ABCA3, a 150 kDa protein belonging to the ATP-Binding Cassette (ABC) transporter superfamily, has been shown to play a role in surfactant production in humans. However, in bacteria, ABC transporters are known to mediate the flux of nitrite. Biotransformation of glyceryl trinitrate (GTN), a drug used in the treatment of heart conditions such as angina pectoris and heart failure, yields the inorganic nitrite anion, and the intracellular oxidation of this ion may lead to the formation of tyrosine-nitrated proteins and cellular damage. Immunohistochemical studies indicate the presence of ABCA3 in rat aortic smooth muscle and endothelial cells. My objective was to assess whether changes in either ABCA3 mRNA expression or nitrite accumulation occur during chronic exposure to GTN. Accordingly, male Sprague-Dawley rats were exposed to 0.4 mg/hr GTN for 48 hours to induce GTN tolerance, and the aortas removed. Nitrite and ABCA3 mRNA levels were assessed using the Greiss colorimetric assay, and real-time or semi-quantitative RT-PCR, respectively. In control aortas, endothelium removal resulted in an apparent 25-35% decrease in ABCA3 mRNA levels, indicating that the transporter is expressed in endothelial cells more abundantly than in smooth muscle cells since the ratio of endothelial cells to smooth muscle cells in the rat aorta is approximately 10:1. Furthermore, ABCA3 mRNA levels were decreased by 70% in aortas from GTN-tolerant animals, whereas the mRNA levels of a related transporter, ABCA1, remained at control levels. An inverse correlation between nitrite and ABCA3 mRNA levels occurred after the induction of GTN tolerance, with an apparent redistribution of nitrite to endothelial cells. These findings indicate that chronic GTN exposure results in altered expression of ABCA3, and that this is associated with altered nitrite distribution in blood vessels from GTN-tolerant animals. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2009-06-23 14:18:16.122

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The most common causes of childhood interstitial lung disease (chILD) are autosomal recessive mutations in the gene encoding ATP Binding Cassette A3 (ABCA3) protein, a lamellar body (LB) associated lipid transporter exclusively expressed within the alveolar epithelial type II cells (AEC2s) in the lung. Instability of primary AEC2s in culture has prevented studies of ABCA3 mutations, resulting in limited understanding of disease pathogenesis. To overcome this challenge, we developed AEC2-like cells from human pluripotent stem cells (PSCs) in vitro, allowing study of normal ABCA3 function and perturbations that result from ABCA3 mutations. To develop an AEC2 model that would recapitulate ABCA3 biology, we targeted human PSC lines with a knock-in GFP fusion reporter (ABCA3:GFP). Differentiations of PSCs into AEC2s (iAEC2s) resulted in exclusive expression of ABCA3:GFP in iAEC2s and intracellular localization to LAMP3+ vesicles, reminiscent of endogenous ABCA3. Moreover, we find these ABCA3:GFP+ iAEC2s express LBs, process surfactant proteins, and secret surfactant lipids, indicative of preserved ABCA3 function. To study the effects of ABCA3 mutations using our model, we generated two sets of PSC reporter lines: 1) two patient-derived iPSC lines carrying rare homozygous E690K and W308R ABCA3 mutations predicted to affect ABCA3 function or trafficking, respectively, and their two syngeneic gene-corrected lines each targeted with the AEC2-specific knock-in fluorescent reporter SFTPCtdTomato; and 2) three syngeneic ABCA3:GFP knock-in iPSC lines encoding wildtype, E690K, or W308R proteins. Directed differentiation of patient iPSCs into iAEC2s revealed attenuated secretion of surfactant-specific lipids, recapitulating clinical findings of surfactant deficiency. Examination of ABCA3 protein trafficking using the ABCA3:GFP fusion reporter revealed retained E690K and W308R mutant ABCA3 protein processing and trafficking compared to the wildtype protein by confocal microscopy and western blot analyses, however mutant iAEC2s exhibited smaller LBs, indicative of defective ABCA3-dependent lipid transport. Bulk RNA sequencing of mutant and gene-corrected SFTPCtdTomato- or ABCA3:GFP-expressing iAEC2s revealed enrichment of the TNF𝛼-NF𝜅B pathway in both W308R and E690K mutant iAEC2s, validated by lentiviral reporter assays and secretion of NF𝜅B-driven cytokines. Thus, we provide insights into how ABCA3 mutations alter AEC2 physiology and developed a platform to study other genetic AEC2 diseases through our ABCA3:GFP reporter system. / 2021-05-26T00:00:00Z