Investigation of PDI Involvement in Leukocyte Adhesion to Endothelial Cell in Inflammatory State Using Gene Editing (CRISPR/Cas9) Technology
| dc.contributor.advisor | Fathallah, Mohamed Dahmani | |
| dc.contributor.author | almashhur, wedad awadh ali | |
| dc.date.accessioned | 2025-08-10T05:51:11Z | |
| dc.date.issued | 2025 | |
| dc.description | Inflammation constitutes a complex biological response to infection, tissue injury, or other harmful stimuli, serving as a critical component of host defense. While acute inflammation is self-limiting and resolves upon elimination of the initiating insult, chronic inflammation persists and underlies the pathogenesis of numerous diseases, including rheumatoid arthritis, atherosclerosis, and malignancies. Endothelial activation is a pivotal event during inflammation, facilitating leucocyte adhesion and transmigration across the vascular wall. This process is orchestrated by the upregulation of adhesion molecules, notably intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. Dysregulation of these molecules contributes to endothelial dysfunction and exacerbates the development of inflammatory pathologies. Protein disulfide isomerases (PDIs) are a family of thiol oxidoreductases classically involved in catalyzing disulfide bond formation and isomerization within the endoplasmic reticulum. Emerging evidence, however, implicates several PDIs in extracellular processes, including modulation of thrombosis, vascular integrity, and inflammatory signaling. While PDI1 has been shown to regulate leucocyte-endothelial interactions, the specific contributions of other PDI isoforms, such as PDIA2, PDIA3, PDIA4, and PDIA6, to endothelial activation remain poorly defined. Given their structural conservation yet functional diversity, it is plausible that distinct PDI family members exert differential effects on vascular inflammation. Despite increasing recognition of the extracellular roles of PDIs, the mechanistic involvement of individual isoforms in regulating adhesion molecule expression and leucocyte adhesion under inflammatory conditions has not been systematically investigated. Addressing this knowledge gap is essential for identifying novel therapeutic targets to ameliorate vascular inflammation and related chronic diseases. By employing, CRISPR/Cas9-mediated gene editing was employed to generate PDIA2, PDIA3, PDIA4, and PDIA6 knockout blood outgrowth endothelial cells (BOECs). The functional consequences of PDI deletion were evaluated by assessing the expression of ICAM-1, VCAM-1, and E-selectin, as well as neutrophil adhesion following TNF-α stimulation. By elucidating the roles of specific PDI isoforms in endothelial activation, this work aims to advance the understanding of PDI-mediated regulation of inflammation and support the development of targeted therapeutic interventions. | |
| dc.description.abstract | Leucocyte adhesion to endothelial cells is a critical step in the inflammatory process, influenced by various molecular mediators, including Protein Disulfide Isomerase 1 (PDI1). This study aimed to investigate the broader roles of additional PDI family members in regulating endothelial cell activation and leucocyte adhesion under inflammatory conditions. We isolated endothelial cells with a cobblestone morphology from human peripheral blood and umbilical cords, achieving yields of 1.2 × 10⁵ and 3.5 × 10⁵ cells/mL, respectively. Neutrophils were isolated from blood samples using magnetic bead technology, and their phenotype was confirmed via flow cytometry. To induce inflammation, endothelial cells were stimulated with TNF-α (100 ng/mL). Quantitative real-time PCR was utilized to assess the expression of PDI genes and adhesion molecules, specifically ICAM-1, VCAM-1, and E-selectin, which serve as established markers of endothelial activation. CRISPR/Cas9 technology facilitated the knockout of PDIA2, PDIA3, PDIA4, and PDIA6 in endothelial cells, with validation achieved through sequencing and Western blot analysis. Efficient gene editing was confirmed by the significant reduction in expression levels of the targeted PDIs. The results demonstrated a 62.5% decrease in the expression of ICAM-1 and VCAM-1, while E-selectin expression was reduced by an average of 55% across all knockouts. Functional assays revealed significant reductions in neutrophil adhesion (p < 0.01) to knockout endothelial cells, with specific decreases of 25% in PDIA2, 20% in PDIA3, 35% in PDIA4, and 2% in PDIA6 knockout cells. These findings underscore the essential roles of various PDIs in the activation of endothelial cells and leucocyte adhesion during inflammation. The study illustrates the effectiveness of CRISPR/Cas9 technology in elucidating gene functions and highlights PDIs as potential therapeutic targets for inflammatory vascular diseases. This research provides valuable insights into the molecular mechanisms underlying inflammation and opens avenues for novel therapeutic strategies. | |
| dc.format.extent | 135 | |
| dc.identifier.citation | APA style | |
| dc.identifier.uri | https://hdl.handle.net/20.500.14154/76114 | |
| dc.language.iso | en | |
| dc.publisher | Arabin Gulf university | |
| dc.subject | Inflammation | |
| dc.subject | TNF-α | |
| dc.subject | PDIa2 | |
| dc.subject | PDIA3 | |
| dc.subject | PDIA4 | |
| dc.subject | PDIA6 | |
| dc.subject | Leucocyte Adhesion Molecules | |
| dc.subject | CRISPR/Cas9 | |
| dc.title | Investigation of PDI Involvement in Leukocyte Adhesion to Endothelial Cell in Inflammatory State Using Gene Editing (CRISPR/Cas9) Technology | |
| dc.type | Thesis | |
| sdl.degree.department | Molecular Medicine Program | |
| sdl.degree.discipline | Specializing in Immunology | |
| sdl.degree.grantor | Arabin Gulf university | |
| sdl.degree.name | PHD |