Date of Award

8-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Advisor

Dr. Narendra Vyavahare

Committee Member

Dr. Jeoung Soo Lee

Committee Member

Dr. Alexey Vertegel

Committee Member

Dr. Bruce Z. Gao

Abstract

There are two distinctive types of vascular calcification: atherosclerotic intimal calcification (AIC) and elastin specific medial arterial calcification (MAC). AIC occurs in the context of atherosclerosis, associated with lipids, macrophages, and vascular smooth muscle cells; whereas, MAC can exist independently of atherosclerosis and is associated with elastin and vascular smooth muscle cells. The work presented here focuses on the mechanisms and reversal of MAC. MAC, termed as Monckeberg's sclerosis, is a type of vascular calcification disease mostly occurs as linear deposits along elastic lamellae. Vascular calcification, including medial calcification, is a strong predictor of cardiovascular morbidity and mortality. Currently the only options to treat vascular calcifications are surgical methods like directional atherectomy and placement of stent grafts. These surgical methods are quite invasive and expensive, and currently there is no available alternative to surgical therapy for reversal of vascular calcification. One possible approach is EDTA chelation therapy. EDTA chelation therapy most often involves the injection of disodium ethylene diamine tetraacetic acid (EDTA), a chemical that binds, or chelate ionic calcium, trace elements and other divalent cations so as to reverse calcification. However, chelation therapy is not yet accepted in US and not approved by FDA as there is no clinical proof that it works and improves cardiovascular function. Besides, it has severe side effects such as renal toxicity, hypercalcemia, and bone loss. Overall goal of our research is to understand the mechanisms of elastin specific MAC, to evaluate efficacy of chelating agents to reverse elastin specific MAC, and to develop a targeted delivery system to reverse elastin specific MAC and circumvent side effects. First, we investigated the mechanisms of elastin specific MAC, specifically cause-and-effect relationship between the elastin-specific MAC and osteoblast-like differentiation of vascular smooth muscle cells (VSMCs). It has been accepted for decades that the pathology of vascular calcification resembles physiological bone mineralization. However, the cause-and-effect relationship between calcification and osteoblast-like differentiation of VSMCs is still unclear. We, for the first time, showed that in response to the calcified matrix, for both hydroxyapatite crystals and calcified aortic elastin, VSMCs lost their smooth muscle lineage markers and underwent chondroblast/osteoblast-like differentiation. Interestingly, this phenotypic transition was reversible and VSMCs restored their original linage upon reversal of calcification. Secondly, we evaluated the efficacy of chelating agents to reverse elastin specific MAC. We tested if chelating agents, such as disodium ethylene diamine tetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA) and sodium thiosulfate (STS) can remove calcium from hydroxyapatite (HA) and calcified tissues without damaging the tissue architecture. Our results indicated that both EDTA and DTPA could effectively remove calcium from hydroxyapatite and calcified tissues, while STS was not effective. The tissue architecture was not altered during chelation. Thirdly, we investigated the therapeutic effects of systemic EDTA chelation therapy for reversal of calcification and its possible side effects in vivo. We used CaCl2 mediated abdominal aorta injury in rats to mimic local aortic calcification similar to what is observed in clinical cases. We demonstrated that systemic EDTA chelation therapy did not reverse local aortic calcification. Moreover, systemic EDTA chelation therapy caused adverse events for serum and urine calcium, specifically hypocalcemia and hypercalciuria, but did not cause bone loss. Finally, we developed a targeted delivery system to reverse elastin specific MAC and to avoid side effects associated with systemic EDTA chelation therapy. We have recently shown that the nanoparticles (NPs) coated with elastin-antibody can be targeted to vascular calcification sites. This paved the way to design an elastin antibody coated and EDTA loaded albumin NPs to reverse elastin-specific medial calcification and avoid side effects. Our in vitro and in vivo results demonstrated that elastin antibody coated and EDTA loaded albumin NPs had good therapeutic effect to reverse elastin specific MAC and did not have side effects typically seen in systemic EDTA chelation therapy such as hypocalcemia and bone loss. In conclusion, our work led to enhanced understanding the possible mechanisms of osteogenesis in vascular calcification and possible alternative therapy to reverse elastin specific MAC without systemic side effects.

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