AAV2/8-hSMAD3 gene delivery attenuates aortic atherogenesis, enhances Th2 response without fibrosis, in LDLR-KO mice on high cholesterol diet (Journal of Translational Medicine)

Background: Inflammation is a key etiologic component in atherogenesis and transforming growth factor beta 1 (TGF?1) is a well known anti-inflammatory cytokine which potentially might be used to limit it. Yet TGF?1 is pleiomorphic, causing fibrosis, cell taxis, and under certain circumstances, can even worsen inflammation. SMAD3 is an important member of TGF?1?s signal transduction pathway, but is a fully intracellular protein.ObjectivesWith the hope of attenuating TGF?1?s adverse systemic effects (eg. fibrosis) and accentuating its anti-inflammatory activity, we proposed the use of human (h)SMAD3 as an intracellular substitute for TGF?1.Study designTo test this hypothesis adeno-associated virus type 2/8 (AAV)/hSMAD3 or AAV/Neo (control) was tail vein injected into the low density lipoprotein receptor knockout (LDLR-KO) mice, then placed on a high-cholesterol diet (HCD). Results: The hSMAD3 delivery was associated with significantly lower atherogenesis as measured by larger aortic cross sectional area, thinner aortic wall thickness, and lower aortic systolic blood velocity compared with Neo gene-treated controls. HSMAD3 delivery also resulted in fewer aortic macrophages by immunohistochemistry for CD68 and ITGAM, and quantitative reverse transcriptase polymerase chain reaction analysis of EMR and ITGAM. Overall, aortic cytokine expression showed an enhancement of Th2 response (higher IL-4 and IL-10); while Th1 response (IL-12) was lower with hSMAD3 delivery. While TGF?1 is often associated with increased fibrosis, AAV/hSMAD3 delivery exhibited no increase of collagen 1A2 or significantly lower 2A1 expression in the aorta compared with Neo-delivery. Connective tissue growth factor (CTGF), a mediator of TGF?1/SMAD3-induced fibrosis, was unchanged in hSMAD3-delivered aortas. In the liver, all three of these genes were down-regulated by hSMAD3 gene delivery. Conclusion: These data strongly suggest that AAV/hSMAD3 delivery gave anti-atherosclerosis therapeutic effect without the expected undesirable effect of TGF?1-associated fibrosis.