Mechanical Stretch on Endothelial Cells Promotes Monocyte Differentiation into Immunogenic Dendritic Cells

Abstract

Hypertension is a leading cause of mortality in Western society, attributed in part to the oxidative injury and inflammation. We have shown that dendritic cells (DCs) from hypertensive mice accumulate isolevuglandins (IsoLGs) that adduct to proteins and promote T cell activation. In hypertension, the endothelium is activated to produce reactive oxygen species and to express adhesion molecules and chemokines that attract inflammatory cells. Human monocytes that traverse the endothelium differentiate into monocyte‐derived DCs upon exposure to pro‐inflammatory state. We hypothesized that human endothelial cells exposed to mechanical stretch will promote conversion of human monocytes into activated DCs. We co‐cultured human aortic endothelial cells (HAECs) with monocytes from normotensive human donors and exposed the HAEC monolayer to either normal cyclical stretch (5%) or hypertensive cyclical stretch (10%) using the Flexcell® Tension System for 48 hours. We found that co‐culture of monocytes with HAECs exposed to 10% mechanical stretch have a marked increase in pro‐inflammatory cytokines such as IL‐6, I‐23A and IL‐1β compared to 5% stretch. HAECs exposed to 10% stretch promoted monocytes in culture to differentiate into DCs, as evidenced by the surface expression of CD209, CD83 and the co‐stimulatory marker CD86. These monocytes also had an accumulation of IsoLG‐adducted proteins compared to controls (69.73 ± 5.802 vs 10.79 ± 1.854 respectively, p = 0.0001) as evidenced by intracellular staining and flow cytometry. In addition, these monocytes in culture with endothelial cells exposed to 10% stretch expressed the phosphorylated form of STAT3 (pSTAT3) and this was prevented when cell cultures were treated with STAT3 inhibitor, stattic. Monocytes co‐cultured with 10% stretched HAECs induced a 1,500‐fold increase in CD4+ T cell proliferation and a 1,300‐fold increase in CD8+ T cells proliferation as monitored by CFSE compared to 5% stretch controls. Conversely, monocytes‐HAEC cultures exposed to 10% stretch and treated with STAT3 inhibitor prevented both CD4+ and CD8+ T cell proliferation. Monocytes were plated inside a transwell dish that only allows the exchange of small molecules while the endothelial cells were stretched to either 5% or 10%. We found that monocytes expressed DC markers, pSTAT3, and accumulated IsoLG‐peptides without direct contact with endothelial cells exposed to hypertensive mechanical stretch. These data show that endothelial cells exposed to mechanical stretch cross‐talk with monocytes to promote differentiation into DCs that are immunogenic potentially via the STAT3 pathway and that this is not cell contact‐driven. These findings give insight into a new mechanism of lymphocyte activation in the vascular endothelium during hypertension.Support or Funding InformationRuth L. Kirschstein National Research Service Award (NRSA) Diversity F31HL132526