Hormone-Independent Sex Differences in Pro-Inflammatory and Adhesion Mediators in a Cellular Model of Atherosclerosis

Introduction: Women are underrepresented in both clinical studies and drug development. However, significant sex differences exist in the occurrence and progression of diseases, which are predominantly attributed to hormonal influences. This also applies to cardiovascular diseases. Failure to consider the sex of cells in basic research can lead to erroneous results and false conclusions. Therefore, in this project, we aimed to investigate hormone-independent sex differences at the cellular level using a cell model of atherosclerosis.

Methods: Male and female human umbilical vein endothelial cells (HUVECs) from twin pairs were treated with either TNF-α (10 ng/ml) or with supernatants from pro-inflammatory stimulated male THP-1 and female HL-60 monocyte-like cells. The expression of the focal adhesion proteins paxillin, vinculin, talin, and tensin and the cell adhesion molecules VCAM1 and ICAM-1 was assessed using quantitative real-time PCR or Western blot analysis. In addition, the levels of secreted cytokines and chemokines were measured in the HUVEC cell culture supernatants via a Cytokine Multiplex Assay.

Results: Female HUVECs exhibited increased levels of the secreted proinflammatory cytokines IL-17a and IL-22, the anti-inflammatory cytokine IL13, the pro-angiogenic cytokine TGF-α, and the chemoattractant protein Eotaxin in the supernatant after treatment with TNF-α or with supernatants from pro-inflammatory stimulated monocyte-like cells compared to the controls (p<0,05). Conversely, male supernatants exhibited increased levels of IL17f and RANTES. Moreover, the cell adhesion proteins VCAM-1 and ICAM-1, the chemokine MDC, and the cytokine MIP-1α showed opposite directions of regulation between female and male cells after both stimulations. The gene and protein expressions of the focal adhesion proteins paxillin, vinculin, talin, and tensin showed no significant differences.

Conclusion: These results provide evidence for the differential regulation of adhesion proteins and cytokines in female and male cells. Hormone-independent sex differences in endothelial cell signaling within a pro-inflammatory environment may be of relevance for the development of atherosclerosis.