The thrombin receptor PAR4 supports visceral adipose tissue inflammation.

Thrombin inhibition suppresses adiposity, WAT inflammation and metabolic dysfunction in mice. Protease-activated receptor (PAR)1 does not account for thrombin-driven obesity, so we explored the culprit role of PAR4 in this context. Male WT and PAR-4-/- mice received a high fat diet (HFD) for 8 weeks, WT controls received standard chow. Body fat was quantified by NMR. Epididymal WAT was assessed by histology, immunohistochemistry, qPCR and lipase activity assay. 3T3-L1 preadipocytes were differentiated ± thrombin, acutely stimulated ± PAR4 activating peptide (AP) and assessed by immunoblot, qPCR and U937 monocyte adhesion. Epicardial adipose tissue (EAT) from obese and lean patients was assessed by immunoblot. PAR4 was upregulated in mouse WAT under HFD. PAR4-/- mice developed less visceral adiposity and glucose intolerance under HFD, featuring smaller adipocytes, fewer macrophages and lower expression of adipogenic (leptin, PPARγ) and pro-inflammatory genes (CCL2, IL-1ß) in WAT. HFD-modified activity and expression of lipases or perilipin were unaffected by PAR4 deletion. 3T3-L1 adipocytes differentiated with thrombin retained Ki67 expression, further upregulated IL-1ß and CCL2 and were more adhesive for monocytes. In mature adipocytes, PAR4-AP increased phosphorylated ERK1/2 and AKT, upregulated Ki67, CCl2, IL-ß and hyaluronan synthase 1 but not TNF-α mRNA, and augmented hyaluronidase-sensitive monocyte adhesion. Obese human EAT expressed more PAR4, CD68 and CD54 than lean EAT. PAR4 upregulated in obesity supports adipocyte hypertrophy, WAT expansion and thrombo-inflammation. The emerging PAR4 antagonists provide a therapeutic perspective in this context beyond their canonical antiplatelet action.

Thrombin receptor PAR4 drives canonical NLRP3 inflammasome signaling in the heart.

The deleterious effects of diabetes in the heart are increasingly attributed to inflammatory signaling through the NLRP3 (NOD, LRR and PYD domains-containing protein 3) inflammasome. Thrombin antagonists reduce cardiac remodeling and dysfunction in diabetic mice, in part by suppressing fibrin-driven inflammation. The role of cellular thrombin receptor subtypes in this context is not known. We sought to determine the causal involvement of protease-activated receptors (PAR) in inflammatory signaling of the diabetic heart. Mice with diet-induced diabetes showed increased abundance of pro-caspase-1 and pro-interleukin (IL)-1ß in the left ventricle (LV), indicating transcriptional NLRP3 inflammasome priming, and augmented cleavage of active caspase-1 and IL-1ß, pointing to canonical NLRP3 inflammasome activation. Caspase-11 activation, which mediates non-canonical NLRP3 inflammasome signaling, was not augmented. Formation of the plasma membrane pore-forming protein N-terminal gasdermin D (GDSMD), a prerequisite for IL-1ß secretion, was also higher in diabetic vs. control mouse LV. NLRP3, ASC and IL-18 expression did not differ between the groups, nor did expression of PAR1 or PAR2. PAR3 was nearly undetectable. LV abundance of PAR4 by contrast increased with diabetes and correlated positively with active caspase-1. Genetic deletion of PAR4 in mice prevented the diet-induced cleavage of caspase-1, IL-1ß and GDSMD. Right atrial appendages from patients with type 2 diabetes also showed higher levels of PAR4, but not of PAR1 or PAR2, than non-diabetic atrial tissue, along with increased abundance of cleaved caspase-1, IL-1ß and GSDMD. Human cardiac fibroblasts maintained in high glucose conditions to mimic diabetes also upregulated PAR4 mRNA and protein, and increased PAR4-dependent IL-1ß transcription and secretion in response to thrombin, while PAR1 and PAR2 expressions were unaltered. In conclusion, PAR4 drives caspase-1-dependent IL-1ß production through the canonical NLRP3 inflammasome pathway in the diabetic heart, providing mechanistic insights into diabetes-associated cardiac thromboinflammation. The emerging PAR4-selective antagonists may provide a feasible approach to prevent cardiac inflammation in patients with diabetes.

Evidence for functional PAR-4 thrombin receptor expression in cardiac fibroblasts and its regulation by high glucose: PAR-4 in cardiac fibroblasts.

BACKGROUND: Thrombin promotes cardiac fibroblast proliferation and fibrosis via protease-activated receptor (PAR-1). PAR-4 is reportedly absent in cardiac fibroblasts. In smooth muscle cells, PAR-4 expression is also low but increases upon hyperglycemia and contributes to vascular remodelling in diabetic mice. We examined if PAR-4 is a glucose-responsive gene with remodelling-related functions in cardiac fibroblasts. METHODS AND RESULTS: Cardiac PAR-4 increased in mice with streptozotocin- or diabetogenic diet (DD)-induced diabetes. PAR-4 mRNA and protein were detectable in cardiac fibroblasts from chow-fed mice and increased in high (HG, 25mM) vs. low glucose (LG; 5.5mM) cultures. Conversely PAR-4 mRNA was higher in fibroblasts from DD-fed mice but reduced in LG cultures. Cardiac fibroblasts in HG culture responded more strongly to thrombin or PAR-4 activating peptide in terms of migration (wound-scratch assay), remodelling-associated gene expression (interleukin 6, alpha smooth muscle actin) and oxidative stress (dihydroethidium fluorescence). CONCLUSION: PAR-4 is expressed in mouse cardiac fibroblasts and is dynamically regulated by extracellular glucose in vitro and diabetes in vivo, thereby impacting on fibroblast functions relevant for cardiac remodelling. These findings add further evidence for the usefulness of the recently developed PAR-4 antagonists in clinical settings.