Supplementary Material for: Calreticulin Regulates Neointima Formation and Collagen Deposition following Carotid Artery Ligation Zimmerman K.A. Xing D. Pallero M.A. Lu A. Ikawa M. Black L. Hoyt K.L. Kabarowski J.H. Michalak M. Murphy-Ullrich J.E. 10.6084/m9.figshare.5129380.v1 https://karger.figshare.com/articles/dataset/Supplementary_Material_for_Calreticulin_Regulates_Neointima_Formation_and_Collagen_Deposition_following_Carotid_Artery_Ligation/5129380 <b><i>Background/Aims:</i></b> The endoplasmic reticulum (ER) stress protein, calreticulin (CRT), is required for the production of TGF-β-stimulated extracellular matrix (ECM) by fibroblasts. Since TGF-β regulates vascular fibroproliferative responses and collagen deposition, we investigated the effects of CRT knockdown on vascular smooth-muscle cell (VSMC) fibroproliferative responses and collagen deposition. <b><i>Methods:</i></b> Using a carotid artery ligation model of vascular injury, Cre-recombinase-IRES-GFP plasmid was delivered with microbubbles (MB) to CRT-floxed mice using ultrasound (US) to specifically reduce CRT expression in the carotid artery. <b><i>Results:</i></b> In vitro, Cre-recombinase-mediated CRT knockdown in isolated, floxed VSMCs decreased the CRT transcript and protein, and attenuated the induction of collagen I protein in response to TGF-β. TGF-β stimulation of collagen I was partly blocked by the NFAT inhibitor 11R-VIVIT. Following carotid artery ligation, CRT staining was upregulated with enhanced expression in the neointima 14-21 days after injury. Furthermore, Cre-recombinase-IRES-GFP plasmid delivered by targeted US reduced CRT expression in the neointima of CRT-floxed mice and led to a significant reduction in neointima formation and collagen deposition. The neointimal cell number was also reduced in mice, with a local, tissue-specific knockdown of CRT. <b><i>Conclusions:</i></b> This work establishes a novel role for CRT in mediating VSMC responses to injury through the regulation of collagen deposition and neointima formation. 2016-02-25 00:00:00 Calreticulin Neointima formation Collagen