Fibroblasts: Key Cells in Inflammation and Fibrosis
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Angiotensin II (Ang II) infusion into normolipidimic mice leads to the development of aortic dissections and abdominal aortic aneurysm (AAA). IL-6 is a pro-inflammatory cytokine that is secreted by fibroblasts and macrophages in the aortic adventitia and promotes the development of aortic dissections. In our previous work, analysis of aortic adventitial fibroblast secretions in vitro suggested that aortic fibroblasts were the main producers IL-6. Furthermore, our lab has demonstrated that Ang II activates the transcription factor nuclear factor- kappa B/ RelA, which binds to the IL-6 promoter and promotes transcription. To investigate the role of fibroblast-RelA in Ang II mediated AAA formation, I aimed to generate fibroblast-specific RelA-deficient mice using Cre-Lox technology. Mice containing Cre recombinase under the transcriptional control of collagen 1a2 promoter fused with a modified estrogen receptor, which could be temporally activated with tamoxifen (Col1a2-CreERT), undergo targeted genetic recombination in aortic fibroblasts. Using mT/mG Cre-reporter mice harboring Col1a2-CreERT, I provide evidence that Col1a2-CreERT is activated by tamoxifen in both adventitial fibroblasts and in vascular smooth muscle cells (VSMCs). Interestingly, I observed that deletion of RelA from aortic VSMCs and fibroblasts, using Col1a2-CreERT mice containing RelA-flox alleles, protects from Ang II mediated AAA formation. Furthermore, aortic-RelA deficient animals have decrease production of pro-inflammatory cytokines IL-6 and IL-1 beta, and decrease recruitment of CD11b+F4/80loLy6Chi inflammatory monocytes into the aortic wall during Ang II infusion. Ang II is also a potent inducer of TGFb, a cytokine that promotes fibroblast to myofibroblast transdifferentiation. We hypothesized that TGFb induces myofibroblast transdifferentiation via activation of fibroblast-RelA, an event that may precede the development of vascular inflammation and AAA formation. Using primary human fibroblasts, I demonstrate that TGFb is a potent inducer of Smad2/3 signaling but not of RelA signaling in fibroblasts. Furthermore, RelA is dispensable for myofibroblast transformation. In contrast, Smad3 promotes the transcription of myofibroblast genes including Nox4, SM22a, collagen 1a1 and fibronectin. Investigation of transcriptional co-activators that promote phosphorylation of RNA polymerase II for transcription elongation of target genes suggests that both BRD4 (bromodomain and ET domain containing protein 4) and CDK9 (cyclin dependent kinase 9) are needed for myofibroblast transformation. I further identify a small molecule inhibitor of BRD4, JQ1, which can not only prevent but also reverse the myofibroblast phenotype in cells taken from hypertrophic scars of burn patients. In conclusion, my work suggests that fibroblast-RelA is not necessary for myofibroblast transdifferentiation but it plays pivotal role in Ang II mediated vascular inflammation and AAA formation.