Date of Award
Doctor of Philosophy (PhD)
Vyavahare , Naren
Nagatomi , Jiro
Elastin, a primary component of elastic arteries, maintains structural stability of the cyclically recoiling artery, and critically regulates vascular cell behavior. Accelerated degradation of elastic matrix, such as that seen in vascular pathologies like abdominal aortic aneurysms (AAA), can therefore severely compromise vessel homeostasis. Tissue engineering and in-situ matrix repair strategies evaluated so far are primarily limited in inducing adult vascular cells to replicate the complex elastic matrix assembly process, and restore lost matrix integrity. Previously, our lab established the elastogenic benefits of concurrent delivery of TGF-β1 and HA-oligomers (together termed elastogenic factors, EFs), within 2D cultures of rat aortic smooth muscle cells (SMCs). Since SMCs are known to switch to a synthetic, highly matrix producing phenotype, in a manner that cannot be replicated in vivo, we sought to develop a relevant 3D in vitro model system, where the benefits of EFs can be replicated. We chose a 3D collagen-gel model since the presence of a collagenous matrix is centric to replicating vascular tissue architecture and mechanics. Further, vascular cells, regardless of the choice of scaffolds, robustly synthesize collagen. Examining the impact of a pre-existing collagenous microenvironment on the ability of the SMCs to synthesize fibrous elastic matrix in response to provided EFs, is pertinent to its clinical translation.
In the first set of studies, we examined a dose range of EFs on inducing rat SMCs-seeded within 3D collagen gel constructs. Relative to untreated control, all the three doses tested showed up to a 2-fold up-regulation in gene expression of the elastin crosslinking enzyme, lysyl oxidase, and increased the accumulation of matrix elastin up to 5-fold. The lowest dose combination of 0.1 ng/ml TGF-β1 and 0.2 µg/ml HA-o, was evaluated to be most elastogenic, and this was utilized in subsequent studies. Next, we evaluated the application of cyclic strains at varying frequencies in improving EF-induced elastic matrix output, and to obtain matrix and cell orientation in a manner similar to that required in vivo. Further, we tested this system on human SMCs seeded within tubular collagen-gel constructs, to examine if they respond to EF-treatment similar to rat SMCs. A bimodal trend in elastic matrix output was observed with increasing frequencies. Relative to static controls, constructs treated with EFs at 2.5% strains and 1.5 Hz were found to improve contractile SMC phenotype, up-regulate elastin gene expression up to 7-fold, and increase elastic matrix content by 5-fold. These parameters were therefore chosen for application in subsequent studies. The presence of high concentrations of matrix degrading proteases, such as MMPs-2 and -9, inherent to AAA wall, as well as within our 3D system, can compromise the accumulation and efficient assembly of newly synthesized elastic matrix components. In the next set of studies, we demonstrated that addition of Doxycycline (DOX), a non-specific MMP inhibitor, along with EFs, suppressed MMP-2 gene expression, within static and dynamic (2.5% strain at 1.5 Hz) tubular constructs, and markedly improved overall elastic matrix synthesis.
Since the effects of EFs and DOX are highly dose-dependent, the successful in vivo translation of their benefits relies on their controlled and targeted delivery specifically to the site of disease. In the final set of studies, we tested the effects of TGF-β1 and DOX released from PLGA nanoparticles, incorporated within the cyclically stretched tubular 3D model optimized in previous studies. We were able to successfully demonstrate that such localized delivery was able to induce elastogenesis in a manner similar to exogenous delivery of the same factors.
Overall, these results will be useful towards addressing a fundamental and widely absent aspect in vascular engineering, that of inducing adult vascular cells to replicate biological and structural mimics of native elastic fiber networks.
Venkataraman, Lavanya, "Induced Elastic Matrix Synthesis within 3-Dimensional Collagen Constructs" (2012). All Dissertations. 1016.