Protease Activated Receptor 2 (PAR-2) Promotes Vascular Inflammation in a Mouse Model of Sickle Cell Disease

Abstract 375

Sickle cell disease (SCD) is a hematologic disorder caused by a single nucleotide mutation of the beta-globin gene. It is associated with increased tissue factor (TF) expression, activation of coagulation and chronic vascular inflammation. Using two mouse models of SCD (BERK and Townes mice), we have recently demonstrated that inhibition of TF with a rat anti-mouse TF (1H1) antibody not only abolishes activation of coagulation (measured by plasma levels of thrombin anti-thrombin (TAT) complexes) but also reduces inflammation and endothelial cell (EC) injury, indicated by attenuation of plasma levels of IL-6 and sVCAM-1, respectively. Furthermore, we showed that EC-specific deletion of TF gene significantly reduced plasma levels of IL-6 but had no effect on activation of coagulation (TAT) or EC injury (sVCAM-1). These data suggest that EC-TF is primarily involved in signaling rather than activation of coagulation. Since TF:factor VIIa complex-dependent activation of protease activated receptor-2 (PAR-2) has been shown to promote inflammation, we have now investigated the role of PAR-2 expressed by non-hematopoietic cells in the pathology of SCD.

PAR-2^+/+ and PAR-2^−/− mice were lethally irradiated and transplanted with bone marrow from BERK SS (sickle cell mice) or BERK AA (non-sickle control) mice(n=6–10). Four months after bone marrow transplantation, mice were sacrificed and the reconstitution of bone marrow was confirmed by electrophoretic analysis of the different forms of hemoglobin. PAR-2^+/+ mice transplanted with bone marrow from BERK SS mice had reduced number of red blood cells and hematocrit compared to PAR-2^+/+ mice transplanted with bone marrow from BERK AA mice. PAR-2 deficiency in all non-hematopoietic cells had no effect on these hematologic parameters. Furthermore, PAR-2^+/+ mice transplanted with bone marrow from BERK SS mice had increased number of monocytes (3.1 fold, p<0.0001) and neutrophils (2.5 fold, p<0.05) in the blood. Interestingly, sickle cell mice lacking PAR-2 in non-hematopoietic cells had significantly reduced neutrophil counts compared to the sickle cell mice with normal levels of PAR-2 (1.9+/−0.2 vs. 5.4+/−1.4 X10³/ul; p<0.05), whereas monocytes counts were not affected. Compared to non-sickle controls, sickle cell mice had increased plasma levels of TAT (1.9 fold, p<0.01), IL-6 (6.8 fold, P<0.0001), serum amyloid protein SAP (6.5 fold, p<0.01; mouse homolog of human C reactive protein) and sVCAM-1 (1.4 fold, p<0.01). Moreover, increased levels of myeloperoxidase (MPO) were observed in the livers of sickle cell mice (3 fold, p<0.0001). Importantly, sickle cell mice lacking PAR-2 expression in all non-hematopoietic cells demonstrated significant reduction of plasma levels of IL-6 (9.4+/−0.9 vs. 18.9+/−4.5 pg/ml; p<0.05) and SAP (60.5+/−12.9 vs. 182.8+/−62.5ug/ml; p<0.05) compared to sickle cell mice with normal levels of PAR2 expression. In addition, deletion of PAR-2 also significantly reduced MPO levels in the liver of sickle cell mice (53.7+/−3.5 vs. 117.4+/−16.9 ng/mg protein; p<0.0001). In contrast, PAR-2 deficiency in non-hematopoietic cells had no effect on activation of coagulation (TAT) or EC injury (sVCAM-1) in sickle mice.

Our data demonstrate that vascular inflammation observed in a mouse model of sickle cell disease is mediated, in part, by PAR-2 expressed by non-hematopoietic cells. Activation of EC (sVCAM-1) was not affected by PAR-2 deficiency. Ongoing studies are investigating the possible contribution of the TF-thrombin-PAR1 pathway to the EC activation in SCD.