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F‐actin Assembly And Cytoskeletal Contraction Mediate TNF‐α Induced Hydrogel Retraction, Stiffness, And Nodule Formation By Murine Valvular Cells
Author(s) -
Lim Jina,
Ehsanipour Arshia,
Lu Jinxiu,
Wu Alexander,
Pedego Taylor,
Walthers Chris,
Demer Linda,
Seidlits Stephanie,
Tintut Yin
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.lb457
Subject(s) - chemistry , tumor necrosis factor alpha , microbiology and biotechnology , cytochalasin d , calcification , myosin , actin , cytokine , cytoskeleton , pathology , medicine , immunology , biology , biochemistry , cell
In calcific aortic valve disease (CAVD), a widespread and rapidly fatal disorder, the valve cusps undergo retraction, stiffening, and nodular calcification. Therapeutic options are limited to invasive procedures. The pro‐inflammatory cytokine, tumor necrosis factor‐alpha (TNF‐α), has been shown to contribute to CAVD progression; however, the mechanisms of its actions on cusp retraction and stiffening are not clear. We investigated the effects of TNF‐α on murine aortic valvular interstitial cells (VIC) within a 3‐dimensional, free‐floating, compliant collagen hydrogel to simulate their natural substrate. Results showed that TNF‐α increased retraction (percent diameter), stiffness (Pascal), and formation of calcified nodular structures (Alizarin Red positivity) in the VIC‐laden hydrogels. Effects of TNF‐α on retraction, stiffness, and nodule formation were attenuated by inhibition of myosin II‐mediated cytoskeletal contraction with blebbistatin. Inhibition of actin polymerization with cytochalasin D, but not inhibition of Rho‐kinase with Y27632, blocked TNF‐α‐induced retraction in 3‐D VIC hydrogels, suggesting that actin stress fibers mediate TNF‐α‐induced effects. In the hydrogels, TNF‐α increased levels of phospho‐IκB, and co‐treatment with an NF‐κB inhibitor blocked TNF‐α‐induced retraction and stiffening, suggesting that NF‐kB signaling mediates the effects of TNF‐α. TNF‐α also significantly increased collagen deposition, as visualized by Masson's trichrome staining, and it upregulated expression of discoidin domain‐containing receptor 2, fibronectin, and smooth muscle alpha‐actin, as evidenced by realtime RT‐qPCR. In human aortic valves, we found that stiffness and collagen deposition were greater in calcified than non‐diseased cusps. In conclusion, inflammation may induce valvular cusp retraction, stiffening, and nodule formation by promoting F‐actin assembly and cytoskeletal contraction. Support or Funding Information This work was supported by funding from the National Institutes of Health (DK081346‐S1, HL114709, HL121019). J.L was supported by UCLA Children's Discovery and Innovation Institute Harry Winston Fellowship Award. S.S. and A.E. were supported by the University of California, Los Angeles Henry Samueli School of Engineering and Applied Sciences.