Plasma Kallikrein as a Novel Modulator of Macrophage Dynamics

Abstract

Cardiovascular diseases (CVD) remain the leading cause of death globally, by which more than 17 million individuals die each year, accounting for 30% of all deaths. Moreover, CVD is responsible for 34% of all deaths in the Middle East, whereby the rate of increase in CVD-associated mortalities is one of the highest rates worldwide. Earlier studies have shown that elevated levels of plasma kallikrein is implicated in the perpetuation of CVD. In the cardiovascular system, plasma kallikrein is a key regulator of several proteolytic cascades. One of these cascades includes the kallikrein-kinin system (KKS). Therefore, it has been proposed that several components of the KKS are mediators or modulators of inflammation and that plasma kallikrein in specific plays an essential role in inflammation. Being a vital component of inflammation, monocytes are mobilized to the heart where they differentiate into macrophages. Following MI, monocyte-derived macrophages are able to secrete a wide range of factors that contribute to several processes such as inflammation, angiogenesis, repair system and phagocytosis. Since the plasma kallikrein-mediated inflammatory processes remain ill-defined and are poorly understood, this project seeks to characterize and assess the inflammatory markers involved in the macrophage-regulated pathogenesis of CVD. To achieve these goals, we employed bone marrow-derived macrophages (BMDM) from C57BL/6 mice. Accordingly, we first cultured the aforementioned cells and treated them with plasma kallikrein for twenty-four hours. Lipopolysaccharide (LPS), a component of the gram-negative bacterial cell wall that is known as a pro-inflammatory factor, was used as a positive control in our study. In order to quantify the level of markers at the protein level, Enzyme-Linked Immunosorbent Assay (ELISA) was performed. To further demonstrate the expression of the markers at the transcript level, we extracted RNA from the cells and conducted Next Generation Sequencing (NGS) so as to elucidate gene expression signatures as well as compare and contrast the transcriptome of the treated and untreated BMDM using RNA-sequencing (RNA-seq). This was done in order to demarcate differentially expressed genes and profiles in the course of plasma kallikrein- and bradykinin-mediated inflammatory processes using specific bioinformatics tools. Genes that were differentially expressed, were identified and their expression profiles were validated by Quantitative Reverse Transcription (RT-qPCR). In addition, we studied the biological network of proteins using Pathway Studio software in order to understand the processes involved in inflammation and CVD. Finally, to identify the receptors in plasma kallikrein-mediated inflammatory responses, the secretion of inflammatory cytokines was investigated after-which receptors were inhibited. Preliminary analysis pointed out the upregulation of pro-inflammatory cytokines in BMDM, of which include Tumor Necrosis Factor-Alpha (TNF-α) and Interleukin-6 (IL-6). The following results were observed at the protein level using ELISA. Moreover, RNA-seq data have also demonstrated the up and down-regulation of several inflammatory and fibrotic markers, which were further validated using RT-qPCR. In this project, we expect to find novel mechanisms underlying the plasma kallikrein mediated inflammatory pathway, through monocyte-derived macrophages. Likewise, this project will serve to expand our current limited knowledge on the role of plasma kallikrein during inflammation in CVD and delineate the interplay between multiple factors, hence paving the way to the development of new therapeutic targets.