Toll-like receptors 2 and 4 mediate hyperglycemia induced macrovascular aortic endothelial cell inflammation and perturbation of the endothelial glycocalyx
Introduction
Diabetes leads to 2- to 4-fold higher risk for cardiovascular events, and nearly 80% of diabetes-associated deaths are caused by cardiovascular disease (CVD) (Winer & Sowers, 2004). Endothelial dysfunction is a well-established response to cardiovascular risk factors and precedes plaque formation in atherosclerosis (Funk et al., 2012, Hadi et al., 2005).The diabetic state confers an increased propensity to accelerated atherogenesis and inflammation appears to play a pivotal role in diabetes and its complications (Devaraj, Dasu, & Jialal, 2010). Endothelial cell dysfunction is characterized by enhanced permeability, increased cell adhesion molecules, chemokines, cytokines, glycocalyx dysfunction and reduced antithrombotic properties (Funk et al., 2012).
Hyperglycemia is the hallmark of diabetes and considerable data exist linking hyperglycemia to accelerated atherogenesis (Funk et al., 2012). Hyperglycemia can induce endothelial cell dysfunction through multiple pathways including the increased oxidative stress (ROS), polyol pathway, hexosamine pathway, activation of protein kinase-C (PKC), advanced glycation end product (AGE) formation, and increased inflammation (Funk et al., 2012). Inflammation appears to be pivotal in all phases of atherosclerosis from the nascent lesion to plaque rupture (Devaraj et al., 2010, Libby, 2012).
Toll-like receptors (TLRs) are pattern recognition receptors (PRRs) important to the innate immune response and are responsible for activating specific signaling pathways leading to distinct inflammatory responses in the host cells (Akira & Uematsu, 2006).TLRs are increased in diabetic patients and play a significant role in atherosclerosis (Tobias & Curtiss, 2007). An exciting body of data from our group has reported significantly increased expression of TLR2 and TLR4 and their activated downstream signaling cascades in monocytes from patients with type 1 and type 2 diabetes mellitus (T2DM) with further increase in patients with diabetic microvascular disease (Dasu et al., 2010, Devaraj, Jialal, et al., 2011, Devaraj et al., 2008, Devaraj et al., 2006). Following these reports, many groups also reported increased TLR expression and/or activity in abdominal adipose tissue, muscle biopsies and B-cells in diabetes (Jialal & Kaur, 2012). Furthermore in recent years, human studies and gene deletion experiments in murine models provided substantial evidence for the involvement of TLR2 and 4 in atherosclerosis (Bjorkbacka et al., 2004, Edfeldt et al., 2002, Li and Sun, 2007, Liu et al., 2008, Michelsen et al., 2004, Mullick et al., 2008).
There are accumulating data supporting the role of TLR expression and activation of their signaling pathway in hyperglycemia induced cell damage (Dasu et al., 2008, Dasu et al., 2010, Devaraj, Jialal, et al., 2011, Devaraj et al., 2008, Devaraj et al., 2006, Rajamani and Jialal, 2014). However, the role of TLRs in endothelial dysfunction and their contribution toward an increased inflammatory response under hyperglycemic conditions in human macrovascular aortic endothelial cells (HMAEC), a pivotal cell in atherogenesis, have not been explored. Hence, in the present study we evaluated the effect of hyperglycemia on TLR2 and TLR4 activity, on inflammation and on glycocalyx perturbation in HMAECs.
Section snippets
Source and reagents
The details for source and reagents are included in Supplementary Data.
Cell culture
Human macrovascular aortic endothelial cells (HMAECs) were obtained from Lonza (Walkersville, MD) and cells below passage 4 were used for experiments. They were maintained in endotoxin-free endothelial growth medium (Lonza) supplemented with FBS and other growth factors at 37 °C with 5% CO2. The cells, when confluent, were subsequently treated with normal glucose (5.5 mM) and high glucose (HG; 15 mM and 25 mM) for 24 h. During
High glucose induces TLRs expression through both MyD88 and non-MyD88 pathways
As depicted in Fig. 1A–B, a significant increase in TLR2 and TLR4 mRNA expression was observed with both 15 mM and 25 mM glucose treatments compared to 5.5 mM glucose. Consistent with our mRNA data HG increased TLR-2 and TLR-4 protein levels significantly compared to normal glucose (Fig. 1C–D). Since TLR4 signals via both MyD88 and non-MyD88 pathways while TLR-2 signals through the MyD88 pathway only (Akira and Takeda, 2004, Beutler, 2004, Jialal and Kaur, 2012) we further confirmed the downstream
Discussion
Hyperglycemia-induced inflammation is central in diabetes complications and macrovascular endothelial cells are important in orchestrating these effects (Funk et al., 2012, Inoguchi et al., 2000, Taye et al., 2010). Growing evidence reveals loss of the endothelial glycocalyx, a vascular protective layer, during acute hyperglycemia in ED (Alphonsus & Rodseth, 2014). Recent data demonstrate HG induced TLR signaling with increased secretion of downstream biomediators and NF-κB activity in
Conclusion
The current study points toward a role for TLR-2- and TLR-4-induced inflammation in the development of endothelial dysfunction under hyperglycemic conditions. We show upregulation and increased activity of both TLR-2 and TLR-4 with HG in HMAECs, and also confirm using different inhibitors and gene specific si-RNAs, that inhibition of TLR2 and TLR4 in cells attenuated NF-κB activity and TLR mediated increased inflammation, leukocyte adhesion and glycocalyx dysfunction indicating that activation
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Conflicts of interest: None.