Abstract 063: Mechanical Stretch on Endothelial Cells Promotes Monocyte Activation and Differentiation into Immunogenic Dendritic Cells via STAT3

Abstract

Mechanical stretch activates the endothelium to produce reactive oxygen species and expression of adhesion molecules and cytokines. Human monocytes that traverse the endothelium differentiate into dendritic cells (DCs) upon exposure to a 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 endothelial cells to either normal (5%) or hypertensive (10%) uniaxial cyclical stretch for 48 hours. Co-culturing monocytes with HAECs exposed to 10% stretch showed a marked increase in pro-inflammatory cytokines such as IL-6, IL-23A and IL-1β compared to 5% stretch. HAECs exposed to 10% stretch promoted monocytes in culture to differentiate into DCs. We have shown that DCs from hypertensive mice accumulate isolevuglandins (IsoLGs) that adduct to proteins and promote T cell activation. Thus, we performed intracellular staining and flow cytometry and found that these monocytes significantly accumulate higher levels of IsoLG-adducted proteins compared to 5% stretch (69.7 ± 5.8 vs 10.8 ± 1.85, respectively). In addition, monocytes co-cultured with endothelial cells exposed to 10% stretch expressed phosphorylated STAT3 (pSTAT3), which was blocked by stattic, a STAT3 inhibitor. Similarly, monocytes co-cultured with HAECs exposed to 10% stretch induced a 1,500-fold and a 1,300-fold increase in CD4 + and CD8 + T cell proliferation; while, inhibition of STAT3 prevented this T cell proliferation. To test if this is due to cell-cell contact, we seeded monocytes in a transwell with endothelial cells exposed to either 5% or 10% stretch. We found that these monocytes expressed DC markers, pSTAT3, and accumulated IsoLG-peptides when exposed to hypertensive mechanical stretch. In addition, angiotensin II (490ng/kg/min) infusion of C57Bl/6 mice increased pSTAT3 in monocytes, macrophages, and DCs in the renal and vascular tissue compared to sham controls. Thus, our data indicate that endothelial cells exposed to mechanical stretch cross-talk with monocytes to promote differentiation into DCs known to be immunogenic via STAT3.