG failure. Trombomodulin (TM), a key component of the anticoagulant protein C pathway, is downregulated early after VG implantation and facilitates local thrombus formation. We found that paracrine release of transforming growth factor-beta(1) (TGF-beta(1)), caused by pressure-induced stretch, was a potent negative regulator of TM in rabbit VGs. Administration of a neutralizing anti-TGF-beta antibody effectively prevented TM downregulation and reduced local thrombus generation without adversely affecting long-term VG remodeling. This may represent a novel strategy to improve patency in patients undergoing arterial bypass procedures.