Thrombin-induced endothelial microparticle generation: identification of a novel pathway involving ROCK-II activation by caspase-2

Abstract

Thrombin exerts pleiotropic effects on endothelial cells, including the release of microparticles (EMPs) that disseminate and exchange information with vascular cells. Nevertheless, the mechanisms leading to their generation are not elucidated. We performed microarray analysis to identify genes involved in EMP release by the endothelial cell line HMEC-1 in response to thrombin. We identified a group of genes linked to the cytoskeleton reorganization family. Among these, the Rhokinase ROCK-II presented a high transcription rate. ROCK-I, another Rhokinase isoform, was not modulated by thrombin. Pharmacologic inhibition of Rho-kinases or specific depletion of ROCK-II by short interfering (si) RNA inhibited thrombin-induced EMP release. In contrast, ROCK-I mRNA silencing did not modify EMP generation by thrombin. Exposure of HMEC-1 to thrombin in presence of the caspase-2 selective inhibitor Z-VDVAD-FMK prevented ROCK-II cleavage and inhibited the thrombin-induced EMP release. These events were observed in absence of cell death. Our data clearly identified ROCK-II as a target of thrombin in EMP generation. They indicated that the 2 Rho-kinases did not share identical functions. The involvement of caspase-2 in ROCK-II activation independently of cell death points out a novel signaling pathway that emphasizes the proteolytic activity of caspase in EMP generation in response to cell activation. IntroductionThrombin is a serine protease playing a central role in the coagulation cascade and hemostasis. Generated at sites of vascular injury in the vicinity of a thrombus, thrombin promotes platelet activation, adhesion, and trafficking of inflammatory cells into sites of injury. 1 Moreover, thrombin exerts pleiotropic effects on the endothelium controlling the proliferative/reparative responses to injury and promoting both proinflammatory and procoagulant endothelial phenotypes. 2 Thrombin activities are mediated by the activation of the G-protein-linked protease-activated receptors (PARs), mainly PAR-1 on the endothelial cells. 3 Recently, the gene profiling of primary endothelial cells in response to thrombin indicates changes in the transcription of a multitude of genes, 2,4 which results in modifications of the endothelial cell shape, cytoskeleton rearrangement, and increased permeability. 5 Despite the large body of experiments describing the effects of thrombin on the endothelium, little is known on its capacity to generate endothelial cell microparticles 6 and the mechanisms involved in their release have never been reported.Microparticles found in the extracellular space derive from membrane blebs. Blebbing is a reversible dynamic event that occurs during cell activation or apoptosis. 7 Viable cells display plasma membrane blebbing when spreading, migrating, dividing, or under conditions of stress. Blebbing depends on intracellular forces generated by the actin-myosin cytoskeleton and is driven through the activation of actin-myosin contraction 8,9 by the small GTP-binding protein Rho and its major effectors, the ...

Rho kinase in the regulation of cell death and survival

“…The underlying mechanisms for the apoptotic role of ROCK in these studies are not defined, and effects of ROCK inhibitors in other cellular events such as inflammatory cell infiltration and cytokine production may be related to the anti-apoptotic effect of ROCK inhibitors. Consistent with the studies mentioned above, ROCK is involved in the regulation of inflammatory responses, such as production of inflammatory cytokines, inflammatory cell infiltration and vehicle secretions, in a number of experimental models [48,50,117,133], and these inflammatory responses may subsequently promote apoptosis. Finally, ROCK also regulates oxidative stress [49,125,150], which in turn can induce apoptosis.…”

“…On the other hand, during cytotoxic lymphocyte granule-induced cell death, human ROCK2 can be cleaved by the proapoptotic protease granzyme B at IGLD1131 site, and this site is not present in ROCK1 [118]. Human ROCK2, but not ROCK1, can also be activated by caspase 2-dependent cleavage in endothelial cells in response to thrombin, but the cleavage site remains to be identified [117].…”

“…An apoptotic role for other ROCK activation pathway such as Rho/ROCK [59,89], granzyme B/ROCK2 [118], caspase 2/ROCK2 [117] may be highly cell type-dependent and/or apoptotic stimulus-dependent. Importantly, ROCK1 and ROCK2 can be distinctly activated or inhibited by a number of positive or negative regulators, and in turn can have distinct cellular or physiological functions.…”

Endothelial microparticles activate endothelial cells to facilitate the inflammatory response

“…The mechanism of EMP generation requires the transcription of NF-κB (22,23), but it is unknown whether NF-κB is crucial to EMP function. Wang et al (17) demonstrated that MPs from LPS-treated THP-1 monocytic cells activated intracellular signaling pathways in human endothelial cells, including the NF-κB pathway.…”

Effect of 40 mg Versus 10 mg of Atorvastatin on Oxidized Low‐Density Lipoprotein, High‐Sensitivity C‐Reactive Protein, Circulating Endothelial‐Derived Microparticles, and Endothelial Progenitor Cells in Patients With Ischemic Cardiomyopathy

“…Various factors are involved in the mechanisms of EMP generation. 17,18 Tumor necrosis factor-α, lipopolysaccharide, or oxLDL stimulation of cultured human umbilical vein endothelial cells results in an increase in the release of EMP expressing surface tissue factor. 19,20 At present, the increasing levels of cEMPs can reflect endothelium impairment.…”