All ETDs from UAB

Advisory Committee Chair

Joanne Murphy-Ullrich

Advisory Committee Members

Rosa Serra

Elizabeth Sztul

Janusz Kabarowski

Document Type

Dissertation

Date of Award

2014

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Calreticulin (CRT) is an ER chaperone and regulator of Ca2+ signaling which is increased following ER stress and in fibrotic and vascular fibroproliferative disorders. Previously, we demonstrated that ER CRT regulates type I collagen transcript, trafficking, secretion, and processing into the extracellular matrix (ECM). These studies investigated the role of CRT in regulating ECM production through control TGF-ß dependent signaling pathways. Our studies show that CRT -/- mouse embryonic fibroblasts (MEFs), rat lung fibroblasts, and human IPF lung fibroblasts with siRNA knockdown of CRT had impaired production of type I collagen and fibronectin when stimulated with TGF-ß. Similarly, knockdown of CRT in CRT floxed vascular smooth muscle cells (VSMCs) with Cre-recombinase-IRES-GFP significantly impaired TGF-ß stimulated type I collagen production. We showed that CRT regulates TGF-ß stimulated ECM production through control of ER Ca2+ release and downstream calcineurin dependent NFAT signaling as pretreatment of MEFs or VSMCs with 11R-VIVIT significantly attenuated TGF-ß stimulated ECM production. Artificial induction of ER stress by tunicamycin could not rescue the inability of CRT -/- MEFs to induce ECM production upon TGF-ß treatment, suggesting that CRT may be a critical component of ER stress induced fibrotic and vascular fibroproliferative disease. To address the role of CRT in vascular fibroproliferative disease, we used a carotid artery ligation model of neointimal hyperplasia in CRT floxed mice. We showed that CRT is strongly upregulated following carotid artery ligation induced vascular injury and that we were able to knockdown CRT to 50% of control levels using ultrasound (US) mediated delivery of Cre-recombinase-IRES-GFP plasmid with microbubbles (MB). Furthermore, knockdown of CRT significantly reduced neointimal area and the neointima-to-media ratio as compared to mice receiving GFP with US/MB or mice receiving Cre-recombinase-IRES-GFP with MB but lacking US. Cre-recombinase-IRES-GFP mediated knockdown of neointimal CRT reduced collagen production by approximately 25%. Together, these studies establish that CRT is a critical component of ER stress induced fibrotic and vascular fibroproliferative disease. In vitro studies show that CRT regulates TGF-ß stimulated ECM production through control of ER Ca2+ release and downstream calcineurin/NFAT signaling.

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