Search

Atherosclerosis is a major underlying cause of cardiovascular disease; however, the molecular mechanisms by which cardiovascular risk factors promote the development of atherosclerosis are poorly understood. Recent evidence from our laboratory suggests that endoplasmic reticulum (ER) stress signaling through glycogen synthase kinase (GSK)-3α/β is involved in the activation of pro-atherosclerotic processes. The objective of this study was to examine the role of ER stress-GSK3α/β signaling on the progression and regression of atherosclerosis in a mouse model system. We first investigated the effects of attenuating ER stress or inhibiting GSK3α/β in a low density lipoprotein receptor deficient (Ldlr-/-) mouse model of atherosclerosis. Mice treated with the ER stress alleviator 4-phenylbutyric acid (4PBA) or the GSK3α/β inhibitor valproate (VPA) have significantly reduced lesion areas and volumes, indicating that these treatments attenuated the development of atherosclerosis. Next, we examined the effects of 4PBA or VPA treatment in Ldlr-/- mice with established lesions. These treatments inhibited the progression of atherosclerosis in established plaques, but we did not see any evidence of plaque regression. However, the plaques appear to be more stable and less prone to rupture. Lastly, to help determine the mechanism by which GSK3α/β is involved in atherosclerosis, cell culture experiments were performed to characterize GSK3α or GSK3β deficient macrophages. Bone marrow derived macrophages were obtained from Ldlr-/- mice with myeloid specific GSK3α or GSK3β deficiency, and were cultured in the presence of the ER stress inducing agents thapsigargin (Tg) or tunicamycin (Tm). The absence of GSK3α attenuated M1 polarization, but did not significantly alter cell viability or the expression of genes involved in lipid biosynthesis and uptake. The deletion of GSK3β minimally affected apoptosis induced by Tg or Tm treatment. In contrast, the deletion of GSK3α and GSK3β significantly improved cell viability. These data support the importance of ER stress-GSK3α/β signaling in atherogenesis. The pharmacological attenuation of ER stress or inhibition of GSK3α/β impedes the development of atherosclerosis in Ldlr-/- mice and appears to promote the stabilization of existing lesions. This may be due in part to a reduction in the pro-inflammatory M1 phenotype associated with the inhibition of the GSK3α homolog. Thesis Master of Science (MSc)

Since the outbreak of the COVID-19 pandemic, widespread infections have allowed SARS-CoV-2 to evolve in human, leading to the emergence of multiple circulating variants. Some of these variants show increased resistance to vaccines, convalescent plasma, or monoclonal antibodies. In particular, mutations in the SARS-CoV-2 spike have drawn attention. To facilitate the isolation of neutralizing antibodies and the monitoring the vaccine effectiveness against these variants, we designed and produced biotin-labeled molecular probes of variant SARS-CoV-2 spikes and their subdomains, using a structure-based construct design that incorporated an N-terminal purification tag, a specific amino acid sequence for protease cleavage, the variant spike-based region of interest, and a C-terminal sequence targeted by biotin ligase. These probes could be produced by a single step using in-process biotinylation and purification. We characterized the physical properties and antigenicity of these probes, comprising the N-terminal domain (NTD), the receptor-binding domain (RBD), the RBD and subdomain 1 (RBD-SD1), and the prefusion-stabilized spike ectodomain (S2P) with sequences from SARS-CoV-2 variants of concern or of interest, including variants Alpha, Beta, Gamma, Epsilon, Iota, Kappa, Delta, Lambda, Mu, and Omicron. We functionally validated probes by using yeast expressing a panel of nine SARS-CoV-2 spike-binding antibodies and confirmed sorting capabilities of variant probes using yeast displaying libraries of plasma antibodies from COVID-19 convalescent donors. We deposited these constructs to Addgene to enable their dissemination. Overall, this study describes a matrix of SARS-CoV-2 variant molecular probes that allow for assessment of immune responses, identification of serum antibody specificity, and isolation and characterization of neutralizing antibodies.