Date of Award
Doctor of Philosophy (PhD)
LaBerge , Martine
Toole , Bryan
Vyavahare , Naren
Abdominal aortic aneurysms (AAAs) are typically fusiform (symmetric) dilations of the aortic wall most commonly arising below the renal arteries. The progression is typically associated with an activated smooth muscle cell (SMC) phenotype, diminished density of mature medial elastic fibers, and an elevated presence of matrix-degrading enzymes (e.g., matrix-metalloproteases; MMPs), which may ultimately lead to vessel rupture. Currently, no surgical or non-surgical methods are available to regress AAAs via regeneration of new elastin matrices to regain normal vessel contour, particularly due to the inherently poor elastin synthesis by adult vascular cells and absence of methods to stimulate the same. Previously, the lab showed that extracellular matrix-derived factors based on hyaluronan oligomers (HA-o) and growth factors (TGF-β1) stimulate elastin regeneration by healthy vascular cells. Yet, the utility of these factors for matrix synthesis by AAA-derived SMCs has not been determined. Thus, the objective of this study was to assess the efficacy of these factors on AAA-derived SMCs (peri-adventitial CaCl2 (aRASMCs) and intralumenal elastase-perfusion AAA models (EaRASMCs)) and further compare their responses to human AAA SMCs (aHASMCs). In addition, we assessed how the pre-existing elastic matrix content/quality influenced elastogenic inductability of SMCs. Finally, we evaluated a mode of factor delivery in vitro using a factor-loaded Extracel-HPTM hydrogel coating on AeosTM ePTFE grafts.
We generated rat abdominal aortic expansions (~45% and 120% increases in diameter for CaCl2 exposure and elastase perfusion, respectively) that had characteristics typical of human AAAs, such as medial elastic matrix disruption, medial thinning, and calcification (CaCl2 only) as identified histologically and with scanning electron microscopy, and increased elastolytic MMP activity as quantified by zymography. Primary cells derived from these expansions had similarities to human AAA-derived SMCs in terms of decreased contractile activity (decreased α-actin, SM-22, caldesmon, and calponin expression) enhanced proliferation (2.5-fold and 2.8-fold greater aRASMC and EaRASMC counts), and reduced elastogenic capacity relative to healthy RASMCs. Also, there was significant differential gene expression of EaRASMC relative to RASMCs, supporting our hypothesis that the AAA-derived cells maintain altered gene expression in vitro. Concurrent delivery of HA-o and TGF-β within our tested doses (0, 2, 20 µg/ml HA-o and 0, 1, 5, 10 ng/ml TGF-β yielded a decrease in aRASMC and EaRASMC proliferation and an increase in their tropoelastin (1.5-fold for aRASMC and 1.9-fold for EaRASMC) and matrix elastin production (2.2-fold for aRASMC and 1.5-fold for EaRASMC) relative to their respective aneurysmal controls. Factors attenuated elastolytic MMP-2 activity in aRASMCs; interestingly, no significant decrease was seen in aHASMC cultures, yet an overall decrease in MMP and TIMP (tissue inhibitors of MMPs) expression was seen with factor supplementation. In support of these findings, differential gene expression (e.g., MMP-12, TIMP-3) with supplementation of HA-o and TGF-β was observed, yet further assessment of functional gene groups is necessary. Overall, we believe the elastase perfusion-injury model of AAAs may be suitable as a surrogate in the context of elastin regeneration within advanced human AAAs, yet a shortcoming is the unparalleled increases in elastin production by aHASMCs in response to factor supplementation (5-fold increase in tropoelastin, 8.5-fold increase in matrix elastin relative to aHASMC controls).
Observations from our in vitro elastase-injury studies illustrated the ability of cell layers to self-repair and regenerate elastic matrices following their proteolytic damage is limited, particularly when elastic matrix injury is severe. Our results also provide evidence that HA-o and TGF-β factors together can elastogenically stimulate RASMCs in elastin-degraded cultures to restore both elastic matrix amounts and elastic fiber deposition to levels of accumulation observed in healthy cultures. Doses of the elastogenic factors must be enhanced and optimized based on the severity of elastic matrix damage. In our preliminary in vitro assessment of factor-loaded Extracel-HPTM hydrogel coatings on AeosTM ePTFE grafts we achieved sustained TGF-β delivery over weeks, which is promising. In future work, our delivery approach could be applied to endovascular delivery in vivo at the site of AAA for elastin regeneration, elimination of endoleak, inhibition of AAA growth, and possibly even AAA regression.
Gacchina, Carmen, "CELL-MEDIATED ELASTIC MATRIX REGENERATION TOWARD REGRESSION OF ABDOMINAL AORTIC ANEURYSMS" (2010). All Dissertations. 569.