Endothelial Cell Phenotype, a Major Determinant of Venous Thrombo-Inflammation

Abstract

Reduced blood flow velocity in the vein triggers inflammation and is associated with the release into the extracellular space of alarmins or damage-associated molecular patterns (DAMPs). These molecules include extracellular nucleic acids, extracellular purinergic nucleotides (ATP, ADP), cytokines and extracellular HMGB1. They are recognized as a danger signal by immune cells, platelets and endothelial cells. Hence, endothelial cells are capable of sensing environmental cues through a wide variety of receptors expressed at the plasma membrane. The endothelium is then responding by expressing pro-coagulant proteins, including tissue factor, and inflammatory molecules such as cytokines and chemokines involved in the recruitment and activation of platelets and leukocytes. This ultimately leads to thrombosis, which is an active pro-inflammatory process, tightly regulated, that needs to be properly resolved to avoid further vascular damages. These mechanisms are often dysregulated, which promote fibrinolysis defects, activation of the immune system and irreversible vascular damages further contributing to thrombotic and inflammatory processes. The concept of thrombo-inflammation is now widely used to describe the complex interactions between the coagulation and inflammation in various cardiovascular diseases. In endothelial cells, activating signals converge to multiple intracellular pathways leading to phenotypical changes turning them into inflammatory-like cells. Accumulating evidence suggest that endothelial to mesenchymal transition (EndMT) may be a major mechanism of endothelial dysfunction induced during inflammation and thrombosis. EndMT is a biological process where endothelial cells lose their endothelial characteristics and acquire mesenchymal markers and functions. Endothelial dysfunction might play a central role in orchestrating and amplifying thrombo-inflammation thought induction of EndMT processes. Mechanisms regulating endothelial dysfunction have been only partially uncovered in the context of thrombotic diseases. In the present review, we focus on the importance of the endothelial phenotype and discuss how endothelial plasticity may regulate the interplay between thrombosis and inflammation. We discuss how the endothelial cells are sensing and responding to environmental cues and contribute to thrombo-inflammation with a particular focus on venous thromboembolism (VTE). A better understanding of the precise mechanisms involved and the specific role of endothelial cells is needed to characterize VTE incidence and address the risk of recurrent VTE and its sequelae.

Keywords: endothelial cell; endothelial plasticity; fibrosis; inflammation; venous thromboembolism.

Figure 1

Hallmarks of venous thromboembolism. Healthy endothelial cells provide an anti-coagulant surface by expressing anti-coagulant factors (TM, TFPI, EPCR and PC) limiting thrombin generation. Intact glycocalyx and production of NO and PGI₂ also protect against venous thrombosis. Thrombo-inflammation is triggered by the release in the bloodstream of DAMPs following cell injury. DAMPs interact with the endothelium and promote the release of cytokines, chemokines and WPB content and expression of adhesion molecules (primary hallmarks). This leads to endothelial dysfunction characterized by platelet and leukocyte recruitment that will in turns become activated and secrete pro-inflammatory and pro-coagulant molecules further contributing to thrombosis (secondary hallmarks). Together with the complement system, platelets and endothelial cells induce NET formation and TF expression in monocytes through interactions between P-selectin and PSGL-1. This initiates the coagulation cascade through both the intrinsic and extrinsic pathways and ultimately leads to thrombin-induced fibrin generation and formation of an obstructive clot (integrative hallmarks). APC, activated protein C; TM, thrombomodulin; TFPI, tissue factor pathway inhibitor; EPCR, endothelial protein C receptor; PC, protein C; NO, nitric oxide; PGI₂, prostaglandin I 2; DAMP, damage-associated molecular pattern; WPB, Weibel Palade body; NET, neutrophil extracellular trap; TF, tissue factor; PSGL-1, P-selectin glycoprotein ligand-1; RAGE, receptor for advanced glycation endproducts; HMGB-1, high mobility group box-1.

Figure 2

DAMP sensing by endothelial cells and signaling pathways induced downstream PRRs in sterile inflammation. In sterile inflammation, damage-associated molecular patterns (DAMPs) can activate pattern recognition receptors (PRRs). These extra- or intracellular DAMPs are recognized by many cell types including endothelial cells. Among PPRs, some are membrane receptor such as TLRs, cytokines receptors, purinergic receptor and some are intracellular receptor including cytoplasmic NLRP3 or reticuloplasmic STING. Cytokines (TGFβ, TNFα, IL-1β) activate via their receptors (TGFβR, TNFα R, IL1βR) many signaling pathways such as NFκB, p38 MAPK and SMAD. These pathways promote pro-inflammatory gene activation including IL-6 and IL-8 but also ICAM-1 and VCAM-1 involved in recruitment and adhesion of immune cells on the endothelium. The high-mobility group box 1 protein (HMGB1) mediates its pro-inflammatory effects through binding to RAGE and activation of MAP kinases and NFκB pathways. The assembly of the NLRP3 inflammasome, which allows the cleavage of the cytokines IL-1β and IL-18 into their active form, is activated by several stimuli. Binding of extracellular ATP to P2X7R results in Ca²⁺ influx and K⁺ efflux. K⁺ efflux can induce NLRP3 inflammasome activation. To counteract pro-inflammatory effects of ATP, the ecto-nucleotidase CD39/CD73 hydrolyzes ATP to adenosine thus preventing its binding to P2XR7. The reactive oxygen species (ROS) production by the mitochondria also contribute to NLRP3 inflammasome assembly. Extracellular DNA derived from cell damage can be recognized by TLR9. Intracellular and mitochondrial DNA activate cGAS signaling in the cytoplasm. Activation of cGAS induces the production of cyclic second messenger GMP—AMP (cGAMP), which binds and activates STING at the endoplasmic reticulum.

Figure 3

Signaling pathways and mechanistic drivers of endothelial-to-mesenchymal transition (EndMT). Stimulation with transforming growth factor-β (TGFβ), cytokines, Notch ligands, high glucose and hypoxia induce expression of transcription factors, such as Twist, Slug, Zeb and Snail resulting in EndMT. Epigenetic mechanisms including modifications of histone methylation marks and methylation of CpG island are induced. These processes are mediated by variety of actors such as microRNAs (miRNAs), long non-coding RNA (GATA6-AS and MALAT1), circularRNA (DLGAP4) and signaling pathways including SMAD, STAT, NFκB, and p38, promoting EndMT.