Abstract
Lipopolysaccharide (LPS) could induce apoptosis and dysfunction of endothelial cells. We aimed to reveal the effects of macrophages on cell proliferation and apoptosis in LPS induced human umbilical vein endothelial cells (HUVECs). THP-1 derived macrophages and HUVECs were co-cultured in the presence of LPS. Cell viability was measured by Cell Counting Kit-8 and apoptosis was analyzed by flow cytometry. Expression of Ang1, the NF-κB component p65 was evaluated by western blot and quantitative PCR. Small interfering RNAs (siRNAs) were used to knockdown the expression of proinflammatory cytokines and p65 in HUVECs. Plasmid transfection-mediated overexpression of Ang1 was employed to see its effects on cell proliferation and apoptosis in HUVECs. Macrophages enhanced LPS-induced proliferation impairments and apoptosis in HUVECs, which could be attenuated by siRNA-mediated knockdown of cytokines TNF-α, IL-1β, IL-6 and IL-12p70 in macrophages. The dysfunction of HUVECs was tightly associated with reduced Ang1 expression and increased phosphorylated p65 (p-65). Overexpression of Ang1 in HUVECs significantly decreased p-p65, suggesting negatively regulation of p-p65 by Ang1. Overexpression of Ang1, adding recombinant Ang1 or silencing of p65 substantially attenuated the dysfunction of HUVECs in terms of cell proliferation and apoptosis. In conclusions, THP-1-derived macrophages enhance LPS induced dysfunction of HUVECs via Ang1 and NF-κB pathways, suggesting new therapeutic targets for sepsis.
Highlights
Sepsis represents a series of common diseases in intensive care unit (ICU), which causes many deaths worldwide
In our experiment designs, we set up four groups, including control human umbilical vein endothelial cells (HUVECs) without treatment or co-culturing (HUVEC), HUVEC treated with LPS alone (HUVEC + LPS), HUVEC co-cultured with THP-1-derived macrophages (HUVEC + THP-1) and LPS stimulated HUVECs that co-cultured with THP-1-derived macrophages (HUVEC + THP-1 + LPS), to compare their cellular proliferation and apoptosis
While either LPS stimulation alone or co-culturing with THP-1 cell alone significantly increase the expression of cleaved PARP and cleaved caspase-3 in HUVECs, administration of both (LPS stimulation plus macrophage co-culturing) synergistically further reduced the expression of cleaved PARP and cleaved caspase-3 protein in HUVECs (Fig. 1C)
Summary
Sepsis represents a series of common diseases in intensive care unit (ICU), which causes many deaths worldwide. Since macrophage is capable of mediating cytotoxic effects and is of great importance in aggressive inflammatory response in sepsis, we speculated that macrophages play roles in the dysfunction of endothelial cells during sepsis. The roles of Ang1/2 and NF-κB pathway in macrophages induced dysfunction of endothelial cells have not been clearly elucidated yet. We co-cultured THP-1-derived macrophages with human umbilical vein endothelial cells (HUVECs), and LPS was added to investigate the alterations on cell proliferation and apoptosis of HUVECs. We found that addition of macrophages in the culture significantly enhanced LPS induced proliferation impairment and apoptosis of HUVECs, through soluble factors like TNF- α, IL-1β, IL-6 and IL-12p70. Ang[1] negatively regulated the phosphorylation of p65 in HUVECs. Our study suggested that Ang1/2 and NF-κB pathway play a central role in regulating macrophages mediated and LPS induced dysfunction of HUVECs
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