Abstract P190: Transcriptomic Data Analysis Reveals Sex-Related Differences in the Interplay Between Toll-Like Receptor 4 and Heat-Shock Protein 70 in the Aorta of Type 2 Diabetic Donors

Background: Emerging evidence highlights distinct mechanisms driving cardiovascular diseases in males and females. Particularly important in this context are the actions of the Heat Shock Protein 70 (HSP70) family, which are molecular chaperones that, under pathological conditions, modulate innate immune receptors such as Toll-like receptor 4 (TLR4). Activation of TLR4 triggers oxidative stress and inflammation, key elements of vascular diseases. However, it is still unknown if HSP70 stimulates TLR4 in smooth muscle cells (SMCs) as well as how the expression of HSP70 family members relate to TLR4 in the vasculature of type 2 diabetic (T2D) donors. Methods and Results: We used a proximity ligation assay to investigate the association between TLR4 and HSP70 in SMCs, and fluorescent spots were observed indicating both target proteins within interacting range. Additionally, we exposed SMCs to high glucose (25 mM) in presence or absence of an HSP70 inhibitor (VER155008, 10 -5 M), and we evaluated TLR4 levels, which showed that HSP70 blockade attenuates TLR4 expression (-3.3-fold vs . high glucose). Next, we analyzed a human transcriptomic dataset of T2D donors from the GTEx database, which contains 46 samples from aorta (30 males and 16 females). To reduce the effects of confounders, we matched two covariates (age and race), and we excluded donors with T1D or unknown T1D status. The statsmodels Python package was used to compute the Pearson correlation among the transcripts per million values of RNA for TLR4 and HSP70 family genes [cognate (HSPA8) and inducible (HSPA1A, HSPA1B, HSPA6)] in T2D donors (n=14 samples for each sex). We observed a positive correlation between HSPA6 and TLR4 in males (r = 0.58; p<0.05), but not in females (r = -0.097; p>0.05). On the other hand, the levels of HSPA8 inversely correlate with TLR4 in females (r = -0.55; p<0.05), but not in males (p>0.05). It appears that in males, an increase in the expression of TLR4 induces HSPA6 without affecting the levels of HSPA8 while in females an increase in TLR4 levels does not associate with the inducible genes, but it impairs HSPA8. No association was found in the other analyzed genes. Conclusion: Our results suggest that sex plays a role in the interplay between TLR4 and HSP70 family members in aortic tissue.