Date of Award

12-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Advisor

LaBerge, Martine

Committee Member

Argraves, W. Scott

Committee Member

LaRue, Amanda

Committee Member

Simionescu, Dan

Committee Member

Yao, Hai

Abstract

Background: Particularly important to the mechanical performance of native arterial blood vessels is elastin, an extracellular matrix (ECM) protein deposited by VSMCs in the form of elastic fibers, arranged in concentric lamellae in the media of the vessel wall. In addition to serving as major structural elements of arterial walls, providing extensibility and elastic recoil, elastic fibers also influence vascular cell behaviors. For these reasons tissue engineers are attempting to exploit elastic fiber biology to enhance vascular graft design and patency. Therefore, developing a greater understanding of the molecular mechanisms of elastogenesis may offer opportunities to control elastogenesis in tissue biofabrication.

Approach: To discover genes critical for elastogenesis we performed analysis of gene expression profiles associated with elastogenesis occurring 1) during lung and aorta development, 2) in the lung and skin in response to injury, and 3) in vascular smooth muscle cells (VSMCs) stimulated to produce elastic fibers. On the resulting convergent gene set we employed Promoter Analysis and Interaction Network Toolset (PAINT) to identify transcription factor binding regions. We also mapped binding sites for microRNA (miRNA) within the convergent gene set. Subsequent screening for potential regulators of elastogenesis were performed using pharmacological agonists and antagonists along with plasmid vector transfection to augment expression. Differences in elastin transcription were measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and by anti-elastin immunostaining in the development of a novel elastin ELISA. A 3D proof of concept tissue culture model containing fibroblasts and macroporous gelatin microcarrier beads was also established and immunostained for elastin.

Results: Our transcriptomic studies revealed a set of genes differentially regulated in all five models of elastogenesis tested. Aside from genes that have previously been established to act in the elastogenesis process there are >50 genes that have not been implicated in elastogenesis. Moreover, promoter analysis of clusters of genes from the 63-gene set having a similar pattern of regulation during developmental elastogenesis revealed two potential elastogenesis regulatory network of TFs. We hypothesize that these sets of genes contain novel positive and negative effectors of elastogenesis. Effects of agonists, antagonists, and expression vectors of these genes on elastin expression were quantified in cultured fibroblasts to identify agents that can be employed to accelerate elastogenesis during tissue biofabrication.

Conclusions: The findings highlight a group of genes whose expression is differentially expressed in multiple models of elastin formation and many not previously associated with elastogenesis and thus may represent novel components of elastogenesis. Transcriptional regulatory network analysis revealed potential transcription factor regulators of elastogenesis. Candidate genes and transcription factors were regulated through agonist and antagonist treatment and transfection of plasmid expression vectors in order to augment elastogenesis in vascular tissue biofabrication.

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