Abstract
Cardiovascular diseases (CVD) encompass multiple types of pathological conditions that influence the structure and function of heart or vascular. Atherosclerosis works as a prototypical example of CVD due to its high prevalence and lethality. A plethora of theories have been proposed to uncover the pathogenesis and underlying mechanisms of atherosclerosis, such as lipogenic theory, endothelial cell damage theory, receptor deletion theory, cytokine theory, viral theory, and oncogene theory. It is well-established that atherosclerosis is a multifactorial disease resulting from the complex interplay between genetic predispositions and environmental influences. Genes involved in lipoprotein regulation, lifestyle choices and environmental conditions contribute to the initiation and progression of atherosclerosis. The complexity etiology, combined with insidious lesions onset and the asymptomatic nature in the early stage of atherosclerosis, slowed the progress in underlying its causes and pathogenesis.Endothelial cells (ECs) are the necessary component of the vascular intima that undergo inflammation, altered metabolism, oxidative stress, and endothelial-mesenchymal transition (EndMT) in atherosclerosis. EndMT is an important physiological process that gets involved in organ development, which is dysregulated in atherosclerosis. The overactivity of EndMT results in ECs dysfunction, characterized by a phenotype switch to mesenchymal cells. This transition contributes to abnormal intima structure and leads to increased infiltration of inflammatory cells into the endothelial layer, which recruits more immune cells, thereby exacerbating the inflammatory response, a key driver in the pathogenesis of atherosclerosis.
Transforming growth factor-beta (TGF-β) is a well-characterized family of secreted peptides that gets involved in cardiac related structure formation and development in the early stage of life. Additionally, TGF-β is one of the most investigated molecules that takes part in EndMT process. TGF-β undergoes proteolytic cleavage to become the activated form and then combines with other two peptides. The complex initiates downstream receptors phosphorylation and then transduces the extracellular signaling via canonical and non-canonical pathway.
In this study, we investigated the role of TGF-β2 in the EndMT process. First, we determined ECs markers expression were down-regulated induced by TGF-β2 overexpression while EndMT markers showed up-regulated. Conversely, TGF-β2 knockdown inhibits the EndMT related genes expression at both mRNA and protein level. These results indicated that TGF-β2 had close association with EndMT process. Next, we tried to identify which factors could influence TGF-β2 expression and found that co-treatment with TNF-α (10 ng/mL) and IL-1ꞵ (2 ng/mL) in human ECs significantly increase TGF-β2 and EndMT markers expression after 36 hours or 72 hours. These effects were reversed by TGF-β2 knockdown, shown as decreased EndMT related genes expression.
Subsequently, we explored the effects of pro-inflammatory cytokines and TGF-ꞵ on EndMT process. Both IL-1ꞵ and TGF-ꞵ2 but not TGF-ꞵ1 were capable of inducing EndMT process, shown as increased EndMT related genes expression. Notably, co-treatment with IL-1ꞵ (2 ng/mL) and TGF-ꞵ2 (5 ng/mL) exerted pronounced effect on promoting EndMT related genes expression in human ECs. However, this synergistic effect of IL-1ꞵ and TGF-ꞵ2 was abolished by TGF-ꞵ2 knockdown in vitro. These results suggested that TGF-ꞵ2 and pro-inflammatory cytokines exerted synergistic effects on EndMT, which depended on endogenous TGF-ꞵ2.
We identified that TGF-ꞵ2 had more potent effects on activating fibrosis related genes expression compared with TGF-ꞵ1. To elucidate the underlying mechanism, we treated human ECs with either TGF-ꞵ1 (5 ng/mL) or TGF-ꞵ2 (5 ng/mL) for a short time. We found that TGF-ꞵ2 had more pronounced effect than TGF-ꞵ1 on promoting transforming growth factor-beta receptor 1 (TGꞵR1) phosphorylation, which then initiate the downstream molecules activation.
TGF-ꞵ2 was found to be more potent in activating TGF-ꞵ signaling compared with TGF-ꞵ1. To investigate the underlying mechanism, we pretreated HUVECs with MG132 followed by treatment with TGF-ꞵ1 (5 ng/mL), TGF-ꞵ2 (5 ng/mL) and IL-1ꞵ (2 ng/mL). We found that both TGF-ꞵ1 and TGF-ꞵ2 could mediate TGꞵR1 degradation, while TGF-ꞵ2 had a weaker effect on mediating TGꞵR1 degradation than TGF-ꞵ1.
In all, this study provides the insight that TGF-ꞵ2 is an effective contributor to EndMT development, which ultimately triggers the initiation of atherosclerosis. TGF-ꞵ2 is a promising therapeutic target for CVD treatment and provides us new insights to explore the underlying mechanisms of CVD development.
| Date of Award | 18 Mar 2025 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Yu HUANG (Supervisor) & Li WANG (Co-supervisor) |