Date of Award
Doctor of Philosophy (PhD)
Charles D. Rice
Vascular calcification can be life-threatening, involving calcium phosphate crystal deposits in the blood vessels. The calcification in the intimal layer of the arterial wall is usually associated with atherosclerotic plaque build-up that, in the advanced stage, harbors ‘dispersed’ and ‘spotty’ deposits of calcium phosphate crystals in the necrotic core of the plaque. In contrast, medial arterial calcification (MAC) can form independently of atherosclerosis and appears as ‘Railroad-like’ calcific deposits along degraded elastin lamellae in the medial layer of arteries. The mechanisms of such elastin-associated medial arterial calcification and potential therapies are the key concerns of this dissertation. MAC is common in the aging population and particularly in those with type 2 diabetes and chronic kidney diseases (CKD), which is due to arterial elastin degradation as well as mineral imbalances in the blood, particularly hyperphosphatemia. MAC-associated elastin calcification causes vascular wall stiffening, resulting in increased systolic hypertension and resistance to blood flow. The resultant overload in cardiac work to move blood throughout the body leads to heart failure. Therefore, preventing medial calcification or reversal of existing calcific deposits from the medial layer without imposing any significant side effects is a pressing healthcare challenge.
Overall, our study aims to understand the effects of elastase-mediated injury and elastin degradation products to initiate MAC either in the presence or absence of hyperphosphatemia. We test the efficacy of pentagalloyl glucose (PGG) to block elastase- and hyperphosphatemia-induced MAC in an aortic ring culture model and the effectiveness of targeted PGG nanoparticles to prevent systemic MAC in an animal model of CKD. We further developed targeted etidronate-loaded albumin nanoparticles and tested their ability to suppress MAC in aortic ring cultures. We also studied vascular cell behavior upon reversal of heavy aortic calcification using site-specific ethylene diamine tetraacetic acid (EDTA)-loaded albumin nanoparticles in a condition of CKD.
We first developed an aortic ring culture model to study the effects of high active elastase on MAC development. High circulating elastase is typical in patients with diabetes, CKD, autoimmune disease, and in aging, either genetic or acquired. This ex vivo study demonstrated that high elastase activity is not only a driving factor for vascular inflammation but also initiates MAC via fragmenting elastin networks and stimulating vascular smooth muscle cells (VSMCs) to produce calcific microvesicles (MVs). Combining high phosphate, another clinical feature of CKD or diabetic nephropathy, exacerbated elastase-induced MAC. However, exogenously added elastin degradation products, such as elastin derived peptides and TGFβ1 alone, do not seem to affect calcium accumulation in the arterial media, though promoted VSMCs phenotypic alterations to osteogenesis.
To study if the blockage of damaged sites on elastin or further elastin degradation may prevent high phosphate-induced extensive MAC, PGG was evaluated in an aortic ring culture model. PGG completely abolished MAC development in elastase-injured aortas cultured in high phosphate. By blocking degraded elastin sites, PGG prevented VSMCs-released calcific MVs from depositing onto the arterial media, preventing calcium phosphate crystal growth and development. For in vivo systemic inhibition of calcification with targeted PGG-loaded albumin nanoparticles, we developed a rodent model of progressive CKD combining adenine diet and vitamin D that aggressively caused severe cardiovascular and kidney calcification. Unfortunately, intravenously delivered site-specific PGG nanoparticles were unsuccessful in inhibiting MAC, which could be attributed to the insufficient drug concentration or slow release of PGG from the nanoparticle systems that failed to compete against the severe and aggressive form of calcification in this CKD model.
To prevent or halt calcification progression, we developed another nanoparticle system with etidronate-loaded albumin nanoparticles conjugated with anti-elastin antibody for targeting damaged elastin in vasculatures. Herein, a single micro-dose of formulated nanoparticles significantly delayed the hyperphosphatemia-induced calcification in the aortic ring culture model. The aortic rings treated with etidronate nanoparticles showed less LDH release in media supernatants compared to non-treated aortas, indicating no cytotoxicity from etidronate or other components of nanoparticles.
Finally, to reverse pre-existing heavy arterial calcification developed with the progressive CKD model, we performed targeted chelation therapy with anti-elastin antibody conjugated ethylene diamine tetraacetic acid (EDTA)-loaded albumin nanoparticles that specifically targeted damaged elastin. EDTA delivery removed calcific deposits from abdominal aortas and returned VSMCs from osteogenic to their natural vascular phenotypes. This chelation therapy also removed calcific deposits from the kidneys. More importantly, targeted chelation therapy significantly improved animals’ survivability than blank therapy.
In summary, our research helped us understand MAC in relation to elastin degradation and chronic hyperphosphatemia that mimics CKD. Our nanoparticle targeting system is based on the hypothesis that elastin degradation is the initial step of MAC before calcification; such degraded elastin can be targeted by anti-elastin antibody to deliver drugs to either prevent, or reverse calcification. Here we studied molecular and cellular behavior in response to our targeted nanotherapeutics that have the potential to inhibit/reverse elastin-associated MAC with minimal to no systemic side effects.
Zohora, Fatema Tuj, "Prevention and Reversal of Elastin-Associated and Phosphate-Dependent Arterial Calcification Using Targeted Nanotherapeutics in Pre-clinical Models" (2023). All Dissertations. 3382.
Available for download on Saturday, August 31, 2024
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