Protease activated receptor‐1 antagonist ameliorates the clinical symptoms of experimental autoimmune encephalomyelitis via inhibiting breakdown of blood–brain barrier

Abstract

To evaluate the question of whether protease activated receptor-1 (PAR-1) antagonist is a potential therapeutic target in multiple sclerosis, we treated experimental autoimmune encephalomyelitis (EAE) mice with two PAR-1 antagonists, KC-A0590 and SCH-530348. Treatment with both antagonists resulted in a significant decrease in the clinical characteristics of EAE mice by suppressing demyelination and infiltration of inflammatory cells in the spinal cord and brain, as well as a significantly reducing the increased thrombin and tumor necrosis factor-a. Profound leakage of dextran was observed in the brain of EAE mice. However, treatment with PAR-1 antagonists resulted in the stabilization of vascular endothelial cells and reduced blood-brain barrier breakdown with suppression of inflammatory response. Treatment with PAR-1 antagonists also resulted in down-regulated expression of matrix metalloproteinase-9 and preserved expression of occludin and zonula occludens (ZO)-1 in the brain and their significant expression was confirmed in neurons, astrocytes, and vascular endothelial cells. Finally, endothelial cells and primary cultured astrocytes were treated with PAR-1 antagonists; both antagonists suppressed thrombin-induced breakdown of ZO-1 in endothelial cells and secretion of matrix metalloproteinase-9 in astrocytes. Collectively, our results suggest that PAR-1 antagonist is effective in attenuation of the clinical symptoms of EAE mice by stabilizing the blood-brain barrier and may have therapeutic potential for treatment of multiple sclerosis. Keywords: blood-brain barrier, experimental autoimmune encephalomyelitis, KC-A0590, protease activated receptor-1, SCH-530348. Thrombin, a serine protease, accumulates in the brain parenchyma by increased permeability when breakdown of the blood-brain barrier (BBB) occurs in association with neurodegenerative disorders (Arai et al. 2006;Luo et al. 2007;Manaenko et al. 2013). Thrombin signaling is mediated through a small family of G-protein-coupled protease activated receptors (PAR)s, which convert an extracellular proteolytic cleavage event by thrombin into Received January 28, 2015; revised manuscript received July 10, 2015; accepted August 4, 2015.Address correspondence and reprint requests to Dr Byung-Tae Choi, Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan 626-870, Korea. E-mail: choibt@ pusan.ac.krAbbreviations used: ANOVA, multifactorial analysis of variance; BBB, blood-brain barrier; CFA, adjuvant incomplete freund; CNS, central nervous system; DAB, diaminobenzidin; DAPI, 4 0 ,6-diamidino-2-phenylindole; EAE, experimental autoimmune encephalomyelitis; ECM, endothelial cell medium; FITC, fluorescein isothiocynate; GFAP, glial fibrillary acidic protein; H&E, hematoxylin and eosin; IL-1b, interleukin1b; LFB, luxol fast blue solution; MMP, matrix metalloproteinase; MOG, MOG 35-55 peptide; MS, multiple sclerosis; NeuN, neuronal nuclei; PAR, protease-activated receptor; TNF-a, tumor necrosis factor-a; ZO-1, zonul...

Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes

“…We and others have shown that PAR1, the receptor for thrombin, couples through RhoA to mediate proliferation and inflammatory responses in astrocytes [39, 40]. Thrombin is also increased in the injured brain [81, 82], and an antagonist of protease activated receptor 1 (PAR1) reduces clinical symptoms in EAE mice [83]. Thus, the evidence that S1P 3 and other GPCRs that stimulate RhoA can contribute to sustained inflammatory responses suggests this pathway as a critical target for blocking neuroinflammation in MS and other CNS diseases.…”

Targeting the thrombin receptor modulates inflammation and astrogliosis to improve recovery after spinal cord injury

“…Functional PAR1 expression has been demonstrated in neurons (Niclou et al, 1998; Smirnova et al, 2001; Hamill et al, 2009; Han et al, 2011; Yoon et al, 2013), as well as astrocytes (Junge et al, 2004; Hamill et al, 2005; Nicole et al, 2005; Vandell et al, 2008; Scarisbrick et al, 2012a), and hence our interests in further evaluation of its roles in neurological injury and utility as a target for CNS repair. Excessive PAR1 activation has been implicated in ischemia related pathogenesis (Striggow et al, 2001; Junge et al, 2003; Olson et al, 2004; Chen et al, 2012; Zhang et al, 2012; Rajput et al, 2014), in HIV- (Boven et al, 2003), and experimental autoimmune encephalitis (EAE) (Kim et al, 2015), in neurodegenerative processes, including tau hyperphosphorylation and aggregation (Suo et al, 2003b), in retrograde amnesia following minimal traumatic brain injury (Itsekson-Hayosh et al, 2015), and in traumatic injury to the spinal cord (Citron et al, 2000), the focus of the current study.…”

Single-cell profiling reveals GPCR heterogeneity and functional patterning during neuroinflammation

“…Activation of thrombin receptor PAR 1 , encoded by F2r, has been show to increase endothelial permeability and to enhance surface expression of adhesion molecules, thereby promoting platelet and leukocyte margination (25). Furthermore, PAR 1 antagonism was shown to ameliorate EAE (56). Also, orphan receptor latrophilin-2 (encoded by Lphn2) was upregulated in ECsc derived from EAE mice, but in contrast to F2r and Darc, it was rather associated with a less-activated endothelial phenotype.…”