Adenovirus-mediated gene transfer of Smad3 results in adaptive remodeling following arterial balloon injury

Abstract

Abstract Introduction: Transforming growth factor-beta1 (TGF-b1) has been implicated in injury induced arterial remodeling. Previously, we found that over-expression of Smad3 (a TGF-b1 signaling protein) alters smooth muscle cell behavior in vitro. Here, we explore the role of Smad3 in arterial injury. Methods: AdNull or Smad3 expressing adenoviruses (AdSmad3) were created. Forty-four rats underwent carotid balloon injury receiving either no virus, AdNull or AdSmad3. Immunohistochemistry (IHC) was performed with anti-Smad3, PCNA (proliferating cell nuclear antigen) and alpha-actin antibodies. Results: We first determined the temporal expression of Smad3 following arterial injury. Using IHC, we found Smad3 levels significantly increased at 7 and 14 days following injury, a time frame associated with arterial remodeling (Table1A). We next explored whether AdSmad3 might affect the injury response. Intra-arterial delivery of AdSmad3 (2.5 × 109pfu) resulted in high levels of Smad3 expression in the media at 3 and 7 days post-injury. Despite increased neointimal thickening, AdSmad3 infected vessels showed an increase in lumen area, consistent with adaptive remodeling (Table1B). To understand the mechanism of Smad3-induced remodeling, we investigated the effect of AdSmad3 on cell proliferation and collagen deposition, events known to contribute to remodeling. AdSmad3 increased cell proliferation and collagen deposition in all vessel layers, however, these effect were most prominent in the adventitia. Moreover, AdSmad3 induced a phenotypic shift in adventitial fibroblasts, reflected by an increase in alpha-actin positive cells. Table 1A . Temporal expression of Smad3 following arterial injury (n = 4/group) Un-injured 3 days post-injury 7 days post-injury 14 days post-injury 28 days post-injury Smad3 Expression (% arterial cells expressing Smad3) 5.8 7.2 29.9 49.1 10.1 ANOVA: p Table 1B . Morphometrics; 14 days post-injury, indexed to no virus control (n = 4/group) Vessel size Adventitia size Adventitia collagen content External elastic lamina length Media area Neointima area Lumen area No virus 1 1 1 1 1 1 1 AdNull 1.02 1.2 1.15 1.07 1.81 1.16 1.07 AdSmad3 1.27 1.61 1.7 1.27 2.09 1.52 1.22 p Value (AdSmad 3 versus AdNull) NS Conclusions: Despite reversing TGF-b’s inhibitory effect on proliferation, Smad3 gene transfer leads to vessel and lumen enlargement implicating Smad3 as a mediator of adaptive arterial remodeling.