Cardiopulmonary bypass (CPB) is essential for most cardiac surgeries but often leads to systemic inflammation and multiorgan dysfunction in neonatal and pediatric patients. These adverse inflammatory responses are driven by severe shear stress on the blood, contact with plastic tubing, and rapid cooling/rewarming. However, the molecular mechanisms underlying these complications are poorly understood, creating a significant barrier in improving clinical outcomes. The Nigam Lab has identified Interleukin 8 (IL-8) and Tumor Necrosis Factor alpha (TNF-α) as inflammatory cytokines upregulated in blood cells in response to CPB-associated shear stress. We hypothesize that Lamins (LMNA) play a key role in driving these transcriptional responses, as these structural proteins form the nuclear lamina and can sense mechanical forces acting on the cell. To investigate this, we performed in-vitro experiments using THP-1 human monocytic cells to simulate bypass conditions, applying shear stress and collecting samples at various time points to study the cells’ response and recovery from CPB. Using mass spectrometry-based proteomics (MS), we have identified changes in LMNA phosphorylation between sheared and static cells, providing insight into the mechanisms driving LMNA modifications under CPB conditions. We are also employing techniques such as proximity-dependent biotin identification (BioID) to explore kinase interactions with LMNA. Furthermore, to understand how LMNA influences chromatin organization, transcription factor binding, and regulation of inflammatory genes, we will perform greenCUT&RUN to map LMNA localization on chromatin in both sheared and static THP-1 cells. We aim to uncover the specific molecular mechanisms by which LMNA is altered under shear stress and how it influences chromatin dynamics and transcription of inflammatory genes during CPB. Ultimately, this research will help us understand the underlying causes of systemic inflammation post-CPB and inform novel drug targets and therapeutics to enhance the quality of life for pediatric patients undergoing cardiac surgery.

Cardiopulmonary bypass (CPB) is a conventional way to treat the majority of cardiac surgeries. CPB is used during heart surgeries to circulate blood out of the patient's body in order for surgeons to operate on the heart. However, CPB has led to inflammation and multiorgan dysfunction especially leading to post CPB complications in neonates. Lack of questioning and understanding behind the complications of the technique have posed issues for improvements to clinical outcomes. Specifically, lack of understanding of molecular mechanisms and CPB-associated post surgery inflammation have posed obstacles to improvement of methods in recent years. To better understand these mechanisms, we performed mRNA and ATAC sequencing on circulating leukocytes from neonatal CPB patients. Notably, IL-8 and TNF-α were strongly upregulated in leukocytes. To explore these findings, I performed in-vitro experiments of running THP-1 human monocytic cells to CPB-like conditions, including high shear stress and cooling/rewarming. These experiments were collected and studied at times pre and post shear, and recovery post shear. Experiments regarding blood plasma changes were proposed and this plasma was similarly collected during varying conditions pre and post bypass. ELISA kits were run on antigens AREG and EREG to determine how antigen binding changes with shearing. Sheared then rested samples were found to show a significant increase in antigen binding in both kits AREG and EREG. Sheared and processed samples also showed an increase in binding when compared to the static samples. I have shifted my focus from plasma experiments to investigating the effects of commonly used plasticizers on blood composition. Specifically, I am analyzing how these plasticizers influence changes in blood and plasma using a PIPSeq kit.