Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


36743.pdf (7.53 MB)

ETD Abstract Container

Abstract Header

Novel in vitro and in vivo Methods to Study the Cardiac Fibroblast

Fischesser, Demetria M
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595845515089609

Abstract Details

2020, PhD, University of Cincinnati, Medicine: Molecular Genetics, Biochemistry, and Microbiology.
Cardiac fibroblasts (CFs) are essential to normal heart function through maintenance of extracellular matrix (ECM). Following injury, CFs can undergo acute activation to increase ECM production and support the injured heart, but chronic activation can cause excess ECM deposition resulting in fibrosis. Although the extent of CF activation determines whether a supportive scar or detrimental fibrosis will ensue, the nuances of CF activation and differentiation are understudied. CFs differentiate into myofibroblasts via two mechanisms: biochemical cues, or mechanical cues such as increased environmental rigidity. Gene expression is a widely-used means of documenting CF activation. Tissue resident CFs express genes such as transcription factor-21 (Tcf21). Upon activation, they express genes including Acta2 which encodes smooth muscle a-actin (aSMA) protein. While these expression profiles have been verified in vivo, other presumed CF characteristics are derived from in vitro studies, often conducted on plastic tissue culture plates (TCPs). As TCPs are 6 orders of magnitude stiffer than cardiac tissue, mechanosensitive CFs cultured on TCPs may display non-physiological biology. Therefore, in vitro studies utilizing TCPs may present non-physiologically relevant results. Though studies utilizing TCPs may be flawed, in vitro studies are important for understanding CF biology as they provide a controlled yet easily manipulated environment. To circumvent the issues of TCP stiffness and provide an environment of physiological stiffness, we generated two novel gelatin-based hydrogel cell culture systems containing either HPMA or NIPAAm crosslinking polymers. Both can be made to mimic the stiffness of a healthy or fibrotic heart and later softened through cysteine treatment, which breaks disulfide bonds between polymers. Both hydrogel systems were biocompatible and demonstrated stiffness-dependent CF activation, measured by cell area and aSMA expression. Notably, specific cell morphologies existed on distinct hydrogel stiffnesses. Interestingly, it was found that upon softening of the stiff hydrogels, cell area decreased, and cell morphologies reverted to those found on softer hydrogels. This suggests that environmental softening during cell culture induces CF dedifferentiation, and therefore potentially manipulates the fibrotic response. To confirm that the morphologies observed on hydrogels were physiologically relevant, we developed a novel tissue clearing technique to study 3D CFs in their native in vivo environment. Optically cleared uninjured and injured hearts from mice with a Tcf21-driven fluorescent fibroblast reporter showed that in vivo 3D CF biology is recapitulated in morphologies seen in the in vitro hydrogel systems, strengthening our findings that studies must move beyond TCPs in order to conduct physiologically relevant studies of CFs. Over the course of these studies, we have developed two novel in vitro hydrogel methods that provide new platforms to study CF differentiation and dedifferentiation. We also developed a novel tissue clearing technique to uncover previously unknown details of the in vivo CF and confirm the physiological relevance of findings made using the in vitro hydrogel system. These technologies and resultant findings about the nature of CF differentiation and dedifferentiation introduce new avenues for manipulation and treatment of cardiac fibrosis, and move the field towards more a physiologically relevant understanding of the ever-important CF.
Jeff Molkentin, Ph.D. (Committee Chair)
William Miller, Ph.D. (Committee Member)
David Wieczorek, Ph.D. (Committee Member)
Katherine Yutzey, Ph.D. (Committee Member)
174 p.
Cellular Biology
Cardiac Fibroblast; Differentiation; Hydrogel; Tissue Clearing; Mechanotension; Fibrosis

Recommended Citations

Citations

  • Fischesser, D. M. (2020). Novel in vitro and in vivo Methods to Study the Cardiac Fibroblast [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595845515089609

    APA Style (7th edition)

  • Fischesser, Demetria. Novel in vitro and in vivo Methods to Study the Cardiac Fibroblast. 2020. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595845515089609.

    MLA Style (8th edition)

  • Fischesser, Demetria. "Novel in vitro and in vivo Methods to Study the Cardiac Fibroblast." Doctoral dissertation, University of Cincinnati, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1595845515089609

    Chicago Manual of Style (17th edition)

ucin1595845515089609
247
© 2020, all rights reserved.
This open access ETD is published by University of Cincinnati and OhioLINK.
Release 3.2.12