Blockade of the TLR4-MD2 complex lowers blood pressure and improves vascular function in a murine model of type 1 diabetes.

While the pathogenesis of diabetes-induced high blood pressure (BP) is not entirely clear, current evidence suggests that Toll-like receptor 4 (TLR4) is a key player in the mechanisms associated with hypertension. However, it is unknown whether this receptor affects BP under type 1 diabetes. Likewise, there is insufficient knowledge about the role of TLR4 in diabetes-associated vascular dysfunction of large arteries. To narrow these gaps, in this study, we investigated if blockade of the TLR4-MD2 complex impacts BP and vascular function in diabetic rats. We injected streptozotocin in male Sprague Dawley rats and treated them with a neutralizing anti-TLR4 antibody for 14 days. BP was directly measured in conscious animals at the end of the treatment. In another set of experiments, we excised the aorta from control and diabetic animals, and measured TLR4 and MD2-a co-receptor that confers functionality to TLR4-levels by Western blotting. We also performed functional studies and evaluated ROS levels with and without a pharmacological inhibitor for TLR4 as well as for MD2. Additionally, we scrutinized a large human RNA-Seq dataset of aortic tissue to assess the co-expression of TLR4, MD2, and subunits of the vascular NADPH oxidases under diabetes and hypertension. We report that (a) chronic blockade of the TLR4-MD2 complex lowers BP in diabetic animals; that (b) type 1 diabetes modulates the levels of MD2 expression in the aorta, but not TLR4, at least in the conditions evaluated in this study; and, that (c) acute inhibition of TLR4 or MD2 diminishes vascular contractility and reduces oxidative stress in the aorta of these animals. In summary, we show evidence that the TLR4-MD2 complex is involved in the mechanisms linking type 1 diabetes and hypertension.

Unveiling the Interplay between the TLR4/MD2 Complex and HSP70 in the Human Cardiovascular System: A Computational Approach.

While precise mechanisms underlying cardiovascular diseases (CVDs) are still not fully understood, previous studies suggest that the innate immune system, through Toll-like receptor 4 (TLR4), plays a crucial part in the pathways leading to these diseases, mainly because of its interplay with endogenous molecules. The Heat-shock protein 70 family (HSP70-70kDa) is of particular interest in cardiovascular tissues as it may have dual effects when interacting with TLR4 pathways. Although the hypothesis of the HSP70 family members acting as TLR4 ligands is becoming widely accepted, to date no co-crystal structure of this complex is available and it is still unknown whether this process requires the co-adaptor MD2. In this study, we aimed at investigating the interplay between the TLR4/MD2 complex and HSP70 family members in the human cardiovascular system through transcriptomic data analysis and at proposing a putative interaction model between these proteins. We report compelling evidence of correlated expression levels between TLR4 and MD2 with HSP70 cognate family members, especially in heart tissue. In our molecular docking simulations, we found that HSP70 in the ATP-bound state presents a better docking score towards the TLR4/MD2 complex compared to the ADP-bound state (-22.60 vs. -10.29 kcal/mol, respectively). Additionally, we show via a proximity ligation assay for HSP70 and TLR4, that cells stimulated with ATP have higher formation of fluorescent spots and that MD2 might be required for the complexation of these proteins. The insights provided by our computational approach are potential scaffolds for future in vivo studies investigating the interplay between the TLR4/MD2 complex and HSP70 family members in the cardiovascular system.