Date of Award

Spring 2023

Document Type

Open Access Dissertation

Department

Biomedical Science

First Advisor

Mohamad Azhar

Abstract

The aorta is the largest artery in the body which is directly connected to the left ventricle of the heart and functions as an elastic conduit that transports the ejected blood from the left ventricle to peripheral vessels. There are three layers to the aortic wall, tunica intima, tunica media, and tunica adventitia. The tunica media, the thickest middle layer, is composed of vascular smooth muscle cells (SMC) and the extracellular matrix (ECM) as its essential components. SMCs are circumferentially arranged and embedded in elastic lamellae. They are responsible for producing and maintaining/remodeling the extracellular matrix of the aortic media. The ECM defines the passive mechanical behavior of large elastic arteries, and it is synthesized, organized, and maintained by SMCs in the media and by fibroblasts in the adventitia. Both irreversible dilation (aneurysm), and aortic calcification are common diseases of the aorta that are resulted from pathological changes of the three aortic layers, especially the tunica media. Currently, besides surgical repair and some preventative medications, there is no standard medical treatment for vascular calcification and thoracic aortic aneurysm. So, non-invasive medications are in dire need for these aortopathies. The overall objective of this research was to study the pathogenesis of medial arterial calcification (Project 1), thoracic aortic aneurysm, and dissection (Project 2), and to test the efficacy of nanoparticles based alternative therapeutics. For Project 1, we used an adenine diet mouse model to induce medial arterial calcification (MAC) and evaluated the therapeutic efficacy of ethylenediaminetetraacetic acid (EDTA) nanoparticles (NP) chelation therapy. Double fluorescent reporter mice with SMC promoter Myh11CreERT2 for cell tracing analysis and C57BL/6 mice were used. Twelve weeks of adenine diet induced calcification in the aortic media, the heart, and the kidney. The Adenine diet caused chronic kidney disease (CKD) like condition resulted in phenotypic switch of vascular SMCs into osteochondrogenic-like cells. The resulting phenotype was accompanied by lowered gene expression of SMC related genes and upregulation of osteochondrogenic genes. The change in gene expression was followed by histological and morphological changes in the aortic media, such as fragmentation of elastic fibers, deposition of collagen, proteoglycans, and calcification. Our cell lineage analysis also indicated the calcifying cells in the aortic media are SMCs. The medial calcification was monitored by in vivo micro-computed tomographic (micro-CT) imaging. EDTA-NPs chelation therapy significantly reduced the medial calcification. In Project 2, we used postnatal transforming growth factor beta 2 (Tgfb2) tamoxifen-inducible conditional knockout mice (iCKO) to study thoracic aortic aneurysm and acute aortic dissection (TAAD). Tgfb2 gene was disrupted at 4 weeks of age using SMC-specific tamoxifen inducible Myh11CreERT2 as as a promotor. Tgfb2 conditional knockout mice (iCKO) showed changes in gene expression of genes related to SMCs function and components of the ECM. SMC specific disruption of Tgfb2 resulted in thoracic aortic aneurysm (TAA) and TAAD resulting fatal aortic rupture and internal bleeding. These morphological changes were accompanied by fragmentation of elastic fibers, and increased deposition of collagen and proteoglycans in the medial and adventitial layers. Tgfb2 iCKO mice, especially with advanced disease showed paradoxically increased canonical (SMAD-dependent) and non-canonical (MAP kinase-dependent) TGFβ signaling. We treated Tgfb2 iCKO mice with TGFβ neutralizing antibody (TGFβ NAb) to evaluate if systemic depletion of TGFβ ligands can induce TAAD. We also tested the therapeutic efficacy of a natural product called pentagalloyl glucose (PGG). Our result showed that PGG treatment attenuated TAA and the TGFβ NAb-induced TAAD. Our cell fate mapping study indicated that bone marrow derived immune cells (macrophages and T cells) participate in the pathogenesis of Tgfb2 iCKO induced thoracic aortopathy. Though the causative factors in medial arterial calcification and thoracic aortic aneurysm in our mouse models are different, they have several overlapping pathogenic pathways related to phenotypic state of smooth muscle cells, components of the ECM, and involvement of bone marrow derived cells. It is also important to note that other factors that are not inherited, such as damage to the walls of the aorta from aging, tobacco use, injury, and male sex can contribute to non-genetic cases of MAC and TAAD. Thus, this project will lead to a better understanding of mechanisms involved in the pathogenesis and development of safer therapies of thoracic aortopathies.

Rights

© 2023, Mengistu G. Gebere

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